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- Title
- Activation Profiles of Human Kallikrein-Related Peptidases by Matrix Metalloproteinases.
- Creator
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Yoon, Hyesook, Blaber, Sachiko, Li, Wu, Scarisbrick, Isobel, Blaber, Michael
- Abstract/Description
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Abstract The 15 human kallikrein-related peptidases (KLKs) are clinically important biomarkers and therapeutic targets of interest in inflammation, cancer, and neurodegenerative disease. KLKs are secreted as inactive pro-forms (pro-KLKs) that are activated extracellularly by specific proteolytic release of their amino-terminal pro-peptide, and this is a key step in their functional regulation. Physiologically relevant KLK regulatory cascades of activation have been described in skin...
Show moreAbstract The 15 human kallikrein-related peptidases (KLKs) are clinically important biomarkers and therapeutic targets of interest in inflammation, cancer, and neurodegenerative disease. KLKs are secreted as inactive pro-forms (pro-KLKs) that are activated extracellularly by specific proteolytic release of their amino-terminal pro-peptide, and this is a key step in their functional regulation. Physiologically relevant KLK regulatory cascades of activation have been described in skin desquamation and semen liquefaction, and work by a large number of investigators has elucidated pairwise and autolytic activation relationships among the KLKs with the potential for more extensive activation cascades. More recent work has asked whether functional intersection of KLKs with other types of regulatory proteases exists. Such studies show a capacity for members of the thrombostasis axis to act as broad activators of pro-KLKs. In the present report, we ask whether such functional intersection is possible between the KLKs and the members of the matrix metalloproteinase (MMP) family by evaluating the ability of the MMPs to activate pro-KLKs. The results identify MMP-20 as a broad activator of pro-KLKs, suggesting the potential for intersection of the KLK and MMP axes under pathological dysregulation of MMP-20 expression.
Show less - Date Issued
- 2013
- Identifier
- FSU_migr_biomed_faculty_publications-0042, 10.1515/hsz-2012-0249, PMC3709557
- Format
- Citation
- Title
- Kallikrein 6 is a Novel Molecular Trigger of Reactive Astrogliosis.
- Creator
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Scarisbrick, Isobel, Radulovic, Maja, Burda, Joshua, Larson, Nadya, Blaber, Sachiko, Giannini, Caterina, Blaber, Michael, Vandell, Alexander
- Abstract/Description
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Kallikrein-related peptidase 6 (KLK6) is a trypsin-like serine protease upregulated at sites of central nervous system (CNS) injury, including de novo expression by reactive astrocytes, yet its physiological actions are largely undefined. Taken with recent evidence that KLK6 activates G-protein-coupled protease-activated receptors (PARs), we hypothesized that injury-induced elevations in KLK6 contribute to the development of astrogliosis and that this occurs in a PAR-dependent fashion. Using...
Show moreKallikrein-related peptidase 6 (KLK6) is a trypsin-like serine protease upregulated at sites of central nervous system (CNS) injury, including de novo expression by reactive astrocytes, yet its physiological actions are largely undefined. Taken with recent evidence that KLK6 activates G-protein-coupled protease-activated receptors (PARs), we hypothesized that injury-induced elevations in KLK6 contribute to the development of astrogliosis and that this occurs in a PAR-dependent fashion. Using primary murine astrocytes and the Neu7 astrocyte cell line, we show that KLK6 causes astrocytes to transform from an epitheliod to a stellate morphology and to secrete interleukin 6 (IL-6). By contrast, KLK6 reduced expression of glial fibrillary acidic protein (GFAP). The stellation-promoting activities of KLK6 were shown to be dependent on activation of the thrombin receptor, PAR1, as a PAR1-specific inhibitor, SCH79797, blocked KLK6-induced morphological changes. The ability of KLK6 to promote astrocyte stellation was also shown to be linked to activation of protein kinase C (PKC). These studies indicate that KLK6 is positioned to serve as a molecular trigger of select physiological processes involved in the development of astrogliosis and that this is likely to occur at least in part by activation of the G-protein-coupled receptor, PAR1.
