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Found across all kingdoms of life, 2-keto acid dehydrogenase complexes possess prominent metabolic roles and form major regulatory sites. Although their component structures are known, their higher-order organization is highly heterogeneous, not only across species or tissues but also even within a single cell. Here, we report a cryo-EM structure of the fully active Chaetomium thermophilum pyruvate dehydrogenase complex (PDHc) core scaffold at 3.85 Å resolution (FSC = 0.143) from native cell extracts. By combining cryo-EM with macromolecular docking and molecular dynamics simulations, we resolve all PDHc core scaffold interfaces and dissect the residing transacetylase reaction. Electrostatics attract the lipoyl domain to the transacetylase active site and stabilize the coenzyme A, while apolar interactions position the lipoate in its binding cleft. Our results have direct implications on the structural determinants of the transacetylase reaction and the role of flexible regions in the context of the overall 10 MDa PDHc metabolon architecture. How is acetyl-CoA produced in the context of the endogenous, eukaryotic pyruvate dehydrogenase complex metabolon? Here the authors dissect the embedded transacetylase reaction through biochemical, cryo-EM, HADDOCKing and molecular dynamics methods. [ABSTRACT FROM AUTHOR]

Background: Pyruvate dehydrogenase complex (PDC) plays a central role in carbohydrate metabolism, linking cytoplasmic glycolysis to the mitochondrial tricarboxylic acid (TCA) cycle. PDC is a conserved E1-E2-E3 dehydrogenase with a PDHA1 and PDHB heterotetramer functioning as the E1 subunit. PDHA1 contains three serine residues that can be reversibly phosphorylated by a dedicated family of four inhibitory pyruvate dehydrogenase kinases (PDHK1–4) and two reactivating phosphatases (PDP1, 2). Hypoxia induces the expression of PDHK1 and PDHK3 and hyperphosphorylates PDHA1. The role of PDC in metabolic reprogramming and tumor progression appears to be for the integration of oncogenic and environmental signals which supports tumor growth. Methods: To isolate the function of the serine-dependent regulation of PDC, we engineered MiaPaca2 cells to express PDHA1 protein with either intact serines at positions 232, 293, and 300 or all the combinations of non-phosphorylatable alanine substitution mutations. These lines were compared in vitro for biochemical response to hypoxia by western blot, metabolic activity by biochemical assay and Seahorse XF flux analysis, and growth in media with reduced exogenous metabolites. The lines were also tested for growth in vivo after orthotopic injection into the pancreata of immune-deficient mice. Results: In this family of cells with non-phosphorylatable PDHA1, we found reduced hypoxic phosphorylation of PDHA1, decreased PDH enzymatic activity in normoxia and hypoxia, decreased mitochondrial function by Seahorse flux assay, reduced in vitro growth of cells in media depleted of lipids, and reduced growth of tumors after orthotopic transplantation of cells into the pancreata of immune-deficient mice. Conclusions: We found that any substitution of alanine for serine at regulatory sites generated a hypomorphic PDC. However, the reduced PDC activity was insensitive to further reduction in hypoxia. These cells had a very modest reduction of growth in vitro, but failed to grow as tumors indicating that dynamic PDC adaptation to microenvironmental conditions is necessary to support pancreatic cancer growth in vivo. [ABSTRACT FROM AUTHOR]

The E2 component of the mitochondrial pyruvate dehydrogenase complex (PDC) is the key autoantigen in primary biliary cholangitis (PBC) and STAT3 is an inflammatory modulator that participates in the pathogenesis of many liver diseases. This study investigated whether PDC-E2 interacts with STAT3 in human cholangiocytes (NHC) and hepatocytes (Hep-G2) under cholestatic conditions induced by glyco-chenodeoxycholic acid (GCDC). GCDC induced PDC-E2 expression in the cytoplasmic and nuclear fraction of NHC, whereas in Hep-G2 cells PDC-E2 expression was induced only in the cytoplasmic fraction. GCDC-treatment stimulated phosphorylation of STAT3 in the cytoplasmic fraction of NHC. siRNA-mediated gene silencing of PDC-E2 reduced the expression of pY-STAT3 in NHC but not in HepG2 cells. Immunoprecipitation and a proximity ligation assay clearly demonstrated that GCDC enhanced pY-STAT3 binding to PDC-E2 in the nuclear and cytoplasmic fraction of NHC cells. Staining with Mitotracker revealed mitochondrial co-localization of PDC-E2/pS-STAT3 complexes in NHC and Hep-G2 cells. In cirrhotic PBC livers the higher expression of both PDC-E2 and pY-STAT3 was observed. The immunoblot analysis demonstrated the occurrence of double bands of PDC-E2 protein in control livers, which was associated with a lower expression of pY-STAT3. Our data indicate the interaction between PDC-E2 and phosphorylated STAT3 under cholestatic conditions, which may play a role in the development of PBC. [ABSTRACT FROM AUTHOR]

Dihydrolipoamide dehydrogenase (DLDH) is a homodimeric flavin‐dependent enzyme that catalyzes the NAD+‐dependent oxidation of dihydrolipoamide. The enzyme is part of several multi‐enzyme complexes such as the Pyruvate Dehydrogenase system that transforms pyruvate into acetyl‐co‐A. Concomitantly with its redox activity, DLDH produces Reactive Oxygen Species (ROS), which are involved in cellular apoptotic processes. DLDH possesses several moonlighting functions. One of these is the capacity to adhere to metal‐oxides surfaces. This was first exemplified by the presence of an exocellular form of the enzyme on the cell‐wall surface of Rhodococcus ruber. This capability was evolutionarily conserved and identified in the human, mitochondrial, DLDH. The enzyme was modified with Arg‐Gly‐Asp (RGD) groups, which enabled its interaction with integrin‐rich cancer cells followed by "integrin‐assisted‐endocytosis." This allowed harnessing the enzyme for cancer therapy. Combining the TiO2‐binding property with DLDH's ROS‐production, enabled us to develop several medical applications including improving oesseointegration of TiO2‐based implants and photodynamic treatment for melanoma. The TiO2‐binding sites of both the bacterial and human DLDH's were identified on the proteins' molecules at regions that overlap with the binding site of E3‐binding protein (E3BP). This protein is essential in forming the multiunit structure of PDC. Another moonlighting activity of DLDH, which is described in this Review, is its DNA‐binding capacity that may affect DNA chelation and shredding leading to apoptotic processes in living cells. The typical ROS‐generation by DLDH, which occurs in association with its enzymatic activity and its implications in cancer and apoptotic cell death are also discussed. [ABSTRACT FROM AUTHOR]

BACKGROUND: Styrax tonkinensis is a white‐flowered tree with considerable potential as a feedstock source for biodiesel production from the oily seed contained within its nutlike drupes. Transcriptome changes during oil accumulation have been previously reported, but not concurrent changes in the proteome. RESULTS: Using proteomic analysis of samples collected at 50, 70, 100 and 130 days after flowering (DAF), we identified 1472 differentially expressed proteins (DEPs). Based on their expression patterns, we grouped the DEPs into nine clusters and analyzed the pathway enrichment. Proteins related to starch and sucrose metabolism were most abundant at 50 DAF. Proteins involved in fatty acid (FA) biosynthesis were mainly grouped into a cluster that peaked at 70 DAF. Proteins related to protein processing in endoplasmic reticulum had two major patterns, trending either upwards or downwards, while proteins involved in amino acid biosynthesis showed more complex relationships. We identified 42 key enzymes involved in lipid accumulation during kernel development, including the acetyl‐CoA carboxylase complex (ACC) and the pyruvate dehydrogenase complex (PDC). One oil body membrane protein, oleosin, continuously increased during kernel development. CONCLUSION: A regulatory network of oil accumulation processes was built based on protein and available transcriptome expression data, which were in good temporal agreement. This analysis placed ACC and PDC in the center of the network, suggesting that the glycolytic provision of substrate plays a central regulatory role in FA biosynthesis and oil accumulation. © 2021 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Background: Identification and characterisation of monogenic causes of complex neurological phenotypes are important for genetic counselling and prognostication. Bi-allelic pathogenic variants in the gene encoding GLRX5, a protein involved in the early steps of Fe-S cluster biogenesis, are rare and cause two distinct phenotypes: isolated sideroblastic anemia and a neurological phenotype with variant non-ketotic hyperglycinemia. In this study, we analysed the evolution of clinical and MRI findings and long-term outcome of patients with GLRX5 mutations.Methods: Four patients from three Australian families of Lebanese descent were identified. All patients presented in childhood and were followed up into adult life through multiple clinical assessments. All were prescribed sodium benzoate.Results: All patients (all females, age range 18-56 years) showed a complex neurological phenotype characterised by varying combinations of spastic paraparesis, length-dependent motor/sensory-motor axonal polyneuropathy, and psychiatric disturbances with variable intellectual disability. All had non-ketotic hyperglycinemia and a homozygous pathogenic c.151_153delAAG (p.K51del) change in GLRX5. Motor disability gradually progressed reaching moderate disability during adolescence and moderately severe disability during adult life. The major MRI finding was the upper cervical spinal cord signal changes with contrast enhancement noted in all and additional leukoencephalopathy in one. On follow up MRI, the white matter lesions diminished on a subsequent scan and then remained static over time. The spinal cord showed gliotic changes. Two patients have previously demonstrated low pyruvate dehydrogenase complex deficiency but none had plasma lactate elevation, nor biochemical evidence of branch-chain keto-dehydrogenase deficiency. Glycine levels reduced in patients that tolerated sodium benzoate, possibly stabilising clinical manifestations.Conclusions: This report demonstrates that the p.K51del GLRX5 variant causes a distinct and predictable neurological phenotype. The clinical assessments spanning from childhood to adult life enable physicians to infer the natural history of GLRX5 related neurological disorder. There may be widespread metabolic consequences, and optimal management is unknown. [ABSTRACT FROM AUTHOR]

