Mitochondrial Thiamin Pyrophosphate Transporter Research Articles

MiR-122-5p is involved in post-transcriptional regulation of the mitochondrial thiamin pyrophosphate transporter (SLC25A19) in pancreatic acinar cells.

Thiamin (vitamin B1) plays a vital role in cellular energy metabolism/ATP production. Pancreatic acinar cells (PACs) obtain thiamin from circulation and convert it to thiamin pyrophosphate (TPP) in the cytoplasm. TPP is then taken up by the mitochondria via a carrier-mediated process that involves the mitochondrial TPP transporter (MTPPT; encoded by the gene SLC25A19). We have previously characterized different aspects of the mitochondrial carrier-mediated TPP uptake process, but nothing is known about its possible regulation at the post-transcriptional level. We address this issue in the current investigations focusing on the role of miRNAs in this regulation. First, we subjected the human (and rat) 3'-untranslated region (3'-UTR) of the SLC25A19 to three in-silico programs, and all have identified putative binding sites for miR-122-5p. Transfecting pmirGLO-hSLC25A19 3'-UTR into rat PAC AR42J resulted in a significant reduction in luciferase activity compared to cells transfected with pmirGLO-empty vector. Mutating as well as truncating the putative miR-122-5p binding sites in the hSLC25A19 3'-UTR led to abrogation of inhibition in luciferase activity in PAC AR42J. Further experiments with PAC AR42J and human primary PACs showed that transfecting/transducing these cells with mimic of miR-122-5p to lead to a significant inhibition in the level of expression of the MTPPT mRNA and protein as well as in mitochondrial carrier-mediated TPP uptake. Conversely, transfecting PAC AR42J with an inhibitor of miR-122-5p increased MTPPT expression and function. These findings show, for the first time, that expression and function of the MTPPT in PACs are subject to post-transcriptional regulation by miR-122-5p.

Role of microRNA miR-122-5p in post-transcriptional regulation of the mitochondrial thiamin pyrophosphate transporter (MTPPT; SLC25A19) in pancreatic acinar cells (PACs)

Thiamin (vitamin B1; also known as the “energy vitamin”) plays a critical role in normal energy metabolism and ATP production. Like all other human/mammalian cells, pancreatic acinar cells (PACs) cannot synthesize thiamin, and thus, must obtain the vitamin from circulation. Internalized free thiamin is then converted to TPP in the cytoplasm and transported into mitochondria for utilization via a carrier-mediated process that is mediated by the MTPPT (product of the SLC25A19 gene). Previous studies from our laboratory have characterized different aspects of the mitochondrial TPP uptake process in PACs; we have also delineated aspects of the MTPPT transcriptional regulation. To date, however, there has been nothing known about post-transcriptional regulation of MTPPT expression and function in PACs. We began to address these issues and, in this study, we examined the potential role of miRNAs in this regulation. First, we subjected the 458 bp long hMTPPT-3' untranslated region (UTR) to different in silico analysis (namely: miRDB, Targetscan, and DIANA-microTCDS), which identified multiple putative miRNA binding sites (including sites for miR-122-5p) in this region. These putative miRNA binding sites were found to be conserved in the human and rat MTPPTs. We then examined the effect of transfecting pmirGLO-hMTPPT-3'UTR into PAC (rat-derived AR42J cells) and observed a significant reduction in luciferase activity compared with cells transfected with pmirGLO empty vector. Focusing on the putative miR-122-5p (since it is expressed in normal human pancreatic cells and that its level is induced in pancreatitis), we found that transfecting AR42J cells as well as human native primary PACs (obtained from normal human organ donors) with a mimic of miR-122-5p to lead to a significant inhibition in level of MTPPT mRNA and protein expression as well as in TPP uptake by isolated mitochondria. On the other hand, inhibiting the miR-122-5p with the use of miR-122-5p inhibitor was found to lead to an increase in MTPPT mRNA and protein expression as well as in TPP uptake by isolated mitochondria. Additionally, truncating the region that carries the putative miR-122-5p binding site(s) in the 3'UTR of MTPPT as well as mutating those sites were found to lead to an abrogation in the inhibitory effect of this miRNA on luciferase activity in PACs. In conclusion, our results demonstrate, for the first time, that expression and function of the MTPPT are subject to post-transcriptional regulation by miR-122-5p. Supported by NIH grants AA-018071 & DK-56061, and VA grants 101BX001142 & IK6BX006189 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The mitochondrial thiamine pyrophosphate transporter TptA promotes adaptation to low iron conditions and virulence in fungal pathogen Aspergillus fumigatus