Show less - Date Issued
- 2012
- Identifier
- FSU_migr_biomed_faculty_publications-0030, 10.1515/hsz-2011-0241
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- Citation
- Title
- Pharmacokinetic Properties of 2(nd)-Generation Fibroblast Growth Factor-1 Mutants for Therapeutic Application.
- Creator
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Xia, Xue, Babcock, Joseph, Blaber, Sachiko, Harper, Kathleen, Blaber, Michael
- Abstract/Description
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Fibroblast growth factor-1 (FGF-1) is an angiogenic factor with therapeutic potential for the treatment of ischemic disease. FGF-1 has low intrinsic thermostability and is characteristically formulated with heparin as a stabilizing agent. Heparin, however, adds a number of undesirable properties that negatively impact safety and cost. Mutations that increase the thermostability of FGF-1 may obviate the need for heparin in formulation and may prove to be useful "2nd-generation" forms for...
Show moreFibroblast growth factor-1 (FGF-1) is an angiogenic factor with therapeutic potential for the treatment of ischemic disease. FGF-1 has low intrinsic thermostability and is characteristically formulated with heparin as a stabilizing agent. Heparin, however, adds a number of undesirable properties that negatively impact safety and cost. Mutations that increase the thermostability of FGF-1 may obviate the need for heparin in formulation and may prove to be useful "2nd-generation" forms for therapeutic use. We report a pharmacokinetic (PK) study in rabbits of human FGF-1 in the presence and absence of heparin, as well as three mutant forms having differential effects upon thermostability, buried reactive thiols, and heparin affinity. The results support the hypothesis that heparan sulfate proteoglycan (HSPG) in the vasculature of liver, kidney and spleen serves as the principle peripheral compartment in the distribution kinetics. The addition of heparin to FGF-1 is shown to increase endocrine-like properties of distribution. Mutant forms of FGF-1 that enhance thermostability or eliminate buried reactive thiols demonstrate a shorter distribution half-life, a longer elimination half-life, and a longer mean residence time (MRT) in comparison to wild-type FGF-1. The results show how such mutations can produce useful 2nd-generation forms with tailored PK profiles for specific therapeutic application.
Show less - Date Issued
- 2012
- Identifier
- FSU_migr_biomed_faculty_publications-0041, 10.1371/journal.pone.0048210
- Format
- Citation
- Title
- Expression and Function of the Kallikrein-Related Peptidase 6 in the Human Melanoma Microenvironment.
- Creator
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Krenzer, Stefanie, Peterziel, Heike, Mauch, Cornelia, Blaber, Sachiko, Blaber, Michael, Angel, Peter, Hess, Jochen
- Abstract/Description
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Cutaneous malignant melanoma is an aggressive disease of poor prognosis. Clinical and experimental studies have provided major insight into the pathogenesis of the disease, including the functional interaction between melanoma cells and surrounding keratinocytes, fibroblasts, and immune cells. Nevertheless, patients with metastasized melanoma have a very poor prognosis and are largely refractory to clinical therapies. Hence, diagnostic tools to monitor melanoma development, as well as...
Show moreCutaneous malignant melanoma is an aggressive disease of poor prognosis. Clinical and experimental studies have provided major insight into the pathogenesis of the disease, including the functional interaction between melanoma cells and surrounding keratinocytes, fibroblasts, and immune cells. Nevertheless, patients with metastasized melanoma have a very poor prognosis and are largely refractory to clinical therapies. Hence, diagnostic tools to monitor melanoma development, as well as therapeutic targets, are urgently needed. We investigated the expression pattern of the kallikrein-related peptidase 6 (KLK6) in human melanoma tissue sections throughout tumor development. Although KLK6 was not detectable in tumor cells, we found strong KLK6 protein expression in keratinocytes and stromal cells located adjacent to benign nevi, primary melanomas, and cutaneous metastatic lesions, suggesting a paracrine function of extracellular KLK6 during neoplastic transformation and malignant progression. Accordingly, recombinant Klk6 protein significantly induced melanoma cell migration and invasion accompanied by an accelerated intracellular Ca(2+) flux. We could further demonstrate that KLK6-induced intracellular Ca(2+) flux and tumor cell invasion critically depends on the protease-activated receptor 1 (PAR1). Our data provide experimental evidence that specific inhibition of the KLK6-PAR1 axis may interfere with the deleterious effect of tumor-microenvironment interaction and represent a potential option for translational melanoma research.
