Interaction proteomics identify NEURL4 and the HECT E3 ligase HERC2 as novel modulators of centrosome architecture

Centrosomes are composed of a centriole pair surrounded by an intricate proteinaceous matrix referred to as pericentriolar material. Although the mechanisms underpinning the control of centriole duplication are now well understood, we know relatively little about the control of centrosome size and shape. Here we used interaction proteomics to identify the E3 ligase HERC2 and the neuralized homologue NEURL4 as novel interaction partners of the centrosomal protein CP110. Using high resolution imaging, we find that HERC2 and NEURL4 localize to the centrosome and that interfering with their function alters centrosome morphology through the appearance of aberrant filamentous structures that stain for a subset of pericentriolar material proteins including pericentrin and CEP135. Using an RNA interference-resistant transgene approach in combination with structure-function analyses, we show that the association between CP110 and HERC2 depends on nonoverlapping regions of NEURL4. Whereas CP110 binding to NEURL4 is dispensable for the regulation of pericentriolar material architecture, its association with HERC2 is required to maintain normal centrosome integrity. NEURL4 is a substrate of HERC2, and together these results indicate that the NEURL4-HERC2 complex participates in the ubiquitin-dependent regulation of centrosome architecture.     

TSC1 stabilizes TSC2 by inhibiting the interaction between TSC2 and the HERC1 ubiquitin ligase

Tuberous sclerosis complex (TSC) is an autosomal dominant disease characterized by hamartoma formation in various organs. Two genes responsible for the disease, TSC1 and TSC2, have been identified. The TSC1 and TSC2 proteins, also called hamartin and tuberin, respectively, have been shown to regulate cell growth through inhibition of the mammalian target of rapamycin pathway. TSC1 is known to stabilize TSC2 by forming a complex with TSC2, which is a GTPase-activating protein for the Rheb small GTPase. We have identified HERC1 as a TSC2-interacting protein. HERC1 is a 532-kDa protein with an E3 ubiquitin ligase homology to E6AP carboxyl terminus (HECT) domain. We observed that the interaction of TSC1 with TSC2 appears to exclude TSC2 from interacting with HERC1. Disease mutations in TSC2, which result in its destabilization, allow binding to HERC1 in the presence of TSC1. Our study reveals a potential molecular mechanism of how TSC1 stabilizes TSC2 by excluding the HERC1 ubiquitin ligase from the TSC2 complex. Furthermore, these data reveal a possible biochemical basis of how certain disease mutations inactivate TSC2.     

Physical and functional interaction of the HECT ubiquitin-protein ligases E6AP and HERC2

Deregulation of the ubiquitin-protein ligase E6AP contributes to the development of the Angelman syndrome and to cervical carcinogenesis suggesting that the activity of E6AP needs to be under tight control. However, how E6AP activity is regulated at the post-translational level under non-pathologic conditions is poorly understood. In this study, we report that the giant protein HERC2, which is like E6AP a member of the HECT family of ubiquitin-protein ligases, binds to E6AP. The interaction is mediated by the RCC1-like domain 2 of HERC2 and a region spanning amino acid residues 150-200 of E6AP. Furthermore, we provide evidence that HERC2 stimulates the ubiquitin-protein ligase activity of E6AP in vitro and within cells and that this stimulatory effect does not depend on the ubiquitin-protein ligase activity of HERC2. Thus, the data obtained indicate that HERC2 acts as a regulator of E6AP.     

Network Analysis of UBE3A/E6AP-Associated Proteins Provides Connections to Several Distinct Cellular Processes

Perturbations in activity and dosage of the UBE3A ubiquitin-ligase have been linked to Angelman syndrome and autism spectrum disorders. UBE3A was initially identified as the cellular protein hijacked by the human papillomavirus E6 protein to mediate the ubiquitylation of p53, a function critical to the oncogenic potential of these viruses. Although a number of substrates have been identified, the normal cellular functions and pathways affected by UBE3A are largely unknown. Previously, we showed that UBE3A associates with HERC2, NEURL4, and MAPK6/ERK3 in a high-molecular-weight complex of unknown function that we refer to as the HUN complex (HERC2, UBE3A, and NEURL4). In this study, the combination of two complementary proteomic approaches with a rigorous network analysis revealed cellular functions and pathways in which UBE3A and the HUN complex are involved. In addition to finding new UBE3A-associated proteins, such as MCM6, SUGT1, EIF3C, and ASPP2, network analysis revealed that UBE3A-associated proteins are connected to several fundamental cellular processes including translation, DNA replication, intracellular trafficking, and centrosome regulation. Our analysis suggests that UBE3A could be involved in the control and/or integration of these cellular processes, in some cases as a component of the HUN complex, and also provides evidence for crosstalk between the HUN complex and CAMKII interaction networks. This study contributes to a deeper understanding of the cellular functions of UBE3A and its potential role in pathways that may be affected in Angelman syndrome, UBE3A-associated autism spectrum disorders, and human papillomavirus-associated cancers.     

