Proteome Analysis of Cultured Fibroblasts from Type 1 Diabetic Patients and Normal Subjects

Puricelli, Lucia; Iori, Elisabetta; Millioni, Renato; Arrigoni, Giorgio; James, Peter; Vedovato, Monica; Tessari, Paolo

doi:10.1210/jc.2006-0274

Cited by 23 publications

(18 citation statements)

References 29 publications

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“…However, metabolic changes associated with diabetes are tissue-specific. For example, an increase in glycolysis was reported in skeletal muscle from type 1 and type 2 patients [28,29], while a reduction was demonstrated in diabetic heart [30,33] and in streptozotocin-induced diabetic rat retinas after 3 months of disease [34]. Our results indicate that systemic metabolic changes observed in other tissues affected by diabetes also occur in the pre-retinopathic RPE.…”

Section: Resultssupporting

confidence: 51%

Human retinal pigment epithelium proteome changes in early diabetes

et al. 2008

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Aims/hypothesis Diabetic retinopathy is the most common complication of diabetes and a leading cause of blindness among working-age adults. Anatomical and functional changes occur in the retina and retinal pigment epithelium (RPE) prior to clinical symptoms of the disease. However, the molecular mechanisms responsible for these early changes, particularly in the RPE, remain unclear. To begin defining the molecular changes associated with pre-retinopathic diabetes, we conducted a comparative proteomics study of human donor RPE. Methods The RPE was dissected from diabetic human donor eyes with no clinically apparent diabetic retinopathy (n=6) and from eyes of age-matched control donors (n= 17). Soluble proteins were separated based upon their mass and charge using two-dimensional (2-D) gel electrophoresis. Protein spots were visualised with a fluorescent dye and spot densities were compared between diabetic and control gels. Proteins from spots with significant disease-related changes in density were identified using mass spectrometry.Results Analysis of 325 spots on 2-D gels identified 31 spots that were either up-or downregulated relative to those from age-matched control donors. The protein identity of 18 spots was determined by mass spectrometry. A majority of altered proteins belonged to two major functional groups, metabolism and chaperones, while other affected categories included protein degradation, synthesis and transport, oxidoreductases, cytoskeletal structure and retinoid metabolism. Conclusions/interpretation Changes identified in the RPE proteome of pre-retinopathic diabetic donor eyes compared with age-matched controls suggest specific cellular alterations that may contribute to diabetic retinopathy. Defining the pre-retinopathic changes affecting the RPE could provide important insight into the molecular events that lead to this disease.

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Section: Resultssupporting

confidence: 51%

Human retinal pigment epithelium proteome changes in early diabetes

et al. 2008

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“…We have identified more than 50 proteins of this group (Table 2a), accounting for <40% of all the identified proteins [29]. This percentage of cytoskeletal-related proteins is relative to the total number of identified spots, and not to the total proteins that may be theoretically expressed by skin fibroblast.…”

Section: Cytoskeletal and Cytoskeleton-related Proteinsmentioning

confidence: 94%

Abnormal cytoskeletal protein expression in cultured skin fibroblasts from type 1 diabetes mellitus patients with nephropathy: A proteomic approach

Millioni

Iori

Puricelli

et al. 2008

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Diabetic nephropathy (DN) develops in about 40% of insulin-dependent type 1 diabetes mellitus (T1DM) patients, and is associated not only with diabetes duration and metabolic control, but also with a genetic predisposition. Constitutive alterations of cytoskeletal proteins may play a role in the development of DN. We investigated the expression of these proteins in cultured skin fibroblasts, obtained from long-term T1DM patients with and without DN but comparable metabolic control, and from matched healthy subjects, by means of 2-DE electrophoresis and MS-MALDI analyses. In T1DM with DN, compared to the other two groups, quantitative analyses revealed an altered expression of 17 spots (p<0.05-p<0.01), corresponding to 12 unique proteins. In T1DM with DN, beta-actin and three isoforms of tubulin beta-2 chain, tropomodulin-3, and LASP-1 were decreased, whereas two tubulin beta-4 chain isoforms, one alpha actinin-4 isoform, membrane-organizing extension spike protein (MOESIN), FLJ00279 (corresponding to a fragment of myosin heavy chain, non-muscle type A), vinculin, a tropomyosin isoform, and the macrophage capping protein were increased. A shift in caldesmon isoforms was also detected. These results demonstrate an association between DN and the constitutive expression of cytoskeleton proteins in cultured skin fibroblasts from T1DM with DN, which may retain pathophysiologycal implications.

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“…This could indicate a change in the glycolytic pathway to release more ATP, a possibility supported by similar changes in phosphoglycerate kinase activity which plays a predominant role in the conversion of 3-phosphoglyceroyl phosphate into 3-phosphoglycerate to make ADP into ATP [55]. Pyruvate kinase isozymes M1/M2 (highly active tetrameric form/nearly inactive dimeric form) was up-regulated and this gycolytic enzyme catalyzes the transfer of a phosphoryl group from phosphoenolpyruvate (PEP) to ADP, (subsequently generating ATP) [56] which corresponds with the up-regulation of ATP synthetase subunit beta and alpha chain. For pyruvate kinase isoenzyme M1/M2 the transition between the 2 isozyme forms contributes to the control of glycolysis [57].…”

Section: Resultsmentioning

confidence: 99%

A proteomic study of cMyc improvement of CHO culture

2010

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BackgroundThe biopharmaceutical industry requires cell lines to have an optimal proliferation rate and a high integral viable cell number resulting in a maximum volumetric recombinant protein product titre. Nutrient feeding has been shown to boost cell number and productivity in fed-batch culture, but cell line engineering is another route one may take to increase these parameters in the bioreactor. The use of CHO-K1 cells with a c-myc plasmid allowing for over-expressing c-Myc (designated cMycCHO) gives a higher integral viable cell number. In this study the differential protein expression in cMycCHO is investigated using two-dimensional gel electrophoresis (2-DE) followed by image analysis to determine the extent of the effect c-Myc has on the cell and the proteins involved to give the new phenotype.ResultsOver 100 proteins that were differentially expressed in cMycCHO cells were detected with high statistical confidence, of which 41 were subsequently identified by tandem mass spectrometry (LC-MS/MS). Further analysis revealed proteins involved in a variety of pathways. Some examples of changes in protein expression include: an increase in nucleolin, involved in proliferation and known to aid in stabilising anti-apoptotic protein mRNA levels, the cytoskeleton and mitochondrial morphology (vimentin), protein biosysnthesis (eIF6) and energy metabolism (ATP synthetase), and a decreased regulation of all proteins, indentified, involved in matrix and cell to cell adhesion.ConclusionThese results indicate several proteins involved in proliferation and adhesion that could be useful for future approaches to improve proliferation and decrease adhesion of CHO cell lines which are difficult to adapt to suspension culture.

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Proteome Analysis of Cultured Fibroblasts from Type 1 Diabetic Patients and Normal Subjects

Abstract: 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.

Cited by 23 publications

References 29 publications

Human retinal pigment epithelium proteome changes in early diabetes

Human retinal pigment epithelium proteome changes in early diabetes

Abnormal cytoskeletal protein expression in cultured skin fibroblasts from type 1 diabetes mellitus patients with nephropathy: A proteomic approach

A proteomic study of cMyc improvement of CHO culture

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