Translation elongation factor eEF1A binds to a novel myosin binding protein-C-like protein

Eukaryotic translation elongation factor 1A (eEF1A) is a guanine-nucleotide binding protein, which transports aminoacylated tRNA to the ribosomal A site during protein synthesis. In a yeast two-hybrid screening of a human skeletal muscle cDNA library, a novel eEF1A binding protein, immunoglobulin-like and fibronectin type III domain containing 1 (IGFN1), was discovered, and its interaction with eEF1A was confirmed in vitro. IGFN1 is specifically expressed in skeletal muscle and presents immunoglobulin I and fibronectin III sets of domains characteristic of sarcomeric proteins. IGFN1 shows sequence and structural homology to myosin binding protein-C fast and slow-type skeletal muscle isoforms. IGFN1 is substantially upregulated during muscle denervation. We propose a model in which this increased expression of IGFN1 serves to down-regulate protein synthesis via interaction with eEF1A during denervation.     

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.     

Reconstitution and molecular analysis of the hRad9-hHus1-hRad1 (9-1-1) DNA damage responsive checkpoint complex

DNA damage activates cell cycle checkpoint signaling pathways that coordinate cell cycle arrest and DNA repair. Three of the proteins involved in checkpoint signaling, Rad1, Hus1, and Rad9, have been shown to interact by immunoprecipitation and yeast two-hybrid studies. However, it is not known how these proteins interact and assemble into a complex. In the present study we demonstrated that in human cells all the hRad9 and hHus1 and approximately one-half of the cellular pool of hRad1 interacted as a stable, biochemically discrete complex, with an apparent molecular mass of 160 kDa. This complex was reconstituted by co-expression of all three recombinant proteins in a heterologous system, and the reconstituted complex exhibited identical chromatographic behavior as the endogenous complex. Interaction studies using differentially tagged proteins demonstrated that the proteins did not self-multimerize. Rather, each protein had a binding site for the other two partners, with the N terminus of hRad9 interacting with hRad1, the N terminus of hRad1 interacting with hHus1, and the N terminus of hHus1 interacting with the C terminus of hRad9's predicted PCNA-like region. Collectively, these analyses suggest a model of how these three proteins assemble to form a functional checkpoint complex, which we dubbed the 9-1-1 complex.     

Poly(C)-Binding Protein 1, a Novel Npro-Interacting Protein Involved in Classical Swine Fever Virus Growth

N^pro is a multifunctional autoprotease unique to pestiviruses. The interacting partners of the N^pro protein of classical swine fever virus (CSFV), a swine pestivirus, have been insufficiently defined. Using a yeast two-hybrid screen, we identified poly(C)-binding protein 1 (PCBP1) as a novel interacting partner of the CSFV N^pro protein and confirmed this by coimmunoprecipitation, glutathione S-transferase (GST) pulldown, and confocal assays. Knockdown of PCBP1 by small interfering RNA suppressed CSFV growth, while overexpression of PCBP1 promoted CSFV growth. Furthermore, we showed that type I interferon was downregulated by PCBP1, as well as N^pro. Our results suggest that cellular PCBP1 positively modulates CSFV growth.     

Fishing out proteins that bind to titin.

Another giant protein has been detected in cross-striated muscle cells. Given the name obscurin, it was discovered in a yeast two-hybrid screen in which the bait was a small region of titin that is localized near the Z-band. Obscurin is about 720 kD, similar in molecular weight to nebulin, but present at about one tenth the level (Young et al., 2001). Like titin, obscurin contains multiple immunoglobulin-like domains linked in tandem, but in contrast to titin it contains just two fibronectin-like domains. It also contains sequences that suggest obscurin may have roles in signal transduction. During embryonic development, its localization changes from the Z-band to the M-band. With these intriguing properties, obscurin may not remain obscure for long.     

A functional interaction between CPI-17 and RACK1 proteins in bronchial smooth muscle cells

CPI-17 is a phosphorylation-dependent inhibitor of smooth muscle myosin light chain. Using yeast two-hybrid system, we have identified the receptor for activated C kinase 1 (RACK1) as a novel interaction partner of CPI-17. The direct interaction and co-localization of CPI-17 with RACK1 were confirmed by immunoprecipitation and confocal microscopy analysis, respectively. An in vitro assay system using recombinant/purified proteins revealed that the PKC-mediated phosphorylation of CPI-17 was augmented in the presence of RACK1. These results suggest that RACK1 may play a role in PKC/CPI-17 signaling pathway.     

