Role Of Mammalian Ubiquitin Ligases UBR1 and UBR2 in Cytosolic Protein Quality Control
Item
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Title
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Role Of Mammalian Ubiquitin Ligases UBR1 and UBR2 in Cytosolic Protein Quality Control
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Identifier
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d_2009_2013:6e15bd8912c6:11519
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identifier
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12007
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Creator
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Sultana, Rasheda,
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Contributor
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Avrom J. Caplan
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Date
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2012
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biochemistry | Molecular biology | Geldanamycin | Molecular Chaperone | Protein Quality Control | Steroid Hormone Receptors | Ubiquitin Ligases
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Abstract
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UBR1 and UBR2 ubiquitin ligases function in the N-end rule degradation pathway in lower and higher eukaryotic cells. In yeast, the Ubr1 homologue also functions by N-end rule independent means to promote degradation of misfolded proteins generated via stress or with Hsp90 inhibitor GA. Based on these studies I examined the role of mammalian UBR1 in the degradation of protein kinase clients upon Hsp90 inhibition. I provide evidence that mammalian UBR1 promotes protein kinase quality control and sensitizes the cells to Hsp90 inhibition. The UBR1 deleted MEF cells showed reduced degradation of several protein kinases in the presence of GA. My findings also showed that Akt, p-Akt and Cdk4 the Hsp90 client protein kinases are still degraded in mouse UBR1 -/- cells treated with GA, but their levels recovered within 12-18 hours, in contrast to the wild type cells. The same findings were observed for human BT474 breast cancer cells with knocked down UBR1 by shRNA. These findings correlate with increased induction of Hsp90 expression in the Ubr1-/- cells compared with wild type cells. In addition, deletion of UBR1 and UBR2 showed resistance in terms of cell viability compared to wild type cells in the presence of GA and PU-H71. I also observed a reduction of UBR1 protein levels in GA-treated MEF and BT474 cells, suggesting that UBR1 is an Hsp90 client. I propose the existance of a novel feedback loop, where UBR1 negatively controls Hsp90 expression, while Hsp90 controls UBR1 stability. Further studies with CHIP reveal that CHIP and UBR1 have some functional overlap with respect to their E3 activities while UBR1 also affects the function of the Hsp90 chaperone machinery.;My studies with other Hsp90 clients showed that UBR1 promotes degradation of steroid hormone receptors GR and AR but not the ER-alpha. Co-expression of rUBR1 with hGR led to reduce the levels of hGR in the presence and absence of GA. There is a direct correlation between increasing UBR1 concentration and decreasing GR levels. Further studies addressed the specificity of that function with analysis of hAR and hER-alpha. In this case, there was a significant reduction of the hAR levels when UBR1 was overexpressed, even in the absence of GA. By contrast, similar experiments with transfected hER-alpha suggest that UBR1 does not play a similar role in the degradation of this receptor. My combined findings suggest that UBR1 acts specifically in the clearance of GR and AR but not in ER-alpha.;All of these findings suggest that UBR1 is involved in the cytosolic protein quality control in mammalian system and it also plays a role in determining the sensitivity of the cells to the Hsp90 inhibitors.
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Type
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dissertation
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Source
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2009_2013.csv
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degree
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Ph.D.
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Program
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Biochemistry
