Bardet-Biedl syndrome (BBS) is a rare genetic disease that causes retinal degradation, obesity, kidney dysfunction, polydactyly, and other cilium-related disorders. To date, more than 20 BBS genes, whose mutants cause BBS phenotypes, have been identified, and eight of those (BBS1-2, 4-5, 7-9, and 18) are known to form the BBSome complex. Recent studies have revealed that the BBSome is closely involved in the trafficking of signaling proteins in the primary cilium. Mutations in BBS genes are highly pathogenic because trafficking in the primary cilium is not fully functional when BBS mutations impair assembly of the BBSome. However, the functional links between onset of BBS and BBSome assembly are not well understood. To address this gap in knowledge, we examined the structure of a BBSome assembly intermediate, the BBSome core complex (BBS2, 7, and 9). We employed a combination of chemical crosslinking coupled with mass spectrometry (XL-MS) and electron microscopy (EM) to determine the structure. We applied this structural information to BBS mutations in the core complex to understand how these mutations might cause the disease. These results provide the first structural model of the BBSome core complex and give insight into the molecular basis of Bardet-Biedl syndrome. We have also investigated the mechanism of assembly of the two mTOR kinase complexes (mTORC1 and 2). mTOR is a master regulator of cell metabolism, growth and proliferation. As such, mTOR is a high-value drug target. We investigated the mechanism of assembly of these mTOR complexes and found that the cytosolic chaperonin CCT contributes to mTOR signaling by assisting in the folding of mLST8 and Raptor, components of mTORC1 and mTORC2. To understand the function of CCT in mTOR complex assembly at the molecular level, we have isolated the mLST8-CCT complex and performed a structural analysis using chemical cross-linking couple with mass spectrometry (XL-MS) and cryogenic EM. We found that mLST8 binds CCT deep in its folding cavity, making specific contacts with the CCTα and γ subunits and forming a near-native β-propeller conformation. This information can be used to develop new therapeutics that regulate mTOR activity by controlling mTOR complex assembly.

G protein signaling depends on the ability of the individual subunits of the G protein heterotrimer to assemble into a functional complex. Formation of the G protein βγ (Gβγ) dimer is particularly challenging because it is an obligate dimer in which the individual subunits are unstable on their own. Recent studies have revealed an intricate chaperone system that brings the Gβ and Gγ subunits together. This system includes the cytosolic chaperonin containing TCP-1 (CCT) and a co-chaperone phosducin-like protein 1 (PhLP1). Two key intermediates in the Gβγ assembly process, the Gβ-CCT and the PhLP1-Gβ-CCT complexes, were isolated and their structures determined by cryo-electron microscopy, chemical cross-linking coupled with mass spectrometry, and unnatural amino acid cross-linking. These structures show that Gβ interacts with CCT in a near-native state through interactions of the Gγ-binding region of Gβ with the CCTγ subunit. PhLP1 binding stabilizes the Gβ β-propeller, disrupting interactions with CCT and releasing a PhLP1-Gβ dimer for assembly with Gγ. We also investigated the role of CCT and PhLP1 in folding and assembling mTOR complexes, which regulate cell growth through phosphorylation. We found that the β-propeller protein mLST8 and one of its binding partners called raptor, which is a large protein in which one domain forms a β-propeller, both bind to CCT. PhLP1 forms a ternary complex with mLST8 and CCT and may play a co-chaperone role. Depletion of PhLP1 or CCT reduces assembly of mTOR complexes in the cell. Collectively, this report reveals diversity in the contributions of CCT to the formation of protein complexes in signaling pathways and presents a molecular mechanism of Gβ folding by CCT and PhLP1.

