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Koshizuka, Tetsuo, Yasushi Kawaguchi e Yukihiro Nishiyama. "Herpes simplex virus type 2 membrane protein UL56 associates with the kinesin motor protein KIF1A". Journal of General Virology 86, n.º 3 (1 de março de 2005): 527–33. http://dx.doi.org/10.1099/vir.0.80633-0.
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The herpes simplex virus UL56 gene product is a C-terminal-anchored, type II membrane protein of unknown function. UL56 was found to interact with KIF1A, a member of the kinesin-3 family, in a yeast two-hybrid screen and a GST pull-down assay. KIF1A mediates the transport of synaptic vesicle precursors and is essential for the function and viability of neurons. When overexpressed, KIF1A co-localized with full-sized UL56, but no clear co-localization was observed when co-expressed with the UL56 mutant protein lacking its C-terminal transmembrane domain (TMD). Although the C-terminal TMD was not essential for the interaction with KIF1A in the yeast two-hybrid screen and GST pull-down assays, these results indicate that the C-terminal TMD, as well as aa 69–217, of UL56 are important for the interaction with KIF1A in vivo. The hypothesis that the UL56 protein affects vesicular trafficking in infected cells, potentially by acting as a receptor for motor proteins in neurons, is discussed.
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Zhou, Jin-wu, Man Zhao, Wen-liang Rang, Xiao-yan Zhang, Zhen-ming Liu, Liang-ren Zhang, Tong-xing Wang, Chu-Tse Wu, Xiao-rui Cheng e Wen-xia Zhou. "Proteome Profiling Identified Amyloid-β Protein Precursor as a Novel Binding Partner and Modulator of VGLUT1". Journal of Alzheimer's Disease 81, n.º 3 (1 de junho de 2021): 981–1038. http://dx.doi.org/10.3233/jad-210117.
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Background: The toxicity of excessive glutamate release has been implicated in various acute and chronic neurodegenerative conditions. Vesicular glutamate transporters (VGLUTs) are the major mediators for the uptake of glutamate into synaptic vesicles. However, the dynamics and mechanism of this process in glutamatergic neurons are still largely unknown. Objective: This study aimed to investigate the candidate protein partners of VGLUT1 and their regulatory roles in the vesicles in rat brain. Methods: Pull down assay, co-immunoprecipitation assay, or split-ubiquitin membrane yeast two hybrid screening coupled with nanoRPLC-MS/MS were used to identify the candidate protein partners of VGLUT1 in the vesicles in rat brain. The in vitro and in vivo models were used to test effects of AβPP, Atp6ap2, Gja1, and Synataxin on VGLUT1 expression. Results: A total of 255 and 225 proteins and 172 known genes were identified in the pull down assay, co-immunoprecipitation assay, or split-ubiquitin yeast two-hybrid screening respectively. The physiological interactions of SV2A, Syntaxin 12, Gja1, AβPP, and Atp6ap2 to VGLUT1 were further confirmed. Knockdown of Atp6ap2, Gja1, and Synataxin increased VGLUT1 mRNA expression and only knockdown of AβPP increased both mRNA and protein levels of VGLUT1 in PC12 cells. The regulatory function of AβPP on VGLUT1 expression was further confirmed in the in vitro and in vivo models. Conclusion: These results elucidate that the AβPP and VGLUT1 interacts at vesicular level and AβPP plays a role in the regulation of VGLUT1 expression which is essential for maintaining vesicular activities.
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Derbigny, Wilbert A., Seong K. Kim, Gretchen B. Caughman e Dennis J. O'Callaghan. "The EICP22 Protein of Equine Herpesvirus 1 Physically Interacts with the Immediate-Early Protein and with Itself To Form Dimers and Higher-Order Complexes". Journal of Virology 74, n.º 3 (1 de fevereiro de 2000): 1425–35. http://dx.doi.org/10.1128/jvi.74.3.1425-1435.2000.
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ABSTRACT The EICP22 protein (EICP22P) of Equine herpesvirus 1(EHV-1) is an early protein that functions synergistically with other EHV-1 regulatory proteins to transactivate the expression of early and late viral genes. We have previously identified EICP22P as an accessory regulatory protein that has the ability to enhance the transactivating properties and the sequence-specific DNA-binding activity of the EHV-1 immediate-early protein (IEP). In the present study, we identify EICP22P as a self-associating protein able to form dimers and higher-order complexes during infection. Studies with the yeast two-hybrid system also indicate that physical interactions occur between EICP22P and IEP and that EICP22P self-aggregates. Results from in vitro and in vivo coimmunoprecipitation experiments and glutathioneS-transferase (GST) pull-down studies confirmed a direct protein-protein interaction between EICP22P and IEP as well as self-interactions of EICP22P. Analyses of infected cells by laser-scanning confocal microscopy with antibodies specific for IEP and EICP22P revealed that these viral regulatory proteins colocalize in the nucleus at early times postinfection and form aggregates of dense nuclear structures within the nucleoplasm. Mutational analyses with a battery of EICP22P deletion mutants in both yeast two-hybrid and GST pull-down experiments implicated amino acids between positions 124 and 143 as the critical domain mediating the EICP22P self-interactions. Additional in vitro protein-binding assays with a library of GST-EICP22P deletion mutants identified amino acids mapping within region 2 (amino acids [aa] 65 to 196) and region 3 (aa 197 to 268) of EICP22P as residues that mediate its interaction with IEP.
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Willingham, Field F., Paul Reynolds, Melinda Lewis, Andrew Ross, Shishir K. Maithel e Flavio G. Rocha. "Hybrid Push-Pull Endoscopic and Laparoscopic Full Thickness Resection for the Minimally Invasive Management of Gastrointestinal Stromal Tumors: A Pilot Clinical Study". Gastroenterology Research and Practice 2015 (2015): 1–7. http://dx.doi.org/10.1155/2015/618756.
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Background. Gastric gastrointestinal stromal tumors (GISTs) that are predominantly endophytic or in anatomically complex locations pose a challenge for laparoscopic wedge resection; however, endoscopic resection can be associated with a positive deep margin given the fourth-layer origin of the tumors.Methods. Patients at two tertiary care academic medical centers with gastric GISTs in difficult anatomic locations or with a predominant endophytic component were considered for enrollment. Preoperative esophagogastroduodenoscopy (EGD), endoscopic ultrasound (EUS) with or without fine needle aspiration (FNA), and cross-sectional imaging were performed. Eligible patients were offered and consented for hybrid and standard management.Results. Over ten months, four patients in two institutions with anatomically complex or endophytic GISTs underwent successful, uncomplicated push-pull hybrid procedures. GIST was confirmed in all resection specimens.Conclusion. In a highly selected population, the hybrid push-pull approach was safe and effective in the removal of complex gastric GISTs. Endoscopic resection alone was associated with a positive deep margin, which the push-pull technique manages with a laparoscopic, full thickness, R0 resection. This novel, minimally invasive, hybrid laparoscopic and endoscopic push-pull technique is a safe and feasible alternative in the management of select GISTs that are not amenable to standard laparoscopic resection.
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Cheng, Xiaogang, Michael Belshan e Lee Ratner. "Hsp40 Facilitates Nuclear Import of the Human Immunodeficiency Virus Type 2 Vpx-Mediated Preintegration Complex". Journal of Virology 82, n.º 3 (21 de novembro de 2007): 1229–37. http://dx.doi.org/10.1128/jvi.00540-07.
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ABSTRACT Human immunodeficiency virus type 2 (HIV-2) Vpx is required for nuclear translocation of the viral preintegration complex (PIC) in quiescent cells. In order to decipher the mechanism of action of Vpx, a cDNA library was screened with the yeast two-hybrid assay, resulting in the identification of heat shock protein 40, Hsp40/DnaJB6, as a Vpx-interactive protein. Interaction with Vpx was confirmed by glutathione S-transferase (GST) pull-down and coimmunoprecipitation assays. Overexpression of Hsp40/DnaJB6 enhanced Vpx nuclear import, whereas overexpression of a nuclear localization mutant of Hsp40/DnaJB6 (H31Q) or down-regulation of Hsp40/DnaJB6 by small interfering RNA (siRNA) reduced the nuclear import of Vpx. Hsp40/DnaJB6 competed with the Pr55Gag precursor protein for the binding of Vpx and incorporation into virus-like particles. Overexpression of Hsp40/DnaJB6 promoted viral PIC nuclear import, whereas siRNA down-regulation of Hsp40/DnaJB6 inhibited PIC nuclear import. These results demonstrate a role for Hsp40/DnaJB6 in the regulation of HIV-2 PIC nuclear transport.
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Sharma, Kusum, Taranum Sultana, Tanya E. S. Dahms e Jo-Anne R. Dillon. "CcpN: a moonlighting protein regulating catabolite repression of gluconeogenic genes in Bacillus subtilis also affects cell length and interacts with DivIVA". Canadian Journal of Microbiology 66, n.º 12 (dezembro de 2020): 723–32. http://dx.doi.org/10.1139/cjm-2020-0022.
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CcpN is a transcriptional repressor in Bacillus subtilis that binds to the promoter region of gapB and pckA, downregulating their expression in the presence of glucose. CcpN also represses sr1, which encodes a small noncoding regulatory RNA that suppresses the arginine biosynthesis gene cluster. CcpN has homologues in other Gram-positive bacteria, including Enterococcus faecalis. We report the interaction of CcpN with DivIVA of B. subtilis as determined using bacterial two-hybrid and glutathione S-transferase pull-down assays. Insertional inactivation of CcpN leads to cell elongation and formation of straight chains of cells. These findings suggest that CcpN is a moonlighting protein involved in both gluconeogenesis and cell elongation.
