scholarly journals Interactions between the bud emergence proteins Bem1p and Bem2p and Rho-type GTPases in yeast.

1994 ◽  
Vol 127 (5) ◽  
pp. 1395-1406 ◽  
Author(s):  
J Peterson ◽  
Y Zheng ◽  
L Bender ◽  
A Myers ◽  
R Cerione ◽  
...  
Keyword(s):  
Synthetic Lethality ◽  
Protein Domain ◽  
Nucleotide Exchange ◽  
Phenotypic Data ◽  
Synthetic Lethal ◽  
Bud Emergence ◽  
Tightly Coupled ◽  
Two Hybrid ◽  
Cortical Cytoskeleton

The SH3 domain-containing protein Bem1p is needed for normal bud emergence and mating projection formation, two processes that require asymmetric reorganizations of the cortical cytoskeleton in Saccharomyces cerevisiae. To identify proteins that functionally and/or physically interact with Bem1p, we screened for mutations that display synthetic lethality with a mutant allele of the BEM1 gene and for genes whose products display two-hybrid interactions with the Bem1 protein. CDC24, which is required for bud emergence and encodes a GEF (guanine-nucleotide exchange factor) for the essential Rho-type GTPase Cdc42p, was identified during both screens. The COOH-terminal 75 amino acids of Cdc24p, outside of the GEF domain, can interact with a portion of Bem1p that lacks both SH3 domains. Bacterially expressed Cdc24p and Bem1p bind to each other in vitro, indicating that no other yeast proteins are required for this interaction. The most frequently identified gene that arose from the bem1 synthetic-lethal screen was the bud-emergence gene BEM2 (Bender and Pringle. 1991. Mol. Cell Biol. 11:1295-1395), which is allelic with IPL2 (increase in ploidy; Chan and Botstein, 1993. Genetics. 135:677-691). Here we show that Bem2p contains a GAP (GTPase-activating protein) domain for Rho-type GTPases, and that this portion of Bem2p can stimulate in vitro the GTPase activity of Rho1p, a second essential yeast Rho-type GTPase. Cells deleted for BEM2 become large and multinucleate. These and other genetic, two-hybrid, biochemical, and phenotypic data suggest that multiple Rho-type GTPases control the reorganization of the cortical cytoskeleton in yeast and that the functions of these GTPases are tightly coupled. Also, these findings raise the possibility that Bem1p may regulate or be a target of action of one or more of these GTPases.

scholarly journals Identification of the bud emergence gene BEM4 and its interactions with rho-type GTPases in Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (8) ◽  
pp. 4387-4395 ◽  
Author(s):  
D Mack ◽  
K Nishimura ◽  
B K Dennehey ◽  
T Arbogast ◽  
J Parkinson ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Synthetic Lethality ◽  
Cell Polarization ◽  
Growth Defect ◽  
Nucleotide Exchange ◽  
Loss Of Function ◽  
Multicopy Suppressor ◽  
Bud Emergence ◽  
Multicopy Suppressors ◽  
Multiple Copies

The Rho-type GTPase Cdc42p is required for cell polarization and bud emergence in Saccharomyces cerevisiae. To identify genes whose functions are linked to CDC42, we screened for (i) multicopy suppressors of a Ts- cdc42 mutant, (ii) mutants that require multiple copies of CDC42 for survival, and (iii) mutations that display synthetic lethality with a partial-loss-of-function allele of CDC24, which encodes a guanine nucleotide exchange factor for Cdc42p. In all three screens, we identified a new gene, BEM4. Cells from which BEM4 was deleted were inviable at 37 degrees C. These cells became unbudded, large, and round, consistent with a model in which Bem4p acts together with Cdc42p in polarity establishment and bud emergence. In some strains, the ability of CDC42 to serve as a multicopy suppressor of the Ts- growth defect of deltabem4 cells required co-overexpression of Rho1p, which is an essential Rho-type GTPase necessary for cell wall integrity. This finding suggests that Bem4p also affects Rho1p function. Bem4p displayed two-hybrid interactions with Cdc42p, Rho1p, and two of the three other known yeast Rho-type GTPases, suggesting that Bem4p can interact with multiple Rho-type GTPases. Models for the role of Bem4p include that it serves as a chaperone or modulates the interaction of these GTPases with one or more of their targets or regulators.