The article focuses on ZBP1-MLKL necroptotic signaling potentiates radiation-induced antitumor immunity via intratumoral STING pathway activation. Topics include the necroptosis, a form of regulated necrosis, participates in tumor development and dying cell immunogenicity, and the unclear how tumor cell–intrinsic necroptotic signaling contributes to radiation-induced antitumor immunity.

Background: No curative therapy for mitochondrial disease (MD) exists, prioritizing supportive treatment for symptom relief. In animal and cell models ketones decrease oxidative stress, increase antioxidants and scavenge free radicals, putting ketogenic diets (KDs) on the list of management options for MD. Furthermore, KDs are well-known, safe and effective treatments for epilepsy, a frequent symptom of MD. This systematic review evaluates efficacy and safety of KD for MD. Methods: We searched Pubmed, Cochrane, Embase and Cinahl (November 2020) with search terms linked to MD and KD. From the identified records, we excluded studies on Pyruvate Dehydrogenase Complex deficiency. From these eligible reports, cases without a genetically confirmed diagnosis and cases without sufficient data on KD and clinical course were excluded. The remaining studies were included in the qualitative analysis. Results: Only 20 cases (14 pediatric) from the 694 papers identified met the inclusion criteria (one controlled trial (n = 5), 15 case reports). KD led to seizure control in 7 out of 8 cases and improved muscular symptoms in 3 of 10 individuals. In 4 of 20 cases KD reversed the clinical phenotype (e.g. cardiomyopathy, movement disorder). In 5 adults with mitochondrial DNA deletion(s) related myopathy rhabdomyolysis led to cessation of KD. Three individuals with POLG mutations died while being on KD, however, their survival was not different compared to individuals with POLG mutations without KD. Conclusion: Data on efficacy and safety of KD for MD is too scarce for general recommendations. KD should be considered in individuals with MD and therapy refractory epilepsy, while KD is contraindicated in mitochondrial DNA deletion(s) related myopathy. When considering KD for MD the high rate of adverse effects should be taken into account, but also spectacular improvements in individual cases. KD is a highly individual management option in this fragile patient group and requires an experienced team. To increase knowledge on this—individually—promising management option more (prospective) studies using adequate outcome measures are crucial. [ABSTRACT FROM AUTHOR]

Background: The mitochondrial pyruvate dehydrogenase complex (PDC) link glycolysis to the tricarboxylic acid cycle by decarboxylating pyruvate to acetyl coenzyme A irreversibly. Cancer cells are characterized by a shift in cellular metabolism from mitochondrial respiration to glycolysis. PDC activity inhibition mediated by phosphorylation via pyruvate dehydrogenase kinase (PDK) has been linked to cancer. However, the clinical significance of PDKs in urothelial cancer prognosis is not clear. We investigated the role and prognostic value of PDK3 expression in patients with upper urinary tract urothelial carcinoma (UTUC) and urinary bladder urothelial carcinoma (UBUC). Patients and Methods: We retrospectively analyzed clinical data and pathological features. Formalin-fixed urothelial carcinoma (UC) tissues were collected and embedded in paraffin. The correlation of PDK3 expression with clinical characteristics, pathological findings and patient outcomes, including metastasis-free survival (MFS) and disease-specific survival (DSS) were analyzed by Pearson's chi-square test, Kaplan–Meier analysis, and the multivariate Cox proportional hazards model. Results: Data from 295 patients with UBUC and 340 patients with UTUC were evaluated. High PDK3 expression significantly correlated with several pathologic variables such as high T stage, lymph node metastases, high tumor grade, vascular invasion, and high mitotic rate (all P < 0.001). High PDK3 expression was associated with poor disease-specific survival (DSS) (P < 0.0001) and metastatic free survival (MFS) (P < 0.0001) in a Kaplan–Meier analysis. Additionally, multivariate analysis demonstrated increased PDK3 expression is a significant predictive risk factor for DSS [hazard ratio (HR) in UBUC, 2.79, P = 0.009; in UTUC, 2.561, P = 0.03] and MFS (HR in UBUC, 1.907, P = 0.024; in UTUC, 1.793, P = 0.044). The gene co-expression analysis showed abundant PDK3 co-upregulated genes were involved in the processes of DNA replication and repair through the Gene Ontology classification system. Conclusion: High PDK3 expression has been linked to negative pathologic characteristics and poor oncological outcomes, suggesting that it could be used as a predictive biomarker for UC. PDK3 mRNA levels and its co-upregulated genes were strongly associated with DNA replication and repair. These results suggest that PDK3 may play a key role in tumor proliferation and development. [ABSTRACT FROM AUTHOR]

The pyruvate dehydrogenase complex (PDHc) links glycolysis to the citric acid cycle by converting pyruvate into acetyl-coenzyme A. PDHc encompasses three enzymatically active subunits, namely pyruvate dehydrogenase, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase. Dihydrolipoyl transacetylase is a multidomain protein comprising a varying number of lipoyl domains, a peripheral subunit-binding domain, and a catalytic domain. It forms the structural core of the complex, provides binding sites for the other enzymes, and shuffles reaction intermediates between the active sites through covalently bound lipoyl domains. The molecular mechanism by which this shuttling occurs has remained elusive. Here, we report a cryo-EM reconstruction of the native E. coli dihydrolipoyl transacetylase core in a resting state. This structure provides molecular details of the assembly of the core and reveals how the lipoyl domains interact with the core at the active site. The pyruvate dehydrogenase complex (PDHc) is a large multienzyme complex that converts pyruvate into acetyl-coenzyme A and in E. coli the core of the PDHc is formed by 24 copies of dihydrolipoyl transacetylase. Here, the authors present the cryo-EM structure of the E. coli dihydrolipoyl transacetylase 24-mer core in a native resting state including lipoyl domains, and discuss the mechanism of substrate shuttling by the lipoyl domains. [ABSTRACT FROM AUTHOR]

Global increases in the prevalence of antimicrobial resistance highlight the urgent need for novel strategies to combat infectious diseases. Recent studies suggest that host metabolic pathways play a key role in host control of intracellular bacterial pathogens. In this study we explored the potential of targeting host metabolic pathways for innovative host-directed therapy (HDT) against intracellular bacterial infections. Through gene expression profiling in human macrophages, pyruvate metabolism was identified as potential key pathway involved in Salmonella enterica serovar Typhimurium (Stm) infections. Next, the effect of targeting pyruvate dehydrogenase kinases (PDKs) – which are regulators of the metabolic checkpoint pyruvate dehydrogenase complex (PDC) – on macrophage function and bacterial control was studied. Chemical inhibition of PDKs by dichloroacetate (DCA) induced PDC activation and was accompanied with metabolic rewiring in classically activated macrophages (M1) but not in alternatively activated macrophages (M2), suggesting cell-type specific effects of dichloroacetate on host metabolism. Furthermore, DCA treatment had minor impact on cytokine and chemokine secretion on top of infection, but induced significant ROS production by M1 and M2. DCA markedly and rapidly reduced intracellular survival of Stm , but interestingly not Mycobacterium tuberculosis , in human macrophages in a host-directed manner. In conclusion, DCA represents a promising novel HDT compound targeting pyruvate metabolism for the treatment of Stm infections. [ABSTRACT FROM AUTHOR]

