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New insights into the disease progression control mechanisms by comparing Long-term-non-progressors vs. Normal-progressors among HIV-1positive patients using an ion current-based MS1 proteomic profiling Xiaomeng Shen, Bindukumar Nair, Supriya D Mahajan, Xiaosheng Jiang, Jun Li, Shichen Shen, Chengjian Tu, Chiu-bin Hsiao, Stanley A Schwartz, and Jun Qu J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b00621 • Publication Date (Web): 20 Oct 2015 Downloaded from http://pubs.acs.org on October 31, 2015
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New insights into the disease progression control mechanisms by comparing Long-term-non-progressors vs. Normal-progressors among HIV-1-positive patients using an ion current-based MS1 proteomic profiling #Xiaomeng Shen1,4, #Bindukumar Nair3 , #Supriya D. Mahajan3, Xiaosheng Jiang2,4, Jun Li2,4, Shichen Shen1,4, 2,4
5
1,2,4
Chengjian Tu , Chiu-bin Hsiao , *Stanley A. Schwartz3 and *Jun Qu
Department of Biochemistry
1
,Pharmaceutical Sciences2 , Medicine, Division of Allergy, Immunology, and
3
Rheumatology , State University of New York at Buffalo, South Campus, NY 14214; the State of New York Center for 4
Excellence in Bioinformatics and Life Science
, 701 Ellicott Street, Buffalo, NY 14203; Infectious Disease Division, 5
Department of Medicine, Allegheny General Hospital , Pittsburgh, PA 15212. #The authors contribute equally to this work.
*Corresponding Author: Stanley A. Schwartz, MD, PhD Distinguished Professor of Medicine, Pediatrics, & Microbiology Department of Medicine State University of NY at Buffalo 100 High Street, B816 Buffalo, NY 14203 Phone: 716-859-2260 FAX: 716-895-1471 Email:
[email protected] Jun Qu, Ph.D. The Department of Pharmaceutical Sciences University at Buffalo State University of New York Buffalo, NY 14260-1200 Phone: (716) 645-4821 Fax:
(716) 645-3693
Email:
[email protected] ACS Paragon Plus Environment
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Abstract: For decades, epidemiological studies have found significant differences in the susceptibility to disease progression among HIV-carrying patients. One unique group of HIV-1-positive patients, the Long-term-non-progressors (LTNP), exhibit far superior ability in virus control compared with Normal-progressors(NP), which proceed to Acquired Immune Deficiency Syndrome(AIDS) much more rapidly. Nonetheless, elucidation of the underlying mechanisms of virus control in LTNP is highly valuable in disease management and treatment, but remains poorly understood. Peripheral blood mononuclear cells (PBMC) have been known to play important roles in innate immune responses and thereby would be of great interest for the investigation of the mechanisms of virus defense in LTNP. Here, we described the first comparative proteome analysis of PBMC from LTNP (n=10) and NP (n=10) patients using a reproducible ion-current-based MS1 approach, which includes efficient and reproducible sample preparation and chromatographic separation followed by an optimized pipeline for protein identification and quantification. This strategy enables analysis of many biological samples in one set with high quantitative precision and extremely low missing data. In total 925 unique proteins were quantified under stringent criteria without missing value in any of the 20 subjects, and 87 proteins showed altered expressions between the two patient groups. These proteins are implicated in key processes such as cytoskeleton organization, defense response, apoptosis regulation, intracellular transport etc, which provided novel insights into the control of disease progressions in LTNP vs. NP, and the expression and phosphorylation states of key regulators were further validated by immunoassay. For instance, 1) SAMH1, a potent and “hot” molecule facilitating HIV-1 defense, was for the first time found elevated in LTNP compared with NP or healthy controls; elevated proteins from IFN-α response pathway may also contribute to viral control in LTNP; 2) decreased proapoptotic protein ASC along with the elevation of anti-apoptotic proteins may contribute to the less apoptotic profile in PBMC of LTNP; and 3) elevated actin polymerization and less microtubule assembly that impede viral protein transport were firstly observed in LTNP. These results not only enhanced the understanding of the mechanisms for non-progression of LTNP, but may also afford highly valuable clues to direct therapeutic efforts. Moreover, this work also demonstrated the ion-current-based MS1 approach as a reliable tool for large-scale clinical research.
