RESUMO
Oxidation of the cysteines from ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) leads to inactivation and promotes structural changes that increase the proteolytic sensitivity and membrane association propensity related to its catabolism. To uncover the individual role of the different cysteines, the sequential order of modification under increasing oxidative conditions was determined using chemical labeling and mass spectrometry. Besides, site-directed RubisCO mutants were obtained in Chlamydomonas reinhardtii replacing single conserved cysteines (Cys84, Cys172, Cys192, Cys247, Cys284, Cys427, Cys459 from the large and sCys41, sCys83 from the small subunit) and the redox properties of the mutant enzymes were determined. All mutants retained significant carboxylase activity and grew photoautotrophically, indicating that these conserved cysteines are not essential for catalysis. Cys84 played a noticeable structural role, its replacement producing a structurally altered enzyme. While Cys247, Cys284, and sCys83 were not affected by the redox environment, all other residues were oxidized using a disulfide/thiol ratio of around two, except for Cys172 whose oxidation was distinctly delayed. Remarkably, Cys192 and Cys427 were apparently protective, their absence leading to a premature oxidation of critical residues (Cys172 and Cys459). These cysteines integrate a regulatory network that modulates RubisCO activity and conformation in response to oxidative conditions.
Assuntos
Chlamydomonas reinhardtii/enzimologia , Cisteína/química , Ribulose-Bifosfato Carboxilase/química , Ribulose-Bifosfato Carboxilase/metabolismo , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Sequência Conservada , Cisteína/metabolismo , Regulação Enzimológica da Expressão Gênica , Modelos Moleculares , Mutação , Conformação Proteica , Ribulose-Bifosfato Carboxilase/genéticaRESUMO
The current catalogue of the human proteome is not yet complete, as experimental proteomics evidence is still elusive for a group of proteins known as the missing proteins. The Human Proteome Project (HPP) has been successfully using technology and bioinformatic resources to improve the characterization of such challenging proteins. In this manuscript, we propose a pipeline starting with the mining of the PRIDE database to select a group of data sets potentially enriched in missing proteins that are subsequently analyzed for protein identification with a method based on the statistical analysis of proteotypic peptides. Spermatozoa and the HEK293 cell line were found to be a promising source of missing proteins and clearly merit further attention in future studies. After the analysis of the selected samples, we found 342 PSMs, suggesting the presence of 97 missing proteins in human spermatozoa or the HEK293 cell line, while only 36 missing proteins were potentially detected in the retina, frontal cortex, aorta thoracica, or placenta. The functional analysis of the missing proteins detected confirmed their tissue specificity, and the validation of a selected set of peptides using targeted proteomics (SRM/MRM assays) further supports the utility of the proposed pipeline. As illustrative examples, DNAH3 and TEPP in spermatozoa, and UNCX and ATAD3C in HEK293 cells were some of the more robust and remarkable identifications in this study. We provide evidence indicating the relevance to carefully analyze the ever-increasing MS/MS data available from PRIDE and other repositories as sources for missing proteins detection in specific biological matrices as revealed for HEK293 cells.
Assuntos
Biologia Computacional/métodos , Bases de Dados de Proteínas , Proteoma/análise , Aorta/química , Feminino , Lobo Frontal/química , Células HEK293 , Humanos , Masculino , Placenta/química , Gravidez , Proteômica/métodos , Retina/química , Espermatozoides/química , Espectrometria de Massas em TandemRESUMO
Experimental evidence for the entire human proteome has been defined in the Human Proteome Project, and it is publicly available in the neXtProt database. However, there are still human proteins for which reliable experimental evidence does not exist, and the identification of such information has become one of the overriding objectives in the chromosome-centric study of the human proteome. With this aim and considering the complexity of protein detection using shotgun and targeted proteomics, the research community has addressed the integration of transcriptomics and proteomics landscapes. Here, we describe an analytical pipeline that predicts the probability of a missing protein being expressed in a biological sample based on (1) gene sequence characteristics, (2) the probability of an expressed gene being a coding gene of a missing protein in a certain sample, and (3) the probability of a gene being expressed in a transcriptomic experiment. More than 3400 microarray experiments were analyzed corresponding to three biological sources: cell lines, normal tissues, and cancer samples. A gene classification based on gene expression profiles distinguished among ubiquitous, nonubiquitous, nonexpressed, and coding genes of missing proteins. In addition, a different tissue-specific expression pattern for the coding genes of missing proteins is reported. Our results underline the relevance of selecting an appropriate sample for the detection of missing proteins and provide a comprehensive method to score their expression probability. Testis, brain, and skeletal muscle are the most promising normal tissues.
