scholarly journals Extracellular domain of YWK-II, a human sperm transmembrane protein, interacts with rat Mullerian-inhibiting substance

Reproduction ◽  
2001 ◽  
pp. 873-880 ◽  
Author(s):  
XY Tian ◽  
YS Sha ◽  
SM Zhang ◽  
YB Chen ◽  
SY Miao ◽  
...  
Keyword(s):  
Hybrid System ◽  
Transmembrane Protein ◽  
Human Sperm ◽  
Extracellular Domain ◽  
Human Spermatozoa ◽  
Müllerian Inhibiting Substance ◽  
Sperm Membrane ◽  
Two Hybrid ◽  
Mullerian Inhibiting Substance

The YWK-II protein in human spermatozoa is structurally related to the betaA4-amyloid precursor protein of Alzheimer disease and has high similarity with amyloid precusor homologues. Antibodies to the YWK-II protein agglutinate human spermatozoa and may be a potential cause of infertility. In the present study, a yeast two-hybrid system (MATCHMAKER Two-Hybrid System 2; Clontech, Palo Alto, CA) was used to screen a rat ovary cDNA library for potential ligands capable of interacting with the YWK-II component. Mullerian-inhibiting substance was found to interact with the extracellular domain of YWK-II protein. The interaction was confirmed by a binding experiment in vitro and surface plasmon resonance assays. The recombinant Mullerian-inhibiting substance can significantly increase the viability and longevity of human spermatozoa after 5 and 22 h of incubation, presumably through binding the YWK-II component on the sperm membrane. The results of this study indicate that the YWK-II sperm membrane protein may function as a receptor for Mullerian-inhibiting substance.

Identifications of DNA Sequences Encoding Key Region of SCR Interacting with SRK Extracellular Domain by Using Yeast Two-Hybrid System

2013 ◽  
Vol 38 (9) ◽  
pp. 1583-1591
Author(s):  
Li-Yan XUE ◽  
Bing LUO ◽  
Li-Quan ZHU ◽  
Yong-Jun YANG ◽  
He-Cui ZHANG ◽  
...  
Keyword(s):  
Hybrid System ◽  
Dna Sequences ◽  
Extracellular Domain ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Two Hybrid

Activation of bovine oocytes matured in vitro by injection of bovine and human spermatozoa or their cytosolic fractions

Zygote ◽  
2001 ◽  
Vol 9 (1) ◽  
pp. 89-95 ◽  
Author(s):  
Osamu Okitsu ◽  
Shuji Yamano ◽  
Toshihiro Aono
Keyword(s):  
Human Sperm ◽  
Human Spermatozoa ◽  
Oocyte Activation ◽  
Sham Injection ◽  
Bovine Sperm ◽  
Female Pronucleus ◽  
Sperm Factor ◽  
Bovine Spermatozoa ◽  
Bovine Oocytes

The aim of this study was to investigate whether bovine spermatozoa possess so-called sperm factor in the cytosolic fraction (CF) which activates bovine oocytes, and whether bovine oocytes matured in vitro are activated by microinjection of CF extracted from spermatozoa of other species. In the first experiment, bovine and human spermatozoa were microinjected into ooplasm of bovine oocytes matured in vitro. Secondly, CF from bovine and human spermatozoa were injected into bovine oocytes. In the third, CF from human spermatozoa was injected into human unfertilised oocytes obtained 18-20 h after clinical intracytoplasmic sperm injection (ICSI). We found that microinjection of bovine spermatozoa into bovine oocytes induced oocyte activation, as shown by resumption of meiosis and formation of a female pronucleus, at a significantly higher rate than the bovine sham injection (63.0% vs 43.0%; p < 0.05). On the other hand, there was no significant difference in activation rate between the human sperm injection (35.9%) and the human sham injection (22.9%). Furthermore, microinjection of bovine sperm CF into bovine oocytes induced oocyte activation at a significantly higher rate than the human CF injection or sham injection (75.9% vs 14.8%, 20.4%; p < 0.01). Formation of a single female pronucleus and second polar body extrusion was observed in 95.1% of activated oocytes after bovine sperm CF injection. When human sperm CF was injected into human unfertilised oocytes, the activation rate was significantly higher than following sham injection (76.9% vs 44.0%; p < 0.05). These results indicate the presence of sperm factor in bovine sperm CF which activate bovine oocytes, and suggest the possibility that sperm factor has species-specificity at least between bovine and human.