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Zhou, Qing, Limei Xu, Hui Li, Yi-Peng Qi e Feng Yang. "Four Major Envelope Proteins of White Spot Syndrome Virus Bind To Form a Complex". Journal of Virology 83, n.º 9 (11 de fevereiro de 2009): 4709–12. http://dx.doi.org/10.1128/jvi.02360-08.
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ABSTRACT Early events in white spot syndrome virus (WSSV) morphogenesis, particularly the formation of viral membranes, are poorly understood. The major envelope proteins of WSSV are VP28, VP26, VP24, and VP19. Our previous results indicated that VP28 interacts with VP26 and VP24. In the present study, we used coimmunoprecipitation assays and pull-down assays to confirm that the four major proteins in the WSSV envelope can form a multiprotein complex. Yeast two-hybrid assays were also used to test for interactions among the four proteins. In summary, three pairwise protein interactions (VP19-VP28, VP19-VP24, and VP24-VP26) and one self-association (VP24-VP24) were identified for the first time.
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Ward, Brian M., e Bernard Moss. "Vaccinia Virus A36R Membrane Protein Provides a Direct Link between Intracellular Enveloped Virions and the Microtubule Motor Kinesin". Journal of Virology 78, n.º 5 (1 de março de 2004): 2486–93. http://dx.doi.org/10.1128/jvi.78.5.2486-2493.2004.
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ABSTRACT Previous work demonstrated that intracellular enveloped vaccinia virus virions use microtubules to move from the site of membrane wrapping to the cell periphery. The mechanism and direction of intracellular virion movement predicted that viral proteins directly or indirectly interact with the microtubule motor protein kinesin. The yeast two-hybrid assay was used to test for interactions between the light chain of kinesin and the cytoplasmic tails from five viral envelope proteins. We found that the N-terminal tetratricopeptide repeat region of the kinesin light chain (KLC-TPR) interacted with the cytoplasmic tail of the viral A36R protein. A series of C- and N-terminal truncations of A36R further defined a region from residues 81 to 111 that was sufficient for interaction with KLC-TPR. Interactions were confirmed by using pull-down assays with purified glutathione S-transferase (GST)-A36R and 35S-labeled KLC-TPR. The defined region on A36R for interaction with kinesin overlaps the recently defined region (residues 91 to 111) for interaction with the A33R envelope protein. The yeast three-hybrid system was used to demonstrate that expression of A33R interrupted the interaction between A36R and KLC-TPR, indicating that the binding of A36R is mutually exclusive to either A33R or kinesin. Pull-down assays with purified GST-A36R and 35S-labeled KLC-TPR in the presence of competing A33R corroborated these findings. Collectively, these results demonstrated that the viral A36R protein interacts directly with the microtubule motor protein kinesin and that the viral protein A33R may regulate this interaction.
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Selvakumar, Dakshnamurthy, Marian J. Drescher, Nathan A. Deckard, Neeliyath A. Ramakrishnan, Barbara J. Morley e Dennis G. Drescher. "Dopamine D1A directly interacts with otoferlin synaptic pathway proteins: Ca2+ and phosphorylation underlie an NSF-to-AP2mu1 molecular switch". Biochemical Journal 474, n.º 1 (22 de dezembro de 2016): 79–104. http://dx.doi.org/10.1042/bcj20160690.
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Dopamine receptors regulate exocytosis via protein–protein interactions (PPIs) as well as via adenylyl cyclase transduction pathways. Evidence has been obtained for PPIs in inner ear hair cells coupling D1A to soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE)-related proteins snapin, otoferlin, N-ethylmaleimide-sensitive factor (NSF), and adaptor-related protein complex 2, mu 1 (AP2mu1), dependent on [Ca2+] and phosphorylation. Specifically, the carboxy terminus of dopamine D1A was found to directly bind t-SNARE-associated protein snapin in teleost and mammalian hair cell models by yeast two-hybrid (Y2H) and pull-down assays, and snapin directly interacts with hair cell calcium-sensor otoferlin. Surface plasmon resonance (SPR) analysis, competitive pull-downs, and co-immunoprecipitation indicated that these interactions were promoted by Ca2+ and occur together. D1A was also found to separately interact with NSF, but with an inverse dependence on Ca2+. Evidence was obtained, for the first time, that otoferlin domains C2A, C2B, C2D, and C2F interact with NSF and AP2mu1, whereas C2C or C2E do not bind to either protein, representing binding characteristics consistent with respective inclusion or omission in individual C2 domains of the tyrosine motif YXXΦ. In competitive pull-down assays, as predicted by KD values from SPR (+Ca2+), C2F pulled down primarily NSF as opposed to AP2mu1. Phosphorylation of AP2mu1 gave rise to a reversal: an increase in binding by C2F to phosphorylated AP2mu1 was accompanied by a decrease in binding to NSF, consistent with a molecular switch for otoferlin from membrane fusion (NSF) to endocytosis (AP2mu1). An increase in phosphorylated AP2mu1 at the base of the cochlear inner hair cell was the observed response elicited by a dopamine D1A agonist, as predicted.
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Pandey, A. K., R. A. Mishra e R. K. Nagaria. "Leakage Power Analysis of Domino XOR Gate". ISRN Electronics 2013 (15 de janeiro de 2013): 1–7. http://dx.doi.org/10.1155/2013/271316.
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Two new XOR gates are proposed. First proposed circuits adopt hybrid transistor topology in the pull-down network with all transistors being low threshold voltages. A second proposed circuit adopts hybrid topology with dual threshold voltage transistors. Simulation parameters are measured at 25°C and 110°C. First proposed circuit reduces leakage power consumption up to 50% at 25°C and 58% at 110°C as compared to standard N-type domino XOR gate. It also reduces active mode power consumption by 14% as compared to standard N-type domino XOR gate. Similarly, second proposed circuit reduces leakage power consumption up to 73% at 25°C and 90% at 110°C as compared to standard N-type domino XOR gate. It also reduces active mode power consumption by 39% as compared to standard N-type domino XOR gate.
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Guo, Fen, Yue-Qin Li, Shi-Qian Li, Zhi-Wen Luo, Xin Zhang, Dong-Sheng Tang e Tian-Hong Zhou. "Interaction of Mouse Pem Protein and Cell Division Cycle 37 Homolog". Acta Biochimica et Biophysica Sinica 37, n.º 11 (1 de novembro de 2005): 784–87. http://dx.doi.org/10.1111/j.1745-7270.2005.00102.x.
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Abstract Mouse Pem, a homeobox gene, encodes a protein consisting of 210 amino acid residues. To study the function of mouse Pem protein, we used the yeast two-hybrid system to screen the library of 7-day mouse embryo with full-length mouse Pem cDNA. Fifty-two colonies were obtained after 1.57×108 colonies were screened by nutrition limitation and β-galactosidase assay. Seven individual insert fragments were obtained from the library, and three of them were identified, one of which was confirmed to be the cell division cycle 37 (Cdc37) homolog gene by sequencing. The interaction between mouse Pem and Cdc37 homolog was then confirmed by glutathione S-transferase pull-down assay, and the possible interaction model was suggested.
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MAHONEY, William M., Jeong-Ho HONG, Michael B. YAFFE e Iain K. G. FARRANCE. "The transcriptional co-activator TAZ interacts differentially with transcriptional enhancer factor-1 (TEF-1) family members". Biochemical Journal 388, n.º 1 (10 de maio de 2005): 217–25. http://dx.doi.org/10.1042/bj20041434.
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Members of the highly related TEF-1 (transcriptional enhancer factor-1) family (also known as TEAD, for TEF-1, TEC1, ABAA domain) bind to MCAT (muscle C, A and T sites) and A/T-rich sites in promoters active in cardiac, skeletal and smooth muscle, placenta, and neural crest. TEF-1 activity is regulated by interactions with transcriptional co-factors [p160, TONDU (Vgl-1, Vestigial-like protein-1), Vgl-2 and YAP65 (Yes-associated protein 65 kDa)]. The strong transcriptional co-activator YAP65 interacts with all TEF-1 family members, and, since YAP65 is related to TAZ (transcriptional co-activator with PDZ-binding motif), we wanted to determine if TAZ also interacts with members of the TEF-1 family. In the present study, we show by GST (glutathione S-transferase) pull-down assays, by co-immunoprecipitation and by modified mammalian two-hybrid assays that TEF-1 interacts with TAZ in vitro and in vivo. Electrophoretic mobility-shift assays with purified TEF-1 and GST–TAZ fusion protein showed that TAZ interacts with TEF-1 bound to MCAT DNA. TAZ can interact with endogenous TEF-1 proteins, since exogenous TAZ activated MCAT-dependent reporter promoters. Like YAP65, TAZ interacted with all four TEF-1 family members. GST pull-down assays with increasing amounts of [35S]TEF-1 and [35S]RTEF-1 (related TEF-1) showed that TAZ interacts more efficiently with TEF-1 than with RTEF-1. This differential interaction also extended to the interaction of TEF-1 and RTEF-1 with TAZ in vivo, as assayed by a modified mammalian two-hybrid experiment. These data show that differential association of TEF-1 proteins with transcriptional co-activators may regulate the activity of TEF-1 family members.
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Buckley, Patrick S., Bryson Kemler, Colin Robbins, Zachary S. Aman, Hunter Storaci, Grant Dornan e Robert F. LaPrade. "Biomechanical Comparison of Three Novel Repair Techniques for Radial Tears of the Medial Meniscus". Orthopaedic Journal of Sports Medicine 7, n.º 7_suppl5 (julho de 2019): 2325967119S0035. http://dx.doi.org/10.1177/2325967119s00356.
