L-Aspartate (aspartic acid; C4H7NO4; 2-aminobutanedoic acid) is a non-essential α-amino acid found ubiquitously throughout the body, including in the brain. Aspartate is one of the protein-forming amino acids and the formation of tRNA-aspartate complex is catalysed by aspartyl tRNA synthetase. Free aspartate, which is the main subject of this review, plays key roles in metabolism, as an amino donor and acceptor. It contributes to the synthesis of protein, arginine and nitric oxide, asparagine, N-acetylaspartate and N-methyl-D-aspartate. Its major metabolic role in the brain is recycling reducing equivalents (protons) between the cytoplasm and mitochondrial matrix as part of the malate-aspartate shuttle. L-Aspartate's actions on synaptic receptors, as well as its possible presence in nerve terminals and synaptic vesicles, are, in principle, consistent with a role as an excitatory neurotransmitter. The evidence is far from conclusive and at times controversial. The role of D-aspartate in brain function is even less certain but, it appears that, rather than being a minor neurotransmitter, D-aspartate is more likely to be involved in fine regulation of endocrine and homeostatic processes. Much research remains to be done in this area. The diversity of its functions and chemistry make aspartate a complex molecule to investigate and measure in vivo. Perturbations of aspartate metabolism have been described in a range of neurological deficits, particularly those of white matter. Here, we examine what is known about the various roles of aspartate in brain, its metabolism, transport and compartmentation, its role as a neurotransmitter or a more general signalling molecule, and what is currently known about its role(s) in disease processes.

• MeSH
• Aspartic Acid * metabolism   MeSH
• Humans MeSH
• Brain * metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

Various strategies have been employed to improve the reliability of 2D, 3D, and co-culture in vitro models of nonalcoholic fatty liver disease, including using extracellular matrix proteins such as collagen I to promote cell adhesion. While studies have demonstrated the significant benefits of culturing cells on collagen I, its effects on the HepG2 cell line after exposure to palmitate (PA) have not been investigated. Therefore, this study aimed to assess the effects of PA-induced lipotoxicity in HepG2 cultured in the absence or presence of collagen I. HepG2 cultured in the absence or presence of collagen I was exposed to PA, followed by analyses that assessed cell proliferation, viability, adhesion, cell death, mitochondrial respiration, reactive oxygen species production, gene and protein expression, and triacylglycerol accumulation. Culturing HepG2 on collagen I was associated with increased cell proliferation, adhesion, and expression of integrin receptors, and improved cellular spreading compared to culturing them in the absence of collagen I. However, PA-induced lipotoxicity was greater in collagen I-cultured HepG2 than in those cultured in the absence of collagen I and was associated with increased α2β1 receptors. In summary, the present study demonstrated for the first time that collagen I-cultured HepG2 exhibited exacerbated cell death following exposure to PA through integrin-mediated death. The findings from this study may serve as a caution to those using 2D models or 3D scaffold-based models of HepG2 in the presence of collagen I.

• MeSH
• Cell Adhesion * drug effects   MeSH
• Cell Death drug effects   MeSH
• Hep G2 Cells MeSH
• Integrin alpha2beta1 metabolism   MeSH
• Integrins metabolism   genetics   MeSH
• Collagen Type I * metabolism   genetics   MeSH
• Humans MeSH
• Non-alcoholic Fatty Liver Disease metabolism   pathology   MeSH
• Palmitates toxicity   pharmacology   MeSH
• Cell Proliferation * drug effects   MeSH
• Reactive Oxygen Species metabolism   MeSH
• Cell Survival * drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

• MeSH
• Biochemistry MeSH
• Metabolic Diseases MeSH
• Pathology MeSH

• Conspectus
• Patologie. Klinická medicína
• Učební osnovy. Vyučovací předměty. Učebnice
• NML Fields
• biochemie
• patologie
• NML Publication type
• učebnice vysokých škol

