RESUMO
INTRODUCTION: In the present study, we sought to quantify and contrast the secretome and biomechanical properties of the non-chondrodystrophic (NCD) and chondrodystrophic (CD) canine intervertebral disc (IVD) nucleus pulposus (NP). METHODS: We used iTRAQ proteomic methods to quantify the secretome of both CD and NCD NP. Differential levels of proteins detected were further verified using immunohistochemistry, Western blotting, and proteoglycan extraction in order to evaluate the integrity of the small leucine-rich proteoglycans (SLRPs) decorin and biglycan. Additionally, we used robotic biomechanical testing to evaluate the biomechanical properties of spinal motion segments from both CD and NCD canines. RESULTS: We detected differential levels of decorin, biglycan, and fibronectin, as well as of other important extracellular matrix (ECM)-related proteins, such as fibromodulin and HAPLN1 in the IVD NP obtained from CD canines compared with NCD canines. The core proteins of the vital SLRPs decorin and biglycan were fragmented in CD NP but were intact in the NP of the NCD animals. CD and NCD vertebral motion segments demonstrated significant differences, with the CD segments having less stiffness and a more varied range of motion. CONCLUSIONS: The CD NP recapitulates key elements of human degenerative disc disease. Our data suggest that at least some of the compromised biomechanical properties of the degenerative disc arise from fibrocartilaginous metaplasia of the NP secondary to fragmentation of SLRP core proteins and associated degenerative changes affecting the ECM. This study demonstrates that the degenerative changes that naturally occur within the CD NP make this animal a valuable animal model with which to study IVD degeneration and potential biological therapeutics.
Assuntos
Degeneração do Disco Intervertebral/metabolismo , Disco Intervertebral/metabolismo , Proteoma/análise , Proteômica/métodos , Animais , Biglicano/análise , Biglicano/metabolismo , Fenômenos Biomecânicos , Western Blotting , Decorina/análise , Decorina/metabolismo , Modelos Animais de Doenças , Cães , Proteínas da Matriz Extracelular/análise , Proteínas da Matriz Extracelular/metabolismo , Feminino , Fibromodulina , Fibronectinas/análise , Fibronectinas/metabolismo , Humanos , Imuno-Histoquímica , Disco Intervertebral/fisiopatologia , Degeneração do Disco Intervertebral/fisiopatologia , Masculino , Proteoglicanas/análise , Proteoglicanas/metabolismo , Proteoma/metabolismoRESUMO
There are no serum biomarkers for the accurate diagnosis of clear cell renal cell carcinoma (ccRCC). Diagnosis and decision of nephrectomy rely on imaging which is not always accurate. Non-invasive diagnostic biomarkers are urgently required. In this study, we preformed quantitative proteomics analysis on a total of 199 patients including 30 matched pairs of normal kidney and ccRCC using isobaric tags for relative and absolute quantitation (iTRAQ) labeling and LC-MS/MS analysis to identify differentially expressed proteins. We found 55 proteins significantly dysregulated in ccRCC compared to normal kidney tissue. 54 were previously reported to play a role in carcinogenesis, and 39 are secreted proteins. Dysregulation of alpha-enolase (ENO1), L-lactate dehydrogenase A chain (LDHA), heat shock protein beta-1 (HSPB1/Hsp27), and 10 kDa heat shock protein, mitochondrial (HSPE1) was confirmed in two independent sets of patients by western blot and immunohistochemistry. Pathway analysis, validated by PCR, showed glucose metabolism is altered in ccRCC compared to normal kidney tissue. In addition, we examined the utility of Hsp27 as biomarker in serum and urine. In ccRCC patients, Hsp27 was elevated in the urine and serum and high serum Hsp27 was associated with high grade (Grade 3-4) tumors. These data together identify potential diagnostic biomarkers for ccRCC and shed new light on the molecular mechanisms that are dysregulated and contribute to the pathogenesis of ccRCC. Hsp27 is a promising diagnostic marker for ccRCC although further large-scale studies are required. Also, molecular profiling may help pave the road to the discovery of new therapies.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Renais/metabolismo , Neoplasias Renais/metabolismo , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/urina , Carcinoma de Células Renais/sangue , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/urina , Estudos de Casos e Controles , Feminino , Perfilação da Expressão Gênica , Proteínas de Choque Térmico HSP27/sangue , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/urina , Proteínas de Choque Térmico , Humanos , Imuno-Histoquímica , Neoplasias Renais/sangue , Neoplasias Renais/genética , Neoplasias Renais/urina , Masculino , Chaperonas Moleculares , Proteínas de Neoplasias/sangue , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/urina , Prognóstico , Proteômica/métodosRESUMO