Show less - Date Issued
- 2011
- Identifier
- FSU_migr_biomed_faculty_publications-0024, 10.1038/jid.2011.190
- Format
- Citation
- Title
- Functional Role of Kallikrein 6 in Regulating Immune Cell Survival.
- Creator
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Scarisbrick, Isobel, Epstein, Benjamin, Cloud, Beth, Yoon, Hyesook, Wu, Jianmin, Renner, Danielle, Blaber, Sachiko, Blaber, Michael, Vandell, Alexander, Bryson, Alexandra
- Abstract/Description
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BACKGROUND: Kallikrein 6 (KLK6) is a newly identified member of the kallikrein family of secreted serine proteases that prior studies indicate is elevated at sites of central nervous system (CNS) inflammation and which shows regulated expression with T cell activation. Notably, KLK6 is also elevated in the serum of multiple sclerosis (MS) patients however its potential roles in immune function are unknown. Herein we specifically examine whether KLK6 alters immune cell survival and the...
Show moreBACKGROUND: Kallikrein 6 (KLK6) is a newly identified member of the kallikrein family of secreted serine proteases that prior studies indicate is elevated at sites of central nervous system (CNS) inflammation and which shows regulated expression with T cell activation. Notably, KLK6 is also elevated in the serum of multiple sclerosis (MS) patients however its potential roles in immune function are unknown. Herein we specifically examine whether KLK6 alters immune cell survival and the possible mechanism by which this may occur. METHODOLOGY/PRINCIPAL FINDINGS: Using murine whole splenocyte preparations and the human Jurkat T cell line we demonstrate that KLK6 robustly supports cell survival across a range of cell death paradigms. Recombinant KLK6 was shown to significantly reduce cell death under resting conditions and in response to camptothecin, dexamethasone, staurosporine and Fas-ligand. Moreover, KLK6-over expression in Jurkat T cells was shown to generate parallel pro-survival effects. In mixed splenocyte populations the vigorous immune cell survival promoting effects of KLK6 were shown to include both T and B lymphocytes, to occur with as little as 5 minutes of treatment, and to involve up regulation of the pro-survival protein B-cell lymphoma-extra large (Bcl-XL), and inhibition of the pro-apoptotic protein Bcl-2-interacting mediator of cell death (Bim). The ability of KLK6 to promote survival of splenic T cells was also shown to be absent in cell preparations derived from PAR1 deficient mice. CONCLUSION/SIGNIFICANCE: KLK6 promotes lymphocyte survival by a mechanism that depends in part on activation of PAR1. These findings point to a novel molecular mechanism regulating lymphocyte survival that is likely to have relevance to a range of immunological responses that depend on apoptosis for immune clearance and maintenance of homeostasis.
Show less - Date Issued
- 2011
- Identifier
- FSU_migr_biomed_faculty_publications-0022, 10.1371/journal.pone.0018376
- Format
- Citation
- Title
- Functional Intersection of the Kallikrein-Related Peptidases (KLKs) and Thrombostasis Axis.
- Creator
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Blaber, Michael, Yoon, Hyesook, Juliano, Maria, Scarisbrick, Isobel, Blaber, Sachiko
- Abstract/Description
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A large body of emerging evidence indicates a functional interaction between the kallikrein-related peptidases (KLKs) and proteases of the thrombostasis axis. These interactions appear relevant for both normal health as well as pathologies associated with inflammation, tissue injury, and remodeling. Regulatory interactions between the KLKs and thrombostasis proteases could impact several serious human diseases, including neurodegeneration and cancer. The emerging network of specific...
Show moreA large body of emerging evidence indicates a functional interaction between the kallikrein-related peptidases (KLKs) and proteases of the thrombostasis axis. These interactions appear relevant for both normal health as well as pathologies associated with inflammation, tissue injury, and remodeling. Regulatory interactions between the KLKs and thrombostasis proteases could impact several serious human diseases, including neurodegeneration and cancer. The emerging network of specific interactions between these two protease families appears to be complex, and much work remains to elucidate it. Complete understanding how this functional network resolves over time, given specific initial conditions, and how it might be controllably manipulated, will probably contribute to the emergence of novel diagnostics and therapeutic agents for major diseases.
