RESUMO
Stem cells are capable of unlimited proliferation but can be induced to form brain cells. Factors that specifically regulate human development are poorly understood. We found that human stem cells expressed high levels of the envelope protein of an endogenized human-specific retrovirus (HERV-K, HML-2) from loci in chromosomes 12 and 19. The envelope protein was expressed on the cell membrane of the stem cells and was critical in maintaining the stemness via interactions with CD98HC, leading to triggering of human-specific signaling pathways involving mammalian target of rapamycin (mTOR) and lysophosphatidylcholine acyltransferase (LPCAT1)-mediated epigenetic changes. Down-regulation or epigenetic silencing of HML-2 env resulted in dissociation of the stem cell colonies and enhanced differentiation along neuronal pathways. Thus HML-2 regulation is critical for human embryonic and neurodevelopment, while it's dysregulation may play a role in tumorigenesis and neurodegeneration.
Assuntos
Diferenciação Celular , Retrovirus Endógenos/fisiologia , Neurônios/metabolismo , Transdução de Sinais , Células-Tronco/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Biomarcadores , Diferenciação Celular/genética , Autorrenovação Celular/genética , Cadeia Pesada da Proteína-1 Reguladora de Fusão/metabolismo , Regulação Viral da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurônios/citologia , Ligação Proteica , Células-Tronco/citologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas do Envelope Viral/genéticaRESUMO
OBJECTIVE: To determine the underlying etiology in a patient with progressive dementia with extrapyramidal signs and chronic inflammation referred to the National Institutes of Health Undiagnosed Diseases Program. METHODS: Extensive investigations included metabolic profile, autoantibody panel, infectious etiologies, genetic screening, whole exome sequencing, and the phage-display assay, VirScan, for viral immune responses. An etiological diagnosis was established postmortem. RESULTS: Using VirScan, enrichment of dengue viral antibodies was detected in cerebrospinal fluid as compared to serum. No virus was detected in serum or cerebrospinal fluid, but postmortem analysis confirmed dengue virus in the brain by immunohistochemistry, in situ hybridization, quantitative polymerase chain reaction, and sequencing. Dengue virus was also detectable by polymerase chain reaction and sequencing from brain biopsy tissue collected 33 months antemortem, confirming a chronic infection despite a robust immune response directed against the virus. Immunoprofiling and whole exome sequencing of the patient did not reveal any immunodeficiency, and sequencing of the virus demonstrated wild-type dengue virus in the central nervous system. INTERPRETATION: Dengue virus is the most common arbovirus worldwide and represents a significant public health concern. Infections with dengue virus are usually self-limiting, and chronic dengue infections have not been previously reported. Our findings suggest that dengue virus infections may persist in the central nervous system causing a panencephalitis and should be considered in patients with progressive dementia with extrapyramidal features in endemic regions or with relevant travel history. Furthermore, this work highlights the utility of comprehensive antibody profiling assays to aid in the diagnosis of encephalitis of unknown etiology. ANN NEUROL 2019;86:695-703.
Assuntos
Dengue/complicações , Dengue/patologia , Encefalite Viral/etiologia , Encefalite Viral/patologia , Doença Crônica , Demência , Vírus da Dengue , Evolução Fatal , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
In the sympatho-adrenal system, angiotensin II (Ang II) acts as a key neuromodulatory component. At sympathetic nerve terminals, Ang II influences sympathetic transmission by enhancing norepinephrine (NE) synthesis, facilitating NE release and inhibiting NE uptake. Previously, it was demonstrated that tyrosine hydroxylase (TH) mRNA is trafficked to the distal axons of primary superior cervical ganglia (SCG) neurons, directed by a cis-acting regulatory element (i.e. zipcode) located in the 3'UTR of the transcript. Results of metabolic labeling studies established that the mRNA is locally translated. It was further shown that the axonal trafficking of the mRNA encoding the enzyme plays an important role in mediating dopamine (DA) and NE synthesis and may facilitate the maintenance of axonal catecholamine levels. In the present study, the hypothesis was tested that Ang II induces NE synthesis in rat primary SCG neurons via the modulation of the trafficking of the mRNAs encoding the catecholamine synthesizing enzymes TH and dopamine ß-hydroxylase (DBH). Treatment of SCG neurons with the Ang II receptor type 1 (AT1R) agonist, L-162,313, increases the axonal levels of TH and DBH mRNA and protein and results in elevated NE levels. Conversely, treatment of rat SCG neurons with the AT1R antagonist, Eprosartan, abolished the L-162,313-mediated increase in axonal levels of TH and DBH mRNA and protein. In a first attempt to identify the proteins involved in the Ang II-mediated axonal transport of TH mRNA, we used a biotinylated 50-nucleotide TH RNA zipcode as bait in the affinity purification of TH zipcode-associated proteins. Mass spectrometric analysis of the TH zipcode ribonucleoprotein (RNP) complex immune-purified from SCG neurons led to the identification of 163 somal and 127 axonal proteins functionally involved in binding nucleic acids, the translational machinery or acting as subunits of cytoskeletal and motor proteins. Surprisingly, immune-purification of the TH axonal trafficking complex, results in the acquisition of DBH mRNA, suggesting that these mRNAs maybe transported to the axon together, possibly in the same RNP complex. Taken together, our results point to a novel mechanism by which Ang II participates in the regulation of axonal synthesis of NE by modulating the local trafficking and expression of TH and DBH, two key enzymes involved in the catecholamine biosynthetic pathway.
