scholarly journals Lipid Chaperoning of a Thylakoid Protease Whose Stability is Modified by the Protonmotive Force

2019 ◽  
Author(s):  
Lucas J. McKinnon ◽  
Jeremy Fukushima ◽  
Kentaro Inoue ◽  
Steven M. Theg
Keyword(s):  
Disulfide Bond ◽  
Thylakoid Membrane ◽  
Bond Formation ◽  
Disulfide Bridge ◽  
Signal Peptidase ◽  
Common Mechanism ◽  
Protonmotive Force ◽  
Type I ◽  
Disulfide Bond Formation

AbstractProtein folding is a complex cellular process often assisted by chaperones but can also be facilitated by interactions with lipids. Disulfide bond formation is a common mechanism to stabilize a protein. This can help maintain functionality amidst changes in the biochemical milieu which are especially common across energy-transducing membranes. Plastidic Type I Signal Peptidase 1 (Plsp1) is an integral thylakoid membrane signal peptidase which requires an intramolecular disulfide bond for in vitro activity. We have investigated the interplay between disulfide bond formation, lipids, and pH in the folding and activity of Plsp1. By combining biochemical approaches with a genetic complementation assay, we provide evidence that interactions with lipids in the thylakoid membrane have chaperoning activity towards Plsp1. Further, the disulfide bridge appears to prevent an inhibitory conformational change resulting from proton motive force-mimicking pH conditions. Broader implications related to the folding of proteins in energy-transducing membranes are discussed.

scholarly journals Disulfide Bond Formation at the C Termini of Vaccinia Virus A26 and A27 Proteins Does Not Require Viral Redox Enzymes and Suppresses Glycosaminoglycan-Mediated Cell Fusion

2009 ◽  
Vol 83 (13) ◽  
pp. 6464-6476 ◽  
Author(s):  
Yao-Cheng Ching ◽  
Che-Sheng Chung ◽  
Cheng-Yen Huang ◽  
Yu Hsia ◽  
Yin-Liang Tang ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Cell Fusion ◽  
Disulfide Bond ◽  
Protein Complex ◽  
Disulfide Bonds ◽  
Bond Formation ◽  
In Vitro Mutagenesis ◽  
Disulfide Bond Formation ◽  
Infected Cells

ABSTRACT Vaccinia virus A26 protein is an envelope protein of the intracellular mature virus (IMV) of vaccinia virus. A mutant A26 protein with a truncation of the 74 C-terminal amino acids was expressed in infected cells but failed to be incorporated into IMV (W. L. Chiu, C. L. Lin, M. H. Yang, D. L. Tzou, and W. Chang, J. Virol 81:2149-2157, 2007). Here, we demonstrate that A27 protein formed a protein complex with the full-length form but not with the truncated form of A26 protein in infected cells as well as in IMV. The formation of the A26-A27 protein complex occurred prior to virion assembly and did not require another A27-binding protein, A17 protein, in the infected cells. A26 protein contains six cysteine residues, and in vitro mutagenesis showed that Cys441 and Cys442 mediated intermolecular disulfide bonds with Cys71 and Cys72 of viral A27 protein, whereas Cys43 and Cys342 mediated intramolecular disulfide bonds. A26 and A27 proteins formed disulfide-linked complexes in transfected 293T cells, showing that the intermolecular disulfide bond formation did not depend on viral redox pathways. Finally, using cell fusion from within and fusion from without, we demonstrate that cell surface glycosaminoglycan is important for virus-cell fusion and that A26 protein, by forming complexes with A27 protein, partially suppresses fusion.