Show less - Date Issued
- 2010
- Identifier
- FSU_migr_biomed_faculty_publications-0018, 10.1515/BC.2010.024, PMC3047482
- Format
- Citation
- Title
- A Completed KLK Activome Profile: Investigation of Activation Profiles of KLK9, 10, and 15..
- Creator
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Yoon, Hyesook, Blaber, Sachiko, Debela, Mekdes, Goettig, Peter, Scarisbrick, Isobel, Blaber, Michael
- Abstract/Description
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We previously reported the activation profiles of the human kallikrein-related peptidases (KLKs) as determined from a KLK pro-peptide fusion-protein system. That report described the activity profiles of 12 of the 15 mature KLKs versus the 15 different pro-KLK sequences. The missing profiles in the prior report, involving KLK9, 10, and 15, are now described. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, mass spectrometry, and N-terminal sequence analyses show that KLK9 and 10...
Show moreWe previously reported the activation profiles of the human kallikrein-related peptidases (KLKs) as determined from a KLK pro-peptide fusion-protein system. That report described the activity profiles of 12 of the 15 mature KLKs versus the 15 different pro-KLK sequences. The missing profiles in the prior report, involving KLK9, 10, and 15, are now described. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, mass spectrometry, and N-terminal sequence analyses show that KLK9 and 10 exhibit low hydrolytic activities towards all of the 15 pro-KLK sequences, while KLK15 exhibits significant activity towards both Arg- and Lys-containing KLK pro-sequences. The ability of KLK15 to activate pro-KLK8, 12, and 14 is confirmed using recombinant pro-KLK proteins, and shown to be significant for activation of pro-KLK8 and 14, but not 12. These additional data for KLK9, 10, and 15 now permit a completed KLK activome profile, using a KLK pro-peptide fusion-protein system, to be described. The results suggest that KLK15, once activated, can potentially feed back into additional pro-KLK activation pathways. Conversely, KLK9 and 10, once activated, are unlikely to participate in further pro-KLK activation pathways, although similar to KLK1 they may activate other bioactive peptides.
Show less - Date Issued
- 2009
- Identifier
- FSU_migr_biomed_faculty_publications-0012
- Format
- Citation
- Title
- X-ray Structure and Biophysical Properties of Rabbit Fibroblast Growth Factor 1.
- Creator
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Lee, Jihun, Blaber, Sachiko, Irsigler, Andre, Aspinwall, Eric, Blaber, Michael
- Abstract/Description
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The rabbit is an important and de facto animal model in the study of ischemic disease and angiogenic therapy. Additionally, fibroblast growth factor 1 (FGF-1) is emerging as one of the most important growth factors for novel proangiogenic and pro-arteriogenic therapy. However, despite its significance, the fundamental biophysical properties of rabbit FGF-1, including its X-ray structure, have never been reported. Here, the cloning, crystallization, X-ray structure and determination of the...
Show moreThe rabbit is an important and de facto animal model in the study of ischemic disease and angiogenic therapy. Additionally, fibroblast growth factor 1 (FGF-1) is emerging as one of the most important growth factors for novel proangiogenic and pro-arteriogenic therapy. However, despite its significance, the fundamental biophysical properties of rabbit FGF-1, including its X-ray structure, have never been reported. Here, the cloning, crystallization, X-ray structure and determination of the biophysical properties of rabbit FGF-1 are described. The X-ray structure shows that the amino-acid differences between human and rabbit FGF-1 are solvent-exposed and therefore potentially immunogenic, while the biophysical studies identify differences in thermostability and receptor-binding affinity that distinguish rabbit FGF-1 from human FGF-1.
Show less - Date Issued
- 2009
- Identifier
- FSU_migr_biomed_faculty_publications-0016, 10.1107/S1744309109040287, PMC2777034
- Format
- Citation
- Title
- Engineering an Improved Crystal Contact Across a Solvent-Mediated Interface of Human Fibroblast Growth Factor 1.
