植物蛋白的体外泛素化检测方法

泛素激活酶(E1)、泛素耦联酶(E2)和泛素连接酶(E3)是蛋白质泛素化修饰的关键酶。在真核基因组上有大量基因编码这些泛素化相关的酶类或蛋白。检测这些泛素化修饰酶及其底物蛋白的生化特性和特异性是分析其生物学功能的重要内容。该文提供了一种简便快速检测体外泛素化反应的方法, 不仅可通过检测对DTT敏感的硫酯键的形成来判断E2的活性、检测E3的体外泛素化活性, 而且可以检测E2-E3和E3-底物的特异性。所用蛋白主要来源于拟南芥(Arabidopsis thaliana), 包括分属于绝大多数E2亚家族的成员, 可用于不同RING类型E3的活性检测。该方法不仅可以采用多种E2进行E3活性分析, 而且可以分析不同组合的E2-RING E3、RING E3-底物的泛素化活性等, 亦可应用于真核生物蛋白质尤其是植物蛋白的体外泛素化活性分析。     

植物蛋白的体外泛素化检测方法

泛素激活酶(E1)、泛素耦联酶(E2)和泛素连接酶(E3)是蛋白质泛素化修饰的关键酶。在真核基因组上有大量基因编码这些泛素化相关的酶类或蛋白。检测这些泛素化修饰酶及其底物蛋白的生化特性和特异性是分析其生物学功能的重要内容。该文提供了一种简便快速检测体外泛素化反应的方法, 不仅可通过检测对DTT敏感的硫酯键的形成来判断E2的活性、检测E3的体外泛素化活性, 而且可以检测E2-E3和E3-底物的特异性。所用蛋白主要来源于拟南芥(Arabidopsis thaliana), 包括分属于绝大多数E2亚家族的成员, 可用于不同RING类型E3的活性检测。该方法不仅可以采用多种E2进行E3活性分析, 而且可以分析不同组合的E2-RING E3、RING E3-底物的泛素化活性等, 亦可应用于真核生物蛋白质尤其是植物蛋白的体外泛素化活性分析。     

Brap2 facilitates HsCdc14A Lys-63 linked ubiquitin modification

Protein phosphotase Cdc14 (Cell division cycle gene 14) is a key regulator of late mitotic events in Saccharomyces cerevisiae. However the function of human Cdc14 (HsCdc14A & B) and its regulatory network are still elusive. In this study, we identified a new partner of HsCdc14A named Brap2 (BRCA1 associated protein 2) using yeast two-hybrid screening assay. The interaction between these two proteins is confirmed by co-immunoprecipitation in human HEK 293T cells. Brap2 co-localizes with HsCdc14A on mitotic spindle poles and over-expression of Brap2 causes multiple spindle poles. Furthermore, we found that Brap2, which has intrinsic RING domain dependent E3 ligase activity, facilitates HsCdc14A Lys-63 linked ubiquitin modification, indicating that Brap2 may be the ubiquitin E3 Ligase of HsCdc14A. Our findings imply that Brap2 plays a significant role in cell cycle regulation besides its facilitation of HsCdc14A ubiquitination.     

Inhibition of ubiquitination and stabilization of human ubiquitin E3 ligase PIRH2 by measles virus phosphoprotein

Using a C-terminal domain (PCT) of the measles virus (MV) phosphoprotein (P protein) as bait in a yeast two-hybrid screen, a cDNA identical to the recently described human p53-induced-RING-H2 (hPIRH2) cDNA was isolated. A glutathione S-transferase-hPIRH2 fusion protein expressed in bacteria was able to pull down P protein when mixed with an extract from P-expressing HeLa cells in vitro, and myc-tagged hPIRH2 could be reciprocally co-immunoprecipitated with MV P protein from human cells. Additionally, immunoprecipitation experiments demonstrated that hPIRH2-myc, MV P, and nucleocapsid (N) proteins form a ternary complex. The hPIRH2 binding site was mapped to the C-terminal X domain region of the P protein by using a yeast two-hybrid assay. The PCT binding site was mapped on hPIRH2 by using a novel yeast two-hybrid tagged PCR approach and by co-immunoprecipitation of hPIRH2 cysteine mutants and mouse/human PIRH2 chimeras. The hPIRH2 C terminus could mediate the interaction with MV P which was favored by the RING-H2 motif. When coexpressed with an enhanced green fluorescent protein-tagged hPIRH2 protein, MV P alone or in a complex with MV N was able to redistribute hPIRH2 to outside the nucleus, within intracellular aggregates. Finally, MV P efficiently stabilized hPIRH2-myc expression and prevented its ubiquitination in vivo but had no effect on the stability or ubiquitination of an alternative ubiquitin E3 ligase, Mdm2. Thus, MV P protein is the first protein from a pathogen that is able to specifically interact with and stabilize the ubiquitin E3 ligase hPIRH2 by preventing its ubiquitination.     