Recently, the morbidity and mortality from lung cancer have continued to increase. Mitochondrial dysfunction plays a key role in apoptosis, proliferation, and the bioenergetic reprogramming of cancer cells, especially for energy metabolism. Herein, we investigated the ability of melatonin (MLT) to influence lung cancer growth and explored the association between mitochondrial functions and the progression of lung tumors. The deacetylase, sirtuin 3 (Sirt3), is a pivotal player in maintenance of mitochondrial function, among participating in ATP production by regulating the acetylone and pyruvate dehydrogenase complex (PDH). We initially found that MLT inhibited lung cancer growth in the Lewis mouse model. Similarly, we observed that MLT inhibited the proliferation of lung cancer cells (A549, PC9, and LLC cells), and the underlying mechanism of MLT was related to reprogramming cancer cell metabolism, accompanied by a shift from cytosolic aerobic glycolysis to oxidative phosphorylation (OXPHOS). These changes were accompanied by higher ATP production, an elevated ATP production‐coupled oxygen consumption rate (QCR), higher ROS levels, higher mito‐ROS levels, and lower lactic acid secretion. Additionally, we observed that MLT improved mitochondrial membrane potential and the activities of complexes Ⅰ and Ⅳ in the electron transport chain. Importantly, we also found and verified that the foregoing changes resulted from activation of Sirt3 and PDH. As a result of these changes, MLT significantly enhanced mitochondrial energy metabolism to reverse the Warburg effect via increasing PDH activity with stimulation of Sirt3. Collectively, these findings suggest the potential use of melatonin as an anti‐lung cancer therapy and provide a mechanistic basis for this proposal. [ABSTRACT FROM AUTHOR]

Shc proteins interact with the insulin receptor, indicating a role in regulating glycolysis. To investigate this idea, the activities of key glycolytic regulatory enzymes and metabolites levels were measured in skeletal muscle from mice with low levels of Shc proteins (ShcKO) and wild-type (WT) controls. The activities of hexokinase, phosphofructokinase-1 and pyruvate kinase were decreased in ShcKO versus WT mice under both fed and fasted conditions. Increased alanine transaminase and branched-chain amino acid transaminase activities were also observed in ShcKO mice under both fed and fasting conditions. Protein expression of glycolytic enzymes was unchanged in the ShcKO and WT mice, indicating that decreased activities were not due to changes in their transcription. Changes in metabolite levels were consistent with the observed changes in enzyme activities. In particular, the levels of fructose-2,6-bisphosphate, a potent activator of phosphofructokinase-1, were consistently decreased in the ShcKO mice. Furthermore, the levels of lactate (inhibitor of hexokinase and phosphofructokinase-1) and citrate (inhibitor of phosphofructokinase-1 and pyruvate kinase) were increased in fed and fasted ShcKO versus WT mice. Pyruvate dehydrogenase activity was lower in ShcKO versus WT mice under fed conditions, and showed inhibition under fasting conditions in both ShcKO and WT mice, with ShcKO mice showing less inhibition than the WT mice. Pyruvate dehydrogenase kinase 4 levels were unchanged under fed conditions but were lower in the ShcKO mice under fasting conditions. These studies indicate that decreased levels of Shc proteins in skeletal muscle lead to a decreased glycolytic capacity in both fed and fasted states.

Decabromodiphenyl ether (BDE-209) has been universally detected in environmental media and animals, but its damage to ovarian function and mechanism is still unclear, and melatonin has been shown to improve mammalian ovarian function. This study aimed to investigate the toxic effects of BDE-209 on the ovary and tried to improve ovarian function with melatonin. Herein, BDE-209 was administered orally to female SD rats for 60 days. Enzyme-linked immunosorbent assay, HE staining, transcriptome analysis, qPCR and immunohistochemical staining were used to explore and verify the potential mechanism. We found that BDE-209 exposure had effects on the ovary, as shown by abnormal changes in the estrous cycle, hormone levels and ovarian reserve function in rats, while increasing the proportion of collagen fibres in ovarian tissue. In terms of mechanism, cuproptosis, a form of cell death, was identified to play a crucial role in BDE-209-induced ovarian dysfunction, with the phenotype manifested as copper salt accumulation in ovary, downregulation of glutathione pathway metabolism and copper transfer molecule (ATP7A/B), and upregulation of FDX1 , lipoic acid pathway (LIAS, LIPT1), pyruvate dehydrogenase complex components (DLAT, PDHB, PDHA1), and copper transfer molecule (SLC31A1). Furthermore, possible interventions were explored. Notably, a supplement with melatonin has a repair effect on the damage to ovarian function by reversing the gene expression of cuproptosis-involved molecules. Overall, this study revealed that cuproptosis is involved in BDE-209-induced ovarian damage and the beneficial effect of melatonin on ovarian copper damage, providing evidence for the prevention and control of female reproductive damage induced by BDE-209. [Display omitted] • Chronic exposure to BDE-209 leads to abnormal ovarian function. • Cuproptosis plays an important role in BDE-209-induced ovarian dysfunction. • Melatonin can change the expression of cuproptosis-related molecules. • Melatonin can improve the ovarian damage caused by BDE-209. [ABSTRACT FROM AUTHOR]

However, no mosaicism was identified in the lymphocytes, hair bulb cells, buccal smear cells and sperm cells of the parents from the other nine trios (Figure 1). The mosaicism was not present by NGS in the lymphocytes, hair bulb cells and buccal smear cells of EB1's father, indicating gonadal mosaicism (Figure 1a). Variants were found only in sperm cells of EB1's father (6-64%) and in buccal smear cells of EB2's father (2-26%), and no variant was detected in other cell samples, indicating those mosaicisms must have occurred late during embryonic development and could not develop the EBS phenotype. [Extracted from the article]

Background: Metabolic remodeling precedes most alterations during cardiac hypertrophic growth under hemodynamic stress. The elevation of glucose utilization has been recognized as a hallmark of metabolic remodeling. However, its role in cardiac hypertrophic growth and heart failure in response to pressure overload remains to be fully illustrated. Here, we aimed to dissect the role of cardiac PKM1 (pyruvate kinase muscle isozyme 1) in glucose metabolic regulation and cardiac response under pressure overload.Methods: Cardiac-specific deletion of PKM1 was achieved by crossing the floxed PKM1 mouse model with the cardiomyocyte-specific Cre transgenic mouse. PKM1 transgenic mice were generated under the control of tetracycline response elements, and cardiac-specific overexpression of PKM1 was induced by doxycycline administration in adult mice. Pressure overload was triggered by transverse aortic constriction. Primary neonatal rat ventricular myocytes were used to dissect molecular mechanisms. Moreover, metabolomics and nuclear magnetic resonance spectroscopy analyses were conducted to determine cardiac metabolic flux in response to pressure overload.Results: We found that PKM1 expression is reduced in failing human and mouse hearts. It is important to note that cardiomyocyte-specific deletion of PKM1 exacerbates cardiac dysfunction and fibrosis in response to pressure overload. Inducible overexpression of PKM1 in cardiomyocytes protects the heart against transverse aortic constriction-induced cardiomyopathy and heart failure. At the mechanistic level, PKM1 is required for the augmentation of glycolytic flux, mitochondrial respiration, and ATP production under pressure overload. Furthermore, deficiency of PKM1 causes a defect in cardiomyocyte growth and a decrease in pyruvate dehydrogenase complex activity at both in vitro and in vivo levels.Conclusions: These findings suggest that PKM1 plays an essential role in maintaining a homeostatic response in the heart under hemodynamic stress. [ABSTRACT FROM AUTHOR]