- Creator
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Meher, Akshaya, Blaber, Sachiko, Lee, Jihun, Honjo, Ejiro, Kuroki, Ryota, Blaber, Michael
- Abstract/Description
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Large-volume protein crystals are a prerequisite for neutron diffraction studies and their production represents a bottleneck in obtaining neutron structures. Many protein crystals that permit the collection of high-resolution X-ray diffraction data are inappropriate for neutron diffraction owing to a plate-type morphology that limits the crystal volume. Human fibroblast growth factor 1 crystallizes in a plate morphology that yields atomic resolution X-ray diffraction data but has...
Show moreLarge-volume protein crystals are a prerequisite for neutron diffraction studies and their production represents a bottleneck in obtaining neutron structures. Many protein crystals that permit the collection of high-resolution X-ray diffraction data are inappropriate for neutron diffraction owing to a plate-type morphology that limits the crystal volume. Human fibroblast growth factor 1 crystallizes in a plate morphology that yields atomic resolution X-ray diffraction data but has insufficient volume for neutron diffraction. The thin physical dimension has been identified as corresponding to the b cell edge and the X-ray structure identified a solvent-mediated crystal contact adjacent to position Glu81 that was hypothesized to limit efficient crystal growth in this dimension. In this report, a series of mutations at this crystal contact designed to both reduce side-chain entropy and replace the solvent-mediated interface with direct side-chain contacts are reported. The results suggest that improved crystal growth is achieved upon the introduction of direct crystal contacts, while little improvement is observed with side-chain entropy-reducing mutations alone.
Show less - Date Issued
- 2009
- Identifier
- FSU_migr_biomed_faculty_publications-0017, 10.1107/S1744309109036987, PMC2777043
- Format
- Citation
- Title
- Activation Profiles of Human Kallikrein-Related Peptidases by Proteases of the Thrombostasis Axis.
- Creator
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Yoon, Hyesook, Blaber, Sachiko, Evans, D., Trim, Julie, Juliano, Maria, Scarisbrick, Isobel, Blaber, Michael
- Abstract/Description
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The human kallikrein-related peptidases (KLKs) comprise 15 members (KLK1-15) and are the single largest family of serine proteases. The KLKs are utilized, or proposed, as clinically important biomarkers and therapeutic targets of interest in cancer and neurodegenerative disease. All KLKs appear to be secreted as inactive pro-forms (pro-KLKs) that are activated extracellularly by specific proteolytic release of their N-terminal pro-peptide. This processing is a key step in the regulation of...
Show moreThe human kallikrein-related peptidases (KLKs) comprise 15 members (KLK1-15) and are the single largest family of serine proteases. The KLKs are utilized, or proposed, as clinically important biomarkers and therapeutic targets of interest in cancer and neurodegenerative disease. All KLKs appear to be secreted as inactive pro-forms (pro-KLKs) that are activated extracellularly by specific proteolytic release of their N-terminal pro-peptide. This processing is a key step in the regulation of KLK function. Much recent work has been devoted to elucidating the potential for activation cascades between members of the KLK family, with physiologically relevant KLK regulatory cascades now described in skin desquamation and semen liquefaction. Despite this expanding knowledge of KLK regulation, details regarding the potential for functional intersection of KLKs with other regulatory proteases are essentially unknown. To elucidate such interaction potential, we have characterized the ability of proteases associated with thrombostasis to hydrolyze the pro-peptide sequences of the KLK family using a previously described pro-KLK fusion protein system. A subset of positive hydrolysis results were subsequently quantified with proteolytic assays using intact recombinant pro-KLK proteins. Pro-KLK6 and 14 can be activated by both plasmin and uPA, with plasmin being the best activator of pro-KLK6 identified to date. Pro-KLK11 and 12 can be activated by a broad-spectrum of thrombostasis proteases, with thrombin exhibiting a high degree of selectivity for pro-KLK12. The results show that proteases of the thrombostasis family can efficiently activate specific pro-KLKs, demonstrating the potential for important regulatory interactions between these two major protease families.
