scholarly journals Yeast Two-Hybrid Screen Identifies PKA-Riα Interacting Proteins during Mouse Spermiogenesis

Genes ◽  
2021 ◽  
Vol 12 (12) ◽  
pp. 1941
Author(s):  
Kunyu Shi ◽  
Lele Yang ◽  
Xueqing Zhuang ◽  
Lan Zhang ◽  
Huayu Qi
Keyword(s):  
Protein Synthesis ◽  
Molecular Mechanisms ◽  
Regulation Of Gene Expression ◽  
Protein Translation ◽  
Regulatory Subunit ◽  
Type I ◽  
Interacting Proteins ◽  
Spermatogenic Cells ◽  
Yeast Two Hybrid ◽  
Two Hybrid

cAMP-dependent protein kinase (PKA) signaling plays various roles during mammalian spermatogenesis, ranging from the regulation of gene expression to the modulation of sperm motility. However, the molecular mechanisms that govern the multifaceted functions of PKA during spermatogenesis remain largely unclear. We previously found that PKA regulatory subunit I α (RIα) and catalytic subunit α (Cα) co-sediment with polyribosomal fractions of mouse testis lysate on sucrose gradient and the stimulation of PKA activity facilitates protein synthesis in post-meiotic elongating spermatids, indicating that type I PKA is intricately associated with protein translation machinery and regulates protein synthesis during mouse spermiogenesis. Since PKA activity is often regulated by interacting proteins that form complexes with its regulatory subunits, the identification of PKA-RIα interacting proteins in post-meiotic spermatogenic cells will facilitate our understanding of its regulatory roles in protein synthesis and spermiogenesis. In the present study, we applied a yeast two-hybrid screen to identify PKA-Riα-binding proteins using a cDNA library generated from mouse round and elongating spermatids. Numerous proteins were found to potentially interact with PKA-RIα, including proteostasis modulators, metabolic enzymes, cytoskeletal regulators, and mitochondrial proteins, many of which are specifically expressed in testes. Consistently, the examination of MENA (mouse ENA/VASP homolog) in developing mouse testes suggested that post-meiotic spermatogenic cells express a short isoform of MENA that interacts with PKA-RIα in yeast two-hybrid assay. The identification of PKA-RIα interacting proteins provides us solid basis to further explore how PKA signaling regulates protein synthesis and cellular morphogenesis during mouse spermatogenesis.

scholarly journals FKBPL and FKBP8 regulate DLK degradation and neuronal responses to axon injury

2021 ◽  
Author(s):  
Bohm Lee ◽  
Yeonsoo Oh ◽  
Eunhye Cho ◽  
Aaron DiAntonio ◽  
Valeria Cavalli ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Axon Regeneration ◽  
Kinase Domain ◽  
Interacting Proteins ◽  
Bafilomycin A1 ◽  
Neuronal Responses ◽  
Yeast Two Hybrid ◽  
Two Hybrid ◽  
Hybrid Screening

DLK is a key regulator of axon regeneration and degeneration in response to neuronal injury. To understand the molecular mechanisms controlling the DLK function, we performed yeast two-hybrid screening analysis and identified FKBPL as a DLK-binding protein that bound to the kinase domain and inhibited the kinase enzymatic activity of DLK. FKBPL regulated DLK stability through ubiquitin-dependent DLK degradation. We tested other members in the FKBP protein family and found that FKBP8 also induced DLK degradation as FKBPL did. We found that Lysine 271 residue in the kinase domain of DLK was a major site of ubiquitination and SUMO3-conjugation and responsible for FKBP8-mediated degradation. In vivo overexpression of FKBP8 delayed progression of axon degeneration and neuronal death following axotomy in sciatic and optic nerves, respectively, although axon regeneration efficiency was not enhanced. This research identified FKBPL and FKBP8 as new DLK-interacting proteins that regulated DLK stability by MG-132 or bafilomycin A1-sensitive protein degradation.

scholarly journals Implication of the Whitefly Protein Vps Twenty Associated 1 (Vta1) in the Transmission of Cotton Leaf Curl Multan Virus

