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Title: MECHANISTIC STUDIES OF THE PLP-DEPENDENT ENZYME, METHIONINE GAMMA-LYASE AND THE HEME-DEPENDENT ENZYME, INDOLEAMINE 2,3-DIOXYGENASE.
Creator: Foo, Timothy, Terentis, Andrew C., Florida Atlantic University, Department of Chemistry and Biochemistry, Charles E. Schmidt College of Science
Abstract/Description: Methionine-γ-lyase (MGL) is a pyridoxal-5′-phosphate (PLP) dependent enzyme found in bacteria and protozoa that catalyzes a variety of reactions, including the γ-elimination of L-methionine (L-Met). Besides its physiological roles in organisms, MGL is also a therapeutic target for pathogenic diseases and cancer. Since MGL’s catalytic mechanism remains uncertain, a new spectrophotometric assay was validated for measuring the kinetics of MGL catalyzed reactions. Kinetic data and density...
Show moreMethionine-γ-lyase (MGL) is a pyridoxal-5′-phosphate (PLP) dependent enzyme found in bacteria and protozoa that catalyzes a variety of reactions, including the γ-elimination of L-methionine (L-Met). Besides its physiological roles in organisms, MGL is also a therapeutic target for pathogenic diseases and cancer. Since MGL’s catalytic mechanism remains uncertain, a new spectrophotometric assay was validated for measuring the kinetics of MGL catalyzed reactions. Kinetic data and density functional theory (DFT) data for the γ-elimination reaction of L-Met and several other substrate analogues by MGL from P. gingivalis is reported. A direct correlation between experimental kcat values and DFT-calculated activation energies of the γ-cleavage reaction of the enamine intermediate was identified for various substrates. Considering these data, we propose a catalytic mechanism for MGL catalysis, whereby the γ-cleavage step is rate-limiting. This conclusion has direct implications for the rational design of substrates or inhibitors aimed at regulating MGL activity. PLP-dependent enzymes are present in all organisms and account for ~4% of all discovered enzyme activities. They all possess a common external aldimine and quinonoid intermediate at the start of their reaction pathway but likely diverge thereafter into different reaction pathways based on their substrate(s) and active site environment. To yield insight into the divergent reactivity of the quinonoid intermediate, DFT calculations of several PLP-dependent enzymes with unique reactivity revealed that the charge distribution is modulated on Cα and C4’, which allows for the regioselectivity of the quinonoid intermediate. The mammalian heme enzyme indoleamine 2,3-dioxygenase-1 (IDO1) catalyzes the first reaction of L-tryptophan (L-Trp) oxidation along the kynurenine pathway. IDO1 is a central immunoregulatory enzyme with important implications for inflammation, infectious disease, autoimmune disorders, and cancer. Since IDO1’s catalytic mechanism remains uncertain, kinetic data and DFT data for the dioxygenation reaction of L-Trp and several other substrate analogues by human IDO1 is reported. A direct correlation between experimental kcat values and DFT-calculated activation energies of the C2-O cleavage reaction of the L-Trp-epoxide intermediate was identified for various substrates. This conclusion has direct implications for the rational design of substrates or inhibitors aimed at regulating IDO1 activity and yields insight into heme-dependent dioxygenation chemistry.
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Date Issued: 2021
PURL: http://purl.flvc.org/fau/fd/FA00013870
Subject Headings: Lyases, Enzymes
Format: Document (PDF)