The study aims to elucidate the therapeutic mechanism of Baicalin (BAI) in alleviating cartilage injury in osteoarthritic (OA) rat models, concentrating on its regulation of the miR-766-3p/AIFM1 axis. An OA rat model was developed with unilateral anterior cruciate ligament transection (ACLT). Interventions comprised of BAI treatment and intra-articular administration of miR-766-3p inhibitor. For evaluation, histopathological staining was conducted to investigate the pathological severity of knee cartilage injury. The levels of oxidative stress (OS) indicators including MDA, SOD, and GSH-Px, were quantified using colorimetric assays. Inflammatory factors (IFs; TNF-?, IL-1?, and IL-6) in knee joint lavage fluids were assessed using ELISA, while RT-PCR was employed to quantify miR-766-3p expression. TUNEL apoptosis staining was utilized to detect chondrocyte apoptosis, and western blotting examined autophagy-related markers (LC3, Beclin, p62), extracellular matrix (ECM) synthesis-associated indices (COL2A, ACAN, MMP13), and apoptosis-inducing factor mitochondrion-associated 1 (AIFM1). Histological examination revealed a marked amelioration of cartilage injury in the BAI-treated OA rat models compared to controls. BAI treatment significantly reduced inflammation and OS of knee joint fluid, activated autophagy, and decreased chondrocyte apoptosis and ECM degradation. Interestingly, the inhibitory effects of BAI on these pathological markers were significantly decreased by the miR-766-3p inhibitor. Further assessment revealed that BAI efficiently promoted miR-766-3p expression while inhibiting AIFM1 protein expression. BAI potentially mitigates articular cartilage injury in OA rats, likely through modulation of miR-766-3p/AIFM1 axis. Keywords: Baicalin, microRNA, AIFM1, Osteoarthritisv, Rat.

• MeSH
• Apoptosis drug effects   MeSH
• Apoptosis Inducing Factor metabolism   MeSH
• Flavonoids * pharmacology   therapeutic use   MeSH
• Cartilage, Articular drug effects   metabolism   pathology   MeSH
• Rats MeSH
• MicroRNAs * metabolism   genetics   biosynthesis   MeSH
• Osteoarthritis drug therapy   metabolism   pathology   MeSH
• Oxidative Stress drug effects   MeSH
• Rats, Sprague-Dawley * MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Protein import and genome replication are essential processes for mitochondrial biogenesis and propagation. The J-domain proteins Pam16 and Pam18 regulate the presequence translocase of the mitochondrial inner membrane. In the protozoan Trypanosoma brucei, their counterparts are TbPam16 and TbPam18, which are essential for the procyclic form (PCF) of the parasite, though not involved in mitochondrial protein import. Here, we show that during evolution, the 2 proteins have been repurposed to regulate the replication of maxicircles within the intricate kDNA network, the most complex mitochondrial genome known. TbPam18 and TbPam16 have inactive J-domains suggesting a function independent of heat shock proteins. However, their single transmembrane domain is essential for function. Pulldown of TbPam16 identifies a putative client protein, termed MaRF11, the depletion of which causes the selective loss of maxicircles, akin to the effects observed for TbPam18 and TbPam16. Moreover, depletion of the mitochondrial proteasome results in increased levels of MaRF11. Thus, we have discovered a protein complex comprising TbPam18, TbPam16, and MaRF11, that controls maxicircle replication. We propose a working model in which the matrix protein MaRF11 functions downstream of the 2 integral inner membrane proteins TbPam18 and TbPam16. Moreover, we suggest that the levels of MaRF11 are controlled by the mitochondrial proteasome.

• MeSH
• DNA, Mitochondrial * genetics   metabolism   MeSH
• Mitochondrial Proteins metabolism   genetics   MeSH
• Mitochondria metabolism   genetics   MeSH
• Evolution, Molecular MeSH
• Protozoan Proteins * metabolism   genetics   MeSH
• DNA Replication * MeSH
• Trypanosoma brucei brucei * metabolism   genetics   MeSH
• Publication type
• Journal Article MeSH

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has been associated with the host dysmetabolism of branched-chain amino acids (BCAAs), however, the implications for the role of BCAA metabolism in PDAC development or progression are not clear. The mitochondrial catabolism of valine, leucine, and isoleucine is a multistep process leading to the production of short-chain R-CoA species. They can be subsequently exported from mitochondria as short-chain carnitines (SC-CARs), utilized in anabolic pathways, or released from the cells. METHODS: We examined the specificities of BCAA catabolism and cellular adaptation strategies to BCAA starvation in PDAC cells in vitro. We used metabolomics and lipidomics to quantify major metabolic changes in response to BCAA withdrawal. Using confocal microscopy and flow cytometry we quantified the fluorescence of BODIPY probe and the level of lipid droplets (LDs). We used BODIPY-conjugated palmitate to evaluate transport of fatty acids (FAs) into mitochondria. Also, we have developed a protocol for quantification of SC-CARs, BCAA-derived metabolites. RESULTS: Using metabolic profiling, we found that BCAA starvation leads to massive triglyceride (TG) synthesis and LD accumulation. This was associated with the suppression of activated FA transport into the mitochondrial matrix. The suppression of FA import into mitochondria was rescued with the inhibitor of the acetyl-CoA carboxylase (ACC) and the activator of AMP kinase (AMPK), which both regulate carnitine palmitoyltransferase 1A (CPT1) activation status. CONCLUSIONS: Our data suggest that BCAA catabolism is required for the import of long chain carnitines (LC-CARs) into mitochondria, whereas the disruption of this link results in the redirection of activated FAs into TG synthesis and its deposition into LDs. We propose that this mechanism protects cells against mitochondrial overload with LC-CARs and it might be part of the universal reaction to amino acid perturbations during cancer growth, regulating FA handling and storage.

