Genomes and Genes
Mechanisms of assembly of photoreceptor G protein complexes
Principal Investigator: Barry M Willardson
Abstract: DESCRIPTION (provided by applicant): Proper protein folding is a key issue in maintaining healthy, functional neurons. Neurodegenerative diseases such as Alzheimer's, Parkinson's, Huntington's and Lou Gehrig's disease (amyotrophic lateral sclerosis or ALS) all result from protein misfolding in neurons. Similar disruptions of proteome homeostasis cause neuronal degeneration of the photoreceptor cells of the retina. Many severe retinopathies have been linked to misfolding mutations in rhodopsin and other photoreceptor proteins. A key component of molecular chaperone system that maintains proteome homeostasis in photoreceptor cells is the cytosolic chaperonin complex (CCT, Chaperonin Containing Tailless polypeptide 1) which folds actin, tubulin and dozens of other cytosolic proteins. Among these CCT substrates are the G protein [unreadable] subunits (G[unreadable]) which form the G [unreadable]1?1 and G[unreadable]5-RGS9 (Regulator of G protein Signaling) dimers that are essential components of the phototransduction cascade. All G[unreadable] subunits require CCT and the co-chaperone phosducin-like protein 1 (PhLP1) to fold G[unreadable] and assemble into functional dimers. Very recently, an additional role for CCT in Bardet-Biedl syndrome (BBS) has been demonstrated. BBS is a genetic disease of ciliary dysfunction displaying multiple pathological conditions including retinal degeneration. Mutations in 14 BBS proteins have been associated with the disease. Seven of them (BBS 1-2, 4-5 and 7-9) form a complex, termed the BBSome, which is essential for vesicle trafficking to cilia. Three others (BBS 6, 10 and 12) are homologous to CCT subunits and form complexes with CCT that are required for the folding of BBS2 and 7 and assembly of the BBSome. Together, these G[unreadable] and BBS findings point to an important role for CCT in the assembly of protein complexes that perform key physiological functions in photoreceptor cells. Thus, the underlying goal of the proposed studies is to understand at the molecular level the mechanisms of protein complex assembly by CCT in order to identify therapeutic targets to improve the folding process and treat diseases like BBS for which effective treatments are not available. Specific Aims 1 and 2 examine the structures of the G[unreadable]1-CCT and RGS9-G[unreadable]5-CCT complexes in order to understand how CCT folds both G[unreadable]1 and G[unreadable]5 and recruits RGS9 to form the G[unreadable]5-RGS9 dimer. Specific Aim 3 investigates the structure of the complex between the chaperonin-like BBS proteins and CCT and the mechanism of BBSome assembly. To accomplish these aims, a number of methods are proposed including site-directed mutagenesis and cell-based binding assays, pulse-chase assembly measurements of protein complexes, purification of large protein complexes and cryo-electron microscopy. A strong team of collaborators has been assembled to provide the necessary expertise to successfully execute the proposed experiments. PUBLIC HEALTH RELEVANCE: Many neurodegenerative diseases are associated with defects in protein folding. This is certainly the case in the photoreceptor cells of the retina, in which misfolding mutations in proteins cause photoreceptor cell death, resulting in retinal degeneration and blindness. This proposal investigates the mechanism of folding and assembly of physiologically important protein complexes that result in diseases when their genes are mutated. One such disease is Bardet-Biedl syndrome, a condition that causes blindness usually by adolescence. An understanding of the way these protein complexes are brought together is necessary to develop treatments that would allow the complexes to assemble and function despite the mutations.
Funding Period: 1999-05-03 - 2015-03-31
more information: NIH RePORT
- Function of phosducin-like proteins in G protein signaling and chaperone-assisted protein foldingBarry M Willardson
Department of Chemistry and Biochemistry, C 100 BNSN, Brigham Young University Provo, Utah 84602, USA
Cell Signal 19:2417-27. 2007....
- Role of molecular chaperones in G protein beta5/regulator of G protein signaling dimer assembly and G protein betagamma dimer specificityAlyson C Howlett
Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, USA
J Biol Chem 284:16386-99. 2009..These findings point to a general role for PhLP1 in the assembly of all Gbetagamma combinations and suggest a CCT-dependent mechanism for Gbeta5-RGS7 assembly that utilizes the co-chaperone activity of PhLP1 in a unique way...
- NMR analysis of G-protein betagamma subunit complexes reveals a dynamic G(alpha)-Gbetagamma subunit interface and multiple protein recognition modesAlan V Smrcka
Institute for Research in Biomedicine, Barcelona Science Park, Barcelona, Spain
Proc Natl Acad Sci U S A 107:639-44. 2010..Overall, these data show that Gbetagamma subunits explore a range of conformations that can be exploited during molecular recognition by diverse binding partners...
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Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602, USA
J Immunol 186:3148-55. 2011....
- Phosducin-like protein 1 is essential for G-protein assembly and signaling in retinal rod photoreceptorsChun Wan J Lai
Department of Chemistry, Brigham Young University, Provo, Utah 84602, USA
J Neurosci 33:7941-51. 2013..These findings conclusively demonstrate in vivo that PhLP1 is required for the folding and assembly of both Gβγ and Gβ5-RGS9...
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