RESUMEN
Of those infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), ~ 10% develop the chronic post-viral debilitating condition, long COVID (LC). Although LC is a heterogeneous condition, about half of cases have typical post-viral fatigue with onset and symptoms that are very similar to myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). A key question is whether these conditions are closely related. ME/CFS is a post-stressor fatigue condition that arises from multiple triggers. To investigate the pathophysiology of LC, a pilot study of patients (n = 6) and healthy controls (n = 5) has used quantitative proteomics to discover changes in peripheral blood mononuclear cell (PBMC) proteins. A principal component analysis separated all long COVID patients from healthy controls. Analysis of 3131 proteins identified 162 proteins differentially regulated, of which 37 were related to immune functions, and 21 to mitochondrial functions. Markov cluster analysis identified clusters involved in immune system processes, and two aspects of gene expression-spliceosome and transcription. These results were compared with an earlier dataset of 346 differentially regulated proteins in PBMC's from ME/CFS patients (n = 9) analysed by the same methodology. There were overlapping protein clusters and enriched molecular pathways particularly in immune functions, suggesting the two conditions have similar immune pathophysiology as a prominent feature, and mitochondrial functions involved in energy production were affected in both conditions.
Asunto(s)
COVID-19 , Síndrome de Fatiga Crónica , Virosis , Humanos , Leucocitos Mononucleares/metabolismo , Proteoma/metabolismo , Síndrome Post Agudo de COVID-19 , Proyectos Piloto , SARS-CoV-2 , COVID-19/metabolismo , Virosis/metabolismoRESUMEN
Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a complex condition arising in susceptible people, predominantly following viral infection, but also other stressful events. The susceptibility factors discussed here are both genetic and environmental although not well understood. While the dysfunctional physiology in ME/CFS is becoming clearer, understanding has been hampered by different combinations of symptoms in each affected person. A common core set of mainly neurological symptoms forms the modern clinical case definition, in the absence of an accessible molecular diagnostic test. This landscape has prompted interest in whether ME/CFS patients can be classified into a particular phenotype/subtype that might assist better management of their illness and suggest preferred therapeutic options. Currently, the same promising drugs, nutraceuticals, or behavioral therapies available can be beneficial, have no effect, or be detrimental to each individual patient. We have shown that individuals with the same disease profile exhibit unique molecular changes and physiological responses to stress, exercise and even vaccination. Key features of ME/CFS discussed here are the possible mechanisms determining the shift of an immune/inflammatory response from transient to chronic in ME/CFS, and how the brain and CNS manifests the neurological symptoms, likely with activation of its specific immune system and resulting neuroinflammation. The many cases of the post viral ME/CFS-like condition, Long COVID, following SARS-CoV-2 infection, and the intense research interest and investment in understanding this condition, provide exciting opportunities for the development of new therapeutics that will benefit ME/CFS patients.
Asunto(s)
COVID-19 , Síndrome de Fatiga Crónica , Humanos , Síndrome de Fatiga Crónica/etiología , Síndrome de Fatiga Crónica/terapia , Síndrome de Fatiga Crónica/diagnóstico , Síndrome Post Agudo de COVID-19 , SARS-CoV-2 , CausalidadRESUMEN
Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a complex disease with variable severity. Patients experience frequent relapses where symptoms increase in severity, leaving them with a marked reduction in quality of life. Previous work has investigated molecular differences between ME/CFS patients and healthy controls, but not the dynamic changes specific to each individual patient. We applied precision medicine here to map genomic changes in two selected ME/CFS patients through a period that contained a relapse recovery cycle. DNA was isolated from two patients and a healthy age/gender matched control at regular intervals and captured the patient relapse in each case. Reduced representation DNA methylation sequencing profiles were obtained spanning the relapse recovery cycle. Both patients showed a significantly larger methylome variability (10-20-fold) through the period of sampling compared with the control. During the relapse, changes in the methylome profiles of the two patients were detected in regulatory-active regions of the genome that were associated, respectively, with 157 and 127 downstream genes, indicating disturbed metabolic, immune and inflammatory functions. Severe health relapses in the ME/CFS patients resulted in functionally important changes in their DNA methylomes that, while differing between the two patients, led to very similar compromised physiology. DNA methylation as a signature of disease variability in ongoing ME/CFS may have practical applications for strategies to decrease relapse frequency.
