scholarly journals Reactivity of free thiol groups in type-I inositol trisphosphate receptors

2005 ◽  
Vol 393 (2) ◽  
pp. 575-582 ◽  
Author(s):  
Suresh K. Joseph ◽  
Steven K. Nakao ◽  
Siam Sukumvanich
Keyword(s):  
Splice Variants ◽  
Cysteine Residue ◽  
Cysteine Residues ◽  
Type I ◽  
Thiol Groups ◽  
Release Channel ◽  
Tryptic Fragment ◽  
Reactive Compound ◽  
Thiol Redox ◽  
Trisphosphate Receptor

The IP3R (inositol 1,4,5-trisphosphate receptor) Ca2+-release channel is known to be sensitive to thiol redox state. The present study was undertaken to characterize the number and location of reactive thiol groups in the type-I IP3R. Using the fluorescent thiol-reactive compound monobromobimane we found that approx. 70% of the 60 cysteine residues in the type-I IP3R are maintained in the reduced state. The accessibility of these residues was assessed by covalently tagging the IP3R in membranes with a 5 kDa or 20 kDa MPEG [methoxypoly(ethylene glycol) maleimide]. MPEG reaction caused a shift in the mobility of IP3R on SDS/PAGE that was blocked by pretreatment of the membranes with dithiothreitol, N-ethylmaleimide, mersalyl or thimerosal, indicating that MPEG reactivity was specific to thiol groups on the IP3R. Trypsin cleavage of the type-I IP3R generates five defined domains. In cerebellum membranes, MPEG reacted over a 5 min interval with tryptic fragment I and fragment III, but not fragments II, IV or V. Fragment I appears as a doublet in cerebellum membranes, corresponding to the presence and absence of the SI splice site in this region (SI is a spliced domain corresponding to amino acids 318–332). Only the fragment I band corresponding to the SI(+) splice form shifted after reaction with MPEG. Expression of SI(+) and SI(−) spliced forms in COS cell microsomes confirmed this result. The MPEG-induced shift was not prevented when the cysteine residue present in the SI splice domain (C326A) or the remaining seven cysteine residues in fragment I were individually mutated. Of the combination mutations screened, only the mutation of C206/214/326A blocked MPEG reactivity in fragment I. We conclude that a set of highly reactive cysteine residues in fragment I are differentially accessible in the SI(+) and SI(−) splice variants of the type-I IP3R.

scholarly journals The Carboxy-Terminal Domain of Glycoprotein N of Human Cytomegalovirus Is Required for Virion Morphogenesis

2007 ◽  
Vol 81 (10) ◽  
pp. 5212-5224 ◽  
Author(s):  
Michael Mach ◽  
Karolina Osinski ◽  
Barbara Kropff ◽  
Ursula Schloetzer-Schrehardt ◽  
Magdalena Krzyzaniak ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Critical Role ◽  
Point Mutations ◽  
Cysteine Residue ◽  
Cysteine Residues ◽  
Type I ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal ◽  
Assembly Compartment

ABSTRACT Glycoproteins M and N (gM and gN, respectively) are among the few proteins that are conserved across the herpesvirus family. The function of the complex is largely unknown. Whereas deletion from most alphaherpesviruses has marginal effects on the replication of the respective viruses, both proteins are essential for replication of human cytomegalovirus (HCMV). We have constructed a series of mutants in gN to study the function of this protein. gN of HCMV is a type I glycoprotein containing a short carboxy-terminal domain of 14 amino acids, including two cysteine residues directly adjacent to the predicted transmembrane anchor at positions 125 and 126. Deletion of the entire carboxy-terminal domain as well as substitution with the corresponding region from alpha herpesviruses or mutations of both cysteine residues resulted in a replication-incompetent virus. Recombinant viruses containing point mutations of either cysteine residue could be generated. These viruses were profoundly defective for replication. Complex formation of the mutant gNs with gM and transport of the complex to the viral assembly compartment appeared unaltered compared to the wild type. However, in infected cells, large numbers of capsids accumulated in the cytoplasm that failed to acquire an envelope. Transiently expressed gN was shown to be modified by palmitic acid at both cysteine residues. In summary, our data suggest that the carboxy-terminal domain of gN plays a critical role in secondary envelopment of HCMV and that palmitoylation of gN appears to be essential for function in secondary envelopment of HCMV and virus replication.

