scholarly journals The Metarhizium anisopliae Toxin, Destruxin A, Interacts with the SEC23A and TEME214 Proteins of Bombyx mori

2021 ◽  
Vol 7 (6) ◽  
pp. 460
Author(s):  
Fei Yin ◽  
Miaomiao Xiao ◽  
Alexander Berestetskiy ◽  
Qiongbo Hu
Keyword(s):  
Bombyx Mori ◽  
Protein Interactions ◽  
Metarhizium Anisopliae ◽  
Binding Proteins ◽  
Protein Transport ◽  
Transmembrane Protein ◽  
Single Treatment ◽  
Action Mechanism ◽  
Two Hybrid

Destruxin A (DA), a mycotoxin isolated from the entomopathogenic fungus Metarhizium anisopliae, has good insecticidal and immune-inhibitory activity, but the action mechanism has not yet been elucidated. In order to identify the DA-binding proteins, we conducted drug affinity responsive target stability (DARTS) experiments, which indicated that the silkworm’s (Bombyx mori) transmembrane protein 214 (BmTEME214) and protein transport protein SEC23A isoform X2 (BmSEC23) are the potential DA-binding proteins. The current research was focused on validation of the interaction between DA and these two proteins via bio-layer interferometry (BLI) in vitro, insect two-hybrid (I2H) in Sf9 cells, and RNAi in the insect. The results of the BLI tests showed that DA has strong affinity to bind BmTEME214 and BmSEC23 proteins with a KD value of 0.286 and 0.291 µM, respectively. In the I2H experiments, DA inhibited (at 0.02 µg/mL) and activated (at 0.002–0.0002 µg/mL) the protein interactions of BmSEC23–BmSEC13, but it only inhibited the BmTMEM214–BmSEC13L interaction. Furthermore, in the RNAi tests, an apparent increase in the silkworm’s mortality was recorded in the joint treatment of DA with dsBmSEC23 or dsBmTMEM214 when compared with the single treatment of DA (1.5 µg/g body), 40 µg/g body dsBmSEC23, or dsBmTMEM214. This research confirmed that BmSEC23 and BmTMEM214 are the DA-binding proteins and provided new insights to understand the action mechanism of DA.

scholarly journals Kinetochore Protein Interactions and their Regulation by the Aurora Kinase Ipl1p

2003 ◽  
Vol 14 (8) ◽  
pp. 3342-3355 ◽  
Author(s):  
Ching Shang ◽  
Tony R. Hazbun ◽  
Iain M. Cheeseman ◽  
Jennifer Aranda ◽  
Stanley Fields ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Aurora Kinase ◽  
Open Reading Frames ◽  
Binding Assays ◽  
A Genome ◽  
Physical Interactions ◽  
Dam1 Complex ◽  
Two Hybrid ◽  
Kinetochore Function

Although there has been a recent explosion in the identification of budding yeast kinetochore components, the physical interactions that underlie kinetochore function remain obscure. To better understand how kinetochores attach to microtubules and how this attachment is regulated, we sought to characterize the interactions among kinetochore proteins, especially with respect to the microtubule-binding Dam1 complex. The Dam1 complex plays a crucial role in the chromosome-spindle attachment and is a key target for phospho-regulation of this attachment by the Aurora kinase Ipl1p. To identify protein–protein interactions involving the Dam1 complex, and the effects of Dam1p phosphorylation state on these physical interactions, we conducted both a genome-wide two-hybrid screen and a series of biochemical binding assays for Dam1p. A two-hybrid screen of a library of 6000 yeast open reading frames identified nine kinetochore proteins as Dam1p-interacting partners. From 113 in vitro binding reactions involving all nine subunits of the Dam1 complex and 32 kinetochore proteins, we found at least nine interactions within the Dam1 complex and 19 potential partners for the Dam1 complex. Strikingly, we found that the Dam1p–Ndc80p and Dam1p–Spc34p interactions were weakened by mutations mimicking phosphorylation at Ipl1p sites, allowing us to formulate a model for the effects of phosphoregulation on kinetochore function.

