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Metabolic programming is important for B cell fate, but the bioenergetic requirement for regulatory B (B reg ) cell differentiation and function is unknown. Here we show that B reg cell differentiation, unlike non-B reg cells, relies on mitochondrial electron transport and homeostatic levels of reactive oxygen species (ROS). Single-cell RNA sequencing analysis revealed that TXN, encoding the metabolic redox protein thioredoxin (Trx), is highly expressed by B reg cells, unlike Trx inhibitor TXNIP which was downregulated. Pharmacological inhibition or gene silencing of TXN resulted in mitochondrial membrane depolarization and increased ROS levels, selectively suppressing B reg cell differentiation and function while favoring pro-inflammatory B cell differentiation. Patients with systemic lupus erythematosus (SLE), characterized by B reg cell deficiencies, present with B cell mitochondrial membrane depolarization, elevated ROS and fewer Trx + B cells. Exogenous Trx stimulation restored B reg cells and mitochondrial membrane polarization in SLE B cells to healthy B cell levels, indicating Trx insufficiency underlies B reg cell impairment in patients with SLE., (© 2024. The Author(s).)

The objective of this study was to identify the role of individual amino acid residues in determining the substrate specificity of the yeast mitochondrial citrate transport protein (CTP). Previously, we showed that the CTP contains at least two substrate-binding sites. In this study, utilizing the overexpressed, single-Cys CTP-binding site variants that were functionally reconstituted in liposomes, we examined CTP specificity from both its external and internal surfaces. Upon mutation of residues comprising the more external site, the CTP becomes less selective for citrate with numerous external anions able to effectively inhibit [(14)C]citrate/citrate exchange. Thus, the site 1 variants assume the binding characteristics of a nonspecific anion carrier. Comparison of [(14)C]citrate uptake in the presence of various internal anions versus water revealed that, with the exception of the R189C mutant, the other site 1 variants showed substantial uniport activity relative to exchange. Upon mutation of residues comprising site 2, we observed two types of effects. The K37C mutant displayed a markedly enhanced selectivity for external citrate. In contrast, the other site 2 mutants displayed varying degrees of relaxed selectivity for external citrate. Examination of internal substrates revealed that, in contrast to the control transporter, the R181C variant exclusively functioned as a uniporter. This study provides the first functional information on the role of specific binding site residues in determining mitochondrial transporter substrate selectivity. We interpret our findings in the context of our homology-modeled CTP as it cycles between the outward-facing, occluded, and inward-facing states.

The present investigation utilized the site-directed spin labeling method of electron paramagnetic resonance (EPR) spectroscopy to identify the effect of citrate, the natural ligand, and transport inhibitors on the conformation of the yeast mitochondrial citrate transport protein (CTP) reconstituted in liposomal vesicles. Spin label was placed at six different locations within the CTP in order to monitor conformational changes that occurred near each of the transporter's two substrate binding sites, as well as at more distant domains within the CTP architecture. We observed that citrate caused little change in the EPR spectra. In contrast the transport inhibitors 1,2,3-benzenetricarboxylate (BTC), pyridoxal 5'-phosphate (PLP), and compound 792949 resulted in spectral changes that indicated a decrease in the flexibility of the attached spin label at each of the six locations tested. The rank order of the immobilizing effect was compound 792949 > PLP > BTC. The four spin-label locations that report on the CTP substrate binding sites displayed the greatest changes in the EPR spectra upon addition of inhibitor. Furthermore, we found that when compound 792949 was added vectorially (i.e., extra- and/or intra-liposomally), the immobilizing effect was mediated nearly exclusively by external reagent. In contrast, upon addition of PLP vectorially, the effect was mediated to a similar extent from both the external and the internal compartments. In combination our data indicate that: i) citrate binding to the CTP substrate binding sites does not alter side-chain and/or backbone mobility in a global manner and is consistent with our expectation that both in the absence and presence of substrate the CTP displays the flexibility required of a membrane transporter; and ii) binding of each of the transport inhibitors tested locked multiple CTP domains into more rigid conformations, thereby exhibiting long-range inter-domain conformational communication. The differential vectorial effects of compound 792949 and PLP are discussed in the context of the CTP homology-modeled structure and potential mechanistic molecular explanations are given.

