scholarly journals Saccharomyces cerevisiae Rbg1 Protein and Its Binding Partner Gir2 Interact on Polyribosomes with Gcn1

2009 ◽  
Vol 8 (7) ◽  
pp. 1061-1071 ◽  
Author(s):  
P. K. Wout ◽  
E. Sattlegger ◽  
S. M. Sullivan ◽  
J. R. Maddock
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Yeast Two Hybrid ◽  
Uncharacterized Protein ◽  
General Amino Acid Control ◽  
Binding Partner ◽  
Inhibition Of Growth ◽  
Fractionation Pattern ◽  
Two Hybrid ◽  
Hybrid Screening

ABSTRACT Rbg1 is a previously uncharacterized protein of Saccharomyces cerevisiae belonging to the Obg/CgtA subfamily of GTP-binding proteins whose members are involved in ribosome function in both prokaryotes and eukaryotes. We show here that Rbg1 specifically associates with translating ribosomes. In addition, in this study proteins were identified that interact with Rbg1 by yeast two-hybrid screening and include Tma46, Ygr250c, Yap1, and Gir2. Gir2 contains a GI (Gcn2 and Impact) domain similar to that of Gcn2, an essential factor of the general amino acid control pathway required for overcoming amino acid shortage. Interestingly, we found that Gir2, like Gcn2, interacts with Gcn1 through its GI domain, and overexpression of Gir2, under conditions mimicking amino acid starvation, resulted in inhibition of growth that could be reversed by Gcn2 co-overexpression. Moreover, we found that Gir2 also cofractionated with polyribosomes, and this fractionation pattern was partially dependent on the presence of Gcn1. Based on these findings, we conclude that Rbg1 and its interacting partner Gir2 associate with ribosomes, and their possible biological roles are discussed.

scholarly journals Structural Glycoprotein E2 of Classical Swine Fever Virus Interacts with Host Protein Dynactin Subunit 6 (DCTN6) during the Virus Infectious Cycle

2019 ◽  
Vol 94 (1) ◽  
Author(s):  
M. V. Borca ◽  
E. A. Vuono ◽  
E. Ramirez-Medina ◽  
P. Azzinaro ◽  
K. A. Berggren ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Interaction ◽  
Hybrid Approach ◽  
Classical Swine Fever ◽  
Host Protein ◽  
Yeast Two Hybrid ◽  
Protein Protein Interaction ◽  
Viral Virulence ◽  
Glycoprotein E2 ◽  
Two Hybrid

ABSTRACT The E2 protein in classical swine fever (CSF) virus (CSFV) is the major virus structural glycoprotein and is an essential component of the viral particle. E2 has been shown to be involved in several functions, including virus adsorption, induction of protective immunity, and virulence in swine. Using the yeast two-hybrid system, we previously identified a swine host protein, dynactin subunit 6 (DCTN6) (a component of the cell dynactin complex), as a specific binding partner for E2. We confirmed the interaction between DCTN6 and E2 proteins in CSFV-infected swine cells by using two additional independent methodologies, i.e., coimmunoprecipitation and proximity ligation assays. E2 residues critical for mediating the protein-protein interaction with DCTN6 were mapped by a reverse yeast two-hybrid approach using a randomly mutated E2 library. A recombinant CSFV mutant, E2ΔDCTN6v, harboring specific substitutions in those critical residues was developed to assess the importance of the E2-DCTN6 protein-protein interaction for virus replication and virulence in swine. CSFV E2ΔDCTN6v showed reduced replication, compared with the parental virus, in an established swine cell line (SK6) and in primary swine macrophage cultures. Remarkably, animals infected with CSFV E2ΔDCTN6v remained clinically normal during the 21-day observation period, which suggests that the ability of CSFV E2 to bind host DCTN6 protein efficiently during infection may play a role in viral virulence. IMPORTANCE Structural glycoprotein E2 is an important component of CSFV due to its involvement in many virus activities, particularly virus-host interactions. Here, we present the description and characterization of the protein-protein interaction between E2 and the swine host protein DCTN6 during virus infection. The E2 amino acid residues mediating the interaction with DCTN6 were also identified. A recombinant CSFV harboring mutations disrupting the E2-DCTN6 interaction was created. The effect of disrupting the E2-DCTN6 protein-protein interaction was studied using reverse genetics. It was shown that the same amino acid substitutions that abrogated the E2-DCTN6 interaction in vitro constituted a critical factor in viral virulence in the natural host, domestic swine. This highlights the potential importance of the E2-DCTN6 protein-protein interaction in CSFV virulence and provides possible mechanisms of virus attenuation for the development of improved CSF vaccines.