scholarly journals Cysteine 96 of Ntcp is responsible for NO-mediated inhibition of taurocholate uptake

2013 ◽  
Vol 305 (7) ◽  
pp. G513-G519 ◽  
Author(s):  
Umadevi Ramasamy ◽  
M. Sawkat Anwer ◽  
Christopher M. Schonhoff
Keyword(s):  
Nitric Oxide ◽  
Plasma Membrane ◽  
Cysteine Residue ◽  
Cysteine Residues ◽  
Wild Type ◽  
Reactive Compound

The Na+taurocholate (TC) cotransporting polypeptide Ntcp/NTCP mediates TC uptake across the sinusoidal membrane of hepatocytes. Previously, we demonstrated that nitric oxide (NO) inhibits TC uptake through S-nitrosylation of a cysteine residue. Our current aim was to determine which of the eight cysteine residues of Ntcp is responsible for NO-mediated S-nitrosylation and inhibition of TC uptake. Thus, we tested the effect of NO on TC uptake in HuH-7 cells transiently transfected with cysteine-to-alanine mutant Ntcp constructs. Of the eight mutants tested, only C44A Ntcp displayed decreased total and plasma membrane (PM) levels that were also reflected in decreased TC uptake. C266A Ntcp showed a decrease in TC uptake that was not explained by a decrease in total expression or PM localization, indicating that C266 is required for optimal uptake. We speculated that NO would target C266 since a previous report had shown the thiol reactive compound [2-(trimethylammonium) ethyl] methanethiosulfonate bromide (MTSET) inhibits TC uptake by wild-type NTCP but not by C266A NTCP. We confirmed that MTSET targets C266 of Ntcp, but, surprisingly, we found that C266 was not responsible for NO-mediated inhibition of TC uptake. Instead, we found that C96 was targeted by NO since C96A Ntcp was insensitive to NO-mediated inhibition of TC uptake. We also found that wild-type but not C96A Ntcp is S-nitrosylated by NO, suggesting that C96 is important in regulating Ntcp function in response to elevated levels of NO.

scholarly journals Oxidation of critical cysteine residues of type I adenylyl cyclase by o-iodosobenzoate or nitric oxide reversibly inhibits stimulation by calcium and calmodulin.

1994 ◽  
Vol 269 (10) ◽  
pp. 7290-7296
Author(s):  
R.J. Duhe ◽  
M.D. Nielsen ◽  
A.H. Dittman ◽  
E.C. Villacres ◽  
E.J. Choi ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Adenylyl Cyclase ◽  
Cysteine Residues ◽  
Type I

scholarly journals Molecular and Pharmacological Characterization of the Interaction between Human Geranylgeranyltransferase Type I and Ras-Related Protein Rap1B

2021 ◽  
Vol 22 (5) ◽  
pp. 2501
Author(s):  
Sonja Hinz ◽  
Dominik Jung ◽  
Dorota Hauert ◽  
Hagen S. Bachmann
Keyword(s):  
Protein Function ◽  
Tumor Development ◽  
Cysteine Residue ◽  
Type I ◽  
Pharmacological Characterization ◽  
Anti Cancer ◽  
And Function ◽  
Caax Motif ◽  
Important Drug

Geranylgeranyltransferase type-I (GGTase-I) represents an important drug target since it contributes to the function of many proteins that are involved in tumor development and metastasis. This led to the development of GGTase-I inhibitors as anti-cancer drugs blocking the protein function and membrane association of e.g., Rap subfamilies that are involved in cell differentiation and cell growth. In the present study, we developed a new NanoBiT assay to monitor the interaction of human GGTase-I and its substrate Rap1B. Different Rap1B prenylation-deficient mutants (C181G, C181S, and ΔCQLL) were designed and investigated for their interaction with GGTase-I. While the Rap1B mutants C181G and C181S still exhibited interaction with human GGTase-I, mutant ΔCQLL, lacking the entire CAAX motif (defined by a cysteine residue, two aliphatic residues, and the C-terminal residue), showed reduced interaction. Moreover, a specific, peptidomimetic and competitive CAAX inhibitor was able to block the interaction of Rap1B with GGTase-I. Furthermore, activation of both Gαs-coupled human adenosine receptors, A2A (A2AAR) and A2B (A2BAR), increased the interaction between GGTase-I and Rap1B, probably representing a way to modulate prenylation and function of Rap1B. Thus, A2AAR and A2BAR antagonists might be promising candidates for therapeutic intervention for different types of cancer that overexpress Rap1B. Finally, the NanoBiT assay provides a tool to investigate the pharmacology of GGTase-I inhibitors.