BACKGROUND: A major barrier to effective treatment of glioblastoma multiforme (GBM) is the invasion of glioma cells into the brain parenchyma rendering local therapies such as surgery and radiation therapy ineffective. GBM patients with such highly invasive and infiltrative tumors have poor prognosis with a median survival time of only about a year. However, the mechanisms leading to increased cell migration, invasion and diffused behavior of glioma cells are still poorly understood. METHODS: In the current study, we applied quantitative proteomics for the identification of differentially expressed proteins in GBMs as compared to non-malignant brain tissues. RESULTS: Our study led to the identification of 23 proteins showing overexpression in GBM; these include membrane proteins, moesin and CD44. The results were verified using Western blotting and immunohistochemistry in independent set of GBM and non-malignant brain tissues. Both GBM tissues and glioma cell lines (U87 / U373) demonstrated membranous expression of moesin and CD44, as revealed by immunohistochemistry and immunofluorescence, respectively. Notably, glioma cells transfected with moesin siRNA displayed reduced migration and invasion on treatment with hyaluronan (HA), an important component of the extracellular matrix in GBM. CD44, a transmembrane glycoprotein, acts as a major receptor for hyaluronan (HA). Using co-immunoprecipitation assays, we further demonstrated that moesin interacts with CD44 in glioma cells only after treatment with HA; this implicates a novel role of moesin in HA-CD44 signaling in gliomas. CONCLUSIONS: Our results suggest that development of inhibitors which interfere with CD44-moesin interactions may open a new avenue in the future to mitigate cellular migration in gliomas.
Assuntos
Movimento Celular/efeitos dos fármacos , Glioblastoma/metabolismo , Ácido Hialurônico/farmacologia , Proteínas dos Microfilamentos/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/genética , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Proteínas dos Microfilamentos/genética , Ligação Proteica/efeitos dos fármacos , Proteoma , ProteômicaRESUMO
Various mass-tagging approaches have been developed over the last few years that have enabled mass spectrometry-based relative and absolute quantification of proteins from complex samples. This, in turn, has facilitated proteomics research to address issues ranging from alterations in the proteome of various model systems in response to various stimuli to biomarker discovery studies. Here we describe the use of one such mass-tagging approach, viz., iTRAQ labeling, as applied to cancer biomarker discovery. When applied to a cohort of tens of clinical samples, this technology can provide useful leads that serve as a basis for a more targeted validation-scale study.
Assuntos
Biomarcadores Tumorais/análise , Marcação por Isótopo , Proteínas/análise , Proteoma/análise , Proteômica/métodos , Humanos , Espectrometria de Massas , Neoplasias/metabolismo , Proteínas/químicaRESUMO
Metastatic renal cell carcinoma (RCC) is one of the most treatment-resistant malignancies, and patients have a dismal prognosis, with a <10% five-year survival rate. The identification of markers that can predict the potential for metastases will have a great effect in improving patient outcomes. In this study, we used differential proteomics with isobaric tags for relative and absolute quantitation (iTRAQ) labeling and LC-MS/MS analysis to identify proteins that are differentially expressed in metastatic and primary RCC. We identified 1256 non-redundant proteins, and 456 of these were quantified. Further analysis identified 29 proteins that were differentially expressed (12 overexpressed and 17 underexpressed) in metastatic and primary RCC. Dysregulated protein expressions of profilin-1 (Pfn1), 14-3-3 zeta/delta (14-3-3ζ), and galectin-1 (Gal-1) were verified on two independent sets of tissues by means of Western blot and immunohistochemical analysis. Hierarchical clustering analysis showed that the protein expression profile specific for metastatic RCC can distinguish between aggressive and non-aggressive RCC. Pathway analysis showed that dysregulated proteins are involved in cellular processes related to tumor progression and metastasis. Furthermore, preliminary analysis using a small set of tumors showed that increased expression of Pfn1 is associated with poor outcome and is a potential prognostic marker in RCC. In addition, 14-3-3ζ and Gal-1 also showed higher expression in tumors with poor prognosis than in those with good prognosis. Dysregulated proteins in metastatic RCC represent potential prognostic markers for kidney cancer patients, and a greater understanding of their involved biological pathways can serve as the foundation of the development of novel targeted therapies for metastatic RCC.
