Proteomic and biochemical comparison of the cellular interaction partners of human VPS33A and VPS33B

ABSTRACTMulti-subunit tethering complexes control membrane fusion events in eukaryotic cells. CORVET and HOPS are two such multi-subunit tethering complexes, both containing the Sec1/Munc18 protein subunit VPS33A. Metazoans additionally possess VPS33B, which has considerable sequence similarity to VPS33A but does not integrate into CORVET or HOPS complexes and instead stably interacts with VIPAR. It has been recently suggested that VPS33B and VIPAR comprise two subunits of a novel multi-subunit tethering complex (named ‘CHEVI’), analogous in configuration to CORVET and HOPS. We utilised the BioID proximity biotinylation assay to compare and contrast the interactomes of VPS33A and VPS33B. Overall, few proteins were identified as associating with both VPS33A and VPS33B, suggesting these proteins have distinct sub-cellular localisations. Consistent with previous reports, we observed that VPS33A was co-localised with many components of class III phosphatidylinositol 3-kinase (PI3KC3) complexes: PIK3C3, PIK3R4, NRBF2, UVRAG and RUBICON. Although in this assay VPS33A clearly co-localised with several subunits of CORVET and HOPS, no proteins with the canonical CORVET/HOPS domain architecture were found to co-localise with VPS33B. Instead, we identified two novel VPS33B-interacting proteins, VPS53 and CCDC22. CCDC22 co-immunoprecipitated with VPS33B and VIPAR in over-expression conditions and interacts directly with the VPS33B-VIPAR complex in vitro. However, CCDC22 does not appear to co-fractionate with VPS33B and VIPAR in gel filtration of human cell lysates. We also observed that the protein complex in HEK293T cells which contained VPS33B and VIPAR was considerably smaller than CORVET/HOPS, suggesting that, unlike VPS33A, VPS33B does not assemble into a large stable multi-subunit tethering complex.

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Ca2+-regulated Pool of Phosphatidylinositol-3-phosphate Produced by Phosphatidylinositol 3-Kinase C2α on Neurosecretory Vesicles

Molecular Biology of the Cell ◽

10.1091/mbc.e08-06-0595 ◽

2008 ◽

Vol 19 (12) ◽

pp. 5593-5603 ◽

Cited By ~ 39

Author(s):

Peter J. Wen ◽

Shona L. Osborne ◽

Isabel C. Morrow ◽

Robert G. Parton ◽

Jan Domin ◽

...

Keyword(s):

Critical Role ◽

Neurosecretory Cells ◽

Secretory Vesicles ◽

Endosomal Trafficking ◽

Class Iii ◽

Phosphatidylinositol 3 Kinase ◽

Lipid Product ◽

Phosphatidylinositol 3

Phosphatidylinositol-3-phosphate [PtdIns(3)P] is a key player in early endosomal trafficking and is mainly produced by class III phosphatidylinositol 3-kinase (PI3K). In neurosecretory cells, class II PI3K-C2α and its lipid product PtdIns(3)P have recently been shown to play a critical role during neuroexocytosis, suggesting that two distinct pools of PtdIns(3)P might coexist in these cells. However, the precise characterization of this additional pool of PtdIns(3)P remains to be established. Using a selective PtdIns(3)P probe, we have identified a novel PtdIns(3)P-positive pool localized on secretory vesicles, sensitive to PI3K-C2α knockdown and relatively resistant to wortmannin treatment. In neurosecretory cells, stimulation of exocytosis promoted a transient albeit large increase in PtdIns(3)P production localized on secretory vesicles sensitive to PI3K-C2α knockdown and expression of PI3K-C2α catalytically inactive mutant. Using purified chromaffin granules, we found that PtdIns(3)P production is controlled by Ca2+. We confirmed that PtdIns(3)P production from recombinantly expressed PI3K-C2α is indeed regulated by Ca2+. We provide evidence that a dynamic pool of PtdIns(3)P synthesized by PI3K-C2α occurs on secretory vesicles in neurosecretory cells, demonstrating that the activity of a member of the PI3K family is regulated by Ca2+ in vitro and in living neurosecretory cells.

