Interaction of the proteasome S5a/Rpn10 multiubiquitin-binding protein and the 8 kDa calcium-binding protein of Schistosoma mansoni

Parasitology ◽  
2003 ◽  
Vol 127 (4) ◽  
pp. 337-347 ◽  
Author(s):  
D. RAM ◽  
E. ZIV ◽  
F. LANTNER ◽  
I. SCHECHTER
Keyword(s):  
Calcium Binding ◽  
Binding Protein ◽  
Calcium Binding Protein ◽  
26S Proteasome ◽  
Dependent Manner ◽  
Terminal Portion ◽  
Binding Interaction ◽  
Western Blots ◽  
Free System ◽  
Amino Terminal

A distinct 8 kDa calcium-binding protein (CaBP) is preferentially expressed at the cercarial stage during the life-cycle of the schistosome. Available data indicate that this CaBP may be associated with tissue/organ remodelling (involving protein degradation and synthesis of new proteins) during transformation of the cercariae from free-living form in water to parasitic life in the vertebrate host. Many CaBP molecules (e.g. calmodulin) show Ca++-dependent interaction with target proteins and thus modulate their activity. Accordingly, the parasite 8 kDa CaBP was used as a probe to clone and identify putative target protein(s) directly by binding interaction. Screening of schistosome λgt11 expression library with radio-iodinated CaBP yielded several overlapping clones showing Ca++-dependent binding of the CaBP. Sequence analyses revealed that these clones encode the S5a/Rpn10 multiubiquitin-binding protein which is a component of the regulatory 19S subunit of the 26S proteasome. The schistosome molecule, designated SmS5a, is 420 amino acids long. The nearly full length molecule (Gln3–Ser420) as well as the amino terminal (N-S5a, Gln3–Gly200) and carboxyl-terminal (C-S5a, Asp225–Ser420) portions were synthesized in bacteria, purified, and antibodies to the parasite SmS5a were prepared. Interaction between SmS5a and the 8 kDa CaBP in a Ca++-dependent manner was found under various experimental conditions: CaBP-Sepharose bound soluble SmS5a, immobilized SmS5a bound soluble CaBP, and complex formation was found when both molecules were in solution. Furthermore, it was shown that the C-terminal portion of SmS5a, but not the N-terminal portion of the molecule, reacted with the CaBP. SmS5a synthesized in a cell-free system and Western blots revealed 2 species, conceivably corresponding to the naked molecule (~50 kDa) and the molecule subjected to post-translational modification (~70 kDa). The present studies suggest that proteasome activity may be modulated by calcium, and this modulation is mediated via CaBP molecule(s).

scholarly journals Binding of pEL98 protein, an S100-related calcium-binding protein, to nonmuscle tropomyosin

1994 ◽  
Vol 124 (5) ◽  
pp. 757-768 ◽  
Author(s):  
K Takenaga ◽  
Y Nakamura ◽  
S Sakiyama ◽  
Y Hasegawa ◽  
K Sato ◽  
...  
Keyword(s):  
Calcium Binding ◽  
Binding Protein ◽  
S100 Protein ◽  
Calcium Binding Protein ◽  
Immunofluorescent Staining ◽  
Dependent Manner ◽  
3T3 Cells ◽  
Cell Extracts ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

The cDNA coding for mouse fibroblast tropomyosin isoform 2 (TM2) was placed into a bacterial expression vector to produce a fusion protein containing glutathione-S-transferase (GST) and TM2 (GST/TM2). Glutathione-Sepharose beads bearing GST/TM2 were incubated with [35S]methionine-labeled NIH 3T3 cell extracts and the materials bound to the fusion proteins were analyzed to identify proteins that interact with TM2. A protein of 10 kD was found to bind to GST/TM2, but not to GST. The binding of the 10-kD protein to GST/TM2 was dependent on the presence of Ca2+ and inhibited by molar excess of free TM2 in a competition assay. The 10-kD protein-binding site was mapped to the region spanning residues 39-107 on TM2 by using several COOH-terminal and NH2-terminal truncation mutants of TM2. The 10-kD protein was isolated from an extract of NIH 3T3 cells transformed by v-Ha-ras by affinity chromatography on a GST/TM2 truncation mutant followed by SDS-PAGE and electroelution. Partial amino acid sequence analysis of the purified 10-kD protein, two-dimensional polyacrylamide gel analysis and a binding experiment revealed that the 10-kD protein was identical to a calcium-binding protein derived from mRNA named pEL98 or 18A2 that is homologous to S100 protein. Immunoblot analysis of the distribution of the 10-kD protein in Triton-soluble and -insoluble fractions of NIH 3T3 cells revealed that some of the 10-kD protein was associated with the Triton-insoluble cytoskeletal residue in a Ca(2+)-dependent manner. Furthermore, immunofluorescent staining of NIH 3T3 cells showed that some of the 10-kD protein colocalized with nonmuscle TMs in microfilament bundles. These results suggest that some of the pEL98 protein interacts with microfilament-associated nonmuscle TMs in NIH 3T3 cells.

