Binding of FKBP23 to BiP in ER Shown by Gel Filtration Chromatography

2007 ◽  
Vol 62 (1-2) ◽  
pp. 133-137 ◽  
Author(s):  
Chen Chen ◽  
Hui Ma ◽  
Huaifeng Mi ◽  
Ying Wang
Keyword(s):  
Endoplasmic Reticulum ◽  
Molecular Chaperone ◽  
Binding Sites ◽  
Gel Filtration ◽  
Gel Filtration Chromatography ◽  
Terminal Domain ◽  
Ppiase Domain ◽  
Main Protein ◽  
Chaperone Hsp70

FKBP23 was found in mouse endoplasmic reticulum (ER) in 1998. It consists of an N-terminal peptidyl-prolyl cis/trans isomerase (PPIase) domain and a C-terminal domain with Ca2+ binding sites. Previously, we reported that FKBP23 specifically binds to BiP, the main protein of the molecular chaperone Hsp70 in ER lumen, and the binding is interrelated with the Ca2+ concentration. In this work we have found the existence of the complex FKBP23/BiP by separation of an ER extract using gel filtration chromatography (GFC), and that the existence of this complex is Ca2+-interrelated. This result further verified the Ca2+- interrelated binding of these two proteins in vivo.

De-Novo Cloning of FKBP23 cDNA from Pig ER Using Nested PCR

2009 ◽  
Vol 64 (3-4) ◽  
pp. 297-302
Author(s):  
Ruifang Han ◽  
Ying Wang ◽  
Chen Chen ◽  
Zhuo Zhao ◽  
Huaifeng Mi
Keyword(s):  
Molecular Chaperone ◽  
Nested Pcr ◽  
De Novo ◽  
Predicted Amino Acid Sequence ◽  
High Identity ◽  
Ppiase Domain ◽  
Ef Hand ◽  
Main Protein ◽  
Fk506 Binding Proteins ◽  
Chaperone Hsp70

FK506 binding proteins (FKBPs) in cells are known as immunophilins. We have identifi ed and characterized a cDNA encoding an endoplasmic reticulum (ER) immunophilin, FKBP23, from pig liver by nested PCR. The predicted amino acid sequence of pig FKBP23 shows high identity to those of human FKBP23 and mouse FKBP23. It possesses a conserved FKBP-type peptidylprolyl cis-trans isomerase (PPIase) domain and EF-hand domain. We constructed a plasmid to express pFKBP23. Furthermore, we proved that the recombinant pFKBP23 can specifi cally bind to natural BiP, the main protein of the molecular chaperone Hsp70 in ER lumen; the binding is interrelated with the Ca2+ concentration just as the FKBP23 from mice.

scholarly journals Structural and Functional Conversion of Molecular Chaperone ClpB from the Gram-Positive Halophilic Lactic Acid Bacterium Tetragenococcus halophilus Mediated by ATP and Stress

2006 ◽  
Vol 188 (23) ◽  
pp. 8070-8078 ◽  
Author(s):  
Shinya Sugimoto ◽  
Hiroyuki Yoshida ◽  
Yoshimitsu Mizunoe ◽  
Keigo Tsuruno ◽  
Jiro Nakayama ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacterium ◽  
Molecular Chaperone ◽  
Thermal Inactivation ◽  
Gel Filtration ◽  
Ring Structure ◽  
Stress Conditions ◽  
Gel Filtration Chromatography ◽  
Gram Positive ◽  
Tetragenococcus Halophilus

