Kinesin-13, tubulins and their new roles in DNA damage repair

Les microtubules sont de longs polymères cylindriques de la protéine α, β tubuline, utilisés dans les cellules pour construire le cytosquelette, le fuseau mitotique et les axonèmes. Ces polymères creux sont cruciaux pour de nombreuses fonctions cellulaires, y compris le transport intracellulaire et la ségrégation chromosomique pendant la division cellulaire. Au fur et à mesure que les cellules se développent, se divisent et se différencient, les microtubules passent par un processus, appelé instabilité dynamique, ce qui signifie qu’ils basculent constamment entre les états de croissance et de rétrécissement. Cette caractéristique conservée et fondamentale des microtubules est étroitement régulée par des familles de protéines associées aux microtubules. Les protéines de kinésine-13 sont une famille de facteurs régulateurs de microtubules qui dépolymérisent catalytiquement les extrémités des microtubules. Cette thèse traite d’abord des concepts mécanistiques sur le cycle catalytique de la kinésine-13. Afin de mieux comprendre le mécanisme moléculaire par lequel les protéines de kinésine-13 induisent la dépolymérisation des microtubules, nous rapportons la structure cristalline d’un monomère de kinésine-13 catalytiquement actif (Kif2A) en complexe avec deux hétérodimères αβ-tubuline courbés dans un réseau tête-à-queue. Nous démontrons également l’importance du « cou » spécifique à la classe de kinésine-13 dans la dépolymérisation catalytique des microtubules. Ensuite, nous avons cherché à fournir la base moléculaire de l’hydrolyse tubuline-guanosine triphosphate (GTP) et son rôle dans la dynamique des microtubules. Dans le modèle que nous présentons ici, l’hydrolyse tubuline-GTP pourrait être déclenchée par les changements conformationnels induits par les protéines kinésine-13 ou par l’agent chimique stabilisant paclitaxel. Nous fournissons également des preuves biochimiques montrant que les changements conformationnels des dimères de tubuline précèdent le renouvellement de la tubuline-GTP, ce qui indique que ce processus est déclenché mécaniquement. Ensuite, nous avons identifié la kinésine de microtubule Kif2C comme une protéine associée à des modèles d’ADN imitant la rupture double brin (DSB) et à d’autres protéines de réparation DSB connues dans les extraits d’œufs de Xenope et les cellules de mammifères. Les cassures double brin d’ADN (DSB) sont un type majeur de lésions d’ADN ayant les effets les plus cytotoxiques. En raison de leurs graves impacts sur la survie cellulaire et la stabilité génomique, les DSB d’ADN sont liés à de nombreuses maladies humaines, y compris le cancer. Nous avons constaté que les activités PARP et ATM étaient toutes deux nécessaires pour le recrutement de Kif2C sur les sites de réparation de l’ADN. Kif2C knockout ou inhibition de son activité de dépolymérisation des microtubules a conduit à l’hypersensibilité des dommages à l’ADN et à une réduction de la réparation du DSB via la jonction terminale non homologue et la recombinaison homologue. Dans l’ensemble, notre modèle suggère que les protéines de kinésine-13 peuvent interagir avec les dimères de tubuline aux extrémités microtubules et modifier leurs conformations, moduler l’étendue des extrêmités tubuline-GTP dans les cellules et déclencher le désassemblage des microtubules. Ces deux modèles pourraient être des clés pour démêler les mécanismes impliqués dans le nouveau rôle de Kif2C dans la réparation de l’ADN DSB sans s’associer à des polymères de microtubules.Microtubules are long, cylindrical polymers of the proteins α, β tubulin, used in cells to construct the cytoskeleton, the mitotic spindle and axonemes. These hollow polymers are crucial for many cellular functions including intracellular transport and chromosome segregation during cell division. As cells grow, divide, and differentiate, microtubules go through a process, called dynamic instability, which means they constantly switch between growth and shrinkage states. This conserved and fundamental feature of microtubules is tightly regulated by families of microtubule-associated proteins (MAPs). Kinesin-13 proteins are a family of microtubule regulatory factors that catalytically depolymerize microtubule ends. This thesis first discusses mechanistic insights into the catalytic cycle of kinesin-13. In order to better understand the molecular mechanism by which kinesin-13 proteins induce microtubule depolymerization, we report the crystal structure of a catalytically active kinesin-13 monomer (Kif2A) in complex with two bent αβ-tubulin heterodimers in a head-to-tail array. We also demonstrate the importance of the kinesin-13 class-specific “neck” in modulating Adenosine triphosphate (ATP) turnover and catalytic depolymerization of microtubules. Then, we aimed to provide the molecular basis for tubulin-Guanosine triphosphate (GTP) hydrolysis and its role in microtubule dynamics. Although it has been known for decades that tubulin-GTP turnover is linked to microtubule dynamics, its precise role in the process and how it is driven are now well understood. In the model we are presenting here, tubulin-GTP hydrolysis could be triggered via the conformational changes induced by kinesin-13 proteins or by the stabilizing chemical agent paclitaxel. We also provide biochemical evidence showing that conformational changes of tubulin dimers precedes the tubulin-GTP turnover, which indicates that this process is triggered mechanically. Next, we identified microtubule kinesin Kif2C as a protein associated with double strand break (DSB)-mimicking DNA templates and other known DSB repair proteins in Xenopus egg extracts and mammalian cells. DNA double strand breaks (DSBs) are a major type of DNA lesions with the most cytotoxic effects. Due to their sever impacts on cell survival and genomic stability, DNA DSBs are related to many human diseases including cancer. Here we found that PARP and ATM activities were both required for the recruitment of Kif2C to DNA repair sites. Kif2C knockdown/knockout or inhibition of its microtubule depolymerizing activity led to accumulation of endogenous DNA damage, DNA damage hypersensitivity, and reduced DSB repair via both non-homologous end-joining (NHEJ) and homologous recombination (HR). Interestingly, genetic depletion of KIF2C, or inhibition of its microtubule depolymerase activity, reduced the mobility of DSBs, impaired the formation of DNA damage foci, and decreased the occurrence of foci fusion and resolution. Altogether, our findings shed light on the mechanisms involved in kinesin-13 catalyzed microtubule depolymerization. Our tubulin-GTP hydrolysis model suggests that kinesin-13 proteins may interact with tubulin dimers at microtubules ends and alter their conformations, modulate the extent of the GTP caps in cells and trigger microtubule disassembly. These two models could be keys to unravel the mechanisms involved in the novel role of Kif2C in DNA DSB repair without associating with microtubule polymers

