A CRMP4-dependent retrograde axon-to-soma death signal in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal non-cell-autonomous neurodegenerative disease characterized by the loss of motor neurons (MNs). Mutations in CRMP4 are associated with ALS in patients, and elevated levels of CRMP4 are suggested to affect MN health in the SOD1G93A -ALS mouse model. However, the mechanism by which CRMP4 mediates toxicity in ALS MNs is poorly understood. Here, by using tissue from human patients with sporadic ALS, MNs derived from C9orf72-mutant patients, and the SOD1G93A -ALS mouse model, we demonstrate that subcellular changes in CRMP4 levels promote MN loss in ALS. First, we show that while expression of CRMP4 protein is increased in cell bodies of ALS-affected MN, CRMP4 levels are decreased in the distal axons. Cellular mislocalization of CRMP4 is caused by increased interaction with the retrograde motor protein, dynein, which mediates CRMP4 transport from distal axons to the soma and thereby promotes MN loss. Blocking the CRMP4-dynein interaction reduces MN loss in human-derived MNs (C9orf72) and in ALS model mice. Thus, we demonstrate a novel CRMP4-dependent retrograde death signal that underlies MN loss in ALS.

Leukocyte Cytoskeleton Polarization Is Initiated by Plasma Membrane Curvature from Cell Attachment.

Cell polarization is important for various biological processes. However, its regulation, particularly initiation, is incompletely understood. Here, we investigated mechanisms by which neutrophils break their symmetry and initiate their cytoskeleton polarization from an apolar state in circulation for their extravasation during inflammation. We show here that a local increase in plasma membrane (PM) curvature resulting from cell contact to a surface triggers the initial breakage of the symmetry of an apolar neutrophil and is required for subsequent polarization events induced by chemical stimulation. This local increase in PM curvature recruits SRGAP2 via its F-BAR domain, which in turn activates PI4KA and results in PM PtdIns4P polarization. Polarized PM PtdIns4P is targeted by RPH3A, which directs PIP5K1C90 and subsequent phosphorylated myosin light chain polarization, and this polarization signaling axis regulates neutrophil firm attachment to endothelium. Thus, this study reveals a mechanism for the initiation of cell cytoskeleton polarization.

Robo Ig4 Is a Dimerization Domain.

Robo receptors play pivotal roles in axonal guidance as well as in neurogenesis, angiogenesis, cell migration, and cancer progression and invasiveness. They are considered to be attractive drug targets for the treatment of cancer, ocular neovascular disorders, chronic kidney diseases, and more. Despite their great importance, the mechanisms by which Robo receptors switch from their "off" to "on" states remain obscure. One possibility involves a monomer-to-dimer or dimer-to-monomer transition that facilitates the recruitment and activation of enzymatic effectors to instigate intracellular signaling. However, it is not known which domains mediate Robo dimerization, or the structural properties of the dimeric interactions. Here, we identify the extracellular Ig4 (D4) as a Robo dimerization domain. We have determined the crystal structure of the tandem Ig4-5 domains (D4-5) of human Robo2 and found that a hydrophobic surface on D4 mediates close homotypic contacts with a reciprocal D4. Analytical ultracentrifugation measurements of intact and mutated D4-5 shows that dimerization through the D4 interface is specific and has a dimerization dissociation constant of 16.9µM in solution. Direct fluorescence resonance energy transfer dimerization measurements in HEK293 cells corroborate the dimerization of transmembrane hRobo2 through D4, and a functional COS-7 cell collapse assay links D4-mediated dimerization with Robo intracellular signaling. The high level of conservation in the D4 dimerization interface throughout all Robo orthologs and paralogs implies that D4-mediated dimerization is a central hallmark in Robo activation and signaling.

Adaptive patterns in the p53 protein sequence of the hypoxia- and cancer-tolerant blind mole rat Spalax.

