Proper protein folding is a key issue in maintaining healthy neurons, including the photoreceptor cells of the retina. Stresses on the photoreceptor proteome may be caused by environmental factors such as light damage or by genetic factors such as misfolding mutations in rhodopsin and many other photoreceptor proteins. The proteome is maintained by a class of proteins called molecular chaperones. These chaperones protect proteins from aggregation, channel their folding pathways and facilitate their association into multi-protein assemblies. An important type of molecular chaperone is the type II chaperonin found in the eukaryotic cytosol, termed CCT (Cytosolic Chaperonin containing Tailless complex polypeptide 1, also called TRiC). The number of proteins known to require CCT for their folding is in the hundreds and continues to grow. Among these are the G protein ? subunits (G?) which form the G?? and G?5-RGS (Regulator of G protein Signaling) dimers that are key components in visual signaling. Both G?? and RGS-G?5 also require the CCT co-chaperone, phosducin-like protein, for proper folding and dimer formation. In previous work, we have determined the structures of two intermediates in G?? assembly by cryo-electron microscopy, chemical cross-linking coupled with mass spectrometry, and site-specific cross-linking with unnatural amino acids. These structures have provided molecular detail into the mechanism of G?? assembly.
In Aim 1, we propose to determine the structures of similar intermediates in RGS-G?5 assembly to understand at the molecular level how CCT and PhLP1 assist in RGS-G?5 dimer formation. An additional role for CCT in Bardet-Biedl syndrome (BBS) has also been demonstrated. BBS is a genetic disease of ciliary dysfunction characterized by multiple pathological conditions including retinal degeneration. BBS is caused by an inability to form the BBSome, a complex of eight proteins that is essential for vesicle trafficking to cilia. CCT and a CCT-like complex made up of three proteins whose mutations are known to cause BBS (BBS 6, 10 and 12) are required for the assembly of the BBSome.
In Aim 2, we propose similar structural studies to determine the molecular mechanism of BBSome assembly and BBSome function. The information gained from these studies will be vital in designing chaperone-based methods to treat retinal diseases caused by RGS-G?5 and BBSome malfunctions.

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.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY012287-16
Application #
9402604
Study Section
Biology of the Visual System Study Section (BVS)
Program Officer
Neuhold, Lisa
Project Start
1999-05-03
Project End
2019-12-31
Budget Start
2018-01-01
Budget End
2018-12-31
Support Year
16
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Brigham Young University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
009094012
City
Provo
State
UT
Country
United States
Zip Code
84602
Lord, Nathan P; Plimpton, Rebecca L; Sharkey, Camilla R et al. (2016) A cure for the blues: opsin duplication and subfunctionalization for short-wavelength sensitivity in jewel beetles (Coleoptera: Buprestidae). BMC Evol Biol 16:107
Tracy, Christopher M; Kolesnikov, Alexander V; Blake, Devon R et al. (2015) Retinal cone photoreceptors require phosducin-like protein 1 for G protein complex assembly and signaling. PLoS One 10:e0117129
Xie, Keqiang; Masuho, Ikuo; Shih, Chien-Cheng et al. (2015) Stable G protein-effector complexes in striatal neurons: mechanism of assembly and role in neurotransmitter signaling. Elife 4:
Plimpton, Rebecca L; Cuéllar, Jorge; Lai, Chun Wan J et al. (2015) Structures of the G?-CCT and PhLP1-G?-CCT complexes reveal a mechanism for G-protein ?-subunit folding and G?? dimer assembly. Proc Natl Acad Sci U S A 112:2413-8
Tracy, Christopher M; Gray, Amy J; Cuéllar, Jorge et al. (2014) Programmed cell death protein 5 interacts with the cytosolic chaperonin containing tailless complex polypeptide 1 (CCT) to regulate ?-tubulin folding. J Biol Chem 289:4490-502
Lai, Chun Wan J; Kolesnikov, Alexander V; Frederick, Jeanne M et al. (2013) Phosducin-like protein 1 is essential for G-protein assembly and signaling in retinal rod photoreceptors. J Neurosci 33:7941-51
Zhou, Xueyuan; Shapiro, Leland; Fellingham, Gilbert et al. (2011) HIV replication in CD4+ T lymphocytes in the presence and absence of follicular dendritic cells: inhibition of replication mediated by ?-1-antitrypsin through altered I?B? ubiquitination. J Immunol 186:3148-55
Smrcka, Alan V; Kichik, Nessim; Tarragó, Teresa et al. (2010) NMR analysis of G-protein betagamma subunit complexes reveals a dynamic G(alpha)-Gbetagamma subunit interface and multiple protein recognition modes. Proc Natl Acad Sci U S A 107:639-44
Howlett, Alyson C; Gray, Amy J; Hunter, Jesse M et al. (2009) Role of molecular chaperones in G protein beta5/regulator of G protein signaling dimer assembly and G protein betagamma dimer specificity. J Biol Chem 284:16386-99
Willardson, Barry M; Howlett, Alyson C (2007) Function of phosducin-like proteins in G protein signaling and chaperone-assisted protein folding. Cell Signal 19:2417-27