Background: Gallbladder cancer (GBC) is a malignant cancer with poor prognosis. Evidences have shown that miRNAs are closely related to the occurrence of GBC; thus, we aimed to explore miRNAs, which plays an important role in the occurrence and development of GBC. Methods: Microarray analysis was performed to investigate the differentially expressed miRNAs between five non-neoplastic gallbladder tissues (normal tissues) and five gallbladder tumor tissues (tumor tissues). RT-qPCR was performed to detect the level of miR-181b-5p in cells, and CCK-8 was performed to detect cell viability. Then, glucose assay kit or lactic acid assay kit was performed to detect the level of glucose consumption or lactate production. Next, transwell and wound healing assays were used to assess cell migration. In addition, dual-luciferase reporter assay was used to verify the relationship between miR-181b-5p and PDHX. At last, Western blotting was performed to determine the protein level of PDHX. Results: Microarray analysis suggested miR-181b-5p was significantly upregulated in GBC tumor tissue. KEGG analysis for the protein targets of miR-181b-5p indicates a close relationship existed between miR-181b-5p and glycolysis. In addition, the level of miR-181b-5p was notably increased in GBC-SD or G415 cells, compared with HIBEpiC cells. GBC cell viability was significantly decreased under hypoxia, and these decreases were exacerbated by miR-181b-5p antagomir. Moreover, glucose consumption or lactate production of GBC cells was significantly upregulated under hypoxia, whereas these increases were completely revered by miR-181b-5p antagomir. Further investigation revealed that PDHX was a direct target of miR-181b-5p. Conclusion: In this study, downregulation of miR-181b-5p inhibits the viability, migration, and glycolysis of GBC by upregulating PDHX under hypoxia. This finding suggested that miR-181b-5p might be considered as a novel therapeutic target for the treatment of GBC. [ABSTRACT FROM AUTHOR]

Acetate overflow refers to the metabolism by which a large part of carbon incorporated as glucose into Escherichia coli cells is catabolized and excreted as acetate into the medium. We previously found that mutants for the acetate overflow pathway enzymes phosphoacetyltransferase (Pta) and acetate kinase (AckA) showed significant diauxic growth after glucose depletion in E. coli. Here, we analyzed the underlying mechanism in the pta mutant. Proteomic and other analyses revealed an increase in pyruvate dehydrogenase complex subunits and a decrease in glyoxylate shunt enzymes, which resulted from pyruvate accumulation. Since restoration of these enzyme levels by overexpressing PdhR (pyruvate‐sensing transcription factor) or deleting iclR (gene encoding a pyruvate‐ and glyoxylate‐sensing transcription factor) alleviated the growth lag of the pta mutant after glucose depletion, these changes were considered as the reason for the phenotype. Given the evidence for decreased coenzyme A (HS‐CoA) levels in the pta mutant, the growth inhibition after glucose depletion was partly explained by limited availability of HS‐CoA in the cell. The findings provide insights into the role of acetate overflow in metabolic regulation, which may be useful for biotechnological applications. [ABSTRACT FROM AUTHOR]

Background: Pyruvate dehydrogenase complex (PDHC) deficiency is a common neurodegenerative disease associated with abnormal mitochondrial energy metabolism. The diagnosis of PDHC is difficult because of the lack of a rapid, accurate, and cost‐effective clinical diagnostic method. Methods: A 4‐year‐old boy was preliminarily diagnosed with putative Leigh syndrome based on the clinical presentation. PDHC activity in peripheral blood leukocytes and a corresponding gene analysis were subsequently undertaken. Sodium pyruvate 1‐13C was used for the analysis of PDHC activity in peripheral leukocytes. The genes encoding PDHC were then scanned for mutations. Results: The results showed that the corresponding PDHC activity was dramatically decreased to 10.5 nmol/h/mg protein as compared with that of healthy controls (124.6 ± 7.1 nmol/h/mg). The ratio of PDHC to citrate synthase was 2.1% (control: 425.3 ± 27.1). The mutation analysis led to the identification of a missense mutation, NM_000284.4:g214C>T, in exon 3 of PDHC. Conclusion: The peripheral blood leukocyte PDHC activity assay may provide a practical enzymatic diagnostic method for PDHC‐related mitochondrial diseases. [ABSTRACT FROM AUTHOR]

CB1 cannabinoid receptor is widely expressed in the central nervous system of animals from late prenatal development to adulthood. Appropriate activation and signaling of CB1 cannabinoid receptors in cortical interneurons are crucial during perinatal/postnatal ages and adolescence, when long‐lasting changes in brain activity may elicit subsequent appearance of disorders in the adult brain. Here we used an optimized immunoprecipitation protocol based on specific antibodies followed by shot‐gun proteomics to find CB1 interacting partners in postnatal rat GABAergic cortical neurons in vitro at two different stages of maturation. Besides describing new proteins associated with CB1 like dihydrolipoyllysine‐residue acetyltransferase component of pyruvate dehydrogenase complex (DLAT), fatty acid synthase (FASN), tyrosine 3‐monooxygenase/tryptophan 5‐monooxygenase activation protein zeta (YWHAZ), voltage‐dependent anion channel 1 (VDAC1), myosin phosphatase Rho‐interacting protein (MPRIP) or usher syndrome type‐1C protein‐binding protein 1 (USHBP1), we show that the signaling complex of CB1 is different between maturational stages. Interestingly, the CB1 signaling complex is enriched at the more immature stage in mitochondrial associated proteins and metabolic molecular functions, whereas at more mature stage, CB1 complex is increased in maturation and synaptic‐associated proteins. We describe also interacting partners specifically immunoprecipitated with either N‐terminal or C‐terminal CB1 directed antibodies. Our results highlight new players that may be affected by altered cannabinoid signaling at this critical window of postnatal cortical development. [ABSTRACT FROM AUTHOR]

Animal African Trypanosomiasis (AAT) is a debilitating livestock disease prevalent across sub-Saharan Africa, a main cause of which is the protozoan parasite Trypanosoma congolense. In comparison to the well-studied T. brucei, there is a major paucity of knowledge regarding the biology of T. congolense. Here, we use a combination of omics technologies and novel genetic tools to characterise core metabolism in T. congolense mammalian-infective bloodstream-form parasites, and test whether metabolic differences compared to T. brucei impact upon sensitivity to metabolic inhibition. Like the bloodstream stage of T. brucei, glycolysis plays a major part in T. congolense energy metabolism. However, the rate of glucose uptake is significantly lower in bloodstream stage T. congolense, with cells remaining viable when cultured in concentrations as low as 2 mM. Instead of pyruvate, the primary glycolytic endpoints are succinate, malate and acetate. Transcriptomics analysis showed higher levels of transcripts associated with the mitochondrial pyruvate dehydrogenase complex, acetate generation, and the glycosomal succinate shunt in T. congolense, compared to T. brucei. Stable-isotope labelling of glucose enabled the comparison of carbon usage between T. brucei and T. congolense, highlighting differences in nucleotide and saturated fatty acid metabolism. To validate the metabolic similarities and differences, both species were treated with metabolic inhibitors, confirming that electron transport chain activity is not essential in T. congolense. However, the parasite exhibits increased sensitivity to inhibition of mitochondrial pyruvate import, compared to T. brucei. Strikingly, T. congolense exhibited significant resistance to inhibitors of fatty acid synthesis, including a 780-fold higher EC50 for the lipase and fatty acid synthase inhibitor Orlistat, compared to T. brucei. These data highlight that bloodstream form T. congolense diverges from T. brucei in key areas of metabolism, with several features that are intermediate between bloodstream- and insect-stage T. brucei. These results have implications for drug development, mechanisms of drug resistance and host-pathogen interactions. Author summary: Animal African Trypanosomiasis (AAT), also known as Nagana, is a devastating disease affecting livestock across sub-Saharan Africa. AAT is primarily caused by the parasite Trypanosoma congolense, yet our biological knowledge about this pathogen is poor, especially compared to the related species T. brucei, subspecies of which cause the human disease Sleeping Sickness. Understanding the core metabolism of T. congolense is crucial in order to gain insights into the infection biology of this important pathogen, as well as providing the potential to identify new drug targets. In this work, we addressed the lack of knowledge concerning T. congolense by carrying out a comprehensive analysis of core metabolism, and comparing the data to T. brucei. We then used the findings of metabolic differences to predict differential sensitivity to inhibitors of metabolic function. We show that unlike T. brucei, where glucose metabolism leads to high levels of pyruvate excretion, T. congolense metabolises glucose to other end-products, namely succinate, malate and acetate. Moreover, there are pronounced differences in the way T. congolense uses glucose to feed into other areas of metabolism. Further analysis also suggests that T. congolense mostly scavenges lipids and fatty acids, rather than synthesising them de novo. To validate these findings, we confirm that T. congolense is differentially susceptible to metabolic inhibitors compared to T. brucei, and that, in particular, T. congolense is significantly less sensitive to inhibitors of fatty acid synthesis. Our study provides a foundation of functional metabolic knowledge on T. congolense, with insights into how this parasite fundamentally differs from T. brucei. [ABSTRACT FROM AUTHOR]