Assuntos
Proteínas/metabolismo , Proteoma , Humanos , Análise Serial de Proteínas , Proteínas/química , Proteínas/genética , TranscriptomaRESUMO
The Spanish team of the Human Proteome Project (SpHPP) marked the annotation of Chr16 and data analysis as one of its priorities. Precise annotation of Chromosome 16 proteins according to C-HPP criteria is presented. Moreover, Human Body Map 2.0 RNA-Seq and Encyclopedia of DNA Elements (ENCODE) data sets were used to obtain further information relative to cell/tissue specific chromosome 16 coding gene expression patterns and to infer the presence of missing proteins. Twenty-four shotgun 2D-LC-MS/MS and gel/LC-MS/MS MIAPE compliant experiments, representing 41% coverage of chromosome 16 proteins, were performed. Furthermore, mapping of large-scale multicenter mass spectrometry data sets from CCD18, MCF7, Jurkat, and Ramos cell lines into RNA-Seq data allowed further insights relative to correlation of chromosome 16 transcripts and proteins. Detection and quantification of chromosome 16 proteins in biological matrices by SRM procedures are also primary goals of the SpHPP. Two strategies were undertaken: one focused on known proteins, taking advantage of MS data already available, and the second, aimed at the detection of the missing proteins, is based on the expression of recombinant proteins to gather MS information and optimize SRM methods that will be used in real biological samples. SRM methods for 49 known proteins and for recombinant forms of 24 missing proteins are reported in this study.
Assuntos
Cromossomos Humanos Par 16 , Proteoma , Transcriptoma , Cromatografia Líquida , Humanos , Espectrometria de Massas , Análise de Sequência de RNARESUMO
BACKGROUND: Patient-derived organoids (PDOs) from advanced colorectal cancer (CRC) patients could be a key platform to predict drug response and discover new biomarkers. We aimed to integrate PDO drug response with multi-omics characterization beyond genomics. METHODS: We generated 29 PDO lines from 22 advanced CRC patients and provided a morphologic, genomic, and transcriptomic characterization. We performed drug sensitivity assays with a panel of both standard and non-standard agents in five long-term cultures, and integrated drug response with a baseline proteomic and transcriptomic characterization by SWATH-MS and RNA-seq analysis, respectively. RESULTS: PDOs were successfully generated from heavily pre-treated patients, including a paired model of advanced MSI high CRC deriving from pre- and post-chemotherapy liver metastasis. Our PDOs faithfully reproduced genomic and phenotypic features of original tissue. Drug panel testing identified differential response among PDOs, particularly to oxaliplatin and palbociclib. Proteotranscriptomic analyses revealed that oxaliplatin non-responder PDOs present enrichment of the t-RNA aminoacylation process and showed a shift towards oxidative phosphorylation pathway dependence, while an exceptional response to palbociclib was detected in a PDO with activation of MYC and enrichment of chaperonin T-complex protein Ring Complex (TRiC), involved in proteome integrity. Proteotranscriptomic data fusion confirmed these results within a highly integrated network of functional processes involved in differential response to drugs. CONCLUSIONS: Our strategy of integrating PDOs drug sensitivity with SWATH-mass spectrometry and RNA-seq allowed us to identify different baseline proteins and gene expression profiles with the potential to predict treatment response/resistance and to help in the development of effective and personalized cancer therapeutics.
Assuntos
Antineoplásicos , Neoplasias Colorretais , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Oxaliplatina/farmacologia , Oxaliplatina/uso terapêutico , Proteômica , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , OrganoidesRESUMO
BACKGROUND: SARS-CoV-2 is continuously disseminating worldwide. The development of strategies to break transmission is mandatory. AIM OF THE STUDY: To investigate the potential of cetylpyridinium chloride (CPC) as a viral inhibitor. METHODS: SARS-CoV-2 Virus Like-Particles (VLPs) were incubated with CPC, a potent surfactant routinely included in mouthwash preparations. RESULTS: Concentrations of 0.05% CPC (w/v) commonly used in mouthwash preparations are sufficient to promote the rupture of SARS-CoV-2 VLP membranes. CONCLUSION: Including CPC in mouthwashes could be a prophylactic strategy to keep SARS-CoV-2 from spreading.
