RESUMO
The search for novel molecular markers of tumor invasion is vital if strategies are to become more effective in the diagnostic and prognostic management of transitional cell carcinoma of the bladder. Up to 50% of tumors detected at stage 1 (pT1) progress to a higher grade even after endoscopic surgical resection, and there are currently no protein markers of this aggressive, invasive phenotype. We have combined SELDI-TOF-MS, ClinProt magnetic bead enrichment, Nano-LC-ESI-ion trap tandem mass spectrometry and immunohistochemical analysis to the study of 12 invasive bladder cancer tissue biopsies paired with normal bladder tissue samples obtained from the same patients for the definition and identification of proteins up-regulated in the tumors. We report the inflammation-associated calcium binding protein S100A8 (MRP-8, calgranulin A) to be highly expressed in tumor cells in contrast to normal urothelium in 50% of the samples, as well as two unidentified protein markers at 5.75 and 6.89 kDa that were differentially detected in 9/12 and 10/12 tumor samples, respectively. These new markers, when fully characterized, may contribute to new target proteins for the prediction of aggressive, invasive bladder tumors.
Assuntos
Biomarcadores Tumorais/metabolismo , Calgranulina A/metabolismo , Carcinoma de Células de Transição/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Western Blotting , Carcinoma de Células de Transição/patologia , Progressão da Doença , Eletroforese em Gel Bidimensional , Humanos , Técnicas Imunoenzimáticas , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Bexiga Urinária/metabolismo , Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/patologiaRESUMO
Pseudomonas aeruginosa is able to grow on acyclic monoterpenes (citronellol, citronellate, geraniol and geranylate), and on other methyl-branched compounds such as leucine or isovalerate. The catabolic pathway of citronellol (Atu, acyclic terpene utilization) enters that of leucine/isovalerate (Liu, leucine and isovalerate utilization) at the level of methylcrotonyl-CoA. Key enzymes of the combined pathways are geranyl-CoA carboxylase (GCase) and methylcrotonyl-CoA carboxylase (MCase). In this study, isovalerate-grown cells specifically expressed MCase (apparent molecular mass of the biotin-containing subunit, 74 kDa) only, and the GCase biotin-containing subunit (71 kDa) was not detected. Citronellol- or citronellate-grown cells produced both carboxylases. Biotin-dependent proteins were purified from crude extracts by avidin-affinity chromatography, and assigned to the corresponding coding genes by trypsin fingerprint analysis. The two subunits of MCase corresponded to liuB/liuD (PA2014/PA2012) of the P. aeruginosa genome database, and atuC/atuF (PA2888/PA2891) encoded GCase subunits. This finding is contrary to that reported by others. The identified genes are part of two separate gene clusters [liuRABCDE (PA2011-PA2016) and atuABCDEFGH (PA2886-PA2893)] that are thought to encode most of the genes of the Atu and Liu pathways.
Assuntos
Carbono-Carbono Ligases/metabolismo , Leucina/metabolismo , Ácidos Pentanoicos/metabolismo , Pseudomonas aeruginosa/enzimologia , Terpenos/metabolismo , Monoterpenos Acíclicos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biotina/metabolismo , Carbono-Carbono Ligases/genética , Meios de Cultura , Hemiterpenos , Leucina/química , Monoterpenos/metabolismo , Família Multigênica , Ácidos Pentanoicos/química , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Terpenos/químicaRESUMO
Most cellular processes are carried out by multiprotein complexes. The identification and analysis of their components provides insight into how the ensemble of expressed proteins (proteome) is organized into functional units. We used tandem-affinity purification (TAP) and mass spectrometry in a large-scale approach to characterize multiprotein complexes in Saccharomyces cerevisiae. We processed 1,739 genes, including 1,143 human orthologues of relevance to human biology, and purified 589 protein assemblies. Bioinformatic analysis of these assemblies defined 232 distinct multiprotein complexes and proposed new cellular roles for 344 proteins, including 231 proteins with no previous functional annotation. Comparison of yeast and human complexes showed that conservation across species extends from single proteins to their molecular environment. Our analysis provides an outline of the eukaryotic proteome as a network of protein complexes at a level of organization beyond binary interactions. This higher-order map contains fundamental biological information and offers the context for a more reasoned and informed approach to drug discovery.