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Zafar Nawaz - One of the best experts on this subject based on the ideXlab platform.
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Ubiquitin Pathway Enzymes: Coactivators of Nuclear Hormone Receptor and Their Role in the Development of Breast Cancer
2020Co-Authors: Zafar NawazAbstract:Abstract : Steroid hormones, estrogen and progesterone, and their intracellular receptors play an important role in the development and progression of breast cancer. Coactivator Proteins modulate the biological activity of these hormone receptors. We have cloned an E3 ubiquitin-Protein ligase enzyme, E6-associated Protein (E6-AP) and E2 ubiquitin-conjugating enzyme, UbcH7 as coactivators of steroid hormone receptors. The purpose of this research is to explore the possibility that the altered expression of E6-AP and UbcH7 may contribute to the development of breast cancer. We have examined this possibility by studying the expression patterns of E6-AP, UbcH7 and estrogen receptor-alpha (ER) in various human breast cancer cell lines and breast tumor biopsy samples. Additionally, we have correlated the expression profile of E6-AP and UbcH7 with that of ER in breast tumor biopsies. Todate, we have examined 56 advanced stage human breast cancer biopsy samples for the expression profile of E6-AP, UbcH7 and ER. We found an inverse correlation between the expression of E6-AP and the expression of ER in these tumors. The Spearman Rank Correlation Coefficient is 0.38 and the p value is 0.004, indicating that this correlation is statistically significant. These data suggest a possible role of E6-AP in mammary gland development and tumorigenesis. However, we did not find any statistically significant eorrelation between the expression profile of UbcH7 and ER in these tumor samples. Presently, we are studying the expression profile of E6-AP, UbcH7 and ER in early and intermediate stage tumors. Another goal of this project is to create novel in vitro models in stable cell lines, which will overexpress coactivator Proteins, E6-AP and UbcH7. In order to achieve this goal, we have already constructed the expression vectors for stable cell lines.
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Overexpression of ligase defective E6-associated Protein, E6-AP, results in mammary tumorigenesis
Breast Cancer Research and Treatment, 2011Co-Authors: Sivapriya Ramamoorthy, Rozina Tufail, Jimmy El Hokayem, M. Jorda, Wei Zhao, Zizi Reis, Zafar NawazAbstract:E6-associated Protein (E6-AP) is a dual function Protein. It acts as an E3 ubiquitin-Protein ligase enzyme and coactivator of steroid hormone receptors such as estrogen (ERα) and progesterone (PR) receptors. It promotes the degradation of ERα and PR through the ubiquitin–proteasome pathway. Furthermore, it has been shown that the levels of E6-AP are inversely associated with that of ERα in human breast tumors. But the role of wild-type human E6-AP and its ubiquitin-Protein ligase activity in mammary tumorigenesis is still unknown. To investigate this role, the authors utilized transgenic mice lines that specifically overexpress either the wild-type human E6-AP (E6-APWT) or the ubiquitin-Protein ligase defective E6-AP that contains C833S mutation (E6-APC833S) in the mammary gland. To further substantiate the role of E6-AP in the development of breast tumorigenesis, it was also examined the expression of E6-AP in a large cohort of human breast cancer samples. The transgenic mice that overexpress wild-type E6-AP (E6-APWT) fail to develop mammary tumors. Unlike the E6-APWT mice, the E6-APC833S mice that overexpress ubiquitin-Protein ligase defective E6-AP Protein develop mammary hyperplasia with a median latency of 18 months. These observations suggest that the inactivation of the ubiquitin-Protein ligase function of E6-AP is sufficient to initiate the process of mammary tumor development. Furthermore, the data also suggests that E6-AP exerts its effects on target cells by modulating the Protein levels and functions of ERα and PR. In addition, it was found in human breast cancer patients that the level of E6-AP is decreased in invasive breast tumors compared to normal breast tissue. Moreover, the authors also show that the survival patterns for E6-AP negative patients were worse compared to E6-AP positive patients. Taken together, these data suggests that E6-AP may act as a tumor suppressor in breast.
