scholarly journals Calcium-Dependent Assembly of Centrin-G-Protein Complex in Photoreceptor Cells

2002 ◽  
Vol 22 (7) ◽  
pp. 2194-2203 ◽  
Author(s):  
Alexander Pulvermüller ◽  
Andreas Gießl ◽  
Martin Heck ◽  
Ralf Wottrich ◽  
Angelika Schmitt ◽  
...  
Keyword(s):  
G Protein ◽  
Protein Complex ◽  
Photoreceptor Cells ◽  
Size Exclusion ◽  
Sensory Cells ◽  
Signaling Proteins ◽  
Specific Domain ◽  
Calcium Dependent ◽  
Exclusion Chromatography ◽  
Potential Link

ABSTRACT Photoexcitation of rhodopsin activates a heterotrimeric G-protein cascade leading to cyclic GMP hydrolysis in vertebrate photoreceptors. Light-induced exchanges of the visual G-protein transducin between the outer and inner segment of rod photoreceptors occur through the narrow connecting cilium. Here we demonstrate that transducin colocalizes with the Ca2+-binding protein centrin 1 in a specific domain of this cilium. Coimmunoprecipitation, centrifugation, centrin overlay, size exclusion chromatography, and kinetic light-scattering experiments indicate that Ca2+-activated centrin 1 binds with high affinity and specificity to transducin. The assembly of centrin-G-protein complex is mediated by the βγ-complex. The Ca2+-dependent assembly of a G protein with centrin is a novel aspect of the supply of signaling proteins in sensory cells and a potential link between molecular translocations and signal transduction in general.

scholarly journals Identifying cystogenic paracrine signaling molecules in cyst fluid of patients with polycystic kidney disease

2019 ◽  
Vol 316 (1) ◽  
pp. F204-F213 ◽  
Author(s):  
Annegien T. Kenter ◽  
Sari E. van Rossum-Fikkert ◽  
Mahdi Salih ◽  
Paul C. M. S. Verhagen ◽  
Geert J. L. H. van Leenders ◽  
...  
Keyword(s):  
Cyst Formation ◽  
Liver Cyst ◽  
Signaling Molecules ◽  
Cyst Fluid ◽  
Size Exclusion ◽  
Signaling Proteins ◽  
Paracrine Signaling ◽  
Polycystic Kidney ◽  
Renal Epithelial Cells ◽  
Exclusion Chromatography

In autosomal dominant polycystic kidney disease (ADPKD) paracrine signaling molecules in cyst fluid can induce proliferation and cystogenesis of neighboring renal epithelial cells. However, the identity of this cyst-inducing factor is still unknown. The aim of this study was to identify paracrine signaling proteins in cyst fluid using a 3D in vitro cystogenesis assay. We collected cyst fluid from 15 ADPKD patients who underwent kidney or liver resection (55 cysts from 13 nephrectomies, 5 cysts from 2 liver resections). For each sample, the ability to induce proliferation and cyst formation was tested using the cystogenesis assay (RPTEC/TERT1 cells in Matrigel with cyst fluid added for 14 days). Kidney cyst fluid induced proliferation and cyst growth of renal epithelial cells in a dose-dependent fashion. Liver cyst fluid also induced cystogenesis. Using size exclusion chromatography, 56 cyst fluid fractions were obtained of which only the fractions between 30 and 100 kDa showed cystogenic potential. Mass spectrometry analysis of samples that tested positive or negative in the assay identified 43 candidate cystogenic proteins. Gene ontology analysis showed an enrichment for proteins classified as enzymes, immunity proteins, receptors, and signaling proteins. A number of these proteins have previously been implicated in ADPKD, including secreted frizzled-related protein 4, S100A8, osteopontin, and cysteine rich with EGF-like domains 1. In conclusion, both kidney and liver cyst fluids contain paracrine signaling molecules that drive cyst formation. Using size exclusion chromatography and mass spectrometry, we procured a candidate list for future studies. Ultimately, cystogenic paracrine signaling molecules may be targeted to abrogate cystogenesis in ADPKD.