ABSTRACTAspergillus fumigatus is the most prevalent airborne fungal pathogen that causes invasive fungal infections in immunosuppressed individuals. Adaptation to iron limited conditions is crucial for A. fumigatus virulence. To identify novel genes that play roles in adaptation to low iron conditions we performed an insertional mutagenesis screen in A. fumigatus. Using this approach, we identified the tptA gene in A. fumigatus, which shares homology with the Saccharomyces cerevisiae thiamine pyrophosphate (ThPP) transporter encoding gene tpc1. Heterologous expression of tpc1 in the tptA deletion mutant completely restored the ThPP auxotrophy phenotype, suggesting that Tpc1 and TptA are functional orthologues. Importantly, TptA was required for adaptation to low iron conditions in A. fumigatus. The ΔtptA mutant had decreased resistance to the iron chelator bathophenanthroline disulfonate (BPS) with severe growth defects. Moreover, loss of tptA decreased the expression of hapX, which is a major transcription factor indispensable for adaptation to iron starvation in A. fumigatus. Overexpression of hapX in the ΔtptA strain greatly rescued the growth defect and siderophore production by A. fumigatus in iron-depleted conditions. Mutagenesis experiments demonstrated that the conserved residues related to ThPP uptake in TptA were also required for low iron adaptation. Furthermore, TptA-mediated adaptation to low iron conditions was found to be dependent on carbon sources. Finally, loss of tptA resulted in the attenuation of virulence in a murine model of aspergillosis. Taken together, this study demonstrated that the mitochondrial ThPP transporter TptA promotes low iron adaptation and virulence in A. fumigatus.

Thiamine supplementation facilitates thiamine transporter expression in the rumen epithelium and attenuates high-grain-induced inflammation in low-yielding dairy cows

An experiment was conducted to uncover the effects of increasing dietary grain levels on expression of thiamine transporters in ruminal epithelium, and to assess the protective effects of thiamine against high-grain-induced inflammation in dairy cows. Six rumen-fistulated, lactating Holstein dairy cows (627 ± 16.9 kg of body weight, 180 ± 6 d in milk; mean ± standard deviation) were randomly assigned to a replicated 3 × 3 Latin square design trial. Three treatments were control (20% dietary starch, dry matter basis), high-grain diet (HG, 33.2% dietary starch, DM basis), and HG diet supplemented with 180 mg of thiamine/kg of dry matter intake. On d 19 and 20 of each period, milk performance was measured. On d 21, ruminal pH, endotoxic lipopolysaccharide (LPS), and thiamine contents in rumen and blood, and plasma inflammatory cytokines were detected; a rumen papillae biopsy was taken on d 21 to determine the gene and protein expression of toll-like receptor 4 (TLR4) signaling pathways. The HG diet decreased ruminal pH (5.93 vs. 6.49), increased milk yield from 17.9 to 20.2 kg/d, and lowered milk fat and protein from 4.28 to 3.83%, and from 3.38 to 3.11%, respectively. The HG feeding reduced thiamine content in rumen (2.89 vs. 8.97 μg/L) and blood (11.66 vs. 17.63 μg/L), and the relative expression value of thiamine transporter-2 (0.37-fold) and mitochondrial thiamine pyrophosphate transporter (0.33-fold) was downregulated by HG feeding. The HG-fed cows exhibited higher endotoxin LPS in rumen fluid (134,380 vs. 11,815 endotoxin units/mL), and higher plasma concentrations of lipopolysaccharide binding protein and pro-inflammatory cytokines when compared with the control group. The gene and protein expression of tumor necrosis factor α (TNFα), IL1B, and IL6 in rumen epithelium increased when cows were fed the HG diet, indicating that local inflammation occurred. The depressions in ruminal pH, milk fat, and protein of HG-fed cows were reversed by thiamine supplementation. Thiamine supplementation increased thiamine contents in rumen and blood, and also upregulated the relative expression of thiamine transporters compared with the HG group. Thiamine supplementation decreased ruminal LPS (49,361 vs. 134,380 endotoxin units/mL) and attenuated the HG-induced inflammation response as indicated by a reduction in plasma IL6, and decreasing gene and protein expression of pro-inflammatory cytokines in rumen epithelium. Western bottling analysis showed that thiamine suppressed the protein expression of TLR4 and the phosphorylation of nuclear factor kappa B (NFκB) unit p65. In conclusion, HG feeding inhibits thiamine transporter expression in ruminal epithelium. Thiamine could attenuate the epithelial inflammation during high-grain feeding, and the protective effects may be due to its ability to suppress TLR4-mediated NFκB signaling pathways.