scholarly journals The Zn-finger of Saccharomyces cerevisiae Rad18 and its adjacent region mediate interaction with Rad5

2021 ◽  
Author(s):  
Orsolya Frittmann ◽  
Vamsi K Gali ◽  
Miklos Halmai ◽  
Robert Toth ◽  
Zsuzsanna Gyorfy ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Ubiquitin Ligase ◽  
Dna Lesions ◽  
Template Switching ◽  
Adjacent Region ◽  
Yeast Two Hybrid ◽  
Replication Complex ◽  
Two Hybrid

Abstract DNA damages that hinder the movement of the replication complex can ultimately lead to cell death. To avoid that, cells possess several DNA damage bypass mechanisms. The Rad18 ubiquitin ligase controls error-free and mutagenic pathways that help the replication complex to bypass DNA lesions by monoubiquitylating PCNA at stalled replication forks. In Saccharomyces cerevisiae, two of the Rad18 governed pathways are activated by monoubiquitylated PCNA and they involve translesion synthesis polymerases, whereas a third pathway needs subsequent polyubiquitylation of the same PCNA residue by another ubiquitin ligase the Rad5 protein, and it employs template switching. The goal of this study was to dissect the regulatory role of the multidomain Rad18 in DNA damage bypass using a structure-function based approach. Investigating deletion and point mutant RAD18 variants in yeast genetic and yeast two-hybrid assays we show that the Zn-finger of Rad18 mediates its interaction with Rad5, and the N-terminal adjacent region is also necessary for Rad5 binding. Moreover, results of the yeast two-hybrid and in vivo ubiquitylation experiments raise the possibility that direct interaction between Rad18 and Rad5 might not be necessary for the function of the Rad5 dependent pathway. The presented data also reveal that yeast Rad18 uses different domains to mediate its association with itself and with Rad5. Our results contribute to better understanding of the complex machinery of DNA damage bypass pathways.

scholarly journals Two LIM Domain Proteins and UNC-96 Link UNC-97/PINCH to Myosin Thick Filaments in Caenorhabditis elegans Muscle

2007 ◽  
Vol 18 (11) ◽  
pp. 4317-4326 ◽  
Author(s):  
Hiroshi Qadota ◽  
Kristina B. Mercer ◽  
Rachel K. Miller ◽  
Kozo Kaibuchi ◽  
Guy M. Benian
Keyword(s):  
Caenorhabditis Elegans ◽  
Binding Assays ◽  
Lim Domain ◽  
Yeast Two Hybrid ◽  
Lim Domains ◽  
Thick Filaments ◽  
Vitro Binding ◽  
Two Hybrid ◽  
Hybrid Screening

By yeast two-hybrid screening, we found three novel interactors (UNC-95, LIM-8, and LIM-9) for UNC-97/PINCH in Caenorhabditis elegans. All three proteins contain LIM domains that are required for binding. Among the three interactors, LIM-8 and LIM-9 also bind to UNC-96, a component of sarcomeric M-lines. UNC-96 and LIM-8 also bind to the C-terminal portion of a myosin heavy chain (MHC), MHC A, which resides in the middle of thick filaments in the proximity of M-lines. All interactions identified by yeast two-hybrid assays were confirmed by in vitro binding assays using purified proteins. All three novel UNC-97 interactors are expressed in body wall muscle and by antibodies localize to M-lines. Either a decreased or an increased dosage of UNC-96 results in disorganization of thick filaments. Our previous studies showed that UNC-98, a C2H2 Zn finger protein, acts as a linkage between UNC-97, an integrin-associated protein, and MHC A in myosin thick filaments. In this study, we demonstrate another mechanism by which this linkage occurs: from UNC-97 through LIM-8 or LIM-9/UNC-96 to myosin.

scholarly journals The Saccharomyces cerevisiae 14-3-3 protein Bmh2 is required for regulation of the phosphorylation status of Fin1, a novel intermediate filament protein

2002 ◽  
Vol 365 (1) ◽  
pp. 51-56 ◽  
Author(s):  
Isabel MAYORDOMO ◽  
Pascual SANZ
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Regulation ◽  
Intermediate Filament ◽  
Protein Phosphatase ◽  
Catalytic Subunit ◽  
Protein Phosphatase 1 ◽  
Intermediate Filament Protein ◽  
Filament Protein ◽  
Two Hybrid ◽  
Hybrid Screening