scholarly journals Insulin Promotes the Association of Heat Shock Protein 90 with the Inositol 1,4,5-Trisphosphate Receptor to Dampen Its Ca2+ Release Activity

Endocrinology ◽  
2009 ◽  
Vol 150 (5) ◽  
pp. 2190-2196 ◽  
Author(s):  
Nathalie Nguyen ◽  
Nancy Francoeur ◽  
Valérie Chartrand ◽  
Klaus Klarskov ◽  
Gaétan Guillemette ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Protein Interactions ◽  
Mammalian Target Of Rapamycin ◽  
Src Kinase ◽  
Target Of Rapamycin ◽  
Resting Cells ◽  
Release Channel ◽  
Inositol 1,4,5 Trisphosphate ◽  
Trisphosphate Receptor

The inositol 1,4,5-trisphosphate receptor (IP3R) is a Ca2+ release channel that plays a pivotal role in regulating intracellular Ca2+ levels in resting cells. Three isoforms of IP3Rs have been identified, and they all possess a large regulatory domain that covers about 60% of the protein. This regulation is accomplished by interaction with small molecules, posttranslational modifications, and mostly protein-protein interactions. In our search for new binding partners of the IP3R, we found that 90-kDa heat-shock protein (Hsp90) binds to the IP3R. This interaction increased on stimulation of HEK293T6.11 cells with insulin but not with Gq protein-coupled receptor (GqPCR) agonists. Moreover, the Hsp90 inhibitor geldanamycin (GA) disrupted the interaction between Hsp90 and the IP3R. Pretreatment of HEK293T6.11 cells with GA greatly increased the intracellular Ca2+ release induced by a GqPCR agonist. Insulin alone did not induce any intracellular Ca2+ release. However, insulin diminished the intracellular Ca2+ release induced by a GqPCR agonist. Interestingly, GA abolished the inhibitory effect of insulin on GqPCR-induced intracellular Ca2+ release. Furthermore, in our search for a mechanistic explanation to this phenomenon, we found that inhibition of kinases activated downstream of the insulin receptor greatly increased the interaction between Hsp90 and the IP3R. Of greater interest, we found that the simultaneous inhibition of mammalian target of rapamycin and the Src kinase almost completely disrupted the interaction between Hsp90 and the IP3R. These results demonstrate that insulin promotes the interaction of Hsp90 with the IP3R to dampen its Ca2+ release activity by a complex mechanism involving mammalian target of rapamycin and the Src kinase.

scholarly journals Calcium channel ITPR2 and mitochondria–ER contacts promote cellular senescence and aging

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Dorian V. Ziegler ◽  
David Vindrieux ◽  
Delphine Goehrig ◽  
Sara Jaber ◽  
Guillaume Collin ◽  
...  
Keyword(s):  
Cellular Senescence ◽  
Calcium Release ◽  
Liver Steatosis ◽  
Metabolic Stress ◽  
Calcium Release Channel ◽  
Release Channel ◽  
Age Related ◽  
Trisphosphate Receptor

AbstractCellular senescence is induced by stresses and results in a stable proliferation arrest accompanied by a pro-inflammatory secretome. Senescent cells accumulate during aging, promoting various age-related pathologies and limiting lifespan. The endoplasmic reticulum (ER) inositol 1,4,5-trisphosphate receptor, type 2 (ITPR2) calcium-release channel and calcium fluxes from the ER to the mitochondria are drivers of senescence in human cells. Here we show that Itpr2 knockout (KO) mice display improved aging such as increased lifespan, a better response to metabolic stress, less immunosenescence, as well as less liver steatosis and fibrosis. Cellular senescence, which is known to promote these alterations, is decreased in Itpr2 KO mice and Itpr2 KO embryo-derived cells. Interestingly, ablation of ITPR2 in vivo and in vitro decreases the number of contacts between the mitochondria and the ER and their forced contacts induce premature senescence. These findings shed light on the role of contacts and facilitated exchanges between the ER and the mitochondria through ITPR2 in regulating senescence and aging.