Microtubules dual chemo- and thermo-responsive depolymerization & optimization of noncovalent loading of vinblastine on single-walled carbon nanotube

The effects of the chemotherapeutic agent vinblastine versus the low temperature of 277 K on the structure of αβ-tubulin heterodimer were investigated by means of molecular dynamics simulations. Individual experiments have shown that vinblastine-bound heterodimer, and its apo structure under low temperature of 277 K, both undergo conformational changes toward destabilization of the dimer as compared to the apo tubulin at 300 K. Both factors exhibit weakening the longitudinal interactions of tubulin heterodimer through displacing dimer interfacial segments, resulting in the dominant electrostatic repulsion at the interface of the subunits. The two independent factors of temperature and anti-mitotic agent facilitate folding alterations in the functional segments of H1-S2 loop, H3, H10 helices and T7 loop, which are known to be important in either longitudinal or lateral contacts among αβ-heterodimers in microtubule protofilaments and the depolymerization mechanism of microtubules. Carbon nanotubes have become one of the candidates for transporting drugs to target sites, because of their size scale, huge surface area and high cellular uptake. Many experimental studies of carbon nanotube drug delivery have been performed in the past decade. The delivery studies of vinblastine and its target microtubule are important, because of the significant role of vinblastine in cancer therapy. However, the interactions between vinblastine and carbon nanotubes have yet to be investigated. The computational studies of the interactions between vinblastine and carbon nanotubes under different conditions are presented in this thesis. The vinblastine-carbon nanotube interactions have been studied from the following perspectives: loading capacity (one to three vinblastine molecules loaded); tube structure (armchair, chiral and zigzag tubes); tube functionalization; and temperature variations (277 K and 300 K). The functionalization of carbon nanotubes strengthened the drug-carrier interactions of all systems at 300 K. The functionalized carbon nanotubes of armchair type were identified suitable for drug delivery at both 277 K and 300 K, due to the relatively strong drug-carrier interactions. The functionalized chiral nanotubes were found especially useful for delivery at 277 K due to the enhanced drug-carrier interactions at this temperature