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Objectives: Historically, radial meniscal tears were treated with partial or near- total meniscectomy which usually resulted in with poor outcomes. Radial meniscal tears function similar to a total meniscectomy and are challenging to treat. Repair of radial meniscal tears should be performed to prevent joint deterioration and the need for salvage procedures in the future. The purpose of this study was to compare three novel repair techniques for radial tears of the medial meniscus; the two-tunnel, the hybrid, and the hybrid tunnel techniques. We hypothesized that there would be no difference between the three groups in regards to gapping and ultimate failure strength. Methods: Thirty human male cadaver knees (ten matched pairs (n=20) and ten unpaired (n=10)) were used to compare the two-tunnel, hybrid, and hybrid tunnel repairs. A complete radial tear was made at the midbody of the medial meniscus. Repairs were performed according to the described techniques. Specimens were potted and mounted on a universal Instron testing machine where each specimen was cyclically loaded for 1000 cycles before experiencing a pull-to-failure. Gap distances at the tear site, ultimate failure load, and failure location were measured and recorded. Results: After 1000 cycles of cyclic loading, there was no significant difference in displacement between the two-tunnel repair (3.0 mm ± 1.7 mm), hybrid repair (3.0 mm ± 0.9 mm) or hybrid tunnel repair (2.3 mm ± 1.0 mm) (p=0.4042). On pull-to-failure testing there was also no significant difference in ultimate failure strength when comparing the two-tunnel (259 N ± 103 N), hybrid (349 N ± 149 N), or hybrid tunnel (365 N ± 146 N) repairs (p=0.26). However, the addition of vertical mattress sutures to act as a “rip stop” did significantly reduce the likelihood of the sutures pulling through the meniscus during pull-to-failure testing for the hybrid and hybrid tunnel repairs (4/16=25%) when compared to the two-tunnel repair (7/9=78%) (p=0.017). Conclusion: This study biomechanically evaluated two previously described techniques- the two-tunnel and hybrid repair- as well as one novel repair technique- the hybrid tunnel repair. The results showed equivalent biomechanical testing in regard to gap distance and pull to failure strength among each repair. The addition of the vertical mattress sutures to act as a rip stop suture was effective in preventing meniscal cut out through the meniscus. [Table: see text][Table: see text]
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Yin, Jiang, Ying Liu, Eckard Wimmer e Aniko V. Paul. "Complete protein linkage map between the P2 and P3 non-structural proteins of poliovirus". Journal of General Virology 88, n.º 8 (1 de agosto de 2007): 2259–67. http://dx.doi.org/10.1099/vir.0.82795-0.
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All of the non-structural proteins of poliovirus, including their processing precursors, are involved in the replication of the viral RNA genome. These proteins assemble into a replication complex, which also contains the viral RNA and cellular factors. An understanding of how these viral proteins interact with each other would enhance our understanding of the molecular events occurring during poliovirus infection of the cell. Previously, we have employed the yeast two-hybrid system to construct two separate linkage maps for the polioviral P2 and P3 proteins, respectively. In the present study, we have searched for interacting pairs between the P2 and P3 proteins in a similar inducible yeast two-hybrid system. Although, the primary functions of the proteolytic products of the P2 and P3 domains of the polyprotein in the viral life cycle are different, we observed significant interactions between 2CATPase and 3AB; 2Apro and 3A, 3Cpro or 3Dpol; 2B and 3A or 3AB. All of the interactions were measured in the yeast two-hybrid system by exchanging the interacting pairs on the transcription-activation and DNA-binding constructs. In vitro GST pull-down assay suggested that the 2CATPase/3AB interaction involves both ionic and hydrophobic contacts between the two proteins. The possible biological implication of the interactions observed in the yeast two-hybrid system will be discussed.
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Feng, Pinghui, David N. Everly e G. Sullivan Read. "mRNA Decay during Herpes Simplex Virus (HSV) Infections: Protein-Protein Interactions Involving the HSV Virion Host Shutoff Protein and Translation Factors eIF4H and eIF4A". Journal of Virology 79, n.º 15 (1 de agosto de 2005): 9651–64. http://dx.doi.org/10.1128/jvi.79.15.9651-9664.2005.
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ABSTRACT During lytic infections, the virion host shutoff (Vhs) protein of herpes simplex virus accelerates the degradation of both host and viral mRNAs. In so doing, it helps redirect the cell from host to viral protein synthesis and facilitates the sequential expression of different viral genes. Vhs interacts with the cellular translation initiation factor eIF4H, and several point mutations that abolish its mRNA degradative activity also abrogate its ability to bind eIF4H. In addition, a complex containing bacterially expressed Vhs and a glutathione S-transferase (GST)-eIF4H fusion protein has RNase activity. eIF4H shares a region of sequence homology with eIF4B, and it appears to be functionally similar in that both stimulate the RNA helicase activity of eIF4A, a component of the mRNA cap-binding complex eIF4F. We show that eIF4H interacts physically with eIF4A in the yeast two-hybrid system and in GST pull-down assays and that the two proteins can be coimmunoprecipitated from mammalian cells. Vhs also interacts with eIF4A in GST pull-down and coimmunoprecipitation assays. Site-directed mutagenesis of Vhs and eIF4H revealed residues of each that are important for their mutual interaction, but not for their interaction with eIF4A. Thus, Vhs, eIF4H, and eIF4A comprise a group of proteins, each of which is able to interact directly with the other two. Whether they interact simultaneously as a tripartite complex or sequentially is unclear. The data suggest a mechanism for linking the degradation of an mRNA to its translation and for targeting Vhs to mRNAs and to regions of translation initiation.
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Wang, Nian, Lizhou Zhang, Yuming Chen, Zhen Lu, Li Gao, Yongqiang Wang, Yulong Gao et al. "Cyclophilin A Interacts with Viral VP4 and Inhibits the Replication of Infectious Bursal Disease Virus". BioMed Research International 2015 (2015): 1–10. http://dx.doi.org/10.1155/2015/719454.
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Nonstructural protein VP4, a serine protease of infectious bursal disease virus (IBDV) that catalyzes the hydrolysis of polyprotein pVP2-VP4-VP3 to form the viral proteins VP2, VP4, and VP3, is essential to the replication of IBDV. However, the interacting partners of VP4 in host cells and the effects of the interaction on the IBDV lifecycle remain incompletely elucidated. In this study, using the yeast two-hybrid system, the putative VP4-interacting partner cyclophilin A (CypA) was obtained from a chicken embryo fibroblast (CEF) expression library. CypA was further confirmed to interact with VP4 of IBDV using co-immunoprecipitation (CO-IP), GST pull-down, and confocal microscopy assays. Moreover, we found that the overexpression of CypA suppressed IBDV replication, whereas the knock-down of CypA by small interfering RNAs promoted the replication of IBDV. Taken together, our findings indicate that the host cell protein CypA interacts with viral VP4 and inhibits the replication of IBDV.
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López, Lissett, Ana Urzainqui, Elvira Domínguez e Juan Antonio García. "Identification of an N-terminal domain of the plum pox potyvirus CI RNA helicase involved in self-interaction in a yeast two-hybrid system". Journal of General Virology 82, n.º 3 (1 de março de 2001): 677–86. http://dx.doi.org/10.1099/0022-1317-82-3-677.
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Potyvirus CI RNA helicase is a protein involved in RNA genome replication and virus movement. The protein aggregates in the cytoplasm of infected cells to form typical cylindrical inclusions. A yeast two-hybrid system was used to analyse interactions of the CI RNA helicase from plum pox potyvirus (PPV) with itself and with other viral proteins. No interactions could be detected of full-length CI protein with itself or with PPV P3/6K1, NIa, NIb or CP proteins. However, positive self-interactions were detected for N-terminal fragments of the CI protein, allowing the mapping of a CI–CI binding domain to the N-terminal 177 aa of the protein. Further deletion analysis suggested that several regions of this domain contribute to the interaction. Moreover, pull-down experiments demonstrate that, at least in vitro, full-length PPV CI protein is able to self-interact in the absence of other virus or plant factors.
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Liang, Pei, Yongqi Wan, Yan Yan, Yuequn Wang, Na Luo, Yun Deng, Xiongwei Fan et al. "MVP interacts with YPEL4 and inhibits YPEL4-mediated activities of the ERK signal pathway". Biochemistry and Cell Biology 88, n.º 3 (junho de 2010): 445–50. http://dx.doi.org/10.1139/o09-166.
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Human YPEL4 is a member of YPEL family. It contains a Yippee domain, which is a putative zinc-finger-like, metal-binding domain. The human YPEL4 gene maps to chromosome 11q12.1, is ubiquitously expressed in adult tissues, and encodes a nuclear protein of 127 amino acids, the function of which remains unknown. To gain insights into the cellular function of this protein, we searched for YPEL4-interacting proteins using a yeast two-hybrid screen. The major vault protein (MVP), a lung resistance associated protein, was identified as a binding partner of YPEL4. The interaction between YPEL4 and MVP in mammalian cells was further demonstrated by a series of biochemical assays including the mammalian two-hybrid assay, GST pull-down assay, co-immunoprecipitation assay, and immunocytochemistry. Using a reporter system, we found that MVP can inhibit YPEL4’s ability to activate Elk-1 in the MAPK signaling pathway. This study provides new clues for understanding the molecular mechanism of YPEL4 in cell division and signal transduction pathways and should be helpful for understanding molecular functions of the YPEL family.
