Krishnakumar Menon, Ph.D.
Associate Professor, Centre for Nanosciences & Molecular Medicine, Amrita University, Kochi, India
A Far-Western Clinical Proteomics Approach to Detect Molecules of Clinical and Pathological Significance in the Neurodegenerative Disease Multiple Sclerosis.
Multiple Sclerosis (MS), an autoimmune neurodegenerative disorder of the central nervous system. The disease affects young adults at their prime age leading to severe debilitation over several years. Despite advances in MS research, the cause of the disease remains elusive. Thus, our objective is to identify novel molecules of pathological and diagnostic significance important in the understanding, early diagnosis and treatment of MS. Biological fluids such as cerebrospinal fluid (CSF), that bathe the brain serve as a potential source for the identification of pathologically significant autoantibody reactivity in MS. In this regard, we report the development of an unbiased clinical proteomics approach for the detection of reactive CSF molecules that target brain proteins from patients with MS. Proteins of myelin and myelin-axolemmal complexes were separated by two-dimensional gel electrophoresis, blotted onto membranes and probed separately with biotinylated unprocessed CSF samples. Protein spots that reacted specifically to MS-CSF’s were further analyzed by matrix assisted laser desorption ionization-time-of-flight time-of-flight mass spectrometry. In addition to previously reported proteins found in MS, we have identified several additional molecules involved in mitochondrial and energy metabolism, myelin gene expression and/or cytoskeletal organization. Among these identified molecules, the cellular expression pattern of collapsin response mediator protein-2 and ubiquitin carboxy-terminal hydrolase L1 were investigated in human chronic-active MS lesions by immunohistochemistry. The observation that in multiple sclerosis lesions phosphorylated collapsin response mediator protein-2 was increased, whereas Ubiquitin carboxy-terminal hydrolase L1 was down-regulated, not only highlights the importance of these molecules in the pathology of this disease, but also illustrates the use of our approach in attempting to decipher the complex pathological processes leading to multiple sclerosis and other neurodegenerative diseases. Further, we show that in clinicaly isolated syndrome (CIS), we could identify important molecules that could serve as an early diagnostic biomarker in MS.
K. P. Mohanakumar, Ph.D.
Chief Scientist, Cell Biology & Physiology Division, Indian Institute of Chemical Biology, Kolkata
Neuroprotective and neurodestructive effects of Ayurvedic drug constituents: Parkinson’s disease
The present study reports the good and the bad entities in an Indian traditional medicine used for treating Parkinson’s disease (PD). A prospective clinical trial on the effectiveness of Ayurvedic medication in a population of PD patients revealed significant benefits, which has been attributed to L-DOPA present in the herbs [1]. Later studies revealed better benefits with one of the herbs alone, compared to pure L-DOPA in a clinical trial conducted in UK [2], and in several studies conducted on animal models of PD in independent laboratories world over [3-5]. We have adapted strategies to segregate molecules from the herb, and then carefully removed L-DOPA contained therein, and tested each of these sub-fractions for anti-PD activity in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, rotenone and 6-hydroxydopamine -induced parkinsonian animal models, and transgenic mitochondrial cybrids. We report here two classes of molecules contained in the herb, one of which possessed severe pro-parkinsonian (phenolic amine derivatives) and the other having excellent anti-parkinsonian potential (substituted tetrahydroisoquinoline derivatives). The former has been shown to cause severe dopamine depletion in the striatum of rodents, when administered acutely or chronically. It also caused significant behavioral aberrations, leading to anxiety and depression [6]. The latter class of molecules administered in PD animal model [7], caused reversal of behavioral dysfunctions and significant attenuation of striatal dopamine loss. These effects were comparable or better than the effects of the anti-PD drugs, selegiline or L-DOPA. The mechanism of action of the molecule has been found to be novel, at the postsynaptic receptor signaling level, as well as cellular α-synuclein oligomerization and specifically at mitochondria. The molecule helped in maintaining mitochondrial ETC complex activity and stabilized cellular respiration, and mitochondrial fusion-fission machinery with specific effect on the dynamin related protein 1. Although there existed significant medical benefits that could be derived to patients due to the synergistic actions of several molecules present in a traditional preparation, accumulated data in our hands suggest complicated mechanisms of actions of Ayurvedic medication. Our results also provide great hope for extracting, synthesizing and optimizing the activity of anti-parkinsonian molecules present in traditional Ayurvedic herbs, and for designing novel drugs with novel mechanisms of action.
