Advancing the high throughput identification of liver fibrosis protein signatures using multiplexed ion mobility spectrometry.

Rapid diagnosis of disease states using less invasive, safer, and more clinically acceptable approaches than presently employed is a crucial direction for the field of medicine. While MS-based proteomics approaches have attempted to meet these objectives, challenges such as the enormous dynamic range of protein concentrations in clinically relevant biofluid samples coupled with the need to address human biodiversity have slowed their employment. Herein, we report on the use of a new instrumental platform that addresses these challenges by coupling technical advances in rapid gas phase multiplexed ion mobility spectrometry separations with liquid chromatography and MS to dramatically increase measurement sensitivity and throughput, further enabling future high throughput MS-based clinical applications. An initial application of the liquid chromatography--ion mobility spectrometry-MS platform analyzing blood serum samples from 60 postliver transplant patients with recurrent fibrosis progression and 60 nontransplant patients illustrates its potential utility for disease characterization.

Early transcriptional programming links progression to hepatitis C virus-induced severe liver disease in transplant patients.

UNLABELLED: Liver failure resulting from chronic hepatitis C virus (HCV) infection is a major cause for liver transplantation worldwide. Recurrent infection of the graft is universal in HCV patients after transplant and results in a rapid progression to severe fibrosis and end-stage liver disease in one third of all patients. No single clinical variable, or combination thereof, has, so far, proven accurate in identifying patients at risk of hepatic decompensation in the transplant setting. A combination of longitudinal, dimensionality reduction and categorical analysis of the transcriptome from 111 liver biopsy specimens taken from 57 HCV-infected patients over time identified a molecular signature of gene expression of patients at risk of developing severe fibrosis. Significantly, alterations in gene expression occur before histologic evidence of liver disease progression, suggesting that events that occur during the acute phase of infection influence patient outcome. Additionally, a common precursor state for different severe clinical outcomes was identified. CONCLUSION: Based on this patient cohort, incidence of severe liver disease is a process initiated early during HCV infection of the donor organ. The probable cellular network at the basis of the initial transition to severe liver disease was identified and characterized.

Proteome and computational analyses reveal new insights into the mechanisms of hepatitis C virus-mediated liver disease posttransplantation.

UNLABELLED: Liver transplant tissues offer the unique opportunity to model the longitudinal protein abundance changes occurring during hepatitis C virus (HCV)-associated liver disease progression in vivo. In this study, our goal was to identify molecular signatures, and potential key regulatory proteins, representative of the processes influencing early progression to fibrosis. We performed global protein profiling analyses on 24 liver biopsy specimens obtained from 15 HCV(+) liver transplant recipients at 6 and/or 12 months posttransplantation. Differentially regulated proteins associated with early progression to fibrosis were identified by analysis of the area under the receiver operating characteristic curve. Analysis of serum metabolites was performed on samples obtained from an independent cohort of 60 HCV(+) liver transplant patients. Computational modeling approaches were applied to identify potential key regulatory proteins of liver fibrogenesis. Among 4,324 proteins identified, 250 exhibited significant differential regulation in patients with rapidly progressive fibrosis. Patients with rapid fibrosis progression exhibited enrichment in differentially regulated proteins associated with various immune, hepatoprotective, and fibrogenic processes. The observed increase in proinflammatory activity and impairment in antioxidant defenses suggests that patients who develop significant liver injury experience elevated oxidative stresses. This was supported by an independent study demonstrating the altered abundance of oxidative stress-associated serum metabolites in patients who develop severe liver injury. Computational modeling approaches further highlight a potentially important link between HCV-associated oxidative stress and epigenetic regulatory mechanisms impacting on liver fibrogenesis. CONCLUSION: Our proteome and metabolome analyses provide new insights into the role for increased oxidative stress in the rapid fibrosis progression observed in HCV(+) liver transplant recipients. These findings may prove useful in prognostic applications for predicting early progression to fibrosis.

Systems virology identifies a mitochondrial fatty acid oxidation enzyme, dodecenoyl coenzyme A delta isomerase, required for hepatitis C virus replication and likely pathogenesis.