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Graff, Joel W., Dana N. Mitzel, Carla M. Weisend, Michelle L. Flenniken e Michele E. Hardy. "Interferon Regulatory Factor 3 Is a Cellular Partner of Rotavirus NSP1". Journal of Virology 76, n.º 18 (15 de setembro de 2002): 9545–50. http://dx.doi.org/10.1128/jvi.76.18.9545-9550.2002.
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ABSTRACT The rotavirus nonstructural protein NSP1 is the least conserved protein in the rotavirus genome, and its function in the replication cycle is not known. We employed NSP1 as bait in the yeast two-hybrid interaction trap to identify candidate cellular partners of NSP1 that may provide clues to its function. Interferon regulatory factor 3 (IRF-3) was identified as an NSP1 interactor. NSP1 synthesized in rotavirus-infected cells bound IRF-3 in a glutathione S-transferase pull-down assay, indicating that the interaction was not unique to the two-hybrid system. NSP1 of murine rotavirus strain EW also interacted with IRF-3. NSP1 deletion and point mutants were constructed to map domains important in the interaction between NSP1 and IRF-3. The data suggest that a binding domain resides in the C terminus of NSP1 and that the N-terminal conserved zinc finger is important but not sufficient to mediate binding to IRF-3. We predict that a role for NSP1 in rotavirus-infected cells is to inhibit activation of IRF-3 and diminish the cellular interferon response.
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Zhang, Yu, Tiantian Gu, Yang Chen, Guoqiang Zhu, Wanwipa Vongsangnak, Qi Xu e Guohong Chen. "Screening and identification of SipC-interacting proteins in Salmonella enteritidis using Gal4 yeast two-hybrid system in duck". PeerJ 7 (13 de setembro de 2019): e7663. http://dx.doi.org/10.7717/peerj.7663.
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The zoonotic pathogen Salmonella not only reduces the production performance in ducks, but also poses a serious threat to human health through eggs and pollutes water bodies through feces. SipC, an effector protein of type III secretion systems (T3SS) in Salmonella, mediates translocation of effectors into the eukaryotic host. However, the precise role of SipC effectors remains unknown in ducks. In this study, the SipC from duck granulosa cells (dGCs) was selected as bait, and the SipC-interacting proteins in Salmonella enteritidis (SE) were screened using Gal4 yeast two-hybrid system in duck. Twelve SipC-interacting proteins were identified. Among those, the p53-effector related to PMP-22 (PERP) and TGF-β activated kinase 1-binding protein 2 (TAB2) were selected to further confirm the function by GST pull-down in vitro. Over-expression of PERP resulted in not only increasing SE adhesion and invasion but also triggering the production of IL-1β and IFN-α in SE infected dGCs, while knock-down PERP showed the opposite tendency (P < 0.01). In addition, TAB2 significantly induced the production of IL-6, IL-1β, IFN-α, and INF-γ in SE infected dGCs (P < 0.05), but did not cause obvious changes in SE adhesion and invasion. When the sipC in SE was deleted, the activities of duck PERP and TAB2 were abolished because they could not bind to SipC. Taken together, although the protein of PERP and TAB2 can interact with SipC, their mechanisms were different in duck challenged by SE. Therefore, PERP was involved in SE invasion and inflammatory response of dGC ovaries, and TAB2 only contributed to dGCs inflammatory response, which provided critical insights about the mechanism in host- bacterium protein interactions during Salmonella invasion in duck.
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Barth, Sandra, Jutta Nesper, Philippe A. Hasgall, Renato Wirthner, Katarzyna J. Nytko, Frank Edlich, Dörthe M. Katschinski, Daniel P. Stiehl, Roland H. Wenger e Gieri Camenisch. "The Peptidyl Prolyl cis/trans Isomerase FKBP38 Determines Hypoxia-Inducible Transcription Factor Prolyl-4-Hydroxylase PHD2 Protein Stability". Molecular and Cellular Biology 27, n.º 10 (12 de março de 2007): 3758–68. http://dx.doi.org/10.1128/mcb.01324-06.
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ABSTRACT The heterodimeric hypoxia-inducible transcription factors (HIFs) are central regulators of the response to low oxygenation. HIF-α subunits are constitutively expressed but rapidly degraded under normoxic conditions. Oxygen-dependent hydroxylation of two conserved prolyl residues by prolyl-4-hydroxylase domain-containing enzymes (PHDs) targets HIF-α for proteasomal destruction. We identified the peptidyl prolyl cis/trans isomerase FK506-binding protein 38 (FKBP38) as a novel interactor of PHD2. Yeast two-hybrid, glutathione S-transferase pull-down, coimmunoprecipitation, colocalization, and mammalian two-hybrid studies confirmed specific FKBP38 interaction with PHD2, but not with PHD1 or PHD3. PHD2 and FKBP38 associated with their N-terminal regions, which contain no known interaction motifs. Neither FKBP38 mRNA nor protein levels were regulated under hypoxic conditions or after PHD inhibition, suggesting that FKBP38 is not a HIF/PHD target. Stable RNA interference-mediated depletion of FKBP38 resulted in increased PHD hydroxylation activity and decreased HIF protein levels and transcriptional activity. Reconstitution of FKBP38 expression abolished these effects, which were independent of the peptidyl prolyl cis/trans isomerase activity. Downregulation of FKBP38 did not affect PHD2 mRNA levels but prolonged PHD2 protein stability, suggesting that FKBP38 is involved in PHD2 protein regulation.
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Mehrsai, Afshin, Hamid-Reza Karimi, Klaus-Dieter Thoben e Bernd Scholz-Reiter. "Using Metaheuristic and Fuzzy System for the Optimization of Material Pull in a Push-Pull Flow Logistics Network". Mathematical Problems in Engineering 2013 (2013): 1–19. http://dx.doi.org/10.1155/2013/359074.
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Alternative material flow strategies in logistics networks have crucial influences on the overall performance of the networks. Material flows can follow push, pull, or hybrid systems. To get the advantages of both push and pull flows in networks, the decoupling-point strategy is used as coordination mean. At this point, material pull has to get optimized concerning customer orders against pushed replenishment-rates. To compensate the ambiguity and uncertainty of both dynamic flows, fuzzy set theory can practically be applied. This paper has conceptual and mathematical parts to explain the performance of the push-pull flow strategy in a supply network and to give a novel solution for optimizing the pull side employing Conwip system. Alternative numbers of pallets and their lot-sizes circulating in the assembly system are getting optimized in accordance with a multi-objective problem; employing a hybrid approach out of meta-heuristics (genetic algorithm and simulated annealing) and fuzzy system. Two main fuzzy sets as triangular and trapezoidal are applied in this technique for estimating ill-defined waiting times. The configured technique leads to smoother flows between push and pull sides in complex networks. A discrete-event simulation model is developed to analyze this thesis in an exemplary logistics network with dynamics.
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Guo, Caixia, Tie-Shan Tang, Marzena Bienko, Joanne L. Parker, Aleksandra B. Bielen, Eiichiro Sonoda, Shunichi Takeda, Helle D. Ulrich, Ivan Dikic e Errol C. Friedberg. "Ubiquitin-Binding Motifs in REV1 Protein Are Required for Its Role in the Tolerance of DNA Damage". Molecular and Cellular Biology 26, n.º 23 (18 de setembro de 2006): 8892–900. http://dx.doi.org/10.1128/mcb.01118-06.
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ABSTRACT REV1 protein is a eukaryotic member of the Y family of DNA polymerases involved in the tolerance of DNA damage by replicative bypass. The precise role(s) of REV1 in this process is not known. Here we show, by using the yeast two-hybrid assay and the glutathione S-transferase pull-down assay, that mouse REV1 can physically interact with ubiquitin. The association of REV1 with ubiquitin requires the ubiquitin-binding motifs (UBMs) located at the C terminus of REV1. The UBMs also mediate the enhanced association between monoubiquitylated PCNA and REV1. In cells exposed to UV radiation, the association of REV1 with replication foci is dependent on functional UBMs. The UBMs of REV1 are shown to contribute to DNA damage tolerance and damage-induced mutagenesis in vivo.
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Peng, Bo, Xia-Yu Tian, Zi-Yue Liu, Juan Peng, Meng-Yang Zheng, Xiao-Hua Song, Ya-Qin Huang et al. "The Application of Modern Biotechnology in Protein Interaction Research-A Review". Journal of Agriculture and Crops, n.º 72 (2 de fevereiro de 2021): 39–47. http://dx.doi.org/10.32861/jac.72.39.47.
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Intermolecular interaction is the material basis for cells to achieve their functions, and protein-protein interaction is an important approach to illuminate the regulation network of biological molecules and has important theoretical significance and potential application value for revealing the activity law of life in nature. This paper mainly summarizes and analyzes the new advances and applications of modern biotechnologies in the study of protein-protein interactions, including local surface plasmon resonance (LSPR), yeast two-hybrid, GST-Pull-down, bimolecular fluorescence complementation, and coimmunoprecipitation. At the same time, the principles of different research methods for protein-protein interaction and their other applications in the field of life sciences are also discussed, all of these will provide a reference value for the analysis of protein-protein interaction and the molecular regulation mechanism of biomacromolecules.
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Li, Xiao-Dong, Tomi P. Mäkelä, Deyin Guo, Rabah Soliymani, Vesa Koistinen, Olli Vapalahti, Antti Vaheri e Hilkka Lankinen. "Hantavirus nucleocapsid protein interacts with the Fas-mediated apoptosis enhancer Daxx". Journal of General Virology 83, n.º 4 (1 de abril de 2002): 759–66. http://dx.doi.org/10.1099/0022-1317-83-4-759.
