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Title: Matrix metalloproteinase-9 (MMP-9) in vivo substrates in left ventricle remodeling process.
Creator: Tokmina-Roszyk, Dorota, Iyer, R.P., Lindsey, M.L., Graduate College, Fields, Gregg B.
Abstract/Description: Matrix metalloproteinase-9 MMP-9 is involved in the early stages of wound healing, including the inflammatory reaction that follows myocardial infarction and neovascularization. However, its overexpression in the infarct zone leads to deleterious effects. Understanding MMP-9 function and modulation of its activity provides an opportunity to prevent excessive remodeling of the left ventricle. To assess the role of MMP-9 in remodeling process we employed a broad search of in vivo substrates....
Show moreMatrix metalloproteinase-9 MMP-9 is involved in the early stages of wound healing, including the inflammatory reaction that follows myocardial infarction and neovascularization. However, its overexpression in the infarct zone leads to deleterious effects. Understanding MMP-9 function and modulation of its activity provides an opportunity to prevent excessive remodeling of the left ventricle. To assess the role of MMP-9 in remodeling process we employed a broad search of in vivo substrates. Based on comparative analysis of MMP-9 null and wild type mice, several peptides mimicking putative substrates were synthesized. The cleavage sites in the substrates were identified using high performance liquid chromatography and mass spectrometry. Peptide mapping studies revealed MMP-9 cleavage sites in several proteins, potential biomarkers of excessive remodeling. Specifically, osteopontin, thrombospondin and C-terminal telopeptide regions of type I collagen were susceptible to proteolysis by MMP-9. The best target for MMP-9 was fibronectin, which has multiple cleavage sites in its sequence. In addition to in vivo substrate screening, a selective triple-helical peptide inhibitor MMP- 9i has been designed, synthesized, and utilized as an MMP-9 probe. The sequence of inhibitor was derived from the known MMP-9 substrate type V collagen. In the MMP-9i construct, the G~V scissile bond has been replaced with phosphinate moiety that mimics the transition state of hydrolysis but cannot be cleaved. MMP-9i's effect on MMP-9 activity in serum was tested in a mouse model. The administration of MMP-9i resulted in 30 loss of MMP-9 activity suggesting that MMP-9i can be utilized to regulate activity of MMP-9 in vivo.
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Date Issued: 2015
PURL: http://purl.flvc.org/fau/fd/FA00005917
Format: Document (PDF)

Title: Inhibition of matrix metalloproteinase-9 reduces clinical severity in a murine model of Multiple Sclerosis.
Creator: Onwuha-Ekpete, Lillian C., Tokmina-Roszyk, Dorota, Fields, Gregg B., Graduate College
Abstract/Description: Matrix metalloproteinases MMPs are a family of proteolytic enzymes that mediate the degradation of various components of the extracellular matrix. Their functions are essential for normal physiological processes such as wound healing, but their dysregulation is associated with various pathologies including autoimmune diseases such as Multiple Sclerosis MS. Experimental Autoimmune Encephalomyelitis EAE is a well-established murine model of MS that is mediated by CD4 T-cells. These cells...
Show moreMatrix metalloproteinases MMPs are a family of proteolytic enzymes that mediate the degradation of various components of the extracellular matrix. Their functions are essential for normal physiological processes such as wound healing, but their dysregulation is associated with various pathologies including autoimmune diseases such as Multiple Sclerosis MS. Experimental Autoimmune Encephalomyelitis EAE is a well-established murine model of MS that is mediated by CD4 T-cells. These cells penetrate the blood-brain-barrier BBB, recruit other immune cells, initiate destruction of the myelin sheath, and cause axonal loss. MMP-9 is a hallmark enzyme in progression of MS that is required for penetration of the BBB and generation of autoantigens in EAE. In addition, recent studies have demonstrated that MMP-9 contributes to normal intracellular function of various cell types including antigen activated T-cells; however, the intracellular role of MMP-9 in immune cell activation during EAE pathogenesis is not known. In this study, we used a highly selective MMP-9 triple-helical peptide inhibitor THPI that is a phosphinate transition state analog to examine antigen specific T-cell responses. We found that selective inhibition of MMP-9 can mitigate pathogenic T-cell activity and cellular trafficking as well as the clinical severity of EAE, suggesting that selective MMP-9 inhibition in MS can be a potent therapeutic option.
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Date Issued: 2015
PURL: http://purl.flvc.org/fau/fd/FA00005904
Format: Document (PDF)

Title: Conopeptides and methods of use.
Creator: Mari, Frank, Fields, Gregg B., Florida Atlantic University
Date Issued: 2004-09
PURL: http://purl.flvc.org/fcla/dt/15821
Format: Document (PDF)

