Search Results (31)

Colorectal cancer (CRC) progression is driven by dysregulated signaling networks that promote proliferation and metastasis. While SLC25A5 is a well-characterized mitochondrial ADP/ATP transporter, its potential non-canonical roles in cancer remain unclear. This study investigated whether SLC25A5 exerts tumor-suppressive functions in CRC. Using transcriptomic datasets and clinical cohorts, we found that SLC25A5 is significantly downregulated in CRC tissues, and low expression is associated with poor patient survival. Restoration of SLC25A5 suppressed CRC cell proliferation, epithelial–mesenchymal transition (EMT), and metastasis in vitro and in vivo. Mechanistically, co-immunoprecipitation and protein stability assays suggested an association between SLC25A5 and EIF3A and indicated that SLC25A5 may promote EIF3A destabilization through the ubiquitin–proteasome pathway without altering its mRNA levels. Subcellular fractionation further suggested the presence of a cytoplasmic pool of SLC25A5, providing a potential basis for this interaction. Rescue experiments showed that EIF3A overexpression partially reversed the tumor-suppressive effects of SLC25A5. In addition, SLC25A5 expression was associated with reduced PI3K/AKT signaling activity, and pharmacological activation of AKT partially restored invasive phenotypes. Collectively, these findings suggest an SLC25A5–EIF3A–PI3K/AKT regulatory axis and reveal a potential non-canonical role for this mitochondrial carrier in tumor progression. This study provides insight into how mitochondrial proteins may influence cytoplasmic signaling pathways in cancer. Full article

Suramin is a century-old polysulfonated naphthylurea that remains a first-line treatment for early-stage human African trypanosomiasis (HAT). Remarkably, despite its age, suramin continues to draw attention because of its unusually broad spectrum of biological activities. Historically known as an antagonist of purinergic (P2) receptors and an inhibitor of extracellular enzymes, suramin has more recently been shown to interact with a range of intracellular and mitochondrial proteins. These include succinate dehydrogenase, the ADP/ATP carrier (AAC), the aspartate/glutamate carriers AGC1 and AGC2, carnitine O-acetyltransferase (CRAT), and the ATP-Mg/Pi carrier (APC2). Across these targets, suramin displays sub-micromolar to low-micromolar potencies, largely driven by electrostatic complementarity between its highly anionic sulfonate groups and basic nucleotide- or anion-binding regions of proteins. This extensive polypharmacology helps explain the diverse biological effects reported for suramin and supports its use as a valuable pharmacological probe of mitochondrial transport and metabolism. At the same time, its largeness and high negative charge limit oral bioavailability and brain penetration, prompting efforts to develop simplified analogues. This review brings together chemical, biological, and structural perspectives on suramin, highlighting opportunities for drug repurposing, transporter-focused drug design, and a better understanding of mitochondrial toxicity. Full article

The inner mitochondrial membrane proteins ATP/ADP carrier protein 1 (AAC1) and Uncoupling protein 1 (UCP1) belong to the SLC25 mitochondrial carrier family. AAC1 is responsible for ATP/ADP exchange, while UCP1-dependent proton transport, which also requires small molecules known as activators, is the basis of brown fat thermogenesis. Arachidonic acid (AA) is an endogenous activator capable of inducing proton transport in both proteins. As such, both AAC1- and UCP1-dependent proton transport are potential targets of weight loss drugs. While AAC1 structures have long been available, only recently have structures of UCP1 been determined. Unfortunately, no AA-bound structure of either protein is available. To explore their interactions with AA, we performed molecular dynamics (MD) simulations of both proteins. Six parallel simulations of each protein were run with an average length of just over 6 μs, for a total of 75 μs of aggregate simulation across both proteins. AA bound deeply between transmembrane helix (TM) helices or in the central cavity of AAC1 in 14 events and between TM helices of UCP1 in 6 events. All AA involved in these deep binding events came from the intermembrane space-facing (C) leaflet. In AAC1, AA most often bound between TM1/TM2 and TM5/TM6. In four cases the fatty acid bound at the bottom of the central cavity rather than in an interhelical groove. In UCP1, all but one deeply bound AA sat between TM5 and TM6. No AA fully entered the cavity as observed in AAC1. In addition to entering the proteins, AAs were enriched around them in the surrounding membrane adjacent to the TM helices. While both protein structures exhibit hydrophobic stretches separating the intermembrane space (IMS) from the matrix, water wires formed through both AAC1 and UCP1, connecting the bulk water in both regions. Grotthuss shuttling along water wires has been proposed as a possible mechanism of AAC1/UCP1-dependent proton transport, but water wires are not present in experimental structures and have not previously been reported in MD simulations. Calculations of electric potentials along these water wires find a large 0.75–1 V electrostatic barrier along water wires through AAC1 and a substantially smaller such barrier of ~0.5 V through UCP1. Full article