In order to identify proteins that interact with Bmh2, a yeast member of the 14-3-3 protein family, we performed a two-hybrid screening using LexA-Bmh2 as bait. We identified Fin1, a novel intermediate filament protein, as the protein that showed the highest degree of interaction. We also identified components of the vesicular transport machinery such as Gic2 and Msb3, proteins involved in transcriptional regulation such as Mbf1, Gcr2 and Reg2, and a variety of other different proteins (Ppt1, Lre1, Rps0A and Ylr177w). We studied the interaction between Bmh2 and Fin1 in more detail and found that Bmh2 only interacted with phosphorylated forms of Fin1. In addition, we showed that Glc7, the catalytic subunit of the protein phosphatase 1 complex, was also able to interact with Fin1.

Specific inhibition of transcriptional activity of the constitutive androstane receptor (CAR) by the splicing factor SF3a3

2008 ◽  
Vol 389 (10) ◽  
Author(s):  
Hye Jin Yun ◽  
Jungsun Kwon ◽  
Wongi Seol
Keyword(s):  
Transcriptional Activity ◽  
Constitutive Androstane Receptor ◽  
Splicing Factor ◽  
Yeast Two Hybrid ◽  
Interacting Protein ◽  
Functional Studies ◽  
Reporter Activity ◽  
Inhibitory Function ◽  
Two Hybrid ◽  
Hybrid Screening

Abstract The constitutive androstane receptor (CAR) is a member of the nuclear receptor superfamily and plays an important role in the degradation of xenobiotics in the liver. Using yeast two-hybrid screening, we identified SF3a3, a 60-kDa subunit of the splicing factor 3a complex, as a specific CAR-interacting protein. We further confirmed their interaction by both co-immunoprecipitation and GST pull-down assay. Functional studies showed that overexpression of SF3a3 inhibited the reporter activity driven by a promoter containing CAR binding sequences by up to 50%, whereas reduced expression of SF3a3 activated the same reporter activity by approximately three-fold. The inhibitory function of SF3a3 is independent of the presence of TCPOBOP, a CAR ligand. These data suggest that SF3a3 functions as a co-repressor of CAR transcriptional activity, in addition to its canonical function.

Yeast Two-Hybrid Screening for Proteins that Interact with Nuclear Hormone Receptors

2003 ◽  
pp. 227-248 ◽  
Author(s):  
Bertrand Le Douarin ◽  
David M. Heery ◽  
Claudine Gaudon ◽  
Elmar vom Baur ◽  
Régine Losson
Keyword(s):  
Hormone Receptors ◽  
Nuclear Hormone Receptors ◽  
Yeast Two Hybrid ◽  
Two Hybrid ◽  
Hybrid Screening

Abstract 3600: HspA12B Promotes Angiogenesis through Suppressing AKAP12 and Up-Regulating VEGF Pathway

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Rebecca J Steagall ◽  
Fang Hua ◽  
Mahesh Thirunazukarasu ◽  
Lijun Zhan ◽  
Chuanfu Li ◽  
...  
Keyword(s):  
Cancer Metastasis ◽  
Yeast Two Hybrid ◽  
Vegf Expression ◽  
Tubule Formation ◽  
Interacting Protein ◽  
Over Expression ◽  
Vegf Pathway ◽  
Two Hybrid ◽  
Hybrid Screening

We have previously shown that HspA12B, a member of HspA70 family subfamily 12, is a novel angiogenesis regulator that is preferentially expressed in endothelial cells (ECs) and required for angiogenesis in vitro . The mechanism by which HspA12B regulates angiogenesis, however, is unknown. In this study we identified AKAP12/SSeCKS as a HSPA12B-interacting protein through a yeast two-hybrid screening and confirmed the interaction by co-immunoprecipitation and co-localization. We observed that HspA12B negatively regulated the expression of AKAP12/SSeCKS, a cancer metastasis repressor that inhibits VEGF expression and angiogen-esis. In HUVEC, HspA12B knockdown increased AKAP12 levels, decreased VEGF by more than 75%, and down-regulated Akt and pAkt; whereas HspA12B over expression decreased AKAP12 and more than doubled VEGF levels. We further identified a 32-AA domain in AKAP12 that was capable of interacting with HspA12B. Overexpression of this 32-AA domain in HUVEC disrupted the HspA12B-AKAP12 interaction and decreased VEGF expression by more than 70%, suggesting the importance of HspA12B-AKAP12 interaction in regulating VEGF. We also observed that HspA12B expression was increased more than 2 folds in ECs by hypoxia or shearing stress, and induced in ischemic rat heart. Inhibition of HspA12B abolished hypoxia-induced tubule formation. Adeno-HspA12B promoted angiogenesis in DIVAA assay. We concluded that this is the first evidence that HspA12B promotes angiogenesis through regulating VEGF by way of suppressing AKAP12. Our finding is the first example of an EC-specific molecular chaperone acting as the regulator of angiogenesis.

