# Proteomic Analysis of Serum and Urine of HIV-Monoinfected and HIV/HCV-Coinfected Patients Undergoing Long Term Treatment with Nevirapine

Authors: Jeerang Wongtrakul; Thananya Thongtan; Sittiruk Roytrakul; Benjawan Kumrapich; Kanokwan Janphen; Jutarat Praparattanapan; Khuanchai Supparatpinyo; Duncan R. Smith
Journal: Disease Markers
 (2014)

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## Abstract

Nevirapine (NVP) is an effective nonnucleoside reverse transcriptase inhibitor (NNRTI) of particular interest as it is often used in resource limited countries.  However, one of the main concerns with the use of NVP is hepatotoxicity and elevation of liver enzymes as a consequence of highly active antiretroviral therapy (HAART) containing NVP is more often reported in HIV patients coinfected with hepatitis C virus than in HIV-monoinfected patients.  To discover possible markers of NVP induced hepatotoxicity, serum and urine samples from twenty-five HIV or HIV/HCV patients, all of whom had received NVP continuously for at least four months, and healthy controls were subjected to in-solution or in-gel proteomic analysis. A total of 83 differentially regulated proteins consisted of 34 proteins identified in serum by in-solution analysis, 2 proteins identified from serum in a 2D gel electrophoresis analysis, and 47 proteins identified in urine in an in-solution analysis.  Three proteins, namely, haptoglobin, Rho-related BTB domain containing protein 3, and death-associated protein kinase 3, were selected for further validation by Western blot analysis and results showed that haptoglobin has potential for further development as an additional marker of NVP induced hepatotoxicity.


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## Body

## 1. Introduction

HIV infection in the absence of therapy is almost invariably fatal with few exceptions, but the introduction of combination antiretroviral therapy (ART) or highly active antiretroviral therapy (HAART) resulted in a dramatic decline in morbidity and mortality that has significantly changed the course of acquired HIV infection [1]. Approximately 35 million people worldwide are living with HIV and an estimated 15% to 30% are coinfected with hepatitis C virus (HCV) [2, 3], although in Thailand the prevalence of HCV coinfection with HIV has been reported as 7.8% [4]. Several studies have reported that HIV induced immunosuppression accelerates the natural history of HCV-related liver disease, and coinfected patients are 3- to 5-fold more likely to develop cirrhosis [5, 6]. Nevirapine (NVP), a nonnucleoside reverse transcriptase inhibitor (NNRTI), is frequently used in the HAART regimen for HIV-infected patients in resource limited settings despite the high risk of hepatotoxicity which occurs in approximately 12–15% of HIV- and HIV/HCV-coinfected patients [7–9].HIV/HCV-coinfected patients are more likely to develop hepatotoxicity caused by HAART than HIV-monoinfected individuals, and HIV/HCV is associated with a 2–10-fold change of aspartate aminotransferase (AST) and/or alanine aminotransferase (ALT) values above the upper level of normality after starting HAART, compared with HIV infection alone [10]. This is supported by studies that show that HCV is associated with a 2.46 increased relative risk (RR) for liver enzyme elevation (5 × upper limit of normal) [11] and that grades 3-4 liver enzyme elevation was detected in 20.8% of HIV/HCV-coinfected patients who continuously use NVP [12].NVP hepatotoxicity is believed to be caused by two mechanisms: an early onset reaction characterized by skin reactions and elevated ALT/AST that occurs within the first two to three weeks of treatment [13–15] and a delayed onset that normally starts some 4-5 months after commencement of treatment [15, 16]. While the mechanism of hepatotoxicity remains poorly characterized, NVP metabolites such as 12-OH-NVP and quinone methide have been strongly implicated in the process [17, 18] and evidence for the metabolic activation of NVP through the detection of mercapturates in urine has been previously presented [19].The early diagnosis of liver toxicity in patients undergoing long term treatment with NVP is critical as the condition can be lethal, and currently this is generally assessed by evaluation of alanine transaminase (ALT) levels [20]. This study aimed to use a proteomic analysis to identify possible biomarkers that are more specific to NVP toxicity than ALT in serum and urine fraction of patients with liver toxicity due to long term nevirapine use.


## 2. Methods

### 2.1. Patients and Samples

#### 2.1.1. Study Design and Participants

The patients included in the study consisted of 18 patients monoinfected with HIV and 13 patients coinfected with HIV and HCV who were being followed up at Maharaj Nakorn Chiang Mai Hospital. Additional five healthy individuals were recruited as controls. All patients received a HAART regimen containing NVP for at least four months. The study was approved by the Research Ethics Committee 2, Faculty of Medicine, Chiang Mai University (RIH-12-985-FB), and the Human Experimentation Committee, Research Institute for Health Sciences (RIHES), Chiang Mai University (4/55). Written informed consent was obtained from all participants.Blood (10 mL each) and urine samples were obtained on the day of scheduled patient follow-up. Serum samples were prepared and screened for aspartate aminotransferase (AST), alanine transaminase (ALT), hepatitis B surface antigen (HBS-Ag), hepatitis B surface antibody (anti-HBS), anti-hepatitis-C antibodies (anti-HCV), and HIV virus antibody (anti-HIV). All serum and urine samples were stored at −80°C until analysis.Participants with HIV/HCV with high ALT were eligible to participate if they had received a HAART regimen containing NVP for at least four months, have positive anti-HCV, have positive anti-HIV, were negative for HBS-Ag, were positive/negative for anti-HBS, and have serum ALT more than 1.25-fold of upper normal limit.Participants with HIV/HCV with normal ALT were eligible to participate if they received a HAART regimen containing NVP for at least four months, have positive anti-HCV, have positive anti-HIV, were negative for HBS-Ag, were positive/negative for anti-HBS, and have serum ALT 0–40 U/L.Participants with HIV monoinfection with high ALT were eligible to participate if they received a HAART regimen containing NVP for at least four months, have positive anti-HIV, have negative anti-HCV, were negative for HBS-Ag, were positive/negative for anti-HBS, and have serum ALT more than 1.25-fold of upper normal limit.Participants with HIV monoinfection with normal ALT were eligible to participate if they received a HAART regimen containing NVP for at least four months, have positive anti-HIV, have negative anti-HCV, were negative for HBS-Ag, were positive/negative for anti-HBS, and have serum ALT 0–40 U/L.The exclusion criteria included positive anti-HBsAg, negative anti-HCV in HIV/HCV coinfection, and less than four months of NVP treatment.


#### 2.1.2. Sample Preparation

Urine samples were thawed at room temperature and centrifuged at 17,000 ×g for 15 min at room temperature to sediment cellular fragments. The supernatants were transferred into new 50 mL tubes and 2 volumes of cold acetone (−20°C) were added to each tube. The tubes were vortexed and subsequently incubated at −20°C overnight after which the samples were centrifuged for 15 minutes at 13,000 ×g. The protein pellets were dissolved in 0.5% SDS and the protein concentration was determined by the Lowry method [21]. Serum samples were diluted 1 : 10 with sterile nano water prior to protein measurement.Protein samples from serum and urine were subsequently pooled into five groups, namely, HIV/HCV-coinfected patients with high ALT (group 1), HIV/HCV-coinfected patients with normal ALT (group 2), HIV-monoinfected patients with high ALT (group 3), HIV-monoinfected patients with normal ALT (group 4), and healthy individuals (Ctl).


#### 2.1.3. Sub-10 kDa Peptidome Analysis

Approximately 240μg of pooled proteins from serum or urine was applied directly onto a Nanosep 10 K ultrafiltration device (Pall Life Sciences, Pall Corporation, Port Washington, NY) and the sub-10 kDa fraction removed for protein quantitation by the Lowry assay [21]. A total of 10 μg of protein was transferred into a new tube and the volume adjusted to 10 μL with 10 mM ammonium bicarbonate. The proteins were reduced with 10 mM dithiothreitol in 10 mM ammonium bicarbonate at room temperature for 1 hr and subsequently alkylated with 100 mM iodoacetamide in 10 mM ammonium bicarbonate at room temperature in the dark for 1 hr. Samples were digested with 10 ng trypsin in 50% acetonitrile/10 mM ammonium bicarbonate at room temperature for 3 hrs. The samples were dried under vacuum until the final volume was 10 μL. Then the sample volume was adjusted to 15 μL with 0.1% FA and subjected to LC-MS/MS exactly as described previously [22].