The full-length and N-terminal deletion of ORF2 protein of hepatitis E virus can dimerize

Hepatitis E virus is a human RNA virus containing three open reading frames. Of these ORF2 encodes, the major capsid protein (pORF2), may possess regulatory functions, in addition to a structural one. In this study, we have shown using the yeast two-hybrid system and in vitro immobilization experiments that full-length pORF2 is capable of self-association, thus forming a homodimer. Using mutational analysis we have studied dimerization of various truncated versions of the ORF2 capsid protein using the yeast two-hybrid system and supported our findings with in vitro immobilization experiments. Deletions of pORF2 reveal a loss of the dimerization potential for all deletions except an N-terminal 127-amino-acid deletion. Our studies suggest that the dimerization property of pORF2 may not be amino-acid sequence-dependent but instead a complex formation of a specific tertiary structure that imparts pORF2 its property to self-associate.     

A new scheme to discover functional associations and regulatory networks of E3 ubiquitin ligases

Background

Protein ubiquitination catalyzed by E3 ubiquitin ligases play important modulatory roles in various biological processes. With the emergence of high-throughput mass spectrometry technology, the proteomics research community embraced the development of numerous experimental methods for the determination of ubiquitination sites. The result is an accumulation of ubiquitinome data, coupled with a lack of available resources for investigating the regulatory networks among E3 ligases and ubiquitinated proteins. In this study, by integrating existing ubiquitinome data, experimentally validated E3 ligases and established protein-protein interactions, we have devised a strategy to construct a comprehensive map of protein ubiquitination networks.

Results

In total, 41,392 experimentally verified ubiquitination sites from 12,786 ubiquitinated proteins of humans have been obtained for this study. Additional 494 E3 ligases along with 1220 functional annotations and 28588 protein domains were manually curated. To characterize the regulatory networks among E3 ligases and ubiquitinated proteins, a well-established network viewer was utilized for the exploration of ubiquitination networks from 40892 protein-protein interactions. The effectiveness of the proposed approach was demonstrated in a case study examining E3 ligases involved in the ubiquitination of tumor suppressor p53. In addition to Mdm2, a known regulator of p53, the investigation also revealed other potential E3 ligases that may participate in the ubiquitination of p53.

Conclusion

Aside from the ability to facilitate comprehensive investigations of protein ubiquitination networks, by integrating information regarding protein-protein interactions and substrate specificities, the proposed method could discover potential E3 ligases for ubiquitinated proteins. Our strategy presents an efficient means for the preliminary screen of ubiquitination networks and overcomes the challenge as a result of limited knowledge about E3 ligase-regulated ubiquitination.

     

The role of the coiled-coil motif in interactions mediated by TPD52

TPD52 (D52)-like proteins are small coiled-coil motif-bearing proteins first identified through their expression in human breast carcinoma that mutually interact in hetero- and homomeric fashions. However, it has been unclear whether the coiled-coil motif is sufficient, or even necessary, for these interactions to occur. We have therefore examined the binding activities of a panel of C-terminally deleted D52 proteins in both the yeast two-hybrid system and pull-down assays. In the yeast two-hybrid system, interactions were only detected when regions C-terminal to the coiled-coil motif were also present. However, using pull-down assays, interactions were detected for all deletion mutants which included the coiled-coil motif. This suggests that the coiled-coil motif is indeed necessary for interactions mediated by D52 proteins, but that C-terminal protein regions facilitate and/or stabilize these interactions.     

