RESEARCH PROGRAMS AND AFFILIATIONS
Intracellular signaling via InsP3 receptors and their regulation by the ubiquitin/proteasome pathway and Bcl-2 family proteins.
Current Graduate Students are: Laura Szczesniak (MD/PhD), Jenson Gao (PhD), Fanghui Hua (PhD) and Caden Bonzerato (PhD)
IP3 receptor regulation, the ubiquitin / proteasome pathway, the Bcl-2 protein family and intracellular signaling
IP3 is an intracellular messenger molecule formed at the plasma membrane when hormones, neurotransmitters or drugs stimulate cells. The effects of IP3 are mediated by proteins called IP3 receptors, channels that govern the release of calcium ions from the endoplasmic reticulum into the cell cytosol: this "calcium mobilization" is a central part of many cellular functions. My laboratory has been studying various aspects of IP3 receptor biochemistry and molecular biology for some time. Our primary focus at the moment is analyzing IP3 receptor down-regulation - a remarkable phenomenon by which IP3 receptors are rapidly depleted from cells when they are stimulated (see diagram). This is a classic adaptive response that enables cells to adjust to their external environment and occurs during chronic exposure to drugs and perhaps in physiological and pathological situations. We are currently investigating the mechanism of IP3 receptor down-regulation and have discovered that it occurs because IP3 receptors are tagged with ubiquitin and then degraded by the proteosome. This is exciting because the ubiquitin / proteosome pathway is currently one of the "hot areas" of cell biology - is it becoming increasingly apparent that this pathway is the mechanism by which many important cellular proteins and misfolded proteins in the endoplasmic reticulum are degraded and is of major relevance to diseases (e.g. cancer, neurodegeneration, diabetes). Our immediate goals, then, are to define at the molecular level the pathway that leads to IP3 receptor degradation via the ubiquitin / proteasome pathway and to begin to build a picture of when, why and how cellular proteins are tagged with ubiquitin. As depicted in the diagram, in recent years we have discovered that IP3 receptor ubiquitination is mediated by a novel complex composed of the proteins erlin1 and erlin2, and the ubiquitin ligase RNF170, and that two ubiquitin chain types (K48-linked and K63-linked) are coupled to activated IP3 receptors. Using advanced techniques (e.g. cryo-EM) we are now defining at the structural level how IP3 receptors and the erlin1/2 complex interact. A second focus is characterizing the interaction of Bcl-2 family proteins (that control apoptosis) with IP3 receptors. We recently discovered that Bok (“Bcl-2-Related Ovarian Killer”) binds constitutively to IP3 receptors and are defining the significance of this interaction. Because of the importance of IP3 receptors, the ubiquitin / proteasome pathway, and the Bcl-2 protein family to cell biology, this work is significant to both our understanding of normal physiology, and also to diseases, such as cancer and neurodegeneration.
58. Pearce, M.M., Wang, Y., Kelley, G.G. and Wojcikiewicz, R.J.H. (2007) SPFH2 mediates the ERAD of IP3 receptors and other substrates in mammalian cells. J. Biol. Chem. 282, 20104-20115.
59. Hanson, C.J., Bootman, M.D., Distelhorst, C.W., Wojcikiewicz, R.J.H. and Roderick, H.L. (2008) Bcl-2 suppresses Ca2+ release through inositol 1,4,5-trisphosphate receptors and inhibits Ca2+ uptake by mitochondria without affecting ER calcium store content. Cell Calcium 44, 324-338.
60. Ito, J., Yoon, S-Y., Lee, B., Vanderhayden, V., Vermassen, E., Wojcikiewicz, R.J.H., Alfandari, D., De Smedt, H., Parys, J.B. and Fissore, R.A. (2008) Inositol 1,4,5-trisphosphate receptor 1, a widespread Ca2+ channel, is a novel substrate of polo-like kinase 1 in eggs. Dev. Biol. 320, 402-413.
61. Kuo, I.Y., Chan-Ling, T., Wojcikiewicz, R.J.H. and Hill, C.E. (2008) Limited intravascular coupling in the rodent brainstem and retina supports a role for glia in regional blood flow. J. Comp. Neurol. 511, 773-787.
62. Sliter, D., Kirkpatrick, D.S., Alzayady, K., Kubota, K., Gygi, S.P. and Wojcikiewicz, R.J.H. (2008) Mass spectral analysis of type I inositol 1,4,5-trisphosphate receptor ubiquitination. J. Biol. Chem. 283, 35319-35328.
