Pavithra Rajagopalan

A draft map of the human proteome

The availability of human genome sequence has transformed biomedical research over the past decade. However, an equivalent map for the human proteome with direct measurements of proteins and peptides does not exist yet. Here we present a draft map of the human proteome using high-resolution Fourier-transform mass spectrometry. In-depth proteomic profiling of 30 histologically normal human samples, including 17 adult tissues, 7 fetal tissues and 6 purified primary haematopoietic cells, resulted in identification of proteins encoded by 17,294 genes accounting for approximately 84% of the total annotated protein-coding genes in humans. A unique and comprehensive strategy for proteogenomic analysis enabled us to discover a number of novel protein-coding regions, which includes translated pseudogenes, non-coding RNAs and upstream open reading frames. This large human proteome catalogue (available as an interactive web-based resource at http://www.humanproteomemap.org) will complement available human genome and transcriptome data to accelerate biomedical research in health and disease.

Population movement and malaria persistence in Rameswaram Island: Foreword

During 1982-84, the Vector Control Research Center (VCRC) of the Indian Council for Medical Research (ICMR) at Pondicherry studied the role of population movement in the persistence of malaria transmission in Rameswaram Island, Tamil Nadu, between mainland India and Sri Lanka. While the island supports a population of 56,000, mostly fishermen, there is also a periodic, back and forth migration of fishermen between mainland villages and Rameswaram. This population movement greatly contributes to the high prevalence of malaria in both areas, since fishermen can be either donor or recipient of malaria in either place. The VCRC monitered and recorded the movement of fishermen in various seasonal camps by questioning them and by the VCRC staff accompanying them when possible. In 9 fishing camps 412 of 1098 families had migrated from mainland villages; 686 families had migrated from villages within Rameswaram Island. A mass blood survey found 138 of 4073 individuals examined positive for malaria; 107 of 680 fever cases examined were positive for malaria. Mosquito collections, the lack of permanent treatment facilities for the transient population, and ecological factors indicate a high receptivity for malaria on Rameswaram Island. With the island attracting between 1000-4000 tourists daily and over 200,000 travelers annually between India and Sri Lanka, evidence exists for considerable danger from the importation of chloroquinine resistant malaria strains into Rameswaram. Adequate attention to human ecology will be needed for malaria control in this area.

LC–MS-based serum metabolomic analysis reveals dysregulation of phosphatidylcholines in esophageal squamous cell carcinoma

UNLABELLED Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive cancers with poor prognosis. Here, we carried out liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS)-based untargeted metabolomic analysis of ESCC serum samples. Statistical analysis resulted in the identification of 652 significantly dysregulated molecular features in serum from ESCC patients as compared to the healthy subjects. Phosphatidylcholines were identified as a major class of dysregulated metabolites in this study suggesting potential perturbation of phosphocholine metabolism in ESCC. By using a targeted MS/MS approach both in positive and negative mode, we were able to characterize and confirm the structure of seven metabolites. Our study describes a quantitative LC-MS approach for characterizing dysregulated lipid metabolism in ESCC. BIOLOGICAL SIGNIFICANCE Altered metabolism is a hallmark of cancer. We carried out (LC-MS)-based untargeted metabolomic profiling of serum from esophageal squamous cell carcinoma (ESCC) patients to characterize dysregulated metabolites. Phosphatidylcholine metabolism was found to be significantly altered in ESCC. Our study illustrates the use of mass spectrometry-based metabolomic analysis to characterize molecular alterations associated with ESCC. This article is part of a Special Issue entitled: Proteomics in India.

