RESUMEN
Protein-protein interactions are fundamental to various aspects of cell biology with many protein complexes participating in numerous fundamental biological processes such as transcription, translation and cell cycle. MS-based proteomics techniques are routinely applied for characterising the interactome, such as affinity purification coupled to mass spectrometry that has been used to selectively enrich and identify interacting partners of a bait protein. In recent years, many orthogonal MS-based techniques and approaches have surfaced including proximity-dependent labelling of neighbouring proteins, chemical cross-linking of two interacting proteins, as well as inferring PPIs from the co-behaviour of proteins such as the co-fractionating profiles and the thermal solubility profiles of proteins. This review discusses the underlying principles, advantages, limitations and experimental considerations of these emerging techniques. In addition, a brief account on how MS-based techniques are used to investigate the structural and functional properties of protein complexes, including their topology, stoichiometry, copy number and dynamics, are discussed.
Asunto(s)
Cromatografía de Afinidad/métodos , Espectrometría de Masas/métodos , Mapeo de Interacción de Proteínas/métodos , Proteínas/metabolismo , Proteoma/metabolismo , Animales , Humanos , Proteoma/análisisRESUMEN
Ubiquitination is an important post-translational event responsible for half-life and turnover of proteins inside the cell. Proteins are ubiquitinated by forming an iso-peptide bond between their lysine residue and C-terminal glycine residue of ubiquitin leading to rapid degradation of proteins by 26S proteosome complex. Deregulation of ubiquitination is manifested by aberrant expression of E3-ligase activity or mutation in the surroundings of ubiquitination sites. Many new experimentally validated ubiquitinated lysines have been recently identified that motivated the study of the environments surrounding the ubiquitinated lysines. With the help of known ubiquitinated proteins, here we present a comprehensive study of sequence and spatial environment of ubiquitination sites of human and yeast proteins. To identify position-specific features, this work distinguishes the spatial environments as proximity and distal regions. Certain amino acids specific to these regions, well differentiate the ubiquitination sites from non-ubiquitination sites are revealed. Additionally, amino acid signatures that contribute for protein disordered regions and solvent accessibility of amino acids are found to be contributing factors in ubiquitination sites. These results suggest that the ubiquitination site environment of the substrate determines the recognition and unfolding of substrate to facilitate the entry into 26S proteosomal complex. We believe that these findings will help in better prediction of ubiquitination sites using the sequence and spatial information.
Asunto(s)
Ubiquitina/metabolismo , Proteínas Ubiquitinadas/metabolismo , Ubiquitinación , Secuencia de Aminoácidos , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Humanos , Conformación Proteica , Proteínas Ubiquitinadas/química , Levaduras/química , Levaduras/metabolismoRESUMEN
OBJECTIVE: CRP gene polymorphism is common in inflammatory diseases, but such association has not been reported in periodontitis. Our objective was to interrogate SNPs of crp in chronic periodontitis in a case-control manner. METHODS: DNAs were extracted from mouthwash samples of 116 volunteers using salting-out method. Selected 12 5'UTR SNPs of crp were genotyped using ARMS-PCR. RESULTS: TC genotype of - 757T > C polymorphism (rs3093059) showed protective association (OR- 0.29, 95%CI-0.12-0.68, and p-0.004), and wild type - 757TT showed susceptible association with a p value of 0.008 (OR-3.09, 95%CI-1.33-7.15). CONCLUSION: The observation of protective and susceptible association of crp - 757T > C polymorphism may be useful for better management and prophylaxis of periodontitis.
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Proteína C-Reactiva/genética , Periodontitis Crónica/genética , Adulto , Anciano , Pueblo Asiatico/genética , Estudios de Casos y Controles , Predisposición Genética a la Enfermedad , Genotipo , Humanos , India , Persona de Mediana Edad , Oportunidad Relativa , Polimorfismo de Nucleótido SimpleRESUMEN
A full description of the human proteome relies on the challenging task of detecting mature and changing forms of protein molecules in the body. Large-scale proteome analysis has routinely involved digesting intact proteins followed by inferred protein identification using mass spectrometry. This 'bottom-up' process affords a high number of identifications (not always unique to a single gene). However, complications arise from incomplete or ambiguous characterization of alternative splice forms, diverse modifications (for example, acetylation and methylation) and endogenous protein cleavages, especially when combinations of these create complex patterns of intact protein isoforms and species. 'Top-down' interrogation of whole proteins can overcome these problems for individual proteins, but has not been achieved on a proteome scale owing to the lack of intact protein fractionation methods that are well integrated with tandem mass spectrometry. Here we show, using a new four-dimensional separation system, identification of 1,043 gene products from human cells that are dispersed into more than 3,000 protein species created by post-translational modification (PTM), RNA splicing and proteolysis. The overall system produced greater than 20-fold increases in both separation power and proteome coverage, enabling the identification of proteins up to 105 kDa and those with up to 11 transmembrane helices. Many previously undetected isoforms of endogenous human proteins were mapped, including changes in multiply modified species in response to accelerated cellular ageing (senescence) induced by DNA damage. Integrated with the latest version of the Swiss-Prot database, the data provide precise correlations to individual genes and proof-of-concept for large-scale interrogation of whole protein molecules. The technology promises to improve the link between proteomics data and complex phenotypes in basic biology and disease research.