scholarly journals Mapping the human translation elongation factor eEF1H complex using the yeast two-hybrid system

2002 ◽  
Vol 365 (3) ◽  
pp. 669-676 ◽  
Author(s):  
Francisco MANSILLA ◽  
Irene FRIIS ◽  
Mandana JADIDI ◽  
Karen M. NIELSEN ◽  
Brian F.C. CLARK ◽  
...  
Keyword(s):  
Elongation Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Translation Elongation Factor ◽  
Yeast Two Hybrid ◽  
Translation Elongation ◽  
Guanine Nucleotide Exchange ◽  
Yeast Two Hybrid System ◽  
Two Hybrid

In eukaryotes, the eukaryotic translation elongation factor eEF1A responsible for transporting amino-acylated tRNA to the ribosome forms a higher-order complex, eEF1H, with its guanine-nucleotide-exchange factor eEF1B. In metazoans, eEF1B consists of three subunits: eEF1Bα, eEF1Bβ and eEF1Bγ. The first two subunits possess the nucleotide-exchange activity, whereas the role of the last remains poorly defined. In mammals, two active tissue-specific isoforms of eEF1A have been identified. The reason for this pattern of differential expression is unknown. Several models on the basis of in vitro experiments have been proposed for the macromolecular organization of the eEF1H complex. However, these models differ in various aspects. This might be due to the difficulties of handling, particularly the eEF1Bβ and eEF1Bγ subunits in vitro. Here, the human eEF1H complex is for the first time mapped using the yeast two-hybrid system, which is a powerful in vivo technique for analysing protein—protein interactions. The following complexes were observed: eEF1A1:eEF1Bα, eEF1A1:eEF1Bβ, eEF1Bβ:eEF1Bβ, eEF1Bα:eEF1Bγ, eEF1Bβ:eEF1Bγ and eEF1Bα:eEF1Bγ:eEF1Bβ, where the last was observed using a three-hybrid approach. Surprisingly, eEF1A2 showed no or only little affinity for the guanine-nucleotide-exchange factors. Truncated versions of the subunits of eEF1B were used to orientate these subunits within the resulting model. The model unit is a pentamer composed of two molecules of eEF1A, each interacting with either eEF1Bα or eEF1Bβ held together by eEF1Bγ. These units can dimerize via eEF1Bβ. Our model is compared with other models, and structural as well as functional aspects of the model are discussed.

scholarly journals Synthetic lethality-based prediction of anti-SARS-CoV-2 targets

2021 ◽  
Author(s):  
Lipika R. Pal ◽  
Kuoyuan Cheng ◽  
Nishanth U Nair ◽  
Laura Martin-Sancho ◽  
Sanju Sinha ◽  
...  
Keyword(s):  
Drug Targets ◽  
Synthetic Lethality ◽  
Host Cells ◽  
Synthetic Lethal ◽  
Viral Propagation ◽  
In Vivo Testing ◽  
Altered Gene ◽  
Host Genes

Novel strategies are needed to identify drug targets and treatments for the COVID-19 pandemic. The altered gene expression of virus-infected host cells provides an opportunity to specifically inhibit viral propagation via targeting the synthetic lethal (SL) partners of such altered host genes. Pursuing this antiviral strategy, here we comprehensively analyzed multiple in vitro and in vivo bulk and single-cell RNA-sequencing datasets of SARS-CoV-2 infection to predict clinically relevant candidate antiviral targets that are SL with altered host genes. The predicted SL-based targets are highly enriched for infected cell inhibiting genes reported in four SARS-CoV-2 CRISPR-Cas9 genome-wide genetic screens. Integrating our predictions with the results of these screens, we further selected a focused subset of 26 genes that we experimentally tested in a targeted siRNA screen using human Caco-2 cells. Notably, as predicted, knocking down these targets reduced viral replication and cell viability only under the infected condition without harming non-infected cells. Our results are made publicly available, to facilitate their in vivo testing and further validation.

