ABSTRACT
Glycoprotein 3 (GP3) serves as a structural protein in equine arteritis virus (EAV), forming a heterotrimeric complex that plays a pivotal role in virus tropism. In this study, we tested the membrane topology of GP3, both when expressed separately and during infection with recombinant tagged EAV GP3-HA. In our antibody accessibility experiment, we made a noteworthy discovery: GP3, when expressed separately, exhibits a dual topology. We introduced an additional N-glycosylation site, which was only partially used, providing further evidence for the dual topology of GP3. Intriguingly, this mutated GP3 was secreted into the medium, a result of the disruption of the ER retention motif RXR. The additional glycosylation site was not used when we examined the recombinant EAV virus with the same mutation. Despite the fact of higher expression levels of mutant GP3-HA, the protein was not secreted, and the recombinant mutant virus did not have growth delay compared to the EAV wild-type virus. This finding suggests that GP3 has a single type one membrane topology in virus infected cells, whereas the expression of GP3 in trans results in the dual topology of this protein. The RXR motif in the C-terminus is a co-factor of ER retention of the protein, but the main retention signal remains elusive.
Subject(s)
Amino Acid Motifs , Endoplasmic Reticulum , Equartevirus , Equartevirus/genetics , Equartevirus/metabolism , Animals , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/virology , Arginine/metabolism , Arginine/genetics , Viral Envelope Proteins/genetics , Viral Envelope Proteins/metabolism , Viral Envelope Proteins/chemistry , Glycosylation , Cell Line , Horses , HumansABSTRACT
Objective: To examine whether the DDAH2 promoter polymorphisms -1415G/A (rs2272592), -1151A/C (rs805304) and -449G/C (rs805305), and their haplotypes, are associated with PE compared with normotensive pregnant women, and whether they affect ADMA levels in these groups. Methods: A total of 208 pregnant women were included in the study and classified as early-onset (N=57) or late-onset PE (N =49), and as normotensive pregnant women (N = 102). Results: Pregnant with early-onset PE carrying the GC and GG genotypes for the DDAH2 -449G/C polymorphism had increased ADMA levels (P=0.01). No association of DDAH2 polymorphisms with PE in single-locus analysis was found. However, the G-C-G haplotype was associated with the risk for late-onset PE. Conclusion: It is suggested that DDAH2 polymorphisms could affect ADMA levels in PE, and that DDAH2 haplotypes may affect the risk for PE.
Subject(s)
Amidohydrolases , Arginine , Haplotypes , Polymorphism, Genetic , Pre-Eclampsia , Humans , Female , Amidohydrolases/genetics , Pre-Eclampsia/genetics , Pre-Eclampsia/blood , Pregnancy , Adult , Arginine/analogs & derivatives , Arginine/blood , Arginine/genetics , Young AdultABSTRACT
Emerging evidence indicates that arginine methylation promotes the stability of arginine-glycine-rich (RGG) motif-containing RNA-binding proteins (RBPs) and regulates gene expression. Here, we report that post-translational modification of FXR1 enhances the binding with mRNAs and is involved in cancer cell growth and proliferation. Independent point mutations in arginine residues of FXR1's nuclear export signal (R386 and R388) and RGG (R453, R455 and R459) domains prevent it from binding to RNAs that form G-quadruplex (G4) RNA structures. Disruption of G4-RNA structures by lithium chloride failed to bind with FXR1, indicating its preference for G4-RNA structure containing mRNAs. Furthermore, loss-of-function of PRMT5 inhibited FXR1 methylation both in vivo and in vitro, affecting FXR1 protein stability, inhibiting RNA-binding activity and cancer cell growth and proliferation. Finally, the enhanced crosslinking and immunoprecipitation (eCLIP) analyses reveal that FXR1 binds with the G4-enriched mRNA targets such as AHNAK, MAP1B, AHNAK2, HUWE1, DYNC1H1 and UBR4 and controls its mRNA expression in cancer cells. Our findings suggest that PRMT5-mediated FXR1 methylation is required for RNA/G4-RNA binding, which promotes gene expression in cancer cells. Thus, FXR1's structural characteristics and affinity for RNAs preferentially G4 regions provide new insights into the molecular mechanism of FXR1 in oral cancer cells.