HERC3 binding to and regulation by ubiquitin

Members of the HERC (domain homologous to E6 associated protein carboxy-terminus and RCC1 domain protein) family may function both as guanine nucleotide exchange factors and E3 ubiquitin ligases. Here we identify an unstudied member, HERC3. This protein was recognized by specific antibodies in different cell types. HERC3 was located in the cytosol and in vesicular-like structures containing beta-COP, ARF and Rab5 proteins. Involvement of HERC3 in the ubiquitin system was suggested by its ability to interact with ubiquitin. The conserved cysteine in HECT proteins was not essential for this non-covalent binding. Moreover, HERC3 was a substrate of ubiquitination being degraded by the proteasome. These observations indicate a fine regulation of HERC3 and suggest a role in vesicular traffic and ubiquitin-dependent processes.     

The E3 ubiquitin ligase HOIL-1 induces the polyubiquitination and degradation of SOCS6 associated proteins

The suppressor of cytokine signaling (SOCS) proteins are thought to exert their function through the recruitment of interacting-proteins to the ubiquitin/proteasome degradation pathway. All SOCS proteins bind an Elongin BC E3 ubiquitin ligase complex through the common Socs-box. Here, we show that haem-oxidized IRP2 ubiquitin ligase-1 (HOIL-1), another E3 ubiquitin ligase, interacts with SOCS6. The Ubl domain of HOIL-1 and the SH2 and Socs-box domains of SOCS6 are required for the interaction. HOIL-1 expression stabilizes SOCS6 and induces the ubiquitination and degradation of proteins associated with SOCS6. These data suggest that SOCS proteins may interact with different E3 ubiquitin ligases in addition to a common Elongin BC E3 complex.     

Parkin facilitates the elimination of expanded polyglutamine proteins and leads to preservation of proteasome function

Parkin, the most commonly mutated gene in familial Parkinson's disease, encodes an E3 ubiquitin ligase. A number of candidate substrates have been identified for parkin ubiquitin ligase action including CDCrel-1, o-glycosylated alpha-synuclein, Pael-R, and synphilin-1. We now show that parkin promotes the ubiquitination and degradation of an expanded polyglutamine protein. Overexpression of parkin reduces aggregation and cytotoxicity of an expanded polyglutamine ataxin-3 fragment. Using a cellular proteasome indicator system based on a destabilized form of green fluorescent protein, we demonstrate that parkin reduces proteasome impairment and caspase-12 activation induced by an expanded polyglutamine protein. Parkin forms a complex with the expanded polyglutamine protein, heat shock protein 70 (Hsp70) and the proteasome, which may be important for the elimination of the expanded polyglutamine protein. Hsp70 enhances parkin binding and ubiquitination of expanded polyglutamine protein in vitro suggesting that Hsp70 may help to recruit misfolded proteins as substrates for parkin E3 ubiquitin ligase activity. We speculate that parkin may function to relieve endoplasmic reticulum stress by preserving proteasome activity in the presence of misfolded proteins. Loss of parkin function and the resulting proteasomal impairment may contribute to the accumulation of toxic aberrant proteins in neurodegenerative diseases including Parkinson's disease.     

Herpes simplex virus type 1 immediate-early protein ICP0 and is isolated RING finger domain act as ubiquitin E3 ligases in vitro.

Proteasome-dependent degradation of ubiquitinated proteins plays a key role in many important cellular processes. Ubiquitination requires the E1 ubiquitin activating enzyme, an E2 ubiquitin conjugating enzyme, and frequently a substrate-specific ubiquitin protein ligase (E3). One class of E3 ubiquitin ligases has been shown to contain a common zinc-binding RING finger motif. We have previously shown that herpes simplex virus type 1 ICP0, itself a RING finger protein, induces the proteasome-dependent degradation of several cellular proteins and induces the accumulation of colocalizing conjugated ubiquitin in vivo. We now report that both full-length ICP0 and its isolated RING finger domain induce the accumulation of polyubiquitin chains in vitro in the presence of E1 and the E2 enzymes UbcH5a and UbcH6. Mutations within the RING finger region that abolish the in vitro ubiquitination activity also cause severe reductions in ICP0 activity in other assays. We conclude that ICP0 has the potential to act as an E3 ubiquitin ligase during viral infection and to target specific cellular proteins for destruction by the 26S proteasome.     