scholarly journals Transcriptional repression in Saccharomyces cerevisiae by a SIN3-LexA fusion protein

1993 ◽  
Vol 13 (3) ◽  
pp. 1805-1814
Author(s):  
H Wang ◽  
D J Stillman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Fusion Protein ◽  
Protein Interactions ◽  
Transcriptional Repression ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Predicted Amino Acid Sequence

The yeast SIN3 gene (also known as SDI1, UME4, RPD1, and GAM2) has been identified as a transcriptional regulator. Previous work has led to the suggestion that SIN3 regulates transcription via interactions with DNA-binding proteins. Although the SIN3 protein is located in the nucleus, it does not bind directly to DNA in vitro. We have expressed a LexA-SIN3 fusion protein in Saccharomyces cerevisiae and show that this fusion protein represses transcription from heterologous promoters that contain lexA operators. The predicted amino acid sequence of the SIN3 protein contains four copies of a paired amphipathic helix (PAH) motif, similar to motifs found in HLH (helix-loop-helix) and TPR (tetratricopeptide repeat) proteins, and these motifs are proposed to be involved in protein-protein interactions. We have conducted a deletion analysis of the SIN3 gene and show that the PAH motifs are required for SIN3 activity. Additionally, the C-terminal region of the SIN3 protein is sufficient for repression activity in a LexA-SIN3 fusion, and deletion of a PAH motif in this region inactivates this repression activity. A model is presented in which SIN3 recognizes specific DNA-binding proteins in vivo in order to repress transcription.

scholarly journals High-Throughput Identification of Nuclear Envelope Protein Interactions in Schizosaccharomyces pombe Using an Arrayed Membrane Yeast-Two Hybrid Library

2020 ◽  
Vol 10 (12) ◽  
pp. 4649-4663 ◽  
Author(s):  
Joseph M. Varberg ◽  
Jennifer M. Gardner ◽  
Scott McCroskey ◽  
Snehabala Saravanan ◽  
William D. Bradford ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Nuclear Envelope ◽  
Schizosaccharomyces Pombe ◽  
High Throughput ◽  
Protein Interactions ◽  
Transmembrane Protein ◽  
Integral Membrane Proteins ◽  
Model Organisms ◽  
Yeast Two Hybrid ◽  
Two Hybrid

The nuclear envelope (NE) contains a specialized set of integral membrane proteins that maintain nuclear shape and integrity and influence chromatin organization and gene expression. Advances in proteomics techniques and studies in model organisms have identified hundreds of proteins that localize to the NE. However, the function of many of these proteins at the NE remains unclear, in part due to a lack of understanding of the interactions that these proteins participate in at the NE membrane. To assist in the characterization of NE transmembrane protein interactions we developed an arrayed library of integral and peripheral membrane proteins from the fission yeast Schizosaccharomyces pombe for high-throughput screening using the split-ubiquitin based membrane yeast two -hybrid system. We used this approach to characterize protein interactions for three conserved proteins that localize to the inner nuclear membrane: Cut11/Ndc1, Lem2 and Ima1/Samp1/Net5. Additionally, we determined how the interaction network for Cut11 is altered in canonical temperature-sensitive cut11-ts mutants. This library and screening approach is readily applicable to characterizing the interactomes of integral membrane proteins localizing to various subcellular compartments.

scholarly journals Protein-protein interactions in the synaptonemal complex.