Title: INVASION ASSAY OF MELANOMA CELLS WM115 AND WM266-4.
Creator: Davis, Dominique, Fields, Gregg B., Florida Atlantic University, Harriet L. Wilkes Honors College
Abstract/Description: Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that play an important role in tumor growth and invasion (Gialeli, 2010). They can degrade cell-adhesion molecules that allow for cell-cell and cell-extracellular matrix interactions, which is necessary for cancer cells to degrade physical barriers (Gialeli, 2010). The presence of MMPs in cancer cells help promote cell invasion. In this project, melanoma cell lines WM115 (primary) and WM266-4 (metastatic) were used...
Show moreMatrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that play an important role in tumor growth and invasion (Gialeli, 2010). They can degrade cell-adhesion molecules that allow for cell-cell and cell-extracellular matrix interactions, which is necessary for cancer cells to degrade physical barriers (Gialeli, 2010). The presence of MMPs in cancer cells help promote cell invasion. In this project, melanoma cell lines WM115 (primary) and WM266-4 (metastatic) were used to conduct the invasion assay. The invasion assay was designed and optimized to test the ability of the cell to cut and pass through the collagen membrane. Next, a general MMP inhibitor was tested for inhibition of invasion. Varying inhibitor concentrations were used to see the effects. Following that, a more specific inhibitor targeting MT-1 MMP was tested with WM266-4 cells and inhibition of invasion was observed. MT1-MMP was the target of the inhibitor because of its involvement in cell invasion and its ability to cleave the collagen membrane.
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Date Issued: 2017
PURL: http://purl.flvc.org/fau/fd/FA00012615
Format: Document (PDF)

Title: Assay development for lysyl hydroxylase.
Creator: Patel, Deepak A., Florida Atlantic University, Fields, Gregg B.
Abstract/Description: Hydroxylysine is produced as a posttranslational modification mainly in collagens, the most abundant protein in mammals. Lysyl hydroxylase (LH) is the enzyme that catalyzes the formation of hydroxylysyl residues in collagen by hydroxylation of -X-Lys-Gly- sequences, for which it requires Fe 2+, 2-oxoglutarate, O2 and ascorbate. In order to study the hydroxylation reaction catalysed by LH, we have synthesized 4 different peptides [for example, GFP*GLP*GAKGE (P*=hydroxyproline) and the...
Show moreHydroxylysine is produced as a posttranslational modification mainly in collagens, the most abundant protein in mammals. Lysyl hydroxylase (LH) is the enzyme that catalyzes the formation of hydroxylysyl residues in collagen by hydroxylation of -X-Lys-Gly- sequences, for which it requires Fe 2+, 2-oxoglutarate, O2 and ascorbate. In order to study the hydroxylation reaction catalysed by LH, we have synthesized 4 different peptides [for example, GFP*GLP*GAKGE (P*=hydroxyproline) and the corresponding hydroxylated (hydroxylysine-containing) peptide] using Fmoc solid-phase methodology. Peptides have been characterized by HPLC, MALDI-TOF mass spectrometry and CD spectroscopy. A new method for efficient separation of lysine- from hydroxylysine-containing peptides by HPLC has been developed in both organic phase (1-anthroylnitrile as derivatizating reagent) and aqueous phase (dansyl chloride as derivatizating reagent). These reagents have been used to derivatize peptides prior to HPLC analysis. The products (di- and tetra-substituted lysine- and hydroxylysine-containing peptides) have been fully separated by HPLC and their structure confirmed by MALDI-TOF MS analysis. Efficient separation of derivatized peptides will allow for the convenient and rapid measurement of LH activity by HPLC methods.
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Date Issued: 2006
PURL: http://purl.flvc.org/fcla/dt/13384
Subject Headings: Biological transport, Proteins--Metabolism, Peptides--Analysis, Coenzymes, Bioorganic chemistry
Format: Document (PDF)

Title: Construction of mini collagen ligands recognized by alpha2beta1 integrin and CD44/CSPG melanoma receptors: New method for the study of signaling pathways.
Creator: Al-Ghoul, Mohammad A., Florida Atlantic University, Fields, Gregg B.
Abstract/Description: The metastatic process involves tumor cell adhesion to basement membrane components, such as type IV collagen. A specific mitogen activated protein kinase cascade is activated by cell adhesion to type IV collagen. This activation causes the expression of proteolytic enzymes. These enzymes will then participate in compromising extracellular matrix components and enhance cell movement through them. To better understand tumor invasion of type IV collagen, we have constructed triple-helical...
Show moreThe metastatic process involves tumor cell adhesion to basement membrane components, such as type IV collagen. A specific mitogen activated protein kinase cascade is activated by cell adhesion to type IV collagen. This activation causes the expression of proteolytic enzymes. These enzymes will then participate in compromising extracellular matrix components and enhance cell movement through them. To better understand tumor invasion of type IV collagen, we have constructed triple-helical peptide (THP) ligands for melanoma cell receptors, and used these ligands to determine if receptors such as CD44/CSPG and the alpha2beta1 integrin have unique matrix metalloproteinase (MMP) signaling pathways affected by the tyrosine kinase inhibitor genistein. MMP protein expression profiles were evaluated using the alpha2beta1 integrin ligand, and CD44/CSPG ligand. Results were indicative of specific activation sequences that tumor cells undergo upon binding to select regions of type IV collagen.
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Date Issued: 2003
PURL: http://purl.flvc.org/fcla/dt/13076
Subject Headings: Collagen, Metalloproteinases, Proteolytic enzymes, Melanoma
Format: Document (PDF)

