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Title: The 2.8 Angstrom Electron Microscopy Structure Of Adeno-associated Virus-dj Bound By A Heparinoid Pentasaccharide.
Creator: Xie, Qing, Spear, John M., Noble, Alex J., Sousa, Duncan R., Meyer, Nancy L., Davulcu, Omar, Zhang, Fuming, Linhardt, Robert J., Stagg, Scott M., Chapman, Michael S.
Abstract/Description: Atomic structures of adeno-associated virus (AAV)-DJ, alone and in complex with fondaparinux, have been determined by cryoelectron microscopy at 3 angstrom resolution. The gene therapy vector, AAV-DJ, is a hybrid of natural serotypes that was previously derived by directed evolution, selecting for hepatocyte entry and resistance to neutralization by human serum. The structure of AAV-DJ differs from that of parental serotypes in two regions where neutralizing antibodies bind, so immune escape...
Show moreAtomic structures of adeno-associated virus (AAV)-DJ, alone and in complex with fondaparinux, have been determined by cryoelectron microscopy at 3 angstrom resolution. The gene therapy vector, AAV-DJ, is a hybrid of natural serotypes that was previously derived by directed evolution, selecting for hepatocyte entry and resistance to neutralization by human serum. The structure of AAV-DJ differs from that of parental serotypes in two regions where neutralizing antibodies bind, so immune escape appears to have been the primary driver of AAV-DJ's directed evolution. Fondaparinux is an analog of cell surface heparan sulfate to which several AAVs bind during entry. Fondaparinux interacts with viral arginines at a known heparin binding site, without the large conformational changes whose presence was controversial in low-resolution imaging of AAV2-heparin complexes. The glycan density suggests multimodal binding that could accommodate sequence variation and multivalent binding along a glycan polymer, consistent with a role in attachment, prior to more specific interactions with a receptor protein mediating entry.
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Date Issued: 2017-06
Identifier: FSU_libsubv1_wos_000406299600001, 10.1016/j.omtm.2017.02.004
Format: Citation

Title: The 2.8 Å Electron Microscopy Structure of Adeno-Associated Virus-DJ Bound by a Heparinoid Pentasaccharide.
Creator: Xie, Qing, Spear, John M, Noble, Alex J, Sousa, Duncan R, Meyer, Nancy L, Davulcu, Omar, Zhang, Fuming, Linhardt, Robert J, Stagg, Scott M, Chapman, Michael S
Abstract/Description: Atomic structures of adeno-associated virus (AAV)-DJ, alone and in complex with fondaparinux, have been determined by cryoelectron microscopy at 3 Å resolution. The gene therapy vector, AAV-DJ, is a hybrid of natural serotypes that was previously derived by directed evolution, selecting for hepatocyte entry and resistance to neutralization by human serum. The structure of AAV-DJ differs from that of parental serotypes in two regions where neutralizing antibodies bind, so immune escape appears...
Show moreAtomic structures of adeno-associated virus (AAV)-DJ, alone and in complex with fondaparinux, have been determined by cryoelectron microscopy at 3 Å resolution. The gene therapy vector, AAV-DJ, is a hybrid of natural serotypes that was previously derived by directed evolution, selecting for hepatocyte entry and resistance to neutralization by human serum. The structure of AAV-DJ differs from that of parental serotypes in two regions where neutralizing antibodies bind, so immune escape appears to have been the primary driver of AAV-DJ's directed evolution. Fondaparinux is an analog of cell surface heparan sulfate to which several AAVs bind during entry. Fondaparinux interacts with viral arginines at a known heparin binding site, without the large conformational changes whose presence was controversial in low-resolution imaging of AAV2-heparin complexes. The glycan density suggests multi-modal binding that could accommodate sequence variation and multivalent binding along a glycan polymer, consistent with a role in attachment, prior to more specific interactions with a receptor protein mediating entry.
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Date Issued: 2017-03-08
Identifier: FSU_pmch_28480299, 10.1016/j.omtm.2017.02.004, PMC5415311, 28480299, 28480299, S2329-0501(17)30044-X
Format: Citation

Title: Advancing NMDA Receptor Physiology by Integrating Multiple Approaches.
Creator: Zhou, Huan-Xiang, Wollmuth, Lonnie P
Abstract/Description: NMDA receptors (NMDARs) are ion channels activated by the excitatory neurotransmitter glutamate and are essential to all aspects of brain function, including learning and memory formation. Missense mutations distributed throughout NMDAR subunits have been associated with an array of neurological disorders. Recent structural, functional, and computational studies have generated many insights into the activation process connecting glutamate binding to ion-channel opening, which is central to...
Show moreNMDA receptors (NMDARs) are ion channels activated by the excitatory neurotransmitter glutamate and are essential to all aspects of brain function, including learning and memory formation. Missense mutations distributed throughout NMDAR subunits have been associated with an array of neurological disorders. Recent structural, functional, and computational studies have generated many insights into the activation process connecting glutamate binding to ion-channel opening, which is central to NMDAR physiology and pathophysiology. The field appears poised for breakthroughs, including the exciting prospect of resolving the conformations and energetics of elementary steps in the activation process, and atomic-level modeling of the effects of missense mutations on receptor function. The most promising strategy going forward is through strong integration of multiple approaches.
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Date Issued: 2017-03-01
Identifier: FSU_pmch_28187950, 10.1016/j.tins.2017.01.001, PMC5339030, 28187950, 28187950, S0166-2236(17)30002-4
Format: Citation

Title: AIM2 inflammasome activation and regulation: A structural perspective..
Creator: Wang, Bing, Yin, Qian
Abstract/Description: Absent in melanoma 2 (AIM2) inflammasome is a multi-protein platform that recognizes aberrant cytoplasmic dsDNA and induces cytokine maturation, release and pyroptosis. It is composed of AIM2, apoptosis-associated speck-like protein containing a CARD (ASC), and caspase-1. Recent X-ray crystallographic and high resolution cryo-electron microscopic (cryo-EM) studies have revealed a series of structures in AIM2 inflammasome activation and regulation. One prominent feature common in multiple...
Show moreAbsent in melanoma 2 (AIM2) inflammasome is a multi-protein platform that recognizes aberrant cytoplasmic dsDNA and induces cytokine maturation, release and pyroptosis. It is composed of AIM2, apoptosis-associated speck-like protein containing a CARD (ASC), and caspase-1. Recent X-ray crystallographic and high resolution cryo-electron microscopic (cryo-EM) studies have revealed a series of structures in AIM2 inflammasome activation and regulation. One prominent feature common in multiple steps is the assembly of high-order structures, especially helical filaments nucleated by upstream molecules, rather than stoichiometric complexes. In this review, we track the AIM2 inflammasome activation process step by step, using high-resolution structures to illustrate the overall architecture of AIM2 inflammasome and its assembly and regulatory mechanisms.
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Date Issued: 2017-12-01
Identifier: FSU_pmch_28813641, 10.1016/j.jsb.2017.08.001, PMC5733693, 28813641, 28813641, S1047-8477(17)30132-6
Format: Citation

Title: Allosteric activation of SENP1 by SUMO1 β-grasp domain involves a dock-and-coalesce mechanism.
Creator: Guo, Jingjing, Zhou, Huan-Xiang
Abstract/Description: Small ubiquitin-related modifiers (SUMOs) are conjugated to proteins to regulate a variety of cellular processes. SENPs are cysteine proteases with a catalytic center located within a channel between two subdomains that catalyzes SUMO C-terminal cleavage for processing of SUMO precursors and de-SUMOylation of target proteins. The β-grasp domain of SUMOs binds to an exosite cleft, and allosterically activates SENPs via an unknown mechanism. Our molecular dynamics simulations showed that...
Show moreSmall ubiquitin-related modifiers (SUMOs) are conjugated to proteins to regulate a variety of cellular processes. SENPs are cysteine proteases with a catalytic center located within a channel between two subdomains that catalyzes SUMO C-terminal cleavage for processing of SUMO precursors and de-SUMOylation of target proteins. The β-grasp domain of SUMOs binds to an exosite cleft, and allosterically activates SENPs via an unknown mechanism. Our molecular dynamics simulations showed that binding of the β-grasp domain induces significant conformational and dynamic changes in SENP1, including widening of the exosite cleft and quenching of nanosecond dynamics in all but a distal region. A dock-and-coalesce mechanism emerges for SENP-catalyzed SUMO cleavage: the wedging of the β-grasp domain enables the docking of the proximal portion of the C-terminus and the strengthened cross-channel motional coupling initiates inter-subdomain correlated motions to allow for the distal portion to coalesce around the catalytic center.
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Date Issued: 2016-08-31
Identifier: FSU_pmch_27576863, 10.7554/eLife.18249, PMC5030089, 27576863, 27576863
Format: Citation

Title: Awake, long-term intranasal insulin treatment does not affect object memory, odor discrimination, or reversal learning in mice.
Creator: Bell, Genevieve A, Fadool, Debra Ann
Abstract/Description: Intranasal insulin delivery is currently being used in clinical trials to test for improvement in human memory and cognition, and in particular, for lessening memory loss attributed to neurodegenerative diseases. Studies have reported the effects of short-term intranasal insulin treatment on various behaviors, but less have examined long-term effects. The olfactory bulb contains the highest density of insulin receptors in conjunction with the highest level of insulin transport within the...
Show moreIntranasal insulin delivery is currently being used in clinical trials to test for improvement in human memory and cognition, and in particular, for lessening memory loss attributed to neurodegenerative diseases. Studies have reported the effects of short-term intranasal insulin treatment on various behaviors, but less have examined long-term effects. The olfactory bulb contains the highest density of insulin receptors in conjunction with the highest level of insulin transport within the brain. Previous research from our laboratory has demonstrated that acute insulin intranasal delivery (IND) enhanced both short- and long-term memory as well as increased two-odor discrimination in a two-choice paradigm. Herein, we investigated the behavioral and physiological effects of chronic insulin IND. Adult, male C57BL6/J mice were intranasally treated with 5μg/μl of insulin twice daily for 30 and 60days. Metabolic assessment indicated no change in body weight, caloric intake, or energy expenditure following chronic insulin IND, but an increase in the frequency of meal bouts selectively in the dark cycle. Unlike acute insulin IND, which has been shown to cause enhanced performance in odor habituation/dishabituation and two-odor discrimination tasks in mice, chronic insulin IND did not enhance olfactometry-based odorant discrimination or olfactory reversal learning. In an object memory recognition task, insulin IND-treated mice did not perform differently than controls, regardless of task duration. Biochemical analyses of the olfactory bulb revealed a modest 1.3 fold increase in IR kinase phosphorylation but no significant increase in Kv1.3 phosphorylation. Substrate phosphorylation of IR kinase downstream effectors (MAPK/ERK and Akt signaling) proved to be highly variable. These data indicate that chronic administration of insulin IND in mice fails to enhance olfactory ability, object memory recognition, or a majority of systems physiology metabolic factors - as reported to elicit a modulatory effect with acute administration. This leads to two alternative interpretations regarding long-term insulin IND in mice: 1) It causes an initial stage of insulin resistance to dampen the behaviors that would normally be modulated under acute insulin IND, but ability to clear a glucose challenge is still retained, or 2) There is a lack of behavioral modulation at high concentration of insulin attributed to the twice daily intervals of hyperinsulinemia caused by insulin IND administration without any insulin resistance, per se.
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Date Issued: 2017-05-15
Identifier: FSU_pmch_28259806, 10.1016/j.physbeh.2017.02.044, PMC5639911, 28259806, 28259806, S0031-9384(16)30820-4
Format: Citation

Title: Ca2+ Effects on ATP Production and Consumption Have Regulatory Roles on Oscillatory Islet Activity.
Creator: McKenna, Joseph P, Ha, Joon, Merrins, Matthew J, Satin, Leslie S, Sherman, Arthur, Bertram, Richard
Abstract/Description: Pancreatic islets respond to elevated blood glucose by secreting pulses of insulin that parallel oscillations in β-cell metabolism, intracellular Ca(2+) concentration, and bursting electrical activity. The mechanisms that maintain an oscillatory response are not fully understood, yet several models have been proposed. Only some can account for experiments supporting that metabolism is intrinsically oscillatory in β-cells. The dual oscillator model (DOM) implicates glycolysis as the source of...
Show morePancreatic islets respond to elevated blood glucose by secreting pulses of insulin that parallel oscillations in β-cell metabolism, intracellular Ca(2+) concentration, and bursting electrical activity. The mechanisms that maintain an oscillatory response are not fully understood, yet several models have been proposed. Only some can account for experiments supporting that metabolism is intrinsically oscillatory in β-cells. The dual oscillator model (DOM) implicates glycolysis as the source of oscillatory metabolism. In the companion article, we use recently developed biosensors to confirm that glycolysis is oscillatory and further elucidate the coordination of metabolic and electrical signals in the insulin secretory pathway. In this report, we modify the DOM by incorporating an established link between metabolism and intracellular Ca(2+) to reconcile model predictions with experimental observations from the companion article. With modification, we maintain the distinguishing feature of the DOM, oscillatory glycolysis, but introduce the ability of Ca(2+) influx to reshape glycolytic oscillations by promoting glycolytic efflux. We use the modified model to explain measurements from the companion article and from previously published experiments with islets.
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Date Issued: 2016-02-02
Identifier: FSU_pmch_26840737, 10.1016/j.bpj.2015.11.3526, PMC4744176, 26840737, 26840737, S0006-3495(15)04814-6
Format: Citation

