Cells transduce signals from the external environment to the inside through phosphatidylinositol-3,4,5-phosphate (PIP3), a major signaling lipid on the plasma membrane. PIP3 is generated by the action of a family of lipid kinases called Class I phosphoinositide 3-kinases (PI3Ks) and controls an array of essential cellular functions including growth, proliferation, survival, metabolism and cytoskeletal architecture. PI3Ks are large heterodimeric complexes composed of a catalytic p110 subunit and a regulatory subunit. Crucial to healthy PIP3 production is the interpretation of diverse activating inputs arising from signaling proteins on the membrane by these subunits. A member of the PI3K family, PI3Kγ is a master regulator of immune functions with therapeutic implications in cancer immunity and inflammatory disease. PI3Kγ is distinct from other well studied PI3Ks due to the presence of unique regulatory mechanisms that control its ability to integrate signals from G-protein coupled receptors, small GTPases, immunoglobulin receptors and toll-like receptors. However, unlike the other well characterized PI3Ks, there are significant gaps in understanding of the molecular details of these mechanisms and how regulatory processes are translated into functions elicited by PI3Kγ in its unique milieu within the immune system. To understand PI3Kγ regulation, I utilized a synergy of cutting-edge approaches including protein biochemistry, X-ray crystallography, cryo-electron microscopy and hydrogen-deuterium exchange mass spectrometry to elucidate the unique regulatory features within its catalytic and regulatory subunits and how these features are disrupted in disease. These studies significantly advanced our understanding of how this enzyme functions and provided novel avenues for potentially targeting the enzyme better in therapy. This dissertation will consist of an introduction chapter summarizing PI3Kγ regulation and its role in disease, followed by three data chapters investigating previously uncharacterized regulatory mechanisms that control its function and how these mechanisms are implicated in disease. These data chapters are followed by a final chapter describing conclusions and future directions. In summary, the work presented in this thesis provides novel insights into the unique regulatory features in the catalytic and regulatory subunits of PI3Kγ that mediate its stimulation by upstream activating partners and the mechanisms by which these features are disrupted in disease. Further, these studies have facilitated the effective characterization of potent molecules that can specifically target PI3Kγ in disease. Altogether, the findings of this dissertation constitute a major advancement in our understanding of PI3K regulation. / Graduate

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第18121号 / 理博第3999号 / 新制||理||1576(附属図書館) / 30979 / 京都大学大学院理学研究科生物科学専攻 / (主査)准教授 土井 知子, 教授 七田 芳則, 教授 高田 彰二 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

The aim of this study is the three-dimensional structural characterization of the genome packaging inside viral capsids via cryo-electron microscopy and three-dimensional reconstruction. The genome of some single-stranded viruses can be densely packaged within their capsid shells. Several stretches of the genome are known to adopt stable secondary structures, however, to date, little is known about the three-dimensional organization of the genome inside their capsid shells. Two techniques have been developed to facilitate the structural elucidation of genome packaging: the asymmetric random-model method, and the symmetry-mismatch, random model method. Both techniques were successfully tested with model and experimental data. The new algorithms were applied to study the genome structure of poliovirus and satellite tobacco mosaic virus. We have not yet found a consistent structure for the two genomes. Nevertheless, we have found that the genome of satellite tobacco mosaic genome is very stable, supporting a model where the RNA acts as a scaffold, with potential implications in capsid stability and assembly.