The mitochondrial citrate transport protein (CTP) is critical to energy metabolism in eukaryotic cells. We demonstrate that 1,2,3-benzenetricarboxylate (BTC), the classic and defining inhibitor of the mitochondrial CTP, is a mixed inhibitor of the reconstituted Cys-less CTP, with a strong competitive component [i.e., a competitive inhibition constant (K(ic)) of 0.12 +/- 0.02 mM and an uncompetitive inhibition constant (K(iu)) of 3.04 +/- 0.74 mM]. Based on docking calculations, a model for BTC binding has been developed. We then determined the K(ic) values for each of the eight substrate binding site cysteine substitution mutants and observed increases of 62- to 261-fold relative to the Cys-less control, thereby substantiating the importance of each of these residues in BTC binding. It is noteworthy that we observed parallel increases in the K(m) for citrate transport with each of these binding site mutants, thereby confirming that with these CTP variants, K(m) approximates the K(d) (for citrate) and is therefore a measure of substrate affinity. To further substantiate the importance of these binding site residues, in silico screening of a database of commercially available compounds has led to discovery of the first purely competitive inhibitor of the CTP. Docking calculations indicate that this inhibitor spans and binds to both substrate sites simultaneously. Finally, we propose a kinetic model for citrate transport in which the citrate molecule sequentially binds to the external and internal binding sites (per CTP monomer) before transport.

The present investigation identifies the molecular basis for the well-documented inhibition of the mitochondrial inner membrane citrate transport protein (CTP) function by the lysine-selective reagent pyridoxal 5'-phosphate. Kinetic analysis indicates that PLP is a linear mixed inhibitor of the Cys-less CTP, with a predominantly competitive component. We have previously concluded that the CTP contains at least two substrate binding sites which are located at increasing depths within the substrate translocation pathway and which contain key lysine residues. In the present investigation, the roles of Lys-83 in substrate binding site one, Lys-37 and Lys-239 in substrate binding site two, and four other off-pathway lysines in conferring PLP-inhibition of transport was determined by functional characterization of seven lysine to cysteine substitution mutants. We observed that replacement of Lys-83 with cysteine resulted in a 78% loss of the PLP-mediated inhibition of CTP function. In contrast, replacement of either Lys-37 or Lys-239 with cysteine caused a modest reduction in the inhibition caused by PLP (i.e., 31% and 20% loss of inhibition, respectively). Interestingly, these losses of PLP-mediated inhibition could be rescued by covalent modification of each cysteine with MTSEA, a reagent that adds a lysine-like moiety (i.e. SCH(2)CH(2)NH(3) (+)) to the cysteine sulfhydryl group. Importantly, the replacement of non-binding site lysines (i.e., Lys-45, Lys-48, Lys-134, Lys-141) with cysteine resulted in little change in the PLP inhibition. Based upon these results, we conducted docking calculations with the CTP structural model leading to the development of a physical binding model for PLP. In combination, our data support the conclusion that PLP exerts its main inhibitory effect by binding to residues located within the two substrate binding sites of the CTP, with Lys-83 being the primary determinant of the total PLP effect since the replacement of this single lysine abolishes nearly all of the observed inhibition by PLP.

The objective of the present investigation was to identify the substrate binding site(s) within the yeast mitochondrial citrate transport protein (CTP). Our strategy involved kinetically characterizing 30 single-Cys CTP mutants that we had previously constructed based on their hypothesized importance in the structure-based mechanism of this carrier. As part of these studies, a modified transport assay was developed that permitted, for the first time, the accurate determination of K(m) values that were elevated >100-fold compared with the Cys-less control value. We identified 10 single-Cys CTP mutants that displayed sharply elevated K(m) values (i.e. 5 to >300-fold). Each of these mutants displayed V(max) values that were reduced by > or = 98% and resultant catalytic efficiencies that were reduced by > or = 99.9%. Importantly, superposition of this functional data onto the three-dimensional homology-modeled CTP structure, which we previously had developed, revealed that nine of these ten residues form two topographically distinct clusters. Additional modeling showed that: (i) each cluster is capable of forming numerous hydrogen bonds with citrate and (ii) the two clusters are sufficiently distant from one another such that citrate is unlikely to interact with all of these residues at the same time. We deduced from these findings that the CTP contains at least two citrate binding sites per monomer, which are located at increasing depths within the translocation pathway. The identification of these sites, combined with an initial assessment of the citrate-amino acid side-chain interactions that may occur at these sites, substantially extends our understanding of CTP functioning at the molecular level.