Title: Cas6 processes tight and relaxed repeat RNA via multiple mechanisms: A hypothesis..
Creator: Sefcikova, Jana, Roth, Mitchell, Yu, Ge, Li, Hong
Abstract/Description: RNA molecules are flexible yet foldable. Proteins must cope with this structural duality when forming biologically active complexes with RNA. Recent studies of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs)-mediated RNA immunity illustrate some remarkable mechanisms with which proteins interact with RNA. Currently known structures of CRISPR-Cas6 endoribonucleases bound with RNA suggest a conserved protein recognition mechanism mediated by RNA stem-loops. However, a...
Show moreRNA molecules are flexible yet foldable. Proteins must cope with this structural duality when forming biologically active complexes with RNA. Recent studies of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs)-mediated RNA immunity illustrate some remarkable mechanisms with which proteins interact with RNA. Currently known structures of CRISPR-Cas6 endoribonucleases bound with RNA suggest a conserved protein recognition mechanism mediated by RNA stem-loops. However, a survey of CRISPR RNA reveals that many repeats either lack a productive stem-loop (Relaxed) or possess stable but inhibitory structures (Tight), which raises the question of how the enzyme processes structurally diverse RNA. In reviewing recent literature, we propose a bivalent trapping and an unwinding mechanism for CRISPR-Cas6 to interact with the Relaxed and the Tight repeat RNA, respectively. Both mechanisms aim to create an identical RNA conformation at the cleavage site for accurate processing.
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Date Issued: 2017-06-01
Identifier: FSU_pmch_28493337, 10.1002/bies.201700019, PMC5699886, 28493337, 28493337
Format: Citation

Title: Challenges in structural approaches to cell modeling.
Creator: Im, Wonpil, Liang, Jie, Olson, Arthur, Zhou, Huan-Xiang, Vajda, Sandor, Vakser, Ilya A
Abstract/Description: Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales. Adequate understanding of biomolecular mechanisms inherently involves our ability to model them. Structural modeling of individual biomolecules and their interactions has been rapidly progressing. However, in terms of the broader picture, the focus is shifting toward larger systems, up to the level of a cell. Such modeling involves a more dynamic and realistic...
Show moreComputational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales. Adequate understanding of biomolecular mechanisms inherently involves our ability to model them. Structural modeling of individual biomolecules and their interactions has been rapidly progressing. However, in terms of the broader picture, the focus is shifting toward larger systems, up to the level of a cell. Such modeling involves a more dynamic and realistic representation of the interactomes in vivo, in a crowded cellular environment, as well as membranes and membrane proteins, and other cellular components. Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations, graph models, and other techniques to model biological networks, imaging data, etc. Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine. A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology. Studies in several related areas are covered: biological networks; automated construction of three-dimensional cell models using experimental data; modeling of protein complexes; prediction of non-specific and transient protein interactions; thermodynamic and kinetic effects of crowding; cellular membrane modeling; and modeling of chromosomes. The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology, and the prospects of future developments in this emerging field.
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Date Issued: 2016-07-31
Identifier: FSU_pmch_27255863, 10.1016/j.jmb.2016.05.024, PMC4976022, 27255863, 27255863, S0022-2836(16)30193-0
Format: Citation

Title: Comparative exploration of hydrogen sulfide and water transmembrane free energy surfaces via orthogonal space tempering free energy sampling.
Creator: Lv, Chao, Aitchison, Erick W, Wu, Dongsheng, Zheng, Lianqing, Cheng, Xiaolin, Yang, Wei
Abstract/Description: Hydrogen sulfide (H2 S), a commonly known toxic gas compound, possesses unique chemical features that allow this small solute molecule to quickly diffuse through cell membranes. Taking advantage of the recent orthogonal space tempering (OST) method, we comparatively mapped the transmembrane free energy landscapes of H2 S and its structural analogue, water (H2 O), seeking to decipher the molecular determinants that govern their drastically different permeabilities. As revealed by our OST...
Show moreHydrogen sulfide (H2 S), a commonly known toxic gas compound, possesses unique chemical features that allow this small solute molecule to quickly diffuse through cell membranes. Taking advantage of the recent orthogonal space tempering (OST) method, we comparatively mapped the transmembrane free energy landscapes of H2 S and its structural analogue, water (H2 O), seeking to decipher the molecular determinants that govern their drastically different permeabilities. As revealed by our OST sampling results, in contrast to the highly polar water solute, hydrogen sulfide is evidently amphipathic, and thus inside membrane is favorably localized at the interfacial region, that is, the interface between the polar head-group and nonpolar acyl chain regions. Because the membrane binding affinity of H2 S is mainly governed by its small hydrophobic moiety and the barrier height inbetween the interfacial region and the membrane center is largely determined by its moderate polarity, the transmembrane free energy barriers to encounter by this toxic molecule are very small. Moreover when H2 S diffuses from the bulk solution to the membrane center, the above two effects nearly cancel each other, so as to lead to a negligible free energy difference. This study not only explains why H2 S can quickly pass through cell membranes but also provides a practical illustration on how to use the OST free energy sampling method to conveniently analyze complex molecular processes. © 2015 Wiley Periodicals, Inc.
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Date Issued: 2016-03-05
Identifier: FSU_pmch_26119423, 10.1002/jcc.23982, PMC4959446, 26119423, 26119423
Format: Citation

Title: A Conserved Glycine Harboring Disease-associated Mutations Permits Nmda Receptor Slow Deactivation And High Ca2+ Permeability.
Creator: Amin, Johansen B., Leng, Xiaoling, Gochman, Aaron, Zhou, Huan-Xiang, Wollmuth, Lonnie P.
Abstract/Description: A variety of de novo and inherited missense mutations associated with neurological disorders are found in the NMDA receptor M4 transmembrane helices, which are peripheral to the pore domain in eukaryotic ionotropic glutamate receptors. Subsets of these mutations affect receptor gating with dramatic effects, including in one instance halting it, occurring at a conserved glycine near the extracellular end of M4. Functional experiments and molecular dynamic simulations of constructs with and...
Show moreA variety of de novo and inherited missense mutations associated with neurological disorders are found in the NMDA receptor M4 transmembrane helices, which are peripheral to the pore domain in eukaryotic ionotropic glutamate receptors. Subsets of these mutations affect receptor gating with dramatic effects, including in one instance halting it, occurring at a conserved glycine near the extracellular end of M4. Functional experiments and molecular dynamic simulations of constructs with and without substitutions at this glycine indicate that it acts as a hinge, permitting the intracellular portion of the ion channel to laterally expand. This expansion stabilizes long-lived open states leading to slow deactivation and high Ca2+ permeability. Our studies provide a functional and structural framework for the effect of missense mutations on NMDARs at central synapses and highlight how the M4 segment may represent a pathway for intracellular modulation of NMDA receptor function.
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Date Issued: 2018-09-14
Identifier: FSU_libsubv1_wos_000444554800005, 10.1038/s41467-018-06145-w
Format: Citation

Title: The Dead-box Protein Rok1 Orchestrates 40s And 60s Ribosome Assembly By Promoting The Release Of Rrp5 From Pre-40s Ribosomes To Allow For 60s Maturation.
Creator: Khoshnevis, Sohail, Askenasy, Isabel, Johnson, Matthew C., Dattolo, Maria D., Young-Erdos, Crystal L., Stroupe, M. Elizabeth, Karbstein, Katrin
Abstract/Description: DEAD-box proteins are ubiquitous regulators of RNA biology. While commonly dubbed "helicases," their activities also include duplex annealing, adenosine triphosphate (ATP)-dependent RNA binding, and RNA-protein complex remodeling. Rok1, an essential DEAD-box protein, and its cofactor Rrp5 are required for ribosome assembly. Here, we use in vivo and in vitro biochemical analyses to demonstrate that ATP-bound Rok1, but not adenosine diphosphate (ADP)-bound Rok1, stabilizes Rrp5 binding to 40S...
Show moreDEAD-box proteins are ubiquitous regulators of RNA biology. While commonly dubbed "helicases," their activities also include duplex annealing, adenosine triphosphate (ATP)-dependent RNA binding, and RNA-protein complex remodeling. Rok1, an essential DEAD-box protein, and its cofactor Rrp5 are required for ribosome assembly. Here, we use in vivo and in vitro biochemical analyses to demonstrate that ATP-bound Rok1, but not adenosine diphosphate (ADP)-bound Rok1, stabilizes Rrp5 binding to 40S ribosomes. Interconversion between these two forms by ATP hydrolysis is required for release of Rrp5 from pre-40S ribosomes in vivo, thereby allowing Rrp5 to carry out its role in 60S subunit assembly. Furthermore, our data also strongly suggest that the previously described accumulation of snR30 upon Rok1 inactivation arises because Rrp5 release is blocked and implicate a previously undescribed interaction between Rrp5 and the DEAD-box protein Has1 in mediating snR30 accumulation when Rrp5 release from pre-40S subunits is blocked.
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Date Issued: 2016-06
Identifier: FSU_libsubv1_wos_000378611200007, 10.1371/journal.pbio.1002480
Format: Citation

Title: Differential serotonergic modulation across the main and accessory olfactory bulbs.
Creator: Huang, Zhenbo, Thiebaud, Nicolas, Fadool, Debra Ann
Abstract/Description: There are serotonergic projections to both the main (MOB) and the accessory olfactory bulb (AOB). Current-clamp experiments demonstrate that serotonergic afferents are largely excitatory for mitral cells (MCs) in the MOB where 5-HT receptors mediate a direct excitatory action. Serotonergic afferents are predominately inhibitory for MCs in the AOB. There are two types of inhibition: indirect inhibition mediated through the 5-HT receptors on GABAergic interneurons and direct inhibition via the...
Show moreThere are serotonergic projections to both the main (MOB) and the accessory olfactory bulb (AOB). Current-clamp experiments demonstrate that serotonergic afferents are largely excitatory for mitral cells (MCs) in the MOB where 5-HT receptors mediate a direct excitatory action. Serotonergic afferents are predominately inhibitory for MCs in the AOB. There are two types of inhibition: indirect inhibition mediated through the 5-HT receptors on GABAergic interneurons and direct inhibition via the 5-HT receptors on MCs. Differential 5-HT neuromodulation of MCs across the MOB and AOB could contribute to select behaviours such as olfactory learning or aggression. Mitral cells (MCs) contained in the main (MOB) and accessory (AOB) olfactory bulb have distinct intrinsic membrane properties but the extent of neuromodulation across the two systems has not been widely explored. Herein, we investigated a widely distributed CNS modulator, serotonin (5-HT), for its ability to modulate the biophysical properties of MCs across the MOB and AOB, using an in vitro, brain slice approach in postnatal 15-30 day mice. In the MOB, 5-HT elicited three types of responses in 93% of 180 cells tested. Cells were either directly excited (70%), inhibited (10%) or showed a mixed response (13%)- first inhibition followed by excitation. In the AOB, 82% of 148 cells were inhibited with 18% of cells showing no response. Albeit located in parallel partitions of the olfactory system, 5-HT largely elicited MC excitation in the MOB while it evoked two different kinetic rates of MC inhibition in the AOB. Using a combination of pharmacological agents, we found that the MC excitatory responses in the MOB were mediated by 5-HT receptors through a direct activation. In comparison, 5-HT-evoked inhibitory responses in the AOB arose due to a polysynaptic, slow-onset inhibition attributed to 5-HT receptor activation exciting GABAergic interneurons. The second type of inhibition had a rapid onset as a result of direct inhibition mediated by the 5-HT class of receptors. The distinct serotonergic modulation of MCs between the MOB and AOB could provide a molecular basis for differential chemosensory behaviours driven by the brainstem raphe nuclei into these parallel systems.
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Date Issued: 2017-06-01
Identifier: FSU_pmch_28229459, 10.1113/JP273945, PMC5451723, 28229459, 28229459
Format: Citation

Title: Divergent roles of a peripheral transmembrane segment in AMPA and NMDA receptors.
Creator: Amin, Johansen B, Salussolia, Catherine L, Chan, Kelvin, Regan, Michael C, Dai, Jian, Zhou, Huan-Xiang, Furukawa, Hiro, Bowen, Mark E, Wollmuth, Lonnie P
Abstract/Description: Ionotropic glutamate receptors (iGluRs), including AMPA receptor (AMPAR) and NMDA receptor (NMDAR) subtypes, are ligand-gated ion channels that mediate signaling at the majority of excitatory synapses in the nervous system. The iGluR pore domain is structurally and evolutionarily related to an inverted two-transmembrane K channel. Peripheral to the pore domain in eukaryotic iGluRs is an additional transmembrane helix, the M4 segment, which interacts with the pore domain of a neighboring...
Show moreIonotropic glutamate receptors (iGluRs), including AMPA receptor (AMPAR) and NMDA receptor (NMDAR) subtypes, are ligand-gated ion channels that mediate signaling at the majority of excitatory synapses in the nervous system. The iGluR pore domain is structurally and evolutionarily related to an inverted two-transmembrane K channel. Peripheral to the pore domain in eukaryotic iGluRs is an additional transmembrane helix, the M4 segment, which interacts with the pore domain of a neighboring subunit. In AMPARs, the integrity of the alignment of a specific face of M4 with the adjacent pore domain is essential for receptor oligomerization. In contrast to AMPARs, NMDARs are obligate heterotetramers composed of two GluN1 and typically two GluN2 subunits. Here, to address the function of the M4 segments in NMDARs, we carry out a tryptophan scan of M4 in GluN1 and GluN2A subunits. Unlike AMPARs, the M4 segments in NMDAR subunits makes only a limited contribution to their biogenesis. However, the M4 segments in both NMDAR subunits are critical for receptor activation, with mutations at some positions, most notably at the extreme extracellular end, completely halting the gating process. Furthermore, although the AMPAR M4 makes a minimal contribution to receptor desensitization, the NMDAR M4 segments have robust and subunit-specific effects on desensitization. These findings reveal that the functional roles of the M4 segments in AMPARs and NMDARs have diverged in the course of their evolution and that the M4 segments in NMDARs may act as a transduction pathway for receptor modulation at synapses.
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Date Issued: 2017-06-05
Identifier: FSU_pmch_28507080, 10.1085/jgp.201711762, PMC5460951, 28507080, 28507080, jgp.201711762
Format: Citation