63. Ellis, A., Goto, K., Brackenbury, T.D., Meaney, K.R., Falck, J.R., Wojcikiewicz, R.J.H. and Hill, C.E. (2009) Angiotensin II-dependency of the role of EETs and gap junctions in mediating EDHF activity in rat mesenteric arteries. J. Pharmacol. Exp. Ther. 330, 413-422.
64. Pearce, M.M.P., Wormer, D.B., Wilkens, S. and Wojcikiewicz, R.J.H. (2009) An ER membrane complex composed of SPFH1 and SPFH2 mediates the ER-associated degradation of IP3 receptors. J. Biol. Chem. 284, 10433-10445.
65. Brodsky, J.L. and Wojcikiewicz R.J.H. (2009) Substrate specific mediators of ER associated degradation (ERAD). Curr. Opin. Cell Biol. 21, 516-21.
66. Wojcikiewicz, R.J.H., Pearce, M.M.P., Sliter, D. and Wang. Y. (2009) When worlds collide: IP3 receptors and the ERAD pathway. Cell Calcium 46, 147-153.
67. Wang, Y., Pearce, M.M.P., Sliter, D., Olzmann, J.A., Christianson, J.C., Kopito, R.R., Boeckmann, S., Gagen, C., Leichner, G., Roitelman, J. and Wojcikiewicz, R.J.H. (2009) SPFH1 and SPFH2 mediate the ubiquitination and degradation of inositol 1,4,5-trisphosphate receptors in muscarinic receptor-expressing HeLa cells. BBA 1793, 1710-1718
68. Sliter D.A., Aguiar, M., Gygi, S.P. and Wojcikiewicz, R.J.H. (2011) Activated inositol 1,4,5-trisphosphate receptors are modified by homogeneous LYS48- and LYS63-linked ubiquitin chains, but only LYS48-linked chains are required for degradation. J. Biol. Chem. 286, 1074-1082.
69. Lu, J.P., Wang, Y., Sliter, D.A., Pearce, M.M.P. and Wojcikiewicz, R.J.H. (2011) RNF170, an endoplasmic reticulum membrane ubiquitin ligase, mediates inositol 1,4,5-trisphosphate receptor ubiquitination and degradation. J. Biol. Chem. 286, 24426-24433.
70. Pednekar, D., Wang, Y., Fedotova, T.V. and Wojcikiewicz, R.J.H. (2011) Clustered hydrophobic amino acids in amphipathic helices mediate erlin 1 / 2 complex assembly. Biochem. Biophys. Res. Commun. 415, 135-140.
71. Wojcikiewicz, R.J.H. (2012) Inositol 1,4,5-trisphosphate receptor degradation pathways. WIREs Membr. Transp. Signal. 1, 126-135.
72. Tsai, Y.C., Leichner, G.S., Pearce, M.M., Wilson, G.L., Wojcikiewicz, R.J., Roitelman, J. and Weissman, A.M. (2012) Differential regulation of HMG-CoA reductase and Insig-1 by enzymes of the ubiquitin-proteasome system. Mol. Biol. Cell. 23, 4484-4494.
73. Hirose, M., Kamoshita, M., Fujiwara, K., Kato, T., Nakamura, A., Wojcikiewicz, R.J.H., Parys, J.B., Ito, J. and Kashiwazaki, N. (2013) Vitrification procedure decreases inositol 1,4,5-trisphosphate receptor expression, resulting in low fertility of pig oocytes. Animal Sci. J. (in press).
74. Schulman, J.J., Wright, F.A., Kaufmann, T and Wojcikiewicz, R.J.H. (2013) The Bcl-2 protein family member Bok binds to the coupling domain of inositol 1,4,5-trisphosphate receptors and protects them from proteolytic cleavage. J. Biol. Chem. 288, 25340-25349.
75. Sathanawongs, A., Fujiwara, K., Kato, T., Hirose, M., Kamoshita, M., Wojcikiewicz, R.J.H., Parys, J.B., Ito, J. and Kashiwazaki, N. (2015) The effect of M-phase stage-dependent kinase inhibitors on inositol 1,4,5-triphosphate receptor 1 (IP3R1) expression and localization in pig oocytes. Animal Sci. J. 86, 138-147.