Asunto(s)
Síndrome de Fatiga Crónica , Epigénesis Genética , Epigenómica , Síndrome de Fatiga Crónica/genética , Síndrome de Fatiga Crónica/metabolismo , Humanos , Calidad de Vida , RecurrenciaRESUMEN
Frameshifting during translation of viral or in rare cases cellular mRNA results in the synthesis of proteins from two overlapping reading frames within the same mRNA. In HIV-1 the protease, reverse transcriptase, and integrase enzymes are in a second reading frame relative to the structural group-specific antigen (gag), and their synthesis is dependent upon frameshifting. This ensures that a strictly regulated ratio of structural proteins and enzymes, which is critical for HIV-1 replication and viral infectivity, is maintained during protein synthesis. The frameshift element in HIV-1 RNA is an attractive target for the development of a new class of anti HIV-1 drugs. However, a number of examples are now emerging of human genes using -1 frameshifting, such as PEG10 and CCR5. In this study we have compared the HIV-1 and PEG10 frameshift elements and shown they have distinct functional characteristics. Frameshifting occurs at several points within each element. Moreover, frameshift modulators that were isolated by high-throughput screening of a library of 114,000 lead-like compounds behaved differently with the PEG10 frameshift element. The most effective compounds affecting the HIV-1 element enhanced frameshifting by 2.5-fold at 10 µM in two different frameshift reporter assay systems. HIV-1 protease:gag protein ratio was affected by a similar amount in a specific assay of virally-infected cultured cell, but the modulation of frameshifting of the first-iteration compounds was not sufficient to show significant effects on viral infectivity. Importantly, two compounds did not affect frameshifting with the human PEG10 element, while one modestly inhibited rather than enhanced frameshifting at the human element. These studies indicate that frameshift elements have unique characteristics that may allow targeting of HIV-1 and of other viruses specifically for development of antiviral therapeutic molecules without effect on human genes like PEG10 that use the same generic mechanism.
Asunto(s)
VIH-1/genética , Proteínas/metabolismo , ARN Viral/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Proteínas Reguladoras de la Apoptosis , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Proteínas de Unión al ADN , Mutación del Sistema de Lectura , Genes Reporteros , Células HEK293 , VIH-1/metabolismo , Humanos , Espectrometría de Masas , Conformación de Ácido Nucleico , Biosíntesis de Proteínas , Proteínas de Unión al ARNRESUMEN
Symbiosis islands are integrative and conjugative mobile genetic elements that convert nonsymbiotic rhizobia into nitrogen-fixing symbionts of leguminous plants. Excision of the Mesorhizobium loti symbiosis island ICEMlSym(R7A) is indirectly activated by quorum sensing through TraR-dependent activation of the excisionase gene rdfS. Here we show that a +1 programmed ribosomal frameshift (PRF) fuses the coding sequences of two TraR-activated genes, msi172 and msi171, producing an activator of rdfS expression named Frameshifted excision activator (FseA). Mass-spectrometry and mutational analyses indicated that the PRF occurred through +1 slippage of the tRNA(phe) from UUU to UUC within a conserved msi172-encoded motif. FseA activated rdfS expression in the absence of ICEMlSym(R7A), suggesting that it directly activated rdfS transcription, despite being unrelated to any characterized DNA-binding proteins. Bacterial two-hybrid and gene-reporter assays demonstrated that FseA was also bound and inhibited by the ICEMlSym(R7A)-encoded quorum-sensing antiactivator QseM. Thus, activation of ICEMlSym(R7A) excision is counteracted by TraR antiactivation, ribosomal frameshifting, and FseA antiactivation. This robust suppression likely dampens the inherent biological noise present in the quorum-sensing autoinduction circuit and ensures that ICEMlSym(R7A) transfer only occurs in a subpopulation of cells in which both qseM expression is repressed and FseA is translated. The architecture of the ICEMlSym(R7A) transfer regulatory system provides an example of how a set of modular components have assembled through evolution to form a robust genetic toggle that regulates gene transcription and translation at both single-cell and cell-population levels.