Title: The dock-and-coalesce mechanism for the association of a WASP disordered region with the Cdc42 GTPase.
Creator: Ou, Li, Matthews, Megan, Pang, Xiaodong, Zhou, Huan-Xiang
Abstract/Description: Intrinsically disordered proteins (IDPs) play key roles in signaling and regulation. Many IDPs undergo folding upon binding to their targets. We have proposed that coupled folding and binding of IDPs generally follow a dock-and-coalesce mechanism, whereby a segment of the IDP, through diffusion, docks to its cognate subsite and, subsequently, the remaining segments coalesce around their subsites. Here, by a combination of experiment and computation, we determined the precise form of dock-and...
Show moreIntrinsically disordered proteins (IDPs) play key roles in signaling and regulation. Many IDPs undergo folding upon binding to their targets. We have proposed that coupled folding and binding of IDPs generally follow a dock-and-coalesce mechanism, whereby a segment of the IDP, through diffusion, docks to its cognate subsite and, subsequently, the remaining segments coalesce around their subsites. Here, by a combination of experiment and computation, we determined the precise form of dock-and-coalesce operating in the association between the intrinsically disordered GTPase-binding domain (GBD) of the Wiskott-Aldrich Syndrome protein and the Cdc42 GTPase. The association rate constants (k ) were measured by stopped-flow fluorescence under various solvent conditions. k reached 10 m ·s at physiological ionic strength and had a strong salt dependence, suggesting that an electrostatically enhanced, diffusion-controlled docking step may be rate limiting. Our computation, based on the transient-complex theory, identified the N-terminal basic region of the GBD as the docking segment. However, several other changes in solvent conditions provided strong evidence that the coalescing step also contributed to determining the magnitude of k . Addition of glucose and trifluoroethanol and an increase in temperature all produced experimental k values much higher than expected from the effects on the docking rate alone. Conversely, addition of urea led to k values much lower than expected if only the docking rate was affected. These results all pointed to k being approximately two-thirds of the docking rate constant under physiological solvent conditions.
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Date Issued: 2017-10-01
Identifier: FSU_pmch_28805312, 10.1111/febs.14197, PMC5643242, 28805312, 28805312
Format: Citation

Title: Dual Detection System for Simultaneous Measurement of Intracellular Fluorescent Markers and Cellular Secretion.
Creator: Yi, Lian, Bandak, Basel, Wang, Xue, Bertram, Richard, Roper, Michael G
Abstract/Description: Glucose-stimulated insulin secretion from pancreatic β-cells within islets of Langerhans plays a critical role in maintaining glucose homeostasis. Although this process is essential for maintaining euglycemia, the underlying intracellular mechanisms that control it are still unclear. To allow simultaneous correlation between intracellular signal transduction events and extracellular secretion, an analytical system was developed that integrates fluorescence imaging of intracellular probes with...
Show moreGlucose-stimulated insulin secretion from pancreatic β-cells within islets of Langerhans plays a critical role in maintaining glucose homeostasis. Although this process is essential for maintaining euglycemia, the underlying intracellular mechanisms that control it are still unclear. To allow simultaneous correlation between intracellular signal transduction events and extracellular secretion, an analytical system was developed that integrates fluorescence imaging of intracellular probes with high-speed automated insulin immunoassays. As a demonstration of the system, intracellular [Ca] ([Ca]) was measured by imaging Fura-2 fluorescence simultaneously with insulin secretion from islets exposed to elevated glucose levels. Both [Ca] and insulin were oscillatory during application of 10 mM glucose with temporal and quantitative profiles similar to what has been observed elsewhere. In previous work, sinusoidal glucose levels have been used to test the entrainment of islets while monitoring either [Ca] or insulin levels; using this newly developed system, we show unambiguously that oscillations of both [Ca] and insulin release are entrained to oscillatory glucose levels and that the temporal correlation of these are maintained throughout the experiment. It is expected that the developed analytical system can be expanded to investigate a number of other intracellular messengers in islets or other stimulus-secretion pathways in different cells.
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Date Issued: 2016-11-01
Identifier: FSU_pmch_27712062, 10.1021/acs.analchem.6b02404, PMC5089909, 27712062, 27712062
Format: Citation

Title: Electrostatic effects on the folding stability of FKBP12.
Creator: Batra, Jyotica, Tjong, Harianto, Zhou, Huan-Xiang
Abstract/Description: The roles of electrostatic interactions in protein folding stability have been a matter of debate, largely due to the complexity in the theoretical treatment of these interactions. We have developed computational methods for calculating electrostatic effects on protein folding stability. To rigorously test and further refine these methods, here we carried out experimental studies into electrostatic effects on the folding stability of the human 12-kD FK506 binding protein (FKBP12). This...
Show moreThe roles of electrostatic interactions in protein folding stability have been a matter of debate, largely due to the complexity in the theoretical treatment of these interactions. We have developed computational methods for calculating electrostatic effects on protein folding stability. To rigorously test and further refine these methods, here we carried out experimental studies into electrostatic effects on the folding stability of the human 12-kD FK506 binding protein (FKBP12). This protein has a close homologue, FKBP12.6, with amino acid substitutions in only 18 of their 107 residues. Of the 18 substitutions, 8 involve charged residues. Upon mutating FKBP12 residues at these 8 positions individually into the counterparts in FKBP12.6, the unfolding free energy (ΔGu) of FKBP12 changed by -0.3 to 0.7 kcal/mol. Accumulating stabilizing substitutions resulted in a mutant with a 0.9 kcal/mol increase in stability. Additional charge mutations were grafted from a thermophilic homologue, MtFKBP17, which aligns to FKBP12 with 31% sequence identity over 89 positions. Eleven such charge mutations were studied, with ΔΔGu varying from -2.9 to 0.1 kcal/mol. The predicted electrostatic effects by our computational methods with refinements herein had a root-mean-square deviation of 0.9 kcal/mol from the experimental ΔΔGu values on 16 single mutations of FKBP12. The difference in ΔΔGu between mutations grafted from FKBP12.6 and those from MtFKBP17 suggests that more distant homologues are less able to provide guidance for enhancing folding stability.
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Date Issued: 2016-08-01
Identifier: FSU_pmch_27381026, 10.1093/protein/gzw014, PMC4955870, 27381026, 27381026, gzw014
Format: Citation

Title: Elevated Resistin Gene Expression in African American Estrogen and Progesterone Receptor Negative Breast Cancer.
Creator: Vallega, Karin A, Liu, NingNing, Myers, Jennifer S, Yu, Kaixian, Sang, Qing-Xiang Amy
Abstract/Description: African American (AA) women diagnosed with breast cancer are more likely to have aggressive subtypes. Investigating differentially expressed genes between patient populations may help explain racial health disparities. Resistin, one such gene, is linked to inflammation, obesity, and breast cancer risk. Previous studies indicated that resistin expression is higher in serum and tissue of AA breast cancer patients compared to Caucasian American (CA) patients. However, resistin expression levels...
Show moreAfrican American (AA) women diagnosed with breast cancer are more likely to have aggressive subtypes. Investigating differentially expressed genes between patient populations may help explain racial health disparities. Resistin, one such gene, is linked to inflammation, obesity, and breast cancer risk. Previous studies indicated that resistin expression is higher in serum and tissue of AA breast cancer patients compared to Caucasian American (CA) patients. However, resistin expression levels have not been compared between AA and CA patients in a stage- and subtype-specific context. Breast cancer prognosis and treatments vary by subtype. This work investigates differential resistin gene expression in human breast cancer tissues of specific stages, receptor subtypes, and menopause statuses in AA and CA women. Differential gene expression analysis was performed using human breast cancer gene expression data from The Cancer Genome Atlas. We performed inter-race resistin gene expression level comparisons looking at receptor status and stage-specific data between AA and CA samples. DESeq was run to test for differentially expressed resistin values. Resistin RNA was higher in AA women overall, with highest values in receptor negative subtypes. Estrogen-, progesterone-, and human epidermal growth factor receptor 2- negative groups showed statistically significant elevated resistin levels in Stage I and II AA women compared to CA women. In inter-racial comparisons, AA women had significantly higher levels of resistin regardless of menopause status. In whole population comparisons, resistin expression was higher among Stage I and III estrogen receptor negative cases. In comparisons of molecular subtypes, resistin levels were significant higher in triple negative than in luminal A breast cancer. Resistin gene expression levels were significantly higher in receptor negative subtypes, especially estrogen receptor negative cases in AA women. Resistin may serve as an early breast cancer biomarker and possible therapeutic target for AA breast cancer.
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Date Issued: 2016-06-17
Identifier: FSU_pmch_27314854, 10.1371/journal.pone.0157741, PMC4912107, 27314854, 27314854, PONE-D-16-17121
Format: Citation

Title: Evidence for a Proapoptotic Role of Matrix Metalloproteinase-26 in Human Prostate Cancer Cells and Tissues.
Creator: Khamis, Zahraa I., Iczkowski, Kenneth A., Man, Yan-Gao, Bou-Dargham, Mayassa J., Sang, Qing-Xiang Amy
Abstract/Description: Matrix metalloproteinases (MMPs) play intricate roles in cancer progression; some promote invasion and angiogenesis while others suppress tumor growth. For example, human MMP-26/endometase/matrilysin-2 was reported to be either protective or pro-tumorigenic. Our previous reports suggested pro-invasion and anti-inflammation properties in prostate cancer. Here, we provide evidence for a protective role of MMP-26 in the prostate. MMP-26 expression levels in androgen-repressed human prostate...
Show moreMatrix metalloproteinases (MMPs) play intricate roles in cancer progression; some promote invasion and angiogenesis while others suppress tumor growth. For example, human MMP-26/endometase/matrilysin-2 was reported to be either protective or pro-tumorigenic. Our previous reports suggested pro-invasion and anti-inflammation properties in prostate cancer. Here, we provide evidence for a protective role of MMP-26 in the prostate. MMP-26 expression levels in androgen-repressed human prostate cancer (ARCaP) cells, transfected with sense or anti-sense MMP-26 cDNA, are directly correlated with those of the pro-apoptotic marker Bax. Immunohistochemical staining of prostate cancer tissue samples shows similar protein expression patterns, correlating the expression levels of MMP-26 and Bax in benign, neoplastic, and invasive prostate cancer tissues. The MMP-26 protein levels were upregulated in high grade prostate intraepithelial neoplasia (HGPIN) and decreased during the course of disease progression. Further analysis using an indirect terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay showed that many tumor cells expressing MMP-26 were undergoing apoptosis. This study showed that the high level of MMP-26 expression is positively correlated with the presence of apoptotic cells. This pro-apoptotic role of MMP-26 in human prostate cancer cells and tissues may enhance our understanding of the paradoxical roles of MMP-26 in tumor invasion and progression.
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Date Issued: 2016
Identifier: FSU_libsubv1_wos_000366281500011, 10.7150/jca.13067
Format: Citation

Title: Evidence for a Proapoptotic Role of Matrix Metalloproteinase-26 in Human Prostate Cancer Cells and Tissues.
Creator: Khamis, Zahraa I, Iczkowski, Kenneth A, Man, Yan-Gao, Bou-Dargham, Mayassa J, Sang, Qing-Xiang Amy
Abstract/Description: Matrix metalloproteinases (MMPs) play intricate roles in cancer progression; some promote invasion and angiogenesis while others suppress tumor growth. For example, human MMP-26/endometase/matrilysin-2 was reported to be either protective or pro-tumorigenic. Our previous reports suggested pro-invasion and anti-inflammation properties in prostate cancer. Here, we provide evidence for a protective role of MMP-26 in the prostate. MMP-26 expression levels in androgen-repressed human prostate...
Show moreMatrix metalloproteinases (MMPs) play intricate roles in cancer progression; some promote invasion and angiogenesis while others suppress tumor growth. For example, human MMP-26/endometase/matrilysin-2 was reported to be either protective or pro-tumorigenic. Our previous reports suggested pro-invasion and anti-inflammation properties in prostate cancer. Here, we provide evidence for a protective role of MMP-26 in the prostate. MMP-26 expression levels in androgen-repressed human prostate cancer (ARCaP) cells, transfected with sense or anti-sense MMP-26 cDNA, are directly correlated with those of the pro-apoptotic marker Bax. Immunohistochemical staining of prostate cancer tissue samples shows similar protein expression patterns, correlating the expression levels of MMP-26 and Bax in benign, neoplastic, and invasive prostate cancer tissues. The MMP-26 protein levels were upregulated in high grade prostate intraepithelial neoplasia (HGPIN) and decreased during the course of disease progression. Further analysis using an indirect terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay showed that many tumor cells expressing MMP-26 were undergoing apoptosis. This study showed that the high level of MMP-26 expression is positively correlated with the presence of apoptotic cells. This pro-apoptotic role of MMP-26 in human prostate cancer cells and tissues may enhance our understanding of the paradoxical roles of MMP-26 in tumor invasion and progression.
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Date Issued: 2016-01-01
Identifier: FSU_pmch_26722363, 10.7150/jca.13067, PMC4679384, 26722363, 26722363, jcav07p0080
Format: Citation

Title: Fast Method for Computing Chemical Potentials and Liquid-Liquid Phase Equilibria of Macromolecular Solutions.
Creator: Qin, Sanbo, Zhou, Huan-Xiang
Abstract/Description: Chemical potential is a fundamental property for determining thermodynamic equilibria involving exchange of molecules, such as between two phases of molecular systems. Previously, we developed the fast Fourier transform (FFT)-based method for Modeling Atomistic Protein-crowder interactions (FMAP) to calculate excess chemical potentials according to the Widom insertion. Intermolecular interaction energies were expressed as correlation functions and evaluated via FFT. Here, we extend this...
Show moreChemical potential is a fundamental property for determining thermodynamic equilibria involving exchange of molecules, such as between two phases of molecular systems. Previously, we developed the fast Fourier transform (FFT)-based method for Modeling Atomistic Protein-crowder interactions (FMAP) to calculate excess chemical potentials according to the Widom insertion. Intermolecular interaction energies were expressed as correlation functions and evaluated via FFT. Here, we extend this method to calculate liquid-liquid phase equilibria of macromolecular solutions. Chemical potentials are calculated by FMAP over a wide range of molecular densities, and the condition for coexistence of low- and high-density phases is determined by the Maxwell equal-area rule. When benchmarked on Lennard-Jones fluids, our method produces an accurate phase diagram at 18% of the computational cost of the current best method. Importantly, the gain in computational speed increases dramatically as the molecules become more complex, leading to many orders of magnitude in speed up for atomistically represented proteins. We demonstrate the power of FMAP by reporting the first results for the liquid-liquid coexistence curve of γII-crystallin represented at the all-atom level. Our method may thus open the door to accurate determination of phase equilibria for macromolecular mixtures such as protein-protein mixtures and protein-RNA mixtures, that are known to undergo liquid-liquid phase separation, both in vitro and in vivo.
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Date Issued: 2016-08-25
Identifier: FSU_pmch_27327881, 10.1021/acs.jpcb.6b01607, PMC5011432, 27327881, 27327881
Format: Citation

