RESUMEN
Major histocompatibility complex class I (MHC-I) molecules, which are dimers of a glycosylated polymorphic transmembrane heavy chain and the small-protein ß2-microglobulin (ß2m), bind peptides in the endoplasmic reticulum that are generated by the cytosolic turnover of cellular proteins. In virus-infected cells, these peptides may include those derived from viral proteins. Peptide-MHC-I complexes then traffic through the secretory pathway and are displayed at the cell surface where those containing viral peptides can be detected by CD8+ T lymphocytes that kill infected cells. Many viruses enhance their in vivo survival by encoding genes that down-regulate MHC-I expression to avoid CD8+ T cell recognition. Here, we report that two accessory proteins encoded by SARS-CoV-2, the causative agent of the ongoing COVID-19 pandemic, down-regulate MHC-I expression using distinct mechanisms. First, ORF3a, a viroporin, reduces the global trafficking of proteins, including MHC-I, through the secretory pathway. The second, ORF7a, interacts specifically with the MHC-I heavy chain, acting as a molecular mimic of ß2m to inhibit its association. This slows the exit of properly assembled MHC-I molecules from the endoplasmic reticulum. We demonstrate that ORF7a reduces antigen presentation by the human MHC-I allele HLA-A*02:01. Thus, both ORF3a and ORF7a act post-translationally in the secretory pathway to lower surface MHC-I expression, with ORF7a exhibiting a specific mechanism that allows immune evasion by SARS-CoV-2.
Asunto(s)
COVID-19 , Antígenos de Histocompatibilidad Clase I , SARS-CoV-2 , Proteínas Reguladoras y Accesorias Virales , Humanos , Presentación de Antígeno , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Antígenos HLA , Péptidos , SARS-CoV-2/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismoRESUMEN
The TAP translocates peptide Ags into the lumen of the endoplasmic reticulum for loading onto MHC class I molecules. MHC class I acquires its peptide cargo in the peptide loading complex, an oligomeric complex that the chaperone tapasin organizes by bridging TAP to MHC class I and recruiting accessory molecules such as ERp57 and calreticulin. Three tapasin binding sites on TAP have been described, two of which are located in the N-terminal domains of TAP1 and TAP2. The third binding site is present in the core transmembrane (TM) domain of TAP1 and is used only by the unassembled subunits. Tapasin is required to promote TAP stability, but through which binding site(s) it is acting is unknown. In particular, the role of tapasin binding to the core TM domain of TAP1 single chains is mysterious because this interaction is lost upon TAP2 association. In this study, we map the respective binding site in TAP1 to the polar face of the amphipathic TM helix TM9 and identify key residues that are essential to establish the interaction. We find that this interaction is dispensable for the peptide transport function but essential to achieve full stability of human TAP1. The interaction is also required for proper heterodimerization of the transporter. Based on similar results obtained using TAP mutants that lack tapasin binding to either N-terminal domain, we conclude that all three tapasin-binding sites in TAP cooperate to achieve high transporter stability and efficient heterodimerization.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Multimerización de Proteína/inmunología , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Miembro 3 de la Subfamilia B de Transportadores de Casetes de Unión a ATP , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Sitios de Unión , Calreticulina/química , Calreticulina/genética , Calreticulina/inmunología , Calreticulina/metabolismo , Línea Celular Tumoral , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/inmunología , Proteína Disulfuro Isomerasas/genética , Proteína Disulfuro Isomerasas/inmunología , Estabilidad Proteica , Estructura Secundaria de ProteínaRESUMEN
BACKGROUND: The development of colorectal cancer (CRC) is accompanied by extensive epigenetic changes, including frequent regional hypermethylation particularly of gene promoter regions. Specific genes, including SEPT9, VIM1 and TMEFF2 become methylated in a high fraction of cancers and diagnostic assays for detection of cancer-derived methylated DNA sequences in blood and/or fecal samples are being developed. There is considerable potential for the development of new DNA methylation biomarkers or panels to improve the sensitivity and specificity of current cancer detection tests. METHODS: Combined epigenomic methods - activation of gene expression in CRC cell lines following DNA demethylating treatment, and two novel methods of genome-wide methylation assessment - were used to identify candidate genes methylated in a high fraction of CRCs. Multiplexed amplicon sequencing of PCR products from bisulfite-treated DNA of matched CRC and non-neoplastic tissue as well as healthy donor peripheral blood was performed using Roche 454 sequencing. Levels of DNA methylation in colorectal tissues and blood were determined by quantitative methylation specific PCR (qMSP). RESULTS: Combined analyses identified 42 candidate genes for evaluation as DNA methylation biomarkers. DNA methylation profiles of 24 of these genes were characterised by multiplexed bisulfite-sequencing in ten matched tumor/normal tissue samples; differential methylation in CRC was confirmed for 23 of these genes. qMSP assays were developed for 32 genes, including 15 of the sequenced genes, and used to quantify methylation in tumor, adenoma and non-neoplastic colorectal tissue and from healthy donor peripheral blood. 