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Death from mechanical asphyxia (DMA) refers to death from acute respiratory disorder caused by mechanical violence. Due to the absence of characteristic signs in corpses, it has been rather challenging to achieve the precise authentication of DMA. In this research, human pulmonary samples were collected and grouped according to different causes of death in search of potential biomarkers of DMA. Hydroxyacyl-CoA dehydrogenase (HADH) was identified significantly up-regulated in DMA group. Cell experiments were conducted to figure out the mechanism of the up-regulation of HADH. According to the results, we assumed acute and severe hypoxia caused by mechanical asphyxia contributed to the expression change of HADH, which could be a self-saving reaction of cells that are forced to adjust energy metabolism. Generally, HADH can be biomarker of DMA and help the precise authentication of DMA., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025. Published by Elsevier B.V.)

The butyrate biosynthetic pathway not only contributes to electron management and energy generation in butyrate forming bacteria, but also confers evolutionary advantages to the host by inhibiting the growth of surrounding butyrate-sensitive microbes. While high butyrate levels induce toxic stress, effects of non-toxic levels on cell growth, health, metabolism, and sporulation remain unclear. Here, we show that butyrate stimulates cellular processes of Clostridium acetobutylicum, a model butyrate forming Firmicute. First, we deleted the 3-hydroxybutyryl-CoA dehydrogenase gene (hbd) from the C. acetobutylicum chromosome to eliminate the butyrate synthetic pathway and thus butyrate formation. A xylose inducible Cas9 cassette was chromosomally integrated and utilized for the one-step markerless gene deletions. Non-toxic butyrate levels significantly affected growth, health, and sporulation of C. acetobutylicum. After deleting spo0A, the gene encoding the master regulator of sporulation, Spo0A, and conducting butyrate addition experiments, we conclude that butyrate affects cellular metabolism through both Spo0A-dependent and independent mechanisms. We also deleted the hbd gene from the chromosome of the asporogenous C. acetobutylicum M5 strain lacking the pSOL1 plasmid to examine the potential involvement of pSOL1 genes on the observed butyrate effects. Addition of crotonate, the precursor of butyrate biosynthesis, to the hbd deficient M5 strain was used to probe the role of butyrate biosynthesis pathway in electron and metabolic fluxes. Finally, we found that butyrate addition can enhance the growth of the non-butyrate forming Clostridium saccharolyticum. Our data suggest that butyrate functions as a stimulator of cellular processes, like a growth factor, in C. acetobutylicum and potentially evolutionarily related Clostridium organisms., (Copyright © 2024 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Atopic dermatitis (AD) is characterized by a T-helper cell type 2 (Th2) inflammatory response leading to skin damage with erythema and edema. Comparative fecal sample analysis has uncovered a strong correlation between AD and Faecalibacterium prausnitzii strain A2-165, specifically associated with butyrate production. Therefore, understanding the functional mechanisms of crucial enzymes in the butyrate pathway, such as 3-hydroxybutyryl-CoA dehydrogenase of A2-165 (A2HBD), is imperative. Here, we have successfully elucidated the three-dimensional structure of A2HBD in complex with acetoacetyl-CoA and NAD + at a resolution of 2.2Å using the PAL-11C beamline (third generation). Additionally, X-ray data of A2HBD in complex with acetoacetyl-CoA at a resolution of 1.9 Å were collected at PAL-XFEL (fourth generation) utilizing Serial Femtosecond Crystallography (SFX). The monomeric structure of A2HBD consists of two domains, N-terminal and C-terminal, with cofactor binding occurring at the N-terminal domain, while the C-terminal domain facilitates dimerization. Our findings elucidate the binding mode of NAD + to A2HBD. Upon acetoacetyl-CoA binding, the crystal structure revealed a significant conformational change in the Clamp-roof domain (root-mean-square deviation of 2.202 Å). Notably, residue R143 plays a critical role in capturing the adenine phosphate ring, underlining its significance in substrate recognition and catalytic activity. The binding mode of acetoacetyl-CoA was also clarified, indicating its lower stability compared to NAD + . Furthermore, the conformational change of hydrophobic residues near the catalytic cavity upon substrate binding resulted in cavity shrinkage from an open to closed conformation. This study confirms the conformational changes of catalytic triads involved in the catalytic reaction and presents a proposed mechanism for substrate reduction based on structural observations.

The Mycobacterium tuberculosis trifunctional enzyme (MtTFE) is an α 2 β 2 tetrameric enzyme in which the α-chain harbors the 2E-enoyl-CoA hydratase (ECH) and 3S-hydroxyacyl-CoA dehydrogenase (HAD) active sites, and the β-chain provides the 3-ketoacyl-CoA thiolase (KAT) active site. Linear, medium-chain and long-chain 2E-enoyl-CoA molecules are the preferred substrates of MtTFE. Previous crystallographic binding and modeling studies identified binding sites for the acyl-CoA substrates at the three active sites, as well as the NAD binding pocket at the HAD active site. These studies also identified three additional CoA binding sites on the surface of MtTFE that are different from the active sites. It has been proposed that one of these additional sites could be of functional relevance for the substrate channeling (by surface crawling) of reaction intermediates between the three active sites. Here, 226 fragments were screened in a crystallographic fragment-binding study of MtTFE crystals, resulting in the structures of 16 MtTFE-fragment complexes. Analysis of the 121 fragment-binding events shows that the ECH active site is the `binding hotspot' for the tested fragments, with 41 binding events. The mode of binding of the fragments bound at the active sites provides additional insight into how the long-chain acyl moiety of the substrates can be accommodated at their proposed binding pockets. In addition, the 20 fragment-binding events between the active sites identify potential transient binding sites of reaction intermediates relevant to the possible channeling of substrates between these active sites. These results provide a basis for further studies to understand the functional relevance of the latter binding sites and to identify substrates for which channeling is crucial., (open access.)

