Biomedical subjects

V Rybin

Publications and source records attributed to V Rybin.

18 recordsLinked to original sources

PubMed2001

Vps9, Rabex-5 and DSS4: proteins with weak but distinct nucleotide-exchange activities for Rab proteins.

The activities of three Rab-specific factors with GDP/GTP exchange activity, Vps9p, Rabex-5 and DSS4, with their cognate GTPases, Ypt51p, Rab5 and Ypt1p, have been analysed quantitatively. In contrast to other exchange factors examined and to DSS4, Vps9p, and by analogy probably Rabex-5, have considerably lower affinity than GDP to the respective GTPases. In keeping with this, they are relatively weak exchangers, with a maximal rate constant for GDP release from the ternary complex between exchange factor, GTPase and GDP of ca 0.01 s(-1), which is several orders of magnitude lower than for other exchange factors examined. If interaction with these proteins is a mandatory aspect of the Rab cycle, this suggests that the overall rate of cycling might be controlled at this point of the cycle. Surprisingly, DSS4, which has the thermodynamic potential to displace GDP effectively from Ypt1p, also does this very slowly, again with a maximal rate constant of ca 0.01 s(-1). An additional, and based on present knowledge, unique, feature of the Ypt1p.DSS4 complex, is that the association of GTP (or GDP) is more than 10(3)-fold slower than to Ypt1p, thus leading to a long life-time of the binary complex between the two proteins, even at the high nucleotide concentrations that prevail in the cell. This leads to the conclusion that the protein-protein complex is likely to have an important biological significance in addition to its probable role in GTP/GDP exchange.

Carrier Proteins↗

PubMed2001

Mice with a homozygous gene trap vector insertion in mgcRacGAP die during pre-implantation development.

In a phenotypic screen in mice using a gene trap approach in embryonic stem cells, we have identified a recessive loss-of-function mutation in the mgcRacGAP gene. Maternal protein is present in the oocyte, and mgcRacGAP gene transcription starts at the four-cell stage and persists throughout mouse pre-implantation development. Total mgcRacGAP deficiency results in pre-implantation lethality. Such E3.5 embryos display a dramatic reduction in cell number, but undergo compaction and form a blastocoel. At E3.0-3.5, binucleated blastomeres in which the nuclei are partially interconnected are frequently observed, suggesting that mgcRacGAP is required for normal mitosis and cytokinesis in the pre-implantation embryo. All homozygous mutant blastocysts fail to grow out on fibronectin-coated substrates, but a fraction of them can still induce decidual swelling in vivo. The mgcRacGAP mRNA expression pattern in post-implantation embryos and adult mouse brain suggests a role in neuronal cells. Our results indicate that mgcRacGAP is essential for the earliest stages of mouse embryogenesis, and add evidence that CYK-4-like proteins also play a role in microtubule-dependent steps in the cytokinesis of vertebrate cells. In addition, the severe phenotype of null embryos indicates that mgcRacGAP is functionally non-redundant and cannot be substituted by other GAPs during early cleavage of the mammalian embryo.

Animals↗

PubMed2001

ATP-dependent membrane assembly of F-actin facilitates membrane fusion.

We recently established an in vitro assay that monitors the fusion between latex-bead phagosomes and endocytic organelles in the presence of J774 macrophage cytosol (). Here, we show that different reagents affecting the actin cytoskeleton can either inhibit or stimulate this fusion process. Because the membranes of purified phagosomes can assemble F-actin de novo from pure actin with ATP (), we focused here on the ability of membranes to nucleate actin in the presence of J774 cytosolic extracts. For this, we used F-actin sedimentation, pyrene actin assays, and torsional rheometry, a biophysical approach that could provide kinetic information on actin polymerization and gel formation. We make two major conclusions. First, under our standard in vitro conditions (4 mg/ml cytosol and 1 mM ATP), the presence of membranes actively catalyzed the assembly of cytosolic F-actin, which assembled into highly viscoelastic gels. A model is discussed that links these results to how the actin may facilitate fusion. Second, cytosolic actin paradoxically polymerized more under ATP depletion than under high-ATP conditions, even in the absence of membranes; we discuss these data in the context of the well described, large increases in F-actin seen in many cells during ischemia.