Inhibition of Intestinal Thiamin Transport in Rat Model of Sepsis.

Thiamin deficiency is highly prevalent in patients with sepsis, but the mechanism by which sepsis induces thiamin deficiency is unknown. This study aimed to determine the influence of various severity of sepsis on carrier-mediated intestinal thiamin uptake, level of expressions of thiamin transporters (thiamin transporter-1 and thiamin transporter-2), and mitochondrial thiamin pyrophosphate transporter. Randomized controlled study. Research laboratory at a Veterans Affairs Medical Center. Twenty-four Sprague-Dawley rats were randomized into controls, mild, moderate, and severe sepsis with equal number of animals in each group. Sepsis was induced by cecal ligation and puncture with the cecum ligated below the cecal valve at 25%, 50%, and 75% of cecal length, defined as severe, moderate, and mild sepsis, respectively. Control animals underwent laparotomy only. After 2 days of induced sepsis, carrier-mediated intestinal thiamin uptake was measured using [H]thiamin. Expressions of thiamin transporter-1, thiamin transporter-2, and mitochondrial thiamin pyrophosphate transporter proteins and messenger RNA were measured. Proinflammatory cytokines (interleukin-1β and interleukin-6) and adenosine triphosphate were also measured. Sepsis inhibited [H]thiamin uptake, and the inhibition was a function of sepsis severity. Both cell membrane thiamin transporters and mitochondrial thiamin pyrophosphate transporter expression levels were suppressed; also levels of adenosine triphosphate in the intestine of animals with moderate and severe sepsis were significantly lower than that of sham-operated controls. For the first time, we demonstrated that sepsis inhibited carrier-mediated intestinal thiamin uptake as a function of sepsis severity, suppressed thiamin transporters and mitochondrial thiamin pyrophosphate transporter, leading to adenosine triphosphate depletion.

Structure-function characterization of the human mitochondrial thiamin pyrophosphate transporter (hMTPPT; SLC25A19): Important roles for Ile33, Ser34, Asp37, His137 and Lys291