Early-onset long-chain 3-hydroxyacyl-coenzyme A dehydrogenase (LCHAD) deficiency is a fatty acid β-oxidation disorder with a poor prognosis. Triheptanoin, an anaplerotic oil with odd-chain fatty acids can improve the disease course. The female patient presented here was diagnosed at the age of 4 months, and treatment was started as fat restriction, frequent feeding, and standard medium-chain triglyceride supplementation. In follow-up, she had frequent rhabdomyolysis episodes (∼8 per year). At the age of six, she had 13 episodes in 6 months, and triheptanoin was started as part of a compassionate use program. Following unrelated hospital stays due to multisystem inflammatory syndrome in children and a bloodstream infection, she had only 3 rhabdomyolysis episodes, and hospitalized days decreased from 73 to 11 during her first year with triheptanoin. Triheptanoin drastically decreased the frequency and severity of rhabdomyolysis, but progression of retinopathy was not altered., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Compared with other species, freshwater fish are more capable of synthesizing DHA via same biosynthetic pathways. Freshwater fish have a "Sprecher" pathway to biosynthesize DHA in a peroxisome-dependent manner. Enoyl-CoA hydratase/3-hydroxyacyl CoA dehydrogenase (Ehhadh) is involved in the hydration and dehydrogenation reactions of fatty acid β-oxidation in peroxisomes. However, the role of Ehhadh in the synthesis of DHA in freshwater fish remains largely unclear. In this study, the knockout of Ehhadh significantly inhibited DHA synthesis in zebrafish. Liver transcriptome analysis showed that Ehhadh deletion significantly inhibited SREBF and PPAR signaling pathways and decreased the expression of PUFA synthesis-related genes. Our results from the analysis of transgenic zebrafish (Tg:Ehhadh) showed that Ehhadh overexpression significantly increased the DHA content in the liver and significantly upregulated the expression of genes related to PUFA synthesis. In addition, the DHA content in the liver of Tg:Ehhadh fed with linseed oil was significantly higher than that of wildtype, but the expression of PUFA synthesis-related genes fads2 and elovl2 were significantly lower, indicating that Ehhadh had a direct effect on DHA synthesis. In conclusion, our results showed that Ehhadh was essential for DHA synthesis in the "Sprecher" pathway, and Ehhadh overexpression could promote DHA synthesis. This study provides insight into the role of Ehhadh in freshwater fish., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Multifunctional proteins are challenging as it can be difficult to confirm pathomechanisms associated with disease-causing genetic variants. The human 17β-hydroxysteroid dehydrogenase 10 (HSD10) is a moonlighting enzyme with at least two structurally and catalytically unrelated functions. HSD10 disease was originally described as a disorder of isoleucine metabolism, but the clinical manifestations were subsequently shown to be linked to impaired mtDNA transcript processing due to deficient function of HSD10 in the mtRNase P complex. A surprisingly large number of other, mostly enzymatic and potentially clinically relevant functions have been attributed to HSD10. Recently, HSD10 was reported to exhibit phospholipase C-like activity towards cardiolipins (CL), important mitochondrial phospholipids. To assess the physiological role of the proposed CL-cleaving function, we studied CL architectures in living cells and patient fibroblasts in different genetic backgrounds and lipid environments using our well-established LC-MS/MS cardiolipidomic pipeline. These experiments revealed no measurable effect on CLs, indicating that HSD10 does not have a physiologically relevant function towards CL metabolism. Evolutionary constraints could explain the broad range of reported substrates for HSD10 in vitro. The combination of an essential structural with a non-essential enzymatic function in the same protein could direct the evolutionary trajectory towards improvement of the former, thereby increasing the flexibility of the binding pocket, which is consistent with the results presented here., (© 2022. The Author(s).)

Alzheimer's disease (AD) is the most common dementia affecting one in nine people over 65. Only a handful of small-molecule drugs and the anti-β amyloid (Aβ) antibody aducanumab are approved to treat AD. However, they only serve to reduce symptoms of advanced disease. Novel treatments administered early in disease progression before the accumulation of Aβ and tau reaches the threshold where neuroinflammation is triggered and irreversible neuronal damage occurs are more likely to provide effective therapy. There is a growing body of evidence implying that mitochondrial dysfunction occurs at an early stage of AD pathology. The mitochondrial enzyme amyloid-binding alcohol dehydrogenase (ABAD) binds to Aβ potentiating toxicity. Moreover, ABAD has been shown to be overexpressed in the same areas of the brain most affected by AD. Inhibiting the Aβ-ABAD protein-protein interaction without adversely affecting normal enzyme turnover is hypothesized to be a potential treatment strategy for AD. Herein, we conduct structure-activity relationship studies across a series of functionalized allopurinol derivatives to determine their ability to inhibit Aβ-mediated reduction of estradiol production from ABAD. The lead compound resulting from these studies possesses potent activity with no toxicity up to 100 μM, and demonstrates an ability to rescue defective mitochondrial metabolism in human SH-SY5Y cells and rescue both defective mitochondrial metabolism and morphology ex vivo in primary 5XFAD AD mouse model neurons.

Hydroxyacyl-CoA dehydrogenase (HADH) catalyzes the third reaction of mitochondrial β-oxidation cascade, while the regulation of its expression and function remains to be elucidated. Using the quantitative translation initiation sequencing (QTI-seq), we have identified that murine Hadh mRNA has two alternative translation start codons. We demonstrated that translation from upstream start codon encodes the mitochondrial isoform of HADH, while translation from downstream start codon produces a short isoform (HADH-S) with predominant nuclear localization. Moreover, overexpression of HADH-S inhibits the proliferation of mouse embryonic fibroblasts. Overall, our results identify a novel isoform of HADH participating in cell proliferation., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Scalability, process control, and modularity are some of the advantages that make flow biocatalysis a key-enabling technology for green and sustainable chemistry. In this context, rigid porous solid membranes hold the promise to expand the toolbox of flow biocatalysis due to their chemical stability and inertness. Yttrium-stabilized zirconia (YSZ) fulfills these properties; however, it has been scarcely exploited as a carrier for enzymes. Here, we discovered an unprecedented interaction between YSZ materials and His-tagged enzymes that enables the fabrication of multifunctional biocatalytic membranes for bioredox cascades. X-ray photoelectron spectroscopy suggests that enzyme immobilization is driven by coordination interactions between the imidazole groups of His-tags and both Zr and Y atoms. As model enzymes, we coimmobilized in-flow a thermophilic hydroxybutyryl-CoA dehydrogenase (TtHBDH-His) and a formate dehydrogenase (His-CbFDH) for the continuous asymmetric reduction of ethyl acetoacetate with in situ redox cofactor recycling. Fluorescence confocal microscopy deciphered the spatial organization of the two coimmobilized enzymes, pointing out the importance of the coimmobilization sequence. Finally, the coimmobilized system succeeded in situ , recycling the redox cofactor, maintaining the specific productivity using only 0.05 mM NADH, and accumulating a total enzyme turnover number of 4000 in 24 h. This work presents YSZ materials as ready-to-use carriers for the site-directed enzyme in-flow immobilization and the application of the resulting heterogeneous biocatalysts for continuous biomanufacturing.

