The conditional nature of gene essentiality.

Essential genes are classically defined as required for cellular viability and reproductive success. Despite this deceptively simple definition, several lines of evidence suggest that gene essentiality is instead a conditional trait. Indeed, gene essentiality has been shown to depend on the environmental and genetic context as well as the variable ability of cells to acquire adaptive mutations to survive inactivation of seemingly essential genes. Here, we will discuss these findings and highlight the mechanisms underlying the ability of cells to survive an essential gene deletion. Also, since essential genes are prioritized as targets for anticancer therapy, we discuss emergence of bypass resistance mechanisms toward targeted therapies as the result of the conditional nature of gene essentiality. To identify targets associated to a lower risk of relapse (i.e. the return of cancer following remission), we finally call for a coordinated effort to quantify the variable nature of gene essentiality across species, cell types, and growth conditions.

Disferlinopatía, una causa de falsa polimiositis refractaria / Dysferlinopathy masquerading as a refractory polymyositis

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Inhibition of microRNA122 decreases SREBP1 expression by modulating suppressor of cytokine signaling 3 expression.

While inhibition of microRNA122 (miR122) function in vivo results in reduced serum cholesterol and fatty acid levels, the molecular mechanisms underlying the link between miR122 function and lipid metabolism remains unclear. Because the expression of SREBP1, a central transcription factor involved in lipid metabolism, is known to be increased by suppressor of cytokine signaling 3 (SOCS3) expression, and because we previously found that SOCS3 expression is regulated by miR122, in this study, we examined the correlation between miR122 status and the expression levels of SOCS3 and SREBP1. SREBP1 expression decreased when SOCS3 expression was reduced by miR122 silencing in vitro. Conversely, SREBP1 expression in miR122-silenced cells was restored by enforced expression of SOCS3. Such correlations were observed in human liver tissues with different miR122 expression levels. These signaling links may explain one of the molecular mechanisms linking inhibition of miR122 function or decreased expression of miR122 to decreased fatty acid and cholesterol levels, in the inhibition of miR122 function, or in pathological status in chronic liver diseases.

Gestational diastolic hypertension with gene mutation-related pheochromocytoma positive at 18F-DOPA PET/CT: Diagnostic and therapeutic implications / Hipertensión diastólica gestacional con feocromocitoma relacionado a mutación genética y PET/TC con 18F-DOPA positiva: implicaciones diagnósticas y terapéuticas

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A screen for genetic suppressor elements of hepatitis C virus identifies a supercharged protein inhibitor of viral replication.

Genetic suppressor elements (GSEs) are biomolecules derived from a gene or genome of interest that act as transdominant inhibitors of biological functions presumably by disruption of critical biological interfaces. We exploited a cell death reporter cell line for hepatitis C virus (HCV) infection, n4mBid, to develop an iterative selection/enrichment strategy for the identification of anti-HCV GSEs. Using this approach, a library of fragments of an HCV genome was screened for sequences that suppress HCV infection. A 244 amino acid gene fragment, B1, was strongly enriched after 5 rounds of selection. B1 derives from a single-base frameshift of the enhanced green fluorescent protein (eGFP) which was used as a filler during fragment cloning. B1 has a very high net positive charge of 43 at neutral pH and a high charge-to-mass (kDa) ratio of 1.5. We show that B1 expression specifically inhibits HCV replication. In addition, five highly positively charged B1 fragments produced from progressive truncation at the C-terminus all retain the ability to inhibit HCV, suggesting that a high positive charge, rather than a particular motif in B1, likely accounts for B1's anti-HCV activity. Another supercharged protein, +36GFP, was also found to strongly inhibit HCV replication when added to cells at the time of infection. This study reports a new methodology for HCV inhibitor screening and points to the anti-HCV potential of positively charged proteins/peptides.

