个人简介

BA, Brigham Young University (1984) Ph.D., Purdue University (1990) Postdoctoral Fellow, University of California, Los Alamos National Lab (1990-92) Staff Scientist, University of California, Los Alamos National Lab (1992-96)

研究领域

Biochemistry

Mechanisms of Assembly of Signaling Complexes − Our lab studies the role of molecular chaperones in assisting the folding and assembly of protein complexes involved in cell signaling. One of these is the G protein heterotrimeric complex. G proteins play an essential role in cellular responses to hormones, neurotransmitters, chemokines and sensory signals. Assembly of the G protein complex begins with the association of the G protein b subunit (Gb) with the g subunit (Gg) to form the Gbg dimer. We have found that Gbg cannot assemble on its own but requires the help of the cytosolic chaperonin complex (CCT) and a co-chaperone phosducin-like protein 1 (PhLP1). We are investigating the molecular mechanism by which these chaperones bring Gbg together in an effort to find ways to control this process and develop new therapeutics to treat some of the many diseases associated with defects in G protein signaling. Most cellular functions are performed by proteins associated together into complexes. In fact, many proteins cannot even exist in the cell without their binding partners. These protein complexes often require the help of other proteins, called chaperones, to bring the complexes together. This is certainly the case for protein complexes involved in cell signaling processes. Our work has focused on one of these signaling complexes, the G protein heterotrimer. It is through the G protein complex and its associated receptors and effectors that cells detect hormones, neurotransmitters, chemokines and sensory signals such as odorants, taste molecules and even photons of light. G proteins regulate almost every aspect of cellular physiology and as a result more than a third of current therapeutic drugs target G protein signaling pathways. In recent years, our lab has described the initial steps in the assembly of the G protein complex. This process begins with the association of the G protein b subunit (Gb) with the G protein g subunit (Gg) into the Gbg dimer. Gbg is an obligate dimer, meaning that neither subunit is stable in the cell without the other. As a result, Gb and Gg must be brought together by chaperones. The way in which chaperone-mediated Gbg assembly occurs is as follows. At some point during or after translation, the nascent Gb subunit binds to the cytosolic chaperonin complex (CCT). CCT is a large protein-folding machine made up of a double-ring structure with eight different chaperonin subunits in each ring. Like other proteins folded by CCT, Gb associates in the folding cavity in the center of the ring and is folded into a near-native conformation. However, Gb cannot achieve its native fold and release from CCT without Gg nor can Gb associate with Gg until it is released from CCT. This conundrum is resolved by the CCT co-chaperone, phosducin-like protein 1 (PhLP1). PhLP1 binds Gb in the CCT folding cavity and initiates the release of Gb from CCT. Once released, Gg is able to bind Gb in the PhLP1-Gb complex and form the stable Gbg dimer. The G protein a subunit then associates with Gbg, forming the active Gabg heterotrimer and simultaneously releasing PhLP1. All four of the typical Gb subunits are assembled with their 12 associated Gg subunits by this same mechanism involving CCT and PhLP1. The atypical Gb5 subunit forms an obligate dimer with regulators of G protein signaling (RGS) proteins of the RGS7 subfamily. These dimers have a different function than Gbg dimers. They turn off G protein signaling in neurons by accelerating the rate of GTP hydrolysis on the Ga subunit. We have found that CCT and PhLP1 also assist in the assembly of these Gb5-RGS complexes. In fact, the conditional deletion of the PhLP1 gene in the rod photoreceptor cells of mice results in the loss of the Gb5-RGS9 dimer from these cells in addition to the loss of Gbg dimers. Consequently, G protein-dependent responses to light by rod photoreceptors were diminished and their recovery was slow. These findings have confirmed the importance of PhLP1 in Gbg and Gb5-RGS dimer formation in vivo. Some of our more recent work has focused on the structural mechanism by which CCT and PhLP1 mediate Gbg assembly. We have isolated two intermediates in the process of Gbg assembly, the Gb-CCT and the PhLP1-Gb-CCT complexes and have determined their structures by cryo-EM in collaboration with the lab of Jose M. Valpuesta at the Centro National de Biotecnologia in Madrid, Spain. The cryo-EM structures show that Gb folds into a near-native conformation within the CCT folding cavity and associates with the apical domain of the CCTb subunit. PhLP1 binds Gb from above the folding cavity, disrupting its interact with CCTb and allowing PhLP1-Gb to release from CCT for association with Gg.

近期论文

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Smrcka, A. V., Kichik, N., Tarrago, T., Burroughs, M., Park, M., Stern, H., Itoga, N. K. Willardson, B. M. and Giralt, E. (2010) “NMR Analysis of G Protein βγ Subunit Complexes Reveals a Dynamic Gα-Gβγ Subunit Interface and Multiple Protein Recognition Modes” Proc. Natl. Acad. Sci. U. S. A. 107, 639-644. Howlett, A. C., Gray, A. J., Hunter, J. M. and Willardson, B. M. (2009) “Role of Molecular Chaperones in G protein β5/Regulator of G protein Signaling Dimer Assembly and G protein βγ Dimer Specificity” J. Biol. Chem. 284, 16386-16399. Lukov, G. L., Baker, C. M., Ludtke, P. J., Hu, T., Carter, M. D., Hackett, R. A., Thulin, C. D. and Willardson, B. M. (2006) “Mechanism of Assembly of G Protein βγ subunits by Protein Kinase CK2-phosphorylated Phosducin-like Protein and the Cytosolic Chaperonin Complex” J. Biol. Chem. 281, 22261-22274. Lukov, G. L., Hu, T., McLaughlin, J. N., Hamm, H. E. and Willardson, B. M. (2005) “Phosducin-like protein acts as a molecular chaperone for G protein βγ dimer assembly” EMBO J. 24, 1965-1975. Martin-Benito, J., Bertrand, S., Hu, T., Ludtke, P., McLaughlin, J.N., Willardson, B.M., Carrascosa, J. L. and Valpuesta, J.M. (2004) “Structure of the complex between phosduin-like protein and the cytosolic chaperonin complex” Proc. Natl. Acad. Sci. 101, 17410-17415. J.N. McLaughlin, C.D. Thulin, S.J. Hart, K.A. Resing, N.G. Ahn and B.M. Willardson (2002) “Regulatory Interaction of Phosducin-like Protein with the Cytosolic Chaperonin Complex” Proc. Natl. Acad. Sci. U.S.A. 99, 7962-7967.