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Substrate Targeting of the Yeast Cyclin-Dependent Kinase Pho85p by the Cyclin Pcl10p

Molecular and Cellular Biology ◽

10.1128/mcb.19.10.7020 ◽

1999 ◽

Vol 19 (10) ◽

pp. 7020-7030 ◽

Cited By ~ 41

Author(s):

Wayne A. Wilson ◽

Alan M. Mahrenholz ◽

Peter J. Roach

Keyword(s):

Yeast Cell ◽

Glycogen Synthase ◽

Sequence Similarity ◽

Cell Cycle Control ◽

Gel Filtration ◽

Substrate Selection ◽

Cell Lysates ◽

Dependent Protein ◽

Molecular Weight Fractions

ABSTRACT In Saccharomyces cerevisiae, PHO85 encodes a cyclin-dependent protein kinase (Cdk) catalytic subunit with multiple regulatory roles thought to be specified by association with different cyclin partners (Pcls). Pcl10p is one of four Pcls with little sequence similarity to cyclins involved in cell cycle control. It has been implicated in specifying the phosphorylation of glycogen synthase (Gsy2p). We report that recombinant Pho85p and Pcl10p produced inEscherichia coli reconstitute an active Gsy2p kinase in vitro. Gsy2p phosphorylation required Pcl10p, occurred at physiologically relevant sites, and resulted in inactivation of Gsy2p. The activity of the reconstituted enzyme was even greater than Pho85p-Pcl10p isolated from yeast, and we conclude that, unlike many Cdks, Pho85p does not require phosphorylation for activity. Pcl10p formed complexes with Gsy2p, as judged by (i) gel filtration of recombinant Pcl10p and Gsy2p, (ii) coimmunoprecipitation from yeast cell lysates, and (iii) enzyme kinetic behavior consistent with Pcl10p binding the substrate. Synthetic peptides modeled on the sequences of known Pho85p sites were poor substrates with highKm values, and we propose that Pcl10p-Gsy2p interaction is important for substrate selection. Gel filtration of yeast cell lysates demonstrated that most Pho85p was present as a monomer, although a portion coeluted in high-molecular-weight fractions with Pcl10p and Gsy2p. Overexpression of Pcl10p sequestered most of the Pho85p into association with Pcl10p. We suggest a model for Pho85p function in the cell whereby cyclins like Pcl10p recruit Pho85p from a pool of monomers, both activating the kinase and targeting it to substrate.

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Biochemical characterization and pharmacological properties of a phospholipase A2 myotoxin inhibitor from the plasma of the snake Bothrops asper

Biochemical Journal ◽

10.1042/bj3260853 ◽

1997 ◽

Vol 326 (3) ◽

pp. 853-859 ◽

Cited By ~ 46

Author(s):

Sergio LIZANO ◽

Bruno LOMONTE ◽

Jay W. FOX ◽

José Maréa GUTIÉRREZ

Keyword(s):

Phospholipase A2 ◽

Molecular Mass ◽

Biochemical Characterization ◽

Biological Activities ◽

Sequence Similarity ◽

Gel Filtration ◽

Cytolytic Activity ◽

Bothrops Asper

A protein that neutralizes the biological activities of basic phospholipase A2 (PLA2) myotoxin isoforms from the venom of the snake Bothrops asper was isolated from its blood by affinity chromatography with Sepharose-immobilized myotoxins. Biochemical characterization of this B. asper myotoxin inhibitor protein (BaMIP) indicated a subunit molecular mass of 23–25 kDa, an isoelectric point of 4, and glycosylation. Gel-filtration studies revealed a molecular mass of 120 kDa, suggesting that BaMIP possesses an oligomeric structure composed of five 23–25 kDa subunits. Functional studies indicated that BaMIP inhibits the PLA2 activity of B. asper basic myotoxins I and III, as well as the myotoxicity and edema-forming activity in vivo and cytolytic activity in vitro towards cultured endothelial cells, of all four myotoxin isoforms (I–IV) tested. Sequence analysis of the first 63 amino acid residues from the N-terminus of BaMIP indicated more than 65% sequence similarity to the PLA2 inhibitors isolated from the blood of the crotalid snakes Trimeresurus flavoviridis and Agkistrodon blomhoffii siniticus. These inhibitors also share sequences similar to the carbohydrate-recognition domains of human and rabbit cellular PLA2 receptors, suggesting a common domain evolution among snake plasma PLA2 inhibitors and mammalian PLA2 receptors. Despite this similarity, this is the first description of a natural anti-myotoxic factor from snake blood.