Title: Biophysical characterization of bioactive peptide amphiphiles.
Creator: Fishel, Ayala, Florida Atlantic University, Fields, Gregg B.
Abstract/Description: In this present work we have examined the biophysical characterization of two peptides. One alpha-helical (SPARC) and the other triple-helical (collagen). We have compared the effect of lipidation on stabilizing of the alpha-helical and triple-helical peptides. For the first peptide, amino acids from the angiogenesis-inducing region of SPARC were incorporated into alpha-helical peptide sequence. A Tyr was placed between the alpha-helical sequence and the peptide to provide a chromophore; Lys...
Show moreIn this present work we have examined the biophysical characterization of two peptides. One alpha-helical (SPARC) and the other triple-helical (collagen). We have compared the effect of lipidation on stabilizing of the alpha-helical and triple-helical peptides. For the first peptide, amino acids from the angiogenesis-inducing region of SPARC were incorporated into alpha-helical peptide sequence. A Tyr was placed between the alpha-helical sequence and the peptide to provide a chromophore; Lys-Ala-Glu-Ile-Glu-Ala-Leu-Lys-Ala-Glu-Ile-Glu-Ala-Leu-Lys-Ala-Tyr-Lys-His-Gly-Lys-NH 2 was the final sequence. For the second peptide, the sequence chosen to mimic the alpha2beta1 integrin binding site in type I collagen was (Gly-Pro-Hyp)4-Gly-Pro-Ala-Gly-Lys-Asp-Gly-Glu-Ala-Gly-Ala-Gln-(Gly-Pro-Hyp) 4-NH2. Next, each peptide was lipidated with a C-16 acid and was biophysically characterized to determine physiological compatibility. Techniques used included circular dichroism spectroscopy, gel electrophoresis, and Fourier transform infrared spectroscopy. Lastly, one of the peptide amphiphiles was examined as a biomaterial modifier.
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Date Issued: 2000
PURL: http://purl.flvc.org/fcla/dt/15780
Subject Headings: Peptides--Synthesis, Lipids, Bioactive compounds
Format: Document (PDF)

Title: Proteome Analysis of Melanoma Progression.
Creator: Al-Ghoul, Mohammad A., Fields, Gregg B., Florida Atlantic University
Abstract/Description: Melanoma starts on the surface of the skin where it is easily seen. It is curable when detected early, but can be fatal if allowed to progress and spread. Melanoma can spread downwards through the skin, ultimately reaching the blood and lymphatic vessels, and metastasize. Thus, one goal is to detect melanoma early before it metastasizes. A high throughput proteomics approach has been applied to better understand the processes that underlie tumor formation and progression. Three studies were...
Show moreMelanoma starts on the surface of the skin where it is easily seen. It is curable when detected early, but can be fatal if allowed to progress and spread. Melanoma can spread downwards through the skin, ultimately reaching the blood and lymphatic vessels, and metastasize. Thus, one goal is to detect melanoma early before it metastasizes. A high throughput proteomics approach has been applied to better understand the processes that underlie tumor formation and progression. Three studies were pursued: I) proteome comparison of the matched primary WM-115 and metastatic WM-266-4 melanoma cell lines; II) proteome comparison between the matched melanoma Hs 895.T and fibroblast Hs 895Sk cell lines; and III) comprehensive proteome cataloging of two metastatic melanoma cell lines Hs 895.T and SK-MEL-2. From these studies we identified proteins that are involved in cellular functions such as metabolism, signal transduction, and DNA binding, as well as structural and heat shock proteins. We hypothesized about a possible oxidative stress pathway involved in melanoma progression, initiated the creation of a melanoma proteome database, and also identified some proteins not previously studied in melanoma (such as cyclophilin A, ADP-ribosylation factor-1, 14-3-3 zeta ATP syntase, Rho GTPase, Plastin T, galectin 1 and 3, annex in II, enolase 1, cofilin, RhoGDI, Rap 1, G6PG, GAPDH, TKT, HK, and nuclear chloride channel protein). These results mark a step forward in the development of a metstatic melanoma protein database, the understanding of the chemical pathways that are involved in metastatic melanoma development, and identification of possible new targets for inhibitor development.
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Date Issued: 2007
PURL: http://purl.flvc.org/fau/fd/FA00000846
Subject Headings: Proteolytic enzymes, Melanoma--Research, Proteomics, Pharmacogenetics
Format: Document (PDF)