• Publication type
• Journal Article MeSH

The long slender bloodstream form Trypanosoma brucei maintains its essential mitochondrial membrane potential (ΔΨm) through the proton-pumping activity of the FoF1-ATP synthase operating in the reverse mode. The ATP that drives this hydrolytic reaction has long been thought to be generated by glycolysis and imported from the cytosol via an ATP/ADP carrier (AAC). Indeed, we demonstrate that AAC is the only carrier that can import ATP into the mitochondrial matrix to power the hydrolytic activity of the FoF1-ATP synthase. However, contrary to expectations, the deletion of AAC has no effect on parasite growth, virulence or levels of ΔΨm. This suggests that ATP is produced by substrate-level phosphorylation pathways in the mitochondrion. Therefore, we knocked out the succinyl-CoA synthetase (SCS) gene, a key mitochondrial enzyme that produces ATP through substrate-level phosphorylation in this parasite. Its absence resulted in changes to the metabolic landscape of the parasite, lowered virulence, and reduced mitochondrial ATP content. Strikingly, these SCS mutant parasites become more dependent on AAC as demonstrated by a 25-fold increase in their sensitivity to the AAC inhibitor, carboxyatractyloside. Since the parasites were able to adapt to the loss of SCS in culture, we also analyzed the more immediate phenotypes that manifest when SCS expression is rapidly suppressed by RNAi. Importantly, when performed under nutrient-limited conditions mimicking various host environments, SCS depletion strongly affected parasite growth and levels of ΔΨm. In totality, the data establish that the long slender bloodstream form mitochondrion is capable of generating ATP via substrate-level phosphorylation pathways.

• MeSH
• Adenosine Triphosphate metabolism   MeSH
• Phosphorylation MeSH
• Mitochondria metabolism   MeSH
• Trypanosoma brucei brucei * metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

• MeSH
• Biology MeSH
• Cytology MeSH
• Molecular Biology MeSH

• Conspectus
• Biochemie. Molekulární biologie. Biofyzika
• Učební osnovy. Vyučovací předměty. Učebnice
• NML Fields
• biologie
• NML Publication type
• učebnice vysokých škol

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.

• MeSH
• Adenosine Triphosphate metabolism   MeSH
• Anions metabolism   MeSH
• Lipid Bilayers * MeSH
• Fatty Acids metabolism   MeSH
• Mitochondrial ADP, ATP Translocases metabolism   MeSH
• Protons * MeSH
• Publication type
• Journal Article MeSH

We have generated a high-confidence mitochondrial proteome (MitoTag) of the Trypanosoma brucei procyclic stage containing 1,239 proteins. For 337 of these, a mitochondrial localization had not been described before. We use the TrypTag dataset as a foundation and take advantage of the properties of the fluorescent protein tag that causes aberrant but fortuitous accumulation of tagged matrix and inner membrane proteins near the kinetoplast (mitochondrial DNA). Combined with transmembrane domain predictions, this characteristic allowed categorization of 1,053 proteins into mitochondrial sub-compartments, the detection of unique matrix-localized fucose and methionine synthesis, and the identification of new kinetoplast proteins, which showed kinetoplast-linked pyrimidine synthesis. Moreover, disruption of targeting signals by tagging allowed mapping of the mode of protein targeting to these sub-compartments, identifying a set of C-tail anchored outer mitochondrial membrane proteins and mitochondrial carriers likely employing multiple target peptides. This dataset represents a comprehensive, updated mapping of the mitochondrion.

• MeSH
• Biology MeSH
• Mitochondrial Proteins metabolism   MeSH
• Mitochondria metabolism   MeSH
• Parasites * metabolism   MeSH
• Protozoan Proteins metabolism   MeSH
• Trypanosoma brucei brucei * metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH