RESUMO
Here, we describe a detailed step-by-step protocol for the detection of phosphoproteins in two-dimensional difference gel electrophoresis (2D-DIGE) gels. A standard 2D-DIGE protocol is combined with subsequent post-staining with phosphospecific fluorescent dye. The combination of these two methods complements 2D-DIGE-based proteome profiling by fluorescence detection of phosphoproteins in the same gel providing additional possibility for sensitive and accurate quantification of the differentially regulated phosphoproteins in biological samples.
Assuntos
Fosfoproteínas , Proteômica , Proteômica/métodos , Eletroforese em Gel Diferencial Bidimensional/métodos , Coloração e Rotulagem , Corantes Fluorescentes , Eletroforese em Gel Bidimensional/métodos , ProteomaRESUMO
Seizure threshold 2 (SZT2) is a component of the KICSTOR complex which, under catabolic conditions, functions as a negative regulator in the amino acid-sensing branch of mTORC1. Mutations in this gene cause a severe neurodevelopmental and epileptic encephalopathy whose main symptoms include epilepsy, intellectual disability, and macrocephaly. As SZT2 remains one of the least characterized regulators of mTORC1, in this work we performed a systematic interactome analysis under catabolic and anabolic conditions. Besides numerous mTORC1 and AMPK signaling components, we identified clusters of proteins related to autophagy, ciliogenesis regulation, neurogenesis, and neurodegenerative processes. Moreover, analysis of SZT2 ablated cells revealed increased mTORC1 signaling activation that could be reversed by Rapamycin or Torin treatments. Strikingly, SZT2 KO cells also exhibited higher levels of autophagic components, independent of the physiological conditions tested. These results are consistent with our interactome data, in which we detected an enriched pool of selective autophagy receptors/regulators. Moreover, preliminary analyses indicated that SZT2 alters ciliogenesis. Overall, the data presented form the basis to comprehensively investigate the physiological functions of SZT2 that could explain major molecular events in the pathophysiology of developmental and epileptic encephalopathy in patients with SZT2 mutations.
Assuntos
Complexos Multiproteicos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Mapas de Interação de Proteínas , Aminoácidos/deficiência , Animais , Proteínas Sanguíneas/farmacologia , Cílios/efeitos dos fármacos , Cílios/metabolismo , Cães , Células HEK293 , Humanos , Células Madin Darby de Rim Canino , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Organogênese/efeitos dos fármacos , Análise de Componente Principal , Mapas de Interação de Proteínas/efeitos dos fármacos , Sirolimo/farmacologiaRESUMO
The diagnosis of COVID-19 relies on the direct detection of SARS-CoV-2 RNA in respiratory specimens by RT-PCR. The pandemic spread of the disease caused an imbalance between demand and supply of materials and reagents needed for diagnostic purposes including swab sets. In a comparative effectiveness study, we conducted serial follow-up swabs in hospitalized laboratory-confirmed COVID-19 patients. We assessed the diagnostic performance of an in-house system developed according to recommendations by the US CDC. In a total of 96 serial swabs, we found significant differences in the accuracy of the different swab systems to generate a positive result in SARS-CoV-2 RT-PCR, ranging from around 50 to 80%. Of note, an in-house swab system was superior to most commercially available sets as reflected by significantly lower Ct values of viral genes. Thus, a simple combination of broadly available materials may enable diagnostic laboratories to bypass global limitations in the supply of swab sets.