Synthetic-lethal interactions between ATR inhibition and functional genetic and proteomic biomarkers to predict responses in endometriosis-associated cancers.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e18000-e18000
Author(s):  
Oren Gilad ◽  
Dansu Li ◽  
Erin George ◽  
Rakesh Chettier ◽  
Fiona Simpkins ◽  
...  
Keyword(s):  
Synthetic Lethality ◽  
In Vivo Studies ◽  
Multiple Cancer ◽  
Driver Genes ◽  
Synthetic Lethal ◽  
Cancer Driver ◽  
Synthetic Lethal Interactions ◽  
Synthetically Lethal

e18000 Background: Endometriosis is a common gynecologic disorder proven to be a precursor to several cancer types. We developed a potent and selective inhibitor (ATRN-119) of a critical DNA damage response (DDR) protein kinase: the ataxia telangiectasia and Rad3-related protein (ATR). Treatment with ATRN-119 is synthetically lethal with multiple cancer-associated changes in DDR pathways, representing a new and effective strategy to treat cancer. The objective of this study is to evaluate the overlap of DDR genes that respond to ATRN-119 and those mutated in endometriosis. Methods: We sequenced the exomes of 2,932 unrelated women with surgically-confirmed endometriosis (GERMLINE) and 274 tissue blocks containing endometriosis lesions (LESION). DNA was extracted using standard methods. Missense and truncation variants were analyzed. These data were compared to analysis of a whole proteome screen for factors that respond to exposure to ATRN-119 and may influence responsiveness to treatment. Factors observed in both methods were considered high-priority biomarker candidates and were experimentally tested for synthetic lethality with ATRN-119 treatment. Results: Analysis of endometriosis patients found 89% of the LESION samples had 2 or more DDR mutations vs 83% of the GERMLINE samples. There is an excess of DDR mutations per sample in LESION (5.5 mutations) vs GERMLINE (3.89 mutations) [p = 4.66x10-6, Mann Whitney test]. In parallel, we identified 92 genes as protein responders to ATRN-119 treatment. Mutations in 21 of these 92 genes show nominal association with surgical endometriosis (p < 0.05). However, of these responsive genes, 18 are known TIER 1 cancer-driver genes and well-characterized mutations were found in three dominant genes in the LESION tissue (ATM, DDB1, and ARID1A). Overall 20% of the patients who’s LESION we examined subsequently developed an endometriosis-associated cancer. Both in vitro and in vivo studies confirmed synthetic-lethal interactions between ATRN-119 treatment and alteration of these genes. Conclusions: The overlap between DDR genes responding to ATRN-119 and those mutated in endometriosis-associated cancer suggest that genetic markers underlying response and resistance will be critical to extend the use of these drugs while increasing efficacy and minimizing toxicities. Furthermore, our data support the inclusion of endometriosis-associated cancer patients in planned ATRN-119 clinical trials.

scholarly journals The Rice Tungro Bacilliform Virus Gene II Product Interacts with the Coat Protein Domain of the Viral Gene III Polyprotein

2000 ◽  
Vol 74 (5) ◽  
pp. 2073-2083 ◽  
Author(s):  
Etienne Herzog ◽  
Orlene Guerra-Peraza ◽  
Thomas Hohn
Keyword(s):  
Coat Protein ◽  
Protein Domain ◽  
Rice Tungro Bacilliform Virus ◽  
Viral Gene ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Genes Encoding ◽  
Two Hybrid