ralGDS family members interact with the effector loop of ras p21

1994 ◽  
Vol 14 (11) ◽  
pp. 7483-7491
Author(s):  
A Kikuchi ◽  
S D Demo ◽  
Z H Ye ◽  
Y W Chen ◽  
L T Williams
Keyword(s):  
Hybrid System ◽  
Family Members ◽  
Dominant Negative Mutant ◽  
Yeast Two Hybrid ◽  
Amino Acid Homology ◽  
Yeast Two Hybrid System ◽  
Ras P21 ◽  
Two Hybrid ◽  
Interacting Domain

Using a yeast two-hybrid system, we identified a novel protein which interacts with ras p21. This protein shares 69% amino acid homology with ral guanine nucleotide dissociation stimulator (ralGDS), a GDP/GTP exchange protein for ral p24. We designated this protein RGL, for ralGDS-like. Using the yeast two-hybrid system, we found that an effector loop mutant of ras p21 was defective in interacting with the ras p21-interacting domain of RGL, suggesting that this domain binds to ras p21 through the effector loop of ras p21. Since ralGDS contained a region highly homologous with the ras p21-interacting domain of RGL, we examined whether ralGDS could interact with ras p21. In the yeast two-hybrid system, ralGDS failed to interact with an effector loop mutant of ras p21. In insect cells, ralGDS made a complex with v-ras p21 but not with a dominant negative mutant of ras p21. ralGDS interacted with the GTP-bound form of ras p21 but not with the GDP-bound form in vitro. ralGDS inhibited both the GTPase-activating activity of the neurofibromatosis gene product (NF1) for ras p21 and the interaction of Raf with ras p21 in vitro. These results demonstrate that ralGDS specifically interacts with the active form of ras p21 and that ralGDS can compete with NF1 and Raf for binding to the effector loop of ras p21. Therefore, ralGDS family members may be effector proteins of ras p21 or may inhibit interactions between ras p21 and its effectors.

scholarly journals Genetic Analysis of theEscherichia coliFtsZ·ZipA Interaction in the Yeast Two-hybrid System

2001 ◽  
Vol 276 (15) ◽  
pp. 11980-11987 ◽  
Author(s):  
Steven A. Haney ◽  
Elizabeth Glasfeld ◽  
Cynthia Hale ◽  
David Keeney ◽  
Zhizhen He ◽  
...  
Keyword(s):  
Hybrid System ◽  
Enzyme Linked Immunosorbent Assay ◽  
Wild Type ◽  
Yeast Two Hybrid ◽  
Conserved Sequence ◽  
Yeast Two Hybrid System ◽  
C Terminus ◽  
Two Hybrid

The recruitment of ZipA to the septum by FtsZ is an early, essential step in cell division inEscherichia coli. We have used polymerase chain reaction-mediated random mutagenesis in the yeast two-hybrid system to analyze this interaction and have identified residues within a highly conserved sequence at the C terminus of FtsZ as the ZipA binding site. A search for suppressors of a mutation that causes a loss of interaction (ftsZD373G) identified eight different changes at two residues within this sequence.In vitro, wild type FtsZ interacted with ZipA with a high affinity in an enzyme-linked immunosorbent assay, whereas FtsZD373Gfailed to interact. Two mutant proteins examined restored this interaction significantly.In vivo, the alleles tested are significantly more toxic than the wild typeftsZand cannot complement a deletion. We have shown that a fusion, which encodes the last 70 residues of FtsZ in the two-hybrid system, is sufficient for the interaction with FtsA and ZipA. However, when the wild type sequence is compared with one that encodes FtsZD373G, no interaction was seen with either protein. Mutations surrounding Asp-373 differentially affected the interactions of FtsZ with ZipA and FtsA, indicating that these proteins bind the C terminus of FtsZ differently.

scholarly journals In vivo evaluation of the interaction between the Escherichia coli IGP synthase subunits using the Bacterial Two-Hybrid system

