京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第18918号 / 薬科博第32号 / 新制||薬||4(附属図書館) / 31869 / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 松﨑 勝巳, 教授 加藤 博章, 教授 石濱 泰 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21339号 / 医博第4397号 / 新制||医||1031(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 井上 治久, 教授 宮本 享, 教授 林 康紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The type VI secretion system (T6SS) is a protein export pathway that mediates competition between Gram-negative bacteria by facilitating the injection of toxic effector proteins from attacking cells into target cells. To function properly, many T6SSs require at least one protein that possesses a proline-alanine-alanine-arginine (PAAR) domain. These PAAR domains are often found within large, multi-domain effectors that possess additional N- and C-terminal extension domains whose function in type VI secretion is not well understood. The work described herein uncovers the function of these accessory domains across multiple PAAR-containing effectors. First, I demonstrated that thousands of PAAR effectors possess N-terminal transmembrane domains (TMDs) and that these effectors require a family of molecular chaperones for stability in the cell prior to their export by the T6SS. Our findings are corroborated by co-crystal structures of chaperones in complex with the TMDs of their cognate effectors, capturing the first high-resolution structural snapshots of T6SS chaperone-effector interactions. Second, I characterize a previously undescribed prePAAR effector named Tas1. My work shows that the C-terminus of Tas1 possesses a toxin domain that pyrophosphorylates ADP and ATP to synthesize the nucleotides adenosine penta- and tetraphosphate (hereafter referred to as (p)ppApp). Delivery of Tas1 into competitor cells drives the rapid accumulation of (p)ppApp, depletion of ADP and ATP, and widespread dysregulation of essential metabolic pathways, resulting in target cell death. These findings reveal a new mechanism of interbacterial antagonism, the first characterization of a (p)ppApp synthetase and the first demonstration of a role for (p)ppApp in bacterial physiology. TMD- and toxin-containing PAAR proteins constitute a large family of over 6,000 T6SS effectors found in Gram-negative bacteria. My work on these proteins has uncovered that different regions found within effectors have distinct roles in trafficking between bacterial cells and in the growth inhibition of the target cell. / Dissertation / Doctor of Philosophy (PhD) / Bacteria constantly compete with their neighbours for resources and space. The type VI secretion system is a protein complex that facilitates competition between Gram-negative bacteria by facilitating the injection of protein toxins, also known as effectors, from attacking cells into target cells. In this work, I characterize several members of a large family of membrane protein effectors. First, I showed that these effectors require a novel family of chaperone proteins for stability and recruitment to the type VI secretion system apparatus. Second, I characterized the growth-inhibitory properties of one of these effectors in-depth and showed that it possesses a toxin domain that depletes the essential nucleotides ATP and ADP in target cells by synthesizing the nucleotides adenosine penta- and tetraphosphate, (p)ppApp. Together, these studies revealed a new mechanism for the intercellular delivery of membrane protein toxins and uncovered the first known physiological role of a (p)ppApp-synthesizing enzyme in bacteria.

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.

Many proteins cannot fold into their native state without the assistance of one or more molecular chaperones. Chaperonins are an essential class of chaperones that provide an isolated chamber for proteins to fold. CCT, a group II chaperonin found in eukaryotes assists in the folding of actins, tubulins, and many other cellular proteins. PhLP1 is a member of the phosducin protein family that assists CCT in the folding of Gβ and its subsequent assembly with Gγ. However, previous studies have not addressed the scope of PhLP1 and CCT-mediated Gβγ; assembly. The data presented in Chapter 2 shows that PhLP1 plays a vital role in the assembly of all Gγ subunits that form dimers with Gβ2 and the assembly of Gγ2 with Gβ1-4, without affecting the specificity of the Gβγ interactions. These findings suggest that PhLP1 has a general role for the assembly of all Gβγ combinations. Although the role of PhLP1 as a co-chaperone for Gβγ assembly has been established, other possible functions for PhLP1 either as a co-chaperone or otherwise are yet to be investigated. A known tumor suppressor protein, PDCD5, was found to interact with PhLP1 in a co-immunoprecipitation proteomics screen. The data presented in Chapter 3 show that PDCD5 binds PhLP1 indirectly through a ternary complex with CCT. Our results signify that the apoptotic function of PDCD5 is cytosolic, is phosphorylation dependent, and most likely involves CCT. Moreover, structural analysis suggests that over-expressed PDCD5 blocks β-actin from entering the CCT folding cavity, suggesting a co-chaperone role for PDCD5 in inhibiting or enhancing folding of yet-to-be determined CCT substrates. Compared to PhLP1, the functions of other members of the phosducin family, PhLP2A, PhLP2B, and PhLP3, are poorly understood. They have no role in G-protein signaling, but appear to assist CCT in the folding of actin, tubulin and proteins involved in cell cycle progression. Chapter 4 investigates the possibility of PhLP2 and/or PhLP3 acting as co-chaperones in the folding and assembly of actins and tubulins. In addition, another mediator of cellular signaling, 14-3-3ε, was found to interact with PhLP2A in a phosphorylation dependent manner and relieve the inhibition of β-actin folding caused by PhLP2A over-expression.