Assuntos
Angiotensina II/metabolismo , Axônios/metabolismo , Dopamina beta-Hidroxilase/metabolismo , Norepinefrina/biossíntese , Tirosina 3-Mono-Oxigenase/metabolismo , Fibras Adrenérgicas/metabolismo , Animais , Transporte Axonal/fisiologia , Células Cultivadas , Neurônios/metabolismo , Transporte Proteico/fisiologia , RNA Mensageiro , Ratos , Ratos Sprague-Dawley , Gânglio Cervical Superior/metabolismoRESUMO
In previous studies, we identified a putative 38-nucleotide stem-loop structure (zipcode) in the 3' untranslated region of the cytochrome c oxidase subunit IV (COXIV) mRNA that was necessary and sufficient for the axonal localization of the message in primary superior cervical ganglion (SCG) neurons. However, little is known about the proteins that interact with the COXIV-zipcode and regulate the axonal trafficking and local translation of the COXIV message. To identify proteins involved in the axonal transport of the COXIV mRNA, we used the biotinylated 38-nucleotide COXIV RNA zipcode as bait in the affinity purification of COXIV zipcode binding proteins. Gel-shift assays of the biotinylated COXIV zipcode indicated that the putative stem-loop structure functions as a nucleation site for the formation of ribonucleoprotein complexes. Mass spectrometric analysis of the COXIV zipcode ribonucleoprotein complex led to the identification of a large number RNA binding proteins, including fused in sarcoma/translated in liposarcoma (FUS/TLS), and Y-box protein 1 (YB-1). Validation experiments, using western analyses, confirmed the presence of the candidate proteins in the COXIV zipcode affinity purified complexes obtained from SCG axons. Immunohistochemical studies show that FUS, and YB-1 are present in SCG axons. Importantly, RNA immunoprecipitation studies show that FUS, and YB-1 interact with endogenous axonal COXIV transcripts. siRNA-mediated downregulation of the candidate proteins FUS and YB-1 expression in the cell-bodies diminishes the levels of COXIV mRNA in the axon, suggesting functional roles for these proteins in the axonal trafficking of COXIV mRNA.
Assuntos
Axônios/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Neurônios/citologia , RNA Mensageiro/metabolismo , Gânglio Cervical Superior/citologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Mitocôndrias/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Neuroblastoma/patologia , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ratos , Ratos Sprague-Dawley , Transfecção , Tretinoína/farmacologia , Proteína 1 de Ligação a Y-Box/genética , Proteína 1 de Ligação a Y-Box/metabolismoRESUMO
MicroRNAs (miRNAs) selectively localize to subcompartments of the neuron, such as dendrites, axons, and presynaptic terminals, where they regulate the local protein synthesis of their putative target genes. In addition to mature miRNAs, precursor miRNAs (pre-miRNAs) have also been shown to localize to somatodendritic and axonal compartments. miRNA-338 (miR-338) regulates the local expression of several nuclear-encoded mitochondrial mRNAs within axons of sympathetic neurons. Previous work has shown that precursor miR-338 (pre-miR-338) introduced into the axon can locally be processed into mature miR-338, where it can regulate local ATP synthesis. However, the mechanisms underlying the localization of pre-miRNAs to the axonal compartment remain unknown. In this study, we investigated the axonal localization of pre-miR-338. Using proteomic and biochemical approaches, we provide evidence for the localization of pre-miR-338 to distal neuronal compartments and identify several constituents of the pre-miR-338 ribonucleoprotein complex. Furthermore, we found that pre-miR-338 is associated with the mitochondria in axons of superior cervical ganglion (SCG) neurons. The maintenance of mitochondrial function within axons requires the precise spatiotemporal synthesis of nuclear-encoded mRNAs, some of which are regulated by miR-338. Therefore, the association of pre-miR-338 with axonal mitochondria could serve as a reservoir of mature, biologically active miRNAs, which could coordinate the intra-axonal expression of multiple nuclear-encoded mitochondrial mRNAs.
