Proteomic Comparison of Various Hepatic Cell Cultures for Preclinical Safety Pharmacology.

Experimental drugs need to be screened for safety within time constraints. Hepatotoxicity is one concerning contributor to the failure of investigational new drugs and a major rationale for postmarketing withdrawal decisions. Ethical considerations in preclinical research force the requirement for highly predictive in vitro assays using human tissue which retains functionality reflective of primary tissue. Here, the proteome of cells commonly used to assess preclinical hepatotoxicity was compared. Primary human hepatocytes (PHHs), hepatocyte-like cells (HLCs) differentiated from human pluripotent stem cells, HepG2 cell monolayers and HepG2 cell 3D spheroids were cultured and collected as whole cell lysates. Over 6000 proteins were identified and quantified in terms of relative abundance in replicate proteomic experiments using isobaric tagging methods. Comparison of these quantitative data provides biological insight into the feasibility of using HLCs, HepG2 monolayers, and HepG2 3D spheroids for hepatotoxicity testing. Collectively these data reveal how HLCs differentiated for 35 days and HepG2 cells proteomes differ from one another and that of PHHs. HepG2 cells possess a strong cancer cell signature and do not adequately express key metabolic proteins which mark the hepatic phenotype, this was not substantially altered by culturing as 3D spheroids. These data suggest that while no single hepatic model reflects the diverse array of outcomes required to mimic the in vivo liver functions, that HLCs are the most suitable investigational avenue for replacing PHHs in vitro.

LC-MS/MS methods for absolute quantification and identification of proteins associated with chimeric plant oil bodies.

Oil bodies (OBs) are plant cell organelles that consist of a lipid core surrounded by a phospholipid monolayer embedded with specialized proteins such as oleosins. Recombinant proteins expressed in plants can be targeted to OBs as fusions with oleosin. This expression strategy is attractive because OBs are easily enriched and purified from other cellular components, based on their unique physicochemical properties. For recombinant OBs to be a potential therapeutic agent in biomedical applications, it is necessary to comprehensively analyze and quantify both endogenous and heterologously expressed OB proteins. In this study, a mass spectrometry (MS)-based method was developed to accurately quantify an OB-targeted heterologously expressed fusion protein that has potential as a therapeutic agent. The effect of the chimeric oleosin expression upon the OB proteome in transgenic plants was also investigated, and the identification of new potential OB residents was pursued through a variety of liquid chromatography (LC)-MS/MS approaches. The results showed that the accumulation of the fusion protein on OBs was low. Moreover, no significant differences in the accumulation of OB proteins were revealed between transgenic and wild-type seeds. The identification of five new putative components of OB proteome was also reported.

Two-dimensional differential in gel electrophoresis (2D-DIGE) analysis of grape berry proteome during postharvest withering.

The practice of postharvest withering is commonly used to correct quality traits and sugar concentration of high quality wines. To date, changes in the metabolome during the berry maturation process have been well documented; however, the biological events which occur at the protein level have yet to be fully investigated. To gain insight into the postharvest withering process, we studied the protein expression profiles of grape (Corvina variety) berry development focusing on withering utilizing a two-dimensional differential in gel electrophoresis (2D-DIGE) proteomics approach. Comparative analysis revealed changes in the abundance of numerous soluble proteins during the maturation and withering processes. On a total of 870 detected spots, 90 proteins were differentially expressed during berry ripening/withering and 72 were identified by MS/MS analysis. The majority of these proteins were related to stress and defense activity (30%), energy and primary metabolism (25%), cytoskeleton remodelling (7%), and secondary metabolism (5%). Moreover, this study demonstrates an active modulation of metabolic pathways throughout the slow dehydration process, including de novo protein synthesis in response to the stress condition and further evolution of physiological processes originated during ripening. These data represent an important insight into the withering process in terms of both Vitis germplasm characterization and knowledge which can assist quality improvement.

Nutrient control of eukaryote cell growth: a systems biology study in yeast.