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The 50 kDa protein subunit of assembly polypeptide (AP) AP-2 adaptor from clathrin-coated vesicles is phosphorylated on threonine-156 by AP-1 and a soluble AP50 kinase which co-purifies with the assembly polypeptides

Biochemical Journal ◽

10.1042/bj2960409 ◽

1993 ◽

Vol 296 (2) ◽

pp. 409-415 ◽

Cited By ~ 24

Author(s):

A Pauloin ◽

C Thurieau

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Protein Complex ◽

Kinase Activity ◽

Phosphorylation Site ◽

Gel Filtration ◽

Coated Vesicles ◽

Protein Subunit

AP50 is a subunit of the assembly polypeptide (AP) subclass AP-2 from bovine brain coated vesicles. It can be phosphorylated in vivo and in vitro on a threonine residue by means of the AP50 kinase activity associated with AP. We have undertaken an analysis of the amino acid sequence around the AP50 phosphorylation site. After phosphorylation in vitro of AP50 followed by tryptic cleavage, only one radioactive peptide was isolated following Mono-Q ion-exchange f.p.l.c. and reverse-phase h.p.l.c. The amino acid sequence of this peptide: Glu146-Glu-Gln-Ser-Gln-Ile-Thr-Ser-Gln-Val-Thr*-Gly-Gly-Ile-Gly-Tr p-Arg162, displayed two threonine residues. Analysis of the yield and radioactivity of the product from automated Edman degradation indicated that only Thr-156 was phosphorylated, reflecting the presence of a single phosphorylation site in AP50. AP phosphorylated the corresponding synthetic peptide on the same threonyl residue. We demonstrated that AP50 was a phosphorylation substrate unable to autophosphorylate. The enzyme involved in the AP50 phosphorylation was shown to be associated with AP-1 and with a soluble protein complex co-purified with APs but resolved from the latter by hydroxyapatite-column exclusion chromatography. This AP50 kinase activity corresponded to a 280 kDa protein complex according to gel-filtration data.

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1442: CXCL12 Over-Expression and Secretion by Aging Fibroblasts Stimulates Human Prostate Epithelial Proliferation in Vitro

The Journal of Urology ◽

10.1016/s0022-5347(18)33646-2 ◽

2006 ◽

Vol 175 (4S) ◽

pp. 466-466

Author(s):

Jill A. Macoska ◽

Lesa Begley ◽

Christine Monteleon ◽

James W. MacDonald ◽

Rajal B. Shah

Keyword(s):

Human Prostate ◽

Epithelial Proliferation ◽

Over Expression ◽

Proliferation In Vitro

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Purification of the Antihemophilic Factor by Gel Filtration on Agarose

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651394 ◽

1969 ◽

Vol 22 (03) ◽

pp. 577-583 ◽

Cited By ~ 3

Author(s):

M.M.P Paulssen ◽

A.C.M.G.B Wouterlood ◽

H.L.M.A Scheffers

Keyword(s):

Factor Viii ◽

Plasma Proteins ◽

Large Scale ◽

Gel Filtration ◽

Large Scale Preparation ◽

Scale Preparation ◽

Antihemophilic Factor

SummaryFactor VIII can be isolated from plasma proteins, including fibrinogen by chromatography on agarose. The best results were obtained with Sepharose 6B. Large scale preparation is also possible when cryoprecipitate is separated by chromatography. In most fractions containing factor VIII a turbidity is observed which may be due to the presence of chylomicrons.The purified factor VIII was active in vivo as well as in vitro.

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A Comparison of the Effect of Decorsin and Two Disintegrins, Albolabrin and Eristostatin, on Platelet Function

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649933 ◽

1995 ◽

Vol 74 (05) ◽

pp. 1316-1322 ◽

Cited By ~ 13

Author(s):

Mary Ann McLane ◽

Jagadeesh Gabbeta ◽

A Koneti Rao ◽

Lucia Beviglia ◽

Robert A Lazarus ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Sequence Similarity ◽