2020 ◽  
Vol 367 (14) ◽  
Author(s):  
Sofia Chioccioli ◽  
Patrizia Bogani ◽  
Sara Del Duca ◽  
Lara Mitia Castronovo ◽  
Alberto Vassallo ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Hybrid System ◽  
De Novo ◽  
Glycerol Phosphate ◽  
In Vivo Evaluation ◽  
Igp Synthase ◽  
Imidazole Glycerol Phosphate ◽  
Two Hybrid

ABSTRACT Histidine biosynthesis is one of the most characterized metabolic routes for its antiquity and its central role in cellular metabolism; indeed, it represents a cross-road between nitrogen metabolism and de novo synthesis of purines. This interconnection is due to the activity of imidazole glycerol phosphate synthase, a heterodimeric enzyme constituted by the products of two his genes, hisH and hisF, encoding a glutamine amidotransferase and a cyclase, respectively. Despite their interaction was suggested by several in vitro experiments, their in vivo complex formation has not been demonstrated. On the contrary, the analysis of the entire Escherichia coli interactome performed using the yeast two hybrid system did not suggest the in vivo interaction of the two IGP synthase subunits. The aim of this study was to demonstrate the interaction of the two proteins using the Bacterial Adenylate Cyclase Two-Hybrid (BACTH) system. Data obtained demonstrated the in vivo interaction occurring between the proteins encoded by the E. coli hisH and hisF genes; this finding might also open the way to pharmaceutical applications through the design of selective drugs toward this enzyme.

scholarly journals Association of a RING finger protein with the cytoplasmic domain of the human type-2 tumour necrosis factor receptor

1995 ◽  
Vol 309 (3) ◽  
pp. 825-829 ◽  
Author(s):  
H Y Song ◽  
D B Donner
Keyword(s):  
Tumour Necrosis Factor ◽  
Hybrid System ◽  
Tumour Necrosis ◽  
Ring Finger ◽  
Tnf Receptor ◽  
Intracellular Domain ◽  
Two Hybrid ◽  
Necrosis Factor

A human gene encoding a protein that specifically binds to the intracellular domain of the 75 kDa type-2 tumour necrosis factor (TNF) receptor (TNFR-2IC) has been identified using the yeast-based two-hybrid system. The N-terminal half of the TNF receptor-associated protein (TRAP) contains RING finger and zinc finger motifs often found in DNA-binding proteins including transcription factors. The 2.4 kb TRAP mRNA was barely detectable, if present at all, in lung, and variably expressed in heart, liver, placenta, brain, skeletal muscle, kidney and the pancreas; interestingly, the TRAP was more highly expressed in transformed cell lines than in normal tissues. This observation may be consistent with a role for this TRAP in promoting or regulating cellular proliferation. After in vitro transcription/translation and 35S labelling the TRAP was precipitated using a fusion protein consisting of glutathione S-transferase and the intracellular domain of TNFR-2 (TNFR-2IC), which showed that the two proteins directly interact in a mammalian cell-free system and also that identification of the TRAP was not an artifact of the two-hybrid system. By using truncated TNFR-2ICs for in vitro precipitation of 35S-TRAP, it was shown that the C-terminal half of the TNFR-2IC contains the domain necessary for interaction with TRAP. The TRAP identified in the present study shares considerable homology with, and may be the human homologue of, a mouse protein, TNF receptor-associated factor 2 (TRAF2), that binds mouse TNFR-2.

THE PRESENCE OF MULLERIAN INHIBITING SUBSTANCE BINDING SITES IN HUMAN SPERM

1998 ◽  
pp. 2210-2214
Author(s):  
MARY E. FALLAT ◽  
YONG SIOW ◽  
ELIZABETH A. KLAR ◽  
ARNOLD M. BELKER ◽  
DAVID T. MACLAUGHLIN
Keyword(s):  
Binding Sites ◽  
Human Sperm ◽  
Müllerian Inhibiting Substance ◽  
Mullerian Inhibiting Substance

scholarly journals Neoadjuvant Treatment With Müllerian-Inhibiting Substance Synchronizes Follicles and Enhances Superovulation Yield