A novel p53 target gene, S100A9, induces p53-dependent cellular apoptosis and mediates the p53 apoptosis pathway

2009 ◽  
Vol 422 (2) ◽  
pp. 363-372 ◽  
Author(s):  
Chunsun Li ◽  
Hongyan Chen ◽  
Fang Ding ◽  
Yu Zhang ◽  
Aiping Luo ◽  
...  
Keyword(s):  
Calcium Binding ◽  
Binding Protein ◽  
Calcium Binding Protein ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Apoptosis Pathway ◽  
Cellular Apoptosis ◽  
Reporter Assays ◽  
P53 Activation ◽  
Acute And Chronic Inflammation

S100A9 (S100 calcium-binding protein A9) is a calcium-binding protein of the S100 family, and its differential expression has been associated with acute and chronic inflammation and several human cancers. Our previous work showed that S100A9 was severely down-regulated in human ESCC (oesophageal squamous cell carcinoma). To further investigate the transcriptional regulation of S100A9, we analysed the S100A9 promoter region and found several putative p53BS (p53-binding sites). Luciferase reporter assays showed that constructs carrying the p53BS exhibited enhanced luciferase activity in response to wild-type p53 activation. Further study demonstrated that S100A9 mRNA and protein expression could be positively regulated in a p53-dependent manner and p53 could bind to p53BS on the S100A9 promoter. Overexpression of S100A9 could induce cellular apoptosis, and this was partly p53-dependent. Knockdown of S100A9 impaired the apoptosis induced by p53. Thus we conclude that a gene down-regulated in ESCC, S100A9, is a novel p53 transcriptional target, induces cellular apoptosis in a partly p53-dependent manner and mediates the p53 apoptosis pathway.

scholarly journals Tat-Binding Protein-1 (TBP-1), an ATPase of 19S Regulatory Particles of the 26S Proteasome, Enhances Androgen Receptor Function in Cooperation with TBP-1-Interacting Protein/Hop2

Endocrinology ◽  
2009 ◽  
Vol 150 (7) ◽  
pp. 3283-3290 ◽  
Author(s):  
Tetsurou Satoh ◽  
Takahiro Ishizuka ◽  
Takuya Tomaru ◽  
Satoshi Yoshino ◽  
Yasuyo Nakajima ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Transcriptional Activation ◽  
Leucine Zipper ◽  
Binding Protein ◽  
26S Proteasome ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Lncap Cells ◽  
Interacting Protein ◽  
Amino Terminal

The 26S proteasome, which degrades ubiquitinated proteins, appears to contribute to the cyclical loading of androgen receptor (AR) to androgen response elements of target gene promoters; however, the mechanism whereby the 26S proteasome modulates AR recruitment remains unknown. Using yeast two-hybrid screening, we previously identified Tat-binding protein-1 (TBP-1), an adenosine triphosphatase of 19S regulatory particles of the 26S proteasome, as a transcriptional coactivator of thyroid hormone receptor. Independently, TBP-1-interacting protein (TBPIP) was also identified as a coactivator of several nuclear receptors, including AR. Here, we investigated whether TBP-1 could interact with and modulate transcriptional activation by AR cooperatively with TBPIP. TBP-1 mRNA was ubiquitously expressed in human tissues, including the testis and prostate, as well as in LNCaP cells. TBP-1 directly bound TBPIP through the amino-terminal domain possessing the leucine zipper structure. AR is physically associated with TBP-1 and TBPIP in vitro and in LNCaP cells. TBP-1 similarly and additively augmented AR-mediated transcription upon coexpression with TBPIP, and the ATPase domain, as well as leucine zipper structure in TBP-1, was essential for transcriptional enhancement. Overexpression of TBP-1 did not alter AR protein and mRNA levels. In the chromatin immunoprecipitation assay, TBP-1 was transiently recruited to the proximal androgen response element of the prostate-specific antigen gene promoter in a ligand-dependent manner in LNCaP cells. These findings suggest that a component of 19S regulatory particles directly binds AR and might participate in AR-mediated transcriptional activation in cooperation with TBPIP.

scholarly journals Messenger ribonucleic acids from pig intestinal mucosa direct synthesis of calcium-binding protein in a cell-free translation system

