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Showing 1 to 6 of 6 (0.01 seconds)| 1 |
THE STRUCTURAL AND BIOCHEMICAL CHARACTERIZATION OF DEUBIQUITINATING ENZYMES LOTA AND UCHL1 R178QKwame J Brown (7033259) 13 August 2019 (has links)
<p>The Deubiquitinating (DUB) enzymes, LotA and UCHL1 R178Q, were examined
biochemically and also structurally in the case of UCHL1 R178Q. LotA is a bacterial
effector of <i>Legionella pneumophila</i>
that enables the pathogen to establish a replicative niche. LotA has two Deubiquitinase
(DUB) domains specific to different substrates. Here, I report biochemical
examinations the first DUB domain that is specific to Lys6-linked di-ubiquitin.
Michaelis- Menten kinetic parameters were determined for this domain. Through
activity assays of various truncations a series of residues were discerned that
contribute to interaction of the distal binding site of ubiquitin chain.</p>
UCHL1 mutant R178Q displays
enhanced activity when compared to wild type (WT). The mutant was crystallized
for structural analysis to gain insights into the higher catalytic activity of
the mutant. The structure revealed that the catalytic histidine maintains a
misaligned orientation similar to the WT enzyme. Biochemical analysis was done
to ascertain the role of key residues that interact with the catalytic histidine.
The residue type at position 178 in the structure plays a key role in enhancing
the enzyme activity.
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Functional And Biochemical Analysis Of A Novel Deubiquitinating Enzyme, Usp32Sapmaz, Aysegul 01 September 2012 (has links) (PDF)
Ubiquitylation is an important post-translational modification and can be reversed by the action of deubiquitinating (DUB) enzymes. The ubiquitylation and deubiquitylation of target proteins are significant in terms of regulating cellular events such as protein degradation, signal transduction, vesicle trafficking, DNA repair and apoptosis. Chromosomal band 17q23 is frequently amplified in breast cancers and harbors a predicted ubiquitin specific protease gene, USP32 (ubiquitin specific protease 32). Given its potential role in breast cancer, we aimed to characterize USP32 for its potential DUB activity. Bioinformatic analysis of USP32 and known yeast and mouse DUBs suggested presence of Cys-His domains which are common in active DUBs of the USP superfamily. Our in vivo and in vitro DUB activity assays revealed that USP32 was indeed an active deubiquitinating enzyme. To investigate its substrate specificity and kinetic properties, USP32 was expressed in insect cell culture to be isolated and purified. Using isolated USP32 protein, diubiquitin assay was performed with all seven types of diubiquitin (K6, K11, K27, K29, K33, K48 and K63) as well as linear diubiquitin. Results showed that USP32 was able to cleave all seven types of ubiquitin linkages with higher cleavage efficiency for K6, K11, K48 and K63-linked diubiquitin. Moreover, kinetic parameters, Km, kcat and kcat/ Km, suggested that full length protein had lower affinity for potential substrates and lower catalytic activity compared to the catalytic domain alone. These data suggested the importance of USP32 tertiary structure and possible role of other non DUB domains (e.g. EF hand domain) which may be regulated by an as of unknown mechanism in cells. Further investigations are underway to understand the functions of USP32 in cells and how it may contribute to breast tumorigenesis.
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Functional Characterization Of Two Potential Breast Cancer Related GenesAkhavantabasi, Shiva 01 April 2012 (has links) (PDF)
Cancer may arise as a result of deregulation of oncogenes and/or tumor suppressors. Although much progress has been made for the identification of such cancer related genes, our understanding of the complex tumorigenesis pathways is still not complete. Therefore, to improve our understanding of how certain basic mechanisms work in normal and in cancer cells, we aimed to characterize two different breast cancer related genes. First part of the study focused on subcellular localization USP32 (Ubiquitin Specific Protease 32) to help understand the function of this uncharacterized gene. USP32 is a member of deubiquitinating enzymes (DUBs) and the gene maps to a gene rich region on 17q23. Genes on 17q23 are known to undergo amplification and overexpression in a subset of breast cancer cells and tumors. DUBs are known to be implicated in a variety of cellular functions including protein degradation, receptor endocytosis and vesicle trafficking. Therefore to elucidate the function of USP32, we localized the full length USP32 protein fused to GFP, in HeLa cells, using Fluorescence Protease Protection (FPP) assay and confocal microscopy. Results suggested a Golgi localization for USP32 as confirmed by co-localization study via BODIPY-TR, a Golgi specific marker. Additional investigations to find the role of USP32 in Golgi will further clarify the function of this candidate oncogene. Second part of the study focused on a potential tumor suppressor. For this purpose, we functionally characterized miR-125b, a microRNA gene as a potential tumor suppressor in breast cancer. microRNAs are regulators of gene expression and their deregulation is detected in cancer cells. miR-125b is reported as a down regulated microRNA in breast cancers. In this study, we investigated the expression, function and possible targets of miR-125b in breast cancer cell lines (BCCLs). Our results revealed a dramatic down regulation of miR-125b in a panel of BCCLs. Restoring the expression of miR-125b in low miR-125b expressing cells decreased the cell proliferation and migration as well as cytoplasmic protrusions, detected by staining of actin filaments. While connection of miR-125b and cell motility based on ERBB2 targeting has been reported earlier, here we present data on ERBB2 independent effects of miR-125b on cell migration in non-ERBB2 overexpressing breast cancer cells. Our results showed involvement of a miR-125b target, ARID3B, in cell motility and migration. Our findings showed miR-125b to be an important regulator of cell proliferation and migration in ERBB2 negative breast cancer cells, possibly through regulating multiple targets.
