RESUMEN
The Saccharomyces cerevisiae Agp2 is a plasma membrane protein initially reported to be an uptake transporter for L-carnitine. Agp2 was later rediscovered, together with three additional proteins, Sky1, Ptk2, and Brp1, to be involved in the uptake of the polyamine analogue bleomycin-A5, an anticancer drug. Mutants lacking either Agp2, Sky1, Ptk2, or Brp1 are extremely resistant to polyamines and bleomycin-A5, suggesting that these four proteins act in the same transport pathway. We previously demonstrated that pretreating cells with the protein synthesis inhibitor cycloheximide (CHX) blocked the uptake of fluorescently labelled bleomycin (F-BLM), raising the possibility that CHX could either compete for F-BLM uptake or alter the transport function of Agp2. Herein, we showed that the agp2Δ mutant displayed striking resistance to CHX as compared to the parent, suggesting that Agp2 is required to mediate the physiological effect of CHX. We examined the fate of Agp2 as a GFP tag protein in response to CHX and observed that the drug triggered the disappearance of Agp2 in a concentration- and time-dependent manner. Immunoprecipitation analysis revealed that Agp2-GFP exists in higher molecular weight forms that were ubiquitinylated, which rapidly disappeared within 10 min of treatment with CHX. CHX did not trigger any significant loss of Agp2-GFP in the absence of the Brp1 protein; however, the role of Brp1 in this process remains elusive. We propose that Agp2 is degraded upon sensing CHX to downregulate further uptake of the drug and discuss the potential function of Brp1 in the degradation process.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Cicloheximida/farmacología , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Bleomicina/farmacología , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
The base excision repair (BER) pathway is essential for maintaining the stability of DNA in all organisms and defects in this process are associated with life-threatening diseases. It is involved in removing specific types of DNA lesions that are induced by both exogenous and endogenous genotoxic substances. BER is a multi-step mechanism that is often initiated by the removal of a damaged base leading to a genotoxic intermediate that is further processed before the reinsertion of the correct nucleotide and the restoration of the genome to a stable structure. Studies in human and yeast cells, as well as fruit fly and nematode worms, have played important roles in identifying the components of this conserved DNA repair pathway that maintains the integrity of the eukaryotic genome. This review will focus on the components of base excision repair, namely, the DNA glycosylases, the apurinic/apyrimidinic endonucleases, the DNA polymerase, and the ligases, as well as other protein cofactors. Functional insights into these conserved proteins will be provided from humans, Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans, and the implications of genetic polymorphisms and knockouts of the corresponding genes.
Asunto(s)
Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Drosophila melanogaster/genética , Saccharomyces cerevisiae/genética , Animales , Proteínas de Unión al ADN/genética , Drosophila melanogaster/metabolismo , Humanos , Saccharomyces cerevisiae/metabolismoRESUMEN
Cisplatin (cis-diamminedichloroplatinum (II)) is the oldest known chemotherapeutic agent. Since the identification of its anti-tumour activity, it earned a remarkable place as a treatment of choice for several cancer types. It remains effective against testicular, bladder, lung, head and neck, ovarian, and other cancers. Cisplatin treatment triggers different cellular responses. However, it exerts its cytotoxic effects by generating inter-strand and intra-strand crosslinks in DNA. Tumour cells often develop tolerance mechanisms by effectively repairing cisplatin-induced DNA lesions or tolerate the damage by adopting translesion DNA synthesis. Cisplatin-associated nephrotoxicity is also a huge challenge for effective therapy. Several preclinical and clinical studies attempted to understand the major limitations associated with cisplatin therapy, and so far, there is no definitive solution. As such, a more comprehensive molecular and genetic profiling of patients is needed to identify those individuals that can benefit from platinum therapy. Additionally, the treatment regimen can be improved by combining cisplatin with certain molecular targeted therapies to achieve a balance between tumour toxicity and tolerance mechanisms. In this review, we discuss the importance of various biological processes that contribute to the resistance of cisplatin and its derivatives. We aim to highlight the processes that can be modulated to suppress cisplatin resistance and provide an insight into the role of uptake transporters in enhancing drug efficacy.