Asunto(s)
Sistema de Lectura Ribosómico , Secuencias Repetitivas Esparcidas , Percepción de Quorum , Ribosomas/ultraestructura , Secuencia de Bases , Sitios de Unión , Técnicas de Transferencia de Gen , Islas Genómicas , Espectrometría de Masas , Mesorhizobium/metabolismo , Plantas/microbiología , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Biosíntesis de Proteínas , Rhizobium/metabolismo , Ribosomas/química , Simbiosis , Factores de Transcripción , Transcripción Genética , Técnicas del Sistema de Dos Híbridos , beta-Galactosidasa/metabolismoRESUMEN
The two codon-specific eubacterial release factors (RF1: UAA/UAG and RF2: UAA/UGA) have specific tripeptide motifs (PXT/SPF) within an exposed recognition loop shown in recent structures to interact with stop codons during protein synthesis termination. The motifs have been inferred to be critical for codon specificity, but this study shows that they are insufficient to determine specificity alone. Swapping the motifs or the entire loop between factors resulted in a loss of codon recognition rather than a switch of codon specificity. From a study of chimeric eubacterial RF1/RF2 recognition loops and an atypical shorter variant in Caenorhabditis elegans mitochondrial RF1 that lacks the classical tripeptide motif PXT, key determinants throughout the whole loop have been defined. It reveals that more than one configuration of the recognition loop based on specific sequence and size can achieve the same desired codon specificity. This study has provided unexpected insight into why a combination of the two factors is necessary in eubacteria to exclude recognition of UGG as stop.
Asunto(s)
Bacterias/metabolismo , Bacterias/genética , Secuencia de Bases , Codón/metabolismo , Codón de Terminación/metabolismo , Eubacterium/genética , Eubacterium/metabolismo , Oligopéptidos/genética , Oligopéptidos/metabolismo , Biosíntesis de Proteínas , Estructura Terciaria de Proteína/genética , Sensibilidad y EspecificidadRESUMEN
Vertebrate mitochondria use stop codons UAA and UAG decoded by the release factor (RF) MTRF1L and two reassigned arginine codons, AGA and AGG. A second highly conserved RF-like factor, MTRF1, which evolved from a gene duplication of an ancestral mitochondrial RF1 and not a RF2, is a good candidate for recognizing the nonstandard codons. MTRF1 differs from other RFs by having insertions in the two external loops important for stop codon recognition (tip of helix alpha5 and recognition loop) and by having key substitutions that are involved in stop codon interactions in eubacterial RF/ribosome structures. These changes may allow recognition of the larger purine base in the first position of AGA/G and, uniquely for RFs, only of G at position 2. In contrast, residues that support A and G recognition in the third position in RF1 are conserved as would be required for recognition of AGA and AGG. Since an assay with vertebrate mitochondrial ribosomes has not been established, we modified Escherichia coli RF1 at the helix alpha5 and recognition loop regions to mimic MTRF1. There was loss of peptidyl-tRNA hydrolysis activity with standard stop codons beginning with U (e.g., UAG), but a gain of activity with codons beginning with A (AAG in particular). A lower level of activity with AGA could be enhanced by solvent modification. These observations imply that MTRF1 has the characteristics to recognize A as the first base of a stop codon as would be required to decode the nonstandard codons AGA and AGG.