Platelet Aggregation Inhibitors ◽

Antiplatelet Drug ◽

Rgd Sequence ◽

Fibrinogen Receptor ◽

Activated Platelets

SummaryNaturally-occurring fibrinogen receptor antagonists and platelet aggregation inhibitors that are found in snake venom (disintegrins) and leeches share many common features, including an RGD sequence, high cysteine content, and low molecular weight. There are, however, significant selectivity and potency differences. We compared the effect of three proteins on platelet function: albolabrin, a 7.5 kDa disintegrin, eristostatin, a 5.4 kDa disintegrin in which part of the disintegrin domain is deleted, and decorsin, a 4.5 kDa non-disintegrin derived from the leech Macrobdella decora, which has very little sequence similarity with either disintegrin. Decorsin was about two times less potent than albolabrin and six times less potent than eristostatin in inhibiting ADP- induced human platelet aggregation. It had a different pattern of interaction with glycoprotein IIb/IIIa as compared to the two disintegrins. Decorsin bound with a low affinity to resting platelets (409 nM) and to ADP-activated platelets (270 nM), and with high affinity to thrombin- activated platelets (74 nM). At concentrations up to 685 nM, it did not cause expression of a ligand-induced binding site epitope on the (β3 subunit of the GPIIb/IIIa complex. It did not significantly inhibit isolated GPIIb/IIIa binding to immobilized von Willebrand Factor. At low doses (1.5-3.0 μg/mouse), decorsin protected mice against death from pulmonary thromboembolism, showing an effect similar to eristostatin. This suggested that decorsin is a much more potent inhibitor of platelet aggregation in vivo than in vitro, and it may have potential as an antiplatelet drug.

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Half-Life of Platelet Factor 4 (PF-4) in Plasma and Platelets from Macaca Mulatta

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649203 ◽

1977 ◽

Vol 37 (01) ◽

pp. 073-080 ◽

Cited By ~ 5

Author(s):

Knut Gjesdal ◽

Duncan S. Pepper

Keyword(s):

Macaca Mulatta ◽

Half Life ◽

Human Platelet ◽

Gel Filtration ◽

Platelet Factor 4 ◽

Active Protein ◽

Platelet Factor ◽

Membrane Bound

SummaryHuman platelet factor 4 (PF-4) showed a reaction of complete identity with PF-4 from Macaca mulatta when tested against rabbit anti-human-PF-4. Such immunoglobulin was used for quantitative precipitation of in vivo labelled PF-4 in monkey serum. The results suggest that the active protein had an intra-platelet half-life of about 21 hours. In vitro 125I-labelled human PF-4 was injected intravenously into two monkeys and isolated by immuno-precipita-tion from platelet-poor plasma and from platelets disrupted after gel-filtration. Plasma PF-4 was found to have a half-life of 7 to 11 hours. Some of the labelled PF-4 was associated with platelets and this fraction had a rapid initial disappearance rate and a subsequent half-life close to that of plasma PF-4. The results are compatible with the hypothesis that granular PF-4 belongs to a separate compartment, whereas membrane-bound PF-4 and plasma PF-4 may interchange.

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Co-purification of bone resorbing activity and adenylate cyclase stimulating activity from human tumours associated with the humoral hypercalcaemia of malignancy

Acta Endocrinologica ◽

10.1530/acta.0.1140018 ◽

1987 ◽

Vol 114 (1) ◽

pp. 18-26 ◽

Cited By ~ 5

Author(s):

Chohei Shigeno ◽

Itsuo Yamamoto ◽

Shegiharu Dokoh ◽

Megumu Hino ◽

Jun Aoki ◽

...

Keyword(s):

Bone Resorption ◽

High Performance ◽

Basic Protein ◽

Gel Filtration ◽

Exchange Chromatography ◽

Protein Factor ◽

Human Tumours ◽

Acid Stable ◽

Bone Resorbing Activity

Abstract. We have partially purified a tumour factor capable of stimulating both bone resorption in vitro and cAMP accumulation in osteoblastic ROS 17/2 cells from three human tumours associated with humoral hypercalcaemia of malignancy. Purification of tumour factor by sequential acid urea extraction, gel filtration and cation-exchange chromatography, reverse-phase high performance liquid chromatography followed by analytical isoelectric focussing provided a basic protein (pI > 9.3) with a molecular weight of approximately 13 000 as a major component of the final preparation which retained both the two bioactivities. Bone resorbing activity and cAMP-increasing activity in purified factor correlated with each other. cAMP-increasing activity of the factor was heat- and acid-stable, but sensitive to alkaline ambient pH. Treatment with trypsin destroyed cAMP-increasing activity of the factor. Synthetic parathyroid hormone (PTH) antagonist, human PTH-(3– 34) completely inhibited the cAMP-increasing activity of the factor. The results suggest that this protein factor, having its effects on both osteoclastic and osteoblastic functions, may be involved in development of enhanced bone resorption in some patients with humoral hypercalcaemia of malignancy.

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