Forkhead-box-O1 locus y marcadores metabólicos en los ancianos: estudio de asociación / Forkhead-box-O1 locus and metabolic markers in the elderly: An association study

Introducción. Las mutaciones del gen forkhead-box-O1 (FoxO1) en el locus 13q14.1 provocan alteraciones en los parámetros bioquímicos conduciendo al envejecimiento prematuro. La proteína FoxO1 participa en la regulación de procesos bioquímicos, que influyen en la regulación del perfil lipídico y glucémico. Estos parámetros son un factor de riesgo de mortalidad en la población anciana. El objeto del estudio fue investigar la relación entre el locus FoxO1 y los marcadores metabólico-nutricionales. Material y métodos. Se investigaron los polimorfismos de nucleótido único (SNP, del inglés single-nucleotide polymorphisms) rs2721069, rs4943794 y rs7981045 en 594 ancianos hospitalizados (65-99 años) en una sección geriátrica, probando la asociación con los marcadores biológicos mediante el análisis de covarianza (ANCOVA) y del modelo estadístico Genotype Score. Resultados. El análisis de ANCOVA bajo distintos modelos genéticos reveló una asociación significativa. Asumiendo un modelo genético dominante se observó una asociación significativa con los niveles de la glucosa para rs2721069 (p=0,034) y rs4943794 (p=0,012). Para rs4943794 se observó también una asociación significativa si considerado libre de modelos genéticos (p=0,039) confirmada en el modelo aditivo (p=0,012). El modelo estadístico Genotype Score confirmó una asociación significativa entre FoxO1 SNP y la glucosa, teniendo en cuenta los SNP rs2721069 y rs4943794 en conjunto (p=0,048; Beta=3,198). Conclusiones. El envejecimiento es un proceso complejo, resultante de la interacción entre varios factores, como los ambientales y los genéticos. Nuestros hallazgos sugieren que el locus FoxO1 puede influir en los niveles séricos de glucemia en pacientes hospitalizados mayores, siendo entonces uno de los factores genéticos que contribuyen a un envejecimiento saludable(AU)

Introduction. Mutations of forkhead-box-O1 (FOXO1) gene at locus 13q14.1 cause changes in biochemical parameters leading to premature aging. Protein FoxO1 participates in the regulation of biochemical pathways, including those influencing the regulation of lipid profile and glucose metabolism. These parameters are a risk factor for all-cause mortality in the elderly population. The aim of this study was to investigate the relationship between FOXO1 locus and metabolic-nutritional markers. Material and methods. Single-nucleotide polymorphisms (SNP) rs2721069, rs4943794 and rs7981045 were determined in 594 hospitalized elderly (65-99 years), patients consecutively admitted to a geriatric ward, and tested the association of FOXO1 variants with biological markers by the analyses of co-variance (ANCOVA) and by Genotype Score Model statistic. Results. The ANCOVA analysis, under different genetic models, revealed significant associations. In particular, assuming a dominant genetic model, a significant association with serum levels of fasting glucose was observed for rs2721069 (P=.034) and rs4943794 (P=.012). For rs4943794 a significant association assuming a free genetic model (P=.039) and an additive one (P=.012) was also observed. No significant relationship was observed between rs7981045 and the analyzed markers. The Genotype Score Model analysis confirmed a significant association between FOXO1 SNP and fasting glucose, taking the SNP rs2721069 and rs4943794 together (P=.048; Beta=3.198). Conclusions. Aging is a complex process, resulting from the interaction between several factors, including environmental and genetic ones. Our findings suggest that FOXO1 locus may influence blood glucose levels in hospitalized older patients, thus being one of the genetic factors contributing to healthy aging(AU)

Cdc14 phosphatase promotes segregation of telomeres through repression of RNA polymerase II transcription.

Kinases and phosphatases regulate messenger RNA synthesis through post-translational modification of the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (ref. 1). In yeast, the phosphatase Cdc14 is required for mitotic exit(2,3) and for segregation of repetitive regions(4). Cdc14 is also a subunit of the silencing complex RENT (refs 5,6), but no roles in transcriptional repression have been described. Here we report that inactivation of Cdc14 causes silencing defects at the intergenic spacer sequences of ribosomal genes during interphase and at Y' repeats in subtelomeric regions during mitosis. We show that the role of Cdc14 in silencing is independent of the RENT deacetylase subunit Sir2. Instead, Cdc14 acts directly on RNA polymerase II by targeting CTD phosphorylation at Ser 2 and Ser 5. We also find that the role of Cdc14 as a CTD phosphatase is conserved in humans. Finally, telomere segregation defects in cdc14 mutants(4) correlate with the presence of subtelomeric Y' elements and can be rescued by transcriptional inhibition of RNA polymerase II.

