Proteomic analysis in endometrial cancer and endometrial hyperplasia tissues by 2D-DIGE technique.

OBJECTIVE: To compare the protein expression of complex atypical endometrial hyperplasia, endometrial carcinoma and healthy endometrial tissues, and by this way, to identify proteins that can be used for diagnosis, prognosis and therapeutic targets. METHODS: Histopathological examination of the D&C material had reported "benign endometrial changes", "complex atypical endometrial hyperplasia" and "endometrioid adenocarcinoma" and 30 patients ,who underwent surgery with these diagnosis, were studied. Protein profiles of the study groups were detected using 2D-DIGE technique and compared to the control group. Protein spots which showing different expression, were defined by MALDI TOF/TOF-MS method. RESULTS: In the present study, significant elevations were observed in the levels of K2C8, UAP56, ENOA, ACTB, GRP78, GSTP1, PSME1, CALR, PPIA, PDIA3 and IDHc proteins when comparisons were made among the cancer cases and the healthy and complex atypical hyperplasia cases. We determined that the induction of CALR activity may be a factor that progresses apoptosis, thus, may be a hope for postoperative new chemotherapy treatment methods. Moreover, when the expressions of the CAH1 and PPIB proteins are compared to complex atypical hyperplasia and endometrial adenocarcinoma stages, we determined that the CAH1 and PPIB levels increased in more advanced stages. Among these indicators, the proteins that had the closest relation to advanced stage cancer were determined as K2C8, UAP56 and GRP78. CONCLUSION: We think that it would be useful to determine the diagnosis, prediction of prognosis and identifying therapeutic targets of the highlighted proteins of our study that are K2C8, UAP56, GRP78 and CALR in endometrial cancer.

Comparative physiological and leaf proteome analysis between drought-tolerant chickpea Cicer reticulatum and drought-sensitive chickpea C. arietinum.

Comparative physiological and proteomic analysis were performed to understand the stress responses of two chickpea species (C. reticulatum and C. arietinum) against drought. Our study revealed that drought stress reduced root length, leaf water content, and enhanced free proline content in both species. Effect of drought stress appeared to be greater in C. arietinum compared to C. reticulatum. A total of 24 differently expressed proteins were identified by using MALDI-TOF/ TOF-MS/MS in response to drought. The proteins involved in photosynthesis and energy mechanisms were up-regulated in C. reticulatum and down-regulated in C. arietinum under drought. Our results suggest that the photosynthesis capacity of C. reticulatum is greater than that of C. arietinum under drought stress. Abundance of proline and sucrose biosynthesis related proteins, glutamine synthetase and cyctosolic fructose-bisphosphate aldolase, respectively, also increased in C. reticulatum under drought stress. The findings of this proteome analysis will help in understanding the mechanism of drought resistance in chickpea and may be also helpful in developing drought-resistant transgenic plants.

Assessment of hazelnut husk as a lignocellulosic feedstock for the production of fermentable sugars and lignocellulolytic enzymes.

The present work focuses firstly on the evaluation of the effect of laccase on enzymatic hydrolysis of hazelnut husk which is one of the most abundant lignocellulosic agricultural residues generated in Turkey. In this respect, the co-enzymatic treatment of hazelnut husk by cellulase and laccase, without a conventional pretreatment step is evaluated. Using 2.75 FPU/g substrate (40 g/L substrate) and a ratio of 131 laccase U/FPU achieved the highest reducing sugars concentration. Gas chromatography mass spectrometry confirmed that the hydrolysate was composed of glucose, xylose, mannose, arabinose and galactose. The inclusion of laccase in the enzyme mixture [carboxymethyl cellulase (CMCase) and ß-glucosidase] increased the final glucose content of the reducing sugars from 20 to 50%. Therefore, a very significant increase in glucose content of the final reducing sugars concentration was obtained by laccase addition. Furthermore, the production of cellulases and laccase by Pycnoporus sanguineus DSM 3024 using hazelnut husk as substrate was also investigated. Among the hazelnut husk concentrations tested (1.5, 6, 12, 18 g/L), the highest CMCase concentration was obtained using 12 g/L husk concentration on the 10th day of fermentation. Besides CMCase, P. sanguineus DSM 3024 produced ß-glucosidase and laccase using hazelnut husk as carbon source. In addition to CMCase and ß-glucosidase, the highest laccase activity measured was 2240 ± 98 U/L (8.89 ± 0.39 U/mg). To the best of our knowledge, this is the first study to report hazelnut husk hydrolysis in the absence of pretreatment procedures.

Exogenous Expressions of FTO Wild-Type and R316Q Mutant Proteins Caused an Increase in HNRPK Levels in 3T3-L1 Cells as Demonstrated by DIGE Analysis.

Fat mass and obesity-associated protein is an enzyme that oxidatively demethylates DNA. Although there are numerous studies regarding the catalytic function of FTO, the overall existence or absence of FTO on cellular proteome has not been investigated. This study investigated the changes in the soluble proteome of 3T3-L1 cells upon expression of the WT and the mutant (R316Q) FTO proteins. Protein extracts prepared from 3T3-L1 cells expressing either the WT or the mutant FTO proteins were used in DIGE experiments. Analysis of the data revealed the number of spots matched to every member and there were 350 ± 20 spots with 30.5% overall mean coefficient of variation. Eleven regulated protein spots were excised from the gels and identified by MALDI-TOF/TOF. One of the identified proteins was heterogeneous nuclear ribonucleoprotein K, which displayed more than 2.6- and 3.7-fold increases in its abundance in the WT and the mutant FTO expressing cells, respectively. Western blot analysis validated these observations. This is the first study revealing the presence of a parallel increase in expressions of FTO and HNRNPK proteins. This increase may codictate the metabolic changes occurring in the cell and may attribute a significance to HNRNPK in FTO-associated transformations.

