RESUMO
Melanoma progression is generally associated with increased transcriptional activity mediated by the Yes-associated protein (YAP). Mechanical signals from the extracellular matrix are sensed by YAP, which then activates the expression of proliferative genes, promoting melanoma progression and drug resistance. Which extracellular signals induce mechanotransduction, and how this is mediated, is not completely understood. Here, using secretome analyses, we reveal the extracellular accumulation of amyloidogenic proteins, i.e. premelanosome protein (PMEL), in metastatic melanoma, together with proteins that assist amyloid maturation into fibrils. We also confirm the accumulation of amyloid-like aggregates, similar to those detected in Alzheimer disease, in metastatic cell lines, as well as in human melanoma biopsies. Mechanistically, beta-secretase 2 (BACE2) regulates the maturation of these aggregates, which in turn induce YAP activity. We also demonstrate that recombinant PMEL fibrils are sufficient to induce mechanotransduction, triggering YAP signaling. Finally, we demonstrate that BACE inhibition affects cell proliferation and increases drug sensitivity, highlighting the importance of amyloids for melanoma survival, and the use of beta-secretase inhibitors as potential therapeutic approach for metastatic melanoma.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Melanoma , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Amiloidogênicas , Humanos , Mecanotransdução Celular , Melanoma/tratamento farmacológico , Melanoma/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Cells in the tumor microenvironment may be reprogrammed by tumor-derived metabolites. Cholesterol-oxidized products, namely oxysterols, have been shown to favor tumor growth directly by promoting tumor cell growth and indirectly by dampening antitumor immune responses. However, the cellular and molecular mechanisms governing oxysterol generation within tumor microenvironments remain elusive. We recently showed that tumor-derived oxysterols recruit neutrophils endowed with protumoral activities, such as neoangiogenesis. Here, we show that hypoxia inducible factor-1a (HIF-1α) controls the overexpression of the enzyme Cyp46a1, which generates the oxysterol 24-hydroxycholesterol (24S-HC) in a pancreatic neuroendocrine tumor (pNET) model commonly used to study neoangiogenesis. The activation of the HIF-1α-24S-HC axis ultimately leads to the induction of the angiogenic switch through the positioning of proangiogenic neutrophils in proximity to Cyp46a1+ islets. Pharmacologic blockade or genetic inactivation of oxysterols controls pNET tumorigenesis by dampening the 24S-HC-neutrophil axis. Finally, we show that in some human pNET samples Cyp46a1 transcripts are overexpressed, which correlate with the HIF-1α target VEGF and with tumor diameter. This study reveals a layer in the angiogenic switch of pNETs and identifies a therapeutic target for pNET patients.
Assuntos
Transformação Celular Neoplásica/metabolismo , Hidroxicolesteróis/metabolismo , Tumores Neuroendócrinos/etiologia , Tumores Neuroendócrinos/metabolismo , Neoplasias Pancreáticas/etiologia , Neoplasias Pancreáticas/metabolismo , Animais , Transformação Celular Neoplásica/genética , Colestanotriol 26-Mono-Oxigenase/genética , Colestanotriol 26-Mono-Oxigenase/metabolismo , Colesterol 24-Hidroxilase , Citocinas/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Ativação Enzimática , Feminino , Imunofluorescência , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Transgênicos , Neovascularização Patológica/genética , Tumores Neuroendócrinos/patologia , Neoplasias Pancreáticas/patologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Human up-frameshift 1 (UPF1) is an ATP-dependent RNA helicase and phosphoprotein implicated in several biological processes but is best known for its key function in nonsense-mediated mRNA decay (NMD). Here we employed a combination of stable isotope labeling of amino acids in cell culture experiments to determine by quantitative proteomics UPF1 interactors. We used this approach to distinguish between RNA-mediated and protein-mediated UPF1 interactors and to determine proteins that preferentially bind the hypo- or the hyper-phosphorylated form of UPF1. Confirming and expanding previous studies, we identified the eukaryotic initiation factor 3 (eIF3) as a prominent protein-mediated interactor of UPF1. However, unlike previously reported, eIF3 binds to UPF1 independently of UPF1's phosphorylation state. Furthermore, our data revealed many nucleus-associated RNA-binding proteins that preferentially associate with hyper-phosphorylated UPF1 in an RNase-sensitive manner, suggesting that UPF1 gets recruited to mRNA and becomes phosphorylated before being exported to the cytoplasm as part of the mRNP.
