Current Proteomics - Volume 11, Issue 1, 2014

Heat shock protein 90 (Hsp90) is highly conserved chaperone protein which plays vital roles in stabilization, regulation and folding of client proteins in cancer cells. Client proteins are key macromolecules for carcinogenesis and their maintenance (stabilization and protection from aggregation and misfolding) is provided by Hsp90 chaperone activity. Hsp90 allows proliferation of cancer cells by keeping misfolded client proteins in their proper functional folded form and suppresses apoptotic pathways for cancer cell survival. For this purpose, Hsp90 inhibitors have become an important growing class of antitumor agents in pharmaceutical industry. To date, numerous compounds have been tested as anticancer drugs in preclinical and clinical studies. Hsp90 inhibitors may be categorized into two main classes: natural and synthetic inhibitors. In this review, we will discuss the general properties and structure of both natural inhibitors (geldanamycin, 17-AAG, 17-DMAG, herbimycin, radicicol, novobiocin, (-)-EGCG, derrubone, gedunin, celastrol and their derivatives) and synthetic inhibitors (purine scaffold inhibitors, pyrazole scaffold inhibitors, SNX-2112, STA9090 and their derivatives) along with their therapeutic strategies in many different cancer types.

Numerous genome sequence projects of various organisms have resulted in generation of large amount of data on genes and proteins sequence information. Functional annotation of these proteins is important to bridge gap between sequence information and functional characterization. As experimental approaches for characterizing the functional class of an enzyme are expensive and time consuming, computational prediction methods are an effective alternative. Various approaches like homology-based function transfer and machine learning methods have been utilized for in silico enzyme functional classifications in terms of Enzyme Commission number (EC number) for a protein. Different types of features have been used in various machine learning techniques and each has its own advantages and limitations. The critical evaluation of performance measure in terms of predictive ability of these methods is necessary. Here, a systematic review on the various approaches used by different research groups, their utility and inference is presented.

Purified chitin binding protein the CBP50 from Bacillus thuringiensis and three bacterial chitinases (Chi74, Chi39 and ChiA) from two different genome sources i.e. Bacillus thuringiensis (Bt) and Serratia proteamaculans, were used to study the crystalline chitin degradation using different combinations of chitinases with and without chitin binding protein CBP50. Overall 2-4 folds increase in enzymatic activity of all three chitinases was observed when used in combination with the CBP50. But the highest increase, 4.64 folds, was observed when combination Chi74+Chi39+CBP50 was used. Gene loci of all proteins involved in Bt-chitin degradation and protein association network were also analyzed. This is the first report of synergistic action of Bt-proteins on chitin degradation. Present study will lead us to develop a technology for environmental friendly conversion of chitin to valuable products.

Cadmium (Cd) is a well known environmental and industrial heavy metal with multi-organ toxic effects. In this study, we examined the effects of Cd as cadmium chloride (CdCl2) on the proteomic profiles of exposed HEK 293, HepG2 and 1321N1 cells in an attempt to develop suitable biomarkers for Cd toxicity. 2D-gel electrophoresis (2DE) was performed on the cell extracts after 24 hr exposure to 5, 10 and 50 µM Cd and protein spots were compared using Phoretix TM 2D analysis software. Comparisons were made between Cd treated and untreated cells and spots were identified by mass spectroscopy using peptide-mass fingerprinting and database searching. The results show that the different concentrations (5-50 µM) of CdCl2 used in this study caused at least 1-5-fold induction in some proteins in the three cell lines. A common feature in the proteomic profile was identified in HepG2 and HEK 293 cells after exposure to 5 µM CdCl2 and this was the induction of one of the subunits of ATP synthase. 2DE analysis shows a 2.95- and 2.54-fold induction in ATP synthase in HEK 293 and HepG2 cells, respectively after 24 hr exposure to 5 µM CdCl2. However, western blot validation shows 4.8- and 3.54-fold induction in ATPase in HEK 293 and HepG2 cells respectively. Both 2DE and western blot analysis shows a 2.2-fold induction in calreticulin, a Ca2-binding protein, in HepG2 cells after 24 hr exposure to 5 µM CdCl2.Though 2DE analysis shows a 1.7-fold induction in Albumin (ALB) protein in HEK 293 cells exposed to 50 µM CdCl2, western blot analysis, however, shows a 10-fold increase. Exposure to 5 µM Cd also induced (1.8-fold) C-protein expression in 1321N1 cells. However, western blot analysis shows a 4.5-fold increase. These results suggest that Cd drastically alters the proteomic profiles of exposed cells, which include alterations in the expressions of proteins, involve in metabolism and intracellular Ca2 homeostasis. These alterations may be important hallmarks in identifying Cd toxicity.

