RESUMEN
During the early studies of N-terminal arginylation, Edman degradation was widely used to identify N-terminally added Arg on protein substrates. This old method is reliable, but highly depends on the purity and abundance of samples and can become misleading unless a highly purified highly arginylated protein can be obtained. Here, we report a mass spectrometry-based method that utilizes Edman degradation chemistry to identify arginylation in more complex and less abundant protein samples. This method can also apply to the analysis of other posttranslational modifications.
Asunto(s)
Arginina , Péptidos , Arginina/metabolismo , Péptidos/metabolismo , Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Espectrometría de Masas/métodosRESUMEN
Here, we describe the method for the identification of arginylated proteins by mass spectrometry. This method has been originally applied to the identification of N-terminally added Arg on proteins and peptides and then expanded to the side chain modification which has been recently described by our groups. The key steps in this method include the use of the mass spectrometry instruments that can identify peptides with very high pass accuracy (Orbitrap) and apply stringent mass cutoffs during automated data analysis, followed by manual validation of the identified spectra. These methods can be used with both complex and purified protein samples and, to date, constitute the only reliable way to confirm arginylation at a particular site on a protein or peptide.
Asunto(s)
Arginina , Procesamiento Proteico-Postraduccional , Arginina/química , Proteínas/química , Péptidos/química , Espectrometría de Masas/métodosRESUMEN
Reciprocal deletion and duplication of the 16p11.2 region is the most common copy number variation (CNV) associated with autism spectrum disorders. We generated cortical organoids from skin fibroblasts of patients with 16p11.2 CNV to investigate impacted neurodevelopmental processes. We show that organoid size recapitulates macrocephaly and microcephaly phenotypes observed in the patients with 16p11.2 deletions and duplications. The CNV dosage affects neuronal maturation, proliferation, and synapse number, in addition to its effect on organoid size. We demonstrate that 16p11.2 CNV alters the ratio of neurons to neural progenitors in organoids during early neurogenesis, with a significant excess of neurons and depletion of neural progenitors observed in deletions. Transcriptomic and proteomic profiling revealed multiple pathways dysregulated by the 16p11.2 CNV, including neuron migration, actin cytoskeleton, ion channel activity, synaptic-related functions, and Wnt signaling. The level of the active form of small GTPase RhoA was increased in both, deletions and duplications. Inhibition of RhoA activity rescued migration deficits, but not neurite outgrowth. This study provides insights into potential neurobiological mechanisms behind the 16p11.2 CNV during neocortical development.
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Trastorno del Espectro Autista , Trastorno Autístico , Trastorno del Espectro Autista/genética , Trastorno Autístico/genética , Encéfalo , Deleción Cromosómica , Cromosomas Humanos Par 16/genética , Variaciones en el Número de Copia de ADN/genética , Humanos , Neurogénesis/genética , Organoides , ProteómicaRESUMEN
The accuracy in pairing tRNAs with correct amino acids by aminoacyl-tRNA synthetases (aaRSs) dictates the fidelity of translation. To ensure fidelity, multiple aaRSs developed editing functions that remove a wrong amino acid from tRNA before it reaches the ribosome. However, no specific mechanism within an aaRS is known to handle the scenario where a cognate amino acid is mischarged onto a wrong tRNA, as exemplified by AlaRS mischarging alanine to G4:U69-containing tRNAThr. Here, we report that the mischargeable G4:U69-containing tRNAThr are strictly conserved in vertebrates and are ubiquitously and abundantly expressed in mammalian cells and tissues. Although these tRNAs are efficiently mischarged, no corresponding Thr-to-Ala mistranslation is detectable. Mistranslation is prevented by a robust proofreading activity of ThrRS towards Ala-tRNAThr. Therefore, while wrong amino acids are corrected within an aaRS, a wrong tRNA is handled in trans by an aaRS cognate to the mischarged tRNA species. Interestingly, although Ala-tRNAThr mischarging is not known to occur in bacteria, Escherichia coli ThrRS also possesses robust cross-editing ability. We propose that the cross-editing activity of ThrRS is evolutionarily conserved and that this intrinsic activity allows G4:U69-containing tRNAThr to emerge and be preserved in vertebrates to have alternative functions without compromising translational fidelity.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Edición de ARN , ARN de Transferencia/metabolismo , Alanina/genética , Animales , Evolución Molecular , Células HEK293 , Humanos , ARN de Transferencia/genética , Treonina/genética , Vertebrados/genéticaRESUMEN
The aberrant activation of hedgehog (HH) signaling is a leading cause of the development of medulloblastoma, a pediatric tumor of the cerebellum. The FDAapproved HH inhibitor, Vismodegib, which targets the transmembrane transducer SMO, has shown limited efficacy in patients with medulloblastoma, due to compensatory mechanisms that maintain an active HHGLI signaling status. Thus, the identification of novel actionable mechanisms, directly affecting the activity of the HHregulated GLI transcription factors is an important goal for these malignancies. In this study, using gene expression and reporter assays, combined with biochemical and cellular analyses, we demonstrate that mitogenactivated kinase kinase kinase 1 (MEKK1), the most upstream kinase of the mitogenactivated protein kinase (MAPK) phosphorylation modules, suppresses HH signaling by associating and phosphorylating GLI1, the most potent HHregulated transcription factor. Phosphorylation occurred at multiple residues in the Cterminal region of GLI1 and was followed by an increased association with the cytoplasmic proteins 1433. Of note, the enforced expression of MEKK1 or the exposure of medulloblastoma cells to the MEKK1 activator, Nocodazole, resulted in a marked inhibitory effect on GLI1 activity and tumor cell proliferation and viability. Taken together, the results of this study shed light on a novel regulatory mechanism of HH signaling, with potentially relevant implications in cancer therapy.
