Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2001. / Vita. Bibliography: 139-169.

Thesis (Ph. D.)--Virginia Commonwealth University, 2008. / Prepared for: Dept. of Biochemistry. Title from thesis description page. Includes bibliographical references.

Thesis (M. Phil.)--University of Hong Kong, 2005. / Title proper from title frame. Also available in printed format.

Voltage-gated Cav2.1 (P/Q-type) and Cav2.2 (N-type) channels are two closely related calcium channels that play indispensable roles in signal transduction pathways by regulating neurotransmitter release. Despite having highly conserved amino acid sequences, they are differentially modulated by calmodulin, which mediate two important feedback mechanisms known as Ca2+-dependent inactivation (CDI) and Ca2+-dependent facilitation (CDF). These dual regulatory mechanisms contribute to synaptic plasticity, but only CDI is observed in Cav2.2 channel, while both CDI and CDF are present in Cav2.1 channel. Previously, it was hypothesized that the lack of CDF in Cav2.2 channel is due to the pre-IQ-IQ domain of the channel’s lower binding affinity for calmodulin compared to that of Cav2.1 channel. Now that the EF-hand domain of calcium channels is identified as one of the two minimally required molecular determinants that are responsible for supporting CDF in Cav2.1 channel and preventing CDF in Cav2.2 channel, it was necessary to determine the role of EF-hand domain in calmodulin binding of Cav2.1 and Cav2.2 channels. Using pull-down binding assays, this study finds that the EF-hand domain enhances calmodulin binding to the proximal C-terminal domain of Cav2.2 channel, which suggests that the lack of CDF in Cav2.2 does not result from the channel’s weak interaction with CaM, but from the EF-pre-IQ-IQ domain of the channel’s inability to allow calmodulin from fully exerting its effects.

Dans leur environnement naturel, les plantes sont constamment exposées aux stress de nature biotique et abiotiques. Aussi, pour maintenir leur croissance et achever leur cycle de développement, les plantes ont développé des mécanismes rapides et efficaces leur permettant de percevoir, de décoder ces signaux de l'environnement et de mettre en places des réponses adaptées. S'il est à présent admis que le calcium joue un rôle crucial en tant que second messager, les mécanismes de décodage jouent eux aussi des rôles indispensables. Parmi les calcium sensors les mieux caractérisés, la calmoduline (CaM) est certainement la plus étudiée. La CaM est retrouvée chez tous les eucaryotes et contribue à la signalisation cellulaire en interagissant avec de nombreuses protéines cibles au cours du développement et en réponse au stress. Cependant, contrairement aux autres eucaryotes, les plantes se caractérisent par la présence dans leur génome de nombreux gènes codant des protéines apparentées à la CaM appelées CalModulin-Like (CMLs) dont les rôles restent encore à révéler. Au cours de ce travail, une analyse moléculaire des CaM et des CML a été réalisée au sein de la lignée verte (des algues aux plantes supérieures). Nous avons montré que l'émergence des CaM et des CMLs mais aussi leur nombre ont évolué au cours des processus de colonisation de la terre par les plantes et que l'émergence de nouvelles classes de CMLs est concomitante de l'apparition de nouvelles fonctions ou organes. Le travail s'est ensuite focalisé sur l'analyse fonctionnelle de CML8 et de CML11 et il a été montré que CML8 est impliqué dans le développement des plantes en particulier dans le développement des racines latérales et la ramification mais également dans l'immunité des plantes contre Pseudomonas syringae et Ralstonia solanacearum en tant que régulateur positif. Les mécanismes moléculaires restent encore à préciser mais CML8 serait impliquée dans l'ETS (Effector Triggered Susceptibility) en lien avec la voie de l'acide salicylique plutôt qu'avec les processus de PTI (PAMP Triggered Immunity) en réponse à Pseudomonas. Concernant CML11, cette CML se caractérise par la présence d'un domaine PolyQ. Si le profil d'expression de CML11 est différent de CML8, l'analyse fonctionnelle n'a pu être qu'initiée pendant ce travail avec un intérêt particulier pour le rôle joué par le domaine polyQ. / Plants are continuously exposed to a variety of unfavorable environmental conditions including biotic and abiotic stresses. To maintain their growth and achieve their development cycle, plant evolved efficient and rapid mechanisms to perceive, transduce and respond to these signals. It is now well-known that calcium plays a crucial role as a secondary messenger in the implementation of adaptative responses. However, the calcium signals need to be decoded and relayed by calcium sensors such as calmodulin to downstram target proteins to trigger specific responses. Calmodulin (CaM) is a well-studied calcium sensor which is ubiquitous in all eukaryotes and that contributes to signaling during developmental processes and stress responses. However, compared to other eukaryotes, plants possess a remarkable variety of CaM-like proteins (CMLs) for which the role remain to be elucidated. CaM and CML evolution analysis among the green lineage from algae to land plants shows that CaM and CMLs evolved during the terrestrial colonization of plants and that the emergence of new CML classes appeared throughout the green lineage and correlated with the acquisition of novel biological traits. The functional analysis of two closely relative protein CML8 and CML11 showed that CML8 is involved in plant development by affecting lateral root formation and shoot branching but also as a positive regulator in plant immunity against Pseudomonas syringae and Ralstonia solanacearum phytopathogenic bacteria. The molecular mechanisms controlled by CML8 are still unsolved but we propose that CML8 may participate in ETS (Effector Triggered Susceptibility) through a salicylic acid dependent pathway rather than in PTI (PAMP Triggered Immunity) in response to Pst inoculation. Concerning CML11, this protein is characterized by the presence of a polyQ domain. While its gene expression profile is different from CML8, its functional analysis was initiated during this work with a particular focus on the polyQ domain.