#### 2.1.4. Two-Dimensional Gel Electrophoresis (2-DE) and Liquid Chromatography-Mass Spectrometry Analysis

Approximately 60μL of each of the pooled serum samples was taken and processed to deplete albumin using the ProteoExtract Albumin removal kit (Merck KGaA, Darmstadt, Germany) according to the manufacturers’ recommendations and protein concentrations were determined as described previously. Two-dimensional gel electrophoresis and LC-MS/MS analysis were performed essentially as described previously [23]. Briefly, 200 μg of each pooled albumin depleted fraction was mixed with 340 μL of rehydration buffer (8 M urea, 4% CHAPS, 0.001% bromophenol blue, and 3 mM dithiothreitol) containing 1% 3–10 NL IPG buffer. Samples were loaded onto 18 cm IPG strips with pH range of 3–10 NL of an isoelectric focusing system (EttanIPGphoreIII). Strips were rehydrated (20°C, 16 h) and isoelectric focused (500 volts for 500 volt-h, 1,000 volts for 800 volt-h, and 10,000 volts to reach 36,000 volt-h). The maximum current was maintained at 75 μA per strip. Strips were subsequently equilibrated twice (15 min each) in equilibration buffer (50 mM Tris pH 8.8, 6 M urea, 30% glycerol, 2% SDS, and 0.03% bromophenol blue) supplemented with 65 mM DTT and 135 mM iodoacetamide. The strips were subjected to the second dimensional separation (Ettan DALTsix) using a SDS-polyacrylamide gel (12.5%) run under an applied voltage of 10 watts per gel at 20°C until the bromophenol blue dye front reached 0.5 cm from the bottom of the gel. The gels were subsequently stained with colloidal Coomassie blue. Spots identified as differentially regulated were removed from the blot and subjected to “in-gel” tryptic digestion as described previously [23]. Peptides were identified by LC-MS/MS using a Dionex Ultimate 3000 (Thermo Scientific) in combination with an electrospray ionization (ESI)/quadrupole ion trap mass spectrometer (amaZon SL, Bruker Daltonik, Germany). The LC separation was performed on a reverse phase column (Hypersil GoLD 50 × 0.5 mm, 5 μm C18), protected by a guard column, and eluted at a flow rate of 100 μL/min under gradient conditions of 5–80%B over 50 min. The mobile phase A consisted of water/formic acid (99.9 : 0.1, v/v), and mobile phase B consists of acetonitrile (100, v). Mass spectral data from 150 to 1500m/z was collected in the positive ionization mode. The MS/MS spectrometry data were searched against the NCBInr database using the MASCOT search engine as described previously [22, 23].


#### 2.1.5. Protein Preparation and Western Blotting

Sera were individually albumin depleted as above and concentrated using a Vivaspin 2 ultrafiltration column with a 10 kDa molecular weight cut-off (GE Healthcare, Buckinghamshire, UK) according to the manufacturer’s recommendations. Protein concentration then was determined as before and samples were stored at −80°C until required. SDS-PAGE and Western blot analysis were employed to determine the enrichment of serum. Approximately 50μg proteins were subjected to electrophoresis on 10% SDS-polyacrylamide gels and transferred to PVDF solid matrix support (Amersham Hybond-P, GE Healthcare) and analyzed as previously described [23]. The primary antibodies used were anti-RhoBTB3 (Sigma-Aldrich Co., St. Louis, MO), anti-ZIP kinase antibody (DAPK3) (Abcam plc, Cambridge, UK), anti-haptoglobin (Abcam plc), anti-transferrin (Abcam plc), and anti-GAPDH (Cell Signaling Technology, Inc., Danvers, MA). The secondary antibodies used were a goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology Inc., Santa Cruz, CA) and a goat anti-mouse IgG-peroxidase (Sigma-Aldrich, Milwaukee, WI). Blots were visualized using Luminata Forte Western HRP Substrate (Merck KGaA, Darmstadt, Germany) detection system, according to the manufacturer’s instructions. Antibodies against transferrin and/or GAPDH were employed as internal controls. Image J software was employed to determine optical density values of bands for relative comparisons. The experiment was performed in triplicate.


### 2.2. Statistical Analysis

The data were expressed as mean ± SD. To compare clinical parameters and the expression level of proteins among patients and control, statistical significance of bands intensity was determined using one-way ANOVA with Bonferroni’s multiple comparison test using Prism 5.P values less than 0.05 were considered statistically significant.


## 2.1. Patients and Samples

### 2.1.1. Study Design and Participants

The patients included in the study consisted of 18 patients monoinfected with HIV and 13 patients coinfected with HIV and HCV who were being followed up at Maharaj Nakorn Chiang Mai Hospital. Additional five healthy individuals were recruited as controls. All patients received a HAART regimen containing NVP for at least four months. The study was approved by the Research Ethics Committee 2, Faculty of Medicine, Chiang Mai University (RIH-12-985-FB), and the Human Experimentation Committee, Research Institute for Health Sciences (RIHES), Chiang Mai University (4/55). Written informed consent was obtained from all participants.Blood (10 mL each) and urine samples were obtained on the day of scheduled patient follow-up. Serum samples were prepared and screened for aspartate aminotransferase (AST), alanine transaminase (ALT), hepatitis B surface antigen (HBS-Ag), hepatitis B surface antibody (anti-HBS), anti-hepatitis-C antibodies (anti-HCV), and HIV virus antibody (anti-HIV). All serum and urine samples were stored at −80°C until analysis.Participants with HIV/HCV with high ALT were eligible to participate if they had received a HAART regimen containing NVP for at least four months, have positive anti-HCV, have positive anti-HIV, were negative for HBS-Ag, were positive/negative for anti-HBS, and have serum ALT more than 1.25-fold of upper normal limit.Participants with HIV/HCV with normal ALT were eligible to participate if they received a HAART regimen containing NVP for at least four months, have positive anti-HCV, have positive anti-HIV, were negative for HBS-Ag, were positive/negative for anti-HBS, and have serum ALT 0–40 U/L.Participants with HIV monoinfection with high ALT were eligible to participate if they received a HAART regimen containing NVP for at least four months, have positive anti-HIV, have negative anti-HCV, were negative for HBS-Ag, were positive/negative for anti-HBS, and have serum ALT more than 1.25-fold of upper normal limit.Participants with HIV monoinfection with normal ALT were eligible to participate if they received a HAART regimen containing NVP for at least four months, have positive anti-HIV, have negative anti-HCV, were negative for HBS-Ag, were positive/negative for anti-HBS, and have serum ALT 0–40 U/L.The exclusion criteria included positive anti-HBsAg, negative anti-HCV in HIV/HCV coinfection, and less than four months of NVP treatment.


### 2.1.2. Sample Preparation

Urine samples were thawed at room temperature and centrifuged at 17,000 ×g for 15 min at room temperature to sediment cellular fragments. The supernatants were transferred into new 50 mL tubes and 2 volumes of cold acetone (−20°C) were added to each tube. The tubes were vortexed and subsequently incubated at −20°C overnight after which the samples were centrifuged for 15 minutes at 13,000 ×g. The protein pellets were dissolved in 0.5% SDS and the protein concentration was determined by the Lowry method [21]. Serum samples were diluted 1 : 10 with sterile nano water prior to protein measurement.Protein samples from serum and urine were subsequently pooled into five groups, namely, HIV/HCV-coinfected patients with high ALT (group 1), HIV/HCV-coinfected patients with normal ALT (group 2), HIV-monoinfected patients with high ALT (group 3), HIV-monoinfected patients with normal ALT (group 4), and healthy individuals (Ctl).


### 2.1.3. Sub-10 kDa Peptidome Analysis

Approximately 240μg of pooled proteins from serum or urine was applied directly onto a Nanosep 10 K ultrafiltration device (Pall Life Sciences, Pall Corporation, Port Washington, NY) and the sub-10 kDa fraction removed for protein quantitation by the Lowry assay [21]. A total of 10 μg of protein was transferred into a new tube and the volume adjusted to 10 μL with 10 mM ammonium bicarbonate. The proteins were reduced with 10 mM dithiothreitol in 10 mM ammonium bicarbonate at room temperature for 1 hr and subsequently alkylated with 100 mM iodoacetamide in 10 mM ammonium bicarbonate at room temperature in the dark for 1 hr. Samples were digested with 10 ng trypsin in 50% acetonitrile/10 mM ammonium bicarbonate at room temperature for 3 hrs. The samples were dried under vacuum until the final volume was 10 μL. Then the sample volume was adjusted to 15 μL with 0.1% FA and subjected to LC-MS/MS exactly as described previously [22].