We previously employed systems biology approaches to identify the mitochondrial fatty acid oxidation enzyme dodecenoyl coenzyme A delta isomerase (DCI) as a bottleneck protein controlling host metabolic reprogramming during hepatitis C virus (HCV) infection. Here we present the results of studies confirming the importance of DCI to HCV pathogenesis. Computational models incorporating proteomic data from HCV patient liver biopsy specimens recapitulated our original predictions regarding DCI and link HCV-associated alterations in cellular metabolism and liver disease progression. HCV growth and RNA replication in hepatoma cell lines stably expressing DCI-targeting short hairpin RNA (shRNA) were abrogated, indicating that DCI is required for productive infection. Pharmacologic inhibition of fatty acid oxidation also blocked HCV replication. Production of infectious HCV was restored by overexpression of an shRNA-resistant DCI allele. These findings demonstrate the utility of systems biology approaches to gain novel insight into the biology of HCV infection and identify novel, translationally relevant therapeutic targets.

Temporal proteome and lipidome profiles reveal hepatitis C virus-associated reprogramming of hepatocellular metabolism and bioenergetics.

Proteomic and lipidomic profiling was performed over a time course of acute hepatitis C virus (HCV) infection in cultured Huh-7.5 cells to gain new insights into the intracellular processes influenced by this virus. Our proteomic data suggest that HCV induces early perturbations in glycolysis, the pentose phosphate pathway, and the citric acid cycle, which favor host biosynthetic activities supporting viral replication and propagation. This is followed by a compensatory shift in metabolism aimed at maintaining energy homeostasis and cell viability during elevated viral replication and increasing cellular stress. Complementary lipidomic analyses identified numerous temporal perturbations in select lipid species (e.g. phospholipids and sphingomyelins) predicted to play important roles in viral replication and downstream assembly and secretion events. The elevation of lipotoxic ceramide species suggests a potential link between HCV-associated biochemical alterations and the direct cytopathic effect observed in this in vitro system. Using innovative computational modeling approaches, we further identified mitochondrial fatty acid oxidation enzymes, which are comparably regulated during in vitro infection and in patients with histological evidence of fibrosis, as possible targets through which HCV regulates temporal alterations in cellular metabolic homeostasis.

Genomic analysis reveals a potential role for cell cycle perturbation in HCV-mediated apoptosis of cultured hepatocytes.

The mechanisms of liver injury associated with chronic HCV infection, as well as the individual roles of both viral and host factors, are not clearly defined. However, it is becoming increasingly clear that direct cytopathic effects, in addition to immune-mediated processes, play an important role in liver injury. Gene expression profiling during multiple time-points of acute HCV infection of cultured Huh-7.5 cells was performed to gain insight into the cellular mechanism of HCV-associated cytopathic effect. Maximal induction of cell-death-related genes and appearance of activated caspase-3 in HCV-infected cells coincided with peak viral replication, suggesting a link between viral load and apoptosis. Gene ontology analysis revealed that many of the cell-death genes function to induce apoptosis in response to cell cycle arrest. Labeling of dividing cells in culture followed by flow cytometry also demonstrated the presence of significantly fewer cells in S-phase in HCV-infected relative to mock cultures, suggesting HCV infection is associated with delayed cell cycle progression. Regulation of numerous genes involved in anti-oxidative stress response and TGF-beta1 signaling suggest these as possible causes of delayed cell cycle progression. Significantly, a subset of cell-death genes regulated during in vitro HCV infection was similarly regulated specifically in liver tissue from a cohort of HCV-infected liver transplant patients with rapidly progressive fibrosis. Collectively, these data suggest that HCV mediates direct cytopathic effects through deregulation of the cell cycle and that this process may contribute to liver disease progression. This in vitro system could be utilized to further define the cellular mechanism of this perturbation.

Use of ProteinChip array surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) to identify thymosin beta-4, a differentially secreted protein from lymphoblastoid cell lines.