Vitamin A is an essential nutrient; it is not synthesized by mammals and therefore must be obtained through the diet. During times of fasting or dietary vitamin A insufficiency, retinol, the alcohol form of the vitamin is released from the liver, its main storage tissue, for circulation in complex with retinol-binding protein 4 (RBP) to provide an adequate supply to peripheral tissues. Stimulated by Retinoic Acid 6 (STRA6), the transmembrane RBP receptor, mediates retinol uptake across blood-tissue barriers such as the retinal pigment epithelium of the eye, the placenta and the choroid plexus of the brain. Our understanding as to how this protein functions has been greatly enhanced by the high-resolution 3D structure of zebrafish STRA6 in complex with calmodulin (CaM) solved by single-particle cryogenic-electron microscopy. However, the nature of the interaction of STRA6 with retinol remains unclear. Here, I present the high-resolution structures of zebrafish and sheep STRA6 reconstituted in nanodisc lipid bilayers in the presence and absence of retinol. The nanodisc reconstitution system has allowed us to study this protein in a close to physiological environment and examine its interaction with the cell membrane and relationship with its ligand, retinol. We also present the structure of sheep STRA6 in complex with human RBP. The structure of the STRA6-RBP complex confirms predictions in the literature as to the nature of the protein-protein interaction needed for retinol transport. Calcium-bound CaM is bound to STRA6 in the RBP-STRA6 structure, consistent with a regulatory role of this calcium binding protein in STRA6-RBP interaction. The analysis of the three states of STRA6 – pre, post and during interaction with retinol – provide unique insights into the mechanism of STRA6-mediated cellular retinol uptake.