Asunto(s)
Antineoplásicos , Neoplasias , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Cisplatino/farmacología , Cisplatino/uso terapéutico , ADN/uso terapéutico , Reparación del ADN , Resistencia a Antineoplásicos , Humanos , Neoplasias/tratamiento farmacológicoRESUMEN
The human organic cation uptake transporter OCT1, encoded by the SLC22A1 gene, is highly expressed in the liver and reported to possess a broad substrate specificity. OCT1 operates by facilitated diffusion and allows the entry of nutrients into cells. Recent findings revealed that OCT1 can mediate the uptake of drugs for treating various diseases such as cancers. The levels of OCT1 expression correlate with the responses towards many drugs and functionally defective OCT1 lead to drug resistance. It has been recently proposed that OCT1 should be amongst the crucial drug targets used for pharmacogenomic analyses. Several single nucleotide polymorphisms exist and are distributed across the entire OCT1 gene. While there are differences in the OCT1 gene polymorphisms between populations, there are at least five variants that warrant consideration in any genetic screen. To date, and despite two decades of research into OCT1 functional role, it still remains uncertain what are the define substrates for this uptake transporter, although studies from mice revealed that one of the substrates is vitamin B1. It is also unclear how OCT1 recognizes a broad array of ligands and whether this involves specific modifications and interactions with other proteins. In this review, we highlight the current findings related to OCT1 with the aim of propelling further studies on this key uptake transporter.
Asunto(s)
Factor 1 de Transcripción de Unión a Octámeros/metabolismo , Preparaciones Farmacéuticas/metabolismo , Secuencia de Aminoácidos , Animales , Sistemas de Liberación de Medicamentos , Humanos , Factor 1 de Transcripción de Unión a Octámeros/antagonistas & inhibidores , Factor 1 de Transcripción de Unión a Octámeros/biosíntesis , Factor 1 de Transcripción de Unión a Octámeros/genética , FarmacocinéticaRESUMEN
The transmembrane protein Agp2, initially shown as a transporter of L-carnitine, mediates the high-affinity transport of polyamines and the anticancer drug bleomycin-A5. Cells lacking Agp2 are hyper-resistant to polyamine and bleomycin-A5. In these earlier studies, we showed that the protein synthesis inhibitor cycloheximide blocked the uptake of bleomycin-A5 into the cells suggesting that the drug uptake system may require de novo synthesis. However, our recent findings demonstrated that cycloheximide, instead, induced rapid degradation of Agp2, and in the absence of Agp2 cells are resistant to cycloheximide. These observations raised the possibility that the degradation of Agp2 may allow the cell to alter its drug resistance network to combat the toxic effects of cycloheximide. In this study, we show that membrane extracts from agp2Δ mutants accentuated several proteins that were differentially expressed in comparison to the parent. Mass spectrometry analysis of the membrane extracts uncovered the pleiotropic drug efflux pump, Pdr5, involved in the efflux of cycloheximide, as a key protein upregulated in the agp2Δ mutant. Moreover, a global gene expression analysis revealed that 322 genes were differentially affected in the agp2Δ mutant versus the parent, including the prominent PDR5 gene and genes required for mitochondrial function. We further show that Agp2 is associated with the upstream region of the PDR5 gene, leading to the hypothesis that cycloheximide resistance displayed by the agp2Δ mutant is due to the derepression of the PDR5 gene.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Cicloheximida , Inhibidores de la Síntesis de la Proteína , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Cicloheximida/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Regulación hacia Arriba/efectos de los fármacos , Farmacorresistencia Fúngica/genética , Farmacorresistencia Fúngica/efectos de los fármacos , Regulación Fúngica de la Expresión Génica/efectos de los fármacosRESUMEN
In yeast Saccharomyces cerevisiae, the immunosuppressant rapamycin mimics starvation by inhibiting the kinase Tor1. We recently documented that this treatment triggers a rapid degradation of Sgs1, a helicase involved in several biological processes such as the prevention of genomic instability. Herein, we show that yeast strains deleted for genes ATG2, ATG9, and PEP4, encoding components of the autophagy pathway, prevent rapamycin-induced degradation of Sgs1. We propose that defects in the autophagy pathway prevent degradation of key proteins in the rapamycin response pathway and as a consequence cause resistance to the drug.