Assuntos
RNA Mensageiro/metabolismo , Transativadores/metabolismo , Células HeLa , Humanos , Imunoprecipitação , Fosforilação , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Proteômica , RNA HelicasesRESUMO
The function of the intracellular protein hematopoietic cell-specific Lyn substrate-1 (HS1) in B lymphocytes is poorly defined. To investigate its role in migration, trafficking, and homing of leukemic B lymphocytes we have used B cells from HS1(-/-) mice, the HS1-silenced human chronic lymphocytic leukemia (CLL) MEC1 cell line and primary leukemic B cells from patients with CLL. We have used both in vitro and in vivo models and found that the lack of expression of HS1 causes several important functional effects. In vitro, we observed an impaired cytoskeletal remodeling that resulted in diminished cell migration, abnormal cell adhesion, and increased homotypic aggregation. In vivo, immunodeficient Rag2(-/-)γ(c)(-/-) mice injected with HS1-silenced CLL B cells showed a decreased organ infiltration with the notable exception of the bone marrow (BM). The leukemic-prone Eµ-TCL1 transgenic mice crossed with HS1-deficient mice were compared with Eµ-TCL1 mice and showed an earlier disease onset and a reduced survival. These findings show that HS1 is a central regulator of cytoskeleton remodeling that controls lymphocyte trafficking and homing and significantly influences the tissue invasion and infiltration in CLL.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linfócitos B/patologia , Proteínas Sanguíneas/metabolismo , Movimento Celular , Proteínas de Ligação a DNA/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linfócitos B/citologia , Proteínas Sanguíneas/genética , Medula Óssea/patologia , Adesão Celular , Linhagem Celular Tumoral , Citoesqueleto/patologia , Citoesqueleto/ultraestrutura , Proteínas de Ligação a DNA/genética , Regulação Leucêmica da Expressão Gênica , Inativação Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Tumorais CultivadasRESUMO
Lipidomics is the comprehensive analysis of lipids in a given biological system. This investigation is often limited by the low amount and high complexity of biological samples, therefore highly sensitive lipidomics methods are required. Nanoflow-LC/MS offers extremely high sensitivity; however, it is challenging as a more demanding maintenance is often needed compared to conventional microflow-LC approaches. Here, we developed a sensitive and reproducible lipidomics LC method, termed Opti-nQL, which can be applied to any biological system. Opti-nQL has been validated with cellular lipid extracts of human and mouse origin and with different lipid extraction methods. Among the resulting 4000 detected features, 700 and even more unique lipid molecular species have been identified covering 16 lipid sub-classes, while 400 lipids were uniquely structure defined by MS/MS. These results were obtained by analyzing an amount of lipids extract equivalent to 40 ng of proteins, being highly suitable for low abundant samples. MS analysis showed that theOpti-nQL method increases the number of identified lipids, which is evidenced by injecting 20 times less material than in microflow based chromatography, being more reproducible and accurate thus enhancing robustness of lipidomics analysis.