Asunto(s)
Isoformas de Proteínas/análisis , Isoformas de Proteínas/química , Proteoma/análisis , Proteoma/química , Proteómica/métodos , Empalme Alternativo , Línea Celular , Senescencia Celular/genética , Daño del ADN , Bases de Datos de Proteínas , Proteína HMGA1a/análisis , Proteína HMGA1b/análisis , Células HeLa , Humanos , Fenotipo , Procesamiento Proteico-Postraduccional , Proteolisis , Proteómica/instrumentaciónRESUMEN
BACKGROUND: Androgens play a crucial role in prostate cancer, hence the androgenic pathway has become an important target of therapeutic intervention. Previously we discovered that gene fusions between the 5'-untranslated region of androgen regulated gene TMPRSS2 and the ETS transcription factor family members were present in a majority of the prostate cancer cases. The resulting aberrant overexpression of ETS genes drives tumor progression. METHODS: Here, we evaluated the expression levels of 5α-reductase isoenzymes in prostate cancer cell lines and tissues. We tested the effect of dutasteride, a 5α-reductase inhibitor, in TMPRSS2-ERG fusion-positive VCaP cell proliferation and cell invasion. We also evaluated the effect of dutasteride on the TMPRSS2-ERG fusion gene expression. Finally, we tested dutasteride alone or in combination with an anti-androgen in VCaP cell xenografts tumor model. RESULTS: Our data showed that 5α-reductase SRD5A1 and SRD5A3 isoenzymes that are responsible for the conversion of testosterone to DHT, are highly expressed in metastatic prostate cancer compared to benign and localized prostate cancer. Dutasteride treatment attenuated VCaP cell proliferation and invasion. VCaP cells pre-treated with dutasteride showed a reduction in ERG and PSA expression. In vivo studies demonstrated that dutasteride in combination with the anti-androgen bicalutamide significantly decreased tumor burden in VCaP cell xenograft model. CONCLUSIONS: Our findings suggest that dutasteride can inhibit ERG fusion-positive cell growth and in combination with anti-androgen, significantly reduce the tumor burden. Our study suggests that anti-androgens used in combination with dutasteride could synergistically augment the therapeutic efficacy in the treatment of ETS-positive prostate cancer.
Asunto(s)
Inhibidores de 5-alfa-Reductasa/farmacología , Azaesteroides/farmacología , Proteínas de Fusión Oncogénica/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Dutasterida , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Isoenzimas/farmacología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias Hormono-Dependientes/tratamiento farmacológico , Neoplasias Hormono-Dependientes/enzimología , Neoplasias Hormono-Dependientes/genética , Neoplasias Hormono-Dependientes/metabolismo , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Proteínas de Fusión Oncogénica/genética , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , ARN Neoplásico/química , ARN Neoplásico/genética , Distribución Aleatoria , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Despite the availability of ultra-high-resolution mass spectrometers, methods for separation and detection of intact proteins for proteome-scale analyses are still in a developmental phase. Here we report robust protocols for online LC-MS to drive high-throughput top-down proteomics in a fashion similar to that of bottom-up proteomics. Comparative work on protein standards showed that a polymeric stationary phase led to superior sensitivity over a silica-based medium in reversed-phase nanocapillary LC, with detection of proteins >50 kDa routinely accomplished in the linear ion trap of a hybrid Fourier transform mass spectrometer. Protein identification was enabled by nozzle-skimmer dissociation and detection of fragment ions with <10 ppm mass accuracy for highly specific database searching using tailored software. This overall approach led to identification of proteins up to 80 kDa, with 10-60 proteins identified in single LC-MS runs of samples from yeast and human cell lines prefractionated by their molecular mass using a gel-based sieving system.
