A systematic strategy for proteomic analysis of chloroplast protein complexes in wheat

A systematic strategy was developed for the proteomic analysis of wheat chloroplast protein complexes. First, comprehensive centrifugation methods were utilized for the exhaustive isolation of thylakoid, envelope, and stromal fractions. Second, 1% n-dodecyl-β-D-maltoside was selected from a series of detergents as the optimal detergent to dissolve protein complexes effectively from membranes. Then, blue native polyacrylamide gel electrophoresis (BN-PAGE) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were improved to separate and analyze the protein complexes. By this systematic strategy, envelopes, thylakoids, and stromata were enriched effectively from chloroplasts in the same process, and more than 18 complexes were obtained simultaneously by BN-PAGE. Finally, thylakoid protein complexes were further analyzed by BN/SDS-PAGE, and nine complex bands and 40 protein spots were observed on BN-PAGE and SDS-PAGE respectively. Our results indicate that this new strategy can be used efficiently to analyze the proteome of chloroplast protein complexes and can be applied conveniently to the analysis of other subcellular protein complexes.     

Induced pluripotent stem cells as a tool for gaining new insights into Fanconi anemia

Induced pluripotent stem cells (iPSC) hold significant promise for advancing biomedical research. In the case of monogenic diseases, patient-iPSC and their derivatives contain the disease-causing mutation, suggesting the possibility of recapitulating salient disease features in vitro. Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. The etiology of bone marrow failure in FA remains largely unclear, but limited studies on patient bone marrow cells indicate cell intrinsic defects as causative. We examined the feasibility of modeling FA in a system based on hematopoietic differentiation of patient-specific iPSC. An informative iPSC-based model is predicated on the ability to derive disease-specific (uncorrected) patient iPSC that contain the disease-causing mutation, are pluripotent, maintain a normal karyotype and are capable of hematopoietic differentiation. Careful analysis of hematopoietic differentiation of such iPSC holds the promise of uncovering new insights into bone marrow failure and may enable high-throughput screening with the goal of identifying compounds that ameliorate hematopoietic failure. Ultimately, genetic correction, molecular characterization and successful engraftment of iPSC-derived cells may provide an attractive alternative to current hematopoietic stem cell-targeted gene therapy in some monogenic diseases, including FA.     

Induced pluripotent stem cells as a tool for gaining new insights into Fanconi anemia

Induced pluripotent stem cells (iPSC) hold significant promise for advancing biomedical research. In the case of monogenic diseases, patient-iPSC and their derivatives contain the disease-causing mutation, suggesting the possibility of recapitulating salient disease features in vitro. Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. The etiology of bone marrow failure in FA remains largely unclear, but limited studies on patient bone marrow cells indicate cell intrinsic defects as causative. We examined the feasibility of modeling FA in a system based on hematopoietic differentiation of patient-specific iPSC. An informative iPSC-based model is predicated on the ability to derive disease-specific (uncorrected) patient iPSC that contain the disease-causing mutation, are pluripotent, maintain a normal karyotype and are capable of hematopoietic differentiation. Careful analysis of hematopoietic differentiation of such iPSC holds the promise of uncovering new insights into bone marrow failure and may enable high-throughput screening with the goal of identifying compounds that ameliorate hematopoietic failure. Ultimately, genetic correction, molecular characterization and successful engraftment of iPSC-derived cells may provide an attractive alternative to current hematopoietic stem cell-targeted gene therapy in some monogenic diseases, including FA.     

Novel insights into the architecture and protein interaction network of yeast eIF3

Translation initiation in eukaryotes is a multistep process requiring the orchestrated interaction of several eukaryotic initiation factors (eIFs). The largest of these factors, eIF3, forms the scaffold for other initiation factors, promoting their binding to the 40S ribosomal subunit. Biochemical and structural studies on eIF3 need highly pure eIF3. However, natively purified eIF3 comprise complexes containing other proteins such as eIF5. Therefore we have established in vitro reconstitution protocols for Saccharomyces cerevisiae eIF3 using its five recombinantly expressed and purified subunits. This reconstituted eIF3 complex (eIF3^rec) exhibits the same size and activity as the natively purified eIF3 (eIF3^nat). The homogeneity and stoichiometry of eIF3^rec and eIF3^nat were confirmed by analytical size exclusion chromatography, mass spectrometry, and multi-angle light scattering, demonstrating the presence of one copy of each subunit in the eIF3 complex. The reconstituted and native eIF3 complexes were compared by single-particle electron microscopy showing a high degree of structural conservation. The interaction network between eIF3 proteins was studied by means of limited proteolysis, analytical size exclusion chromatography, in vitro binding assays, and isothermal titration calorimetry, unveiling distinct protein domains and subcomplexes that are critical for the integrity of the protein network in yeast eIF3. Taken together, the data presented here provide a novel procedure to obtain highly pure yeast eIF3, suitable for biochemical and structural analysis, in addition to a detailed picture of the network of protein interactions within this complex.     