Assuntos
Teste de Ácido Nucleico para COVID-19/instrumentação , COVID-19/diagnóstico , Equipamentos Descartáveis/provisão & distribuição , Técnicas de Diagnóstico Molecular/instrumentação , SARS-CoV-2/isolamento & purificação , Teste de Ácido Nucleico para COVID-19/métodos , Técnicas de Laboratório Clínico , Testes Diagnósticos de Rotina , Genes Virais , Humanos , Técnicas de Diagnóstico Molecular/métodos , Controle de Qualidade , RNA Viral/análise , Reprodutibilidade dos Testes , Alocação de Recursos , Manejo de EspécimesRESUMO
The mechanistic target of rapamycin complex 1 (mTORC1) is a key metabolic hub that controls the cellular response to environmental cues by exerting its kinase activity on multiple substrates1-3. However, whether mTORC1 responds to diverse stimuli by differentially phosphorylating specific substrates is poorly understood. Here we show that transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy4,5, is phosphorylated by mTORC1 via a substrate-specific mechanism that is mediated by Rag GTPases. Owing to this mechanism, the phosphorylation of TFEB-unlike other substrates of mTORC1, such as S6K and 4E-BP1- is strictly dependent on the amino-acid-mediated activation of RagC and RagD GTPases, but is insensitive to RHEB activity induced by growth factors. This mechanism has a crucial role in Birt-Hogg-Dubé syndrome, a disorder that is caused by mutations in the RagC and RagD activator folliculin (FLCN) and is characterized by benign skin tumours, lung and kidney cysts and renal cell carcinoma6,7. We found that constitutive activation of TFEB is the main driver of the kidney abnormalities and mTORC1 hyperactivity in a mouse model of Birt-Hogg-Dubé syndrome. Accordingly, depletion of TFEB in kidneys of these mice fully rescued the disease phenotype and associated lethality, and normalized mTORC1 activity. Our findings identify a mechanism that enables differential phosphorylation of mTORC1 substrates, the dysregulation of which leads to kidney cysts and cancer.
Assuntos
Síndrome de Birt-Hogg-Dubé/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/química , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/deficiência , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Síndrome de Birt-Hogg-Dubé/genética , Síndrome de Birt-Hogg-Dubé/patologia , Linhagem Celular , Modelos Animais de Doenças , Ativação Enzimática , Células HeLa , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Camundongos , Camundongos Knockout , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Proteína Enriquecida em Homólogo de Ras do Encéfalo/metabolismo , Especificidade por Substrato , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genéticaRESUMO
Late endosomal/lysosomal adaptor and MAPK and mTOR activator (LAMTOR/Ragulator) is a scaffold protein complex that anchors and regulates multiprotein signaling units on late endosomes/lysosomes. To identify LAMTOR-modulated endolysosomal proteins, primary macrophages were derived from bone marrow of conditional knockout mice carrying a specific deletion of LAMTOR2 in the monocyte/macrophage cell lineage. Affymetrix-based transcriptomic analysis and quantitative iTRAQ-based organelle proteomic analysis of endosomes derived from macrophages were performed. Further analyses showed that LAMTOR could be a novel regulator of foam cell differentiation. The lipid droplet formation phenotype observed in macrophages was additionally confirmed in MEFs, where lipidomic analysis identified cholesterol esters as specifically downregulated in LAMTOR2 knockout cells. The data obtained indicate a function of LAMTOR2 in lipid metabolism.
Assuntos
Diferenciação Celular , Células Espumosas/metabolismo , Metabolismo dos Lipídeos , Macrófagos/metabolismo , Proteínas/metabolismo , Animais , Células Cultivadas , Ésteres do Colesterol/metabolismo , Células Espumosas/citologia , Gotículas Lipídicas/metabolismo , Macrófagos/citologia , Camundongos , Proteínas/genética , TranscriptomaRESUMO
Mechanisms that control lysosomal function are essential for cellular homeostasis. Lysosomes adapt in size and number to cellular needs but little is known about the underlying molecular mechanism. We demonstrate that the late endosomal/lysosomal multimeric BLOC-1-related complex (BORC) regulates the size of these organelles via PIKfyve-dependent phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2 ] production. Deletion of the core BORC component Diaskedin led to increased levels of PI(3,5)P2 , suggesting activation of PIKfyve, and resulted in enhanced lysosomal reformation and subsequent reduction in lysosomal size. This process required AMP-activated protein kinase (AMPK), a known PIKfyve activator, and was additionally dependent on the late endosomal/lysosomal adaptor, mitogen-activated protein kinases and mechanistic target of rapamycin activator (LAMTOR/Ragulator) complex. Consistently, in response to glucose limitation, AMPK activated PIKfyve, which induced lysosomal reformation with increased baseline autophagy and was coupled to a decrease in lysosomal size. These adaptations of the late endosomal/lysosomal system reversed under glucose replete growth conditions. In summary, our results demonstrate that BORC regulates lysosomal reformation and size in response to glucose availability.
