Regulation of Aerobic Succinate Transporter dctA of E. coli by cAMP-CRP, DcuS-DcuR, and EIIAGlc: Succinate as a Carbon Substrate and Signaling Molecule.

INTRODUCTION: C4-dicarboxylates (C4-DC) have emerged as significant growth substrates and signaling molecules for various Enterobacteriaceae during their colonization of mammalian hosts. Particularly noteworthy is the essential role of fumarate respiration during colonization of pathogenic bacteria. To investigate the regulation of aerobic C4-DC metabolism, the study explored the transcriptional control of the main aerobic C4-DC transporter, dctA, under different carbohydrate conditions. In addition, mutants related to carbon catabolite repression (CCR) and C4-DC regulation (DcuS-DcuR) were examined to better understand the regulatory integration of aerobic C4-DC metabolism into CCR. For initial insight into posttranslational regulation, the interaction between the aerobic C4-DC transporter DctA and EIIAGlc from the glucose-specific phosphotransferase system was investigated. METHODS: The expression of dctA was characterized in the presence of various carbohydrates and regulatory mutants affecting CCR. This was accomplished by fusing the dctA promoter (PdctA) to the lacZ reporter gene. Additionally, the interaction between DctA and EIIAGlc of the glucose-specific phosphotransferase system was examined in vivo using a bacterial two-hybrid system. RESULTS: The dctA promoter region contains a class I cAMP-CRP-binding site at position -81.5 and a DcuR-binding site at position -105.5. DcuR, the response regulator of the C4-DC-activated DcuS-DcuR two-component system, and cAMP-CRP stimulate dctA expression. The expression of dctA is subject to the influence of various carbohydrates via cAMP-CRP, which differently modulate cAMP levels. Here we show that EIIAGlc of the glucose-specific phosphotransferase system strongly interacts with DctA, potentially resulting in the exclusion of C4-DCs when preferred carbon substrates, such as sugars, are present. In contrast to the classical inducer exclusion known for lactose permease LacY, inhibition of C4-DC uptake into the cytoplasm affects only its role as a substrate, but not as an inducer since DcuS detects C4-DCs in the periplasmic space ("substrate exclusion"). The work shows an interplay between cAMP-CRP and the DcuS-DcuR regulatory system for the regulation of dctA at both transcriptional and posttranslational levels. CONCLUSION: The study highlights a hierarchical interplay between global (cAMP-CRP) and specific (DcuS-DcuR) regulation of dctA at the transcriptional and posttranslational levels. The integration of global and specific transcriptional regulation of dctA, along with the influence of EIIAGlc on DctA, fine-tunes C4-DC catabolism in response to the availability of other preferred carbon sources. It attributes DctA a central role in the control of aerobic C4-DC catabolism and suggests a new role to EIIAGlc on transporters (control of substrate uptake by substrate exclusion).

Asuc_0142 of Actinobacillus succinogenes 130Z is the l-aspartate/C4-dicarboxylate exchanger DcuA.