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e3 ubiquitin Protein ligase E6 associated Protein E6 ap regulates pi3k akt signaling and prostate cell growth
Biochimica et Biophysica Acta, 2011Co-Authors: Sathish Srinivasan, Zafar NawazAbstract:Abstract This study elucidates the role of E6-associated Protein, E6-AP (a dual function steroid hormone receptor coactivator and ubiquitin–Protein ligase) in the regulation of PI3K-Akt signaling pathway, prostate gland growth and proliferation. Here, we report the generation of transgenic mice and prostate cancer cell line, LNCaP cells that overexpress E6-AP Protein. Using these models we show that the levels of total Akt and phosphorylated Akt (active Akt) are increased in E6-AP overexpressing prostate gland and LNCaP cells suggesting that E6-AP regulates the PI3K-Akt signaling pathway. The prostate glands in our transgenic mice are ~ 20% larger and produce preneoplastic lesions at the age of 18 months. Our data also suggest that E6-AP modulates PI3K-Akt signaling pathway by both androgen-independent and -dependent mechanisms. In the androgen-independent mechanism, E6-AP modulates PI3K-Akt signaling by regulating the Protein levels of RhoA, a small GTPase, which is a negative regulator of the Akt signaling pathway. Further, we show that E6-AP, a known coactivator of AR, amplifies the androgen-dependent activation of PI3K-Akt signaling pathway. In addition, we show that stable overexpression of E6-AP in prostate cancer cells results in increased cell size and proliferation. Overall our data suggests that E6-AP regulates both the positive and negative modulators of the PI3K-Akt pathway in prostate cells which results in increased prostate cell growth, proliferation and decreased apoptosis.This article is part of a Special Issue entitled The 26S Proteasome: When degradation is just not enough!
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E6 associated Protein E6 ap is a dual function coactivator of steroid hormone receptors
Nuclear Receptor Signaling, 2008Co-Authors: Sivapriya Ramamoorthy, Zafar NawazAbstract:: Steroid hormone receptors (SHR) belong to a large family of ligand-activated transcription factors that perform their biological functions by enhancing the transcription of specific target genes. The transactivation functions of SHRs are regulated by a specialized group of Proteins called coactivators. The SHR coactivators represent a growing class of Proteins with various enzymatic activities that serve to modify the chromatin to facilitate the transcription of SHR target genes. The ubiquitin-proteasome pathway enzymes have also been added to the growing list of enzymatic activities that are recruited to the SHR target gene promoters during transcription. One such ubiquitin-proteasome pathway enzyme to be identified and characterized as a SHR coactivator was E6-associated Protein (E6-AP). E6-AP is a hect (homologous to E6-associated Protein carboxy-terminal domain) domain containing E3 ubiquitin ligase that possesses two independent separable functions; a coactivation function and an ubiquitin-Protein ligase activity. Being a component of the ubiquitin-proteasome pathway, it is postulated that E6-AP may orchestrate the dynamics of steroid hormone receptor-mediated transcription by regulating the degradation of the transcriptional complexes. E6-AP has also been shown to be involved in the regulation of various aspects of reproduction such as prostate and mammary gland development. Furthermore, it has been demonstrated that E6-AP expression is down-regulated in breast and prostate tumors and that the expression of E6-AP is inversely associated with that of estrogen and androgen receptors. This review summarizes our current knowledge about the structures, molecular mechanisms, spatiotemporal expression patterns and biological functions of E6-AP.
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E6-Associated Protein (E6-AP): A Potential Tumor Suppressor Protein for Non-Hodgkin Lymphoma.