Title: Fully reduced granulin-B is intrinsically disordered and displays concentration-dependent dynamics.
Creator: Ghag, Gaurav, Wolf, Lauren M, Reed, Randi G, Van Der Munnik, Nicholas P, Mundoma, Claudius, Moss, Melissa A, Rangachari, Vijayaraghavan
Abstract/Description: Granulins (Grns) are a family of small, cysteine-rich proteins that are generated upon proteolytic cleavage of their precursor, progranulin (Pgrn). All seven Grns (A-G) contain 12 conserved cysteines that form 6 intramolecular disulfide bonds, rendering this family of proteins unique. Grns are known to play multi-functional roles, including wound healing, embryonic growth, and inflammation and are implicated in neurodegenerative diseases. Despite their manifold functions, there exists a...
Show moreGranulins (Grns) are a family of small, cysteine-rich proteins that are generated upon proteolytic cleavage of their precursor, progranulin (Pgrn). All seven Grns (A-G) contain 12 conserved cysteines that form 6 intramolecular disulfide bonds, rendering this family of proteins unique. Grns are known to play multi-functional roles, including wound healing, embryonic growth, and inflammation and are implicated in neurodegenerative diseases. Despite their manifold functions, there exists a dearth of information regarding their structure-function relationship. Here, we sought to establish the role of disulfide bonds in promoting structure by investigating the fully reduced GrnB (rGrnB). We report that monomeric rGrnB is an intrinsically disordered protein (IDP) at low concentrations. rGrnB undergoes dimerization at higher concentrations to form a fuzzy complex without a net gain in the structure-a behavior increasingly identified as a hallmark of some IDPs. Interestingly, we show that rGrnB is also able to activate NF-κB in human neuroblastoma cells in a concentration-dependent manner. This activation correlates with the observed monomer-dimer dynamics. Collectively, the presented data establish that the intrinsic disorder of rGrnB governs conformational dynamics within the reduced form of the protein, and suggest that the overall structure of Grns could be entirely dictated by disulfide bonds.
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Date Issued: 2016-05-01
Identifier: FSU_pmch_26957645, 10.1093/protein/gzw005, PMC4830411, 26957645, 26957645, gzw005
Format: Citation

Title: Gating Motions and Stationary Gating Properties of Ionotropic Glutamate Receptors: Computation Meets Electrophysiology..
Creator: Zhou, Huan-Xiang
Abstract/Description: Ionotropic glutamate receptors (iGluRs) are tetrameric ligand-gated ion channels essential to all aspects of brain function, including higher order processes such as learning and memory. For decades, electrophysiology was the primary means for characterizing the function of iGluRs and gaining mechanistic insight. Since the turn of the century, structures of isolated water-soluble domains and transmembrane-domain-containing constructs have provided the basis for formulating mechanistic...
Show moreIonotropic glutamate receptors (iGluRs) are tetrameric ligand-gated ion channels essential to all aspects of brain function, including higher order processes such as learning and memory. For decades, electrophysiology was the primary means for characterizing the function of iGluRs and gaining mechanistic insight. Since the turn of the century, structures of isolated water-soluble domains and transmembrane-domain-containing constructs have provided the basis for formulating mechanistic hypotheses. Because these structures only represent sparse, often incomplete snapshots during iGluR activation, significant gaps in knowledge remain regarding structures, energetics, and dynamics of key substates along the functional processes. Some of these gaps have recently been filled by molecular dynamics simulations and theoretical modeling. In this Account, I describe our work in the latter arena toward characterizing iGluR gating motions and developing a formalism for calculating thermodynamic and kinetic properties of stationary gating. The structures of iGluR subunits have a highly modular architecture, in which the ligand-binding domain and the transmembrane domain are well separated and connected by flexible linkers. The ligand-binding domain in turn is composed of two subdomains. During activation, agonist binding induces the closure of the intersubdomain cleft. The cleft closure leads to the outward pulling of a linker tethered to the extracellular terminus of the major pore-lining helix of the transmembrane domain, thereby opening the channel. This activation model based on molecular dynamics simulations was validated by residue-specific information from electrophysiological data on cysteine mutants. A further critical test was made through introducing glycine insertions in the linker. Molecular dynamics simulations showed that, with lengthening by glycine insertions, the linker became less effective in pulling the pore-lining helix, leading to weaker stabilization of the channel-open state. In full agreement, single-channel recordings showed that the channel open probability decreased progressively as the linker was lengthened by glycine insertions. Crystal structures of ligand-binding domains showing different degrees of cleft closure between full and partial agonists suggested a simple mechanism for one subtype of iGluRs, but mysteries surrounded a second subtype, where the ligand-binding domains open to similar degrees when bound with either full or partial agonists. Our free energy simulations now suggest that broadening of the free energy basin for cleft closure is a plausible solution. A theoretical basis for these mechanistic hypotheses on partial agonisms was provided by a model for the free energy surface of a full receptor, where the stabilization by cleft closure is transmitted via the linker to the channel-open state. This model can be implemented by molecular dynamics simulations to predict thermodynamic and kinetics properties of stationary gating that are amenable to direct test by single-channel recordings. Close integration between computation and electrophysiology holds great promises in revealing the conformations of key substates in functional processes and the mechanisms of disease-associated mutations.
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Date Issued: 2017-04-18
Identifier: FSU_pmch_28186717, 10.1021/acs.accounts.6b00598, PMC5398286, 28186717, 28186717
Format: Citation

Title: Generalized Ensemble Sampling of Enzyme Reaction Free Energy Pathways.
Creator: Wu, D, Fajer, M I, Cao, L, Cheng, X, Yang, W
Abstract/Description: Free energy path sampling plays an essential role in computational understanding of chemical reactions, particularly those occurring in enzymatic environments. Among a variety of molecular dynamics simulation approaches, the generalized ensemble sampling strategy is uniquely attractive for the fact that it not only can enhance the sampling of rare chemical events but also can naturally ensure consistent exploration of environmental degrees of freedom. In this review, we plan to provide a...
Show moreFree energy path sampling plays an essential role in computational understanding of chemical reactions, particularly those occurring in enzymatic environments. Among a variety of molecular dynamics simulation approaches, the generalized ensemble sampling strategy is uniquely attractive for the fact that it not only can enhance the sampling of rare chemical events but also can naturally ensure consistent exploration of environmental degrees of freedom. In this review, we plan to provide a tutorial-like tour on an emerging topic: generalized ensemble sampling of enzyme reaction free energy path. The discussion is largely focused on our own studies, particularly ones based on the metadynamics free energy sampling method and the on-the-path random walk path sampling method. We hope that this minipresentation will provide interested practitioners some meaningful guidance for future algorithm formulation and application study.
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Date Issued: 2016-01-01
Identifier: FSU_pmch_27498634, 10.1016/bs.mie.2016.05.012, PMC4978182, 27498634, 27498634, S0076-6879(16)30047-7
Format: Citation

Title: Glucose Oscillations Can Activate an Endogenous Oscillator in Pancreatic Islets.
Creator: McKenna, Joseph P, Dhumpa, Raghuram, Mukhitov, Nikita, Roper, Michael G, Bertram, Richard
Abstract/Description: Pancreatic islets manage elevations in blood glucose level by secreting insulin into the bloodstream in a pulsatile manner. Pulsatile insulin secretion is governed by islet oscillations such as bursting electrical activity and periodic Ca2+ entry in β-cells. In this report, we demonstrate that although islet oscillations are lost by fixing a glucose stimulus at a high concentration, they may be recovered by subsequently converting the glucose stimulus to a sinusoidal wave. We predict with...
Show morePancreatic islets manage elevations in blood glucose level by secreting insulin into the bloodstream in a pulsatile manner. Pulsatile insulin secretion is governed by islet oscillations such as bursting electrical activity and periodic Ca2+ entry in β-cells. In this report, we demonstrate that although islet oscillations are lost by fixing a glucose stimulus at a high concentration, they may be recovered by subsequently converting the glucose stimulus to a sinusoidal wave. We predict with mathematical modeling that the sinusoidal glucose signal's ability to recover islet oscillations depends on its amplitude and period, and we confirm our predictions by conducting experiments with islets using a microfluidics platform. Our results suggest a mechanism whereby oscillatory blood glucose levels recruit non-oscillating islets to enhance pulsatile insulin output from the pancreas. Our results also provide support for the main hypothesis of the Dual Oscillator Model, that a glycolytic oscillator endogenous to islet β-cells drives pulsatile insulin secretion.
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Date Issued: 2016-10-27
Identifier: FSU_pmch_27788129, 10.1371/journal.pcbi.1005143, PMC5082885, 27788129, 27788129, PCOMPBIOL-D-16-00306
Format: Citation

Title: The Impact of DNA Topology and Guide Length on Target Selection by a Cytosine-Specific Cas9.
Creator: Tsui, Tsz Kin Martin, Hand, Travis H, Duboy, Emily C, Li, Hong
Abstract/Description: Cas9 is an RNA-guided DNA cleavage enzyme being actively developed for genome editing and gene regulation. To be cleaved by Cas9, a double stranded DNA, or the protospacer, must be complementary to the guide region, typically 20-nucleotides in length, of the Cas9-bound guide RNA, and adjacent to a short Cas9-specific element called Protospacer Adjacent Motif (PAM). Understanding the correct juxtaposition of the protospacer- and PAM-interaction with Cas9 will enable development of versatile...
Show moreCas9 is an RNA-guided DNA cleavage enzyme being actively developed for genome editing and gene regulation. To be cleaved by Cas9, a double stranded DNA, or the protospacer, must be complementary to the guide region, typically 20-nucleotides in length, of the Cas9-bound guide RNA, and adjacent to a short Cas9-specific element called Protospacer Adjacent Motif (PAM). Understanding the correct juxtaposition of the protospacer- and PAM-interaction with Cas9 will enable development of versatile and safe Cas9-based technology. We report identification and biochemical characterization of Cas9 from Acidothermus cellulolyticus (AceCas9). AceCas9 depends on a 5'-NNNCC-3' PAM and is more efficient in cleaving negative supercoils than relaxed DNA. Kinetic as well as in vivo activity assays reveal that AceCas9 achieves optimal activity when combined with a guide RNA containing a 24-nucleotide complementarity region. The cytosine-specific, DNA topology-sensitive, and extended guide-dependent properties of AceCas9 may be explored for specific genome editing applications.
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Date Issued: 2017-06-16
Identifier: FSU_pmch_28277645, 10.1021/acssynbio.7b00050, PMC5706465, 28277645, 28277645
Format: Citation

Title: The incretin hormone glucagon-like peptide 1 increases mitral cell excitability by decreasing conductance of a voltage-dependent potassium channel.
Creator: Thiebaud, Nicolas, Llewellyn-Smith, Ida J, Gribble, Fiona, Reimann, Frank, Trapp, Stefan, Fadool, Debra Ann
Abstract/Description: The gut hormone called glucagon-like peptide 1 (GLP-1) is a strong moderator of energy homeostasis and communication between the peripheral organs and the brain. GLP-1 signalling occurs in the brain; using a newly developed genetic reporter line of mice, we have discovered GLP-synthesizing cells in the olfactory bulb. GLP-1 increases the firing frequency of neurons (mitral cells) that encode olfactory information by decreasing activity of voltage-dependent K channels (Kv1.3). Modifying GLP-1...
Show moreThe gut hormone called glucagon-like peptide 1 (GLP-1) is a strong moderator of energy homeostasis and communication between the peripheral organs and the brain. GLP-1 signalling occurs in the brain; using a newly developed genetic reporter line of mice, we have discovered GLP-synthesizing cells in the olfactory bulb. GLP-1 increases the firing frequency of neurons (mitral cells) that encode olfactory information by decreasing activity of voltage-dependent K channels (Kv1.3). Modifying GLP-1 levels, either therapeutically or following the ingestion of food, could alter the excitability of neurons in the olfactory bulb in a nutrition or energy state-dependent manner to influence olfactory detection or metabolic sensing. The results of the present study uncover a new function for an olfactory bulb neuron (deep short axon cells, Cajal cells) that could be capable of modifying mitral cell activity through the release of GLP-1. This might be of relevance for the action of GLP-1 mimetics now widely used in the treatment of diabetes. The olfactory system is intricately linked with the endocrine system where it may serve as a detector of the internal metabolic state or energy homeostasis in addition to its classical function as a sensor of external olfactory information. The recent development of transgenic mGLU-yellow fluorescent protein mice that express a genetic reporter under the control of the preproglucagon reporter suggested the presence of the gut hormone, glucagon-like peptide (GLP-1), in deep short axon cells (Cajal cells) of the olfactory bulb and its neuromodulatory effect on mitral cell (MC) first-order neurons. A MC target for the peptide was determined using GLP-1 receptor binding assays, immunocytochemistry for the receptor and injection of fluorescence-labelled GLP-1 analogue exendin-4. Using patch clamp recording of olfactory bulb slices in the whole-cell configuration, we report that GLP-1 and its stable analogue exendin-4 increase the action potential firing frequency of MCs by decreasing the interburst interval rather than modifying the action potential shape, train length or interspike interval. GLP-1 decreases Kv1.3 channel contribution to outward currents in voltage clamp recordings as determined by pharmacological blockade of Kv1.3 or utilizing mice with Kv1.3 gene-targeted deletion as a negative control. Because fluctuations in GLP-1 concentrations monitored by the olfactory bulb can modify the firing frequency of MCs, olfactory coding could change depending upon nutritional or physiological state. As a regulator of neuronal activity, GLP-1 or its analogue may comprise a new metabolic factor with a potential therapeutic target in the olfactory bulb (i.e. via intranasal delivery) for controlling an imbalance in energy homeostasis.
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Date Issued: 2016-05-15
Identifier: FSU_pmch_26931093, 10.1113/JP272322, PMC4865572, 26931093, 26931093
Format: Citation