Metabolic changes that correlate to cognitive changes are well-known in Alzheimer's disease (AD). Metabolism is often linked to functional changes in proteins by post-translational modifications. The importance of the regulation of transcription by acetylation is well documented. Advanced mass spectrometry reveals hundreds of acetylated proteins in multiple tissues, but the acetylome of human brain, its functional significance, and the changes with disease are unknown. Filling this gap is critical for understanding the pathophysiology and development of therapies. To fill this gap, we assessed the human brain acetylome in human brain and its changes with AD. More than 5% of the 4,442 proteins from the human brain global proteome were acetylated. Acetylated proteins were primarily found in the cytosol (148), mitochondria (100), nucleus (91), and plasma membrane (58). The comparison of the brain acetylome in controls to that of patients with AD revealed striking and selective differences in terms of its abundances of acetylated peptides/sites. Acetylation of 18 mitochondrial proteins decreased, while acetylation of two cytosolic proteins, tau and GFAP, increased. Our experiments demonstrate that acetylation at some specific lysine sites alters enzyme function. The results indicate that general activation of de-acetylases (i.e., sirtuins) is not an appropriate therapeutic approach for AD. [ABSTRACT FROM AUTHOR]

Aims/Introduction: Brown adipose tissue (BAT) utilizes large amounts of fuel for thermogenesis, but the mechanism by which fuel substrates are switched in response to changes in energy status is poorly understood. We have now investigated the role of Kruppel‐like factor 15 (KLF15), a transcription factor expressed at a high level in adipose tissue, in the regulation of fuel utilization in BAT. Materials and Methods: Depletion or overexpression of KLF15 in HB2 differentiated brown adipocytes was achieved by adenoviral infection. Glucose and fatty acid oxidation were measured with radioactive substrates, pyruvate dehydrogenase complex activity was determined with a colorimetric assay, and gene expression was examined by reverse transcription and real‐time polymerase chain reaction analysis. Results: Knockdown of KLF15 in HB2 cells attenuated fatty acid oxidation in association with downregulation of the expression of genes related to this process including Acox1 and Fatp1, whereas it increased glucose oxidation. Expression of the gene for pyruvate dehydrogenase kinase 4 (PDK4), a negative regulator of pyruvate dehydrogenase complex, was increased or decreased by KLF15 overexpression or knockdown, respectively, in HB2 cells, with these changes being accompanied by a respective decrease or increase in pyruvate dehydrogenase complex activity. Chromatin immunoprecipitation showed that Pdk4 is a direct target of KLF15 in HB2 cells. Finally, fasting increased expression of KLf15, Pdk4 and genes involved in fatty acid utilization in BAT of mice, whereas refeeding suppressed Klf15 and Pdk4 expression. Conclusions: Our results implicate KLF15 in the regulation of fuel switching between glucose and fatty acids in response to changes in energy status in BAT. [ABSTRACT FROM AUTHOR]

Altering metabolic flux at a key branch point in metabolism has commonly been accomplished through gene knockouts or by modulating gene expression. An alternative approach to direct metabolic flux preferentially toward a product is decreasing the activity of a key enzyme through protein engineering. In Escherichia coli, pyruvate can accumulate from glucose when carbon flux through the pyruvate dehydrogenase complex is suppressed. Based on this principle, 16 chromosomally expressed AceE variants were constructed in E. coli C and compared for growth rate and pyruvate accumulation using glucose as the sole carbon source. To prevent conversion of pyruvate to other products, the strains also contained deletions in two nonessential pathways: lactate dehydrogenase (ldhA) and pyruvate oxidase (poxB). The effect of deleting phosphoenolpyruvate synthase (ppsA) on pyruvate assimilation was also examined. The best pyruvate-accumulating strains were examined in controlled batch and continuous processes. In a nitrogen-limited chemostat process at steady-state growth rates of 0.15 to 0.28 h21, an engineered strain expressing the AceE[H106V] variant accumulated pyruvate at a yield of 0.59 to 0.66 g pyruvate/g glucose with a specific productivity of 0.78 to 0.92 g pyruvate/g cells·h. These results provide proof of concept that pyruvate dehydrogenase complex variants can effectively shift carbon flux away from central carbon metabolism to allow pyruvate accumulation. This approach can potentially be applied to other key enzymes in metabolism to direct carbon toward a biochemical product. [ABSTRACT FROM AUTHOR]

Influenza A virus (IAV) infection alters lung epithelial cell metabolism in vitro by promoting a glycolytic shift. We hypothesized that this shift benefits the virus rather than the host and that inhibition of glycolysis would improve infection outcomes. A/WSN/33 IAV-inoculated C57BL/6 mice were treated daily from 1 day post-inoculation (d.p.i.) with 2-deoxy- d -glucose (2-DG) to inhibit glycolysis and with the pyruvate dehydrogenase kinase (PDK) inhibitor dichloroacetate (DCA) to promote flux through the TCA cycle. To block OXPHOS, mice were treated every other day from 1 d.p.i. with the Complex I inhibitor rotenone (ROT). 2-DG significantly decreased nocturnal activity, reduced respiratory exchange ratios, worsened hypoxemia, exacerbated lung dysfunction, and increased humoral inflammation at 6 d.p.i. DCA and ROT treatment normalized oxygenation and airway resistance and attenuated IAV-induced pulmonary edema, histopathology, and nitrotyrosine formation. None of the treatments altered viral replication. These data suggest that a shift to glycolysis is host-protective in influenza. • Influenza infection causes a glycolytic shift in respiratory epithelial cells. • Effects of influenza on whole body metabolism and nocturnal activity were measured. • Systemic inhibition of glycolysis exacerbates influenza severity. • Blockade of pyruvate dehydrogenase kinase or Complex I improves outcomes. • Complex I blockade attenuates nitrotyrosine formation in the lungs. [ABSTRACT FROM AUTHOR]

Metabolic changes that correlate to cognitive changes are well‐known in Alzheimer's disease (AD). Metabolism is often linked to functional changes in proteins by post‐translational modifications. The importance of the regulation of transcription by acetylation is well documented. Advanced mass spectrometry reveals hundreds of acetylated proteins in multiple tissues, but the acetylome of human brain, its functional significance, and the changes with disease are unknown. Filling this gap is critical for understanding the pathophysiology and development of therapies. To fill this gap, we assessed the human brain acetylome in human brain and its changes with AD. More than 5% of the 4,442 proteins from the human brain global proteome were acetylated. Acetylated proteins were primarily found in the cytosol (148), mitochondria (100), nucleus (91), and plasma membrane (58). The comparison of the brain acetylome in controls to that of patients with AD revealed striking and selective differences in terms of its abundances of acetylated peptides/sites. Acetylation of 18 mitochondrial proteins decreased, while acetylation of two cytosolic proteins, tau and GFAP, increased. Our experiments demonstrate that acetylation at some specific lysine sites alters enzyme function. The results indicate that general activation of de‐acetylases (i.e., sirtuins) is not an appropriate therapeutic approach for AD. [ABSTRACT FROM AUTHOR]