Title: Kinetics and mechanistic studies of molybdenum(2) oxygen(3) (diethyl dtc)(2) (THF)(2) iodine(2).
Creator: Abburi, Chakravarthi., Florida Atlantic University, Baird, Donald M.
Abstract/Description: A study of the kinetic behavior of the oxygen atom transfer reaction of Mo2o3 (Et2dtc)2 (THF) 2I2, (I), with a variety of derivatives of pyridine N-oxides has been performed. The reaction behaves in pseudo-first-order fashion under conditions of excess N-oxide. Plots of kobs vs. [N-oxide] do not give straight lines, while plots of 1/kobs vs. 1/[N-oxide] are linear. The rate constant k is taken as the inverse of the slope of this latter plot. There is a marked dependence of the rate on the...
Show moreA study of the kinetic behavior of the oxygen atom transfer reaction of Mo2o3 (Et2dtc)2 (THF) 2I2, (I), with a variety of derivatives of pyridine N-oxides has been performed. The reaction behaves in pseudo-first-order fashion under conditions of excess N-oxide. Plots of kobs vs. [N-oxide] do not give straight lines, while plots of 1/kobs vs. 1/[N-oxide] are linear. The rate constant k is taken as the inverse of the slope of this latter plot. There is a marked dependence of the rate on the functional group in the 4-position of the pyridine ring with the relative rates -CH3 > -H > -Cl > -CN. The above observations can be used to support a mechanism in which a rapid pre-equilibrium, with a large equilibrium constant, is followed by a rate-determining breaking of the Mo-O-Mo bridge.
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Date Issued: 1992
PURL: http://purl.flvc.org/fcla/dt/14828
Subject Headings: Enzyme kinetics, Molybdenum enzymes, Chemical kinetics
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: Development of methods for the immobilization of enzymes used in 9,11-secosteroid biosynthesis.
Creator: Chen, Ying, Florida Atlantic University, Kerr, Russell G.
Abstract/Description: 9,11-Secosteroids exhibit novel biological activity and interesting pharmacological properties. A biosynthetic study of 9,11-secogorgosterol and other novel 9,11-secosteroids using an enzyme extract from the gorgonian Pseudopterogorgia americana demonstrated that this system is an efficient catalyst for a wide range 9,11-secosteroid production. Two methods to recover the enzyme from 9,11-secosteroid synthesis have been developed: (1) membrane-enclosed enzymatic catalysis, which combines...
Show more9,11-Secosteroids exhibit novel biological activity and interesting pharmacological properties. A biosynthetic study of 9,11-secogorgosterol and other novel 9,11-secosteroids using an enzyme extract from the gorgonian Pseudopterogorgia americana demonstrated that this system is an efficient catalyst for a wide range 9,11-secosteroid production. Two methods to recover the enzyme from 9,11-secosteroid synthesis have been developed: (1) membrane-enclosed enzymatic catalysis, which combines certain of the advantages of soluble enzymes and immobilized enzymes, shows high activity, simple recovery and at least 5 cycles or 23 days; (2) covalently bonded enzyme-agarose gel, attached by cyanogen bromide via the amino groups of enzymes, show 80% activity of free enzymes and at least 7 cycles or 46 days. Based on stability and kinetic studies of these immobilized enzymes in 9,11-secosteroid synthesis, a continuous flow column reactor for scale-up of 9,11-secosteroid production has been developed with great efficiency, which represents the first enzymatic production of a bioactive compound from a marine invertebrate on a large scale.
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Date Issued: 1998
PURL: http://purl.flvc.org/fcla/dt/15521
Subject Headings: Octocorallia, Enzymes--Synthesis, Steroids
Format: Document (PDF)

Title: Biosynthesis and enzymology of secosteroids from Pseudopterogorgia americana.
Creator: Head, Kevin, Florida Atlantic University, Kerr, Russell G.
Abstract/Description: 9,11-Secosteroids are biologically active secondary metabolites from the marine invertebrate, Pseudopterogorgia americana. They are produced in vitro through an incubation of the steroid substrate with a cell-free extract of P. americana containing the necessary enzymes. Further optimization of this incubation was attempted through adjustment of incubation reagents and cofactors. The enzymes responsible for secosteroid production were partially purified through chromatography in an effort to...
Show more9,11-Secosteroids are biologically active secondary metabolites from the marine invertebrate, Pseudopterogorgia americana. They are produced in vitro through an incubation of the steroid substrate with a cell-free extract of P. americana containing the necessary enzymes. Further optimization of this incubation was attempted through adjustment of incubation reagents and cofactors. The enzymes responsible for secosteroid production were partially purified through chromatography in an effort to isolate and purify these enzymes. Finally, experiments with radiolabeled gorgosterol led to the elucidation and isolation of a key secosteroid precursor, 9,11-dihydroxygorgosterol.
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Date Issued: 1999
PURL: http://purl.flvc.org/fcla/dt/15711
Subject Headings: Alcyonacea, Enzymes--Synthesis, Sterols
Format: Document (PDF)