Assuntos
Endossomos/metabolismo , Proteínas de Membrana Lisossomal/metabolismo , Lisossomos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Animais , Autofagia , Células HEK293 , Células HeLa , Humanos , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Proteínas de Membrana Lisossomal/genética , Sistema de Sinalização das MAP Quinases , Camundongos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Quinases/metabolismo , Proteínas/genética , Proteínas/metabolismoRESUMO
Here, we describe the detailed step-by-step protocol for detection of phosphoproteins in two-dimensional difference gel electrophoresis (DIGE) gels. A standard DIGE protocol is combined with subsequent post-staining with phosphospecific fluorescent dye. The combination of these two methods complements DIGE-based proteome profiling by fluorescence detection of phosphoproteins in the same gel providing additional possibility for sensitive and accurate quantification of the differentially regulated phosphoproteins in biological samples.
Assuntos
Fosfoproteínas , Proteômica , Eletroforese em Gel Diferencial Bidimensional , Processamento de Imagem Assistida por Computador , Proteoma , Proteômica/métodos , Eletroforese em Gel Diferencial Bidimensional/métodosRESUMO
Signaling from lysosomes controls cellular clearance and energy metabolism. Lysosomal malfunction has been implicated in several pathologies, including neurodegeneration, cancer, infection, immunodeficiency, and obesity. Interestingly, many functions are dependent on the organelle position. Lysosomal motility requires the integration of extracellular and intracellular signals that converge on a competition between motor proteins that ultimately control lysosomal movement on microtubules. Here, we identify a novel upstream control mechanism of Arl8b-dependent lysosomal movement toward the periphery of the cell. We show that the C-terminal domain of lyspersin, a subunit of BLOC-1-related complex (BORC), is essential and sufficient for BORC-dependent recruitment of Arl8b to lysosomes. In addition, we establish lyspersin as the linker between BORC and late endosomal/lysosomal adaptor and mitogen activated protein kinase and mechanistic target of rapamycin activator (LAMTOR) complexes and show that epidermal growth factor stimulation decreases LAMTOR/BORC association, thereby promoting BORC- and Arl8b-dependent lysosomal centrifugal transport.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Proteínas de Transporte/metabolismo , Endossomos/metabolismo , Lisossomos/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fatores de Ribosilação do ADP/genética , Proteínas de Transporte/genética , Endossomos/efeitos dos fármacos , Endossomos/ultraestrutura , Fator de Crescimento Epidérmico/farmacologia , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lisossomos/efeitos dos fármacos , Lisossomos/ultraestrutura , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Movimento , Complexos Multiproteicos/genética , Proteínas do Tecido Nervoso/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico , Transdução de SinaisRESUMO
The LAMTOR [late endosomal and lysosomal adaptor and MAPK (mitogen-activated protein kinase) and mTOR (mechanistic target of rapamycin) activator] complex, also known as "Ragulator," controls the activity of mTOR complex 1 (mTORC1) on the lysosome. The crystal structure of LAMTOR consists of two roadblock/LC7 domain-folded heterodimers wrapped and apparently held together by LAMTOR1, which assembles the complex on lysosomes. In addition, the Rag guanosine triphosphatases (GTPases) associated with the pentamer through their carboxyl-terminal domains, predefining the orientation for interaction with mTORC1. In vitro reconstitution and experiments with site-directed mutagenesis defined the physiological importance of LAMTOR1 in assembling the remaining components to ensure fidelity of mTORC1 signaling. Functional data validated the effect of two short LAMTOR1 amino acid regions in recruitment and stabilization of the Rag GTPases.