Regulation of synaptophysin degradation by mammalian homologues of seven in absentia.

Synaptophysin is an integral membrane protein of synaptic vesicles characterized by four transmembrane domains with both termini facing the cytoplasm. Although synaptophysin has been implicated in neurotransmitter release, and decreased synaptophysin levels have been associated with several neurodegenerative diseases, the molecular mechanism that regulates the degradation of synaptophysin remains unsolved. Using the cytoplasmic C terminus of synaptophysin as bait in a yeast two-hybrid screen, we identified two synaptophysin-binding proteins, Siah-1A and Siah-2, which are rat homologues of Drosophila Seven in Absentia. We demonstrated that Siah-1A and Siah-2 associate with synaptophysin both in vitro and in vivo and defined the binding domains of synaptophysin and Siah that mediate their association. Siah proteins exist in both cytosolic and membrane-associated pools and co-localize with synaptophysin on synaptic vesicles and early endosomes. In addition, Siah proteins interact specifically with the brain-enriched E2 ubiquitin-conjugating enzyme UbcH8 and facilitate the ubiquitination of synaptophysin. Furthermore, overexpression of Siah proteins promotes the degradation of synaptophysin via the ubiquitin-proteasome pathway. Our findings indicate that Siah proteins function as E3 ubiquitin-protein ligases to regulate the ubiquitination and degradation of synaptophysin.     

Unfolding of apomyoglobin examined by synchrotron footprinting

Hepatitis E virus is a human RNA virus containing three open reading frames. Of these, ORF2 encodes the major capsid protein (pORF2) and may possess regulatory functions, in addition to a structural one. In this study, we have shown using the yeast two-hybrid system and in vitro immobilization experiments that full-length pORF2 is capable of self-association, thus forming a homodimer. Using mutational analysis we have studied dimerization of various truncated versions of the ORF2 capsid protein using the yeast two-hybrid system and supported our findings with in vitro immobilization experiments. Deletions of pORF2 reveal a loss of the dimerization potential for all deletions except an N-terminal 127-amino-acid deletion. Our studies suggest that the dimerization property of pORF2 may not be amino-acid sequence dependent but instead a complex formation of a specific tertiary structure that imparts pORF2 its property to self-associate.     

酵母双杂交系统筛选GATA-1相互作用蛋白质及功能验证

目的 利用酵母双杂交技术从人脑cDNA文库中筛选与人GATA-1相互作用的蛋白质.方法 从人K562细胞中扩增出全长GATA1基因,设计引物将其3段截断体亚克隆入酵母表达载体pDBLeu中,转化至AH109感受态酵母中,利用酵母双杂交技术筛选人脑cDNA文库中与其相互作用的蛋白质,阳性克隆通过回转及免疫共沉淀试验进行验证,利用3xGATA荧光素酶报告基因对相互作用蛋白质进行功能验证.结果 成功构建出酵母诱饵蛋白表达质粒pDBLeu-GATA1（1）,pDBLeu-GATA1（2）,pDBLeu-GATA1（3）,筛到34个阳性克隆,用生物信息学分析及回转验证得到5个与GATA-1相互作用的候选蛋白,通过免疫共沉淀试验进一步验证,获得3个蛋白质能与GATA-1相互作用,分别是ECSIT,EFEMP1和GPS2.荧光素酶试验表明这3个蛋白质均能对GATA1的转录活性产生影响,证实它们之间的相互作用具有影响GATA1转录的功能.结论 应用酵母双杂交技术及免疫共沉淀试验,从人脑cDNA文库中成功获得3个与GATA-1相互作用并对其转录活性具有调节作用的蛋白质,为研究GATA1蛋白质的功能提供了新的线索.     

