Jinsook Chang

Proteomics Analysis Identifies Molecular Targets Related to Diabetes Mellitus-associated Bladder Dysfunction

Protein expression profiles in rat bladder smooth muscle were compared between animal models of streptozotocin-induced diabetes mellitus (STZ-DM) and age-matched controls at 1 week and 2 months after induction of hyperglycemia with STZ treatment. At each time point, protein samples from four STZ-DM and four age-matched control rat bladder tissues were prepared independently and analyzed together across multiple DIGE gels using a pooled internal standard sample to quantify expression changes with statistical confidence. A total of 100 spots were determined to be significantly changing among the four experimental groups. A subsequent mass spectrometry analysis of the 100 spots identified a total of 56 unique proteins. Of the proteins identified by two-dimensional DIGE/MS, 10 exhibited significant changes 1 week after STZ-induced hyperglycemia, whereas the rest showed differential expression after 2 months. A network analysis of these proteins using MetaCore™ suggested induction of transcriptional factors that are too low to be detected by two-dimensional DIGE and identified an enriched cluster of down-regulated proteins that are involved in cell adhesion, cell shape control, and motility, including vinculin, intermediate filaments, Ppp2r1a, and extracellular matrix proteins. The proteins that were up-regulated include proteins involved in muscle contraction (e.g. Mrlcb and Ly-GDI), in glycolysis (e.g. α-enolase and Taldo1), in mRNA processing (e.g. heterogeneous nuclear ribonucleoprotein A2/B1), in inflammatory response (e.g. S100A9, Annexin 1, and apoA-I), and in chromosome segregation and migration (e.g. Tuba1 and Vil2). Our results suggest that the development of diabetes-related complications in this model involves the down-regulation of structural and extracellular matrix proteins in smooth muscle that are essential for normal muscle contraction and relaxation but also induces proteins that are associated with cell proliferation and inflammation that may account for some of the functional deficits known to occur in diabetic complications of bladder.

Proteomic changes during intestinal cell maturation in vivo.

Intestinal epithelial cells undergo progressive cell maturation as they migrate along the crypt-villus axis. To determine molecular signatures that define this process, proteins differentially expressed between the crypt and villus were identified by 2D-DIGE and MALDI-MS. Forty-six differentially expressed proteins were identified, several of which were validated by immunohistochemistry. Proteins upregulated in the villus were enriched for those involved in brush border assembly and lipid uptake, established features of differentiated intestinal epithelial cells. Multiple proteins involved in glycolysis were also upregulated in the villus, suggesting increased glycolysis is a feature of intestinal cell differentiation. Conversely, proteins involved in nucleotide metabolism, and protein processing and folding were increased in the crypt, consistent with functions associated with cell proliferation. Three novel paneth cell markers, AGR2, HSPA5 and RRBP1 were also identified. Notably, significant correlation was observed between overall proteomic changes and corresponding gene expression changes along the crypt-villus axis, indicating intestinal cell maturation is primarily regulated at the transcriptional level. This proteomic profiling analysis identified several novel proteins and functional processes differentially induced during intestinal cell maturation in vivo. Integration of proteomic, immunohistochemical, and parallel gene expression datasets demonstrate the coordinated manner in which intestinal cell maturation is regulated.

Molecular Targets for Diabetes Mellitus-associated Erectile Dysfunction

Protein expression profiles in rat corporal smooth muscle tissue were compared between animal models of streptozotocin-induced diabetes mellitus (STZ-DM) and age-matched controls (AMCs) at 1 week and 2 months after induction of hyperglycemia with STZ treatment. At each time point, protein samples from four STZ-DM and four AMC rat corpora tissues were prepared independently and analyzed together across multiple quantitative two-dimensional gels using a pooled internal standard sample to quantify expression changes with statistical confidence. A total of 170 spots were differential expressed among the four experimental groups. A subsequent mass spectrometry analysis of the 170 spots identified a total of 57 unique proteins. Network analysis of these proteins using MetaCoreTM suggested altered activity of transcriptional factors that are of too low abundance to be detected by the two-dimensional gel method. The proteins that were down-regulated with diabetes include isoforms of collagen that are precursors to fibril-forming collagen type 1; Hsp47, which assists and mediates the proper folding of procollagen; and several proteins whose abundance is controlled by sex hormones (e.g. CRP1 and A2U). On the other hand, proteins seen or predicted to be up-regulated include proteins involved in cell apoptosis (e.g. p53, 14-3-3-γ, Serpinf1, Cct4, Cct5, and Sepina3n), proteins that neutralize the biological activity of nerve growth factor (e.g. anti-NGF 30), and proteins involved in lipid metabolism (e.g. apoA-I and apoA-IV). Subsequent Western blot validation analysis of p53, 14-3-3-γ, and Hsp47 confirmed increased p53 and 14-3-3-γ and decreased Hsp47 levels in separate samples. According to the results from the Western blot analysis, Hsp47 protein showed a ∼3-fold decrease at 1 week and was virtually undetectable at 2 months in diabetic versus control. Taken together, our results identify novel candidate proteins playing a role in erectile dysfunction in diabetes resulting from STZ treatment.