RESUMO
The somatic extract of Zygocotyle lunata (Trematoda: Paramphistomidae) adults collected from experimentally infected mice was investigated using a proteomic approach to separate and identify tryptic peptides from the somatic extract of Z. lunata adult worms. A shot-gun liquid chromatography/tandem mass spectrometry procedure was used. We used the MASCOT search engine (Matrix-Science) and ProteinPilot software v2.0 (Applied Biosystems) for the database search. A total of 36 proteins were accurately identified from the worms. The largest protein family consisted of metabolic enzymes. Structural, motor and receptor binding proteins and proteins related to oxygen transport were identified in the somatic extract of Z. lunata. This is the first study that attempts to identify the proteome of Z. lunata. However, more work is needed to improve our knowledge of trematodiasis in general and more specifically to have a better understanding about host-parasite relationships in infections with paramphistomes.
Assuntos
Proteínas de Helminto/análise , Paramphistomatidae/química , Proteoma/análise , Infecções por Trematódeos/parasitologia , Animais , Cromatografia Líquida , Bases de Dados de Proteínas , Proteínas de Helminto/genética , Interações Hospedeiro-Parasita , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Paramphistomatidae/genética , Paramphistomatidae/fisiologia , Proteoma/genética , Espectrometria de Massas em TandemRESUMO
The excretory/secretory proteome of Echinostoma caproni (Trematoda: Echinostomatidae) adults collected from experimentally infected mice was investigated using a proteomic approach. We performed a shot-gun liquid chromatography/tandem mass spectrometry for the separation and identification of tryptic peptides from the excretory/secretory products of E. caproni adult worms. Database search was performed using MASCOT search engine (Matrix-Science) and ProteinPilot software v2.0 (Applied Biosystems). A total of 39 parasite proteins were accurately identified. Strikingly, metabolic enzymes, and particularly glycolytic enzymes, constituted the largest protein family in the excretory/secretory proteome of E. caproni adult worms. Moreover, representative proteins involved in parasite structure, response against stress, chaperones, calcium-binding, and signal transduction were also identified. This work extends our knowledge of host-parasite relationships in the E. caproni-rodent model that is extensively used to analyze the factors determining the intestinal helminth rejection. Consequently, information on many proteins may be useful to better understand the molecular basis that determines the survival of this parasite in the definitive host.
Assuntos
Echinostoma/fisiologia , Equinostomíase/parasitologia , Proteínas de Helminto/metabolismo , Proteoma , Animais , Biomphalaria/parasitologia , Cromatografia Líquida , Modelos Animais de Doenças , Echinostoma/metabolismo , Echinostoma/patogenicidade , Perfilação da Expressão Gênica , Proteínas de Helminto/química , Interações Hospedeiro-Parasita , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos ICRRESUMO
MTAP (5'-methylthioadenosine phosphorylase) catalyses the reversible phosphorolytic cleavage of methylthioadenosine leading to the production of methylthioribose-1-phosphate and adenine. Deficient MTAP activity has been correlated with human diseases including cirrhosis and hepatocellular carcinoma. In the present study we have investigated the regulation of MTAP by ROS (reactive oxygen species). The results of the present study support the inactivation of MTAP in the liver of bacterial LPS (lipopolysaccharide)-challenged mice as well as in HepG2 cells after exposure to t-butyl hydroperoxide. Reversible inactivation of purified MTAP by hydrogen peroxide results from a reduction of V(max) and involves the specific oxidation of Cys(136) and Cys(223) thiols to sulfenic acid that may be further stabilized to sulfenyl amide intermediates. Additionally, we found that Cys(145) and Cys(211) were disulfide bonded upon hydrogen peroxide exposure. However, this modification is not relevant to the mediation of the loss of MTAP activity as assessed by site-directed mutagenesis. Regulation of MTAP by ROS might participate in the redox regulation of the methionine catabolic pathway in the liver. Reduced MTA (5'-deoxy-5'-methylthioadenosine)-degrading activity may compensate for the deficient production of the precursor S-adenosylmethionine, allowing maintenance of intracellular MTA levels that may be critical to ensure cellular adaptation to physiopathological conditions such as inflammation.