Anaerobic bacteria often use antiporters DcuB (malate/succinate antiport) or DcuA (l-aspartate/succinate antiport) for the excretion of succinate during fumarate respiration. The rumen bacterium Actinobacillus succinogenes is able to produce large amounts of succinate by fumarate respiration, using the DcuB-type transporter DcuE for l-malate/succinate antiport. Asuc_0142 was annotated as a second DcuB-type transporter. Deletion of Asuc_0142 decreased the uptake rate for l-[14C]aspartate into A. succinogenes cells. Properties of transport by heterologously expressed Asuc_0142 were investigated in an Escherichia coli mutant deficient of anaerobic C4DC transporters. Expression of Asuc_0142 resulted in high uptake activity for l-[14C]fumarate or l-[14C]aspartate, but the former showed a strong competitive inhibition by l-aspartate. In E. coli loaded with l-[14C]aspartate, [14C]succinate or [14C]fumarate, extracellular C4DCs initiated excretion of the intracellular substrates, with a preference for l-aspartateex/succinatein or l-aspartateex/fumaratein antiport. These findings indicate that Asuc_0142 represents a DcuA-type transporter for l-aspartate uptake and l-aspartateex/C4DCin antiport, differentiating it from the DcuB-type transporter DcuE for l-malateex/succinatein antiport. Sequence analysis and predicted structural characteristics confirm structural similarity of Asuc_0142 to DcuA, and Asuc_0142 was thus re-named as DcuAAs. The bovine rumen fluid contains l-aspartate (99.6 µM), whereas fumarate and l-malate are absent. Therefore, bovine rumen colonisers depend on l-aspartate as an exogenous substrate for fumarate respiration. A. succinogenes encodes HemG (protoporphyrinogen oxidase) and PyrD (dihydroorotate dehydrogenase) for haem and pyrimidine biosynthesis. The enzymes require fumarate as an electron acceptor, suggesting an essential role for l-aspartate, DcuAAs, and fumarate respiration for A. succinogenes growing in the bovine rumen.

Fumarate, a central electron acceptor for Enterobacteriaceae beyond fumarate respiration and energy conservation.

C4-dicarboxylates (C4-DCs) such as fumarate, l-malate and l-aspartate are key substrates for Enterobacteria such as Escherichia coli or Salmonella typhimurium during anaerobic growth. In general, C4-DCs are oxidants during biosynthesis, e.g., of pyrimidine or heme, acceptors for redox balancing, a high-quality nitrogen source (l-aspartate) and electron acceptor for fumarate respiration. Fumarate reduction is required for efficient colonization of the murine intestine, even though the colon contains only small amounts of C4-DCs. However, fumarate can be produced endogenously by central metabolism, allowing autonomous production of an electron acceptor for biosynthesis and redox balancing. Bacteria possess a complex set of transporters for the uptake (DctA), antiport (DcuA, DcuB, TtdT) and excretion (DcuC) of C4-DCs. DctA and DcuB exert regulatory functions and link transport to metabolic control through interaction with regulatory proteins. The sensor kinase DcuS of the C4-DC two-component system DcuS-DcuR forms complexes with DctA (aerobic) or DcuB (anaerobic), representing the functional state of the sensor. Moreover, EIIAGlc from the glucose phospho-transferase system binds to DctA and presumably inhibits C4-DC uptake. Overall, the function of fumarate as an oxidant in biosynthesis and redox balancing explains the pivotal role of fumarate reductase for intestinal colonization, while the role of fumarate in energy conservation (fumarate respiration) is of minor importance.

Dissecting key residues of a C4-dicarboxylic acid transporter to accelerate malate export in Myceliophthora.

Efficient transporters are necessary for high concentration and purity of desired products during industrial production. In this study, we explored the mechanism of substrate transport and preference of the C4-dicarboxylic acid transporter AoMAE in the fungus Myceliophthora thermophila, and investigated the roles of 18 critical amino acid residues within this process. Among them, the residue Arg78, forming a hydrogen bond network with Arg23, Phe25, Thr74, Leu81, His82, and Glu94 to stabilize the protein conformation, is irreplaceable for the export function of AoMAE. Furthermore, varying the residue at position 100 resulted in changes to the size and shape of the substrate binding pocket, leading to alterations in transport efficiencies of both malic acid and succinic acid. We found that the mutation T100S increased malate production by 68%. Using these insights, we successfully generated an AoMAE variant with mutation T100S and deubiquitination, exhibiting an 81% increase in the selective export activity of malic acid. Simply introducing this version of AoMAE into M. thermophila wild-type strain increased production of malic acid from 1.22 to 54.88 g/L. These findings increase our understanding of the structure-function relationships of organic acid transporters and may accelerate the process of engineering dicarboxylic acid transporters with high efficiency. KEY POINTS: • This is the first systematical analysis of key residues of a malate transporter in fungi. • Protein engineering of AoMAE led to 81% increase of malate export activity. • Arg78 was essential for the normal function of AoMAE in M. thermophila. • Substitution of Thr100 affected export efficiency and substrate selectivity of AoMAE.