Assuntos
Carcinoma de Células Renais/genética , Neoplasias Renais/genética , Proteínas de Neoplasias/análise , Proteoma/análise , Proteínas 14-3-3/metabolismo , Biomarcadores Tumorais/análise , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/mortalidade , Carcinoma de Células Renais/secundário , Cromatografia Líquida , Progressão da Doença , Galectina 1/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/mortalidade , Metástase Neoplásica , Profilinas/metabolismo , Prognóstico , Proteômica , Espectrometria de Massas em TandemRESUMO
OBJECTIVE: This review describes the most common methods currently used for mass spectrometry-based quantification in proteomic applications. DESIGN AND METHODS: Quantification can be performed using either labeled or unlabeled approaches. Labeled approaches rely on mass-tagging of peptide reference standards either by direct synthesis or through chemical or metabolic means. Following labeling, samples are pooled and analyzed as one, thereby circumventing any potential error that could result from run-to-run variability. Unlabeled approaches rely on run-to-run comparisons between test and reference samples and assume a highly reproducible separation and similar sample compositions. RESULTS: A number of commercial labeling reagents are now available and each of these are described along with their strengths and limitations. CONCLUSIONS: The choice of method will ultimately depend, not just on financial considerations, but also on the context of the experiment.
Assuntos
Cromatografia Líquida/métodos , Proteínas , Proteômica , Espectrometria de Massas em Tandem/métodos , Técnicas de Laboratório Clínico , Biologia Computacional , Perfilação da Expressão Gênica , Humanos , Marcação por Isótopo , Ciência de Laboratório Médico , Proteínas/isolamento & purificação , Padrões de ReferênciaRESUMO
Cerebral hyaline astrocytic inclusions have been observed in a subset of patients with early onset epilepsy, brain structural anomalies, and developmental delay, which indicates that it may represent a unique clinicopathologic entity. To further characterize this condition we use proteomics to investigate differentially expressed proteins in epileptic brain tissue from three pediatric epileptic patients with cerebral hyaline astrocytic inclusions, ranging in age from 5-13 years, and compare to brain tissue from two normal controls. Catalase and carbonic anhydrase I both exhibited increased expression in epileptic brain tissue compared to controls. These findings were confirmed by Western blot analysis. Furthermore, both proteins were localized to astrocytes and in epileptic brain were located within the cerebral hyaline astrocytic inclusions, suggesting a potential role in the generation of this pathologic feature of early onset epilepsy with cerebral hyaline astrocytic inclusions.
Assuntos
Astrócitos/patologia , Epilepsia/metabolismo , Epilepsia/patologia , Corpos de Inclusão/patologia , Proteômica/métodos , Adolescente , Astrócitos/metabolismo , Anidrases Carbônicas/metabolismo , Catalase/metabolismo , Pré-Escolar , Epilepsia/diagnóstico , Feminino , Humanos , Hialina/química , Corpos de Inclusão/metabolismoRESUMO
Equilibrative Nucleoside Transporters (SLC29) are a family of proteins that transport nucleosides, nucleobases and nucleoside analogue drugs across cellular membranes. ENT1 is expressed ubiquitously in mammalian tissues and responsible for a significant portion of nucleoside analog drug uptake in humans. Despite the important clinical role of ENT1, many aspects of the regulation of this protein remain unknown. A major outstanding question in this field is the whether ENT1 is phosphorylated directly. To answer this question, we overexpressed tagged human (h) and mouse (m) ENT1, affinity purified protein using the tag, conducted phosphoamino acid analysis and found that m/hENT1 is predominantly phosphorylated at serine residues. The large intracellular loop of ENT1, between transmembrane domains 6 and 7, has been suggested to be a site of regulation by phosphorylation, therefore we generated His/Ubiquitin tagged peptides of this region and used them for in vitro kinase assays to identify target serines. Our data support a role for PKA and PKC in the phosphorylation of ENT1 within the intracellular loop and show that PKA can phosphorylate multiple sites within this loop while PKC specifically targets serines 279 and 286 and threonine 274. These data demonstrate, for the first time, that ENT1 is a phosphoprotein that can be directly phosphorylated at several sites by more than one kinase. The presence of multiple kinase targets within the loop suggests that ENT1 phosphorylation is considerably more complex than previously thought and thus ENT1 may be subject to phosphorylation by multiple pathways.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Proteína Quinase C/metabolismo , Animais , Células COS , Chlorocebus aethiops , Transportador Equilibrativo 1 de Nucleosídeo/química , FosforilaçãoRESUMO