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USP5 enhances SGTA mediated protein quality control.02 August 2022 (has links)
Yes / Mislocalised membrane proteins (MLPs) present a risk to the cell due to exposed hydrophobic amino acids which cause MLPs to aggregate. Previous studies identified SGTA as a key component of the machinery that regulates the quality control of MLPs. Overexpression of SGTA promotes deubiqutination of MLPs resulting in their accumulation in cytosolic inclusions, suggesting SGTA acts in collaboration with deubiquitinating enzymes (DUBs) to exert these effects. However, the DUBs that play a role in this process have not been identified. In this study we have identified the ubiquitin specific peptidase 5 (USP5) as a DUB important in regulating the quality control of MLPs. We show that USP5 is in complex with SGTA, and this association is increased in the presence of an MLP. Overexpression of SGTA results in an increase in steady-state levels of MLPs suggesting a delay in proteasomal degradation of substrates. However, our results show that this effect is strongly dependent on the presence of USP5. We find that in the absence of USP5, the ability of SGTA to increase the steady state levels of MLPs is compromised. Moreover, knockdown of USP5 results in a reduction in the steady state levels of MLPs, while overexpression of USP5 increases the steady state levels. Our findings suggest that the interaction of SGTA with USP5 enables specific MLPs to escape proteasomal degradation allowing selective modulation of MLP quality control. These findings progress our understanding of aggregate formation, a hallmark in a range of neurodegenerative diseases and type II diabetes, as well as physiological processes of aggregate clearance.
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Interplay of the COP9 signalosome deneddylase and the UspA deubiquitinase to coordinate fungal development and secondary metabolismMeister, Cindy 06 June 2018 (has links)
No description available.
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SCF cdc4 regulates msn2 and msn4 dependent gene expression to counteract hog1 induced lethalityVendrell Arasa, Alexandre 16 January 2009 (has links)
L'activació sostinguda de Hog1 porta a una inhibició del creixement cel·lular. En aquest treball, hem observat que el fenotip de letalitat causat per l'activació sostinguda de Hog1 és parcialment inhibida per la mutació del complexe SCFCDC4. La inhibició de la mort causada per l'activació sostinguda de Hog1 depèn de la via d'extensió de la vida. Quan Hog1 s'activa de manera sostinguda, la mutació al complexe SCFCDC4 fa que augmenti l'expressió gènica depenent de Msn2 i Msn4 que condueix a una sobreexpressió del gen PNC1 i a una hiperactivació de la deacetilassa Sir2. La hiperactivació de Sir2 és capaç d'inhibir la mort causada per l'activació sostinguda de Hog1. També hem observat que la mort cel·lular causada per l'activació sostinguda de Hog1 és deguda a una inducció d'apoptosi. L'apoptosi induïda per Hog1 és inhibida per la mutació al complexe SCFCDC4. Per tant, la via d'extensió de la vida és capaç de prevenir l'apoptosi a través d'un mecanisme desconegut. / Sustained Hog1 activation leads to an inhibition of cell growth. In this work, we have observed that the lethal phenotype caused by sustained Hog1 activation is prevented by SCFCDC4 mutants. The prevention of Hog1-induced cell death by SCFCDC4 mutation depends on the lifespan extension pathway. Upon sustained Hog1 activation, SCFCDC4 mutation increases Msn2 and Msn4 dependent gene expression that leads to a PNC1 overexpression and a Sir2 deacetylase hyperactivation. Then, hyperactivation of Sir2 is able to prevent cell death caused by sustained Hog1 activation. We have also observed that cell death upon sustained Hog1 activation is due to an induction of apoptosis. The apoptosis induced by Hog1 is decreased by SCFCDC4 mutation. Therefore, lifespan extension pathway is able to prevent apoptosis by an unknown mechanism.
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