Vadim A. Klenchin

PIP Kinase Iγ Is the Major PI(4,5)P2 Synthesizing Enzyme at the Synapse

Abstract Disruption of the presynaptically enriched polyphosphoinositide phosphatase synaptojanin 1 leads to an increase of clathrin-coated intermediates and of polymerized actin at endocytic zones of nerve terminals. These changes correlate with elevated levels of PI(4,5)P 2 in neurons. We report that phosphatidylinositol phosphate kinase type Iγ (PIPKIγ), a major brain PI(4)P 5-kinase, is concentrated at synapses. Synaptojanin 1 and PIPKIγ antagonize each other in the recruitment of clathrin coats to lipid membranes. Like synaptojanin 1 and other proteins involved in endocytosis, PIPKIγ undergoes stimulation-dependent dephosphorylation. These results implicate PIPKIγ in the synthesis of a PI(4,5)P 2 pool that acts as a positive regulator of clathrin coat recruitment and actin function at the synapse.

The Gain of Rod Phototransduction: Reconciliation of Biochemical and Electrophysiological Measurements

We have resolved a central and long-standing paradox in understanding the amplification of rod phototransduction by making direct measurements of the gains of the underlying enzymatic amplifiers. We find that under optimized conditions a single photoisomerized rhodopsin activates transducin molecules and phosphodiesterase (PDE) catalytic subunits at rates of 120-150/s, much lower than indirect estimates from light-scattering experiments. Further, we measure the Michaelis constant, Km, of the rod PDE activated by transducin to be 10 microM, at least 10-fold lower than published estimates. Thus, the gain of cGMP hydrolysis (determined by kcat/Km) is at least 10-fold higher than reported in the literature. Accordingly, our results now provide a quantitative account of the overall gain of the rod cascade in terms of directly measured factors.

CAPS Acts at a Prefusion Step in Dense-Core Vesicle Exocytosis as a PIP2 Binding Protein

CAPS-1 is required for Ca2+-triggered fusion of dense-core vesicles with the plasma membrane, but its site of action and mechanism are unknown. We analyzed the kinetics of Ca2+-triggered exocytosis reconstituted in permeable PC12 cells. CAPS-1 increased the initial rate of Ca2+-triggered vesicle exocytosis by acting at a rate-limiting, Ca2+-dependent prefusion step. CAPS-1 activity depended upon prior ATP-dependent priming during which PIP2 synthesis occurs. CAPS-1 activity and binding to the plasma membrane depended upon PIP2. Ca2+ was ineffective in triggering vesicle fusion in the absence of CAPS-1 but instead promoted desensitization to CAPS-1 resulting from decreased plasma membrane PIP2. We conclude that CAPS-1 functions following ATP-dependent priming as a PIP2 binding protein to enhance Ca2+-dependent DCV exocytosis. Essential prefusion steps in dense-core vesicle exocytosis involve sequential ATP-dependent synthesis of PIP2 and the subsequent PIP2-dependent action of CAPS-1. Regulation of PIP2 levels and CAPS-1 activity would control the secretion of neuropeptides and monoaminergic transmitters.

Actin-targeting natural products: structures, properties and mechanisms of action

Abstract.Natural small-molecule inhibitors of actin cytoskeleton dynamics have long been recognized as valuable molecular probes for dissecting complex mechanisms of cellular function. More recently, their potential use as chemotherapeutic drugs has become a focus of scientific investigation. The primary focus of this review is the molecular mechanism by which different actin-targeting natural products function, with an emphasis on structural considerations of toxins for which high-resolution structural information of their interaction with actin is available. By comparing the molecular interactions made by different toxin families with actin, the structural themes of those that alter filament dynamics in similar ways can be understood. This provides a framework for novel synthetic-compound designs with tailored functional properties that could be applied in both research and clinical settings.

Yeast Sec14p Deficient in Phosphatidylinositol Transfer Activity Is Functional In Vivo

Yeast phosphatidylinositol transfer protein (Sec14p) is essential for Golgi secretory function. It is widely accepted, though unproven, that phosphatidylinositol transfer between membranes represents the physiological activity of phosphatidylinositol transfer proteins (PITPs). We report that Sec14pK66,239A is inactivated for phosphatidylinositol, but not phosphatidylcholine (PC), transfer activity. As expected, Sec14pK66,239A fails to meet established criteria for a PITP in vitro and fails to stimulate phosphoinositide production in vivo. However, its expression efficiently rescues the lethality and Golgi secretory defects associated with sec14-1ts and sec14 null mutations. This complementation requires neither phospholipase D activation nor the involvement of a novel class of minor yeast PITPs. These findings indicate that PI binding/transfer is remarkably dispensable for Sec14p function in vivo.