In search of blood-based biomarkers that would enhance the ability to diagnose head and neck/oral squamous cell carcinoma (HNOSCC) in early stages or predict its prognosis, we analyzed the HNOSCC secretome (ensemble of proteins secreted and/or shed from the tumor cells) for potential biomarkers using proteomic technologies. LC-MS/MS was used to identify proteins in the conditioned media of four HNOSCC cell lines (SCC4, HSC2, SCC38, and AMOSIII); 140 unique proteins were identified on the basis of 5% global false discovery rate, 122 of which were secretory proteins, with 29 being previously reported to be overexpressed in HNOSCC in comparison to normal head and neck tissues. Of these, five proteins including α-enolase, peptidyl prolyl isomerase A/cyclophilin A, 14-3-3 ζ, heterogeneous ribonucleoprotein K, and 14-3-3 σ were detected in the sera of HNOSCC patients by Western blot analysis. Our study provides the evidence that analysis of head and neck cancer cells' secretome is a viable strategy for identifying candidate serological biomarkers for HNOSCC. In future, these biomarkers may be useful in predicting the likelihood of transformation of oral pre-malignant lesions, prognosis of HNOSCC patients and evaluate response to therapy using minimally invasive tests.
Assuntos
Biomarcadores/sangue , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/genética , Meios de Cultivo Condicionados/química , Neoplasias de Cabeça e Pescoço/diagnóstico , Neoplasias de Cabeça e Pescoço/genética , Proteínas de Neoplasias/genética , Carcinoma de Células Escamosas/sangue , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Cromatografia Líquida , Diagnóstico Precoce , Neoplasias de Cabeça e Pescoço/sangue , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Espectrometria de Massas , Proteínas de Neoplasias/sangue , Proteínas de Neoplasias/metabolismo , Estadiamento de Neoplasias , Prognóstico , Proteômica , Espectrometria de Massas em TandemRESUMO
BACKGROUND: The number of patients with endometrial carcinoma (EmCa) with advanced stage or high histological grade is increasing and prognosis has not improved for over the last decade. There is an urgent need for the discovery of novel molecular targets for diagnosis, prognosis and treatment of EmCa, which will have the potential to improve the clinical strategy and outcome of this disease. METHODOLOGY AND RESULTS: We used a "drill-down" proteomics approach to facilitate the identification of novel molecular targets for diagnosis, prognosis and/or therapeutic intervention for EmCa. Based on peptide ions identified and their retention times in the first LC-MS/MS analysis, an exclusion list was generated for subsequent iterations. A total of 1529 proteins have been identified below the Proteinpilot® 5% error threshold from the seven sets of iTRAQ experiments performed. On average, the second iteration added 78% new peptides to those identified after the first run, while the third iteration added 36% additional peptides. Of the 1529 proteins identified, only 40 satisfied our criteria for significant differential expression in EmCa in comparison to normal proliferative tissues. These proteins included metabolic enzymes (pyruvate kinase M2 and lactate dehydrogenase A); calcium binding proteins (S100A6, calcyphosine and calumenin), and proteins involved in regulating inflammation, proliferation and invasion (annexin A1, interleukin enhancer-binding factor 3, alpha-1-antitrypsin, macrophage capping protein and cathepsin B). Network analyses revealed regulation of these molecular targets by c-myc, Her2/neu and TNF alpha, suggesting intervention with these pathways may be a promising strategy for the development of novel molecular targeted therapies for EmCa. CONCLUSIONS: Our analyses revealed the significance of drill-down proteomics approach in combination with iTRAQ to overcome some of the limitations of current proteomics strategies. This study led to the identification of a number of novel molecular targets having therapeutic potential for targeted molecular therapies for endometrial carcinoma.