Subject(s)
Arginine , Cell Proliferation , Protein-Arginine N-Methyltransferases , RNA-Binding Proteins , Humans , Protein-Arginine N-Methyltransferases/metabolism , Protein-Arginine N-Methyltransferases/genetics , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , Arginine/metabolism , Arginine/genetics , Methylation , RNA, Messenger/metabolism , RNA, Messenger/genetics , Cell Line, Tumor , Protein Binding , G-Quadruplexes , Gene Expression Regulation, Neoplastic , Repressor Proteins/metabolism , Repressor Proteins/genetics , Repressor Proteins/chemistry , Protein Processing, Post-Translational , Neoplasms/genetics , Neoplasms/metabolism , HEK293 Cells , Protein StabilityABSTRACT
Recent studies have revealed that arginine is the most favorable target of amino acid alteration in most cancer types and it has been suggested that the high preference for arginine mutations reflects the critical roles of this amino acid in the function of proteins. High rates of mutations of arginine residues in cancer, however, might also be due to increased mutability of arginine codons of the CGN family as the CpG dinucleotides of these codons may be methylated. In the present work we have analyzed spectra of single base substitutions of cancer genes (oncogenes, tumor suppressor genes) and passenger genes in cancer tissues to assess the contributions of CpG hypermutability and selection to arginine mutations. Our studies have shown that arginines encoded by the CGN codon family display higher rates of mutation in both cancer genes and passenger genes than arginine codons AGA and AGG that are devoid of CpG dinucleotide, suggesting that the predominance of arginine mutations in cancer is primarily due to CpG hypermutability, rather than selection for arginine replacement. Nevertheless, our results also suggest that CGN codons for arginines may serve as Achilles' heels of cancer genes. CpG hypermutability of key arginines of proto-oncogenes, leading to high rates of recurrence of driver mutations, contributes significantly to carcinogenesis. Similarly, our results indicate that hypermutability of the CpG dinucleotide of CGA codons (converting them to TGA stop codons) contributes significantly to recurrent truncation and inactivation of tumor suppressor genes.
Subject(s)
Arginine , Codon , CpG Islands , Neoplasms , Arginine/genetics , Arginine/chemistry , Humans , Codon/genetics , Neoplasms/genetics , CpG Islands/genetics , Mutation , Oncogenes/genetics , Genes, Tumor SuppressorABSTRACT
N-terminal arginine (NTR) methylation is a conserved feature of PIWI proteins, which are central components of the PIWI-interacting RNA (piRNA) pathway. The significance and precise function of PIWI NTR methylation in mammals remains unknown. In mice, PIWI NTRs bind Tudor domain containing proteins (TDRDs) that have essential roles in piRNA biogenesis and the formation of the chromatoid body. Using mouse MIWI (PIWIL1) as paradigm, we demonstrate that the NTRs are essential for spermatogenesis through the regulation of transposons and gene expression. The loss of TDRD5 and TDRKH interaction with MIWI results in attenuation of piRNA amplification. We find that piRNA amplification is necessary for transposon control and for sustaining piRNA levels including select, nonconserved, pachytene piRNAs that target specific mRNAs required for spermatogenesis. Our findings support the notion that the vast majority of pachytene piRNAs are dispensable, acting as self-serving genetic elements that rely for propagation on MIWI piRNA amplification. MIWI-NTRs also mediate interactions with TDRD6 that are necessary for chromatoid body compaction. Furthermore, MIWI-NTRs promote stabilization of spermiogenic transcripts that drive nuclear compaction, which is essential for sperm formation. In summary, the NTRs underpin the diversification of MIWI protein function.