BACKGROUND: The subterranean blind mole rat, Spalax (genus Nannospalax) endures extreme hypoxic conditions and fluctuations in oxygen levels that threaten DNA integrity. Nevertheless, Spalax is long-lived, does not develop spontaneous cancer, and exhibits an outstanding resistance to carcinogenesis in vivo, as well as anti-cancer capabilities in vitro. We hypothesized that adaptations to similar extreme environmental conditions involve common mechanisms for overcoming stress-induced DNA damage. Therefore, we aimed to identify shared features among species that are adapted to hypoxic stress in the sequence of the tumor-suppressor protein p53, a master regulator of the DNA-damage response (DDR). RESULTS: We found that the sequences of p53 transactivation subdomain 2 (TAD2) and tetramerization and regulatory domains (TD and RD) are more similar among hypoxia-tolerant species than expected from phylogeny. Specific positions in these domains composed patterns that are more frequent in hypoxia-tolerant species and have proven to be good predictors of species' classification into stress-related categories. Some of these positions, which are known to be involved in the interactions between p53 and critical DDR proteins, were identified as positively selected. By 3D modeling of p53 interactions with the coactivator p300 and the DNA repair protein RPA70, we demonstrated that, compared to humans, these substitutions potentially reduce the binding of these proteins to Spalax p53. CONCLUSIONS: We conclude that extreme hypoxic conditions may have led to convergent evolutionary adaptations of the DDR via TAD2 and TD/RD domains of p53.

The strength and cooperativity of KIT ectodomain contacts determine normal ligand-dependent stimulation or oncogenic activation in cancer.

The receptor tyrosine kinase KIT plays an important role in development of germ cells, hematopoietic cells, and interstitial pacemaker cells. Oncogenic KIT mutations play an important "driver" role in gastrointestinal stromal tumors, acute myeloid leukemias, and melanoma, among other cancers. Here we describe the crystal structure of a recurring somatic oncogenic mutation located in the C-terminal Ig-like domain (D5) of the ectodomain, rendering KIT tyrosine kinase activity constitutively activated. The structural analysis, together with biochemical and biophysical experiments and detailed analyses of the activities of a variety of oncogenic KIT mutations, reveals that the strength of homotypic contacts and the cooperativity in the action of D4D5 regions determines whether KIT is normally regulated or constitutively activated in cancers. We propose that cooperative interactions mediated by multiple weak homotypic contacts between receptor molecules are responsible for regulating normal ligand-dependent or oncogenic RTK activation via a "zipper-like" mechanism for receptor activation.

Expression, purification and crystallization of the phosphate-binding PstS protein from Pseudomonas aeruginosa.

Pseudomonas aeruginosa (PA) infections pose a serious threat to human health. PA is a leading cause of fatal lung infections in cystic fibrosis and immune-suppressed patients, of sepsis in burn victims and of nosocomial infections. An important element in PA virulence is its ability to establish biofilms that evade suppression by the host's immune system and antibiotics. PstS, a periplasmic subunit of the Pst phosphate-transport system of PA, plays a critical role in the establishment of biofilms. In some drug-resistant PA strains, PstS is secreted in large quantities from the bacteria, where it participates in the assembly of adhesion fibres that enhance bacterial virulence. In order to understand the dual function of PstS in biofilm formation and phosphate transport, the crystal structure of PA PstS was determined. Here, the overexpression in Escherichia coli and purification of PA PstS in the presence of phosphate are described. Two crystal forms were obtained using the vapour-diffusion method at 20°C and X-ray diffraction data were collected. The first crystal form belonged to the centred orthorhombic space group C2221, with unit-cell parameters a=67.5, b=151.3, c=108.9 Å. Assuming the presence of a dimer in the asymmetric unit gives a crystal volume per protein weight (VM) of 2.09 Å3 Da(-1) and a solvent content of 41%. The second crystal form belonged to the primitive orthorhombic space group P212121, with unit-cell parameters a=35.4, b=148.3, c=216.7 Å. Assuming the presence of a tetramer in the asymmetric unit gives a crystal volume per protein weight (VM) of 2.14 Å3 Da(-1) and a solvent content of 42.65%. A pseudo-translational symmetry is present in the P212121 crystal form which is consistent with a filamentous arrangement of PstS in the crystal lattice.