The identification of proteins differentially expressed between cancer and normal cells is vital for the development of cancer diagnostics, therapeutics and vaccines. Using a ProteinChip Biomarker System (Ciphergen Biosystems, Fremont, CA) which combines ProteinChip technology with time-of-flight mass spectrometry, we have developed a simple method to screen and identify differentially secreted proteins from tumor cell lines. Mass spectra of the range of proteins secreted from normal B-cells were generated along with those secreted from Epstein-Barr virus transformed B-cells. A mass peak at m/z = 4972.1 that was highly over-represented in the transformed B-cell line was chosen for identification and purified by reversed phase chromatography with concomitant monitoring of fractions by SELDI-TOF MS. The resulting purified protein was digested with trypsin and the peptide masses derived from the SELDI-TOF spectrum were used to search the public databases for protein identification. Fragment matching of the resulting peptides identified the protein as thymosin beta-4. Using LC-electrospray ionization MS/MS, the identity of this protein was confirmed. Thymosin beta-4 is a known marker in LCLs establishing the utility of this method to discover and identify proteins differentially expressed between cancers and their matched normal counterparts.

Exogenous bone morphogenetic protein-7 fails to attenuate renal fibrosis in rats with overload proteinuria.

BACKGROUND: Bone morphogenetic protein-7 (BMP-7) plays a critical role in renal development, accelerates recovery from acute renal injury, and more recently it has been shown to delay progressive renal disease. The present study was designed to investigate the effect of BMP-7 on interstitial fibrosis in the rat protein-overloaded model. METHODS: Renal disease was induced in 26 rats by daily intraperitoneal injections of bovine serum albumin (BSA); controls (n = 28) were injected with saline. Half of the rats in each group were treated with human recombinant BMP-7 (300 microg/kg i.p. 3 times weekly) and half with placebo. Animals were killed after 3 or 6 weeks. RESULTS: Compared to the saline control groups, the BSA groups had evidence of chronic renal disease: significantly increased urinary protein excretion rates; total kidney collagen content, and increased fibronectin and collagen III interstitial areas. By 6 weeks the BSA + BMP-7 group compared to the BSA + placebo group had a nonsignificant decrease in blood urea nitrogen (40 +/- 13 vs. 46 +/- 11 mg/dl), total kidney collagen (10.8 +/- 2.1 vs. 12.2 +/- 3.5 microg/kidney), fibronectin interstitial area (23 +/- 4 vs. 25 +/- 8%) and collagen III interstitial area (22 +/- 6 vs. 28 +/- 7%). Despite these results, renal gene expression profiles actually predicted worse fibrosis in the BSA + BMP-7 group with significantly higher total kidney mRNA levels for alpha(1)(III) procollagen (2.8 +/- 0.5 vs. 1.6 +/- 0.6, p < 0.05) and fibronectin at 6 weeks (1.9 +/- 0.3 vs. 1.2 +/- 0.5, p < 0.05). Renal BMP-7 mRNA levels at 6 weeks were significantly increased in the BSA + placebo group compared to the saline + placebo group with no difference between the BSA + BMP-7 and the BSA + placebo groups. Both cortical and medullary tubules expressed BMP-7 protein but BMP-7 was only detected in the tubular lumina and urine of proteinuric animals. CONCLUSIONS: In rats with protein-overload proteinuria, renal tubules continue to express BMP-7 but some of the endogenous protein is secreted into the urinary space. Administration of exogenous recombinant BMP-7 had no effect on proteinuria but was associated with a nonsignificant trend towards less interstitial fibrosis at 6 weeks despite significantly higher kidney extracellular matrix gene mRNA levels. These findings suggest that BMP-7 treatment may have anti-fibrotic effects through enhancement of matrix turnover, although overall these effects are modest in proteinuric states in the absence of significant tubular epithelial cell apoptosis and epithelial-mesenchymal transition.

Molecular Signatures of Recurrent Hepatocellular Carcinoma Secondary to Hepatitis C Virus following Liver Transplantation.