Subject(s)
Arginine , Argonaute Proteins , Pachytene Stage , RNA, Small Interfering , Spermatogenesis , Animals , Male , Mice , Arginine/metabolism , Arginine/genetics , Argonaute Proteins/metabolism , Argonaute Proteins/genetics , DNA Transposable Elements , Piwi-Interacting RNA , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , RNA-Binding Proteins , Tudor DomainABSTRACT
Perilipin-2 (PLIN2) can anchor to lipid droplets (LDs) and play a crucial role in regulating nascent LDs formation. Bimolecular fluorescence complementation (BiFC) and flow cytometry were examined to verify the PLIN2-CGI-58 interaction efficiency in bovine adipocytes. GST-Pulldown assay was used to detect the key site arginine315 function in PLIN2-CGI-58 interaction. Experiments were also examined to research these mutations function of PLIN2 in LDs formation during adipocytes differentiation, LDs were measured after staining by BODIPY, lipogenesis-related genes were also detected. Results showed that Leucine (L371A, L311A) and glycine (G369A, G376A) mutations reduced interaction efficiencies. Serine (S367A) mutations enhanced the interaction efficiency. Arginine (R315A) mutations resulted in loss of fluorescence in the cytoplasm and disrupted the interaction with CGI-58, as verified by pulldown assay. R315W mutations resulted in a significant increase in the number of LDs compared with wild-type (WT) PLIN2 or the R315A mutations. Lipogenesis-related genes were either up- or downregulated when mutated PLIN2 interacted with CGI-58. Arginine315 in PLIN2 is required for the PLIN2-CGI-58 interface and could regulate nascent LD formation and lipogenesis. This study is the first to study amino acids on the PLIN2 interface during interaction with CGI-58 in bovine and highlight the role played by PLIN2 in the regulation of bovine adipocyte lipogenesis.
Subject(s)
Arginine , Lipid Droplets , Animals , Cattle , Perilipin-2/genetics , Perilipin-2/chemistry , Perilipin-2/metabolism , Arginine/genetics , Arginine/metabolism , Lipid Droplets/metabolism , Mutation , Adipocytes/metabolism , Lipid MetabolismABSTRACT
ALKBH5 is a master regulator of N6-methyladenosine (m6A) modification, which plays a crucial role in many biological processes. Here, we show that ALKBH5 is required for breast tumor growth. Interestingly, PRMT6 directly methylates ALKBH5 at R283, which subsequently promotes breast tumor growth. Furthermore, arginine methylation of ALKBH5 by PRMT6 increases LDHA RNA stability via m6A demethylation, leading to increased aerobic glycolysis. Moreover, PRMT6-mediated ALKBH5 arginine methylation is confirmed in PRMT6-knockout mice. Collectively, these findings identify a PRMT6-ALKBH5-LDHA signaling axis as a novel target for the treatment of breast cancer.
Subject(s)
AlkB Homolog 5, RNA Demethylase , Arginine , Breast Neoplasms , Glycolysis , Protein-Arginine N-Methyltransferases , Protein-Arginine N-Methyltransferases/metabolism , Protein-Arginine N-Methyltransferases/genetics , Animals , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Female , Humans , Mice , AlkB Homolog 5, RNA Demethylase/metabolism , AlkB Homolog 5, RNA Demethylase/genetics , Methylation , Arginine/metabolism , Arginine/analogs & derivatives , Arginine/genetics , Carcinogenesis/genetics , Mice, Knockout , Cell Line, Tumor , Nuclear ProteinsABSTRACT
Hexanucleotide repeat expansion in C9ORF72 (C9) is the most prevalent mutation among amyotrophic lateral sclerosis (ALS) patients. The patients carry over ~30 to hundreds or thousands of repeats translated to dipeptide repeats (DPRs) where poly-glycine-arginine (GR) and poly-proline-arginine (PR) are most toxic. The structure-function relationship is still unknown. Here, we examined the minimal neurotoxic repeat number of poly-GR and found that extension of the repeat number led to a loose helical structure disrupting plasma and nuclear membrane. Poly-GR/PR bound to nucleotides and interfered with transcription. We screened and identified a sulfated disaccharide that bound to poly-GR/PR and rescued poly-GR/PR-induced toxicity in neuroblastoma and C9-ALS-iPSC-derived motor neurons. The compound rescued the shortened life span and defective locomotion in poly-GR/PR expressing Drosophila model and improved motor behavior in poly-GR-injected mouse model. Overall, our results reveal structural and toxicity mechanisms for poly-GR/PR and facilitate therapeutic development for C9-ALS.