24 of the 32 genes were methylated in >50% of neoplastic samples, including 11 genes that were methylated in 80% or more CRCs and a similar fraction of adenomas. CONCLUSIONS: This study has characterised a panel of 23 genes that show elevated DNA methylation in >50% of CRC tissue relative to non-neoplastic tissue. Six of these genes (SOX21, SLC6A15, NPY, GRASP, ST8SIA1 and ZSCAN18) show very low methylation in non-neoplastic colorectal tissue and are candidate biomarkers for stool-based assays, while 11 genes (BCAT1, COL4A2, DLX5, FGF5, FOXF1, FOXI2, GRASP, IKZF1, IRF4, SDC2 and SOX21) have very low methylation in peripheral blood DNA and are suitable for further evaluation as blood-based diagnostic markers.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias Colorrectales/genética , Metilación de ADN/genética , Regulación Neoplásica de la Expresión Génica , Estudios de Asociación Genética/métodos , Biomarcadores de Tumor/metabolismo , Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales/metabolismo , Células HCT116 , Células HT29 , HumanosRESUMEN
Major histocompatibility complex class I (MHC-I) molecules, which are dimers of a glycosylated polymorphic transmembrane heavy chain and the small protein ß 2 -microglobulin (ß 2 m), bind peptides in the endoplasmic reticulum that are generated by the cytosolic turnover of cellular proteins. In virus-infected cells these peptides may include those derived from viral proteins. Peptide-MHC-I complexes then traffic through the secretory pathway and are displayed at the cell surface where those containing viral peptides can be detected by CD8 + T lymphocytes that kill infected cells. Many viruses enhance their in vivo survival by encoding genes that downregulate MHC-I expression to avoid CD8 + T cell recognition. Here we report that two accessory proteins encoded by SARS-CoV-2, the causative agent of the ongoing COVID-19 pandemic, downregulate MHC-I expression using distinct mechanisms. One, ORF3a, a viroporin, reduces global trafficking of proteins, including MHC-I, through the secretory pathway. The second, ORF7a, interacts specifically with the MHC-I heavy chain, acting as a molecular mimic of ß 2 m to inhibit its association. This slows the exit of properly assembled MHC-I molecules from the endoplasmic reticulum. We demonstrate that ORF7a reduces antigen presentation by the human MHC-I allele HLA-A*02:01. Thus, both ORF3a and ORF7a act post-translationally in the secretory pathway to lower surface MHC-I expression, with ORF7a exhibiting a novel and specific mechanism that allows immune evasion by SARS-CoV-2. Significance Statement: Viruses may down-regulate MHC class I expression on infected cells to avoid elimination by cytotoxic T cells. We report that the accessory proteins ORF7a and ORF3a of SARS-CoV-2 mediate this function and delineate the two distinct mechanisms involved. While ORF3a inhibits global protein trafficking to the cell surface, ORF7a acts specifically on MHC-I by competing with ß 2 m for binding to the MHC-I heavy chain. This is the first account of molecular mimicry of ß 2 m as a viral mechanism of MHC-I down-regulation to facilitate immune evasion.
RESUMEN
The pigment cell-specific protein PMEL forms a functional amyloid matrix in melanosomes onto which the pigment melanin is deposited. The amyloid core consists of a short proteolytic fragment, which we have termed the core-amyloid fragment (CAF) and perhaps additional parts of the protein, such as the PKD domain. A highly O-glycosylated repeat (RPT) domain also derived from PMEL proteolysis associates with the amyloid and is necessary to establish the sheet-like morphology of the assemblies. Excluded from the aggregate is the regulatory N-terminus, which nevertheless must be linked in cis to the CAF in order to drive amyloid formation. The domain is then likely cleaved away immediately before, during, or immediately after the incorporation of a new CAF subunit into the nascent amyloid. We had previously identified a 21 amino acid long region, which mediates the regulatory activity of the N-terminus towards the CAF. However, many mutations in the respective segment caused misfolding and/or blocked PMEL export from the endoplasmic reticulum, leaving their phenotype hard to interpret. Here, we employ a saturating mutagenesis approach targeting the motif at single amino acid resolution. Our results confirm the critical nature of the PMEL N-terminal region and identify several residues essential for PMEL amyloidogenesis.