Show less - Date Issued
- 2008
- Identifier
- FSU_migr_biomed_faculty_publications-0009
- Format
- Citation
- Title
- Kallikreins are Associated with Secondary Progressive Multiple Sclerosis and Promote Neurodegeneration.
- Creator
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Scarisbrick, Isobel, Linbo, Rachel, Vandell, Alexander, Keegan, Mark, Blaber, Sachiko, Blaber, Michael, Sneve, Diane, Lucchinetti, Claudia F., Rodriguez, Moses, Diamandis,...
Show moreScarisbrick, Isobel, Linbo, Rachel, Vandell, Alexander, Keegan, Mark, Blaber, Sachiko, Blaber, Michael, Sneve, Diane, Lucchinetti, Claudia F., Rodriguez, Moses, Diamandis, Eleftherios P.
Show less - Abstract/Description
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Tissue kallikrein KLK1 and the kallikrein-related peptidases KLK2-15 are a subfamily of serine proteases that have defined or proposed roles in a range of central nervous system (CNS) and non-CNS pathologies. To further understand their potential activity in multiple sclerosis (MS), serum levels of KLK1, 6, 7, 8 and 10 were determined in 35 MS patients and 62 controls by quantitative fluorometric ELISA. Serum levels were then correlated with Expanded Disability Status Scale (EDSS) scores...
Show moreTissue kallikrein KLK1 and the kallikrein-related peptidases KLK2-15 are a subfamily of serine proteases that have defined or proposed roles in a range of central nervous system (CNS) and non-CNS pathologies. To further understand their potential activity in multiple sclerosis (MS), serum levels of KLK1, 6, 7, 8 and 10 were determined in 35 MS patients and 62 controls by quantitative fluorometric ELISA. Serum levels were then correlated with Expanded Disability Status Scale (EDSS) scores determined at the time of serological sampling or at last clinical follow-up. Serum levels of KLK1 and KLK6 were elevated in MS patients (p
Show less - Date Issued
- 2008
- Identifier
- FSU_migr_biomed_faculty_publications-0008
- Format
- Citation
- Title
- S(1)' and S(2)' Subsite Specificities of Human Plasma Kallikrein and Tissue Kallikrein 1 for the Hydrolysis of Peptides Derived from the Bradykinin Domain of Human Kininogen.
- Creator
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Lima, Aurelio, Alves, Fabiana, Angelo, Pedro, Andrade, Douglas, Blaber, Sachiko, Blaber, Michael, Juliano, Luiz, Juliano, Maria
- Abstract/Description
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The S(1)' and S(2)' subsite specificities of human tissue kallikrein 1 (KLK1) and human plasma kallikrein (HPK) were examined with the peptide series Abz-GFSPFRXSRIQ-EDDnp and Abz-GFSPFRSXRIQ-EDDnp [X=natural amino acids or S(PO(3)H(2))]. KLK1 efficiently hydrolyzed most of the peptides except those containing negatively charged amino acids at P(1)' and P(2)' positions. Abz-GFSPFRSSRIQ-EDDnp, as in human kininogen, is the best substrate for KLK1 and exclusively cleaved the R-S bond. All other...
Show moreThe S(1)' and S(2)' subsite specificities of human tissue kallikrein 1 (KLK1) and human plasma kallikrein (HPK) were examined with the peptide series Abz-GFSPFRXSRIQ-EDDnp and Abz-GFSPFRSXRIQ-EDDnp [X=natural amino acids or S(PO(3)H(2))]. KLK1 efficiently hydrolyzed most of the peptides except those containing negatively charged amino acids at P(1)' and P(2)' positions. Abz-GFSPFRSSRIQ-EDDnp, as in human kininogen, is the best substrate for KLK1 and exclusively cleaved the R-S bond. All other peptides were cleaved also at the F-R bond. The synthetic human kininogen segment Abz-MISLMKRPPGFSPFRS(390)S(391)RI-NH(2) was hydrolyzed by KLK1 first at R-S and then at M-K bonds, releasing Lys-bradykinin. In the S(390) and S(391) phosphorylated analogs, this order of hydrolysis was inverted due to the higher resistance of the R-S bond. Abz-MISLMKRPPG-FSPFRSS(PO(3)H(2))(391)RI-NH(2) was hydrolyzed by KLK1 at M-K and mainly at the F-R bond, releasing des-(Arg(9))-Lys-Bk which is a B1 receptor agonist. HPK cleaved all the peptides at R and showed restricted specificity for S in the S(1)' subsite, with lower specificity for the S(2)' subsite. Abz-MISLMKRPPGFSPFRSSRI-NH(2) was efficiently hydrolyzed by HPK under bradykinin release, while the analogs containing S(PO(3)H(2)) were poorly hydrolyzed. In conclusion, S(1)' and S(2)' subsite specificities of KLK1 and HPK showed peculiarities that were observed with substrates containing the amino acid sequence of human kininogen.
