A conserved, buried cysteine near the P-site is accessible to cysteine modifications and increases ROS stability in the P-type plasma membrane H+-ATPase

2021 ◽  
Vol 478 (3) ◽  
pp. 619-632
Author(s):  
Marcel Welle ◽  
Jesper T. Pedersen ◽  
Tina Ravnsborg ◽  
Maki Hayashi ◽  
Sandra Maaß ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Plasma Membrane ◽  
Peptide Identification ◽  
Cysteine Residue ◽  
Amino Acid Residues ◽  
Wild Type ◽  
P Type ◽  
Kinetics Of ◽  
Aspartate Residue ◽  
Sulfur Containing

Sulfur-containing amino acid residues function in antioxidative responses, which can be induced by the reactive oxygen species generated by excessive copper and hydrogen peroxide. In all Na+/K+, Ca2+, and H+ pumping P-type ATPases, a cysteine residue is present two residues upstream of the essential aspartate residue, which is obligatorily phosphorylated in each catalytic cycle. Despite its conservation, the function of this cysteine residue was hitherto unknown. In this study, we analyzed the function of the corresponding cysteine residue (Cys-327) in the autoinhibited plasma membrane H+-ATPase isoform 2 (AHA2) from Arabidopsis thaliana by mutagenesis and heterologous expression in a yeast host. Enzyme kinetics of alanine, serine, and leucine substitutions were identical with those of the wild-type pump but the sensitivity of the mutant pumps was increased towards copper and hydrogen peroxide. Peptide identification and sequencing by mass spectrometry demonstrated that Cys-327 was prone to oxidation. These data suggest that Cys-327 functions as a protective residue in the plasma membrane H+-ATPase, and possibly in other P-type ATPases as well.

scholarly journals Molecular Alterations in the Stomach of Tff1-Deficient Mice: Early Steps in Antral Carcinogenesis

2020 ◽  
Vol 21 (2) ◽  
pp. 644 ◽  
Author(s):  
Eva B. Znalesniak ◽  
Franz Salm ◽  
Werner Hoffmann
Keyword(s):  
Cysteine Residue ◽  
Molecular Forms ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Rt Pcr ◽  
Molecular Alterations ◽  
Protein Levels ◽  
Increased Sensitivity ◽  
A Minor ◽  
Deficient Mice

TFF1 is a peptide of the gastric mucosa co-secreted with the mucin MUC5AC. It plays a key role in gastric mucosal protection and repair. Tff1-deficient (Tff1KO) mice obligatorily develop antropyloric adenoma and about 30% progress to carcinomas. Thus, these mice represent a model for gastric tumorigenesis. Here, we compared the expression of selected genes in Tff1KO mice and the corresponding wild-type animals (RT-PCR analyses). Furthermore, we systematically investigated the different molecular forms of Tff1 and its heterodimer partner gastrokine-2 (Gkn2) in the stomach (Western blot analyses). As a hallmark, a large portion of murine Tff1 occurs in a monomeric form. This is unexpected because of its odd number of seven cysteine residues. Probably the three conserved acid amino acid residues (EEE) flanking the 7th cysteine residue allow monomeric secretion. As a consequence, the free thiol of monomeric Tff1 could have a protective scavenger function, e.g., for reactive oxygen/nitrogen species. Furthermore, a minor subset of Tff1 forms a disulfide-linked heterodimer with IgG Fc binding protein (Fcgbp). Of special note, in Tff1KO animals a homodimeric form of Gkn2 was observed. In addition, Tff1KO animals showed strongly reduced Tff2 transcript and protein levels, which might explain their increased sensitivity to Helicobacter pylori infection.

scholarly journals NADPH Oxidase-Mediated Reactive Oxygen Species Production: Subcellular Localization and Reassessment of Its Role in Plant Defense

2009 ◽  
Vol 22 (7) ◽  
pp. 868-881 ◽  
Author(s):  
Jeannine Lherminier ◽  
Taline Elmayan ◽  
Jérôme Fromentin ◽  
Khadija Tantaoui Elaraqui ◽  
Simona Vesa ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Hydrogen Peroxide ◽  
Plasma Membrane ◽  
Nadph Oxidase ◽  
Subcellular Localization ◽  
Acquired Resistance ◽  
Reactive Oxygen ◽  
Oxygen Species ◽  
Wild Type ◽  
Hydrogen Peroxide Production

Chemiluminescence detection of reactive oxygen species (ROS) triggered in tobacco BY-2 cells by the fungal elicitor cryptogein was previously demonstrated to be abolished in cells transformed with an antisense construct of the plasma membrane NADPH oxidase, NtrbohD. Here, using electron microscopy, it has been confirmed that the first hydrogen peroxide production occurring a few minutes after challenge of tobacco cells with cryptogein is plasma membrane located and NtrbohD mediated. Furthermore, the presence of NtrbohD in detergent-resistant membrane fractions could be associated with the presence of NtrbohD-mediated hydrogen peroxide patches along the plasma membrane. Comparison of the subcellular localization of ROS in wild-type tobacco and in plants transformed with antisense constructs of NtrbohD revealed that this enzyme is also responsible for the hydrogen peroxide production occurring at the plasma membrane after infiltration of tobacco leaves with cryptogein. Finally, the reactivity of wild-type and transformed plants to the elicitor and their resistance against the pathogenic oomycete Phytophthora parasitica were examined. NtrbohD-mediated hydrogen peroxide production does not seem determinant for either hypersensitive response development or the establishment of acquired resistance but it is most likely involved in the signaling pathways associated with the protection of the plant cell.