Asunto(s)
Aminoácidos/química , Dominios Proteicos , Antígeno gp100 del Melanoma/química , Secuencia de Aminoácidos , Retículo Endoplásmico/metabolismo , Humanos , Melanosomas/metabolismo , Mutación , Pliegue de Proteína , Transporte de Proteínas , Fracciones Subcelulares/metabolismo , Antígeno gp100 del Melanoma/metabolismoRESUMEN
PMEL is a pigment cell-specific protein that forms a functional amyloid matrix in melanosomes. The matrix consists of well-separated fibrillar sheets on which the pigment melanin is deposited. Using electron tomography, we demonstrate that this sheet architecture is governed by the PMEL repeat (RPT) domain, which associates with the amyloid as an accessory proteolytic fragment. Thus, the RPT domain is dispensable for amyloid formation as such but shapes the morphology of the matrix, probably in order to maximize the surface area available for pigment adsorption. Although the primary amino acid sequence of the RPT domain differs vastly among various vertebrates, we show that it is a functionally conserved, interchangeable module. RPT domains of all species are predicted to be very highly O-glycosylated, which is likely the common defining feature of this domain. O-glycosylation is indeed essential for RPT domain function and the establishment of the PMEL sheet architecture. Thus, O-glycosylation, not amino acid sequence, appears to be the major factor governing the characteristic PMEL amyloid morphology.
Asunto(s)
Proteínas Amiloidogénicas/química , Melanosomas/metabolismo , Dominios Proteicos , Antígeno gp100 del Melanoma/química , Animales , Línea Celular Tumoral , Pollos , Colubridae , Glicosilación , Humanos , Ratones , Polisacáridos/química , Xenopus laevis , Pez CebraRESUMEN
Over the last few years a number of restriction enzymes that cut DNA only if cytosines within their recognition sequences are methylated have been characterized and become commercially available. Cleavage with these enzymes to release DNA fragments in a methylation-dependent manner can be combined with a novel method of amplification, Helper Dependent Chain Reaction (HDCR), to selectively amplify these fragments. HDCR uses "Helper" oligonucleotides as templates for extension of the free 3' end of target fragments to incorporate tag sequences at the ends of fragments. These tag sequences are then used for priming of amplification of target fragments. Modifications to the amplification primers (Drivers) and the Helpers ensure that there is selection for the sequences within target fragments with each cycle of amplification. The combination of methylation-dependent enzymes and HDCR allows the sensitive and selective amplification of methylated DNA sequences without the need for bisulfite treatment.
Asunto(s)
Metilación de ADN , Enzimas de Restricción del ADN/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Células CACO-2 , Línea Celular , Cartilla de ADN/genética , Epigénesis Genética , Humanos , SulfitosRESUMEN
Selective amplification in PCR is principally determined by the sequence of the primers and the temperature of the annealing step. We have developed a new PCR technique for distinguishing related sequences in which additional selectivity is dependent on sequences within the amplicon. A 5' extension is included in one (or both) primer(s) that corresponds to sequences within one of the related amplicons. After copying and incorporation into the PCR product this sequence is then able to loop back, anneal to the internal sequences and prime to form a hairpin structure-this structure is then refractory to further amplification. Thus, amplification of sequences containing a perfect match to the 5' extension is suppressed while amplification of sequences containing mismatches or lacking the sequence is unaffected. We have applied Headloop PCR to DNA that had been bisulphite-treated for the selective amplification of methylated sequences of the human GSTP1 gene in the presence of up to a 10(5)-fold excess of unmethylated sequences. Headloop PCR has a potential for clinical application in the detection of differently methylated DNAs following bisulphite treatment as well as for selective amplification of sequence variants or mutants in the presence of an excess of closely related DNA sequences.