Assuntos
Hepatócitos/enzimologia , Purina-Núcleosídeo Fosforilase/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Dissulfetos/química , Dissulfetos/metabolismo , Ativação Enzimática/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/farmacologia , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oxirredução , Purina-Núcleosídeo Fosforilase/química , Tionucleosídeos/metabolismoRESUMO
The role of hydrophobic amino acids in the formation of hydrophobic cores as one of the major driving forces in protein folding has been extensively studied. However, the implication of neutral solvent-exposed amino acids is less clear and available information is scarce. We have used a combinatorial approach to study the structural relevance of three solvent-exposed residues (Tyr(327), Thr(329), and Gln(331)) located in thebeta-sheet of the tetramerization domain of the tumor suppressor p53 (p53TD). A conformationally defined peptide library was designed where these three positions were randomized. The library was screened for tetramer stability. A set of p53TD mutants containing putative stabilizing or destabilizing residue combinations was synthesized for a thermodynamic characterization. Unfolding experiments showed a wide range of stabilities, with T(m) values between 27 and 83 degrees C. Wild type p53TD and some highly destabilized and stabilized mutants were further characterized. Thermodynamic and biophysical data indicated that these proteins were folded tetramers, with the same overall structure, in equilibrium with unfolded monomers. An NMR study confirmed that the main structural features of p53TD are conserved in all the mutants analyzed. The thermodynamic stability of the different p53TD mutants showed a strong correlation with parameters that favor formation and stabilization of the beta-sheet. We propose that stabilization through hydrophobic interactions of key secondary structure elements might be the underlying mechanism for the strong influence of solvent-exposed residues in the stability of p53TD.
Assuntos
Aminoácidos/química , Técnicas de Química Combinatória/métodos , Solventes/farmacologia , Proteína Supressora de Tumor p53/química , Sequência de Aminoácidos , Aminoácidos/genética , Biofísica/métodos , Dicroísmo Circular , Dimerização , Glicina/química , Guanidina/farmacologia , Temperatura Alta , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ressonância Magnética Nuclear Biomolecular , Biblioteca de Peptídeos , Peptídeos/síntese química , Peptídeos/química , Peptídeos/isolamento & purificação , Conformação Proteica , Desnaturação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Temperatura , Termodinâmica , Treonina/química , Proteína Supressora de Tumor p53/genética , Tirosina/químicaRESUMO
Virions are often described as virus-only entities with no cellular components with the exception of the lipids in their membranes. However, advances in proteomics are revealing substantial amounts of host proteins in the viral particles. In the case of Nipah virus (NiV), the viral components in the virion have been known for some time. Nonetheless, no information has been obtained regarding the cellular proteins in the viral particles. To address this question, we produced Virus-Like Particles (VLPs) for NiV by expressing the F, G and M proteins in human-derived cells. Next, the proteomic content in these VLPs was analyzed by LC-MS/MS. We identified 67 human proteins including soluble and membrane-bound proteins involved in vesicle sorting and transport. Interestingly, many of them have been reported to interact with other viruses. Finally, thanks to the semi-quantitative nature of our data we were able to estimate the ratio among F, G and M proteins and also the ratio between cellular and viral proteins in the VLPs. We believe our data contribute to the better understanding of NiV life cycle and might facilitate future attempts for developing antiviral agents and the design of further experimental studies for this deadly infection. BIOLOGICAL SIGNIFICANCE: Traditionally viral particles have been described as pure entities carrying only viral-derived proteins. Advances in proteomics are changing this simplified view. Host proteins have been identified in many viruses (especially in enveloped viruses). These cell-derived proteins participate in multiple steps in the viral life cycle and might be as important for the survival of the virus as any other viral-encoded protein. In this work, we analyze utilizing LC-MS/MS the cellular proteins incorporated or bound to the virions of Nipah virus (NiV), an emerging, highly pathogenic, zoonotic virus from the Paramyxoviridiae family. Furthermore, we analyzed the ratio between cellular and viral proteins and among the viral F, G and M proteins in the viral particles. The characterization of the Nipah virus-human interactions occurring in the virion might facilitate the development of new therapeutic and prophylactic therapies for this viral illness.