Blood, 2006Co-Authors: Shazia Zafar, Sathish Srinivasan, Zafar NawazAbstract:Over the past decade considerable progress has been made in cloning and characterization of potential tumor suppressor genes. Tumor suppressors have a repressive effect on the regulation of the cell cycle or promote apoptosis and sometimes do both. The function of tumor suppressor Proteins fall into several categories, tumor suppressor genes are presumed to encode negative regulator of proliferation and inhibit mitotic activity. Loss of tumor suppressor Protein or function of a tumor suppressor Protein has been shown to be associated with the cancer formation. Continued investigation into the biochemical and cell biological functions of the tumor suppressor is critical to elucidate the mechanisms by which they normally inhibit proliferation/tumor development and to provide a molecular explanation for their frequent inactivation in cancer. Our laboratory has previously shown that the expression of E6-associated Protein (E6-AP), which is an E3 ubiquitin-Protein ligase and a coactivator of nuclear hormone receptors, is significantly reduced in human cancers having epithelial cell origin such as breast cancer. In this prospective study, we want to extend our observation to the cancers originating from lymphoid tissue. Non-Hodgkin lymphoma is a cancer of lymphoid tissue. The main cell type found in lymphoid tissue is the lymphocyte. The 2 main types of lymphocytes are B-lymphocytes (B-cells) and T-lymphocytes (T-cells). B-cell lymphomas are much more common than T-cell lymphomas. In the U. S., 85% of all cases of non-Hodgkin lymphoma come from B lymphocytes (B-cell) and 15% from T lymphocytes (T-cell). We performed immunohistochemistry analysis to investigate the expression pattern of E6-AP in normal lymph nodes and lymphoid tumors. Tissue micro arrays representing samples from 60 different patients were analyzed in this study. Our analysis suggest that on an average there was about 55 % reduction in E6-AP Protein levels in B-cell lymphomas ( P =0.0001) and 98.5 % reduction in E6-AP levels in T-cell lymphomas ( P =0.0002) compared to normal lymph node. Based on our previous studies in breast and prostate tumors and considering our current finding of reduced/loss of E6-AP in lymphoid tumors, we propose that E6-AP may act as a potential tumor suppressor Protein. This proposed idea is consistent with our in vivo data generated from E6-AP null mice which shows that the number of B- and T-cells are significantly increased in spleen compared to normal wild-type animals. Taken together our data establish the role of E6-AP as a potential growth and tumor suppressor Protein.
Martin Scheffner - One of the best experts on this subject based on the ideXlab platform.
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the ubiquitin Protein ligase E6 associated Protein E6 ap serves as its own substrate
FEBS Journal, 1998Co-Authors: Ulrike Nuber, Sylvia E Schwarz, Martin ScheffnerAbstract:Recognition of substrate Proteins by the ubiquitin-conjugation system is a highly specific and regulated event and involves the action of ubiquitin-conjugating enzymes (E2) and ubiquitin-Protein ligases (E3). However, the E2 and E3 involved in the recognition of particular substrates have been identified in only a few cases. The ubiquitin-Protein ligase E6-associated Protein (E6-AP) was originally identified as a Protein involved in the human papillomavirus E6-oncoProtein-induced degradation of p53. The substrate Proteins of E6-AP in the absence of the E6 oncoProtein, however, have not been identified. We show here that E6-AP can target itself for ubiquitination in vitro and provide evidence that, under conditions of overexpression, E6-AP efficiently promotes its own degradation in vivo. Autoubiquitination of E6-AP is mediated mainly by intermolecular transfer of ubiquitin. In addition, highly ubiquitinated forms of E6-AP cannot bind to p53 in the presence of the E6 oncoProtein and, conversely, binding of E6-AP to p53 interferes with ubiquitination of E6-AP. These results suggest that autoubiquitination and subsequent degradation of E6-AP represents a mechanism to control intracellular E6-AP levels by inactivating E6-AP molecules that are not bound to substrate Proteins.