Assuntos
Proteínas de Transporte/química , Lisossomos/enzimologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas de Transporte/ultraestrutura , Cristalografia por Raios X , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/ultraestrutura , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Domínios Proteicos , Transdução de SinaisRESUMO
Chronic kidney disease (CKD) is a progressive pathological condition marked by a gradual loss of kidney function. Treatment of CKD is most effective when diagnosed at an early stage and patients are still asymptomatic. However, current diagnostic biomarkers (e.g., serum creatinine and urine albumin) are insufficient for prediction of the pathogenesis of the disease. To address this need, we applied a cell-SELEX (systematic evolution of ligands by exponential enrichment) approach and identified a series of DNA aptamers, which exhibit high affinity and selectivity for cytokine-stimulated cells, resembling some aspects of a CKD phenotype. The cell-SELEX approach was driven toward the enrichment of aptamers that internalize via the endosomal pathway by isolating the endosomal fractions in each selection cycle. Indeed, we demonstrated co-localization of selected aptamers with lysosomal-associated membrane protein 1 (LAMP-1), a late endosomal and lysosomal marker protein, by fluorescence in situ hybridization. These findings are consistent with binding and subsequent internalization of the aptamers into cytokine-stimulated cells. Thus, our study sets the stage for applying selected DNA aptamers as theragnostic reagents for the development of targeted therapies to combat CKD.
RESUMO
The endo/lysosomal system in cells provides membranous platforms to assemble specific signaling complexes and to terminate signal transduction, thus, is essential for physiological signaling. Endocytic organelles can significantly extend signaling of activated cell surface receptors, and may additionally provide distinct locations for the generation of specific signaling outputs. Failures of regulation at different levels of endocytosis, recycling, degradation as well as aberrations in specific endo/lysosomal signaling pathways, such as mTORC1, might lead to different diseases including cancer. Therefore, a better understanding of spatio-temporal compartmentalization of sub-cellular signaling might provide an opportunity to interfere with aberrant signal transduction in pathological processes by novel combinatorial therapeutic approaches.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/genética , Endossomos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/genética , Neoplasias/tratamento farmacológico , Biomarcadores Tumorais/metabolismo , Compartimento Celular , Linhagem Celular Tumoral , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Endocitose/efeitos dos fármacos , Endossomos/metabolismo , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Terapia de Alvo Molecular , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas de Neoplasias/sangue , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Transdução de Sinais , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Cell growth and proliferation are tightly linked to nutrient availability. The mechanistic target of rapamycin complex 1 (mTORC1) integrates the presence of growth factors, energy levels, glucose and amino acids to modulate metabolic status and cellular responses. mTORC1 is activated at the surface of lysosomes by the RAG GTPases and the Ragulator complex through a not fully understood mechanism monitoring amino acid availability in the lysosomal lumen and involving the vacuolar H(+)-ATPase. Here we describe the uncharacterized human member 9 of the solute carrier family 38 (SLC38A9) as a lysosomal membrane-resident protein competent in amino acid transport. Extensive functional proteomic analysis established SLC38A9 as an integral part of the Ragulator-RAG GTPases machinery. Gain of SLC38A9 function rendered cells resistant to amino acid withdrawal, whereas loss of SLC38A9 expression impaired amino-acid-induced mTORC1 activation. Thus SLC38A9 is a physical and functional component of the amino acid sensing machinery that controls the activation of mTOR.
Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/metabolismo , Lisossomos/metabolismo , Complexos Multiproteicos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Linhagem Celular , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Nucleotídeos/metabolismoRESUMO