Contribution of cysteine residue to the properties of Listeria monocytogenes listeriolysin O

2009 ◽  
Vol 55 (10) ◽  
pp. 1153-1159 ◽  
Author(s):  
Radosław Stachowiak ◽  
Jarosław Wiśniewski ◽  
Olga Osińska ◽  
Jacek Bielecki
Keyword(s):  
Listeria Monocytogenes ◽  
Hemolytic Activity ◽  
Cysteine Residue ◽  
Listeriolysin O ◽  
Wild Type ◽  
Gram Positive Bacteria ◽  
Culture Model ◽  
The Family ◽  
Kinetics Of

Listeriolysin (LLO) is the key virulence factor critical for Listeria monocytogenes pathogenesis. Listerial cytolysin belongs to the family of cholesterol-dependent cytolysins (CDCs), a group of pore-forming toxins produced by related gram-positive bacteria. Most CDCs contain a cysteine residue in the conserved undecapeptide — a sequence that is highly preserved among this group of proteins. Substitutions of cysteine do not always lead to loss of hemolytic activity, questioning the purpose of such strong conservation of this amino acid in the sequence of CDC. The properties of 3 L. monocytogenes strains, a wild type and 2 mutants expressing modified LLO within the cysteine residue, were analyzed in this work. The first of these mutants producing a toxin with cysteine to alanine substitution showed similar features to the wild type except that a thiol-reducing agent was not necessary for hemolytic activity. Another strain secreting LLO containing serine instead of cysteine exhibited strikingly different properties than the wild type. Modified toxin is independent of the reducing reagents, less stable, and shows accelerated kinetics of cytolysis in comparison with the unchanged protein. However, both mutant strains are less invasive in the cell culture model showing the important role of cysteine in L. monocytogenes virulence.

scholarly journals Yeast P4-ATPases Drs2p and Dnf1p Are Essential Cargos of the NPFXD/Sla1p Endocytic Pathway

2007 ◽  
Vol 18 (2) ◽  
pp. 487-500 ◽  
Author(s):  
Ke Liu ◽  
Zhaolin Hua ◽  
Joshua A. Nepute ◽  
Todd R. Graham
Keyword(s):  
Plasma Membrane ◽  
Protein Transport ◽  
Endocytic Pathway ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Sensitive Mutant ◽  
C Terminus ◽  
P Type ◽  
Golgi Network ◽  
Endocytic Pathways

Drs2p family P-type ATPases (P4-ATPases) are required in multiple vesicle-mediated protein transport steps and are proposed to be phospholipid translocases (flippases). The P4-ATPases Drs2p and Dnf1p cycle between the exocytic and endocytic pathways, and here we define endocytosis signals required by these proteins to maintain a steady-state localization to internal organelles. Internalization of Dnf1p from the plasma membrane uses an NPFXD endocytosis signal and its recognition by Sla1p, part of an endocytic coat/adaptor complex with clathrin, Pan1p, Sla2p/End4p, and End3p. Drs2p has multiple endocytosis signals, including two NPFXDs near the C terminus and PEST-like sequences near the N terminus that may mediate ubiquitin (Ub)-dependent endocytosis. Drs2p localizes to the trans-Golgi network in wild-type cells and accumulates on the plasma membrane when both the Ub- and NPFXD-dependent endocytic mechanisms are inactivated. Surprisingly, the pan1-20 temperature-sensitive mutant is constitutively defective for Ub-dependent endocytosis but is not defective for NPFXD-dependent endocytosis at the permissive growth temperature. To sustain viability of pan1-20, Drs2p must be endocytosed through the NPFXD/Sla1p pathway. Thus, Drs2p is an essential endocytic cargo in cells compromised for Ub-dependent endocytosis. These results demonstrate an essential role for endocytosis in retrieving proteins back to the Golgi, and they define critical cargos of the NPFXD/Sla1p system.

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.