Asunto(s)
Autofagia , RecQ Helicasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Sirolimus/farmacología , Secuencia de Bases , Cartilla de ADN , Proteolisis , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/inmunologíaRESUMEN
In yeast Saccharomyces cerevisiae, the immunosuppressant rapamycin inhibits the TORC1 kinase causing rapid alteration in gene expression and leading to G1 arrest. We recently reported the isolation and characterization from the histone mutant collection of a histone H2B R95A mutant that displays resistance to rapamycin. This mutant is defective in the expression of several genes belonging to the pheromone response pathway including STE5 encoding a scaffold protein that promotes the activation of downstream MAP kinases. Cells lacking Ste5 cannot arrest the cell cycle in response to rapamycin and as a consequence exhibit similar resistance to rapamycin as the H2B R95A mutant. Herein, we show that the H2B R95A mutation weakens the association of H2B with Spt16 a component of the FACT complex (FAcilitates Chromatin Transcription), and an essential factor that interacts with the histone H2A-H2B dimer to promote transcription and preserve chromatin integrity. From a collection of spt16 mutants, spt16 E857K and spt16-11 showed striking sensitivity to rapamycin as compared to the parent strain. spt16 E857K and spt16-11 expressed distinct forms of Ste5, while a suppressor mutation H2B A84D of the spt16-11 mutant prevents the expression of Ste5 and confers marked resistance to rapamycin. We interpret these findings to suggest that the Arg95 residue of histone H2B is required to recruit Spt16 to maintain the expression of STE5, which performs a role to arrest cells in the G1 phase in response to rapamycin.
Asunto(s)
Histonas , Proteínas de Saccharomyces cerevisiae , Histonas/genética , Histonas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Nucleosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Elongación Transcripcional/metabolismo , Cromatina/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Feromonas/farmacología , Feromonas/metabolismo , Sirolimus/farmacología , Sirolimus/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismoRESUMEN
Rapid, highly specific, and robust diagnostic kits to detect viruses and pathogens are needed to control disease spread and transmission globally. Of the many different methods proposed to diagnose COVID-19 infection, CRISPR-based detection of nucleic acids tests are among the most prominent. Here, we describe a new way of using CRISPR/Cas systems as a rapid and highly specific tool to detect the SARS-CoV-2 virus using the in vitro dCas9-sgRNA-based technique. As a proof of concept, we used a synthetic DNA of the M gene, one of the SARS-CoV-2 virus genes, and demonstrated that we can specifically inactivate unique restriction enzyme sites on this gene using CRISPR/Cas multiplexing of dCas9-sgRNA-BbsI and dCas9-sgRNA-XbaI. These complexes recognize and bind to the target sequence spanning the BbsI and XbaI restriction enzyme sites, respectively, and protect the M gene from digestion by BbsI and/or XbaI. We further demonstrated that this approach can be used to detect the M gene when expressed in human cells and from individuals infected with SARS-CoV-2. We refer to this approach as dead Cas9 Protects Restriction Enzyme Sites, and believe that it has the potential to be applied as a diagnostic tool for many DNA/RNA pathogens.
