Heterogeneous nuclear ribonucleoprotein K is over expressed, aberrantly localised and is associated with poor prognosis in colorectal cancer.

Heterogeneous ribonucleoprotein K (hnRNP K) is a member of the hnRNP family which has several different cellular roles including transcription, mRNA shuttling, RNA editing and translation. Several reports implicate hnRNP K having a role in tumorigenesis, for instance hnRNP K increases transcription of the oncogene c-myc and hnRNP K expression is regulated by the p53/MDM 2 pathway. In this study comparing normal colon to colorectal cancer by proteomics, hnRNP K was identified as being overexpressed in this type of cancer. Immunohistochemistry with a monoclonal antibody to hnRNP K (which we developed) on colorectal cancer tissue microarray, confirmed that hnRNP K was overexpressed in colorectal cancer (P<0.001) and also showed that hnRNP K had an aberrant subcellular localisation in cancer cells. In normal colon hnRNP K was exclusively nuclear whereas in colorectal cancer the protein localised both in the cytoplasm and the nucleus. There were significant increases in both nuclear (P=0.007) and cytoplasmic (P=0.001) expression of hnRNP K in Dukes C tumours compared with early stage tumours. In Dukes C patient's good survival was associated with increased hnRNP K nuclear expression (P=0.0093). To elaborate on the recent observation that hnRNP K is regulated by p53, the expression profiles of these two proteins were also analysed. There was no correlation between hnRNP K and p53 expression, however, patients who presented tumours that were positive for hnRNP K and p53 had a poorer survival outcome (P=0.045).

Characterization and over-expression of chaperonin t-complex proteins in colorectal cancer.

The chaperonins are key molecular complexes, which are essential in the folding of proteins to produce stable and functionally competent protein conformations. One member of the chaperonin group of proteins is TCP1 (chaperonin containing t-complex polypeptide 1, or CCT), but little is known about this protein in tumours. In this study, we used comparative proteomic analysis to show that t-complex protein subunits TCP1 beta and TCP1 epsilon are over-expressed in colorectal adenocarcinomas. Monoclonal antibodies to these proteins were developed and the expression and cellular localization of these two proteins in colorectal cancer were analysed by immunohistochemistry on a colorectal cancer tissue microarray. In colorectal cancer, TCP1 beta cellular localization was exclusively cytoplasmic, whereas TCP1 epsilon staining was seen in both the nucleus and the cytoplasm. Both cytoplasmic TCP1 beta and cytoplasmic TCP1 epsilon were significantly over-expressed (p < 0.001 for each protein) in primary colorectal cancer and also showed increased expression with advancing Dukes' stage (p = 0.018 for TCP1 beta and p = 0.045 for TCP1 epsilon). A trend was also identified between over-expression of cytoplasmic TCP1 beta and reduced patient survival (p = 0.05). These results show that both TCP1 beta and TCP1 epsilon are over-expressed in colorectal cancer and indicate a role for TCP1 beta and TCP1 epsilon in colorectal cancer progression.

Liver fatty acid binding protein expression in colorectal neoplasia.

Liver fatty acid binding protein is a member of the fatty acid binding group of proteins that are involved in the intracellular transport of bioactive fatty acids and participate in intracellular signalling pathways, cell growth and differentiation. In this study we have used proteomics and immunohistochemistry to determine the changes in liver fatty acid binding protein in colorectal neoplasia. Comparative proteome analysis of paired samples colorectal cancer and normal colon identified consistent loss of liver fatty acid binding protein (L-FABP) in colorectal cancer compared with normal colon. To identify the changes in liver fatty acid binding protein expression during colorectal cancer development and progression the cell-specific expression of L-FABP was determined by immunohistochemistry in a series of colorectal cancers and colorectal adenomas. Decreased L-FABP immunoreactivity was significantly associated with poorly differentiated cancers (P<0.001). In colorectal adenomas there was a significant trend towards decreased staining of L-FABP in the larger adenomas (P<0.001). There was consistent L-FABP immunostaining of normal surface colonocytes. This study demonstrates that loss of L-FABP occurs at the adenoma stage of colorectal tumour development and also indicates that L-FABP is a marker of colorectal cancer differentiation.