ABSTRACT Rice tungro bacilliform virus (RTBV) is a plant pararetrovirus whose DNA genome contains four genes encoding three proteins and a large polyprotein. The function of most of the viral proteins is still unknown. To investigate the role of the gene II product (P2), we searched for interactions between this protein and other RTBV proteins. P2 was shown to interact with the coat protein (CP) domain of the viral gene III polyprotein (P3) both in the yeast two-hybrid system and in vitro. Domains involved in the P2-CP association have been identified and mapped on both proteins. To determine the importance of this interaction for viral multiplication, the infectivity of RTBV gene II mutants was investigated by agroinoculation of rice plants. The results showed that virus viability correlates with the ability of P2 to interact with the CP domain of P3. This study suggests that P2 could participate in RTBV capsid assembly.

scholarly journals New Synthetic Lethality Re-Sensitizing Platinum-Refractory Cancer Cells to Cisplatin In Vitro: The Rationale to Co-Use PARP and ATM Inhibitors

2021 ◽  
Vol 22 (24) ◽  
pp. 13324
Author(s):  
Watson P. Folk ◽  
Alpana Kumari ◽  
Tetsushi Iwasaki ◽  
Erica K. Cassimere ◽  
Slovénie Pyndiah ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Cancer Cells ◽  
Cisplatin Resistance ◽  
Synthetic Lethality ◽  
Lethal Effect ◽  
Parp Inhibition ◽  
Synthetic Lethal ◽  
Cisplatin Sensitivity ◽  
Platinum Refractory

The pro-apoptotic tumor suppressor BIN1 inhibits the activities of the neoplastic transcription factor MYC, poly (ADP-ribose) polymerase-1 (PARP1), and ATM Ser/Thr kinase (ATM) by separate mechanisms. Although BIN1 deficits increase cancer-cell resistance to DNA-damaging chemotherapeutics, such as cisplatin, it is not fully understood when BIN1 deficiency occurs and how it provokes cisplatin resistance. Here, we report that the coordinated actions of MYC, PARP1, and ATM assist cancer cells in acquiring cisplatin resistance by BIN1 deficits. Forced BIN1 depletion compromised cisplatin sensitivity irrespective of Ser15-phosphorylated, pro-apoptotic TP53 tumor suppressor. The BIN1 deficit facilitated ATM to phosphorylate the DNA-damage-response (DDR) effectors, including MDC1. Consequently, another DDR protein, RNF8, bound to ATM-phosphorylated MDC1 and protected MDC1 from caspase-3-dependent proteolytic cleavage to hinder cisplatin sensitivity. Of note, long-term and repeated exposure to cisplatin naturally recapitulated the BIN1 loss and accompanying RNF8-dependent cisplatin resistance. Simultaneously, endogenous MYC was remarkably activated by PARP1, thereby repressing the BIN1 promoter, whereas PARP inhibition abolished the hyperactivated MYC-dependent BIN1 suppression and restored cisplatin sensitivity. Since the BIN1 gene rarely mutates in human cancers, our results suggest that simultaneous inhibition of PARP1 and ATM provokes a new BRCAness-independent synthetic lethal effect and ultimately re-establishes cisplatin sensitivity even in platinum-refractory cancer cells.

scholarly journals Arx1 Is a Nuclear Export Receptor for the 60S Ribosomal Subunit in Yeast

2008 ◽  
Vol 19 (2) ◽  
pp. 735-744 ◽  
Author(s):  
Nai-Jung Hung ◽  
Kai-Yin Lo ◽  
Samir S. Patel ◽  
Kara Helmke ◽  
Arlen W. Johnson
Keyword(s):  
Nuclear Export ◽  
Ribosomal Subunit ◽  
In Vitro Assays ◽  
Dependent Manner ◽  
Yeast Two Hybrid ◽  
Synthetic Lethal ◽  
Nuclear Export Receptor ◽  
Export Receptors ◽  
Two Hybrid

