Biomedical subjects

Peter M Howley

Publications and source records attributed to Peter M Howley.

At least 19 recordsLinked to original sources

PubMed2006

E6AP ubiquitin ligase mediates ubiquitylation and degradation of hepatitis C virus core protein.

Hepatitis C virus (HCV) core protein is a major component of viral nucleocapsid and a multifunctional protein involved in viral pathogenesis and hepatocarcinogenesis. We previously showed that the HCV core protein is degraded through the ubiquitin-proteasome pathway. However, the molecular machinery for core ubiquitylation is unknown. Using tandem affinity purification, we identified the ubiquitin ligase E6AP as an HCV core-binding protein. E6AP was found to bind to the core protein in vitro and in vivo and promote its degradation in hepatic and nonhepatic cells. Knockdown of endogenous E6AP by RNA interference increased the HCV core protein level. In vitro and in vivo ubiquitylation assays showed that E6AP promotes ubiquitylation of the core protein. Exogenous expression of E6AP decreased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected Huh-7 cells. Furthermore, knockdown of endogenous E6AP by RNA interference increased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected cells. Taken together, our results provide evidence that E6AP mediates ubiquitylation and degradation of HCV core protein. We propose that the E6AP-mediated ubiquitin-proteasome pathway may affect the production of HCV particles through controlling the amounts of viral nucleocapsid protein.

Cell Line↗

PubMed2006

Requirement of E7 oncoprotein for viability of HeLa cells.

Most human papillomavirus (HPV)-positive cervical cancers contain integrated copies of the viral genome in their chromosomes and express the viral oncoproteins E6 and E7. A virus-encoded transcription factor, E2, is known to repress E6/E7 expression in HPV-positive cancer cells, leading to growth inhibition, which indicates that E6/E7 is required for the survival of the cells. We found that the E2-mediated growth inhibition of HeLa cells, an HPV18-positive cancer cell line, was coupled with a reduction in telomerase activity, an effect which was rescued by the complementation of E7 expression, but not E6 expression, indicating that the cell viability and the telomerase activity in HeLa cells are maintained by an E7-associated function. Analysis of E7 mutants suggested that the binding to the pRB family of pocket proteins was involved in the ability of E7 to rescue the growth potential and telomerase activity inhibited by E2 expression. We also showed that the telomerase activity upregulated by E7 expression was determined by the hTERT promoter activity, and that c-Myc upregulation caused by pRB inactivation could account for the promoter activity. The activation of p53 and consequent accumulation of p21Cip1, which were triggered by the downregulation of E6, appeared not to be essential for the E2-mediated growth arrest.

Cell Cycle Proteins↗

PubMed2006

Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen interacts with bromodomain protein Brd4 on host mitotic chromosomes.

The latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV) is required for viral episome maintenance in host cells during latent infection. Two regions of the protein have been implicated in tethering LANA/viral episomes to the host mitotic chromosomes, and LANA chromosome-binding sites are subjects of high interest. Because previous studies had identified bromodomain protein Brd4 as the mitotic chromosome anchor for the bovine papillomavirus E2 protein, which tethers the viral episomes to host mitotic chromosomes (J. You, J. L. Croyle, A. Nishimura, K. Ozato, and P. M. Howley, Cell 117:349-360, 2004, and J. You, M. R. Schweiger, and P. M. Howley, J. Virol. 79:14956-14961, 2005), we examined whether KSHV LANA interacts with Brd4. We found that LANA binds Brd4 in vivo and in vitro and that the binding is mediated by a direct protein-protein interaction between the ET (extraterminal) domain of Brd4 and a carboxyl-terminal region of LANA previously implicated in chromosome binding. Brd4 associates with mitotic chromosomes throughout mitosis and demonstrates a strong colocalization with LANA and the KSHV episomes on host mitotic chromosomes. Although another bromodomain protein, RING3/Brd2, binds to LANA in a similar fashion in vitro, it is largely excluded from the mitotic chromosomes in KSHV-uninfected cells and is partially recruited to the chromosomes in KSHV-infected cells. These data identify Brd4 as an interacting protein for the carboxyl terminus of LANA on mitotic chromosomes and suggest distinct functional roles for the two bromodomain proteins RING3/Brd2 and Brd4 in LANA binding. Additionally, because Brd4 has recently been shown to have a role in transcription, we examined whether Brd4 can regulate the CDK2 promoter, which can be transactivated by LANA.