Abbreviations:
Acquired Immune Deficiency Syndrome(AIDS); AntiRetroviral Therapy(ART); Area-Under-
Curve(AUC); Experimental Null(EN); False Altered protein Discovery Rate (FADR); False Discovery Rate (FDR); Genome-wide Association Study(GWAS); Human Immunodeficiency Virus Type I (HIV-1); Human Leukocyte ACS Paragon Plus Environment
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Antigen(HLA); Ion Current(IC); Long Term None Progressors(LTNP); Normal Progressors(NP); Peripheral Blood Mononuclear Cells (PBMC); Reactive Oxygen Species (ROS); Spectral Counting(SpC). Key words: HIV; LTNP; PBMC; Proteomics; Label-free; ion current-based quantification
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1. Introduction For years, epidemiological studies reveal high variability in patients’ susceptibility to disease progression after infection of Human Immunodeficiency Virus Type I (HIV-1), presumably rooting from innate genetic and phenotypic variations1. The vast majority of patients (termed as normal-progressors, NP) suffer from progressive virus replication and loss of CD4+ T cells that lead to Acquired Immune Deficiency Syndrome(AIDS) within several years, especially for those without antiretroviral therapy(ART). On the contrary, a small percentage (~5%) of HIV-1-infected individuals maintain normal counts of CD4+ T cells and controlled viremia without ART for many years, which are defined as “long-term-nonprogressors(LTNP)” or “elite controllers”, owing to the effective viral replication control and high survival rate of immune cells in these patients 1, 2. LTNP cannot be differentiated from NP based on regular background parameters such as gender, age, ethnic group or mode of contamination3. The LTNP is of high interest clinically because it provides opportunities to study how human innate system control disease progression in HIV-positive patients. Identification of the factors responsible for non-progression in LTNP would greatly contribute to the understanding of HIV pathogenesis and can thereby shed light on therapeutic efforts through the modulation of the innate immune system4. Unfortunately, though emerging evidences postulated that host response5 and HLA Class I alleles6 may contribute to the restraint of viremia, the mechanisms for disease progression control in LTNP remain largely unclear7. To gain novel insights into the mechanisms of virus control in LTNP, many clinical studies employed discovery-based Genome-wide Association Study(GWAS) and transcriptomics analysis to compare LTNP vs. NP groups. GWAS aims to identify genetic variations responsible for none progression; e.g. it was speculated in recent studies that CCR5∆32 inherited mutation and protective Human Leukocyte Antigen(HLA) alleles such as B27 and B57 may be closely associated with non-progression in LTNP individuals8. However, genetic factors provide very limited explanation for the ability of virus control, and most subjects carrying these alleles are not LTNP7. Transcriptomics analysis in this regard emerged since 2004, which suggested some new clues for the mechanisms responsible of non-progression in LTNP. Some studies discovered a number of altered mRNA in LTNP that are implicated in categories such as cytoskeleton organization, apoptosis, cell cycle and cytokine-involved networks 9. As the majority of pathway modulators are proteins and an increasing number of work showed poor correlation between changes on transcriptional vs. translational levels10, 11, using genomics- and transcriptomics- based method alone may not accurately and comprehensively reveal mechanisms ACS Paragon Plus Environment
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underlying LTNP. In this regard, proteomic profiling is capable of comparing the protein levels in LTNP vs. NP cohorts, and therefore providing directly relevant biological information that substantially contributes to elucidation of virus defense mechanisms in LTNP. Nonetheless, proteomics study on clinical sets is quite challenging as it is necessary to analyze many clinical replicates so as to alleviate the high biological variability, which renders accurate and extensive quantification difficult12, 13. Moreover, missing data among the biological replicates represent a severe problem for current techniques14. To our knowledge, the only proteomics study of LTNP vs. NP conducted so far was carried out with two-dimensional gel electrophoresis, which compared serum from LTNP and NP and identified 10) without missing data in any replicate on protein level. Protein Quantification and Altered Proteins Selection. The quantitative analysis by IC was performed by two steps: procurement of area-under-curve data for peptides using SIEVE® v2.1 (Thermo Scientific, San Jose, CA) and then a sumintensity method to aggregate the quantitative data from peptide level to protein level by a homemade R data analysis package. SIEVE® is a label-free proteomics quantification software that performs chromatographic alignment and global intensity-based MS1 feature extraction. The software processes chromatographic alignment between among sequential LC/MS runs using the ChromAlign algorithm 24. Then quantitative frames containing the group of area-under-curve(AUC) data from each peptide were defined using a set of stringent criteria, such as the requirement of S/N>10 for frame detection and the threshold windows of m/z< ±0.01 amu and retention time =1.4 (i.e. log2 ratio threshold is 0.4854 in both direction) and p-value