Proteomics and Pathway Mapping Analyses Reveal Phase-Dependent Over-Expression of Proteins Associated with Carbohydrate Metabolic Pathways in Candida albicans Biofilms

Candida albicans is the most commonly isolated fungus associated with biofilms, which are extracellular ma- trix (ECM)-encased, drug-resistant microbial communities formed on indwelling medical devices. Protein profiles of fun- gal biofilms have not been investigated in detail, although such profiles are believed to play critical roles in fungal biofilm formation. In this study, we used two-dimensional difference-in-gel electrophoresis (DIGE)-based proteomics to identify differentially expressed proteins in C. albicans biofilms grown to early and mature phases, compared to planktonic cells. The resulting proteomic data set was subjected to pathway mapping to reveal phase-specific pathways that were differen- tially expressed in biofilm cell walls and extracellular matrix (ECM). Our analyses showed 107 proteins to be differen- tially expressed in ECM, while 44 were differentially expressed in cell walls during biofilm formation, compared to planktonic controls. Furthermore, 95% (102/107) and 68% (30/44) of these differentially expressed proteins were upregu- lated in ECM and cell walls of biofilms, respectively. These proteins were mapped to cellular pathways, which revealed that these differentially expressed proteins were associated with several metabolic pathways, in a phase-dependent man- ner. For example, among ECM-associated proteins, proteins within 18 pathways were differentially expressed, with two pathways (glutamate and nitrogen metabolism) unique to early phase, and four pathways (purine, Gly/Ser/Thr, and inosi- tol metabolism, and carbon fixation) unique to mature phase biofilms. Such differences were also observed in cell wall- associated proteins, where proteins associated with 14 specific pathways were differentially regulated. We also found gly- colytic enzymes including the key enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were overexpressed in biofilms at both early and mature phases, compared to planktonic controls. Iodoacetate-mediated inhibition of this enzyme completely abrogated the ability of C. albicans to form biofilms, indicating the role of glycolysis/gluconeogenesis path- ways in biofilm formation. Taken together, we demonstrate that ECM and cell walls of C. albicans biofilms express in- creased levels of specific proteins within pathways in a phase-dependent manner, suggesting that these pathways, espe- cially glycolysis/gluconeogenesis, play critical roles in fungal biofilm formation and maintenance.

Proteomics, bioinformatics and targeted gene expression analysis reveals up-regulation of cochlin and identifies other potential biomarkers in the mouse model for deafness in usher syndrome type 1F

Proteins and protein networks associated with cochlear pathogenesis in the Ames waltzer (av) mouse, a model for deafness in Usher syndrome 1F (USH1F), were identified. Cochlear protein from wild-type and av mice at postnatal day 30, a time point in which cochlear pathology is well established, was analyzed by quantitative 2D gel electrophoresis followed by mass spectrometry (MS). The analytic gel resolved 2270 spots; 69 spots showed significant changes in intensity in the av cochlea compared with the control. The cochlin protein was identified in 20 peptide spots, most of which were up-regulated, while a few were down-regulated. Analysis of MS sequence data showed that, in the av cochlea, a set of full-length isoforms of cochlin was up-regulated, while isoforms missing the N-terminal FCH/LCCL domain were down-regulated. Protein interaction network analysis of all differentially expressed proteins was performed with Metacore software. That analysis revealed a number of statistically significant candidate protein networks predicted to be altered in the affected cochlea. Quantitative PCR (qPCR) analysis of select candidates from the proteomic and bioinformatic investigations showed up-regulation of Coch mRNA and those of p53, Brn3a and Nrf2, transcription factors linked to stress response and survival. Increased mRNA of Brn3a and Nrf2 has previously been associated with increased expression of cochlin in human glaucomatous trabecular meshwork. Our report strongly suggests that increased level of cochlin is an important etiologic factor leading to the degeneration of cochlear neuroepithelia in the USH1F model.