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The ubiquitin‐Protein ligase E6‐associated Protein (E6‐AP) serves as its own substrate
FEBS Journal, 1998Co-Authors: Ulrike Nuber, Sylvia E Schwarz, Martin ScheffnerAbstract:Recognition of substrate Proteins by the ubiquitin-conjugation system is a highly specific and regulated event and involves the action of ubiquitin-conjugating enzymes (E2) and ubiquitin-Protein ligases (E3). However, the E2 and E3 involved in the recognition of particular substrates have been identified in only a few cases. The ubiquitin-Protein ligase E6-associated Protein (E6-AP) was originally identified as a Protein involved in the human papillomavirus E6-oncoProtein-induced degradation of p53. The substrate Proteins of E6-AP in the absence of the E6 oncoProtein, however, have not been identified. We show here that E6-AP can target itself for ubiquitination in vitro and provide evidence that, under conditions of overexpression, E6-AP efficiently promotes its own degradation in vivo. Autoubiquitination of E6-AP is mediated mainly by intermolecular transfer of ubiquitin. In addition, highly ubiquitinated forms of E6-AP cannot bind to p53 in the presence of the E6 oncoProtein and, conversely, binding of E6-AP to p53 interferes with ubiquitination of E6-AP. These results suggest that autoubiquitination and subsequent degradation of E6-AP represents a mechanism to control intracellular E6-AP levels by inactivating E6-AP molecules that are not bound to substrate Proteins.
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the hpv 16 E6 and E6 ap complex functions as a ubiquitin Protein ligase in the ubiquitination of p53
Cell, 1993Co-Authors: Martin Scheffner, Jon M Huibregtse, Richard D Vierstra, Peter M. HowleyAbstract:Abstract The ubiquitin-dependent proteolytic pathway plays a major role in selective Protein degradation. Ubiquitination of Proteins requires the sequential action of the ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzymes (E2), and in some cases ubiquitin-Protein ligases (E3s). The oncogenic human papillomavirus (HPV) types 16 and 18 utilize this cellular proteolytic system to target the tumor suppressor Protein p53. The HPV E6 oncoProtein binds to a cellular Protein of 100 kd, termed E6-associated Protein (E6-AP). The E6-E6-AP complex specifically interacts with p53, resulting in the rapid ubiquitin-dependent degradation of p53. Here we report the purification and identification of the factors necessary for the E6-E6-AP-mediated ubiquitination of p53. The ubiquitination of p53 requires the E1 enzyme and a novel E2 in mammalian cells, while E3 activity is conferred by the E6-E6-AP complex. Furthermore, E6-AP appears to have ubiquitin-Protein ligase activity in the absence of E6.
Ulrike Nuber - One of the best experts on this subject based on the ideXlab platform.
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the ubiquitin Protein ligase E6 associated Protein E6 ap serves as its own substrate
FEBS Journal, 1998Co-Authors: Ulrike Nuber, Sylvia E Schwarz, Martin ScheffnerAbstract:Recognition of substrate Proteins by the ubiquitin-conjugation system is a highly specific and regulated event and involves the action of ubiquitin-conjugating enzymes (E2) and ubiquitin-Protein ligases (E3). However, the E2 and E3 involved in the recognition of particular substrates have been identified in only a few cases. The ubiquitin-Protein ligase E6-associated Protein (E6-AP) was originally identified as a Protein involved in the human papillomavirus E6-oncoProtein-induced degradation of p53. The substrate Proteins of E6-AP in the absence of the E6 oncoProtein, however, have not been identified. We show here that E6-AP can target itself for ubiquitination in vitro and provide evidence that, under conditions of overexpression, E6-AP efficiently promotes its own degradation in vivo. Autoubiquitination of E6-AP is mediated mainly by intermolecular transfer of ubiquitin. In addition, highly ubiquitinated forms of E6-AP cannot bind to p53 in the presence of the E6 oncoProtein and, conversely, binding of E6-AP to p53 interferes with ubiquitination of E6-AP. These results suggest that autoubiquitination and subsequent degradation of E6-AP represents a mechanism to control intracellular E6-AP levels by inactivating E6-AP molecules that are not bound to substrate Proteins.