Computational design of second-site suppressor mutations at protein-protein interfaces.

The importance of a protein-protein interaction to a signaling pathway can be established by showing that amino acid mutations that weaken the interaction disrupt signaling, and that additional mutations that rescue the interaction recover signaling. Identifying rescue mutations, often referred to as second-site suppressor mutations, controls against scenarios in which the initial deleterious mutation inactivates the protein or disrupts alternative protein-protein interactions. Here, we test a structure-based protocol for identifying second-site suppressor mutations that is based on a strategy previously described by Kortemme and Baker. The molecular modeling software Rosetta is used to scan an interface for point mutations that are predicted to weaken binding but can be rescued by mutations on the partner protein. The protocol typically identifies three types of specificity switches: knob-in-to-hole redesigns, switching hydrophobic interactions to hydrogen bond interactions, and replacing polar interactions with nonpolar interactions. Computational predictions were tested with two separate protein complexes; the G-protein Galpha(i1) bound to the RGS14 GoLoco motif, and UbcH7 bound to the ubiquitin ligase E6AP. Eight designs were experimentally tested. Swapping a buried hydrophobic residue with a polar residue dramatically weakened binding affinities. In none of these cases were we able to identify compensating mutations that returned binding to wild-type affinity, highlighting the challenges inherent in designing buried hydrogen bond networks. The strongest specificity switches were a knob-in-to-hole design (20-fold) and the replacement of a charge-charge interaction with nonpolar interactions (55-fold). In two cases, specificity was further tuned by including mutations distant from the initial design. Proteins 2010. (c) 2009 Wiley-Liss, Inc.

Novel repression of Kcc2 transcription by REST-RE-1 controls developmental switch in neuronal chloride.

Transcriptional upregulation of Kcc2b, the gene variant encoding the major isoform of the KCC2 chloride transporter, underlies a rapid perinatal decrease in intraneuronal chloride concentration (chloride shift), which is necessary for GABA to act inhibitory. Here we identify a novel repressor element-1 (RE-1) site in the 5' regulatory region of Kcc2b. In primary cortical neurons, which recapitulate the chloride shift in culture, the novel upstream RE-1 together with a known intronic RE-1 site function in concerted interaction to suppress Kcc2b transcription. With critical relevance for the chloride shift, only in the presence of the dual RE-1 site could inhibition of REST upregulate Kcc2b transcription. For this, we confirmed increased KCC2 protein expression and decreased intraneuronal chloride. Kcc2b developmental upregulation was potentiated by BDNF application, which was fully dependent on the presence of dual RE-1. In addition, the developmental chloride shift and GABA switch, from excitatory to inhibitory action, was accelerated by REST inhibition and slowed by REST overexpression. These results identify the REST-dual RE-1 interaction as a novel mechanism of transcriptional Kcc2b upregulation that significantly contributes to the ontogenetic shift in chloride concentration and GABA action in cortical neurons, which is fundamental for brain function in health and disease. Thus, we present here a new logic for the perinatal chloride shift, which is critical for establishment of GABAergic cortical inhibitory neurotransmission.

C-terminal flap endonuclease (rad27) mutations: lethal interactions with a DNA ligase I mutation (cdc9-p) and suppression by proliferating cell nuclear antigen (POL30) in Saccharomyces cerevisiae.