A fundamental question in biology is how proteins, which are synthesized by the ribosome as a linear sequence of amino acids, fold into their native functional state. Many proteins require the assistance of molecular chaperones to maneuver through the folding process to protect them from aggregation and to help them reach their native state in the very concentrated protein environment of the cell. This study focuses on the roles of Phosducin-like Protein 1 (PhLP1) and Programmed Cell Death Protein 5 (PDCD5) as molecular co-chaperones of the Cytosolic Chaperonin Complex (CCT).Signaling in retinal photoreceptors is mediated by canonical G protein pathways. Previous in vitro studies have demonstrated that Gβ subunits rely on CCT and its co-chaperone PhLP1 to fold and assemble into Gβγ and RGS-Gβ5 heterodimers. The importance of PhLP1 in the assembly process was first demonstrated in vivo in a retinal rod photoreceptor-specific deletion of PhLP1. To test whether this mechanism applied to other cell types, we prepared a second mouse line that specifically disrupts the PhLP1 gene in cone photoreceptor cells and measured the effects on G-protein expression and cone visual signal transduction. In PhLP1 depleted cones, Gt2 and RGS9-Gβ5 levels were dramatically reduced, resulting a 60-fold decrease in cone sensitivity and a 50-fold increase in cone photoresponse recovery time. These results demonstrate a common mechanism of Gβγ and RGS9-Gβ5 assembly in rods and cones, underlining the significance of PhLP1/CCT-mediated folding in G protein signaling.PDCD5 has been proposed to act as a pro-apoptotic factor and tumor suppressor. However, the mechanisms underlying its apoptotic function are largely unknown. A proteomics search for PhLP1 binding partners revealed a robust interaction between PDCD5 and CCT. PDCD5 formed a complex with CCT and β-tubulin, a key CCT folding substrate, and specifically inhibited β-tubulin folding. Cryo-electron microscopy studies of the PDCD5-CCT complex suggested a possible mechanism of inhibition of β-tubulin folding. PDCD5 binds the apical domain of the CCTβ subunit, projecting above the folding cavity without entering it. Like PDCD5, β-tubulin also interacts with the CCTβ apical domain, but a second site is found at the sensor loop deep within the folding cavity. These orientations of PDCD5 and β-tubulin suggest that PDCD5 sterically interferes with β-tubulin binding to the CCTβ apical domain and inhibits β-tubulin folding. Given the importance of tubulins in cell division and proliferation, PDCD5 might exert its apoptotic function at least in part through inhibition of β-tubulin folding.

In order for G protein signaling to occur, the G protein heterotrimer must be assembled from its nascent polypeptides. The most difficult step in this process is the formation of the Gβγ dimer from the free subunits since both are unstable in the absence of the other. Recent studies have shown that phosducin-like protein (PhLP1) works as a co-chaperone with the cytosolic chaperonin complex (CCT) to fold Gβ and mediate its interaction with Gγ. However, these studies did not address questions concerning the scope of PhLP1 and CCT-mediated Gβγ assembly, which are important questions given that there are four Gβs that form various dimers with 12 Gγs and a 5th Gβ that dimerizes with the four regulator of G protein signaling (RGS) proteins of the R7 family. The data presented in Chapter 2 shows that PhLP1 plays a vital role in the assembly of Gγ2 with all four Gβ1-4 subunits and in the assembly of Gβ2 with all twelve Gγ subunits, without affecting the specificity of the Gβγ interactions. The results of Chapter 3 show that Gβ5-RGS7 assembly is dependent on CCT and PhLP1, but the apparent mechanism is different from that of Gβγ. PhLP1 seems to stabilize the interaction of Gβ5 with CCT until Gβ5 is folded, after which it is released to allow Gβ5 to interact with RGS7. These findings point to a general role for PhLP1 in the assembly of all Gβγ combinations, and suggest a CCT-dependent mechanism for Gβ5-RGS7 assembly that utilizes the co-chaperone activity of PhLP1 in a unique way. Chapter 4 discusses PhLP2, a recently discovered essential protein, and member of the Pdc family that does not play a role in G protein signaling. Several studies have indicated that PhLP2 acts as a co-chaperone with CCT in the folding of actin, tubulin, and several cell cycle and pro-apoptotic proteins. In a proteomics screen for PhLP2A interacting partners, α-tubulin, 14-3-3, elongation factor 1α, and ribosomal protein L3 were found. Further proteomics studies indicated that PhLP2A is a phosphoprotein that is phosphorylated by CK2 at threonines 47 and 52.