Assuntos
Axônios/metabolismo , MicroRNAs/metabolismo , Mitocôndrias/metabolismo , Precursores de RNA/metabolismo , Transporte de RNA , Animais , Proteínas do Citoesqueleto/metabolismo , Redes Reguladoras de Genes , MicroRNAs/genética , Ligação Proteica , Ratos Sprague-Dawley , Ribonuclease III/metabolismo , Gânglio Cervical Superior/metabolismoRESUMO
The bacterial ribosomal protein S12 contains a universally conserved D88 residue on a loop region thought to be critically involved in translation due to its proximal location to the A site of the 30S subunit. While D88 mutants are lethal this residue has been found to be post-translationally modified to ß-methylthioaspartic acid, a post-translational modification (PTM) identified in S12 orthologs from several phylogenetically distinct bacteria. In a previous report focused on characterizing this PTM, our results provided evidence that this conserved loop region might be involved in forming multiple proteins-protein interactions ( Strader , M. B. ; Costantino , N. ; Elkins , C. A. ; Chen , C. Y. ; Patel , I. ; Makusky , A. J. ; Choy , J. S. ; Court , D. L. ; Markey , S. P. ; Kowalak , J. A. A proteomic and transcriptomic approach reveals new insight into betamethylthiolation of Escherichia coli ribosomal protein S12. Mol. Cell. Proteomics 2011 , 10 , M110 005199 ). To follow-up on this study, the D88 containing loop was probed to identify candidate binders employing a two-step complementary affinity purification strategy. The first involved an endogenously expressed S12 protein containing a C-terminal tag for capturing S12 binding partners. The second strategy utilized a synthetic biotinylated peptide representing the D88 conserved loop region for capturing S12 loop interaction partners. Captured proteins from both approaches were detected by utilizing SDS-PAGE and one-dimensional liquid chromatography-tandem mass spectrometry. The results presented in this report revealed proteins that form direct interactions with the 30S subunit and elucidated which are likely to interact with S12. In addition, we provide evidence that two proteins involved in regulating ribosome and/or mRNA transcript levels under stress conditions, RNase R and Hfq, form direct interactions with the S12 conserved loop, suggesting that it is likely part of a protein binding interface.
Assuntos
Proteínas de Escherichia coli/metabolismo , Proteômica , Proteínas Ribossômicas/metabolismo , Sequência de Aminoácidos , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/isolamento & purificação , Dados de Sequência Molecular , Ligação Proteica , Proteínas Ribossômicas/química , Espectrometria de Massas em TandemRESUMO
ß-methylthiolation is a novel post-translational modification mapping to a universally conserved Asp 88 of the bacterial ribosomal protein S12. This S12 specific modification has been identified on orthologs from multiple bacterial species. The origin and functional significance was investigated with both a proteomic strategy to identify candidate S12 interactors and expression microarrays to search for phenotypes that result from targeted gene knockouts of select candidates. Utilizing an endogenous recombinant E. coli S12 protein with an affinity tag as bait, mass spectrometric analysis identified candidate S12 binding partners including RimO (previously shown to be required for this post-translational modification) and YcaO, a conserved protein of unknown function. Transcriptomic analysis of bacterial strains with deleted genes for RimO and YcaO identified an overlapping transcriptional phenotype suggesting that YcaO and RimO likely share a common function. As a follow up, quantitative mass spectrometry additionally indicated that both proteins dramatically impacted the modification status of S12. Collectively, these results indicate that the YcaO protein is involved in ß-methylthiolation of S12 and its absence impairs the ability of RimO to modify S12. Additionally, the proteomic data from this study provides direct evidence that the E. coli specific ß-methylthiolation likely occurs when S12 is assembled as part of a ribosomal subunit.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Perfilação da Expressão Gênica/métodos , Proteômica/métodos , Proteínas Ribossômicas/metabolismo , Compostos de Sulfidrila/metabolismo , Sequência de Aminoácidos , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/química , Espectrometria de Massas , Dados de Sequência Molecular , Mutação/genética , Peptídeos/química , Peptídeos/metabolismo , Fenótipo , Ligação Proteica , Ribonucleoproteínas/metabolismo , Proteínas Ribossômicas/química , Transcrição GênicaRESUMO