During lagging-strand DNA replication in eukaryotic cells primers are removed from Okazaki fragments by the flap endonuclease and DNA ligase I joins nascent fragments. Both enzymes are brought to the replication fork by the sliding clamp proliferating cell nuclear antigen (PCNA). To understand the relationship among these three components, we have carried out a synthetic lethal screen with cdc9-p, a DNA ligase mutation with two substitutions (F43A/F44A) in its PCNA interaction domain. We recovered the flap endonuclease mutation rad27-K325* with a stop codon at residue 325. We created two additional rad27 alleles, rad27-A358* with a stop codon at residue 358 and rad27-pX8 with substitutions of all eight residues of the PCNA interaction domain. rad27-pX8 is temperature lethal and rad27-A358* grows slowly in combination with cdc9-p. Tests of mutation avoidance, DNA repair, and compatibility with DNA repair mutations showed that rad27-K325* confers severe phenotypes similar to rad27Delta, rad27-A358* confers mild phenotypes, and rad27-pX8 confers phenotypes intermediate between the other two alleles. High-copy expression of POL30 (PCNA) suppresses the canavanine mutation rate of all the rad27 alleles, including rad27Delta. These studies show the importance of the C terminus of the flap endonuclease in DNA replication and repair and, by virtue of the initial screen, show that this portion of the enzyme helps coordinate the entry of DNA ligase during Okazaki fragment maturation.

Spp382p interacts with multiple yeast splicing factors, including possible regulators of Prp43 DExD/H-Box protein function.

Prp43p catalyzes essential steps in pre-mRNA splicing and rRNA biogenesis. In splicing, Spp382p stimulates the Prp43p helicase to dissociate the postcatalytic spliceosome and, in some way, to maintain the integrity of the spliceosome assembly. Here we present a dosage interference assay to identify Spp382p-interacting factors by screening for genes that when overexpressed specifically inhibit the growth of a conditional lethal prp38-1 spliceosome assembly mutant in the spp382-1 suppressor background. Identified, among others, are genes encoding the established splicing factors Prp8p, Prp9p, Prp11p, Prp39p, and Yhc1p and two poorly characterized proteins with possible links to splicing, Sqs1p and Cwc23p. Sqs1p copurifies with Prp43p and is shown to bind Prp43p and Spp382p in the two-hybrid assay. Overexpression of Sqs1p blocks pre-mRNA splicing and inhibits Prp43p-dependent steps in rRNA processing. Increased Prp43p levels buffer Sqs1p cytotoxicity, providing strong evidence that the Prp43p DExD/H-box protein is a target of Sqs1p. Cwc23p is the only known yeast splicing factor with a DnaJ motif characteristic of Hsp40-like chaperones. We show that similar to SPP382, CWC23 activity is critical for efficient pre-mRNA splicing and intron metabolism yet, surprisingly, this activity does not require the canonical DnaJ/Hsp40 motif. These and related data establish the value of this dosage interference assay for finding genes that alter cellular splicing and define Sqs1p and Cwc23p as prospective modulators of Spp382p-stimuated Prp43p function.

[Overexpression of gene PPZ1 in the yeast Saccharomyces cerevisiae affects the efficiency of nonsense suppression].

The phenomenon of nonsense suppression, which leads to the reading of stop codons as sense codons, may be related to disturbances in the operation of various components of the translation apparatus and the proteins interacting with them. The phosphatase Ppzlp is one of the factors affecting the nonsense suppression efficiency in the saccharomycete yeast. In this work, the impact of the overexpression of gene PPZ1 and its mutant allele PPZ1-R451L on the phenotypic expression of various mutant alleles of genes SUP35 and SUP45 or the yeast prion [PSI+] was analyzed. On the basis of the data obtained, a suggestion about the possible role of proteins Sup35p and Sup45p in the processes mediating the influence of gene PPZ1 overexpression on the efficiency of nonsense suppression is made.

Point mutations in helper component protease of clover yellow vein virus are associated with the attenuation of RNA-silencing suppression activity and symptom expression in broad bean.