Trisoxazole macrolide toxins mimic the binding of actin-capping proteins to actin

Marine macrolide toxins of trisoxazole family target actin with high affinity and specificity and have promising pharmacological properties. We present X-ray structures of actin in complex with two members of this family, kabiramide C and jaspisamide A, at a resolution of 1.45 and 1.6 Å, respectively. The structures reveal the absolute stereochemistry of these toxins and demonstrate that their trisoxazole ring interacts with actin subdomain 1 while the aliphatic side chain is inserted into the hydrophobic cavity between actin subdomains 1 and 3. The binding site is essentially the same as the one occupied by the actin-capping domain of the gelsolin superfamily of proteins. The structural evidence suggests that actin filament severing and capping by these toxins is also analogous to that of gelsolin. Consequently, these macrolides may be viewed as small molecule biomimetics of an entire class of actin-binding proteins.

Construction and Use of New Cloning Vectors for the Rapid Isolation of Recombinant Proteins from Escherichia coli

We describe the construction and use of two sets of vectors for the over-expression and purification of protein from Escherichia coli. The set of pTEV plasmids (pTEV3, 4, 5) directs the synthesis of a recombinant protein with a N-terminal hexahistidine (His(6)) tag that is removable by the tobacco etch virus (TEV) protease. The set of pKLD plasmids (pKLD66, 116) directs the synthesis of a recombinant protein that contains a N-terminal His(6) and maltose-binding protein tag in tandem, which can also be removed with TEV protease. The usefulness of these plasmids is illustrated by the rapid, high-yield purification of the 2-methylcitrate dehydratase (PrpD) protein of Salmonella enterica, and the 2-methylaconitate isomerase (PrpF) protein of Shewanella oneidensis, two enzymes involved in the catabolism of propionate to pyruvate via the 2-methylcitric acid cycle.

Rhodopsin Kinase Inhibition by Recoverin FUNCTION OF RECOVERIN MYRISTOYLATION

Recoverin is a Ca-binding protein that may play a role in vertebrate photoreceptor light adaptation by imparting Ca sensitivity to rhodopsin kinase. It is heterogeneously acylated (mostly myristoylated) at its amino-terminal glycine. Recent studies have shown that recoverin myristoylation is necessary for its Ca-dependent membrane association and cooperative Ca binding. We have addressed several issues concerning the role of recoverin myristoylation with respect to inhibition of rhodopsin kinase. We find that 1) myristoylation of recoverin is not necessary for inhibition of rhodopsin kinase, 2) myristoylation of recoverin induces a cooperative Ca-dependence for rhodopsin kinase inhibition, and 3) each Ca-binding site on the nonmyristoylated recoverin partially inhibits rhodopsin kinase. The available data suggest that the functions of recoverin myristoylation in the living rod are to induce a sharp Ca dependence of rhodopsin kinase inhibition and to bring this dependence into the rods physiological Ca concentration range.

Biomolecular mimicry in the actin cytoskeleton: Mechanisms underlying the cytotoxicity of kabiramide C and related macrolides

This study characterizes the interactions between kabiramide C (KabC) and related macrolides and actin and establishes the mechanisms that underlie their inhibition of actin filament dynamics and cytotoxicity. The G-actin–KabC complex is formed through a two-step binding reaction and is extremely stable and long-lived. Competition-binding studies show that KabC binds to the same site on G-actin as Gelsolin domain 1 and CapG. KabC also binds to protomers within F-actin and results in the severing and capping of the (+) end; these studies suggest that free KabC and related macrolides act as biomimetics of Gelsolin. The G-actin–KabC complex binds to the (+) end of a growing filament, where it functions as a novel, unregulated, (+)-end capper and is largely responsible for the inhibition of motility and cytokinesis in ≈10 –100 nM KabC-treated cells. KabC and related macrolides are useful probes to study the regulation of the actin filament (+) end and may lead to new therapies to treat diseases of the actin cytoskeleton.

Structure of the tropomyosin overlap complex from chicken smooth muscle: insight into the diversity of N-terminal recognition .

Tropomyosin is a stereotypical alpha-helical coiled coil that polymerizes to form a filamentous macromolecular assembly that lies on the surface of F-actin. The interaction between the C-terminal and N-terminal segments on adjacent molecules is known as the overlap region. We report here two X-ray structures of the chicken smooth muscle tropomyosin overlap complex. A novel approach was used to stabilize the C-terminal and N-terminal fragments. Globular domains from both the human DNA ligase binding protein XRCC4 and bacteriophage varphi29 scaffolding protein Gp7 were fused to 37 and 28 C-terminal amino acid residues of tropomyosin, respectively, whereas the 29 N-terminal amino acids of tropomyosin were fused to the C-terminal helix bundle of microtubule binding protein EB1. The structures of both the XRCC4 and Gp7 fusion proteins complexed with the N-terminal EB1 fusion contain a very similar helix bundle in the overlap region that encompasses approximately 15 residues. The C-terminal coiled coil opens to allow formation of the helix bundle, which is stabilized by hydrophobic interactions. These structures are similar to that observed in the NMR structure of the rat skeletal overlap complex [Greenfield, N. J., et al. (2006) J. Mol. Biol. 364, 80-96]. The interactions between the N- and C-terminal coiled coils of smooth muscle tropomyosin show significant curvature, which differs somewhat between the two structures and implies flexibility in the overlap complex, at least in solution. This is likely an important attribute that allows tropomyosin to assemble around the actin filaments. These structures provide a molecular explanation for the role of N-acetylation in the assembly of native tropomyosin.