Asunto(s)
Proteínas Hedgehog/genética , Quinasa 1 de Quinasa de Quinasa MAP/genética , Meduloblastoma/genética , Proteína con Dedos de Zinc GLI1/genética , Anilidas/administración & dosificación , Animales , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/patología , Ratones , Células 3T3 NIH , Fosforilación/efectos de los fármacos , Piridinas/administración & dosificación , ARN Mensajero/genética , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/genéticaRESUMEN
The vitellogenin receptor (VgR), which belongs to the low-density lipoprotein receptors (LDLR) family, regulates the absorption of yolk protein accumulated in developing oocytes during oogenesis. In the present study, the full sequence of Rhipicephalus microplus VgR (RmVgR) and the partial sequence of Rhipicephalus appendiculatus VgR (RaVgR) ORF were determined and cloned. The RmVgR amino acid sequence contains the five highly conserved structural motifs characteristic of LDLR superfamily members, the same overall structure as observed in other species. Phylogenetic analysis separated VgRs in two major groups, corresponding to receptors from acarines and insects. Consistent with observations from other arthropods, RmVgR was specifically expressed in the ovarian tissue and its peak of expression occurs in females that are detaching from the host. Silencing with RmVgR dsRNA reduced VgR expression, which resulted in reduced fertility, evidenced by a decrease in the number of larvae. The present study confirms RmVgR is a specific receptor involved in yolk protein uptake and oocyte maturation in R. microplus, playing an important role in tick reproduction.
Asunto(s)
Proteínas de Artrópodos/genética , Proteínas del Huevo/genética , Oogénesis/genética , Receptores de Superficie Celular/genética , Rhipicephalus/genética , Transcriptoma , Animales , Proteínas de Artrópodos/metabolismo , Proteínas del Huevo/metabolismo , Femenino , Larva/genética , Larva/crecimiento & desarrollo , Larva/fisiología , Óvulo/crecimiento & desarrollo , Óvulo/fisiología , Receptores de Superficie Celular/metabolismo , Rhipicephalus/crecimiento & desarrollo , Rhipicephalus/fisiología , Análisis de Secuencia de ProteínaRESUMEN
Fasciola hepatica is the agent of fasciolosis, a foodborne zoonosis that affects livestock production and human health. Although flukicidal drugs are available, re-infection and expanding resistance to triclabendazole demand new control strategies. Understanding the molecular mechanisms underlying the complex interaction with the mammalian host could provide relevant clues, aiding the search for novel targets in diagnosis and control of fasciolosis. Parasite survival in the mammalian host is mediated by parasite compounds released during infection, known as excretory/secretory (E/S) products. E/S products are thought to protect parasites from host responses, allowing them to survive for a long period in the vertebrate host. This work provides in-depth proteomic analysis of F. hepatica intra-mammalian stages, and represents the largest number of proteins identified to date for this species. Functional classification revealed the presence of proteins involved in different biological processes, many of which represent original findings for this organism and are important for parasite survival within the host. These results could lead to a better comprehension of host-parasite relationships, and contribute to the development of drugs or vaccines against this parasite.
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Fasciola hepatica/crecimiento & desarrollo , Proteínas del Helminto/metabolismo , Hígado/parasitología , Proteómica/métodos , Animales , Fasciola hepatica/metabolismo , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Interacciones Huésped-ParásitosRESUMEN
Guanosine monophosphate synthetase (GMPS), encoded by guaA gene, is a key enzyme for guanine nucleotide biosynthesis in Mycobacterium tuberculosis. The guaA gene from several bacterial pathogens has been shown to be involved in virulence; however, no information about the physiological effect of direct guaA deletion in M. tuberculosis has been described so far. Here, we demonstrated that the guaA gene is essential for M. tuberculosis H37Rv growth. The lethal phenotype of guaA gene disruption was avoided by insertion of a copy of the ortholog gene from Mycobacterium smegmatis, indicating that this GMPS protein is functional in M. tuberculosis. Protein validation of the guaA essentiality observed by PCR was approached by shotgun proteomic analysis. A quantitative method was performed to evaluate protein expression levels, and to check the origin of common and unique peptides from M. tuberculosis and M. smegmatis GMPS proteins. These results validate GMPS as a molecular target for drug design against M. tuberculosis, and GMPS inhibitors might prove to be useful for future development of new drugs to treat human tuberculosis.