Background: Mitochondrial 17β-hydroxysteroid dehydrogenase type 10 (17β-HSD10) is necessary for brain cognitive function, but its studies were confounded by reports of Aβ-peptide binding alcohol dehydrogenase (ABAD), formerly endoplasmic reticulum-associated Aβ-peptide binding protein (ERAB), for two decades so long as ABAD serves as the alternative term of 17β-HSD10., Objective: To determine whether those ABAD reports are true or false, even if they were published in prestigious journals., Methods: 6xHis-tagged 17β-HSD10 was prepared and characterized by well-established experimental procedures., Results: The N-terminal 6xHis tag did not significantly interfere with the dehydrogenase activities of 17β-HSD10, but the kinetic constants of its 3-hydroxyacyl-CoA dehydrogenase activity are drastically distinct from those of ABAD, and it was not involved in ketone body metabolism as previously reported for ABAD. Furthermore, it was impossible to measure its generalized alcohol dehydrogenase activities underlying the concept of ABAD because the experimental procedures described in ABAD reports violated basic chemical and/or biochemical principles. More incredibly, both authors and journals had not yet agreed to make any corrigenda of ABAD reports., Conclusion: Brain 17β-HSD10 plays a key role in neurosteroid metabolism and further studies in this area may lead to potential treatments of neurodegeneration including AD.

The Mycobacterium tuberculosis trifunctional enzyme (MtTFE) is an α 2 β 2 tetrameric enzyme. The α-chain harbors the 2E-enoyl-CoA hydratase (ECH) and 3S-hydroxyacyl-CoA dehydrogenase (HAD) activities and the β-chain provides the 3-ketoacyl-CoA thiolase (KAT) activity. Enzyme kinetic data reported here show that medium and long chain enoyl-CoA molecules are preferred substrates for MtTFE. Modelling studies indicate how the linear medium and long acyl chains of these substrates can bind to each of the active sites. In addition, crystallographic binding studies have identified three new CoA binding sites which are different from the previously known CoA binding sites of the three TFE active sites. Structure comparisons provide new insights into the properties of ECH, HAD and KAT active sites of MtTFE. The interactions of the adenine moiety of CoA with loop-2 of the ECH active site cause a conformational change of this loop by which a competent ECH active site is formed. The NAD + binding domain (domain C) of the HAD part of MtTFE has only a few interactions with the rest of the complex and adopts a range of open conformations, whereas the A-domain of the ECH part is rigidly fixed with respect to the HAD part. Two loops, the CB1-CA1 region and the catalytic CB4-CB5 loop, near the thiolase active site and the thiolase dimer interface, have high B-factors. Structure comparisons suggest that a competent and stable thiolase dimer is formed only when complexed with the α-chains, highlighting the importance of the assembly for the proper functioning of the complex., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Human mitochondrial transcripts contain messenger and ribosomal RNAs flanked by transfer RNAs (tRNAs), which are excised by mitochondrial RNase (mtRNase) P and Z to liberate all RNA species. In contrast to nuclear or bacterial RNase P, mtRNase P is not a ribozyme but comprises three protein subunits that carry out RNA cleavage and methylation by unknown mechanisms. Here, we present the cryo-EM structure of human mtRNase P bound to precursor tRNA, which reveals a unique mechanism of substrate recognition and processing. Subunits TRMT10C and SDR5C1 form a subcomplex that binds conserved mitochondrial tRNA elements, including the anticodon loop, and positions the tRNA for methylation. The endonuclease PRORP is recruited and activated through interactions with its PPR and nuclease domains to ensure precise pre-tRNA cleavage. The structure provides the molecular basis for the first step of RNA processing in human mitochondria., (© 2021. The Author(s).)

Objective: The immediate signals that couple exercise to metabolic adaptations are incompletely understood. Nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) produces reactive oxygen species (ROS) and plays a significant role in metabolic and vascular adaptation during stress conditions. Our objective was to determine the role of Nox4 in exercise-induced skeletal muscle metabolism., Methods: Mice were subjected to acute exercise to assess their immediate responses. mRNA and protein expression responses to Nox4 and hydrogen peroxide (H 2 O 2 ) were measured by qPCR and immunoblotting. Functional metabolic flux was measured via ex vivo fatty acid and glucose oxidation assays using 14 C-labeled palmitate and glucose, respectively. A chronic exercise regimen was also utilized and the time to exhaustion along with key markers of exercise adaptation (skeletal muscle citrate synthase and beta-hydroxyacyl-coA-dehydrogenase activity) were measured. Endothelial-specific Nox4-deficient mice were then subjected to the same acute exercise regimen and their subsequent substrate oxidation was measured., Results: We identified key exercise-responsive metabolic genes that depend on H 2 O 2 and Nox4 using catalase and Nox4-deficient mice. Nox4 was required for the expression of uncoupling protein 3 (Ucp3), hexokinase 2 (Hk2), and pyruvate dehydrogenase kinase 4 (Pdk4), but not the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc-1α). Global Nox4 deletion resulted in decreased UCP3 protein expression and impaired glucose and fatty acid oxidization in response to acute exercise. Furthermore, Nox4-deficient mice demonstrated impaired adaptation to chronic exercise as measured by the time to exhaustion and activity of skeletal muscle citrate synthase and beta-hydroxyacyl-coA-dehydrogenase. Importantly, mice deficient in endothelial-Nox4 similarly demonstrated attenuated glucose and fatty acid oxidation following acute exercise., Conclusions: We report that H 2 O 2 and Nox4 promote immediate responses to exercise in skeletal muscle. Glucose and fatty acid oxidation were blunted in the Nox4-deficient mice post-exercise, potentially through regulation of UCP3 expression. Our data demonstrate that endothelial-Nox4 is required for glucose and fatty acid oxidation, suggesting inter-tissue cross-talk between the endothelium and skeletal muscle in response to exercise., (Copyright © 2021 The Authors. Published by Elsevier GmbH.. All rights reserved.)

A series of tacrine - benzothiazole hybrids incorporate inhibitors of acetylcholinesterase (AChE), amyloid β (Aβ) aggregation and mitochondrial enzyme ABAD, whose interaction with Aβ leads to mitochondrial dysfunction, into a single molecule. In vitro, several of 25 final compounds exerted excellent anti-AChE properties and interesting capabilities to block Aβ aggregation. The best derivative of the series could be considered 10w that was found to be highly potent and selective towards AChE with the IC 50 value in nanomolar range. Moreover, the same drug candidate exerted absolutely the best results of the series against ABAD, decreasing its activity by 23% at 100 µM concentration. Regarding the cytotoxicity profile of highlighted compound, it roughly matched that of its parent compound - 6-chlorotacrine. Finally, 10w was forwarded for in vivo scopolamine-induced amnesia experiment consisting of Morris Water Maze test, where it demonstrated mild procognitive effect. Taking into account all in vitro and in vivo data, highlighted derivative 10w could be considered as the lead structure worthy of further investigation., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Endurance exercise training enhances muscle fat oxidation while concomitantly reducing carbohydrate (glycogen) utilization during exercise, thereby delaying the onset of fatigue. This study examined the effects of dietary fat restriction on endurance training-induced metabolic adaptations in rat skeletal muscle. Male Sprague-Dawley rats were placed on either a control diet (CON: 19.2% protein, 21.6% fat, and 59.2% carbohydrate as a percentage of total energy) or a fat-restricted diet (FR: 21.5% protein, 2.4% fat, and 76.1% carbohydrate as a percentage of total energy) for 4 wks. Half the rats in each dietary group performed daily 6-h swimming exercise (two 3-h sessions separated by 45 min of rest) on 5 days each wk. Endurance training significantly increased the expression of β-hydroxyacyl CoA dehydrogenase (βHAD), a key enzyme of fat oxidation, and pyruvate dehydrogenase kinase 4 (PDK4), an inhibitory regulator of glycolytic flux, in the skeletal muscle of rats fed the CON diet. However, such endurance training-induced increases in muscle βHAD and PDK4 were partially suppressed by the FR diet, suggesting that a FR diet may diminish the endurance training-induced enhancement of fat oxidation and reduction in glycogen utilization during exercise. We then assessed the muscle glycogen utilization rate during an acute bout of swimming exercise in the trained rats fed either the CON or the FR diet and consequently found that rats fed the FR diet had a significantly higher muscle glycogen utilization rate during exercise compared with rats fed the CON diet. In conclusion, dietary fat restriction may attenuate the endurance training-induced metabolic adaptations in skeletal muscle.

