Gillian Murphy, Ph.D.
Professor, Department of Oncology, University of Cambridge, UK
A novel strategy for targeting metalloproteinases in cancer
Epithelial tumours evolve in a multi-step manner, involving both inflammatory and mesenchymal cells. Although intrinsic factors drive malignant progression, the influence of the micro-environment of neoplastic cells is a major feature of tumorigenesis. Extracellular proteinases, notably the metalloproteinases, are key players in the regulation of this cellular environment, acting as major effectors of both cell-cell and cell-extracellular matrix (ECM) interactions. They are involved in modifying ECM integrity, growth factor availability and the function of cell surface signalling systems, with consequent effects on cellular differentiation, proliferation and apoptosis.This has made metalloproteinases important targets for therapeutic interventions in cancer and small molecule inhibitors focussed on chelation of the active site zinc and binding within the immediate active site pocket were developed. Â These were not successful in early clinical trials due to the relative lack of specificity and precise knowledge of the target proteinase(s) in specific cancers. We can now appreciate that it is essential that we understand the relative roles of the different enzymes (of which there are over 60) in terms of their pro and anti tumour activity and their precise sites of expression The next generations of metalloproteinase inhibitors need the added specificity that might be gained from an understanding of the structure of individual active sites and the role of extra catalytic domains in substrate binding and other aspects of their biology. We have prepared scFv antibodies to the extra catalytic domains of two membrane metalloproteinases, MMP-14 and ADAM17, that play key roles in the tumour microenvironment. Our rationale and experiences with these agents will be presented in more detail.
Sanjeeva Srivastava, Ph.D.
Assistant Professor, Proteomics Lab, IIT-Bombay, India
Identification of Potential Early Diagnostic Biomarkers for Gliomas and Various Infectious Diseases using Proteomic TechnologiesÂ
The spectacular advancements achieved in the field of proteomics research during the last decade have propelled the growth of proteomics for clinical research. Recently, comprehensive proteomic analyses of different biological samples such as serum or plasma, tissue, CSF, urine, saliva etc. have attracted considerable attention for the identification of protein biomarkers as early detection surrogates for diseases (Ray et al., 2011). Biomarkers are biomolecules that can be used for early disease detection, differentiation between closely related diseases with similar clinical manifestations as well as aid in scrutinizing disease progression. Our research group is performing in-depth analysis of alteration in human proteome in different types of brain tumors and various pathogenic infections to obtain mechanistic insight about the disease pathogenesis and host immune responses, and identification of surrogate protein markers for these fatal human diseases.
Applying 2D-DIGE in combination with MALDI-TOF/TOF MS we have analyzed the serum and tissue proteome profiles of glioblastoma multiforme; the most common and lethal adult malignant brain tumor (Gollapalli et al., 2012) (Figure 1). Results obtained were validated by employing different immunoassay-based approaches. In serum proteomic analysis we have identified some interesting proteins like haptoglobin, ceruloplasmin, vitamin-D binding protein etc. Moreover, proteomic analysis of different grades (grade-I to IV) of gliomas and normal brain tissue was performed and differential expressions of quite a few proteins such as SIRT2, GFAP, SOD, CDC42 have been identified, which have significant correlation with the tumor growth. While proteomic analysis of cerebrospinal fluid from low grade (grade I & II) vs. high grade (grade III & IV) gliomas revealed modulation of CSF levels of apolipoprotein E, dickkopf related protein 3, vitamin D binding protein and albumin in high grade gliomas. The prospective candidates identified in our studies provide a mechanistic insight of glioma pathogenesis and identification of potential biomarkers. We are also studying the role of JAK/STAT interactome and therapeutic potential of STAT3 inhibitors in gliomas using proteomics approach. Several candidates of the JAK/STAT interactome were identified with altered expression and a significant correlation was observed between STAT3 and PDK1 transcript expression level.
We have also investigated the changes in human serum proteome in different infectious diseases including falciparum and vivax malaria (Ray et al., 2012a; Ray et al., 2012b), dengue (Ray et al., 2012c) and leptospirosis (Srivastava et al., 2012). Although, quite a few serum proteins were found to be commonly altered in different infectious diseases and might be a consequence of inflammation mediated acute phase response signaling, uniquely modulated candidates were identified in each pathogenic infection indicating the some inimitable responses. Further, a panel of identified proteins consists of six candidates; serum amyloid A, hemopexin, apolipoprotein E, haptoglobin, retinol-binding protein and apolipoprotein A-I was used to build statistical sample class prediction models employing PLSDA and other classification methods to predict the clinical phenotypic classes and 91.37% overall prediction accuracy was achieved (Figure 2). ROC curve analysis was carried out to evaluate the individual performance of classifier proteins. The excellent discrimination among the different disease groups on the basis of differentially expressed proteins demonstrates the potential diagnostic implications of this analytical approach.