Assuntos
Neoplasias do Endométrio/química , Terapia de Alvo Molecular/métodos , Proteínas de Neoplasias/análise , Proteômica/métodos , Cromatografia Líquida , Neoplasias do Endométrio/diagnóstico , Neoplasias do Endométrio/tratamento farmacológico , Feminino , Humanos , Prognóstico , Espectrometria de Massas em TandemRESUMO
Ribosome inactivating proteins are glycosidases synthesized by many plants and have been hypothesized to serve in defence against pathogens. These enzymes catalytically remove a conserved purine from the sarcin/ricin loop of the large ribosomal RNA, which has been shown in vitro to limit protein synthesis. The resulting toxicity suggests that plants may possess a mechanism to protect their ribosomes from depurination during the synthesis of these enzymes. For example, pokeweed antiviral protein (PAP) is cotranslationally inserted into the lumen of the endoplasmic reticulum and travels via the endomembrane system to be stored in the cell wall. However, some PAP may retrotranslocate across the endoplasmic reticulum membrane to be released back into the cytosol, thereby exposing ribosomes to depurination. In this work, we isolated and characterized a complexed form of the enzyme that exhibits substantially reduced activity. We showed that this complex is a homodimer of PAP and that dimerization involves a peptide that contains a conserved aromatic amino acid, tyrosine 123, located in the active site of the enzyme. Bimolecular fluorescence complementation demonstrated that the homodimer may form in vivo and that dimerization is prevented by the substitution of tyrosine 123 for alanine. The homodimer is a minor form of PAP, observed only in the cytosol of cells and not in the apoplast. Taken together, these data support a novel mechanism for the limitation of depurination of autologous ribosomes by molecules of the protein that escape transport to the cell wall by the endomembrane system.
Assuntos
Phytolacca americana/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 1/metabolismo , Ribossomos/metabolismo , Immunoblotting , Espectrometria de Massas , Proteínas de Plantas/metabolismo , Multimerização ProteicaRESUMO
Mass spectrometry (MS)-based proteomics is a rapidly developing technology for both qualitative and quantitative analyses of proteins, and investigations into protein posttranslational modifications, subcellular localization, and interactions. Recent advancements in MS have made tremendous impact on the throughput and comprehensiveness of cancer proteomics, paving the way to unraveling deregulated cellular pathway networks in human malignancies. In turn, this knowledge is rapidly being translated into the discovery of novel potential cancer markers (PCMs) and targets for molecular therapeutics. Head-and-neck cancer is one of the most morbid human malignancies with an overall poor prognosis and severely compromised quality of life. Early detection and novel therapeutic strategies are urgently needed for more effective disease management. The characterizations of protein profiles of head-and-neck cancers and non-malignant tissues, with unprecedented sensitivity and precision, are providing technology platforms for identification of novel PCMs and drug targets. Importantly, low-abundance proteins are being identified and characterized, not only from the tumor tissues, but also from bodily fluids (plasma, saliva, and urine) in a high-throughput and unbiased manner. This review is a critical appraisal of recent advances in MS-based proteomic technologies and platforms for facilitating the discovery of biomarkers and novel drug targets in head-and-neck cancer. A major challenge in the discovery and verification of these cancer biomarkers is the typically limited availability of well-characterized and adequately stored clinical samples in tumor and sera banks, collected using recommended procedures, and with detailed information on clinical, pathological parameters, and follow-up. Most biomarker discovery studies use limited number of clinical samples and verification of cancer markers in large number of samples is beyond the scope of a single laboratory. The validation of these potential markers in large sample cohorts in multicentric studies is needed for their translation from the bench to the bedside.
Assuntos
Antineoplásicos/farmacologia , Neoplasias de Cabeça e Pescoço/diagnóstico , Proteômica/instrumentação , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais , Cromatografia Líquida de Alta Pressão , Sistemas de Liberação de Medicamentos , Eletroforese em Gel Bidimensional , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Humanos , Espectrometria de Massas , Metanálise como Assunto , Microdissecção , Proteômica/métodosRESUMO
Patients diagnosed in advanced stages of head and neck squamous cell carcinoma often show limited response to chemotherapeutic agents. Recently, we reported the overexpression of 14-3-3ζ protein in head and neck premalignant and cancer tissues using liquid chromatography-tandem mass spectrometry with isotopic labeling and revealed its significance as a prognostic marker using immunohistochemical analysis. In this study, we determined the potential of 14-3-3ζ as a therapeutic target for head and neck cancer. Small interfering RNA (siRNA) targeting 14-3-3ζ was used to downregulate its expression in head and neck cancer cells in culture. Cell cycle analysis showed that head and neck cancer cells transfected with siRNA targeting 14-3-3ζ showed G(2)-M arrest. These siRNA transfectants also showed increased cell death on treatment with any one of the following chemotherapeutic agents: cisplatin, 5-fluorouracil, paclitaxel, or doxorubicin in comparison with the no transfection controls. Flow cytometric analysis using propidium iodide staining showed increased sub-G(0) fraction in siRNA-transfected cells treated with any of these chemotherapeutic agents, suggesting cell death; in addition, Annexin V staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay revealed increased apoptosis. Taken together, our results strongly showed that downregulation of 14-3-3ζ expression may serve to improve the sensitivity of head and neck cancer cells to chemotherapeutic agents.