Actins↗

PubMed2001

Functional synergy between Rab5 effector Rabaptin-5 and exchange factor Rabex-5 when physically associated in a complex.

Rab GTPases are central elements of the vesicular transport machinery. An emerging view is that downstream effectors of these GTPases are multiprotein complexes that include nucleotide exchange factors to ensure coupling between GTPase activation and effector function. We have previously shown that Rab5, which regulates various steps of transport along the early endocytic pathway, is activated by a complex consisting of Rabex-5, a Rab5 nucleotide exchange factor, and the effector Rabaptin-5. We postulated that the physical association of these two proteins is necessary for their activity in Rab5-dependent endocytic membrane transport. To evaluate the functional implications of such complex formation, we have reconstituted it with the use of recombinant proteins and characterized its properties. First, we show that Rabaptin-5 increases the exchange activity of Rabex-5 on Rab5. Second, Rab5-dependent recruitment of Rabaptin-5 to early endosomes is completely dependent on its physical association with Rabex-5. Third, complex formation between Rabaptin-5 and Rabex-5 is essential for early endosome homotypic fusion. These results reveal a functional synergy between Rabaptin-5 and Rabex-5 in the complex and have implications for the function of analogous complexes for Rab and Rho GTPases.

Animals↗

PubMed2000

The Eps8 protein coordinates EGF receptor signalling through Rac and trafficking through Rab5.

How epidermal growth factor receptor (EGFR) signalling is linked to EGFR trafficking is largely unknown. Signalling and trafficking involve small GTPases of the Rho and Rab families, respectively. But it remains unknown whether the signalling relying on these two classes of GTPases is integrated, and, if it is, what molecular machinery is involved. Here we report that the protein Eps8 connects these signalling pathways. Eps8 is a substrate of the EGFR, which is held in a complex with Sos1 by the adaptor protein E3bl (ref. 2), thereby mediating activation of Rac. Through its src homology-3 domain, Eps8 interacts with RN-tre. We show that RN-tre is a Rab5 GTPase-activating protein, whose activity is regulated by the EGFR. By entering in a complex with Eps8, RN-tre acts on Rab5 and inhibits internalization of the EGFR. Furthermore, RN-tre diverts Eps8 from its Rac-activating function, resulting in the attenuation of Rac signalling. Thus, depending on its state of association with E3b1 or RN-tre, Eps8 participates in both EGFR signalling through Rac, and trafficking through Rab5.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Crystal structure of the protein disulfide bond isomerase, DsbC, from Escherichia coli.

DsbC is one of five Escherichia coli proteins required for disulfide bond formation and is thought to function as a disulfide bond isomerase during oxidative protein folding in the periplasm. DsbC is a 2 x 23 kDa homodimer and has both protein disulfide isomerase and chaperone activity. We report the 1.9 A resolution crystal structure of oxidized DsbC where both Cys-X-X-Cys active sites form disulfide bonds. The molecule consists of separate thioredoxin-like domains joined via hinged linker helices to an N-terminal dimerization domain. The hinges allow relative movement of the active sites, and a broad uncharged cleft between them may be involved in peptide binding and DsbC foldase activities.

Amino Acid Sequence↗

PubMed1999

Oligomeric complexes link Rab5 effectors with NSF and drive membrane fusion via interactions between EEA1 and syntaxin 13.

SNAREs and Rab GTPases cooperate in vesicle transport through a mechanism yet poorly understood. We now demonstrate that the Rab5 effectors EEA1 and Rabaptin-5/Rabex-5 exist on the membrane in high molecular weight oligomers, which also contain NSF. Oligomeric assembly is modulated by the ATPase activity of NSF. Syntaxin 13, the t-SNARE required for endosome fusion, is transiently incorporated into the large oligomers via direct interactions with EEA1. This interaction is required to drive fusion, since both dominant-negative EEA1 and synthetic peptides encoding the FYVE Zn2+ finger hinder the interaction and block fusion. We propose a novel mechanism whereby oligomeric EEA1 and NSF mediate the local activation of syntaxin 13 upon membrane tethering and, by analogy with viral fusion proteins, coordinate the assembly of a fusion pore.

