Education:

Graduate:

Ph.D. in Biochemistry; University of Bhopal and University of Bonn, 1997
Advisor: Dr. P.S. Bisen
Thesis: Calcium dinitrogen fixation and calmodulin in cyanobacteria

Postgraduate:

• NIH Postdoctoral Fellow, Gene Therapy and Therapeutics Branch, National Institute of Dental and Craniofacial Research. The National Institutes of Health, Bethesda, MD. 1999-2002.
• Postdoctoral Research Associate, Department of Pharmacology, Medical College of Wisconsin, Milwaukee, WI. 1998-1999.
• DAAD Fellow, Department of Molecular Biochemistry. University of Bonn. Germany. 1996-1998.

Research:

My research has been focused towards identification and regulation of the molecular mechanisms involved in Ca²⁺ signaling, which is activated by the emptying of intracellular Ca²⁺ stores, referred to as store-operated Ca²⁺ entry (SOCE). This influx pathway is present in all cell types. However, the mechanism by which the depletion of the internal Ca²⁺ store is communicated to the plasma membrane in order to activate or inactivate Ca²⁺ influx is not yet fully understood. A major drawback in understanding the mechanism of SOCE is the lack of information regarding the identity of the channel. Recently, homologs of the Drosophila trp genes have been suggested as components of the store operated calcium channel (SOCC). Presently, seven mammalian trps have been identified, which appear to be members of a large multi-gene family, including polycystins, vanilloid receptors and mucolipin. During the last three years, my work has primarily involved in identifying the physiological function of Trp1 and Trp3 in the regulation of SOCE. We have studied the regulation of the Trp proteins in terms of their assembly, in the plasma membrane and structure- function relationship.

Towards this we expressed Trp1 in cultured cell lines as well as in vivo in rat salivary glands using adenoviral system. Cell's expressing Trp1 showed higher level of Trp1 protein and displayed increased SOCE. Transfection of HSG cells with antisence htrp1, decreased endogenous Trp1 protein and significantly reduced calcium influx. Importantly, three days post-infection of the glands with AdTrp1, showed a 5-fold increase in philocarpine-stimulated fluid secretion from SMGs. Further, there was a corresponding increase in hTrp1 expression and the protein was localized in the basolateral region of acinar cells. Measurement of [Ca²⁺]i mobilization in dispersed acini isolated from SMG infected with AdTrp1 showed an increase in SOCE in cells expressing Trp1, suggesting a role of Trp1 and [Ca²⁺]i in salivary gland fluid secretion. These studies represented the first "in vivo" assessments of Trp1 function.

Our studies also demonstrated that like Drosophila Trp, mammalian Trp1 and Trp3 were assembled in signaling complexes associated with caveolin-scaffolding lipid raft domain(s). We suggested that caveolar microdomains provide a scaffold for assembly of key Ca²⁺ signaling proteins into a complex and coordination of the molecular interactions could lead to the activation of SOCE. We have also identified that the c-terminus of Trp1 is involved in Ca²⁺-dependent feedback inactivation of SOCE. Trp1 expressing cells showed a Ca²⁺ dependent inactivation, whereas, cells expressing Trp1DC cells showed no inactivation. CaM overlay assays, demonstrated that calmodulin interacted with full length Trp1 protein, but not with Trp1DC. Expression of mutant calmodulin, lacking calcium-binding properties also decreased Ca²⁺ dependent inactivation of SOCE, suggesting that CaM functions as the calcium sensor for SOCE. Thus we concluded that binding of calmodulin to the c-terminus of Trp1 mediates Ca²⁺ dependent inactivation of SOCE.

Ongoing studies address the interaction and regulation of Trp1 and Trp3 with other proteins. We have shown that Trp3 is also associated with a signalplex including key Ca2+ signaling proteins and caveolin. Further, it demonstrates that conditions, which stabilize cortical actin, induce loss of Trp3 activity due to internalization of the Trp3-signaling complex. We are using yeast-two hybrid system to further study these interactions. Moreover, future studies include biochemical and functional assays to test the physiological relevance of the interaction.