The yeast prion protein Ure2: insights into the mechanism of amyloid formation

Ure2, a regulator of nitrogen metabolism, is the protein determinant of the [URE3] prion state in Saccharomyces cerevisiae. Upon conversion into the prion form, Ure2 undergoes a heritable conformational change to an amyloid-like aggregated state and loses its regulatory function. A number of molecular chaperones have been found to affect the prion properties of Ure2. The studies carried out in our laboratory have been aimed at elucidating the structure of Ure2 fibrils, the mechanism of amyloid formation and the effect of chaperones on the fibril formation of Ure2.     

Ecm11 protein of yeast Saccharomyces cerevisiae is regulated by sumoylation during meiosis

Damaged regulation of the small ubiquitin-like modifier (SUMO) system contributes to some human diseases; therefore, it is very important to identify the SUMO targets and to determine the function of their sumoylation. In this study, it is shown that Ecm11 protein in Saccharomyces cerevisiae is modified by SUMO during meiosis. It is known that Ecm11 is required in the early stages of yeast meiosis where its function is related to DNA replication and crossing over. Here it is shown that the level of Ecm11 protein is low in mitosis, but high in meiosis. The highest level of Ecm11 is in the early-middle phase of sporulation. A specific site of sumoylation was identified in Ecm11 at Lys5 and evidence is provided that sumoylation at this site directly regulates Ecm11 function in meiosis. On the other hand, no relationship was observed between sumoylation of Ecm11 and its role during vegetative growth. It was shown that Ecm11 interacts with Siz2 SUMO ligase in a two-hybrid system; although Siz2 is not essential for the Ecm11 sumoylation.     

A data-analytic strategy for protein biomarker discovery: profiling of high-dimensional proteomic data for cancer detection

With recent advances in mass spectrometry techniques, it is now possible to investigate proteins over a wide range of molecular weights in small biological specimens. This advance has generated data-analytic challenges in proteomics, similar to those created by microarray technologies in genetics, namely, discovery of 'signature' protein profiles specific to each pathologic state (e.g. normal vs. cancer) or differential profiles between experimental conditions (e.g. treated by a drug of interest vs. untreated) from high-dimensional data. We propose a data-analytic strategy for discovering protein biomarkers based on such high-dimensional mass spectrometry data. A real biomarker-discovery project on prostate cancer is taken as a concrete example throughout the paper: the project aims to identify proteins in serum that distinguish cancer, benign hyperplasia, and normal states of prostate using the Surface Enhanced Laser Desorption/Ionization (SELDI) technology, a recently developed mass spectrometry technique. Our data-analytic strategy takes properties of the SELDI mass spectrometer into account: the SELDI output of a specimen contains about 48,000 (x, y) points where x is the protein mass divided by the number of charges introduced by ionization and y is the protein intensity of the corresponding mass per charge value, x, in that specimen. Given high coefficients of variation and other characteristics of protein intensity measures (y values), we reduce the measures of protein intensities to a set of binary variables that indicate peaks in the y-axis direction in the nearest neighborhoods of each mass per charge point in the x-axis direction. We then account for a shifting (measurement error) problem of the x-axis in SELDI output. After this pre-analysis processing of data, we combine the binary predictors to generate classification rules for cancer, benign hyperplasia, and normal states of prostate. Our approach is to apply the boosting algorithm to select binary predictors and construct a summary classifier. We empirically evaluate sensitivity and specificity of the resulting summary classifiers with a test dataset that is independent from the training dataset used to construct the summary classifiers. The proposed method performed nearly perfectly in distinguishing cancer and benign hyperplasia from normal. In the classification of cancer vs. benign hyperplasia, however, an appreciable proportion of the benign specimens were classified incorrectly as cancer. We discuss practical issues associated with our proposed approach to the analysis of SELDI output and its application in cancer biomarker discovery.     

NMR insights into protein allostery

Allosterism is one of nature's principal methods for regulating protein function. Allosterism utilizes ligand binding at one site to regulate the function of the protein by modulating the structure and dynamics of a distant binding site. In this review, we first survey solution NMR techniques and how they may be applied to the study of allostery. Subsequently, we describe several examples of application of NMR to protein allostery and highlight the unique insight provided by this experimental technique.     