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Hantaviruses cause two severe diseases, haemorrhagic fever with renal syndrome in Eurasia and hantavirus pulmonary syndrome in the Americas. To understand more about the molecular mechanisms that lead to these diseases, the associations of Puumala virus nucleocapsid protein (PUUV-N) with cellular proteins were studied by yeast two-hybrid screening. Daxx, known as an apoptosis enhancer, was identified from a HeLa cDNA library and its interaction with PUUV-N was confirmed by GST pull-down assay, co-immunoprecipitation and co-localization studies. Furthermore, domains of interaction were mapped to the carboxyl-terminal region of 142 amino acids in Daxx and the carboxyl-terminal 57 residues in PUUV-N, respectively. In pepscan assays, the binding sites of Daxx to PUUV-N were mapped further to two lysine-rich regions, of which one overlaps the sequence of the predicted nuclear localization signal of Daxx. These data suggest a direct link between host cell machinery and a hantavirus structural component.
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Lee, Soo-Kyung, Hueng-Sik Choi, Mi-Ryoung Song, Mi-Ock Lee e Jae Woon Lee. "Estrogen Receptor, a Common Interaction Partner for a Subset of Nuclear Receptors". Molecular Endocrinology 12, n.º 8 (1 de agosto de 1998): 1184–92. http://dx.doi.org/10.1210/mend.12.8.0146.
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Abstract Nuclear receptors regulate transcription by binding to specific DNA response elements as homodimers or heterodimers. Herein, the yeast and mammalian two-hybrid tests as well as glutathione-S-transferase pull-down assays were exploited to demonstrate that estrogen receptor (ER) directly binds to a subset of nuclear receptors through protein-protein interactions between ligand-binding domains. These receptors include hepatocyte nuclear factor 4, thyroid hormone receptor (TR), retinoic acid receptor (RAR), ERβ, and retinoid X receptor (RXR). In yeast cells, a LexA fusion protein to the human ER ligand-binding domain (LexA/ER-LBD) was an inert transactivator of a LacZ reporter gene controlled by upstream LexA-binding sites. However, LexA/ER-LBD differentially modulated the LacZ reporter gene expression when coexpressed with native TRs, RARs, or RXRs. Similarly, cotransfection of these receptors in CV1 cells up- or down-regulated transactivations by ER. From these results, we propose that ER is a common interaction partner for a subset of receptors, and these interactions should mediate novel signaling pathways in vivo.
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TAKAHASHI, Kyoko, Chiyuki MATSUMOTO e Chisei RA. "FHL3 negatively regulates human high-affinity IgE receptor β-chain gene expression by acting as a transcriptional co-repressor of MZF-1". Biochemical Journal 386, n.º 1 (8 de fevereiro de 2005): 191–200. http://dx.doi.org/10.1042/bj20040775.
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The high-affinity IgE receptor FcεRI plays a key role in triggering allergic reactions. We recently reported that human FcεRI β-chain gene expression was down-regulated by a transcription factor, MZF-1, through an element in the fourth intron. In the present study, we found that this transcriptional repression by MZF-1 required FHL3 (four and a half LIM domain protein 3) as a cofactor. Yeast two-hybrid and immunoprecipitation assays demonstrated that FHL3 bound MZF-1 in vitro and in vivo. Overexpression of FHL3 in KU812 cells suppressed the β-chain promoter activity through the element in the fourth intron in an MZF-1-dependent manner. Furthermore, results from pull-down assays and gel-filtration chromatography employing nuclear extracts indicated that MZF-1 and FHL3 formed a complex of high molecular mass with some additional proteins in the nucleus. Granulocyte–macrophage colony-stimulating factor, which was reported to decrease FcεRI expression, induced the accumulation of FHL3 in the nucleus, in accordance with the repressive role of FHL3 in β-chain gene expression.
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Daruliza Kernain e Shaharum Shamsuddin. "Interaction between Two Transcriptional Factors CTCF and YB-1 – Truncated domains in Brain Cancer Cell line". International Journal of Research in Pharmaceutical Sciences 10, n.º 4 (16 de outubro de 2019): 3332–38. http://dx.doi.org/10.26452/ijrps.v10i4.1642.
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CTCF is a protein involved in the regulation of transcription, insulator function, and the X-chromosome inactivation. It is an 11 ZF transcriptional factor which is highly conserved between the species. Identification of proteins interacting with CTCF can help to elucidate the function in the cell. Previously reported studies had identified numerous CTCF protein interacting partners, and one of the interacting partners chosen in this study is YB-1. Brain cancer cell –RGBM was selected as a model to study the interaction between CTCF and YB-1. Firstly, proteins were transformed and expressed in the bacterial expression system, and these proteins were chosen to further map the interaction via pull-down assay. Results showed CTCF-ZF was the only domain able to binds to YB-1 CSD. Other truncated areas did not show any interaction hence demonstrating the interaction between these two proteins took place at the ZF for CTCF and CSD for YB-1. Next, the significant of the interaction was further characterized using the mammalian two-hybrid system. Results show strong interaction when both we co-transfected into RGBM cells. Thus, this study shows a significant binding between CTCF/YB-1 interaction in the brain cell line.
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Dong, Bo, Wei Gao, Huijun Lu, Kui Zhao, Ning Ding, Wenfeng Liu, Jiakuan Zhao et al. "A Small Region of Porcine Hemagglutinating Encephalomyelitis Virus Spike Protein Interacts with the Neural Cell Adhesion Molecule". Intervirology 58, n.º 2 (2015): 130–37. http://dx.doi.org/10.1159/000381060.
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Objective: The spike (S) protein of porcine hemagglutinating encephalomyelitis virus (PHEV) may mediate infection by binding to a cellular neural cell adhesion molecule (NCAM). This study aimed to identify the crucial domain of the S1 subunit of the S protein that interacts with NCAM. Methods: Three truncated segments (S1-291, S277-794 and S548-868) of the S gene of PHEV and the NCAM gene were cloned individually into the Escherichia coli expression vectors and yeast two-hybrid expression vectors. The interaction between S1-291, S277-794, S548-868 and NCAM were detected by a GST pull-down experiment and yeast two-hybrid assay. Results: Three fusion proteins (S1-291, S277-794 and S548-868) were screened for their interactions with NCAM by protein-protein interaction assays. The results of these assays clarified that S277-794 interacted with NCAM, while S1-291 and S548-868 did not. Conclusions: A small fragment (258-amino-acid fragment, residues 291-548) on the PHEV S protein was posited to be the minimum number of amino acids necessary to interact with NCAM. This fragment may be the receptor-binding domain that mediates PHEV binding to NCAM.
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Haim, M., A. Trost, C. J. Maier, G. Achatz, S. Feichtner, H. Hintner, J. W. Bauer e K. önder. "Cytokeratin 8 interacts with clumping factor B: a new possible virulence factor target". Microbiology 156, n.º 12 (1 de dezembro de 2010): 3710–21. http://dx.doi.org/10.1099/mic.0.034413-0.
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Staphylococcus aureus is a human pathogen of growing clinical significance, owing to its increasing levels of resistance to most antibiotics. Infections range from mild wound infections to severe infections such as endocarditis, osteomyelitis and septic shock. Adherence of S. aureus to human host cells is an important step, leading to colonization and infection. Adherence is mediated by a multiplicity of proteins expressed on the bacterial surface, including clumping factor B. In this study, we aimed to identify new targets of clumping factor B in human keratinocytes by undertaking a genome-wide yeast two-hybrid screen of a human keratinocyte cDNA library. We show that clumping factor B is capable of binding cytokeratin 8 (CK8), a type II cytokeratin. Using a domain-mapping strategy we identified amino acids 437–464 as necessary for this interaction. Recombinantly expressed fragments of both proteins were used in pull-down experiments and confirmed the yeast two-hybrid studies. Analysis with S. aureus strain Newman deficient in clumping factor B showed the clumping factor B-dependence of the interaction with CK8. We postulate that the clumping factor B–CK8 interaction is a novel factor in S. aureus infections.
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Subramanian, Veedamali S., Svetlana M. Nabokina, Joseph R. Patton, Jonathan S. Marchant, Hamid Moradi e Hamid M. Said. "Glyoxalate reductase/hydroxypyruvate reductase interacts with the sodium-dependent vitamin C transporter-1 to regulate cellular vitamin C homeostasis". American Journal of Physiology-Gastrointestinal and Liver Physiology 304, n.º 12 (15 de junho de 2013): G1079—G1086. http://dx.doi.org/10.1152/ajpgi.00090.2013.
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The human sodium-dependent vitamin C transporter 1 (hSVCT1) contributes to cellular uptake of ascorbic acid (AA). Although different aspects of hSVCT1 cell biology have been extensively studied, nothing is currently known about the broader hSVCT1 interactome that modulates its role in cellular physiology. Here, we identify the enzyme human glyoxalate reductase/hydroxypyruvate reductase (hGR/HPR) as an hSVCT1 associated protein by yeast two-hybrid (Y2H) screening of a human liver cDNA library. The interaction between hSVCT1 and hGR/HPR was further confirmed by in vitro GST pull-down assay, in vivo coimmunoprecipitation and mammalian two-hybrid firefly luciferase assays. This interaction had functional significance as coexpression of hGR/HPR with hSVCT1 led to an increase in AA uptake. Reciprocally, siRNA-mediated knockdown of endogenous hGR/HPR led to an inhibition of AA uptake. Given that oxalate is a degradation product of vitamin C and hGR/HPR acts to limit cellular oxalate levels, this association physically couples two independent regulators of cellular oxalate production. Furthermore, confocal imaging of human liver HepG2 cells coexpressing GFP-hSVCT1 and hGR/HPR-mCherry demonstrated that these two proteins colocalize within a subpopulation of intracellular organelles. This provides a possible molecular basis for organellar AA transport and regulation of local glyoxylate/glycolate concentration in the vicinity of organelle membranes.