Title: OPTIMIZATION OF STABLE CELL-BASED OVEREXPRESSION OF MATRIX METALLOPROTEINASE 14.
Creator: Estrada, Christie-Anne, Fields, Gregg B., Florida Atlantic University, Harriet L. Wilkes Honors College
Abstract/Description: Matrix Metalloproteinase-14 (MMP-14) functions as a protease that assists in the digestion of type 1 collagen, a significant component of the extracellular matrix (ECM). MMP-14 may be essential in the metastases and invasion of cancers like lung, melanoma, and colon as well as diseases of the connective tissue. The progression of certain cancers may be constricted by developing specific enzymatic inhibitors that can target the action of this enzyme. In order to study the role of MMP-14...
Show moreMatrix Metalloproteinase-14 (MMP-14) functions as a protease that assists in the digestion of type 1 collagen, a significant component of the extracellular matrix (ECM). MMP-14 may be essential in the metastases and invasion of cancers like lung, melanoma, and colon as well as diseases of the connective tissue. The progression of certain cancers may be constricted by developing specific enzymatic inhibitors that can target the action of this enzyme. In order to study the role of MMP-14 mediated invasion, we focused on the creation of a human breast adenocarcinoma (MCF-7) cell line stably overexpressing the MMP-14 protein. This cell line will be used for future diagnostic research of cell-based MMP-14 kinetic activity in order to eventually develop an effective and specific inhibitor for this protease.
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Date Issued: 2017
PURL: http://purl.flvc.org/fau/fd/FA00012616
Format: Document (PDF)

Title: SYNTHESIS OF FMOC-GLY-VAL PHOSPHINIC PSEUDODIPEPTIDE BUILDING BLOCK FOR PRODUCTION OF TRIPLE-HELICAL PEPTIDE INHIBITORS OF MATRIX METALLOPROTEINASE-2 AND -9.
Creator: Deutsch, Michael H., Fields, Gregg B., Harriet L. Wilkes Honors College, Florida Atlantic University
Abstract/Description: Capable of reshaping the extracellular matrix, matrix metalloproteinases (MMPs) are of enormous consequence to human health. The pathologies of cancers and diseases of the skeletal, central nervous, and cardiovascular systems often owe to the overactivity of MMPs. While efforts to produce therapeutic inhibitors have been largely unsuccessful, triple-helical peptide inhibitors (THPIs) of MMPs show tremendous potential. The synthesis of phosphinic pseudodipeptide building blocks needed for...
Show moreCapable of reshaping the extracellular matrix, matrix metalloproteinases (MMPs) are of enormous consequence to human health. The pathologies of cancers and diseases of the skeletal, central nervous, and cardiovascular systems often owe to the overactivity of MMPs. While efforts to produce therapeutic inhibitors have been largely unsuccessful, triple-helical peptide inhibitors (THPIs) of MMPs show tremendous potential. The synthesis of phosphinic pseudodipeptide building blocks needed for THPIs is entirely replicable and convenient. Here we replicate a crucial step in the synthesis, the cascade bis-deprotection, and formation of Fmoc-amine. The procedure’s feasibility is demonstrated through a 77% yield of the Fmoc-Gly-Val phosphinic pseudodipeptide building block to be incorporated into THPIs of the gelatinases. In the future, it is hoped that such procedures will culminate in large-scale production of refined THPIs, enabling in-depth biochemical studies, further optimization, clinical trials, and novel therapeutics.
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Date Issued: 2022
PURL: http://purl.flvc.org/fau/fd/FAUHT00194
Format: Document (PDF)