Exposure to extremely low-frequency magnetic fields (ELF-MF) can induce biological alterations in human cells, including peripheral blood mononuclear cells (PBMCs). However, the molecular mechanisms and key regulatory factors underlying this cellular response remain largely unknown. In this study, we analyzed the proteomic profiles of PBMCs isolated from three human subjects. PBMCs were exposed to 50 Hz, 1 mT of ELF-MF for 24 h and compared to unexposed PBMCs from the same individuals. ELF-MF exposure altered the expression levels of several PBMC proteins without affecting cell proliferation, cell viability, or cell cycle progression. A total of 51 proteins were upregulated, 36 of which were intercorrelated and associated with the Cellular Metabolic Process (GO:0044237) and Metabolic Process (GO:0008152). Among them, solute carrier family 25 member 4 (SLC25A4), which catalyzes the exchange of cytoplasmic ADP for mitochondrial ATP across the inner mitochondrial membrane, was consistently upregulated in all ELF-MF–exposed samples. Additionally, 67 proteins were downregulated, many of which are linked to T cell costimulation (GO:0031295), Cell activation (GO:0001775), and Immune system processes (GO:0002376) included ASPSCR1, PCYT1A, PCYT2, QRAS, and REPS1. In conclusion, ELF-MF exposure induces metabolic reprogramming in human PBMCs, characterized by the upregulation of mitochondrial proteins and downregulation of immune-activation-related proteins, without compromising cell viability or proliferation. Full article

The carnitine/acylcarnitine carrier (CAC) is a crucial protein for cellular energy metabolism, facilitating the exchange of acylcarnitines and free carnitine across the mitochondrial membrane, thereby enabling fatty acid β-oxidation and oxidative phosphorylation (OXPHOS). Although CAC has not been crystallised, structural insights are derived from the mitochondrial ADP/ATP carrier (AAC) structures in both cytosolic and matrix conformations. These structures underpin a single binding centre-gated pore mechanism, a common feature among mitochondrial carrier (MC) family members. The functional implications of this mechanism are well-supported, yet the structural organization of the CAC, particularly the formation of dimeric or oligomeric assemblies, remains contentious. Recent investigations employing biochemical techniques on purified and reconstituted CAC, alongside molecular modelling based on crystallographic AAC dimeric structures, suggest that CAC can indeed form dimers. Importantly, this dimerization does not alter the transport mechanism, a phenomenon observed in various other membrane transporters across different protein families. This observation aligns with the ping–pong kinetic model, where the dimeric form potentially facilitates efficient substrate translocation without necessitating mechanistic alterations. The presented findings thus contribute to a deeper understanding of CAC’s functional dynamics and its structural parallels with other MC family members. Full article

Mitochondrial adenine nucleotide translocase (ANT) exchanges ADP for ATP to maintain energy production in the cell. Its protonophoric function in the presence of long-chain fatty acids (FA) is also recognized. Our previous results imply that proton/FA transport can be best described with the FA cycling model, in which protonated FA transports the proton to the mitochondrial matrix. The mechanism by which ANT1 transports FA anions back to the intermembrane space remains unclear. Using a combined approach involving measurements of the current through the planar lipid bilayers reconstituted with ANT1, site-directed mutagenesis and molecular dynamics simulations, we show that the FA anion is first attracted by positively charged arginines or lysines on the matrix side of ANT1 before moving along the positively charged protein–lipid interface and binding to R79, where it is protonated. We show that R79 is also critical for the competitive binding of ANT1 substrates (ADP and ATP) and inhibitors (carboxyatractyloside and bongkrekic acid). The binding sites are well conserved in mitochondrial SLC25 members, suggesting a general mechanism for transporting FA anions across the inner mitochondrial membrane. Full article