Previous examination of the accessibility of a panel of single-Cys mutants in transmembrane domain III (TMDIII) of the yeast mitochondrial citrate transport protein to the hydrophilic, cysteine-specific methanethiosulfonate reagent MTSES enabled identification of the water-accessible surface of this TMD. Further studies on the effect of citrate on MTS reagent accessibility, indicated eight sites within TMD III at which citrate conferred temperature-independent protection, thus providing strong evidence for participation of these residues in the formation of a portion of the substrate translocation pathway. Unexpectedly, citrate did not protect against inhibition of the Leu120Cys variant, despite its location on a water- and citrate-accessible surface of the TMDIII helix. This led to the hypothesis that in the 3-dimensional CTP structure, TMDIV packs against TMDIII in a manner such that the Leu120 side-chain folds behind the side-chain of Gln182. The present investigations addressed this hypothesis by examining the properties of the Gln182Cys single mutant and the Leu120Cys/Gln182Ala double mutant. We observed that in contrast to our findings with the Leu120Cys mutant, citrate did protect the Gln182Cys variant against MTSES-mediated inhibition. Importantly, truncation of the Gln182 side-chain to Ala enabled citrate to protect the Leu120Cys double mutant against inhibition. In combination these data support the idea that the Gln182 side-chain lines the transport path and sterically blocks access of citrate to the Leu120 side-chain. In a parallel series of investigations, we constructed 24 single-Cys substitution mutants that were chosen based on their hypothesized importance in substrate binding and/or translocation. We observed that substitution of Cys for residues E34, K37, K83, R87, Y148, D236, K239, T240, R276, and R279 resulted in > or =98% inactivation of CTP function, suggesting an essential structural and/or mechanistic role for these native residues. Superposition of this functional data onto a detailed 3-dimensional homology model of the CTP structure indicates that the side-chains of each of these residues project into the putative transport pathway. We hypothesize that a subset of these residues, in combination with four previously identified essential residues, define the citrate binding site(s) within the CTP.

Previous examination of the accessibility of a panel of single-Cys mutants in transmembrane domain III (TMDIII) of the yeast mitochondrial citrate transport protein to hydrophilic, cysteine-specific methanethiosulfonate reagents, enabled identification of the water-accessible surface of this domain and suggested its potential participation in the formation of a portion of the substrate translocation pathway. To evaluate this idea, we conducted a detailed characterization of the functional properties of 20 TMDIII single-Cys substitution mutants. Kinetic studies indicate that the A118C, S123C, and K134C mutants displayed a 3- to 7-fold increase in K(m). Moreover, the A118C mutation caused a doubling of the V(max) value, whereas the S123C, E131C, and K134C mutations caused V(max) to dramatically decrease, resulting in a reduction of the catalytic efficiencies of these three mutants by >97%. Examination of the ability of citrate to protect against the inhibition mediated by sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) indicated that citrate conferred significant protection of cysteines substituted at eight water-accessible locations (i.e. Gly-115, Leu-116, Gly-117, Leu-121, Ser-123, Val-127, Glu-131, and Thr-135), but not at other sites. Importantly, similar levels of protection were observed at both 4 degrees C and 20 degrees C. The temperature independence of the protection indicates that substrate binding and/or occupancy of the transport pathway sterically blocks the access of MTSES to these sites, thereby providing direct protection, without involvement of a major protein conformational change. The significance of these extensive functional investigations is discussed in terms of the three-dimensional CTP homology model that we previously developed and a new model of the dimer interface.

We have used homology modeling to construct a three-dimensional model of the yeast mitochondrial citrate transport protein (CTP), based on the recently published x-ray crystal structure of another mitochondrial transport protein, the ADP/ATP carrier. Superposition of the backbone traces of the homology-modeled CTP onto the crystallographically determined ADP carrier structure indicates that the CTP transmembrane domains are well modeled (i.e., root mean square deviation of 0.94 A), whereas the loops facing the intermembrane space and the mitochondrial matrix are less certain (i.e., root mean square deviation values of 0.72-2.06 A). The homology-modeled CTP is consistent with our earlier de novo models of the transporter's transmembrane domains, with respect to residues which face into the transport path. Importantly, the resulting model is consistent with our previous experimental data obtained from measuring reactivity of 34 single cysteine mutants in transmembrane domains 3 and 4 with methanethiosulfonate reagents. The model also points to a likely dimer interface region. In conclusion, our data help to define the substrate translocation pathway in both the modeled CTP structure and the crystallographic ADP carrier structure.