2021 ◽  
Vol 9 (2) ◽  
pp. 304
Author(s):  
Yao Chi ◽  
Li-Long Pan ◽  
Shu-Sheng Liu ◽  
Shahid Mansoor ◽  
Xiao-Wei Wang
Keyword(s):  
Coat Protein ◽  
Molecular Mechanisms ◽  
Cotton Leaf ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Vacuolar Protein ◽  
Leaf Curl Disease ◽  
Two Hybrid ◽  
Asia Ii 1 ◽  
Leaf Curl

Cotton leaf curl Multan virus (CLCuMuV) is one of the major casual agents of cotton leaf curl disease. Previous studies show that two indigenous whitefly species of the Bemisia tabaci complex, Asia II 1 and Asia II 7, are able to transmit CLCuMuV, but the molecular mechanisms underlying the transmission are poorly known. In this study, we attempted to identify the whitefly proteins involved in CLCuMuV transmission. First, using a yeast two-hybrid system, we identified 54 candidate proteins of Asia II 1 that putatively can interact with the coat protein of CLCuMuV. Second, we examined interactions between the CLCuMuV coat protein and several whitefly proteins, including vacuolar protein sorting-associated protein (Vps) twenty associated 1 (Vta1). Third, using RNA interference, we found that Vta1 positively regulated CLCuMuV acquisition and transmission by the Asia II 1 whitefly. In addition, we showed that the interaction between the CLCuMuV coat protein and Vta1 from the whitefly Middle East-Asia Minor (MEAM1), a poor vector of CLCuMuV, was much weaker than that between Asia II 1 Vta1 and the CLCuMuV coat protein. Silencing of Vta1 in MEAM1 did not affect the quantity of CLCuMuV acquired by the whitefly. Taken together, our results suggest that Vta1 may play an important role in the transmission of CLCuMuV by the whitefly.

scholarly journals Protein phosphatase type 1 interacts with proteins required for meiosis and other cellular processes in Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (8) ◽  
pp. 4199-4206 ◽  
Author(s):  
J Tu ◽  
W Song ◽  
M Carlson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Hybrid System ◽  
Protein Phosphatase ◽  
Regulatory Subunit ◽  
Specific Substrate ◽  
Type I ◽  
Cellular Processes ◽  
Cell Extracts ◽  
Two Hybrid ◽  
Protein Phosphatase Type

Protein phosphatase type I (PP1) is involved in diverse cellular processes, and its activity toward specific substrates is thought to be controlled by different regulatory or targeting subunits. To identify regulatory subunits and substrates of the Saccharomyces cerevisiae PP1, encoded by GLC7, we used the two-hybrid system to detect interacting proteins. Among the many proteins identified were Gac1, a known glycogen regulatory subunit, and a protein with homology to Gac1. We also characterized a new gene designated GIP1, for Glc7-interacting protein. We show that a Gip1 fusion protein coimmunoprecipitates with PP1 from cell extracts. Molecular and genetic analyses indicate that GIP1 is expressed specifically during meiosis, affects transcription of late meiotic genes, and is essential for sporulation. Thus, the Gip1 protein is a candidate for a meiosis-specific substrate or regulator of PP1. Finally, we recovered two genes, RED1 and SCD5, with roles in meiosis and the vesicular secretory pathway, respectively. These results provide strong evidence implicating PP1 function in meiosis. In addition, this study indicates that the two-hybrid system offers a promising approach to understanding the multiple roles and interactions of PP1 in cellular regulation.

scholarly journals Evaluation of Interactions of Human Cytomegalovirus Immediate-Early IE2 Regulatory Protein with Small Ubiquitin-Like Modifiers and Their Conjugation Enzyme Ubc9

2001 ◽  
Vol 75 (8) ◽  
pp. 3859-3872 ◽  
Author(s):  
Jin-Hyun Ahn ◽  
Yixun Xu ◽  
Won-Jong Jang ◽  
Michael J. Matunis ◽  
Gary S. Hayward
Keyword(s):  
Hcmv Infection ◽  
Dependent Manner ◽  
Interacting Proteins ◽  
Binding Assays ◽  
Yeast Two Hybrid ◽  
Vitro Binding ◽  
Lysine Residues ◽  
Infected Cells ◽  
Two Hybrid