Chronic hepatitis C virus (HCV) induced hepatocellular carcinoma (HCC) is a primary indication for liver transplantation (LT). In western countries, the estimated rate of HCC recurrence following LT is between 15% and 20% and is a major cause of mortality. Currently, there is no standard method to treat patients who are at high risk for HCC recurrence. The aim of this study was to investigate the molecular signatures underlying HCC recurrence that may lead to future studies on gene regulation contributing to new therapeutic options. Two groups of patients were selected, one including patients with HCV who developed HCC recurrence (HCC-R) ≤3 years from LT and the second group including patients with HCV who did not have recurrent HCC (HCC-NR). Microarray analysis containing more than 29,000 known genes was performed on formalin-fixed-paraffin-embedded (FFPE) liver tissue from explanted livers. Gene expression profiling revealed 194 differentially regulated genes between the two groups. These genes belonged to cellular networks including cell cycle G1/S checkpoint regulators, RAN signaling, chronic myeloid leukemia signaling, molecular mechanisms of cancer, FXR/RXR activation and hepatic cholestasis. A subset of molecular signatures associated with HCC recurrence was found. The expression levels of these genes were validated by quantitative PCR analysis.

Topological analysis of protein co-abundance networks identifies novel host targets important for HCV infection and pathogenesis.

BACKGROUND: High-throughput methods for obtaining global measurements of transcript and protein levels in biological samples has provided a large amount of data for identification of 'target' genes and proteins of interest. These targets may be mediators of functional processes involved in disease and therefore represent key points of control for viruses and bacterial pathogens. Genes and proteins that are the most highly differentially regulated are generally considered to be the most important. We present topological analysis of co-abundance networks as an alternative to differential regulation for confident identification of target proteins from two related global proteomics studies of hepatitis C virus (HCV) infection. RESULTS: We analyzed global proteomics data sets from a cell culture study of HCV infection and from a clinical study of liver biopsies from HCV-positive patients. Using lists of proteins known to be interaction partners with pathogen proteins we show that the most differentially regulated proteins in both data sets are indeed enriched in pathogen interactors. We then use these data sets to generate co-abundance networks that link proteins based on similar abundance patterns in time or across patients. Analysis of these co-abundance networks using a variety of network topology measures revealed that both degree and betweenness could be used to identify pathogen interactors with better accuracy than differential regulation alone, though betweenness provides the best discrimination. We found that though overall differential regulation was not correlated between the cell culture and liver biopsy data, network topology was conserved to an extent. Finally, we identified a set of proteins that has high betweenness topology in both networks including a protein that we have recently shown to be essential for HCV replication in cell culture. CONCLUSIONS: The results presented show that the network topology of protein co-abundance networks can be used to identify proteins important for viral replication. These proteins represent targets for further experimental investigation that will provide biological insight and potentially could be exploited for novel therapeutic approaches to combat HCV infection.

Chronic immune activation is a distinguishing feature of liver and PBMC gene signatures from HCV/HIV coinfected patients and may contribute to hepatic fibrogenesis.

Hepatitis C virus/human immunodeficiency virus (HCV/HIV) coinfected patients demonstrate accelerated progression to severe liver injury in comparison to HCV monoinfected patients, although the underlying mechanisms are unclear owing to infection of separate tissue compartments with two distinct viral pathogens. Microarray analysis of paired liver biopsy and peripheral blood mononuclear cell (PBMC) specimens from HCV/HIV coinfected and HCV monoinfected patients identified a gene expression signature associated with increased inflammation and immune activation that was present only in liver and PBMC samples from coinfected patients. We also identified in these samples liver- and PBMC-specific signatures enriched with fibrogenic/hepatic stellate activation and proinflammatory genes, respectively. Finally, Bayesian networks were constructed by assimilating these data with existing data from liver and PBMC samples from other cohorts, augmenting enrichment of biologically important pathways and further indicating that chronic immune activation in HCV/HIV coinfection may exacerbate liver disease progression in coinfected patients.

Virus-host interactions: from systems biology to translational research.