The mitochondrial citrate transport protein (CTP) has been investigated by mutating 28 consecutive residues within transmembrane domain III (TMDIII), one at a time, to cysteine. A cysteine-less CTP that retains wild-type functional properties, served as the starting template. The single Cys CTP mutants were abundantly expressed in Escherichia coli, isolated, and functionally reconstituted in a liposomal system. The accessibility of each single Cys mutant to two methanethiosulfonate reagents was evaluated by determining the rate constants for inhibition of CTP function. These rate constants varied by over five orders of magnitude. With two independent data sets we observed peaks and troughs in the rate constant data at identical amino acid positions and a periodicity of 4 was observed from residues 123-137. Based on the pattern of accessibility we conclude that residues 123-137 exist as an alpha-helix. Although less certain, a combination of the rate constant data and the specific activity data with the single Cys mutants suggests that the alpha-helical secondary structure may extend to residue 113. Furthermore, the rate constant data define water-accessible and water-inaccessible faces of the helix. We infer that the water-accessible face comprises a portion of the substrate translocation pathway through the CTP, whereas the water-inaccessible surface faces the lipid bilayer. Finally, based on a combination of the CTP inhibition rate constant data and the existence of significant sequence identity with a transmembrane segment within glycophorin A that forms a portion of its dimer interface, a model for a putative CTP TMDIII-TMDIII' dimer interface has been developed.

To explore the spatial organization and functional dynamics of the citrate transport protein (CTP), a nitroxide scan was carried out along 22 consecutive residues within the fourth transmembrane domain (TMDIV). This domain has been implicated as being of unique importance to the CTP mechanism due to (i) the presence of two intramembranous positive charges that are essential for CTP function and (ii) the existence of a transmembrane aqueous surface within this domain which likely corresponds to a portion of the citrate translocation pathway. The sequence-specific variation in the mobilities of the introduced nitroxides and their accessibilities to molecular O(2) reveal an alpha-helical conformation along the sequence. The accessibilities to NiEDDA are out of phase with accessibilites to O(2), indicating that one face of the helix is solvated by the lipid bilayer while the other is solvated by an aqueous environment. A gradient of NiEDDA accessibility is observed along the helix surface facing the aqueous phase, and the EPR spectral line shapes at these sites indicate considerable motional restriction. In the context of the model where TMDIV lines the translocation pathway, these data suggest a barrier to passive diffusion through the pathway. This paper reports the first use of site-directed spin labeling to study mitochondrial transporter structure.

The mitochondrial citrate transport protein (CTP) has been investigated by replacing 22 consecutive residues within transmembrane domain IV, one at a time, with cysteine. A cysteine-less CTP retaining wild-type functional properties served as the starting template. The single Cys CTP variants were overexpressed in Escherichia coli, isolated, and functionally reconstituted in a liposomal system. The accessibility of each single Cys mutant to three methanethiosulfonate reagents was evaluated by determining the pseudo first order rate constants for inhibition of CTP function. These rate constants varied by seven orders of magnitude. With three independent data sets we observed peaks and troughs in the rate constant data at identical amino acid positions and a periodicity of four was observed from residues 177-193. Based on the pattern of accessibility we conclude that residues 177-193 exist as an alpha-helix. Furthermore, a water-accessible face of the helix has been defined consisting of Pro-177, Val-178, Arg-181, Gln-182, Asn-185, Gln-186, Arg-189, Leu-190, and Tyr-193, and a water-inaccessible face has been delineated consisting of Ser-179, Met-180, Ala-183, Ala-184, Ala-187, Val-188, Gly-191, and Ser-192. We infer that the water-accessible face comprises a portion of the substrate translocation pathway through the CTP, whereas the water-inaccessible surface faces the lipid bilayer.