Cell Regulation by Phosphotyrosine-Targeted Ubiquitin Ligases

Three classes of E3 ubiquitin ligases, members of the Cbl, Hakai, and SOCS-Cul5-RING ligase families, stimulate the ubiquitination of phosphotyrosine-containing proteins, including receptor and nonreceptor tyrosine kinases and their phosphorylated substrates. Because ubiquitination frequently routes proteins for degradation by the lysosome or proteasome, these E3 ligases are able to potently inhibit tyrosine kinase signaling. Their loss or mutational inactivation can contribute to cancer, autoimmunity, or endocrine disorders, such as diabetes. However, these ligases also have biological functions that are independent of their ubiquitination activity. Here we review relevant literature and then focus on more-recent developments in understanding the structures, substrates, and pathways through which the phosphotyrosine-specific ubiquitin ligases regulate diverse aspects of cell biology.     

Two-hybrid reporter vectors for gap repair cloning

Yeast two-hybrid analysis is a valuable approach to the discovery and characterization of protein interactions. We have developed vectors that can indicate the presence of an insert when used in two-hybrid bait and prey construction by gap repair cloning. The strategy uses a recombination cloning site flanked by sequences encoding the GAL4 activation and binding domains. After gap repair cloning in standard hosts carrying an ADE2 reporter gene, disruption of GAL4 by an insert can be identified by the development of red colony color, while empty vector plasmids produce white colonies. Function in yeast two-hybrid applications was initially validated using known interacting proteins in pair-wise analyses, and subsequently, the bait vectors were used in library screens with the mouse Mad212 and human Mccd1 proteins, identifying a number of putative new interactions for these proteins. These vectors should facilitate high-throughput yeast two-hybrid screens in which large numbers of bait and prey constructs may be required.     

RNF5, a RING finger protein that regulates cell motility by targeting paxillin ubiquitination and altered localization

RNF5 is a RING finger protein found to be important in the growth and development of Caenorhabditis elegans. The search for RNF5-associated proteins via a yeast two-hybrid screen identified a LIM-containing protein in C. elegans which shows homology with human paxillin. Here we demonstrate that the human homologue of RNF5 associates with the amino-terminal domain of paxillin, resulting in its ubiquitination. RNF5 requires intact RING and C-terminal domains to mediate paxillin ubiquitination. Whereas RNF5 mediates efficient ubiquitination of paxillin in vivo, protein extracts were required for in vitro ubiquitination, suggesting that additional modifications and/or an associated E3 ligase assist RNF5 targeting of paxillin ubiquitination. Mutant Ubc13 efficiently inhibits RNF5 ubiquitination, suggesting that RNF5 generates polychain ubiquitin of the K63 topology. Expression of RNF5 increases the cytoplasmic distribution of paxillin while decreasing its localization within focal adhesions, where it is primarily seen under normal growth. Concomitantly, RNF5 expression results in inhibition of cell motility. Via targeting of paxillin ubiquitination, which alters its localization, RNF5 emerges as a novel regulator of cell motility.     

Dimerization of the Epstein-Barr virus ZEBRA protein in the yeast two-hybrid system. Comparison Of a ZEBRA variant with the B95-8 form

Epstein-Barr virus (EBV) is a herpes virus associated with several human tumors. The EBV protein, ZEBRA, is a transactivator of the basic leucine zipper family (bZip). It binds to specific sequences on DNA and is able to interact with cellular proteins such as p53. The interaction of the ZEBRA protein with its cognate DNA sequences is stable as long as the dimerization domain is functional. Recent work from this laboratory identified a ZEBRA variant (Z206) with a single amino acid change at residue 206. An alanine is substituted for a serine, and this replacement is present in 72% of nasopharyngeal carcinoma from Europe and North Africa. As amino acid 206 lies within the dimerization domain it could be instrumental in interactions with other proteins. The yeast two-hybrid system was used to study ZEBRA-protein interactions. As ZEBRA by itself is a transactivator in yeast, it cannot be used directly in this assay. This paper describes modifications in ZEBRA amino acid sequences, rendering it usable in the yeast two-hybrid assay. We compared the dimerization capacity of the Z206 variant to that of ZEBRA from B95-8 (Z95) and observed that reporter gene activity with Z206 was consistently lower than that of Z95 (P < 0.05). Furthermore, no interaction was found to occur between either form of ZEBRA (Z206 or Z95) and the tumor suppressor, p53 in the yeast two-hybrid system.     