Cigarette smoke and chewing tobacco alter expression of different sets of miRNAs in oral keratinocytes

Carcinogenic effect of tobacco in oral cancer is through chewing and/or smoking. Significant differences exist in development of oral cancer between tobacco users and non-users. However, molecular alterations induced by different forms of tobacco are yet to be fully elucidated. We developed cellular models of chronic exposure to chewing tobacco and cigarette smoke using immortalized oral keratinocytes. Chronic exposure to tobacco resulted in increased cell scattering and invasiveness in immortalized oral keratinocytes. miRNA sequencing using Illumina HiSeq 2500 resulted in the identification of 10 significantly dysregulated miRNAs (4 fold; p ≤ 0.05) in chewing tobacco treated cells and 6 in cigarette smoke exposed cells. We integrated this data with global proteomic data and identified 36 protein targets that showed inverse expression pattern in chewing tobacco treated cells and 16 protein targets that showed inverse expression in smoke exposed cells. In addition, we identified 6 novel miRNAs in chewing tobacco treated cells and 18 novel miRNAs in smoke exposed cells. Integrative analysis of dysregulated miRNAs and their targets indicates that signaling mechanisms leading to oncogenic transformation are distinct between both forms of tobacco. Our study demonstrates alterations in miRNA expression in oral cells in response to two frequently used forms of tobacco.

Proteome-wide changes in primary skin keratinocytes exposed to diesel particulate extract—A role for antioxidants in skin health

BACKGROUND Skin acts as a protective barrier against direct contact with pollutants but inhalation and systemic exposure have indirect effect on keratinocytes. Exposure to diesel exhaust has been linked to increased oxidative stress. OBJECTIVE To investigate global proteomic alterations in diesel particulate extract (DPE)/its vapor exposed skin keratinocytes. METHODS We employed Tandem Mass Tag (TMT)-based proteomics to study effect of DPE/DPE vapor on primary skin keratinocytes. RESULTS We observed an increased expression of oxidative stress response protein NRF2, upon chronic exposure of primary keratinocytes to DPE/its vapor which includes volatile components such as polycyclic aromatic hydrocarbons (PAHs). Mass spectrometry-based quantitative proteomics led to identification 4490 proteins of which 201 and 374 proteins were significantly dysregulated (≥1.5 fold, p≤0.05) in each condition, respectively. Proteins involved in cellular processes such as cornification (cornifin A), wound healing (antileukoproteinase) and differentiation (suprabasin) were significantly downregulated in primary keratinocytes exposed to DPE/DPE vapor. These results were corroborated in 3D skin models chronically exposed to DPE/DPE vapor. Bioinformatics analyses indicate that DPE and its vapor affect distinct molecular processes in skin keratinocytes. Components of mitochondrial oxidative phosphorylation machinery were seen to be exclusively overexpressed upon chronic DPE vapor exposure. In addition, treatment with an antioxidant like vitamin E partially restores expression of proteins altered upon exposure to DPE/DPE vapor. CONCLUSIONS Our study highlights distinct adverse effects of chronic exposure to DPE/DPE vapor on skin keratinocytes and the potential role of vitamin E in alleviating adverse effects of environmental pollution.

Molecular alterations associated with chronic exposure to cigarette smoke and chewing tobacco in normal oral keratinocytes.

ABSTRACT Tobacco usage is a known risk factor associated with development of oral cancer. It is mainly consumed in two different forms (smoking and chewing) that vary in their composition and methods of intake. Despite being the leading cause of oral cancer, molecular alterations induced by tobacco are poorly understood. We therefore sought to investigate the adverse effects of cigarette smoke/chewing tobacco exposure in oral keratinocytes (OKF6/TERT1). OKF6/TERT1 cells acquired oncogenic phenotype after treating with cigarette smoke/chewing tobacco for a period of 8 months. We employed whole exome sequencing (WES) and quantitative proteomics to investigate the molecular alterations in oral keratinocytes chronically exposed to smoke/ chewing tobacco. Exome sequencing revealed distinct mutational spectrum and copy number alterations in smoke/ chewing tobacco treated cells. We also observed differences in proteomic alterations. Proteins downstream of MAPK1 and EGFR were dysregulated in smoke and chewing tobacco exposed cells, respectively. This study can serve as a reference for fundamental damages on oral cells as a consequence of exposure to different forms of tobacco.

