scholarly journals Characterization of synapsin I fragments produced by cysteine-specific cleavage: a study of their interactions with F-actin.

1989 ◽  
Vol 108 (5) ◽  
pp. 1841-1849 ◽  
Author(s):  
M Bähler ◽  
F Benfenati ◽  
F Valtorta ◽  
A J Czernik ◽  
P Greengard
Keyword(s):  
Synaptic Vesicles ◽  
Function Analysis ◽  
Actin Binding ◽  
Structural Basis ◽  
Synapsin I ◽  
Dependent Manner ◽  
Tail Region ◽  
Head Region ◽  
Parent Molecule

Synapsin I is a neuron-specific phosphoprotein that is concentrated in the presynaptic nerve terminal in association with the cytoplasmic surface of synaptic vesicles. It has been demonstrated to bundle F-actin in a phosphorylation-dependent manner in vitro, a property consistent with its proposed role in linking synaptic vesicles to the cytoskeleton and its involvement in the regulation of neurotransmitter release. Synapsin I is composed of two distinct domains, a COOH terminal, collagenase-sensitive, hydrophilic, and strongly basic tail region, and an NH2 terminal, collagenase-resistant head region relatively rich in hydrophobic amino acids. To elucidate the structural basis for the interactions between synapsin I and F-actin and how it relates to other characteristics of synapsin I, we have performed a structure-function analysis of fragments of synapsin I produced by cysteine-specific cleavage with 2-nitro-5-thiocyanobenzoic acid. The fragments were identified and aligned with the parent molecule using the deduced primary structure of synapsin I and the known phosphorylation sites as markers. We have purified these fragments and examined their interactions with F-actin. Two distinct fragments, a 29-kD NH2-terminal fragment and a 15-kD middle fragment, were shown to contain F-actin binding sites. A 51/54-kD middle/tail fragment retained the F-actin binding and bundling activity of synapsin I, but the isolated tail fragment did not retain either activity. In contrast to phosphorylation of sites two and three in intact synapsin I, which abolishes F-actin bundling activity, phosphorylation of these sites in the middle/tail fragment failed to abolish this activity. In conclusion, three domains of synapsin I appear to be involved in F-actin binding and bundling.

scholarly journals Moss Kinesin-14 KCBP accelerates chromatid motility in anaphase

2019 ◽  
Author(s):  
Mari Yoshida ◽  
Moé Yamada ◽  
Gohta Goshima
Keyword(s):  
Physcomitrella Patens ◽  
Function Analysis ◽  
Protein S ◽  
Dependent Manner ◽  
Tail Region ◽  
Cargo Transport ◽  
Direct Binding ◽  
Knockout Line

KCBP is a microtubule (MT) minus-end-directed kinesin widely conserved in plants. It was shown in Arabidopsis that KCBP controls trichome cell shape by orchestrating MT and actin cytoskeletons using its tail and motor domains. In contrast, the KCBP knockout (KO) line in the moss Physcomitrella patens showed a defect in nuclear and organelle positioning in apical stem cells. Moss KCBP is postulated to transport the nucleus and chloroplast via direct binding to their membranes, since it binds to and transports liposomes composed of phospholipids in vitro. However, domains required for cargo transport in vivo have not been mapped. Here, we performed a structure-function analysis of moss KCBP. We found that the FERM domain in the tail region, which is known to bind to lipids as well as other proteins, is essential for both nuclear and chloroplast positioning, whereas the proximal MyTH4 domain plays a supporting role in chloroplast transport. After anaphase but prior to nuclear envelope re-formation, KCBP accumulates on the chromosomes, in particular at the centromeric region in a FERM-dependent manner. In the KCBP knockout line, poleward chromosome motility in anaphase was reduced and lagging chromosomes occasionally appeared. These results suggest that KCBP binds to non-membranous naked chromosomes via an unidentified protein(s) for their transport. Finally, the liverwort orthologue of KCBP rescued the chromosome/chloroplast mis-positioning of the moss KCBP KO line, suggesting that the cargo transport function is conserved at least in bryophytes.