RESUMEN
Quinoxaline is a heterocyclic compound with a two-membered ring structure that undergoes redox cycling to produce toxic free radicals. It has antiviral, antibacterial, antifungal, and antitumor activities. However, the biological functions that are involved in mounting a response against the toxic effects of quinoxaline have not been investigated. Herein, we performed a genome-wide screen using the yeast haploid mutant collection and reported the identification of 12 mutants that displayed varying sensitivity towards quinoxaline. No mutant was recovered that showed resistance to quinoxaline. The quinoxaline-sensitive mutants were deleted for genes that encode cell cycle function, as well as genes that belong to other physiological pathways such as the vacuolar detoxification process. Three of the highly sensitive gene-deletion mutants lack the DDC1, DUN1, and MFT1 genes. While Ddc1 and Dun1 are known to perform roles in the cell cycle arrest pathway, the role of Mft1 remains unclear. We show that the mft1Δ mutant is as sensitive to quinoxaline as the ddc1Δ mutant. However, the double mutant ddc1Δ mft1Δ lacking the DDC1 and MFT1 genes, is extremely sensitive to quinoxaline, as compared to the ddc1Δ and mft1Δ single mutants. We further show that the mft1Δ mutant is unable to arrest in the G2/M phase in response to the drug. We conclude that Mft1 performs a unique function independent of Ddc1 in the cell cycle arrest pathway in response to quinoxaline exposure. This is the first demonstration that quinoxaline exerts its toxic effect likely by inducing oxidative DNA damage causing cell cycle arrest. We suggest that clinical applications of quinoxaline and its derivatives should entail targeting cancer cells with defective cell cycle arrest.
RESUMEN
Targeting tumour metabolism through glucose transporters is an attractive approach. However, the role these transporters play through interaction with other signalling proteins is not yet defined. The glucose transporter SLC2A3 (GLUT3) is a member of the solute carrier transporter proteins. GLUT3 has a high affinity for D-glucose and regulates glucose uptake in the neurons, as well as other tissues. Herein, we show that GLUT3 is involved in the uptake of arsenite, and its level is regulated by peroxiredoxin 1 (PRDX1). In the absence of PRDX1, GLUT3 mRNA and protein expression levels are low, but they are increased upon arsenite treatment, correlating with an increased uptake of glucose. The downregulation of GLUT3 by siRNA or deletion of the gene by CRISPR cas-9 confers resistance to arsenite. Additionally, the overexpression of GLUT3 sensitises the cells to arsenite. We further show that GLUT3 interacts with PRDX1, and it forms nuclear foci, which are redistributed upon arsenite exposure, as revealed by immunofluorescence analysis. We propose that GLUT3 plays a role in mediating the uptake of arsenite into cells, and its homeostatic and redox states are tightly regulated by PRDX1. As such, GLUT3 and PRDX1 are likely to be novel targets for arsenite-based cancer therapy.
Asunto(s)
Arsenitos , Transportador de Glucosa de Tipo 3 , Arsenitos/toxicidad , Glucosa/metabolismo , Transportador de Glucosa de Tipo 3/genética , Transportador de Glucosa de Tipo 3/metabolismo , Linfocitos Nulos/efectos de los fármacos , Linfocitos Nulos/metabolismo , Peroxirredoxinas/metabolismo , Humanos , Células HEK293RESUMEN
Polyamines are nitrogenous compounds found in all eukaryotic and prokaryotic cells and absolutely essential for cell viability. In plants, they regulate several growth and developmental processes and the levels of polyamines are also correlated with the plant responses to various biotic and abiotic stresses. In plant cells, polyamines are synthesized in plastids and cytosol. This biosynthetic compartmentation indicates that the specific transporters are essential to transport polyamines between the cellular compartments. In the present study, a phylogenetic analysis was used to identify candidate polyamine transporters in rice. A full-length cDNA rice clone AK068055 was heterologously expressed in the Saccharomyces cerevisiae spermidine uptake mutant, agp2∆. Radiological uptake and competitive inhibition studies with putrescine indicated that rice gene encodes a protein that functioned as a spermidine-preferential transporter. In competition experiments with several amino acids at 25-fold higher levels than spermidine, only methionine, asparagine, and glutamine were effective in reducing uptake of spermidine to 60% of control rates. Based on those observations, this rice gene was named polyamine uptake transporter 1 (OsPUT1). Tissue-specific expression of OsPUT1 by semiquantitative RT-PCR showed that the gene was expressed in all tissues except seeds and roots. Transient expression assays in onion epidermal cells and rice protoplasts failed to localize to a cellular compartment. The characterization of the first plant polyamine transporter sets the stage for a systems approach that can be used to build a model to fully define how the biosynthesis, degradation, and transport of polyamines in plants mediate developmental and biotic responses.