RESUMO
Although human nucleoporin Tpr is frequently deregulated in cancer, its roles are poorly understood. Here we show that Tpr depletion generates transcription-dependent replication stress, DNA breaks, and genomic instability. DNA fiber assays and electron microscopy visualization of replication intermediates show that Tpr deficient cells exhibit slow and asymmetric replication forks under replication stress. Tpr deficiency evokes enhanced levels of DNA-RNA hybrids. Additionally, complementary proteomic strategies identify a network of Tpr-interacting proteins mediating RNA processing, such as MATR3 and SUGP2, and functional experiments confirm that their depletion trigger cellular phenotypes shared with Tpr deficiency. Mechanistic studies reveal the interplay of Tpr with GANP, a component of the TREX-2 complex. The Tpr-GANP interaction is supported by their shared protein level alterations in a cohort of ovarian carcinomas. Our results reveal links between nucleoporins, DNA transcription and replication, and the existence of a network physically connecting replication forks with transcription, splicing, and mRNA export machinery.
Assuntos
Replicação do DNA , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo , Sobrevivência Celular , Dano ao DNA , Instabilidade Genômica , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Mapas de Interação de Proteínas , Proteínas Proto-Oncogênicas/genética , Transporte de RNARESUMO
OBJECTIVES: GDI1 gene encodes for αGDI, a protein controlling the cycling of small GTPases, reputed to orchestrate vesicle trafficking. Mutations in human GDI1 are responsible for intellectual disability (ID). In mice with ablated Gdi1, a model of ID, impaired working and associative short-term memory was recorded. This cognitive phenotype worsens if the deletion of αGDI expression is restricted to neurons. However, whether astrocytes, key homeostasis providing neuroglial cells, supporting neurons via aerobic glycolysis, contribute to this cognitive impairment is unclear. METHODS: We carried out proteomic analysis and monitored [18F]-fluoro-2-deoxy-d-glucose uptake into brain slices of Gdi1 knockout and wild type control mice. d-Glucose utilization at single astrocyte level was measured by the Förster Resonance Energy Transfer (FRET)-based measurements of cytosolic cyclic AMP, d-glucose and L-lactate, evoked by agonists selective for noradrenaline and L-lactate receptors. To test the role of astrocyte-resident processes in disease phenotype, we generated an inducible Gdi1 knockout mouse carrying the Gdi1 deletion only in adult astrocytes and conducted behavioural tests. RESULTS: Proteomic analysis revealed significant changes in astrocyte-resident glycolytic enzymes. Imaging [18F]-fluoro-2-deoxy-d-glucose revealed an increased d-glucose uptake in Gdi1 knockout tissue versus wild type control mice, consistent with the facilitated d-glucose uptake determined by FRET measurements. In mice with Gdi1 deletion restricted to astrocytes, a selective and significant impairment in working memory was recorded, which was rescued by inhibiting glycolysis by 2-deoxy-d-glucose injection. CONCLUSIONS: These results reveal a new astrocyte-based mechanism in neurodevelopmental disorders and open a novel therapeutic opportunity of targeting aerobic glycolysis, advocating a change in clinical practice.
Assuntos
Desoxiglucose/farmacologia , Glicólise/efeitos dos fármacos , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Deficiência Intelectual/genética , Transtornos da Memória/prevenção & controle , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Células Cultivadas , Desoxiglucose/uso terapêutico , Regulação para Baixo/efeitos dos fármacos , Glucose/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina/deficiência , Deficiência Intelectual/tratamento farmacológico , Deficiência Intelectual/metabolismo , Deficiência Intelectual/patologia , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Memória/efeitos dos fármacos , Transtornos da Memória/genética , Camundongos , Camundongos KnockoutRESUMO