Asunto(s)
Metilación de ADN , Reacción en Cadena de la Polimerasa/métodos , Secuencia de Bases , Islas de CpG , Cartilla de ADN/química , Genes Bacterianos , Genómica , Gutatión-S-Transferasa pi , Glutatión Transferasa/genética , Humanos , Isoenzimas/genética , Magnesio/química , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Ribosómico 16S/genética , Sulfitos/químicaRESUMEN
PMEL is a pigment cell protein that forms physiological amyloid in melanosomes. Many amyloids and/or their oligomeric precursors are toxic, causing or contributing to severe, incurable diseases including Alzheimer's and prion diseases. Striking similarities in intracellular formation pathways between PMEL and various pathological amyloids including Aß and PrPSc suggest PMEL is an excellent model system to study endocytic amyloid. Learning how PMEL fibrils assemble without apparent toxicity may help developing novel therapies for amyloid diseases. Here we identify the critical PMEL domain that forms the melanosomal amyloid core (CAF). An unbiased alanine-scanning screen covering the entire region combined with quantitative electron microscopy analysis of the full set of mutants uncovers numerous essential residues. Many of these rely on aromaticity for function suggesting a role for π-stacking in melanosomal amyloid assembly. Various mutants are defective in amyloid nucleation. This extensive data set informs the first structural model of the CAF and provides insights into how the melanosomal amyloid core forms.
Asunto(s)
Proteínas Amiloidogénicas/metabolismo , Melanosomas/metabolismo , Antígeno gp100 del Melanoma/metabolismo , Proteínas Amiloidogénicas/química , Línea Celular , Humanos , Antígeno gp100 del Melanoma/químicaRESUMEN
Solid tumors shed DNA into circulation, and there is growing evidence that the detection of circulating tumor DNA (ctDNA) has broad clinical utility, including monitoring of disease, prognosis, response to chemotherapy and tracking tumor heterogeneity. The appearance of ctDNA in the circulating cell-free DNA (ccfDNA) isolated from plasma or serum is commonly detected by identifying tumor-specific features such as insertions, deletions, mutations and/or aberrant methylation. Methylation is a normal cell regulatory event, and since the majority of ccfDNA is derived from white blood cells (WBC), it is important that tumour-specific DNA methylation markers show rare to no methylation events in WBC DNA. We have used a novel approach for assessment of low levels of DNA methylation in WBC DNA. DNA methylation in 29 previously identified regions (residing in 17 genes) was analyzed in WBC DNA and eight differentially-methylated regions (DMRs) were taken through to testing in clinical samples using methylation specific PCR assays. DMRs residing in four genes, BCAT1, GRASP, IKZF1 and IRF4, exhibited low positivity, 3.5% to 7%, in the plasma of colonoscopy-confirmed healthy subjects, with the sensitivity for detection of ctDNA in colonoscopy-confirmed patients with colorectal cancer being 65%, 54.5%, 67.6% and 59% respectively.
RESUMEN
The CAHM gene (Colorectal Adenocarcinoma HyperMethylated), previously LOC100526820, is located on chromosome 6, hg19 chr6:163 834 097-163 834 982. It lacks introns, encodes a long non-coding RNA (lncRNA) and is located adjacent to the gene QKI, which encodes an RNA binding protein. Deep bisulphite sequencing of ten colorectal cancer (CRC) and matched normal tissues demonstrated frequent hypermethylation within the CAHM gene in cancer. A quantitative methylation-specific PCR (qMSP) was used to characterize additional tissue samples. With a threshold of 5% methylation, the CAHM assay was positive in 2/26 normal colorectal tissues (8%), 17/21 adenomas (81%), and 56/79 CRC samples (71%). A reverse transcriptase-qPCR assay showed that CAHM RNA levels correlated negatively with CAHM % methylation, and therefore CAHM gene expression is typically decreased in CRC. The CAHM qMSP assay was applied to DNA isolated from plasma specimens from 220 colonoscopy-examined patients. Using a threshold of 3 pg methylated genomic DNA per mL plasma, methylated CAHM sequences were detected in the plasma DNA of 40/73 (55%) of CRC patients compared with 3/73 (4%) from subjects with adenomas and 5/74 (7%) from subjects without neoplasia. Both the frequency of detection and the amount of methylated CAHM DNA released into plasma increased with increasing cancer stage. Methylated CAHM DNA shows promise as a plasma biomarker for use in screening for CRC.