The role of PYCR1 in inhibiting 5-fluorouracil-induced ferroptosis and apoptosis through SLC25A10 in colorectal cancer.

Although PYCR1 is a well-recognized oncogenic gene for malignant tumors, the causal relationship of its expression with malignant growth and cytotoxic chemotherapeutics remains unclear. Therefore, this study aimed to clarify the role of PYCR1 and its interaction with SLC25A10 in a chemotherapeutic agent 5-fluorouracil (5-FU)'s toxicity to colorectal cancer cells. PYCR1 and SLC25A10 expressions were detected in The Cancer Genome Atlas database and colon adenocarcinoma (COAD) clinical samples. PYCR1 upregulation was associated with SLC25A10 expression and poor prognosis, and its high expression indicated decreased survival rates in patients with COAD. PYCR1 overexpression inhibited lipid reactive oxygen species production and promoted SLC25A10 expression in colorectal cancer cells. PYCR1 silencing enhanced the antitumor effects of 5-FU. Ferroptosis inhibitor deferoxamine suppressed the antitumor effects of PYCR1 silencing, whereas ferroptosis inducer erastin inhibited the protumor effects of PYCR1 overexpression. SLC25A10 overexpression reversed the antitumor effects of PYCR1 silencing in vitro and inhibited the antitumor effects of erastin in vivo. Therefore, PYCR1 is an oncogenic gene that promotes colorectal tumor growth and desensitizes colorectal cancer cells to 5-FU cytotoxicity by preventing apoptosis and ferroptosis.

Identification and validation of a gene-based signature reveals SLC25A10 as a novel prognostic indicator for patients with ovarian cancer.

BACKGROUND: Ovarian cancer is a common gynecological cancer with poor prognosis and poses a serious threat to woman life and health. In this study, we aimed to establish a prognostic signature for the risk assessment of ovarian cancer. METHODS: The Cancer Genome Atlas (TCGA) dataset was used as the training set and the International Cancer Genome Consortium (ICGC) dataset was set as an independent external validation. A multi-stage screening strategy was used to determine the prognostic features of ovarian cancer with R software. The relationship between the prognosis of ovarian cancer and the expression level of SLC25A10 was selected for further analysis. RESULTS: A total of 16 prognosis-associated genes were screened to construct the risk score signature. Survival analysis showed that patients in the high-risk score group had a poor prognosis compared to the low-risk group. Accuracy of this prognostic signature was confirmed by the receiver operating characteristic (ROC) curve and decision curve analysis (DCA), and validated with ICGC cohort. This signature was identified as an independent factor for predicting overall survival (OS). Nomogram constructed by multiple clinical parameters showed excellent performance for OS prediction. Finally, it's found that patients with low expression of SLC25A10 generally had poor survival and higher resistance to most chemotherapeutic drugs. CONCLUSIONS: In sum, we developed a 16-gene prognostic signature, which could serve as a promising tool for the prognostic prediction of ovarian cancer, and the expression level of SLC25A10 was tightly associated with OS of the patients.

Role of Plasma Membrane Dicarboxylate Transporters in the Uptake and Toxicity of Diglycolic Acid, a Metabolite of Diethylene Glycol, in Human Proximal Tubule Cells.