Subject(s)
Amyotrophic Lateral Sclerosis , Animals , Mice , Humans , Amyotrophic Lateral Sclerosis/drug therapy , Amyotrophic Lateral Sclerosis/genetics , Dipeptides/pharmacology , Arginine/genetics , Sulfates , Drosophila/genetics , DNA Damage , DNA Repeat Expansion , C9orf72 Protein/genetics , C9orf72 Protein/metabolismABSTRACT
Salinity, a key limiting factor, affects the distribution and survival of marine species. The Hong Kong oyster (Crassostrea hongkongensis), a euryhaline species found along the coast of the South China Sea, has become a major aquaculture bivalve species. To determine the molecular mechanism by which oysters respond to coastal waters with varying salinity levels, we used RNA-seq to sequence the gill samples of oysters exposed to normal (25 , S25), low (5 , S5) and high (35 , S35) salinity conditions for one month. The results revealed different expression transcriptome levels among oysters living under low and high salinity conditions. Using high-throughput sequencing, we identified 811 up-regulated genes and 769 down-regulated genes. As determined by KEGG pathway mapping, the differentially expressed genes (DEGs) were significantly enriched in the prion diseases, histidine metabolism, arginine and proline metabolism, and beta-alanine metabolism pathways in both the S5 vs. S25 and S35 vs. S25 group comparison. Several DEGs including heat shock 70 kDa protein 12B-like, poly (ADP-ribose) polymerase (PARP), and tripartite motif-containing protein 2 (TRIM2), and low-density lipoprotein receptor-like, as well as KEGG pathways, including arginine and proline metabolism, apoptosis, PPAR signaling pathway, the thyroid hormone signaling pathway, were concerning response to salinity stress. Additionally, eight DEGs involved in salinity adaptation were selected for RT-qPCR validation, and the results confirmed the credibility of the transcriptome sequencing data. Overall, we designed a one-month, medium-term experiment to examine the responses of C. hongkongensis exposed to different levels of salinity stress and performed transcriptome analysis using high-throughput sequencing. Our results enhance current understanding of the molecular mechanisms of salinity stress responses in C. hongkongensis and provided insights into the osmotic biology of oysters.
Subject(s)
Crassostrea , Animals , Crassostrea/metabolism , Transcriptome , Salt Stress , Arginine/genetics , Arginine/metabolism , Proline/genetics , Proline/metabolism , SalinityABSTRACT
The MT-TL2 m.12315G>A pathogenic variant has previously been reported in five individuals with mild clinical phenotypes. Herein we report the case of a 5-year-old child with heteroplasmy for this variant who developed neurological regression and stroke-like episodes similar to those observed in mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). Biochemical evaluation revealed depletion of arginine on plasma amino acid analysis and low z-scores for citrulline on untargeted plasma metabolomics analysis. These findings suggested that decreased availability of nitric oxide may have contributed to the stroke-like episodes. The use of intravenous arginine during stroke-like episodes and daily enteral L-citrulline supplementation normalized her biochemical values of arginine and citrulline. Untargeted plasma metabolomics showed the absence of nicotinamide and 1-methylnicotinamide, and plasma total glutathione levels were low; thus, nicotinamide riboside and N-acetylcysteine therapies were initiated. This report expands the phenotype associated with the rare mitochondrial variant MT-TL2 m.12315G>A to include neurological regression and a MELAS-like phenotype. Individuals with this variant should undergo in-depth biochemical analysis to include untargeted plasma metabolomics, plasma amino acids, and glutathione levels to help guide a targeted approach to treatment.
