VDAC PROPERTIES ARE INFLUENCED BY THE SOURCE OF ITS PURIFICATION

  • Ashvini Kumar Dubey National Centre for Biological Sciences (NCBS-TIFR), GKVK Campus, Bellary Road, Bangalore-65 Department of Biotechnology, University of Mysore, Mysore 570006, India.
  • Ashwini Godbole Centre for Pharmacognosy, Pharmaceutics and Pharmacology, Institute of Ayurveda and Integrative Medicine, No.74/2, JarakbandeKaval, Post: Attur, Via Yelahanka, Bangalore - 560 064, India.
  • Sujitha Srinivasan Department of Genetics, School of Biological Sciences, Madurai Kamaraj University.
  • M. K. Mathew National Centre for Biological Sciences (NCBS-TIFR), GKVK Campus, Bellary Road, Bangalore-65

Abstract

Objectives: The Voltage Dependent Anion-Selective Channel (VDAC), the most abundant protein of the outer mitochondrial membrane (OMM), forms the major conduit for metabolite transport across this membrane. It has also been shown to be involved in cell death signalling through interaction with other proteins like Hexokinase and by mediating release of apoptogenic proteins like cyt c from mitochondria. As in case of other channel proteins, functional characterization of purified reconstituted protein by using electrophysiological techniques can be used in development of VDAC targeted drugs. Here we report electrophysiological properties of VDACs (one of the target for cancerous cells) purified from different sources.

Methods: Human VDAC1 and rice VDAC4 were heterologously expressed and purified from E. coli BL21 (DE3)-pLysS, while rat and yeast VDACs were purified from mitochondria. Electrophysiological studies of all VDACs were done by using BLM and the data was analysed by using pCLAMP 10 (Axon Instruments).

Results: VDACs purified from both the sources showed conserved voltage dependence and channel conductance, however they showed significant difference in dynamics. VDAC purified from mitochondria had relatively short occupancy of each electrophysiological state compared to protein purified from inclusion bodies.

Conclusion: Our results suggest that the source of purified protein could be critical for some aspects of channel function.

Keyword: VDAC, BLM, Mitochondria, Bacterial expression, Purification

Keywords: VDAC, BLM, Mitochondria, Bacterial expression, Purification

Downloads

Download data is not yet available.

Author Biographies

Ashvini Kumar Dubey, National Centre for Biological Sciences (NCBS-TIFR), GKVK Campus, Bellary Road, Bangalore-65 Department of Biotechnology, University of Mysore, Mysore 570006, India.
Department of Biochemistry, Biophysics and Bioinformatics
M. K. Mathew, National Centre for Biological Sciences (NCBS-TIFR), GKVK Campus, Bellary Road, Bangalore-65
Department of Biochemistry, Biophysics and Bioinformatics