ATR responds to mechanical stress at the nuclear envelope and mediates envelope-associated repair of aberrant topological DNA states. By combining microscopy, electron microscopic analysis, biophysical and in vivo models, we report that ATR-defective cells exhibit altered nuclear plasticity and YAP delocalization. When subjected to mechanical stress or undergoing interstitial migration, ATR-defective nuclei collapse accumulating nuclear envelope ruptures and perinuclear cGAS, which indicate loss of nuclear envelope integrity, and aberrant perinuclear chromatin status. ATR-defective cells also are defective in neuronal migration during development and in metastatic dissemination from circulating tumor cells. Our findings indicate that ATR ensures mechanical coupling of the cytoskeleton to the nuclear envelope and accompanying regulation of envelope-chromosome association. Thus the repertoire of ATR-regulated biological processes extends well beyond its canonical role in triggering biochemical implementation of the DNA damage response.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Núcleo Celular/metabolismo , Estresse Mecânico , Citoesqueleto de Actina , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Encéfalo , Cromatina , Citoplasma , Citoesqueleto/metabolismo , Dano ao DNA , Camundongos Knockout , Metástase Neoplásica , Neurogênese , Membrana Nuclear/metabolismoRESUMO
UNLABELLED: Sixteen different amino acids (Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, Val) have been separately linked to chromatographic beads and used for studying the mechanism of binding of such baits to proteins, as represented by the cytoplasmic proteome of the human red blood cell (RBC). The 16 different amino acid columns were confronted with equal amounts of RBC lysate, washed to remove unbound material, and eluted with denaturing agents. All eluates were analyzed by nanoLC-MS/MS. THE RESULTS: there appears to be a dichotomy between a class of "Grand Catchers" (Arg, His, Ile, Lys, Phe, Trp, Tyr, Val), all able to bind from 330 up to 441 unique gene products, and the "Petite Catchers" (Asn, Asp, Gln, Glu, Gly, Pro, Ser, Thr), that bind in general half as much, with the notable exception of Glu that under the described conditions seems to bind only traces of proteins. By comparing homogeneous classes of amino acids (e.g., the basic, the hydrophobic aromatic, the neutral hydrophilic, etc.), it is found that, in general, more than half as many proteins are held in common among the members of each family. In a 16-way comparison, 72 proteins (less than 10% of the total amount, which amounts to 800 unique, nonredundant, identified proteins) appear to be the common catch of all 16 amino acids, suggesting that such proteins might have either multiple binding sites or general motifs recognized by any generic bait. By far, it would appear that the strongest interactions and thus the strongest catches occur with the three aromatic moieties of Phe, Trp, and Tyr, all able to capture a practically identical number of proteins. Ionic interactions, which in principle should be the strongest ones, appear to behave in a peculiar way: they are quite strong with the three basic amino acids (Arg, His, Lys) but almost inexistent with their acidic counterparts. This suggests a peculiar mechanism of interaction: upon formation of the ion pair, the linkage between the protein and the bait is stabilized by the hydrophobicity of the underlying chain (e.g., a butyl in the case of Lys).
Assuntos
Aminoácidos/química , Proteínas Sanguíneas/isolamento & purificação , Técnicas de Química Combinatória , Eritrócitos/química , Biblioteca de Peptídeos , Proteoma , Proteínas Sanguíneas/química , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , HumanosRESUMO
Coordination of the multiple processes underlying DNA replication is key for maintaining genome stability and preventing tumorigenesis. CLASPIN, a critical player in replication fork stabilization and checkpoint responses, must be tightly regulated during the cell cycle to prevent the accumulation of DNA damage. In this study, we used a quantitative proteomics approach and identified USP9X as a novel CLASPIN-interacting protein. USP9X is a deubiquitinase involved in multiple signaling and survival pathways whose tumor suppressor or oncogenic activity is highly context dependent. We found that USP9X regulated the expression and stability of CLASPIN in an S-phase-specific manner. USP9X depletion profoundly impairs the progression of DNA replication forks, causing unscheduled termination events with a frequency similar to CLASPIN depletion, resulting in excessive endogenous DNA damage. Importantly, restoration of CLASPIN expression in USP9X-depleted cells partially suppressed the accumulation of DNA damage. Furthermore, USP9X depletion compromised CHK1 activation in response to hydroxyurea and UV, thus promoting hypersensitivity to drug-induced replication stress. Taken together, our results reveal a novel role for USP9X in the maintenance of genomic stability during DNA replication and provide potential mechanistic insights into its tumor suppressor role in certain malignancies. Cancer Res; 76(8); 2384-93. ©2016 AACR.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Dano ao DNA/fisiologia , Replicação do DNA/fisiologia , Fase S , Ubiquitina Tiolesterase/fisiologia , Linhagem Celular Tumoral , HumanosRESUMO