### 2.1.4. Two-Dimensional Gel Electrophoresis (2-DE) and Liquid Chromatography-Mass Spectrometry Analysis

Approximately 60μL of each of the pooled serum samples was taken and processed to deplete albumin using the ProteoExtract Albumin removal kit (Merck KGaA, Darmstadt, Germany) according to the manufacturers’ recommendations and protein concentrations were determined as described previously. Two-dimensional gel electrophoresis and LC-MS/MS analysis were performed essentially as described previously [23]. Briefly, 200 μg of each pooled albumin depleted fraction was mixed with 340 μL of rehydration buffer (8 M urea, 4% CHAPS, 0.001% bromophenol blue, and 3 mM dithiothreitol) containing 1% 3–10 NL IPG buffer. Samples were loaded onto 18 cm IPG strips with pH range of 3–10 NL of an isoelectric focusing system (EttanIPGphoreIII). Strips were rehydrated (20°C, 16 h) and isoelectric focused (500 volts for 500 volt-h, 1,000 volts for 800 volt-h, and 10,000 volts to reach 36,000 volt-h). The maximum current was maintained at 75 μA per strip. Strips were subsequently equilibrated twice (15 min each) in equilibration buffer (50 mM Tris pH 8.8, 6 M urea, 30% glycerol, 2% SDS, and 0.03% bromophenol blue) supplemented with 65 mM DTT and 135 mM iodoacetamide. The strips were subjected to the second dimensional separation (Ettan DALTsix) using a SDS-polyacrylamide gel (12.5%) run under an applied voltage of 10 watts per gel at 20°C until the bromophenol blue dye front reached 0.5 cm from the bottom of the gel. The gels were subsequently stained with colloidal Coomassie blue. Spots identified as differentially regulated were removed from the blot and subjected to “in-gel” tryptic digestion as described previously [23]. Peptides were identified by LC-MS/MS using a Dionex Ultimate 3000 (Thermo Scientific) in combination with an electrospray ionization (ESI)/quadrupole ion trap mass spectrometer (amaZon SL, Bruker Daltonik, Germany). The LC separation was performed on a reverse phase column (Hypersil GoLD 50 × 0.5 mm, 5 μm C18), protected by a guard column, and eluted at a flow rate of 100 μL/min under gradient conditions of 5–80%B over 50 min. The mobile phase A consisted of water/formic acid (99.9 : 0.1, v/v), and mobile phase B consists of acetonitrile (100, v). Mass spectral data from 150 to 1500m/z was collected in the positive ionization mode. The MS/MS spectrometry data were searched against the NCBInr database using the MASCOT search engine as described previously [22, 23].


### 2.1.5. Protein Preparation and Western Blotting

Sera were individually albumin depleted as above and concentrated using a Vivaspin 2 ultrafiltration column with a 10 kDa molecular weight cut-off (GE Healthcare, Buckinghamshire, UK) according to the manufacturer’s recommendations. Protein concentration then was determined as before and samples were stored at −80°C until required. SDS-PAGE and Western blot analysis were employed to determine the enrichment of serum. Approximately 50μg proteins were subjected to electrophoresis on 10% SDS-polyacrylamide gels and transferred to PVDF solid matrix support (Amersham Hybond-P, GE Healthcare) and analyzed as previously described [23]. The primary antibodies used were anti-RhoBTB3 (Sigma-Aldrich Co., St. Louis, MO), anti-ZIP kinase antibody (DAPK3) (Abcam plc, Cambridge, UK), anti-haptoglobin (Abcam plc), anti-transferrin (Abcam plc), and anti-GAPDH (Cell Signaling Technology, Inc., Danvers, MA). The secondary antibodies used were a goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology Inc., Santa Cruz, CA) and a goat anti-mouse IgG-peroxidase (Sigma-Aldrich, Milwaukee, WI). Blots were visualized using Luminata Forte Western HRP Substrate (Merck KGaA, Darmstadt, Germany) detection system, according to the manufacturer’s instructions. Antibodies against transferrin and/or GAPDH were employed as internal controls. Image J software was employed to determine optical density values of bands for relative comparisons. The experiment was performed in triplicate.


## 2.1.1. Study Design and Participants

The patients included in the study consisted of 18 patients monoinfected with HIV and 13 patients coinfected with HIV and HCV who were being followed up at Maharaj Nakorn Chiang Mai Hospital. Additional five healthy individuals were recruited as controls. All patients received a HAART regimen containing NVP for at least four months. The study was approved by the Research Ethics Committee 2, Faculty of Medicine, Chiang Mai University (RIH-12-985-FB), and the Human Experimentation Committee, Research Institute for Health Sciences (RIHES), Chiang Mai University (4/55). Written informed consent was obtained from all participants.Blood (10 mL each) and urine samples were obtained on the day of scheduled patient follow-up. Serum samples were prepared and screened for aspartate aminotransferase (AST), alanine transaminase (ALT), hepatitis B surface antigen (HBS-Ag), hepatitis B surface antibody (anti-HBS), anti-hepatitis-C antibodies (anti-HCV), and HIV virus antibody (anti-HIV). All serum and urine samples were stored at −80°C until analysis.Participants with HIV/HCV with high ALT were eligible to participate if they had received a HAART regimen containing NVP for at least four months, have positive anti-HCV, have positive anti-HIV, were negative for HBS-Ag, were positive/negative for anti-HBS, and have serum ALT more than 1.25-fold of upper normal limit.Participants with HIV/HCV with normal ALT were eligible to participate if they received a HAART regimen containing NVP for at least four months, have positive anti-HCV, have positive anti-HIV, were negative for HBS-Ag, were positive/negative for anti-HBS, and have serum ALT 0–40 U/L.Participants with HIV monoinfection with high ALT were eligible to participate if they received a HAART regimen containing NVP for at least four months, have positive anti-HIV, have negative anti-HCV, were negative for HBS-Ag, were positive/negative for anti-HBS, and have serum ALT more than 1.25-fold of upper normal limit.Participants with HIV monoinfection with normal ALT were eligible to participate if they received a HAART regimen containing NVP for at least four months, have positive anti-HIV, have negative anti-HCV, were negative for HBS-Ag, were positive/negative for anti-HBS, and have serum ALT 0–40 U/L.The exclusion criteria included positive anti-HBsAg, negative anti-HCV in HIV/HCV coinfection, and less than four months of NVP treatment.


## 2.1.2. Sample Preparation

Urine samples were thawed at room temperature and centrifuged at 17,000 ×g for 15 min at room temperature to sediment cellular fragments. The supernatants were transferred into new 50 mL tubes and 2 volumes of cold acetone (−20°C) were added to each tube. The tubes were vortexed and subsequently incubated at −20°C overnight after which the samples were centrifuged for 15 minutes at 13,000 ×g. The protein pellets were dissolved in 0.5% SDS and the protein concentration was determined by the Lowry method [21]. Serum samples were diluted 1 : 10 with sterile nano water prior to protein measurement.Protein samples from serum and urine were subsequently pooled into five groups, namely, HIV/HCV-coinfected patients with high ALT (group 1), HIV/HCV-coinfected patients with normal ALT (group 2), HIV-monoinfected patients with high ALT (group 3), HIV-monoinfected patients with normal ALT (group 4), and healthy individuals (Ctl).


## 2.1.3. Sub-10 kDa Peptidome Analysis

Approximately 240μg of pooled proteins from serum or urine was applied directly onto a Nanosep 10 K ultrafiltration device (Pall Life Sciences, Pall Corporation, Port Washington, NY) and the sub-10 kDa fraction removed for protein quantitation by the Lowry assay [21]. A total of 10 μg of protein was transferred into a new tube and the volume adjusted to 10 μL with 10 mM ammonium bicarbonate. The proteins were reduced with 10 mM dithiothreitol in 10 mM ammonium bicarbonate at room temperature for 1 hr and subsequently alkylated with 100 mM iodoacetamide in 10 mM ammonium bicarbonate at room temperature in the dark for 1 hr. Samples were digested with 10 ng trypsin in 50% acetonitrile/10 mM ammonium bicarbonate at room temperature for 3 hrs. The samples were dried under vacuum until the final volume was 10 μL. Then the sample volume was adjusted to 15 μL with 0.1% FA and subjected to LC-MS/MS exactly as described previously [22].


## 2.1.4. Two-Dimensional Gel Electrophoresis (2-DE) and Liquid Chromatography-Mass Spectrometry Analysis

Approximately 60μL of each of the pooled serum samples was taken and processed to deplete albumin using the ProteoExtract Albumin removal kit (Merck KGaA, Darmstadt, Germany) according to the manufacturers’ recommendations and protein concentrations were determined as described previously. Two-dimensional gel electrophoresis and LC-MS/MS analysis were performed essentially as described previously [23]. Briefly, 200 μg of each pooled albumin depleted fraction was mixed with 340 μL of rehydration buffer (8 M urea, 4% CHAPS, 0.001% bromophenol blue, and 3 mM dithiothreitol) containing 1% 3–10 NL IPG buffer. Samples were loaded onto 18 cm IPG strips with pH range of 3–10 NL of an isoelectric focusing system (EttanIPGphoreIII). Strips were rehydrated (20°C, 16 h) and isoelectric focused (500 volts for 500 volt-h, 1,000 volts for 800 volt-h, and 10,000 volts to reach 36,000 volt-h). The maximum current was maintained at 75 μA per strip. Strips were subsequently equilibrated twice (15 min each) in equilibration buffer (50 mM Tris pH 8.8, 6 M urea, 30% glycerol, 2% SDS, and 0.03% bromophenol blue) supplemented with 65 mM DTT and 135 mM iodoacetamide. The strips were subjected to the second dimensional separation (Ettan DALTsix) using a SDS-polyacrylamide gel (12.5%) run under an applied voltage of 10 watts per gel at 20°C until the bromophenol blue dye front reached 0.5 cm from the bottom of the gel. The gels were subsequently stained with colloidal Coomassie blue. Spots identified as differentially regulated were removed from the blot and subjected to “in-gel” tryptic digestion as described previously [23]. Peptides were identified by LC-MS/MS using a Dionex Ultimate 3000 (Thermo Scientific) in combination with an electrospray ionization (ESI)/quadrupole ion trap mass spectrometer (amaZon SL, Bruker Daltonik, Germany). The LC separation was performed on a reverse phase column (Hypersil GoLD 50 × 0.5 mm, 5 μm C18), protected by a guard column, and eluted at a flow rate of 100 μL/min under gradient conditions of 5–80%B over 50 min. The mobile phase A consisted of water/formic acid (99.9 : 0.1, v/v), and mobile phase B consists of acetonitrile (100, v). Mass spectral data from 150 to 1500m/z was collected in the positive ionization mode. The MS/MS spectrometry data were searched against the NCBInr database using the MASCOT search engine as described previously [22, 23].