Parallel and comparative analysis of the proteome and transcriptome of sorbic acid-stressed Saccharomyces cerevisiae.

Exposure of Saccharomyces cerevisiae to 0.9 mM sorbic acid at pH 4.5 resulted in the upregulation of 10 proteins; Hsp42, Atp2, Hsp26, Ssa1 or Ssa2, Ssb1 or Ssb2, Ssc1, Ssa4, Ach1, Zwf1 and Tdh1; and the downregulation of three proteins; Ade16, Adh3 and Eno2. In parallel, of 6144 ORFs, 94 (1.53%) showed greater than a 1.4-fold increase in transcript level after exposure to sorbic acid and five of these were increased greater than two-fold; MFA1, AGA2, HSP26, SIP18 and YDR533C. Similarly, of 6144 ORFs, 72 (1.17%) showed greater than a 1.4-fold decrease in transcript level and only one of these, PCK1, was decreased greater than two-fold Functional categories of genes that were induced by sorbic acid stress included cell stress (particularly oxidative stress), transposon function, mating response and energy generation. We found that proteomic analysis yielded distinct information from transcript analysis. Only the upregulation of Hsp26 was detected by both methods. Subsequently, we demonstrated that a deletion mutant of Hsp26 was sensitive to sorbic acid. Thus, the induction of Hsp26, which occurs during adaptation to sorbic acid, confers resistance to the inhibitory effects of this compound.

The human oesophageal squamous epithelium exhibits a novel type of heat shock protein response.

The human oesophageal epithelium is subject to damage from thermal stresses and low extracellular pH that can play a role in the cancer progression sequence, thus identifying a physiological model system that can be used to determine how stress responses control carcinogenesis. The classic heat shock protein HSP70 is not induced but rather is down-regulated after thermal injury to squamous epithelium ex vivo; this prompted a longer-term study to address the nature of the heat shock response in this cell type. An ex vivo epithelial culture system was subsequently used to identify three major proteins of 78, 70, and 58 kDa, whose steady-state levels are elevated after heat shock. Two of the three heat shock proteins were identified by mass spectrometric sequencing to be the calcium-calmodulin homologue transglutaminase-3 (78 kDa) and a recently cloned oesophageal-specific gene called C1orf10, which encodes a 53-kDa putative calcium binding protein we have named squamous epithelial heat shock protein 53 (SEP53). The 70-kDa heat shock protein (we have named SEP70) was not identifiable by mass spectrometry, but it was purified and studied immunochemically to demonstrate that it is distinct from HSP70 protein. Monoclonal antibodies to SEP70 protein were developed to indicate that: (a) SEP70 is induced by exposure of cultured cells to low pH or glucose starvation, under conditions where HSP70 protein was strikingly down-regulated; and (b) SEP70 protein exhibits variable expression in preneoplastic Barrett's epithelium under conditions where HSP70 protein is not expressed. These results indicate that human oesophageal squamous epithelium exhibits an atypical heat shock protein response, presumably due to the evolutionary adaptation of cells within this organ to survive in an unusual microenvironment exposed to chemical, thermal and acid reflux stresses.

Characterization of crosslinking sites in fibrinogen for plasminogen activator inhibitor 2 (PAI-2).