Antigens, Viral↗

PubMed2006

Bromodomain protein 4 mediates the papillomavirus E2 transcriptional activation function.

The papillomavirus E2 regulatory protein has essential roles in viral transcription and the initiation of viral DNA replication as well as for viral genome maintenance. Brd4 has recently been identified as a major E2-interacting protein and, in the case of the bovine papillomavirus type 1, serves to tether E2 and the viral genomes to mitotic chromosomes in dividing cells, thus ensuring viral genome maintenance. We have explored the possibility that Brd4 is involved in other E2 functions. By analyzing the binding of Brd4 to a series of alanine-scanning substitution mutants of the human papillomavirus type 16 E2 N-terminal transactivation domain, we found that amino acids required for Brd4 binding were also required for transcriptional activation but not for viral DNA replication. Functional studies of cells expressing either the C-terminal domain of Brd4 that can bind E2 and compete its binding to Brd4 or short interfering RNA to knock down Brd4 protein levels revealed a role for Brd4 in the transcriptional activation function of E2 but not for its viral DNA replication function. Therefore, these studies establish a broader role for Brd4 in the papillomavirus life cycle than as the chromosome tether for E2 during mitosis.

Cell Cycle Proteins↗

PubMed2005

E6AP and calmodulin reciprocally regulate estrogen receptor stability.

Estrogen promotes the proliferation of human breast epithelial cells by interacting with the estrogen receptor (ER). Physiological responses of cells to estrogen are regulated in part by degradation of the ER. Previous studies revealed that calmodulin binds directly to the ER, thereby enhancing its stability. Consistent with these findings, cell-permeable calmodulin antagonists dramatically reduced the number of ER in MCF-7 human breast epithelial cells. Here we investigated the molecular mechanism by which calmodulin attenuates ER degradation. MG132 and lactacystin, inhibitors of the ubiquitin-proteasome pathway, prevented the calmodulin antagonist CGS9343B from reducing the amount of ER in MCF-7 cells. In contrast, protease inhibitors afforded no protection. Moreover, CGS9343B enhanced ER ubiquitination. A point mutant ER construct that is unable to bind calmodulin, termed ERDeltaCaM, is ubiquitinated to a greater extent than wild type ER. The ubiquitin-protein isopeptide ligase E6-associated protein (E6AP) associated with and promoted the degradation of ER. The possible convergence of calmodulin and E6AP on ER degradation was examined. ERDeltaCaM bound E6AP with higher affinity than that of wild type ER. Moreover, calmodulin attenuated the in vitro interaction between ER and E6AP in a Ca(2+)-dependent manner. Collectively, our data reveal that E6AP is a component of ER degradation via the ubiquitin-proteasome pathway and that Ca(2+)/calmodulin modulates this degradation mechanism. These results have potential implications for the development of selectively targeted therapeutic agents for breast cancer.

Animals↗

PubMed2005

Bovine papillomavirus E7 transformation function correlates with cellular p600 protein binding.

The E7 oncoprotein of bovine papillomavirus type 1 (BPV-1) is required for the full transformation activity of the virus. However, the mechanism by which E7 contributes to cellular transformation is unknown. To address this question, we used the proteomic approach of tandem affinity purification to identify cellular proteins that are in complex with E7, and identified the 600-kDa protein, p600, as a binding partner of E7. The ability of E7 to complex with p600 correlated with its ability to enhance anchorage independence of BPV-1 E6-expressing cells. Furthermore, E7 mutant proteins impaired in their ability to bind p600 were transformation defective. Additionally, knockdown of p600 reduced transformation of cells expressing both BPV-1 E6 and E7, as well as E6 alone, suggesting that the ability of E7 to transformed cells is mediated, at least in part, through its ability to bind p600. These data complement work that shows that HPV16 E7 also interacts with p600, and that this interaction correlates with the ability of HPV16 E7 to transform cells. These studies thus identify p600 as a shared target of the E7 proteins of multiple papillomaviruses.

Amino Acid Sequence↗

PubMed2005

Association of the human papillomavirus type 16 E7 oncoprotein with the 600-kDa retinoblastoma protein-associated factor, p600.