In mammalian cells, nutrients and growth factors signal through an array of upstream proteins to regulate the mTORC1 growth control pathway. Because the full complement of these proteins has not been systematically identified, we developed a FACS-based CRISPR-Cas9 genetic screening strategy to pinpoint genes that regulate mTORC1 activity. Along with almost all known positive components of the mTORC1 pathway, we identified many genes that impact mTORC1 activity, including DCAF7 , CSNK2B , SRSF2 , IRS4 , CCDC43 , and HSD17B10 Using the genome-wide screening data, we generated a focused sublibrary containing single guide RNAs (sgRNAs) targeting hundreds of genes and carried out epistasis screens in cells lacking nutrient- and stress-responsive mTORC1 modulators, including GATOR1, AMPK, GCN2, and ATF4. From these data, we pinpointed mitochondrial function as a particularly important input into mTORC1 signaling. While it is well appreciated that mitochondria signal to mTORC1, the mechanisms are not completely clear. We find that the kinases AMPK and HRI signal, with varying kinetics, mitochondrial distress to mTORC1, and that HRI acts through the ATF4-dependent up-regulation of both Sestrin2 and Redd1. Loss of both AMPK and HRI is sufficient to render mTORC1 signaling largely resistant to mitochondrial dysfunction induced by the ATP synthase inhibitor oligomycin as well as the electron transport chain inhibitors piericidin and antimycin. Taken together, our data reveal a catalog of genes that impact the mTORC1 pathway and clarify the multifaceted ways in which mTORC1 senses mitochondrial dysfunction., Competing Interests: The authors declare no competing interest.

Emerging evidence indicates that non-mutational drug tolerance mechanisms underlie the survival of residual cancer "persister" cells. Here, we find that BRAF(V600E) mutant melanoma persister cells tolerant to BRAF/MEK inhibitors switch their metabolism from glycolysis to oxidative respiration supported by peroxisomal fatty acid β-oxidation (FAO) that is transcriptionally regulated by peroxisome proliferator-activated receptor alpha (PPARα). Knockdown of the key peroxisomal FAO enzyme, acyl-CoA oxidase 1 (ACOX1), as well as treatment with the peroxisomal FAO inhibitor thioridazine, specifically suppresses the oxidative respiration of persister cells and significantly decreases their emergence. Consistently, a combination treatment of BRAF/MEK inhibitors with thioridazine in human-melanoma-bearing mice results in a durable anti-tumor response. In BRAF(V600E) melanoma samples from patients treated with BRAF/MEK inhibitors, higher baseline expression of FAO-related genes and PPARα correlates with patients' outcomes. These results pave the way for a metabolic strategy to overcome drug resistance., Competing Interests: Declaration of Interests C.R. is an occasional consultant for Roche, BMS, MSD, Merck, Sanofi, Pierre Fabre, Biothera, CureVac, and Novartis., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

17β-hydroxysteroid dehydrogenase (17β-HSD10) is a multifunctional human enzyme with important roles both as a structural component and also as a catalyst of many metabolic pathways. This mitochondrial enzyme has important functions in the metabolism, development and aging of the neural system, where it is involved in the homeostasis of neurosteroids, especially in regard to estradiol, changes in which make it an essential part of neurodegenerative pathology. These roles therefore, indicate that 17β-HSD10 may be a possible druggable target for neurodegenerative diseases including Alzheimer's disease (AD), and in hormone-dependent cancer. The objective of this review was to provide a summary about physiological functions and pathological roles of 17β-HSD10 and the modulators of its activity., (© 2020 International Society for Neurochemistry.)

Background: The plasma acylcarnitine profile is frequently used as a biochemical assessment for follow-up in diagnosed patients with fatty acid oxidation disorders (FAODs). Disease specific acylcarnitine species are elevated during metabolic decompensation but there is clinical and biochemical heterogeneity among patients and limited data on the utility of an acylcarnitine profile for routine clinical monitoring., Methods: We evaluated plasma acylcarnitine profiles from 30 diagnosed patients with long-chain FAODs (carnitine palmitoyltransferase-2 (CPT2), very long-chain acyl-CoA dehydrogenase (VLCAD), and long-chain 3-hydroxy acyl-CoA dehydrogenase or mitochondrial trifunctional protein (LCHAD/TFP) deficiencies) collected after an overnight fast, after feeding a controlled low-fat diet, and before and after moderate exercise. Our purpose was to describe the variability in this biomarker and how various physiologic states effect the acylcarnitine concentrations in circulation., Results: Disease specific acylcarnitine species were higher after an overnight fast and decreased by approximately 60% two hours after a controlled breakfast meal. Moderate-intensity exercise increased the acylcarnitine species but it varied by diagnosis. When analyzed for a genotype/phenotype correlation, the presence of the common LCHADD mutation (c.1528G > C) was associated with higher levels of 3-hydroxyacylcarnitines than in patients with other mutations., Conclusions: We found that feeding consistently suppressed and that moderate intensity exercise increased disease specific acylcarnitine species, but the response to exercise was highly variable across subjects and diagnoses. The clinical utility of routine plasma acylcarnitine analysis for outpatient treatment monitoring remains questionable; however, if acylcarnitine profiles are measured in the clinical setting, standardized procedures are required for sample collection to be of value., (Copyright © 2020 Elsevier Inc. All rights reserved.)