scholarly journals In vitro characterization of native mammalian smooth-muscle protein synaptopodin 2

2008 ◽  
Vol 28 (4) ◽  
pp. 195-203 ◽  
Author(s):  
Mechthild M. Schroeter ◽  
Brent Beall ◽  
Hans W. Heid ◽  
Joseph M. Chalovich
Keyword(s):  
Smooth Muscle ◽  
Muscle Protein ◽  
Actin Binding ◽  
Dependent Manner ◽  
In Vitro Characterization ◽  
Purification Scheme ◽  
Synaptopodin 2 ◽  
Muscle Myosin

An analysis of the primary structure of the actin-binding protein fesselin revealed it to be the avian homologue of mammalian synaptopodin 2 [Schroeter, Beall, Heid, and Chalovich (2008) Biochem. Biophys. Res. Commun. 371, 582–586]. We isolated two synaptopodin 2 isoforms from rabbit stomach that corresponded to known types of human synaptopodin 2. The purification scheme used was that developed for avian fesselin. These synaptopodin 2 forms shared several key functions with fesselin. Both avian fesselin and mammalian synaptopodin 2 bound to Ca2+–calmodulin, α-actinin and smooth-muscle myosin. In addition, both proteins stimulated the polymerization of actin in a Ca2+–calmodulin-dependent manner. Synaptopodin 2 has never before been shown to polymerize actin in the absence of α-actinin, to polymerize actin in a Ca2+–calmodulin-dependent manner, or to bind to Ca2+–calmodulin or myosin. These properties are consistent with the proposed function of synaptopodin 2 in organizing the cytoskeleton.

scholarly journals The effects of phosphorylation of smooth-muscle caldesmon

1987 ◽  
Vol 244 (2) ◽  
pp. 417-425 ◽  
Author(s):  
P K Ngai ◽  
M P Walsh
Keyword(s):  
Smooth Muscle ◽  
Physiological Role ◽  
Actin Binding ◽  
Dependent Manner ◽  
Binding Studies ◽  
Independent Manner ◽  
Thin Filaments ◽  
Atpase Reaction ◽  
Dependent Protein

Caldesmon is a major calmodulin- and actin-binding protein of smooth muscle which interacts with calmodulin in a Ca2+-dependent manner or with actin in a Ca2+-independent manner. Isolated caldesmon is capable of inhibiting the actin-activated Mg2+-ATPase of smooth-muscle myosin, suggesting a possible physiological role for caldesmon in regulating the contractile state of smooth-muscle. Caldesmon can be phosphorylated in vitro by a co-purifying Ca2+/calmodulin-dependent protein kinase and dephosphorylated by a protein phosphatase, both of which are present in smooth muscle. We investigated further the phosphorylation of caldesmon and the effects which phosphorylation has on the functional properties of the protein. The kinetics of caldesmon phosphorylation were similar whether the caldesmon substrate was free or bound to actin, actin/tropomyosin or thin filaments. Caldesmon containing endogenous kinase activity was rapidly phosphorylated (to approx. 1 mol of Pi/mol of caldesmon in 5 min) when reconstituted with actin, myosin, tropomyosin, calmodulin and myosin light-chain kinase in the presence of Ca2+ and MgATP2-. Under conditions in which unphosphorylated caldesmon showed substantial inhibition of the actin-activated myosin Mg2+-ATPase, no inhibition was observed with phosphorylated caldesmon. This was the case whether caldesmon was phosphorylated before addition to the actomyosin Mg2+-ATPase system, or phosphorylation was allowed to take place during the ATPase reaction. Binding studies revealed maximal binding of 1 mol of unphosphorylated caldesmon/9.5 mol of actin and 1 mol of phosphorylated caldesmon/11.7 mol of actin. All the bound phosphorylated caldesmon could be released by Ca2+/calmodulin, with half-maximal release at 0.11 microM-Ca2+, whereas only 62% of the bound unphosphorylated caldesmon could be removed, with half-maximal release at 0.16 microM-Ca2+. However, under conditions in which inhibition of actomyosin Mg2+-ATPase activity by non-phosphorylated but not by phosphorylated caldesmon was observed, both forms of caldesmon would remain bound to the thin filament. These observations suggest a possible mechanism whereby caldesmon phosphorylation may prevent its inhibitory action on the actomyosin Mg2+-ATPase.