Mechanistic insights into the dual inhibition strategy for checking Leishmaniasis

Leishmaniasis (1) is an endemic disease mainly caused by the protozoan Leishmania donovani (Ld). Polyamines have been identified as essential organic compounds for the growth and survival of Ld. These are synthesized in Ld by polyamine synthesis pathway comprising of many enzymes such as ornithine decarboxylase (ODC), spermidine synthase (SS), and S-adenosylmethionine decarboxylase. Inhibition of these enzymes in Ld offers a viable prospect to check its growth and development. In the present work, we used computational approaches to search natural inhibitors against ODC and SS enzymes. We predicted three-dimensional structures of ODC and SS using comparative modeling and molecular dynamics (MD) simulations. Thousands of natural compounds were virtually screened against target proteins using high throughput approach. MD simulations were then performed to examine molecular interactions between the screened compounds and functional residues of the active sites of the enzymes. Herein, we report two natural compounds of dual inhibitory nature active against the two crucial enzymes of polyamine pathway of Ld. These dual inhibitors have the potential to evolve as lead molecules in the development of antileishmanial drugs. (1)These authors contributed equally.     

Mechanistic insights into the dual inhibition strategy for checking Leishmaniasis

Leishmaniasis ^{1 1}These authors contributed equally.Communicated by Ramaswamy H. SarmaCommunicated by Ramaswamy H. Sarma is an endemic disease mainly caused by the protozoan Leishmania donovani (Ld). Polyamines have been identified as essential organic compounds for the growth and survival of Ld. These are synthesized in Ld by polyamine synthesis pathway comprising of many enzymes such as ornithine decarboxylase (ODC), spermidine synthase (SS), and S-adenosylmethionine decarboxylase. Inhibition of these enzymes in Ld offers a viable prospect to check its growth and development. In the present work, we used computational approaches to search natural inhibitors against ODC and SS enzymes. We predicted three-dimensional structures of ODC and SS using comparative modeling and molecular dynamics (MD) simulations. Thousands of natural compounds were virtually screened against target proteins using high throughput approach. MD simulations were then performed to examine molecular interactions between the screened compounds and functional residues of the active sites of the enzymes. Herein, we report two natural compounds of dual inhibitory nature active against the two crucial enzymes of polyamine pathway of Ld. These dual inhibitors have the potential to evolve as lead molecules in the development of antileishmanial drugs.     

Comparative proteomic analysis provides new insights into chilling stress responses in rice

Low temperature is one of the major abiotic stresses limiting the productivity and the geographical distribution of many important crops. To gain a better understanding of chilling stress responses in rice (Oryza sativa L. cv. Nipponbare), we carried out a comparative proteomic analysis. Three-week-old rice seedlings were treated at 6 degrees C for 6 or 24 h and then recovered for 24 h. Chilling treatment resulted in stress phenotypes of rolling leaves, increased relative electrolyte leakage, and decreased net photosynthetic rate. The temporal changes of total proteins in rice leaves were examined using two-dimensional electrophoresis. Among approximately 1,000 protein spots reproducibly detected on each gel, 31 protein spots were down-regulated, and 65 were up-regulated at least at one time point. Mass spectrometry analysis allowed the identification of 85 differentially expressed proteins, including well known and novel cold-responsive proteins. Several proteins showed enhanced degradation during chilling stress, especially the photosynthetic proteins such as Rubisco large subunit of which 19 fragments were detected. The identified proteins are involved in several processes, i.e. signal transduction, RNA processing, translation, protein processing, redox homeostasis, photosynthesis, photorespiration, and metabolisms of carbon, nitrogen, sulfur, and energy. Gene expression analysis of 44 different proteins by quantitative real time PCR showed that the mRNA level was not correlated well with the protein level. In conclusion, our study provides new insights into chilling stress responses in rice and demonstrates the advantages of proteomic analysis.     

New insights into the mechanism of homologous recombination in yeast

Genome stability is of primary importance for the survival and proper functioning of all organisms. Double-strand breaks (DSBs) arise spontaneously during growth, or can be created by external insults. Repair of DSBs by homologous recombination provides an efficient and fruitful pathway to restore chromosomal integrity. Exciting new work in yeast has lately provided insights into this complex process. Many of the proteins involved in recombination have been isolated and the details of the repair mechanism are now being unraveled at the molecular level. In this review, we focus on recent studies which dissect the recombinational repair of a single broken chromosome. After DSB formation, a decision is made regarding the mechanism of repair (recombination or non-homologous end-joining). This decision is under genetic control. Once committed to the recombination pathway, the broken chromosomal ends are resected by a still unclear mechanism in which the DNA damage checkpoint protein Rad24 participates. At this stage several proteins are recruited to the broken ends, including Rad51p, Rad52p, Rad55p, Rad57p, and possibly Rad54p. A genomic search for homology ensues, followed by strand invasion, promoted by the Rad51 filament with the participation of Rad55p, Rad57p and Rad54p. DNA synthesis then takes place, restoring the resected ends. Crossing-over formation depends on the length of the homologous recombining sequences, and is usually counteracted by the activity of the mismatch repair system. Given the conservation of the repair mechanisms and genes throughout evolution, these studies have profound implications for other eukaryotic organisms.     