Complexes [Ru(η6-pcym)(bpydca)Cl]PF6 (Rudca) and [Ir(η5-Cp*)(bpydca)Cl]PF6 (Irdca) were developed as model compounds for the investigation of multi-targeted ester-functionalized half-sandwich ruthenium(II) and iridium(III) complexes; pcym = 1-methyl-4-(propan-2-yl)benzene (p-cymene), bpydca = 2,2′-bipyridine-4,4′-diyldimethanediyl bis(dichloroacetate), Cp* = pentamethylcyclopentadienyl. Aiming to understand the in-solution behaviour of these first-in-class complexes containing the pyruvate dehydrogenase kinase inhibitor dichloroacetate (dca) as the terminal bioactive substituent, several experiments were performed under aqueous conditions for Rudca and Irdca, as well as for compounds [Ru(η6-pcym)(bpyOH)Cl]PF6 (RuOH) and [Ir(η5-Cp*)(bpyOH)Cl]PF6 (IrOH), and acetyl analogues [Ru(η6-pcym)(bpyac)Cl]PF6 (Ruac) and [Ir(η5-Cp*)(bpyac)Cl]PF6 (Irac) bearing a different (biologically inactive) terminal substituent; bpyOH = 2,2′-bipyridine-4,4′-diyldimethanol, bpyac = 2,2′-bipyridine-4,4′-diyldimethanediyl diacetate. The experiments were also conducted in the presence of porcine liver esterase (PLE). All the six complexes were characterized by relevant techniques (e.g., NMR and mass spectrometry), including a single-crystal X-ray analysis of complexes Rudca, Ruac, RuOH and IrOH. Although designed as model compounds, Rudca, Irdca, RuOH and IrOH were also screened for their antiproliferative activity in four human cancer cell lines (HCT116 colon carcinoma, MDA-MB-231 and MCF-7 breast adenocarcinomas, DU145 prostate carcinoma), where the tested complexes did not show any effect (IC50 > 100 μM). [ABSTRACT FROM AUTHOR]

Summary: Primary biliary cholangitis (PBC) is characterized by the presence of serum anti‐mitochondrial autoantibodies (AMAs). To date, four antigens among the 2‐oxo‐acid dehydrogenase complex family, which commonly have lipoyl domains as an epitope, have been identified as AMA‐corresponding antigens (AMA‐antigens). It has recently been reported that AMAs react more strongly with certain chemically modified mimics than with the native lipoyl domains in AMA‐antigens. Moreover, high concentrations of circulating immune complexes (ICs) in PBC patients have been reported. However, the existence of ICs formed by AMAs and their antigens has not been reported to date. We hypothesized that AMAs and their antigens formed ICs in PBC sera, and analyzed sera of PBC and four autoimmune diseases (Sjögren's syndrome, systemic lupus erythematosus, systemic scleroderma, and rheumatoid arthritis) using immune complexome analysis, in which ICs are separated from serum and are identified by nano‐liquid chromatography‐tandem mass spectrometry. To correctly assign MS/MS spectra to peptide sequences, we used a protein‐search algorithm that including lipoylation and certain xenobiotic modifications. We found three AMA‐antigens, the E2 subunit of the pyruvate dehydrogenase complex (PDC‐E2), the E2 subunit of the 2‐oxo‐glutarate dehydrogenase complex (OGDC‐E2) and dihydrolipoamide dehydrogenase binding protein (E3BP), by detecting peptides containing lipoylation and xenobiotic modifications from PBC sera. Although the lipoylated sites of these peptides were different from the well‐known sites, abnormal lipoylation and xenobiotic modification may lead to production of AMAs and the formation ICs. Further investigation of the lipoylated sites, xenobiotic modifications, and IC formation will lead to deepen our understanding of PBC pathogenesis. [ABSTRACT FROM AUTHOR]

Many current cancer vaccine strategies suffer from the inability to mount a CD8 T cell response that is strong enough to overcome the low immunogenicity of tumors. Viruses naturally possess the sizes, geometries, and physical properties for which the immune system has evolved to recognize, and mimicking those properties with nanoparticles can produce robust platforms for vaccine design. Using the nonviral E2 core of pyruvate dehydrogenase, we have engineered a viral-mimicking vaccine platform capable of encapsulating dendritic cell (DC)-activating CpG molecules in an acid-releasable manner and displaying MHC I-restricted SIINFEKL peptide epitopes. Encapsulated CpG activated bone marrow-derived DCs at a 25-fold lower concentration in vitro when delivered with the E2 nanoparticle than with unbound CpG alone. Combining CpG and SIINFEKL within a single multifunctional particle induced ∼3-fold greater SIINFEKL display on MHC I by DCs over unbound peptide. Importantly, combining CpG and SIINFEKL to the E2 nanoparticle for simultaneous temporal and spatial delivery to DCs showed increased and prolonged CD8 T cell activation, relative to free peptide or peptide-bound E2. By codelivering peptide epitopes and CpG activator in a particle of optimal DC-uptake size, we demonstrate the ability of a noninfectious protein nanoparticle to mimic viral properties and facilitate enhanced DC activation and cross-presentation.

Ferroptosis is a non-accidental, regulated form of cell death operated by lipid peroxidation under strict control of GPx4 activity. This is consistent with the notion that lipid peroxidation is initiated by radicals produced from decomposition of traces of pre-existing lipid hydroperoxides. The question, therefore, emerges about the formation of these traces of lipid hydroperoxides interacting with Fe2+. In the most realistic option, they are produced by oxygen activated species generated during aerobic metabolism. Screening for metabolic sources of superoxide supporting ferroptosis induced by GSH depletion, we failed to detect, in our cell model, a role of respiratory chain. We observed instead that the pyruvate dehydrogenase complex -as other α keto acid dehydrogenases already known as a major source of superoxide in mitochondria- supports ferroptosis. The opposite effect on ferroptosis by silencing either the E1 or the E3 subunit of the pyruvate dehydrogenase complex pointed out the autoxidation of dihydrolipoamide as the source of superoxide. We finally observed that GSH depletion activates superoxide production, seemingly through the inhibition of the specific kinase that inhibits pyruvate dehydrogenase. In summary, this set of data is compatible with a scenario where the more electrophilic status produced by GSH depletion not only activates ferroptosis by preventing GPx4 activity, but also favors the formation of lipid hydroperoxides. In an attractive perspective of tissue homeostasis, it is the activation of energetic metabolism associated to a decreased nucleophilic tone that, besides supporting energy demanding proliferation, also sensitizes cells to a regulated form of death. [Display omitted] • Ferroptosis is activated by the synergy between low GSH and glucose aerobic metabolism. • GSH depletion enhances superoxide production by the pyruvate dehydrogenase complex. • Superoxide is produced by autoxidation of dihydrolipoamide. • Protonated superoxide is proposed generating traces of lipid hydroperoxides. [ABSTRACT FROM AUTHOR]

Background: Leigh syndrome, the most common mitochondrial syndrome in pediatrics, has diverse clinical manifestations and is genetically heterogeneous. Pathogenic mutations in more than 75 genes of two genomes (mitochondrial and nuclear) have been identified. PDHA1 encoding the E1 alpha subunit is an X‐chromosome gene whose mutations cause pyruvate dehydrogenase complex deficiency. Methods: Here, we have described a 12‐year‐old boy with lethal neuropathy who almost died of a sudden loss of breathing and successive cardiac arrest. Extracorporeal membrane oxygenation rescued his life. His diagnosis was corrected from Guillain–Barré syndrome to Leigh syndrome 1 month later by clinical exome sequencing. Furthermore, we used software to predict the protein structure caused by frameshift mutations. We treated the boy with vitamin B1, coenzyme Q10, and a ketogenic diet. Results: A PDHA1 mutation (NM_000284.4:c.1167_1170del) was identified as the underlying cause. The amino acid mutation was p.Ser390LysfsTer33. Moreover, the protein structure prediction results suggested that the protein structure has changed. The parents of the child were negative, so the mutation was de novo. The comprehensive assessment of the mutation was pathogenic. His condition gradually improved after receiving treatment. Conclusion: This case suggests that gene detection should be popularized to improve diagnosis accuracy, especially in developing countries such as China. [ABSTRACT FROM AUTHOR]

Pyruvate dehydrogenase complex (PDC) catalyzes the oxidative decarboxylation of pyruvate to acetyl-coenzyme A, hinging glycolysis and the tricarboxylic acid cycle. PDC deficiency, an inborn error of metabolism, has a broad phenotypic spectrum. Symptoms range from fatal lactic acidosis or progressive neuromuscular impairment in the neonatal period, to chronic neurodegeneration. Most disease-causing mutations in PDC deficiency affect the PDHA1 gene, encoding the α subunit of the PDC-E1 component. Detailed biophysical analysis of pathogenic protein variants is a challenging approach to support the design of therapies based on improving and correcting protein structure and function. Herein, we report the characterization of clinically relevant PDC-E1α variants identified in Portuguese PDC deficient patients. These variants bear amino acid substitutions in different structural regions of PDC-E1α. The structural and functional analyses of recombinant heterotetrameric (αα'ββ') PDC-E1 variants, combined with molecular dynamics (MD) simulations, show a limited impact of the amino acid changes on the conformational stability, apart from the increased propensity for aggregation of the p.R253G variant as compared to wild-type PDC-E1. However, all variants presented a functional impairment in terms of lower residual PDC-E1 enzymatic activity and ≈3–100 × lower affinity for the thiamine pyrophosphate (TPP) cofactor, in comparison with wild-type PDC-E1. MD simulations neatly showed generally decreased stability (increased flexibility) of all variants with respect to the WT heterotetramer, particularly in the TPP binding region. These results are discussed in light of disease severity of the patients bearing such mutations and highlight the difficulty of developing chaperone-based therapies for PDC deficiency. [Display omitted] • Pyruvate dehydrogenase complex (PDC) links glycolysis and the tricarboxylic acid cycle. • PDC deficiency results from mutations in the genes encoding PDC subunits, mostly PDHA1. • Missense PDHA1 mutations originate functionally impaired PDC-E1 variants with decreased affinity for TPP. • PDC-E1 p.R253G variant has increased propensity for aggregation. • Molecular dynamics simulations reveal decreased stability of PDC-E1 variants near the TPP binding site. [ABSTRACT FROM AUTHOR]