Title: First report of the little-known scyphomedusa Drymonema dalmatinum in the Caribbean Sea, with notes on its biology.
Creator: Larson, R. J., Harbor Branch Oceanographic Institute
Date Issued: 1987
PURL: http://purl.flvc.org/FCLA/DT/3172776
Subject Headings: Scyphozoa, Jellyfishes, Pelagic fishes, Proteolytic enzymes, Hyperiidae
Format: Document (PDF)

Title: Chemoenzymatic synthesis of 9,11-secosteroids using an enzyme extract from a marine coral.
Creator: Rodriguez, Lesbeth Caridad., Florida Atlantic University, Kerr, Russell G.
Abstract/Description: 9,11-Secogorgosterol, a secondary metabolite from the gorgonian Pseudopterogorgia americana, exhibits inhibitory activity against protein kinase C, and potent anti-proliferative and anti-inflammatory activity. An efficient method for the production of 9,11-secogorgosterol has been developed and optimized using an enzyme extract from the gorgonian P. americana. The gorgonian also produces two other 9,11-secosteroids which have marked differences in their side chains and nuclei, which suggested...
Show more9,11-Secogorgosterol, a secondary metabolite from the gorgonian Pseudopterogorgia americana, exhibits inhibitory activity against protein kinase C, and potent anti-proliferative and anti-inflammatory activity. An efficient method for the production of 9,11-secogorgosterol has been developed and optimized using an enzyme extract from the gorgonian P. americana. The gorgonian also produces two other 9,11-secosteroids which have marked differences in their side chains and nuclei, which suggested that the enzymes responsible for their production were likely relatively nonspecific. Novel 9,11-secosteroids have been synthesized using the enzyme extract from the gorgonian.
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Date Issued: 1997
PURL: http://purl.flvc.org/fcla/dt/15431
Subject Headings: Steroids, Enzymes--Synthesis, Alcyonacea, Corals
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: Polyketide synthase genes from marine dinoflagellates.
Creator: Snyder, Richard V., Gibbs, P. D. L., Palacios, A., Abiy, L., Dickey, R., Lopez, Jose V., Rein, Kathleen S., Harbor Branch Oceanographic Institute
Date Issued: 2003
PURL: http://purl.flvc.org/FCLA/DT/2795618
Subject Headings: Dinoflagellates --Physiology, Carbonyl compounds, Polyketides, Cell biology, Enzyme activation
Format: Document (PDF)

Title: Discorhabdin P, a new enzyme inhibitor from a deep-water Caribbean sponge of the genus Batzella.
Creator: Gunasekera, Sarath P., McCarthy, Peter J., Longley, Ross E., Pomponi, Shirley A., Wright, Amy E., Lobkovsky, E., Clardy, J.
Date Issued: 1999
PURL: http://purl.flvc.org/FCLA/DT/3319100
Subject Headings: Sponges --Research, Marine natural products, discorhabdin P, Enzyme inhibitors
Format: Document (PDF)