Show less - Date Issued
- 2008
- Identifier
- FSU_migr_biomed_faculty_publications-0011
- Format
- Citation
- Title
- Protease-Activated Receptor Dependent and Independent Signaling by Kallikreins 1 and 6 in CNS Neuron and Astroglial Cell Lines.
- Creator
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Vandell, Alexander, Larson, Nadya, Laxmikanthan, Gurunathan, Panos, Michael, Blaber, Sachiko, Blaber, Michael, Scarisbrick, Isobel
- Abstract/Description
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While protease-activated receptors (PARs) are known to mediate signaling events in CNS, contributing both to normal function and pathogenesis, the endogenous activators of CNS PARs are poorly characterized. In this study, we test the hypothesis that kallikreins (KLKs) represent an important pool of endogenous activators of CNS PARs. Specifically, KLK1 and KLK6 were examined for their ability to evoke intracellular Ca(2+) flux in a PAR-dependent fashion in NSC34 neurons and Neu7 astrocytes....
Show moreWhile protease-activated receptors (PARs) are known to mediate signaling events in CNS, contributing both to normal function and pathogenesis, the endogenous activators of CNS PARs are poorly characterized. In this study, we test the hypothesis that kallikreins (KLKs) represent an important pool of endogenous activators of CNS PARs. Specifically, KLK1 and KLK6 were examined for their ability to evoke intracellular Ca(2+) flux in a PAR-dependent fashion in NSC34 neurons and Neu7 astrocytes. Both KLKs were also examined for their ability to activate mitogen-activated protein kinases (extracellular signal-regulated kinases, C-Jun N-terminal kinases, and p38) and protein kinase B (AKT) intracellular signaling cascades. Cumulatively, these studies show that KLK6, but not KLK1, signals through PARs. KLK6 evoked intracellular Ca(2+) flux was mediated by PAR1 in neurons and both PAR1 and PAR2 in astrocytes. Importantly, both KLK1 and KLK6 altered the activation state of mitogen-activated protein kinases and AKT, suggestive of important roles for each in CNS neuron and glial differentiation, and survival. The cellular specificity of CNS-KLK activity was underscored by observations that both proteases promoted AKT activation in astrocytes, but inhibited such signaling in neurons. PAR1 and bradykinin receptor inhibitors were used to demonstrate that KLK1-mediated activation of extracellular signal-regulated kinases in neurons occurred in a non-PAR, bradykinin 2 (B2) receptor-dependent fashion, while similar signaling by KLK6 was mediated by the combined activation of PAR1 and B2. Cumulatively results indicate KLK6, but not KLK1 is an activator of CNS PARs, and that both KLKs are poised to signal in a B2 receptor-dependent fashion to regulate multiple signal transduction pathways relevant to CNS physiologic function and dysfunction.
Show less - Date Issued
- 2008
- Identifier
- FSU_migr_biomed_faculty_publications-0010
- Format
- Citation
- Title
- Substrate Specificity of Human Kallikreins 1 and 6 Determined by Phage Display.