Title: A Study to Elucidate Matrix Metalloproteinase 14 in Pancreatic Cancer Cell Lines Under Both Normoxic and Hypoxic Conditions.
Creator: Martin, Kathryn, Fields, Gregg B., Harriet L. Wilkes Honors College, Florida Atlantic University
Abstract/Description: Various key players have been implicated in the development of pancreatic cancer, among these is a member of the matrix metalloproteinase (MMP) family of proteolytic enzymes, MMP 14. This enzyme’s proteolytic activities have been implicated in cancer proliferation, invasion, and metastasis; however, little is known about its non-proteolytic and/or intracellular roles. Furthermore, research to date has focused on in vitro cell culture conditions under normoxic conditions, yet cancer exists...
Show moreVarious key players have been implicated in the development of pancreatic cancer, among these is a member of the matrix metalloproteinase (MMP) family of proteolytic enzymes, MMP 14. This enzyme’s proteolytic activities have been implicated in cancer proliferation, invasion, and metastasis; however, little is known about its non-proteolytic and/or intracellular roles. Furthermore, research to date has focused on in vitro cell culture conditions under normoxic conditions, yet cancer exists physiologically under hypoxic conditions. Under physiological hypoxic conditions members of the MMP family have been associated with altered cellular behavior. Thus, there is a need to elucidate MMP-14’s roles under both normoxic and hypoxic conditions. This study seeks to: (1) characterize the expression of MMP-14 in representative pancreatic cancer cell lines in relation to other cancer associated MMPs; (2) elucidate the impact of hypoxic conditions on MMP-14 expression and/or functionality; and (3) monitor the differences in MMP-14 at both the gene and protein expression levels.
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Date Issued: 2023
PURL: http://purl.flvc.org/fau/fd/FAUHT00263
Format: Document (PDF)

Title: DELINEATING THE INTRACELLULAR AND EXTRACELLULAR ROLE OF MATRIX METALLOPROTEINASE 14 IN PRIMARY AND METASTATIC PANCREATIC CANCERS BxPC-3 AND HPAF-II.
Creator: Gopi, Nihasika, Fields, Gregg B., Harriet L. Wilkes Honors College, Florida Atlantic University
Abstract/Description: Using flow cytometry and two forms of hypoxic induction, chemical (CoCl2) and gaseous (Tri-gas chamber), this study highlighted the advantages of quantitative analyses of MMP-14 expression (intracellularly/extracellularly) for one primary (BxPC-3) and one metastatic (HPAF-II) pancreatic cancer line. No significant changes in MMP-14 expression were observed for pancreatic cancer lines using CoCl2. Increased expression levels of MMP-14 were observed using tri-gas incubation maintaining oxygen...
Show moreUsing flow cytometry and two forms of hypoxic induction, chemical (CoCl2) and gaseous (Tri-gas chamber), this study highlighted the advantages of quantitative analyses of MMP-14 expression (intracellularly/extracellularly) for one primary (BxPC-3) and one metastatic (HPAF-II) pancreatic cancer line. No significant changes in MMP-14 expression were observed for pancreatic cancer lines using CoCl2. Increased expression levels of MMP-14 were observed using tri-gas incubation maintaining oxygen levels at 2% intracellularly for HPAF-II, but not BxPC-3. A combination of 2D/3D cell culturing techniques were also used to examine changes in cellular behavior/morphology after hypoxic exposure and a 24-hour reoxygenation cycle. BxPC-3 cells showed a greater propensity toward oxidative damage caused by reoxygenation using 2D culturing techniques. Using 3D biomimetic culturing techniques, reoxygenated BxPC-3 cells did not undergo significant apoptosis or necrosis. These results suggest that changes in cellular metabolism and behavior depend on both phenotype and culture scaffolding.
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Date Issued: 2023
PURL: http://purl.flvc.org/fau/fd/FAUHT00284
Format: Document (PDF)

Title: SYNTHESIS AND EVALUATION OF NOVEL MEMBRANE TYPE 1 MATRIX METALLOPROTEINASE (MT1-MMP) INHIBITORS.
Creator: Borges, Nicholas, Fields, Gregg B., Harriet L. Wilkes Honors College, Florida Atlantic University
Abstract/Description: Membrane type 1 matrix metalloproteinase (MT1-MMP)/(MMP-14) is a type I transmembrane metalloproteinase involved in degradation of extracellular matrix (ECM) and non-ECM substrates. MT1-MMP facilitates cell migration, and is associated with multiple physiological processes, such as morphogenesis, skeletal development, and wound healing. However, MT1-MMP overexpression causes cancer cell invasion, metastasis, and growth. Therefore, MT1-MMP activities should be regulated either through...
Show moreMembrane type 1 matrix metalloproteinase (MT1-MMP)/(MMP-14) is a type I transmembrane metalloproteinase involved in degradation of extracellular matrix (ECM) and non-ECM substrates. MT1-MMP facilitates cell migration, and is associated with multiple physiological processes, such as morphogenesis, skeletal development, and wound healing. However, MT1-MMP overexpression causes cancer cell invasion, metastasis, and growth. Therefore, MT1-MMP activities should be regulated either through endogenous tissue inhibitors of MMPs (TIMPs) or novel synthetic MT1-MMP inhibitors. The specific aim of this thesis work is the synthesis and evaluation of novel MT1-MMP C6 and C12 hit inhibitors obtained from one-bead one-compound (OBOC) libraries in order to aid in the discovery and development of effective treatments for many cancers, including melanoma, small cell lung cancer, head and neck carcinoma, bladder cancer, breast cancer, etc.
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Date Issued: 2024
PURL: http://purl.flvc.org/fau/fd/FAUHT00291
Format: Document (PDF)

