Browsing by Subject "Cell Adhesion Molecules"
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Item Adhesion molecules-potential targets of new anti-inflammatory therapies(1993-11-04) Lipsky, Peter E.Item Adhesion molecules: overview and approach to inhibition(1997-08-07) Kavanaugh, ArthurItem Dissecting the Function of FARP1 and Rho GTPases in Semaphorin-Plexin Signaling: Structural Perspective(2017-11-30) Kuo, Yi-Chun; Albanesi, Joseph P.; Huang, Jun-Shen; Rizo-Rey, José; Sternweis, Paul C.The Semaphorin-Plexin signaling is important for regulating axon guidance. Binding of Semaphorin to the Plexin receptor induces the dimerization of Plexin and stimulates its cytoplasmic GAP activity towards Rap, resulting in cytoskeleton re-organization. The growth cone of an axon then turns around, which ultimately leads to the repulsive guidance of the axon. Structural studies by our laboratory and others have revealed how the RapGAP activity of Plexin cytoplasmic region is stimulated by dimerization, how the Semaphorin ligand interacts with Plexin ectodomain, and how extracellular domains prevent Plexin from premature activation. Several downstream effectors interacting with Plexin cytoplasmic domain were found; however, the molecular mechanisms by which these effectors regulate Plexin signaling remain largely unknown. Our laboratory was interested in one of the effectors named FARP. Previous studies in our laboratory determined the crystal structures of the functional units of FARP1 and FARP2 but failed to detect any GEF activity in vitro. A recent screening with yeast two-hybrid identified a RhoGTPase, Rif, as a novel interacting protein for FARP1. To investigate how Rif/FARP1 interaction is involved in Plexin signaling, I determined the crystal structure of Rif-bound FARP1. This complex structure explains the functional roles that FARP1 and Rif might exert in dendritic spine formation and neurite outgrowth. Also, the N-terminal FERM domain of FARPs is known to interact with the cytoplasmic domain of Plexin. To interrogate how FERM interacts with Plexin and how this interaction might regulate Plexin signaling, I characterized the interaction of FERM with Plexin both biophysically and biochemically. The last question that I attempted to address in this dissertation is how a RhoGTPase, RhoD, inhibits activation of Plexin. RhoD was known for its remarkable inhibitory effect on Plexin signaling presumably through binding to the defined RhoGTPase binding domain of Plexin. The structure of Rac1 or Rnd1 in complex with Plexin did not explain how this binding regulates Plexin activity. I solved the crystal structure of the RhoD/Plexin complex. Modeling of this structure with that of Plexin active dimer in the context of the plasma membrane reveals a mechanism by which RhoD inhibits Plexin activation. Structure studies in this dissertation added another layer of comprehension on how the Semaphorin-Plexin signaling is regulated.Item Regulatory Mechanisms of Semaphorin/Plexin/Mical-Mediated F-actin Disassembly and Cellular Remodeling(2017-04-14) Rich, Shannon Kay Good; Johnson, Jane E.; Terman, Jonathan R.; Krämer, Helmut; Alto, NealDynamic changes to the actin cytoskeleton modify the shape of cells and their membranous extensions, and underlie diverse developmental and functional events in multiple tissues including migration, navigation, and connectivity. Semaphorins, together with their Plexin receptors, are a large family of extracellular cues that trigger complex cytoskeletal rearrangements to direct these cellular phenomena, but the mechanisms regulating their effects are poorly understood. Emerging evidence identifies Mical, a conserved oxidoreductase (Redox) enzyme, as a critical component in Semaphorin/Plexin signaling through its post-translational oxidation of F-actin, which promotes actin instability and disassembly. How this Mical-mediated redox regulation of actin dynamics is locally positioned and coordinated with the activity of other actin regulatory proteins to achieve specific, targeted effects on the cytoskeleton remains unknown. Therefore, as a part of my dissertation research, I used a genetic assay to begin to address these questions and search for proteins that could alter Semaphorin/Plexin/Mical signaling effects on the cytoskeleton. In this dissertation, I present my discovery of a functional interplay between Mical and two critical new interactors - cofilin, a well-known ubiquitous F-actin regulatory protein, and Sisyphus, an unconventional class XV myosin. With regards to cofilin, my in vivo genetic/functional assays reveal that cofilin activity is required for and enhances Semaphorin/Plexin/Mical-dependent cytoskeletal rearrangements and morphological changes. Additionally, in vitro biochemical assays demonstrate that cofilin preferentially binds Mical-oxidized actin and accelerates its disassembly. Together, these findings indicate that cofilin and Mical act as a functional pair in both neuronal and non-neuronal cells to rapidly and efficiently disassemble actin filaments. Similarly, my results reveal that Sisyphus is necessary and sufficient for triggering Semaphorin/Plexin/Mical-dependent