## 2.1.5. Protein Preparation and Western Blotting

Sera were individually albumin depleted as above and concentrated using a Vivaspin 2 ultrafiltration column with a 10 kDa molecular weight cut-off (GE Healthcare, Buckinghamshire, UK) according to the manufacturer’s recommendations. Protein concentration then was determined as before and samples were stored at −80°C until required. SDS-PAGE and Western blot analysis were employed to determine the enrichment of serum. Approximately 50μg proteins were subjected to electrophoresis on 10% SDS-polyacrylamide gels and transferred to PVDF solid matrix support (Amersham Hybond-P, GE Healthcare) and analyzed as previously described [23]. The primary antibodies used were anti-RhoBTB3 (Sigma-Aldrich Co., St. Louis, MO), anti-ZIP kinase antibody (DAPK3) (Abcam plc, Cambridge, UK), anti-haptoglobin (Abcam plc), anti-transferrin (Abcam plc), and anti-GAPDH (Cell Signaling Technology, Inc., Danvers, MA). The secondary antibodies used were a goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology Inc., Santa Cruz, CA) and a goat anti-mouse IgG-peroxidase (Sigma-Aldrich, Milwaukee, WI). Blots were visualized using Luminata Forte Western HRP Substrate (Merck KGaA, Darmstadt, Germany) detection system, according to the manufacturer’s instructions. Antibodies against transferrin and/or GAPDH were employed as internal controls. Image J software was employed to determine optical density values of bands for relative comparisons. The experiment was performed in triplicate.


## 2.2. Statistical Analysis

The data were expressed as mean ± SD. To compare clinical parameters and the expression level of proteins among patients and control, statistical significance of bands intensity was determined using one-way ANOVA with Bonferroni’s multiple comparison test using Prism 5.P values less than 0.05 were considered statistically significant.


## 3. Results

### 3.1. Study Participants and Clinical Parameters of Participants

Between June 22, 2012, and August 1, 2012, a total of 31 HIV/HCV and HIV adults were screened for enrollment in the study (Figure1). A total of 11 patients (35.5%) were excluded due to the presence of normal ALT (1), negative anti-HCV (2), borderline ALT level (4), and a further 4 were not included in the analysis to give equal N values in the four patient groups. In addition, five healthy controls were recruited. All patients had had normal baseline ALT levels before commencement of NVP therapy. Based upon levels of ALT at the time of sample collection, patients were divided into four groups, HIV/HCV-coinfected patients with high ALT (group 1), HIV/HCV-coinfected patients with normal ALT (group 2), HIV-monoinfected patients with high ALT (group 3), HIV-monoinfected patients with normal ALT (group 4), and healthy controls represented a fifth group (Ctl). Figure 2 shows the characteristics of each group for age, ALT, AST, period of using NVP, and CD4+ cell count. No significant difference was observed between all groups for age distribution, and similarly no difference was observed in the patient groups for the period of NVP usage. Group 1 had significantly higher ALT and AST levels than the control group as well as groups 2 and 4. Group 4 had a significantly higher mean CD4+ cell count than groups 1, 2, and 3.Figure 1
Schematic of patient inclusion and exclusion.Means ± SD of clinical parameters. The information was obtained from 5 HIV/HCV-coinfected patients with high ALT (G1), 5 HIV/HCV-coinfected patients with normal ALT (G2), 5 HIV-monoinfected patients with high ALT (G3), 5 HIV-monoinfected patients with normal ALT (G4), and 5 healthy controls (Ctl). ALT: alanine transaminase; AST: aspartate aminotransferase. All values are reported as means ± SD. One-way ANOVA with Bonferroni’s multiple comparison test was performed; statistical significance is shown by# for P
<
0.05.
(a)
(b)
(c)
(d)
(e)


### 3.2. Urine Proteomic Analysis

Sub-10 kDa protein fractions prepared from pooled urine samples of the five groups were subjected to in-solution tryptic digestion followed by LC-MS/MS analysis. A total of 318 proteins were identified, of which 47 were differentially expressed between groups (Table1). The majority of differentially expressed proteins were identified as being involved in transcription (24%), signal transduction (13%), transport (11%), and immune responses (11%). The remaining proteins were associated with a range of diverse functions. Some 32% of the identified proteins were cytoplasmic, with 21% being membrane proteins and a further 24% were identified as nuclear proteins.Table 1
List of proteins detected as differentially regulated in urine of patients undergoing long term NVP treatment and normal controls.
Protein name
Database ID number
Protein ID score
Function
Localization
Mass (Da)
Peptide sequence
ADAMTS12
gi∣124297123
3.21
Protein metabolism
Unknown
1036.6585
KCNQQACK
Sacsin
gi∣327200585
2.49
Immune response
Membrane
911.5248
FTSEVAXR
NADPH oxidase
gi∣34810991
2.66
Immune response
Cytoplasm
722.5438
YTVVXK
Ubiquitin-conjugating enzyme E2
gi∣269914374
2.24
Ubiquitin metabolism
Cytoplasm
849.2900
AGSMALKR
Carboxy-terminal domain RNA polymerase II polypeptide A small phosphatase 3
gi∣2289786
12.59
Transcription
Nucleus
672.2483
LPGAGEK
dynein
gi∣119627741
4.40
Single
Cytoplasm
733.7764
LFAQVR
Dystroglycan
gi∣294997282
9.68
Cytoskeleton
Cytoplasm
679.6234
KPPLPK
E3 ubiquitin-protein ligase HECTD1
gi∣118498337
22.38
Ubiquitin metabolism
Cytoplasm
870.7503
KASLALIR
FL100154
gi∣18676514
6.78
Others
Unknown
1532.2699
PEMTGVTVSGLTPAR
hCG1783738
gi∣119606481
1.35
Others
Unknown
756.2752
KPALGNR
hCG2042782
gi∣119574283
5.61
Others
Unknown
972.8246
VAMTVIKPV
hCG2045758
gi∣119626212
5.75
Others
Unknown
867.1841
RVASAYLS
Protein HEG homolog 1
gi∣33871486
8.09
Cell development
Membrane
850.1389
LFDLADR
hydroxyacylglutathione hydrolase
gi∣119606016
3.62
Carbohydrate metabolism
Mitochondria
782.3398
VYGGDDR
Hypothetical protein
gi∣60219224
7.34
Others
Unknown
1568.0209
DMQMGPGLFLRMR
IgA nephropathy-related protein 1
gi∣307133516
5.16
Single
Membrane
795.7409
GPVSPPNK
Immunoglobulin heavy chain VHDJ region
gi∣21669823
1.48
Immune response
Cytoplasm
773.9408
RGGVGTTK
Immunoglobulin kappa light chain
gi∣62860960
3.36
Immune response
Cytoplasm
920.3153
TVAAPSVFK
Keratin
gi∣32567786
17.72
Cell development
Cytoplasm
701.4250
MDLHGK
KIAA0591
gi∣29421178
4.69
Apoptosis
Mitochondria
833.9171
SYTMMGK
KIAA0895
gi∣4240279
5.22
Others
Unknown
835.4904
DIGRFVK
Patched domain containing protein
gi∣326205357
3.36
Transport
Membrane
674.6955
LSKNGR
KIAA1409
gi∣119601937
26.30
Response to stress
Membrane
856.7432
IAVSAIQR
KIAA2010
gi∣24899184
1.10
Protein metabolism
Cytoplasm
902.6365
EDTLPLSK
Metabotropic glutamate receptor 5
gi∣4504143
7.70
Single
Nucleus
1406.2405
YEIMNFKEMGK
MHC class 1 antigen
gi∣123278215
7.54
Immune response
Membrane
688.0275
QAPTDR
Myosin light chain kinase 2
gi∣14043538
3.01
Transport
Cytoplasm
1434.2871
DFVSNLIVKDQR
Peroxisome proliferator-activated receptor alpha
gi∣1514595
1.01
Transcription
Nucleus
758.7757
FDFAMK
Progesterone-induced-blocking factor 1
gi∣3925685
6.64
Transcription
Cytoplasm
986.7480
QNSLIXKR
Pleckstrin homology domain containing family A member 6
gi∣37595548
2.93
Lipid metabolism
Cytoplasm
757.5746
SVPQAVR
Poly(ADP-ribose) polymerase 1
gi∣119590191
7.36
Transcription
Nucleus
1475.5247
LQGHVPQTSLQLR
Suppressor of cytokine signaling 5
gi∣23273934
3.30
Single
Cytoplasm
783.9406
RDSYSR
REST corepressor
gi∣344925845
5.18
Transcription
Nucleus
673.1253
GPEVSGK
REST corepressor 3
gi∣21595082
7.97
Transcription
Nucleus
714.2440
GPELLGK
Uncharacterized protein FLJ37004
gi∣74729710
3.79
Others
Unknown
1519.9062
ATAAAARGSEVSGEGGK
Serine/threonine protein kinase RIO3
gi∣119621558
2.00
Transport
Membrane
1263.2434
ENVGDGIGMDLK
Serine/threonine protein kinase PLK4
gi∣2125814
2.52
Single
Cytoplasm
866.4060
DRASFNR
SEC14-like 4
gi∣119580300
7.04
Transport
Membrane
819.6610
SSATLWR
SH2 domain containing protein 3C
gi∣4704739
3.24
Single
Membrane
658.6454
TAVGRR
Sorting nexin-3
gi∣3126979
2.35
Transport
Membrane
1017.4337
AETVADTRR
NEDD8-activating enzyme E1 catalytic subunit
gi∣3342564
1.45
Transcription
Nucleus
744.0996
LTQGVVK
Uncharacterized protein
gi∣254692913
2.93
Others
Unknown
1000.3987
RLLNELDK
Ribosomal RNA upstream binding transcription factor
gi∣1916615
2.03
Transcription
Nucleus
780.6412
LRGPNPK
Vacuolar protein sorting-associated protein 45
gi∣18105063
5.13
Transcription
Nucleus
838.4481
KMSGTVSK
WD repeat-containing protein 46
gi∣256773176
7.41
Transcription
Nucleus
696.2765
KPQVPK
Coiled-coil domain containing protein 105
gi∣226492892
2.14
Transcription
Nucleus
540.5465
APTPR
17-beta-Hydroxysteroid dehydrogenase 14
gi∣59889578
7.76
Cell division
Cytoplasm
506.0660
ATGTROf the 47 differentially expressed proteins, 33 were found in all groups while 14 proteins were identified only in some groups. Of the 14 proteins found in only some groups, seven (Sacsin, carboxy-terminal domain RNA polymerase II polypeptide A small phosphatase 3, KIAA0591 protein, metabotropic glutamate receptor 5 isoform b precursor, UBA3, vacuolar protein sorting-associated protein 45, and E3 ubiquitin-protein ligase HECTD1) were only found in patients groups and were absent from controls, although the level of the 7 proteins was similar between the patient groups (data not shown). The remaining 7 proteins showed no discernible trend for association with NVP treatment and liver damage.