Cell migration is mediated by the dynamic remodeling of focal adhesions (FAs). Recently, an important role of endosomal signaling in regulation of cell migration was recognized. Here, we show an essential function for late endosomes carrying the p14-MP1 (LAMTOR2/3) complex in FA dynamics. p14-MP1-positive endosomes move to the cell periphery along microtubules (MTs) in a kinesin1- and Arl8b-dependent manner. There they specifically target FAs to regulate FA turnover, which is required for cell migration. Using genetically modified fibroblasts from p14-deficient mice and Arl8b-depleted cells, we demonstrate that MT plus end-directed traffic of p14-MP1-positive endosomes triggered IQGAP1 disassociation from FAs. The release of IQGAP was required for FA dynamics. Taken together, our results suggest that late endosomes contribute to the regulation of cell migration by transporting the p14-MP1 scaffold complex to the vicinity of FAs.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Movimento Celular/fisiologia , Endossomos/metabolismo , Adesões Focais/metabolismo , Proteínas/metabolismo , Fatores de Ribosilação do ADP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linhagem Celular , Fibroblastos/citologia , Células HeLa , Humanos , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Células NIH 3T3 , Proteínas/genética , Transdução de Sinais/fisiologia , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/metabolismoRESUMO
LAMTOR3 (MP1) and LAMTOR2 (p14) form a heterodimer as part of the larger Ragulator complex that is required for MAPK and mTOR1 signaling from late endosomes/lysosomes. Here, we show that loss of LAMTOR2 (p14) results in an unstable cytosolic monomeric pool of LAMTOR3 (MP1). Monomeric cytoplasmic LAMTOR3 is rapidly degraded in a proteasome-dependent but lysosome-independent manner. Mutational analyses indicated that the turnover of the protein is dependent on ubiquitination of several lysine residues. Similarly, other Ragulator subunits, LAMTOR1 (p18), LAMTOR4 (c7orf59), and LAMTOR5 (HBXIP), are degraded as well upon the loss of LAMTOR2. Thus the assembly of the Ragulator complex is monitored by cellular quality control systems, most likely to prevent aberrant signaling at the convergence of mTOR and MAPK caused by a defective Ragulator complex.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Multimerização Proteica , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Western Blotting , Células Cultivadas , Embrião de Mamíferos/citologia , Endossomos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Knockout , Microscopia Confocal , Estabilidade Proteica , Proteínas/genética , Proteínas/metabolismo , Proteólise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ubiquitina/metabolismoRESUMO
In this study, a new type of immobilized metal-ion affinity chromatography (IMAC) resin for the isolation of phosphopeptides was synthesized which is based on the specific interaction between phosphate groups and chelated lanthanide metal ions. In this regard trivalent lanthanum, holmium and erbium ions were chelated to a highly porous phosphonate polymer which was prepared by radical polymerization of vinylphosphonic acid (VPA) and divinylbenzene (DVB). The developed method was evaluated with peptide mixtures from digested standard proteins (α-casein, ß-casein and ovalbumin) as well as with bovine milk, egg white and a spiked HeLa cell lysate. Compared to the commonly used TiO2 approach, the presented method showed higher selectivity for phosphorylated peptides. This can be explained by the strong preference of trivalent lanthanide ions for phosphates with which they form very tight ionic bonds. Mono- and multiply phosphorylated peptides could be enriched and released in a single basic elution step, while non-phosphorylated peptides remained on the resin. Ab initio quantum mechanical energy minimizations of model complexes for polymer-ion-ligand interactions provided geometries, binding energies and charges which are discussed in conjunction with the observed experimental properties, leading to the most satisfying agreement. The presented lanthanide-IMAC resins represent promising affinity materials for the selective isolation of phosphopeptides from biological samples.
Assuntos
Quelantes/química , Cromatografia de Afinidade , Elementos da Série dos Lantanídeos/química , Fosfopeptídeos/isolamento & purificação , Quelantes/síntese química , Íons/química , Estrutura Molecular , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
Members of the Nogo66 receptor family (NgR) are closely associated with nerve growth inhibition and plasticity in the CNS. All three members, NgR1, NgR2 and NgR3, are GPI anchored and highly glycosylated proteins. The binding and signaling properties of NgR1 are well described, but largely unknown for NgR2. At present the only known ligands are myelin associated glycoprotein (MAG) and amyloid beta precursor protein (APP). Despite the requirement of co-receptors for signaling no other binding partner has been uncovered. To learn more about the interactome of NgR2 we performed pull down experiments and were able to identify F-box protein that recognizes sugar chain 1 (Fbs1) as binding partner. We confirmed this finding with co-immunoprecipitations and in vitro binding assays and showed that the binding is mediated by the substrate recognition domain of Fbs1. As a substrate recognition protein of the SCF complex, Fbs1 binding leads to polyubiquitination and finally degradation of its substrates. This is the first time a member of the Nogo receptor family has been connected with an intracellular degradation pathway, which has not only implications for its production, but also for amyloid deposition in Alzheimer's disease.