Future Directions:

Calcium is a major intracellular messenger in excitable and non-excitable cells. In excitable tissues like brain, endocrine glands and neurosensory tissues, calcium serves numerous function. The physiological function of all these tissues is uniquely regulated by changes in cytosolic calcium, which are achieved both via Ca²⁺ influx across the plasma membrane and Ca²⁺ release from internal stores. Ca²⁺ also plays a direct role in controlling the transcriptional events. The role of Ca²⁺ in differential gene expression is still in its infancy but is rapidly developing into an active area of research. The role of Ca²⁺ influx via voltage-gated Ca²⁺ channels in the activation of various early genes has been described in neuronal cells. However, the role of Trps in neuronal cells remains to be investigated. All Trp channels are expressed in relatively high levels in excitable cells, but their functional role has not yet been characterized. Trp3 for example, has been associated with BDNF mediated signaling in brain and Trp7 showed a higher expression level in the eye. Therefore I would like to focus my research on the role of Trps channels in retinal function and gene regulation. The work I have described above provides me, with sufficient expertise to study calcium signaling in the vertebrate retina. This is an important but an as yet not fully characterized. I believe an understanding of the calcium signaling and calcium dependent gene expression will provide a better understanding of retinal dysfunction such as retinal degeneration, retinopathy, macular dystrophy and retinitis pigmentosa. Importantly, I will be having an opportunity to draw on resources and expertise from my present lab at NIH, along with my previous labs and will also collaborate with people in this field.

Based on the established role of Trp in Drosophila vision, I propose to study the role of Trps in neuronal tissues specifically in vision. In Drosophila rhabdomeres, (equivalent to eyes in mammals) light response leads to generation of IP₃ thereby; depleting internal calcium stores and followed by the activation of Trp channels. In human vision, calcium also plays a key role, but the role of SOCE is not yet established. Although reports about the identification of Trps in neuronal tissues are increasing, still not much is known about their possible function. Therefore a detail study using various Trps will be helpful in understanding the role of SOCC in neuronal tissues. Previously, I had identified that a domain of Ran binding protein-2 (RanBP2) mediates the docking of the nuclear export tranporter, exportin-1. Also another RanBP2 functional domain was found to specifically interact with microtubule-based motor proteins, kinesins. This indicates that RanBP2 is a dynamic scaffold protein and can act as an integrator (and modulator) of signaling pathways. Based on Drosophila signalplex model, it is possible that Trps could interact with RanBP2, as like INAD, RanBP2 is also known to interact with rhodopsin. Presently we have constructed adenovirus for most of Trps, which can be used to identify the role of Trps in vivo in mammalian retina. Also Trp knockout animals as well as knockout animals for rhodopsin are available, which will help, in further characterization of the role of Trp in vision.

The second major area, which I propose to study, is the expression of Trps and Trp -dependent gene regulation. To study the regulation of Trps, I would like to identify the promoter regions of different Trps and further study its regulation and expression pattern in adult retina during its development and in retinal diseases. Calcium is known to activate transcription factors. Calcium stimulates Ca²⁺-sensitive protein phosphatases calcineurin, which dephosphorylates other signaling proteins. Therefore it is possible that Trps can play a direct role in the activation of transcription factors. To investigate this major Trps in retinal tissues will be identified and Trp dependent gene regulation will be studied using gene array technologies.

In spite of growing interest in the role of Trps in SOCE, major questions still remain unanswered regarding the SOCE mechanism. One important question that I will continue to investigate is to identify key proteins that are needed for the regulation of Trps. In Drosophila rhabdomeres Trp is known to interact with a scaffolding protein INAD. INAD consists of 5 PDZ domains and interacts to a minimum of seven target proteins, which facilitate rapid activation of phototransduction. Studies from our lab and by others have shown that mammalian Trps also form multimeric complexes similar to Drosophila signalplex. However still the scaffold protein, which can tether these key regulatory proteins is not yet identified. To address this key question we have used multiple approach including yeast-two hybrid, GST pull down assay, micro array and other biochemical methods such as mass spectroscopy. Based on our initial screen we had identified few proteins, which specifically interact with Trp1 and Trp3 and could be important for their regulation. However the exact physiological function of these interactions needs to be investigated. These studies not only provide a better understanding of Trp function but will also help to understand the neurosensory mechanism in the retina, which is critical for vision.