### 3.3. Serum Proteomic Analysis: In-Solution

LC-MS/MS analysis of the sub-10 kDa fraction of pooled serum samples revealed a total of 251 identified proteins, of which 34 were found to be differentially expressed. Details of the 34 proteins and their biological function are presented in Table2. The majority of differentially expressed proteins were associated with transport (14%), cell development (12%), apoptosis (9%), cell adhesion (9%), lipid metabolism (9%), signal transduction (9%), and transcription (9%). The identified proteins were primarily identified as cytoplasmic (47%) and membrane proteins (20%).Table 2
List of proteins detected as differentially regulated in serum of patients undergoing long term NVP treatment and normal controls.
Protein name
Database ID number
Protein ID score
Function
Localization
Mass (Da)
Peptide sequence
BAI 1
gi∣2653432
10.04
Cell adhesion
Membrane
857.1950
NATGLILR
BCL2/adenovirus E1B 19 kDa interacting protein
gi∣119573869
2.14
Apoptosis
Nucleus
878.1002
VFRMGPR
Catenin alpha-1
gi∣21411495
6.22
Cell adhesion
Cytoplasm
1492.7295
EYAQVFREHANK
Death-associated protein kinase 3
gi∣4557511
1.79
Apoptosis
Cytoplasm
617.4699
QKGTGK
Zinc finger protein 174
gi∣159163639
7.90
Transcription
Nucleus
742.0348
CLMSSK
Collagen type X11 alpha-1
gi∣1846005
15.77
Cell adhesion
Unknown
1554.9274
GGNTMTGDAIDYLVK
Corin
gi∣4884872
2.91
Protein metabolism
Membrane
1474.3988
WVLTVAHCFEGR
Chondroitin sulfate proteoglycan 4
gi∣124504610
3.64
Carbohydrate metabolism
Membrane
1551.0338
TGKHDVQVLTAKPR
FYVE and coiled domain containing protein 1
gi∣13276231
4.84
Transport
Cytoplasm
874.5961
AALDDQDK
hCG1811779
gi∣119578948
2.12
Others
Unknown
701.1700
LTGPAGGK
hCG2018146
gi∣119595992
1.36
Others
Unknown
815.1358
CLLRPR
hCG2033821
gi∣119618839
2.35
Others
Unknown
707.6396
TATEMR
Obscurin isoform B
gi∣89143259
1.77
Apoptosis
Cytoplasm
1002.3102
KKPGLASFR
Eukaryotic translation initiation factor 4B
gi∣48146033
3.18
Translation
Cytoplasm
828.0649
EPSNPER
Coiled-coil domain containing 146
gi∣23271918
4.84
Cytoskeleton
Cytoplasm
955.2563
ELVVNLLR
Sodium dependent glucose transporter 1
gi∣154937330
5.29
Transport
Membrane
982.5936
HLPETRTK
Microtubule associated protein 1a
gi∣1790878
2.47
Cytoskeleton
Cytoplasm
849.2162
DKDLEXK
Myosin VI
gi∣9280816
1.43
Cell development
Cytoplasm
519.0438
AAAGTK
Peroxisomaltrans-2-enoyl-coA reductase
gi∣19923817
7.82
Lipid metabolism
Cytoplasm
846.0755
GAGDLSVVK
Phospholipase B domain containing protein 1
gi∣15029600
3.14
Lipid metabolism
Cytoplasm
927.4162
MMADSGKR
Calmodulin-regulated spectrin-associated protein 2
gi∣44955929
20.38
Cytoskeleton
Cytoplasm
701.6011
AGSLILK
C-type lectin domain family 4 member K
gi∣305410858
12.79
Immune response
Cytoplasm
702.7117
ASALNTK
Zinc finger and BTB domain containing protein 44
gi∣30047807
20.92
Transcription
Nucleus
905.7966
VQDKIFR
RNA exonuclease 1 homolog
gi∣145199237
10.68
DNA replication
Nucleus
821.9409
ASSRDER
Rhesus blood group D antigen
gi∣28629452
6.69
Transport
Membrane
691.7388
VVITLF
Rho-related BTB domain containing 3
gi∣27371219
5.84
Singal
Membrane
1897.7174
CEVMAAMFNGNYMEAK
Solute carrier family 4, sodium bicarbonate cotransporter, member 5
gi∣119620095
24.26
Transport
Membrane
856.6955
SPSSLLPR
Tyrosine-protein phosphate non receptor type 23
gi∣24308073
3.39
Single
Cytoplasm
1008.7161
HVEQVLQR
Zinc finger protein 556
gi∣13376460
4.83
Transcription
Nucleus
710.6905
CGKCGK
Dynactin 1
gi∣4139121
3.52
Transport
Cytoplasm
515.6265
APTAR
Casein kinase I isoform gamma-1
gi∣98986450
7.18
Single
Cytoplasm
685.1477
HIPYR
hCG2045456
gi∣119605796
2.79
Others
Unknown
671.2872
EFNNF
Group IIF secretory phospholipase A2
gi∣145553989
3.89
Lipid metabolism
Cytoplasm
462.4213
ADGAK
hCG2038093
gi∣119602010
3.27
Others
Unknown
613.7015
HVSRDTwenty-seven of the differentially expressed proteins were found in all groups. Seven proteins (Rho-related BTB domain containing protein 3 (RhoBTB3), zinc finger and BTB domain containing protein 44 (ZTBT44), obscurin isoform B, BAI 1, RNA exonuclease 1 homolog, death-associated protein kinase 3 (DAPK3), and chondroitin sulfate proteoglycan 4 protein (CSPG4)) showed restricted expression in 1 to 3 groups.The peptide intensity analysis showed (Table3) that RhoBTB3 was detected only in the high ALT groups from both HIV/HCV coinfection and HIV monoinfection and was not detected in the normal ALT groups or in normal control samples. The peptide intensity in the HIV/HCV-coinfected group (group 1) was approximately double that found in the monoinfected group (group 3). DAPK3 and CSPG4 were both highly expressed in control and greatly reduced or absent in the patient groups. The remaining peptide intensity distributions were largely uninformative, and so RhoBTB3 and DAPK3 were selected for validation by Western blot analysis.Table 3
Peptide intensities of 7 proteins showing differential expression of serum proteins between groups.
Protein
Group 1
Group 2
Group 3
Group 4
Control
RhoBTB3
20.03
0
9.20
0
0
ZTBT44
0
5.49
8.48
16.72
0
Obscurin isoform B
17.40
5.76
3.58
0
28.70
BAI 1
17.30
4.69
1.60
0
28.86
RNA exonuclease 1 homolog
19.05
0
9.10
16.11
29.70
DAPK3
0
7.61
10.61
14.59
30.44
CSPG4
0
6.15
0
12.80
30.66
Group 1: HIV/HCV-coinfected patients with high ALT; Group 2: HIV/HCV-coinfected patients with normal ALT; Group 3: HIV-monoinfected patients with high ALT; Group 4: HIV-monoinfected patients with normal ALT; Control:  healthy controls. Seven proteins RhoBTB3: Rho-related BTB domain containing protein 3; ZTBT44: zinc finger and BTB domain containing protein 44; DAPK3: death-associated protein kinase 3; CSPG4: chondroitin sulfate proteoglycan 4. Peptide intensity values are shown to 2 decimal places.