Dynamics and Regulation of Cytoskeletal Proteins

In this dissertation, I apply molecular dynamics (MD) simulations to improve our understanding of the dynamics, and hence, function and regulation of cytoskeletal proteins. Microtubules and kinesin motor proteins play a critical role in the cytoskeleton of the cell. They provide structural support, facilitate cellular transport, and are involved in beating of cilia and flagella, and in separation of chromosomes during the cell cycle. The importance of tubulin as a vital therapeutic target is exemplified by the widely prescribed paclitaxel (Taxol), an anti-cancer drug that prevents cancer cells from undergoing cell division by arresting tubulin dynamics. Furthermore, the importance of understanding the structural, dynamical, and functional aspects of kinesin motor domains and their modifications is demonstrated by efforts in developing small-molecule inhibitors as antimitotic therapeutic agents in various cancers. However, despite strong conservation of the motor domain across the kinesin superfamily, how various kinesins have tailored their motility characteristics to best meet their functional needs in cells remains unclear. Detailed comparison of structures from large heterogeneous protein families, such as kinesin motors, can inform on structural dynamic mechanisms critical for protein function including ligand binding, enzymatic catalysis, allosteric regulation and bimolecular recognition. However, existing tools for quantitative analyses of their sequence, structure and dynamics often require significant computational expertise and typically remain accessible only to expert users with relevant programming skills. In the first section of my dissertation, I describe the development of Bio3D-web, a free and open-source online application for interactive investigation of protein sequence-structure-dynamic relationships. Bio3D-web requires no programming knowledge and thus decreases the entry barrier to performing advanced comparative structural bioinformatics analyses. In the second part, I discuss a method for analyzing experimental structures and dynamical data generated with MD simulations. The ensemble distance difference matrix method (eDDM) analyzes changes in residue-residue distances in protein structures and dynamical data to identify residues critical for protein regulation and function. I apply eDDM to three families of kinesin motor proteins in the following case studies: First, I elucidate the effect of a posttranslational modification in kinesin 5 mitotic motor Eg5. I show that acetylation of residue K146 in Eg5 alters its mechanochemical properties, wherein it acts as a “brake” during spindle separation in cells during mitosis. Second, I identify residues critical for force generation in kinesin 1 transport motor KIF5C. Mutating these residues in two important structural elements—A5G and S8G in the cover strand and N334A in the neck linker—severely cripple the ability of motors in ensemble to generate force during intracellular transport. Third, I characterize the allosteric effects of disease-associated variants in kinesin 3 neuronal transport motor KIF1A. KIF1A-associated neurological disorder (KAND) is associated with cognitive disability, spasticity, and cerebellar atrophy, typically with a progressive course. In the third part, I highlight the divergent mechanism of tubulin polymerization in C. elegans. Through comparative analysis of MD simulations of C. elegans and B. taurus tubulin dimers, I found that sequence changes in the C. elegans tubulin lead to additional secondary structure formation in the lateral contact loops, and this changes the polymerization behavior as well as the structure of the microtubule. Finally, I also map the inter-conformer relationships of experimentally determined structures of tubulin through principal component analysis (PCA), enabling comparison of the intrinsic dynamics of tubulin heterodimers, such as different isoforms, nucleotide states, and disease-associated mutations.PHDBioinformaticsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/153354/1/jari_1.pd

