Loss of the yeast transporter Agp2 upregulates the pleiotropic drug-resistant pump Pdr5 and confers resistance to the protein synthesis inhibitor cycloheximide.

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.

Altered Regulation of the Glucose Transporter GLUT3 in PRDX1 Null Cells Caused Hypersensitivity to Arsenite.

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.

A CRISPR-based approach using dead Cas9-sgRNA to detect SARS-CoV-2.

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.

The histone H2B Arg95 residue efficiently recruits the transcription factor Spt16 to mediate Ste5 expression of the pheromone response pathway.

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.

The Yeast Permease Agp2 Senses Cycloheximide and Undergoes Degradation That Requires the Small Protein Brp1-Cellular Fate of Agp2 in Response to Cycloheximide.

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.

Mft1, identified from a genome-wide screen of the yeast haploid mutants, mediates cell cycle arrest to counteract quinoxaline-induced toxicity.

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.

C. elegans ribosomal protein S3 protects against H2O2-induced DNA damage and suppresses spontaneous mutations in yeast.

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.

Can Cisplatin Therapy Be Improved? Pathways That Can Be Targeted.

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.

The histone H2B Arg95 residue links the pheromone response pathway to rapamycin-induced G1 arrest in yeast.

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.

A simple protocol to isolate a single human cell PRDX1 knockout generated by CRISPR-Cas9 system.

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.

A Screening Method to Identify Essential Yeast Genes for Responses Towards Spermine.

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.

The base excision repair process: comparison between higher and lower eukaryotes.

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.

Dead Cas9-sgRNA Complex Shelters Vulnerable DNA Restriction Enzyme Sites from Cleavage for Cloning Applications.

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.

The Base Excision Repair Pathway in the Nematode Caenorhabditis elegans.

Exogenous and endogenous damage to the DNA is inevitable. Several DNA repair pathways including base excision, nucleotide excision, mismatch, homologous and non-homologous recombinations are conserved across all organisms to faithfully maintain the integrity of the genome. The base excision repair (BER) pathway functions to repair single-base DNA lesions and during the process creates the premutagenic apurinic/apyrimidinic (AP) sites. In this review, we discuss the components of the BER pathway in the nematode Caenorhabditis elegans and delineate the different phenotypes caused by the deletion or the knockdown of the respective DNA repair gene, as well as the implications. To date, two DNA glycosylases have been identified in C. elegans, the monofunctional uracil DNA glycosylase-1 (UNG-1) and the bifunctional endonuclease III-1 (NTH-1) with associated AP lyase activity. In addition, the animal possesses two AP endonucleases belonging to the exonuclease-3 and endonuclease IV families and in C. elegans these enzymes are called EXO-3 and APN-1, respectively. In mammalian cells, the DNA polymerase, Pol beta, that is required to reinsert the correct bases for DNA repair synthesis is not found in the genome of C. elegans and the evidence indicates that this role could be substituted by DNA polymerase theta (POLQ), which is known to perform a function in the microhomology-mediated end-joining pathway in human cells. The phenotypes observed by the C. elegans mutant strains of the BER pathway raised many challenging questions including the possibility that the DNA glycosylases may have broader functional roles, as discuss in this review.

CRISPR FokI Dead Cas9 System: Principles and Applications in Genome Engineering.

The identification of the robust clustered regularly interspersed short palindromic repeats (CRISPR) associated endonuclease (Cas9) system gene-editing tool has opened up a wide range of potential therapeutic applications that were restricted by more complex tools, including zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Nevertheless, the high frequency of CRISPR system off-target activity still limits its applications, and, thus, advanced strategies for highly specific CRISPR/Cas9-mediated genome editing are continuously under development including CRISPR-FokI dead Cas9 (fdCas9). fdCas9 system is derived from linking a FokI endonuclease catalytic domain to an inactive Cas9 protein and requires a pair of guide sgRNAs that bind to the sense and antisense strands of the DNA in a protospacer adjacent motif (PAM)-out orientation, with a defined spacer sequence range around the target site. The dimerization of FokI domains generates DNA double-strand breaks, which activates the DNA repair machinery and results in genomic edit. So far, all the engineered fdCas9 variants have shown promising gene-editing activities in human cells when compared to other platforms. Herein, we review the advantages of all published variants of fdCas9 and their current applications in genome engineering.

Yeast Lacking the PP2A Phosphatase Regulatory Subunit Rts1 Sensitizes rad51 Mutants to Specific DNA Damaging Agents.

Rts1 is a regulatory subunit of the trimeric protein phosphatase 2A phosphatase and it participates in many biological processes by modulating the phosphorylation status of proteins. Consistent with its role, mutants lacking Rts1 display multiple phenotypes. We have previously performed a high throughput screen to search for yeast haploid mutants with altered sensitivity to the anticancer drug bleomycin, which acts by damaging the DNA to produce single and double strand breaks. RTS1 was among the genes that when singly deleted cause sensitivity to bleomycin. We investigate whether Rts1 plays a role in the repair of bleomycin-induced DNA lesions. We show that deletion of the RTS1 gene in the rad51 null background, lacking Rad51 known to be involved in the repair of bleomycin-induced DNA lesions, resulted in double mutants that were sensitized to bleomycin and not to other DNA damaging agents that creates DNA adducts. We further show that Rts1 has the ability to bind to DNA and in its absence cells displayed an increase in the frequency of both spontaneous and bleomycin-induced mutations compared to the parent. This is the first report implicating Rts1 with a role in DNA damage and repair, perhaps regulating the phosphorylation status of one or more proteins involved in the repair of DNA strand breaks.

The human organic cation transporter OCT1 and its role as a target for drug responses.

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.

UNG-1 and APN-1 are the major enzymes to efficiently repair 5-hydroxymethyluracil DNA lesions in C. elegans.

In Caenorhabditis elegans, two DNA glycosylases, UNG-1 and NTH-1, and two AP endonucleases, APN-1 and EXO-3, have been characterized from the base-excision repair (BER) pathway that repairs oxidatively modified DNA bases. UNG-1 removes uracil, while NTH-1 can remove 5-hydroxymethyluracil (5-hmU), an oxidation product of thymine, as well as other lesions. Both APN-1 and EXO-3 can incise AP sites and remove 3'-blocking lesions at DNA single strand breaks, and only APN-1 possesses 3'- to 5'-exonulease and nucleotide incision repair activities. We used C. elegans mutants to study the role of the BER pathway in processing 5-hmU. We observe that ung-1 mutants exhibited a decrease in brood size and lifespan, and an elevated level of germ cell apoptosis when challenged with 5-hmU. These phenotypes were exacerbated by RNAi downregulation of apn-1 in the ung-1 mutant. The nth-1 or exo-3 mutants displayed wild type phenotypes towards 5-hmU. We show that partially purified UNG-1 can act on 5-hmU lesion in vitro. We propose that UNG-1 removes 5-hmU incorporated into the genome and the resulting AP site is cleaved by APN-1 or EXO-3. In the absence of UNG-1, the 5-hmU is removed by NTH-1 creating a genotoxic 3'-blocking lesion that requires the action of APN-1.