Title: Development of a convenient peptide-based assay for lysyl hydroxylase.
Creator: Cudic, Mare, Patel, Deepak A., Lauer-Fields, Janelle L., Brew, Keith, Fields, Gregg B.
Date Issued: 2007-07-03
PURL: http://purl.flvc.org/fau/flvc_fau_islandoraimporter_10.1002_bip.20799_1638210547
Format: Document (PDF)

Title: Engineered Sarafotoxins as Tissue Inhibitor of Metalloproteinases-like Matrix Metalloproteinase Inhibitors.
Creator: Lauer-Fields, Janelle L., Cudic, Mare, Wei, Shuo, Mari, Frank, Fields, Gregg B., Brew, Keith
Date Issued: 2007-09
PURL: http://purl.flvc.org/fau/flvc_fau_islandoraimporter_10.1074_jbc.M611612200_1638212835
Format: Document (PDF)

Title: Bilayer Membrane Modulation of Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) Structure and Proteolytic Activity.
Creator: Cerofolini, Linda, Amar, Sabrina, Lauer, Janelle L., Martelli, Tommaso, Fragai, Marco, Luchinat, Claudio, Fields, Gregg B.
Abstract/Description: Cell surface proteolysis is an integral yet poorly understood physiological process. The present study has examined how the pericellular collagenase membrane-type 1 matrix metalloproteinase (MT1-MMP) and membrane-mimicking environments interplay in substrate binding and processing. NMR derived structural models indicate that MT1-MMP transiently associates with bicelles and cells through distinct residues in blades III and IV of its hemopexin-like domain, while binding of collagen-like triple...
Show moreCell surface proteolysis is an integral yet poorly understood physiological process. The present study has examined how the pericellular collagenase membrane-type 1 matrix metalloproteinase (MT1-MMP) and membrane-mimicking environments interplay in substrate binding and processing. NMR derived structural models indicate that MT1-MMP transiently associates with bicelles and cells through distinct residues in blades III and IV of its hemopexin-like domain, while binding of collagen-like triple-helices occurs within blades I and II of this domain. Examination of simultaneous membrane interaction and triple-helix binding revealed a possible regulation of proteolysis due to steric effects of the membrane. At bicelle concentrations of 1%, enzymatic activity towards triple-helices was increased 1.5-fold. A single mutation in the putative membrane interaction region of MT1-MMP (Ser466Pro) resulted in lower enzyme activation by bicelles. An initial structural framework has thus been developed to define the role(s) of cell membranes in modulating proteolysis.
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Date Issued: 2016-09-13
PURL: http://purl.flvc.org/fau/fd/FAUIR0000004
Format: Citation

Title: Synthesis of Fluorogenic Probes Specific for Matrix Metalloproteinase 13.
Creator: Ibrahim, Mariam, Fields, Gregg B., Leventouri, Theodora, Florida Atlantic University, Department of Chemistry and Biochemistry, Charles E. Schmidt College of Science
Abstract/Description: Matrix Metalloproteinase-13 (MMP-13) belongs to a large family of proteolytic enzymes which are characterized by their ability to degrade the extracellular matrix components. MMP-13 appears to have a critical role in tumor invasion and metastasis. In this study, several fluorogenic probes specific for MMP-13 were designed and characterized. These synthesized probes could be modified with chelators to be applied for imaging MMP-13 in breast cancer and/or multiple myeloma models. The activity...
Show moreMatrix Metalloproteinase-13 (MMP-13) belongs to a large family of proteolytic enzymes which are characterized by their ability to degrade the extracellular matrix components. MMP-13 appears to have a critical role in tumor invasion and metastasis. In this study, several fluorogenic probes specific for MMP-13 were designed and characterized. These synthesized probes could be modified with chelators to be applied for imaging MMP-13 in breast cancer and/or multiple myeloma models. The activity and selectivity of MMP-13 and other MMPs against these probes were studied through two approaches. It was found that these probes were cleaved by all MMPs, but MMP-13 showed the highest activity and selectivity towards these peptides.
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Date Issued: 2020
PURL: http://purl.flvc.org/fau/fd/FA00013507
Subject Headings: Matrix Metalloproteinases, Peptides, Fluorogenic probes
Format: Document (PDF)