### 3.4. Serum Proteomic Analysis: In-Gel

Albumin depleted pooled serum samples from the five groups were subjected to 2D gel electrophoresis and the gels were stained with Coomassie blue (data not shown). Two spots were identified, one of which was only expressed in group 1 while the second was observed to be significantly downregulated in group 1 as compared to the other groups, including control. The two spots were excised from the gels, subjected to tryptic digestion, and the resultant peptides analyzed by LC-MS/MS. Resultant data was searched against the SwissProt database for protein identification using the Mascot software (http://www.matrixscience.com/). The proteins were identified as transthyretin and haptoglobin, respectively.


## 3.1. Study Participants and Clinical Parameters of Participants

Between June 22, 2012, and August 1, 2012, a total of 31 HIV/HCV and HIV adults were screened for enrollment in the study (Figure1). A total of 11 patients (35.5%) were excluded due to the presence of normal ALT (1), negative anti-HCV (2), borderline ALT level (4), and a further 4 were not included in the analysis to give equal N values in the four patient groups. In addition, five healthy controls were recruited. All patients had had normal baseline ALT levels before commencement of NVP therapy. Based upon levels of ALT at the time of sample collection, patients were divided into four groups, HIV/HCV-coinfected patients with high ALT (group 1), HIV/HCV-coinfected patients with normal ALT (group 2), HIV-monoinfected patients with high ALT (group 3), HIV-monoinfected patients with normal ALT (group 4), and healthy controls represented a fifth group (Ctl). Figure 2 shows the characteristics of each group for age, ALT, AST, period of using NVP, and CD4+ cell count. No significant difference was observed between all groups for age distribution, and similarly no difference was observed in the patient groups for the period of NVP usage. Group 1 had significantly higher ALT and AST levels than the control group as well as groups 2 and 4. Group 4 had a significantly higher mean CD4+ cell count than groups 1, 2, and 3.Figure 1
Schematic of patient inclusion and exclusion.Means ± SD of clinical parameters. The information was obtained from 5 HIV/HCV-coinfected patients with high ALT (G1), 5 HIV/HCV-coinfected patients with normal ALT (G2), 5 HIV-monoinfected patients with high ALT (G3), 5 HIV-monoinfected patients with normal ALT (G4), and 5 healthy controls (Ctl). ALT: alanine transaminase; AST: aspartate aminotransferase. All values are reported as means ± SD. One-way ANOVA with Bonferroni’s multiple comparison test was performed; statistical significance is shown by# for P
<
0.05.
(a)
(b)
(c)
(d)
(e)


## 3.2. Urine Proteomic Analysis

Sub-10 kDa protein fractions prepared from pooled urine samples of the five groups were subjected to in-solution tryptic digestion followed by LC-MS/MS analysis. A total of 318 proteins were identified, of which 47 were differentially expressed between groups (Table1). The majority of differentially expressed proteins were identified as being involved in transcription (24%), signal transduction (13%), transport (11%), and immune responses (11%). The remaining proteins were associated with a range of diverse functions. Some 32% of the identified proteins were cytoplasmic, with 21% being membrane proteins and a further 24% were identified as nuclear proteins.Table 1
List of proteins detected as differentially regulated in urine of patients undergoing long term NVP treatment and normal controls.
Protein name
Database ID number
Protein ID score
Function
Localization
Mass (Da)
Peptide sequence
ADAMTS12
gi∣124297123
3.21
Protein metabolism
Unknown
1036.6585
KCNQQACK
Sacsin
gi∣327200585
2.49
Immune response
Membrane
911.5248
FTSEVAXR
NADPH oxidase
gi∣34810991
2.66
Immune response
Cytoplasm
722.5438
YTVVXK
Ubiquitin-conjugating enzyme E2
gi∣269914374
2.24
Ubiquitin metabolism
Cytoplasm
849.2900
AGSMALKR
Carboxy-terminal domain RNA polymerase II polypeptide A small phosphatase 3
gi∣2289786
12.59
Transcription
Nucleus
672.2483
LPGAGEK
dynein
gi∣119627741
4.40
Single
Cytoplasm
733.7764
LFAQVR
Dystroglycan
gi∣294997282
9.68
Cytoskeleton
Cytoplasm
679.6234
KPPLPK
E3 ubiquitin-protein ligase HECTD1
gi∣118498337
22.38
Ubiquitin metabolism
Cytoplasm
870.7503
KASLALIR
FL100154
gi∣18676514
6.78
Others
Unknown
1532.2699
PEMTGVTVSGLTPAR
hCG1783738
gi∣119606481
1.35
Others
Unknown
756.2752
KPALGNR
hCG2042782
gi∣119574283
5.61
Others
Unknown
972.8246
VAMTVIKPV
hCG2045758
gi∣119626212
5.75
Others
Unknown
867.1841
RVASAYLS
Protein HEG homolog 1
gi∣33871486
8.09
Cell development
Membrane
850.1389
LFDLADR
hydroxyacylglutathione hydrolase
gi∣119606016
3.62
Carbohydrate metabolism
Mitochondria
782.3398
VYGGDDR
Hypothetical protein
gi∣60219224
7.34
Others
Unknown
1568.0209
DMQMGPGLFLRMR
IgA nephropathy-related protein 1
gi∣307133516
5.16
Single
Membrane
795.7409
GPVSPPNK
Immunoglobulin heavy chain VHDJ region
gi∣21669823
1.48
Immune response
Cytoplasm
773.9408
RGGVGTTK
Immunoglobulin kappa light chain
gi∣62860960
3.36
Immune response
Cytoplasm
920.3153
TVAAPSVFK
Keratin
gi∣32567786
17.72
Cell development
Cytoplasm
701.4250
MDLHGK
KIAA0591
gi∣29421178
4.69
Apoptosis
Mitochondria
833.9171
SYTMMGK
KIAA0895
gi∣4240279
5.22
Others
Unknown
835.4904
DIGRFVK
Patched domain containing protein
gi∣326205357
3.36
Transport
Membrane
674.6955
LSKNGR
KIAA1409
gi∣119601937
26.30
Response to stress
Membrane
856.7432
IAVSAIQR
KIAA2010
gi∣24899184
1.10
Protein metabolism
Cytoplasm
902.6365
EDTLPLSK
Metabotropic glutamate receptor 5
gi∣4504143
7.70
Single
Nucleus
1406.2405
YEIMNFKEMGK
MHC class 1 antigen
gi∣123278215
7.54
Immune response
Membrane
688.0275
QAPTDR
Myosin light chain kinase 2
gi∣14043538
3.01
Transport
Cytoplasm
1434.2871
DFVSNLIVKDQR
Peroxisome proliferator-activated receptor alpha
gi∣1514595
1.01
Transcription
Nucleus
758.7757
FDFAMK
Progesterone-induced-blocking factor 1
gi∣3925685
6.64
Transcription
Cytoplasm
986.7480
QNSLIXKR
Pleckstrin homology domain containing family A member 6
gi∣37595548
2.93
Lipid metabolism
Cytoplasm
757.5746
SVPQAVR
Poly(ADP-ribose) polymerase 1
gi∣119590191
7.36
Transcription
Nucleus
1475.5247
LQGHVPQTSLQLR
Suppressor of cytokine signaling 5
gi∣23273934
3.30
Single
Cytoplasm
783.9406
RDSYSR
REST corepressor
gi∣344925845
5.18
Transcription
Nucleus
673.1253
GPEVSGK
REST corepressor 3
gi∣21595082
7.97
Transcription
Nucleus
714.2440
GPELLGK
Uncharacterized protein FLJ37004
gi∣74729710
3.79
Others
Unknown
1519.9062
ATAAAARGSEVSGEGGK
Serine/threonine protein kinase RIO3
gi∣119621558
2.00
Transport
Membrane
1263.2434
ENVGDGIGMDLK
Serine/threonine protein kinase PLK4
gi∣2125814
2.52
Single
Cytoplasm
866.4060
DRASFNR
SEC14-like 4
gi∣119580300
7.04
Transport
Membrane
819.6610
SSATLWR
SH2 domain containing protein 3C
gi∣4704739
3.24
Single
Membrane
658.6454
TAVGRR
Sorting nexin-3
gi∣3126979
2.35
Transport
Membrane
1017.4337
AETVADTRR
NEDD8-activating enzyme E1 catalytic subunit
gi∣3342564
1.45
Transcription
Nucleus
744.0996
LTQGVVK
Uncharacterized protein
gi∣254692913
2.93
Others
Unknown
1000.3987
RLLNELDK
Ribosomal RNA upstream binding transcription factor
gi∣1916615
2.03
Transcription
Nucleus
780.6412
LRGPNPK
Vacuolar protein sorting-associated protein 45
gi∣18105063
5.13
Transcription
Nucleus
838.4481
KMSGTVSK
WD repeat-containing protein 46
gi∣256773176
7.41
Transcription
Nucleus
696.2765
KPQVPK
Coiled-coil domain containing protein 105
gi∣226492892
2.14
Transcription
Nucleus
540.5465
APTPR
17-beta-Hydroxysteroid dehydrogenase 14
gi∣59889578
7.76
Cell division
Cytoplasm
506.0660
ATGTROf the 47 differentially expressed proteins, 33 were found in all groups while 14 proteins were identified only in some groups. Of the 14 proteins found in only some groups, seven (Sacsin, carboxy-terminal domain RNA polymerase II polypeptide A small phosphatase 3, KIAA0591 protein, metabotropic glutamate receptor 5 isoform b precursor, UBA3, vacuolar protein sorting-associated protein 45, and E3 ubiquitin-protein ligase HECTD1) were only found in patients groups and were absent from controls, although the level of the 7 proteins was similar between the patient groups (data not shown). The remaining 7 proteins showed no discernible trend for association with NVP treatment and liver damage.