BACKGROUND: To elucidate the biological processes affected by changes in growth rate and nutrient availability, we have performed a comprehensive analysis of the transcriptome, proteome and metabolome responses of chemostat cultures of the yeast, Saccharomyces cerevisiae, growing at a range of growth rates and in four different nutrient-limiting conditions. RESULTS: We find significant changes in expression for many genes in each of the four nutrient-limited conditions tested. We also observe several processes that respond differently to changes in growth rate and are specific to each nutrient-limiting condition. These include carbohydrate storage, mitochondrial function, ribosome synthesis, and phosphate transport. Integrating transcriptome data with proteome measurements allows us to identify previously unrecognized examples of post-transcriptional regulation in response to both nutrient and growth-rate signals. CONCLUSIONS: Our results emphasize the unique properties of carbon metabolism and the carbon substrate, the limitation of which induces significant changes in gene regulation at the transcriptional and post-transcriptional level, as well as altering how many genes respond to growth rate. By comparison, the responses to growth limitation by other nutrients involve a smaller set of genes that participate in specific pathways. See associated commentary http://www.biomedcentral.com/1741-7007/8/62.

DsbA plays a critical and multifaceted role in the production of secreted virulence factors by the phytopathogen Erwinia carotovora subsp. atroseptica.

Erwinia carotovora subsp. atroseptica is an enterobacterial phytopathogen causing economically significant soft rot disease. Pathogenesis is mediated by multiple secreted virulence factors, many of which are secreted by the type II (Out) secretion system. DsbA catalyzes the introduction of disulfide bonds into periplasmic and secreted proteins. In this study, the extracellular proteome (secretome) of wild type E. carotovora subsp. atroseptica SCRI1043, and dsbA and out mutants, was analyzed by spectral counting mass spectrometry. This revealed that dsbA inactivation had a huge impact on the secretome and identified diverse DsbA- and Out-dependent secreted proteins, representing known, predicted, and novel candidate virulence factors. Further characterization of the dsbA mutant showed that secreted enzyme activities, motility, production of the quorum-sensing signal, and virulence were absent or substantially reduced. The impact of DsbA on secreted virulence factor production was mediated at multiple levels, including impacting on the Out secretion system and the virulence gene regulatory network. Transcriptome analyses revealed that the abundance of a broad, but defined, set of transcripts, including many virulence factors, was altered in the dsbA mutant, identifying a new virulence regulon responsive to extracytoplasmic conditions. In conclusion, DsbA plays a crucial, multifaceted role in the pathogenesis of E. carotovora subsp. atroseptica.

SETH1 and SETH2, two components of the glycosylphosphatidylinositol anchor biosynthetic pathway, are required for pollen germination and tube growth in Arabidopsis.

Glycosylphosphatidylinositol (GPI) anchoring provides an alternative to transmembrane domains for anchoring proteins to the cell surface in eukaryotes. GPI anchors are synthesized in the endoplasmic reticulum via the sequential addition of monosaccharides, fatty acids, and phosphoethanolamines to phosphatidylinositol. Deficiencies in GPI biosynthesis lead to embryonic lethality in animals and to conditional lethality in eukaryotic microbes by blocking cell growth, cell division, or morphogenesis. We report the genetic and phenotypic analysis of insertional mutations disrupting SETH1 and SETH2, which encode Arabidopsis homologs of two conserved proteins involved in the first step of the GPI biosynthetic pathway. seth1 and seth2 mutations specifically block male transmission and pollen function. This results from reduced pollen germination and tube growth, which are associated with abnormal callose deposition. This finding suggests an essential role for GPI anchor biosynthesis in pollen tube wall deposition or metabolism. Using transcriptomic and proteomic approaches, we identified 47 genes that encode potential GPI-anchored proteins that are expressed in pollen and demonstrated that at least 11 of these proteins are associated with pollen membranes by GPI anchoring. Many of the identified candidate proteins are homologous with proteins involved in cell wall synthesis and remodeling or intercellular signaling and adhesion, and they likely play important roles in the establishment and maintenance of polarized pollen tube growth.