Diethylene glycol (DEG) mass poisonings have resulted from ingestion of pharmaceuticals mistakenly adulterated with DEG, typically leading to proximal tubular necrosis and acute kidney injury. The metabolite, diglycolic acid (DGA) accumulates greatly in kidney tissue and its direct administration results in toxicity identical to that in DEG-treated rats. DGA is a dicarboxylic acid, similar in structure to metabolites like succinate. These studies have assessed the mechanism for cellular accumulation of DGA, specifically whether DGA is taken into primary cultures of human proximal tubule (HPT) cells via sodium dicarboxylate transporters (NaDC-1 or NaDC-3) like those responsible for succinate uptake. When HPT cells were cultured on membrane inserts, sodium-dependent succinate uptake was observed from both apical and basolateral directions. Pretreatment with the NaDC-1 inhibitor N-(p-amylcinnamoyl)anthranilic acid (ACA) markedly reduced apical uptakes of both succinate and DGA. Basolateral uptake of both succinate and DGA were decreased similarly following combined treatment with ACA and the NaDC-3 inhibitor 2,3-dimethylsuccinate. When the cells were pretreated with siRNA to knockdown NaDC-1 function, apical uptake of succinate and toxicity of apically applied DGA were reduced, while the reduction in basolateral succinate uptake and basolateral DGA toxicity was marginal with NaDC-3 knockdown. DGA reduced apical uptake of succinate but not basolateral uptake. This study confirmed that primary HPT cells retain sodium dicarboxylate transport functionality and that DGA was taken up by these transporters. This study identified NaDC-1 as a likely and NaDC-3 as a possible molecular target to reduce uptake of this toxic metabolite by the kidney.

A mutation in the putative CRP binding site of the dctA promoter of Salmonella enterica serovar Typhimurium enables growth with low orotate concentrations.

Salmonella enterica and Escherichia coli use the inner membrane transporter DctA to import the pyrimidine biosynthetic pathway intermediate orotate from the environment. To study the regulation of dctA expression, we used an S. enterica serovar Typhimurium pyrimidine auxotroph to select a mutant that could grow in an otherwise nonpermissive culture medium containing glucose and a low concentration of orotate. Whole genome sequencing revealed a point mutation upstream of dctA in the putative cyclic AMP receptor protein (CRP) binding site. The C→T transition converted the least favourable base to the most favourable base for CRP-DNA affinity. A dctA::lux transcriptional fusion confirmed that the mutant dctA promoter gained responsiveness to CRP even in the presence of glucose. Moreover, dctA expression was higher in the mutant than the wild type in the presence of alternative carbon sources that activate CRP.

C4-dicarboxylate metabolons: interaction of C4-dicarboxylate transporters of Escherichia coli with cytosolic enzymes.

Metabolons represent the structural organization of proteins for metabolic or regulatory pathways. Here, the interaction of fumarase FumB, aspartase AspA, and L-tartrate dehydratase TtdAB with the C4-dicarboxylate (C4-DC) transporters DcuA, DcuB, DcuC, and the L-tartrate transporter TtdT of Escherichia coli was tested by a bacterial two-hybrid (BACTH) assay in situ, or by co-chromatography using mSPINE (membrane Streptavidin protein interaction experiment). From the general C4-DC transporters, DcuB interacted with FumB and AspA, DcuA with AspA, whereas DcuC interacted with neither FumB nor AspA. Moreover, TtdT did not interact with TtdAB. The fumB-dcuB, the dcuA-aspA, and the ttdAB-ttdT genes encoding the respective proteins colocalize on the genome and each pair of genes forms cotranscripts, whereas the dcuC gene lies alone. The data suggest the formation of DcuB/FumB and DcuB/AspA metabolons for the uptake of L-malate, or L-aspartate, and their conversion to fumarate for fumarate respiration and excretion of the product succinate. The DcuA/AspA metabolon catalyzes uptake and conversion of L-aspartate to fumarate coupled to succinate excretion. The DcuA/AspA metabolon provides ammonia at the same time for nitrogen assimilation (ammonia shuttle). On the other hand, TtdT and TtdAB are not organized in a metabolon. Reasons for the formation (DcuA/AspA, DcuB/FumB, and DcuB/AspA) or nonformation (DcuC, TtdT, and TtdAB) of metabolons are discussed based on their metabolic roles.

Tight Complex Formation of the Fumarate Sensing DcuS-DcuR Two-Component System at the Membrane and Target Promoter Search by Free DcuR Diffusion.