The human papillomavirus type 16 (HPV-16) E7 gene encodes a multifunctional oncoprotein that can subvert multiple cellular regulatory pathways. The best-known cellular targets of the HPV-16 E7 oncoprotein are the retinoblastoma tumor suppressor protein pRB and the related pocket proteins p107 and p130. However, there is ample evidence that E7 has additional cellular targets that contribute to its transforming potential. We isolated HPV-16 E7 associated cellular protein complexes by tandem affinity purification and mass spectrometry and identified the 600-kDa retinoblastoma protein associated factor, p600, as a cellular target of E7. Association of E7 with p600 is independent of the pocket proteins and is mediated through the N terminal E7 domain, which is related to conserved region 1 of the adenovirus E1A protein and importantly contributes to cellular transformation independent of pRB binding. Depletion of p600 protein levels by RNA interference substantially decreased anchorage-independent growth in HPV-positive and -negative human cancer cells. Therefore, p600 is a cellular target of E7 that regulates cellular pathways that contribute to anchorage-independent growth and cellular transformation.

Calmodulin-Binding Proteins↗

PubMed2005

Interaction of tSNARE syntaxin 18 with the papillomavirus minor capsid protein mediates infection.

The papillomavirus capsid mediates binding to the cell surface and passage of the virion to the perinuclear region during infection. To better understand how the virus traffics across the cell, we sought to identify cellular proteins that bind to the minor capsid protein L2. We have identified syntaxin 18 as a protein that interacts with bovine papillomavirus type 1 (BPV1) L2. Syntaxin 18 is a target membrane-associated soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (tSNARE) that resides in the endoplasmic reticulum (ER). The ectopic expression of FLAG-tagged syntaxin 18, which disrupts ER trafficking, blocked BPV1 pseudovirion infection. Furthermore, the expression of FLAG-syntaxin 18 prevented the passage of BPV1 pseudovirions to the perinuclear region that is consistent with the ER. Genetic studies identified a highly conserved L2 domain, DKILK, comprising residues 40 to 44 that mediated BPV1 trafficking through the ER during infection via an interaction with the tSNARE syntaxin 18. Mutations within the DKILK motif of L2 that did not significantly impact virion morphogenesis or binding at the cell surface prevented the L2 interaction with syntaxin 18 and disrupted BPV1 infection.

Amino Acid Motifs↗

PubMed2005

Identification of TRAIL as an interferon regulatory factor 3 transcriptional target.

Interferon production and apoptosis in virus-infected cells are necessary to prevent progeny virus production and to eliminate infected cells. Paramyxovirus infection induces apoptosis through interferon regulatory factor 3 (IRF-3), but the exact mechanism of how IRF-3 functions is unknown. We show that IRF-3 is involved in the transcriptional induction of TRAIL, a key player in the apoptosis pathway. IRF-3 upregulates TRAIL transcription following viral infection and binds an interferon-stimulated response element in the TRAIL promoter. The mRNA for TRAIL and its receptor, DR5, are induced following viral infection. These studies identify TRAIL as a novel IRF-3 transcriptional target.

Apoptosis↗

PubMed2005

Inhibition of E2 binding to Brd4 enhances viral genome loss and phenotypic reversion of bovine papillomavirus-transformed cells.

The bovine papillomavirus E2 protein tethers the viral genomes to mitotic chromosomes in dividing cells through binding to the C-terminal domain (CTD) of Brd4. Expression of the Brd4-CTD competes the binding of E2 to endogenous Brd4 in cells. Here we extend our previous study that identified Brd4 as the E2 mitotic chromosome receptor to show that Brd4-CTD expression released the viral DNA from mitotic chromosomes in BPV-1 transformed cells. Furthermore, stable expression of Brd4-CTD enhanced the frequency of morphological reversion of BPV-1 transformed C127 cells resulting in the complete elimination of the viral DNA in the resulting flat revertants.

Animals↗

PubMed2004

Ubiquitin family proteins and their relationship to the proteasome: a structural perspective.