Asunto(s)
Poliaminas Biogénicas/metabolismo , Oryza/genética , Oryza/metabolismo , Transporte Biológico , Células Cultivadas , ADN Complementario , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Ingeniería Genética , Variación Genética , Datos de Secuencia Molecular , Filogenia , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Putrescina/metabolismo , Espermidina/metabolismo , Estrés FisiológicoRESUMEN
We exploited the yeast DAmP mutant collection to identify essential genes that play a role in polyamine resistance. Herein, we described in details the methodology to obtain these genes. This approach is applicable for screening many nontoxic and toxic drugs.
Asunto(s)
Saccharomyces cerevisiae , Genes Esenciales , Poliaminas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , EsperminaRESUMEN
Here, we describe a protocol for human PRDX1 gene knockout cells using the CRISPR-Cas9 system. The protocol describes all the steps sequentially: (1) single-guide RNA design, cloning, and transfection; (2) gene editing evaluation by T7EI assay; (3) single-cell isolation; and (4) knockout verification to determine indels in one or both alleles by Sanger sequencing. This strategy is based on the efficiency of DNA editing, avoids antibiotic selection, and bypasses the need for cell sorting.
Asunto(s)
Sistemas CRISPR-Cas , ARN Guía de Kinetoplastida , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Técnicas de Inactivación de Genes , Humanos , Peroxirredoxinas/genética , ARN Guía de Kinetoplastida/genética , TransfecciónRESUMEN
Rapamycin is an immunosuppressant used for treating many types of diseases such as kidney carcinomas. In yeast, rapamycin inhibits the TORC1 kinase signaling pathway causing rapid alteration in gene expression and ultimately cell cycle arrest in G1 through mechanisms that are not fully understood. Herein, we screened a histone mutant collection and report that one of the mutants, H2B R95A, is strikingly resistant to rapamycin due to a defective cell cycle arrest. We show that the H2B R95A causes defects in the expression of a subset of genes of the pheromone pathway required for α factor-induced G1 arrest. The expression of the STE5 gene and its encoded scaffold protein Ste5, required for the sequential activation of the MAPKs of the pheromone pathway, is greatly reduced in the H2B R95A mutant. Similar to the H2B R95A mutant, cells devoid of Ste5 are also resistant to rapamycin. Rapamycin-induced G1 arrest does not involve detectable phosphorylation of the MAPKs, Kss1, and Fus3, as reported for α factor-induced G1 arrest. However, we observed a sharp induction of the G1 cyclin Cln2 (~ 3- to 4-fold) in the ste5Δ mutant within 30 min of exposure to rapamycin. Our data provide a new insight whereby rapamycin signaling via the Torc1 kinase may exploit the pheromone pathway to arrest cells in the G1 phase.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Ciclinas/metabolismo , Proteínas Fúngicas/genética , Histonas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Feromonas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sirolimus/metabolismo , Sirolimus/farmacologíaRESUMEN
Carcinogenicity and cytotoxicity are severe consequences of DNA damage. Base Excision Repair (BER) is a conserved DNA repair pathway that replaces many damaged bases caused by oxidation. Aberrations in BER are associated with carcinogenesis, neurodegeneration, and aging. The nematode C. elegans is an attractive model system for studying BER. However, in this organism, the complete pathway is not fully delineated. To further explore the BER process in C. elegans, we used affinity tag chromatography and mass spectrometry to identify the interactome of uracil DNA glycosylase-1 (CeUNG-1), an enzyme that acts during the first step of the BER pathway. Our analysis identified that CeUNG-1 is associated with the 40 S ribosomal protein S3 (CeRPS-3), homologs of which have been shown to process 8-oxoguanine and abasic site lesions in other organisms. We report a strong in silico association between CeUNG-1 and CeRPS-3 and confirmed this interaction using the yeast two-hybrid system. Downregulation of the Cerps-3 gene reduced the viability of wild-type worms upon exposure to the chemical oxidant hydrogen peroxide. Further analysis shows that Cerps-3 knockdown significantly sensitized the AP endonuclease APN-1-deficient strain, apn-1, but to a lesser extent exo-3, to the lethal effects of hydrogen peroxide. A cross-species complementation experiment reveals that the expression of CeRPS-3 rescued the hydrogen peroxide sensitivity, and suppressed the high mutation frequency of the yeast AP endonuclease-deficient strain lacking Apn1 and Apn2. We propose that CeRPS-3 may function as an auxiliary DNA repair enzyme in C. elegans to process oxidative DNA lesions.