scholarly journals Identification of persulfide-binding and disulfide-forming cysteine residues in the NifS-like domain of the molybdenum cofactor sulfurase ABA3 by cysteine-scanning mutagenesis

2012 ◽  
Vol 441 (3) ◽  
pp. 823-839 ◽  
Author(s):  
Markus Lehrke ◽  
Steffen Rump ◽  
Torsten Heidenreich ◽  
Josef Wissing ◽  
Ralf R. Mendel ◽  
...  
Keyword(s):  
Structural Model ◽  
Bond Distance ◽  
Aldehyde Oxidase ◽  
Disulfide Bridge ◽  
Cysteine Residue ◽  
Molybdenum Cofactor ◽  
Cysteine Residues ◽  
Xanthine Oxidoreductase ◽  
Cysteine Scanning Mutagenesis ◽  
Molybdenum Cofactor Sulfurase

The Moco (molybdenum cofactor) sulfurase ABA3 from Arabidopsis thaliana catalyses the sulfuration of the Moco of aldehyde oxidase and xanthine oxidoreductase, which represents the final activation step of these enzymes. ABA3 consists of an N-terminal NifS-like domain that exhibits L-cysteine desulfurase activity and a C-terminal domain that binds sulfurated Moco. The strictly conserved Cys430 in the NifS-like domain binds a persulfide intermediate, which is abstracted from the substrate L-cysteine and finally needs to be transferred to the Moco of aldehyde oxidase and xanthine oxidoreductase. In addition to Cys430, another eight cysteine residues are located in the NifS-like domain, with two of them being highly conserved among Moco sulfurase proteins and, at the same time, being in close proximity to Cys430. By determination of the number of surface-exposed cysteine residues and the number of persulfide-binding cysteine residues in combination with the sequential substitution of each of the nine cysteine residues, a second persulfide-binding cysteine residue, Cys206, was identified. Furthermore, the active-site Cys430 was found to be located on top of a loop structure, formed by the two flanking residues Cys428 and Cys435, which are likely to form an intramolecular disulfide bridge. These findings are confirmed by a structural model of the NifS-like domain, which indicates that Cys428 and Cys435 are within disulfide bond distance and that a persulfide transfer from Cys430 to Cys206 is indeed possible.

Modification of a novel x-type high-molecular-weight glutenin subunit gene from Aegilops markgrafii to improve dough strength of wheat flour

2018 ◽  
Vol 69 (9) ◽  
pp. 873
Author(s):  
Xin Ma ◽  
Xuye Du ◽  
Cunyao Bo ◽  
Hongwei Wang ◽  
Anfei Li ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Wheat Flour ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Dough Strength ◽  
Typical Structure ◽  
Dough Quality

High-molecular-weight glutenin subunits (HMW-GS) in bread wheat are major determinants of dough viscoelastic properties and the end-use quality of wheat flour. Cysteine residues, which form intermolecular disulphide bonds in HMW-GS, could improve the strength of gluten. To our knowledge, the number and position of cysteine residues in HMW-GS are conserved between wheat (Triticum aestivum) and Aegilops markgrafii. In the present study, we modified a gene (1Cx1.1) from Ae. markgrafii for an HMW-GS that possessed the typical structure and conserved number of cysteines. Site-directed mutagenesis was carried out in 1Cx1.1 to investigate how the position of cysteine residues in HMW-GS affects the mixing properties of dough. Six HMW-GS containing an extra cysteine residue were expressed in Escherichia coli, and the proteins were purified at sufficient scale for incorporation into flour to test dough quality. There were large differences in dough property among samples containing different modified subunits. Cysteine substituting in the N-terminal or repetitive-domain of HMW-GS could significantly improve dough quality. The results showed that the strategy was useful for providing genetic resources for gene engineering, and hence could be valuable for improving the processing quality of wheat.

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