Many biological processes rely on targeted protein degradation, the dysregulation of which contributes to the pathogenesis of various diseases. Ubiquitin plays a well-established role in this process, in which the covalent attachment of polyubiquitin chains to protein substrates culminates in their degradation via the proteasome. The three-dimensional structural topology of ubiquitin is highly conserved as a domain found in a variety of proteins of diverse biological function. Some of these so-called "ubiquitin family proteins" have recently been shown to bind components of the 26S proteasome via their ubiquitin-like domains, thus implicating proteasome activity in pathways other than protein degradation. In this chapter, we provide a structural perspective of how the ubiquitin family of proteins interacts with the proteasome.

Amino Acid Sequence↗

PubMed2004

Biochemical analysis of Angelman syndrome-associated mutations in the E3 ubiquitin ligase E6-associated protein.

Angelman syndrome is a severe neurological disorder characterized by mental retardation, absent speech, ataxia, seizures, and hyperactivity. The gene affected in this disorder is UBE3A, the gene encoding the E6-associated protein (E6AP) ubiquitin-protein ligase. Most patients have chromosomal deletions that remove the entire maternal allele of UBE3A. However, a small subset of patients have E6AP point mutations that result in single amino acid changes or short in-frame deletions that still allow translation of a full-length protein. By studying these point mutations in E6AP, we found a strong correlation between Angelman-associated mutations and a loss of E3 ubiquitin ligase activity. Interestingly the point mutations affect E6AP activity in different ways. Some mutant proteins cannot form thiol ester intermediates with ubiquitin, others retain the thiol ester formation activity but cannot efficiently transfer ubiquitin to a substrate, and still others are unstable in cells. Our results suggest that the loss of E6AP catalytic activity and likely the improper regulation of E6AP substrate(s) are important in the development of Angelman syndrome.

Angelman Syndrome↗

PubMed2004

Interaction of the bovine papillomavirus E2 protein with Brd4 tethers the viral DNA to host mitotic chromosomes.

The papillomavirus E2 protein tethers viral genomes to host mitotic chromosomes to ensure genome maintenance. We have identified the bromodomain protein Brd4 as a major cellular interacting partner of the bovine papillomavirus E2. Brd4 associates with mitotic chromosomes and colocalizes with E2 on mitotic chromosomes. The site of E2 binding maps to the C-terminal domain of Brd4. Expression of this C-terminal Brd4 domain functions in a dominant-negative manner to abrogate the colocalization of E2 with Brd4 on mitotic chromosomes, to block association of the viral episomes with Brd4, and to inhibit BPV-1 DNA-mediated cellular transformation. Brd4 also associates with HPV16 E2, indicating that Brd4 binding may be a shared property of all papillomavirus E2 proteins. The interaction of E2 with Brd4 is required to ensure the tethering of viral genomes to the host mitotic chromosomes for persistence of viral episomes in PV-infected cells.

Adenovirus E2 Proteins↗

PubMed2003

Ubiquitin recognition by the DNA repair protein hHR23a.

Ubiquitin is a prominent regulatory protein in numerous biological processes, including targeted protein degradation, endocytic sorting, transcriptional control, intranuclear localization, and retroviral virion budding. Ubiquitin-associated (UBA) domains, ubiquitin interacting motifs (UIM), and coupling of ubiquitin conjugation to ER degradation (CUE) motifs have been identified as ubiquitin receptors. The DNA repair protein hHR23a has two UBA domains that can each bind ubiquitin in addition to an N-terminal UBL domain that binds S5a and S2, two components of the 26S proteasome. Here we reveal hHR23a recognizes ubiquitin through a predominately hydrophobic surface formed by residues within alpha1 and alpha3 of each of its UBA domains. These two UBA surfaces bind a region on ubiquitin that includes K48. These findings have implications for published studies revealing that hHR23a inhibits K48-linked polyubiquitin chain formation. In addition, by using (15)N NMR relaxation experiments, we find that binding ubiquitin requires a structural change in hHR23a. HHR23 proteins are hypothesized to link ubiquitin to S5a, and we provide direct evidence that hHR23 could form a ternary complex with ubiquitin and S5a.

Carrier Proteins↗

PubMed2003

DNA-repair protein hHR23a alters its protein structure upon binding proteasomal subunit S5a.