17-beta-hydroxysteroid dehydrogenase 10 (HSD17B10) plays an important role in mitochondrial fatty acid metabolism and is also involved in mitochondrial tRNA maturation. HSD17B10 missense mutations cause HSD10 mitochondrial disease (HSD10MD). HSD17B10 with mutations identified from cases of HSD10MD show loss of function in dehydrogenase activity and mitochondrial tRNA maturation, resulting in mitochondrial dysfunction. It has also been implicated to play roles in the development of Alzheimer disease (AD) and tumorigenesis. Here, we found that HSD17B10 is a new substrate of NAD-dependent deacetylase Sirtuin 3 (SIRT3). HSD17B10 is acetylated at lysine residues K79, K99 and K105 by the acetyltransferase CBP, and the acetylation is reversed by SIRT3. HSD17B10 acetylation regulates its enzymatic activity and the formation of mitochondrial RNase P. Furthermore, HSD17B10 acetylation regulates the intracellular functions, affecting cell growth and cell resistance in response to stresses. Our results demonstrated that acetylation is an important regulation mechanism for HSD17B10 and may provide insight into interrupting the development of AD.

Chemotherapy is the first-tier treatment regime for gastric cancer (GC) patients at advance stages. Mesenchymal stem cell (MSC) cam affect drug-resistance of GC cells in tumor microenvironment, but the detailed mechanism remains poorly understood. Present study aimed to investigate the regulation of MSC-induced long non-coding RNA (lncRNA) in GC. Dysregulated lncRNAs in GC were analyzed based on GEO data. Stemness and drug-resistance of GC cells were detected by sphere formation, colony formation, CCK-8, and flow cytometry analyses. MicroRNA (miRNA)-related pathways were analyzed by online KEGG analysis tool DAVID6.8. Molecular interactions were determined by luciferase reporter assay, pulldown, RNA immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP), and co-immunoprecipitation (CoIP). Results revealed that MSC co-culture improved stemness and drug-resistance of GC cells. LncRNA histocompatibility leukocyte antigen complex P5 (HCP5) was induced in GC cells by MSC co-culture, contributing to stemness and drug-resistance. Mechanistically, HCP5 sequestered miR-3619-5p and upregulated PPARG coactivator 1 alpha (PPARGC1A), increasing transcription complex Peroxisome proliferator activated receptor (PPAR) coactivator-1α (PGC1α)/CEBPB and transcriptionally inducing carnitine palmitoyltransferase 1 (CPT1), which prompted the fatty acid oxidation (FAO) in GC cells. In conclusion, MSC-induced lncRNA HCP5 drove FAO through miR-3619-5p/AMPK/PGC1α/CEBPB axis to promote stemness and chemo-resistance of GC, indicating that targeting HCP5 was a novel approach to enhancing the efficacy of chemotherapy in GC.

Chilling and freezing are essential for poultry meat preservation. Fresh poultry, visually indistinguishable from frozen-thawed poultry, presents an attractive target for adulteration. Activity levels of the mitochondrial enzyme β-hydroxyacyl-CoA-dehydrogenase (HADH) can indicate previous freezing. This study aims to optimize the analysis method and determine a more sensitive cut-off limit distinguishing fresh from frozen poultry. 125 chicken breasts were sampled from the Lebanese market. Two sets of chilled breasts were analyzed before and after freezing at -22 °C for 5 days, with/without defrosting at 4 °C for 24 h. Frozen breasts samples were studied before and after refreezing at -22 °C. HADH activity was measured by spectrophotometry and found to be significantly higher for frozen samples. Using a cut off limit of 0.3, resulted in the correct identification of all frozen poultry samples instead of 93%. Greater control over incorrectly labelled poultry meat and increased consumer protection can be achieved using the modified thermal identification method., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Selective toxicity among cancer cells of the same lineage is a hallmark of targeted therapies. As such, identifying compounds that impair proliferation of a subset of non-small-cell lung cancer (NSCLC) cell lines represents one strategy to discover new drugs for lung cancer. Previously, phenotypic screens of 202 103 compounds led to the identification of 208 selective NSCLC toxins ( McMillan , E. A. , Cell , 2018 , 173 , 864 ). The mechanism of action for the majority of these compounds remains unknown. Here, we discovered the target for a series of quinazoline diones (QDC) that demonstrate selective toxicity among 96 NSCLC lines. Using photoreactive probes, we found that the QDC binds to both mitochondrial complex I of the electron transport chain and hydroxyacyl CoA dehydrogenase subunit alpha (HADHA), which catalyzes long-chain fatty acid oxidation. Inhibition of complex I is the on-target activity for QDC, while binding to HADHA is off-target. The sensitivity profile of the QDC across NSCLC lines correlated with the sensitivity profiles of six additional structurally distinct compounds. The antiproliferative activity of these compounds is also the consequence of binding to mitochondrial complex I, reflecting significant structural diversity among complex I inhibitors. Small molecules targeting complex I are currently in clinical development for the treatment of cancer. Our results highlight complex I as a target in NSCLC and report structurally diverse scaffolds that inhibit complex I.

Alzheimer's disease (AD) is a multifactorial neurodegenerative disease and has become a major socioeconomic issue in many developed countries. Currently available therapeutic agents for AD provide only symptomatic treatments, mainly because the complete mechanism of the AD pathogenesis is still unclear. Although several different hypotheses have been proposed, mitochondrial dysfunction has gathered interest because of its profound effect on brain bioenergetics and neuronal survival in the pathophysiology of AD. Various therapeutic agents targeting the mitochondrial pathways associated with AD have been developed over the past decade. Although most of these agents are still early in the clinical development process, they are used to restore mitochondrial function, which provides an alternative therapeutic strategy that is likely to slow the progression of the disease. In this mini review, we will survey the AD-related mitochondrial pathways and their small-molecule modulators that have therapeutic potential. We will focus on recently reported examples, and also overview the current challenges and future perspectives of ongoing research. [BMB Reports 2020; 53(1): 47-55].