RESUMEN
Differential expression of proteins between tissues underlies organ-specific functions. Under certain pathological conditions, this may also lead to tissue vulnerability. Furthermore, post-translational modifications exist between different cell types and pathological conditions. We employed SILAM (Stable Isotope Labeling in Mammals) combined with mass spectrometry to quantify the proteome between mammalian tissues. Using (15)N labeled rat tissue, we quantified 3742 phosphorylated peptides in nuclear extracts from liver and brain tissue. Analysis of the phosphorylation sites revealed tissue specific kinase motifs. Although these tissues are quite different in their composition and function, more than 500 protein identifications were common to both tissues. Specifically, we identified an up-regulation in the brain of the phosphoprotein, ZFHX1B, in which a genetic deletion causes the neurological disorder Mowat-Wilson syndrome. Finally, pathway analysis revealed distinct nuclear pathways enriched in each tissue. Our findings provide a valuable resource as a starting point for further understanding of tissue specific gene regulation and demonstrate SILAM as a useful strategy for the differential proteomic analysis of mammalian tissues.
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Regulación de la Expresión Génica , Fosfoproteínas/análisis , Proteómica/métodos , Animales , Encéfalo , Facies , Enfermedad de Hirschsprung , Discapacidad Intelectual , Hígado , Microcefalia , Procesamiento Proteico-Postraduccional , Ratas , Ratas Sprague-Dawley , Proteínas Represoras , Distribución TisularRESUMEN
PatternLab for proteomics is a one-stop shop computational environment for analyzing shotgun proteomic data. Its modules provide means to pinpoint proteins/peptides that are differentially expressed and those that are unique to a state. It can also cluster the ones that share similar expression profiles in time-course experiments, as well as help in interpreting results according to Gene Ontology. PatternLab is user-friendly, simple, and provides a graphical user interface.
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Bases de Datos de Proteínas , Proteómica/métodos , Programas Informáticos , Análisis por Conglomerados , Guías como Asunto , Proteínas/metabolismo , Homología de Secuencia de Aminoácido , Interfaz Usuario-ComputadorRESUMEN
The quantitative proteomic analysis of complex protein mixtures is emerging as a technically challenging but viable systems-level approach for studying cellular function. This study presents a large-scale comparative analysis of protein abundances from yeast protein lysates derived from both wild-type yeast and yeast strains lacking key components of the Snf1 kinase complex. Four different strains were grown under well-controlled chemostat conditions. Multidimensional protein identification technology followed by quantitation using either spectral counting or stable isotope labeling approaches was used to identify relative changes in the protein expression levels between the strains. A total of 2388 proteins were relatively quantified, and more than 350 proteins were found to have significantly different expression levels between the two strains of comparison when using the stable isotope labeling strategy. The stable isotope labeling based quantitative approach was found to be highly reproducible among biological replicates when complex protein mixtures containing small expression changes were analyzed. Where poor correlation between stable isotope labeling and spectral counting was found, the major reason behind the discrepancy was the lack of reproducible sampling for proteins with low spectral counts. The functional categorization of the relative protein expression differences that occur in Snf1-deficient strains uncovers a wide range of biological processes regulated by this important cellular kinase.
Asunto(s)
Proteínas Fúngicas/análisis , Perfilación de la Expresión Génica , Proteoma/análisis , Proteómica/métodos , Mezclas Complejas/análisis , Métodos , Mutación , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas de Saccharomyces cerevisiae/análisis , LevadurasRESUMEN
Genome integrity is protected by Cds1 (Chk2), a checkpoint kinase that stabilizes arrested replication forks. How Cds1 accomplishes this task is unknown. We report that Cds1 interacts with Rad60, a protein required for recombinational repair in fission yeast. Cds1 activation triggers Rad60 phosphorylation and nuclear delocalization. A Rad60 mutant that inhibits regulation by Cds1 renders cells specifically sensitive to replication fork arrest. Genetic and biochemical studies indicate that Rad60 functions codependently with Smc5 and Smc6, subunits of an SMC (structural maintenance of chromosomes) complex required for recombinational repair. These studies indicate that regulation of Rad60 is an important part of the replication checkpoint response controlled by Cds1. We propose that control of Rad60 regulates recombination events at stalled forks.