ABSTRACT The human cytomegalovirus (HCMV) major immediate-early protein IE2 is a nuclear phosphoprotein that is believed to be a key regulator in both lytic and latent infections. Using yeast two-hybrid screening, small ubiquitin-like modifiers (SUMO-1, SUMO-2, and SUMO-3) and a SUMO-conjugating enzyme (Ubc9) were isolated as IE2-interacting proteins. In vitro binding assays with glutathioneS-transferase (GST) fusion proteins provided evidence for direct protein-protein interaction. Mapping data showed that the C-terminal end of SUMO-1 is critical for interaction with IE2 in both yeast and in vitro binding assays. IE2 was efficiently modified by SUMO-1 or SUMO-2 in cotransfected cells and in cells infected with a recombinant adenovirus expressing HCMV IE2, although the level of modification was much lower in HCMV-infected cells. Two lysine residues at positions 175 and 180 were mapped as major alternative SUMO-1 conjugation sites in both cotransfected cells and an in vitro sumoylation assay and could be conjugated by SUMO-1 simultaneously. Although mutations of these lysine residues did not interfere with the POD (or ND10) targeting of IE2, overexpression of SUMO-1 enhanced IE2-mediated transactivation in a promoter-dependent manner in reporter assays. Interestingly, many other cellular proteins identified as IE2 interaction partners in yeast two-hybrid assays also interact with SUMO-1, suggesting that either directly bound or covalently conjugated SUMO moieties may act as a bridge for interactions between IE2 and other SUMO-1-modified or SUMO-1-interacting proteins. When we investigated the intracellular localization of SUMO-1 in HCMV-infected cells, the pattern changed from nuclear punctate to predominantly nuclear diffuse in an IE1-dependent manner at very early times after infection, but with some SUMO-1 protein now associated with IE2 punctate domains. However, at late times after infection, SUMO-1 was predominantly detected within viral DNA replication compartments containing IE2. Taken together, these results show that HCMV infection causes the redistribution of SUMO-1 and that IE2 both physically binds to and is covalently modified by SUMO moieties, suggesting possible modulation of both the function of SUMO-1 and protein-protein interactions of IE2 during HCMV infection.

Identification of Connexin-Interacting Proteins: Application of the Yeast Two-Hybrid Screen

Methods ◽  
2000 ◽  
Vol 20 (2) ◽  
pp. 219-231 ◽  
Author(s):  
Chengshi Jin ◽  
Alan F. Lau ◽  
Kendra Dean Martyn
Keyword(s):  
Interacting Proteins ◽  
Yeast Two Hybrid ◽  
Two Hybrid

Yeast Two-Hybrid Assay to Identify Interacting Proteins

2018 ◽  
Vol 95 (1) ◽  
pp. e70 ◽  
Author(s):  
Aurora Paiano ◽  
Azzurra Margiotta ◽  
Maria De Luca ◽  
Cecilia Bucci
Keyword(s):  
Interacting Proteins ◽  
Yeast Two Hybrid ◽  
Two Hybrid

scholarly journals Ebolavirus VP35 Interacts with the Cytoplasmic Dynein Light Chain 8

2009 ◽  
Vol 83 (13) ◽  
pp. 6952-6956 ◽  
Author(s):  
Toru Kubota ◽  
Mayumi Matsuoka ◽  
Tsung-Hsien Chang ◽  
Mike Bray ◽  
Steven Jones ◽  
...  
Keyword(s):  
Life Cycle ◽  
Light Chain ◽  
Viral Protein ◽  
Cytoplasmic Dynein ◽  
Type I ◽  
Dynein Light Chain ◽  
Yeast Two Hybrid ◽  
Binding Partners ◽  
Mapping Analysis ◽  
Two Hybrid

ABSTRACT The viral protein VP35 of ebolavirus (EBOV) is implicated to have diverse roles in the viral life cycle. We employed a yeast two-hybrid screen to search for VP35 binding partners and identified the cytoplasmic dynein light chain (DLC8) as a protein that interacts with VP35. Mapping analysis unraveled a consensus motif, SQTQT, within VP35 through which VP35 binds to DLC8. The disruption of DLC8 binding does not affect the ability of VP35 to inhibit type I IFN production. Given that VP35 from various EBOV species interacts with DLC8, this interaction may have a role in regulating the EBOV life cycle.

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