ABSTRACT In this study, we report the purification, initial structural characterization, and functional analysis of the molecular chaperone ClpB from the gram-positive, halophilic lactic acid bacterium Tetragenococcus halophilus. A recombinant T. halophilus ClpB (ClpB Tha ) was overexpressed in Escherichia coli and purified by affinity chromatography, hydroxyapatite chromatography, and gel filtration chromatography. As demonstrated by gel filtration chromatography, chemical cross-linking with glutaraldehyde, and electron microscopy, ClpB Tha forms a homohexameric single-ring structure in the presence of ATP under nonstress conditions. However, under stress conditions, such as high-temperature (>45°C) and high-salt concentrations (>1 M KCl), it dissociated into dimers and monomers, regardless of the presence of ATP. The hexameric ClpB Tha reactivated heat-aggregated proteins dependent upon the DnaK system from T. halophilus (KJE Tha ) and ATP. Interestingly, the mixture of dimer and monomer ClpB Tha , which was formed under stress conditions, protected substrate proteins from thermal inactivation and aggregation in a manner similar to those of general molecular chaperones. From these results, we hypothesize that ClpB Tha forms dimers and monomers to function as a holding chaperone under stress conditions, whereas it forms a hexamer ring to function as a disaggregating chaperone in cooperation with KJE Tha and ATP under poststress conditions.

scholarly journals The binding of spermine to the ribosomes and ribosomal ribonucleic acid from Bacillus stearothermophilus

1969 ◽  
Vol 113 (1) ◽  
pp. 117-121 ◽  
Author(s):  
L. Stevens
Keyword(s):  
Ionic Strength ◽  
Ribosomal Rna ◽  
Ribonucleic Acid ◽  
Binding Sites ◽  
Bacillus Stearothermophilus ◽  
Gel Filtration ◽  
Ribosomal Ribonucleic Acid ◽  
Number Of Binding Sites ◽  
Filtration Technique

1. The total intracellular concentrations of Na+, K+, Mg2+, spermine, spermidine and RNA were measured in Bacillus stearothermophilus. 2. The binding of spermine to ribosomes and to ribosomal RNA from B. stearothermophilus was studied under various conditions by using a gel-filtration technique. 3. The affinity of spermine for ribosomes and for ribosomal RNA decreased with increasing ionic strength of the medium in which they were suspended. 4. The extent of spermine binding did not change appreciably in the temperature range 4–60°. 5. Optimum binding occurred at about pH7·0. 6. The number of binding sites for spermine on either ribosomes or ribosomal RNA was 0·10–0·13/RNA phosphate group. 7. A high proportion of the intracellular spermine is likely to be bound to the ribosomes in vivo; spermine competes with Mg2+ on equal terms for sites on the ribosomes.

scholarly journals Activities of virE1 and the VirE1 Secretion Chaperone in Export of the Multifunctional VirE2 Effector via an Agrobacterium Type IV Secretion Pathway

2001 ◽  
Vol 183 (13) ◽  
pp. 3855-3865 ◽  
Author(s):  
Zhenming Zhao ◽  
Evgeniy Sagulenko ◽  
Zhiyong Ding ◽  
Peter J. Christie
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Higher Order ◽  
Effector Proteins ◽  
Type Iv Secretion ◽  
Gel Filtration Chromatography ◽  
Type Iv ◽  
Self Association ◽  
Secretion Chaperone

ABSTRACT Agrobacterium tumefaciens uses a type IV secretion system to deliver oncogenic nucleoprotein particles and effector proteins, such as the multifunctional VirE2 protein, to plant cells. In this study, we examined the function of virE1 and its product, the VirE1 secretion chaperone, in mediating VirE2 export. A nonpolar virE1 null mutant accumulated low levels of VirE2, and trans expression of virE1 in this mutant only partially restored VirE2 abundance. Deletion of virE1did not affect transcription but decreased translation ofvirE2, as shown by analysis of lacZtranscriptional and translational fusions. VirE2 was stable for a prolonged period, more than 6 h, when it was expressed incis with virE1, and it exhibited half-lives of about 2 h when it was expressed in trans withvirE1 and less than 10 min when it was expressed in the absence of virE1, as shown by pulse-chase experiments. VirE1 stabilized VirE2 via an interaction with a domain near the N terminus of VirE2, as shown by analyses of VirE2 truncation and insertion mutants synthesized in A. tumefaciens. VirE1 self-association was demonstrated by using bacteriophage λ cI repressor fusion and pull-down assays, and evidence of VirE1 homomultimerization in vivo was obtained by native polyacrylamide gel electrophoresis and gel filtration chromatography. A putative VirE1-VirE2 complex with a molecular mass of about 70 to 80 kDa was detected by gel filtration chromatography of extracts from wild-type cells, whereas higher-order VirE2 complexes or aggregates were detected in extracts from a virE1 mutant. Taken together, our findings show that virE1 contributes in several ways to VirE2 export:(i) virE1 regulates efficientvirE2 translation in the context of expression from the native PvirE promoter; (ii) the VirE1 secretion chaperone stabilizes VirE2, most probably via an interaction with an N-terminal domain; and (iii) VirE1 forms a VirE1-VirE2 complex with a predicted 2:1 stoichiometry that inhibits assembly of higher-order VirE2 complexes or aggregates.

