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

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.

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Individual Palmitoyl Residues Serve Distinct Roles in H-Ras Trafficking, Microlocalization, and Signaling

Molecular and Cellular Biology ◽

10.1128/mcb.25.15.6722-6733.2005 ◽

2005 ◽

Vol 25 (15) ◽

pp. 6722-6733 ◽

Cited By ~ 142

Author(s):

Sandrine Roy ◽

Sarah Plowman ◽

Barak Rotblat ◽

Ian A. Prior ◽

Cornelia Muncke ◽

...

Keyword(s):

Plasma Membrane ◽

Golgi Apparatus ◽

Lipid Rafts ◽

Lipid Bilayer ◽

Spatial Organization ◽

Cysteine Residues ◽

Wild Type ◽

Membrane Affinity ◽

Lateral Segregation

ABSTRACT H-ras is anchored to the plasma membrane by two palmitoylated cysteine residues, Cys181 and Cys184, operating in concert with a C-terminal S-farnesyl cysteine carboxymethylester. Here we demonstrate that the two palmitates serve distinct biological roles. Monopalmitoylation of Cys181 is required and sufficient for efficient trafficking of H-ras to the plasma membrane, whereas monopalmitoylation of Cys184 does not permit efficient trafficking beyond the Golgi apparatus. However, once at the plasma membrane, monopalmitoylation of Cys184 supports correct GTP-regulated lateral segregation of H-ras between cholesterol-dependent and cholesterol-independent microdomains. In contrast, monopalmitoylation of Cys181 dramatically reverses H-ras lateral segregation, driving GTP-loaded H-ras into cholesterol-dependent microdomains. Intriguingly, the Cys181 monopalmitoylated H-ras anchor emulates the GTP-regulated microdomain interactions of N-ras. These results identify N-ras as the Ras isoform that normally signals from lipid rafts but also reveal that spacing between palmitate and prenyl groups influences anchor interactions with the lipid bilayer. This concept is further supported by the different plasma membrane affinities of the monopalmitoylated anchors: Cys181-palmitate is equivalent to the dually palmitoylated wild-type anchor, whereas Cys184-palmitate is weaker. Thus, membrane affinity of a palmitoylated anchor is a function both of the hydrophobicity of the lipid moieties and their spatial organization. Finally we show that the plasma membrane affinity of monopalmitoylated anchors is absolutely dependent on cholesterol, identifying a new role for cholesterol in promoting interactions with the raft and nonraft plasma membrane.

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Probing why trypanosomes assemble atypical cytochrome c with an AxxCH haem-binding motif instead of CxxCH

Biochemical Journal ◽

10.1042/bj20120757 ◽

2012 ◽

Vol 448 (2) ◽

pp. 253-260 ◽

Cited By ~ 7

Author(s):

Michael L. Ginger ◽

Katharine A. Sam ◽

James W. A. Allen

Keyword(s):

Cytochrome C ◽

Cysteine Residue ◽

Cysteine Residues ◽

Binding Motif ◽

Wild Type ◽

Post Translational Modification ◽

Cytochrome C Biogenesis ◽

Genes Encoding ◽

Trypanosomatid Parasites ◽

Core Components

Mitochondrial cytochromes c and c1 are core components of the respiratory chain of all oxygen-respiring eukaryotes. These proteins contain haem, covalently bound to the polypeptide in a catalysed post-translational modification. In all eukaryotes, except members of the protist phylum Euglenozoa, haem attachment is to the cysteine residues of a CxxCH haem-binding motif. In the Euglenozoa, which include medically relevant trypanosomatid parasites, haem attachment is to a single cysteine residue in an AxxCH haem-binding motif. Moreover, genes encoding known c-type cytochrome biogenesis machineries are all absent from trypanosomatid genomes, indicating the presence of a novel biosynthetic apparatus. In the present study, we investigate expression and maturation of cytochrome c with a typical CxxCH haem-binding motif in the trypanosomatids Crithidia fasciculata and Trypanosoma brucei. Haem became attached to both cysteine residues of the haem-binding motif, indicating that, in contrast with previous hypotheses, nothing prevents formation of a CxxCH cytochrome c in euglenozoan mitochondria. The cytochrome variant was also able to replace the function of wild-type cytochrome c in T. brucei. However, the haem attachment to protein was not via the stereospecifically conserved linkage universally observed in natural c-type cytochromes, suggesting that the trypanosome cytochrome c biogenesis machinery recognized and processed only the wild-type single-cysteine haem-binding motif. Moreover, the presence of the CxxCH cytochrome c resulted in a fitness cost in respiration. The level of cytochrome c biogenesis in trypanosomatids was also found to be limited, with the cells operating at close to maximum capacity.