PAI-2 is a serpin that can be crosslinked to fibrin(ogen) via the Gln-Gln-Ile-Gln sequence (residues 83-86). We have characterized the lysine residues in fibrinogen to which PAI-2 is crosslinked by tissue transglutaminase and factor XIIIa. There was no competition with the crosslinking of alpha 2-antiplasmin, another inhibitor of fibrinolysis, which was specific for Lys 303 in the A alpha chain. PAI-2 was crosslinked to several lysine residues, all in the A alpha chain, 148, 176, 183, 230, 413, and 457, but not to Lys 303. The contrast with alpha 2-antiplasmin was clear from studies with truncated fibrinogens and competition by peptides. This was confirmed and extended by mass spectrometry of peptides after protease digestion of crosslinked products, which identified the lysine residues to which the inhibitors were crosslinked. PAI-2 remained active after cross-linking and inhibited fibrin breakdown, even by two-chain t-PA. Thus, a second inhibitor of fibrinolysis, in addition to alpha 2-antiplasmin, is crosslinked to fibrin and protects it from lysis.

Application of laser capture microdissection and proteomics in colon cancer.

AIMS: Laser capture microdissection is a recent development that enables the isolation of specific cell types for subsequent molecular analysis. This study describes a method for obtaining proteome information from laser capture microdissected tissue using colon cancer as a model. METHODS: Laser capture microdissection was performed on toluidine blue stained frozen sections of colon cancer. Tumour cells were selectively microdissected. Conditions were established for solubilising proteins from laser microdissected samples and these proteins were separated by two dimensional gel electrophoresis. Individual protein spots were cut from the gel, characterised by mass spectrometry, and identified by database searching. These results were compared with protein expression patterns and mass spectroscopic data obtained from bulk tumour samples run in parallel. RESULTS: Proteins could be recovered from laser capture microdissected tissue in a form suitable for two dimensional gel electrophoresis. The solubilised proteins retained their expected electrophoretic mobility in two dimensional gels as compared with bulk samples, and mass spectrometric analysis was also unaffected. CONCLUSION: A method for performing two dimensional gel electrophoresis and mass spectrometry using laser capture microdissected tissue has been developed.

Spot the differences: proteomics in cancer research.

Separation of thousands of cellular proteins by two-dimensional electrophoresis allows the detailed comparison of proteins from normal and diseased tissue. Mass spectrometry provides a fast and reliable way of characterising proteins of interest, particularly when the gene sequence of the source organism is known. The availability of the human genome sequence has opened up the possibility of identifying protein differences between normal and diseased tissue, thus providing the opportunity to search for tumour markers or for therapeutic targets. This new technology will give much-needed insight into the molecular mechanisms of tumour development and progression.

Proteomics: a new approach to the study of disease.

The global analysis of cellular proteins has recently been termed proteomics and is a key area of research that is developing in the post-genome era. Proteomics uses a combination of sophisticated techniques including two-dimensional (2D) gel electrophoresis, image analysis, mass spectrometry, amino acid sequencing, and bio-informatics to resolve comprehensively, to quantify, and to characterize proteins. The application of proteomics provides major opportunities to elucidate disease mechanisms and to identify new diagnostic markers and therapeutic targets. This review aims to explain briefly the background to proteomics and then to outline proteomic techniques. Applications to the study of human disease conditions ranging from cancer to infectious diseases are reviewed. Finally, possible future advances are briefly considered, especially those which may lead to faster sample throughput and increased sensitivity for the detection of individual proteins.

Processing of synthetic pro-islet amyloid polypeptide (proIAPP) 'amylin' by recombinant prohormone convertase enzymes, PC2 and PC3, in vitro.

Islet amyloid polypeptide (IAPP), amylin, is the constituent peptide of pancreatic islet amyloid deposits which form in islets of Type 2 diabetic subjects. Human IAPP is synthesized as a 67-residue propeptide in islet beta-cells and colocalized with insulin in beta-cell granules. The mature 37-amino acid peptide is produced by proteolysis at pairs of basic residues at the C- and N-termini of the mature peptide. To determine the enzymes responsible for proteolysis and their activity at the potential cleavage sites, synthetic human proIAPP was incubated (0.5-16 h) with recombinant prohormone convertases, PC2 or PC3 at appropriate conditions of calcium and pH. The products were analysed by MS and HPLC. Proinsulin was used as a control and was cleaved by both recombinant enzymes resulting in intermediates. PC3 was active initially at the N-terminal-IAPP junction and later at the C-terminus, whereas initial PC2 activity was at the IAPP-C-terminal junction. Processing at the basic residues within the C-terminal flanking peptide rarely occurred. There was no evidence for substantial competition for the processing enzymes when the combined substrates proinsulin and proIAPP were incubated with both PC2 and PC3. As proinsulin cleavage is sequential in vivo (PC3 active at the B-chain-C-peptide junction, followed by PC2 at A chain-C-peptide junction), these data suggest that proteolysis of proIAPP and proinsulin is coincident in secretory granules and increased proinsulin secretion in diabetes could be accompanied by increased production of proIAPP.

