Aafaque Ahmad Khan

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.

Plasma Proteome Database as a resource for proteomics research: 2014 update.

Plasma Proteome Database (PPD; http://www.plasmaproteomedatabase.org/) was initially described in the year 2005 as a part of Human Proteome Organization’s (HUPO’s) pilot initiative on Human Plasma Proteome Project. Since then, improvements in proteomic technologies and increased throughput have led to identification of a large number of novel plasma proteins. To keep up with this increase in data, we have significantly enriched the proteomic information in PPD. This database currently contains information on 10 546 proteins detected in serum/plasma of which 3784 have been reported in two or more studies. The latest version of the database also incorporates mass spectrometry-derived data including experimentally verified proteotypic peptides used for multiple reaction monitoring assays. Other novel features include published plasma/serum concentrations for 1278 proteins along with a separate category of plasma-derived extracellular vesicle proteins. As plasma proteins have become a major thrust in the field of biomarkers, we have enabled a batch-based query designated Plasma Proteome Explorer, which will permit the users in screening a list of proteins or peptides against known plasma proteins to assess novelty of their data set. We believe that PPD will facilitate both clinical and basic research by serving as a comprehensive reference of plasma proteins in humans and accelerate biomarker discovery and translation efforts.

Pancreatic Cancer Database: An integrative resource for pancreatic cancer

Pancreatic cancer is the fourth leading cause of cancer-related death in the world. The etiology of pancreatic cancer is heterogeneous with a wide range of alterations that have already been reported at the level of the genome, transcriptome, and proteome. The past decade has witnessed a large number of experimental studies using high-throughput technology platforms to identify genes whose expression at the transcript or protein levels is altered in pancreatic cancer. Based on expression studies, a number of molecules have also been proposed as potential biomarkers for diagnosis and prognosis of this deadly cancer. Currently, there are no repositories which provide an integrative view of multiple Omics data sets from published research on pancreatic cancer. Here, we describe the development of a web-based resource, Pancreatic Cancer Database (http://www.pancreaticcancerdatabase.org), as a unified platform for pancreatic cancer research. PCD contains manually curated information pertaining to quantitative alterations in miRNA, mRNA, and proteins obtained from small-scale as well as high-throughput studies of pancreatic cancer tissues and cell lines. We believe that PCD will serve as an integrative platform for scientific community involved in pancreatic cancer research.

A multicellular signal transduction network of AGE/RAGE signaling.