Mitochondria and releasable endoplasmic reticulum (ER) calcium modulate neuronal calcium signaling, and both change in Alzheimer's disease (AD). The releasable calcium stores in the ER are exaggerated in fibroblasts from AD patients and in multiple models of AD. The activity of the alpha‐ketoglutarate dehydrogenase complex (KGDHC), a key mitochondrial enzyme complex, is diminished in brains from AD patients, and can be plausibly linked to plaques and tangles. Our previous studies in cell lines and mouse neurons demonstrate that reductions in KGDHC increase the ER releasable calcium stores. The goal of these studies was to test whether the relationship was true in human iPSC‐derived neurons. Inhibition of KGDHC for one or 24 hr increased the ER releasable calcium store in human neurons by 69% and 144%, respectively. The effect was mitochondrial enzyme specific because inhibiting the pyruvate dehydrogenase complex, another key mitochondrial enzyme complex, diminished the ER releasable calcium stores. The link of KGDHC to ER releasable calcium stores was cell type specific as the interaction was not present in iPSC or neural stem cells. Thus, these studies in human neurons verify a link between KGDHC and releasable ER calcium stores, and support the use of human neurons to examine mechanisms and potential therapies for AD. [ABSTRACT FROM AUTHOR]

Early detection of prion protein aggregation with a fluorescent pentameric oligothiophene pro... I A. Stepanchuk, W. Tahir, K. P. R. Nilsson, H. M. Schatzl and P. K. Stys i Early detection of misfolded and aggregated prion protein might provide useful insights into pathophysiology of prion diseases. Identification of truncated C-terminal fragments of the Alzheimer's disease amyloid protein p... I S. Mosser, H. Gerber and P. C. Fraering i Recent evidence suggests that the amyloid- precursor protein C-terminal fragments (APP-CTFs) contribute to Alzheimer's disease (AD). I D. Trinh, A. R. Israwi, L. R. Arathoon, J. A. Gleave and J. E. Nash i In Parkinson's disease (PD), mitochondria of dopaminergic neurons in the SNc are more susceptible to cellular stress than other neurons, which contribute to neurodegeneration. The stability of the complexes (C1- -Syn amyloid fibril or C2- -Syn amyloid fibril) formed by these fluorophores is three orders of magnitude larger than the adduct formed by ThT. [Extracted from the article]

Pyruvate dehydrogenase kinase (PDK) can regulate the catalytic activity of pyruvate decarboxylation oxidation via the mitochondrial pyruvate dehydrogenase complex, and it further links glycolysis with the tricarboxylic acid cycle and ATP generation. This review seeks to elucidate the regulation of PDK activity in different species, mainly mammals, and the role of PDK inhibitors in preventing increased blood glucose, reducing injury caused by myocardial ischemia, and inducing apoptosis of tumor cells. Regulations of PDKs expression or activity represent a very promising approach for treatment of metabolic diseases including diabetes, heart failure, and cancer. The future research and development could be more focused on the biochemical understanding of the diseases, which would help understand the cellular energy metabolism and its regulation by pharmacological effectors of PDKs. [ABSTRACT FROM AUTHOR]

The metabolism of Chinese hamster ovary (CHO) cell lines is typically characterized by high rates of aerobic glycolysis with increased lactate formation, known as the "Warburg" effect. Although this metabolic state can switch to lactate consumption, the involved regulations of the central metabolism have only been partially studied so far. An important reaction transferring the lactate precursor, pyruvate, into the tricarboxylic acid cycle is the decarboxylation reaction catalyzed by the pyruvate dehydrogenase enzyme complex (PDC). Among other mechanisms, PDC is mainly regulated by phosphorylation–dephosphorylation at the three sites Ser232, Ser293, and Ser300. In this work, the PDC phosphorylation in antibody‐producing CHO DP‐12 cell culture is investigated during the lactate switch. Batch cultivations were carried out with frequent sampling (every 6 h) during the transition from lactate formation to lactate uptake, and the PDC phosphorylation levels were quantified using a novel indirect flow cytometry protocol. Contrary to the expected activation of PDC (i.e., reduced PDC phosphorylation) during lactate consumption, Ser293 and Ser300 phosphorylation levels were 33% higher compared to the phase of glucose excess. At the same time, the relative phosphorylation level of Ser232 increased steadily throughout the cultivation (66% increase overall). The intracellular pyruvate was found to accumulate only during the period of high lactate production, while acetyl‐CoA showed nearly no accumulation. These results indicate a deactivation of PDC and reduced oxidative metabolism during lactate switch even though the cells undergo a metabolic transition to lactate‐based cell growth and metabolism. Overall, this study provides a unique view on the regulation of PDC during the lactate switch, which contributes to an improved understanding of PDC and its interaction with the bioprocess. [ABSTRACT FROM AUTHOR]

Pyruvate dehydrogenase complex (PDHC) deficiency is a mitochondrial disorder. We report two cases of PDHC deficiency with clinical symptoms and brain imaging findings reminiscent of FOXG1 syndrome, suggesting a phenotypic overlap of these disorders. [ABSTRACT FROM AUTHOR]

The contribution of altered mitochondrial Ca2+ handling to metabolic and functional defects in type 2 diabetic (T2D) mouse hearts is not well understood. In this study, we show that the T2D heart is metabolically inflexible and almost exclusively dependent on mitochondrial fatty acid oxidation as a consequence of mitochondrial calcium uniporter complex (MCUC) inhibitory subunit MCUb overexpression. Using a recombinant endonuclease-deficient Cas9-based gene promoter pulldown approach coupled with mass spectrometry, we found that MCUb is upregulated in the T2D heart due to loss of glucose homeostasis regulator nuclear receptor corepressor 2 repression, and chromatin immunoprecipitation assays identified peroxisome proliferator-activated receptor α as a mediator of MCUb gene expression in T2D cardiomyocytes. Upregulation of MCUb limits mitochondrial matrix Ca2+ uptake and impairs mitochondrial energy production via glucose oxidation by depressing pyruvate dehydrogenase complex activity. Gene therapy displacement of endogenous MCUb with a dominant-negative MCUb transgene (MCUbW246R/V251E) in vivo rescued T2D cardiomyocytes from metabolic inflexibility and stimulated cardiac contractile function and adrenergic responsiveness by enhancing phospholamban phosphorylation via protein kinase A. We conclude that MCUb represents one newly discovered molecular effector at the interface of metabolism and cardiac function, and its repression improves the outcome of the chronically stressed diabetic heart. [ABSTRACT FROM AUTHOR]

Hyperbaric exposure (1.3 atmospheres absolute with 20.9% O2) for 1 h a day was shown to improve exercise capacity. The present study was designed to reveal whether the daily exposure time affects exercise performance and metabolism in skeletal and cardiac muscles. Male mice in the training group were housed in a cage with a wheel activity device for 7 weeks from 5 weeks old. Trained mice were then subjected to hybrid training (HT, endurance exercise for 30 min followed by sprint interval exercise for 30 min). Hyperbaric exposure was applied following daily HT for 15 min (15HT), 30 min (30HT), or 60 min (60HT) for 4 weeks. In the endurance capacity test, maximal work values were significantly increased by 30HT and 60HT. In the left ventricle (LV), activity levels of 3‐hydroxyacyl‐CoA‐dehydrogenase, citrate synthase, and carnitine palmitoyl transferase (CPT) 2 were significantly increased by 60HT. CPT2 activity levels were markedly increased by hyperbaric exposure in red gastrocnemius (Gr) and plantaris muscle (PL). Pyruvate dehydrogenase complex activity values in PL were enhanced more by 30HT and 60HT than by HT. Protein levels of N‐terminal isoform of PGC1α (NT‐PGC1α) protein were significantly enhanced in three hyperbaric exposed groups in Gr, but not in LV. These results indicate that hyperbaric exposure for 30 min or longer has beneficial effects on endurance, and 60‐min exposure has the potential to further increase performance by facilitating fatty acid metabolism in skeletal and cardiac muscles in highly trained mice. NT‐PGC1α may have important roles for these adaptations in skeletal muscle. [ABSTRACT FROM AUTHOR]