Assuntos
Doença de Alzheimer/metabolismo , Proteínas F-Box/metabolismo , Receptores de Superfície Celular/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Receptor Nogo 2 , Especificidade por SubstratoRESUMO
Abnormal protein phosphorylation is implicated in a variety of diseases, but until recently the complexity of tissue material, technical limitations, and the substantial volume of required data processing did not allow large-scale phosphoproteomic analysis of patient material, despite tremendous progress in developing mass spectrometry technologies. Phosphoproteomic approaches were primarily developed using model systems such as transformed cell lines, but technological advances in proteomics now make it feasible to analyze thousands of phosphorylation sites in a quantitative manner in patient materials or complex animal and cellular model systems to identify signaling abnormalities. This review summarizes very recent phosphoproteomic studies on complex tissue material, including tissue samples in biobanks, to complement recent reviews that focus primarily on technical advances in instrumentation and methods. Several successful examples reviewed here suggest it is now possible to apply phosphoproteomic techniques to address more challenging medical questions such as mapping within patient samples signal transduction defects that are relevant for diagnosis and individualized treatment development.
Assuntos
Fosfoproteínas/metabolismo , Proteômica/métodos , Transdução de Sinais , Animais , Humanos , Espectrometria de Massas , Fosfoproteínas/químicaRESUMO
The proteome analysis of endocytic compartments has been constrained by the limited purity of the organelle fractions obtained by current biochemical methods. Duclos and coworkers have developed a novel method to isolate highly purified endosomal organelles based on small latex beads internalization followed by gradient centrifugation and successfully combined it with a redundant peptide counting method to compare the relative abundance of proteins in organelles. The presence of bona fide markers in their respective subcellular organelles and the identification of several new endosomal-associated proteins, attested the applicability of their combinatory approach. Future applications of this strategy may deliver a comprehensive endosomal proteome chart: from the identification of the key players to the determination of time and signaling-dependent proteome changes. As a long-term perspective, such an approach may unveil new clues to the molecular mechanisms underlining human diseases associated with endosomal biogenesis defects.
RESUMO
Scaffold proteins regulate intracellular MAP kinase signaling by providing critical spatial and temporal specificities. We have shown previously that the scaffold protein MEK1 partner (MP1) is localized to late endosomes by the adaptor protein p14. Using conditional gene disruption of p14 in livers of mice (p14(Δhep) ) we analyzed protein and transcript signatures in tissue samples. Further biological network analysis predicted that the differentially expressed transcripts and proteins are involved in cell cycle progression and regulation of cellular proliferation. Although some of the here identified signatures were previously linked to phospho-ERK activity, most of them were novel targets of the late endosomal p14/MP1/MEK/ERK signaling module. Finally, the proliferation defect was confirmed in a chemically induced liver regeneration model in p14(Δhep) knockout mice.
Assuntos
Biomarcadores/metabolismo , Perfilação da Expressão Gênica , Proteínas/fisiologia , Proteoma/metabolismo , Animais , Biomarcadores/análise , Eletroforese em Gel Bidimensional , Feminino , Immunoblotting , Integrases/metabolismo , Regeneração Hepática , Masculino , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Proteoma/análise , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Proteomics screening methods for the identification of diagnostic and prognostic biomarkers in cancer are still lagging behind DNA- or RNA-based analysis. We used two-dimensional differential gel electrophoresis (2D-DIGE) in combination with laser capture microdissection (LCM) and MALDI-TOF/TOF mass spectrometry to determine differentially abundant proteins and candidate biomarkers in prostate cancer. Paired (benign and tumor) samples were isolated from 23 Gleason Score 6 (GS 6) and 23 Gleason Score 8 and higher (GS 8+) radical prostatectomy specimens and subjected to 2D-DIGE analysis. Minimal fluorescent dye labeling was applied and electrophoresis performed with triple samples (paired benign and tumor; internal control) for each case of tumor. Nineteen differently abundant proteins were identified by mass spectrometry and further validated. One half of them were associated with glycolysis and the Warburg effect; these were upregulated in tumors. The upregulation correlated with tumor dedifferentiation and might be relevant for selection of therapeutic strategies. Among the other proteins, heat shock protein 60 (HSP60) was significantly upregulated in tumor tissue compared to its benign counterpart. Furthermore, lamin A was statistically highly discriminatory between low and high Gleason score tumors and might serve as a new biomarker of tumor differentiation and prognosis.