Atypical Teratoid Rhabdoid Tumor (AT/RT) is a rare pediatric central nervous system cancer often characterized by deletion or mutation of SMARCB1, a tumor suppressor gene. In this study, we found that SMARCB1 regulates Human Endogenous Retrovirus K (HERV-K, subtype HML-2) expression. HML-2 is a repetitive element scattered throughout the human genome, encoding several intact viral proteins that have been associated with stem cell maintenance and tumorigenesis. We found HML-2 env expression in both the intracellular and extracellular compartments in all AT/RT cell lines (n = 4) and in 95% of AT/RT patient tissues (n = 37) evaluated. SMARCB1 knock-down in neural stem cells (NSCs) led to an upregulation of HML-2 transcription. We found that SMARCB1 binds adjacent to the HML-2 promoter, repressing its transcription via chromatin immunoprecipitation; restoration of SMARCB1 expression in AT/RT cell lines significantly downregulated HML-2 expression. Further, targeted downregulation of HML-2 transcription via CRISPR-dCas9 coupled with suppressor proteins led to cellular dispersion, decreased proliferation, and cell death in vitro. HML-2 knock-down with shRNA, siRNA, and CRISPR-dCas9 significantly decreased Ras expression as measured by qRT-PCR, suggesting that HML-2 modulates MAPK/ERK signaling in AT/RT cells. Overexpression of NRAS was sufficient to restore cellular proliferation, and MYC, a transcription factor downstream of NRAS, was bound to the HERV-K LTR significantly more in the absence of SMARCB1 expression in AT/RT cells. We show a mechanism by which these undifferentiated tumors remain pluripotent, and we demonstrate that their formation is aided by aberrant HML-2 activation, which is dependent on SMARCB1 and its interaction with MYC.
Assuntos
Transformação Celular Neoplásica/genética , Retrovirus Endógenos/genética , Tumor Rabdoide/etiologia , Tumor Rabdoide/patologia , Proteína SMARCB1/deficiência , Deleção de Sequência , Ativação Viral/genética , Biomarcadores Tumorais , Linhagem Celular Tumoral , Proliferação de Células , Micropartículas Derivadas de Células/metabolismo , Suscetibilidade a Doenças , GTP Fosfo-Hidrolases/metabolismo , Regulação da Expressão Gênica , Humanos , Proteínas de Membrana/metabolismo , Sequências Repetitivas de Ácido Nucleico , Transdução de SinaisRESUMO
PURPOSE: Senescent Ccl2(-/-) mice are reported to develop cardinal features of human age-related macular degeneration (AMD). Loss-of-function single-nucleotide polymorphisms within CX3CR1 are also found to be associated with AMD. The authors generated Ccl2(-/-)/Cx3cr1(-/-) mice to establish a more characteristic and reproducible AMD model. METHODS: Single Ccl2- and Cx3cr1-deficient mice were crossbred to obtain Ccl2(-/-)/Cx3cr1(-/-) mice. Funduscopy, histopathology, retinal A2E quantification, proteomics, RT-PCR gene expression assay, immunochemistry, and Western blotting were used to examine the retina and to evaluate gene expression within the retinal tissue. RESULTS: By 6 weeks of age, all Ccl2(-/-)/Cx3cr1(-/-) mice developed AMD-like retinal lesions, including drusen, retinal pigment epithelium alteration, and photoreceptor degeneration. Furthermore, choroidal neovascularization occurred in 15% of the mice. These degenerative lesions progressed with age. A2E, a major lipofuscin fluorophore that accumulated during AMD progression, was significantly higher in the Ccl2(-/-)/Cx3cr1(-/-) retina than in the wild-type retina. Complement cofactor was higher in the Ccl2(-/-)/Cx3cr1(-/-) RPE. Proteomics data indicated that four proteins were differentially expressed in Ccl2(-/-)/Cx3cr1(-/-) retina compared with control. One of these proteins, ERp29, an endoplasmic reticulum protein, functions as an escort chaperone and in protein folding. CONCLUSIONS: The authors concluded that Ccl2(-/-)/Cx3cr1(-/-) mice develop a broad spectrum of AMD abnormalities with early onset and high penetrance. These observations implicate certain chemokines and endoplasmic reticulum proteins in AMD pathogenesis. Similar to the mechanism of neurodegeneration caused by dysfunction of endoplasmic reticulum proteins, decreased chaperoning may cause misfolded protein accumulation, leading to drusen formation and retinal degeneration.