1997 ◽  
Vol 8 (8) ◽  
pp. 1405-1414 ◽  
Author(s):  
M Tarsounas ◽  
R E Pearlman ◽  
P J Gasser ◽  
M S Park ◽  
P B Moens
Keyword(s):  
Synaptonemal Complex ◽  
Protein Interactions ◽  
Coiled Coil ◽  
Protein Protein Interactions ◽  
Late Prophase ◽  
Yeast Saccharomyces Cerevisiae ◽  
Heterotypic Interactions ◽  
Two Hybrid ◽  
Homotypic Interactions

In mammalian systems, an approximately M(r) 30,000 Cor1 protein has been identified as a major component of the meiotic prophase chromosome cores, and a M(r) 125,000 Syn1 protein is present between homologue cores where they are synapsed and form the synaptonemal complex (SC). Immunolocalization of these proteins during meiosis suggests possible homo- and heterotypic interactions between the two as well as possible interactions with yet unrecognized proteins. We used the two-hybrid system in the yeast Saccharomyces cerevisiae to detect possible protein-protein associations. Segments of hamsters Cor1 and Syn1 proteins were tested in various combinations for homo- and heterotypic interactions. In the cause of Cor1, homotypic interactions involve regions capable of coiled-coil formation, observation confirmed by in vitro affinity coprecipitation experiments. The two-hybrid assay detects no interaction of Cor1 protein with central and C-terminal fragments of Syn1 protein and no homotypic interactions involving these fragments of Syn1. Hamster Cor1 and Syn1 proteins both associate with the human ubiquitin-conjugation enzyme Hsubc9 as well as with the hamster Ubc9 homologue. The interactions between SC proteins and the Ubc9 protein may be significant for SC disassembly, which coincides with the repulsion of homologs by late prophase I, and also for the termination of sister centromere cohesiveness at anaphase II.

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.

scholarly journals Subunit Oligomerization and Substrate Recognition of the Escherichia coli ClpYQ (HslUV) Protease Implicated by In Vivo Protein-Protein Interactions in the Yeast Two-Hybrid System

2003 ◽  
Vol 185 (8) ◽  
pp. 2393-2401 ◽  
Author(s):  
Yi-Ying Lee ◽  
Chiung-Fang Chang ◽  
Chueh-Ling Kuo ◽  
Meng-Ching Chen ◽  
Chien Hung Yu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Hybrid System ◽  
Protein Interactions ◽  
Large Subunit ◽  
Protein Protein Interactions ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Two Hybrid

ABSTRACT The Escherichia coli ClpYQ (HslUV) is an ATP-dependent protease that consists of an ATPase large subunit with homology to other Clp family ATPases and a peptidase small subunit related to the proteasomal β-subunits of eukaryotes. Six identical subunits of both ClpY and ClpQ self-assemble into an oligomeric ring, and two rings of each subunit, two ClpQ rings surrounded by single ClpY rings, form a dumbbell shape complex. The ClpYQ protease degrades the cell division inhibitor, SulA, and a positive regulator of capsule transcription, RcsA, as well as RpoH, a heat shock sigma transcription factor. Using the yeast-two hybrid system, we explored the in vivo protein-protein interactions of the individual subunits of the ClpYQ protease involved in self-oligomerization, as well as in recognition of specific substrates. Interactions were detected with ClpQ/ClpQ, ClpQ/ClpY, and ClpY/SulA. No interactions were observed in experiments with ClpY/ClpY, ClpQ/RcsA, and ClpQ/SulA. However, ClpY, lacking domain I (ClpYΔI) was able to interact with itself and with intact ClpY. The C-terminal region of ClpY is important for interaction with other ClpY subunits. The previously defined PDZ-like domains at the C terminus of ClpY, including both D1 and D2, were determined to be indispensable for substrate binding. Various deletion and random point mutants of SulA were also made to verify significant interactions with ClpY. Thus, we demonstrated in vivo hetero- and homointeractions of ClpQ and ClpY molecules, as well as a direct association between ClpY and substrate SulA, thereby supporting previous in vitro biochemical findings.