Keywords: Diagnostic biomarkers, Gliomas, Infectious Diseases, Proteomics, Serum proteome
Acknowledgments: This disease biomarker discovery research was supported by Department of Biotechnology, India grant (No. BT/PR14359/MED/30/916/2010), Board of Research in Nuclear Sciences (BRNS) DAE young scientist award (2009/20/37/4/BRNS) and a startup grant 09IRCC007 from the IIT Bombay. The active support from Advanced Center for Treatment Research and Education in Cancer (ACTREC), Tata Memorial Hospital (TMH), and Seth GS Medical College and KEM Hospital Mumbai, India in clinical sample collection process is gratefully acknowledged.
References :
- Ray S, Reddy PJ, Jain R, Gollapalli K. Moiyadi A, Srivastava S. Proteomic technologies for the identification of disease biomarkers in serum: advances and challenges ahead. Proteomics 11: 2139-61, 2011.
- Gollapalli K, Ray S, Srivastava R, Renu D, Singh P, Dhali S, Dikshit JB, Srikanth R, Moiyadi A, Srivastava S. Investigation of serum proteome alterations in human glioblastoma multiforme. Proteomics 12(14): 2378-90, 2012.
- Ray S, Renu D, Srivastava R, Gollapalli K, Taur S, Jhaveri T, Dhali S, Chennareddy S, Potla A, Dikshit JB, Srikanth R, Gogtay N, Thatte U, Patankar S, Srivastava S. Proteomic investigation of falciparum and vivax malaria for identification of surrogate protein markers. PLoS One 7(8): e41751, 2012a.
- Ray S, Kamath KS, Srivastava R, Raghu D, Gollapalli K, Jain R, Gupta SV, Ray S, Taur S, Dhali S, Gogtay N, Thatte U, Srikanth R, Patankar S, Srivastava S. Serum proteome analysis of vivax malaria: An insight into the disease pathogenesis and host immune response. J Proteomics 75(10): 3063-80, 2012b.
- Srivastava R, Ray S, Vaibhav V, Gollapalli K, Jhaveri T, Taur S, Dhali S, Gogtay N, Thatte U, Srikanth R, Srivastava S. Serum profiling of leptospirosis patients to investigate proteomic alterations. J Proteomics 76: 56-68, 2012.
- Ray S, Srivastava R, Tripathi K, Vaibhav V, Srivastava S. Serum proteome changes in dengue virus-infected patients from a dengue-endemic area of India: towards new molecular targets? OMICS 16(10): 527-36, 2012c.
* Correspondence: Dr. Sanjeeva Srivastava, Department of Biosciences and Bioengineering, IIT Bombay, Mumbai 400 076, India: E-mail: sanjeeva@iitb.ac.in; Phone: +91-22-2576-7779, Fax: +91-22-2572-3480
Andrey Panteleyev, Ph.D.
Vice Chair, Division of Molecular Biology, NBICS Centre-Kurchatov Institute, Moscow, Russia
The system of PAS proteins (HIF and AhR) as an interface between environment and skin homeostasis
Regulation of normal skin functions as well as etiology of many skin diseases are both tightly linked to the environmental impact. Nevertheless, molecular aspects of skin-environment communication and mechanisms coordinating skin response to a plurality of environmental stressors remain poorly understood.
Our studies along with the work of other groups have identified the family of PAS dimeric transcription factors as an essential sensory and regulatory component of communication between skin and the environment. This protein family comprises a number of hypoxia-induced factors (HIF-alpha proteins), aryl hydrocarbon receptor (AhR), AhR nuclear translocator (ARNT), and several proteins implicated in control of rhythmic processes (Clock, Period, and Bmal proteins). Together, various PAS proteins (and first of all ARNT – as the central dimerization partner in the family) control such pivotal aspects of cell physiology as drug/xenobiotic metabolism, hypoxic and UV light response, ROS activity, pathogen defense, overall energy balance and breathing pathways.