In the heart, sodium (Na+) influx via NaV1.5 channels initiates the action potential, and calcium (Ca2+) influx via CaV1.2 channels has a key role in excitation-contraction coupling and determining the plateau phase of the action potential. Mutations in the genes that encode these ion channels or in proteins that modulate them are linked to arrhythmias and cardiomyopathy, underscoring the need for characterizing mechanisms of regulation. The work presented in this thesis is subdivided into three different chapters, each with a distinct focus on ion channel modulation. The first chapter details our investigation of the functional PKA phosphorylation target for β-adrenergic regulation of CaV1.2. Physiologic β-adrenergic activation of PKA during the sympathetic “fight or flight” response increases Ca2+ influx through CaV1.2 in cardiomyocytes, leading to increased cardiac contractility. The molecular mechanisms of β-adrenergic regulation of CaV1.2 in cardiomyocytes are incompletely known, but activation of PKA is required for this process. Recent data suggest that β-adrenergic regulation of CaV1.2 does not require any combination of PKA phosphorylation sites conserved in human, guinea pig, rabbit, rat, and mouse α1C subunits. To test if any non-conserved sites are required for regulation, we generated mice with inducible cardiac-specific expression of α1C with mutations at both conserved and non- conserved predicted PKA phosphorylation sites (35-mutant α1C). Additionally, we createdanother mouse with inducible cardiac-specific expression of β2 with mutations at predicted PKA phosphorylation sites (28-mutant β2B). In each of these mice, β-adrenergic stimulation of Ca²⁺ current was unperturbed. Finally, to test the hypothesis that redundant functional PKA phosphorylation sites exist on the α1C subunit and β2 subunit or that several sites confer incremental regulation, we crossed the 35-mutant α1C mice with the 28-mutant β2B mice to generate offspring expressing both mutant subunits. In these offspring, intact regulation was observed. These results provide the definitive answer that phosphorylation of the α1C subunit or β2 subunit is not required for β-adrenergic regulation of CaV1.2 in the heart. In the second chapter, we study the influence of calmodulin and fibroblast growth homologous factor (FHF) FGF13 on late Na+ current. Studies in heterologous expression systems show that the Ca²⁺-binding protein calmodulin plays a key role in decreasing late Na⁺ current. The effect of loss of calmodulin binding to NaV1.5 on late Na+ current has yet to be resolved in native cardiomyocytes. We created transgenic mice with cardiac-specific expression of human NaV1.5 channels with alanine substitutions for the IQ motif (IQ/AA), disrupting calmodulin binding to the C-terminus. Surprisingly, we found that the IQ/AA mutation did not cause an increase late Na⁺ current in cardiomyocytes. These findings suggest the existence of endogenous protective mechanisms that counteract the increase in late Na+ current that occurs with loss of calmodulin binding. We reasoned that FGF13, a known modulator of late Na+ current that is endogenously expressed in cardiomyocytes but not HEK cells, might play a protective role in limiting late Na+ current. Finally, we coexpressed the IQ/AA mutant NaV1.5 channel in HEK293 cells with FGF13 and found that FGF13 diminished the late Na⁺ currentcompared to cells without FGF13, suggesting that endogenous FHFs may serve to prevent late Na⁺ current in mouse cardiomyocytes. The third chapter of this thesis focuses on the use of proximity labeling and multiplexed quantitative proteomics to define changes in the NaV1.5 macromolecular complex in Duchenne muscular dystrophy (DMD), in which the absence of dystrophin predisposes affected individuals to arrhythmias and cardiac dysfunction.. Standard methods to characterize macromolecular complexes have relied on candidate immunoprecipitation or immunocytochemistry techniques that fall short of providing a comprehensive view of the numbers and types of interactors, as well as the potential dynamic nature of the interactions that may be perturbed by disease states. To provide an inclusive understanding of NaV1.5 macromolecular complexes, we utilize live-cell APEX2 proximity labeling in cardiomyocytes. We identify several proximal changes that align with the electrophysiological NaV1.5 phenotype of young dystrophin-deficient mice, including a decrease in Ptpn3 and Gdp1l and an increase in proteasomal machinery. Whole-cell protein expression fold-change results were used to reveal the altered global expression profile and to place context behind NaV1.5-proximal changes. Finally, we leveraged the neighborhood- specificity of proteins at the lateral membrane, intercalated disc, and transverse tubules of cardiomyocytes to demonstrate that NaV1.5 channels can traffic to all three membrane compartments even in the absence of dystrophin. Thus, the approach of proximity labeling in cardiomyocytes from an animal model of human disease offers new insights into molecular mechanisms of NaV1.5 dysfunction in DMD and provides a template for similar investigations in other cardiac diseases.