Appropriate diet is essential for the regulation of age-related macular degeneration (AMD). In particular the type of dietary polyunsaturated fatty acids (PUFA) and poor antioxidant status including carotenoid levels concomitantly contribute to AMD risk. Build-up of oxidative stress in AMD induces PUFA oxidation, and a mix of lipid oxidation products (LOPs) are generated. However, LOPs are not comprehensively evaluated in AMD. LOPs are considered biomarkers of oxidative stress but also contributes to inflammatory response. In this cross-sectional case-control study, plasma omega-6/omega-3 PUFA ratios and antioxidant status (glutathione, superoxide dismutase and catalase), and plasma and urinary LOPs (41 types) were determined to evaluate its odds-ratio in the risk of developing exudative AMD (n = 99) compared to age-gender-matched healthy controls (n = 198) in adults with Chinese diet. The odds ratio of developing exudative AMD increased with LOPs from omega-6 PUFA and decreased from those of omega-3 PUFA. These observations were associated with a high plasma omega-6/omega-3 PUFA ratio and low carotenoid levels. In short, poor PUFA and antioxidant status increased the production of omega-6 PUFA LOPs such as dihomo-isoprostane and dihomo-isofuran, and lowered omega-3 PUFA LOPs such as neuroprostanes due to the high omega-6/omega-3 PUFA ratios; they were also correlated to the risk of AMD development. These findings indicate the generation of specific LOPs is associated with the development of exudative AMD., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Silent information regulator 1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD + )-dependent deacetylase, and the function is linked to cellular metabolism including mitochondrial biogenesis. Hepatic L-serine concentration is decreased significantly in fatty liver disease. We reported that the supplementation of the amino acid ameliorated the alcoholic fatty liver by enhancing L-serine-dependent homocysteine metabolism. In this study, we hypothesized that the metabolic production of NAD + from L-serine and thus activation of SIRT1 contribute to the action of L-serine. To this end, we evaluated the effects of L-serine on SIRT1 activity and mitochondria biogenesis in C2C12 myotubes. L-Serine increased intracellular NAD + content and led to the activation of SIRT1 as determined by p53 luciferase assay and western blot analysis of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) acetylation. L-Serine treatment increased the expression of the genes associated with mitochondrial biogenesis and enhanced mitochondrial mass and function. In addition, L-serine reversed cellular insulin resistance determined by insulin-induced phosphorylation of Akt and GLUT4 expression and membrane translocation. L-Serine-induced mitochondrial gene expression, fatty acid oxidation, and insulin sensitization were mediated by enhanced SIRT1 activity, which was verified by selective SIRT1 inhibitor (Ex-527) and siRNA directed to SIRT1. L-Serine effect on cellular NAD + level is dependent on the L-serine metabolism to pyruvate that is subsequently converted to lactate by lactate dehydrogenase. In summary, these data suggest that L-serine increases cellular NAD + level and thus SIRT1 activity in C2C12 myotubes.

Background: Epicardial adipose tissue (EAT) is a risk factor for cardiovascular diseases. Glucagon-like peptide 1 analogs (GLP-1A) may have beneficial cardiovascular effects and reduce EAT, possibly throughout targeting GLP-1 receptor (GLP-1R). Nevertheless, the role of EAT GLP-1R, GLP-2R and their interplay with EAT genes involved in adipogenesis and fatty acid (FA) metabolism are unknown. We analyzed whether EAT transcriptome is related to GLP-1R/GLP-2R gene expression, and GLP-1/GLP-2 plasma levels in coronary artery disease patients (CAD)., Methods: EAT was collected from 17 CAD patients undergoing CABG for microarray analysis of GLP-1R, GLP-2R and genes involved in FA metabolism and adipogenesis. EAT thickness was measured by echocardiography. GLP-1 and GLP-2 levels were quantified by ELISA in CAD and healthy subjects (CTR)., Results: EAT GLP-1R was directly correlated with genes promoting beta-oxidation and white-to-brown adipocyte differentiation, and inversely with pro-adipogenic genes. GLP-2R was positively correlated with genes involved in adipogenesis and lipid synthesis, and inversely with genes promoting beta-oxidation. GLP-1 and GLP-2 levels were higher in CAD than CTR and in patients with greater EAT thickness., Conclusions: GLP-1 analogs may target EAT GLP-1R and therefore reduce local adipogenesis, improve fat utilization and induce brown fat differentiation. As EAT lies in direct contiguity to myocardium and coronary arteries, the beneficial effects of GLP-1 activation may extent to the heart. The increased levels of circulating GLP-1 and GLP-2 and EAT GLP-2R may be compensatory mechanisms related to CAD and also EAT expansion, but the meaning of these observations needs to be further investigated., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Exercise training performed with lowered muscle glycogen stores can amplify adaptations related to oxidative metabolism, but it is not known if this is affected by the "train-low" strategy used (i.e., once-daily versus twice-a-day training). Fifteen healthy men performed 3 wk of an endurance exercise (100-min) followed by a high-intensity interval exercise 2 (twice-a-day group, n = 8) or 14 h (once-daily group, n = 7) later; therefore, the second training session always started with low muscle glycogen in both groups. Mitochondrial efficiency (state 4 respiration) was improved only for the twice-a-day group (group × training interaction, P < 0.05). However, muscle citrate synthase activity, mitochondria, and lipid area in intermyofibrillar and subsarcolemmal regions, and PGC1α, PPARα, and electron transport chain relative protein abundance were not altered with training in either group ( P > 0.05). Markers of aerobic fitness (e.g., peak oxygen uptake) were increased, and plasma lactate, O 2 cost, and rating of perceived exertion during a 100-min exercise task were reduced in both groups, although the reduction in rating of perceived exertion was larger in the twice-a-day group (group × time × training interaction, P < 0.05). These findings suggest similar training adaptations with both training low approaches; however, improvements in mitochondrial efficiency and perceived effort seem to be more pronounced with twice-a-day training. NEW & NOTEWORTHY We assessed, for the first time, the differences between two "train-low" strategies (once-daily and twice-a-day) in terms of training-induced molecular, functional, and morphological adaptations. We found that both strategies had similar molecular and morphological adaptations; however, only the twice-a-day strategy increased mitochondrial efficiency and had a superior reduction in the rating of perceived exertion during a constant-load exercise compared with once-daily training. Our findings provide novel insights into skeletal muscle adaptations using the "train-low" strategy.