Assuntos
Vírus Nipah/química , Proteômica/métodos , Proteínas Virais/análise , Vírion/química , Cromatografia Líquida , Interações Hospedeiro-Patógeno , Humanos , Ligação Proteica , Espectrometria de Massas em TandemRESUMO
Alzheimer׳s disease is one of the main causes of dementia in the elderly and its frequency is on the rise worldwide. It is considered the result of complex interactions between genetic and environmental factors, being many of them unknown. Therefore, there is a dire necessity for the identification of novel molecular players for the understanding of this disease. In this data article we determined the protein expression profiles of whole protein extracts from cortex regions of brains from patients with Alzheimer׳s disease in comparison to a normal brain. We identified 721 iTRAQ-labeled polypeptides with more than 95% in confidence. We analyzed all proteins that changed in their expression level and located them in the KEGG metabolic pathways, as well as in the mitochondrial complexes of the electron transport chain and ATP synthase. In addition, we analyzed the over- and sub-expressed polypeptides through IPA software, specifically Core I and Biomarkers I modules. Data in this article is related to the research article "Identification of proteins that are differentially expressed in brains with Alzheimer's disease using iTRAQ labeling and tandem mass spectrometry" (Minjarez et al., 2016) [1].
RESUMO
Breast cancer is the principal cancer in women worldwide. Although there are serum tumor markers such as CEA and HER2, they are detected in advanced stages of the disease and used as progression and recurrence markers. Therefore, there is a necessity for the identification of new markers that might lead to an early detection and also provide evidence of an effective treatment. The aim of this work was to determine the differential protein expression profiles of four breast cancer cell lines in comparison to a normal control cell line by iTRAQ labelling and tandem mass spectrometry, in order to identify putative biomarkers of the disease. We identified 1,020 iTRAQ-labelled polypeptides with at least one peptide identified with more than 95% in confidence. Overexpressed polypeptides in all cancer cell lines were 78, whilst the subexpressed were 128. We categorised them with PANTHER program into biological processes, being the metabolic pathways the most affected. We detected six groups of proteins with the STRING program involved in DNA topology, glycolysis, translation initiation, splicing, pentose pathway, and proteasome degradation. The main subexpressed protein network included mitochondrial proteins involved in oxidative phosphorylation. We propose BAG6, DDX39, ANXA8 and COX4 as putative biomarkers in breast cancer. BIOLOGICAL SIGNIFICANCE: We report a set of differentially expressed proteins in the MCF7 and T47D (Luminal A), MDA-MB-231 (Claudin low) and SK-BR-3 (HER2(+)) breast cancer cell lines that have not been previously reported in breast cancer disease. From these proteins, we propose BAG6, DDX39, ANXA8 and COX4 as putative biomarkers in breast cancer. On the other hand, we propose sets of unique polypeptides in each breast cancer cell line that can be useful in the classification of different subtypes of breast cancer.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Perfilação da Expressão Gênica/métodos , Espectrometria de Massas/métodos , Proteínas de Neoplasias/metabolismo , Mapeamento de Peptídeos/métodos , Biomarcadores Tumorais/química , Neoplasias da Mama/química , Linhagem Celular Tumoral , Humanos , Proteínas de Neoplasias/química , Coloração e Rotulagem/métodosRESUMO
Breast cancer is the most common and the leading cause of mortality in women worldwide. There is a dire necessity of the identification of novel molecules useful in diagnosis and prognosis. In this work we determined the differentially expression profiles of four breast cancer cell lines compared to a control cell line. We identified 1020 polypeptides labelled with iTRAQ with more than 95% in confidence. We analysed the common proteins in all breast cancer cell lines through IPA software (IPA core and Biomarkers). In addition, we selected the specific overexpressed and subexpressed proteins of the different molecular classes of breast cancer cell lines, and classified them according to protein class and biological process. Data in this article is related to the research article "Determination of the protein expression profiles of breast cancer cell lines by Quantitative Proteomics using iTRAQ Labelling and Tandem Mass Spectrometry" (Calderón-González et al. [1] in press).