Suite aux conséquences environnementales provoquées par l’accident nucléaire de Fukushima, il est fondamental d’étudier les mécanismes gouvernant les effets des radionucléides sur la biosphère et ainsi identifier les processus moléculaires responsables du transport et de la déposition d’actinides comme le neptunium et l’uranium. Cependant, les informations concernant l’aspect microscopique des interactions entre actinide et molécules biologiques sont rares. Les données publiées étant majoritairement issue d’études in vivo, la structure des sites de coordination et l’effet de cette complexation sur les fonctions des protéines restent encore à découvrir.La calmoduline (CaM), qui est connue pour son affinité envers les actinides, agit comme un régulateur métabolique du calcium. Cette protéine, qui est présente de manière ubiquitaire dans le corps humain, peut également complexer d’autres métaux comme les actinides. Ainsi, en cas de contamination interne, les actinides complexés à la protéine pourraient l’empêcher de fonctionner correctement et donc avoir des répercussions sur un grand nombre de fonctions vitales pour l’organisme.La complexation du Np et de l’U par la CaM a été étudiée par spectroscopie EXAFS ce qui nous a permis de montrer que les actinides sont incorporés au site de complexation du calcium. Une fois les aspects structuraux et thermodynamiques étudiés, c’est l’impact de cette complexation sur les fonctions de la protéine qui a été étudié.Afin d’évaluer les conséquences de la complexation, une méthode calorimétrique basée sur une réaction enzymatique (Phosphodiesterase) a été développée. Ces expériences réalisées avec des concentrations variables d’actinides (30-500 nM) montrent une diminution de l’activité enzymatique lorsque la concentration d’actinide augmente. Les résultats montrent que le complexe CaM-An agit comme un inhibiteur enzymatique. De plus, on observe qu’à haute concentration en actinide, le complexe CaM-métal agit comme un poison et tue complètement l’activité enzymatique. / Considering the environmental impact of the Fukushima nuclear accident, it is fundamental to study the mechanisms governing the effects of the released radionuclides on the biosphere and thus identify the molecular processes generating the transport and deposition of actinides, such as neptunium and uranium. However, the information about the microscopic aspect of the interaction between actinides and biological molecules (peptides, proteins…) is scarce. The data being mostly reported from a physiological point of view, the structure of the coordination sites remains largely unknown. These microscopic data are indeed essential for the understanding of the interdependency between structural aspect, function and affinity.The Calmodulin (CaM) (abbreviation for CALcium-MODULated proteIN), also known for its affinity towards actinides, acts as a metabolic regulator of calcium. This protein is a Ca carrier, which is present ubiquitously in the human body, may also bind other metals such as actinides. Thus, in case of a contamination, actinides that bind to CaM could avoid the protein to perform properly and lead to repercussions on a large range of vital functions.The complexation of Np and U was studied by EXAFS spectroscopy which showed that actinides were incorporated in a calcium coordination site. Once the thermodynamical and structural aspects studied, the impact of the coordination site distortion on the biological efficiency was analyzed.In order to evaluate these consequences, a calorimetric method based on enzyme kinetics was developed. This experiment, which was conducted with both uranium (50 – 500 nM) and neptunium (30 – 250 nM) showed a decrease of the heat produced by the enzymatic reaction with an increasing concentration of actinides in the medium. Our findings showed that the Calmodulin actinide complex works as an enzymatic inhibitor. Furthermore, at higher neptunium (250 nM) and uranium (500 nM) concentration the metals seem to have a poison-like behavior and “kill” completely the enzymatic activity.

Calmodulin kinase cascade is a signaling pathway which is involved in the response to the increasing intracellular calcium levels. Ca2+ is a ubiquitous second messenger which promotes wide-range of cellular signaling events. Many of these signaling pathways start with the binding of Ca2+ to its primary intracellular receptor calmodulin. Calmodulin in turn binds to its downstream targets in the Ca2+ /calmodulin signaling cascade. One of the most important enzymes of this cascade is a Ca2+ /calmodulin-dependent protein kinase kinase 2 (CaMKK2). CaMKK2 is a serine/threonine protein kinase which regulates for example gene transcription or energy homeostasis by phosphorylation of its downstream targets. Catalytic domain (which provides kinase activity) is located in the middle part of the protein and possesses structure typical for kinases. CaMKK2 consists of 588 amino acids but the secondary structure is known only for the region of the kinase domain (298 residues). The rest of the protein is assumed to be unstructured as long as CaMKK2 is not bound to any interaction partner. The aim of this study was to prepare several constructs of human isoform of CaMKK2 for the further structural and activity studies. It is believed that CaMKK2 is regulated by site-specific phosphorylation. Phosphorylation of some...

1. The role of calmodulin (CaM) in transmitter release was investigated using liposomes to deliver CaM and monoclonal antibodies against CaM (antiCaM) directly into the frog motor nerve terminal. 2. Miniature endplate potentials (MEPPs) were recorded in a high K+ solution, and effects on transmitter release were monitored using estimates of the quantal release parameters m (number of quanta released), n (number of functional transmitter release sites), p (mean probability of release), and vars p (spatial variance in p). 3. Administration of CaM, but not heat-inactivated CaM, encapsulated in liposomes (1000 units ml-1) produced an increase in m (25%) that was due to an increase in n. MEPP amplitude was not altered by CaM. 4. Administration of antiCaM, but not heat-inactivated antiCaM, in liposomes (50 μl ml-1) produced a progressive decrease in m (40%) that was associated with decreases in n and p. MEPP amplitude was decreased (15%) after a 25 min lag time, suggesting a separation in time between the decreases in quantal release and quantal size. 5. Bath application of the membrane-permeable CaM antagonist W7 (28 μM) produced a gradual decrease in m (25%) that was associated with a decrease in n. W7 also produced a decrease in MEPP amplitude that paralleled the decrease in m. The decreases in MEPP size and m produced by W7 were both reversed by addition of CaM. 6. Our results suggest that CaM increases transmitter release by mobilizing synaptic vesicles at the frog motor nerve terminal.