Huperzine A (HupA) is a kind of Lycopodium alkaloid with potential disease-modifying qualities that has been reported to protect against β-amyloid (Aβ)-mediated mitochondrial damage in Alzheimer's disease. However, the fundamental molecular mechanism underlying the protective action of HupA against Aβ-mediated mitochondrial malfunction is not completely understood. Recently, the mitochondrial enzyme amyloid-binding alcohol dehydrogenase (ABAD) protein has been reported to facilitate Aβ-induced mitochondrial damage, resulting in mitochondrial malfunction and cell death. Our study found that HupA, but not the acetylcholinesterase inhibitor tacrine, reduced the deposition of Aβ and the ABAD level, and further reduced Aβ-ABAD complexes, thereby improving cerebral mitochondrial function in APP/PS1 mice. This was accompanied by attenuated reactive oxygen species overload, as well as increases adenosine triphosphate levels. Moreover, HupA decreased the release of cytochrome-c from mitochondria and the level of cleaved caspase-3, thereby increasing dissociated brain cell viability in APP/PS1 mice. Thus, our study demonstrated that a reduction in ABAD was involved in the protective mechanism of HupA on the cerebral mitochondrial function in APP/PS1 mice., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Reduced insulin sensitivity is observed with aging and often explained by decreased physical activity. The mechanisms involved are not clarified, but bioactive lipids may play a role. We aimed to evaluate the influence of age and cardiorespiratory fitness on ceramide and diacylglycerol content in muscle and key proteins in lipid metabolism and insulin signaling. Healthy males were stratified by age into trained and untrained groups including 27 young (23.2 ± 0.3 years) and 33 aged (65.2 ± 0.6 years). Maximal oxygen uptake and body composition were measured and fasting blood samples and muscle biopsies obtained. Muscle ceramide and diacylglycerol were determined by thin-layer and gas-liquid chromatography and proteins by western blotting. We show that HOMA-IR was higher and VO2 peak lower in aged compared with young. Total, saturated, C16:0 and C18:0 ceramide content were lower in muscle from aged compared with young. Intramuscular C18:1n9 and C20:4n6 content were higher in trained versus untrained. Content of total unsaturated and C16:1n7 diacylglycerol fatty acids were higher and C24:0 lower in muscle of aged versus young. Cardiorespiratory fitness had no impact on total diacylglycerol content. In conclusion, these data argue against intramuscular ceramide or diacylglycerol accumulation as driver of age-related insulin resistance in lean individuals., (© The Author(s) 2018. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Alzheimer's disease (AD) is the most common dementia. No cure exists, and current treatment only manages early symptoms. Mitochondrial dysfunction is a hallmark of amyloid-β (Aβ) neurotoxicity, the pathogenic protein implicated in AD. This is due in part to the interaction between Aβ and amyloid-binding alcohol dehydrogenase (ABAD). This mitochondrial protein is a vital energy regulator that, following Aβ binding, activates signaling cascades that lead to neuronal death. One of the most significant roles of ABAD is to maintain the balance of estradiol/estrone in neurons. However, the Aβ-ABAD interaction disrupts this balance and leads to a reduction in levels of estradiol, thus leading to an increase in reactive oxygen species levels and to apoptosis. Two additional proteins, peroxiredoxin-2 and endophilin-1, are implicated in Aβ-ABAD complex-mediated toxicity. Targeting the Aβ-ABAD interaction has emerged as a novel therapeutic strategy for AD. Herein, we review the chemistry and pharmacology of reported ABAD inhibitors.

Ralstonia eutropha H16 contains both NADH- and NADPH-dependent reduction activities to acetoacetyl-CoA, and the NADPH-dependent activity is mediated by PhaB paralogs with (R)-stereospecificity providing (R)-3-hydroxybutyryl (3HB)-CoA monomer for poly((R)-3-hydroxybutyrate) synthesis. In contrast, the gene encoding the NADH-dependent enzyme has not been identified to date. This study focused on the NADH-dependent dehydrogenase with (S)-stereospecificity in R. eutropha, as the (S)-specific reduction of acetoacetyl-CoA potentially competed with the polyester biosynthesis via (R)-3HB-CoA. The NADH-dependent reduction activity decreased to one-half when the gene for H16&#95;A0282 (PaaH1), one of two homologs of clostridial NADH-3HB-CoA dehydrogenase, was deleted. The enzyme responsible for the remaining activity was partially purified and identified as H16&#95;A0602 (Had) belonging to a different family from PaaH1. Gene disruption analysis elucidated that most of the NADH-dependent activity was mediated by PaaH1 and Had. The kinetic analysis using the recombinant enzymes indicated that PaaH1 and Had were both NADH-dependent 3-hydroxyacyl-CoA dehydrogenases with rather broad substrate specificity to 3-oxoacyl-CoAs of C 4 to C 8 . The deletion of had in the R. eutropha strain previously engineered for biosynthesis of poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) led to decrease in the C 6 composition of the copolyester synthesized from soybean oil, suggesting the role of Had in (S)-specific reduction of 3-oxohexanoyl-CoA with reverse β-oxidation direction. Crotonase ((S)-specific enoyl-CoA hydratase) in R. eutropha H16 was also partially purified and identified as H16&#95;A3307., (Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Several neurodegenerative disorders including Alzheimer's disease (AD) have been connected with deregulation of casein kinase 1 (CK1) activity. Inhibition of CK1 therefore presents a potential therapeutic strategy against such pathologies. Recently, novel class of CK1-specific inhibitors with N-(benzo[d]thiazol-2-yl)-2-phenylacetamide structural scaffold has been discovered. 1-(benzo[d]thiazol-2-yl)-3-phenylureas, on the other hand, are known inhibitors amyloid-beta binding alcohol dehydrogenase (ABAD), an enzyme also involved in pathophysiology of AD. Based on their tight structural similarity, we decided to evaluate series of previously published benzothiazolylphenylureas, originally designed as ABAD inhibitors, for their inhibitory activity towards CK1. Several compounds were found to be submicromolar CK1 inhibitors. Moreover, two compounds were found to inhibit both, ABAD and CK1. Such dual-activity could be of advantage for AD treatment, as it would simultaneously target two distinct pathological processes involved in disease's progression. Based on PAMPA testing both compounds were suggested to permeate the blood-brain barrier, which makes them, together with their unique dual activity, interesting lead compounds for further development.

(S)-3-Hydroxybutyryl-CoA dehydrogenase (HBD) has been gaining increased attention recently as it is a key enzyme in the enantiomeric formation of (S)-3-hydroxybutyryl-CoA [(S)-3HB-CoA]. It converts acetoacetyl-CoA to (S)-3HB-CoA in the synthetic metabolic pathway. (S)-3HB-CoA is further modified to form (S)-3-hydroxybutyrate, which is a source of biodegradable polymers. During the course of a study to develop biodegradable polymers, attempts were made to determine the crystal structure of HBD from Clostridium acetobutylicum (CacHBD), and the crystal structures of both apo and NAD + -bound forms of CacHBD were determined. The crystals belonged to different space groups: P2 1 2 1 2 1 and P2 1 . However, both structures adopted a hexamer composed of three dimers in the asymmetric unit, and this oligomerization was additionally confirmed by gel-filtration column chromatography. Furthermore, to investigate the catalytic residues of CacHBD, the enzymatic activities of the wild type and of three single-amino-acid mutants were analyzed, in which the Ser, His and Asn residues that are conserved in the HBDs from C. acetobutylicum, C. butyricum and Ralstonia eutropha, as well as in the L-3-hydroxyacyl-CoA dehydrogenases from Homo sapiens and Escherichia coli, were substituted by alanines. The S117A and N188A mutants abolished the activity, while the H138A mutant showed a slightly lower K m value and a significantly lower k cat value than the wild type. Therefore, in combination with the crystal structures, it was shown that His138 is involved in catalysis and that Ser117 and Asn188 may be important for substrate recognition to place the keto group of the substrate in the correct position for reaction.