Human Plasma Gelsolin Inhibits Cellular Responses to Platelet Activating Factor (PAF).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3568-3568
Author(s):  
Teresia A. Magnuson-Osborn ◽  
Claes Dahlgren ◽  
John H. Hartwig ◽  
Thomas P. Stossel
Keyword(s):  
Platelet Activating Factor ◽  
Actin Binding ◽  
Major Surgery ◽  
Human Neutrophils ◽  
Dependent Manner ◽  
Cellular Activation ◽  
Blood Concentrations ◽  
Plasma Gelsolin ◽  
Molar Excess

Abstract Gelsolin is a highly conserved intracellular actin-binding protein with an extracellular isoform named plasma gelsolin (pGSN). Relatively high (250 mg /L) blood concentrations of pGSN decrease in response to trauma, major surgery, sepsis, burns, ionizing radiation, and hyperoxia. Depletion of pGSN to a critical (~20%) level precedes and predicts complications of primary injuries such as lung permeability changes, ARDS, assisted ventilation and death. Administration of recombinant pGSN ameliorates such complications and reduces mortality in animal models. A proposed mechanism for pGSN’s protective effects is that it inhibits inflammatory mediators generated during primary injuries, since pGSN binds bioactive mediators, including lysophospatidic acid (LPA) and endotoxin in vitro. Because of its structural similarity we hypothesized that plasma gelsolin binds also to the potent lipid mediator platelet activating factor (PAF) and report here on the inhibition of PAF-induced cellular activation. Recombinant pGSN inhibited PAF-induced P-selectin up-regulation by human platelets as measured by flow cytometry. A ten- to 40-fold molar excess (0.5–20 μM) of pGSN over PAF inhibits P-selectin expression by 40 to 80%. The concentrations of plasma gelsolin used approximate the ~2–3 μM concentrations in plasma, and the molar excess of pGSN over PAF is probably greater in biological systems, where PAF has nanomolar affinity for its receptor. pGSN also inhibited PAF-induced superoxide anion (O2-) production (measured by chemiluminescence) of human neutrophils (PMN) in a concentration-dependent manner. The inhibition was up to 80% at a concentration of 10 μM (tenfold molar excess over PAF). A phospholipid-binding peptide derived from pGSN (QRLFQVKGRR) also inhibited PAF-mediated O2- generation by PMN. The inhibition was 65% at a 1:1 molar ratio (1 μM). In conclusion pGSN interferes with PAF-induced cellular activation in vitro, suggesting a mechanism for the protective role of plasma gelsolin that has been observed in vivo.

scholarly journals The toxofilin–actin–PP2C complex of Toxoplasma: identification of interacting domains

2007 ◽  
Vol 401 (3) ◽  
pp. 711-719 ◽  
Author(s):  
Gaelle Jan ◽  
Violaine Delorme ◽  
Violaine David ◽  
Celine Revenu ◽  
Angelita Rebollo ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Actin Filaments ◽  
Function Analysis ◽  
Protozoan Parasite ◽  
Actin Binding ◽  
Protein Protein Interactions ◽  
The Third ◽  
Parasite Motility