Asunto(s)
Proteínas de Caenorhabditis elegans , Proteínas de Saccharomyces cerevisiae , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Daño del ADN , Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Peróxido de Hidrógeno/toxicidad , Mutación , Proteínas Ribosómicas , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Bleomycin is used in combination with other antineoplastic agents to effectively treat lymphomas, testicular carcinomas, and squamous cell carcinomas of the cervix, head, and neck. However, resistance to bleomycin remains a persistent limitation in exploiting the full therapeutic benefit of the drug with other types of cancers. Previously, we documented that the Saccharomyces cerevisiae L-carnitine transporter Agp2 is responsible for the high affinity uptake of polyamines and of the polyamine analogue bleomycin-A5. Herein, we document that the human L-carnitine transporter hCT2 encoded by the SLC22A16 gene is involved in bleomycin-A5 uptake, as well as polyamines. We show that NT2/D1 human testicular cancer cells, which highly express hCT2, are extremely sensitive to bleomycin-A5, whereas HCT116 human colon carcinoma cells devoid of detectable hCT2 expression or MCF-7 human breast cancer cells that only weakly express the permease showed striking resistance to the drug. NT2/D1 cells accumulated fluorescein-labeled bleomycin-A5 to substantially higher levels than HCT116 cells. Moreover, L-carnitine protected NT2/D1 cells from the lethal effects of bleomycin-A5 by preventing its influx, and siRNA targeted to hCT2 induced resistance to bleomycin-A5-dependent genotoxicity. Furthermore, hCT2 overexpression induced by transient transfection of a functional hCT2-GFP fusion protein sensitized HCT116 cells to bleomycin-A5. Collectively, our data strongly suggest that hCT2 can mediate bleomycin-A5 and polyamine uptake, and that the rate of bleomycin-A5 accumulation may account for the differential response to the drug in patients.
Asunto(s)
Bleomicina/análogos & derivados , Proteínas de Transporte de Catión Orgánico/metabolismo , Antibióticos Antineoplásicos/farmacocinética , Bleomicina/farmacocinética , Neoplasias de la Mama , Línea Celular Tumoral , Neoplasias del Colon , Resistencia a Antineoplásicos , Femenino , Humanos , Masculino , Farmacocinética , Poliaminas/farmacocinética , Neoplasias TesticularesRESUMEN
In Saccharomyces cerevisiae , rapamycin exposure inhibits the target of rapamycin (TOR) signaling pathway, causing a profound alteration in the transcription pattern of many genes, including those involved in ribosome biogenesis and nutritional changes. Deletion of the RRD1 gene encoding a peptidyl prolyl isomerase resulted in mutants that are resistant to rapamycin. These rrd1Δ mutants are unable to efficiently downregulate genes such as ribosomal protein genes, or to upregulate genes involved in diauxic shift. It is believed that the isomerase function of Rrd1 plays a role in changing the transcriptional profile upon rapamycin exposure. Herein, we set out to search for genes that when deleted in the rrd1Δ mutant would suppress the rapamycin-resistant phenotype. The analysis revealed that deletion of the SGS1 gene in the rrd1Δ mutant partially suppresses the rapamycin-resistant phenotype of the single rrd1Δ mutant. SGS1 encodes a helicase that functions in many biological processes, including transcriptional regulation. We further show, and for the first time, that Sgs1 is rapidly lost in the parent cells in response to rapamycin, but not by other agents. Interestingly, Sgs1 reduction was completely blocked in the rrd1Δ mutant, suggesting that Rrd1 is required to mediate this process. Genes such as PUT4 and HSP42, known to be upregulated in the parent in response to rapamycin, were not induced in the rrd1Δ mutant if the SGS1 gene was deleted. Since Sgs1 plays a role in transcriptional regulation, we propose that it acts as a repressor of a subset of rapamycin responsive genes. Thus, the observed Rrd1-dependent reduction in Sgs1 level may promote expression of specific classes of genes in response to rapamycin.