Assuntos
Quimiocina CCL2/genética , Degeneração Macular/genética , Degeneração Macular/patologia , Receptores de Quimiocinas/genética , Envelhecimento/patologia , Animais , Receptor 1 de Quimiocina CX3C , Quimiocina CCL2/metabolismo , Complemento C3b/metabolismo , Complemento C4b/metabolismo , Modelos Animais de Doenças , Retículo Endoplasmático/fisiologia , Feminino , Proteínas de Choque Térmico/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/patologia , Microscopia Eletrônica de Transmissão , Compostos de Piridínio/metabolismo , Receptores de Quimiocinas/metabolismo , Retina/patologia , Retina/ultraestrutura , Drusas Retinianas/genética , Drusas Retinianas/patologia , Retinoides/metabolismoRESUMO
Accurate protein identification sometimes requires careful discrimination between closely related protein isoforms that may differ by as little as a single amino acid substitution or post-translational modification. The ABRF Proteomics Research Group sent a mixture of three picomoles each of three closely related proteins to laboratories who requested it in the form of intact proteins, and participating laboratories were asked to identify the proteins and report their results. The primary goal of the ABRF-PRG04 Study was to give participating laboratories a chance to evaluate their capabilities and practices with regards to sample fractionation (1D- or 2D-PAGE, HPLC, or none), protein digestion methods (in-solution, in-gel, enzyme choice), and approaches to protein identification (instrumentation, use of software, and/or manual techniques to facilitate interpretation), as well as determination of amino acid or post-translational modifications. Of the 42 laboratories that responded, 8 (19%) correctly identified all three isoforms and N-terminal acetylation of each, 16 (38%) labs correctly identified two isoforms, 9 (21%) correctly identified two isoforms but also made at least one incorrect identification, and 9 (21%) made no correct protein identifications. All but one lab used mass spectrometry, and data submitted enabled a comparison of strategies and methods used.
Assuntos
Anidrases Carbônicas/classificação , Anidrases Carbônicas/isolamento & purificação , Proteômica , Sequência de Aminoácidos , Anidrases Carbônicas/genética , Isoenzimas/classificação , Isoenzimas/genética , Isoenzimas/isolamento & purificação , Espectrometria de Massas , Dados de Sequência Molecular , Inquéritos e QuestionáriosRESUMO
Nodding syndrome is an epileptic disorder of unknown etiology that occurs in children in East Africa. There is an epidemiological association with Onchocerca volvulus, the parasitic worm that causes onchocerciasis (river blindness), but there is limited evidence that the parasite itself is neuroinvasive. We hypothesized that nodding syndrome may be an autoimmune-mediated disease. Using protein chip methodology, we detected autoantibodies to leiomodin-1 more abundantly in patients with nodding syndrome compared to unaffected controls from the same village. Leiomodin-1 autoantibodies were found in both the sera and cerebrospinal fluid of patients with nodding syndrome. Leiomodin-1 was found to be expressed in mature and developing human neurons in vitro and was localized in mouse brain to the CA3 region of the hippocampus, Purkinje cells in the cerebellum, and cortical neurons, structures that also appear to be affected in patients with nodding syndrome. Antibodies targeting leiomodin-1 were neurotoxic in vitro, and leiomodin-1 antibodies purified from patients with nodding syndrome were cross-reactive with O. volvulus antigens. This study provides initial evidence supporting the hypothesis that nodding syndrome is an autoimmune epileptic disorder caused by molecular mimicry with O. volvulus antigens and suggests that patients may benefit from immunomodulatory therapies.