In the infection process, native poliovirus (160S) first converts to a cell-entry intermediate (135S) particle, which causes the externalization of capsid proteins VP4 and the N-terminus of VP1 (residues 1-53). The externalization of these entities is followed by release of the RNA genome, leaving an empty (80S) particle. Three antibodies were utilized to track the location of VP1 residues in different states of poliovirus by cryogenic electron microscopy (cryo-EM). "P1" antibody binds to N-terminal residues 24-40 of VP1. Three-dimensional reconstruction of 135S-P1 showed that P1 binds to a prominent capsid peak known as the "propeller tip". In contrast, our initial 80S-P1 reconstruction showed P1 Fabs also binding to a second site, ~60 Å distant, at the icosahedral twofold axes. Analysis of 80S-P1 reconstructions showed that the overall population of 80S-P1 particles consisted of three kinds of capsids: those with P1 Fabs bound only at the propeller tips; only at the twofold axes; or simultaneously at both positions. Our results indicate that, in 80S particles, a significant fraction of VP1 can deviate from icosahedral symmetry. Similar deviations from icosahedral symmetry may be biologically significant during other viral transitions. "C3" antibody binds to 93-103 residues (BC loop) of VP1. The C3 epitope shifts outwards in radius by 4.5% and twists through 15° in the 160S-to-135S transition, but appears unchanged in the 135S-to-80S transition. In addition, binding of C3 to either 160S or 135S particles causes residues of the BC loop to move an estimated 5 (±2) Å, indicating flexibility. The flexibility of BC loop may play a role in cell-entry interactions. At 37°C, the structure of poliovirus is dynamic, and internal polypeptides VP4 and the N-terminus of VP1 externalize reversibly. An antibody, binding to the residues 39-55 of VP1, was utilized to track the location of the N-terminus of VP1 in 160S particle in the "breathing" state. The resulting reconstruction showed the capsid expands similarly to the irreversibly altered 135S particle, but the N-terminus of VP1 is located near the twofold axes, instead of the propeller tip as in 135S particles.

No description available.

<p dir="ltr">In life sciences, the determination of macromolecular structures and their functions, particularly proteins and protein complexes, is of paramount importance, as these molecules play critical roles within cells. The specific physical interactions of macromolecules govern molecular and cellular functions, making the 3D structure elucidation of these entities essential for comprehending the mechanisms underlying life processes, diseases, and drug discovery. Cryo-electron microscopy (cryo-EM) has emerged as a promising experimental technique for obtaining 3D macromolecular structures. In the course of my research, I proposed CryoREAD, an innovative AI-based method for <i>de nov</i>o DNA/RNA structure modeling. This novel approach represents the first fully automated solution for DNA/RNA structure modeling from cryo-EM maps at near-atomic resolution. However, as the resolution decreases, structure modeling becomes significantly more challenging. To address this challenge, I introduced Emap2sec+, a 3D deep convolutional neural network designed to identify protein secondary structures, RNA, and DNA information from cryo-EM maps at intermediate resolutions ranging from 5-10 Å. Additionally, I presented Alpha-EM-Multimer, a groundbreaking method for automatically building full protein complexes from cryo-EM maps at intermediate resolution. Alpha-EM-Multimer employs a diffusion model to trace the protein backbone and subsequently fits the AlphaFold predicted single-chain structure to construct the complete protein complex. Notably, this method stands as the first to enable the modeling of protein complexes with more than 10,000 residues for cryo-EM maps at intermediate resolution, achieving an average TM-Score of predicted protein complexes above 0.8, which closely approximates the native structure. Furthermore, I addressed the recognition of local structural errors in predicted and experimental protein structures by proposing DAQ, an evaluation approach for experimental protein structure quality that utilizes detection probabilities derived from cryo-EM maps via a pretrained multi-task neural network. In the pursuit of evaluating protein complexes generated through computational methods, I developed GNN-DOVE and DOVE, leveraging convolutional neural networks and graph neural networks to assess the accuracy of predicted protein complex structures. These advancements in cryo-EM-based structural modeling and evaluation methodologies hold significant promise for advancing our understanding of complex macromolecular systems and their biological implications.</p>

No description available.