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Nabokina, Svetlana M., Veedamali S. Subramanian e Hamid M. Said. "Association of PDZ-containing protein PDZD11 with the human sodium-dependent multivitamin transporter". American Journal of Physiology-Gastrointestinal and Liver Physiology 300, n.º 4 (abril de 2011): G561—G567. http://dx.doi.org/10.1152/ajpgi.00530.2010.
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Intestinal absorption of biotin is mediated via the sodium-dependent multivitamin transporter (SMVT). Studies from our laboratory and others have characterized different aspects of the human SMVT (hSMVT), but nothing is currently known about protein(s) that may interact with hSMVT and affect its physiology/biology. In this study, a PDZ-containing protein PDZD11 was identified as an interacting partner with hSMVT using yeast two-hybrid screen of a human intestinal cDNA library. The interaction between hSMVT and PDZD11 was confirmed by in vitro GST-pull-down assay and in vivo in a mammalian cell environment by a two-hybrid luciferase and coimmunoprecipitation assays. Furthermore, confocal imaging of live human intestinal epithelial HuTu-80 cells expressing hSMVT-GFP and DsRed-PDZD11 demonstrated colocalization of these two proteins. We also examined the functional consequence of the interaction between hSMVT and PDZD11 in HuTu-80 cells and observed significant induction in [3H]biotin uptake upon coexpression of hSMVT and PDZD11. In contrast, knocking down of PDZD11 with gene-specific small interfering RNA led to a significant decrease in biotin uptake; biotinylation assay showed this to be associated with a marked decrease in level of expression of hSMVT at the cell membrane. By truncation approach, we also demonstrated that the PDZ binding domain that is located in the COOH-terminal tail of hSMVT polypeptide is involved in the interaction with PDZD11. These results demonstrate for the first time that PDZD11 is an interacting partner with hSMVT in intestinal epithelial cells and that this interaction affects hSMVT function and cell biology.
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Chen, Xu, Tinghui Hu, Gang Liang, Maojun Yang, Shudong Zong, Shiying Miao, Samuel S. Koide e Linfang Wang. "A novel testis protein, RSB-66, interacting with INCA1 (inhibitor of Cdk interacting with cyclin A1)". Biochemistry and Cell Biology 86, n.º 4 (agosto de 2008): 345–51. http://dx.doi.org/10.1139/o08-072.
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rsb-66 is a novel gene from a suppression subtracted hybridization (SSH) library of round spermatid-specific cDNAs against those of primary spermatocytes. It was found to be specifically expressed in round spermatids. To explore the function of RSB-66, a yeast two-hybrid system was used to screen for potential interacting partners in a human testis cDNA library. HSD45, also known as INCA1 (inhibitor of Cdk interacting with cyclin A1), was identified as one of the positive clones. The interaction between RSB-66 and INCA1 was demonstrated to occur by GST pull down and coimmunoprecipitation. Using immunofluorescence, RSB-66 was found to be specifically expressed in round spermatids, mainly in the cytoplasm. When being transfected into HeLa cells, RSB-66 and INCA1 were found to be co-localized principally in the cytoplasm. The α helix in the RSB-66 C terminal and two amino acid residues (tyr117 and his119) appear to be crucial for its function.
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Kabisch, Ute, Angelika Landgraf, Jana Krause, Ulla Bonas e Jens Boch. "Type III secretion chaperones ShcS1 and ShcO1 from Pseudomonas syringae pv. tomato DC3000 bind more than one effector". Microbiology 151, n.º 1 (1 de janeiro de 2005): 269–80. http://dx.doi.org/10.1099/mic.0.27491-0.
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The hrp-type III secretion (TTS) system is a key pathogenicity factor of the plant pathogen Pseudomonas syringae pv. tomato DC3000 that translocates effector proteins into the cytosol of the eukaryotic host cell. The translocation of a subset of effectors is dependent on specific chaperones. In this study an operon encoding a TTS chaperone (ShcS1) and the truncated effector HopS1′ was characterized. Yeast two-hybrid analysis and pull-down assays demonstrated that these proteins interact. Using protein fusions to AvrRpt2 it was shown that ShcS1 facilitates the translocation of HopS1′, suggesting that ShcS1 is a TTS chaperone for HopS1′ and that amino acids 1 to 118 of HopS1′ are required for translocation. P. syringae pv. tomato DC3000 carries two shcS1 homologues, shcO1 and shcS2, which are located in different operons, and both operons include additional putative effector genes. Transcomplementation experiments showed that ShcS1 and ShcO1, but not ShcS2, can facilitate the translocation of HopS1′ : : AvrRpt2. To characterize the specificities of the putative chaperones, yeast two-hybrid interaction studies were performed between the three chaperones and putative target effectors. These experiments showed that both ShcS1 and ShcO1 bind to two different effectors, HopS1′ and HopO1-1, that share only 16 % amino acid sequence identity. Using gel filtration it was shown that ShcS1 forms homodimers, and this was confirmed by yeast two-hybrid experiments. In addition, ShcS1 is also able to form heterodimers with ShcO1. These data demonstrate that ShcS1 and ShcO1 are exceptional class IA TTS chaperones because they can bind more than one target effector.
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Lazarczyk, Maciej, Christian Pons, José-Andrès Mendoza, Patricia Cassonnet, Yves Jacob e Michel Favre. "Regulation of cellular zinc balance as a potential mechanism of EVER-mediated protection against pathogenesis by cutaneous oncogenic human papillomaviruses". Journal of Experimental Medicine 205, n.º 1 (24 de dezembro de 2007): 35–42. http://dx.doi.org/10.1084/jem.20071311.
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Epidermodysplasia verruciformis (EV) is a genodermatosis associated with skin cancers that results from a selective susceptibility to related human papillomaviruses (EV HPV). Invalidating mutations in either of two genes (EVER1 and EVER2) with unknown functions cause most EV cases. We report that EVER1 and EVER2 proteins form a complex and interact with the zinc transporter 1 (ZnT-1), as shown by yeast two-hybrid screening, GST pull-down, and immunoprecipitation experiments. In keratinocytes, EVER and ZnT-1 proteins do not influence intracellular zinc concentration, but do affect intracellular zinc distribution. EVER2 was found to inhibit free zinc influx to nucleoli. Keratinocytes with a mutated EVER2 grew faster than wild-type keratinocytes. In transiently and stably transfected HaCaT cells, EVER and ZnT-1 down-regulated transcription factors stimulated by zinc (MTF-1) or cytokines (c-Jun and Elk), as detected with luciferase assays. To get some insight into the control of EV HPV infection, we searched for interaction between EVER and ZnT-1 and oncoproteins of cutaneous (HPV5) and genital (HPV16) genotypes. HPV16 E5 protein binds to EVER and ZnT-1 and blocks their negative regulation. The lack of a functional E5 protein encoded by EV HPV genome may account for host restriction of these viruses.
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MAGIERA, Maria M., Mona GUPTA, Catherine J. RUNDELL, Nilima SATISH, Isabelle ERNENS e Stephen J. YARWOOD. "Exchange protein directly activated by cAMP (EPAC) interacts with the light chain (LC) 2 of MAP1A". Biochemical Journal 382, n.º 3 (7 de setembro de 2004): 803–10. http://dx.doi.org/10.1042/bj20040122.
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Using EPAC1 (exchange protein directly activated by cAMP 1) as bait in two-hybrid screens of foetal and adult human brain libraries, we identified the LC2 (light chain 2) of MAP1A (microtubule-associated protein 1A) as a protein capable of interaction with EPAC1. We applied an immunoprecipitation assay to demonstrate protein interaction between EPAC1 and LC2 in co-transfected human embryonic kidney 293 cells. EPAC2 also co-immunoprecipitated with LC2 from extracts of rat cerebellum. Immunolocalization in co-transfected human embryonic kidney 293 cells revealed that EPAC1 co-localizes with LC2 throughout the cell body. We found that endogenous EPAC2 is also immunolocalized with LC2 in PC12 cells. Immunolocalization of EPAC1 in transfected COS1 cells showed that EPAC1 is associated with the perinuclear region surrounding the nucleus and filamentous structures throughout the cell. Removal of the cAMP-binding domain of EPAC1 (ΔcAMP-EPAC1) appeared to disrupt targeting of EPAC1 in cells resulting in a more dispersed staining pattern. Using two-hybrid assay, we tested the ability of LC2 to interact with ΔcAMP-EPAC1 and ΔDEP-EPAC1, which lacks a DEP domain (dishevelled, Egl-10 and pleckstrin homology domain). We found that deletion of the cAMP-binding domain inhibited interaction between EPAC1 and LC2 in a two-hybrid assay, but removal of the DEP domain had little effect. LC2 was found to interact with a glutathione-S-transferase-fusion protein of the cAMP-binding domain of EPAC1 in a pull-down assay, but not the DEP, REM (Ras exchange motif) or CAT (catalytic) domains. Together with our two-hybrid results, this suggests that the cAMP-binding domain of EPAC1 mediates interaction with LC2.
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Weger, Stefan, Eva Hammer e Regine Heilbronn. "Topors, a p53 and topoisomerase I binding protein, interacts with the adeno-associated virus (AAV-2) Rep78/68 proteins and enhances AAV-2 gene expression". Journal of General Virology 83, n.º 3 (1 de março de 2002): 511–16. http://dx.doi.org/10.1099/0022-1317-83-3-511.