References

1. Akl H, Bultynck G. Altered Ca(2+) signaling in cancer cells:proto-oncogenes and tumor suppressors targeting IP3 receptors. J Biochim Biophys Acta 2013;1835(2):180-93.
2. Israelson A, Arbel N, Da Cruz S, Ilieva H, Yamanaka K, Shoshan-Barmatz V, et al. Misfolded mutant SOD1 directly inhibits VDAC1 conductance in a mouse model of inherited ALS. J Neuron 2010;67(4):575-87.
3. Karachitos A, García Del Pozo JS, de Groot PWJ, Kmita H, Jordán J. Minocycline mediated mitochondrial cytoprotection:premises for therapy of cerebrovascular and neurodegenerative diseases. J Curr Drug Targets 2013;14(1):47-55.
4. Krasnov GS, Dmitriev AA, Lakunina VA, Kirpiy AA, Kudryavtseva AV. Targeting VDAC-bound hexokinase II:a promising approach for concomitant anti-cancer therapy. J Expert Opin Ther Targets 2013;17(10):1221-33.
5. Leanza L, Biasutto L, Managò A, Gulbins E, Zoratti M, Szabò I. Intracellular ion channels and cancer. J Front Physiol 2013;4:227.
6. Peixoto PM, Dejean LM, Kinnally KW. The therapeutic potential of mitochondrial channels in cancer, ischemia-reperfusion injury, and neurodegeneration. J Mitochondrion 2012;12(1):14-23.
7. Shoshan-Barmatz V, Mizrachi V. D. from structure to cancer therapy. Frontiers in oncology 2 p 164. 2012;1.
8. Veenman L, Shandalov Y, Gavish M. VDAC activation by the 18 kDa translocator protein (TSPO), implications for apoptosis. J Bioenerg Biomembr 2008;40(3):199-205.
9. Yadav, N. and D. Chandra, Mitochondrial and postmitochondrial survival signaling in cancer. J Mitochondrion 2013.
10. Goldin N, Arzoine L, Heyfets A, Israelson A, Zaslavsky Z, Bravman T, et al. Methyl jasmonate binds to and detaches mitochondria-bound hexokinase. J Oncogene 2008;27(34):4636-43.
11. Katsetos CD, Anni H, Dráber P. Mitochondrial dysfunction in gliomas. J Semin Pediatr Neurol 2013;20(3):216-27.
12. Kim JE, He Q, Chen Y, Shi C, Yu K. mTOR-targeted therapy:differential perturbation to mitochondrial membrane potential and permeability transition pore plays a role in therapeutic response. J Biochem Biophys Res Commun 2014;447(1):184-91.
13. Nakashima RA, Mangan PS, Colombini M, Pedersen PL. Hexokinase receptor complex in hepatoma mitochondria:evidence from N,N'-dicyclohexylcarbodiimide-labeling studies for the involvement of the pore-forming protein VDAC. J Biochemistry 1986;25(5):1015-21.
14. Pastorino JG, Hoek JB. Regulation of hexokinase binding to VDAC. J Bioenerg Biomembr 2008;40(3):171-82.
15. Plotz M. Disruption of the VDAC2-Bak interaction by Bcl-x(S) mediates efficient induction of apoptosis in melanoma cells. Cell death and differentiation p. 2012;19(12):1928-38.
16. Stiles BL. PI-3-K and AKT:Onto the mitochondria. J Adv Drug Delivery Rev 2009;61(14):1276-82.
17. Tan W. VDAC blockage by phosphorothioate oligonucleotides and its implication in apoptosis. J Biochim Biophys Acta 2012;1818(6):1555-61.
18. Pastorino JG, Hoek JB, Shulga N. Activation of glycogen synthase kinase 3beta disrupts the binding of hexokinase II to mitochondria by phosphorylating voltage-dependent anion channel and potentiates chemotherapy-induced cytotoxicity. J Cancer Res 2005;65(22):10545-54.
19. Pedersen PL. Warburg, me and Hexokinase 2:Multiple discoveries of key molecular events underlying one of cancers' most common phenotypes, the "Warburg Effect", i.e., elevated glycolysis in the presence of oxygen. J Bioenerg Biomembr 2007;39(3):211-22.
20. Pedersen PL. Voltage dependent anion channels (VDACs):a brief introduction with a focus on the outer mitochondrial compartment's roles together with hexokinase-2 in the "Warburg effect" in cancer. J Bioenerg Biomembr 2008;40(3):123-6.