Precociously disseminated cancer cells may seed quiescent sites of future metastasis if they can protect themselves from immune surveillance. However, there is little knowledge about how such sites might be achieved. Here, we present evidence that prostate cancer stem-like cells (CSC) can be found in histopathologically negative prostate draining lymph nodes (PDLN) in mice harboring oncogene-driven prostate intraepithelial neoplasia (mPIN). PDLN-derived CSCs were phenotypically and functionally identical to CSC obtained from mPIN lesions, but distinct from CSCs obtained from frank prostate tumors. CSC derived from either PDLN or mPIN used the extracellular matrix protein Tenascin-C (TNC) to inhibit T-cell receptor-dependent T-cell activation, proliferation, and cytokine production. Mechanistically, TNC interacted with α5ß1 integrin on the cell surface of T cells, inhibiting reorganization of the actin-based cytoskeleton therein required for proper T-cell activation. CSC from both PDLN and mPIN lesions also expressed CXCR4 and migrated in response to its ligand CXCL12, which was overexpressed in PDLN upon mPIN development. CXCR4 was critical for the development of PDLN-derived CSC, as in vivo administration of CXCR4 inhibitors prevented establishment in PDLN of an immunosuppressive microenvironment. Taken together, our work establishes a pivotal role for TNC in tuning the local immune response to establish equilibrium between disseminated nodal CSC and the immune system.
Assuntos
Células-Tronco Neoplásicas/imunologia , Neoplasias da Próstata/imunologia , Linfócitos T/imunologia , Tenascina/fisiologia , Evasão Tumoral , Animais , Movimento Celular , Proliferação de Células , Humanos , Integrina alfa5beta1/metabolismo , Metástase Linfática , Ativação Linfocitária , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias da Próstata/patologia , Fibras de Estresse/metabolismo , Células Tumorais CultivadasRESUMO
The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease's biology and, as a consequence, for the clinical management of patients. In the present work we will describe an optimized proteomic approach for the identification of molecules involved in the progression of Chronic Lymphocytic Leukemia (CLL). In detail, leukemic cell lysates are resolved by 2-dimensional Electrophoresis (2DE) and visualized as "spots" on the 2DE gels. Comparative analysis of proteomic maps allows the identification of differentially expressed proteins (in terms of abundance and post-translational modifications) that are picked, isolated and identified by Mass Spectrometry (MS). The biological function of the identified candidates can be tested by different assays (i.e. migration, adhesion and F-actin polymerization), that we have optimized for primary leukemic cells.
Assuntos
Proteínas Sanguíneas/metabolismo , Eletroforese em Gel Bidimensional/métodos , Leucemia Linfocítica Crônica de Células B/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Linfócitos B/patologia , Proteínas Sanguíneas/análise , Movimento Celular/fisiologia , Citoesqueleto/metabolismo , Humanos , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/patologia , Dados de Sequência Molecular , Fosforilação , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
Lifelong blood cell production is governed through the poorly understood integration of cell-intrinsic and -extrinsic control of hematopoietic stem cell (HSC) quiescence and activation. MicroRNAs (miRNAs) coordinately regulate multiple targets within signaling networks, making them attractive candidate HSC regulators. We report that miR-126, a miRNA expressed in HSC and early progenitors, plays a pivotal role in restraining cell-cycle progression of HSC in vitro and in vivo. miR-126 knockdown by using lentiviral sponges increased HSC proliferation without inducing exhaustion, resulting in expansion of mouse and human long-term repopulating HSC. Conversely, enforced miR-126 expression impaired cell-cycle entry, leading to progressively reduced hematopoietic contribution. In HSC/early progenitors, miR-126 regulates multiple targets within the PI3K/AKT/GSK3ß pathway, attenuating signal transduction in response to extrinsic signals. These data establish that miR-126 sets a threshold for HSC activation and thus governs HSC pool size, demonstrating the importance of miRNA in the control of HSC function.