Publications:

1. Pani B., Ong, H.L., Liu, X., Rauser, C., Ambudkar, I.S. and Singh, B.B (2008) Lipid Rafts Determine Clustering of STIM1 in ER-Plasma Membrane Junctions and regulation of SOCE. Journal of Biological Chemistry. Published April 22  2008
2. Pani, B and Singh, B.B. (2008) Dariers disease: a calcium-signaling perspective. Cellular and Molecular Life Sciences. 65(2):205-211.
3. Liu, X., Cheng, O., Bandyopadhyay B.C, Pani B., Dietrich, A., Paria, B., Swaim, W., Birnbaumer, L., Singh, B.B., and Ambudkar, I.S. (2007) An essential role for TRPC1 in store-operated Ca2+ entry and regulation of salivary gland fluid secretion. Proceedings National Academy of Science, USA. Oct 30; 104(44): 17542-17547.
4. Pani, B and Singh, B.B. (2007) Principles and applications of Proteomics: Molecular determination of TRPC1 target proteins. Text book on Molecular Biotechnology (Ajit Varma and Neeraj Verma eds) IK International Publishing house. (In press).
5. Ong, H.L., Liu, X, Atanasova, K-T., Singh, B.B., Bandyopadhyay, B., Swaim, W.D., Hegde, R.S., Sherman, A, and Ambudkar, I.S. (2007) Relocalization of STIM1 for activation of store-operated Ca2+ entry is determined by the depletion of subplasma membrane endoplasmic reticulum Ca2+ store. Journal of Biological Chemistry. 282, 12176-12185.
6. Labyed, Y., Kaabouch, N., Schultz, R.R., and Singh, B.B. (2007). “Automatic segmentation and band detection of protein images based on the standard deviation profile and its derivative,” IEEE Electro/Information Technology Proceedings, ISBN: 1424409411, pp. 669-673.
7. Ong, H.L., Cheng, K.T., Liu, X., Bandyopadhyay, B., Paria, B.C., Soboloff, J., Pani B., Gwasck, Y., Srikanth, S., Gill, D., Singh, B.B., and Ambudkar, I.S (2007). Dynamic assembly of TRPC1/STIM1/Orai1 ternary complex is involved in store-operated Ca2+ influx. Journal of Biological Chemistry. 282, 9105-9116.
8. Jara, J.H, Singh, B.B., Floden, A.M, Hansen, J, and Combs, C.K. (2007) Tumor necrosis factor alpha stimulates NMDA receptor activity in mouse cortical neurons resulting in ERK-dependent death. Journal of Neurochemistry. 100; 1407-1420.
9. Pani B., Cornatzer E., Cornatzer W., Shin DM., Pittelkow MR., Hovnanian, A., Ambudkar IS., and Singh B.B. (2006) Upregulation of TRPC1 following SERCA2 gene silencing promotes cell survival. Molecular Biology of the Cell. Aug 9; [Epub ahead of print].
10. Bollimuntha, S., Ebadi, M., and Singh, B.B (2006) TRPC1 protects human SH-SY5Y cells against salsolinol induced cytotoxicity by inhibiting apoptosis. Brain Research. 1099. 141-149.
11. Liu, X., Bandyopadhyay, B., Nakamoto, T., Singh, B.B., Liedtke, W., Melvin, J.E., and Ambudkar, I.S. (2006) A role of AQP5 in activation of TRPV4 by hypotonocity: concerted involvement of AQP5 and TRPV4 in regulation of cell volume recovery. Journal of Biological Chemistry. 281, 15485-15495.
12. Liu, X., Bandyopadhyay, B., Singh, B.B., Groschner, K., and Ambudkar, I.S. (2005) Molecular analysis of a store-operated and OAG sensitive non-selective cation channel: Heteromeric assembly of TRPC1- TRPC3. Journal of Biological Chemistry. 280, 21600-21606.
13. Bollimuntha, S., Cornatzer, E, and Singh, B.B (2005) Plasma membrane localization and function of TRPC1 depend on its interaction with tubulin in epithelium cells. Visual Neuroscience. 22 163-170.
14. Bollimuntha, S., Shavali, S., Sharma, S.K., Ebadi. M., and Singh, B.B (2005) TRPC1-mediated inhibition of MPP+ neurotoxicity in human SH-SY5Y neuroblastoma cells. Journal of Biological Chemistry. 280, 2132-2140.