Title: In search of MMP specific inhibitors: protein engineering of TIMPs.
Creator: Bahudhanapati, Harinathachari., Charles E. Schmidt College of Science, Department of Biomedical Science
Abstract/Description: The tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of the matrix metalloproteinases (MMPs). Since unregulated MMP activities are linked to arthritis, cancer, and atherosclerosis, TIMP variants that are selective inhibitors of disease-related MMPs have potential therapeutic value. The structures of TIMP/MMP complexes reveal that most interactions with the MMP involve the N-terminal region of TIMP and the C-D B-strand connector which occupy the primed (right side of...
Show moreThe tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of the matrix metalloproteinases (MMPs). Since unregulated MMP activities are linked to arthritis, cancer, and atherosclerosis, TIMP variants that are selective inhibitors of disease-related MMPs have potential therapeutic value. The structures of TIMP/MMP complexes reveal that most interactions with the MMP involve the N-terminal region of TIMP and the C-D B-strand connector which occupy the primed (right side of the active site) and unprimed (left side) regions of the active site. Substitutions for Thr2 of N-TIMP- 1 strongly influence MMP selectivity. In this study we found that Arg and Gly, which generally reduce MMP affinity, have less effect on binding to MMP-9. When the Arg mutation is added to the NTIMP-1 mutant with AB loop of TIMP-2, it produced a gelatinase-specific inhibitor with Ki values of 2.8 and 0.4 nM for MMP-2 and MMP-9, respectively. The Gly mutant has a Ki of 2.1 nM for MMP-9 and > 40 uM for MMP-2, indicating that engineered TIMPs can discriminate between MMPs in the same subfamily. In collaboration with Dr. Yingnan Zhang at Genentech, we have developed a protocol for the phage display of full-length human TIMP-2 to identify high-affinity selective inhibitors of human MMP-1, a protease that plays a role in cleaving extracellular matrix (ECM) components, connective tissue remodeling during development, angiogenesis, and apoptosis. We have generated a library containing 2x1010 variants of TIMP-2 randomized at residues 2-6 (L1), at residues 34-40 (L2) and 67-70 (L3)., The L1 library yielded a positive signal for MMP-1 binding. Clones from the L1 library, designated TM1, TM8, TM13, and TM14, were isolated after 5 rounds of selection on immobilized MMP-1 and MMP-3 and found to show a greater selectivity for MMP-1 relative to MMP-3. TM8, which has Ser2 to Asp and Ser4 to Ala substitutions, showed the greatest apparent selectivity of 10-fold toward MMP-1 compared to MMP-3. The various mutations identified by phage display were introduced into recombinant Nterminal TIMP-2 and the variants characterized as inhibitors of an array of MMP catalytic domains. The TM8-based mutant showed pronounced selectivity (> 1000-fold for MMP-1 vs. MMP-3) and may be a step towards the generation of MMP-1-specific inhibitors. Molecular modeling was used to rationalize the structural basis of MMP selectivity in the mutants.
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Date Issued: 2009
PURL: http://purl.flvc.org/FAU/221942
Subject Headings: Metalloproteinases, Inhibitors, Apoptosis, Extracellular matrix proteins, Proteolytic enzymes
Format: Document (PDF)