Comparative proteomic analysis provides new insights into the fiber elongating process in cotton

A comparative proteomic analysis was performed to explore the mechanism of cell elongation in developing cotton fibers. The temporal changes of global proteomes at five representative development stages (5-25 days post-anthesis [dpa]) were examined using 2-D electrophoresis. Among approximately 1800 stained protein spots reproducibly detected on each gel, 235 spots were differentially expressed with significant dynamics in elongating fibers. Of these, 120 spots showed a more than 2-fold change in at least one stage point, and 21 spots appeared to be specific to developmental stages. Furthermore, 106 differentially expressed proteins were identified from mass spectrometry to match 66 unique protein species. These proteins involve different cellular and metabolic processes with obvious functional tendencies toward energy/carbohydrate metabolism, protein turnover, cytoskeleton dynamics, cellular responses and redox homeostasis, indicating a good correlation between development-dependent proteins and fiber biochemical processes, as well as morphogenesis. Newly identified proteins such as phospholipase D alpha, vf14-3-3 protein, small ras-related protein, and GDP dissociation inhibitor will advance our knowledge of the complicated regulatory network. Identification of these proteins, combined with their changes in abundance, provides a global view of the development-dependent protein changes in cotton fibers, and offers a framework for further functional research of target proteins associated with fiber development.     

Novel insights into the osmotic stress response of yeast

Response to hyperosmolarity in the baker's yeast Saccharomyces cerevisiae has attracted a great deal of attention of molecular and cellular biologists in recent years, from both the fundamental scientific and applied viewpoint. Indeed the underlying molecular mechanisms form a clear demonstration of the intricate interplay of (environmental) signalling events, regulation of gene expression and control of metabolism that is pivotal to any living cell. In this article we briefly review the cellular response to conditions of hyperosmolarity, with focus on the high-osmolarity glycerol mitogen-activated protein kinase pathway as the major signalling route governing cellular adaptations. Special attention will be paid to the recent finding that in the yeast cell also major structural changes occur in order to ensure maintenance of cell integrity. The intriguing role of glycerol in growth of yeast under (osmotic) stress conditions is highlighted.     

The potential impact of recent insights into proteomic changes associated with glaucoma

Introduction: Glaucoma, a major ocular neuropathy, is still far from being understood on a molecular scale. Proteomic workflows revealed glaucoma associated alterations in different eye components. By using state-of-the-art mass spectrometric (MS) based discovery approaches large proteome datasets providing important information about glaucoma related proteins and pathways could be generated. Corresponding proteomic information could be retrieved from various ocular sample species derived from glaucoma experimental models or from original human material (e.g. optic nerve head or aqueous humor). However, particular eye tissues with the potential for understanding the disease’s molecular pathomechanism remains underrepresented.

Areas covered: The present review provides an overview of the analysis depth achieved for the glaucomatous eye proteome. With respect to different eye regions and biofluids, proteomics related literature was found using PubMed, Scholar and UniProtKB. Thereby, the review explores the potential of clinical proteomics for glaucoma research.

Expert commentary: Proteomics will provide important contributions to understanding the molecular processes associated with glaucoma. Sensitive discovery and targeted MS approaches will assist understanding of the molecular interplay of different eye components and biofluids in glaucoma. Proteomic results will drive the comprehension of glaucoma, allowing a more stringent disease hypothesis within the coming years.     

Mechanistic insights into Sky1p,a yeast homologue of the mammalian SR protein kinases

The SRPK family is distinguished from typical eukaryotic protein kinases by several unique structural features recently elucidated by X-ray diffraction methods [Nolen et al. (2001) Nat. Struct. Biol. 8, 176-183]. To determine whether these features impart unique catalytic function, the phosphorylation of the physiological Sky1p substrate, Npl3p, was monitored using steady-state and pre-steady-state kinetic techniques. While Sky1p has a low apparent affinity for ATP compared to other protein kinases, it binds Npl3p with very high affinity. The latter is achieved through a combination of local and distal factors in the protein substrate. The phosphoryl donor ATP has access to the nucleotide pocket in the absence or presence of Npl3p, indicating that a large protein substrate does not enforce an ordered addition of ligands. Sky1p binds two Mg(2+)-the first is essential whereas the second further enhances catalysis. While the turnover number is low (0.5 s(-1)), Npl3p is rapidly phosphorylated in the active site (40 s(-1)) based on single turnover experiments. These results indicate that Sky1p employs a catalytic pathway involving fast phosphoryl transfer followed by slow net release of products. These studies represent the first kinetic investigation of a member of the SRPK family and the first pre-steady-state kinetic study of a protein kinase using a natural protein substrate.