Comparative Analysis of Proteome Patterns of Francisella tularensis Isolates from Patients and the Environment.

Francisella tularensis is the causative agent of tularemia. Although major contributors and the main mechanism of the virulence are well known, some of the molecular details are still missing. Proteomics studies regarding F. tularensis have provided snapshot pictures of the organism grown under different culture conditions to understand the mechanism of virulence. In general, such studies were carried out with standard strains e.g., LVS and did not involve comparisons of F. tularensis isolates from either clinical or environmental sources. In this study, we performed two-dimensional gel electrophoresis (2DE)-based proteomic analysis and compared the protein profiles of the F. tularensis subsp. holarctica strains isolated from the clinical and the environmental samples. Regulations were detected in 14 spots when twofold regulation criteria were applied. The regulated protein spots were subjected to MALDI-TOF/TOF analysis and identified. Classification of the identified proteins based on metabolic functions revealed that the translation machinery was the most varying metabolic processes among the isolates. Using normalized protein spot intensities, PCA analysis was also performed. The results indicated that the strain isolated from water source was different then the strains isolated from the patients. Most interestingly, the isolates were strikingly distinguishable from the standard NCTC 10857 strain.

Proteomic Analysis of Kidney Preservation Solutions Prior to Renal Transplantation.

One of the main issues in kidney transplantation is the optimal functional preservation of the organ until its transplantation into the appropriate recipient. Despite intensive efforts, the functional preservation period remains limited to hours. During this time, as a result of cellular injury, various proteins, peptides, and other molecules are released by the organ into the preservation medium. In this study, we used proteomic techniques to analyze the protein profiles of preservation solutions in which organs had been preserved prior to their transplantation. Samples were obtained from the preservation solutions of 25 deceased donor kidneys scheduled for transplantation. The protein profiles of the solutions were analyzed using 2D gel electrophoresis/MALDI-TOF and LC-MS/MS. We identified and quantified 206 proteins and peptides belonging to 139 different groups. Of these, 111 proteins groups were belonging to kidney tissues. This study used proteomic techniques to analyze the protein profiles of organ preservation solutions. These findings will contribute to the development of improved preservation solutions to effectively protect organs for transplantation.

Linking a compound-heterozygous Parkin mutant (Q311R and A371T) to Parkinson's disease by using proteomic and molecular approaches.

Parkin is an E3-protein ubiquitin ligase, which plays an important role as a scavenger in cell metabolism. Since the discovery of the link between Parkin and Parkinson's disease, Parkin was placed in the center of Parkinson's disease research. Previously, we isolated a mutant form of the Parkin protein (Q311R and A371T) from a Parkinson's disease patient. In this study, we aimed at characterizing this mutant Parkin protein by using biochemical and proteomic approaches. We used neuroblastoma cells (SH-SY5Y) as our model and created two inducible cell lines that expressed the wild type and the mutant Parkin proteins. We first investigated the effect of expressing both the wild type and the mutant Parkin proteins on the overall proteome by using 2D-DIGE approach. The experiments yielded the identification of 22 differentially regulated proteins, of which 13 were regulated in the mutant Parkin expressing cells. Classification of the identified proteins based on biological process and molecular function revealed that the majority of the regulated proteins belonged to protein folding and energy metabolism. Ingenuity Pathway Analysis predicted the presence of a link between the regulated proteins of the mutant Parkin expressing cells and Parkinson's disease. We also performed biochemical characterization studies on the wild type and the mutant Parkin proteins to make sense out of the differences observed at the proteome level. Both proteins displayed biological activity, had similar stabilities and localized similarly to the cytoplasm and the nucleus in SH-SY5Y cells. The mutant protein, however, was cut by a protease and subjected to a post-translational modification. The observed differences at the proteome level might be due to the differences in processing of the mutant Parkin protein. Overall, we were able to create a possible link between a pair of Parkin mutations to its pertinent disease by using 2D-DIGE in combination with biochemical and molecular approaches.

Assessment of berberine as a multi-target antimicrobial: a multi-omics study for drug discovery and repositioning.

Postgenomics drug development is undergoing major transformation in the age of multi-omics studies and drug repositioning. Rather than applications solely in personalized medicine, omics science thus additionally offers a better understanding of a broader range of drug targets and drug repositioning. Berberine is an isoquinoline alkaloid found in many medicinal plants. We report here a whole genome microarray study in tandem with proteomics techniques for mining the plethora of targets that are putatively involved in the antimicrobial activity of berberine against Escherichia coli. We found DNA replication/repair and transcription to be triggered by berberine, indicating that nucleic acids, in general, are among its targets. Our combined transcriptomics and proteomics multi-omics findings underscore that, in the presence of berberine, cell wall or cell membrane transport and motility-related functions are also specifically regulated. We further report a general decline in metabolism, as seen by repression of genes in carbohydrate and amino acid metabolism, energy production, and conversion. An involvement of multidrug efflux pumps, as well as reduced membrane permeability for developing resistance against berberine in E. coli was noted. Collectively, these findings offer original and significant leads for omics-guided drug discovery and future repositioning approaches in the postgenomics era, using berberine as a multi-omics case study.