Title: Thermodynamic Origins of Selectivity in the Interactions of N- TIMP Variants and Metalloproteinases Catalytic Domains.
Creator: Zou, Haiyin, Brew, Keith, Florida Atlantic University, Charles E. Schmidt College of Medicine, Department of Biomedical Science
Abstract/Description: Matrix metalloproteinases (MMPs) constitute the major class of enzymes capable of degrading all protein components of extracellular matrix (ECM) and have important roles in normal physiologic processes of maintaining tissue integrity and remodeling. However, excess MMP activities are associated with many diseases including rheumatoid arthritis and osteoarthritis, cardiomyopathy, and macular degeneration. The activity of MMPs is regulated by their endogenous protein inhibitors, the tissue...
Show moreMatrix metalloproteinases (MMPs) constitute the major class of enzymes capable of degrading all protein components of extracellular matrix (ECM) and have important roles in normal physiologic processes of maintaining tissue integrity and remodeling. However, excess MMP activities are associated with many diseases including rheumatoid arthritis and osteoarthritis, cardiomyopathy, and macular degeneration. The activity of MMPs is regulated by their endogenous protein inhibitors, the tissue inhibitors of metalloproteinases (TIMPs) which are avid broad-spectrum inhibitors of numerous human matrixins (MMPs and ADAMs). Uncontrolled matrix degradation occurs when the balance between TIMPs and MMPs is disrupted, resulting in serious diseases such as cancer, arthritis and chronic tissue ulcers. Thus, the engineering of TIMPs to produce highly selective and efficacious inhibitors of individual MMPs may be utilized for future treatment of diseases. Such engineering requires detailed analysis for the structural and biophysical information of MMP-TIMP interaction. Changes in the dynamics of proteins and solvent that accompany their associations with different binding partners, influence the specificity of binding through entropic effects. From the current studies it appears that the interactions of the inhibitory domains of TIMPs-1 and -2 (N-TIMPs) with MT1-MMP are driven by entropy increases that are partitioned between solvent and conformational entropy (ΔSsolv and ΔSconf), and a large conformational entropy penalty is responsible for the weak inhibition of MT1-MMP by NT1.We investigated how mutations that modify N-TIMP selectivity affect the thermodynamics of interactions with MMP1, MMP3 and MT1-MMP. The weak inhibition of MT1-MMP by N-TIMP-1 is enhanced by mutation of threonine 98, on the edge of the binding ridge, to leucine. This mutation increases the large ΔSconf cost for binding to MT1-MMP but this is offset by a greater increase in ΔSsolv. In contrast, this mutation enhances binding to MMP3 by increasing ΔSconf for the interaction. ΔSsolv and ΔSconf show mutual compensation for all interactions, with characteristic ranges for each MMP. Distinct electrostatic and dynamic features of MMPs are key factors in their selective inhibition.
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Date Issued: 2016
PURL: http://purl.flvc.org/fau/fd/FA00004643
Subject Headings: Metalloproteinases -- Inhibitors., Proteolytic enzymes., Extracellular matrix proteins., Apoptosis.
Format: Document (PDF)

Title: Thermodynamics-structure correlations of interactions between metalloproteinases and tissue inhibitors of metalloproteinase variants.
Creator: Wu, Ying., Charles E. Schmidt College of Science, Department of Biological Sciences
Abstract/Description: The 23 matrix metalloproteinases (MMPs) in humans catalyze the turnover of all protein components of the extracellular matrix (ECM) and have important roles in tissue remodeling, wound healing, embryo implantation, cell migration and shedding of cell surface proteins. Excess MMP activities are associated with many diseases including arthritis, heart disease and cancer. The activities of MMPs are regulated by a family of four protein inhibitors, the tissue inhibitors of metalloproteinases ...
Show moreThe 23 matrix metalloproteinases (MMPs) in humans catalyze the turnover of all protein components of the extracellular matrix (ECM) and have important roles in tissue remodeling, wound healing, embryo implantation, cell migration and shedding of cell surface proteins. Excess MMP activities are associated with many diseases including arthritis, heart disease and cancer. The activities of MMPs are regulated by a family of four protein inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), that are endogenous inhibitors of matrix metalloproteinases (MMPs), ADAMs (A Disintegrin And Metalloproteinase) and ADAMTS (disintegrin-metalloproteinase with thrombospmdin motifs) .... The balance between TIMPs and active metzinicins is very important and imbalances are linked to human diseases such as arthritis, cancer, and atherosclerosis. The engineering of TIMPs to produce specific inhibitors of individual MPs could provide new therapeutic principles for disease treatment, but this requires a detailed understanding of the biophysical and structural basis of the interactions of TIMPs and MMPs and ADAMs.
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Date Issued: 2012
PURL: http://purl.flvc.org/FAU/3356904
Subject Headings: Proteolytic enzymes, Metalloproteinase, Inhibitors, Apoptosis, Extracellular matrix proteins
Format: Document (PDF)