Assuntos
Doenças Autoimunes/parasitologia , Síndrome do Cabeceio/imunologia , Síndrome do Cabeceio/parasitologia , Onchocerca volvulus/fisiologia , Sequência de Aminoácidos , Animais , Autoanticorpos/sangue , Autoanticorpos/líquido cefalorraquidiano , Autoantígenos/química , Autoantígenos/imunologia , Doenças Autoimunes/sangue , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Criança , Pré-Escolar , Reações Cruzadas/imunologia , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/imunologia , Feminino , Proteínas de Helminto/metabolismo , Humanos , Masculino , Síndrome do Cabeceio/sangue , Síndrome do Cabeceio/líquido cefalorraquidianoRESUMO
OBJECTIVE: Hemangioblastomas are benign CNS tumors that occur sporadically or in patients with von Hippel-Lindau (VHL) disease. These tumors are characteristically associated with formation of intra- or peritumoral cysts. Hemangioblastoma cyst formation is a major cause of morbidity and mortality with these tumors. While peritumoral cysts have been suggested to result from vascular leakage, the mechanism of intratumoral cyst formation is not understood. METHODS: To elucidate the origin of intratumoral hemangioblastoma cyst fluid, we characterized its biochemical composition by two-dimensional (2D) proteomic profiling followed by sequencing of several proteins. The proteomic pattern of intratumoral cyst fluid was furthermore compared to the proteomic pattern of serum, hemangioblastoma tumor tissue, and hemangioblastoma peritumoral cyst fluid. RESULTS: We show that proteomic patterns of intra- and peritumoral cyst fluid are identical Both are highly similar to serum and not to tumor. CONCLUSIONS: Intratumoral hemangioblastoma cyst fluid originates from serum. Cyst formation associated with hemangioblastoma, whether peri- or intratumoral, is a consequence of vascular leakage. Anti-VEGF therapy may effectively control hemangioblastoma cyst formation.
Assuntos
Neoplasias Cerebelares/metabolismo , Líquido Cístico/metabolismo , Hemangioblastoma/metabolismo , Proteoma/análise , Biomarcadores Tumorais/metabolismo , Neoplasias Cerebelares/patologia , Cromatografia Líquida , Eletroforese em Gel Bidimensional , Hemangioblastoma/patologia , Humanos , Espectrometria de MassasRESUMO
A one- or two-dimensional high performance liquid chromatography system for electrospray ionization mass spectrometers has been developed that is optimized for ion exchange and reversed phase separations. A unique and simple valve configuration permits the use of a variety of non-volatile salts; ammonium sulfate was used in an example of strong cation exchange separations. The system was designed and evaluated for both micro- and nanoflow chromatography. The peptide detection limit was approximately 100 fmol for micro- and 20 fmol for nanoflow, demonstrating the concentration and mass sensitivity improvements expected with nanoelectrospray ionization. The 1D/2D-HPLC MS system is fully automated for routine peptide analyses, compatible with direct injection of proteolytic digests, and exhibits chromatographic reproducibility and sensitivity. Software permits operator selection of either a 1D or 2D configuration with corresponding system parameters as required for individual samples. The hardware elements and resulting performance are described in this paper.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Cromatografia por Troca Iônica/métodos , Sais/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Automação , Nanotecnologia , Peptídeos/análise , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
A rugged, reproducible, multi-dimensional LC-MS system was developed to identify and characterize proteins involved in protein-protein interactions and/or protein complexes. Our objective was to optimize chromatographic parameters for complex protein mixture analyses using automated peptide sequence recognition as an analytical end-point. The chromatographic system uses orthogonal separation mechanisms by employing strong cation exchange (SCX) in the first dimension and reversed phase (RP) in the second dimension. The system is fully automated and sufficiently robust to handle direct injections of protein digests. This system incorporates a streamlined post analysis results comparison, called DBParser, which permitted comprehensive evaluation of sample loading and chromatographic conditions to optimize the performance and reproducibility. Peptides obtained from trypsin digestion of a yeast soluble extract provided an open-ended model system containing a wide variety and dynamic range of components. Conditions are described that resulted in an average (n = 4) of 1489 unique peptide identifications, corresponding to 459 non-redundant protein sequence database records (SDRs) in the 20 microg soluble fraction digest.
