Laura Lawrie

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. Copyright

Mortalin is over-expressed by colorectal adenocarcinomas and correlates with poor survival

Using comparative proteomic analysis we have identified over‐expression of mortalin in colorectal adenocarcinomas. Mortalin, also known as mitochondrial heat‐shock protein 70 (mhsp 70), is involved in cell cycle regulation with important roles in cellular senescence and immortalization pathways. It is known to bind to and inactivate wild‐type tumour suppressor protein p53 and influences the Ras‐Raf‐MAPK pathway. By immunostaining a colorectal cancer tissue microarray linked to a patient database, we further found that mortalin over‐expression correlates with poor patient survival and, in multivariate analysis, is independent of standard prognostic variables (p = 0.04). Our findings demonstrate that mortalin over‐expression may predict outcome in colorectal cancer and suggest that this protein is involved in colorectal neoplasia. Our experimental approach emphasises the analytical power of combining proteomics with tissue microarray analysis in the context of a well‐defined tumour database. Copyright

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 patients 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).

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. Copyright

The Barrett’s Antigen Anterior Gradient-2 Silences the p53 Transcriptional Response to DNA Damage

The esophageal epithelium is subject to damage from bile acid reflux that promotes normal tissue injury resulting in the development of Barrett’s epithelium. There is a selection pressure for mutating p53 in this preneoplastic epithelium, thus identifying a physiologically relevant model for discovering novel regulators of the p53 pathway. Proteomic technologies were used to identify such p53 regulatory factors by identifying proteins that were overexpressed in Barrett’s epithelium. A very abundant polypeptide selectively expressed in Barrett’s epithelium was identified as anterior gradient-2. Immunochemical methods confirmed that anterior gradient-2 is universally up-regulated in Barrett’s epithelium, relative to normal squamous tissue derived from the same patient. Transfection of the anterior gradient-2 gene into cells enhances colony formation, similar to mutant oncogenic p53 encoded by the HIS175 allele, suggesting that anterior gradient-2 can function as a survival factor. Deletion of the C-terminal 10 amino acids of anterior gradient-2 neutralizes the colony enhancing activity of the gene, suggesting a key role for this domain in enhancing cell survival. Constitutive overexpression of anterior gradient-2 does not alter cell-cycle parameters in unstressed cells, suggesting that this gene is not directly modifying the cell cycle. However, cells overexpressing anterior gradient-2 attenuate p53 phosphorylation at both Ser15 and Ser392 and silence p53 transactivation function in ultraviolet (UV)-damaged cells. Deletion of the C-terminal 10 amino acids of anterior gradient-2 permits phosphorylation at Ser15 in UV-damaged cells, suggesting that the C-terminal motif promoting colony survival also contributes to suppression of the Ser15 kinase pathway. These data identify anterior gradient-2 as a novel survival factor whose study may shed light on cellular pathways that attenuate the tumor suppressor p53.

Cross-linking of Plasminogen Activator Inhibitor 2 and α2-Antiplasmin to Fibrin(ogen)

In this study, we identified lysine residues in the fibrinogen Aα chain that serve as substrates during transglutaminase (TG)-mediated cross-linking of plasminogen activator inhibitor 2 (PAI-2). Comparisons were made with α2-antiplasmin (α2-AP), which is known to cross-link to lysine 303 of the Aα chain. A 30-residue peptide containing Lys-303 specifically competed with fibrinogen for cross-linking to α2-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-αC fibrinogens, which lack 100 and 390 amino acids from the C terminus of the Aα chain, respectively. PAI-2 or α2-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 Aα chain. α2-AP was cross-linked only to lysine 303. Cross-linking of PAI-2 to fibrinogen did not compete with α2-AP, and the two proteins utilized different lysines in the Aα chain. Therefore, PAI-2 and α2-AP can cross-link simultaneously to the α polymers of a fibrin clot and promote resistance to lysis.

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.

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.

Laser capture microdissection and colorectal cancer proteomics.

The ability to define protein profiles of normal and diseased cells is important in understanding cell function. Laser capture microdissection permits the isolation of specific cell types for subsequent molecular analysis. In this study we have established conditions for obtaining proteomic information from laser capture microdissected colorectal cancer cells. Laser capture microdissection was performed on toluidine blue-stained frozen sections of colorectal cancer. Proteins were solubilized from microdissected cells and the solubilized proteins were separated by two-dimensional gel electrophoresis: protein spots were characterized by peptide mass mapping using matrix assisted laser desorption ionization-time of flight mass spectrometry. Proteins isolated from laser capture microdissected tissue retained their expected electrophoretic mobility and peptide mass mapping was also unaffected. The ability to study the protein expression profile of specific cell types will allow for the identification of novel disease markers and therapeutic targets and also provide for the enhanced understanding of pathogenetic mechanisms.

Characterization of Crosslinking Sites in Fibrinogen for Plasminogen Activator Inhibitor 2 (PAI‐2)

Abstract: 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 α2‐antiplasmin, another inhibitor of fibrinolysis, which was specific for Lys 303 in the Aα chain. PAI‐2 was crosslinked to several lysine residues, all in the Aα chain, 148, 176, 183, 230, 413, and 457, but not to Lys 303. The contrast with α2‐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 α2‐antiplasmin, is crosslinked to fibrin and protects it from lysis.