In his presentation Dr. Panteleyev will focus on the role of ARNT activity and local hypoxia in control of keratinocyte differentiation and cornification. His recent work revealed that ARNT negatively regulates expression of late differentiation genes through modulation of amphiregulin expression and downstream alterations in activity of EGFR pathway. All these effects are highly dependent on epigenetic mechanisms such as histone deacetylation. Characterisation of hypoxia as a key microenvironmental factor in the skin and the role of HIF pathway in control of dermal vasculature and epidermal functions is another major focus of Dr. Panteleyev’s presentation.
In general, the studies of Dr. Panteleyev’s laboratory provide an insight into the PAS-dependent maintenance of skin homeostasis and point to the potential role of these proteins in pathogenesis of environmentally-modulated skin diseases such as barrier defects, desquamation abnormalities, psoriasis, etc.
John Stanley, Satheesh Babu, Ramacahandran T and Bipin Nair
Pt-Pd decorated TiO2 nanotube array for the non-enzymatic determination of glucose in neutral medium
Rapidly expanding diabetic population and the complications associated with elevated glycemic levels necessitates the need for a highly sensitive, selective and stable blood glucose measurement strategy. The high sensitivity and selectivity of enzymatic sensors together with viable manufacturing technologies such as screen-printing have made a great social and economic impact. However, the intrinsic nature of the enzymes leads to lack of stability and consequently reduces shelf life and imposes the need for stringent storage conditions. As a result much effort has been directed towards the development of ‘enzyme-free’ glucose sensors (Park et al. 2006). In this paper, a non-enzymatic amperometric sensor for selective and sensitive direct electrooxidation of glucose in neutral medium was fabricated based on Platinum-Palladium (Pt–Pd) nanoparticle decorated titanium dioxide (TiO2) nanotube arrays. Highly ordered TiO2 nanotube arrays were obtained using a single step anodization process (Grimes C A and Mor G K 2009) over which Pt–Pd nanoparticles where electrochemically deposited. Scanning Electron Microscopy (SEM) analysis revealed the diameter of the TiO2 nanotubes to be approximately 40 nm. Elemental analysis after electrochemical deposition confirms the presence of Pt–Pd. Electrochemical characterization of the sensor was carried out using cyclic voltammetry technique (−1.0 to +1.0V) in phosphate buffer saline (PBS) pH 7.4. All further glucose oxidation studies were performed in PBS (pH 7.4). The sensor exhibited good linear response towards glucose for a concentration range of 1 μM to 20mM with a linear regression coefficient of R = 0.998. The electrodes are found to be selective in the presence of other commonly interfering molecules such as ascorbic acid, uric acid, dopamine and acetamidophenol. Thus a nonenzymatic sensor with good selectivity and sensitivity towards glucose in neutral medium has been developed.
Akhilesh Pandey, Ph.D.
Professor, Johns Hopkins University School of Medicine, Baltimore, USA
A draft map of the human proteome
We have generated a draft map of the human proteome through a systematic and comprehensive analysis of normal human adult tissues, fetal tissues and hematopoietic cells as an India-US initiative. This unique dataset was generated from 30 histologically normal adult tissues, fetal tissues and purified primary hematopoietic cells that were analyzed at high resolution in the MS mode and by HCD fragmentation in the MS/MS mode on LTQ-Orbitrap Velos/Elite mass spectrometers. This dataset was searched against a 6-frame translation of the human genome and RNA-Seq transcripts in addition to standard protein databases. In addition to confirming a large majority (>16,000) of the annotated protein-coding genes in humans, we obtained novel information at multiple levels: novel protein-coding genes, unannotated exons, novel splice sites, proof of translation of pseudogenes (i.e. genes incorrectly annotated as pseudogenes), fused genes, SNPs encoded in proteins and novel N-termini to name a few. Many proteins identified in this study were identified by proteomic methods for the first time (e.g. hypothetical proteins or proteins annotated based solely on their chromosomal location). We have generated a catalog of proteins that show a more tissue-restricted pattern of expression, which should serve as the basis for pursuing biomarkers for diseases pertaining to specific organs. This study also provides one of the largest sets of proteotypic peptides for use in developing MRM assays for human proteins. Identification of several novel protein-coding regions in the human genome underscores the importance of systematic characterization of the human proteome and accurate annotation of protein-coding genes. This comprehensive dataset will complement other global HUPO initiatives using antibody-based as well as MRM mass spectrometry-based strategies. Finally, we believe that this dataset will become a reference set for use as a spectral library as well as for interesting interrogations pertaining to biomedical as well as bioinformatics questions.