FREE ENERGIES IN BIOMOLECULAR SIMULATIONS: FROM PROTEIN-PROTEIN INTERACTIONS TO UNFOLDING INHIBITION

Part I - Microtubules are polymeric structures formed by the self association of tubulin dimers. They are extremely dynamical structures, that can undergo phases of growing and shrinking, playing a key role during cells proliferation process. Due to its importance for mitosis, tubulin is the target of many anticancer drugs currently in use or under clinical trial. The success of these molecules, however, is limited by the onset of resistant tumor cells during the treatment, so new resistance-proof compounds need to be developed. We analyze the protein-protein interactions allowing microtubules formation using molecular dynamics and free energy calculations. We were able to identify the most important amino acids for tubulin-tubulin binding and thus to design peptides, corresponding to tubulin subsequences. These peptides, able to interfere with microtubules formations, were proved to exhibit antitumoral activity. Part II - Understanding the molecular mechanisms that allow some organisms to survive in extremely harsh conditions is an important achievement that might disclose a wide range of applications and that is constantly drawing the attention of many research fields. The simple small organic molecules, called osmolytes, responsible for the high adaptability of these living creatures are well known and of common use; nevertheless a full disclosure of the machinery behind their activity is still to be obtained. We developed a computational approach that, taking advantage of advanced simulation techniques, allowed to fully describe the effects of osmo-protectants on a small hairpin peptide and on a full mini-protein. The computational study allowed to highlight interesting new features and to develop a theory on the \u201cosmoprotection driving force\u201d

The model of local axon homeostasis - Explaining the role and regulation of microtubule bundles in axon maintenance and pathology

Axons are the slender, cable-like, up to meter-long projections of neurons that electrically wire our brains and bodies. In spite of their challenging morphology, they usually need to be maintained for an organism's lifetime. This makes them key lesion sites in pathological processes of ageing, injury and neurodegeneration. The morphology and physiology of axons crucially depends on the parallel bundles of microtubules (MTs), running all along to serve as their structural backbones and highways for life-sustaining cargo transport and organelle dynamics. Understanding how these bundles are formed and then maintained will provide important explanations for axon biology and pathology. Currently, much is known about MTs and the proteins that bind and regulate them, but very little about how these factors functionally integrate to regulate axon biology. As an attempt to bridge between molecular mechanisms and their cellular relevance, we explain here the model of local axon homeostasis, based on our own experiments in Drosophila and published data primarily from vertebrates/mammals as well as C. elegans. The model proposes that (1) the physical forces imposed by motor protein-driven transport and dynamics in the confined axonal space, are a life-sustaining necessity, but pose a strong bias for MT bundles to become disorganised. (2) To counterbalance this risk, MT-binding and -regulating proteins of different classes work together to maintain and protect MT bundles as necessary transport highways. Loss of balance between these two fundamental processes can explain the development of axonopathies, in particular those linking to MT-regulating proteins, motors and transport defects. With this perspective in mind, we hope that more researchers incorporate MTs into their work, thus enhancing our chances of deciphering the complex regulatory networks that underpin axon biology and pathology

Mechanical inhibition of microtubule depolymerisation by kinesin

Kinesin-driven transport of molecular cargo along microtubules is central to the self-organisation of eukaryotic cells. We investigated the effect of kinesin-1 on microtubule stability using in vitro techniques. We found that kinesin-1, which was previously reported to have no influence on microtubule dynamics, to reduce shrinkage rates by approximately two orders of magnitude if maintained in a nucleotide-free or ATP-bound state. No effect was observed in the presence of high ADP concentrations, indicating that the microtubule-stabilising ability of kinesin-1 is constrained to a subset of the kinetic states of its ATPase cycle. By decorating just one side of the microtubule lattice with kinesin, we were able to gain additional insights into the mechanics of microtubules. By stabilising just 2-3 protofilaments with kinesin, the structural integrity of most of the microtubule could be maintained. Curiously, in such circumstances the microtubule would split at its ends. We further showed that microtubule curvature induced by hydrodynamic flow is trapped or even increased by nucleotide-free kinesin. We propose a mechanism whereby kinesin-1 drives the conformation of polymerised GDP-tubulin into a slightly elongated and shrinkage-resistant conformation. This is essentially the converse mechanism of that reported for the kinesin-13, MCAK, which supports tubulin in a curved conformation that is incompatible with the microtubule lattice