Human immunodeficiency virus (HIV) Rev and Hepatitis B virus (HBV) e-antigen are both viral proteins that have key functions in their respective viral replication cycles. Both have evaded crystallization for decades due to their tendency to aggregate and/or form higher-order species. In this thesis the structure determination of HIV Rev and HBV e-antigen is presented—achieved via complexing with monoclonal antibody Fab fragments—and their structures are analysed. HIV Rev is a small regulatory protein that mediates the nuclear export of viral mRNAs, an essential step in the HIV replication cycle. In this process, Rev cooperatively oligomerises onto a highly structured RNA motif, the Rev response element. The structure of Rev (complexed with Fab), determined to 2.3 Å resolution, reveals a molecular dimer where the ordered portion of each subunit (N-terminal domain; NTD; residues 9-65) contains two coplanar a-helices arranged in hairpin fashion. Rev subunits dimerise via interaction of identical hydrophobic patches that overlap to form a V-shaped assembly. Mating of hydrophobic patches on the outer surface of the dimer promotes higher order interactions. Cryo-electron microscopy and helical image reconstruction of in vitro assembled Rev filaments were performed to better understand higher-order Rev oligomerisation. Reconstructions of Rev filaments were determined to ~13 Å resolution, permitting docking of the Rev NTD structure. Conformational variability of the Rev dimer subunits and use of a third ligomerisation interface engender filaments that can expand and contract. Both characteristics were also observed in the crystal structures of Rev. Surface features of the Rev filaments are altered in different expansion states, which may have implications for the assembled forms that Rev adopts during nuclear export of RNA and subsequent re-import into the nucleus. Various models for Rev oligomerisation onto the viral RNA are proposed. Chronic Hepatitis B virus (HBV) infection afflicts millions worldwide with cirrhosis and liver cancer. HBV e-antigen (HBeAg), a clinical marker for disease severity, is a soluble variant of the protein (core antigen, HBcAg) that forms the building-blocks of capsids. HBeAg is not required for virion production, but is implicated in establishing immune tolerance and chronic infection. The crystal structure of HBeAg clarifies how the short N-terminal propeptide of HBeAg induces a radically altered mode of dimerisation relative to HBcAg (~140 rotation), which is locked into place through formation of intramolecular disulfide bridges. This structural switch precludes capsid assembly and engenders a distinct antigenic repertoire, explaining why the two antigens are cross-reactive at the T-cell level (through sequence identity) but not at the B-cell level (through conformation). The structure offers insight into how HBeAg may establish immune tolerance for HBcAg while evading its robust immunogenicity.

In eukaryotes, cell proliferation and cell cycle transitions are strictly controlled by the anaphase-promoting complex/cyclosome (APC/C). The APC/C is an E3 ubiquitin ligase that regulates chromatid segregation at the metaphase to anaphase transition, exit from mitosis and the establishment and maintenance of G1. The APC/C’s catalytic activity and substrate specificity are controlled by its interactions with two coactivators, Cdc20 and Cdh1. In contrast to Cdh1, APC/C activation by Cdc20 during mitosis requires hyper-phosphorylation of APC/C subunits by cyclin-dependent kinase (Cdk) and polo kinase. The aim of the first part of this thesis was to understand how mitotic phosphorylation regulates APC/C activity. Using cryo-electron microscopy and biochemical analysis, we found that an auto-inhibitory segment of the Apc1 subunit acts as a molecular switch that in apo unphosphorylated APC/C interacts with a coactivator-binding site (C-box binding site), thereby obstructing engagement of Cdc20. Phosphorylation of the auto-inhibitory segment displaces it from the C-box binding site to relieve APC/C auto-inhibition. Efficient phosphorylation of the auto-inhibitory segment requires the recruitment of the kinase Cdk-cyclin-Cks to a hyper-phosphorylated loop of Apc3. In addition to regulation of APC/C activity by phosphorylation, ordered cell progression is ensured by the ability of the APC/C to target substrate degradation in a defined order. At mitosis onset, degradation of securin and cyclin B1 is inhibited by the spindle assembly checkpoint, exerted by the mitotic checkpoint complex (MCC), whereas both cyclin A2 and Nek2A are not subject to MCC inhibition. The aim of the second part of the thesis was to elucidate the mechanism of how the APC/C achieves its substrate specificity. Our biochemical analysis showed that the resistance of cyclin A2 to MCC inhibition is due to its ABBA motif and the Cdk-associated Cks2 subunit. Furthermore, we found that it is the Cdc20 molecule of the MCC that binds to the ABBA motif to allow for cyclin A2 ubiquitination. Strikingly, mutating all three known degrons (KEN box, D box and ABBA motif) of cyclin A did not affect its ubiquitination by APC/CCdc20. Deletion of a potential novel degron found within residues 60-80 of cyclin A2 impaired cyclin A2 ubiquitination.

No description available.