Assuntos
Peptídeos/análise , Saccharomyces cerevisiae/química , Cromatografia Líquida , Bases de Dados Genéticas , Indicadores e Reagentes , Hidrolisados de Proteína/análise , Saccharomyces cerevisiae/crescimento & desenvolvimento , Espectrometria de Massas por Ionização por Electrospray , Tripsina/químicaRESUMO
Mammalian transient receptor potential canonical (TRPC) channels are a family of nonspecific cation channels that are activated in response to stimulation of phospholipase C (PLC)-dependent hydrolysis of the membrane lipid phosphatidylinositol 4,5-bisphosphate. Despite extensive studies, the mechanism(s) involved in regulation of mammalian TRPC channels remains unknown. Presence of various protein-interacting domains in TRPC channels have led to the suggestion that they associate with proteins that are involved in their function and regulation. This study was directed toward identifying the proteins associated with native TRPC3 using a shotgun proteomic approach. Anti-TRPC3 antibody was used to immunoprecipitate TRPC3 from solubilized rat brain crude membranes under conditions that allow retention of TRPC3 function. Proteins in the TRPC3 (using anti-TRPC3 antibody) and control (using rabbit IgG) immunoprecipitates were separated by SDS-PAGE, the gel was sectioned, and the resolved proteins were digested by trypsin in situ. After extraction of the peptides, the peptides were separated by HPLC and sequences derived by MS/MS. Analysis of the data revealed 64 specific TRPC3-associated proteins which can be grouped in terms of their cellular location and involvement in specific cellular function. Many of the proteins identified have been previously reported as TRPC3-regulatory proteins, such as IP3Rs and vesicle trafficking proteins. In addition, we report novel putative TRPC3-interacting proteins, including those involved in protein endocytosis and neuronal growth. To our knowledge, this is the first comprehensive proteomic analysis of a native TRPC channel. These data reveal potential TRPC3 regulatory proteins and provide novel insights of the mechanism(s) regulating TRPC3 channels as well as the possible cellular functions where the channel might be involved.
Assuntos
Canais de Cátion TRPC/metabolismo , Espectrometria de Massas em Tandem/métodos , Animais , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Imunoprecipitação , Ligação Proteica , RatosRESUMO
A common request of proteomics core facilities is protein identification. However, in some instances primary sequence information for the protein in question is not present in public databases. In other cases, the amino acid sequence of a protein may differ in some way from the sequence predicted from the gene sequence in a database as a result of gene mutation, gene splicing, and/or multiple posttranslational modifications. Thus, it may be necessary to determine the sequence of one or more peptides de novo in order to identify and/or adequately characterize the protein of interest. The primary goal of this study was to give participating laboratories an opportunity to evaluate their proficiency in sequencing unknown peptides that are not included in any published database. Samples containing 3-6 pmol each of five synthetic peptides with amino acid sequences that were not present in public databases were sent to 106 laboratories. One nonstandard amino acid was present in one of the peptides. From a comparison of the results obtained by different strategies, participating laboratories will be able to gauge their own capabilities and establish realistic expectations for the approaches that can be used for this determination.
Assuntos
Peptídeos/química , Proteômica/métodos , Análise de Sequência de Proteína/métodos , Biotecnologia , Cromatografia Líquida de Alta Pressão , Laboratórios , Espectrometria de Massas/métodos , Oligopeptídeos/síntese química , Oligopeptídeos/química , Peptídeos/síntese química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The determination of differences in relative protein abundance is a critical aspect of proteomics research that is increasingly used to answer diverse biological questions. The Association of Biomolecular Resource Facilities Proteomics Research Group 2006 study was a quantitative proteomics project in which the aim was to determine the identity and the relative amounts of eight proteins in two mixtures. There are numerous methodologies available to study the relative abundance of proteins between samples, but to date, there are few examples of studies that have compared these different approaches. For the 2006 Proteomics Research Group study, there were 52 participants who used a wide variety of gel electrophoresis-, HPLC-, and mass spectrometry-based methods for relative quantitation. The quantitative data arising from this study were evaluated along with several other experimental details relevant to the methodologies used.