Utilizing site-directed mutagenesis in combination with chemical modification of mutated residues, we have studied the roles of cysteine and arginine residues in the mitochondrial citrate transport protein (CTP) from Saccharomyces cerevisiae. Our strategy consisted of the sequential replacement of each of the four endogenous cysteine residues with Ser or in the case of Cys(73) with Val. Wild-type and mutated forms of the CTP were overexpressed in Escherichia coli, purified, and reconstituted in phospholipid vesicles. During the sequential replacement of each Cys, the effects of both hydrophilic and hydrophobic sulfhydryl reagents were examined. The data indicate that Cys(73) and Cys(256) are primarily responsible for inhibition of the wild-type CTP by hydrophilic sulfhydryl reagents. Experiments conducted with triple Cys replacement mutants (i.e. Cys(192) being the only remaining Cys) indicated that sulfhydryl reagents no longer inhibit but in fact stimulate CTP function 2-3-fold. Following the simultaneous replacement of all four endogenous Cys, the functional properties of the resulting Cys-less CTP were shown to be quite similar to those of the wild-type protein. Finally, utilizing the Cys-less CTP as a template, the roles of Arg(181) and Arg(189), two positively charged residues located within transmembrane domain IV, in CTP function were examined. Replacement of either residue with a Cys abolishes function, whereas replacement with a Lys or a Cys that is subsequently covalently modified with (2-aminoethyl)methanethiosulfonate hydrobromide, a reagent that restores positive charge at this site, supports CTP function. The results clearly show that positive charge at these two positions is essential for CTP function, although the chemistry of the guanidinium residue is not. Finally, these studies: (i) definitely demonstrate that Cys residues do not play an important role in the mechanism of the CTP; (ii) prove the utility of the Cys-less CTP for studying structure/function relationships within this metabolically important protein; and (iii) have led to the hypothesis that the polar face of alpha-helical transmembrane domain IV, within which Arg(181), Arg(189), and Cys(192) are located, constitutes an essential portion of the citrate translocation pathway through the membrane.

Experiments have been conducted to determine the oligomeric state of the mitochondrial citrate transport protein (CTP) from the yeast Saccharomyces cerevisiae. Both wild-type and cysteine-less (Cys-less) CTPs were overexpressed in E. coli and solubilized with sarkosyl. The purity of the solubilized material is approximately 75%. Upon incorporation into phospholipid vesicles, a high specific transport activity is obtained with both the wild-type and Cys-less CTPs, thereby demonstrating the structural and functional integrity of the preparations. Two independent approaches were utilized to determine native molecular weight. First, CTP molecular weight was determined via nondenaturing size-exclusion chromatography. With this methodology we obtained molecular weight values of 70,961 and 70,118 for the wild-type and Cys-less CTPs, respectively. Second, charge-shift native gel electrophoresis was carried out utilizing a low concentration of the negatively charged detergent sarkosyl, which served to both impart a charge shift to the CTP and the protein standards, as well as to promote protein solubility. Via the second method, we obtained molecular weight values of 69,122 and 74,911 for the wild-type and Cys-less CTPs, respectively. Both methods clearly indicate that following solubilization, the wild-type and the Cys-less CTPs exist exclusively as dimers. Furthermore, disulfide bonds are not required for either dimer formation or stabilization. The dimeric state of the CTP has important implications for the structural basis underlying the CTP translocation mechanism.

We investigated Ca2+ handling in airway smooth muscle (SM) using fura 2 fluorescence, ion currents, and contractions as indexes of intracellular Ca2+ concentration ([Ca2+]i). Carbachol evoked a transient elevation of [Ca2+]i, the magnitude of which was smaller and the rate of decay faster at 37 degrees C, indicating that some temperature-sensitive mechanism contributed to recovery. Removal of external Na+ had no effect on agonist-evoked Ca2+ transients or contractions or on spontaneous Ca(2+)-dependent K+ currents. Cyclopiazonic acid, a selective inhibitor of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, evoked a transient elevation of [Ca2+]i and contraction, markedly slowed recovery of the cholinergic Ca2+ transient, and depleted the SR. Sodium vanadate evoked a sustained elevation of [Ca2+]i and markedly slowed the decay of the cholinergic Ca2+ transient. We conclude that, in canine airway SM, 1) Na+/Ca2+ exchange makes at best only a minor contribution to Ca2+ homeostasis, 2) the SR Ca(2+)-ATPase compensates for spontaneous and agonist-triggered release of Ca2+, and 3) [Ca2+]i homeostasis involves some other extrusion pathway, likely the plasmalemmal Ca(2+)-ATPase.