The Rad23 family of proteins, including the human homologs hHR23a and hHR23b, stimulates nucleotide excision repair and has been shown to provide a novel link between proteasome-mediated protein degradation and DNA repair. In this work, we illustrate how the proteasomal subunit S5a regulates hHR23a protein structure. By using NMR spectroscopy, we have elucidated the structure and dynamic properties of the 40-kDa hHR23a protein and show it to contain four structured domains connected by flexible linker regions. In addition, we reveal that these domains interact in an intramolecular fashion, and by using residual dipolar coupling data in combination with chemical shift perturbation analysis, we present the hHR23a structure. By itself, hHR23a adopts a closed conformation defined by the interaction of an N-terminal ubiquitin-like domain with two ubiquitin-associated domains. Interestingly, binding of the proteasomal subunit S5a disrupts the hHR23a interdomain interactions and thereby causes it to adopt an opened conformation.

Amino Acid Sequence↗

PubMed2003

The ubiquitin-associated domain of hPLIC-2 interacts with the proteasome.

The ubiquitin-like hPLIC proteins can associate with proteasomes, and hPLIC overexpression can specifically interfere with ubiquitin-mediated proteolysis (Kleijnen et al., 2000). Because the hPLIC proteins can also interact with certain E3 ubiquitin protein ligases, they may provide a link between the ubiquitination and proteasomal degradation machineries. The amino-terminal ubiquitin-like (ubl) domain is a proteasome-binding domain. Herein, we report that there is a second proteasome-binding domain in hPLIC-2: the carboxyl-terminal ubiquitin-associated (uba) domain. Coimmunoprecipitation experiments of wild-type and mutant hPLIC proteins revealed that the ubl and uba domains each contribute independently to hPLIC-2-proteasome binding. There is specificity for the interaction of the hPLIC-2 uba domain with proteasomes, because uba domains from several other proteins failed to bind proteasomes. Furthermore, the binding of uba domains to polyubiquitinated proteins does not seem to be sufficient for the proteasome binding. Finally, the uba domain is necessary for the ability of full-length hPLIC-2 to interfere with the ubiquitin-mediated proteolysis of p53. The PLIC uba domain has been reported to bind and affect the functions of proteins such as GABAA receptor and presenilins. It is possible that the function of these proteins may be regulated or mediated through proteasomal degradation pathways.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Transcriptome signature of irreversible senescence in human papillomavirus-positive cervical cancer cells.

A frequent characteristic of human papillomavirus (HPV)-positive cervical cancers is the loss of viral E2 gene expression in HPV-infected cervical epithelial cells as a consequence of viral DNA integration into the cellular genome. The expression of E2 in HPV-positive cancer cells results in the repression of the viral E6/E7 oncogenes, activation of the p53 and pRB pathways, and a G1 cell cycle arrest, followed by induction of cellular senescence. The transcriptional consequences of E2-mediated cell cycle arrest that lead to senescence currently are unknown. Using conditional senescence induction in HeLa cells and microarray analysis, we describe here the expression profile of cells irreversibly committed to senescence. Our results provide insight into the molecular anatomy of senescence pathways and its regulation by HPV on-coproteins. These include the induction of the RAB vesicular transport machinery and a general down-regulation of chromatin regulatory molecules. The repression of tumor-specific G antigens during E2 senescence supports a reversal of the tumorigenic phenotype by E2 and the potential approach of tumor-specific G antigen-specific immunotherapy for cervical cancer.

Adenoviridae↗

PubMed2003

The ER-lumenal domain of the HHV-7 immunoevasin U21 directs class I MHC molecules to lysosomes.

Like all members of the herpesvirus family, human herpesvirus-7 has evolved mechanisms to evade immune detection. The human herpesvirus-7 gene product U21 encodes an immunoevasin that binds to class I major histocompatibility complex molecules and diverts them to a lysosomal compartment. Here we show that the cytoplasmic tail of U21, although sufficient to sequester a heterologous membrane protein (CD4 chimera), has no effect on U21's ability to redirect class I major histocompatibility complex molecules to lysosomes. Instead, the ER-lumenal domain of U21 is sufficient to redirect class I major histocompatibility complex molecules to the lysosomal compartment. These observations demonstrate a novel viral immunoevasive mechanism for U21, and implicate the ER-lumenal domain of a type I transmembrane protein in lysosomal sorting.

Amino Acid Motifs↗