## 3.3. Serum Proteomic Analysis: In-Solution

LC-MS/MS analysis of the sub-10 kDa fraction of pooled serum samples revealed a total of 251 identified proteins, of which 34 were found to be differentially expressed. Details of the 34 proteins and their biological function are presented in Table2. The majority of differentially expressed proteins were associated with transport (14%), cell development (12%), apoptosis (9%), cell adhesion (9%), lipid metabolism (9%), signal transduction (9%), and transcription (9%). The identified proteins were primarily identified as cytoplasmic (47%) and membrane proteins (20%).Table 2
List of proteins detected as differentially regulated in serum of patients undergoing long term NVP treatment and normal controls.
Protein name
Database ID number
Protein ID score
Function
Localization
Mass (Da)
Peptide sequence
BAI 1
gi∣2653432
10.04
Cell adhesion
Membrane
857.1950
NATGLILR
BCL2/adenovirus E1B 19 kDa interacting protein
gi∣119573869
2.14
Apoptosis
Nucleus
878.1002
VFRMGPR
Catenin alpha-1
gi∣21411495
6.22
Cell adhesion
Cytoplasm
1492.7295
EYAQVFREHANK
Death-associated protein kinase 3
gi∣4557511
1.79
Apoptosis
Cytoplasm
617.4699
QKGTGK
Zinc finger protein 174
gi∣159163639
7.90
Transcription
Nucleus
742.0348
CLMSSK
Collagen type X11 alpha-1
gi∣1846005
15.77
Cell adhesion
Unknown
1554.9274
GGNTMTGDAIDYLVK
Corin
gi∣4884872
2.91
Protein metabolism
Membrane
1474.3988
WVLTVAHCFEGR
Chondroitin sulfate proteoglycan 4
gi∣124504610
3.64
Carbohydrate metabolism
Membrane
1551.0338
TGKHDVQVLTAKPR
FYVE and coiled domain containing protein 1
gi∣13276231
4.84
Transport
Cytoplasm
874.5961
AALDDQDK
hCG1811779
gi∣119578948
2.12
Others
Unknown
701.1700
LTGPAGGK
hCG2018146
gi∣119595992
1.36
Others
Unknown
815.1358
CLLRPR
hCG2033821
gi∣119618839
2.35
Others
Unknown
707.6396
TATEMR
Obscurin isoform B
gi∣89143259
1.77
Apoptosis
Cytoplasm
1002.3102
KKPGLASFR
Eukaryotic translation initiation factor 4B
gi∣48146033
3.18
Translation
Cytoplasm
828.0649
EPSNPER
Coiled-coil domain containing 146
gi∣23271918
4.84
Cytoskeleton
Cytoplasm
955.2563
ELVVNLLR
Sodium dependent glucose transporter 1
gi∣154937330
5.29
Transport
Membrane
982.5936
HLPETRTK
Microtubule associated protein 1a
gi∣1790878
2.47
Cytoskeleton
Cytoplasm
849.2162
DKDLEXK
Myosin VI
gi∣9280816
1.43
Cell development
Cytoplasm
519.0438
AAAGTK
Peroxisomaltrans-2-enoyl-coA reductase
gi∣19923817
7.82
Lipid metabolism
Cytoplasm
846.0755
GAGDLSVVK
Phospholipase B domain containing protein 1
gi∣15029600
3.14
Lipid metabolism
Cytoplasm
927.4162
MMADSGKR
Calmodulin-regulated spectrin-associated protein 2
gi∣44955929
20.38
Cytoskeleton
Cytoplasm
701.6011
AGSLILK
C-type lectin domain family 4 member K
gi∣305410858
12.79
Immune response
Cytoplasm
702.7117
ASALNTK
Zinc finger and BTB domain containing protein 44
gi∣30047807
20.92
Transcription
Nucleus
905.7966
VQDKIFR
RNA exonuclease 1 homolog
gi∣145199237
10.68
DNA replication
Nucleus
821.9409
ASSRDER
Rhesus blood group D antigen
gi∣28629452
6.69
Transport
Membrane
691.7388
VVITLF
Rho-related BTB domain containing 3
gi∣27371219
5.84
Singal
Membrane
1897.7174
CEVMAAMFNGNYMEAK
Solute carrier family 4, sodium bicarbonate cotransporter, member 5
gi∣119620095
24.26
Transport
Membrane
856.6955
SPSSLLPR
Tyrosine-protein phosphate non receptor type 23
gi∣24308073
3.39
Single
Cytoplasm
1008.7161
HVEQVLQR
Zinc finger protein 556
gi∣13376460
4.83
Transcription
Nucleus
710.6905
CGKCGK
Dynactin 1
gi∣4139121
3.52
Transport
Cytoplasm
515.6265
APTAR
Casein kinase I isoform gamma-1
gi∣98986450
7.18
Single
Cytoplasm
685.1477
HIPYR
hCG2045456
gi∣119605796
2.79
Others
Unknown
671.2872
EFNNF
Group IIF secretory phospholipase A2
gi∣145553989
3.89
Lipid metabolism
Cytoplasm
462.4213
ADGAK
hCG2038093
gi∣119602010
3.27
Others
Unknown
613.7015
HVSRDTwenty-seven of the differentially expressed proteins were found in all groups. Seven proteins (Rho-related BTB domain containing protein 3 (RhoBTB3), zinc finger and BTB domain containing protein 44 (ZTBT44), obscurin isoform B, BAI 1, RNA exonuclease 1 homolog, death-associated protein kinase 3 (DAPK3), and chondroitin sulfate proteoglycan 4 protein (CSPG4)) showed restricted expression in 1 to 3 groups.The peptide intensity analysis showed (Table3) that RhoBTB3 was detected only in the high ALT groups from both HIV/HCV coinfection and HIV monoinfection and was not detected in the normal ALT groups or in normal control samples. The peptide intensity in the HIV/HCV-coinfected group (group 1) was approximately double that found in the monoinfected group (group 3). DAPK3 and CSPG4 were both highly expressed in control and greatly reduced or absent in the patient groups. The remaining peptide intensity distributions were largely uninformative, and so RhoBTB3 and DAPK3 were selected for validation by Western blot analysis.Table 3
Peptide intensities of 7 proteins showing differential expression of serum proteins between groups.
Protein
Group 1
Group 2
Group 3
Group 4
Control
RhoBTB3
20.03
0
9.20
0
0
ZTBT44
0
5.49
8.48
16.72
0
Obscurin isoform B
17.40
5.76
3.58
0
28.70
BAI 1
17.30
4.69
1.60
0
28.86
RNA exonuclease 1 homolog
19.05
0
9.10
16.11
29.70
DAPK3
0
7.61
10.61
14.59
30.44
CSPG4
0
6.15
0
12.80
30.66
Group 1: HIV/HCV-coinfected patients with high ALT; Group 2: HIV/HCV-coinfected patients with normal ALT; Group 3: HIV-monoinfected patients with high ALT; Group 4: HIV-monoinfected patients with normal ALT; Control:  healthy controls. Seven proteins RhoBTB3: Rho-related BTB domain containing protein 3; ZTBT44: zinc finger and BTB domain containing protein 44; DAPK3: death-associated protein kinase 3; CSPG4: chondroitin sulfate proteoglycan 4. Peptide intensity values are shown to 2 decimal places.