Asunto(s)
Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Isomerasa de Peptidilprolil/metabolismo , RecQ Helicasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Regulación hacia Abajo , Estudios de Asociación Genética , Genotipo , Péptidos y Proteínas de Señalización Intracelular/genética , Isomerasa de Peptidilprolil/genética , Fenotipo , RecQ Helicasas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Eliminación de Secuencia , Transducción de Señal/genética , Transcripción GenéticaRESUMEN
In Saccharomyces cerevisiae, the immunosuppressor rapamycin engenders the degradation of excessive RNA polymerase II leading to growth arrest but the regulation of this process is not known yet. Here, we show that this mechanism is dependent on the peptidyl prolyl cis/trans isomerase Rrd1. Strikingly this degradation is independent of RNA polymerase II polyubiquitylation and does not require the elongation factor Elc1. Our data reveal that there are at least two alternative pathways to degrade RNA polymerase II that depend on different type of stresses.
Asunto(s)
Inmunosupresores/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Isomerasa de Peptidilprolil/metabolismo , ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Sirolimus/farmacología , Péptidos y Proteínas de Señalización Intracelular/genética , Isomerasa de Peptidilprolil/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , UbiquitinaciónRESUMEN
The creation of the nuclease-dead Cas protein (dCas9) offers a new platform for a plethora of new discoveries. Diverse dCas9 tools have been developed for transcription regulation, epigenetic engineering, base editing, genome imaging, genetic screens, and chromatin immunoprecipitation. Here, we show that dCas9 and single-guide RNA preassembled to form ribonucleoprotein dCas9-sgRNA (referred to as dRNP) is capable of specifically and reversibly blocking the activity of DNA cleavage by restriction enzymes (REs). We show that the inhibition of RE activities occurs when the recognition or the cleavage site of the DNA is overlapped by the sgRNA or the protospacer adjacent motif sequence. Furthermore, we show that multiple dRNPs can be used simultaneously to inhibit more than one RE sites. As such, we exploited this novel finding as a method to demonstrate that inserts can be ligated into vectors, and vice versa, whereby selective RE sites are temporarily sheltered to allow the desired cloning.
Asunto(s)
Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Enzimas de Restricción del ADN/genética , Enzimas de Restricción del ADN/metabolismo , ARN Guía de Kinetoplastida/genética , Sistemas CRISPR-Cas , Inmunoprecipitación de Cromatina , Clonación Molecular , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , División del ADN , Daño del ADN , Desoxirribonucleasa BamHI , Epigenómica , Edición Génica , Genoma , RibonucleoproteínasRESUMEN
BACKGROUND: In Saccharomyces cerevisiae, the immunosuppressant rapamycin engenders a profound modification in the transcriptional profile leading to growth arrest. Mutants devoid of Rrd1, a protein possessing in vitro peptidyl prolyl cis/trans isomerase activity, display striking resistance to the drug, although how Rrd1 activity is linked to the biological responses has not been elucidated. RESULTS: We now provide evidence that Rrd1 is associated with the chromatin and it interacts with RNA polymerase II. Circular dichroism revealed that Rrd1 mediates structural changes onto the C-terminal domain (CTD) of the large subunit of RNA polymerase II (Rpb1) in response to rapamycin, although this appears to be independent of the overall phosphorylation status of the CTD. In vitro experiments, showed that recombinant Rrd1 directly isomerizes purified GST-CTD and that it releases RNA polymerase II from the chromatin. Consistent with this, we demonstrated that Rrd1 is required to alter RNA polymerase II occupancy on rapamycin responsive genes. CONCLUSION: We propose as a mechanism, that upon rapamycin exposure Rrd1 isomerizes Rpb1 to promote its dissociation from the chromatin in order to modulate transcription.