Toxofilin is a 27 kDa protein isolated from the human protozoan parasite Toxoplasma gondii, which causes toxoplasmosis. Toxofilin binds to G-actin, and in vitro studies have shown that it controls elongation of actin filaments by sequestering actin monomers. Toxofilin affinity for G-actin is controlled by the phosphorylation status of its Ser53, which depends on the activities of a casein kinase II and a type 2C serine/threonine phosphatase (PP2C). To get insights into the functional properties of toxofilin, we undertook a structure–function analysis of the protein using a combination of biochemical techniques. We identified a domain that was sufficient to sequester G-actin and that contains three peptide sequences selectively binding to G-actin. Two of these sequences are similar to sequences present in several G- and F-actin-binding proteins, while the third appears to be specific to toxofilin. Additionally, we identified two toxofilin domains that interact with PP2C, one of which contains the Ser53 substrate. In addition to characterizing the interacting domains of toxofilin with its partners, the present study also provides information on an in vivo-based approach to selectively and competitively disrupt the protein–protein interactions that are important to parasite motility.

scholarly journals Interactions of synapsin I with phospholipids: possible role in synaptic vesicle clustering and in the maintenance of bilayer structures.

1993 ◽  
Vol 123 (6) ◽  
pp. 1845-1855 ◽  
Author(s):  
F Benfenati ◽  
F Valtorta ◽  
M C Rossi ◽  
F Onofri ◽  
T Sihra ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
High Surface ◽  
Resonance Spectroscopy ◽  
Synapsin I ◽  
Hydrophobic Core ◽  
Membrane Phospholipids ◽  
Head Region ◽  
Vesicle Membrane ◽  
Liposome Fusion

Synapsin I is a synaptic vesicle-specific phosphoprotein composed of a globular and hydrophobic head and of a proline-rich, elongated and basic tail. Synapsin I binds with high affinity to phospholipid and protein components of synaptic vesicles. The head region of the protein has a very high surface activity, strongly interacts with acidic phospholipids and penetrates the hydrophobic core of the vesicle membrane. In the present paper, we have investigated the possible functional effects of the interaction between synapsin I and vesicle phospholipids. Synapsin I enhances both the rate and the extent of Ca(2+)-dependent membrane fusion, although it has no detectable fusogenic activity per se. This effect, which appears to be independent of synapsin I phosphorylation and localized to the head region of the protein, is attributable to aggregation of adjacent vesicles. The facilitation of Ca(2+)-induced liposome fusion is maximal at 50-80% of vesicle saturation and then decreases steeply, whereas vesicle aggregation does not show this biphasic behavior. Association of synapsin I with phospholipid bilayers does not induce membrane destabilization. Rather, 31P-nuclear magnetic resonance spectroscopy demonstrated that synapsin I inhibits the transition of membrane phospholipids from the bilayer (L alpha) to the inverted hexagonal (HII) phase induced either by increases in temperature or by Ca2+. These properties might contribute to the remarkable selectivity of the fusion of synaptic vesicles with the presynaptic plasma membrane during exocytosis.

Dose-dependent linkage, assembly inhibition and disassembly of vimentin and cytokeratin 5/14 filaments through plectin's intermediate filament-binding domain

2000 ◽  
Vol 113 (3) ◽  
pp. 483-491 ◽  
Author(s):  
F.A. Steinbock ◽  
B. Nikolic ◽  
P.A. Coulombe ◽  
E. Fuchs ◽  
P. Traub ◽  
...  
Keyword(s):  
Intermediate Filament ◽  
Ectopic Expression ◽  
Type I ◽  
Dependent Manner ◽  
Tail Region ◽  
Concentration Dependent Manner ◽  
Native Proteins ◽  
Dose Dependent