Research embracing systems biology approaches and careful analysis of the critical host response has greatly expanded our understanding of infectious diseases. First-generation studies based on genomics and proteomics have made significant progress in establishing the foundation for network-based investigations on virus-host interactions. More recently, data from complementary high-throughput technologies, such as siRNA and microRNA screens and next-generation sequencing, are augmenting systems level analyses and are providing a more detailed and insightful multidimensional view of virus-host networks. Together with advances in data integration, systems biology approaches now have the potential to provide profound impacts on translational research, leading to the more rapid development of new therapeutics and vaccines for infectious diseases. In this review, we highlight new high-throughput technologies, a new philosophy for studying virus-host interactions, and discuss the potential of systems biology to facilitate bench-to-bedside research and create novel strategies to combat disease. Can we save the world using these approaches? Read on.

Is systems biology the key to preventing the next pandemic?

Sporadic outbreaks of epizootics including SARS coronavirus and H5N1 avian influenza remind us of the potential for communicable diseases to quickly spread into worldwide epidemics. To confront emerging viral threats, nations have implemented strategies to prepare for pandemics and to control virus spread. Despite improved surveillance and quarantine measures, we find ourselves in the midst of a H1N1 influenza pandemic. Effective therapeutics and vaccines are essential to protect against current and future pandemics. The best route to effective therapeutics and vaccines is through a detailed and global view of virus-host interactions that can be achieved using a systems biology approach. Here, we provide our perspective on the role of systems biology in deepening our understanding of virus-host interactions and in improving drug and vaccine development. We offer examples from influenza virus research, as well as from research on other pandemics of our time - HIV/AIDS and HCV - to demonstrate that systems biology offers one possible key to stopping the cycle of viral pandemics.

Quantitative analysis of human immunodeficiency virus type 1-infected CD4+ cell proteome: dysregulated cell cycle progression and nuclear transport coincide with robust virus production.

Relatively little is known at the functional genomic level about the global host response to human immunodeficiency virus type 1 (HIV-1) infection. Microarray analyses by several laboratories, including our own, have revealed that HIV-1 infection causes significant changes in host mRNA abundance and regulation of several cellular biological pathways. However, it remains unclear what consequences these changes bring about at the protein level. Here we report the expression levels of approximately 3,200 proteins in the CD4(+) CEMx174 cell line after infection with the LAI strain of human immunodeficiency virus type 1 (HIV-1); the proteins were assessed using liquid chromatography-mass spectrometry coupled with stable isotope labeling and the accurate mass and time tag approach. Furthermore, we found that 687 (21%) proteins changed in abundance at the peak of virus production at 36 h postinfection. Pathway analysis revealed that the differential expression of proteins was concentrated in select biological pathways, exemplified by ubiquitin-conjugating enzymes in ubiquitination, carrier proteins in nucleocytoplasmic transport, cyclin-dependent kinase in cell cycle progression, and pyruvate dehydrogenase of the citrate cycle pathways. Moreover, we observed changes in the abundance of proteins with known interactions with HIV-1 viral proteins. Our proteomic analysis captured changes in the host protein milieu at the time of robust virus production, depicting changes in cellular processes that may contribute to virus replication. Continuing analyses are expected to focus on blocking virus replication by targeting these pathways and their effector proteins.

Proteomic profiling of human liver biopsies: hepatitis C virus-induced fibrosis and mitochondrial dysfunction.

UNLABELLED: Liver biopsies from hepatitis C virus (HCV)-infected patients offer the unique opportunity to study human liver biology and disease in vivo. However, the low protein yields associated with these small samples present a significant challenge for proteomic analysis. In this study we describe the application of an ultrasensitive proteomics platform for performing robust quantitative proteomic studies on microgram amounts of HCV-infected human liver tissue from 15 patients at different stages of fibrosis. A high-quality liver protein database containing 5,920 unique protein identifications supported high throughput quantitative studies using (16)O/(18)O stable isotope labeling in combination with the accurate mass and time (AMT) tag approach. A total of 1,641 liver biopsy proteins were quantified, and analysis of variance (ANOVA) identified 210 proteins exhibiting statistically significant differences associated with fibrosis stage. Hierarchical clustering showed that biopsies representative of later fibrosis stages (for example, Batts-Ludwig stages 3-4) exhibited a distinct protein expression profile, indicating an apparent down-regulation of many proteins when compared with samples from earlier fibrosis stages (for example, Batts-Ludwig stages 0-2). Functional analysis of these signature proteins suggests that impairment of key mitochondrial processes including fatty acid oxidation and oxidative phosphorylation, and response to oxidative stress and reactive oxygen species occurs during advanced stage 3 to 4 fibrosis. CONCLUSION: The results reported here represent a significant advancement in clinical proteomics providing to our knowledge, the first demonstration of global proteomic alterations accompanying liver disease progression in patients chronically infected with HCV. Our findings contribute to a generally emerging theme associating oxidative stress and hepatic mitochondrial dysfunction with HCV pathogenesis.