- N, Nagashayana, P Sankarankutty, MRV Nampoothiri, PK Mohan and KP Mohanakumar, J Neurol Sci. 176, 124-7, 2000.
- Katzenschlager R, Evans A, Manson A, Patsalos PN, Ratnaraj N, Watt H, Timmermann L, Van der Giessen R, Lees AJ. J Neurol Neurosurg Psychiatry.75, 1672-7, 2004.
- Manyam BV, Dhanasekaran M, Hare TA. Phytother Res. 18, 706-12, 2004.
- Kasture S, Pontis S, Pinna A, Schintu N, Spina L, Longoni R, Simola N, Ballero M, Morelli M. Neurotox Res. 15, 111-22, 2009.
- Lieu CA, Kunselman AR, Manyam BV, Venkiteswaran K, Subramanian T. Parkinsonism Relat Disord.16, 458-65, 2010.
- T Sengupta and KP Mohanakumar, Neurochem Int. 57, 637-46, 2010.
- T Sengupta, J Vinayagam, N Nagashayana, B Gowda, P Jaisankar and KP Mohanakumar, Neurochem Res 36, 177-86, 2011
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.
Tejaswini Subbannayya, Nandini A. Sahasrabuddhe, Arivusudar Marimuthu, Santosh Renuse, Gajanan Sathe, Srinivas M. Srikanth, Mustafa A. Barbhuiya, Bipin Nair, Juan Carlos Roa, Rafael Guerrero-Preston, H. C. Harsha, David Sidransky, Akhilesh Pandey, T. S. Keshava Prasad and Aditi Chatterjee
Proteomic profiling of gallbladder cancer secretome – a source for circulatory biomarker discovery
Gallbladder cancer (GBC) is the fifth most common cancer of the gastrointestinal tract and one of the common malignancies that occur in the biliary tract (Misra et al. 2006; Lazcano-Ponce et al. 2001). It has a poor prognosis with survival of less than 5 years in 90% of the cases (Misra et al. 2003). The etiology is ill-defined. Several risk factors have been reported including cholelithiasis, obesity, female gender and exposure to carcinogens (Eslick 2010; Kumar et al. 2006). Poor prognosis in GBC is mainly due to late presentation of the disease and lack of reliable biomarkers for early diagnosis. This emphasizes the need to identify and characterize cancer biomarkers to aid in the diagnosis and prognosis of GBC. Secreted proteins are an important class of molecules which can be detected in body fluids and has been targeted for biomarker discovery. There are challenges faced in the proteomic interrogation of body fluids especially plasma such as low abundance of tumor secreted proteins, high complexity and high abundance of other proteins that are not released by the tumor cells (Tonack et al. 2009). Profiling of conditioned media from the cancer cell lines can be used as an alternate means to identify secreted proteins from tumor cells (Kashyap et al. 2010; Marimuthu et al. 2012). We analyzed the invasive property of 7 GBC cell lines (SNU-308, G-415, GB-d1, TGBC2TKB, TGBC24TKB, OCUG-1 and NOZ). Four cell lines were selected for analysis of the cancer secretome based on the invasive property of the cells. We employed isobaric tags for relative and absolute quantitation (iTRAQ) labeling technology coupled with high resolution mass spectrometry to identify and characterize secretome from the panel of 4GBC cancer cells mentioned above. In total, we have identified around 2,000 proteins of which 175 were secreted at differential abundance across all the four cell lines. This secretome analysis will act as a reservoir of candidate biomarkers. Currently, we are investigating and validating these candidate markers from GBC cell secretome. Through this study, we have shown mass spectrometry-based quantitative proteomic analysis as a robust approach to investigate secreted proteins in cancer cells.