1980 ◽  
Vol 185 (3) ◽  
pp. 601-607 ◽  
Author(s):  
H Mellersh ◽  
S Tomlinson ◽  
A Pollock
Keyword(s):  
Calcium Binding ◽  
Binding Protein ◽  
Direct Synthesis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Duodenal Mucosa ◽  
Exchange Chromatography ◽  
Calcium Binding Protein ◽  
Translation System ◽  
Free System

mRNA from pig duodenal mucosa directs synthesis, in a wheat-germ cell-free system, of two products precipitated by antiserum to pure calcium-binding protein. One of these products has a higher molecular weight than authentic calcium-binding protein, but, like the authentic protein, is heat-stable. The other protein co-migrates with pure calcium-binding protein on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, is stable on heating to 70 degrees C for 15 min and alters its elution position on ion-exchange chromatography depending on whether Ca2+ is present in or absent from the elution buffer. The synthesized protein has these properties in common with authentic calcium-binding protein.

scholarly journals Calcium-binding protein S100A6 interaction with VEGF receptors integrates signaling and trafficking pathways

2021 ◽  
Author(s):  
Sreenivasan Ponnambalam ◽  
Leyuan Bao ◽  
Gareth W Fearnley ◽  
Chi-Chuan Lin ◽  
Adam F Odell ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Calcium Binding ◽  
Mapk Pathway ◽  
Binding Protein ◽  
Calcium Binding Protein ◽  
Mitogen Activated Protein Kinase ◽  
Tyrosine Kinase Domain ◽  
Dependent Manner ◽  
Endothelial Cell Function

The mammalian endothelium which lines all blood vessels responds to soluble factors which control vascular development and sprouting. Endothelial cells bind to vascular endothelial growth factor A via two different receptor tyrosine kinases (VEGFR1, VEGFR2) which regulate such cellular responses. The integration of VEGFR signal transduction and membrane trafficking is not well understood. Here, we used a yeast-based membrane protein screen to identify VEGFR-interacting factor(s) which modulate endothelial cell function. By screening a human endothelial cDNA library, we identified a calcium-binding protein, S100A6, which can interact with either VEGFR. We found that S100A6 binds in a calcium-dependent manner to either VEGFR1 or VEGFR2. S100A6 binding was mapped to the VEGFR2 tyrosine kinase domain. Depletion of S100A6 impacts on VEGF-A-regulated signaling through the canonical mitogen-activated protein kinase (MAPK) pathway. Furthermore, S100A6 depletion caused contrasting effects on biosynthetic VEGFR delivery to the plasma membrane. Co-distribution of S100A6 and VEGFRs on tubular profiles suggest the presence of transport carriers that facilitate VEGFR trafficking. We propose a mechanism whereby S100A6 acts as a calcium regulated switch which facilitates biosynthetic VEGFR trafficking from the TGN-to-plasma membrane. VEGFR-S100A6 interactions thus enable integration of signaling and trafficking pathways in controlling the endothelial response to VEGF-A.

scholarly journals Stimulation of intestinal calcium-binding-protein mRNA synthesis in the nucleus of vitamin D-deficient chicks by 1,25-dihydroxycholecalciferol

1978 ◽  
Vol 175 (3) ◽  
pp. 1089-1094 ◽  
Author(s):  
R. Spencer ◽  
M. Charman ◽  
D. E. M. Lawson
Keyword(s):  
Vitamin D ◽  
Calcium Binding ◽  
Binding Protein ◽  
Calcium Binding Protein ◽  
Free System ◽  
Rna Molecules ◽  
Pulse Dose ◽  
A Cell ◽  
Active Calcium ◽  
Stimulation Of

Stimulation of intestinal calcium transport by the hormone 1,25-dihydroxycholecalciferol appears to involve RNA transcriptions and the synthesis of new proteins. Although one of these proteins has been identified as calcium-binding protein, no RNA molecules specifically induced by the hormone in the nucleus have been identified. Nuclear RNA from intestine of vitamin D-deficient chicks before and at various time intervals after treatment with the hormone or cholecalciferol was tested for its ability to code for calcium-binding protein in a cell-free system. Calcium-binding-protein mRNA could only just be detected in the intestinal nuclei 2h after dosing with these steroids which is the same time that it was first observed in the polyribosomes. Thus 1,25-dihydroxycholecalciferol induces the production of new calcium-binding protein by stimulating the formation and rapid release from the nucleus of new mRNA molecules for this protein. Polyribosomal translation of the mRNA continued only as long as it was being synthesized, and the maximum rate of synthesis following a pulse dose of 125ng of the hormone was the same as that observed after prolonged stimulation with cholecalciferol. The possibility that other 1,25-dihydroxycholecalciferol-dependent events may be occurring in the nucleus in the lag period between accumulation of the hormone in the intestine and the appearance of active calcium-binding-protein mRNA, and that these may ultimately control the synthesis of that mRNA, is discussed.

Sign in / Sign up

Export Citation Format

Share Document