Rice serine/threonine kinase 1 is required for the stimulation of OsNug2 GTPase activity

Several GTPases are required for ribosome biogenesis and assembly. We recently identified rice (Oryza sativa) nuclear/nucleolar GTPase 2 (OsNug2), a YlqF/YawG family GTPase, as having a role in pre-60S ribosomal subunit maturation. To investigate the potential factors involved in regulating OsNug2 function, yeast two-hybrid screens were performed using OsNug2 as bait. Rice serine/threonine kinase 1 (OsSTK1) was identified as a candidate interacting protein. OsSTK1 appeared to interact with OsNug2 both in vitro and in vivo. OsSTK1 was found to have no effect on the GTP-binding activity of OsNug2; however, the presence of recombinant OsSTK1 in OsNug2 assay reaction mixtures increased OsNug2 GTPase activity. A kinase assay showed that OsSTK1 had weak autophosphorylation activity and strongly phosphorylated serine 209 of OsNug2. Using yeast complementation testing, we identified a GAL::OsNug2(S209N) mutation-harboring yeast strain that exhibited a growth-defective phenotype on galactose medium at 39 °C, which was divergent from that of a yeast strain harboring GAL::OsNug2. The intrinsic GTPase activity of OsNug2(S209N), which was found to be similar to that of OsNug2, was not fully enhanced upon weak binding of OsSTK1. Our findings indicate that OsSTK1 functions as a positive regulator of OsNug2 by enhancing OsNug2 GTPase activity. In addition, phosphorylation of OsNug2 serine 209 is essential for its complete function in biological functional pathway.     

Cullin,a component of the SCF complex,interacts with TaRMD5 during wheat spike development

Cullin, a major component of the SKP1-cullin-F box protein (SCF) complex, is a scaffold protein that binds to both SKP1 and RBX1 for selective protein degradation through the ubiquitin proteasome system. In order to study the role of cullin in common wheat, we isolated TaCullin (Cullin gene from Triticum aestivum) from wheat spike cDNA. TaCullin was expressed during all spike/grain developmental stages and in high amounts during early spike/grain development. The TaCullin gene is located on the chromosome arm 2DL. Our results suggest that unneddylated TaCullin is located in the nucleus. Based on previous proposals of Cullin-SKP1 interactions, we examined the interaction between TaCullin and SKP1-like protein (TaSKP) families by using a yeast two-hybrid approach. Yeast cotransformation demonstrated that the N-terminus of TaCullin physically interacts with TaSKP proteins. Using the yeast two-hybrid screen, we identified potential TaCullin-interacting proteins in a wheat spike library. Among the 9 clones that were identified as potential interacting partners of TaCullin, we identified E3-like ubiquitin ligase, targeting fructose-1,6-bisphosphatase (RMD5) homolog A-like protein. The interaction between TaCullin and the TaRMD5 homolog A-like protein was specifically mediated through the C-terminus of TaCullin. The results of bimolecular fluorescence complementation assay indicated that TaCullin-TaRMD5 is localized in the plasma membrane and cytoplasm. In this study, we present that TaRMD5, such as RING box protein 1 (RBX1), has the potential to interact with TaCullin, depending on the developmental stage and particular organ tissues analyzed.     

The Golgi matrix protein GM130: a specific interacting partner of the small GTPase rab1b

To detect specific partners of the small Golgi-localized GTPase rab1b we generated rab1b mutants and used them as bait proteins in yeast two-hybrid screens. We isolated several specifically interacting clones. Two of them encode large protein fragments highly homologous to rat GM130 and to human Golgin95. The full-length human GM130 cDNA was cloned and its interaction with rab1b was characterized in detail by yeast two-hybrid and in vitro binding assays. Here we report for the first time that the rab1b protein interacts specificially with GM130 in a GTP-dependent manner and therefore needs the hypervariable regions of the N- and C-termini. We mapped the rab1b binding site of GM130 and provide evidence that it is different to the previously described p115 and Grasp65 binding sites of the GM130 protein.     

The Nucleolar PICT-1/GLTSCR2 Protein Forms Homo-Oligomers

The human “protein interacting with carboxyl terminus 1” (PICT-1), also designated as the “glioma tumor suppressor candidate region 2 gene product”, GLTSCR2, is a nucleolar protein whose activity is, as yet, unknown. Contradictory results regarding the role of PICT-1 in cancer have been reported, and PICT-1 has been suggested to function either as a tumor suppressor protein or as an oncogene. In this study, we demonstrate self-association of PICT-1. Through yeast two-hybrid assay, we identified PICT-1 as its own interaction partner. We confirmed the interaction of PICT-1 with itself by direct yeast two-hybrid assay and also showed self-association of PICT-1 in mammalian cells by co-immunoprecipitation and fluorescence resonance energy transfer assays. Furthermore, we confirmed direct self-association of PICT-1 by using in vitro microfluidic affinity binding assays. The later assay also identified the carboxy-terminal domain as mediating self-interaction of PICT-1. Glutaraldehyde cross-linking and gel-filtration assays suggest that PICT-1 forms dimers, though it may form higher-order complexes as well. Our findings add another layer of complexity in understanding the different functions of PICT-1 and may help provide insights regarding the activities of this protein.     