2019 ◽  
Vol 3 (11) ◽  
pp. 2123-2134 ◽  
Author(s):  
Motohiro Kano ◽  
Jennifer Y Hsu ◽  
Hatice D Saatcioglu ◽  
Nicholas Nagykery ◽  
LiHua Zhang ◽  
...  
Keyword(s):  
Neoadjuvant Treatment ◽  
Reproductive Hormones ◽  
Negative Regulator ◽  
Poor Responders ◽  
Ovarian Suppression ◽  
Vitro Fertilization ◽  
Antral Follicles ◽  
Müllerian Inhibiting Substance ◽  
Mullerian Inhibiting Substance

Abstract Müllerian-inhibiting substance (MIS), also known as anti-Müllerian hormone, is thought to be a negative regulator of primordial follicle activation. We have previously reported that treatment with exogenous MIS can induce complete ovarian suppression within 5 weeks of treatment in mice. To investigate the kinetics of the return of folliculogenesis following the reversal of suppression, we treated animals with recombinant human MIS (rhMIS) protein for 40 days in adult female Nu/Nu mice and monitored the recovery of each follicle type over time. Following cessation of MIS therapy, secondary, and antral follicles returned within 30 days, along with the normalization of reproductive hormones, including LH, FSH, MIS, and Inhibin B. Furthermore, 30 days following MIS pretreatment, the number of antral follicles were significantly higher than controls, and superovulation with timed pregnant mare serum gonadotropin and human chorionic gonadotropin stimulation at this time point resulted in an approximately threefold increased yield of eggs. Use of the combined rhMIS-gonadotropin superovulation regimen in a diminished ovarian reserve (DOR) mouse model, created by 4-vinylcyclohexene dioxide treatment, also resulted in a twofold improvement in the yield of eggs. In conclusion, treatment with rhMIS can induce a reversible ovarian suppression, following which a rapid and synchronized large initial wave of growing follicles can be harnessed to enhance the response to superovulation. Therapies modulating MIS signaling may therefore augment the response to current ovarian stimulation protocols and could be particularly useful to women with DOR or poor responders to controlled ovarian hyperstimulation during in vitro fertilization.

scholarly journals Cytosolic adaptor protein Dab2 is an intracellular ligand of endocytic receptor gp600/megalin

2000 ◽  
Vol 347 (3) ◽  
pp. 613-621 ◽  
Author(s):  
Andrew V. OLEINIKOV ◽  
Jun ZHAO ◽  
Sudesh P. MAKKER
Keyword(s):  
Signal Transduction ◽  
Hybrid System ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Adaptor Protein ◽  
Human Kidney ◽  
Negative Regulator ◽  
Interaction Domain ◽  
Two Hybrid

gp600/megalin, an endocytic receptor, belongs to the low-density lipoprotein receptor family. It is most abundant in the renal proximal tubular cells, where it is implicated in the reabsorption of a number of molecules filtered through the glomerulus. The cytoplasmic tail (CT) of gp600/megalin contains a number of sequence similarities, which indicate that gp600/megalin might be involved in signal transduction. To find intracellular proteins that would interact with the gp600/megalin CT, a human kidney cDNA library was screened by using the yeast two-hybrid system. The phosphotyrosine interaction domain (PID) of the Disabled protein 2 (Dab2), a mammalian structural analogue of Drosophila Disabled, was found to bind to the gp600/megalin CT in this system. The interaction between these two proteins was confirmed by a binding assay in vitro and by the co-immunoprecipitation of both proteins from renal cell lysates. The gp600/megalin CT contains three ΨXNPXY motifs (in which Ψ represents a hydrophobic residue) that are potentially able to interact with PID. Analysis of the CT deletion and point-mutation variants of gp600/megalin by the two-hybrid system revealed that the third ΨXNPXY motif is most probably involved in this interaction. Dab2 is a mitogen-responsive phosphoprotein thought to be an adaptor molecule involved in signal transduction, and a suggested negative regulator of cell growth. Dab2 is the first intracellular ligand identified for gp600/megalin; gp600/megalin is the first known transmembrane receptor that interacts with the cytosolic protein Dab2. We speculate that their interaction might involve gp600/megalin in signal transduction pathways or might mediate the intracellular trafficking of this receptor.

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