RESUMO
One of the features of liver cirrhosis is an abnormal metabolism of methionine--a characteristic that was described more than a half a century ago. Thus, after an oral load of methionine, the rate of clearance of this amino acid from the blood is markedly impaired in cirrhotic patients compared with that in control subjects. Almost 15 y ago we observed that the failure to metabolize methionine in cirrhosis was due to an abnormally low activity of the enzyme methionine adenosyltransferase (EC 2.5.1.6). This enzyme converts methionine, in the presence of ATP, to S-adenosyl-L-methionine (SAMe), the main biological methyl donor. Since then, it has been suspected that a deficiency in hepatic SAMe may contribute to the pathogenesis of the liver in cirrhosis. The studies reviewed here are consistent with this hypothesis.
Assuntos
Cirrose Hepática/etiologia , S-Adenosilmetionina/deficiência , Animais , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Metionina Adenosiltransferase/genética , Metionina Adenosiltransferase/metabolismo , Óxido Nítrico/fisiologia , Espécies Reativas de Oxigênio/metabolismo , S-Adenosilmetionina/biossíntese , S-Adenosilmetionina/uso terapêuticoRESUMO
Anti-silencing function 1 (Asf1) is a conserved key eukaryotic histone H3/H4 chaperone that participates in a variety of DNA and chromatin-related processes. These include the assembly and disassembly of histones H3 and H4 from chromatin during replication, transcription, and DNA repair. In addition, Asf1 is required for H3K56 acetylation activity dependent on histone acetyltransferase Rtt109. Thus, Asf1 impacts on many aspects of DNA metabolism. To gain insights into the functional links of Asf1 with other cellular machineries, we employed mass spectrometry coupled to tandem affinity purification (TAP) to investigate novel physical interactions of Asf1. Under different TAP-MS analysis conditions, we describe a new repertoire of Asf1 physical interactions and novel Asf1 post-translational modifications as ubiquitination, methylation and acetylation that open up new ways to regulate Asf1 functions. Asf1 co-purifies with several subunits of the TREX-2, SAGA complexes, and with nucleoporins Nup2, Nup60, and Nup57, which are all involved in transcription coupled to mRNA export in eukaryotes. Reciprocally, Thp1 and Sus1 interact with Asf1. Albeit mRNA export and GAL1 transcription are not affected in asf1Δ a strong genetic interaction exists between ASF1 and SUS1. Notably, supporting a functional link between Asf1 and TREX-2, both Sus1 and Thp1 affect the levels of Asf1-dependent histone H3K56 acetylation and histone H3 and H4 incorporation onto chromatin. Additionally, we provide evidence for a role of Asf1 in histone H2B ubiquitination. This work proposes a functional link between Asf1 and TREX-2 components in histone metabolism at the vicinity of the nuclear pore complex.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Chaperonas de Histonas/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Acetilação , Regulação Fúngica da Expressão Gênica/genética , Histonas/metabolismo , Metilação , Processamento de Proteína Pós-Traducional , Transporte de RNA/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/metabolismo , Transcrição Gênica/genéticaRESUMO
Alzheimer's disease (AD) is the most common cause of dementia in the elderly. AD brains are characterized by the presence of neurofibrillary tangles (NFTs) and neuritic plaques. NFTs are constituted of paired helical filaments, which are structurally composed by assembled hyperphosphorylated and truncated tau polypeptides. To date, the integral constituents of NFTs remain unknown mainly due to the high insolubility of NFTs. The aim of this study was to identify by tandem mass spectrometry, the polypeptides contained in both isolated NFTs by laser capture microdissection and total homogenates, using tissue sections from paraformaldehyde-fixed AD brains. In the first case, we isolated 2,000 NFTs from tissue samples of hippocampus from each of the three Mexican AD brains used in our study. These were previously stained with anti-hyperphosphorylated tau AT-100 antibodies. After the removal of paraformaldehyde and delipidation with organic solvents, we tested three solubilization methods. We identified 102 polypeptides from total homogenates and 41 from isolated NFTs. We selected UCH-L1, transferrin, and GAPDH polypeptides to be studied by immunofluorescence and confocal microscopy. Only UCH-L1 and GAPDH colocalized with hyperphosphorylated tau in NFTs.