We previously showed that nuclear export of the large (60S) ribosomal subunit relies on Nmd3 in a Crm1-dependent manner. Recently the general mRNA export factor, the Mtr2/Mex67 heterodimer, was shown to act as an export receptor in parallel with Crm1. These observations raise the possibility that nuclear export of the 60S subunit in Saccharomyces cerevisiae requires multiple export receptors. Here, we show that the previously characterized 60S subunit biogenesis factor, Arx1, also acts as an export receptor for the 60S subunit. We found that deletion of ARX1 was synthetic lethal with nmd3 and mtr2 mutants and was synthetic sick with several nucleoporin mutants. Deletion of ARX1 led to accumulation of pre-60S particles in the nucleus that were enriched for Nmd3, Crm1, Mex67, and Mtr2, suggesting that in the absence of Arx1, 60S export is impaired even though the subunit is loaded with export receptors. Finally, Arx1 interacted with several nucleoporins in yeast two-hybrid as well as in vitro assays. These results show that Arx1 can directly bridge the interaction between the pre-60S particle and the NPC and thus is a third export receptor for the 60S subunit in yeast.

scholarly journals The Identification of Wos2, a p23 Homologue That Interacts With Wee1 and Cdc2 in the Mitotic Control of Fission Yeasts

Genetics ◽  
1999 ◽  
Vol 153 (4) ◽  
pp. 1561-1572 ◽  
Author(s):  
Manuel J Muñoz ◽  
Eduardo R Bejarano ◽  
Rafael R Daga ◽  
Juan Jimenez
Keyword(s):  
Fission Yeast ◽  
Synthetic Lethality ◽  
In Vitro Assays ◽  
Proliferating Cells ◽  
Mutant Proteins ◽  
Synthetic Lethal ◽  
Multicopy Suppressors ◽  
Hsp90 Activity ◽  
Vitro Analysis

Abstract The Wee1 kinase inhibits entry into mitosis by phosphorylation of the Cdc2 kinase. Searching for multicopy suppressors that abolish this inhibition in the fission yeast, we have identified a novel gene, here named wos2, encoding a protein with significant homology to human p23, an Hsp90-associated cochaperone. The deletion mutant has a modest phenotype, being heat-shock sensitive. Using antibodies raised against bacterially produced protein, we determined that Wos2 is very abundant, ubiquitously distributed in the yeast cell, and its expression dropped drastically as cells entered into early stationary phase, indicating that its function is associated with cell proliferation. In proliferating cells, the amount of Wos2 protein was not subjected to cell cycle regulation. However, in vitro assays demonstrated that this Hsp90 cochaperone is potentially regulated by phosphorylation. In addition to suppressing Wee1 activity, overproduction of Wos2 displayed synthetic lethality with Cdc2 mutant proteins, indicating that this Hsp90 cochaperone functionally interacts with Cdc2. The level of Cdc2 protein and its associated H1 kinase activity under synthetic lethal conditions suggested a regulatory role for this Wos2-Cdc2 interaction. Hsp90 complexes are required for CDK regulation; the synergy found between the excess of Wos2 and a deficiency in Hsp90 activity suggests that Wos2 could specifically interfere with the Hsp90-dependent regulation of Cdc2. In vitro analysis indicated that the above genetic interactions could take place by physical association of Wos2 with the single CDK complex of the fission yeast. Expression of the budding yeast p23 protein (encoded by the SBA1 gene) in the fission yeast indicated that Wos2 and Sba1 are functionally exchangeable and therefore that properties described here for Wos2 could be of wide significance in understanding the biological function of cochaperone p23 in eukaryotic cells.

scholarly journals Characterization of Synthetic-Lethal Mutants Reveals a Role for the Saccharomyces cerevisiae Guanine-Nucleotide Exchange Factor Cdc24p in Vacuole Function and Na+ Tolerance