Signaling of two-component systems by phosphoryl transfer requires interaction of the sensor kinase with the response regulator. Interaction of the C4-dicarboxylate-responsive and membrane-integral sensor kinase DcuS with the response regulator DcuR was studied. In vitro, the cytoplasmic part of DcuS (PASC-Kin) was employed. Stable complexes were formed, when either DcuS or DcuR were phosphorylated (Kd 22 ± 11 and 28 ± 7 nM, respectively). The unphosphorylated proteins produced a more labile complex (Kd 1380 ± 395 nM). Bacterial two-hybrid studies confirm interaction of DcuR with DcuS (and PASC-Kin) in vivo. The absolute contents of DcuR (197-979 pmol mg-1 protein) in the bacteria exceeded those of DcuS by more than 1 order of magnitude. According to the Kd values, DcuS exists in complex, with phosphorylated but also unphosphorylated DcuR. In live cell imaging, the predominantly freely diffusing DcuR becomes markedly less mobile after phosphorylation and activation of DcuS by fumarate. Portions of the low mobility fraction accumulated at the cell poles, the preferred location of DcuS, and other portions within the cell, representing phosphorylated DcuR bound to promoters. In the model, acitvation of DcuS increases the affinity toward DcuR, leading to DcuS-P × DcuR formation and phosphorylation of DcuR. The complex is stable enough for phosphate-transfer, but labile enough to allow exchange between DcuR from the cytosol and DcuR-P of the complex. Released DcuR-P diffuses to target promoters and binds. Uncomplexed DcuR-P in the cytosol binds to nonactivated DcuS and becomes dephosphorylated. The lower affinity between DcuR and DcuS avoids blocking of DcuS and allows rapid exchange of DcuR. IMPORTANCE Complex formation of membrane-bound sensor kinases with the response regulators represents an inherent step of signaling from the membrane to the promoters on the DNA. In the C4-dicarboxylate-sensing DcuS-DcuR two-component system, complex formation is strengthened by activation (phosphorylation) in vitro and in vivo, with trapping of the response regulator DcuR at the membrane. Single-molecule tracking of DcuR in the bacterial cell demonstrates two populations of DcuR with decreased mobility in the bacteria after activation: one at the membrane, but a second in the cytosol, likely representing DNA-bound DcuR. The data suggest a model with binding of DcuR to DcuS-P for phosphorylation, and of DcuR-P to DcuS for dephosphorylation, allowing rapid adaptation of the DcuR phosphorylation state. DcuR-P is released and transferred to DNA by 3D diffusion.

Genomic rearrangements in the aspA-dcuA locus of Propionibacterium freudenreichii are associated with aspartase activity.

Propionibacterium freudenreichii is crucial in Swiss-type cheese manufacture. Classic propionic acid fermentation yields the nutty flavor and the typical eyes. Co-metabolism of aspartate pronounces the flavor of the cheese; however, it also increases the size of the eyes, which can induce splitting and reduce the cheese quality. Aspartase (EC 4.3.1.1) catalyzes the deamination of aspartate, yielding fumarate and ammonia. The aspartase activity varies considerably among P. freudenreichii strains. Here, the correlation between aspartase activity and the locus of aspartase-encoding genes (aspA ) and dcuA encoding the C4-dicarboxylate transporter was investigated in 46 strains to facilitate strain selection for cheese culture. Low aspartase activity was correlated with a particular genomic rearrangement: low in vitro aspartase activity always occurred in strains with gene clusters aspA - dcuA where the dcuA was frameshifted, producing a stop codon or was disrupted by an ISL3-like element. The low aspartase activity could be due to the protein sequence of the aspartase or a dysfunctional DcuA. The highest values of aspartase activity were detected in strains with aspA1 - aspA2-dcuA with a DcuA sequence sharing 99.07 - 100% identity with the DcuA sequence of strain DSM 20271 T and an additional C4-dicarboxylate transporter belonging to the DcuAB family.

