Mechanisms of assembly of photoreceptor G protein complexes

Summary

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

Top Publications

  1. pmc Function of phosducin-like proteins in G protein signaling and chaperone-assisted protein folding
    Barry M Willardson
    Department of Chemistry and Biochemistry, C 100 BNSN, Brigham Young University Provo, Utah 84602, USA
    Cell Signal 19:2417-27. 2007
  2. pmc Role of molecular chaperones in G protein beta5/regulator of G protein signaling dimer assembly and G protein betagamma dimer specificity
    Alyson C Howlett
    Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, USA
    J Biol Chem 284:16386-99. 2009
  3. pmc NMR analysis of G-protein betagamma subunit complexes reveals a dynamic G(alpha)-Gbetagamma subunit interface and multiple protein recognition modes
    Alan V Smrcka
    Institute for Research in Biomedicine, Barcelona Science Park, Barcelona, Spain
    Proc Natl Acad Sci U S A 107:639-44. 2010
  4. pmc HIV replication in CD4+ T lymphocytes in the presence and absence of follicular dendritic cells: inhibition of replication mediated by α-1-antitrypsin through altered IκBα ubiquitination
    Xueyuan Zhou
    Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602, USA
    J Immunol 186:3148-55. 2011
  5. pmc Phosducin-like protein 1 is essential for G-protein assembly and signaling in retinal rod photoreceptors
    Chun Wan J Lai
    Department of Chemistry, Brigham Young University, Provo, Utah 84602, USA
    J Neurosci 33:7941-51. 2013

Detail Information

Publications5

  1. pmc Function of phosducin-like proteins in G protein signaling and chaperone-assisted protein folding
    Barry M Willardson
    Department of Chemistry and Biochemistry, C 100 BNSN, Brigham Young University Provo, Utah 84602, USA
    Cell Signal 19:2417-27. 2007
    ....
  2. pmc Role of molecular chaperones in G protein beta5/regulator of G protein signaling dimer assembly and G protein betagamma dimer specificity
    Alyson 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...
  3. pmc NMR analysis of G-protein betagamma subunit complexes reveals a dynamic G(alpha)-Gbetagamma subunit interface and multiple protein recognition modes
    Alan 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...
  4. pmc HIV replication in CD4+ T lymphocytes in the presence and absence of follicular dendritic cells: inhibition of replication mediated by α-1-antitrypsin through altered IκBα ubiquitination
    Xueyuan Zhou
    Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602, USA
    J Immunol 186:3148-55. 2011
    ....
  5. pmc Phosducin-like protein 1 is essential for G-protein assembly and signaling in retinal rod photoreceptors
    Chun 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...

Research Grants30

  1. Neuronal Cell Cycle and Survival
    Nicholas E Baker; Fiscal Year: 2013
    ....
  2. Hsp110 protein chaperone function in yeast
    KEVIN ANTHONY MORANO; Fiscal Year: 2013
    ....
  3. Slow axonal transport of cytosolic cargoes by dynamic-recruitment - a new traffic
    Subhojit Roy; Fiscal Year: 2013
    ..Upon completion, these studies would answer long-standing questions about the transport of these proteins and also open the door for investigation of their transport in pathologic states. ..
  4. Impaired Protein Degradation Pathways in Cardiac Proteinopathy
    Patrick M McLendon; Fiscal Year: 2013
    ..abstract_text> ..
  5. Expanding the National Health Accounts
    David M Cutler; Fiscal Year: 2013
    ..Establishment of a set of national health accounts will allow us as a society to understand which medical interventions improve the health of the U.S. population most efficiently. ..
  6. HSF1 as a therapeutic target in neurodegenerative disease
    Dennis J Thiele; Fiscal Year: 2013
    ....
  7. MOLECULAR STUDIES OF RETINAL DEGENERATION IN DROSOPHILA
    Nansi J Colley; Fiscal Year: 2013
    ..We anticipate that this work will greatly impact our understanding of the fundamental mechanisms of protein trafficking and also provide important insights into retinal diseases such as RP and AMD. ..
  8. Engineering inhibitable kinesin motors to study axonal transport
    Kristen J Verhey; Fiscal Year: 2013
    ..This work will provide exciting new insights into how kinesin motors give rise to coordinated transport of protein complexes in cells and will suggest therapeutic targets in human disease. ..
  9. Celllular mechanisms of neuronal metal transport and toxicity
    Victor Faundez; Fiscal Year: 2013
    ..Our studies will impact our understanding and possibly treatment of acute and chronic neurological disease processes where zinc play a role such as epilepsy and Alzheimer's disease. ..
  10. Defining the mechanistic basis of a prion disaggregase
    James Shorter; Fiscal Year: 2013
    ....
  11. Molecular Basis of Protein Transport in Photoreceptor
    Ching Hwa Sung; Fiscal Year: 2013
    ..Transfected rodent rods, conditional knockout mice, cell culture models, and several innovative techniques will be employed to comprehensively investigate these questions. ..
  12. Analysis and Therapy of Age-Dependent Proteostasis Failure in Neurodegeneration
    ROBERT JOSEPH SHMOOKLER REIS; Fiscal Year: 2013
    ..Proteins or modifications in either soluble or insoluble aggregates, which vary concordantly with neurotoxicity, become functional candidates ..
  13. EINSTEIN AGING STUDY
    Richard B Lipton; Fiscal Year: 2013
    ..Together, these Projects will help disentangle the multifactorial processes that lead to cognitive and locomotor decline and dementia. ..
  14. Alerations of Sleep and Circadian Timing in Aging
    Eve Van Cauter; Fiscal Year: 2013
    ..Core B (Methods and Analysis) will standard operating procedures for data collection, archival and analysis. Core C will assay peripheral levels of hormones, cytokines and other blood constituents. ..