Genetics ◽  
1997 ◽  
Vol 147 (1) ◽  
pp. 43-55 ◽  
Author(s):  
William H White ◽  
Douglas I Johnson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Polarity ◽  
Synthetic Lethality ◽  
Guanine Nucleotide Exchange Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Synthetic Lethal ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

Abstract Cdc24p is the guanine-nucleotide exchange factor for the Cdc42p GTPase, which controls cell polarity in Saccharomyces cerevisiae. To identify new genes that may affect cell polarity, we characterized six UV-induced csl (CDC24  synthetic-lethal) mutants that exhibited synthetic-lethality with cdc24-4ts at 23°. Five mutants were not complemented by plasmid-borne CDC42, RSRI, BUD5, BEM1, BEM2, BEM3 or CLA4 genes, which are known to play a role in cell polarity. The csl3 mutant displayed phenotypes similar to those observed with calcium-sensitive, Pet−  vma mutants defective in vacuole function. CSL5 was allelic to VMA5, the vacuolar H+-ATPase subunit C, and one third of cs15 cdc24-4ts cells were elongated or had misshapen buds. A cdc24-4ts Δvma5::LEU2 double mutant did not exhibit synthetic lethality, suggesting that the csl5/vma5 cdc24-4ts synthetic-lethality was not simply due to altered vacuole function. The cdc24-4ts mutant, like Δvma5::LEU2 and cs13 mutants, was sensitive to high levels of Ca2+ as well as Na+ in the growth media, which did not appear to be a result of a fragile cell wall because the phenotypes were not remedied by 1 m sorbitol. Our results indicated that Cdc24p was required in one V-ATPase mutant and another mutant affecting vacuole morphology, and also implicated Cdc24p in Na+ tolerance.

scholarly journals EZH2 Inhibition Sensitizes BRCA1-Deficient Breast Cancer to Synthetic Lethal Therapy with ATM Inhibitors

2021 ◽  
Author(s):  
Leonie Ratz ◽  
Chiara Brambillasca ◽  
Leandra Bartke ◽  
Marieke van de Ven ◽  
Natalie Proost ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Synthetic Lethality ◽  
Molecular Subtype ◽  
Combined Treatment ◽  
Breast Tumors ◽  
Mammary Tumors ◽  
Genotoxic Stress ◽  
Synthetic Lethal

Abstract Background: The majority of BRCA1-mutant breast cancers are characterized by a triple-negative phenotype (TNBC) and a basal-like molecular subtype, associated with aggressive clinical behavior. Current treatment options are limited, highlighting the need for the development of novel targeted therapies for this tumor subtype. Methods: Our group previously showed that EZH2 is functionally relevant in BRCA1-deficient breast tumors and blocking EZH2 enzymatic activity could be a potent treatment strategy. To validate the role of EZH2 as a therapeutic target and to identify new synergistic drug combinations, we performed a high-throughput drug combination screen in various cell lines derived from BRCA1-proficient and deficient - mouse mammary tumors. Results: We identified the combined inhibition of EZH2 and the proximal DNA damage response kinase ATM as a novel synthetic lethality-based therapy for the treatment of BRCA1-deficient breast tumors. We show that the combined treatment with the EZH2 inhibitor GSK126 and the ATM inhibitor AZD1390 led to reduced colony formation, increased genotoxic stress, and apoptosis-mediated cell death in BRCA1-deficient mammary tumor cells in vitro. These findings were corroborated by in vivo experiments showing that simultaneous inhibition of EZH2 and ATM significantly increased anti-tumor activity in mice bearing BRCA1-deficient mammary tumors. Conclusion: Taken together, we identified a synthetic lethal interaction between EZH2 and ATM and propose this synergistic interaction as a novel molecular combination for the treatment of BRCA1-mutant breast cancer.

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