PSD-93 mediate the dialogue between neuron and microglia and facilitate acute ischemic stroke by binding 357-395 amino acid sequence of CX3CL1

2020 ◽  
Author(s):  
Qingxiu Zhang ◽  
Lei He ◽  
Mo Chen ◽  
Hui Yang ◽  
Xiaowei Cao ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Ischemia Reperfusion ◽  
Yeast Two Hybrid ◽  
Neuron Death ◽  
Specific Amino Acid ◽  
Ischemic Brain ◽  
Two Hybrid

Abstract Background: Our previous experiments demonstrated that PSD-93 mediates glutamate excitotoxicity induced by ischemic brain injury, which promotes the release of inflammatory cytokines in early ischemic brain injury by activating the NMDA receptor. Glutamate activity is the key to neuronal excitatory toxicity and microglial cell inflammatory response in the joints. However, the underlying mechanisms of how does PSD-93 mediate the dialogue between neurons and microglia in the postsynaptic dense region remain elusive. And CX3 chemokine ligand 1 (CX3CL1) is a chemokine that is specifically expressed in neurons. Its only receptor CX3CR1 is highly expressed in microglia and its main forms are membrane binding and soluble. In this study, we aim to clarify the specific amino acid sequence of the binding of psd-93 and CX3CL1 and investigate role of PSD-93 on regulating the crosstalk between neuron and microglia in acute ischemic stroke. Methods: In this study, male C57BL/6 mice aged 8-12 weeks and weighted 22-26g were applied with Middle Cerebral Artery Occlusion (MCAO) model and randomly divided into different groups. Firstly, co-immunoprecipitation and immunoblotting were used to detect the binding of PSD-93 and CX3CL1 at different time points 3h, 6h, 12h 24h, 48h and 72h following cerebral ischemic/reperfusion. Meanwhile, ELISA was used to investigate the expression of soluble CX3CL1 at the same time points to confirm the relationship between of the expression of soluble CX3CL1 and the combination of PSD-93 and CX3CL1. Secondly, two bait plasmids pSos-PSD-93-full length, pSos-CX3CL1-full length and five mutant plasmids: pMyr-PSD-93-mut1, pMyr-PSD-93-mut2, pMyr-PSD-93-mut3, pMyr-PSD-93-mut4, and pMyr-CX3CL1-mut, were constructed and used a yeast two-hybrid system to screen and identify positive clones and to determine the sequence in which the two proteins bind to each other. Thirdly, the proteins corresponding to the three positive clones obtained in the yeast two-hybrid experiment were used to construct plasmids for transfection of eukaryotic cells and the protein expression binding was verified again by in vitro co-immunoprecipitation. Finally, a specific fusion small peptide Tat-CX3CL1 were designed according to above experiment to inhibit the integration of PSD-93 and CX3CL1 and to explore their role on neuron death following reperfusion. Results: We found that the binding capacity of PSD-93 and CX3CL1 proteins peaked at 6h after ischemia/reperfusion and then decreased gradually. The specific amino acid sequence of PSD-93 and CX3CL1 binding was obtained by yeast double hybridization and in vitro immunoprecipitation. We identified that their binding sites are located in the 420-535 amino acid sequence of PSD-93 and 357-395 amino acid sequence of CX3CL1. And a specific fusion small peptide Tat-CX3CL1 (357-395aa) were designed to inhibit the integration of PSD-93 and CX3CL1 and perform neuroprotection on neuron death following reperfusion. Conclusions: Our results suggest that PSD-93 promotes the formation of its soluble form by binding to CX3CL1, which is recruited to the surface of microglia to bind to CX3CR1, thereby activating microglia to initiate inflammation. Thus, specific blockade of PSD-93-CX3CL1 coupling can reduce ischemia-reperfusion induced neuronal cell death, which provide a new target to treat ischemic stroke.

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