76. Wright, F.A., Lu, J.P., Sliter, D.A., Dupré, N., Rouleau, G.A. and Wojcikiewicz, R.J.H. (2015) A point mutation in the ubiquitin ligase RNF170 that causes autosomal dominant sensory ataxia destabilizes the protein and impairs inositol 1,4,5-trisphosphate receptor-mediated Ca2+ signaling. J. Biol. Chem. 290, 13948-13957.
77. Schulman, J.J., Wright, F.A., Han, X., Zluhan, E.J., Szczesniak, L.M. and Wojcikiewicz, R.J.H. (2016) The stability and expression level of Bok binds are governed by binding to inositol 1,4,5-trisphosphate receptors. J. Biol. Chem. 291, 11820-11828.
78. Wright, F.A. and Wojcikiewicz, R.J.H. (2016) Inositol 1,4,5-trisphosphate receptor ubiquitination. Prog. Mol. Biol. Trans. Sci. 141, 141-159.
79. Wang, L., Shi, C., Wright, F.A., Guo, D., Wang, X., Wang, D., Wojcikiewicz, R.J.H. and Luo, J. (2017) Multifunctional Telodendrimer Nanocarriers Restore Synergy of Bortezomib and Doxorubicin in Ovarian Cancer Treatment. Cancer Res. 77, 3293 - 3305.
80. Wojcikiewicz, R.J.H. (2018) The making and breaking of inositol 1, 4, 5 - trisphosphate receptor tetramers. Messenger 6, 45-49.
81. Wright, F.A., Bonzerato, C.G., Sliter, D.A. and Wojcikiewicz, R.J.H. (2018) The erlin2 T65I mutation inhibits erlin1/2 complex-mediated inositol 1,4,5-trisphosphate receptor ubiquitination and phosphatidylinositol 3-phosphate binding. J. Biol. Chem. 293, 15706-15714.
82. Schulman, J.J., Szczesniak, L.M., Bunker, E.N., Nelson, H.A., Roe, M.W., Wagner II, L.A., Yule, D.I. and Wojcikiewicz, R.J.H. (2019) Bok regulates mitochondrial fusion and morphology. Cell Death and Diff. doi: 10.1038/s41418-019-0327-4.83.
83. Gao, X. and Wojcikiewicz, R.J.H. (2020) The emerging link between IP3 receptor turnover and Hereditary Spastic Paraplegia. Cell Calcium 86:102142. doi: 10.1016/j.ceca.2019.102142.
84. Dong, Y., Lee, Y., Cui, K., He, M., Wang, B., Bhattacharjee, S., Zhu, B., Yago, T., Zhang, K., Deng, L., Ouyang, K., Wen, A., Cowan, D.B., Song, K., Yu, L., Brophy, M.L., Liu, X., Wylie-Sears, J., Wu, H., Wong, S., Cui, G., Kawashima, Y., Matsumoto, H., Kodera, Y., Wojcikiewicz, R.J.H., Srivastava, S., Bischoff, J., Wang, D.Z., Ley, K., Chen, H. (2020) Epsin-mediated degradation of IP3R1 fuels atherosclerosis. Nat. Commun. 11(1):3984. doi: 10.1038/s41467-020-17848-4.
85. Szczesniak, L.M., Bonzerato, C.G., Schulman, J.J., Bah, A. and Wojcikiewicz, R.J.H. (2021) Bok binds to a largely disordered loop in the coupling domain of type 1 inositol 1,4,5-trisphosphate receptor. Biochem. Biophys. Res Commun. 553, 180-186.
86. Szczesniak, L.M., Bonzerato, C.G. and Wojcikiewicz, R.J.H. (2021) Identification of the Bok interactome using proximity labeling. Front. Cell Dev. Biol. 9:689951. doi: 10.3389/fcell.2021.689951.
87. Gao, X., Bonzerato, C.G. and Wojcikiewicz, R.J.H. (2022) Binding of the erlin1/2 complex to the third intralumenal loop of IP3R1 triggers its ubiquitin-proteasomal degradation. J. Biol. Chem. 298, 102026.
88. Bonzerato, C.G., Keller, K.R., Schulman, J.J., Gao, X., Szczesniak, L.M. and Wojcikiewicz, R.J.H. (2022) Endogenous Bok is stable at the endoplasmic reticulum and does not mediate proteasome inhibitor-induced apoptosis. Frontiers in Cell and Dev Biol. 10, 1094302. doi: 10.3389/fcell.2022.1094302.