Regulation of the human papillomavirus type 18 E6/E6AP ubiquitin ligase complex by the HECT domain-containing protein EDD

Human papillomavirus (HPV) E6 oncoproteins target many cellular proteins for ubiquitin-mediated proteasomal degradation. In the case of p53, this is mediated principally by the E6AP ubiquitin ligase. Several studies have reported that E6 can target certain of its substrates in an apparently E6AP-independent fashion and that several of these substrates vary in the degree to which they are degraded by E6 at different stages of malignancy. To more fully understand the regulation of the E6AP/E6 proteolytic targeting complex, we performed a mass spectroscopic analysis of HPV type 18 (HPV-18) E6 protein complexes and identified the HECT domain-containing ubiquitin ligase EDD as a new HPV-18 E6 binding partner. We show that EDD can interact independently with both E6 and E6AP. Furthermore, EDD appears to regulate E6AP expression levels independently of E6, and loss of EDD stimulates the proteolytic activity of the E6/E6AP complex. Conversely, higher levels of EDD expression protect a number of substrates from E6-induced degradation, partly as a consequence of lower levels of E6 and E6AP expression. Intriguingly, reduction in EDD expression levels in HPV-18-positive HeLa cells enhances cell resistance to apoptotic and growth arrest stimuli. These studies suggest that changes in the levels of EDD expression during different stages of the viral life cycle or during malignancy could have a profound effect upon the ability of E6 to target various substrates for proteolytic degradation and thereby directly influence the development of HPV-induced malignancy.     

Identification of annexin A1 as a novel substrate for E6AP‐mediated ubiquitylation

E6‐associated protein (E6AP) is a cellular ubiquitin protein ligase that mediates ubiquitylation and degradation of p53 in conjunction with the high‐risk human papillomavirus E6 proteins. However, the physiological functions of E6AP are poorly understood. To identify a novel biological function of E6AP, we screened for binding partners of E6AP using GST pull‐down and mass spectrometry. Here we identified annexin A1, a member of the annexin superfamily, as an E6AP‐binding protein. Ectopic expression of E6AP enhanced the degradation of annexin A1 in vivo. RNAi‐mediated downregulation of endogenous E6AP increased the levels of endogenous annexin A1 protein. E6AP interacted with annexin A1 and induced its ubiquitylation in a Ca²⁺‐dependent manner. GST pull‐down assay revealed that the annexin repeat domain III of annexin A1 is important for the E6AP binding. Taken together, our data suggest that annexin A1 is a novel substrate for E6AP‐mediated ubiquitylation. Our findings raise the possibility that E6AP may play a role in controlling the diverse functions of annexin A1 through the ubiquitin‐proteasome pathway. J. Cell. Biochem. 106: 1123–1135, 2009. © 2009 Wiley‐Liss, Inc.     

Control of mRNA export by adenovirus E4orf6 and E1B55K proteins during productive infection requires E4orf6 ubiquitin ligase activity

During the adenovirus infectious cycle, the early proteins E4orf6 and E1B55K are known to perform several functions. These include nuclear export of late viral mRNAs, a block of nuclear export of the bulk of cellular mRNAs, and the ubiquitin-mediated degradation of selected proteins, including p53 and Mre11. Degradation of these proteins occurs via a cellular E3 ubiquitin ligase complex that is assembled through interactions between elongins B and C and BC boxes present in E4orf6 to form a cullin 5-based ligase complex. E1B55K, which has been known for some time to associate with the E4orf6 protein, is thought to bind to specific substrate proteins to bring them to the complex for ubiquitination. Earlier studies with E4orf6 mutants indicated that the interaction between the E4orf6 and E1B55K proteins is optimal only when E4orf6 is able to form the ligase complex. These and other observations suggested that most if not all of the functions ascribed to E4orf6 and E1B55K during infection, including the control of mRNA export, are achieved through the degradation of specific substrates by the E4orf6 ubiquitin ligase activity. We have tested this hypothesis through the generation of a virus mutant in which the E4orf6 product is unable to form a ligase complex and indeed have found that this mutant behaves identically to an E4orf6⁻ virus in production of late viral proteins, growth, and export of the late viral L5 mRNA.     