Structural basis of ion - substrate coupling in the Na+-dependent dicarboxylate transporter VcINDY.

The Na+-dependent dicarboxylate transporter from Vibrio cholerae (VcINDY) is a prototype for the divalent anion sodium symporter (DASS) family. While the utilization of an electrochemical Na+ gradient to power substrate transport is well established for VcINDY, the structural basis of this coupling between sodium and substrate binding is not currently understood. Here, using a combination of cryo-EM structure determination, succinate binding and site-directed cysteine alkylation assays, we demonstrate that the VcINDY protein couples sodium- and substrate-binding via a previously unseen cooperative mechanism by conformational selection. In the absence of sodium, substrate binding is abolished, with the succinate binding regions exhibiting increased flexibility, including HPinb, TM10b and the substrate clamshell motifs. Upon sodium binding, these regions become structurally ordered and create a proper binding site for the substrate. Taken together, these results provide strong evidence that VcINDY's conformational selection mechanism is a result of the sodium-dependent formation of the substrate binding site.

Compound Heterozygous Mutations Presented with Quadriparesis and Menopause. A Case Report.

Mitochondrion regulates cellular metabolism with the aid of its respiratory complexes; any defect within these complexes can result in mitochondrial malfunction and various conditions. One such mutation can occur in SLC25A10, resulting in mitochondrial DNA depletion syndrome. It should be noted that the pattern of inheritance of this syndrome is autosomal recessive. However, we present a case with compound heterozygous mutations within this gene resulting in disease. An 18-year-old female was referred to our clinic due to menopause with a medical history of hearing loss, spasticity, hypotonia and quadriparesis. The child's birth and development were uneventful until the initiation of movement reduction and hypotonia when she was 12 months old. Afterward, the hypotonia progressed to quadriparesis and spasticity throughout the years. Our patient became completely quadriplegic up to the age of 3 and became completely deaf at 10. Her puberty onset was at the age of 9, and no significant event took place until she was 17 years old when suddenly her periods, which were regular until that time, became irregular and ceased after a year; hence, a thorough evaluation began, but similar to her previous evaluations all tests were insignificant. Nonetheless, we suspected an underlying metabolic or genetic defect; thus, we ordered a whole-exome sequencing (WES) workup and found simultaneous heterozygous mutations within SLC25A10, HFE and TTN genes that could explain her condition. When all other tests fail, and we suspect an underlying genetic or metabolic cause, WES can be of great value.

Gliomas with Downregulation of lncRNA SLC25A21-AS1 Carry a Dismal Prognosis and an Accelerated Progression in Cell Proliferation, Migration and Invasion.

Glioma is one type of primary intracranial carcinoma with a relatively poor prognosis. We investigated the level of SLC25A21-AS1 in gliomas and the association with survival and progression in patients with glioma. Specimens of gliomas from patients were assessed by quantitative real-time polymerase chain reaction analysis of the SLC25A21-AS1 level (117 specimens). For prognostic value assessment, χ2 test, Kaplan-Meier method with the log-rank test, and Multivariate survival analysis were performed. The direct targets for SLC25A21-AS1 were explored. The biological roles of SLC25A21-AS1 were investigated by manipulating the expression level of SLC25A21-AS1 in glioma cells. SLC25A21-AS1 was significantly downregulated in glioma specimens and cell lines compared to non-cancerous ones. Significant associations were found between SLC25A21-AS1 downregulation and WHO stage, IDH status, poor disease-free survival/overall survival. miR-221-3p/miR-222-3p were the target miRNAs for SLC25A21-AS1. Overexpression of SLC25A21-AS1 inhibited glioma cell growth, invasion, and migration while miR-221-3p/miR-222-3p-overexpressed groups could offset this effect. Downregulation of SLC25A21-AS1 in gliomas carries a universally poor prognosis. Overexpression of SLC25A21-AS1 inhibited glioma progression via miR-221-3p/miR-222-3p.