Title: Liquid-liquid Phase Separation Of Patchy Particles Illuminates Diverse Effects Of Regulatory Components On Protein Droplet Formation.
Creator: Nguemaha, Valery, Zhou, Huan-Xiang
Abstract/Description: Recently many cellular functions have been associated with membraneless organelles, or protein droplets, formed by liquid-liquid phase separation (LLPS). Proteins in these droplets often contain RNA-binding domains, but the effects of RNA on LLPS have been controversial. To gain better understanding on the roles of RNA and other macromolecular regulators, here we used Gibbs-ensemble simulations to determine phase diagrams of two-component patchy particles, as models for mixtures of proteins...
Show moreRecently many cellular functions have been associated with membraneless organelles, or protein droplets, formed by liquid-liquid phase separation (LLPS). Proteins in these droplets often contain RNA-binding domains, but the effects of RNA on LLPS have been controversial. To gain better understanding on the roles of RNA and other macromolecular regulators, here we used Gibbs-ensemble simulations to determine phase diagrams of two-component patchy particles, as models for mixtures of proteins with regulatory components. Protein-like particles have four patches, with attraction strength epsilon(PP); regulatory particles experience mutual steric repulsion but have two attractive patches toward proteins, with the strength epsilon(PR) tunable. At low epsilon(PR), the regulator, due to steric repulsion, preferentially partitions in the dispersed phase, thereby displacing the protein into the droplet phase and promoting LLPS. At moderate epsilon(PR), the regulator starts to partition and displace the protein in the droplet phase, but only to weaken bonding networks and thereby suppress LLPS. At epsilon(PR) > epsilon(PP), the enhanced bonding ability of the regulator initially promotes LLPS, but at higher amounts, the resulting displacement of the protein suppresses LLPS. These results illustrate how RNA can have disparate effects on LLPS, thus able to perform diverse functions in different organelles.
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Date Issued: 2018-04-30
Identifier: FSU_libsubv1_wos_000431104900029, 10.1038/s41598-018-25132-1
Format: Citation

Title: Margatoxin-bound quantum dots as a novel inhibitor of the voltage-gated ion channel Kv1.3.
Creator: Schwartz, Austin B, Kapur, Anshika, Wang, Wentao, Huang, Zhenbo, Fardone, Erminia, Palui, Goutam, Mattoussi, Hedi, Fadool, Debra Ann
Abstract/Description: Venom-derived ion channel inhibitors have strong channel selectivity, potency, and stability; however, tracking delivery to their target can be challenging. Herein, we utilized luminescent quantum dots (QDs) conjugated to margatoxin (MgTx) as a traceable vehicle to target a voltage-dependent potassium channel, Kv1.3, which has a select distribution and well-characterized role in immunity, glucose metabolism, and sensory ability. We screened both unconjugated (MgTx) and conjugated MgTx (QD...
Show moreVenom-derived ion channel inhibitors have strong channel selectivity, potency, and stability; however, tracking delivery to their target can be challenging. Herein, we utilized luminescent quantum dots (QDs) conjugated to margatoxin (MgTx) as a traceable vehicle to target a voltage-dependent potassium channel, Kv1.3, which has a select distribution and well-characterized role in immunity, glucose metabolism, and sensory ability. We screened both unconjugated (MgTx) and conjugated MgTx (QD-MgTx) for their ability to inhibit Shaker channels Kv1.1 to Kv1.7 using patch-clamp electrophysiology in HEK293 cells. Our data indicate that MgTx inhibits 79% of the outward current in Kv1.3-transfected cells and that the QD-MgTx conjugate is able to achieve a similar level of block, albeit a slightly reduced efficacy (66%) and at a slower time course (50% block by 10.9 ± 1.1 min, MgTx; vs. 15.3 ± 1.2 min, QD-MgTx). Like the unbound peptide, the QD-MgTx conjugate inhibits both Kv1.3 and Kv1.2 at a 1 nM concentration, whereas it does not inhibit other screened Shaker channels. We tested the ability of QD-MgTx to inhibit native Kv1.3 expressed in the mouse olfactory bulb (OB). In brain slices of the OB, the conjugate acted similarly to MgTx to inhibit Kv1.3, causing an increased action potential firing frequency attributed to decreased intraburst duration rather than interspike interval. Our data demonstrate a retention of known biophysical properties associated with block of the vestibule of Kv1.3 by QD-MgTx conjugate compared to that of MgTx, inferring QDs could provide a useful tool to deliver ion channel inhibitors to targeted tissues in vivo.
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Date Issued: 2017-02-01
Identifier: FSU_pmch_27861889, 10.1111/jnc.13891, PMC5250575, 27861889, 27861889
Format: Citation

Title: Mechanism and rate constants of the Cdc42 GTPase binding with intrinsically disordered effectors.
Creator: Pang, Xiaodong, Zhou, Huan-Xiang
Abstract/Description: Intrinsically disordered proteins (IDPs) are often involved in signaling and regulatory functions, through binding to cellular targets. Many IDPs undergo disorder-to-order transitions upon binding. Both the binding mechanisms and the magnitudes of the binding rate constants can have functional importance. Previously we have found that the coupled binding and folding of any IDP generally follows a sequential mechanism that we term dock-and-coalesce, whereby one segment of the IDP first docks...
Show moreIntrinsically disordered proteins (IDPs) are often involved in signaling and regulatory functions, through binding to cellular targets. Many IDPs undergo disorder-to-order transitions upon binding. Both the binding mechanisms and the magnitudes of the binding rate constants can have functional importance. Previously we have found that the coupled binding and folding of any IDP generally follows a sequential mechanism that we term dock-and-coalesce, whereby one segment of the IDP first docks to its subsite on the target surface and the remaining segments subsequently coalesce around their respective subsites. Here we applied our TransComp method within the framework of the dock-and-coalesce mechanism to dissect the binding kinetics of two Rho-family GTPases, Cdc42 and TC10, with two intrinsically disordered effectors, WASP and Pak1. TransComp calculations identified the basic regions preceding the GTPase binding domains (GBDs) of the effectors as the docking segment. For Cdc42 binding with both WASP and Pak1, the calculated docking rate constants are close to the observed overall binding rate constants, suggesting that basic-region docking is the rate-limiting step and subsequent conformational coalescence of the GBDs on the Cdc42 surface is fast. The possibility that conformational coalescence of the WASP GBD on the TC10 surface is slow warrants further experimental investigation. The account for the differences in binding rate constants among the three GTPase-effector systems and mutational effects therein yields deep physical and mechanistic insight into the binding processes. Our approach may guide the selection of mutations that lead to redesigned binding pathways.
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Date Issued: 2016-05-01
Identifier: FSU_pmch_26879470, 10.1002/prot.25018, PMC4840055, 26879470, 26879470
Format: Citation

Title: Mitochondrial Ultrastructure And Glucose Signaling Pathways Attributed To The Kv1.3 Ion Channel.
Creator: Kovach, Christopher P., Al Koborssy, Dolly, Huang, Zhenbo, Chelette, Brandon M., Fadool, James M., Fadool, Debra A.
Abstract/Description: Gene-targeted deletion of the potassium channel Kv1.3 (Kv1.3(-/-)) results in "Super-smeller" mice with a sensory phenotype that includes an increased olfactory ability linked to changes in olfactory circuitry, increased abundance of olfactory cilia, and increased expression of odorant receptors and the G-protein, G(olf). Kv1.3(-/-) mice also have a metabolic phenotype including lower body weight and decreased adiposity, increased total energy expenditure (TEE), increased locomotor activity,...
Show moreGene-targeted deletion of the potassium channel Kv1.3 (Kv1.3(-/-)) results in "Super-smeller" mice with a sensory phenotype that includes an increased olfactory ability linked to changes in olfactory circuitry, increased abundance of olfactory cilia, and increased expression of odorant receptors and the G-protein, G(olf). Kv1.3(-/-) mice also have a metabolic phenotype including lower body weight and decreased adiposity, increased total energy expenditure (TEE), increased locomotor activity, and resistance to both diet- and genetic-induced obesity. We explored two cellular aspects to elucidate the mechanism by which loss of Kv1.3 channel in the olfactory bulb (OB) may enhance glucose utilization and metabolic rate. First, using in situ hybridization we find that Kv1.3 and the insulin-dependent glucose transporter type 4 (GLUT4) are co-localized to the mitral cell layer of the OB. Disruption of Kv1.3 conduction via construction of a pore mutation (W386F Kv1.3) was sufficient to independently translocate GLUT4 to the plasma membrane in HEK 293 cells. Because olfactory sensory perception and the maintenance of action potential (AP) firing frequency by mitral cells of the OB is highly energy demanding and Kv1.3 is also expressed in mitochondria, we next explored the structure of this organelle in mitral cells. We challenged wildtype (WT) and Kv1.3(-/-) male mice with a moderately high-fat diet (MHF, 31.8 % kcal fat) for 4 months and then examined OB ultrastructure using transmission electron microscopy. In WT mice, mitochondria were significantly enlarged following diet-induced obesity (DIO) and there were fewer mitochondria, likely due to mitophagy. Interestingly, mitochondria were significantly smaller in Kv1.3(-/-) mice compared with that of WT mice. Similar to their metabolic resistance to DIO, the Kv1.3(-/-) mice had unchanged mitochondria in terms of cross sectional area and abundance following a challenge with modified diet. We are very interested to understand how targeted disruption of the Kv1.3 channel in the OB can modify TEE. Our study demonstrates that Kv1.3 regulates mitochondrial structure and alters glucose utilization; two important metabolic changes that could drive whole system changes in metabolism initiated at the OB.
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Date Issued: 2016-05-19
Identifier: FSU_libsubv1_wos_000376059000001, 10.3389/fphys.2016.00178
Format: Citation

Title: Mitochondrial Ultrastructure and Glucose Signaling Pathways Attributed to the Kv1.3 Ion Channel.
Creator: Kovach, Christopher P, Al Koborssy, Dolly, Huang, Zhenbo, Chelette, Brandon M, Fadool, James M, Fadool, Debra A
Abstract/Description: Gene-targeted deletion of the potassium channel Kv1.3 (Kv1.3(-∕-)) results in "Super-smeller" mice with a sensory phenotype that includes an increased olfactory ability linked to changes in olfactory circuitry, increased abundance of olfactory cilia, and increased expression of odorant receptors and the G-protein, Golf. Kv1.3(-∕-) mice also have a metabolic phenotype including lower body weight and decreased adiposity, increased total energy expenditure (TEE), increased locomotor activity,...
Show moreGene-targeted deletion of the potassium channel Kv1.3 (Kv1.3(-∕-)) results in "Super-smeller" mice with a sensory phenotype that includes an increased olfactory ability linked to changes in olfactory circuitry, increased abundance of olfactory cilia, and increased expression of odorant receptors and the G-protein, Golf. Kv1.3(-∕-) mice also have a metabolic phenotype including lower body weight and decreased adiposity, increased total energy expenditure (TEE), increased locomotor activity, and resistance to both diet- and genetic-induced obesity. We explored two cellular aspects to elucidate the mechanism by which loss of Kv1.3 channel in the olfactory bulb (OB) may enhance glucose utilization and metabolic rate. First, using in situ hybridization we find that Kv1.3 and the insulin-dependent glucose transporter type 4 (GLUT4) are co-localized to the mitral cell layer of the OB. Disruption of Kv1.3 conduction via construction of a pore mutation (W386F Kv1.3) was sufficient to independently translocate GLUT4 to the plasma membrane in HEK 293 cells. Because olfactory sensory perception and the maintenance of action potential (AP) firing frequency by mitral cells of the OB is highly energy demanding and Kv1.3 is also expressed in mitochondria, we next explored the structure of this organelle in mitral cells. We challenged wildtype (WT) and Kv1.3(-∕-) male mice with a moderately high-fat diet (MHF, 31.8 % kcal fat) for 4 months and then examined OB ultrastructure using transmission electron microscopy. In WT mice, mitochondria were significantly enlarged following diet-induced obesity (DIO) and there were fewer mitochondria, likely due to mitophagy. Interestingly, mitochondria were significantly smaller in Kv1.3(-∕-) mice compared with that of WT mice. Similar to their metabolic resistance to DIO, the Kv1.3(-∕-) mice had unchanged mitochondria in terms of cross sectional area and abundance following a challenge with modified diet. We are very interested to understand how targeted disruption of the Kv1.3 channel in the OB can modify TEE. Our study demonstrates that Kv1.3 regulates mitochondrial structure and alters glucose utilization; two important metabolic changes that could drive whole system changes in metabolism initiated at the OB.
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Date Issued: 2016-05-19
Identifier: FSU_pmch_27242550, 10.3389/fphys.2016.00178, PMC4871887, 27242550, 27242550
Format: Citation