From transcriptional landscapes to the identification of biomarkers for robustness

Background

Recombinant antibodies can be produced in different formats and different expression systems. Single chain variable fragments (scFvs) represent an attractive alternative to full-length antibodies and they can be easily produced in bacteria or yeast. However, the scFvs exhibit monovalent antigen-binding properties and short serum half-lives. The stability and avidity of the scFvs can be improved by their multimerization or fusion with IgG Fc domain. The aim of the current study was to investigate the possibilities to produce in yeast high-affinity scFv-Fc proteins neutralizing the cytolytic activity of vaginolysin (VLY), the main virulence factor of Gardnerella vaginalis.

Results

The scFv protein derived from hybridoma cell line producing high-affinity neutralizing antibodies against VLY was fused with human IgG1 Fc domain. Four different variants of anti-VLY scFv-Fc fusion proteins were constructed and produced in yeast Saccharomyces cerevisiae. The non-tagged scFv-Fc and hexahistidine-tagged scFv-Fc proteins were found predominantly as insoluble aggregates and therefore were not suitable for further purification and activity testing. The addition of yeast α-factor signal sequence did not support secretion of anti-VLY scFv-Fc but increased the amount of its intracellular soluble form. However, the purified protein showed a weak VLY-neutralizing capability. In contrast, the fusion of anti-VLY scFv-Fc molecules with hamster polyomavirus-derived VP2 protein and its co-expression with VP1 protein resulted in an effective production of pseudotype virus-like particles (VLPs) that exhibited strong VLY-binding activity. Recombinant scFv-Fc molecules displayed on the surface of VLPs neutralized VLY-mediated lysis of human erythrocytes and HeLa cells with high potency comparable to that of full-length antibody.

Conclusions

Recombinant scFv-Fc proteins were expressed in yeast with low efficiency. New approach to display the scFv-Fc molecules on the surface of pseudotype VLPs was successful and allowed generation of multivalent scFv-Fc proteins with high VLY-neutralizing potency. Our study demonstrated for the first time that large recombinant antibody molecule fused with hamster polyomavirus VP2 protein and co-expressed with VP1 protein in the form of pseudotype VLPs was properly folded and exhibited strong antigen-binding activity. The current study broadens the potential of recombinant VLPs as a highly efficient carrier for functionally active complex proteins.     

GAL4 Is a Substrate for Caspases: Implications for Two-Hybrid Screening and Other GAL4-Based Assays

Yeast two-hybrid technology as well as mammalian reporter assays use fusions between a protein of interest and the GAL4 DNA-binding domain (GAL4DB). We demonstrate that expression of a GAL4DB/caspase-1 chimeric protein in yeast leads to autoproteolytic cleavage of GAL4DB. Moreover, recombinant GAL4DB is a good in vitro substrate for recombinant caspase-1 and several other caspases. Cleavage sites map at the C-terminus of GAL4DB and result in release of the fused protein. The finding that GAL4DB can be cleaved by caspases has important implications for the use of caspases in two-hybrid analysis and in the interpretation of mammalian assays based on GAL4-dependent reporter gene expression.     

A tuftelin-interacting protein (TIP39) localizes to the apical secretory pole of mouse ameloblasts

Enamel biomineralization is a complex process that involves interactions between extracellular matrix proteins. To identify proteins interacting with tuftelin, a potential nucleator of enamel crystallites, the yeast two-hybrid system was applied to a mouse tooth expression library and a tuftelin-interacting protein (TIP) was isolated for further characterization. Polyclonal antibodies were prepared against two recombinant variants of this protein. Both antibodies identified a major protein product in tooth organs at 39 kDa, and this protein has been called TIP39. Northern analysis showed TIP39 messenger RNA in multiple organs, a pattern similar to that of tuftelin messenger RNA. In situ hybridization of mandibles of 1-day-old mice detected TIP39 RNA in secretory ameloblasts and odontoblasts. Immunolocalization of TIP39 and tuftelin in cultured ameloblast-like cells showed that these two proteins colocalize. Within the developing tooth organ, TIP39 and tuftelin immunolocalized to the apical pole of secretory ameloblasts (Tomes' processes) and to the newly secreted extracellular enamel matrix. TIP39 amino acid sequence appears to be highly conserved with similarities to proteins in species as diverse as yeast and primates. Available sequence data and the findings reported here suggest a role for TIP39 in the secretory pathway of extracellular proteins.     