C4-Dicarboxylates as Growth Substrates and Signaling Molecules for Commensal and Pathogenic Enteric Bacteria in Mammalian Intestine.

The C4-dicarboxylates (C4-DC) l-aspartate and l-malate have been identified as playing an important role in the colonization of mammalian intestine by enteric bacteria, such as Escherichia coli and Salmonella enterica serovar Typhimurium, and succinate as a signaling molecule for host-enteric bacterium interaction. Thus, endogenous and exogenous fumarate respiration and related functions are required for efficient initial growth of the bacteria. l-Aspartate represents a major substrate for fumarate respiration in the intestine and a high-quality substrate for nitrogen assimilation. During nitrogen assimilation, DcuA catalyzes an l-aspartate/fumarate antiport and serves as a nitrogen shuttle for the net uptake of ammonium only, whereas DcuB acts as a redox shuttle that catalyzes the l-malate/succinate antiport during fumarate respiration. The C4-DC two-component system DcuS-DcuR is active in the intestine and responds to intestinal C4-DC levels. Moreover, in macrophages and in mice, succinate is a signal that promotes virulence and survival of S. Typhimurium and pathogenic E. coli. On the other hand, intestinal succinate is an important signaling molecule for the host and activates response and protective programs. Therefore, C4-DCs play a major role in supporting colonization of enteric bacteria and as signaling molecules for the adaptation of host physiology.

The ups and downs of elevator-type di-/tricarboxylate membrane transporters.

The divalent anion sodium symporter (DASS) family contains both sodium-driven anion cotransporters and anion/anion exchangers. The family belongs to a broader ion transporter superfamily (ITS), which comprises 24 families of transporters, including those of AbgT antibiotic efflux transporters. The human proteins in the DASS family play major physiological roles and are drug targets. We recently determined multiple structures of the human sodium-dependent citrate transporter (NaCT) and the succinate/dicarboxylate transporter from Lactobacillus acidophilus (LaINDY). Structures of both proteins show high degrees of structural similarity to the previously determined VcINDY fold. Conservation between these DASS protein structures and those from the AbgT family indicates that the VcINDY fold represents the overall protein structure for the entire ITS. The new structures of NaCT and LaINDY are captured in the inward- or outward-facing conformations, respectively. The domain arrangements in these structures agree with a rigid body elevator-type transport mechanism for substrate translocation across the membrane. Two separate NaCT structures in complex with a substrate or an inhibitor allowed us to explain the inhibition mechanism and propose a detailed classification scheme for grouping disease-causing mutations in the human protein. Structural understanding of multiple kinetic states of DASS proteins is a first step toward the detailed characterization of their entire transport cycle.

Adiposity associated DNA methylation signatures in adolescents are related to leptin and perinatal factors.

Epigenetics links perinatal influences with later obesity. We identifed differentially methylated CpG (dmCpG) loci measured at 17 years associated with concurrent adiposity measures and examined whether these were associated with hsCRP, adipokines, and early life environmental factors. Genome-wide DNA methylation from 1192 Raine Study participants at 17 years, identified 29 dmCpGs (Bonferroni corrected p < 1.06E-07) associated with body mass index (BMI), 10 with waist circumference (WC) and 9 with subcutaneous fat thickness. DmCpGs within Ras Association (RalGDS/AF-6), Pleckstrin Homology Domains 1 (RAPH1), Musashi RNA-Binding Protein 2 (MSI2), and solute carrier family 25 member 10 (SLC25A10) are associated with both BMI and WC. Validation by pyrosequencing confirmed these associations and showed that MSI2 , SLC25A10 , and RAPH1 methylation was positively associated with serum leptin. These were  also associated with the early environment; MSI2 methylation (ß = 0.81, p = 0.0004) was associated with pregnancy maternal smoking, SLC25A10 (CpG2 ß = 0.12, p = 0.002) with pre- and early pregnancy BMI, and RAPH1 (ß = -1.49, p = 0.036) with gestational weight gain. Adjusting for perinatal factors, methylation of the dmCpGs within MSI2, RAPH1, and SLC25A10 independently predicted BMI, accounting for 24% of variance. MSI2 methylation was additionally associated with BMI over time (17 years old ß = 0.026, p = 0.0025; 20 years old ß = 0.027, p = 0.0029) and between generations (mother ß = 0.044, p = 7.5e-04). Overall findings suggest that DNA methylation in MSI2, RAPH1, and SLC25A10 in blood may be robust markers, mediating through early life factors.