HepatoProteomics: applying proteomic technologies to the study of liver function and disease.

The wealth of human genome sequence information now available, coupled with technological advances in robotics, nanotechnology, mass spectrometry, and information systems, has given rise to a method of scientific inquiry known as functional genomics. By using these technologies to survey gene expression and protein production on a near global scale, the goal of functional genomics is to assign biological function to genes with currently unknown roles in physiology. This approach carries particular appeal in disease research, where it can uncover the function of previously unknown genes and molecular pathways that are directly involved in disease progression. With this knowledge may come improved diagnostic techniques, prognostic capabilities, and novel therapeutic approaches. In this regard, the continuing evolution of proteomic technologies has resulted in an increasingly greater impact of proteome studies in many areas of research and hepatology is no exception. Our laboratory has been extremely active in this area, applying both genomic and proteomic technologies to the analysis of virus-host interactions in several systems, including the study of hepatitis C virus (HCV) infection and HCV-associated liver disease. Since proteomic technologies are foreign to many hepatologists (and to almost everyone else), this article will provide an overview of proteomic methods and technologies and describe how they are being used to study liver function and disease.

Analysis of prostate cancer by proteomics using tissue specimens.

PURPOSE: Prostate cancer cells differ from their normal counterpart in gene expression. Therefore, cancer and normal tissue/cells would show differences in their proteomes. From a diagnostic standpoint differences in the composition of secreted protein species are especially relevant. We used ProteinChip Array (Ciphergen Biosystems, Fremont, California) surface enhanced laser desorption/ionization (SELDI) time of flight mass spectrometry to profile prostate tissue samples to generate phenomic fingerprints. We used quantitative proteomics based on glycopeptide capture followed by tandem mass spectrometry to identify differentially expressed proteins. MATERIALS AND METHODS: Patient matched cancer and noncancer specimens were digested by collagenase to single cells. After digestion the cells were pelleted and the cell-free supernatant was used for analysis. A reversed phase hydrophobic ProteinChip Array was used to generate SELDI patterns from 43 primary prostate tumors, including 26 with matched noncancer specimens. Quantitative proteomics was applied to 1 specimen and the expression pattern was verified by Western blotting and immunohistochemistry. RESULTS: SELDI profiles showed that cancers of similar TNM stages were more likely to have similar profiles. On quantitative proteomics tissue metalloproteinase inhibitor-1 was identified to be down-regulated in cancer. Tissue metalloproteinase inhibitor-1 expression was localized to secretory cells. CONCLUSIONS: Protein profiling by SELDI is relatively easy to perform and it has great potential in prostate cancer diagnosis through pattern recognition. Quantitative proteomics can potentially determine the identity of many biomarkers specific for prostate cancer.

Identification of serum amyloid A as a biomarker to distinguish prostate cancer patients with bone lesions.