Cross-linking of plasminogen activator inhibitor 2 and alpha 2-antiplasmin to fibrin(ogen).

In this study, we identified lysine residues in the fibrinogen Aalpha chain that serve as substrates during transglutaminase (TG)-mediated cross-linking of plasminogen activator inhibitor 2 (PAI-2). Comparisons were made with alpha(2)-antiplasmin (alpha(2)-AP), which is known to cross-link to lysine 303 of the Aalpha chain. A 30-residue peptide containing Lys-303 specifically competed with fibrinogen for cross-linking to alpha(2)-AP but not for cross-linking to PAI-2. Further evidence that PAI-2 did not cross-link via Lys-303 was the cross-linking of PAI-2 to I-9 and des-alphaC fibrinogens, which lack 100 and 390 amino acids from the C terminus of the Aalpha chain, respectively. PAI-2 or alpha(2)-AP was cross-linked to fibrinogen and digested with trypsin or endopeptidase Glu-C, and the resulting peptides analyzed by mass spectrometry. Peptides detected were consistent with tissue TG (tTG)-mediated cross-linking of PAI-2 to lysines 148, 176, 183, 457 and factor XIIIa-mediated cross-linking of PAI-2 to lysines 148, 230, and 413 in the Aalpha chain. alpha(2)-AP was cross-linked only to lysine 303. Cross-linking of PAI-2 to fibrinogen did not compete with alpha(2)-AP, and the two proteins utilized different lysines in the Aalpha chain. Therefore, PAI-2 and alpha(2)-AP can cross-link simultaneously to the alpha polymers of a fibrin clot and promote resistance to lysis.

A proteomic analysis of erythromycin resistance in Streptococcus pneumoniae.

Streptococcus pneumoniae is a significant human pathogen which is an important cause of pneumonia and bacteraemia. Over the past few years the incidence of antibiotic resistance among clinical isolates of S. pneumoniae has increased. Penicillin resistance is now widespread and the frequency of isolates that are resistant to erythromycin has risen. Erythromycin resistance in S. pneumoniae follows two basic patterns. The MLS erythromycin-resistant phenotype is due to the enzymatic methylation of ribosomal RNA that blocks erythromycin binding to the ribosome. Alternatively, in isolates of the M phenotype, a more recently documented mechanism, resistance is associated with an active efflux process that reduces intracellular levels of erythromycin. We used two-dimensional electrophoresis to examine the proteins synthesised by erythromycin-susceptible and -resistant S. pneumoniae. Erythromycin-resistant S. pneumoniae with the M phenotype showed a significantly increased synthesis of a 38,500 Dalton (pI 6.27) protein compared to susceptible isolates. Peptide mass mapping was used to identify the 38,500 Dalton protein as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). It was demonstrated that S. pneumoniae synthesised at least three forms of GAPDH that differed in their isoelectric points. The form of GAPDH possessing the most basic pI showed the increased synthesis in the erythromycin-resistant S. pneumoniae isolates. Alterations in the synthesis of GAPDH were only found for those erythromycin-resistant isolates possessing the M phenotype. S. pneumoniae isolates with the MLS phenotype were indistinguishable from the susceptible strains using the analytical conditions employed for the current study. The possible role of GAPDH in erythromycin resistance of S. pneumoniae is considered.