Crystal structure of the extracellular juxtamembrane region of Robo1.

Robo receptors play pivotal roles in neurodevelopment, and their deregulation is implicated in several neuropathological conditions and cancers. To date, the mechanism of Robo activation and regulation remains obscure. Here we present the crystal structure of the juxtamembrane (JM) domains of human Robo1. The structure exhibits unexpectedly high backbone similarity to the netrin and RGM binding region of neogenin and DCC, which are functionally related receptors of Robo1. Comparison of these structures reveals a conserved surface that overlaps with a cluster of oncogenic and neuropathological mutations found in all Robo isoforms. The structure also reveals the intricate folding of the JM linker, which points to its role in Robo1 activation. Further experiments with cultured cells demonstrate that exposure or relief of the folded JM linker results in enhanced shedding of the Robo1 ectodomain.

Structure, domain organization, and different conformational states of stem cell factor-induced intact KIT dimers.

Using electron microscopy and fitting of crystal structures, we present the 3D reconstruction of ligand-induced dimers of intact receptor tyrosine kinase, KIT. We observe that KIT protomers form close contacts throughout the entire structure of ligand-bound receptor dimers, and that the dimeric receptors adopt multiple, defined conformational states. Interestingly, the homotypic interactions in the membrane proximal Ig-like domain of the extracellular region differ from those observed in the crystal structure of the unconstrained extracellular regions. We observe two prevalent conformations in which the tyrosine kinase domains interact asymmetrically. The asymmetric arrangement of the cytoplasmic regions may represent snapshots of molecular interactions occurring during trans autophosphorylation. Moreover, the asymmetric arrangements may facilitate specific intermolecular interactions necessary for trans phosphorylation of different KIT autophosphorylation sites that are required for stimulation of kinase activity and recruitment of signaling proteins by activated KIT.

Structural basis for activation of the receptor tyrosine kinase KIT by stem cell factor.

Stem Cell Factor (SCF) initiates its multiple cellular responses by binding to the ectodomain of KIT, resulting in tyrosine kinase activation. We describe the crystal structure of the entire ectodomain of KIT before and after SCF stimulation. The structures show that KIT dimerization is driven by SCF binding whose sole role is to bring two KIT molecules together. Receptor dimerization is followed by conformational changes that enable lateral interactions between membrane proximal Ig-like domains D4 and D5 of two KIT molecules. Experiments with cultured cells show that KIT activation is compromised by point mutations in amino acids critical for D4-D4 interaction. Moreover, a variety of oncogenic mutations are mapped to the D5-D5 interface. Since key hallmarks of KIT structures, ligand-induced receptor dimerization, and the critical residues in the D4-D4 interface, are conserved in other receptors, the mechanism of KIT stimulation unveiled in this report may apply for other receptor activation.

The voltage-dependent calcium channel beta subunit contains two stable interacting domains.

Voltage-dependent calcium channels selectively enable Ca2+ ion movement through cellular membranes. These multiprotein complexes are involved in a wide spectrum of biological processes such as signal transduction and cellular homeostasis. alpha1 is the membrane pore-forming subunit, whereas beta is an intracellular subunit that binds to alpha1, facilitating and modulating channel function. We have expressed, purified, and characterized recombinant beta3 and beta2a using both biochemical and biophysical methods, including electrophysiology, to better understand the beta family's protein structural and functional correlates. Our results indicate that the beta protein is composed of two distinct domains that associate with one another in a stable manner. The data also suggest that the polypeptide regions outside these domains are not structured when beta is not in complex with the channel. In addition, the beta structural core, comprised of just these two domains without other sequences, binds tightly to the alpha interaction domain (AID) motif, a sequence derived from the alpha1 subunit and the principal anchor site of beta. Domain II is responsible for this binding, but domain I enhances it.