15. Ebadi, M., Brown-Borg, H., Refaey, H.E., Singh, B.B., Garrett, S., Shavali, S and Sharma, SK (2005) Metallothionein-mediated neuroprotection in genetically engineered mice models of Parkinson’s disease and aging. Molecular Brain Research. 134, 67-75.
16. Singh, B.B., Lockwich, T., Bandyopadhyay, B., Liu, X., Bollimuntha, S., Brazer, SC., Combs, C., Das, S., Leenders, M., Sheng, Z., Knepper, M., Ambudkar, SV., and Indu S. Ambudkar.(2004) VAMP-2-dependent exocytosis is involved in plasma membrane insertion of TRPC3 channels and contributes to agonist-stimulated Ca,²⁺ influx. Molecular Cell 15, 635-646. A summary of this article was published in Science and Nature Cell Biology.
17. Itagaki, K., Kannan, K.B., Singh B.B. and Hauser C.J. (2004) Cytoskeletal reorganization internalizes multiple Transient Receptor Potential channels and blocks calcium entry into human neutrophils. Journal of Immunology 172, 601-607.
18. Ambudkar IS, Brazer SC, Liu X, Lockwich T, Singh B.B. (2004) Plasma membrane localization of TRPC channels: role of caveolar lipid rafts. Novartis Foundation Symposium, 258: 63-70.
19. Brazer, S.W., Singh B.B., Liu, X., Swaim, W & Ambudkar I.S (2003) caveolin-1 contributes to assembly of store-operated Ca²⁺ influx channels in regulating plasma membrane localization of TRPC1. Journal of Biological Chemistry. 278. 27208-15.
20. Singh, B.B*., Liu, X* & Ambudkar, I.S. (2003) Acidic amino acid residues in the S5-S6 region of TRPC1 contributes to store-operated Ca²⁺ influx. Journal of Biological Chemistry. 278. 11337-11343.
21. Singh, B.B., Liu, X., Tang, J., Zhu, M.X and Ambudkar, I.S. (2002) Calmodulin regulates Ca²⁺ -dependent feedback inhibition of store-operated Ca²⁺ influx by interaction with a site in the C-terminus of Trp1. Molecular Cell. 9. 739-750.
22. Singh, B.B., Zheng, C., Liu, X., Lockwich, T., Liao, D., Zhu, M., Birnbaumer, L and Ambudkar, I.S. (2001). Trp1-dependent enhancement of salivary gland fluid secretion: Role of store-operated calcium entry. FASEB Journal. 15 (9), 1652-1654. Singh, B.B., Lockwich, T., Liu, X and Ambudkar, I.S. (2001) Stabilization of cortical actin induces internalization of Trp3-associated caveolar Ca²⁺signaling complex and loss of Ca²⁺ influx without disruption of Trp3-IP₃R association. Journal of Biological Chemistry. 276: 45, 42401-42408.
23. Yunfei, C., Singh, B.B., Aslanukov, A., Zhao, H and Ferreira, P.A. (2001) The docking of heterotetrameric kinesins to RanBP2 is mediated via a novel RanBP2 domain. Journal of Biological Chemistry. 276: 45, 41594-41602
24. Singh, B.B; Curdt, I; Shomburg, D; Bisen, P.S and Bohme, H (2001) Valine 77 of heterocystous ferredoxin FdxH2 in Anabaena variabilis ATCC 29413 is critical for its oxygen sensitivity. Molecular and Cellular Biochemistry. 217, 137-142.
25. Singh, B.B., Liu, X., and Ambudkar, I.S (2000) Expression of truncated Trp1a: Evidence that the Trp1 C-terminus modulates store-operated Ca²⁺ entry. Journal of Biological Chemistry. 275, 36483 - 36486.
26. Lockwich, T., Liu, X., Singh, B.B., Jadlowiec, J. Weiland, S and Ambudkar I.S (2000). Assembly of Trp1 in a Signaling Complex Associated with Caveolin-Scaffolding Lipid Raft Domains. Journal of Biological Chemistry. 275, 11934-11942
27. Liu, X., Wang, W., Singh, B.B., Lockwich, T., Jadlowiec J., Connell, B.O., Wellner, R., Zhu, X and Ambudkar, I.S (2000) Trp1 a candidate protein for the store-operated Ca²⁺ influx Mechanism in salivary gland cells Journal of Biological Chemistry. 275, 3403-3411
28. Curdt, I., Singh, B.B., Jakoby, M., Hachtel, W and Bohme, H (2000) Identification of amino acid residues of nitrite reductase from Anabaena sp. PCC 7120 involved in ferredoxin binding. BBA Bioenergetics. 1543, 60-68.