Thiamin plays a critical role in cellular energy metabolism. Mammalian cells obtain the vitamin from their surroundings, converted it to thiamin pyrophosphate (TPP) in the cytoplasm, followed by uptake of TPP by mitochondria via a carrier-mediated process that involves the MTPPT (product of the SLC25A19 gene). Previous studies have characterized different physiological/biological aspects of the human MTPPT (hMTPPT), but less is known about structural features that are important for its function. Here, we used a protein-docking model (“Phyre2” and “DockingServer”) to predict residues that may be important for function (substrate recognition) of the hMTPPT; we also examined the role of conserved positively-charged residues predicted (“PRALINE”) to be in the trans-membrane domains (TMDs) in uptake of the negatively-charged TPP. Among the six residues predicted by the docking model (i.e., Thr29, Arg30, Ile33, Ser34, Asp37 and Phe298), only Ile33, Ser34 and Asp37 were found to be critical for function. While no change in translational efficiency/protein stability of the Ser34 mutant was observed, both the Ile33 and Asp37 mutants showed a decrease in this parameter(s); there was also a decrease in the expression of the latter two mutants in mitochondria. A need for a polar residue at position 34 of the hMTPPT was evident. Our findings with the positively-charged residues (i.e., His82, His137, Lys231 and Lys291) predicted in the TMD showed that His137 and Lys291 are important for function (via a role in proper delivery of the protein to mitochondria). These investigations provide important information about the structure-function relationship of the hMTPPT.

Mitochondrial Uptake of Thiamin Pyrophosphate: Physiological and Cell Biological Aspects

Mammalian cells obtain vitamin B1 (thiamin) from their surrounding environment and convert it to thiamin pyrophosphate (TPP) in the cytoplasm. Most of TPP is then transported into the mitochondria via a carrier-mediated process that involves the mitochondrial thiamin pyrophosphate transporter (MTPPT). Knowledge about the physiological parameters of the MTPP-mediated uptake process, MTPPT targeting and the impact of clinical mutations in MTPPT in patients with Amish lethal microcephaly and neuropathy and bilateral striatal necrosis are not fully elucidated, and thus, were addressed in this study using custom-made 3H-TPP as a substrate and mitochondria isolated from mouse liver and human-derived liver HepG2 cells. Results showed 3H-TPP uptake by mouse liver mitochondria to be pH-independent, saturable (Km = 6.79±0.53 µM), and specific for TPP. MTPPT protein was expressed in mouse liver and HepG2 cells, and confocal images showed a human (h)MTPPT-GFP construct to be targeted to mitochondria of HepG2 cells. A serial truncation analysis revealed that all three modules of hMTPPT protein cooperated (although at different levels of efficiency) in mitochondrial targeting rather than acting autonomously as independent targeting module. Finally, the hMTPPT clinical mutants (G125S and G177A) showed proper mitochondrial targeting but displayed significant inhibition in 3H-TPP uptake and a decrease in level of expression of the MTPPT protein. These findings advance our knowledge of the physiology and cell biology of the mitochondrial TPP uptake process. The results also show that clinical mutations in the hMTPPT system impair its functionality via affecting its level of expression with no effect on its targeting to mitochondria.

Characterization of the human mitochondrial thiamine pyrophosphate transporter SLC25A19 minimal promoter: A role for NF-Y in regulating basal transcription

Transcriptional regulation of expression of the human mitochondrial thiamine pyrophosphate transporter (the product of the SLC25A19 gene) is unknown. To understand this regulation, we cloned and characterized the 5′-regulatory region of the SLC25A19 gene (1080bp). The cloned fragment was found to possess promoter activity in transiently transfected human-derived liver HepG2 cells. 5′- and 3′-deletion analysis has identified the minimal region required for basal SLC25A19 promoter activity to be between −131 and +20 (using the distal transcriptional start site as +1). The minimal promoter lacks typical TATA motif and contains two inverted CCAAT boxes (binding sites for NF-Y transcriptional factor). By means of mutational analysis, the critical role of both the upstream and downstream CCAAT boxes in basal SLC25A19 promoter activity was established; however, each of these boxes alone was found to be unable to support promoter activity. EMSA and supershift EMSA (with the use of specific antibodies against NF-Y subunits) studies, as well as chromatin immunoprecipitation assay, demonstrated the binding of NF-Y to both CCAAT boxes in vitro and in vivo, respectively. The requirement for NF-Y in SLC25A19 promoter activity in vivo was directly confirmed by the use of a dominant negative NF-YA mutant in transiently transfected HepG2 cells. These studies report for the first time the characterization of the SLC25A19 promoter and demonstrate an essential role for NF-Y in its basal activity.