Subject(s)
Acidosis, Lactic , MELAS Syndrome , Mitochondrial Encephalomyopathies , Stroke , Child, Preschool , Female , Humans , Arginine/genetics , Citrulline , Glutathione/metabolism , MELAS Syndrome/diagnosis , MELAS Syndrome/genetics , MELAS Syndrome/complications , Nitric Oxide Donors/metabolism , Stroke/complications , Stroke/drug therapyABSTRACT
Histone H4 asymmetrically dimethylated at arginine 3 (H4R3me2a) is an active histone mark catalyzed by protein arginine methyltransferase 1 (PRMT1), a major arginine methyltransferase in vertebrates catalyzing asymmetric dimethylation of arginine. H4R3me2a stimulates the activity of lysine acetyltransferases such as CBP/p300, which catalyze the acetylation of H3K27, a mark of active enhancers, super-enhancers, and promoters. There are a few studies on the genomic location of H4R3me2a. In chicken polychromatic erythrocytes, H4R3me2a is found in introns and intergenic regions and binds to the globin locus control region (a super-enhancer) and globin regulatory regions. In this report, we analyzed chromatin immunoprecipitation sequencing data for the genomic location of H4R3me2a in the breast cancer cell line MCF7. As in avian cells, MCF7 H4R3me2a is present in intronic and intergenic regions. Nucleosomes with H4R3me2a and H3K27ac next to nucleosome-free regions are found at super-enhancers, enhancers, and promoter regions of expressed genes. Genes with critical roles in breast cancer cells have broad domains of nucleosomes with H4R3me2a, H3K27ac, and H3K4me3. Our results are consistent with PRMT1-mediated H4R3me2a playing a key role in the chromatin organization of regulatory regions of vertebrate genomes.
Subject(s)
Histones , Nucleosomes , Animals , Histones/genetics , Histones/metabolism , Arginine/genetics , DNA, Intergenic , Globins/genetics , Globins/metabolism , Chromatin , AcetylationABSTRACT
Cadmium (Cd), which is a nonessential heavy metal element for organisms, can have a severe impact on the growth and development of organisms that absorb excessive Cd. Studies have shown that Brassica carinata, a semiwild oil crop, has strong tolerance to various abiotic stresses, and RNA-seq has revealed that the B. carinata superoxide dismutase gene (BcaSOD1) likely responds to Cd stress. To elucidate the BcaSOD1 function involved in tolerance of Cd stress, we cloned the coding sequences of BcaSOD1 from a purple B. carinata accession and successfully transferred it into Arabidopsis thaliana. The subcellular localization results demonstrated that BcaSOD1 was primarily located in the plasma membrane, mitochondria and nucleus. Overexpression of BcaSOD1 in transgenic Arabidopsis (OE) effectively decreased the toxicity caused by Cd stress. Compared to the WT (wild type lines), the OE lines exhibited significantly increased activities of antioxidant enzymes (APX, CAT, POD, and SOD) after exposure to 2.5 mM CdCl2. The Cd content of underground (root) in the OE line was dominantly higher than that in the WT; however, the Cd content of aboveground (shoot) was comparable between the OE and WT types. Moreover, the qRTâPCR results showed that several heavy metal detoxification-related genes (AtIREG2, AtMTP3, AtHMA3, and AtNAS4) were significantly upregulated in the roots of OE lines under Cd treatment, suggesting that these genes are likely involved in Cd absorption in the roots of OE lines. In addition, both comparable transcriptome and qRT-PCR analyses revealed that exogenous BcaSOD1 noticeably facilitates detoxification by stimulating the expression of two arginine (Arg) biosynthesis genes (AtGDH1 and AtGDH2) while inhibiting the expression of AtARGAH1, a negative regulator in biosynthesis of Arg. The Arg content was subsequently confirmed to be significantly enhanced in OE lines under Cd treatment, indicating that BcaSOD1 likely strengthened Cd tolerance by regulating the expression of Arg-related genes. This study demonstrates that BcaSOD1 can enhance Cd tolerance and reveals the molecular mechanism of this gene, providing valuable insights into the molecular mechanism of Cd tolerance in plants.