Tmem27 dimerization, deglycosylation, plasma membrane depletion, and the extracellular Phe-Phe motif are negative regulators of cleavage by Bace2

2012 ◽  
Vol 393 (6) ◽  
pp. 473-484 ◽  
Author(s):  
Daria Esterházy ◽  
Pinar Akpinar ◽  
Markus Stoffel
Keyword(s):  
Plasma Membrane ◽  
Mutational Analysis ◽  
Cell Mass ◽  
Surface Protein ◽  
Cysteine Residue ◽  
Cleavage Sites ◽  
Amino Acid Residues ◽  
Β Cell ◽  
Glucose Stimulation ◽  
Disease States

Abstract The pancreatic β-cell surface protein Tmem27 is promotes the preservation of functional β-cell mass. It is a selective substrate of the protease Bace2, yet the intramolecular features of Tmem27 that regulate its processing by this sheddase have not been characterized. In particular, the importance of homodimerization, glycosylation, trafficking to the plasma membrane (PM), the existence of multiple cleavage sites, and the amino acid residues that govern these features are currently unknown. Using Tmem27 mutational analysis and multiple biochemical approaches, we here show that Tmem27 dimerization is a dynamic process mediated by its intracellular cysteine residue and that prevents Tmem27 cleavage, that extracellular asparagine glycosylation is essential for Tmem27 trafficking to the PM and its processing by Bace2, that the amount of Tmem27 at the PM is proportional to its total cell levels upon glucose stimulation and Bace2 inhibition, and that the double phenylalanine motif in the Tmem27 cleavage site is an intramolecular Bace2 inhibitor. These findings define structural properties of Tmem27 that affect the susceptibility to its protease Bace2 and have implications for the efficiency with which Tmem27 and other Bace2 substrates are cleaved in normal and disease states.

scholarly journals Comparison of the Dynamics of Resistance-Associated Mutations to Nucleoside Reverse Transcriptase Inhibitors, Nonnucleoside Reverse Transcriptase Inhibitors, and Protease Inhibitors after Cessation of Antiretroviral Combination Therapy

2004 ◽  
Vol 48 (2) ◽  
pp. 644-647 ◽  
Author(s):  
Marc Wirden ◽  
Constance Delaugerre ◽  
Anne Genevieve Marcelin ◽  
Nadine Ktorza ◽  
Hocine Ait Mohand ◽  
...  
Keyword(s):  
Amino Acid ◽  
Reverse Transcriptase ◽  
Protease Inhibitors ◽  
Antiretroviral Drugs ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Reverse Transcriptase Inhibitors ◽  
Nucleoside Reverse Transcriptase Inhibitors ◽  
Nonnucleoside Reverse Transcriptase Inhibitors ◽  
Kinetics Of

ABSTRACT The dynamics of mutations associated with resistance to antiretroviral drugs were analyzed after cessation of therapy. The results showed that the kinetics of the shift to wild-type amino acid residues were significantly faster for protease inhibitors, intermediate for nonnucleoside reverse transcriptase inhibitors, and slower for nucleoside reverse transcriptase inhibitors.

scholarly journals Structural studies of domain movement in active-site mutants of porphobilinogen deaminase fromBacillus megaterium

2017 ◽  
Vol 73 (11) ◽  
pp. 612-620 ◽  
Author(s):  
Jingxu Guo ◽  
Peter Erskine ◽  
Alun R. Coker ◽  
Steve P. Wood ◽  
Jonathan B. Cooper
Keyword(s):  
Active Site ◽  
Cysteine Residue ◽  
Porphobilinogen Deaminase ◽  
Structural Studies ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Domain Movement ◽  
Covalently Linked ◽  
Active Site Mutants ◽  
Aspartate Residue

The enzyme porphobilinogen deaminase (PBGD) is one of the key enzymes in tetrapyrrole biosynthesis. It catalyses the formation of a linear tetrapyrrole from four molecules of the substrate porphobilinogen (PBG). It has a dipyrromethane cofactor (DPM) in the active site which is covalently linked to a conserved cysteine residue through a thioether bridge. The substrate molecules are linked to the cofactor in a stepwise head-to-tail manner during the reaction, which is catalysed by a conserved aspartate residue: Asp82 in theB. megateriumenzyme. Three mutations have been made affecting Asp82 (D82A, D82E and D82N) and their crystal structures have been determined at resolutions of 2.7, 1.8 and 1.9 Å, respectively. These structures reveal that whilst the D82E mutant possesses the DPM cofactor, in the D82N and D82A mutants the cofactor is likely to be missing, incompletely assembled or disordered. Comparison of the mutant PBGD structures with that of the wild-type enzyme shows that there are significant domain movements and suggests that the enzyme adopts `open' and `closed' conformations, potentially in response to substrate binding.

scholarly journals Kinetics of inhibitory effect of hydrogen peroxide on activity of plasma membrane transporting Cа2+, Mg2+-ATPase of sperm cells

2018 ◽  
Vol 12 (1) ◽  
pp. 17-24
Author(s):  
R. V. Fafula ◽  
◽  
O. I. Meskalo ◽  
E. I. Lychkovskyy ◽  
Z. D. Vorobets ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Plasma Membrane ◽  
Inhibitory Effect ◽  
Sperm Cells ◽  
Kinetics Of
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