Target molecules of molecular chaperone (HSP70 family) in injured gastric mucosa in vivo

Life Sciences ◽  
2009 ◽  
Vol 84 (19-20) ◽  
pp. 664-667 ◽  
Author(s):  
Michiro Otaka ◽  
Masaru Odashima ◽  
Yuko Izumi ◽  
Akihito Nagahara ◽  
Taro Osada ◽  
...  
Keyword(s):  
Gastric Mucosa ◽  
Molecular Chaperone ◽  
Hsp70 Family ◽  
Target Molecules ◽  
Chaperone Hsp70

scholarly journals Molecular Chaperones in the Yeast Endoplasmic Reticulum Maintain the Solubility of Proteins for Retrotranslocation and Degradation

2001 ◽  
Vol 153 (5) ◽  
pp. 1061-1070 ◽  
Author(s):  
Shuh-ichi Nishikawa ◽  
Sheara W. Fewell ◽  
Yoshihito Kato ◽  
Jeffrey L. Brodsky ◽  
Toshiya Endo
Keyword(s):  
Endoplasmic Reticulum ◽  
Molecular Chaperone ◽  
Molecular Chaperones ◽  
Elevated Temperatures ◽  
Carboxypeptidase Y ◽  
Mutant Form ◽  
Genes Encoding

Endoplasmic reticulum (ER)-associated degradation (ERAD) is the process by which aberrant proteins in the ER lumen are exported back to the cytosol and degraded by the proteasome. Although ER molecular chaperones are required for ERAD, their specific role(s) in this process have been ill defined. To understand how one group of interacting lumenal chaperones facilitates ERAD, the fates of pro–α-factor and a mutant form of carboxypeptidase Y were examined both in vivo and in vitro. We found that these ERAD substrates are stabilized and aggregate in the ER at elevated temperatures when BiP, the lumenal Hsp70 molecular chaperone, is mutated, or when the genes encoding the J domain–containing proteins Jem1p and Scj1p are deleted. In contrast, deletion of JEM1 and SCJ1 had little effect on the ERAD of a membrane protein. These results suggest that one role of the BiP, Jem1p, and Scj1p chaperones is to maintain lumenal ERAD substrates in a retrotranslocation-competent state.

scholarly journals A high-affinity oestrogen-binding protein in cockerel liver cytosol

1979 ◽  
Vol 180 (2) ◽  
pp. 347-353 ◽  
Author(s):  
C B Lazier ◽  
A J Haggarty
Keyword(s):  
Binding Sites ◽  
Proteinase Inhibitors ◽  
Specific Binding ◽  
Binding Protein ◽  
Gel Filtration ◽  
Binding Activity ◽  
Single Class ◽  
High Affinity ◽  
Significant Concentration