Title: Topological Specificity in Inhibitor Recognition by Matrix Metalloproteinases.
Creator: Lauer-Fields, Janelle, Florida Atlantic University, Brew, Keith, Charles E. Schmidt College of Medicine, Department of Biomedical Science
Abstract/Description: Alterations in activities of one family of proteases, the metzincins have been implicated in an array of physiological and pathological processes. In the present study, metzincin inhibitors were developed by utilizing topologically constrained peptides and pseudopeptides. The endothelin-family framework was used to develop a disulfideconstrained topology. This framework was chosen due to its three-dimensional similarity with a family of endogenous metzincin inhibitors, the tissue inhibitors...
Show moreAlterations in activities of one family of proteases, the metzincins have been implicated in an array of physiological and pathological processes. In the present study, metzincin inhibitors were developed by utilizing topologically constrained peptides and pseudopeptides. The endothelin-family framework was used to develop a disulfideconstrained topology. This framework was chosen due to its three-dimensional similarity with a family of endogenous metzincin inhibitors, the tissue inhibitors of metalloproteases (TIMPs). The collagenous triple-helix was chosen as a second framework, because only a subset of proteolytic enzymes have the capacity to bind and hydrolyze a triple-helix. Both templates were successfully modified to generate an array of inhibitors. These inhibitors displayed subnanomolar to micromolar apparent Ki values, while being moderately selective metzincin inhibitors. In both cases the threedimensional structure was determined to be important for activity. This work encourages the further development of both frameworks as metzincin inhibitors.
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Date Issued: 2007
PURL: http://purl.flvc.org/fau/fd/FA00000867
Subject Headings: Metalloproteinases--Inhibitors, Proteolytic enzymes, Extracellular matrix proteins
Format: Document (PDF)

Title: Characterization and optimization of in vitro assay conditions for (1,3)β-glucan synthase activity from Aspergillus fumigatus and Candida albicans for enzyme inhibition screening.
Creator: Wood, R. L., Miller, T. K., Wright, Amy E., McCarthy, Peter J., Taft, C. S., Pomponi, Shirley A., Selitrennikoff, Claude, Harbor Branch Oceanographic Institute
Date Issued: 1998
PURL: http://purl.flvc.org/FCLA/DT/3351974
Subject Headings: Aspergillus fumigatus, Candida albicans, Glucosyltransferases, In vitro, Glucan synthase, Enzyme inhibitors
Format: Document (PDF)

Title: Aplysillin A, a thrombin receptor antagonist from the marine sponge, Aplysina fistularis fulva.
Creator: Gulavita, N. K., Pomponi, Shirley A., Wright, Amy E., Garay, M., Sills, Matthew A.
Date Issued: 1995
PURL: http://purl.flvc.org/FCLA/DT/3319084
Subject Headings: Sponges --Research, Marine natural products, Receptors, Thrombin, Aplysillin A, Antagonists, Enzyme, Marine pharmacology
Format: Document (PDF)

Title: Proteomic identification of N86-hnRNPU-interacting proteins involved in HIV-1 inhibition.
Creator: Hoxha, Sarah., Harriet L. Wilkes Honors College
Abstract/Description: HIV-1 is the human immunodeficiency virus that can lead to acquired immune deficiency syndrome, or AIDS. Multiple cellular proteins have been identified as playing a critical role in all steps of HIV-1 replication. The heterogeneous nuclear ribonuclear protein U, or hnRNPU is a RNA and DNA binding protein known to influence pre-mRNA processsing, transport to the cytoplasm, intracellular localization, translation and turnover of mRNAs. Recently, the expression of N86-hnRNPU, an N-terminal...
Show moreHIV-1 is the human immunodeficiency virus that can lead to acquired immune deficiency syndrome, or AIDS. Multiple cellular proteins have been identified as playing a critical role in all steps of HIV-1 replication. The heterogeneous nuclear ribonuclear protein U, or hnRNPU is a RNA and DNA binding protein known to influence pre-mRNA processsing, transport to the cytoplasm, intracellular localization, translation and turnover of mRNAs. Recently, the expression of N86-hnRNPU, an N-terminal fragment of hnRNPU, was found to inhibit HIV-1 mRNA export (6). This study primarily aims at identifying proteins that associate with the fragment (N86-hnRNPU) also called H1, and secondarily aims to exclude the possibility that N86-hnRNPU transcripts act as microRNAs.
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Date Issued: 2012
PURL: http://purl.flvc.org/FAU/3359314
Subject Headings: Enzyme inhibitors, Proteinase, Inhibitors, HIV infections, Prevention, HIV infections, Pathogenesis, Antiviral agents
Format: Document (PDF)