Amylase-binding proteins A (AbpA) and B (AbpB) differentially affect colonization of rats' teeth by Streptococcus gordonii

Microbiology ◽  
2003 ◽  
Vol 149 (9) ◽  
pp. 2653-2660 ◽  
Author(s):  
J. M. Tanzer ◽  
L. Grant ◽  
A. Thompson ◽  
L. Li ◽  
J. D. Rogers ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Binding Proteins ◽  
Complex Nature ◽  
Streptococcus Gordonii ◽  
Wild Type ◽  
Carious Lesions ◽  
Specific Pathogen ◽  
Colonization Factors

Streptococcus gordonii produces two α-amylase-binding proteins, AbpA and AbpB, that have been extensively studied in vitro. Little is known, however, about their significance in oral colonization and cariogenicity (virulence). To clarify these issues, weanling specific pathogen-free Osborne-Mendel rats, TAN : SPFOM(OM)BR, were inoculated either with wild-type strains FAS4-S or Challis-S or with strains having isogenic mutations of abpA, abpB, or both, to compare their colonization abilities and persistence on the teeth. Experiments were done with rats fed a sucrose-rich diet containing low amounts of starch or containing only starch. The mutants and wild-types were quantified in vivo and carious lesions were scored. In 11 experiments, S. gordonii was a prolific colonizer of the teeth when rats were fed the sucrose (with low starch)-supplemented diet, often dominating the flora. Sucrose-fed rats had several-fold higher recoveries of inoculants than those eating the sucrose-free, starch-supplemented diet, regardless of inoculant type. The strain defective in AbpB could not colonize teeth of starch-only-eating rats, but could colonize rats if sucrose was added to the diet. Strains defective in AbpA surprisingly colonized better than their wild-types. A double mutant deficient in both AbpA and AbpB (abpA/abpB) colonized like its wild-type. Wild-types FAS4-S and Challis-S had no more than marginal cariogenicity. Notably, in the absence of AbpA, cariogenicity was slightly augmented. Both the rescue of colonization by the AbpB− mutant and the augmentation of colonization by AbpA− mutant in the presence of dietary sucrose suggested additional amylase-binding protein interactions relevant to colonization. Glucosyltransferase activity was greater in mutants defective in abpA and modestly increased in the abpB mutant. It was concluded that AbpB is required for colonization of teeth of starch-eating rats and its deletion is partially masked if rats eat a sucrose-starch diet. AbpA appears to inhibit colonization of the plaque biofilm in vivo. This unexpected effect in vivo may be associated with interaction of AbpA with glucosyltransferase or with other colonization factors of these cells. These data illustrate that the complex nature of the oral environment may not be adequately modelled by in vitro systems.

scholarly journals Antiproliferative Properties of Scandium Exopolysaccharide Complexes on Several Cancer Cell Lines

Marine Drugs ◽  
2021 ◽  
Vol 19 (3) ◽  
pp. 174
Author(s):  
Javier Muñoz-Garcia ◽  
Mattia Mazza ◽  
Cyrille Alliot ◽  
Corinne Sinquin ◽  
Sylvia Colliec-Jouault ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Protein Interactions ◽  
Transmembrane Protein ◽  
Biological Effects ◽  
Growth Factor Receptor ◽  
Predictive Biomarker ◽  
Biological Properties ◽  
Osteosarcoma Cell

Antimetastatic properties on both murine and human osteosarcoma cell lines (POS-1 and KHOS) have been evidenced using exopolysaccharide (EPS) derivatives, produced by Alteromonas infernus bacterium. These derivatives had no significant effect on the cell cycle neither a pro-apoptotic effect on osteosarcoma cells. Based on this observation, these EPSs could be employed as new drug delivery systems for therapeutic uses. A theranostic approach, i.e., combination of a predictive biomarker with a therapeutic agent, has been developed notably by combining with true pair of theranostic radionuclides, such as scandium 47Sc/44Sc. However, it is crucial to ensure that, once complexation is done, the biological properties of the vector remain intact, allowing the molecular tropism of the ligand to recognize its molecular target. It is important to assess if the biological properties of EPS evidenced on osteosarcoma cell lines remain when scandium is complexed to the polymers and can be extended to other cancer cell types. Scandium-EPS complexes were thus tested in vitro on human cell lines: MNNG/HOS osteosarcoma, A375 melanoma, A549 lung adenocarcinoma, U251 glioma, MDA231 breast cancer, and Caco2 colon cancer cells. An xCELLigence Real Cell Time Analysis (RTCA) technology assay was used to monitor for 160 h, the proliferation kinetics of the different cell lines. The tested complexes exhibited an anti-proliferative effect, this effect was more effective compared to EPS alone. This increase of the antiproliferative properties was explained by a change in conformation of EPS complexes due to their polyelectrolyte nature that was induced by complexation. Alterations of both growth factor-receptor signaling, and transmembrane protein interactions could be the principal cause of the antiproliferative effect. These results are very promising and reveal that EPS can be coupled to scandium for improving its biological effects and also suggesting that no major structural modification occurs on the ligand.