Ageing causes cytoplasmic retention of MaxiK channels in rat corporal smooth muscle cells

The MaxiK channel plays a critical role in the regulation of corporal smooth muscle tone and thereby erectile function. Given that ageing results in a decline in erectile function, we determined changes in the expression of MaxiK, which might impact erectile function. Quantitative-polymerase chain reaction demonstrated that although there is no significant change in transcription of the α- and β-subunits that comprise the MaxiK channel, there are significant changes in the expression of transcripts encoding different splice variants. One transcript, SV1, is 13-fold increased in expression in the ageing rat corpora. SV1 has previously been reported to trap other isoforms of the MaxiK channel in the cytoplasm. Correlating with increased expression of SV1, we observed in older rats there is approximately a 13-fold decrease in MaxiK protein in the corpora cell membrane and a greater proportion is retained in the cytoplasm (approximately threefold). These experiments demonstrate that ageing of the corpora is accompanied by changes in alternative splicing and cellular localization of the MaxiK channel.

Pharmacologic intervention targeting glycolytic-related pathways protects against retinal injury due to ischemia and reperfusion.

Retinal ischemia contributes to multiple ocular diseases while aminoguanidine (AMG) treatment significantly inhibits the neuronal and vascular degeneration due to acute retinal ischemia and reperfusion (I/R) injury. In the present study, 2‐D DIGE was applied to profile global protein expression changes due to retinal I/R injury, and the protection effects mediated by AMG. Retinal ischemia was induced by elevated intraocular pressure to 80–90 mmHg for 2 h, and reperfusion was established afterward. Retinal tissues were collected 2 days after I/R injury. After 2‐D DIGE analysis, a total of 96 proteins were identified. Among them, 28 proteins were identified within gel spots whose intensities were normalized by AMG pretreatment, pathway analysis indicated that most were involved in glycolysis and carbohydrate metabolism. Selected enzymes identified by MS/MS within these pathways, including transketolase, triosephosphate isomerase 1, aldolase C, total enolase, and pyruvate kinase were validated by quantitative Western blots. Glycolytic enzymes and other differentially regulated proteins likely play previously unrecognized roles in retinal degeneration after I/R injury, and inhibition of the resulting metabolic changes, using pharmacologically agents such as AMG, serve to inhibit the changes in metabolism and mitigate retinal degeneration. Select glycolytic enzymes may provide novel therapeutic targets for inhibiting the neuronal and vascular degeneration after retinal I/R injury.

Comparison of Label Free and 18 O Labeling Mass Spectrometry in Relative Protein Quantification

Mass–spectrometry-based quantification methods have been increasingly applied to measure proteomic changes in biological systems between different physiological states. In this report, we compared two popular mass-spectrometry-based quantification strategies, stable isotope labeling and label free approaches. We spiked known amounts of standard peptides into a complex biological sample and analyzed this mixture with both stable isotope 18O labeling and label free mass spectrometry methods. We optimized data pre-processing and normalization algorithms for each method, and compared their sensitivities and accuracies. We found that both methods gave relatively accurate results, and the label free methods provided higher proteome coverage.

Erratum: Ageing causes cytoplasmic retention of MaxiK channels in rat corporal smooth muscle cells (International Journal of Impotence Research (2007) 19, (371-377) DOI: 10.1038/sj.ijir.3901541)

The MaxiK channel plays a critical role in the regulation of corporal smooth muscle tone and thereby erectile function. Given that ageing results in a decline in erectile function, we determined changes in the expression of MaxiK, which might impact erectile function. Quantitative-polymerase chain reaction demonstrated that although there is no significant change in transcription of the alpha- and beta-subunits that comprise the MaxiK channel, there are significant changes in the expression of transcripts encoding different splice variants. One transcript, SV1, is 13-fold increased in expression in the ageing rat corpora. SV1 has previously been reported to trap other isoforms of the MaxiK channel in the cytoplasm. Correlating with increased expression of SV1, we observed in older rats there is approximately a 13-fold decrease in MaxiK protein in the corpora cell membrane and a greater proportion is retained in the cytoplasm (approximately threefold). These experiments demonstrate that ageing of the corpora is accompanied by changes in alternative splicing and cellular localization of the MaxiK channel.