Title: Cleavage of brain glutamic acid decarboxylase 65 by calpain under pathological conditions.
Creator: Buddhala, Chandana, Charles E. Schmidt College of Science, Department of Biological Sciences
Abstract/Description: Brain glutamic acid decarboxylase 65 (GAD65) catalyzes the rate-limiting step in the biosynthesis of the major inhibitory neurotransmitter-amino butyric acid (GABA) from the substrate L-glutamic acid. Severe lapse in GABA neurotransmission is one of the etiologies documented in the manifestation of certain neurodegenerative diseases such as epilepsy, Parkinson's disease, Huntington's disease etc. Because GAD65 synthesizes GABA, any modulation of GAD65, therefore, has direct implications on...
Show moreBrain glutamic acid decarboxylase 65 (GAD65) catalyzes the rate-limiting step in the biosynthesis of the major inhibitory neurotransmitter-amino butyric acid (GABA) from the substrate L-glutamic acid. Severe lapse in GABA neurotransmission is one of the etiologies documented in the manifestation of certain neurodegenerative diseases such as epilepsy, Parkinson's disease, Huntington's disease etc. Because GAD65 synthesizes GABA, any modulation of GAD65, therefore, has direct implications on the quanta of GABA released at the synapse. Hence, the major objective of this study was to focus on the regulation of GAD65, with special emphasis on investigating the proteolytic cleavage of fGAD65. Previously, we have shown in vitro that GAD65 was cleaved to form its truncated form (tGAD65), which was more active than the full length form (fGAD65). The enzyme responsible for cleavage was later identified as calpain. Calpain is known to cleave its substrates either under a transient physiologica l stimulus or upon a sustained pathological insult. However, the precise role of calpain cleavage of fGAD65 is poorly understood. In this study, we examined the cleavage of fGAD65 under a range of conditions encompassing both physiological and pathological aspects, including rats under ischemia/reperfusion insult, rat brain synaptosomes or primary neuronal cultures subjected to excitotoxic stimulation with KCl. It was observed that the formation of tGAD65 progressively increased with increasing stimulus concentration. More importantly, cleavage of synaptic vesicle (SV) - associated fGAD65 by calpain was demonstrated, and the resulting tGAD65 harboring the active site of the enzyme was detached from the SVs. Vesicular uptake of the newly synthesized GABA into the SVs was found to be reduced in calpain treated SVs. Furthermore, we also observed that the levels of tGAD65 in the focal cerebral ischemic rat brain tissue increased corresponding to the elevation of local glutamate indica, d by in vivo micro dialysis. Based on these observations, we conclude that calpain cleavage of fGAD65 occurs under pathological conditions.
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Date Issued: 2012
PURL: http://purl.flvc.org/FAU/3342053
Subject Headings: Glutamic acids, Antagonists, Proteolytic enzymes, Research, Cellular signal transduction, Calpain, Glutamic acid, Metabolism
Format: Document (PDF)