Title: A Muts Beta-dependent Contribution Of Muts Alpha To Repeat Expansions In Fragile X Premutation Mice?.
Creator: Zhao, Xiao-Nan, Lokanga, Rachel, Allette, Kimaada, Gazy, Inbal, Wu, Di, Usdin, Karen
Abstract/Description: The fragile X-related disorders result from expansion of a CGG/CCG microsatellite in the 5' UTR of the FMR1 gene. We have previously demonstrated that the MSH2/MSH3 complex, MutS beta, that is important for mismatch repair, is essential for almost all expansions in a mouse model of these disorders. Here we show that the MSH2/MSH6 complex, MutS alpha also contributes to the production of both germ line and somatic expansions as evidenced by the reduction in the number of expansions observed in...
Show moreThe fragile X-related disorders result from expansion of a CGG/CCG microsatellite in the 5' UTR of the FMR1 gene. We have previously demonstrated that the MSH2/MSH3 complex, MutS beta, that is important for mismatch repair, is essential for almost all expansions in a mouse model of these disorders. Here we show that the MSH2/MSH6 complex, MutS alpha also contributes to the production of both germ line and somatic expansions as evidenced by the reduction in the number of expansions observed in Msh6(-/-) mice. This effect is not mediated via an indirect effect of the loss of MSH6 on the level of MSH3. However, since MutS beta is required for 98% of germ line expansions and almost all somatic ones, MutS alpha is apparently not able to efficiently substitute for MutS beta in the expansion process. Using purified human proteins we demonstrate that MutS alpha, like MutS beta, binds to substrates with loop-outs of the repeats and increases the thermal stability of the structures that they form. We also show that MutS alpha facilitates binding of MutS beta to these loop-outs. These data suggest possible models for the contribution of MutS alpha to repeat expansion. In addition, we show that unlike MutS beta, MutS alpha may also act to protect against repeat contractions in the Fmr1 gene.
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Date Issued: 2016-07
Identifier: FSU_libsubv1_wos_000381050100049, 10.1371/journal.pgen.1006190
Format: Citation

Title: A new synthetic matrix metalloproteinase inhibitor reduces human mesenchymal stem cell adipogenesis.
Creator: Bosco, Dale B, Roycik, Mark D, Jin, Yonghao, Schwartz, Martin A, Lively, Ty J, Zorio, Diego A R, Sang, Qing-Xiang Amy
Abstract/Description: Development of adipose tissue requires the differentiation of less specialized cells, such as human mesenchymal stem cells (hMSCs), into adipocytes. Since matrix metalloproteinases (MMPs) play critical roles in the cell differentiation process, we conducted investigations to determine if a novel mercaptosulfonamide-based MMP inhibitor (MMPI), YHJ-7-52, could affect hMSC adipogenic differentiation and lipid accumulation. Enzyme inhibition assays, adipogenic differentiation experiments, and...
Show moreDevelopment of adipose tissue requires the differentiation of less specialized cells, such as human mesenchymal stem cells (hMSCs), into adipocytes. Since matrix metalloproteinases (MMPs) play critical roles in the cell differentiation process, we conducted investigations to determine if a novel mercaptosulfonamide-based MMP inhibitor (MMPI), YHJ-7-52, could affect hMSC adipogenic differentiation and lipid accumulation. Enzyme inhibition assays, adipogenic differentiation experiments, and quantitative PCR methods were employed to characterize this inhibitor and determine its effect upon adipogenesis. YHJ-7-52 reduced lipid accumulation in differentiated cells by comparable amounts as a potent hydroxamate MMPI, GM6001. However, YHJ-7-82, a non-inhibitory structural analog of YHJ-7-52, in which the zinc-binding thiol group is replaced by a hydroxyl group, had no effect on adipogenesis. The two MMPIs (YHJ-7-52 and GM6001) were also as effective in reducing lipid accumulation in differentiated cells as T0070907, an antagonist of peroxisome-proliferator activated receptor gamma (PPAR-gamma), at a similar concentration. PPAR-gamma is a typical adipogenic marker and a key regulatory protein for the transition of preadiopocyte to adipocyte. Moreover, MMP inhibition was able to suppress lipid accumulation in cells co-treated with Troglitazone, a PPAR-gamma agonist. Our results indicate that MMP inhibitors may be used as molecular tools for adipogenesis and obesity treatment research.
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Date Issued: 2017-02-24
Identifier: FSU_pmch_28234995, 10.1371/journal.pone.0172925, PMC5325569, 28234995, 28234995, PONE-D-16-48797
Format: Citation

Title: A Non-Stem-Loop CRISPR RNA Is Processed by Dual Binding Cas6.
Creator: Shao, Yaming, Richter, Hagen, Sun, Shengfang, Sharma, Kundan, Urlaub, Henning, Randau, Lennart, Li, Hong
Abstract/Description: A subclass of recently discovered CRISPR repeat RNA in bacteria contains minimally recognizable structural features that facilitate an unknown mechanism of recognition and processing by the Cas6 family of endoribonucleases. Cocrystal structures of Cas6 from Methanococcus maripaludis (MmCas6b) bound with its repeat RNA revealed a dual site binding structure and a cleavage site conformation poised for phosphodiester bond breakage. Two non-interacting MmCas6b bind to two separate AAYAA motifs...
Show moreA subclass of recently discovered CRISPR repeat RNA in bacteria contains minimally recognizable structural features that facilitate an unknown mechanism of recognition and processing by the Cas6 family of endoribonucleases. Cocrystal structures of Cas6 from Methanococcus maripaludis (MmCas6b) bound with its repeat RNA revealed a dual site binding structure and a cleavage site conformation poised for phosphodiester bond breakage. Two non-interacting MmCas6b bind to two separate AAYAA motifs within the same repeat, one distal and one adjacent to the cleavage site. This bound structure potentially competes with a stable but non-productive RNA structure. At the cleavage site, MmCas6b supplies a base pair mimic to stabilize a short 2 base pair stem immediately upstream of the scissile phosphate. Complementary biochemical analyses support the dual-AAYAA binding model and a critical role of the protein-RNA base pair mimic. Our results reveal a previously unknown method of processing non-stem-loop CRISPR RNA by Cas6.
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Date Issued: 2016-04-05
Identifier: FSU_pmch_26996962, 10.1016/j.str.2016.02.009, PMC4823167, 26996962, 26996962, S0969-2126(16)00068-X
Format: Citation

Title: Pathogenic Tfg Mutations Underlying Hereditary Spastic Paraplegia Impair Secretory Protein Trafficking And Axon Fasciculation.
Creator: Slosarek, Erin L., Schuh, Amber L., Pustova, Iryna, Johnson, Adam, Bird, Jennifer, Johnson, Matthew, Frankel, E. B., Bhattacharya, Nilakshee, Hanna, Michael G., Burke, Jordan E....
Show moreSlosarek, Erin L., Schuh, Amber L., Pustova, Iryna, Johnson, Adam, Bird, Jennifer, Johnson, Matthew, Frankel, E. B., Bhattacharya, Nilakshee, Hanna, Michael G., Burke, Jordan E., Ruhl, David A., Quinney, Kyle, Block, Samuel, Peotter, Jennifer L., Chapman, Edwin R., Sheets, Michael D., Butcher, Samuel E., Stagg, Scott M., Audhya, Anjon
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Abstract/Description: Length-dependent axonopathy of the corticospinal tract causes lower limb spasticity and is characteristic of several neurological disorders, including hereditary spastic paraplegia (HSP) and amyotrophic lateral sclerosis. Mutations in Trk-fused gene (TFG) have been implicated in both diseases, but the pathomechanisms by which these alterations cause neuropathy remain unclear. Here, we biochemically and genetically define the impact of a mutation within the TFG coiled-coil domain, which...
Show moreLength-dependent axonopathy of the corticospinal tract causes lower limb spasticity and is characteristic of several neurological disorders, including hereditary spastic paraplegia (HSP) and amyotrophic lateral sclerosis. Mutations in Trk-fused gene (TFG) have been implicated in both diseases, but the pathomechanisms by which these alterations cause neuropathy remain unclear. Here, we biochemically and genetically define the impact of a mutation within the TFG coiled-coil domain, which underlies early-onset forms of HSP. We find that the TFG (p.R106C) mutation alters compaction of TFG ring complexes, which play a critical role in the export of cargoes from the endoplasmic reticulum (ER). Using CRISPR-mediated genome editing, we engineered human stem cells that express the mutant form of TFG at endogenous levels and identified specific defects in secretion from the ER and axon fasciculation following neuronal differentiation. Together, our data highlight a key role for TFG-mediated protein transport in the pathogenesis of HSP.
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Date Issued: 2018-08-28
Identifier: FSU_libsubv1_wos_000442923900005, 10.1016/j.celrep.2018.07.081
Format: Citation

Title: Prediction of homoprotein and heteroprotein complexes by protein docking and template-based modeling: A CASP-CAPRI experiment..
Creator: Lensink, Marc F, Velankar, Sameer, Kryshtafovych, Andriy, Huang, Shen-You, Schneidman-Duhovny, Dina, Sali, Andrej, Segura, Joan, Fernandez-Fuentes, Narcis, Viswanath, Shruthi,...
Show moreLensink, Marc F, Velankar, Sameer, Kryshtafovych, Andriy, Huang, Shen-You, Schneidman-Duhovny, Dina, Sali, Andrej, Segura, Joan, Fernandez-Fuentes, Narcis, Viswanath, Shruthi, Elber, Ron, Grudinin, Sergei, Popov, Petr, Neveu, Emilie, Lee, Hasup, Baek, Minkyung, Park, Sangwoo, Heo, Lim, Rie Lee, Gyu, Seok, Chaok, Qin, Sanbo, Zhou, Huan-Xiang, Ritchie, David W, Maigret, Bernard, Devignes, Marie-Dominique, Ghoorah, Anisah, Torchala, Mieczyslaw, Chaleil, Raphaël A G, Bates, Paul A, Ben-Zeev, Efrat, Eisenstein, Miriam, Negi, Surendra S, Weng, Zhiping, Vreven, Thom, Pierce, Brian G, Borrman, Tyler M, Yu, Jinchao, Ochsenbein, Françoise, Guerois, Raphaël, Vangone, Anna, Rodrigues, João P G L M, van Zundert, Gydo, Nellen, Mehdi, Xue, Li, Karaca, Ezgi, Melquiond, Adrien S J, Visscher, Koen, Kastritis, Panagiotis L, Bonvin, Alexandre M J J, Xu, Xianjin, Qiu, Liming, Yan, Chengfei, Li, Jilong, Ma, Zhiwei, Cheng, Jianlin, Zou, Xiaoqin, Shen, Yang, Peterson, Lenna X, Kim, Hyung-Rae, Roy, Amit, Han, Xusi, Esquivel-Rodriguez, Juan, Kihara, Daisuke, Yu, Xiaofeng, Bruce, Neil J, Fuller, Jonathan C, Wade, Rebecca C, Anishchenko, Ivan, Kundrotas, Petras J, Vakser, Ilya A, Imai, Kenichiro, Yamada, Kazunori, Oda, Toshiyuki, Nakamura, Tsukasa, Tomii, Kentaro, Pallara, Chiara, Romero-Durana, Miguel, Jiménez-García, Brian, Moal, Iain H, Férnandez-Recio, Juan, Joung, Jong Young, Kim, Jong Yun, Joo, Keehyoung, Lee, Jooyoung, Kozakov, Dima, Vajda, Sandor, Mottarella, Scott, Hall, David R, Beglov, Dmitri, Mamonov, Artem, Xia, Bing, Bohnuud, Tanggis, Del Carpio, Carlos A, Ichiishi, Eichiro, Marze, Nicholas, Kuroda, Daisuke, Roy Burman, Shourya S, Gray, Jeffrey J, Chermak, Edrisse, Cavallo, Luigi, Oliva, Romina, Tovchigrechko, Andrey, Wodak, Shoshana J
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Abstract/Description: We present the results for CAPRI Round 30, the first joint CASP-CAPRI experiment, which brought together experts from the protein structure prediction and protein-protein docking communities. The Round comprised 25 targets from amongst those submitted for the CASP11 prediction experiment of 2014. The targets included mostly homodimers, a few homotetramers, and two heterodimers, and comprised protein chains that could readily be modeled using templates from the Protein Data Bank. On average 24...
Show moreWe present the results for CAPRI Round 30, the first joint CASP-CAPRI experiment, which brought together experts from the protein structure prediction and protein-protein docking communities. The Round comprised 25 targets from amongst those submitted for the CASP11 prediction experiment of 2014. The targets included mostly homodimers, a few homotetramers, and two heterodimers, and comprised protein chains that could readily be modeled using templates from the Protein Data Bank. On average 24 CAPRI groups and 7 CASP groups submitted docking predictions for each target, and 12 CAPRI groups per target participated in the CAPRI scoring experiment. In total more than 9500 models were assessed against the 3D structures of the corresponding target complexes. Results show that the prediction of homodimer assemblies by homology modeling techniques and docking calculations is quite successful for targets featuring large enough subunit interfaces to represent stable associations. Targets with ambiguous or inaccurate oligomeric state assignments, often featuring crystal contact-sized interfaces, represented a confounding factor. For those, a much poorer prediction performance was achieved, while nonetheless often providing helpful clues on the correct oligomeric state of the protein. The prediction performance was very poor for genuine tetrameric targets, where the inaccuracy of the homology-built subunit models and the smaller pair-wise interfaces severely limited the ability to derive the correct assembly mode. Our analysis also shows that docking procedures tend to perform better than standard homology modeling techniques and that highly accurate models of the protein components are not always required to identify their association modes with acceptable accuracy. Proteins 2016; 84(Suppl 1):323-348. © 2016 Wiley Periodicals, Inc.
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Date Issued: 2016-09-01
Identifier: FSU_pmch_27122118, 10.1002/prot.25007, PMC5030136, 27122118, 27122118
Format: Citation