21. Zaid H, Abu-Hamad S, Israelson A, Nathan I, Shoshan-Barmatz V. The voltage-dependent anion channel-1 modulates apoptotic cell death. J Cell Death Differ. 2005;12(7):751-60.
22. Beurdeley-Thomas A, Miccoli L, Oudard S, Dutrillaux B, Poupon MF. The peripheral benzodiazepine receptors:a review. J Neurooncol 2000;46(1):45-56.
23. Galiegue S, Tinel N, Casellas P. The peripheral benzodiazepine receptor:a promising therapeutic drug target. J Curr Med Chem 2003;10(16):1563-72.
24. Levin E, Premkumar A, Veenman L, Kugler W, Leschiner S, Spanier I, et al. The peripheral-type benzodiazepine receptor and tumorigenicity:isoquinoline binding protein (IBP) antisense knockdown in the C6 glioma cell line. J Biochemistry 2005;44(29):9924-35.
25. Mukherjee S, Das SK. Translocator protein (TSPO) in breast cancer. J Curr Mol Med 2012;12(4):443-57.
26. Rahman MA. Mitochondria:Insight Target of Drug Development in Cancer Cells. Int J of Pharm Sci and Res 2012;3(9).
27. Godbole A, Dubey AK, Reddy PS, Udayakumar M, Mathew MK. Mitochondrial VDAC and hexokinase together modulate plant programmed cell death. J Protoplasma 2013;250(4):875-84.
28. Godbole A, Varghese J, Sarin A, Mathew MK. VDAC is a conserved element of death pathways in plant and animal systems. J Biochim Biophys Acta 2003;1642(1-2):87-96.
29. Arbel N, Ben-Hail D, Shoshan-Barmatz V. Mediation of the antiapoptotic activity of Bcl-xL protein upon interaction with VDAC1 protein. J of Biological Chemistry 2012;287(27):23152-61.
30. Shoshan-Barmatz V. D. Mizrachi, and N. Keinan Oligomerization of the mitochondrial protein VDAC from structure to function and cancer therapy Progress in molecular biology and translational science 117 p. 2013;1:303-34.
31. Pedersen PL. 3-Bromopyruvate (3BP) a fast acting, promising, powerful, specific, and effective "small molecule" anti-cancer agent taken from labside to bedside:introduction to a special issue. J Bioenerg Biomembr 2012;44(1):1-6.
32. Shao L, Kinnally KW, Mannella CA. Circular dichroism studies of the mitochondrial channel, VDAC, from Neurospora crassa. J Biophys 1996;71(2):778-86.
33. Malia TJ, Wagner G. NMR structural investigation of the mitochondrial outer membrane protein VDAC and its interaction with antiapoptotic Bcl-xL. J Biochemistry 2007;46(2):514-25.
34. Colombini M, Blachly-Dyson E, Forte M. VDAC, a channel in the outer mitochondrial membrane. J Ion Channels 1996;4:169-202.
35. Doring C, Colombini M. The mitochondrial voltage-dependent channel, VDAC, is modified asymmetrically by succinic anhydride. J of Membrane Biology 1985;83(1-2):87-94.
36. Godbole A, Mitra R, Dubey AK, Reddy PS, Mathew MK. Bacterial expression, purification and characterization of a rice voltage-dependent, anion-selective channel isoform, OsVDAC4. J of Membrane Biology 2011;244(2):67-80.
37. Maldonado EN, Sheldon KL, DeHart DN, Patnaik J, Manevich Y, Townsend DM, et al. Voltage-dependent anion channels modulate mitochondrial metabolism in cancer cells:regulation by free tubulin and erastin. J of Biological Chemistry 2013;288(17):11920-9.
38. Noskov SY, Rostovtseva TK, Bezrukov SM. ATP transport through VDAC and the VDAC-tubulin complex probed by equilibrium and nonequilibrium MD simulations. J Biochemistry 2013;52(51):9246-56.
39. Rostovtseva TK, Bezrukov SM. VDAC inhibition by tubulin and its physiological implications. J Biochim Biophys Acta 2012;1818(6):1526-35.