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.     

人泛素连接酶AREL1催化结构域的晶体结构

泛素化是一种重要的翻译后修饰,几乎调控着生命活动的所有方面.泛素连接酶是泛素化过程中唯一对底物蛋白质有特异性识别能力的一类酶,它们在泛素化过程中是不可或缺的,起到非常关键的作用.人抗凋亡E3泛素连接酶(AREL1)是HECT泛素连接酶家族成员之一,它能够泛素化促凋亡蛋白SMAC、HtrA2和ARTS,并通过蛋白酶体将它们降解,从而发挥抵抗细胞凋亡的作用.本文解析了3.2?分辨率的人AREL1蛋白催化结构域(AREL1HECT)的晶体结构,并将其与HECT家族中其他成员的结构进行了比对.尺寸排阻色谱和X射线小角散射的结果表明,AREL1HECT在溶液中是以多种聚集状态形式存在的,小角散射的3D模型进一步表明AREL1HECT在溶液中会发生二聚化.这些结果将为AREL1HECT与泛素复合物结构的解析及功能的分析提供坚实的结构基础,为揭示AREL1泛素化底物蛋白质的分子机制提供重要的依据.     

Degradation of tyrosine phosphatase PTPN3 (PTPH1) by association with oncogenic human papillomavirus E6 proteins

Oncoproteins from DNA tumor viruses associate with critical cellular proteins to regulate cell proliferation, survival, and differentiation.Human papillomavirus (HPV) E6 oncoproteins have been previously shown to associate with a cellular HECT domain ubiquitin ligase termed E6AP (UBE3A). Here we show that the E6-E6AP complex associates with and targets the degradation of the protein tyrosine phosphatase PTPN3 (PTPH1) in vitro and in living cells. PTPN3 is a membrane-associated tyrosine phosphatase with FERM, PDZ, and PTP domains previously implicated in regulating tyrosine phosphorylation of growth factor receptors and p97 VCP (valosin-containing protein, termed Cdc48 in Saccharomyces cerevisiae) and is mutated in a subset of colon cancers. Degradation of PTPN3 by E6 requires E6AP, the proteasome, and an interaction between the carboxy terminus of E6 and the PDZ domain of PTPN3. In transduced keratinocytes, E6 confers reduced growth factor requirements, a function that requires the PDZ ligand of E6 and that can in part be replicated by inhibiting the expression of PTPN3. This report demonstrates the potential of E6 to regulate phosphotyrosine metabolism through the targeted degradation of a tyrosine phosphatase.     

Deubiquitinase FAM/USP9X Interacts with the E3 Ubiquitin Ligase SMURF1 Protein and Protects It from Ligase Activity-dependent Self-degradation

Ubiquitination is an essential post-translational modification that mediates diverse cellular functions. SMAD-specific E3 ubiquitin protein ligase 1 (SMURF1) belongs to the Nedd4 family of HECT ubiquitin ligases that directly catalyzes ubiquitin conjugation onto diverse substrates. As a result, SMURF1 regulates a great variety of cellular physiologies including bone morphogenetic protein (BMP) signaling, cell migration, and planar cell polarity. Structurally, SMURF1 consists of a C2 domain, two WW domain repeats, and a catalytic HECT domain essential for its E3 ubiquitin ligase activity. This modular architecture allows for interactions with other proteins, which are either substrates or adaptors of SMURF1. Despite the increasing number of SMURF1 substrates identified, current knowledge regarding regulatory proteins and their modes of action on controlling SMURF1 activity is still limited. In this study, we employed quantitative mass spectrometry to analyze SMURF1-associated cellular complexes, and identified the deubiquitinase FAM/USP9X as a novel interacting protein for SMURF1. Through domain mapping study, we found the second WW domain of SMURF1 and the carboxyl terminus of USP9X critical for this interaction. SMURF1 is autoubiquitinated through its intrinsic HECT E3 ligase activity, and is degraded by the proteasome. USP9X association antagonizes this activity, resulting in deubiquitination and stabilization of SMURF1. In MDA-MB-231 breast cancer cells, SMURF1 expression is elevated and is required for cellular motility. USP9X stabilizes endogenous SMURF1 in MDA-MB-231 cells. Depletion of USP9X led to down-regulation of SMURF1 and significantly impaired cellular migration. Taken together, our data reveal USP9X as an important regulatory protein of SMURF1 and suggest that the association between deubiquitinase and E3 ligase may serve as a common strategy to control the cellular protein dynamics through modulating E3 ligase stability.