Advanced glycation end products (AGEs) are heterogeneous glycated products of proteins, lipids and nucleotides. The major receptor for AGEs, known as receptor for advanced glycation end products (RAGE or AGER), is a multi-ligand transmembrane receptor of immunoglobulin superfamily. It has an extracellular region, a transmembrane domain and a short cytoplasmic domain. Extracellular region of RAGE consists of one V type (critical for ligand binding) and two C type immunoglobulin domains (Schmidt et al. 1994a, b). Although the short cytoplasmic tail of 43 amino acid residues is found to be important for the signaling events mediated by RAGE, it does not have any known domain or motif (Neeper et al. 1992). The other cell surface receptors for AGEs include dolichyl-diphosphooligosaccharide-protein glycosyltransferase (AGE-R1) (Li et al. 1996), protein kinase C substrate, 80KH phosphoprotein (AGE-R2) (Goh et al. 1996), galectin-3 (AGE-R3) (Vlassara et al. 1995), and class A macrophage scavenger receptors type I and II. RAGE is also considered as a pattern recognition receptor due to its ability to bind different AGEs. RAGE has numerous extracellular ligands in addition to AGEs, which include extracellular high mobility group box-1 (HMGB1), S100 family of calcium binding proteins and amyloid-beta peptide (Fritz 2011). RAGE is expressed in diverse tissues such as lung, heart, kidney, brain, and skeletal muscle and in a variety of cells including endothelial cells, macrophages/monocytes, neutrophils, and lymphocytes (Brett et al. 1993; Ding and Keller 2005; Neeper et al. 1992). RAGE has been implicated in the pathogenesis of diverse diseases such as diabetes, cardiovascular disorders, arthritis, cancers and neurological disorders (Yan et al. 2009). Interactions of AGEs with their receptors alter cell function through the generation of free radicals (Schmidt et al. 1994a, b). In diabetes, interaction of AGEs with RAGE induces oxidative stress and inflammatory reactions thereby resulting in vascular damage and related complications (Yamagishi 2011). RAGE also plays an important role in the progression of atherosclerosis through oxidative stress and proinflammatory responses (Sun et al. 2009). Expression of RAGE in synovial tissue, T cells, B cells and macrophages of arthritic patients implies its significance in inflammatory joint disorders (Drinda et al. 2005). Overexpression of RAGE has also been reported in various types of cancers such as pancreatic, gastric, breast, lung cancers and lymphoma (Logsdon et al. 2007). Knockdown of RAGE expression was shown to inhibit ductal neoplasia in an animal model of pancreatic cancer (DiNorcia et al. 2012). A recent study by Liang et al. reported that the inactivation of RAGE in colorectal cancer cells reduced angiogenesis (Liang et al. 2011). The signaling events mediated by RAGE are complex due to the diversity of its ligands and their diverse effects mediated in different cell types. AGE/RAGE signaling in endothelial cells is reported to modulate oxidative stress, inflammation, apoptosis, autophagy, endothelial-mesenchymal-transition, endothelial permeability and dysfunction (Toma et al. 2009; Xu et al. 2010; Li et al. 2011; Xie et al. 2011; Ma et al. 2010; Del Turco et al. 2011). In smooth muscle cells, AGE/RAGE interaction leads to generation of reactive oxygen species, autophagy, proliferation and calcification (Yoon et al. 2009; Hu et al. 2012; Yuan et al. 2011; Tanikawa et al. 2009). AGE/RAGE signaling is reported to mediate proliferation in lymphocytes (Takahashi et al. 2010). In fibroblasts, it induces migration, inflammation and apoptosis (Liu et al. 2010; Shimoda et al. 2011). A diverse array of molecules and signaling modules were identified to be activated by RAGE depending on the intensity and duration of RAGE ligation. Specific signaling modules such as ERK1/2 (Lander et al. 1997), p38 MAPK (Lander et al. 1997), CDC42/RAC (Bondeva et al. 2011), SAPK/JNK (Hu et al. 2012) and NF-κB (Liu et al. 2010) have been shown to be triggered by AGE/RAGE interaction in different cell types. Currently, there are no resources, which contain RAGE signaling pathway data for visualization and analysis. Therefore, we have gathered signaling pathway reactions induced by AGE/RAGE interaction in diverse cell types and tissues from literature. We have also cataloged genes transcriptionally regulated by AGE/RAGE system in humans along with their transcriptional regulators. We have provided the AGE/RAGE signaling pathway data to scientific community through NetPath (http://www.netpath.org), a resource of signaling pathways developed by us (Kandasamy et al. 2010).

A pathway map of glutamate metabolism

Glutamate metabolism plays a vital role in biosynthesis of nucleic acids and proteins. It is also associated with a number of different stress responses. Deficiency of enzymes involved in glutamate metabolism is associated with various disorders including gyrate atrophy, hyperammonemia, hemolytic anemia, γ-hydoxybutyric aciduria and 5-oxoprolinuria. Here, we present a pathway map of glutamate metabolism representing metabolic intermediates in the pathway, 107 regulator molecules, 9 interactors and 3 types of post-translational modifications. This pathway map provides detailed information about enzyme regulation, protein-enzyme interactions, post-translational modifications of enzymes and disorders due to enzyme deficiency. The information included in the map was based on published experimental evidence reported from mammalian systems.

A knowledgebase resource for interleukin-17 family mediated signaling.

Interleukin-17 (IL-17) belongs to a relatively new family of cytokines that has garnered attention as the signature cytokine of Th17 cells. This cytokine family consists of 6 ligands, which bind to 5 receptor subtypes and induce downstream signaling. Although the receptors are ubiquitously expressed, cellular responses to ligands vary across tissues. The cytokine family is associated with various autoimmune disorders including rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, asthma and psoriasis in addition to being implicated in the pathogenesis of cancer. In addition, this family plays a role in host defense against bacterial and fungal infections. The signaling mechanisms of the IL-17 family of proinflammatory cytokines are not well explored. In this study, we present a resource of literature-annotated reactions induced by IL-17. The reactions are catalogued under 5 categories, namely; molecular association, catalysis, transport, activation/inhibition and gene regulation. A total of 93 molecules and 122 reactions have been annotated. The IL-17 pathway is freely available through NetPath, a resource of signal transduction pathways previously developed by our group.