Adenosine Triphosphatases↗

PubMed1998

Two distinct effectors of the small GTPase Rab5 cooperate in endocytic membrane fusion.

Using the yeast two-hybrid system, we have identified a novel 62 kDa coiled-coil protein that specifically interacts with the GTP-bound form of Rab5, a small GTPase that regulates membrane traffic in the early endocytic pathway. This protein shares 42% sequence identity with Rabaptin-5, a previously identified effector of Rab5, and we therefore named it Rabaptin-5beta. Like Rabaptin-5, Rabaptin-5beta displays heptad repeats characteristic of coiled-coil proteins and is recruited on the endosomal membrane by Rab5 in a GTP-dependent manner. However, Rabaptin-5beta has features that distinguish it from Rabaptin-5. The relative expression levels of the two proteins varies in different cell types. Rabaptin-5beta does not heterodimerize with Rabaptin-5, and forms a distinct complex with Rabex-5, the GDP/GTP exchange factor for Rab5. Immunodepletion of the Rabaptin-5beta complex from cytosol only partially inhibits early endosome fusion in vitro, whereas the additional depletion of the Rabaptin-5 complex has a stronger inhibitory effect. Fusion activity can mostly be recovered by addition of the Rabaptin-5 complex alone, but maximal fusion efficiency requires the presence of both Rabaptin-5 and Rabaptin-5beta complexes. Our results suggest that Rab5 binds to at least two distinct effectors which cooperate for optimal endocytic membrane docking and fusion.

Amino Acid Sequence↗

PubMed1998

Distinct Rab-binding domains mediate the interaction of Rabaptin-5 with GTP-bound Rab4 and Rab5.

Rabaptin-5 functions as an effector for the small GTPase Rab5, a regulator of endocytosis and early endosome fusion. We have searched for structural determinants that confer functional specificity on Rabaptin-5. Here we report that native cytosolic Rabaptin-5 is present in a homodimeric state and dimerization depends upon the presence of its coiled-coil predicted sequences. A 73 residue C-terminal region of Rabaptin-5 is necessary and sufficient both for the interaction with Rab5 and for Rab5-dependent recruitment of the protein on early endosomes. Surprisingly, we uncovered the presence of an additional Rab-binding domain at the N-terminus of Rabaptin-5. This domain mediates the direct interaction with the GTP-bound form of Rab4, a small GTPase that has been implicated in recycling from early endosomes to the cell surface. Based on these results, we propose that Rabaptin-5 functions as a molecular linker between two sequentially acting GTPases to coordinate endocytic and recycling traffic.

Amino Acid Sequence↗

PubMed1997

A novel Rab5 GDP/GTP exchange factor complexed to Rabaptin-5 links nucleotide exchange to effector recruitment and function.

The small GTPase Rab5 plays an essential role in endocytic traffic. Rab GDP dissociation inhibitor delivers Rab5 to the membrane, where a nucleotide exchange activity allows recruitment of an effector protein, Rabaptin-5. Here we uncovered a novel 60 kDa Rab5-binding protein, Rabex-5. Rabex-5 forms a tight physical complex with Rabaptin-5, and this complex is essential for endocytic membrane fusion. Sequencing of mammalian Rabex-5 by nanoelectrospray mass spectrometry and cloning revealed striking homology to Vps9p, a yeast protein implicated in endocytic traffic. Rabex-5 displays GDP/GTP exchange activity on Rab5 upon delivery of the GTPase to the membrane. This demonstrates that a soluble exchange factor coupled to a Rab effector translocates from cytosol to the membrane, where the complex stabilizes the GTPase in the active state.

Animals↗

PubMed1997

Do adult rat ventricular myocytes express protein kinase C-alpha?