Assuntos
Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , MicroRNAs/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Técnicas de Silenciamento de Genes , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Hematopoese/genética , Células-Tronco Hematopoéticas/enzimologia , Humanos , Camundongos , MicroRNAs/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Transplante HeterólogoRESUMO
The pre-treatment of biological extracts with the aim of detecting very low-abundance proteins generates complexity requiring a proper fractionation. Therefore the success of identifying all newly detectable species depends on the selected fractionation methods. In this context and starting from a human serum, where the dynamic concentration range was reduced by means of a preliminary treatment with a combinatorial hexapeptide ligand library, we fractionated the sample using a novel method based on the differences in isoelectric points of proteins by means of Solid-State Buffers (SSB) associated with cation exchangers. The number of fractions was limited to four and was compared to a classical anion exchange method generating the same number of fractions. What was observed is that when using SSB technology the protein redundancy between fractions was significantly reduced compared to ion exchange fractionation allowing thus a better detection of novel species. The analysis of trypsinized protein fractions by nanoLC-MS/MS confirmed that the SSB technology used is more discriminant than anion exchange chromatography fractionation. A sample fractionation by SSB after the reduction of dynamic concentration range can be accomplished without either adjustment of pH and ionic strength or protein concentration and cleanup. Both advantages over either classical chromatography or isoelectric fractionations allow approaching the discovery of markers of interest under easier conditions applicable in a variety of fields of investigation.
Assuntos
Proteínas Sanguíneas/química , Biblioteca de Peptídeos , Proteômica/métodos , Soluções Tampão , Cátions , Cromatografia por Troca Iônica/métodos , Cromatografia Líquida/métodos , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Humanos , Focalização Isoelétrica/métodos , Ligantes , Espectrometria de Massas/métodos , Peptídeos/químicaRESUMO
The use of combinatorial peptide ligand libraries (CPLLs), containing hexapeptides terminating with a primary amine, or modified with a terminal carboxyl group, or with a terminal tertiary amine, allowed discovering and identifying a large number of previously unreported egg yolk proteins. Whereas the most comprehensive list up to date [K. Mann, M. Mann, Proteomics, 8 (2008) 178-191] tabulated about 115 unique gene products in the yolk plasma, our findings have more than doubled this value to 255 unique protein species. From the initial non-treated egg yolk it was possible to find 49 protein species; the difference was generated thanks to the use of the three combined CPLLs. The aberrant behaviour of some proteins, upon treatment via the CPLL method, such as proteins that do not interact with the library, is discussed and evaluated. Simplified elution protocols from the CPLL beads are taken into consideration, of which direct elution in a single step via sodium dodecyl sulphate desorption seems to be quite promising. Alternative methods are suggested. The list of egg yolk components here reported is by far the most comprehensive at present and could serve as a starting point for isolation and functional characterization of proteins possibly having novel pharmaceutical and biomedical applications.
Assuntos
Técnicas de Química Combinatória , Citoplasma/química , Gema de Ovo/química , Biblioteca de Peptídeos , Proteoma , Animais , Galinhas , Eletroforese em Gel Bidimensional , Espectrometria de Massas em TandemRESUMO
S-nitrosylation is emerging as an important signaling mechanism that regulates a broad range of cellular functions. The recognition of Cysteine residues that undergo S-nitrosylation is crucial to elucidate how this modification modulates protein activity. We report here a novel strategy, defined His-tag switch, which allows the purification and identification of S-nitrosylated proteins and the unambiguous localization of the modified cysteine residues by mass spectrometry analysis.