## 3.4. Serum Proteomic Analysis: In-Gel

Albumin depleted pooled serum samples from the five groups were subjected to 2D gel electrophoresis and the gels were stained with Coomassie blue (data not shown). Two spots were identified, one of which was only expressed in group 1 while the second was observed to be significantly downregulated in group 1 as compared to the other groups, including control. The two spots were excised from the gels, subjected to tryptic digestion, and the resultant peptides analyzed by LC-MS/MS. Resultant data was searched against the SwissProt database for protein identification using the Mascot software (http://www.matrixscience.com/). The proteins were identified as transthyretin and haptoglobin, respectively.


## 4. Validation of Serum Biomarkers Using Western Blotting

From the results of the in-solution and in-gel proteomic analyses of serum, three proteins (DAPK3, RhoBTB3, and haptoglobin) were selected for confirmation by Western blotting analysis. Each of the 25 serum samples was individually albumin depleted and proteins were separated by electrophoresis before transfer to solid matrix support and Western blot analysis. Levels of GAPDH or transferrin were additionally determined as an internal control. Results showed that haptoglobin (Figure3) was significantly reduced in group 1 (HIV/HCV-coinfected patients with high ALT). Levels of serum haptoglobin in group 4 (HIV-monoinfected patients with normal ALT) were similar to levels in the control group and significantly above the levels seen in the three remaining patients groups (HIV/HCV-coinfected patients with high ALT, HIV/HCV-coinfected patients with normal ALT, and HIV-monoinfected patients with high ALT). The largest difference in haptoglobin levels was seen between group 1 (HIV/HCV-coinfected patients with high ALT) and group 4 (HIV-monoinfected patients with normal ALT). While some slight differences were noted between the groups for both RhoBTB3 and DAPK3, the differences did not reach statistical significance.Western blot analysis and quantitation of haptoglobin expression in serum of normal controls and HIV patients undergoing long term NVP treatment. (a) Serum samples were individually depleted for albumin and proteins run as triplicate replicates before transfer to solid matrix support and western analysis to determine haptoglobin expression. Filters were reprobed after stripping with an antibody against transferrin to provide a loading control. G1 (group 1; HIV/HCV-coinfected patients with high ALT), G2 (group 2; HIV/HCV-coinfected patients with normal ALT), G3 (group 3; HIV-monoinfected patients with high ALT), G4 (group 4; HIV-monoinfected patients with normal ALT), and control (healthy controls). (b) Quantitation was determined by image analysis and is expressed as relative intensity (haptoglobin/transferrin). Data is plotted as means ± SD. Statistical significance was determined using one-way ANOVA with Bonferroni’s multiple comparison test.# designates P
<
0.05.
(a)
(b)


## 5. Discussion

A highly active antiretroviral treatment (HAART) regimen containing NVP appears to be associated with a higher risk for increased liver damage in HIV-1-infected patients who are coinfected with HCV as compared to monoinfected HIV patients, and as such specific biomarkers that can be used in conjunction with ALT would be useful as an adjunct marker to ALT alone. Towards this end, this study undertook a proteomic analysis of serum and urine from HIV-monoinfected and HIV/HCV-coinfected patients undergoing long term NVP treatment in parallel with control samples.The patient samples were analyzed in four groups, namely, HIV/HCV coinfection with high ALT (group 1), HIV/HCV coinfection with normal ALT (group 2), HIV monoinfection with high ALT (group 3), and HIV monoinfection with normal ALT (group 4). The patients in the high ALT cohorts all had grade 1 liver elevation except two patients in group 1 who had grade 2 elevation. No higher ALT grade patients were available as their regimes are normally altered to prevent further liver damage. As such, this study was somewhat limited in the range of liver hepatotoxicity studied and the study should be further cautiously interpreted in light of the small number of patients in each group.A total of 83 proteins were identified through the three arms of the study (in-solution analysis of serum and urine and in-gel analysis of serum), although no protein was identified consistently in all three arms of the study, presumably as a result of differing sample and methodological approach. The two proteins selected for further validation from those identified from the in-solution proteomic analysis (RhoBTB3 and DAPK3) showed only slight and nonsignificant differences between the four patient groups. The third protein, haptoglobin, identified from the in-gel 2D electrophoresis assay showed slight but not statistically significant elevation in monoinfected HIV patients with normal ALT and statistically significantly reduced levels compared to normal controls in HIV/HCV-coinfected patients with high ALT. Levels of haptoglobin were slightly reduced in HIV/HCV-coinfected patients with high ALT (group 1) as compared to HIV/HCV-coinfected patients with normal ALT (group 2) although this was not statistically significant. However, as noted previously, the absence of grades 3 and 4 ALT levels probably reduces the discriminating ability of the analysis.The plasma protein, haptoglobin, is synthesized by hepatocytes and functions to noncovalently bind oxidized hemoglobin generated by hemolysis [24]. The hemoglobin-haptoglobin complex is taken up by the reticuloendothelial system both to scavenge iron for recycling and to prevent adverse oxidative effects [24]. A significant decrease in haptoglobin levels during ART has been previously reported [25], and the decrease was unrelated to hemophilia or HCV status, and was not associated with the presence of increased markers of hemolysis. In our study, we observed a significant decrease in haptoglobin levels in patients with HIV/HCV coinfection and high ALT and nonsignificant reductions in patients with HIV/HCV coinfection and normal ALT and in monoinfected HIV patients with normal and high ALT. A significant difference was seen between levels of haptoglobin in monoinfected HIV patients with high and normal ALT, and while it did not reach statistical significance, the levels of haptoglobin were reduced in HIV/HCV-coinfected patients with high ALT as compared to HIV/HCV-coinfected patients with normal ALT. These results suggest that reduced levels of haptoglobin may reflect increased liver damage. In this regard, haptoglobin has previously been reported as a serum marker of fibrosis in chronic hepatitis C patients [26], where it is believed that liver fibrosis leads to increased expression of hepatocyte growth factor promoting a subsequent decrease in haptoglobin levels.Studies have suggested that serum haptoglobin predicts liver fibrosis in hepatitis C patients where it was found that haptoglobin was one of the top candidate proteins for discriminating grade 0 from grades 1–4 liver fibrosis as well as discriminating grade 3 from grade 4 [27], while another study reported that haptoglobin serum levels were negatively correlated to scores of fibrosis (P
<
0.001) and suggested that determination of haptoglobin serum level may be useful in the follow-up of patients with chronic hepatitis C [28].Although the patients in this project were not yet in fibrotic stages, a correlation between liver status and haptoglobin was observed in group 1 in comparison with group 2 HCV patients. The patients in group 1 have more severe grade 2 liver injury as compared to the patients in group 2 that contained only grade 1 liver injury and showed lower haptoglobin levels. However, this was not statistically significant, possibly since only two out of five patients in group 1 were grade 2 for liver injury. Interestingly, patient group 3 (monoinfected with HIV with high ALT) had relatively low level of haptoglobin compared to group 4 who had normal ALT suggesting that haptoglobin may be an earlier marker for liver injury caused by NVP in HIV patients. Haptoglobin is one of five parameters (alpha2-macroglobulin, haptoglobin, apolipoprotein A1, gamma glutamyl transpeptidase, and bilirubin) used to evaluate liver fibrosis stage in the Fibrotest [29] and an inverse relationship between haptoglobin and bilirubin levels have been reported in hepatitis patients although the relationship was dependent upon the type of liver disease [30].Overall, this study identified a number of possible candidate biomarkers of NVP induced liver toxicity, one of which (haptoglobin) was successfully validated. Two potential biomarkers failed Western blot validation (RhoBTB3 and DAPK3) but remain of interest for possible future validation in larger cohorts of patients.



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*Source: 315824-2014-12-17.xml*