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The adeno-associated virus type 2 (AAV-2) Rep proteins are essential for AAV DNA replication and regulation of AAV gene expression. We have identified a cellular protein interacting with Rep78 and Rep68 in yeast two-hybrid analysis and in GST pull-down assays. This protein has recently been described as both a p53 (p53BP3) and a topoisomerase I interacting protein (Topors). It contains an arginine/serine-rich domain, a RING finger domain and five PEST sequences. A minimal sequence sufficient for interaction with Rep was mapped to Topors amino acids 871 to 917. We show that the same region is also involved in the interaction with p53. Rep sequences involved in interaction with Topors were mapped to Rep amino acids 172 to 481. Overexpression of Topors stimulated AAV gene expression in the absence of helper virus, suggesting a function of Topors as a transcriptional regulator.
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REILLY, John F., Shawndra D. MARTINEZ, Gregory MICKEY e Pamela A. MAHER. "A novel role for farnesyl pyrophosphate synthase in fibroblast growth factor-mediated signal transduction". Biochemical Journal 366, n.º 2 (1 de setembro de 2002): 501–10. http://dx.doi.org/10.1042/bj20020560.
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Farnesyl pyrophosphate synthase (FPPS) catalyses the formation of a key cellular intermediate in isoprenoid metabolic pathways. Here we describe a novel role for this enzyme in fibroblast growth factor (FGF)-mediated signalling. We demonstrate the binding of FPPS to FGF receptors (FGFRs) using the yeast two-hybrid assay, pull-down assays and co-immunoprecipitation. The interaction between FPPS and FGFR is regulated by the cellular metabolic state and by treatment with FGF-2. Overexpression of FPPS inhibits FGF-2-induced cell proliferation, accompanied by a failure of the FGF-2-mediated induction of cyclins D1 and E. Overexpression of FPPS in fibroblasts also promotes increased farnesylation of Ras, and temporally extends FGF-2-stimulated activation of the Ras/ERK (extracellular-signal-regulated kinase) cascade. These data suggest that, in addition to its role in isoprenoid biosynthesis, FPPS may function as a modulator of the cellular response to FGF treatment.
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Umareddy, Indira, Alex Chao, Aruna Sampath, Feng Gu e Subhash G. Vasudevan. "Dengue virus NS4B interacts with NS3 and dissociates it from single-stranded RNA". Journal of General Virology 87, n.º 9 (1 de setembro de 2006): 2605–14. http://dx.doi.org/10.1099/vir.0.81844-0.
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Dengue virus, a member of the family Flaviviridae of positive-strand RNA viruses, has seven non-structural proteins: NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5. Except for enzymic activities contained within NS3 and NS5, the roles of the other proteins in virus replication and pathogenesis are not well defined. In this study, a physical interaction between NS4B and the helicase domain of NS3 was identified by using a yeast two-hybrid assay. This interaction was further confirmed by biochemical pull-down and immunoprecipitation assays, both with purified proteins and with dengue virus-infected cell lysates. NS4B co-localized with NS3 in the perinuclear region of infected human cells. Furthermore, NS4B dissociated NS3 from single-stranded RNA and consequently enhanced the helicase activity of NS3 in an in vitro unwinding assay. These results suggest that NS4B modulates dengue virus replication via its interaction with NS3.
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Vittone, Valerio, Eve Diefenbach, Damian Triffett, Mark W. Douglas, Anthony L. Cunningham e Russell J. Diefenbach. "Determination of Interactions between Tegument Proteins of Herpes Simplex Virus Type 1". Journal of Virology 79, n.º 15 (1 de agosto de 2005): 9566–71. http://dx.doi.org/10.1128/jvi.79.15.9566-9571.2005.
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ABSTRACT The aim of this study was to elucidate protein-protein interactions between tegument proteins of herpes simplex virus type 1 (HSV-1). To do so, we have cloned and expressed in the LexA yeast (Saccharomyces cerevisiae) two-hybrid system, 13 of the 21 currently known tegument proteins of HSV-1. These included the tegument proteins essential for replication in cell lines, UL17, UL36, UL37, UL48, and UL49, and the nonessential tegument proteins US11, UL11, UL14, UL16, UL21, UL41, UL46, and UL47. A total of 104 combinations were screened in the yeast two-hybrid assay, with 9 interactions identified. These included: UL11-UL16, UL36-UL37, UL36-UL48, UL46-UL48, UL47-UL48, and UL48-UL49. The remaining interactions consisted of self-associations that were observed for US11, UL37, and UL49. The interactions UL36-UL37, UL36-UL48, UL37-UL37, UL46-UL48, and UL47-UL48 have not been previously reported for HSV-1. The interaction of UL46-UL48 was verified using an in vitro pull-down assay. The interactions of UL36-UL37 and UL37-UL37 were verified with a coimmunoprecipitation assay. Knowledge of HSV-1 tegument protein-protein interactions will provide insights into the pathways of tegument assembly, and the identified interactions are potential targets for new antiviral drugs.
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Krajčíková, Daniela, Magda Lukáčová, Denisa Müllerová, Simon M. Cutting e Imrich Barák. "Searching for Protein-Protein Interactions within the Bacillus subtilis Spore Coat". Journal of Bacteriology 191, n.º 10 (20 de março de 2009): 3212–19. http://dx.doi.org/10.1128/jb.01807-08.
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ABSTRACT The capability of endospores of Bacillus subtilis to withstand extreme environmental conditions is secured by several attributes. One of them, the protein shell that encases the spore and is known as the coat, provides the spore with its characteristic resistance to toxic chemicals, lytic enzymes, and predation by unicellular and multicellular eukaryotes. Despite most of the components of the spore coat having been identified, we have only a vague understanding of how such a complex structure is assembled. Using the yeast two-hybrid system, we attempted to identify direct contacts among the proteins allocated to the insoluble fraction of the spore coat: CotV, CotW, CotX, CotY, and CotZ. We also examined whether they could interact with CotE, one of the most crucial morphogenetic proteins governing outer coat formation and also present in the insoluble fraction. Out of all 21 possible interactions we tested, 4 were found to be positive. Among these interactions, we confirmed the previous observation that CotE forms homo-oligomers. In addition, we observed homotypic interactions of CotY, strong interactions between CotZ and CotY, and relatively weak, yet significant, interactions between CotV and CotW. The results of this yeast two-hybrid analysis were confirmed by size exclusion chromatography of recombinant coat proteins and a pull-down assay.
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Wang, Yicheng, Jingjing Sun, Nan Wang, Haifeng Xu, Changzhi Qu, Shenghui Jiang, Hongcheng Fang, Mengyu Su, Zongying Zhang e Xuesen Chen. "MdMYBL2 helps regulate cytokinin-induced anthocyanin biosynthesis in red-fleshed apple (Malus sieversii f. niedzwetzkyana) callus". Functional Plant Biology 46, n.º 2 (2019): 187. http://dx.doi.org/10.1071/fp17216.
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Anthocyanin biosynthesis is induced by cytokinins, and is regulated by MYB transcription factors. However, the underlying molecular mechanisms have not been fully characterised. In the present study, red-fleshed apple callus were induced from the leaves of an R6/R6 homozygous line, which was the hybrid offspring of Malus sieversii f. niedzwetzkyana and ‘Fuji’. We analysed the callus anthocyanin contents in response to different cytokinin concentrations. We observed that cytokinin treatments upregulated the expression of anthocyanin structural genes MdDFR and MdUFGT and transcription factor genes MdMYB10 and MdbHLH3. Additionally, the expression of MdMYBL2, which encodes the bHLH and EAR motifs, was inhibited by cytokinin treatments. The MdMYBL2-overexpressing callus had lower anthocyanin contents than the wild-type controls. We noted that the expression levels of anthocyanin biosynthesis structural genes MdDFR and MdUFGT and transcription factor genes MdMYB10 and MdbHLH3 were strongly suppressed in the transgenic callus. Subsequent yeast two-hybrid, bimolecular fluorescence complementation, and pull-down assays indicated that MdMYBL2 interacts with MdbHLH3, which may influence the expression of anthocyanin biosynthesis-related genes. Our findings may provide new insights into how MYB transcription factors influence the cytokinin-regulated anthocyanin biosynthesis in red-fleshed apples.
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Fruehn, Juergen, Ian F. Jones, Victoria Valler, Pranaya Sangvai, Ajoy Biswal e Mohit Mathur. "Resolving near-seabed velocity anomalies: Deep water offshore eastern India". GEOPHYSICS 73, n.º 5 (setembro de 2008): VE235—VE241. http://dx.doi.org/10.1190/1.2957947.
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Imaging in deep-water environments poses a specific set of challenges, both in data preconditioning and velocity model building. These challenges include scattered, complex 3D multiples, aliased noise, and low-velocity shallow anomalies associated with channel fills and gas hydrates. We describe an approach to tackling such problems for data from deep water off the east coast of India, concentrating our attention on iterative velocity model building, and more specifically the resolution of near-surface and other velocity anomalies. In the region under investigation, the velocity field is complicated by narrow buried canyons ([Formula: see text] wide) filled with low-velocity sediments, which give rise to severe pull-down effects; possible free-gas accumulation below an extensive gas-hydrate cap, causing dimming of the image below (perhaps as a result of absorption); and thin-channel bodies with low-velocity fill. Hybrid gridded tomography using a conjugate gradient solver (with [Formula: see text] vertical cell size) was applied to resolve small-scale velocity anomalies (with thicknesses of about [Formula: see text]). Manual picking of narrow-channel features was used to define bodies too small for the tomography to resolve. Prestack depth migration, using a velocity model built with a combination of these techniques, could resolve pull-down and other image distortion effects in the final image. The resulting velocity field shows high-resolution detail useful in identifying anomalous geobodies of potential exploration interest.