Title: DNAJC25 Pro90Leu J-domain mutation demonstrates decreased chaperone activity in vitro.
Creator: Chauss, Daniel C., Charles E. Schmidt College of Medicine, Department of Biomedical Science
Abstract/Description: Molecular chaperones guide peptide fold conformation throughout the lifetime of the peptide. One network of chaperone proteins involved in this activity, Heat shock protein 70s (Hsp70s), are well characterized at restoring peptide fold, utilizing J-domain containing protein chaperone cofactors to activate Hsp70 activity. DnaJ (Hsp40) homolog, subfamily C, member 25 (DNAJC25) is a class III transmembrane J-domain containing protein that to date is underrepresented in the literature. Recently,...
Show moreMolecular chaperones guide peptide fold conformation throughout the lifetime of the peptide. One network of chaperone proteins involved in this activity, Heat shock protein 70s (Hsp70s), are well characterized at restoring peptide fold, utilizing J-domain containing protein chaperone cofactors to activate Hsp70 activity. DnaJ (Hsp40) homolog, subfamily C, member 25 (DNAJC25) is a class III transmembrane J-domain containing protein that to date is underrepresented in the literature. Recently, Hejtmancik et al. 2012. (unpublished data) have revealed that missense mutation to DNACJ25 at Pro90Leu (P90L) is strongly correlated with inherited Closed-Angle Glaucoma. Inherited mutations are well characterized for Open-Angle Glaucoma, however, prior to this finding, were unknown for Closed-Angle Glaucoma. In this report, analysis of the in vitro chaperone activity of DNAJC25 w+ and P90L is assessed utilizing an Hsp70 mediated Glucose-6-Phosphate Dehydrogenase refolding system, SWISS-MODEL predictions are performed for the J-domain structure of DNAJC25 w+ and P90L with consequent analysis of DNAJC25 Pro90 conservation relative to other type I, II, and III J-domain containing proteins. DNAJC25 P90L demonstrated decreased chaperone activity in vitro compared to w+ DNAJC25.
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Date Issued: 2012
PURL: http://purl.flvc.org/FAU/3342040
Subject Headings: Cell physiology, Methodology, Molecular chaperones, Physiological effect, Cellular signal transduction, Proteolytic enzymes
Format: Document (PDF)

Title: Engineered and natural TIMP mutations.
Creator: Hamze, Asmaa Bilal., Charles E. Schmidt College of Science, Department of Biological Sciences
Abstract/Description: Tissue inhibitors of metalloproteinases (TIMPs) comprise a family of four proteins in humans that modulate the turnover of the extracellular matrix by regulating the activities of endopeptidases that catalyze its degradation, especially the matrix metalloproteinases (MMP). In general, the four TIMPs are broad-spectrum tight binding inhibitors of MMPs with individual differences in specificity. In this study, we attempted to understand the basis of such variation by using membrane type-1 MMP ...
Show moreTissue inhibitors of metalloproteinases (TIMPs) comprise a family of four proteins in humans that modulate the turnover of the extracellular matrix by regulating the activities of endopeptidases that catalyze its degradation, especially the matrix metalloproteinases (MMP). In general, the four TIMPs are broad-spectrum tight binding inhibitors of MMPs with individual differences in specificity. In this study, we attempted to understand the basis of such variation by using membrane type-1 MMP (MT1-MMP) as a model, since it is inefficiently inhibited by TIMP-1 in contrast with the other TIMPs. We designed and engineered mutations in the N-domain of TIMP-1, based on current knowledge of TIMP interactions. By measuring inhibition levels of each mutant against several MMPs, including MT1-MMP, we were able to obtain a triple mutant with an vii improved affinity for MT1-MMP., Our results, along with previous data, confirm that multiple residues in the critical interface segments between TIMPs and MMPs, namely at positions 2, 4, 5, 6, and 98, are key in determining the basic interaction between the two molecules. The second part of this work focused on naturally occurring mutations in TIMP-3 which cause an early form of macular degeneration called Sorsby's Fundus Dystrophy (SFD). The TIMP-3 mutants identified so far share certain features but the mechanism by which they result in macular disease is not yet understood. As an initial step, we expressed recombinant TIMP-3 carrying a truncation mutation, glutamic acid 139 to a stop codon (E139X), and assessed its activity towards representative MMPs and tumor necrosis factor-(Sa (Bconverting enzyme, another metalloproteinase normally inhibited by TIMP-3. Our results indicate that this mutation does not impair the inhibitory activity of TIMP-3., Expression of this mutant in mammalian retinal cells revealed a difference in localization between wild-type and E139X mutant TIMP-3. Therefore, we concluded that the SFD mutations may actually influence the processing and/or binding properties of TIMP-3 in the retina.
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Date Issued: 2008
PURL: http://purl.flvc.org/FAU/186296
Subject Headings: Proteolytic enzymes, Extracellular matrix proteins, Metalloproteinases, Inhibitors, Apoptosis, Retinal degeneration, Research
Format: Document (PDF)

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