Subject(s)
Arabidopsis , Metals, Heavy , Cadmium/toxicity , Cadmium/metabolism , Arabidopsis/metabolism , Superoxide Dismutase/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Metals, Heavy/toxicity , Metals, Heavy/metabolism , Arginine/genetics , Arginine/metabolismABSTRACT
Cell-penetrating peptides (CPPs), for example, arginine (Arg) rich peptides, are used for the intracellular delivery of nucleic acids. In this study, oligosarcosine-conjugated Arg-rich peptides were designed as plasmid DNA (pDNA) carriers, and the physicochemical parameters and transfection efficiency of the peptide/pDNA complexes were evaluated. Oligosarcosine with different lengths were conjugated to a base sequence composed of arginine and α-aminoisobutyric acid (Aib) [(Aib-Arg-Arg)3]. Oligosarcosine conjugation inhibited the aggregation of the complexes after mixing with pDNA, shielded the positive charge of the complexes, and provided efficient pDNA transfection in cultured cells. The efficiency of the pDNA transfection was improved by varying the length of the oligosarcosine moiety (10-15 units were optimal). The cellular uptake efficiency and intracellular distribution of pDNA were the same regardless of oligosarcosine conjugation. These results implied that intracellular processes, including the decondensation of pDNA, contributed to the efficiency of the protein expression from pDNA. This study demonstrated the advantages of oligosarcosine conjugation to Arg-rich CPPs and provided valuable insight into the future design of CPPs.
Subject(s)
Arginine , Cell-Penetrating Peptides , Arginine/genetics , DNA/chemistry , DNA/genetics , Plasmids/genetics , Transfection , Cell-Penetrating Peptides/pharmacology , Cell-Penetrating Peptides/chemistry , Cell-Penetrating Peptides/geneticsABSTRACT
BACKGROUND: Prostate cancer is a common solid tumor that affects a significant number of men worldwide. Conventional androgen deprivation therapy (ADT) increases the risk of developing castration-resistant prostate cancer (CRPC). Effective clinical management of patients with CRPC is challenging due to the limited understanding. METHODS: In this study, transcriptomic and metabolomic profiles of androgen-dependent prostate cancer cell line LNCaP and the androgen-independent cells developed from LNCaP cells (LNCaP-ADR) were investigated using RNA-sequencing and LC-MS/MS, respectively. The differentially expressed genes and metabolites were analyzed, and integrative analysis of transcriptomic and metabolomic data was further conducted to obtain a comprehensive understanding of the metabolic characteristics in LNCaP-ADR cells. Quantitative real-time PCR (QPCR) was employed to ascertain the mRNA expression levels of the selected differentially expressed genes. RESULTS: The arginine and proline metabolism pathway was identified as a commonly altered pathway at both the transcriptional and metabolic levels. In the LNCaP-ADR cells, significant upregulation was observed for metabolites including 5-Aminopentanoic acid, L-Arginine, L-Glutamic acid, N-Acetyl-L-alanine, and Pyrrole-2-carboxylic acid at the metabolic level. At the transcriptional level, MAOA, ALDH3A2, ALDH2, ARG1, CKMT2, and CNDP1 were found to be significantly upregulated in the LNCaP-ADR cells. Gene set enrichment analysis (GSEA) identified various enriched gene sets in the LNCaP-ADR cells, encompassing inflammatory response, 9plus2 motile cilium, motile cilium, ciliary plasm, cilium or flagellum-dependent cell motility, cilium movement, cilium, response to endoplasmic reticulum stress, PTEN DN.V1 DN, SRC UP.V1 UP, IL15 UP.V1 DN, RB DN.V1 DN, AKT UP MTOR DN.V1 UP, VEGF A UP.V1 UP, and KRAS.LUNG.BREAST UP.V1 UP. CONCLUSIONS: These findings highlight the substantial association between the arginine and proline metabolism pathway and CRPC, emphasizing the need to prioritize strategies that target dysregulated metabolites and differentially expressed genes as essential interventions in the clinical management of CRPC.