Plectin, the largest and most versatile member of the cytolinker/plakin family of proteins characterized to date, has a tripartite structure comprising a central 200 nm-long (α)-helical rod domain flanked by large globular domains. The C-terminal domain comprises a short tail region preceded by six highly conserved repeats (each 28–39 kDa), one of which (repeat 5) contains plectin's intermediate filament (IF)-binding site. We used recombinant and native proteins to assess the effects of plectin repeat 5-binding to IF proteins of different types. Quantitative Eu(3+)-based overlay assays showed that plectin's repeat 5 domain bound to type III IF proteins (vimentin) with preference over type I and II cytokeratins 5 and 14. The ability of both types of IF proteins to self-assemble into filaments in vitro was impaired by plectin's repeat 5 domain in a concentration-dependent manner, as revealed by negative staining and rotary shadowing electron microscopy. This effect was much more pronounced in the case of vimentin compared to cytokeratins 5/14. Preassembled filaments of both types became more and more crosslinked upon incubation with increasing concentrations of plectin repeat 5. However, at high proportions of plectin to IF proteins, disassembly of filaments occurred. Again, vimentin filaments proved considerably more sensitive towards disassembly than those composed of cytokeratins 5 and 14. In general, IFs formed from recombinant proteins were found to be slightly more responsive towards plectin influences than their native counterparts. A dose-dependent plectin-inflicted collapse and putative disruption of IFs was also observed in vivo after ectopic expression of vimentin and plectin's repeat 5 domain in cotransfected vimentin-deficient SW13 (vim(-)) cells. Our results suggest an involvement of plectin not only in crosslinking and stabilization of cytoskeletal IF networks, but also in regulation of their dynamics.

Exogenous NO inhibits basal NO release from vascular endothelium in vitro and in vivo

1996 ◽  
Vol 271 (5) ◽  
pp. H2045-H2051 ◽  
Author(s):  
X. L. Ma ◽  
B. L. Lopez ◽  
T. A. Christopher ◽  
D. S. Birenbaum ◽  
J. Vinten-Johansen
Keyword(s):  
In Vivo Studies ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
No Donor ◽  
Parent Molecule ◽  
Arterial Blood ◽  
No Release ◽  
Basal Release

This study tested the hypothesis that exogenous nitric oxide (NO) inhibits basal release of NO in isolated rat aortic rings and in vivo. Thoracic aortic rings were suspended in organ chambers with Krebs-Henseleit solution. In untreated rings, the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) markedly increased basal vascular tone by 34.6 +/- 5.2% of maximal force produced by 100 nM thromboxane A2 mimetic U-46619, indicating a basal release of NO. Other rings were pretreated with the exogenous NO donor S-nitroso-N-acetylpenicillamine (SNAP) for 20 min and then washed free of drug. In these rings, L-NAME-induced vasoconstriction was significantly attenuated in a concentration-dependent manner (from 34.6 +/- 5.2 to 25.7 +/- 2.9% at SNAP = 0.5 microM, 15.2 +/- 3.1% at 1 microM, and 11.9 +/- 2.5% at 5 microM), while having no effect on NO-independent phenylephrine-induced vasoconstriction (35.4 +/- 4.7 untreated vs. 41.3 +/- 4.3% SNAP pretreated, not significant). In addition, the nonnitrosylated parent molecule of SNAP, acetylpenicillamine, had no effect on the vasoconstriction induced by L-NAME. In the in vivo studies in anesthetized rats, L-NAME caused significant hypertensive responses (34 +/- 4-mmHg increase in mean arterial blood pressure). Subvasoactive doses of SNAP attenuated these hypertensive responses in a dose-dependent manner (20 +/- 3-mmHg increase with 10 micrograms/kg SNAP pretreatment and 16 +/- 4-mmHg increase with 20 micrograms/kg SNAP pretreatment), but any dose of acetylpenicillamine studied had no effect. Coadministration of superoxide dismutase and SNAP significantly potentiated the inhibitory effect of the NO donor on vasocontraction responses to L-NAME. Furthermore, SNAP did not attenuate the hypertensive responses to phenylephrine. These results indicate that exogenous NO significantly inhibits basal NO release both in vitro and in vivo, suggesting that NO plays an important negative-feedback regulatory role under physiological conditions.