Assuntos
Doença de Alzheimer/patologia , Encéfalo/metabolismo , Emaranhados Neurofibrilares/metabolismo , Peptídeos/metabolismo , Espectrometria de Massas em Tandem , Idoso , Idoso de 80 Anos ou mais , Encéfalo/ultraestrutura , Cromatografia Líquida de Alta Pressão , Biologia Computacional , Feminino , Humanos , Microdissecção e Captura a Laser , Masculino , Microscopia Eletrônica de Transmissão , Emaranhados Neurofibrilares/ultraestruturaRESUMO
The lipase produced by Burkholderia glumae folds spontaneously into an inactive near-native state and requires a periplasmic chaperone to reach its final active and secretion-competent fold. The B. glumae lipase-specific foldase (Lif) is classified as a member of the steric-chaperone family of which the propeptides of α-lytic protease and subtilisin are the best known representatives. Steric chaperones play a key role in conferring kinetic stability to proteins. However, until present there was no solid experimental evidence that Lif-dependent lipases are kinetically trapped enzymes. By combining thermal denaturation studies with proteolytic resistance experiments and the description of distinct folding intermediates, we demonstrate that the native lipase has a kinetically stable conformation. We show that a newly discovered molten globule-like conformation has distinct properties that clearly differ from those of the near-native intermediate state. The folding fingerprint of Lif-dependent lipases is put in the context of the protease-prodomain system and the comparison reveals clear differences that render the lipase-Lif systems unique. Limited proteolysis unveils structural differences between the near-native intermediate and the native conformation and sets the stage to shed light onto the nature of the kinetic barrier.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Burkholderia/enzimologia , Lipase/química , Cinética , Lipase/metabolismo , Chaperonas Moleculares/metabolismo , Conformação Proteica , Dobramento de Proteína , ProteóliseRESUMO
Proteomics is an expanding technology with potential applications in many research fields. Even though many research groups do not have direct access to its main analytical technique, mass spectrometry, they can interact with proteomics core facilities to incorporate this technology into their projects. Protein identification is the analysis most frequently performed in core facilities and is, probably, the most robust procedure. Here we discuss a few chemical reactions that are easily implemented within the conventional protein identification workflow. Chemical modification of proteins with N-hydroxysuccinimide esters, 4-sulfophenyl isothiocyanate, O-methylisourea or through ß-elimination/Michael addition can be easily performed in any laboratory. The reactions are quite specific with almost no side reactions. These chemical tools increase considerably the number of applications and have been applied to characterize protein-protein interactions, to determine the N-terminal residues of proteins, to identify proteins with non-sequenced genomes or to locate phosphorylated and O-glycosylated.
Assuntos
Proteínas/análise , Proteômica/métodos , Glicosilação , Fosforilação , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , Proteômica/tendênciasRESUMO
Methionine adenosyl transferase (MAT) is an essential enzyme that synthesizes AdoMet. The liver-specific MAT isoform, MAT III, is a homodimer of a 43.7-kDa subunit that organizes in three nonsequential alpha-beta domains. Although MAT III structure has been recently resolved, little is known about its folding mechanism. Equilibrium unfolding and refolding of MAT III, and the monomeric mutant R265H, have been monitored using different physical parameters. Tryptophanyl fluorescence showed a three-state folding mechanism. The first unfolding step was a folding/association process as indicated by its dependence on protein concentration. The monomeric folding intermediate produced was the predominant species between 1.5 and 3 m urea. It had a relatively compact conformation with tryptophan residues and hydrophobic surfaces occluded from the solvent, although its N-terminal region may be very unstructured. The second unfolding step monitored the denaturation of the intermediate. Refolding of the intermediate showed first order kinetics, indicating the presence of a kinetic intermediate within the folding/association transition. Its presence was confirmed by measuring the 1,8-anilinonaphtalene-8-sulfonic acid binding in the presence of tripolyphosphate. We propose that the folding rate-limiting step is the formation of an intermediate, probably a structured monomer with exposed hydrophobic surfaces, that rapidly associates to form dimeric MAT III.