scholarly journals An insulin receptor peptide (1135-1156) stimulates guanosine 5′-[γ-thio]triphosphate binding to the 67 kDa G-protein associated with the insulin receptor

1993 ◽  
Vol 294 (1) ◽  
pp. 19-24 ◽  
Author(s):  
H Jo ◽  
W Radding ◽  
G M Anantharamaiah ◽  
J M McDonald
Keyword(s):  
Insulin Receptor ◽  
G Protein ◽  
Kinase Domain ◽  
Size Exclusion ◽  
Tyrosine Kinase Domain ◽  
Mutation Site ◽  
Binding Motifs ◽  
Insulin Resistant ◽  
Exclusion Chromatography ◽  
Gamma S

Peptides representing two putative G-protein-binding motifs (GPBP1 and GPBP2) derived from insulin-receptor sequences were tested for their ability to stimulate guanosine 5′-[gamma-thio]-triphosphate (GTP[S]; ‘GTP gamma S’) binding to a preparation containing the 41 and 67 kDa G-proteins that are associated with the insulin receptor [Jo, Cha, Davis and McDonald (1992) Endocrinology (Baltimore) 131, 2855-2861]. GPBP2 (residues 1135-1156) specifically stimulated GTP[S] binding, whereas GPBP1 (1319-1333) did not. Substitution of Arg-1152 with Gln in GPBP2 corresponding to a mutation site in insulin-resistant patients [Cocozza, Porcellini, Riccardi, Monticelli, Condorelli, Ferrera, Pianese, Miele, Capaldo, Beguinot and Varrone (1992) Diabetes 41, 521-526] attenuated the stimulatory potency of GPBP2. Size-exclusion chromatography and studies with purified 67 kDa G-protein revealed that GPBP2 stimulated GTP[S] binding only to the 67 kDa G-protein. These studies provide evidence for a potential regulatory site for G-protein interaction with the insulin receptor in the tyrosine kinase domain.

scholarly journals Crystal structure of IspF from Bacillus subtilis and absence of protein complex assembly amongst IspD/IspE/IspF enzymes in the MEP pathway

2018 ◽  
Vol 38 (1) ◽  
Author(s):  
Zhongchuan Liu ◽  
Yun Jin ◽  
Weifeng Liu ◽  
Yong Tao ◽  
Ganggang Wang
Keyword(s):  
Bacillus Subtilis ◽  
Protein Complex ◽  
Isoprenoid Biosynthesis ◽  
Mep Pathway ◽  
Size Exclusion ◽  
Complex Assembly ◽  
Exclusion Chromatography ◽  
Key Enzyme ◽  
Accessible Surface ◽  
Protein Complex Assembly

2-C-Methyl-d-erythritol 2,4-cyclodiphosphate synthase (IspF) is a key enzyme in the 2-C-Methyl-d-erythritol-4-phosphate (MEP) pathway of isoprenoid biosynthesis. This enzyme catalyzes the 4-diphosphocytidyl-2-C-methyl-d-erythritol 2-phosphate (CDPME2P) to 2-C-methyl-d-erythritol 2,4-cyclodiphosphate (MEcDP) with concomitant release of cytidine 5′-diphospate (CMP). Bacillus subtilis is a potential host cell for the production of isoprenoids, but few studies are performed on the key enzymes of MEP pathway in B. subtilis. In this work, the high-resolution crystal structures of IspF in native and complex with CMP from B. subtilis have been determined. Structural comparisons indicate that there is a looser packing of the subunits of IspF in B. subtilis, whereas the solvent accessible surface of its active pockets is smaller than that in Escherichia coli. Meanwhile, the protein–protein associations of 2-C-Methyl-d-erythritol-4-phosphatecytidyltransferase (IspD), CDPME kinase (IspE) and IspF from B. subtilis and E. coli, which catalyze three consecutive steps in the MEP pathway, are analyzed by native gel shift and size exclusion chromatography methods. The data here show that protein complex assembly is not detectable. These results will be useful for isoprenoid biosynthesis by metabolic engineering.