Title: Protein Allostery and Conformational Dynamics.
Creator: Guo, Jingjing, Zhou, Huan-Xiang
Abstract/Description: The functions of many proteins are regulated through allostery, whereby effector binding at a distal site changes the functional activity (e.g., substrate binding affinity or catalytic efficiency) at the active site. Most allosteric studies have focused on thermodynamic properties, in particular, substrate binding affinity. Changes in substrate binding affinity by allosteric effectors have generally been thought to be mediated by conformational transitions of the proteins or, alternatively,...
Show moreThe functions of many proteins are regulated through allostery, whereby effector binding at a distal site changes the functional activity (e.g., substrate binding affinity or catalytic efficiency) at the active site. Most allosteric studies have focused on thermodynamic properties, in particular, substrate binding affinity. Changes in substrate binding affinity by allosteric effectors have generally been thought to be mediated by conformational transitions of the proteins or, alternatively, by changes in the broadness of the free energy basin of the protein conformational state without shifting the basin minimum position. When effector binding changes the free energy landscape of a protein in conformational space, the change affects not only thermodynamic properties but also dynamic properties, including the amplitudes of motions on different time scales and rates of conformational transitions. Here we assess the roles of conformational dynamics in allosteric regulation. Two cases are highlighted where NMR spectroscopy and molecular dynamics simulation have been used as complementary approaches to identify residues possibly involved in allosteric communication. Perspectives on contentious issues, for example, the relationship between picosecond-nanosecond local and microsecond-millisecond conformational exchange dynamics, are presented.
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Date Issued: 2016-06-08
Identifier: FSU_pmch_26876046, 10.1021/acs.chemrev.5b00590, PMC5011433, 26876046, 26876046
Format: Citation

Title: Protein folding, binding, and droplet formation in cell-like conditions.
Creator: Qin, Sanbo, Zhou, Huan-Xiang
Abstract/Description: The many bystander macromolecules in the crowded cellular environments present both steric repulsion and weak attraction to proteins undergoing folding or binding and hence impact the thermodynamic and kinetic properties of these processes. The weak but nonrandom binding with bystander macromolecules may facilitate subcellular localization and biological function. Weak binding also leads to the emergence of a protein-rich droplet phase, which has been implicated in regulating a variety of...
Show moreThe many bystander macromolecules in the crowded cellular environments present both steric repulsion and weak attraction to proteins undergoing folding or binding and hence impact the thermodynamic and kinetic properties of these processes. The weak but nonrandom binding with bystander macromolecules may facilitate subcellular localization and biological function. Weak binding also leads to the emergence of a protein-rich droplet phase, which has been implicated in regulating a variety of cellular functions. All these important problems can now be addressed by realistic modeling of intermolecular interactions. Configurational sampling of concentrated protein solutions is an ongoing challenge.
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Date Issued: 2017-04-01
Identifier: FSU_pmch_27771543, 10.1016/j.sbi.2016.10.006, PMC5397379, 27771543, 27771543, S0959-440X(16)30169-5
Format: Citation

Title: Proteomic characterization of paired non-malignant and malignant African-American prostate epithelial cell lines distinguishes them by structural proteins.
Creator: Myers, Jennifer S, Vallega, Karin A, White, Jason, Yu, Kaixian, Yates, Clayton C, Sang, Qing-Xiang Amy
Abstract/Description: While many factors may contribute to the higher prostate cancer incidence and mortality experienced by African-American men compared to their counterparts, the contribution of tumor biology is underexplored due to inadequate availability of African-American patient-derived cell lines and specimens. Here, we characterize the proteomes of non-malignant RC-77 N/E and malignant RC-77 T/E prostate epithelial cell lines previously established from prostate specimens from the same African-American...
Show moreWhile many factors may contribute to the higher prostate cancer incidence and mortality experienced by African-American men compared to their counterparts, the contribution of tumor biology is underexplored due to inadequate availability of African-American patient-derived cell lines and specimens. Here, we characterize the proteomes of non-malignant RC-77 N/E and malignant RC-77 T/E prostate epithelial cell lines previously established from prostate specimens from the same African-American patient with early stage primary prostate cancer. In this comparative proteomic analysis of RC-77 N/E and RC-77 T/E cells, differentially expressed proteins were identified and analyzed for overrepresentation of PANTHER protein classes, Gene Ontology annotations, and pathways. The enrichment of gene sets and pathway significance were assessed using Gene Set Enrichment Analysis and Signaling Pathway Impact Analysis, respectively. The gene and protein expression data of age- and stage-matched prostate cancer specimens from The Cancer Genome Atlas were analyzed. Structural and cytoskeletal proteins were differentially expressed and statistically overrepresented between RC-77 N/E and RC-77 T/E cells. Beta-catenin, alpha-actinin-1, and filamin-A were upregulated in the tumorigenic RC-77 T/E cells, while integrin beta-1, integrin alpha-6, caveolin-1, laminin subunit gamma-2, and CD44 antigen were downregulated. The increased protein level of beta-catenin and the reduction of caveolin-1 protein level in the tumorigenic RC-77 T/E cells mirrored the upregulation of beta-catenin mRNA and downregulation of caveolin-1 mRNA in African-American prostate cancer specimens compared to non-malignant controls. After subtracting race-specific non-malignant RNA expression, beta-catenin and caveolin-1 mRNA expression levels were higher in African-American prostate cancer specimens than in Caucasian-American specimens. The "ECM-Receptor Interaction" and "Cell Adhesion Molecules", and the "Tight Junction" and "Adherens Junction" pathways contained proteins are associated with RC-77 N/E and RC-77 T/E cells, respectively. Our results suggest RC-77 T/E and RC-77 N/E cell lines can be distinguished by differentially expressed structural and cytoskeletal proteins, which appeared in several pathways across multiple analyses. Our results indicate that the expression of beta-catenin and caveolin-1 may be prostate cancer- and race-specific. Although the RC-77 cell model may not be representative of all African-American prostate cancer due to tumor heterogeneity, it is a unique resource for studying prostate cancer initiation and progression.
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Date Issued: 2017-07-11
Identifier: FSU_pmch_28697756, 10.1186/s12885-017-3462-7, PMC5504803, 28697756, 28697756, 10.1186/s12885-017-3462-7
Format: Citation

Title: Proteomic Upregulation of Fatty Acid Synthase and Fatty Acid Binding Protein 5 and Identification of Cancer- and Race-Specific Pathway Associations in Human Prostate Cancer Tissues.
Creator: Myers, Jennifer S., von Lersner, Ariana K., Sang, Qing-Xiang Amy
Abstract/Description: Protein profiling studies of prostate cancer have been widely used to characterize molecular differences between diseased and non-diseased tissues. When combined with pathway analysis, profiling approaches are able to identify molecular mechanisms of prostate cancer, group patients by cancer subtype, and predict prognosis. This strategy can also be implemented to study prostate cancer in very specific populations, such as African Americans who have higher rates of prostate cancer incidence...
Show moreProtein profiling studies of prostate cancer have been widely used to characterize molecular differences between diseased and non-diseased tissues. When combined with pathway analysis, profiling approaches are able to identify molecular mechanisms of prostate cancer, group patients by cancer subtype, and predict prognosis. This strategy can also be implemented to study prostate cancer in very specific populations, such as African Americans who have higher rates of prostate cancer incidence and mortality than other racial groups in the United States. In this study, age-, stage-, and Gleason score-matched prostate tumor specimen from African American and Caucasian American men, along with non-malignant adjacent prostate tissue from these same patients, were compared. Protein expression changes and altered pathway associations were identified in prostate cancer generally and in African American prostate cancer specifically. In comparing tumor to non-malignant samples, 45 proteins were significantly cancer-associated and 3 proteins were significantly downregulated in tumor samples. Notably, fatty acid synthase (FASN) and epidermal fatty acid-binding protein (FABP5) were upregulated in human prostate cancer tissues, consistent with their known functions in prostate cancer progression. Aldehyde dehydrogenase family 1 member A3 (ALDH1A3) was also upregulated in tumor samples. The Metastasis Associated Protein 3 (MTA3) pathway was significantly enriched in tumor samples compared to non-malignant samples. While the current experiment was unable to detect statistically significant differences in protein expression between African American and Caucasian American samples, differences in overrepresentation and pathway enrichment were found. Structural components (Cytoskeletal Proteins and Extracellular Matrix Protein protein classes, and Biological Adhesion Gene Ontology (GO) annotation) were overrepresented in African American but not Caucasian American tumors. Additionally, 5 pathways were enriched in African American prostate tumors: the Small Cell Lung Cancer, Platelet-Amyloid Precursor Protein, Agrin, Neuroactive Ligand-Receptor Interaction, and Intrinsic pathways. The protein components of these pathways were either basement membrane proteins or coagulation proteins.
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Date Issued: 2016
Identifier: FSU_libsubv1_wos_000380050300010, 10.7150/jca.15860
Format: Citation

Title: Proteomic Upregulation of Fatty Acid Synthase and Fatty Acid Binding Protein 5 and Identification of Cancer- and Race-Specific Pathway Associations in Human Prostate Cancer Tissues.
Creator: Myers, Jennifer S, von Lersner, Ariana K, Sang, Qing-Xiang Amy
Abstract/Description: Protein profiling studies of prostate cancer have been widely used to characterize molecular differences between diseased and non-diseased tissues. When combined with pathway analysis, profiling approaches are able to identify molecular mechanisms of prostate cancer, group patients by cancer subtype, and predict prognosis. This strategy can also be implemented to study prostate cancer in very specific populations, such as African Americans who have higher rates of prostate cancer incidence...
Show moreProtein profiling studies of prostate cancer have been widely used to characterize molecular differences between diseased and non-diseased tissues. When combined with pathway analysis, profiling approaches are able to identify molecular mechanisms of prostate cancer, group patients by cancer subtype, and predict prognosis. This strategy can also be implemented to study prostate cancer in very specific populations, such as African Americans who have higher rates of prostate cancer incidence and mortality than other racial groups in the United States. In this study, age-, stage-, and Gleason score-matched prostate tumor specimen from African American and Caucasian American men, along with non-malignant adjacent prostate tissue from these same patients, were compared. Protein expression changes and altered pathway associations were identified in prostate cancer generally and in African American prostate cancer specifically. In comparing tumor to non-malignant samples, 45 proteins were significantly cancer-associated and 3 proteins were significantly downregulated in tumor samples. Notably, fatty acid synthase (FASN) and epidermal fatty acid-binding protein (FABP5) were upregulated in human prostate cancer tissues, consistent with their known functions in prostate cancer progression. Aldehyde dehydrogenase family 1 member A3 (ALDH1A3) was also upregulated in tumor samples. The Metastasis Associated Protein 3 (MTA3) pathway was significantly enriched in tumor samples compared to non-malignant samples. While the current experiment was unable to detect statistically significant differences in protein expression between African American and Caucasian American samples, differences in overrepresentation and pathway enrichment were found. Structural components (Cytoskeletal Proteins and Extracellular Matrix Protein protein classes, and Biological Adhesion Gene Ontology (GO) annotation) were overrepresented in African American but not Caucasian American tumors. Additionally, 5 pathways were enriched in African American prostate tumors: the Small Cell Lung Cancer, Platelet-Amyloid Precursor Protein, Agrin, Neuroactive Ligand-Receptor Interaction, and Intrinsic pathways. The protein components of these pathways were either basement membrane proteins or coagulation proteins.
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Date Issued: 2016-07-05
Identifier: FSU_pmch_27471561, 10.7150/jca.15860, PMC4964129, 27471561, 27471561, jcav07p1452
Format: Citation

Title: Rate Constants and Mechanisms of Protein-Ligand Binding.
Creator: Pang, Xiaodong, Zhou, Huan-Xiang
Abstract/Description: Whereas protein-ligand binding affinities have long-established prominence, binding rate constants and binding mechanisms have gained increasing attention in recent years. Both new computational methods and new experimental techniques have been developed to characterize the latter properties. It is now realized that binding mechanisms, like binding rate constants, can and should be quantitatively determined. In this review, we summarize studies and synthesize ideas on several topics in the...
Show moreWhereas protein-ligand binding affinities have long-established prominence, binding rate constants and binding mechanisms have gained increasing attention in recent years. Both new computational methods and new experimental techniques have been developed to characterize the latter properties. It is now realized that binding mechanisms, like binding rate constants, can and should be quantitatively determined. In this review, we summarize studies and synthesize ideas on several topics in the hope of providing a coherent picture of and physical insight into binding kinetics. The topics include microscopic formulation of the kinetic problem and its reduction to simple rate equations; computation of binding rate constants; quantitative determination of binding mechanisms; and elucidation of physical factors that control binding rate constants and mechanisms.
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Date Issued: 2017-05-22
Identifier: FSU_pmch_28375732, 10.1146/annurev-biophys-070816-033639, PMC5592114, 28375732, 28375732
Format: Citation

Title: Reversal Learning Deficits Associated with Increased Frontal Cortical Brain-Derived Neurotrophic Factor Tyrosine Kinase B Signaling in a Prenatal Cocaine Exposure Mouse Model.
Creator: McCarthy, Deirdre M, Bell, Genevieve A, Cannon, Elisa N, Mueller, Kaly A, Huizenga, Megan N, Sadri-Vakili, Ghazaleh, Fadool, Debra A, Bhide, Pradeep G
Abstract/Description: Prenatal cocaine exposure remains a major public health concern because of its adverse impact on cognitive function in children and adults. We report that prenatal cocaine exposure produces significant deficits in reversal learning, a key component of cognitive flexibility, in a mouse model. We used an olfactory reversal learning paradigm and found that the prenatally cocaine-exposed mice showed a marked failure to learn the reversed paradigm. Because brain-derived neurotrophic factor (BDNF)...
Show morePrenatal cocaine exposure remains a major public health concern because of its adverse impact on cognitive function in children and adults. We report that prenatal cocaine exposure produces significant deficits in reversal learning, a key component of cognitive flexibility, in a mouse model. We used an olfactory reversal learning paradigm and found that the prenatally cocaine-exposed mice showed a marked failure to learn the reversed paradigm. Because brain-derived neurotrophic factor (BDNF) is a key regulator of cognitive functions, and because prenatal cocaine exposure increases the expression of BDNF and the phosphorylated form of its receptor, tyrosine kinase B (TrkB), we examined whether BDNF-TrkB signaling is involved in mediating the reversal learning deficit in prenatally cocaine-exposed mice. Systemic administration of a selective TrkB receptor antagonist restored normal reversal learning in prenatally cocaine-exposed mice, suggesting that increased BDNF-TrkB signaling may be an underlying mechanism of reversal learning deficits. Our findings provide novel mechanistic insights into the reversal learning phenomenon and may have significant translational implications because impaired cognitive flexibility is a key symptom in psychiatric conditions of developmental onset.
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Date Issued: 2016-01-01
Identifier: FSU_pmch_27951531, 10.1159/000452739, PMC5360472, 27951531, 27951531, 000452739
Format: Citation