In contrast with several earlier reports, cytosol from cockerel liver contains a significant concentration of a protein that binds oestradiol with high affinity. To demonstrate the activity, certain alterations in the conventional method of preparation of cytosol must be made. Homogenization in sucrose-containing buffer at pH 8.4 in the presence of proteinase inhibitors and rapid fractionation of the cytosol with (NH4)2SO4 enables demonstration of a single class of oestradiol-binding sites with a Kd of about 1 nM and specificity only for oestrogens. The concentration is about 300 sites per cell in liver from 2-week-old cockerels. Oestradiol treatment in vivo decreases the number of exchangeable cytosol oestradiol-binding sites by about 80% for 1–4h, after which time it is gradually restored. Gel filtration of the cytosol preparation in the presence of high salt concentrations reveals that most of the oestradiol-binding activity is in high-molecular-weight aggregates, but a mild trypsin treatment generates a specific binding protein with an approximate mol.wt. of 40 000. This protein may be an oestrogen receptor.

scholarly journals Stimulation of 3′→5′ Exonuclease and 3′-Phosphodiesterase Activities of Yeast Apn2 by Proliferating Cell Nuclear Antigen

2002 ◽  
Vol 22 (18) ◽  
pp. 6480-6486 ◽  
Author(s):  
Ildiko Unk ◽  
Lajos Haracska ◽  
Xavier V. Gomes ◽  
Peter M. J. Burgers ◽  
Louise Prakash ◽  
...  
Keyword(s):  
Proliferating Cell Nuclear Antigen ◽  
Gel Filtration ◽  
Nuclear Antigen ◽  
Binding Motif ◽  
Terminal Domain ◽  
Abasic Sites ◽  
Factor C ◽  
Proliferating Cell

ABSTRACT The Apn2 protein of Saccharomyces cerevisiae contains 3′→5′ exonuclease and 3′-phosphodiesterase activities, and these activities function in the repair of DNA strand breaks that have 3′-damaged termini and which are formed in DNA by the action of oxygen-free radicals. Apn2 also has an AP endonuclease activity and functions in the removal of abasic sites from DNA. Here, we provide evidence for the physical and functional interaction of Apn2 with proliferating cell nuclear antigen (PCNA). As indicated by gel filtration and two-hybrid studies, Apn2 interacts with PCNA both in vitro and in vivo and mutations in the consensus PCNA-binding motif of Apn2 abolish this interaction. Importantly, PCNA stimulates the 3′→5′ exonuclease and 3′-phosphodiesterase activities of Apn2. We have examined the involvement of the interdomain connector loop (IDCL) and of the carboxy-terminal domain of PCNA in Apn2 binding and found that Apn2 binds PCNA via distinct domains dependent upon whether the binding is in the absence or presence of DNA. In the absence of DNA, Apn2 binds PCNA through its IDCL domain, whereas in the presence of DNA, when PCNA has been loaded onto the template-primer junction by replication factor C, the C-terminal domain of PCNA mediates the binding.

Differential secretion of pro-opiomelanocortin peptides by the pars distalis and pars intermedia of beagle dogs

1988 ◽  
Vol 117 (1) ◽  
pp. 91-96 ◽  
Author(s):  
R. J. Kemppainen ◽  
J. L. Sartin
Keyword(s):  
Gel Filtration ◽  
Pars Intermedia ◽  
Dopamine Antagonist ◽  
Test Substance ◽  
Pars Distalis ◽  
Beagle Dogs ◽  
Gel Filtration Chromatography ◽  
Mongrel Dog ◽  
Peptide Secretion