Homozygous missense mutation of NDUFV1 as the cause of infantile bilateral striatal necrosis

Infantile bilateral striatal necrosis (IBSN) includes a heterogeneous group of disorders, characterized by symmetrical degeneration predominantly of the caudate nucleus and the putamen [1]. The clinical features encompass pyramidal and extrapyramidal signs such as choreoathetosis, dystonia, spasticity, dysarthria, and dysphagia in conjunction with developmental regression or mental retardation, and additional neurological symptoms [1]. The differential diagnosis has to take into account a variety of toxic, infectious, metabolic, and neurodegenerative disorders [1]. In familial cases, mutations in the mitochondrial genes ATP6 and ND6, and in the nuclear encoded genes nucleoporin 62 (NUP62) and mitochondrial thiamine pyrophosphate transporter (SLC25A19), have been described [2]. We report two siblings with IBSN and a mutation in NDUFV1, not yet linked to this condition. The patients’ parents are unrelated, but belong to a population of German settlers immigrating to Russia about 250 years ago (Fig. 1c). In both children, gait and speech difficulties were noticed from age 4 years. Neurological examination at age 9 (F3) and 5 years (F5) revealed dysarthria, muscular hypertonia with cogwheel rigidity, exaggerated deep tendon reflexes, and bilateral Babinski signs, while MRI demonstrated symmetric cystic lesions of the putamen (Fig. 1a, b). The patients were treated with biotin and thiamine before the definite diagnosis was established, and additionally, with creatine, riboflavin, and other vitamins thereafter. This resulted in a distinct and sustained improvement of their neurological status until last examination at age 11 and 7 years, respectively. Both still walk and attend regular schools. Biochemical analysis of a muscle biopsy from patient F3 revealed isolated complex I deficiency (0.08 U/U citrate synthetase; normal 0.17–0.56). Therefore, we sequenced the entire mtDNA including the genes ATP6 and ND6 without detecting pathogenic variants [3, 4].

Thiamin uptake by pancreatic acinar cells: effect of chronic alcohol feeding/exposure

Thiamin is important for normal function of pancreatic acinar cells, but little is known about its mechanism of uptake and about the effect of chronic alcohol use on the process. We addressed these issues using freshly isolated rat primary and rat-derived cultured AR42J pancreatic acinar cells as models. Results showed thiamin uptake by both primary and cultured AR42J pancreatic acinar cells to be via a specific carrier-mediated mechanism and that both of the thiamin transporters 1 and 2 (THTR-1 and THTR-2) are expressed in these cells. Chronic alcohol feeding of rats was found to lead to a significant inhibition of carrier-mediated thiamin uptake by pancreatic acinar cells and was associated with a significant reduction in level of expression of THTR-1 and THTR-2 at the protein and mRNA levels. Chronic exposure (96 h) of AR42J cells to alcohol also led to a significant decreased carrier-mediated thiamin uptake, an effect that was associated with a significant decrease in the activity of the human SLC19A2 and SLC19A3 promoters expressed in these cells. We also examined the effect of chronic alcohol feeding of rats on level of expression of key thiamin metabolizing enzymes (thiamin phosphokinase and thiamin pyrophosphatase) as well as on level of expression of the mitochondrial thiamin pyrophosphate transporter of pancreatic acinar cells and observed a significant inhibition in all these parameters. These results demonstrate for the first time that thiamin uptake by pancreatic acinar cells is via a carrier-mediated process and that both the THTR-1 as well as THTR-2 are expressed in these cells. Also, chronic alcohol feeding/exposure inhibits thiamin uptake process and the inhibition is, at least in part, being exerted at the transcriptional level. Furthermore, chronic alcohol feeding also negatively impacts intracellular parameters of thiamin metabolism in pancreatic acinar cells.