Title: MONITORING COLLAGENOLYSIS UTILIZING TRIPLE HELICAL PEPTIDE PROBES.
Creator: Tokmina-Roszyk, Michal, Fields, Gregg B., Florida Atlantic University, Department of Chemistry and Biochemistry, Charles E. Schmidt College of Science
Abstract/Description: Collagen is the major structural scaffold in the body and serves as barrier between tissues, and thus its turnover is tightly regulated. Collagen triple-helical structure renders it resistant to general proteolysis. Several proteases are capable of cleaving the triplehelical regions of collagen, including several mammalian matrix metalloproteinases (MMPs) and bacterial collagenases. MMP-mediated collagenolysis is associated with numerous diseases and some bacterial collagenases have found...
Show moreCollagen is the major structural scaffold in the body and serves as barrier between tissues, and thus its turnover is tightly regulated. Collagen triple-helical structure renders it resistant to general proteolysis. Several proteases are capable of cleaving the triplehelical regions of collagen, including several mammalian matrix metalloproteinases (MMPs) and bacterial collagenases. MMP-mediated collagenolysis is associated with numerous diseases and some bacterial collagenases have found clinical application use due to their efficiency in the hydrolysis of the collagen triple-helix. A selective Förster resonance energy transfer triple-helical peptide (fTHP) probe for monitoring the activity of Clostridial collagenase has been developed. The fTHP [sequence: Gly-mep-Flp-(Glyvi Pro-Hyp)4-Gly-Lys(Mca)-Thr-Gly-Pro-Leu-Gly-Pro-Pro-Gly-Lys(Dnp)-Ser-(Gly-Pro-Hyp)4-NH2] was stable at 37 °C and was efficiently hydrolyzed by bacterial collagenase (kcat/KM = 25,000 s -1 M-1) but not by clostripain, trypsin, neutral protease, thermolysin, or elastase. The bacterial collagenase fTHP assay can be utilized in applications where specific activity towards triple-helical collagen needs to be evaluated, such as isolation of cells from various tissues. An fTHP scaffold was also utilized to evaluate the sequence preferences of eight MMPs. Residues spanning from P3 to P11 investigated using a positional scanning synthetic combinatorial library. Deconvolution of the library data revealed distinct motifs for several MMPs and discrimination among closely related MMPs. The results of this study show that the P10 11 substrate play an important role in the collagenase-substrate interactions and that modifying these residues can drastically affect the affinity of MMPs towards THP substrates. The identified sequence preferences of MMPs will enable the design of selective triple-helical MMP probes that could be used for monitoring in vivo enzyme activity and enzyme-facilitated drug delivery.
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Date Issued: 2019
PURL: http://purl.flvc.org/fau/fd/FA00013422
Subject Headings: Collagen, Peptides, Matrix Metalloproteinases, Microbial Collagenase, Molecular Probes
Format: Document (PDF)

Title: THE ROLE OF MATRIX METALLOPROTEINASE-28 IN HEALTH AND DISEASE.
Creator: Tokmina-Roszyk, Dorota, Fields, Gregg B., Florida Atlantic University, Department of Chemistry and Biochemistry, Charles E. Schmidt College of Science
Abstract/Description: Matrix Metalloproteinase-28 (MMP-28) is the newest and least characterized member of MMP family. To date several potential substrate candidates for MMP-28 have been proposed but no in vivo substrates for this enzyme were confirmed. In the central nervous system (CNS) MMP-28 is believed to be important factor during myelination of the developing nervous system as well as during remyelination that follows neuronal injury. On the other hand, MMP-28 has been found in actively demyelinating...
Show moreMatrix Metalloproteinase-28 (MMP-28) is the newest and least characterized member of MMP family. To date several potential substrate candidates for MMP-28 have been proposed but no in vivo substrates for this enzyme were confirmed. In the central nervous system (CNS) MMP-28 is believed to be important factor during myelination of the developing nervous system as well as during remyelination that follows neuronal injury. On the other hand, MMP-28 has been found in actively demyelinating lesions in both experimental autoimmune encephalopathy (EAE) and multiple sclerosis patients suggesting its possible role in pathological events associated with autoimmune neurodegenerative processes. In addition, MMP-28 has been linked to modulation of immune response and activation of macrophages which presents another role of this enzyme in autoimmune pathologies. In the study described herein, MMP-28 has been shown to affect myelin composition and appearance, mitochondrial protein content, and vesicular transport proteins. Moreover, the decrease in myelin basic protein quantity observed in healthy MMP-28KO animals affected the myelin staining intensity in various brain regions including corpus callous. Cellular energetic studies did not reveal differences in mitochondrial function in MMP-28KO animals and no difference in reactive oxygen species was observed. In the EAE model, MMP-28 deletion increased the occurrence of atypical form of EAE characterized by increased inflammation of arbor vitae of the brain. In addition, MMP-28 deletion decreased the inflammatory infiltrates present in brains obtained from EAE animals. Lastly, MMP-28 has been shown to affect cellular energetics and activation of bone marrow derived macrophages during the initial stages and after 24 h activation. In addition, MMP-28 deletion increased proinflammatory cytokines and receptors CD86 and iNOS found in M1 polarized macrophages.
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Date Issued: 2020
PURL: http://purl.flvc.org/fau/fd/FA00013601
Subject Headings: Matrix Metalloproteinases, Multiple sclerosis, Neurodegenerative disease
Format: Document (PDF)