N-terminal region of rice polycomb group protein OsEZ1 is required for OsEZ1–OsFIE2 protein interaction

Polycomb group (PcG) proteins form Polycomb Repressive Complex 2 (PRC2), which regulates seed development by the epigenetic control of gene expression. Interaction assay among Arabidopsis Fertilization-independent-seed2 (FIS) class PcG proteins showed that Fertilization-independent endosperm (FIE) interacts with Medea (MEA), a SET-domain polycomb protein, of which N-terminal region is crucial for the interaction. In this study, rice SET-domain PcG protein OsEZ1, also known as OsiEZ1 in indica rice, was analyzed to identify an interacting domain of OsEZ1 required for OsEZ1–OsFIE2 protein interaction. A series of OsEZ1 deletions were generated and used to determine an interacting domain of OsEZ1 with OsFIE2 using the yeast two-hybrid system. Among OsEZ1 deletions, only OsEZ1?2 and OsEZ1?3 interacted with OsFIE2, indicating that the 155K–169R or N-proximal region of OsEZ1 is crucial for OsFIE2–OsEZ1 interaction. To examine the physiological roles of OsEZ1, 35S:OsEZ1 Arabidopsis lines were generated. OsEZ1 overexpressors exhibited altered seedling growth and seed size, implying that OsEZ1 may play important roles in seedling and seed development.     

戊型肝炎病毒衣壳蛋白结合CYP 2A6并降低其底物催化能力

肝炎病毒的感染常常导致人体肝脏代谢能力变化,研究表明甲型肝炎病毒(Hepatitis A Virus,HAV)、乙型肝炎病毒(Hepatitis B Virus,HBV)和丙型肝炎病毒(Hepatitis C Virus,HCV)的感染都会影响肝细胞对药物的代谢能力。在本研究中,通过酵母双杂交系统在人肝cDNA文库中筛选与戊型肝炎病毒重组衣壳蛋白E2结合的蛋白,发现了与细胞色素P450 2A6(CYP2A6)部分片段高度同源的蛋白序列。通过pull-down、免疫共沉淀以及特异性底物催化反应评价等方法进一步证实了CYP2A6与重组衣壳蛋白E2、p239间的相互作用,并发现与p239结合可以降低CYP2A6对其特异性底物香豆素的催化能力。上述结果提示CYP2A6可能在戊肝病毒入侵细胞后的细胞病变过程中起作用。     

Directed co-evolution of interacting protein–peptide pairs by compartmentalized two-hybrid replication (C2HR)

Directed evolution methodologies benefit from read-outs quantitatively linking genotype to phenotype. We therefore devised a method that couples protein–peptide interactions to the dynamic read-out provided by an engineered DNA polymerase. Fusion of a processivity clamp protein to a thermostable nucleic acid polymerase enables polymerase activity and DNA amplification in otherwise prohibitive high-salt buffers. Here, we recapitulate this phenotype by indirectly coupling the Sso7d processivity clamp to Taq DNA polymerase via respective fusion to a high affinity and thermostable interacting protein–peptide pair. Escherichia coli cells co-expressing protein–peptide pairs can directly be used in polymerase chain reactions to determine relative interaction strengths by the measurement of amplicon yields. Conditional polymerase activity is further used to link genotype to phenotype of interacting protein–peptide pairs co-expressed in E. coli using the compartmentalized self-replication directed evolution platform. We validate this approach, termed compartmentalized two-hybrid replication, by selecting for high-affinity peptides that bind two model protein partners: SpyCatcher and the large fragment of NanoLuc luciferase. We further demonstrate directed co-evolution by randomizing both protein and peptide components of the SpyCatcher–SpyTag pair and co-selecting for functionally interacting variants.     

Protein-protein interactions in the yeastPKC1 pathway: Pkc1p interacts with a component of the MAP kinase cascade

The two-hybrid system for the identification of protein-protein interactions was used to screen for proteins that interact in vivo with theSaccharomyces cerevisiae Pkc1 protein, a homolog of mammalian protein kinase C. Four positive clones were isolated that encoded portions of the protein kinase Mkk1, which acts downstream of Pkc1p in thePKC1-mediated signalling pathway. Subsequently, Pkc1p and the otherPKC1 pathway components encoding members of a MAP kinase cascade, Bck1p (a MEKK), Mkk1p, Mkk2p (two functionally homologous MEKs), and Mpk1p (a MAP kinase), were tested pairwise for interaction in the two-hybrid assay. Pkc1p interacted specifically with small N-terminal deletions of Mkk1p, and no interaction between Pkc1p and any of the other known pathway components could be detected. Interaction between Pkc1p and Mkk1p, however, was found to be independent of Mkk1p kinase activity. Bck1p was also found to interact with Mkk1p and Mkk2p, and the interaction required only the predicted C-terminal catalytic domain of Mkk1p. Furthermore, we detected protein-protein interactions between two Bck1p molecules via their N-terminal regions. Finally, Mkk2p and Mpk1p also interacted in the two-hybrid assay. These results suggest that the members of thePKC1-mediated MAP kinase cascade form a complex in vivo and that Pkc1p is capable of directly interacting with at least one component of this pathway.