BACKGROUND: Prostate cancer has a propensity to metastasize to the bone. Currently, there are no curative treatments for this stage of the disease. Sensitive biomarkers that can be monitored in the blood to indicate the presence or development of bone metastases and/or response to therapies are lacking. Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) is an affinity-based approach that allows sensitive and high-throughput protein profiling and screening of biological samples. METHODS: We used SELDI-TOF MS for protein profiling of sera from prostate cancer patients (n = 38) with and without bone metastases in our effort to identify individual or multiple serum markers that may be of added benefit to those in current use. Serum was applied to ProteinChip surfaces (H4 and IMAC) to quickly screen samples and detect peaks predominating in the samples obtained from patients with bone metastases. Unique proteins in the bone metastasis cohort observed by SELDI-TOF MS were identified by two-dimensional gel electrophoresis, in-gel trypsin digestion, and tandem MS. The identities of the proteins were confirmed by ELISA and immunodepletion assays. RESULTS: The cluster of unique proteins in the sera of patients with bone metastases was identified as isoforms of serum amyloid A. Machine-learning algorithms were also used to identify patients with bone metastases with a sensitivity and specificity of 89.5%. CONCLUSIONS: SELDI-TOF MS protein profiling in combination with other proteomic approaches may provide diagnostic tools with potential clinical applications and serve as tools to aid in the discovery of biomarkers associated with various diseases.

Proteome analysis of liver cells expressing a full-length hepatitis C virus (HCV) replicon and biopsy specimens of posttransplantation liver from HCV-infected patients.

The development of a reproducible model system for the study of hepatitis C virus (HCV) infection has the potential to significantly enhance the study of virus-host interactions and provide future direction for modeling the pathogenesis of HCV. While there are studies describing global gene expression changes associated with HCV infection, changes in the proteome have not been characterized. We report the first large-scale proteome analysis of the highly permissive Huh-7.5 cell line containing a full-length HCV replicon. We detected >4,200 proteins in this cell line, including HCV replicon proteins, using multidimensional liquid chromatographic (LC) separations coupled to mass spectrometry. Consistent with the literature, a comparison of HCV replicon-positive and -negative Huh-7.5 cells identified expression changes of proteins involved in lipid metabolism. We extended these analyses to liver biopsy material from HCV-infected patients where a total of >1,500 proteins were detected from only 2 mug of liver biopsy protein digest using the Huh-7.5 protein database and the accurate mass and time tag strategy. These findings demonstrate the utility of multidimensional proteome analysis of the HCV replicon model system for assisting in the determination of proteins/pathways affected by HCV infection. Our ability to extend these analyses to the highly complex proteome of small liver biopsies with limiting protein yields offers the unique opportunity to begin evaluating the clinical significance of protein expression changes associated with HCV infection.

High-throughput screening of the yeast kinome: identification of human serine/threonine protein kinases that phosphorylate the hepatitis C virus NS5A protein.

The hepatitis C virus NS5A protein plays a critical role in virus replication, conferring interferon resistance to the virus through perturbation of multiple intracellular signaling pathways. Since NS5A is a phosphoprotein, it is of considerable interest to understand the role of phosphorylation in NS5A function. In this report, we investigated the phosphorylation of NS5A by taking advantage of 119 glutathione S-transferase-tagged protein kinases purified from Saccharomyces cerevisiae to perform a global screening of yeast kinases capable of phosphorylating NS5A in vitro. A database BLAST search was subsequently performed by using the sequences of the yeast kinases that phosphorylated NS5A in order to identify human kinases with the highest sequence homologies. Subsequent in vitro kinase assays and phosphopeptide mapping studies confirmed that several of the homologous human protein kinases were capable of phosphorylating NS5A. In vivo phosphopeptide mapping revealed phosphopeptides common to those generated in vitro by AKT, p70S6K, MEK1, and MKK6, suggesting that these kinases may phosphorylate NS5A in mammalian cells. Significantly, rapamycin, an inhibitor commonly used to investigate the mTOR/p70S6K pathway, reduced the in vivo phosphorylation of specific NS5A phosphopeptides, strongly suggesting that p70S6 kinase and potentially related members of this group phosphorylate NS5A inside the cell. Curiously, certain of these kinases also play a major role in mRNA translation and antiapoptotic pathways, some of which are already known to be regulated by NS5A. The findings presented here demonstrate the use of high-throughput screening of the yeast kinome to facilitate the major task of identifying human NS5A protein kinases for further characterization of phosphorylation events in vivo. Our results suggest that this novel approach may be generally applicable to the screening of other protein biochemical activities by mechanistic class.