Title: Targeted Drug Delivery Utilizing a Mini-Collagen Ligand Recognized by CD44/CSPG Melanoma Receptor.
Creator: Khan, David R., Florida Atlantic University, Fields, Gregg B., Charles E. Schmidt College of Science, Department of Chemistry and Biochemistry
Abstract/Description: Liposomes of varying lipid compositions are currently used as drug carriers. and great efforts have been made to target these vehicles to specific cellular receptors. Previous targeting components include peptides. proteins. antibodies. or vitamins. CD44/CSPG is among the receptors overexpressed in metastatic melanoma. and the sequence to which it binds within the type IV collagen triple-helix has been identified. A triple-helical '·peptide-amphiphile .. (al(JV)1263-1277 PA) which binds CD44...
Show moreLiposomes of varying lipid compositions are currently used as drug carriers. and great efforts have been made to target these vehicles to specific cellular receptors. Previous targeting components include peptides. proteins. antibodies. or vitamins. CD44/CSPG is among the receptors overexpressed in metastatic melanoma. and the sequence to which it binds within the type IV collagen triple-helix has been identified. A triple-helical '·peptide-amphiphile .. (al(JV)1263-1277 PA) which binds CD44/CSPG has been constructed and incorporated into liposomes of differing lipid compositions. Liposomes containing distearoyl phosphatidylcholine (DSPC) as the major bilayer component. in combination with distearoyl phosphatidylglycerol (DSPG) and cholesterol. were more stable than analogous liposomes containing dipalmitoyl phosphatidylcholine (DPPC) instead of DSPC. The presence of the al(JV)1263-1277 PA conferred greater stability to the DPPC liposomal systems. and did not affect the stability of the DSPC liposomes. The addition of either PEG 750 or PEG 2000 (5 mol %) to DSPG/DSPC/Chol liposomes did not affect the stability of this system. Fluorophore delivery of rhodamine loaded liposomes to cells varymg 111 CD44/CSPG expresston was determined usmg fluorescence microscopy. The CD44/CSPG receptor content for two normal fibroblast cell lines, BJ and Hs895Sk, and a highly metastatic melanoma derived cell line, M 14#5. was determined using whole cell ELISA. The results of the ELISA showed varying levels of CD44 receptors amongst the cell lines. with M 14#5 cells having the most. A positive correlation was observed for cellular fluorophore delivery by the ai(IV)1263-1277 PA liposomes and CD44/CSPG receptor content. Conversely, non-targeted liposomes delivered minimal fluorophore to cells regardless of the CD44/CSPG receptor content. When cells were treated with exogeneous a I (IV) 1263-1277, prior to incubation with a I (IV) 1263 -1277 PA liposomes, a dose-dependent decrease in the amount of fluorophore delivered was observed with respect to increasing concentrations of exogeneous al(IV)1263-1277. In addition, we have found a positive correlation between the cytotoxic effect of ai(IV)l263-1277 PA targeted liposomes (with and without PEG) loaded with doxorubicin and the CD44/CSPG content amongst the three different cell lines. This trend was not observed with non-targeted liposomes. These findings provide the possibility of a novel drug carrier system to be used in future clinical applications against metastatic melanoma.
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Date Issued: 2007
PURL: http://purl.flvc.org/fau/fd/FA00000866
Subject Headings: Tumor suppressor proteins, Antioncogenes, Drug targeting, Melanoma--Treatment
Format: Document (PDF)

FAU Theses and Dissertations (9)	+ -
Honors Student Theses (6)	+ -
FAU College Collections (5)	+ -
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Florida Atlantic University Patents (1)	+ -

Matrix Metalloproteinases (3)	+ -
Collagen (2)	+ -
Melanoma--Research (2)	+ -
Peptides (2)	+ -
Proteolytic enzymes (2)	+ -

Antioncogenes (1)	+ -
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Bioorganic chemistry (1)	+ -
Coenzymes (1)	+ -
Drug targeting (1)	+ -
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Lipids (1)	+ -
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Electronic Thesis or Dissertation (9)	+ -
thesis (6)	+ -
article (5)	+ -
poster (2)	+ -
patent (1)	+ -

Fields, Gregg B. (23)	+ -
Florida Atlantic University (16)	+ -
Harriet L. Wilkes Honors College (6)	+ -
Charles E. Schmidt College of Science (5)	+ -
Department of Chemistry and Biochemistry (5)	+ -

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