scholarly journals High-throughput identification of nuclear envelope protein interactions in Schizosaccharomyces pombe using an arrayed membrane yeast-two hybrid library

2020 ◽  
Author(s):  
Joseph M. Varberg ◽  
Jennifer M. Gardner ◽  
Scott McCroskey ◽  
Snehabala Saravanan ◽  
William D. Bradford ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Nuclear Envelope ◽  
Schizosaccharomyces Pombe ◽  
High Throughput ◽  
Protein Interactions ◽  
Transmembrane Protein ◽  
Integral Membrane Proteins ◽  
Model Organisms ◽  
Yeast Two Hybrid ◽  
Two Hybrid

The nuclear envelope (NE) contains a specialized set of integral membrane proteins that maintain nuclear shape and integrity and influence chromatin organization and gene expression. Advances in proteomics techniques and studies in model organisms have identified hundreds of proteins that localize to the NE. However, the function of many of these proteins at the NE remains unclear, in part due to a lack of understanding of the interactions that these proteins participate in at the NE membrane. To assist in the characterization of NE transmembrane protein interactions we developed an arrayed library of integral and peripheral membrane proteins in the fission yeast Schizosaccharomyces pombe for high-throughput screening using the split-ubiquitin based membrane yeast two hybrid sys-tem. We used this approach to characterize protein interactions for three conserved proteins that localize to the inner nu-clear membrane: Cut11/Ndc1, Lem2, and Ima1/Samp1/NET5. Additionally, we determined how the interaction network for Cut11 is altered in canonical temperature-sensitive cut11 mutants. This library and screening approach is readily applicable to characterizing the interactomes of integral membrane proteins localizing to various subcellular compartments.

scholarly journals Transcriptional repression in Saccharomyces cerevisiae by a SIN3-LexA fusion protein.

1993 ◽  
Vol 13 (3) ◽  
pp. 1805-1814 ◽  
Author(s):  
H Wang ◽  
D J Stillman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Fusion Protein ◽  
Protein Interactions ◽  
Transcriptional Repression ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Predicted Amino Acid Sequence

The yeast SIN3 gene (also known as SDI1, UME4, RPD1, and GAM2) has been identified as a transcriptional regulator. Previous work has led to the suggestion that SIN3 regulates transcription via interactions with DNA-binding proteins. Although the SIN3 protein is located in the nucleus, it does not bind directly to DNA in vitro. We have expressed a LexA-SIN3 fusion protein in Saccharomyces cerevisiae and show that this fusion protein represses transcription from heterologous promoters that contain lexA operators. The predicted amino acid sequence of the SIN3 protein contains four copies of a paired amphipathic helix (PAH) motif, similar to motifs found in HLH (helix-loop-helix) and TPR (tetratricopeptide repeat) proteins, and these motifs are proposed to be involved in protein-protein interactions. We have conducted a deletion analysis of the SIN3 gene and show that the PAH motifs are required for SIN3 activity. Additionally, the C-terminal region of the SIN3 protein is sufficient for repression activity in a LexA-SIN3 fusion, and deletion of a PAH motif in this region inactivates this repression activity. A model is presented in which SIN3 recognizes specific DNA-binding proteins in vivo in order to repress transcription.

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