The molecular mechanisms that cause myopia are largely unknown. However, signaling pathways originating in the retina are implicated in regulating refractive development. The study of retinal protein expression profiles ("proteomics") in animal eyes is a powerful approach to unraveling these molecular mechanisms. Previously, we used this proteomics approach in the chick model to identify a number of proteins whose expression was either up or down regulated during myopia development, compared to untreated contralateral "control" eyes. In order to search for mechanisms conserved between animal species, here we applied proteomic techniques to study retinal protein changes induced by form deprivation using the mammalian albino guinea pig model. We identified 8 differentially expressed retinal proteins in myopic eyes: Significant increases in protein levels were found in myopic eyes for Septin-6, Fascin-1, Collapsin response mediator protein, Preproalbumin precursor, Succinate dehydrogenase complex subunit A and Vimentin; whereas protein levels were found to be decreased for β-soluble NSF attachment protein and Phosphoglycerate mutase 1. Four of these proteins (Septin-6, Collapsin response mediator protein and Phosphoglycerate mutase 1 and Vimentin) were found previously to be differentially expressed – in the same direction – in the retina of chicks developing lens-induced myopia, thus providing independent replication of their differential abundance in myopia, and the conservation of molecular changes in the myopic retina between birds and mammals, as well as across myopia-inducing paradigms. The eight differentially expressed proteins identified here are potential candidates with roles in the regulation of myopic eye growth.

Background and Aims: Our previous yeast two-hybrid screening data showed the Fibrinogen alpha chain (FGA) as one of the candidate binding proteins of S regional of the HBsAg (HBs). In this study, we aimed to define that FGA is a binding protein of HBs and to determine its function in Hepatocellular carcinoma (HCC) tumorigenesis. Methods: The binding and co-localization between HBs and FGA were confirmed using co-immunoprecipitation and confocal microscopy was employed flow cytometry to analyze cell apoptosis. The involved mechanisms were investigated using protein array and western blot. Results: Our results indicated that FGA is a binding protein of HBs and is co-localized in the cytoplasm in vitro. Interaction between HBs and FGA significantly induced apoptosis in HepG2 cells. Moreover, knockdown of the FGA protein decreased the expression levels of the pro-survival factors Bcl-XL and Mcl-1, increased the expression of the proapoptotic proteins, and decreased the phosphorylation levels of Akt in this cell. Conclusion: FGA is a binding protein of HBs and their interaction induced the apoptotic capacity of HepG2 cells, suggesting the interactions between viral and host cell proteins are involved in the development of virus-related hepatitis or HCC.

We demonstrate the Binary Identified Contact Domain (BICD) method for predicting binding sites for proteins that starts with amino-acid sequences of several species, finds regions that are evolutionarily conserved, and weights regions by their local hydrophilicity. The measure of conservation takes transversions as significant but transitions as insignificant, in keeping with a theory of original singlet codes. The BICD method correctly predicts several known binding sites for the BRCA2 protein with PABL2 and RAD51 as well as the binding sites of the MDM2-P53 complex.

To understand the cold stress response of soybean, 480 soybean germplasms were used to find the tolerant and sensitivity of germplasm to cold. Then a comparative proteomic analysis between soybean seed treated at chilling and non-chilling temperature was carried out using the tolerant cold soybean germplasm. During the germination of seed, it was possible to discriminate between proteins involved in symptoms and proteins implicated in response to chilling. A proteomic approach, based on two-dimensional electrophoresis and MALDI-ToF-MS peptide mass fingerprinting experiments, was used for this purpose. The comparison of the resulting proteomic maps highlighted proteins showing quantitative variations induced by temperature treatments. Twenty-nine protein spots exhibited significant quantitative changes under at least one stress condition. Identified, differentially represented proteins belonged to two main broad functional groups, namely energy production/carbon metabolism and response to abiotic and oxidative stresses. In addition, our data seem to indicate a possible candidate to be used as a protein marker for further studies on cold stress.

The aleurone layer (AL) is an inner tissue of the wheat grain. It contains micronutrients, vitamins, antioxidants and fibre, and can greatly increase the nutritional quality of flour if it is not removed from the kernel with the bran. The AL of mature kernels of three varieties of the two major cultivated wheat species T. aestivum (genome A, B and D) and T. monococcum (genome A) were manually dissected and analyzed using two-dimensional gel-based proteomics. In T. monococcum although composed of only genome A, the maximum number of Coomassie stained AL spots was close to the number found in the bread wheat varieties (1320 and 1258, respectively). Inter-variety variation in spots was higher in the three T. monococcum varieties (103 spots) than in the three T. aestivum varieties (79 spots). Comparison of the two species revealed that only 88 spots differed significantly either in abundance or presence/absence. The B and D genome did not drastically modify the AL proteome, as demonstrated by the fact that 93% of the spots present in T. Monococcum AL spots were also present in T. aestivum. Proteins which differed within and between species were identified using MALDI-TOF and LC-MS/MS Mass Spectrometry. Among the 182 spots that differed, 115 were identified, 53 differed between the two species and 44 (83%) were globulin (Glo) storage proteins. The remarkable environmental stability of the AL proteome previously observed in T. durum and T. aestivum species was confirmed in the variety T. monococcum DV92, grown for two consecutive years in field conditions. Only 15 proteins (out of 1320 AL spots) exhibited significant quantitative variations.