This post hoc study aimed to determine whether major elective abdominal surgery had any acute impact on mitochondrial pyruvate dehydrogenase complex (PDC) activity and maximal mitochondrial ATP production rates (MAPR) in a large muscle group (vastus lateralis -VL) distant to the site of surgical trauma. Fifteen patients undergoing major elective open abdominal surgery were studied. Muscle biopsies were obtained after the induction of anesthesia from the VL immediately before and after surgery for the determination of PDC and maximal MAPR (utilizing a variety of energy substrates). Muscle PDC activity was reduced by >50% at the end of surgery compared with pre-surgery (p < 0.05). Muscle MAPR were comprehensively suppressed by surgery for the substrate combinations: glutamate + succinate; glutamate + malate; palmitoylcarnitine + malate; and pyruvate + malate (all p < 0.05), and could not be explained by a lower mitochondrial yield. PDC activity and mitochondrial ATP production capacity were acutely impaired in muscle distant to the site of surgical trauma. In keeping with the limited data available, we surmise these events resulted from the general anesthesia procedures employed and the surgery related trauma. These findings further the understanding of the acute dysregulation of mitochondrial function in muscle distant to the site of major surgical trauma in patients, and point to the combination of general anesthesia and trauma related inflammation as being drivers of muscle metabolic insult that warrants further investigation. Registered at (NCT01134809). [ABSTRACT FROM AUTHOR]

We report a 5-year-old male with a PDHA1 variant who presented with alternating hemiplegia of childhood and later developed developmental regression, basal ganglia injury and episodic lactic acidosis. Enzyme assay in lymphocytes confirmed a diagnosis of Pyruvate Dehydrogenase Complex (PDC) deficiency. His mother who was heterozygous for the same variant suffered from ophthalmoplegia, chronic migraine and developed flaccid paralysis at 36 years of age. PDHA1 is the most common genetic cause of PDC deficiency and presents with a myriad of neurological phenotypes including neonatal form with lactic acidosis, non-progressive infantile encephalopathy, Leigh syndrome subtype and intermittent ataxia. The presentations in our 2 patients contribute to the clinical heterogeneity of this neurogenetic condition. [ABSTRACT FROM AUTHOR]

Development of hyperpolarized technology utilizing dynamic nuclear polarization has enabled the measurement of (13)C metabolism in vivo at very high signal-to-noise ratio (SNR). In vivo mitochondrial metabolism can, in principle, be monitored with pyruvate, which is catalyzed to acetyl-CoA via pyruvate dehydrogenase (PDH). The purpose of this work was to determine whether the compound sodium dichloroacetate (DCA) could aid the study of mitochondrial metabolism with hyperpolarized pyruvate. DCA stimulates PDH by inhibiting its inhibitor, pyruvate dehydrogenase kinase. In this work, hyperpolarized [1-(13)C]pyruvate and [2-(13)C]pyruvate were used to probe mitochondrial metabolism in normal rats. Increased conversion to bicarbonate (+181±69%, P=.025) was measured when [1-(13)C]pyruvate was injected after DCA administration, and increased glutamate (+74±23%, P=.004), acetoacetate (+504±281%, P=.009) and acetylcarnitine (+377±157%, P=.003) were detected when [2-(13)C]pyruvate was used.

A functional 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is required for isoprenoid biosynthesis and hence survival in Escherichia coli and most other bacteria. In the first two steps of the pathway, MEP is produced from the central metabolic intermediates pyruvate and glyceraldehyde 3-phosphate via 1-deoxy-D-xylulose 5-phosphate (DXP) by the activity of the enzymes DXP synthase (DXS) and DXP reductoisomerase (DXR). Because the MEP pathway is absent from humans, it was proposed as a promising new target to develop new antibiotics. However, the lethal phenotype caused by the deletion of DXS or DXR was found to be suppressed with a relatively high efficiency by unidentified mutations. Here we report that several mutations in the unrelated genes aceE and ribB rescue growth of DXS-defective mutants because the encoded enzymes allowed the production of sufficient DXP in vivo. Together, this work unveils the diversity of mechanisms that can evolve in bacteria to circumvent a blockage of the first step of the MEP pathway.

Zervopoulos et al. (2022) propose a non-canonical nuclear import pathway for the functional mitochondrial pyruvate dehydrogenase complex (PDC), facilitated by dynamic MFN2-mediated tethering of mitochondria to the nuclear envelope upon exposure to proliferative stimuli. [ABSTRACT FROM AUTHOR]

The Pyruvate Dehydrogenase Complex (PDC), a key enzyme in glucose metabolism, catalyzes an irreversible oxidative decarboxylation reaction of pyruvate to acetyl- CoA, linking the cytosolic glycolytic pathway to mitochondrial tricarboxylic acid cycle and oxidative phosphorylation. Earlier we reported a down-regulation of several key hepatic lipogenic enzymes and their upstream regulators in liver-specific PDC-deficient mouse (L-PDCKO model by deleting the Pdha1 gene). In this study we investigated gene expression profiles of key glycolytic enzymes and other proteins that respond to various metabolic stresses in liver from L-PDCKO mice. Transcripts of several, such as hexokinase 2, phosphoglycerate kinase 1, pyruvate kinase muscletype 2, and lactate dehydrogenase B as well as those for the nonglycolysis-related proteins, CD-36, C/EBP homologous protein, and peroxisome proliferator-activated receptor γ, were up-regulated in L-PDCKO liver whereas hypoxia-induced factor-1α, pyruvate dehydrogenase kinase 1 and Sirtuin 1 transcripts were down-regulated. The protein levels of pyruvate kinase muscle-type 2 and lactate dehydrogenase B were increased whereas that of lactate dehydrogenase A was decreased in PDC-deficient mouse liver. Analysis of endoplasmic reticulum and oxidative stress indicators suggests that the L-PDCKO liver showed evidence of the former but not the latter. These findings indicate that (i) liver-specific PDC deficiency is sufficient to induce "aerobic glycolysis characteristic" in mouse liver, and (ii) the mechanism(s) responsible for these changes appears distinct from that which induces the Warburg effect in some cancer cells. [ABSTRACT FROM AUTHOR]

Good health depends on the maintenance of metabolic flexibility, which in turn is dependent on the maintenance of regulatory flexibility of a large number of regulatory enzymes, but especially the pyruvate dehydrogenase complex (PDC), because of its central role in carbohydrate metabolism. Flexibility in regulation of PDC is dependent on rapid changes in the phosphorylation state of PDC determined by the relative activities of the pyruvate dehydrogenase kinases (PDKs) and the pyruvate dehydrogenase phosphatases. Inactivation of the PDC by overexpression of PDK4 contributes to hyperglycemia, and therefore the serious health problems associated with diabetes. Loss of regulatory flexibility of PDC occurs in other disease states and pathological conditions that have received less attention than diabetes. These include cancers, non‐alcoholic fatty liver disease, cancer‐induced cachexia, diabetes‐induced nephropathy, sepsis and amyotrophic lateral sclerosis. Overexpression of PDK4, and in some situations, the other PDKs, as well as under expression of the pyruvate dehydrogenase phosphatases, leads to inactivation of the PDC, mitochondrial dysfunction and deleterious effects with health consequences. The possible basis for this phenomenon, along with evidence that overexpression of PDK4 results in phosphorylation of "off‐target" proteins and promotes excessive transport of Ca2+ into mitochondria through mitochondria‐associated endoplasmic reticulum membranes are discussed. Recent efforts to find small molecule PDK inhibitors with therapeutic potential are also reviewed. [ABSTRACT FROM AUTHOR]