Metabolic flux from the Krebs cycle to glutamate transmission tunes a neural brake on seizure onset.

Kohlschütter-Tönz syndrome (KTS) manifests as neurological dysfunctions, including early-onset seizures. Mutations in the citrate transporter SLC13A5 are associated with KTS, yet their underlying mechanisms remain elusive. Here, we report that a Drosophila SLC13A5 homolog, I'm not dead yet (Indy), constitutes a neurometabolic pathway that suppresses seizure. Loss of Indy function in glutamatergic neurons caused "bang-induced" seizure-like behaviors. In fact, glutamate biosynthesis from the citric acid cycle was limiting in Indy mutants for seizure-suppressing glutamate transmission. Oral administration of the rate-limiting α-ketoglutarate in the metabolic pathway rescued low glutamate levels in Indy mutants and ameliorated their seizure-like behaviors. This metabolic control of the seizure susceptibility was mapped to a pair of glutamatergic neurons, reversible by optogenetic controls of their activity, and further relayed onto fan-shaped body neurons via the ionotropic glutamate receptors. Accordingly, our findings reveal a micro-circuit that links neural metabolism to seizure, providing important clues to KTS-associated neurodevelopmental deficits.

Expanding the phenotype: Four new cases and hope for treatment in Bachmann-Bupp syndrome.

Bachmann-Bupp syndrome (BABS) is a rare syndrome caused by gain-of-function variants in the C-terminus of ornithine decarboxylase (ODC coded by the ODC1 gene). BABS is characterized by developmental delay, macrocephaly, macrosomia, and an unusual pattern of non-congenital alopecia. Recent diagnosis of four more BABS patients provides further characterization of the phenotype of this syndrome including late-onset seizures in the oldest reported patient at 23 years of age, representing the first report for this phenotype in BABS. Neuroimaging abnormalities continue to be an inconsistent feature of the syndrome. This may be related to the yet unknown impact of ODC/polyamine dysregulation on the developing brain in this syndrome. Variants continue to cluster, providing support to a universal biochemical mechanism related to elevated ODC protein, enzyme activity, and abnormalities in polyamine levels. Recommendations for medical management can now be suggested as well as the potential for targeted molecular or metabolic testing when encountering this unique phenotype. The natural history of this syndrome will evolve with difluoromethylornithine (DFMO) therapy and raise new questions for further study and understanding.

The versatility of plant organic acid metabolism in leaves is underpinned by mitochondrial malate-citrate exchange.

Malate and citrate underpin the characteristic flexibility of central plant metabolism by linking mitochondrial respiratory metabolism with cytosolic biosynthetic pathways. However, the identity of mitochondrial carrier proteins that influence both processes has remained elusive. Here we show by a systems approach that DICARBOXYLATE CARRIER 2 (DIC2) facilitates mitochondrial malate-citrate exchange in vivo in Arabidopsis thaliana. DIC2 knockout (dic2-1) retards growth of vegetative tissues. In vitro and in organello analyses demonstrate that DIC2 preferentially imports malate against citrate export, which is consistent with altered malate and citrate utilization in response to prolonged darkness of dic2-1 plants or a sudden shift to darkness of dic2-1 leaves. Furthermore, isotopic glucose tracing reveals a reduced flux towards citrate in dic2-1, which results in a metabolic diversion towards amino acid synthesis. These observations reveal the physiological function of DIC2 in mediating the flow of malate and citrate between the mitochondrial matrix and other cell compartments.