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The ubiquitin‐Protein ligase E6‐associated Protein (E6‐AP) serves as its own substrate
FEBS Journal, 1998Co-Authors: Ulrike Nuber, Sylvia E Schwarz, Martin ScheffnerAbstract:Recognition of substrate Proteins by the ubiquitin-conjugation system is a highly specific and regulated event and involves the action of ubiquitin-conjugating enzymes (E2) and ubiquitin-Protein ligases (E3). However, the E2 and E3 involved in the recognition of particular substrates have been identified in only a few cases. The ubiquitin-Protein ligase E6-associated Protein (E6-AP) was originally identified as a Protein involved in the human papillomavirus E6-oncoProtein-induced degradation of p53. The substrate Proteins of E6-AP in the absence of the E6 oncoProtein, however, have not been identified. We show here that E6-AP can target itself for ubiquitination in vitro and provide evidence that, under conditions of overexpression, E6-AP efficiently promotes its own degradation in vivo. Autoubiquitination of E6-AP is mediated mainly by intermolecular transfer of ubiquitin. In addition, highly ubiquitinated forms of E6-AP cannot bind to p53 in the presence of the E6 oncoProtein and, conversely, binding of E6-AP to p53 interferes with ubiquitination of E6-AP. These results suggest that autoubiquitination and subsequent degradation of E6-AP represents a mechanism to control intracellular E6-AP levels by inactivating E6-AP molecules that are not bound to substrate Proteins.
D P Lane - One of the best experts on this subject based on the ideXlab platform.
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Ubiquitin-independent degradation of p53 mediated by high-risk human papillomavirus Protein E6
Oncogene, 2007Co-Authors: S Camus, S Menéndez, C F Cheok, L F Stevenson, S Laín, D P LaneAbstract:In vitro , high-risk human papillomavirus E6 Proteins have been shown, in conjunction with E6-associated Protein (E6AP), to mediate ubiquitination of p53 and its degradation by the 26S proteasome by a pathway that is thought to be analogous to Mdm2-mediated p53 degradation. However, differences in the requirements of E6/E6AP and Mdm2 to promote the degradation of p53, both in vivo and in vitro , suggest that these two E3 ligases may promote p53 degradation by distinct pathways. Using tools that disrupt ubiquitination and degradation, clear differences between E6- and Mdm2-mediated p53 degradation are presented. The consistent failure to fully protect p53 Protein from E6-mediated degradation by disrupting the ubiquitin-degradation pathway provides the first evidence of an E6-dependent, ubiquitin-independent, p53 degradation pathway in vivo .
Bert W Omalley - One of the best experts on this subject based on the ideXlab platform.
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the angelman syndrome associated Protein E6 ap is a coactivator for the nuclear hormone receptor superfamily
Molecular and Cellular Biology, 1999Co-Authors: Zafar Nawaz, David M. Lonard, Carolyn L. Smith, Sophia Y. Tsai, Ming-jer Tsai, Efrat Levlehman, Bert W OmalleyAbstract:Steroids, thyroid hormones, vitamin D, and retinoids regulate diverse biological processes including growth, development, and homeostasis through their cognate nuclear hormone receptors, which make up a superfamily of structurally related intracellular ligand-activated transcription factors (18, 34, 40, 47). Nuclear hormone receptors contain common structural motifs which include a poorly conserved amino-terminal activation function (activation factor 1 [AF-1]) that affects transcription efficiency, a central DNA-binding domain, which mediates receptor binding to specific DNA enhancer sequences and determines target gene specificity, and a carboxy-terminal hormone-binding domain. The latter domain contains AF-2, a region which mediates the hormone-dependent activation function of receptors (40). When bound to hormone, these receptors undergo a conformational change, dissociation from heat shock Proteins, receptor dimerization, phosphorylation, DNA binding at an enhancer element of the target gene, interaction with coactivators, and subsequent recruitment of basal transcription factors to form a stable preinitiation complex. These events are followed by either up-regulation or down-regulation of target gene transcription (40). Nuclear hormone receptor coactivators represent a growing class of Proteins which interact with receptors in a ligand-specific manner and serve to enhance their transcriptional activities (33). Prior to their