Asunto(s)
Adenocarcinoma/metabolismo , Adenoma/metabolismo , Biomarcadores de Tumor/genética , Neoplasias Colorrectales/metabolismo , Metilación de ADN , ARN Largo no Codificante/genética , Adenocarcinoma/genética , Adenocarcinoma/patología , Adenoma/genética , Adenoma/patología , Adulto , Anciano , Secuencia de Bases , Biomarcadores de Tumor/metabolismo , Células CACO-2 , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Estadificación de Neoplasias , ARN Largo no Codificante/metabolismoRESUMEN
Differential denaturation during PCR can be used to selectively amplify unmethylated DNA from a methylated DNA background. The use of differential denaturation in PCR is particularly suited to amplification of undermethylated sequences following treatment with bisulphite, since bisulphite selectively converts cytosines to uracil while methylated cytosines remain unreactive. Thus amplicons derived from unmethylated DNA retain fewer cytosines and their lower G + C content allows for their amplification at the lower melting temperatures, while limiting amplification of the corresponding methylated amplicons (Bisulphite Differential Denaturation PCR, BDD-PCR). Selective amplification of unmethylated DNA of four human genomic regions from three genes, GSTP1, BRCA1 and MAGE-A1, is demonstrated with selectivity observed at a ratio of down to one unmethylated molecule in 10(5) methylated molecules. BDD-PCR has the potential to be used to selectively amplify and detect aberrantly demethylated genes, such as oncogenes, in cancers. Additionally BDD-PCR can be effectively utilized in improving the specificity of methylation specific PCR (MSP) by limiting amplification of DNA that is not fully converted, thus preventing misinterpretation of the methylation versus non-conversion.
Asunto(s)
Metilación de ADN , Reacción en Cadena de la Polimerasa/métodos , Sulfitos/química , Proteína BRCA1/genética , ADN/química , ADN/metabolismo , Cartilla de ADN/química , Femenino , Genoma Humano , Gutatión-S-Transferasa pi/genética , Humanos , Desnaturalización de Ácido Nucleico , TemperaturaRESUMEN
Residues 386-423 of the rat brain serotonin transporter (SERT) are predicted to form a hydrophilic loop connecting transmembrane spans 7 and 8 (extracellular loop 4 or EL4). EL4 has been hypothesized to play a role in conformational changes associated with substrate translocation. To more fully investigate EL4 structure and function, we performed cysteine-scanning mutagenesis and methanethiosulfonate (MTS) accessibility studies on these 38 residues. Four EL4 mutants (M386C, R390C, G402C, and L405C) showed very low transport activities, low cell surface expression, and strong inhibition by MTS reagents, indicating high structural and functional importance. Twelve mutants were sensitive to very low MTS concentrations, indicating positions highly exposed to the aqueous environment. Eleven mutants were MTS-insensitive, indicating positions that were either buried in EL4 structure or functionally unimportant. The patterns of sensitivity to mutation and MTS reagents were used to produce a structural model of EL4. Positions 386-399 and 409-421 are proposed to form alpha-helices, connected by nine consecutive MTS-sensitive positions, within which four positions, 402-405, may form a turn or hinge. The presence of serotonin changed the MTS accessibility of cysteines at nine positions, while cocaine, a non-transportable blocker, did not affect accessibility. Serotonin-induced accessibility changes required both Na(+) and Cl(-), indicating that they were associated with active substrate translocation. With the exception of a single mutant, F407C, neither mutation to cysteine nor treatment with MTS reagents affected SERT affinities for serotonin or the cocaine analog beta-CIT. These studies support the role of EL4 in conformational changes occurring during translocation and show that it does not play a direct role in serotonin binding.
Asunto(s)
Proteínas Portadoras/química , Cisteína/química , Glicoproteínas de Membrana/química , Proteínas de Transporte de Membrana , Proteínas del Tejido Nervioso/química , Animales , Sitios de Unión , Transporte Biológico , Biotinilación , Cocaína/farmacología , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Células HeLa , Humanos , Indicadores y Reactivos/farmacología , Iones , Cinética , Mesilatos/farmacología , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Ratas , Serotonina/metabolismo , Serotonina/farmacología , Proteínas de Transporte de Serotonina en la Membrana Plasmática , Relación Estructura-Actividad , Zinc/químicaRESUMEN
Methadone pharmacokinetics were determined in an open-label, within subject study in 16 methadone-maintained, non-HIV-infected subjects prior to and following administration of one lamivudine 150-mg/zidovudine 300-mg combination tablet to determine whether this antiretroviral therapy alters methadone serum concentrations. No significant differences in the mean area under the serum concentration-time curve (AUC(0-24h); 8,753 +/- 4,280 vs. 8,641 +/- 4,431 microg-h/L),oralclearance(CL/F;9.9 +/- 4.9vs. 10.3 +/- 5.5 L/h),oral volume of distribution (Vd/F; 647 +/- 465 vs. 481 +/- 305 L), maximum serum concentration (Cmax; 514 +/- 223 vs. 5,510 +/- 237 microg/L), or terminal elimination half-life (t 1/2; 55.3 +/- 61.0 vs. 35.0 +/- 17.5 h) were detected. These results suggest that methadone dose change is not likely to be necessary for patients treated with lamivudine/zidovudine combination pharmacotherapy.