ABSTRACT Beagle dogs were given saline, insulin or the dopamine antagonist, haloperidol, to examine peripheral concentrations of immunoreactive (ir)-pro-opiomelanocortin (POMC) peptides resulting from pars distalis or pars intermedia stimulation. Six beagles were given each test substance on separate occasions with and without dexamethasone pretreatment. Plasma was assayed directly for glucose, ir-ACTH, ir-α-MSH, cortisol and, after Sephadex G-50 Fine gel filtration chromatography, for ir-β-lipotrophin (ir-β-LPH) and ir-β-endorphin (ir-β-END) content. Injection of 0·5 units insulin/kg lowered (P < 0·001) plasma glucose from 4·9 ± 0·3 mmol/l (mean ± s.d., saline controls) to 2·3 ± 0·5 mmol/l, coincident with increasing ir-ACTH (9·5 ± 3·1 to 106 ± 54 pmol/l), cortisol (52 ± 27 to 221± 27 nmol/l), ir-β-LPH (not detectable to 34 ± 18 pmol/l) and ir-β-END (not detectable to 52 ± 22 pmol/l). Plasma ir-α-MSH concentrations were not affected by insulin. Pretreatment with dexamethasone abolished the ir-ACTH, cortisol, ir-β-LPH and ir-β-END increases in response to 0·75 units insulin/kg. Haloperidol (1 mg/kg) increased (P < 0·01) plasma ir-ACTH (to 103 ± 63 pmol/l), cortisol (to 243 ± 11 nmol/l), ir-β-LPH (to 16 ± 6 pmol/l), ir-β-END (to 136 ± 73 pmol/l) and additionally raised ir-α-MSH (7 ± 8 pmol/l in saline controls to 131 ± 80 pmol/l after haloperidol). Pretreatment with dexamethasone significantly (P < 0·01 ) reduced the plasma ir-ACTH (96%), cortisol (93%), ir-β-LPH (100%) and ir-β-END (55%) response to haloperidol, but the ir-α-MSH increase (117 ± 81 pmol/l) was not affected. The pituitary distribution of ir-β-END-like peptides was determined in tissue obtained from one healthy mongrel dog. Following G-50 gel filtration chromatography of HCl extracts, 40% of the total immunoreactivity determined in an extract of pars distalis eluted near the position of human β-LPH while 60% eluted near human β-END(1–31). In contrast, over 95% of ir-β-END eluted near β-END(1–31) in the tissue extract prepared from the neurointermediate lobe. From the in-vivo data, it appears that insulin-induced hypoglycaemia selectively activates POMC peptide secretion by the pars distalis in dogs and that this effect is totally suppressible by dexamethasone. In contrast, haloperidol appears to activate secretion of POMC peptide from both the pars distalis and pars intermedia. The dog pars distalis secretes a mixture of ir-β-LPH and ir-β-END while the pars intermedia preferentially secretes ir-β-END. J. Endocr. (1988) 117, 91–96

scholarly journals Electrostatics cause the molecular chaperone BiP to preferentially bind oligomerized states of a client protein

2021 ◽  
Author(s):  
Judy L.M. Kotler ◽  
Wei-Shao Wei ◽  
Erin E Deans ◽  
Timothy O. Street
Keyword(s):  
Endoplasmic Reticulum ◽  
Molecular Chaperone ◽  
Binding Sites ◽  
Client Protein ◽  
High Affinity ◽  
Hsp70 Family ◽  
Differential Affinity ◽  
Peptide Region ◽  
Positively Charged ◽  
Micromolar Range

Hsp70-family chaperones bind short monomeric peptides with a weak characteristic affinity in the low micromolar range, but can also bind some aggregates, fibrils, and amyloids, with low nanomolar affinity. While this differential affinity enables Hsp70 to preferentially target potentially toxic aggregates, it is unknown how Hsp70s differentiate between monomeric and oligomeric states of a target protein. Here we examine the interaction of BiP (the Hsp70 paralog in the endoplasmic reticulum) with proIGF2, the pro-protein form of IGF2 that includes a long and mostly disordered E-peptide region that promotes proIGF2 oligomerization. We discover that electrostatic attraction enables the negatively charged BiP to bind positively charged E-peptide oligomers with low nanomolar affinity. We identify the specific BiP binding sites on proIGF2, and although some are positively charged, as monomers they bind BiP with characteristically low affinity in the micromolar range. We conclude that electrostatics enable BiP to preferentially recognize oligomeric states of proIGF2. Electrostatic targeting of Hsp70 to aggregates may be broadly applicable, as all the currently-documented cases in which Hsp70 binds aggregates with high-affinity involve clients that are expected to be positively charged.

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