Asunto(s)
Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Nanotecnología , Proteínas/análisis , Proteínas/química , Secuencia de Aminoácidos , Animales , Bovinos , Células HeLa , Humanos , Datos de Secuencia Molecular , Peso Molecular , Polímeros/química , Porosidad , Proteoma/análisis , Proteoma/química , Proteínas de Saccharomyces cerevisiae/análisis , Proteínas de Saccharomyces cerevisiae/química , Factores de TiempoRESUMEN
Two new peptide antibiotics were secreted by a Gram-positive bacterial strain isolated from fermented tomato fruit. Based on its 99% 16S rDNA sequence similarity with Paenibacillus alvei, the isolate was designated as P. alvei NP75. Among these two peptides, one is active against Gram-positive pathogens while the other against Gram-negative pathogens; thus these peptides were named as paenibacillin P and paenibacillin N, respectively. After the purification of those peptide antibiotics from the cell free culture supernatant by RP-HPLC, they were analyzed for their temperature sensitivity and susceptibility to proteases. Higher-temperature tolerant paenibacillin N was easily degraded by proteinase K, while the temperature sensitive paenibacillin P was not affected by any of the proteases used in this study other than a specific protease that was secreted by the same NP75 strain. Mass-spectrometry analysis of the above peptide antibiotics further confirmed their distinction among the known peptide antibiotics. We are reporting first of its kind the co-production of two different new peptide antibiotics from a single bacterial isolate of P. alvei strain.
Asunto(s)
Antibacterianos/biosíntesis , Péptidos Catiónicos Antimicrobianos/biosíntesis , Bacillus/metabolismo , Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Péptidos Catiónicos Antimicrobianos/farmacología , Bacillus/genética , Bacillus/aislamiento & purificación , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Solanum lycopersicum/microbiología , Espectrometría de MasasRESUMEN
PURPOSE: External beam radiotherapy (RT) is often used in an attempt to cure localized prostate cancer (PCa), but it is only palliative against disseminated disease. Raf kinase inhibitory protein (RKIP) is a metastasis suppressor whose expression is reduced in approximately 50% of localized PCa tissues and is absent in metastases. Chemotherapeutic agents have been shown to induce tumor apoptosis through induction of RKIP expression. Our goal was to test whether RT similarly induces apoptosis through induction of RKIP expression. METHODS AND MATERIALS: The C4-2B PCa cell line was engineered to overexpress or underexpress RKIP. The engineered cells were tested for apoptosis in cell culture and tumor regression in mice after RT. RESULTS: RT induced both RKIP expression and apoptosis of PCa cells. Overexpression of RKIP sensitized PCa cells to radiation-induced apoptosis. In contrast, short-hairpin targeting of RKIP, so that RT could not induce RKIP expression, protected cells from radiation-induced apoptosis. In a murine model, knockdown of RKIP in PCa cells diminished radiation-induced apoptosis. Molecular concept mapping of genes altered on manipulation of RKIP expression revealed an inverse correlation with the concept of genes altered by RT. CONCLUSION: The data presented in this report indicate that the loss of RKIP, as seen in primary PCa tumors and metastases, confers protection against radiation-induced apoptosis. Therefore, it is conceivable that the loss of RKIP confers a growth advantage on PCa cells at distant sites, because the loss of RKIP would decrease apoptosis, favoring proliferation.
Asunto(s)
Apoptosis/efectos de la radiación , Proteínas de Neoplasias/deficiencia , Proteínas de Unión a Fosfatidiletanolamina/deficiencia , Neoplasias de la Próstata/radioterapia , Tolerancia a Radiación/fisiología , Animales , Apoptosis/fisiología , Línea Celular Tumoral , Inducción Enzimática/efectos de la radiación , Masculino , Ratones , Metástasis de la Neoplasia , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Proteínas de Unión a Fosfatidiletanolamina/biosíntesis , Proteínas de Unión a Fosfatidiletanolamina/genética , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/metabolismo , Neoplasias de la Próstata/enzimología , Inmunodeficiencia Combinada GraveRESUMEN
Protein lysine acetylation (LysAc) has recently been demonstrated to be widespread in E. coli and Salmonella, and to broadly regulate bacterial physiology and metabolism. However, LysAc in plant pathogenic bacteria is largely unknown. Here we first report the lysine acetylome of Erwinia amylovora, an enterobacterium causing serious fire blight disease of apples and pears. Immunoblots using generic anti-lysine acetylation antibodies demonstrated that growth conditions strongly affected the LysAc profiles in E. amylovora. Differential LysAc profiles were also observed for two E. amylovora strains, known to have differential virulence in plants, indicating translational modification of proteins may be important in determining virulence of bacterial strains. Proteomic analysis of LysAc in two E. amylovora strains identified 141 LysAc sites in 96 proteins that function in a wide range of biological pathways. Consistent with previous reports, 44% of the proteins are involved in metabolic processes, including central metabolism, lipopolysaccharide, nucleotide and amino acid metabolism. Interestingly, for the first time, several proteins involved in E. amylovora virulence, including exopolysaccharide amylovoran biosynthesis- and type III secretion-associated proteins, were found to be lysine acetylated, suggesting that LysAc may play a major role in bacterial virulence. Comparative analysis of LysAc sites in E. amylovora and E. coli further revealed the sequence and structural commonality for LysAc in the two organisms. Collectively, these results reinforce the notion that LysAc of proteins is widespread in bacterial metabolism and virulence.