Helper component protease (HC-Pro) is a potyvirus-encoded multifunctional protein and a major determinant of symptom expression in a susceptible plant. Here, we show the involvement of clover yellow vein virus (ClYVV) HC-Pro in necrotic symptom expression in broad bean (Vicia faba cv. Wase). In this host, lethal necrosis was induced by ClYVV no. 30, from which a spontaneous, mosaic-inducing mutant (MM) was obtained. Mapping with chimeric viruses between ClYVV no. 30 and MM attributed the symptom attenuation to two mutations at the HC-Pro positions 27 (threonine to isoleucine) and 193 (aspartic acid to tyrosine). Although neither mutant with the single amino acid substitution at position 27 or 193 (ClYVV/T27I or D193Y) induced the lethal necrosis, ClYVV/T27I still retained the ability to induce necrotic symptoms, but ClYVV/D193Y scarcely did so. The virus accumulation of ClYVV/D193Y was also lower than that of ClYVV no. 30. The mutations, T27I and D193Y, are located in a putative zinc finger domain and in one (N-terminal) of the two RNA binding domains, respectively, of HC-Pro. RNA-silencing suppression (RSS) activity of P1/HC-Pro in Nicotiana benthamiana was weakened by both mutations. Our results suggest a correlation between viral virulence and RSS function and the importance of the two domains in HC-Pro.

Functional census of mutation sequence spaces: the example of p53 cancer rescue mutants.

Many biomedical problems relate to mutant functional properties across a sequence space of interest, e.g., flu, cancer, and HIV. Detailed knowledge of mutant properties and function improves medical treatment and prevention. A functional census of p53 cancer rescue mutants would aid the search for cancer treatments from p53 mutant rescue. We devised a general methodology for conducting a functional census of a mutation sequence space by choosing informative mutants early. The methodology was tested in a double-blind predictive test on the functional rescue property of 71 novel putative p53 cancer rescue mutants iteratively predicted in sets of three (24 iterations). The first double-blind 15-point moving accuracy was 47 percent and the last was 86 percent; r = 0.01 before an epiphanic 16th iteration and r = 0.92 afterward. Useful mutants were chosen early (overall r = 0.80). Code and data are freely available (http://www.igb.uci.edu/research/research.html, corresponding authors: R.H.L. for computation and R.K.B. for biology).

Efficient expression of the 15-kDa form of infectious pancreatic necrosis virus VP5 by suppression of a UGA codon.

Infectious pancreatic necrosis virus (IPNV), a member of the Birnaviridae family, encodes a nonstructural VP5 protein from a small open reading frame (ORF), which overlaps with a major ORF encoding pVP2, VP4 and VP3 proteins. In majority of the Sp strains of IPNV sequenced to date, VP5 gene codes for a 15-kDa protein. However, we have shown that in highly virulent strains, there is a premature in-frame stop codon (UGA) at nucleotide (nt) position 427, (preceding the 15-kDa stop codon at nt position 511) which could encode a 12-kDa protein. Using reverse genetics, we recovered recombinant rNVI15, rNVI15-15K and rNVI15-DeltaVP5 viruses (which could encode 12 or 15-kDa VP5 or lack the expression of VP5, respectively) and demonstrated that VP5 is dispensable for viral replication in vivo but is not involved in virulence (Santi, N., Song, H., Vakharia, V. N., Evensen, Ø., 2005a. Infectious pancreatic necrosis virus VP5 is dispensable for virulence and persistence. J. Virol. 79, 9206-9216). Here, we utilized these viruses to investigate the gene expression of VP5 in vitro. Our results indicate that a 15-kDa VP5 is produced in rNVI15-infected cells, albeit at lower levels than in rNVI15-15K-infected cells, suggesting that the opal stop codon at nt 427 is suppressed. Furthermore, to examine translational suppression of the opal stop codon in VP5 gene, we constructed plasmids containing VP5-specific sequence and employed a yeast-based bicistronic dual-luciferase reporter system (Harger, J.W., Dinman, J.D., 2003. An in vivo dual-luciferase assay system for studying translational recoding in the yeast Saccharomyces cerevisiae. RNA 9, 1019-1024). Our results demonstrate that the VP5 sequence (with or without a stop codon) yielded approximately 13% termination suppression and the efficiency is directly related to the base immediately 3' of the termination codon, C>A>U>G.

The conserved ATPase Get3/Arr4 modulates the activity of membrane-associated proteins in Saccharomyces cerevisiae.