identification, coactivators were predicted to exist based on experiments which showed that different receptors compete for a limiting pool of factors required for optimal transcription. Stimulation of one receptor resulted in transrepression of another receptor, indicating the depletion of a common coactivator pool (6, 10, 31, 39). Among the coactivators cloned to date are steroid receptor coactivator 1 (SRC-1) (33), TIF2 (GRIP1) (17, 51), p/CIP (ACTR/RAC3/AIB1/TRAM-1) (2, 9, 28, 46, 48), and ARA70 (54). Coactivators were originally envisioned to serve a bridging role, linking the receptor to the basal transcription machinery (36, 45). Recently, they were shown to possess enzymatic activities which contribute to their ability to enhance receptor-mediated transcription; SRC-1, p300/CBP, and RAC3/ACTR/AIB1 possess histone acetyltransferase activity (HAT) (2, 9, 28, 32, 41). Ligand-activated receptors are thought to bring these HAT activity-containing coactivators to the chromatin surrounding the receptor, disrupting the local repressive chromatin structure by acetylating histones and possibly other chromatin-associated factors (41). Because of their ability to enhance receptor-mediated gene expression, coactivators are thought to play an important role in regulating the magnitude of the biological response to steroids, vitamin D, and retinoids in different tissues or individuals. The level of coactivator expression may contribute to variations in hormone responsiveness seen in the population, and disruption in coactivator expression could lead to the pathological hyper- or hyposensitivity to steroid hormones. Recently, it was shown that disruption of the SRC-1 locus in mice resulted in an attenuated response to steroid hormones, a finding consistent with this hypothesis (53). In this report, we describe the cloning and characterization of E6-associated Protein (E6-AP) (21), a Protein linked to Angelman syndrome (AS) (26, 30, 42), as a progesterone receptor (PR)-interacting Protein. E6-AP was previously identified as a Protein of 100 kDa, present in both the cytoplasm and the nucleus (14). E6-AP mediates the interaction of human papillomavirus type 16 and 18 E6 Proteins with p53, a growth-suppressive and tumor-suppressive Protein (14, 22). Initial in vitro studies suggested that the E6–E6-AP complex specifically interacts with p53 and promotes the degradation of p53 via the ubiquitin-proteasome degradation pathway, but recent in vivo studies show that E6-AP can directly interact with p53 and promote its degradation even in the absence of the papillomavirus E6 Protein (11, 20, 38). E6-AP is a member of a family of Proteins, known as E3 ubiquitin-Protein ligases, which have been proposed to play a role in defining the substrate specificity of the ubiquitin-proteasome degradation system. Protein ubiquitination also involves two other classes of enzymes, namely, E1 ubiquitin-activating enzymes and E2 ubiquitin-conjugating enzymes, which activate ubiquitin moieties and transfer them to target Proteins and E3, respectively (19). The carboxyl-terminal 350 amino acids (aa) of E6-AP constitute a hect (homologous to the E6-AP carboxy terminus) domain which is conserved among many E3 ubiquitin-Protein ligases and E6-AP-related Proteins (19). The extreme carboxyl-terminal 100-aa segment contains the catalytic region of E6-AP, which transfers ubiquitin to the Protein targeted for degradation (19). The E6-binding domain consists of an 18-aa region located within the central portion of the E6-AP Protein (22). Recently, it was shown that a genetic disorder, AS, is caused by the absence of a functional maternal copy of the E6-AP gene (26, 30, 42). AS is a neurological disorder characterized by severe mental retardation, seizures, speech impairment, and other symptoms (5). However, the exact mechanism by which the defective E6-AP gene causes AS remains unknown. Our analysis of mutant E6-AP Proteins from AS patients revealed that the ubiquitin-Protein ligase function of E6-AP was defective, whereas the coactivator function was intact, in the majority of AS patients examined. In this report, we also show that the ubiquitin ligase activity of E6-AP is not required for the coactivation function of E6-AP. Furthermore, our data indicate that the catalytic function located within the hect domain of E6-AP is not necessary for the ability of E6-AP to interact with and coactivate steroid hormone receptor function. These findings suggest that E6-AP possesses two independent functions, as both a coactivator and a ubiquitin-Protein ligase.