THE CLASP FAMILY REGULATES MICROTUBULE DYNAMICS BY USING AN ARRAY OF TOG-LIKE DOMAINS

CLASP is a key regulator of microtubule (MT) dynamics and bipolar mitotic spindle formation, with mutants displaying chromosome aggregation, aberrant monopolar spindle morphologies, and aneuploidy. How CLASP binds the microtubule lattice to regulate MT dynamics and facilitate proper spindle assembly remains unknown; however, it has been postulated that cryptic TOG domains underlie CLASP's ability to regulate MT dynamics. In this work, we report the crystal structure of the first cryptic TOG domain (TOG2) from human CLASP1, confirming the presence of a TOG array in CLASP. CLASP1 TOG2 displays a bent architecture at the tubulin-binding surface that contrasts with the flat tubulin-binding surface from XMAP215 family TOG domains. Mutating key tubulin-binding determinants along the tubulin-binding surface of TOG2 abrogated the ability of CLASP to 1) rescue mitotic bipolar spindle formation in Drosophila S2 cells 2) associate CLASP with the MT lattice, and 3) promote in vitro MT polymerization. These findings highlight the mechanistic use of a cryptic TOG domain in CLASP to facilitate bipolar spindle formation and MT polymerization. Determining the crystal structure of TOG1 and the second cryptic TOG-like domain (TOG3) is ongoing. Structural characterization of CLASP's array of TOG domains we shows that differential TOG domain architecture confers distinct functions for each TOG domain including MT lattice association, MT polymerization, and MT stabilization. In addition, CLASP's C-terminal domain (CTD) associates with the coiled-coil regions of various associating factors to recruit CLASP at specific cellular locations and is also a necessary component for CLASP dimerization. To determine the role of CLASP CTD in promoting dimerization and interacting with known CLASP-associating factors, we are structurally and biochemically characterizing the interaction between CLASP CTD and the coiled-coil (CC) domain of CLIP-170, a known CLASP-associating factor. CLASP CTD and CLIP-170 CC form a complex in SEC-MALS and ITC experiments. In addition, CLASP CTD alone exists as a monomer, suggesting that CLASP CTD is necessary, but not sufficient, for dimerization. Further analysis to structurally characterize the interaction between CLASP CTD and CLIP-170 CC is an ongoing goal for this thesis work.Doctor of Philosoph

INSIGHTS INTO POLYMERISATION OF FTSZ AND OTHER CYTOMOTIVE FILAMENTS

Protein filaments are used in different ways to organise other molecules in space and time within cells. Some proteins form filaments that couple hydrolysis of nucleotides to their polymerisation cycle in order to power the directed movement of other molecules, these filaments are termed cytomotive. Only members of the actin and tubulin superfamilies are known to form cytomotive filaments. The protein FtsZ, a homologue of eukaryotic tubulins, forms cytomotive filaments that are used in almost all bacteria and many archaea to organise cell division. Here I show using X-ray crystallography and electron cryomicroscopy (cryoEM) that FtsZ switches conformation when it polymerises into filaments. I then show using cryoEM that this conformational switch is likely needed for recognition of filaments by the widely conserved filament cross-linking protein ZapA. I also present the development of a high- throughput assay for detection of better FtsZ inhibitors, which uses principles derived from the structural studies. Finally, I demonstrate that the conformational switch upon polymerisation seen in FtsZ is conserved within the tubulin superfamily, that actin superfamily members also exhibit a conserved conformational switch upon polymerisation, and that having such a switch explains the coupling of kinetic and structural polarities required for cytomotivity of the filaments formed by these protein families.I was supported by scholarships from the MRC and the Boehringer Ingelheim Fonds

Intrinsically Disordered Proteins and Chronic Diseases

This book is an embodiment of a series of articles that were published as part of a Special Issue of Biomolecules. It is dedicated to exploring the role of intrinsically disordered proteins (IDPs) in various chronic diseases. The main goal of the articles is to describe recent progress in elucidating the mechanisms by which IDPs cause various human diseases, such as cancer, cardiovascular disease, amyloidosis, neurodegenerative diseases, diabetes, and genetic diseases, to name a few. Contributed by leading investigators in the field, this compendium serves as a valuable resource for researchers, clinicians as well as postdoctoral fellows and graduate student