Adenosine 5′ triphosphate (ATP) is the energy currency of life, which is produced in mitochondria (~90%) and cytosol (less than 10%). Real-time effects of metabolic changes on cellular ATP dynamics remain indeterminate. Here we report the design and validation of a genetically encoded fluorescent ATP indicator that allows for real-time, simultaneous visualization of cytosolic and mitochondrial ATP in cultured cells. This dual-ATP indicator, called smacATPi (simultaneous mitochondrial and cytosolic ATP indicator), combines previously described individual cytosolic and mitochondrial ATP indicators. The use of smacATPi can help answer biological questions regarding ATP contents and dynamics in living cells. As expected, 2-deoxyglucose (2-DG, a glycolytic inhibitor) led to substantially decreased cytosolic ATP, and oligomycin (a complex V inhibitor) markedly decreased mitochondrial ATP in cultured HEK293T cells transfected with smacATPi. With the use of smacATPi, we can also observe that 2-DG treatment modestly attenuates mitochondrial ATP and oligomycin reduces cytosolic ATP, indicating the subsequent changes of compartmental ATP. To evaluate the role of ATP/ADP carrier (AAC) in ATP trafficking, we treated HEK293T cells with an AAC inhibitor, Atractyloside (ATR). ATR treatment attenuated cytosolic and mitochondrial ATP in normoxia, suggesting AAC inhibition reduces ADP import from the cytosol to mitochondria and ATP export from mitochondria to cytosol. In HEK293T cells subjected to hypoxia, ATR treatment increased mitochondrial ATP along with decreased cytosolic ATP, implicating that ACC inhibition during hypoxia sustains mitochondrial ATP but may not inhibit the reversed ATP import from the cytosol. Furthermore, both mitochondrial and cytosolic signals decrease when ATR is given in conjunction with 2-DG in hypoxia. Thus, real-time visualization of spatiotemporal ATP dynamics using smacATPi provides novel insights into how cytosolic and mitochondrial ATP signals respond to metabolic changes, providing a better understanding of cellular metabolism in health and disease. Full article

The Carnitine-Acylcarnitine Carrier is a member of the mitochondrial Solute Carrier Family 25 (SLC25), known as SLC25A20, involved in the electroneutral exchange of acylcarnitine and carnitine across the inner mitochondrial membrane. It acts as a master regulator of fatty acids β-oxidation and is known to be involved in neonatal pathologies and cancer. The transport mechanism, also known as “alternating access”, involves a conformational transition in which the binding site is accessible from one side of the membrane or the other. In this study, through a combination of state-of-the-art modelling techniques, molecular dynamics, and molecular docking, the structural dynamics of SLC25A20 and the early substrates recognition step have been analyzed. The results obtained demonstrated a significant asymmetry in the conformational changes leading to the transition from the c- to the m-state, confirming previous observations on other homologous transporters. Moreover, analysis of the MD simulations’ trajectories of the apo-protein in the two conformational states allowed for a better understanding of the role of SLC25A20 Asp231His and Ala281Val pathogenic mutations, which are at the basis of Carnitine-Acylcarnitine Translocase Deficiency. Finally, molecular docking coupled to molecular dynamics simulations lend support to the multi-step substrates recognition and translocation mechanism already hypothesized for the ADP/ATP carrier. Full article