Although calcium-insensitive protein kinase C (PKC) isoforms (PKC-epsilon and PKC-delta) are consistently detected in adult ventricular myocytes, the evidence that adult ventricular myocytes also express calcium-sensitive PKC-alpha is inconsistent. The current study used four different anti-PKC-alpha-antibodies to resolve some of the uncertainties regarding the immunodetection of PKC-alpha in adult ventricular myocytes. Three of the antibodies used in this study barely (GIBCO-BRL) or rather faintly (Transduction Laboratories and Seikagaku America) recognize PKC-alpha in crude preparations from adult ventricular myocytes. Although each of these antibodies recognizes a prominent 80-kDa band, which is similar in size to PKC-alpha, this represents nonspecific immunoreactivity and should not be confused with PKC-alpha. This conclusion is based on peptide-blocking experiments (GIBCO-BRL), the absence of the requisite sensitivity to calcium- and phorbol 12-myristate 13-acetate-induced translocation (Seikagaku America and Transduction Laboratories), and/or the failure to copurify with PKC-alpha on DEAE-Sephacel chromatography. Nevertheless, an antibody from Upstate Biotechnology clearly recognizes PKC-alpha and not other unrelated nonspecific immunoreactive species in crude preparations from adult ventricular myocytes. Each of the antisera used in this study could detect PKC-alpha immunoreactivity following chromatographic purification of the samples to enrich for PKC-alpha and remove nonspecific immunoreactive proteins. These results suggest that PKC-alpha is expressed by adult ventricular myocytes and argue that differences in the sensitivity and/or specificity of available antisera contribute to at least some of the confusion regarding PKC-alpha expression in adult ventricular myocytes.

Animals↗

PubMed1996

Endosome dynamics regulated by a Rho protein.

Vesicular transport is a dynamic process that requires coordinated interactions between membrane and cytoskeleton. The mechanisms and molecules integrating these interactions are unclear. A Rho protein, RhoD, might provide a molecular link between membrane traffic and the cytoskeleton. Activated RhoD causes rearrangements of the actin cytoskeleton and cell surface, and governs early endosome motility and distribution.

Actins↗

PubMed1996

Crystallization of DsbC, the disulfide bond isomerase of Escherichia coli.

DsbC is a 2 x 23 kDa soluble dimeric protein molecule involved in protein disulfide bond formation in the E. coli periplasm, primarily catalyzing disulfide bond rearrangements. Crystals of both the native and selenomethione protein suitable for structure determination were obtained using the hanging-drop vapour-diffusion method. The best crystals were obtained using 18-22% (v/v) polyethylene glycol 550 monomethyl ether in 100 mM Tris-HCl (pH 8.9). Seeding methods were used to produce large crystals diffracting to 2 A resolution, and the detergent n-octyl-beta-glucoside was used to improve crystal quality. Significant variation in cell dimensions and crystal order was observed. Cell dimensions obtained for frozen crystals were in the range a = 58.8 (0.3), b = 78.9 (0.5), c = 95.2 (5.0) A. The lattice is orthorhombic and systematic absences indicate that the space group is P2(1)2(1)2(1).

Journal Article↗

PubMed1996

GTPase activity of Rab5 acts as a timer for endocytic membrane fusion.

The GTPase cycle is a versatile regulatory mechanism directing many cell functions, and Rab family members use it to regulate intracellular transport. Current models propose that GTP hydrolysis by Rab proteins is either required for membrane fusion or occurs afterwards to allow recycling of the protein. To measure the GTPase activity of Rab5 in endocytic membrane fusion, we engineered a mutant that preferentially binds xanthosine 5'-triphosphate (XTP),Rab5(D136N) and monitored the kinetics of [alpha(32)P]-XTP hydrolysis in situ during endosome fusion in vitro. Surprisingly, nucleotide hydrolysis occurred even in the absence of membrane fusion, indicating that membrane-bound Rab5 undergoes futile cycles of GTP(XTP) binding and hydrolysis. Nucleotide triphosphate hydrolysis by Rab5 is not conditional on membrane fusion and is reduced by its effector Rabaptin-5. Our data reveal that the GTP cycle of Rab proteins differs from that of other GTPases (for example, EF-Tu) and indicate that GTP hydrolysis acts as a timer that determines the frequency of membrane docking/fusion events.

ADP-Ribosylation Factors↗

PubMed1996

Thyroid hormone represses protein kinase C isoform expression and activity in rat cardiac myocytes.

We have previously demonstrated that at least four isoforms of protein kinase C (PKC; alpha, delta, epsilon, and zeta) are expressed in neonatal rat ventricular myocytes and that development is associated with a decline in their expression. The mechanism(s) regulating PKC isoform expression in ventricular myocytes is completely unknown. The developmental decline in PKC expression occurs, in large part, during the first 2 weeks of postnatal life, while thyroid hormone levels are known to be progressively increasing. Accordingly, this study examined the influence of thyroid hormone on PKC isoform expression to determine whether thyroid hormone can be implicated as a potential physiological regulator of PKC gene expression during normal cardiac development. Hypothyroidism was induced in adult rats by surgical thyroidectomy; thyroid status was manipulated in cultured neonatal ventricular myocytes by growth in serum-free medium with varying triiodothyronine (T3) levels. In each case, hypothyroidism was verified by a 10- to 50-fold increase in steady state mRNA for beta-myosin heavy chain. In hypothyroid adult ventricular myocardium, there was a selective 60% increase in the expression of PKC epsilon protein that corresponded to an increase in maximally stimulated PKC enzyme activity with PKC epsilon substrate peptide (epsilon pep) but not with histone as substrate. Northern blot analysis revealed a 70% increase in PKC epsilon mRNA, indicating that the regulatory effects of thyroid hormone are mediated, at least in part, at the message level. In neonatal ventricular myocytes, there was a T3-dependent reduction in immunoreactivity for both PKC alpha and PKC epsilon that was associated with significant reductions in both histone- and epsilon pep-kinase activities. The concentration of T3 that half-maximally repressed PKC alpha and PKC epsilon expression was approximately 0.5 nmol/L. Thyroid hormone had no effect on PKC delta and PKC zeta expression in neonatal or adult ventricular myocytes. PKC isoform expression in cardiac fibroblasts was not influenced by variations in the thyroid hormone concentration during culture. These results provide evidence that thyroid hormone specifically represses PKC alpha and PKC epsilon in the neonatal heart and PKC epsilon in the adult heart. Thyroid hormone-induced changes in PKC may play an important permissive role in the modulation of autonomic responsiveness in ventricular cardiomyocytes.

Animals↗

PubMed1994

Mechanisms for vagal modulation of ventricular repolarization and of coronary occlusion-induced lethal arrhythmias in cats.

Our goal was to better understand the mechanisms underlying muscarinic receptor actions on the ventricle in vivo. Therefore, we studied the effects of vagal stimulation on ventricular repolarization and of vagal tone on lethal arrhythmias induced by 30 minutes of left anterior descending coronary artery ligation in anesthetized cats. Experimental groups included normal control cats subjected only to coronary ligation and cats pretreated with atropine, pertussis toxin (PTX), or propranolol. All cats received bilateral cervical vagal stimulation (Vstim) at 1, 3, and 5 Hz for 1 minute at 10-minute intervals. Before coronary ligation, Vstim slowed sinus rate, prolonged the PR interval, and lowered blood pressure. Most important from the point of view of electrophysiological function was a vagally induced acceleration of ventricular repolarization in paced and unpaced hearts, which could be explained by the effects of acetylcholine (ie, shortening the subepicardial muscle action potentials). The effect on repolarization was blocked by atropine or PTX but not by propranolol. The extent of sinus slowing and acceleration of repolarization was directly related to the level of functional PTX-sensitive G protein (P < .05). Coronary occlusion was performed during atrial pacing such that the heart rate in all groups was equal. The incidence of ventricular fibrillation (VF) was 10% in the control group and 50% and 54% in atropine and PTX groups, respectively (P < .05). During atrial pacing before coronary occlusion, a vagal index was calculated as percent QTc shortening during Vstim. When the vagal index was 13% to 26%, the incidence of VF during occlusion was zero. When the vagal index was 0% to 12%, VF was 52% (P < .01). Conclusions are as follows: (1) Vstim accelerates ventricular repolarization in cats via a pathway that incorporates a PTX-sensitive G protein and involves an altered gradient between epicardium and endocardium. (2) Removal of vagal tone during ischemia favors VF, as predicted by a vagal index.

Acetylcholine↗

PubMed1992

A Gs protein couples P2-purinergic stimulation to cardiac Ca channels without cyclic AMP production.

P2-purinergic stimulation of the L-type Ca current induced by the external application of 100 microM ATP gamma S was investigated in rat ventricular cardiomyocytes using the whole-cell patch-clamp technique. The purinergic-induced increase in ICa was slow and monophasic and reached a steady state within 3 min. In contrast to beta-adrenergic stimulation, after a brief agonist application the current did not continue to increase on washout; recovery started immediately after agonist removal. The P2-purinergic increase in ICa was significantly less in the presence of GDP beta S, but it occurred much faster and was twice as large when a low dose of GTP gamma S (100 microM) was added to a GTP-containing internal medium. This suggests that the ICa increase was mediated by a G protein. Based on electrophoretic mobility and susceptibility to cholera toxin and anti-G alpha s serum, it is proposed that the G protein involved during purinergic-induced ICa stimulation is an isoform of Gs not coupled to the adenylyl cyclase, since the cyclic AMP level was unaffected. High intracellular GTP gamma S (1 mM) maximally activated ICa so that neither beta-adrenergic nor P2-purinergic agonists further increased ICa. In the absence of GTP and an ATP-regenerating system, GTP gamma S was much more potent in increasing basal ICa and supporting purinergic stimulation. This indicates that a nucleoside diphosphate kinase activity might replenish endogenous GTP; GTP exchange with GTP gamma S on the G protein was promoted by the P2-purinergic stimulation and led to a reversible and reproducible increase in ICa. In the presence of 3 mM internal ATP gamma S, the P2-purinergic stimulation was also reversible and reproducible. Moreover, under these conditions (ATP gamma S or GTP gamma S) the increase in ICa was not maintained during prolonged agonist application. Such an inhibition occurred slowly and irreversibly; it might be related to the threefold increase in cyclic GMP. In conclusion, we propose that extracellular ATP induces both a stimulatory and an inhibitory effect on ICa, probably mediated by subtypes of P2-purinergic receptors. An isoform of the Gs protein is likely to mediate the stimulation.

Adenosine Diphosphate↗

PubMedDate not supplied

Cellular signalling by lipoproteins in cultured smooth muscle cells from spontaneously hypertensive rats.

We investigated pivotal signalling responses of cultured aortic smooth muscle cells (VSMC) from the spontaneously hypertensive rat (SHR) and the normotensive Wistar Kyoto rat (WKY) to lipoproteins. Low-density lipoprotein (LDL) and high-density lipoprotein (HDL3) stimulated a time- and dose-dependent accumulation of inositol phosphates in VSMC. SHR and WKY VSMC exhibited comparable half-maximal dose requirements (approximately 13 micrograms/ml for LDL and approximately 14 micrograms/ml for HDL3), although, at any given dose, the response of SHR VSMC was significantly greater than WKY VSMC. Simultaneous addition of LDL and HDL3 to VSMC resulted in additive stimulatory effects on phosphoinositide catabolism. Pertussis toxin pretreatment of VSMC completely negated the stimulatory effects of LDL and HDL3 on IP accumulation. [32P]-ADP ribosylation and immunoblotting studies revealed the guanine nucleotide-binding (G protein) substrate(s) for pertussis toxin to be a Gi protein(s). SHR and WKY VSMC did not differ with respect to levels of Gi alpha or G beta, and thus, the amplified responsiveness in SHR VSMC cannot be attributed to alterations in levels of pertussis toxin-sensitive G protein. The spectrum of signalling responses elicited by LDL and HDL3 are similar to those elicited by vasoactive hormones, and thus lipoproteins may, via stimulation of phosphoinositide catabolism, 45Ca2+ uptake and Na+/H(+)-exchange, directly regulate smooth muscle cell growth and contraction.

Animals↗