Investigation of curcumin-mediated signalling pathways in head and neck squamous cell carcinoma:

Objectives: Curcumin has been shown to exhibit anti-neoplastic effects. However, due to its poor bioavailability, the use of curcumin as an anti-cancer drug is limited. Thus, it is necessary to identify molecules as an alternative to curcumin that could serve as anti-cancer targets. In this study, we attempted to understand the underlying curcumin-mediated signalling pathways contributing to anti-neoplastic effects of curcumin. Methods: We carried out mass spectrometry-based phosphoproteomic analysis of head and neck cancer cell line, CAL 27, treated with and without curcumin to identify curcumin-mediated signalling pathways. Serine/threonine kinases were enriched using titanium dioxide. Results: This resulted in the identification of 5921 phosphopeptides corresponding to 1878 proteins. Of these, 275 and 183 phosphopeptides corresponding to 335 and 242 proteins (≥2.0-fold) were found to be hyper- and hypo-phosphorylated, respectively, in response to curcumin treatment. Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2), a serine/threonine kinase, and its downstream target protein kinase AMP-activated non-catalytic subunit beta 1 (PRKAB1) were found to be hypo-phosphorylated when treated with curcumin. Further, silencing or inhibiting CaMKK2 resulted in decreased invasion and colony forming ability of not only CAL 27 cells but also other head and neck squamous cell carcinoma (HNSCC) cell lines. Further, Western blot analysis showed that curcumin-mediated signalling is corroborated by CaMKK2. Conclusions: Taken together, our results suggest that CaMKK2 could be a novel therapeutic target in HNSCC and can serve as an alternative to curcumin.

Delineating miRNA profile induced by chewing tobacco in oral keratinocytes

The major established etiologic risk factor for oral cancer is tobacco (chewed, smoked and snuffed forms). Chewing form of tobacco is predominantly used in India making it the leading cause of oral cancer. Despite being one of the leading causes of oral cancer, the molecular alterations induced by chewing tobacco remains largely unclear. Carcinogenic effect of chewing tobacco is through chronic and not acute exposure. To understand the molecular alterations induced by chewing tobacco, we developed a cell line model where non-neoplastic oral keratinocytes were chronically exposed to chewing tobacco for a period of 6 months. This resulted in increased cellular proliferation and invasive ability of normal oral keratinocytes. Using this cellular model we studied the differential expression of miRNAs associated with chewing tobacco and the altered signaling pathways through which the aberrantly expressed miRNAs affect tumorigenesis. miRNA sequencing was carried out using Illumina HiSeq 2500 platform which resulted in the identification of 427 annotated miRNAs of which 10 were significantly dysregulated (≥ 4 fold; p-value ≤ 0.05) in tobacco exposed cells compared to untreated parental cells. To study the altered signaling in oral keratinocytes chronically exposed to chewing tobacco, we employed quantitative proteomics to characterize the dysregulated proteins. Integration of miRNA sequencing data with proteomic data resulted in identification of 36 proven protein targets which (≥1.5 fold; p-value ≤ 0.05) showed expression correlation with the 10 significantly dysregulated miRNAs. Pathway analysis of the dysregulated targets revealed enrichment of interferon signaling and mRNA processing related pathways in the chewing tobacco exposed cells. In addition, we also identified 6 novel miRNA in oral keratinocytes chronically exposed to chewing tobacco extract. Our study provides a framework to understand the oncogenic transformation induced by chromic tobacco exposure in normal oral keratinocytes. Citation: Bhat, M.Y., Advani1, J., Rajagopalan, P., Patel, K., Nanjappa, V., Solanki, H.S., Patil, A.H., Bhat, F., Mathur, P.P., Nair, B., Prasad, T.S.K., Sidransky, D., Gowda, H. and Chatterjee, A. Delineating miRNA profile induced by chewing tobacco in oral keratinocytes [Abstract]. In: Abstracts of the NGBT conference; Oct 02-04, 2017; Bhubaneswar, Odisha, India: Can J biotech, Volume 1, Special Issue, Page 59. https://doi.org/10.24870/cjb.2017-a46