In type-1 diabetes mellitus (T1DM) with diabetic nephropathy (DN), accumulation of abnormal proteins in the kidney and other tissues may derive from constitutive alterations of intracellular protein recognition, assembly, and turnover. We characterized the proteins involved in these functions in cultured skin fibroblasts from long-term T1DM patients with [DN+] or without [DN-] nephropathy but similar metabolic control, and from matched healthy subjects. 2-D gel electrophoresis and MS-MALDI analysis were employed. The [DN+] T1DM patients, compared with the two other groups, exhibited increased abundance of a high-molecular weight isoform of protein disulphide-isomerase A3 and a decrease of two low-molecular weight isoforms. They also had increased levels of heat shock protein (HSP) 60 kDa isoform #A4, of HSP71 kDa isoform #A30, and of HSP27 kDa isoform #6, whereas the HSP27 kDa isoforms #A90 and #A71 were decreased. Cathepsin beta-2 (#40), the cation-independent mannose 6-phosphate receptor binding protein 1 (CIMPR) (#A27), and annexin 2 (#A9) were also decreased in the [DN+] T1DM patients, whereas the RNA-binding protein regulatory subunity (#38) and the translationally-controlled tumor protein (TCTP) (#A45) were increased. These changes of chaperone-like proteins in fibroblasts may highlight those of the kidney and be patho-physiologically related to the development of nephropathy in T1DM.

Since type 1 diabetes mellitus (T1DM) patients with nephropathy (DN+) are insulin-resistant, we aimed to identify (new) potential molecular sites involved in the alterations of glucose metabolism in these patients. We examined the expression of glycolytic enzymes in cultured fibroblasts from T1DM(DN+) patients as compared to those from T1DM patients without nephropathy (DN-) and from controls. Pyruvate kinase (PK) activity was also determined. Human skin fibroblasts were grown in normal glucose (6 mM). RNAs and proteins were analyzed, respectively, using cRNA microarray and two-dimensional electrophoresis followed by identification with mass spectrometry. PK activity was measured using a spectrophotometric assay. As compared to controls, increases in the gene expression of hexokinase, phosphoglucomutase, phosphofructokinase, aldolase and triosephosphate isomerase were found in T1DM(DN+) patients, but not in T1DM(DN-) patients. In T1DM(DN+) patients, the protein analysis showed an altered expression of three glycolytic enzymes: triosophosphate isomerase, enolase and PK. In addition, PK activity in fibroblasts from T1DM(DN+) patients was lower than that in T1DM(DN-) and in controls. In conclusion, this study reports novel alterations of enzymes involved in glucose metabolism that may be associated with the pathophysiology of insulin resistance and of renal damage in T1DM(DN+) patients.

During the course of infection, the common human pathogen Streptococcus pyogenes encounters plasma. We show that plasma causes S. pyogenes to rapidly remodel its cellular metabolism and virulence pathways. We also identified a variant of the major virulence factor, M1 protein, lacking 13 amino acids at the NH(2)-terminus in bacteria grown with plasma. The pronounced effect of plasma on protein expression, suggests this is an important adaptive mechanism with implications for S. pyogenes pathogenicity.

Abstract. Some clinical results indicate that somatostatin (sms) might be useful in the treatment of advanced prostate cancer (HRPC). Because of its transient in vivo half-life only more stable derivatives of sms are of interest in this context. Recent studies have shown that natural sms can be conjugated to a carbohydrate (smsdx) with preservation of sms-like effects on the prostatic tumor cell proteome. The present study identifies some of the affected proteins in an effort to elucidate pathways and proteins that might be of importance for the potential usefulness of sms treatment in HRPC. After incubating the LNCaP cell-line with sms14/smsdx, comparative proteomics was used for analysing and identifying affected proteins. Protein expression patterns were analysed with two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. Catalytic mitochondrial and mitochondrial-associated proteins were significantly affected (fold change ~2 or higher) and they were in general up-regulated. Apoptosis-related proteins were both up-regulated (VDAC1, VDAC2) and down-regulated (PRDX2, TCTP). The fold change was >2 for PRDX2 and <2 for the others. There was a strong agreement between sms and smsdx on the up-and down-regulation of proteins. Sms/smsdx triggered up-regulation of catalytic mitochondrial proteins and seemed to affect apoptosis-related proteins. This could indicate important pathways on which smsdx might be able to curb the progression of HRPC. The results encourage a pending clinical phase II study.

We generated 2-DE reference maps of the proteome of human skin fibroblasts from both normal and uncomplicated type 1 diabetic patients. Differences in glycolytic enzymes and structural proteins were found. The functional implications of the identified proteins are discussed.