Signaling Network Map of Endothelial TEK Tyrosine Kinase

TEK tyrosine kinase is primarily expressed on endothelial cells and is most commonly referred to as TIE2. TIE2 is a receptor tyrosine kinase modulated by its ligands, angiopoietins, to regulate the development and remodeling of vascular system. It is also one of the critical pathways associated with tumor angiogenesis and familial venous malformations. Apart from the vascular system, TIE2 signaling is also associated with postnatal hematopoiesis. Despite the involvement of TIE2-angiopoietin system in several diseases, the downstream molecular events of TIE2-angiopoietin signaling are not reported in any pathway repository. Therefore, carrying out a detailed review of published literature, we have documented molecular signaling events mediated by TIE2 in response to angiopoietins and developed a network map of TIE2 signaling. The pathway information is freely available to the scientific community through NetPath, a manually curated resource of signaling pathways. We hope that this pathway resource will provide an in-depth view of TIE2-angiopoietin signaling and will lead to identification of potential therapeutic targets for TIE2-angiopoietin associated disorders.

A network map of Interleukin-10 signaling pathway.

Interleukin-10 (IL-10) is an anti-inflammatory cytokine with important immunoregulatory functions. It is primarily secreted by antigen-presenting cells such as activated T-cells, monocytes, B-cells and macrophages. In biologically functional form, it exists as a homodimer that binds to tetrameric heterodimer IL-10 receptor and induces downstream signaling. IL-10 is associated with survival, proliferation and anti-apoptotic activities of various cancers such as Burkitt lymphoma, non-Hodgkins lymphoma and non-small scell lung cancer. In addition, it plays a central role in survival and persistence of intracellular pathogens such as Leishmania donovani, Mycobacterium tuberculosis and Trypanosoma cruzi inside the host. The signaling mechanisms of IL-10 cytokine are not well explored and a well annotated pathway map has been lacking. To this end, we developed a pathway resource by manually annotating the IL-10 induced signaling molecules derived from literature. The reactions were categorized under molecular associations, activation/inhibition, catalysis, transport and gene regulation. In all, 37 molecules and 76 reactions were annotated. The IL-10 signaling pathway can be freely accessed through NetPath, a resource of signal transduction pathways previously developed by our group.

Phosphotyrosine profiling identifies ephrin receptor A2 as a potential therapeutic target in esophageal squamous‐cell carcinoma

Esophageal squamous‐cell carcinoma (ESCC) is one of the most common malignancies in Asia. Currently, surgical resection of early‐stage tumor is the best available treatment. However, most patients present late when surgery is not an option. Data suggest that chemotherapy regimens are inadequate for clinical management of advanced cancer. Targeted therapy has emerged as one of the most promising approaches to treat several malignancies. A prerequisite for developing targeted therapy is prior knowledge of proteins and pathways that drive proliferation in malignancies. We carried out phosphotyrosine profiling across four different ESCC cell lines and compared it to non‐neoplastic Het‐1A cell line to identify activated tyrosine kinase signaling pathways in ESCC. A total of 278 unique phosphopeptides were identified across these cell lines. This included several tyrosine kinases and their substrates that were hyperphosphorylated in ESCC. Ephrin receptor A2 (EPHA2), a receptor tyrosine kinase, was hyperphosphorylated in all the ESCC cell lines used in the study. EPHA2 is reported to be oncogenic in several cancers and is also known to promote metastasis. Immunohistochemistry‐based studies have revealed EPHA2 is overexpressed in nearly 50% of ESCC. We demonstrated EPHA2 as a potential therapeutic target in ESCC by carrying out siRNA‐based knockdown studies. Knockdown of EPHA2 in ESCC cell line TE8 resulted in significant decrease in cell proliferation and invasion, suggesting it is a promising therapeutic target in ESCC that warrants further evaluation.

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.