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TANG, Zhaohua, Norbert F. KÄUFER e Ren-Jang LIN. "Interactions between two fission yeast serine/arginine-rich proteins and their modulation by phosphorylation". Biochemical Journal 368, n.º 2 (1 de dezembro de 2002): 527–34. http://dx.doi.org/10.1042/bj20021133.
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The unexpected low number of genes in the human genome has triggered increasing attention to alternative pre-mRNA splicing, and serine/arginine-rich (SR) proteins have been correlated with the complex alternative splicing that is a characteristic of metazoans. SR proteins interact with RNA and splicing protein factors, and they also undergo reversible phosphorylation, thereby regulating constitutive and alternative splicing in mammals and Drosophila. However, it is not clear whether the features of SR proteins and alternative splicing are present in simple and genetically tractable organisms, such as yeasts. In the present study, we show that the SR-like proteins Srp1 and Srp2, found in the fission yeast Schizosaccharomyces pombe, interact with each other and the interaction is modulated by protein phosphorylation. By using Srp1 as bait in a yeast two-hybrid analysis, we specifically isolated Srp2 from a random screen. This Srp interaction was confirmed by a glutathione-S-transferase pull-down assay. We also found that the Srp1—Srp2 complex was phosphorylated at a reduced efficiency by a fission yeast SR-specific kinase, Dis1-suppression kinase (Dsk1). Conversely, Dsk1-mediated phosphorylation inhibited the formation of the Srp complex. These findings offer the first example in fission yeast for interactions between SR-related proteins and the modulation of the interactions by specific protein phosphorylation, suggesting that a mammalian-like SR protein function may exist in fission yeast.
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Xiang, Ying, Jing Guo, Feng Li e Jie Xiong. "Tudor domain of histone demethylase KDM4B is a reader of H4K20me3". Acta Biochimica et Biophysica Sinica 52, n.º 8 (15 de junho de 2020): 901–6. http://dx.doi.org/10.1093/abbs/gmaa064.
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Abstract The lysine histone demethylase KDM4B is overexpressed in several types of cancers and plays dual roles in genome stability maintenance. Although KDM4B is able to recognize several histone methylations, the underlying molecular mechanism is still unknown. In this study, we purified the KDM4B chromatin-associated hybrid tudor domains (HTDs) and plant home domains (PHDs) and performed the pull-down assay to screen the tri-methyl modified histone peptides that could be efficiently recognized by KDM4B. Our results showed that both HTD alone and the combination of HTD and PHD were able to specifically bind to H3K4me3 and H4K20me3. Because H4K20me3 is essential for KDM4B’s rapid recruitment to DNA damage site, we further aligned the multiple tudor peptide sequence and identified two conserved residues Y993 and W987 that are critical for KDM4B-H4K20me3 interaction. The surface plasmon resonance analysis revealed that HTD displayed a rapid H4K20me3 bind-dissociate pattern. These findings therefore provided mechanistic insights into the binding of tudor domain of KDM4B protein with H4K20me3.
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Yan, Min, Wen Jing, Ni Xu, Like Shen, Qun Zhang e Wenhua Zhang. "Arabidopsis thaliana constitutively active ROP11 interacts with the NADPH oxidase respiratory burst oxidase homologue F to regulate reactive oxygen species production in root hairs". Functional Plant Biology 43, n.º 3 (2016): 221. http://dx.doi.org/10.1071/fp15090.
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Reactive oxygen species (ROS) play a key signalling role in cells. Plant NADPH oxidases, also known as respiratory burst oxidase homologues (Rbohs), are well characterised ROS-generating systems. In this study, we found that the constitutively active small guanosine triphosphatase (GTPase) ROP11 (CA-ROP11) interacted with RbohF by using a yeast two-hybrid analysis, a pull-down assay and an in vivo bimolecular fluorescence complementation assay. The mutation of amino acid L336 or L337 in RbohF abolished its interaction with CA-ROP11. Coexpression of CA-ROP11 and wild-type RbohF in Nicotiana benthamiana Domin enhanced ROS production compared with coexpression of CA-ROP11 and mutant RbohF or of dominant negative ROP11 and wild-type RbohF. Moreover, CA-ROP11 overexpression resulted in ROS accumulation and a swollen root hair phenotype in Arabidopsis thaliana (L.) Heynh. The deletion of RbohF partially reduced the increase in ROS in Arabidopsis plants overexpressing CA-ROP11. These results suggest that Arabidopsis ROP11 modulates ROS production by interacting with RbohF in root hairs.
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Ahmed, Noveera T., Chunlei Gao, Ben F. Lucker, Douglas G. Cole e David R. Mitchell. "ODA16 aids axonemal outer row dynein assembly through an interaction with the intraflagellar transport machinery". Journal of Cell Biology 183, n.º 2 (13 de outubro de 2008): 313–22. http://dx.doi.org/10.1083/jcb.200802025.
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Formation of flagellar outer dynein arms in Chlamydomonas reinhardtii requires the ODA16 protein at a previously uncharacterized assembly step. Here, we show that dynein extracted from wild-type axonemes can rebind to oda16 axonemes in vitro, and dynein in oda16 cytoplasmic extracts can bind to docking sites on pf28 (oda) axonemes, which is consistent with a role for ODA16 in dynein transport, rather than subunit preassembly or binding site formation. ODA16 localization resembles that seen for intraflagellar transport (IFT) proteins, and flagellar abundance of ODA16 depends on IFT. Yeast two-hybrid analysis with mammalian homologues identified an IFT complex B subunit, IFT46, as a directly interacting partner of ODA16. Interaction between Chlamydomonas ODA16 and IFT46 was confirmed through in vitro pull-down assays and coimmunoprecipitation from flagellar extracts. ODA16 appears to function as a cargo-specific adaptor between IFT particles and outer row dynein needed for efficient dynein transport into the flagellar compartment.
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Vidy, Aurore, Mounira Chelbi-Alix e Danielle Blondel. "Rabies Virus P Protein Interacts with STAT1 and Inhibits Interferon Signal Transduction Pathways". Journal of Virology 79, n.º 22 (15 de novembro de 2005): 14411–20. http://dx.doi.org/10.1128/jvi.79.22.14411-14420.2005.
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ABSTRACT Rabies virus P protein is a cofactor of RNA polymerase. We investigated other potential roles of P (CVS strain) by searching for cellular partners using two-hybrid screening. We isolated a cDNA encoding the signal transducer and activator of transcription 1 (STAT1) that is a critical component of interferon type I (IFN-α/β) and type II (IFN-γ) signaling. We confirmed this interaction by glutathione S-transferase-pull-down assay. Deletion mutant analysis indicated that the carboxy-terminal part of P interacted with a region containing the DNA-binding domain and the coiled-coil domain of STAT1. The expression of P protein inhibits IFN-α- and IFN-γ-induced transcriptional responses, thus impairing the IFN-induced antiviral state. Mechanistic studies indicate that P protein does not induce STAT1 degradation and does not interfere with STAT1 phosphorylation but prevents IFN-induced STAT1 nuclear accumulation. These results indicate that rabies P protein overcomes the antiviral response of the infected cells.
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Cheng, Chuangang, Yunpeng Shao, Lan Su, Yin Zhou e Xiulian Sun. "Interactions among Dendrolimus punctatus cypovirus proteins and identification of the genomic segment encoding its A-spike". Journal of General Virology 95, n.º 7 (1 de julho de 2014): 1532–38. http://dx.doi.org/10.1099/vir.0.064022-0.
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Revealing the interactions among cypovirus proteins would facilitate our understanding of the replication and assembly of this virus. In the present study, interactions among proteins encoded by the 10 segments of Dendrolimus punctatus cypovirus (DpCPV) were identified using yeast two-hybrid (Y2H) and far-Western blotting assays. In total, 24 pairs of interactions were detected. Twelve pairs of one-direction interactions, four pairs of binary interactions and four pairs of self-associations were identified in the Y2H assays. Another four pairs of interactions were identified by far-Western blotting. The interactions between the methyltransferase domain of the turret protein (VP3) and VP4 as well as between polyhedrin and VP4 were further confirmed by far-Western blotting and pull-down assays, respectively. In addition, immunogold labelling showed that the A-spike of DpCPV is formed by VP4. In conclusion, we obtained a protein–protein interaction network of DpCPV and showed that its A-spike is formed by VP4 encoded by genomic segment 6.
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Sevcikova, Beatrica, Bronislava Rezuchova, Dagmar Homerova e Jan Kormanec. "The Anti-Anti-Sigma Factor BldG Is Involved in Activation of the Stress Response Sigma Factor σH in Streptomyces coelicolor A3(2)". Journal of Bacteriology 192, n.º 21 (3 de setembro de 2010): 5674–81. http://dx.doi.org/10.1128/jb.00828-10.
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ABSTRACT The alternative stress response sigma factor σH has a role in regulation of the osmotic stress response and in morphological differentiation in Streptomyces coelicolor A3(2). Its gene, sigH, is located in an operon with the gene that encodes its anti-sigma factor UshX (PrsH). However, no gene with similarity to an anti-anti-sigma factor which may have a role in σH activation by a “partner-switching” mechanism is located in the operon. By using a combination of several approaches, including pull-down and bacterial two-hybrid assays and visualization of the complex by native polyacrylamide electrophoresis, we demonstrated a direct interaction between UshX and the pleiotropic sporulation-specific anti-anti-sigma factor BldG. Osmotic induction of transcription of the sigHp2 promoter that is specifically recognized by RNA polymerase containing σH was absent in an S. coelicolor bldG mutant, indicating a role of BldG in σH activation by a partner-switching-like mechanism.