Classification and interaction modes of 40 rice E2 ubiquitin-conjugating enzymes with 17 rice ARM-U-box E3 ubiquitin ligases

Rice, a monocot model crop, contains at least 48 putative E2 ubiquitin (Ub)-conjugating enzymes. Based on homology comparisons with 40 Arabidopsis E2 proteins and 35 human E2s, 48 rice E2s were classified into 15 different groups. Yeast two-hybrid analyses using the U-box-domain regions of armadillo (ARM)-U-box E3 Ub-ligases and the Ub-conjugating (UBC) domains of E2s showed that, among 40 rice E2s, 11 E2s accounted for 70% of the interactions with 17 ARM-U-box E3s. Thus, a single E2 could interact with multiple ARM-U-box E3s, suggesting the presence of E2 hubs for E2–E3 interactions in rice. Rice SPL11 ARM-U-box E3 displayed distinct self-ubiquitination patterns, including poly-ubiquitination, mono-ubiquitination, or no ubiquitination, depending on different E2 partners. This suggests that the mode of ubiquitination of SPL11 E3 is critically influenced by individual E2s.     

Identification of a S-ribonuclease-binding protein in Petunia hybrida

To investigate protein-protein interactions in gametophytic self-incompatibility, we used a yeast two-hybrid assay to identify proteins that could interact with the S-ribonuclease protein. These assays identified a pollen-expressed protein, which we have named PhSBP1, that appears to bind with a high degree of specificity to the Petunia hybrida S-ribonuclease. Although PhSBP1 activates reporter gene expression only when expressed in tandem with a S-RNAse bait protein, binding is not allele-specific. Sequence analysis demonstrated that PhSBP1 contained a C-terminal cysteine-rich region that includes a RING-HC domain. Because many RING-finger domain proteins appear to function as E3 ubiquitin ligases, our results suggest that ubiquitination and protein degradation may play a role in regulating self-incompatibility interactions. Together, these results suggest that PhSBP1 may be a candidate for the recently proposed general inhibitor (RI) of self-incompatibility ribonucleases.     

Selection and characterization of large collections of peptide aptamers through optimized yeast two-hybrid procedures

Peptide aptamers are combinatorial proteins that specifically bind intracellular proteins and modulate their function. They are powerful tools to study protein function within complex regulatory networks and to guide small-molecule drug discovery. Here we describe methodological improvements that enhance the yeast two-hybrid selection and characterization of large collections of peptide aptamers. We provide a detailed protocol to perform high-efficiency transformation of peptide aptamer libraries, in-depth validation experiments of the bait proteins, high-efficiency mating to screen large numbers of peptide aptamers and streamlined confirmation of the positive clones. We also describe yeast two-hybrid mating assays, which can be used to determine the specificity of the selected aptamers, map their binding sites on target proteins and provide structural insights on their target-binding surface. Overall, 12 weeks are required to perform the protocols. The improvements on the yeast two-hybrid method can be also usefully applied to the screening of cDNA libraries to identify protein interactions.     

A high throughput screen to identify substrates for the ubiquitin ligase Rsp5

Ubiquitin-protein ligases (E3s) are implicated in various human disorders and are attractive targets for therapeutic intervention. Although most cellular proteins are ubiquitinated, ubiquitination cannot be linked directly to a specific E3 for a large fraction of these proteins, and the substrates of most E3 enzymes are unknown. We have developed a luminescent assay to detect ubiquitination in vitro, which is more quantitative, effective, and sensitive than conventional ubiquitination assays. By taking advantage of the abundance of purified proteins made available by genomic efforts, we screened hundreds of purified yeast proteins for ubiquitination, and we identified previously reported and novel substrates of the yeast E3 ligase Rsp5. The relevance of these substrates was confirmed in vivo by showing that a number of them interact genetically with Rsp5, and some were ubiquitinated by Rsp5 in vivo. The combination of this sensitive assay and the availability of purified substrates will enable the identification of substrates for any purified E3 enzyme.