The regulation of cellular membrane dynamics is crucial for maintaining proper cell growth and division. The Cdc48-Npl4-Ufd1 complex is required for several regulated membrane-associated processes as part of the ubiquitin-proteasome system, including ER-associated degradation and the control of lipid composition in yeast. In this study we report the results of a genetic screen in Saccharomyces cerevisiae for extragenic suppressors of a temperature-sensitive npl4 allele and the subsequent analysis of one suppressor, GET3/ARR4. The GET3 gene encodes an ATPase with homology to the regulatory component of the bacterial arsenic pump. Mutants of GET3 rescue several phenotypes of the npl4 mutant and transcription of GET3 is coregulated with the proteasome, illustrating a functional relationship between GET3 and NPL4 in the ubiquitin-proteasome system. We have further found that Get3 biochemically interacts with the trans-membrane domain proteins Get1/Mdm39 and Get2/Rmd7 and that Deltaget3 is able to suppress phenotypes of get1 and get2 mutants, including sporulation defects. In combination, our characterization of GET3 genetic and biochemical interactions with NPL4, GET1, and GET2 implicates Get3 in multiple membrane-dependent pathways.

Ubc9 interacts with SOX4 and represses its transcriptional activity.

SOX4 is a member of SOX transcriptional factor family that is crucial for many cellular processes. In this study, a yeast two-hybrid screening of human mammary cDNA library identified human ubiquitin-conjugating enzyme 9 (hUbc9) that interacted with SOX4. This interaction was confirmed by GST pull-down in vitro and co-immunoprecipitation assays in vivo. Deletion mapping demonstrated that HMG-box domain of SOX4 is required to mediate the interaction with Ubc9 in yeast. Furthermore, confocal microscopy showed that Ubc9 co-localized with SOX4 in the nucleus. Luciferase assays found that Ubc9 specifically repressed SOX4 transcriptional activity in 293T cells. We further demonstrated that Ubc9 could functionally repress the transcriptional activity of endogenous SOX4 induced by progesterone in T47D cells. The C93S mutant of Ubc9, which abrogates SUMO-1 conjugation activity, did not abolish the ability to repress SOX4 activity. It shows that Ubc9 interacts with SOX4 and represses its transcriptional activity independent of its SUMO-1-conjugating activity.

The wound response mutant suppressor of prosystemin-mediated responses6 (spr6) is a weak allele of the tomato homolog of CORONATINE-INSENSITIVE1 (COI1).

The systemic defense response of tomato plant in response to insect attack and wounding is regulated by the 18 amino acid peptide systemin and the phytohormone jasmonic acid (JA). Recent genetic analyses based mainly on spr (suppressors of prosystemin-mediated responses) mutant screens have led to the hypothesis that systemin acts at, or near, the site of wounding to amplify the production of JA, which in turn functions as a mobile signal to promote the systemic defense response. In order to identify more components involved in the systemin/JA-signaled defense response, we carried out a larger scale screen for new spr mutants in tomato. Here we describe the characterization of spr6, a mutant impaired in wound- and systemin-induced defense gene expression. Using a candidate gene approach based on genetic linkage, we demonstrate that spr6 is allelic to jai1-1, which is a loss-of-function allele of the tomato homolog of CORONATINE-INSENSITIVE1 (COI1), an F-box protein that is required for JA-signaled processes in Arabidopsis. We show several aspects of the spr6 mutant phenotype distinct from that of jai1-1. First, the responsiveness of spr6 plants to exogenous JA shows a dosage dependency, i.e. it is more sensitive to JA than jai1-1 while less sensitive to JA than the wild-type. Secondly, unlike the sterile jai1-1, the spr6 plant displays normal fertility and seed set and thus can be maintained as a pure line and does not require selection. Therefore, spr6 provides a valuable tool, which can complement the limitations of jai1-1, to study JA signaling in tomato. The gene identification process of Spr6 we described herein represents an example showing the convenience of a candidate gene approach, based on genetic linkage, to identify gene functions of genetic loci defined by tomato wound response mutants.