Asunto(s)
Erwinia amylovora/metabolismo , Lisina/metabolismo , Acetilación , Proteínas Bacterianas/metabolismo , Cromatografía Liquida , Enfermedades de las Plantas/microbiología , Procesamiento Proteico-Postraduccional , Proteómica , Espectrometría de Masas en Tándem , Virulencia/genéticaRESUMEN
BACKGROUND: The problem of prostate cancer progression to androgen independence has been extensively studied. Several studies systematically analyzed gene expression profiles in the context of biological networks and pathways, uncovering novel aspects of prostate cancer. Despite significant research efforts, the mechanisms underlying tumor progression are poorly understood. We applied a novel approach to reconstruct system-wide molecular events following stimulation of LNCaP prostate cancer cells with synthetic androgen and to identify potential mechanisms of androgen-independent progression of prostate cancer. METHODOLOGY/PRINCIPAL FINDINGS: We have performed concurrent measurements of gene expression and protein levels following the treatment using microarrays and iTRAQ proteomics. Sets of up-regulated genes and proteins were analyzed using our novel concept of "topological significance". This method combines high-throughput molecular data with the global network of protein interactions to identify nodes which occupy significant network positions with respect to differentially expressed genes or proteins. Our analysis identified the network of growth factor regulation of cell cycle as the main response module for androgen treatment in LNCap cells. We show that the majority of signaling nodes in this network occupy significant positions with respect to the observed gene expression and proteomic profiles elicited by androgen stimulus. Our results further indicate that growth factor signaling probably represents a "second phase" response, not directly dependent on the initial androgen stimulus. CONCLUSIONS/SIGNIFICANCE: We conclude that in prostate cancer cells the proliferative signals are likely to be transmitted from multiple growth factor receptors by a multitude of signaling pathways converging on several key regulators of cell proliferation such as c-Myc, Cyclin D and CREB1. Moreover, these pathways are not isolated but constitute an interconnected network module containing many alternative routes from inputs to outputs. If the whole network is involved, a precisely formulated combination therapy may be required to fight the tumor growth effectively.
Asunto(s)
Andrógenos/farmacología , Perfilación de la Expresión Génica , Neoplasias de la Próstata/metabolismo , Proteómica , Línea Celular Tumoral , Humanos , Masculino , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patologíaRESUMEN
Top Down mass spectrometry (MS) has emerged as an alternative to common Bottom Up strategies for protein analysis. In the Top Down approach, intact proteins are fragmented directly in the mass spectrometer to achieve both protein identification and characterization, even capturing information on combinatorial post-translational modifications. Just in the past two years, Top Down MS has seen incremental advances in instrumentation and dedicated software, and has also experienced a major boost from refined separations of whole proteins in complex mixtures that have both high recovery and reproducibility. Combined with steadily advancing commercial MS instrumentation and data processing, a high-throughput workflow covering intact proteins and polypeptides up to 70 kDa is directly visible in the near future.
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Biomarcadores/análisis , Espectrometría de Masas/métodos , Proteínas/análisis , Proteómica/métodos , Células HeLa , HumanosRESUMEN
Chromosomal rearrangements fusing the androgen-regulated gene TMPRSS2 to the oncogenic ETS transcription factor ERG occur in approximately 50% of prostate cancers, but how the fusion products regulate prostate cancer remains unclear. Using chromatin immunoprecipitation coupled with massively parallel sequencing, we found that ERG disrupts androgen receptor (AR) signaling by inhibiting AR expression, binding to and inhibiting AR activity at gene-specific loci, and inducing repressive epigenetic programs via direct activation of the H3K27 methyltransferase EZH2, a Polycomb group protein. These findings provide a working model in which TMPRSS2-ERG plays a critical role in cancer progression by disrupting lineage-specific differentiation of the prostate and potentiating the EZH2-mediated dedifferentiation program.