Assuntos
Proteínas/análise , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas/métodos , Proteômica , Reprodutibilidade dos TestesRESUMO
IDO induction can deplete L-tryptophan in target cells, an effect partially responsible for the antimicrobial activities and antiallogeneic T cell responses of IFN-gamma in human macrophages, dendritic cells, and bone marrow cells. L-tryptophan depletion and NO production are both known to have an antimicrobial effect in macrophages, and the interaction of these two mechanisms is unclear. In this study we found that IDO activity was inhibited by the peroxynitrite generator, 3-(4-morpholinyl)sydnonimine, in PMA-differentiated cytokine-induced THP-1 (acute monocytic leukemia) cells and IFN-gamma-stimulated PBMCs, whereas IDO protein expression was unaffected compared with that in untreated cells. Nitrotyrosine was detected in immunoprecipitated (IP)-IDO from PMA-differentiated cytokine-induced THP-1 cells treated with 3-(4-morpholinyl)sydnonimine, but not from untreated cells. Treatment of IP-IDO and recombinant IDO (rIDO) with peroxynitrite significantly decreased enzyme activity. Nitrotyrosine was detected in both peroxynitrite-treated IP-IDO and rIDO, but not in either untreated IP-IDO or rIDO. Peptide analysis by liquid chromatography/electrospray ionization and tandem mass spectrometry demonstrated that Tyr15, Tyr345, and Tyr353 in rIDO were nitrated by peroxynitrite. The levels of Tyr nitration and the inhibitory effect of peroxynitrite on IDO activity were significantly reduced in the Tyr15-to-Phe mutant. These results indicate that IDO is nitrated and inactivated by peroxynitrite and that nitration of Tyr15 in IDO protein is the most important factor in the inactivation of IDO.
Assuntos
Indolamina-Pirrol 2,3,-Dioxigenase/efeitos dos fármacos , Leucócitos Mononucleares/efeitos dos fármacos , Ácido Peroxinitroso/farmacologia , Sequência de Aminoácidos , Western Blotting , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/química , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Leucócitos Mononucleares/imunologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Espectrometria de Massas por Ionização por Electrospray , Tirosina/análogos & derivados , Tirosina/biossíntese , Tirosina/química , Tirosina/efeitos dos fármacosRESUMO
Alleles at NACP-Rep1, the polymorphic microsatellite repeat located approximately 10 kb upstream of the alpha -synuclein gene (SNCA), are associated, in some reports, with differing risks of sporadic Parkinson disease (PD). We showed previously that NACP-Rep1 acts as a negative modulator of SNCA transcription, with an effect that varied threefold among different NACP-Rep1 alleles. Given that duplications and triplications of SNCA have been implicated in familial Parkinson disease (PD), even a 1.5-2-fold increase in alpha -synuclein expression may, over many decades, contribute to PD. Thus, the association of different NACP-Rep1 alleles with PD may be a consequence of polymorphic differences in transcriptional regulation of SNCA. Here we aimed to identify the factor(s) that bind to NACP-Rep1 and potentially contribute to SNCA transcriptional modulation, by pulling down proteins that bind to NACP-Rep1 and identifying them by mass spectrometry. One of these proteins was poly-(ADP-ribose) transferase/polymerase-1 (PARP-1), a DNA-binding protein and transcriptional regulator. Electrophoresis mobility shift and chromatin immunoprecipitation assays showed specific binding of PARP-1 to NACP-Rep1. Inhibition of PARP-1's catalytic domain increased the endogenous SNCA mRNA levels in cultured SH-SY5Y cells. Furthermore, PARP-1 binding to NACP-Rep1 specifically reduced the transcriptional activity of the SNCA promoter/enhancer in luciferase reporter assays. This down-regulation effect of PARP-1 depended on NACP-Rep1 being present in the construct and was abrogated by inhibiting PARP-1's catalytic activity with 3-aminobenzamide. The association of different NACP-Rep1 alleles with PD may be mediated, in part, by the effect of PARP-1, as well as other factors, on SNCA expression.
Assuntos
Repetições de Microssatélites , Proteínas do Tecido Nervoso/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Alelos , Sequência de Bases , Benzamidas/farmacologia , Sítios de Ligação/genética , Linhagem Celular , DNA/genética , DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Humanos , Proteínas do Tecido Nervoso/metabolismo , Doença de Parkinson/genética , Inibidores de Poli(ADP-Ribose) Polimerases , Polimorfismo Genético , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sinucleínas , alfa-SinucleínaRESUMO
An algorithm is introduced to assess spectral quality for peptide CID spectra acquired by a quadrupole ion trap mass spectrometer. The method employs a quadratic discriminant function calibrated with manually classified 'bad' and 'good' quality spectra, producing a single 'spectral quality' score. Many spectra examined that do not have significant matches are assessed to have good spectral quality, indicating that advances in search methods may yield substantial improvements in results.