Traditional sanitation practices remain the main strategy for controlling Bombyx mori infections caused by microsporidia Nosema bombycis. This actualizes the development of new approaches to increase the silkworm resistance to this parasite. Here, we constructed a mouse scFv library against the outer loops of N. bombycis ATP/ADP carriers and selected nine scFv fragments to the transporter, highly expressed in the early stages of the parasite intracellular growth. Expression of selected scFv genes in Sf9 cells, their infection with different ratios of microsporidia spores per insect cell, qPCR analysis of N. bombycis PTP2 and Spodoptera frugiperda COXI transcripts in 100 infected cultures made it possible to select the scFv fragment most effectively inhibiting the parasite growth. Western blot analysis of 42 infected cultures with Abs against the parasite β-tubulin confirmed its inhibitory efficiency. Since the VL part of this scFv fragment was identified as a human IgG domain retained from the pSEX81 phagemid during library construction, its VH sequence should be a key antigen-recognizing determinant. Along with the further selection of new recombinant Abs, this suggests the searching for its natural mouse VL domain or “camelization” of the VH fragment by introducing cysteine and hydrophilic residues, as well as the randomization of its CDRs. Full article

The control of the duration of the dormancy phase is a significant challenge in the potato industry and for seed producers. However, the proteome landscape involved in the regulation of the length of the dormancy period over potato cultivars remains largely unexplored. In this study, we performed for the first time a comparative proteome profiling of potato cultivars with differential duration of tuber dormancy. More specifically, the proteome profiling of Agata, Kennebec and Agria commercial potato varieties with short, medium and medium-long dormancy, respectively, was assessed at the endodormancy stage using high-resolution two-dimensional electrophoresis (2-DE) coupled to reversed-phase liquid chromatography–tandem mass spectrometry (LC-TripleTOF MS/MS). A total of 11 proteins/isoforms with statistically significant differential abundance among cultivars were detected on 2-DE gels and confidently identified by LC-TripleTOF MS/MS. Identified proteins have known functions related to tuber development, sprouting and the oxylipins biosynthesis pathway. Fructokinase, a mitochondrial ADP/ATP carrier, catalase isozyme 2 and heat shock 70 kDa were the proteins with the strongest response to dormancy variations. To the best of our knowledge, this study reports the first candidate proteins underlying variable dormancy length in potato cultivars. Full article

The ADP/ATP carrier (AAC) plays a central role in oxidative metabolism by exchanging ATP and ADP across the inner mitochondrial membrane. Previous experiments have shown the involvement of the matrix loops of AAC in its function, yet potential mechanisms remain largely elusive. One obstacle is the limited information on the structural dynamics of the matrix loops. In the current work, unbiased all-atom molecular dynamics (MD) simulations were carried out on c-state wild-type AAC and mutants. Our results reveal that: (1) two ends of a matrix loop are tethered through interactions between the residue of triplet 38 (Q38, D143 and Q240) located at the C-end of the odd-numbered helix and residues of the [YF]xG motif located before the N-end of the short matrix helix in the same domain; (2) the initial progression direction of a matrix loop is determined by interactions between the negatively charged residue of the [DE]G motif located at the C-end of the short matrix helix and the capping arginine (R30, R139 and R236) in the previous domain; (3) the two chemically similar residues D and E in the highly conserved [DE]G motif are actually quite different; (4) the N-end of the M3 loop is clamped by the [DE]G motif and the capping arginine of domain 2 from the two sides, which strengthens interactions between domain 2 and domain 3; and (5) a highly asymmetric stable core exists within domains 2 and 3 at the m-gate level. Moreover, our results help explain almost all extremely conserved residues within the matrix loops of the ADP/ATP carriers from a structural point of view. Taken together, the current work highlights asymmetry in the three matrix loops and implies a close relationship between asymmetry and ADP/ATP transport. Full article

Recent data have shown that the mitochondrial permeability transition pore (MPTP) is the complex of the Ca²⁺-modified adenine nucleotide translocase (ANT) and the Ca²⁺-modified ATP synthase. We found in a previous study that ANT conformational changes may be involved in Tl⁺-induced MPTP opening in the inner membrane of Ca²⁺-loaded rat liver mitochondria. In this study, the effects of thiol-modifying agents (eosin-5-maleimide (EMA), fluorescein isothiocyanate (FITC), Cu(o-phenanthroline)₂ (Cu(OP)₂), and embelin (Emb)), and MPTP inhibitors (ADP, cyclosporine A (CsA), n-ethylmaleimide (NEM), and trifluoperazine (TFP)) on MPTP opening were tested simultaneously with increases in swelling, membrane potential (ΔΨ_mito) decline, decreases in state 3, 4, and 3U_DNP (2,4-dinitrophenol-uncoupled) respiration, and changes in the inner membrane free thiol group content. The effects of these thiol-modifying agents on the studied mitochondrial characteristics were multidirectional and showed a clear dependence on their concentration. This research suggests that Tl⁺-induced MPTP opening in the inner membrane of calcium-loaded mitochondria may be caused by the interaction of used reagents (EMA, FITC, Emb, Cu(OP)₂) with active groups of ANT, the mitochondrial phosphate carrier (PiC) and the mitochondrial respiratory chain complexes. This study provides further insight into the causes of thallium toxicity and may be useful in the development of new treatments for thallium poisoning. Full article