Asunto(s)
Proteínas de Unión al ADN/genética , Fusión Génica , Proteínas de Fusión Oncogénica/genética , Neoplasias de la Próstata/patología , Receptores Androgénicos/genética , Factores de Transcripción/genética , Inmunoprecipitación de Cromatina , Progresión de la Enfermedad , Proteína Potenciadora del Homólogo Zeste 2 , Humanos , Masculino , Complejo Represivo Polycomb 2 , Neoplasias de la Próstata/genética , Transducción de SeñalRESUMEN
Prostate cancer remains the most common malignancy among men in United States, and there is no remedy currently available for the advanced stage hormone-refractory cancer. This is partly due to the incomplete understanding of androgen-regulated proteins and their encoded functions. Whole-cell proteomes of androgen-starved and androgen-treated LNCaP cells were analyzed by semi-quantitative MudPIT ESI- ion trap MS/MS and quantitative iTRAQ MALDI- TOF MS/MS platforms, with identification of more than 1300 high-confidence proteins. An enrichment-based pathway mapping of the androgen-regulated proteomic data sets revealed a significant dysregulation of aminoacyl tRNA synthetases, indicating an increase in protein biosynthesis- a hallmark during prostate cancer progression. This observation is supported by immunoblot and transcript data from LNCaP cells, and prostate cancer tissue. Thus, data derived from multiple proteomics platforms and transcript data coupled with informatics analysis provides a deeper insight into the functional consequences of androgen action in prostate cancer.
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Aminoacil-ARNt Sintetasas/química , Andrógenos/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Próstata/patología , Proteómica/métodos , Línea Celular Tumoral , Progresión de la Enfermedad , Humanos , Immunoblotting/métodos , Masculino , Modelos Biológicos , Próstata/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
For fractionation of intact proteins by molecular weight (MW), a sharply improved two-dimensional (2D) separation is presented to drive reproducible and robust fractionation before top-down mass spectrometry of complex mixtures. The "GELFrEE" (i.e., gel-eluted liquid fraction entrapment electrophoresis) approach is implemented by use of Tris-glycine and Tris-tricine gel systems applied to human cytosolic and nuclear extracts from HeLa S3 cells, to achieve a MW-based fractionation of proteins from 5 to >100 kDa in 1 h. For top-down tandem mass spectroscopy (MS/MS) of the low-mass proteome (5-25 kDa), between 5 and 8 gel-elution (GE) fractions are sampled by nanocapillary-LC-MS/MS with 12 or 14.5 tesla Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. Single injections give about 40 detectable proteins, about half of which yield automated ProSight identifications. Reproducibility metrics of the system are presented, along with comparative analysis of protein targets in mitotic versus asynchronous cells. We forward this basic 2D approach to facilitate wider implementation of top-down mass spectrometry and a variety of other protein separation and/or characterization approaches.
Asunto(s)
Biomarcadores de Tumor/análisis , Fraccionamiento Químico/métodos , Electroforesis en Gel Bidimensional/métodos , Proteínas de Neoplasias/análisis , Proteoma/análisis , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Células HeLa , Humanos , Manejo de Especímenes/métodosRESUMEN
Prostate cancer is the most common type of tumor found in American men and is the second leading cause of cancer death in males. To identify biomarkers that distinguish prostate cancer from normal, we compared multiple gene expression profiling studies. Through meta-analysis of expression array data from multiple prostate cancer studies, we identified GOLM1 (Golgi membrane protein 1, Golm 1) as consistently up-regulated in clinically localized prostate cancer. This observation was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and validated at the protein level by immunoblot assay and immunohistochemistry. Prostate epithelial cells were identified as the cellular source of GOLM1 expression using laser capture microdissection. Immunohistochemical staining localized the GOLM1 signal to the subapical cytoplasmic region, typical of a Golgi distribution. Surprisingly, GOLM1 immunoreactivity was detected in the supernatants of prostate cell lines and in the urine of patients with prostate cancer. The mechanism by which intact GOLM1 might be released from cells has not yet been elucidated. GOLM1 transcript levels were measured in urine sediments using quantitative PCR on a cohort of patients presenting for biopsy or radical prostatectomy. We found that urinary GOLM1 mRNA levels were a significant predictor of prostate cancer. Further, GOLM1 outperformed serum prostate-specific antigen (PSA) in detecting prostate cancer. The area under the receiver-operating characteristic curve was 0.622 for GOLM1 (P = .0009) versus 0.495 for serum PSA (P = .902). Our data indicating the up-regulation of GOLM1 expression and its appearance in patients' urine suggest GOLM1 as a potential novel biomarker for clinically localized prostate cancer.