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Identification of an important cysteine residue in human glutamate–cysteine ligase catalytic subunit by site-directed mutagenesis

Biochemical Journal ◽

10.1042/bj3360675 ◽

1998 ◽

Vol 336 (3) ◽

pp. 675-680 ◽

Cited By ~ 37

Author(s):

Zhongheng TU ◽

M. W. ANDERS

Keyword(s):

Expression System ◽

Baculovirus Expression System ◽

Cysteine Residue ◽

Catalytic Subunit ◽

Site Directed Mutagenesis ◽

Regulatory Subunit ◽

Cysteine Residues ◽

Directed Mutagenesis ◽

Wild Type ◽

Glutamate Cysteine Ligase

Glutamate–cysteine ligase (GLCL) catalyses the rate-limiting step in glutathione biosynthesis. To identify cysteine residues in GLCL that are involved in its activity, eight conserved cysteine residues in human GLCL catalytic subunit (hGLCLC) were replaced with glycine residues by PCR-based site-directed mutagenesis. Both recombinant hGLCLC and hGLCL holoenzyme were expressed and purified with a baculovirus expression system. The activity of purified hGLCL holoenzyme with the mutant hGLCLC-C553G was 110±12 µmol/h per mg of protein compared with 370±20 µmol/h per mg of protein for the wild-type. Holoenzymes with hGLCLC-C52G, -C248G, -C249G, -C295G, -C491G, -C501G or -C605G showed activities similar to the wild type. The Km values of hGLCL containing hGLCLC-C553G were slightly lower than those of the wild type, indicating that the replacement of cysteine-553 with Gly in hGLCLC did not significantly affect substrate binding by the enzyme. hGLCLC-C553G was more easily dissociated from hGLCLR than the wild-type hGLCLC. GLCL activity increased by 11% after hGLCLC-C553G was incubated with an equimolar amount of purified hGLCL regulatory subunit (hGLCLR) at room temperature for 30 min, but increased by 110% after wild-type hGLCLC was incubated with hGLCLR for 10 min. These results indicate that cysteine-553 in hGLCLC is involved in heterodimer formation between hGLCLC and hGLCLR.

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Improved Stability of Baeyer–Villiger Mono-Oxygenase from Pseudomonas fluorescens by Substitution of Cysteine Residues

Journal of Biobased Materials and Bioenergy ◽

10.1166/jbmb.2019.1878 ◽

2019 ◽

Vol 13 (4) ◽

pp. 490-497

Author(s):

Luo Liu ◽

Shuaiqi Meng ◽

Ren Wei ◽

Min Jiang ◽

Fang Wang ◽

...

Keyword(s):

Pseudomonas Fluorescens ◽

Enzyme Stability ◽

Sebacic Acid ◽

Cysteine Residue ◽

Cysteine Residues ◽

Carbonyl Groups ◽

Wild Type ◽

Sitedirected Mutagenesis ◽

Improved Stability ◽

The Stability

Baeyer–Villiger mono-oxygenases (BVMOs) are enzymes that could insert an oxygen into carbon skeletons adjacent to carbonyl groups to form lactone or ester. Recently, a new method of sebacic acid production from oleic acid based on Pseudomonas fluorescens DSM 50106 (PfBVMO) catalysis was reported. However, the instable property of PfBVMO limited the further investigation and application of this enzyme. In this study, we aimed to improve the stability of PfBVMO by sitedirected mutagenesis. The sequence of PfBVMO indicates probable factors from the susceptible oxidizing cysteine residue leading to the instability of PfBVMO. Activity and stability of mutants and the wild-type were characterized to investigate the correlation of cysteine residues and activity. The mutant enzyme with entire cysteine residues replaced by serine residues, showed significantly higher stability than the wild type. The result would facilitate further work on the enzyme modification and crystal structure determination. Meanwhile, the results of this study provided potential for other enzyme stability evolution.

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

Biochemical Journal ◽

10.1042/bj20050889 ◽

2005 ◽

Vol 393 (2) ◽

pp. 575-582 ◽

Cited By ~ 33

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.

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A cysteine residue located in the transmembrane domain of CD44 is important in binding of CD44 to hyaluronic acid.