Targeting Oncogenic Interleukein-7 Receptor Signaling With N-Acetylcysteine In T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2535-2535
Author(s):  
Marc R. Mansour ◽  
Casie Reed ◽  
Amy Eisenberg ◽  
Jen-Chieh Tseng ◽  
Akinori Yoda ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Disulfide Bond ◽  
Transmembrane Domain ◽  
Lymphoblastic Leukemia ◽  
Bond Formation ◽  
Disulfide Bond Formation ◽  
Acetaminophen Overdose ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Activating mutations of the interleukin-7 receptor (IL7R) occur in approximately 10% of patients with T-cell acute lymphoblastic leukemia. Most mutations generate a cysteine at the transmembrane domain leading to receptor homodimerization through disulfide bond formation and ligand-independent activation of STAT5. We hypothesized that the reducing agent N-acetylcysteine (NAC), a well-tolerated drug used widely in clinical practice to treat acetaminophen overdose, would disrupt disulfide bond formation, and inhibit mutant IL7R-mediated oncogenic signaling. To first identify a suitable cell model to study mutant IL7R signaling, we sequenced exon 6 of IL7R in 21 T-ALL cell lines. We identified a 4-amino-acid insertion (p.L242_L243insLSRC) in DND-41 cells which is predicted to form IL7R homodimers through disulfide bond formation with the unpaired cysteine of neighboring mutant IL7Rs. We found that treatment with NAC at clinically achievable concentrations disrupted IL7R homodimerization in IL7R-mutant DND-41 cells in vitro (IC50 approximately 150 micromolar) and led to STAT5 dephosphorylation and cell apoptosis. These effects could be rescued in part by a constitutively active allele of STAT5, indicating the mechanism of NAC is mediated predominantly through disruption of IL7R-STAT5 signaling in these cells. In a murine xenograft model of T-ALL, intraperitoneal NAC treatment led to significant inhibition of tumor progression, indicating NAC has activity in vivo. Previous studies of NAC pharmacokinetics in humans have shown steady state plasma levels range from 200 to 900 micromolar when given on standard treatment regimens for acetaminophen overdose, well within the therapeutic range required to kill DND-41 cells in vitro. Targeting leukemogenic IL7R homodimerization with NAC offers a potentially effective, cheap and feasible therapeutic strategy that warrants testing in clinical trials. Disclosures: Rodig: Daiichi-Sankyo/Arqule Inc., Ventana/Roche Inc., Shape Pharmaceuticals Inc.: Consultancy; Ventana/Roche Inc.: Research Funding.

scholarly journals Chloroplast chaperonin-mediated targeting of a thylakoid membrane protein

2020 ◽  
Author(s):  
Laura Klasek ◽  
Kentaro Inoue ◽  
Steven M. Theg
Keyword(s):  
Membrane Protein ◽  
Thylakoid Membrane ◽  
Protein Targeting ◽  
Transmembrane Proteins ◽  
Signal Peptidase ◽  
Aqueous Environment ◽  
Type I ◽  
Isolated Chloroplasts

AbstractPost-translational protein targeting requires chaperone assistance to direct insertion-competent proteins to integration pathways. Chloroplasts integrate nearly all thylakoid transmembrane proteins post-translationally, but mechanisms in the stroma that assist their insertion remain largely undefined. Here, we investigated how the chloroplast chaperonin (Cpn60) facilitated the thylakoid integration of Plastidic type I signal peptidase 1 (Plsp1) using in vitro targeting assays. Cpn60 bound Plsp1 in the stroma. In isolated chloroplasts, the membrane integration of imported Plsp1 correlated with its dissociation from Cpn60. When the Plsp1 residues that interacted with Cpn60 were removed, Plsp1 did not integrate into the membrane. These results suggested Cpn60 was an intermediate in Plsp1’s thylakoid targeting. In isolated thylakoids, the integration of Plsp1 decreased if Cpn60 was present in excess of cpSecA1, the stromal motor of the cpSec1 translocon which inserts unfolded Plsp1 into the thylakoid. An excess of cpSecA1 favored integration. Introducing Cpn60’s obligate substrate RbcL displaced Cpn60-bound Plsp1; then, the released Plsp1 exhibited increased accessibility to cpSec1. These in vitro targeting experiments support a model in which Cpn60 captures and then releases insertion-competent Plsp1, while cpSecA1 recognizes free Plsp1 for integration. Thylakoid transmembrane proteins transiting the stroma can interact with Cpn60 to shield from the aqueous environment.One-sentence summaryThe chloroplast chaperonin captures and releases Plastidic type I signal peptidase 1 during its targeting to the thylakoid membrane.

scholarly journals In Vitro Oxidation of Snap-25 Leads to Double Disulfide Bond Formation and Protein Destabilization

2013 ◽  
Vol 104 (2) ◽  
pp. 622a
Author(s):  
Dixon J. Woodbury ◽  
Nathan S. Doyle ◽  
Nozomi Ogawa ◽  
Ryan M. Taylor ◽  
John T. Prince
Keyword(s):  
Disulfide Bond ◽  
Bond Formation ◽  
Disulfide Bond Formation

Synthesis of bicyclic organo-peptide hybrids via oxime/intein-mediated macrocyclization followed by disulfide bond formation