Title: Role of cardiac troponin I carboxy terminal mobile domain and linker sequence in regulating cardiac contraction.
Creator: Meyer, Nancy L, Chase, P Bryant
Abstract/Description: Inhibition of striated muscle contraction at resting Ca(2+) depends on the C-terminal half of troponin I (TnI) in thin filaments. Much focus has been on a short inhibitory peptide (Ip) sequence within TnI, but structural studies and identification of disease-associated mutations broadened emphasis to include a larger mobile domain (Md) sequence at the C-terminus of TnI. For Md to function effectively in muscle relaxation, tight mechanical coupling to troponin's core-and thus tropomyosin-is...
Show moreInhibition of striated muscle contraction at resting Ca(2+) depends on the C-terminal half of troponin I (TnI) in thin filaments. Much focus has been on a short inhibitory peptide (Ip) sequence within TnI, but structural studies and identification of disease-associated mutations broadened emphasis to include a larger mobile domain (Md) sequence at the C-terminus of TnI. For Md to function effectively in muscle relaxation, tight mechanical coupling to troponin's core-and thus tropomyosin-is presumably needed. We generated recombinant, human cardiac troponins containing one of two TnI constructs: either an 8-amino acid linker between Md and the rest of troponin (cTnILink8), or an Md deletion (cTnI1-163). Motility assays revealed that Ca(2+)-sensitivity of reconstituted thin filament sliding was markedly increased with cTnILink8 (∼0.9 pCa unit leftward shift of speed-pCa relation compared to WT), and increased further when Md was missing entirely (∼1.4 pCa unit shift). Cardiac Tn's ability to turn off filament sliding at diastolic Ca(2+) was mostly (61%), but not completely eliminated with cTnI1-163. TnI's Md is required for full inhibition of unloaded filament sliding, although other portions of troponin-presumably including Ip-are also necessary. We also confirm that TnI's Md is not responsible for superactivation of actomyosin cycling by troponin.
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Date Issued: 2016-07-01
Identifier: FSU_pmch_26971468, 10.1016/j.abb.2016.03.010, PMC4899117, 26971468, 26971468, S0003-9861(16)30062-5
Format: Citation

Title: Semiclosed Conformations of the Ligand-Binding Domains of NMDA Receptors during Stationary Gating.
Creator: Dai, Jian, Zhou, Huan-Xiang
Abstract/Description: NMDA receptors are tetrameric ligand-gated ion channels. In the continuous presence of saturating agonists, NMDA receptors undergo stationary gating, in which the channel stochastically switches between an open state that permits ion conductance and a closed state that prevents permeation. The ligand-binding domains (LBDs) of the four subunits are expected to have closed clefts in the channel-open state. On the other hand, there is little knowledge about the conformational status of the LBDs...
Show moreNMDA receptors are tetrameric ligand-gated ion channels. In the continuous presence of saturating agonists, NMDA receptors undergo stationary gating, in which the channel stochastically switches between an open state that permits ion conductance and a closed state that prevents permeation. The ligand-binding domains (LBDs) of the four subunits are expected to have closed clefts in the channel-open state. On the other hand, there is little knowledge about the conformational status of the LBDs in the channel-closed state during stationary gating. To probe the latter conformational status, Kussius and Popescu engineered interlobe disulfide cross-links in NMDA receptors and found that the cross-linking produced stationary gating kinetics that differed only subtly from that produced by agonist binding. These authors assumed that the cross-linking immobilized the LBDs in cleft-closed conformations, and consequently concluded that throughout stationary gating, agonist-bound LBDs also stayed predominantly in cleft-closed conformations and made only infrequent excursions to cleft-open conformations. Here, by calculating the conformational free energies of cross-linked and agonist-bound LBDs, we assess whether cross-linking actually traps the LBDs in cleft-closed conformations and delineate semiclosed conformations of agonist-bound LBDs that may potentially be thermodynamically and kinetically important during stationary gating. Our free-energy results show that the cross-linked LBDs are not locked in the fully closed form; rather, they sample semiclosed conformations almost as readily as the agonist-bound LBDs. Several lines of reasoning suggest that LBDs are semiclosed in the channel-closed state during stationary gating. Our free-energy simulations suggest possible structural details of such semiclosed LBD conformations, including intra- and intermolecular interactions that serve as alternatives to those in the cleft-closed conformations.
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Date Issued: 2016-10-04
Identifier: FSU_pmch_27705765, 10.1016/j.bpj.2016.08.010, PMC5052434, 27705765, 27705765, S0006-3495(16)30697-X
Format: Citation

Title: The Solution Assembly of Biological Molecules Using Ion Mobility Methods: From Amino Acids to Amyloid β-Protein..
Creator: Bleiholder, Christian, Bowers, Michael T
Abstract/Description: Ion mobility spectrometry-mass spectrometry (IMS-MS) methods are increasingly used to study noncovalent assemblies of peptides and proteins. This review focuses on the noncovalent self-assembly of amino acids and peptides, systems at the heart of the amyloid process that play a central role in a number of devastating diseases. Three different systems are discussed in detail: the 42-residue peptide amyloid-β42 implicated in the etiology of Alzheimer's disease, several amyloid-forming peptides...
Show moreIon mobility spectrometry-mass spectrometry (IMS-MS) methods are increasingly used to study noncovalent assemblies of peptides and proteins. This review focuses on the noncovalent self-assembly of amino acids and peptides, systems at the heart of the amyloid process that play a central role in a number of devastating diseases. Three different systems are discussed in detail: the 42-residue peptide amyloid-β42 implicated in the etiology of Alzheimer's disease, several amyloid-forming peptides with 6-11 residues, and the assembly of individual amino acids. We also discuss from a more fundamental perspective the processes that determine how quickly proteins and their assemblies denature when the analyte ion has been stripped of its solvent in an IMS-MS measurement and how to soften the measurement so that biologically meaningful data can be recorded.
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Date Issued: 2017-06-12
Identifier: FSU_pmch_28375705, 10.1146/annurev-anchem-071114-040304, PMC6287953, 28375705, 28375705
Format: Citation

Title: Structural Heterogeneity in Pre-40S Ribosomes.
Creator: Johnson, Matthew C, Ghalei, Homa, Doxtader, Katelyn A, Karbstein, Katrin, Stroupe, M Elizabeth
Abstract/Description: Late-stage 40S ribosome assembly is a highly regulated dynamic process that occurs in the cytoplasm, alongside the full translation machinery. Seven assembly factors (AFs) regulate and facilitate maturation, but the mechanisms through which they work remain undetermined. Here, we present a series of structures of the immature small subunit (pre-40S) determined by three-dimensional (3D) cryoelectron microscopy with 3D sorting to assess the molecule's heterogeneity. These structures demonstrate...
Show moreLate-stage 40S ribosome assembly is a highly regulated dynamic process that occurs in the cytoplasm, alongside the full translation machinery. Seven assembly factors (AFs) regulate and facilitate maturation, but the mechanisms through which they work remain undetermined. Here, we present a series of structures of the immature small subunit (pre-40S) determined by three-dimensional (3D) cryoelectron microscopy with 3D sorting to assess the molecule's heterogeneity. These structures demonstrate an extensive structural heterogeneity of interface AFs that likely regulates subunit joining during 40S maturation. We also present structural models for the beak and the platform, two regions where the low resolution of previous studies did not allow for localization of AFs and the rRNA, respectively. These models are supported by biochemical analyses using point variants and suggest that maturation of the 18S 3' end is regulated by dissociation of the AF Dim1 from the subunit interface, consistent with previous biochemical analyses.
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Date Issued: 2017-02-07
Identifier: FSU_pmch_28111018, 10.1016/j.str.2016.12.011, PMC5314460, 28111018, 28111018, S0969-2126(16)30401-4
Format: Citation

Title: Structural Influences: Cholesterol, Drug, and Proton Binding to Full-Length Influenza A M2 Protein..
Creator: Ekanayake, E Vindana, Fu, Riqiang, Cross, Timothy A
Abstract/Description: The structure and functions of the M2 protein from Influenza A are sensitive to pH, cholesterol, and the antiinfluenza drug Amantadine. This is a tetrameric membrane protein of 97 amino-acid residues that has multiple functions, among them as a proton-selective channel and facilitator of viral budding, replacing the need for the ESCRT proteins that other viruses utilize. Here, various amino-acid-specific-labeled samples of the full-length protein were prepared and mixed, so that only...
Show moreThe structure and functions of the M2 protein from Influenza A are sensitive to pH, cholesterol, and the antiinfluenza drug Amantadine. This is a tetrameric membrane protein of 97 amino-acid residues that has multiple functions, among them as a proton-selective channel and facilitator of viral budding, replacing the need for the ESCRT proteins that other viruses utilize. Here, various amino-acid-specific-labeled samples of the full-length protein were prepared and mixed, so that only interresidue (13)C-(13)C cross peaks between two differently labeled proteins representing interhelical interactions are observed. This channel is activated at slightly acidic pH values in the endosome when the His(37) residues in the middle of the transmembrane domain take on a +2 or +3 charged state. Changes observed here in interhelical distances in the N-terminus can be accounted for by modest structural changes, and no significant changes in structure were detected in the C-terminal portion of the channel upon activation of the channel. Amantadine, which blocks proton conductance by binding in the aqueous pore near the N-terminus, however, significantly modifies the tetrameric structure on the opposite side of the membrane. The interactions between the juxtamembrane amphipathic helix of one monomer and its neighboring monomer observed in the absence of drug are disrupted in its presence. However, the addition of cholesterol prevents this structural disruption. In fact, strong interactions are observed between cholesterol and residues in the amphipathic helix, accounting for cholesterol binding adjacent to a native palmitoylation site and near to an interhelix crevice that is typical of cholesterol binding sites. The resultant stabilization of the amphipathic helix deep in the bilayer interface facilitates the bilayer curvature that is essential for viral budding.
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Date Issued: 2016-03-29
Identifier: FSU_pmch_27028648, 10.1016/j.bpj.2015.11.3529, PMC4816700, 27028648, 27028648, S0006-3495(16)00152-1
Format: Citation

Title: Structural modeling for the open state of an NMDA receptor.
Creator: Pang, Xiaodong, Zhou, Huan-Xiang
Abstract/Description: NMDA receptors are tetrameric ligand-gated ion channels that are crucial for neurodevelopment and higher order processes such as learning and memory, and have been implicated in numerous neurological disorders. The lack of a structure for the channel open state has greatly hampered the understanding of the normal gating process and mechanisms of disease-associated mutations. Here we report the structural modeling for the open state of an NMDA receptor. Staring from the crystal structure of...
Show moreNMDA receptors are tetrameric ligand-gated ion channels that are crucial for neurodevelopment and higher order processes such as learning and memory, and have been implicated in numerous neurological disorders. The lack of a structure for the channel open state has greatly hampered the understanding of the normal gating process and mechanisms of disease-associated mutations. Here we report the structural modeling for the open state of an NMDA receptor. Staring from the crystal structure of the closed state, we repacked the pore-lining helices to generate an initial open model. This model was modified to ensure tight packing between subunits and then refined by a molecular dynamics simulation in explicit membrane. We identify Cα-H…O hydrogen bonds, between the Cα of a conserved glycine in one transmembrane helix and a carbonyl oxygen of a membrane-parallel helix, at the extracellular side of the transmembrane domain as important for stabilizing the open state. This observation explains why mutations of the glycine are associated with neurological diseases and lead to significant decrease in channel open probability.
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Date Issued: 2017-12-01
Identifier: FSU_pmch_28739483, 10.1016/j.jsb.2017.07.005, PMC5733723, 28739483, 28739483, S1047-8477(17)30121-1
Format: Citation

Institute of Molecular Biophysics (58)	+ -
Department of Physics (20)	+ -
Department of Chemistry and Biochemistry (18)	+ -
Department of Biological Science (12)	+ -
Program in Neuroscience (9)	+ -

Department of Mathematics (4)	+ -
National High Magnetic Field Laboratory (1)	+ -
Center for Brain Repair (1)	+ -
Department of Biomedical Sciences (1)	+ -
Department of Psychology (1)	+ -
Department of Statistics (1)	+ -
USEDiT (1)	+ -

Humans (25)	+ -
Animals (18)	+ -
Models, Molecular (11)	+ -
Protein Binding (11)	+ -
Molecular Dynamics Simulation (9)	+ -

Thermodynamics (9)	+ -
Mice (7)	+ -
Protein Conformation (7)	+ -
Binding Sites (6)	+ -
Male (6)	+ -
Female (5)	+ -
Protein Multimerization (5)	+ -
Computer Simulation (4)	+ -
Kinetics (4)	+ -
Mice, Inbred C57BL (4)	+ -
Models, Biological (4)	+ -
Protein Domains (4)	+ -
Protein Folding (4)	+ -
Protein Structure, Secondary (4)	+ -
Proteins/chemistry (4)	+ -
Receptors, N-Methyl-D-Aspartate/chemistry (4)	+ -
Receptors, N-Methyl-D-Aspartate/metabolism (4)	+ -
Algorithms (3)	+ -
Calcium/metabolism (3)	+ -
Catalytic Domain (3)	+ -
Gene Expression Regulation, Neoplastic (3)	+ -
HEK293 Cells (3)	+ -
Insulin/metabolism (3)	+ -
Intrinsically Disordered Proteins/chemistry (3)	+ -
Intrinsically Disordered Proteins/metabolism (3)	+ -

text (59)	+ -
journal article (52)	+ -
conference poster (1)	+ -

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