Impaired cerebral glucose metabolism is an early event that contributes to the pathogenesis of Alzheimer’s disease (AD). Importantly, restoring glucose availability by pharmacological agents or genetic manipulation has been shown to protect against Aβ toxicity, ameliorate AD pathology, and increase lifespan. Lithium, a therapeutic agent widely used as a treatment for mood disorders, has been shown to attenuate AD pathology and promote glucose metabolism in skeletal muscle. However, despite its widespread use in neuropsychiatric disorders, lithium’s effects on the brain have been poorly characterized. Here we evaluated the effect of lithium on glucose metabolism in hippocampal neurons from wild-type (WT) and APPSwe/PS1ΔE9 (APP/PS1) mice. Our results showed that lithium significantly stimulates glucose uptake and replenishes ATP levels by preferential oxidation of glucose through glycolysis in neurons from WT mice. This increase was also accompanied by a strong increase in glucose transporter 3 (Glut3), the major carrier responsible for glucose uptake in neurons. Similarly, using hippocampal slices from APP-PS1 mice, we demonstrate that lithium increases glucose uptake, glycolytic rate, and the ATP:ADP ratio in a process that also involves the activation of AMPK. Together, our findings indicate that lithium stimulates glucose metabolism and can act as a potential therapeutic agent in AD. Full article

The mitochondrial ADP/ATP carrier (AAC) exports ATP and imports ADP through alternating between cytosol-open (c-) and matrix-open (m-) states. The salt bridge networks near the matrix side (m-gate) and cytosol side (c-gate) are thought to be crucial for state transitions, yet our knowledge on these networks is still limited. In the current work, we focus on more conserved m-gate network in the c-state AAC. All-atom molecular dynamics (MD) simulations on a variety of mutants and the CATR-AAC complex have revealed that: (1) without involvement of other positive residues, the charged residues from the three Px[DE]xx[KR] motifs only are prone to form symmetrical inter-helical network; (2) R235 plays a determinant role for the asymmetry in m-gate network of AAC; (3) R235 significantly strengthens the interactions between H3 and H5; (4) R79 exhibits more significant impact on m-gate than R279; (5) CATR promotes symmetry in m-gate mainly through separating R234 from D231 and fixing R79; (6) vulnerability of the H2-H3 interface near matrix side could be functionally important. Our results provide new insights into the highly conserved yet variable m-gate network in the big mitochondrial carrier family. Full article

Higher concentration of protons in the mitochondrial intermembrane space compared to the matrix results in an electrochemical potential causing the back flux of protons to the matrix. This proton transport can take place through ATP synthase complex (leading to formation of ATP) or can occur via proton transporters of the mitochondrial carrier superfamily and/or membrane lipids. Some mitochondrial proton transporters, such as uncoupling proteins (UCPs), transport protons as their general regulating function; while others are symporters or antiporters, which use the proton gradient as a driving force to co-transport other substrates across the mitochondrial inner membrane (such as phosphate carrier, a symporter; or aspartate/glutamate transporter, an antiporter). Passage (or leakage) of protons across the inner membrane to matrix from any route other than ATP synthase negatively impacts ATP synthesis. The focus of this review is on regulated proton transport by UCPs. Recent findings on the structure and function of UCPs, and the related research methodologies, are also critically reviewed. Due to structural similarity of members of the mitochondrial carrier superfamily, several of the known structural features are potentially expandable to all members. Overall, this report provides a brief, yet comprehensive, overview of the current knowledge in the field. Full article