Assuntos
Proteínas 14-3-3/genética , Antineoplásicos/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , RNA Interferente Pequeno/genética , Sequência de Bases , Western Blotting , Carcinoma de Células Escamosas/patologia , Ciclo Celular , Proliferação de Células , Citometria de Fluxo , Inativação Gênica , Neoplasias de Cabeça e Pescoço/patologia , Marcação In Situ das Extremidades Cortadas , Prognóstico , Transfecção , Células Tumorais CultivadasRESUMO
Formalin-fixed paraffin-embedded (FFPE) tissues are the primary and preferred medium for archiving patients' samples. Here we demonstrate relative quantifications of protein biomarkers in extracts of laser microdissected epithelial cells from FFPE endometrial carcinoma tissues versus those from normal proliferative endometria by means of targeted proteomic analyses using LC-multiple reaction monitoring (MRM) MS with MRM Tags for Relative and Absolute Quantitation (mTRAQ) labeling. Comparable results of differential expressions for pyruvate kinase isoform M2 (PK-M2) and polymeric Ig receptor were observed between analyses on laser microdissected epithelial cells from FFPE tissues and corresponding homogenates from frozen tissues of the same individuals that had previously been analyzed and reported. We also identified PK-M2 in the normal proliferative phase of the endometrium. Other biomarkers in addition to PK-M2 and polymeric Ig receptor were also observed but not consistently and/or were at levels below the threshold for quantification.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias do Endométrio/química , Marcação por Isótopo/métodos , Espectrometria de Massas/métodos , Sequência de Aminoácidos , Biomarcadores Tumorais/metabolismo , Cromatografia por Troca Iônica , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Células Epiteliais/metabolismo , Feminino , Fase Folicular , Formaldeído , Humanos , Microdissecção , Inclusão em Parafina , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/metabolismo , Piruvato Quinase/análise , Piruvato Quinase/metabolismo , Receptores de Imunoglobulina Polimérica/análise , Receptores de Imunoglobulina Polimérica/metabolismoRESUMO
Equilibrative nucleoside transporters (ENTs) are integral membrane proteins that facilitate the movement of nucleosides and hydrophilic nucleoside analog (NA) drugs across cell membranes. ENTs are also targets for cardioprotectant drugs, which block re-uptake of the purine nucleoside adenosine, thereby enhancing purinergic receptor signaling pathways. ENTs are therefore important contributors to drug bioavailability and efficacy. Despite this important clinical role, very little is known about the structure and regulation of ENTs. Biochemical and structural studies on ENT proteins have been limited by their low endogenous expression levels, hydrophobicity and labile nature. To address these issues, we developed an approach whereby tagged mammalian ENT1 protein was over-expressed in mammalian cell lines, confirmed to be functional and isolated by affinity purification to sufficient levels to be analyzed using MALDI-TOF and tandem MS mass spectrometry. This proteomic approach will allow for a more detailed analysis of the structure, function and regulation of ENTs in the future.