40. Parkerson KA, Sontheimer H. Biophysical and pharmacological characterization of hypotonically activated chloride currents in cortical astrocytes. J Glia 2004;46(4):419-36.
41. Israelson A, Zaid H, Abu-Hamad S, Nahon E, Shoshan-Barmatz V. Mapping the ruthenium red-binding site of the voltage-dependent anion channel-1. J Cell Calcium 2008;43(2):196-204.
42. Colombini M. Purification of VDAC (voltage-dependent anion-selective channel) from rat liver mitochondria. J of Membrane Biology 1983;74(2):115-21.
43. Daum G, Böhni PC, Schatz G. Import of proteins into mitochondria. Cytochrome b2 and cytochrome c peroxidase are located in the intermembrane space of yeast mitochondria. J of Biological Chemistry 1982;257(21):13028-33.
44. Koppel DA, Kinnally KW, Masters P, Forte M, Blachly-Dyson E, Mannella CA. Bacterial expression and characterization of the mitochondrial outer membrane channel. Effects of n-terminal modifications. J of Biological Chemistry 1998;273(22):13794-800.
45. Bagal SK, Brown AD, Cox PJ, Omoto K, Owen RM, Pryde DC, et al. Ion channels as therapeutic targets:a drug discovery perspective. J Med Chem 2013;56(3):593-624.
46. Ryan DP, Ptácek LJ. Episodic neurological channelopathies. J Neuron 2010;68(2):282-92.
47. Mugabe C, Matsui Y, So AI, Gleave ME, Baker JHE, Minchinton AI, et al. In vivo evaluation of mucoadhesive nanoparticulate docetaxel for intravesical treatment of non-muscle-invasive bladder cancer. J Clinical Cancer Res 2011;17(9):2788-98.
48. Suginta W, Mahendran KR, Chumjan W, Hajjar E, Schulte A, Winterhalter M, et al. Molecular analysis of antimicrobial agent translocation through the membrane porin BpsOmp38 from an ultraresistant Burkholderia pseudomallei strain. J Biochim Biophys Acta 2011;1808(6):1552-9.
49. Lipton SA. Failures and successes of NMDA receptor antagonists:molecular basis for the use of open-channel blockers like memantine in the treatment of acute and chronic neurologic insults. NeuroRx. J of the American Society for Experimental NeuroTherapeutics 2004;1(1):101-10.
50. Norris CM, Foster TC. MK-801 improves retention in aged rats:implications for altered neural plasticity in age-related memory deficits. J Neurobiol Learn Mem 1999;71(2):194-206.
51. Prezma T, Shteinfer A, Admoni L, Raviv Z, Sela I, Levi I, et al. VDAC1-based peptides:novel pro-apoptotic agents and potential therapeutics for B-cell chronic lymphocytic leukemia. J Cell Death Dis 2013;4:e809.
52. Rostovtseva T, Colombini M. ATP flux is controlled by a voltage-gated channel from the mitochondrial outer membrane. Journal of Biological Chemistry 1996;271(45):28006-8.
53. Rostovtseva TK, Tan W, Colombini M. On the role of VDAC in apoptosis:fact and fiction. J Bioenerg Biomembr 2005;37(3):129-42.
54. Colombini M. VDAC structure, selectivity, and dynamics. J Biochim Biophys Acta 2012;1818(6):1457-65.
55. Shanmugavadivu B, Apell H-J, Meins T, Zeth K, Kleinschmidt JH. Correct folding of the beta-barrel of the human membrane protein VDAC requires a lipid bilayer. J Mol Biol 2007;368(1):66-78.
56. Mertins B, Psakis G, Grosse W, Back KC, Salisowski A, Reiss P, et al. Flexibility of the N-terminal mVDAC1 segment controls the channel's gating behavior. J PLoS One 2012;7(10):47938.
Statistics
330 Views | 866 Downloads
How to Cite
Dubey, A. K., A. Godbole, S. Srinivasan, and M. K. Mathew. “VDAC PROPERTIES ARE INFLUENCED BY THE SOURCE OF ITS PURIFICATION”. International Journal of Pharmacy and Pharmaceutical Sciences, Vol. 6, no. 7, July 2014, pp. 126-30, https://innovareacademics.in/journals/index.php/ijpps/article/view/1332.
Section
Original Article(s)