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Proteome Analysis of Melanoma Progression
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)
Mutant huntingtin reduces palmitoylation of GAD65 and impairs its vesicular trafficking
Title
Mutant huntingtin reduces palmitoylation of GAD65 and impairs its vesicular trafficking.
Creator
Rush, Daniel., Charles E. Schmidt College of Medicine, Department of Biomedical Science
Abstract/Description
Huntington's disease (HD) is caused by an expanded plyglutamine repeat in the huntingtin protein. In this study, I focused on the effect of the mutant huntingtin protein (mhtt) on the subcellular localization of glutamic acid decarboxylase (GAD), the enzyme responsible for synthesizing gama-aminobutyric acid (GABA). Subcellular distribution of GAD65 is significantly altered in two neuronal cell lines that express either the N-terminus or full length mhtt. GAD65 is predominantly associated...
Show moreHuntington's disease (HD) is caused by an expanded plyglutamine repeat in the huntingtin protein. In this study, I focused on the effect of the mutant huntingtin protein (mhtt) on the subcellular localization of glutamic acid decarboxylase (GAD), the enzyme responsible for synthesizing gama-aminobutyric acid (GABA). Subcellular distribution of GAD65 is significantly altered in two neuronal cell lines that express either the N-terminus or full length mhtt. GAD65 is predominantly associated with the Golgi membrane in cells expressing normal huntingtin (Htt). However, it diffuses in the cytosol of cells expressing mhtt. Palmitoylation of GAD65 is required for GAD65 trafficking, and I demonstrated the palmitoylation of GAD65 is reduced in the HD model. Overexpression of huntingtin-interacting protein 14 (HIP14), the enzyme that palmitoylates GAD65, rescues GAD65 palmitoylation and vesicle-associated trafficking. This data suggests that impairment of GAD65 palmitoylation by mhtt may alter its localization and lead to altered inhibitory neurotransmission in HD.
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Date Issued
2012
PURL
http://purl.flvc.org/FAU/3352831
Subject Headings
Glutamic acids, Antagonists, Cellular signal transduction, Proteolytic enzymes, Research, Proteins, Physiological transport, Huntington's chorea, Research
Format
Document (PDF)
Cleavage of brain glutamic acid decarboxylase 65 by calpain under pathological conditions
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)
Engineered and natural TIMP mutations
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)
Taurine inhibits glutamate-induced excitotoxicity through a calpain dependent pathway
Title
Taurine inhibits glutamate-induced excitotoxicity through a calpain dependent pathway.
Creator
Leon, Rebecca, Prentice, Howard, Florida Atlantic University, Charles E. Schmidt College of Science, Department of Biological Sciences
Abstract/Description
Taurine, an endogenous ammo acid and neuromodulator, has been found to be neuroprotective against numerous forms of neurotoxicity including glutamate-induced excitotoxicity. Previously we have shown that taurine inhibits glutamate-induced calcium influx through VGCCs and NMDA receptors. Although the neuroprotective effects of taurine against excitotoxicity have been attributed to its intracellular Ca2+ regulatory functions, the complete mechanism underling taurine neuroprotection has remained...
Show moreTaurine, an endogenous ammo acid and neuromodulator, has been found to be neuroprotective against numerous forms of neurotoxicity including glutamate-induced excitotoxicity. Previously we have shown that taurine inhibits glutamate-induced calcium influx through VGCCs and NMDA receptors. Although the neuroprotective effects of taurine against excitotoxicity have been attributed to its intracellular Ca2+ regulatory functions, the complete mechanism underling taurine neuroprotection has remained unclear. Using primary rat cortical neuronal cell cultures, we have determined key cytosolic components to the mechanism of taurine neuroprotection. In this study we have found that taurine inhibits excitotoxicity by suppressing glutamate-induced elevations in [Ca2+]i, preventing calpain activation, and inhibiting reductions in Bel- 2:Bax ratios and consequently activation of the intrinsic pathway.
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Date Issued
2008
PURL
http://purl.flvc.org/fau/fd/FA00000788
Subject Headings
Cellular signal transduction, Taurine--Physiological effect, Proteolytic enzymes--Research
Format
Document (PDF)