827 Slc25A19 Mutation is a Novel Cause of Neuropathy and Bilateral Striatal Necrosis

Acute basal ganglia necrosis in the pediatric age group is a neurological disorder characterized by symmetrical degeneration of the caudate nucleus, putamen, and occasionally the globus pallidus. The outcome is less favorable for the familial cases where the course is relentlessly progressive or punctuated by recurrent episodes with involvement of additional brain regions and significant residual neurological damage. The differential diagnosis of the latter group includes mitochondrial respiratory chain defects organic acid disorders, Wilson disease, juvenile Huntington's chorea, pantothenate kinaseassociated neurodegeneration, biotin responsive basal ganglia disease, and the striatal necrosis associated with NUP62 mutation.In this study we report on four siblings from a consanguineous Arab Muslim family who suffered from recurrent episodes of flaccid paralysis and encephalopathy and chronic progressive polyneuropathy associated with bilateral striatal necrosis. Using homozygosity mapping the pathogenic homozygous missense mutation c.373G>A was identified. This mutation alters a highly conserved glycine residue at position 125 to serine in the SLC25A19 gene which encodes the mitochondrial thiamine pyrophosphate transporter. SLC25A19 mutation was previously reported only in the Amish congenital lethal microcephaly but our patients' phenotype is markedly different with normal head circumference, early childhood development, and age-appropriate cognitive skills. Based on the role of SLC25A19 in mitochondrial thiamine transport, supplementation with high dose thiamine was initiated in all patients. Preliminary results suggest a favorable effect.Our report suggests an allelic phenotype in SLC25A19 gene defects. Determination of the SLC25A19 sequence may be warranted in patients with bilateral striatal necrosis or progressive polyneuropathy.

Effect of chronic alcohol feeding on physiological and molecular parameters of renal thiamin transport

The renal thiamin reabsorption process plays an important role in regulating thiamin body homeostasis and involves both thiamin transporters-1 and -2 (THTR1 and THTR2). Chronic alcohol use is associated with thiamin deficiency. Although a variety of factors contribute to the development of this deficiency, effects of chronic alcohol use on renal thiamin transport have not been thoroughly examined. We addressed this issue by examining the effect of chronic alcohol feeding of rats with liquid diet on physiological and molecular parameters of renal thiamin transport. Chronic alcohol feeding caused a significant inhibition in carrier-mediated thiamin transport across the renal brush-border membrane and was evident as early as 2 wk after initiation of alcohol feeding. Similarly, thiamin transport across the renal basolateral membrane was significantly inhibited by chronic alcohol feeding. The inhibition in renal thiamin transport was associated with a marked decrease in the level of expression of THTR1 and -2 proteins, mRNAs, and heterogeneous nuclear RNAs. Chronic alcohol feeding also caused a significant reduction in the level of expression of thiamin pyrophosphokinase but not that of the mitochondrial thiamin pyrophosphate transporter. These studies show that chronic alcohol feeding inhibits the entry and exit of thiamin in the polarized renal epithelial cells and that the effect is, at least in part, mediated at the transcriptional level. These findings also suggest that chronic alcohol feeding interferes with the normal homeostasis of thiamin in renal epithelial cells.

SLC25A19 mutation as a cause of neuropathy and bilateral striatal necrosis

Four patients, aged 7-20 years, suffered from recurrent episodes of flaccid paralysis and encephalopathy associated with bilateral striatal necrosis and chronic progressive polyneuropathy. Using homozygosity mapping, a pathogenic missense mutation in the SLC25A19 gene that encodes the mitochondrial thiamine pyrophosphate transporter was identified. An SLC25A19 mutation was previously reported in Amish congenital lethal microcephaly but the present patients' phenotype is markedly different, with normal head circumference, normal early childhood development, age-appropriate cognitive skills, and normal urinary organic acid profile. Determination of the SLC25A19 sequence should be considered in patients with bilateral striatal necrosis and progressive polyneuropathy.