Assuntos
Proteínas de Transporte de Nucleosídeo Equilibrativas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Células COS , Chlorocebus aethiops , Proteínas de Transporte de Nucleosídeo Equilibrativas/genética , Proteínas de Transporte de Nucleosídeo Equilibrativas/metabolismo , Camundongos , Oligopeptídeos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tioinosina/análogos & derivados , Tioinosina/metabolismo , TripsinaRESUMO
Emerging evidence suggests that RNA interference (RNAi)-related processes act both in the cytoplasm and in the nucleus. However, the process by which the RNAi machinery is transported into the nucleus remains poorly understood. The Tetrahymena Argonaute protein Twi1p localizes to the nucleus and is crucial for small RNA-directed programmed DNA elimination. In this study, we identify Giw1p, which binds to Twi1p and is required for its nuclear localization. Furthermore, the endoribonuclease (Slicer) activity of Twi1p plays a vital role in the removal of one of the two strands of Twi1p-associated small interfering RNAs (siRNAs), leading to a functionally mature Twi1p-siRNA complex. Slicer activity is also shown to be required for nuclear localization of Twi1p and for its association with Giw1p. These results suggest that Giw1p senses the state of Twi1p-associated siRNAs and selectively transports the mature Twi1p-siRNA complex into the nucleus.
Assuntos
Núcleo Celular/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Proteínas de Protozoários/metabolismo , RNA Interferente Pequeno/metabolismo , Tetrahymena thermophila/metabolismo , Sequência de Aminoácidos , Conjugação Genética , Citoplasma/metabolismo , Proteínas de Protozoários/química , Tetrahymena thermophila/citologia , Proteína 1 Relacionada a Twist/metabolismoRESUMO
Glioblastoma multiforme (GBM) is the most common and lethal primary human brain tumor. GBMs are characterized by a variety of genetic alterations, among which oncogenic mutations of epidermal growth factor receptor (EGFRvIII) is most common. GBMs harboring EGFRvIII have increased proliferation and invasive characteristics versus those expressing wild-type (wt) EGFR. To identify the molecular basis of this increased tumorgenic phenotype, we used iTRAQ-labeling differential proteomic analysis. Among several differentially expressed proteins, we selected CRMP1, a protein implicated in cellular invasion that was markedly decreased in GBMs expressing EGFRvIII, for further study. The differential expression of CRMP1 was confirmed in a panel of human GBM cell lines and operative specimens that express wtEGFR or mutant EGFRvIII by quantitative real-time PCR, Western blot, and immunohistochemical analysis. In human GBM samples, decreased expression of CRMP1 correlated with EGFRvIII positivity. Knockdown of CRMP1 by siRNA resulted in increased invasion of wtEGFR expressing human GBM cells (U87 and U373) to those found in isogenic GBM cells. Exogenous expression of EGFRvIII in these wtEGFR-expressing GBM cells promoted their ability to invade and was accompanied by decreased expression of CRMP1. Rescuing CRMP1 expression decreased invasion of the EGFRvIII-expressing GBM cells by tilting the balance between Rac and Rho. Collectively, these results show that the loss of CRMP1 contribute to the increased invasive phenotype of human GBMs expressing mutant EGFRvIII.
Assuntos
Neoplasias Encefálicas/patologia , Glioma/patologia , Invasividade Neoplásica/genética , Proteínas do Tecido Nervoso/biossíntese , Western Blotting , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Cromatografia Líquida , Receptores ErbB/genética , Receptores ErbB/metabolismo , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Humanos , Imuno-Histoquímica , Espectrometria de Massas , Mutação , Invasividade Neoplásica/patologia , Proteínas do Tecido Nervoso/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas rac de Ligação ao GTP , Proteína rhoA de Ligação ao GTPRESUMO
Renal cell carcinoma (RCC) is the most common neoplasm in the adult kidney. Unfortunately, there are currently no biomarkers for the diagnosis of RCC. In addition to early detection, biomarkers have a potential use for prognosis, for monitoring recurrence after treatment, and as predictive markers for treatment efficiency. In this study, we identified proteins that are dysregulated in RCC, utilizing a quantitative mass spectrometry analysis. We compared the protein expression of kidney cancer tissues to their normal counterparts from the same patient using LC-MS/MS. iTRAQ labeling permitted simultaneous quantitative analysis of four samples (cancer, normal, and two controls) by separately tagging the peptides in these samples with four cleavable mass-tags (114, 115, 116, and 117 Da). The samples were then pooled, and the tagged peptides resolved first by strong cation exchange chromatography and then by nanobore reverse phase chromatography coupled online to nanoelectrospray MS/MS. We identified a total of 937 proteins in two runs. There was a statistically significant positive correlation of the proteins identified in both runs (r(p) = 0.695, p < 0.001). Using a cutoff value of 0.67 fold for underexpression and 1.5 fold for overexpression, we identified 168 underexpressed proteins and 156 proteins that were overexpressed in RCC compared to normal tissues. These dysregulated proteins in RCC were statistically significantly different from those of transitional cell carcinoma and end-stage glomerulonephritis. We performed an in silico validation of our results using different tools and databases including Serial Analysis of Gene Expression (SAGE), UniGene EST ProfileViewer, Cancer Genome Anatomy Project, and Gene Ontology consortium analysis.