SYK regulates B-cell migration by phosphorylation of the F-actin interacting protein SWAP-70

Blood ◽  
2011 ◽  
Vol 117 (5) ◽  
pp. 1574-1584 ◽  
Author(s):  
Glen Pearce ◽  
Tatsiana Audzevich ◽  
Rolf Jessberger
Keyword(s):  
Cell Migration ◽  
B Cells ◽  
B Cell ◽  
Cell Polarization ◽  
Actin Binding ◽  
Lymphoid Tissues ◽  
Dependent Manner ◽  
Interacting Protein

Abstract B-cell migration into and within lymphoid tissues is not only central to the humoral immune response but also for the development of malignancies and autoimmunity. We previously demonstrated that SWAP-70, an F-actin-binding, Rho GTPase-interacting protein strongly expressed in activated B cells, is necessary for normal B-cell migration in vivo. SWAP-70 regulates integrin-mediated adhesion and cell attachment. Here we show that upon B-cell activation, SWAP-70 is extensively posttranslationally modified and becomes tyrosine phosphorylated by SYK at position 517. This phosphorylation inhibits binding of SWAP-70 to F-actin. Phospho-site mutants of SWAP-70 disrupt B-cell polarization in a dominant-negative fashion in vitro and impair migration in vivo. After CXCL12 stimulation of B cells SYK becomes activated and SWAP-70 is phosphorylated in a SYK-dependent manner. Use of the highly specific SYK inhibitor BAY61-3606 showed SYK activity is necessary for normal chemotaxis and B-cell polarization in vitro and for entry of B cells into lymph nodes in vivo. These findings demonstrate a novel requirement for SYK in migration and polarization of naive recirculating B cells and show that SWAP-70 is an important target of SYK in this pathway.

scholarly journals Comparative Structure-Function Analysis of Mannose-Specific FimH Adhesins from Klebsiella pneumoniae and Escherichia coli

2009 ◽  
Vol 191 (21) ◽  
pp. 6592-6601 ◽  
Author(s):  
Steen G. Stahlhut ◽  
Veronika Tchesnokova ◽  
Carsten Struve ◽  
Scott J. Weissman ◽  
Sujay Chattopadhyay ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
Function Analysis ◽  
Bacterial Species ◽  
Host Cells ◽  
Structural Basis ◽  
Adaptive Mutation ◽  
Single Mutation ◽  
Dependent Manner ◽  
E Coli

ABSTRACT FimH, the adhesive subunit of type 1 fimbriae expressed by many enterobacteria, mediates mannose-sensitive binding to target host cells. At the same time, fine receptor-structural specificities of FimH from different species can be substantially different, affecting bacterial tissue tropism and, as a result, the role of the particular fimbriae in pathogenesis. In this study, we compared functional properties of the FimH proteins from Escherichia coli and Klebsiella pneumoniae, which are both 279 amino acids in length but differ by some ∼15% of residues. We show that K. pneumoniae FimH is unable to mediate adhesion in a monomannose-specific manner via terminally exposed Manα(1-2) residues in N-linked oligosaccharides, which are the structural basis of the tropism of E. coli FimH for uroepithelial cells. However, K. pneumoniae FimH can bind to the terminally exposed Manα(1-3)Manβ(1-4)GlcNAcβ1 trisaccharide, though only in a shear-dependent manner, wherein the binding is marginal at low shear force but enhanced sevenfold under increased shear. A single mutation in the K. pneumoniae FimH, S62A, converts the mode of binding from shear dependent to shear independent. This mutation has occurred naturally in the course of endemic circulation of a nosocomial uropathogenic clone and is identical to a pathogenicity-adaptive mutation found in highly virulent uropathogenic strains of E. coli, in which it also eliminates the dependence of E. coli binding on shear. The shear-dependent binding properties of the K. pneumoniae and E. coli FimH proteins are mediated via an allosteric catch bond mechanism. Thus, despite differences in FimH structure and fine receptor specificity, the shear-dependent nature of FimH-mediated adhesion is highly conserved between bacterial species, supporting its remarkable physiological significance.

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