Subject(s)
Prostatic Neoplasms, Castration-Resistant , Prostatic Neoplasms , Male , Humans , Prostatic Neoplasms/pathology , Androgens/metabolism , Prostatic Neoplasms, Castration-Resistant/drug therapy , Androgen Antagonists/therapeutic use , Chromatography, Liquid , Tandem Mass Spectrometry , Gene Expression Profiling , Transcriptome , Arginine/genetics , Proline/genetics , Cell Line, Tumor , Receptors, Androgen/metabolism , Gene Expression Regulation, Neoplastic , Aldehyde Dehydrogenase, Mitochondrial/geneticsABSTRACT
BACKGROUND: Regulation of alternative splicing is a new therapeutic approach in cancer. The programmed cell death receptor 1 (PD-1) is an immunoinhibitory receptor expressed on immune cells that binds to its ligands, PD-L1 and PD-L2 expressed by cancer cells forming a dominant immune checkpoint pathway in the tumour microenvironment. Targeting this pathway using blocking antibodies (nivolumab and pembrolizumab) is the mainstay of anti-cancer immunotherapies, restoring the function of exhausted T cells. PD-1 is alternatively spliced to form isoforms that are either transmembrane signalling receptors (flPD1) that mediate T cell death by binding to the ligand, PD-L1 or an alternatively spliced, soluble, variant that lacks the transmembrane domain. METHODS: We used PCR and western blotting on primary peripheral blood mononuclear cells (PBMCs) and Jurkat T cells, IL-2 ELISA, flow cytometry, co-culture of melanoma and cholangiocarcinoma cells, and bioinformatics analysis and molecular cloning to examine the mechanism of splicing of PD1 and its consequence. RESULTS: The soluble form of PD-1, generated by skipping exon 3 (∆Ex3PD1), was endogenously expressed in PBMCs and T cells and prevents cancer cell-mediated T cell repression. Multiple binding sites of SRSF1 are adjacent to PD-1 exon 3 splicing sites. Overexpression of phosphomimic SRSF1 resulted in preferential expression of flPD1. Inhibition of SRSF1 phosphorylation both by SRPK1 shRNA knockdown and by a selective inhibitor, SPHINX31, resulted in a switch in splicing to ∆Ex3PD1. Cholangiocarcinoma cell-mediated repression of T cell IL-2 expression was reversed by SPHINX31 (equivalent to pembrolizumab). CONCLUSIONS: These results indicate that switching of the splicing decision from flPD1 to ∆Ex3PD1 by targeting SRPK1 could represent a potential novel mechanism of immune checkpoint inhibition in cancer.
Subject(s)
Alternative Splicing , Cholangiocarcinoma , Humans , Phosphorylation , B7-H1 Antigen/genetics , B7-H1 Antigen/metabolism , Protein Serine-Threonine Kinases/genetics , RNA Splicing Factors/genetics , RNA Splicing Factors/metabolism , Arginine/genetics , Arginine/metabolism , Serine/chemistry , Serine/genetics , Serine/metabolism , T-Cell Exhaustion , Interleukin-2/genetics , Leukocytes, Mononuclear/metabolism , Programmed Cell Death 1 Receptor/metabolism , Serine-Arginine Splicing Factors/genetics , Protein Isoforms/genetics , Protein Isoforms/metabolism , ImmunotherapyABSTRACT
Background: The E6 oncoprotein of HPV plays a crucial role in promoting cell proliferation and inhibiting apoptosis, leading to tumor growth. Non-viral vectors such as nona-arginine (R9) peptides have shown to be potential as carriers for therapeutic molecules. This study aimed to investigate the efficacy of nona-arginine in delivering E6 shRNA and suppressing the E6 gene of HeLa cells in vitro. Methods: HeLa cells carrying E6 gene were treated with a complex of nona-arginine and E6 shRNA. The complex was evaluated using gel retardation assay and FESEM microscopy. The optimal N/P ratio for R9 peptide to transfect HeLa cells with luciferase gene was determined. Relative real-time PCR was used to evaluate the efficiency of mRNA suppression efficiency for E6 shRNA, while the effect of E6 shRNA on cell viability was measured using an MTT assay. Results: The results indicated that R9 efficiently binds to shRNA and effectively transfects E6 shRNA complexes at N/P ratios greater than 30. Transfection with R9 and PEI complexes resulted in a significant toxicity compared to the scrambled plasmid, indicating selective toxicity for HeLa cells. Real-time PCR confirmed the reduction of E6 mRNA expression levels in the cells transfected with anti-E6 shRNA. Conclusion: The study suggests that R9 is a promising non-viral gene carrier for transfecting E6 shRNA in vitro, with significant transfection efficiency and minimal toxicity.