Assuntos
Biomarcadores Tumorais/biossíntese , Carcinoma de Células Renais/metabolismo , Neoplasias Renais/metabolismo , Proteínas de Neoplasias/biossíntese , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Biomarcadores Tumorais/genética , Carcinoma de Células Renais/genética , Cromatografia Líquida/métodos , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Marcação por Isótopo , Neoplasias Renais/genética , Modelos Genéticos , Proteínas de Neoplasias/genética , Reprodutibilidade dos TestesRESUMO
In conjugating Tetrahymena thermophila, massive DNA elimination occurs upon the development of the new somatic genome from the germ line genome. Small, approximately 28-nucleotide scan RNAs (scnRNAs) and Twi1p, an Argonaute family member, mediate H3K27me3 and H3K9me3 histone H3 modifications, which lead to heterochromatin formation and the excision of the heterochromatinized germ line-limited sequences. In our search for new factors involved in developmental DNA rearrangement, we identified two Twi1p-interacting proteins, Wag1p and CnjBp. Both proteins contain GW (glycine and tryptophan) repeats, which are characteristic of several Argonaute-interacting proteins in other organisms. Wag1p and CnjBp colocalize with Twi1p in the parental macronucleus early in conjugation and in the new developing macronucleus during later developmental stages. Around the time DNA elimination occurs, Wag1p forms multiple nuclear bodies in the developing macronuclei that do not colocalize with heterochromatic DNA elimination structures. Analyses of DeltaWAG1, DeltaCnjB, and double DeltaWAG1 DeltaCnjB knockout strains revealed that WAG1 and CnjB genes need to be deleted together to inhibit the downregulation of specific scnRNAs, the formation of DNA elimination structures, and DNA excision. Thus, Wag1p and CnjBp are two novel players with overlapping functions in RNA interference-mediated genome rearrangement in Tetrahymena.
Assuntos
Rearranjo Gênico , Genoma de Protozoário/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Sequências Repetitivas de Aminoácidos , Tetrahymena thermophila/genética , Tetrahymena thermophila/metabolismo , Animais , Sequência de Bases , Núcleo Celular/metabolismo , DNA de Protozoário/metabolismo , Regulação para Baixo/genética , Técnicas de Inativação de Genes , Imunoprecipitação , Fenótipo , Ligação Proteica , Transporte Proteico , Proteínas de Protozoários/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , RNA não Traduzido/metabolismo , Tetrahymena thermophila/citologiaRESUMO
Oral leukoplakia is a heterogeneous lesion with risk of cancer development; there are no biomarkers to predict its potential of malignant transformation. Tissue proteomic analysis of oral leukoplakia using iTRAQ labeling liquid chromatography-mass spectrometry showed overexpression of heterogeneous ribonucleoprotein K (hnRNP K), a transformation-related RNA-binding protein, in leukoplakia in comparison with normal tissue. Herein, we investigated the clinical significance of hnRNP K in identification of oral leukoplakic lesions in early stages and as a prognostic marker in head-and-neck/oral squamous cell carcinomas (HNOSCCs). Immunohistochemical analysis of hnRNP K was performed in 100 HNOSCCs, 199 leukoplakias and 55 nonmalignant tissues and correlated with clinicopathologic parameters and disease prognosis over 6 years for HNOSCCs. hnRNP K nuclear expression increased from normal tissues to leukoplakia, and frank malignancy (p < 0.001). Cytoplasmic hnRNP K increased significantly from leukoplakia to HNOSCCs (p < 0.001) and was associated with poor prognosis of HNOSCCs (p = 0.011) by Kaplan-Meier analysis. The most important finding of our follow-up study is that cytoplasmic hnRNP K is an independent predictor of disease recurrence in HNOSCC patients. In conclusion, nuclear hnRNP K may serve as a potential marker for early diagnosis, whereas its cytoplasmic accumulation can help to identify a subgroup of HNOSCC patients with poor prognosis, suggesting its putative utility in clinical management of HNOSCC.