CHARACTERIZATION OF A NEW LCAT MUTATION CAUSING FAMILIAL LCAT DEFICIENCY (FLD) AND THE ROLE OF APOE AS A MODIFIER GENE OF THE FLD PHENOTYPE

2009 ◽  
Vol 32 (6S) ◽  
pp. 3
Author(s):  
A Baass ◽  
H Wassef ◽  
M Tremblay ◽  
L Bernier ◽  
R Dufour ◽  
...  
Keyword(s):  
Modifier Gene ◽  
Size Exclusion ◽  
Plasma Lipoproteins ◽  
Lipoprotein Profile ◽  
Exclusion Chromatography ◽  
Low Hdl ◽  
Lcat Deficiency ◽  
Apoe Genotypes

Introduction: LCAT (lecithin:cholesterol acyltransferase ) is an enzyme which plays an essential role in cholesterol esterification and reverse cholesterol transport. Familial LCAT deficiency (FLD) is a disease characterized by a defect in LCAT resulting in extremely low HDL-C, premature corneal opacities, anemia as well as proteinuria and renal failure. Method: We have identified two brothers presenting characteristics of familial LCAT deficiency. We sequenced the LCAT gene, measured the lipid profile as well as the LCAT activity in 15 members of this kindred. We also characterized the plasma lipoproteins by agarose gel electrophoresis and size exclusion chromatography and sequenced several candidate genes related to dysbetalipoproteinemia in this family. Results: We have identified the first French Canadian kindred with familial LCAT deficiency. Two brothers affected by FLD, were homozygous for a novel LCAT mutation. This c.102delG mutation occurs at the codon for His35 causing a frameshift that stops transcription at codon 61 abolishing LCAT enzymatic activity both in vivo and in vitro. It has a dramatic effect on the lipoprotein profile, with an important reduction of HDL-C in both heterozygotes (22%) and homozygotes (88%) and a significant decrease in LDL-C in heterozygotes (35%) as well as homozygotes (58%). Furthermore, the lipoprotein profile differed markedly between the two affected brothers who had different APOE genotypes. We propose that APOE could be an important modifier gene explaining heterogeneity in lipoprotein profiles observed among FLD patients. Our results suggest that a LCAT-/- genotype associated with an APOE ?2 allele could be a novel mechanism leading to dysbetalipoproteinemia.

Structure of a shear-thickening polysaccharide extracted from the New Zealand black tree fern, Cyathea medullaris

2020 ◽  
Author(s):  
M Wee ◽  
M Mastrangelo ◽  
Susan Carnachan ◽  
Ian Sims ◽  
K Goh
Keyword(s):  
New Zealand ◽  
Uronic Acid ◽  
Average Molecular Weight ◽  
Shear Thickening ◽  
Water Soluble ◽  
Size Exclusion ◽  
Resonance Spectroscopy ◽  
Tree Fern ◽  
13C Nuclear Magnetic Resonance ◽  
Exclusion Chromatography

A shear-thickening water-soluble polysaccharide was purified from mucilage extracted from the fronds of the New Zealand black tree fern (Cyathea medullaris or 'mamaku' in Māori) and its structure characterised. Constituent sugar analysis by three complementary methods, combined with linkage analysis (of carboxyl reduced samples) and 1H and 13C nuclear magnetic resonance spectroscopy (NMR) revealed a glucuronomannan comprising a backbone of 4-linked methylesterified glucopyranosyl uronic acid and 2-linked mannopyranosyl residues, branched at O-3 of 45% and at both O-3 and O-4 of 53% of the mannopyranosyl residues with side chains likely comprising terminal xylopyranosyl, terminal galactopyranosyl, non-methylesterified terminal glucopyranosyl uronic acid and 3-linked glucopyranosyl uronic acid residues. The weight-average molecular weight of the purified polysaccharide was ~1.9×106Da as determined by size-exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALLS). The distinctive rheological properties of this polysaccharide are discussed in relation to its structure. © 2014 Elsevier B.V.

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