Abstract: 2-Chlorodeoxyadenosine (CldAdo, Cladribine), a nucleoside analog used in the treatment of hairy cell leukemia, is phosphorylated and incorporated into DNA, but is not an absolute chain terminator. We hypothesized that the presence of a chlorine molecule projecting into the DNA minor groove would affect DNA:protein-binding interactions. Here, we investigated recognition of and binding to double-stranded CldAMP-substituted TATA promoter sequences by human TATA-binding protein (TBP) using mobility shift assays. Depending on the site, CldAMP in place of dAMP within a TATA sequence decreased in vitro TBP binding by ∼30% to 55% compared to control sites. When bound to a CldAMP-substituted TATA box, however, the TBP complex was more resistant to polyanions, suggesting enhanced stability. Limited exposure of the TBP:DNA complex to proteases indicated that TBP conformation was altered on CldAMP-substituted DNA compared to control. Further, binding of transcription factor IIB to TBP was diminished on analog-containing TATA sequences. These results suggest normal TBP-binding interactions—specifically recognition, stability, and conformation—are disrupted by CldAMP insertion into eukaryotic promoter sequences. [Copyright &y& Elsevier]

The objective of the present investigation was to identify the substrate binding site(s) within the yeast mitochondrial citrate transport protein (CTP). Our strategy involved kinetically characterizing 30 single-Cys CTP mutants that we had previously constructed based on their hypothesized importance in the structure-based mechanism of this carrier. As part of these studies, a modified transport assay was developed that permitted, for the first time, the accurate determination of Km values that were elevated >100-fold compared with the Cys-less control value. We identified 10 single-Cys CTP mutants that displayed sharply elevated Km values (i.e. 5 to >300-fold). Each of these mutants displayed Vmax values that were reduced by ≥98% and resultant catalytic efficiencies that were reduced by ≥99.9%. Importantly, superposition of this functional data onto the three-dimensional homology-modeled CTP structure, which we previously had developed, revealed that nine of these ten residues form two topographically distinct clusters. Additional modeling showed that: (i) each cluster is capable of forming numerous hydrogen bonds with citrate and (ii) the two clusters are sufficiently distant from one another such that citrate is unlikely to interact with all of these residues at the same time. We deduced from these findings that the CTP contains at least two citrate binding sites per monomer, which are located at increasing depths within the translocation pathway. The identification of these sites, combined with an initial assessment of the citrate-amino acid side-chain interactions that may occur at these sites, substantially extends our understanding of CTP functioning at the molecular level. [ABSTRACT FROM AUTHOR]

The interaction between IgE and its high affinity receptor (Fc∈RI) is a critical step in the development of allergic responses. Detailed characterization of the IgEFc∈RI interaction may offer insights into possible modes of inhibiting the interaction, which could thereby act as a potential therapy for allergy. In this study, NMR, CD, and fluorescence spectroscopies have been used to characterize structurally the C∈3 domain of IgE and its interaction with other protein ligands, namely, C∈2, C∈4, sFc∈RIα, and CD23. We have shown that the recombinant C∈3 domain exists alone in solution as a "molten globule." On interaction with sFc∈RIα, C∈3 adopts a folded tertiary structure, as shown by the release of the fluorescent probe 8-anilinonaphthalene-1-sulfonate and by characteristic changes in the ¹H, 15N heteronuclear single quantum coherence NMR spectrum. However, the interactions between the C∈3 domain and C∈2, C∈4, or CD23 do not induce such folding and would therefore be expected to involve only local interaction surfaces. The conformational flexibility of the C∈3 domain of the whole IgE molecule may play a role in allowing fine tuning of the affinity and specificity of IgE for a variety of different physiological ligands and may be involved in the conformational change of IgE postulated to occur on interaction with Fc∈RI. [ABSTRACT FROM AUTHOR]

Describes the properties of the mitochondrial citrate transport protein. Substrate translocation pathways; Dimer interfaces; Liposomal system; Escherichia coli.