- Creator
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Li, Hai-Xin, Hwang, Bum-Yeol, Laxmikanthan, Gurunathan, Blaber, Sachiko, Blaber, Michael, Golubkov, Pavel, Ren, Pengyu, Iverson, Brent, Georgiou, George
- Abstract/Description
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The human tissue kallikrein (KLK) family contains 15 secreted serine proteases that are expressed in a wide range of tissues and have been implicated in different physiological functions and disease states. Of these, KLK1 has been shown to be involved in the regulation of multiple physiological processes such as blood pressure, smooth muscle contraction, and vascular cell growth. KLK6 is overexpressed in breast and ovarian cancer tissues and has been shown to cleave peptide derived from human...
Show moreThe human tissue kallikrein (KLK) family contains 15 secreted serine proteases that are expressed in a wide range of tissues and have been implicated in different physiological functions and disease states. Of these, KLK1 has been shown to be involved in the regulation of multiple physiological processes such as blood pressure, smooth muscle contraction, and vascular cell growth. KLK6 is overexpressed in breast and ovarian cancer tissues and has been shown to cleave peptide derived from human myelin protein and Abeta amyloid peptide in vitro. Here we analyzed the substrate specificity of KLK1 and KLK6, by substrate phage display using a random octapeptide library. Consistent with earlier biochemical data, KLK1 was shown to exhibit both trypsin- and chymotrypsin-like selectivities with Tyr/Arg preferred at site P1, Ser/Arg strongly preferred at P1', and Phe/Leu at P2. KLK6 displayed trypsin-like activity, with the P1 position occupied only by Arg and a strong preference for Ser in P1'. Docking simulations of consensus peptide provide information on the identity of the enzyme residues that are responsible for substrate binding. Bioinformatic analysis suggested several putative KLK6 protein substrates, such as ionotropic glutamate receptor (GluR) and synphilin.
Show less - Date Issued
- 2008
- Identifier
- FSU_migr_biomed_faculty_publications-0006
- Format
- Citation
- Title
- The Autolytic Regulation of Human Kallikrein-Related Peptidase 6.
- Creator
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Blaber, Sachiko, Yoon, Hyesook, Scarisbrick, Isobel, Juliano, Maria, Blaber, Michael
- Abstract/Description
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Human kallikrein-related peptidase 6 (KLK6) is a member of the kallikrein family of serine-type proteases, characterized as an arginine-specific digestive-type protease capable of degrading a wide-variety of extracellular matrix proteins. KLK6 has been proposed to be a useful biomarker for breast and ovarian cancer prognosis, is abundantly expressed in the CNS and cerebrospinal fluid, and is intimately associated with regions of active inflammatory demyelination in multiple sclerosis (MS)...
Show moreHuman kallikrein-related peptidase 6 (KLK6) is a member of the kallikrein family of serine-type proteases, characterized as an arginine-specific digestive-type protease capable of degrading a wide-variety of extracellular matrix proteins. KLK6 has been proposed to be a useful biomarker for breast and ovarian cancer prognosis, is abundantly expressed in the CNS and cerebrospinal fluid, and is intimately associated with regions of active inflammatory demyelination in multiple sclerosis (MS) lesions. Inhibition of KLK6 results in delayed onset and reduced severity of symptoms associated with experimental autoimmune encephalomyelitis, suggesting a key effector role for this protease in CNS inflammatory disease. KLK6 has been shown to autolytically cleave internally, leading to inactivation and suggesting a negative feedback inhibition control mechanism. Alternatively, the ability of KLK6 to self-activate has also been reported, suggesting a positive feedback activation loop control mechanism. Activation of pro-KLK6 requires hydrolysis after a Lys residue; however, KLK6 exhibits 2 order of magnitude reduced affinity for hydrolysis after Lys versus Arg residues; therefore, the ability to autolytically activate has been called into question. In the present study the catalytic activity of KLK6 toward its pro-sequence and internal autolytic sequence is characterized. The results show that the ability of KLK6 to activate pro-KLK6 is essentially negligible when compared to the rate of the internal autolytic inactivation or to the ability of other proteases to activate pro-KLK6. The results thus show that the primary autolytic regulatory mechanism of KLK6 is negative feedback inhibition, and activation is likely achieved through the action of a separate protease.
Show less - Date Issued
- 2007
- Identifier
- FSU_migr_biomed_faculty_publications-0002, 10.1021/bi6025006, PMC2517904
- Format
- Citation