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.

We studied the usefulness of genetic fusion to TELSAM polymers as an effective protein crystallization strategy. We observed novel properties in crystals of two TELSAM-target protein fusions. TELSAM as a crystallization chaperone shows rapid crystallization when it's fused to target proteins and possibly with a greater propensity. Some TELSAM-target fusions crystallized more rapidly than the same target protein alone. TELSAM-target proteins can be crystallized at relatively low protein concentrations such as 0.1 mg/mL. TELSAM requires no TELSAM polymers touching one another in the crystal lattice in order to form well-diffracting crystals. This lack of crystal contacts has not been observed in previously reported TELSAM crystal structures. Flexible TELSAM-target protein linkers can allow target proteins to find productive binding modes against the TELSAM polymer. This study tested TELSAM linker lengths varying by the number of glycines, such as 2xGly, 4xGly, 6xGly, 8xGly, and 10xGly. Only TELSAM fused to UBA with 2 and 4 glycine linkers were crystalized. TELSAM polymers can adjust their helical rise to allow fused target proteins to make productive crystal contacts, and fusion to TELSAM polymers increases avidity to stabilize weak inter-target protein crystal contacts. In conclusion, we report features of TELSAM-target protein crystal structures and outline future work needed to validate TELSAM as a crystallization chaperone and define the best practices for its use.

Hsp70 molecular chaperones protect proteins from aggregation, assist in their native structure formation, and regulate stress responses in the cell. A mechanistic understanding of Hsp70 function will be necessary to explain its physiological roles and guide the therapeutic modulation of various disease states. To this end, several fundamental features of the Hsp70 structure-function relationship are investigated. The central component of Hsp70 chaperone function is its capacity for allosteric signaling between structural domains and tunable binding of misfolded protein substrates. In order to identify a cooperative network of sites that mediates interdomain allostery within Hsp70, a mutational correlation analysis is performed using genetic data. Evolutionarily correlations that describe an allosteric network are validated by examining roles for implicated sites in cellular fitness and molecular function. In a second component of the Hsp70 molecular mechanism, a novel function is discovered for the disordered C-terminal tail. This region of the protein enhances the refolding efficiency of substrate proteins independently of interdomain allostery and is required in the cell upon depletion of compensatory chaperones, suggesting a previously undescribed mode of chaperone action. Finally, experiments are initiated to assess the dynamic assembly of Hsp70 domains in various allosteric states and how domain orientations may be guided through interaction with partner co-chaperone proteins.

Indiana University-Purdue University Indianapolis (IUPUI) / Heat Shock Protein 60/10 (HSP60/10, or GroEL/ES in bacteria) chaperonin systems play a critical role in protein homeostasis through facilitating proper folding of misfolded or partially folded polypeptides that are otherwise prone to aggregation. HSP60 chaperonins are highly conserved and essential in nearly all organisms studied thus far, making them a promising target for antibiotic development. Early high-throughput screens in the Johnson lab have identified five main scaffolds that, though hit-to-lead development, have been optimized for chaperonin inhibition and antimicrobial effects. While these initial studies have shown promising evidence to support the viability of a chaperonin-targeting antibiotic strategy, it was unclear whether the conservation of human HSP60 (48% identity to bacterial GroEL) would hinder this therapeutic strategy from advancing due to potential toxicity associated with off-target inhibition of the human homolog. Additionally, while chaperonin inhibition often correlated with cytotoxicity to the various pathogens studied, there was a clear need to investigate inhibitor mechanisms to 1) verify on-target effects, and 2) guide future development of more potent and selective chaperonin-targeting antibiotic candidates. Herein, we conduct a medium-throughput screening of known bioactive molecules, approved drugs, and natural products against both bacterial GroEL and human HSP60, demonstrating that most molecules exhibited low-to-no toxicity to human cells in culture, despite being near equipotent inhibitors of human HSP60 and E. coli GroEL in our refolding assays. Thus, sequence conservation between human HSP60 and bacterial GroELs does not necessarily predict toxicity in vivo. We then investigate inhibitory mechanisms of our most well-established inhibitor series, the phenylbenzoxazole (PBZ) series, identifying three binding sites whereby PBZ molecules modulate GroEL folding and ATPase functions in a site-specific manner, predominately through its ability to interact with its co-chaperone GroES. Finally, we demonstrate that two standard of care drugs for T. brucei infections, suramin and nifurtimox, may elicit their trypanocidal effects through inhibiting HSP60. Due to structural similarities, we then screened our N-acylhydrazone (NAH) and α,β-unsaturated ketone (ABK) series of HSP60 inhibitors against T. brucei, finding that they are highly potent and selective trypanocidal agents. Together, these studies further support HSP60 as a viable drug target for antibiotic development. / 2025-07-03

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