2014 ◽  
Vol 12 (7) ◽  
pp. 1135-1142 ◽  
Author(s):  
Jessica M. Smith ◽  
Nicholas C. Hill ◽  
Peter J. Krasniak ◽  
Rudi Fasan
Keyword(s):  
Disulfide Bond ◽  
Bond Formation ◽  
Disulfide Bridge ◽  
Disulfide Bond Formation ◽  
New Strategy ◽  
Recombinant Polypeptides

A new strategy is described to convert recombinant polypeptides into bicyclic organo-peptide hybrids constrained by an intramolecular disulfide bridge.

scholarly journals Oxidant-induced Activation of Type I Protein Kinase A Is Mediated by RI Subunit Interprotein Disulfide Bond Formation

2006 ◽  
Vol 281 (31) ◽  
pp. 21827-21836 ◽  
Author(s):  
Jonathan P. Brennan ◽  
Sonya C. Bardswell ◽  
Joseph R. Burgoyne ◽  
William Fuller ◽  
Ewald Schröder ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Disulfide Bond ◽  
Bond Formation ◽  
Type I ◽  
Disulfide Bond Formation ◽  
Kinase A

Two dileucine motifs mediate late endosomal/lysosomal targeting of transmembrane protein 192 (TMEM192) and a C-terminal cysteine residue is responsible for disulfide bond formation in TMEM192 homodimers

2011 ◽  
Vol 434 (2) ◽  
pp. 219-231 ◽  
Author(s):  
Jörg Behnke ◽  
Eeva-Liisa Eskelinen ◽  
Paul Saftig ◽  
Bernd Schröder
Keyword(s):  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Transmembrane Protein ◽  
Bond Formation ◽  
Immunogold Labelling ◽  
Disulfide Bridge ◽  
Cysteine Residue ◽  
Disulfide Bond Formation ◽  
Transmembrane Segments ◽  
Late Endosomes

TMEM192 (transmembrane protein 192) is a novel constituent of late endosomal/lysosomal membranes with four potential transmembrane segments and an unknown function that was initially discovered by organellar proteomics. Subsequently, localization in late endosomes/lysosomes has been confirmed for overexpressed and endogenous TMEM192, and homodimers of TMEM192 linked by disulfide bonds have been reported. In the present study the molecular determinants of TMEM192 mediating its transport to late endosomes/lysosomes were analysed by using CD4 chimaeric constructs and mutagenesis of potential targeting motifs in TMEM192. Two directly adjacent N-terminally located dileucine motifs of the DXXLL-type were found to be critical for transport of TMEM192 to late endosomes/lysosomes. Whereas disruption of both dileucine motifs resulted in mistargeting of TMEM192 to the plasma membrane, each of the two motifs was sufficient to ensure correct targeting of TMEM192. In order to study disulfide bond formation, mutagenesis of cysteine residues was performed. Mutation of Cys266 abolished disulfide bridge formation between TMEM192 molecules, indicating that TMEM192 dimers are linked by a disulfide bridge between their C-terminal tails. According to the predicted topology, Cys266 would be localized in the reductive milieu of the cytosol where disulfide bridges are generally uncommon. Using immunogold labelling and proteinase protection assays, the localization of the N- and C-termini of TMEM192 on the cytosolic side of the late endosomal/lysosomal membrane was experimentally confirmed. These findings may imply close proximity of the C-termini in TMEM192 dimers and a possible involvement of this part of the protein in dimer assembly.

scholarly journals The Mitochondrial Disulfide Relay System: Roles in Oxidative Protein Folding and Beyond

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
Manuel Fischer ◽  
Jan Riemer
Keyword(s):  
Disulfide Bond ◽  
Protein Import ◽  
Bond Formation ◽  
Mitochondrial Morphology ◽  
Disulfide Bond Formation ◽  
Intermembrane Space ◽  
Intact Cells ◽  
Dependent Protein ◽  
Main Components

Disulfide bond formation drives protein import of most proteins of the mitochondrial intermembrane space (IMS). The main components of this disulfide relay machinery are the oxidoreductase Mia40 and the sulfhydryl oxidase Erv1/ALR. Their precise functions have been elucidated in molecular detail for the yeast and human enzymesin vitroand in intact cells. However, we still lack knowledge on how Mia40 and Erv1/ALR impact cellular and organism physiology and whether they have functions beyond their role in disulfide bond formation. Here we summarize the principles of oxidation-dependent protein import mediated by the mitochondrial disulfide relay. We proceed by discussing recently described functions of Mia40 in the hypoxia response and of ALR in influencing mitochondrial morphology and its importance for tissue development and embryogenesis. We also include a discussion of the still mysterious function of Erv1/ALR in liver regeneration.

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