29. Varma A., Singh B.B., Karnani N., Lichtenberg, H., Hoefer M., Magee B.B. and Prasad R. (2000) Molecular cloning and functional characterization of a glucose transporter, CAHGTI, of Candida albicans. FEMS Microbiology letters.182, 15-21
30. Xibao Liu; Singh, B.B and. Ambudkar, I.S (1999) ATP-dependent activation of K_Ca and ROMK-type K_ATP channels in human submandibular gland ductal cells. Journal of Biological Chemistry. 274, 25121-25129.
31. Singh, B.B., Patel, H.H., Ropeman, R., Schick, D and Ferreira, P.A (1999). The Zinc finger cluster domain of RanBP2 is a specific docking site for Nuclear Export Factor, Exportin-1. Journal of Biological Chemistry. 274, 37370-37378.
32. Singh, B.B; Curdt, I; Jakobs, C; Bisen, P.S and Bohme, H (1999) Identification of amino acids responsible for oxygen sensitivity of ferredoxins from Anabaena variabilis using site-directed mutagenesis BBA Bioenergetics.1412, 288-294.
33. Sengupta, L.K; Singh, B.B ; Mishra, R ; Pandey, P.K ; Singh, S ; Sengupta, S and Bisen, P.S (1998) Calcium- dependent metabolic regulations in prokaryotes indicate conserved nature of calmodulin gene. Indian Journal of Experimental Biology. 36, 136-147.
34. Singh, B.B; Pandey, P.K; Singh, S and Bisen, P.S (1997) Calcium induced physiological and biochemical changes in the cyanobacterium Nostoc MAC. Indian Journal of Experimental Biology. 35, 881-885.
35. Pandey, P.K; Singh, B.B; Mishra, R. and Bisen, P.S. (1996) Ca²⁺ uptake and its regulation in the cyanobacterium Nostoc MAC. Current Microbiology. 32; 332-335.
36. Singh, B.B; Pandey, P.K; Singh, S and Bisen, P.S (1996) Evidence for the nitrate assimilation - dependent nitrite excretion in cyanobacterium Nostoc MAC. World Journal of Microbiology and Biotechnology. 12. 285-287.
37. Singh, B.B; Pandey, P.K; Singh, S and Bisen, P.S (1996) Regulation of nitrate uptake and nitrite efflux in the cyanobacterium Nostoc MAC. Journal of Basic Microbiology. 36; 433-438
38. Singh, B.B; Pandey, P.K; Singh, S and Bisen, P.S. (1996) Calcium / calmodulin antagonist induced changes in the cyanobactrium Nostoc MAC. Physiology & Molecular Biology of Plants. 2; 75 - 78.
39. Singh,S ; Singh,B.B & Bisen, P.S. (1995) Copper induced changes in the urea uptake and urease activity in the cyanobacteria Anabaena doliolum and Anacystis nidulans : Interaction with sulpher containing amino acids. Biomedical and Environmental Science. 8: 158 - 163.
40. Singh, S ; Singh, B.B & Bisen, P.S. (1995) Role of ammonium assimilation in the urea inhibition of nitrogenase activity in cultured cyanobiont Nostoc ANTH. Indian Journal of Experimental Biology.33; 33 -39.
41. Pandey, P.K ; Singh, B.B ; Singh, S & Bisen, P.S. (1995) NO2- efflux and its regulation in the cyanobacterium Nostoc MAC Current Microbiology. 31; 119 -123.
42. Singh, S ; Singh, B.B & Bisen, P.S. (1994) Role of membrane potential in ammonium inhibition of nitrogenase activity in the cultured cyanobiont Nostoc ANTH. World Journal of Microbiology & Biotechnology. 10 : 600

Invited Book Chapters:

1. Bisen, P.S ; Singh, B.B ; Sengupta, L.K ; Mishra, R ; Sengupta, S ; Pandey, P.K & Singh, S (1997) Calcium- calmodulin : An overview with reference to microbes In : Microbiology in India; History Prospects and Applications (edited by A. Varma) IBH & Oxford Publications, New Delhi. pp 125-137.
2. Singh, B.B, S. Singh and P.S. Bisen (1998) who does diazotrophic cyanobacterium in its immobilized state liberate ammonium. (Ed.) V.K. Jain Wisdom Publishing House, New Delhi 352-358.