F-actin disassembly/cellular remodeling. Moreover, using in vivo functional assays, I find that Sisyphus uses its myosin motor activity and the first MyTH4 domain of its C-terminal tail region to modify the subcellular localization of Mical. In this way, Sisyphus spatially controls Mical-dependent F-actin disassembly/cellular remodeling. Therefore, both cofilin and Sisyphus function to promote Mical-mediated F-actin disassembly; thereby, they act as critical regulators of Semaphorin/Plexin/Mical-mediated effects on cytoskeletal and morphological dynamics. Thus, my findings unveil novel molecular and biochemical mechanisms that orchestrate cellular, developmental, and neural biology.Item Structural Bases of Plexin Signal Transduction(2015-07-14) Pascoe, Heath Garrick; Chen, Zhe; Zhang, Xuewu; Jiang, Qiu-Xing; Chook, YuhMinPlexins are a family of transmembrane receptors for the Semaphorin family of repulsive axon guidance molecules. Plexin mediated signal transduction is critical for a variety of cellular processes including regulation of adhesion and actin organization. Aberrant plexin signaling is associated with numerous pathologies. Despite plexin's essential cellular functions, a detailed understanding of the structural basis underlying plexin singling remains elusive. Here, the structural bases for two aspects of plexin signaling are revealed. Plexins relay signals in part by accelerating the GTP hydrolysis reaction catalyzed by the small GTPase Rap. This discovery appears at odds with the known structural features of plexin GAP domains. Plexin GAPs are structurally related to RasGAPs and possess RasGAP catalytic machinery. Conversely, plexins are structurally unrelated to canonical RapGAPs and don't possess their associated catalytic machinery. Here, the structural basis underlying the non-canonical RapGAP activity of plexins is revealed. Plexins induce a unique configuration of Rap's Switch II loop to utilize a non-canonical catalytic residue. Plexins also regulate RhoA activity. The B family plexins recruit two RhoGEFs (PDZ-RhoGEF and Leukemia Associated RhoGEF) by specifically binding to the PDZ domains of these GEFs. Conversely, these PDZ domains bind promiscuously to a variety of ligands. The structural basis by which plexins specifically recognize these two PDZ domains is revealed here. B family plexins interact with the PDZ domain of PDZ-RhoGEF using a secondary interface outside the canonical PDZ binding site. This secondary interface contributes to tight binding and is important for RhoA activation following plexin activation. Secondary interfaces may be a general mechanism utilized by modular protein-protein interaction domains to achieve specificity. Structural and biochemical characterization of plexins can be difficult. Here, two methods for studying plexins are described. These methods were critical to the success of the above studies. First, a method used to generate fusion proteins in-vitro for use in crystallography is described. This method allowed for the successful crystallization of a plexin/Rap complex. Second, a biochemical assay is described for easily measuring Plexin GAP activity in-vitro. This method circumvents the difficulties of many alternative approaches to measuring GAP activity.Item Structural Mechanisms of Semaphorin/Plexin Signaling(2014-06-30) Wang, Yuxiao; Rice, Luke M.; Rosen, Michael K.; Yu, Hongtao; Zhang, XuewuPlexins are cell surface receptors that bind to their ligand semaphorins and transduce signals for regulating processes including neuronal development, angiogenesis and immune response. Deregulations of the plexin pathway are associated with cancers and neurodegenerative diseases. Signaling through plexins has been proposed to rely on their GTPase activating protein (GAP) activity for R-Ras and M-Ras. Activation of this GAP activity requires binding of semaphorin to the plexin extracellular region. However, the GAP activity of plexins eluded detection in several studies, and the mechanisms by which semaphorins activate plexins remained elusive. I discovered that plexins function as GAPs specific for Rap GTPases but not for R-Ras or M-Ras. The RapGAP activity of plexins is stimulated by binding of semaphorins, and is essential for the physiological functions of plexins. I further showed that induced-dimerization of plexin cytoplasmic region leads to activation of the GAP domain. The crystal structure of the active dimer of PlexinC1 was determined, revealing the structural basis for the dimerization-induced allosteric activation of plexins. I also solved a structure of PlexinA4 cytoplasmic region, which suggested the existence of a pre-formed inhibitory dimer. Biochemical and cellular assay showed that although PlexinA4 indeed homodimerizes on cell surface, the pre-formed dimer I crystallized is likely to be physiologically irrelevant. To summarize, these findings define an essential pathway for semaphorin-plexin signaling, and reveal the structural mechanisms for the activation of plexins by semaphorins. We also show that plexins in cells exist as pre-formed dimers, the formation of which likely maintains the autoinhibited state of plexins.