Journal of Experimental Medicine ◽

10.1084/jem.183.5.1987 ◽

1996 ◽

Vol 183 (5) ◽

pp. 1987-1994 ◽

Cited By ~ 44

Author(s):

D Liu ◽

M S Sy

Keyword(s):

Hyaluronic Acid ◽

Transmembrane Domain ◽

Cysteine Residue ◽

Jurkat Cells ◽

Cysteine Residues ◽

Wild Type ◽

Mutant Proteins ◽

Stable Transfectants ◽

Cd44 Antibody

In the transmembrane domain and cytoplasmic domain of human CD44 protein there are two cysteine residues. These two cysteines are conserved in all known mammalian CD44 proteins. The functions of these cysteine residues are not known. Site-specific mutagenesis was used to create CD44 mutant proteins lacking either one or both of these cysteine residues. Wild-type CD44 and mutant CD44 genes were transfected into CD44- Jurkat cells to establish stable transfectants. These transfectants were used to study whether these two cysteine residues are important in the binding of CD44(H) to fluorescein-conjugated hyaluronic acid (F-HA). Jurkat transfectant bearing wild-type CD44 did not bind F-HA, unless they were stimulated in vitro with immobilized anti-CD3 monoclonal antibody. Anti-CD3 antibody also stimulated the binding of F-HA in Jurkat CD44.C295A transfectant in which the cytoplasmic cysteine residue has been replaced with alanine. In contrast, anti-CD3 antibody failed to stimulate the binding of F-HA in Jurkat transfectant (CD44.C286A), in which the transmembrane domain cysteine 286 has been replaced with an alanine, and in Jurkat transfectant CD44.2C2A, in which both of the cysteine residues have been altered. Binding can also be induced with a monoclonal anti-CD44 antibody (F-44-10-2) in Jurkat wild-type CD44 and Jurkat CD44.C295A transfectants but not in CD44. C286A transfectant. These results provide evidence that the transmembrane domain of CD44, more specifically the cysteine residue in the transmembrane domain, is important for both activation-induced and anti-CD44 antibody-induced binding of soluble HA.

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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

Biochemical Journal ◽

10.1042/bcj20200559 ◽

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.

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Role of cysteine 416 in N-ethylmaleimide sensitivity of human equilibrative nucleoside transporter 1 (hENT1)

Biochemical Journal ◽

10.1042/bcj20180543 ◽

2018 ◽

Vol 475 (20) ◽

pp. 3293-3309 ◽

Cited By ~ 2

Author(s):

Sylvia Y.M. Yao ◽

Amy M.L. Ng ◽

Carol E. Cass ◽

James D. Young

Keyword(s):

Expression System ◽

Cysteine Residue ◽

Cysteine Residues ◽

Nucleoside Transporter ◽

Wild Type ◽

Binding Characteristics ◽

Equilibrative Nucleoside Transporter ◽

C Terminus ◽

Nucleoside Drugs

Human equilibrative nucleoside transporter 1 (hENT1), the first identified member of the ENT family of integral membrane proteins, is the primary mechanism for cellular uptake of physiologic nucleosides and many antineoplastic and antiviral nucleoside drugs. hENT1, which is potently inhibited by nitrobenzylthioinosine (NBMPR), possesses 11 transmembrane helical domains with an intracellular N-terminus and an extracellular C-terminus. As a protein with 10 endogenous cysteine residues, it is sensitive to inhibition by the membrane permeable sulfhydryl-reactive reagent N-ethylmaleimide (NEM) but is unaffected by the membrane impermeable sulfhydryl-reactive reagent p-chloromercuriphenyl sulfonate. To identify the residue(s) involved in NEM inhibition, we created a cysteine-less version of hENT1 (hENT1C-), with all 10 endogenous cysteine residues mutated to serine, and showed that it displays wild-type uridine transport and NBMPR-binding characteristics when produced in the Xenopus oocyte heterologous expression system, indicating that endogenous cysteine residues are not essential for hENT1 function. We then tested NEM sensitivity of recombinant wild-type hENT1, hENT1 mutants C1S to C10S (single cysteine residues replaced by serine), hENT1C- (all cysteine residues replaced by serine), and hENT1C- mutants S1C to S10C (single serine residues converted back to cysteine). Mutants C9S (C416S/hENT1) and S9C (S416C/hENT1C-) were insensitive and sensitive, respectively, to inhibition by NEM, identifying Cys416 as the endofacial cysteine residue in hENT1 responsible for NEM inhibition. Kinetic experiments suggested that NEM modification of Cys416, which is located at the inner extremity of TM10, results in the inhibition of hENT1 uridine transport and NBMPR binding by constraining the protein in its inward-facing conformation.

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