Subject(s)
Oncogene Proteins, Viral , Uterine Cervical Neoplasms , Humans , Female , RNA, Small Interfering/genetics , HeLa Cells , Oncogene Proteins, Viral/genetics , Oncogene Proteins, Viral/metabolism , Repressor Proteins/metabolism , Apoptosis/genetics , RNA, Messenger/genetics , Arginine/pharmacology , Arginine/genetics , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/pathology , Transfection , Cell Line, TumorABSTRACT
Arginine is high in hepatocellular carcinoma and promotes tumorigenesis by altering metabolic gene expression.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Humans , Liver Neoplasms/metabolism , Arginine/genetics , Arginine/metabolism , Carcinoma, Hepatocellular/metabolism , Carcinogenesis/pathology , Liver/metabolismABSTRACT
Identifying genetic modifiers of familial amyotrophic lateral sclerosis (ALS) may reveal targets for therapeutic modulation with potential application to sporadic ALS. GGGGCC (G4C2) repeat expansions in the C9orf72 gene underlie the most common form of familial ALS, and generate toxic arginine-containing dipeptide repeats (DPRs), which interfere with membraneless organelles, such as the nucleolus. Here we considered senataxin (SETX), the genetic cause of ALS4, as a modifier of C9orf72 ALS, because SETX is a nuclear helicase that may regulate RNA-protein interactions involved in ALS dysfunction. After documenting that decreased SETX expression enhances arginine-containing DPR toxicity and C9orf72 repeat expansion toxicity in HEK293 cells and primary neurons, we generated SETX fly lines and evaluated the effect of SETX in flies expressing either (G4C2)58 repeats or glycine-arginine-50 [GR(50)] DPRs. We observed dramatic suppression of disease phenotypes in (G4C2)58 and GR(50) Drosophila models, and detected a striking relocalization of GR(50) out of the nucleolus in flies co-expressing SETX. Next-generation GR(1000) fly models, that show age-related motor deficits in climbing and movement assays, were similarly rescued with SETX co-expression. We noted that the physical interaction between SETX and arginine-containing DPRs is partially RNA-dependent. Finally, we directly assessed the nucleolus in cells expressing GR-DPRs, confirmed reduced mobility of proteins trafficking to the nucleolus upon GR-DPR expression, and found that SETX dosage modulated nucleolus liquidity in GR-DPR-expressing cells and motor neurons. These findings reveal a hitherto unknown connection between SETX function and cellular processes contributing to neuron demise in the most common form of familial ALS.
Subject(s)
Amyotrophic Lateral Sclerosis , Frontotemporal Dementia , Humans , Animals , Amyotrophic Lateral Sclerosis/metabolism , Dipeptides/genetics , C9orf72 Protein/genetics , C9orf72 Protein/metabolism , Arginine/genetics , Arginine/metabolism , HEK293 Cells , Motor Neurons/metabolism , Drosophila/metabolism , RNA/metabolism , Frontotemporal Dementia/genetics , DNA Repeat Expansion/genetics , DNA Helicases/genetics , RNA Helicases/genetics , Multifunctional Enzymes/geneticsABSTRACT
The Dam1 complex is essential for mitotic progression across evolutionarily divergent fungi. Upon analyzing amino acid (aa) sequences of Dad2, a Dam1 complex subunit, we identified a conserved 10-aa-long Dad2 signature sequence (DSS). An arginine residue (R126) in the DSS is essential for viability in Saccharomyces cerevisiae that possesses point centromeres. The corresponding arginine residues are functionally important but not essential for viability in Candida albicans and Cryptococcus neoformans; both carry several kilobases long regional centromeres. The purified recombinant Dam1 complex containing either Dad2ΔDSS or Dad2R126A failed to bind microtubules (MTs) or form any visible rings like the WT complex. Intriguingly, functional analysis revealed that the requirement of the conserved arginine residue for chromosome biorientation and mitotic progression reduced with increasing centromere length. We propose that plasticity of the invariant arginine of Dad2 in organisms with regional centromeres is achieved by conditional elevation of the kinetochore protein(s) to enable multiple kinetochore MTs to bind to each chromosome. The capacity of a chromosome to bind multiple kinetochore MTs may mask the deleterious effects of such lethal mutations.