The import of most of peroxisomal proteins into the lumen of their target organelle is driven by C-terminal (PTS1) or N-terminal (PTS2) signals recognized by the Pex5p or Pex7p receptors, respectively. However, some proteins in budding yeast, such as acyl-CoA oxidase (AOx) and carnitine acetyltransferase (Cat2p), are imported into peroxisomes via an alternative route that does not rely on known PTS signals and involves the Pex5p receptor N-terminal region. Here, we show that two other budding yeast peroxisomal proteins, a multifunctional enzyme from the β-oxidation pathway (Fox2p) and catalase A (Cta1p), both of which contain PTS1, can be imported independently of this signal. The I264K amino acid substitution in Pex5p adjacent to its FxxxW diaromatic motif, previously shown to abolish the import of AOx and Cat2p into peroxisomes, also affects Fox2p and Cta1p import. Moreover, we demonstrate that Pex9p, a newly discovered paralog of Pex5p that was recently implicated in the import of malate synthases in budding yeast, also exhibits weak receptor activity towards Fox2p and Cta1p. These findings indicate the need to re-evaluate the peroxisomal import paradigm.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Mitochondrial tRNAs are transcribed as long polycistronic transcripts of precursor tRNAs and undergo posttranscriptional modifications such as endonucleolytic processing and methylation required for their correct structure and function. Among them, 5'-end processing and purine 9 N1-methylation of mitochondrial tRNA are catalyzed by two proteinaceous complexes with overlapping subunit composition. The Mg 2+ -dependent RNase P complex for 5'-end cleavage comprises the methyltransferase domain-containing protein tRNA methyltransferase 10C, mitochondrial RNase P subunit (TRMT10C/MRPP1), short-chain oxidoreductase hydroxysteroid 17β-dehydrogenase 10 (HSD17B10/MRPP2), and metallonuclease KIAA0391/MRPP3. An MRPP1-MRPP2 subcomplex also catalyzes the formation of 1-methyladenosine/1-methylguanosine at position 9 using S -adenosyl-l-methionine as methyl donor. However, a lack of structural information has precluded insights into how these complexes methylate and process mitochondrial tRNA. Here, we used a combination of X-ray crystallography, interaction and activity assays, and small angle X-ray scattering (SAXS) to gain structural insight into the two tRNA modification complexes and their components. The MRPP1 N terminus is involved in tRNA binding and monomer-monomer self-interaction, whereas the C-terminal SPOUT fold contains key residues for S -adenosyl-l-methionine binding and N1-methylation. The entirety of MRPP1 interacts with MRPP2 to form the N1-methylation complex, whereas the MRPP1-MRPP2-MRPP3 RNase P complex only assembles in the presence of precursor tRNA. This study proposes low-resolution models of the MRPP1-MRPP2 and MRPP1-MRPP2-MRPP3 complexes that suggest the overall architecture, stoichiometry, and orientation of subunits and tRNA substrates., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Degradation of acetone by the sulfate-reducing bacterium Desulfococcus biacutus involves an acetone-activation reaction different from that used by aerobic or nitrate-reducing bacteria, because the small energy budget of sulfate-reducing bacteria does not allow for major expenditures into ATP-consuming carboxylation reactions. In the present study, an inducible coenzyme B 12 -dependent conversion of 2-hydroxyisobutyryl-CoA to 3-hydroxybutyryl-CoA was demonstrated in cell-free extracts of acetone-grown D. biacutus cells, together with a NAD + -dependent oxidation of 3-hydroxybutyryl-CoA to acetoacetyl-CoA. Genes encoding two mutase subunits and a dehydrogenase, which were found previously to be strongly induced during growth with acetone, were heterologously expressed in E. coli. The activities of the purified recombinant proteins matched with the inducible activities observed in cell-free extracts of acetone-grown D. biacutus: proteins (IMG locus tags) DebiaDRAFT&#95;04573 and 04574 constituted a B 12 -dependent 2-hydroxyisobutyryl-CoA/3-hydroxybutyryl-CoA mutase, and DebiaDRAFT&#95;04571 was a 3-hydroxybutyryl-CoA dehydrogenase. Hence, these enzymes play key roles in the degradation of acetone and define an involvement of CoA esters in the pathway. Further, the involvement of 2-hydroxyisobutyryl-CoA strongly indicates that the carbonyl-C 2 of acetone is added, most likely, to formyl-CoA through a TDP-dependent enzyme that is co-induced in acetone-grown cells and is encoded in the same gene cluster as the identified mutase and dehydrogenase., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Escherichia coli is commonly used for recombinant protein production with many available host strains. Screening experiments are often performed in batch mode using shake flasks and evaluating only the final product concentration. This conventional approach carries the risk of missing the best strain due to limited monitoring capabilities. Thus, this study focuses on investigating the general suitability of online respiration measurement for selecting expression hosts for heterologous protein production. The oxygen transfer rate (OTR) for different T7-RNA polymerase-dependent Escherichia coli expression strains was compared under inducing and noninducing conditions. As model enzymes, a lipase A from Bacillus subtilis (BSLA) and a 3-hydroxybutyryl-CoA dehydrogenase from Thermus thermophilus (HBD) were chosen. Four strains were compared during expression of both enzymes in autoinduction medium. Additionally, four strains were compared during expression of the BSLA with IPTG induction. It was found that the metabolic burden during recombinant protein production induces a phase of constant OTR, while undisturbed cell growth with no or little product formation is indicated by an exponential increase. This pattern is independent of the host strain, expressed enzyme, and induction method. Furthermore, the OTR gives information about carbon source consumption, biomass formation, and the transition from production to noninduced second growth phase, thereby ensuring a fair comparison of different strains. In conclusion, online monitoring of the respiration activity is suited to qualitatively identify, if a recombinant protein is produced by a strain or not. Furthermore, laborious offline sampling is avoided. Thus, the technique is easier and faster compared to conventional approaches. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:315-327, 2018., (© 2018 American Institute of Chemical Engineers.)

17β-Hydroxysteroid dehydrogenase type 10 is a multifunctional, homotetrameric, mitochondrial protein encoded by the HSD17B10 gene at Xp 11.2. This protein, 17β-HSD10, is overexpressed in brain cells of Alzheimer's disease (AD) patients. It was reported to be involved in AD pathogenesis as the endoplasmic reticulum-associated amyloid-β binding protein (ERAB) and as amyloid-β binding alcohol dehydrogenase (ABAD). However, the exaggerated catalytic efficiencies for ERAB/ABAD in these reports necessitated the re-characterization of the catalytic functions of this brain enzyme. In addition to isoleucine metabolism, 17β-HSD10 is also responsible for the mitochondrial metabolism of neurosteroids such as 5α-androstane-3α,17β-diol and 17β-estradiol. These neurosteroids are inactivated by the oxidation catalyzed by 17β-HSD10. Since neurosteroid homeostasis is presumably essential for cognitive function, analysis of the impact of 17β-HSD10 and its inhibitor, amyloid-β peptide (Aβ), on the metabolism of neuroactive steroids offers a new approach to AD pathogenesis.

MRPP2 (also known as HSD10/SDR5C1) is a multifunctional protein that harbours both catalytic and non-catalytic functions. The protein belongs to the short-chain dehydrogenase/reductases (SDR) family and is involved in the catabolism of isoleucine in vivo and steroid metabolism in vitro. MRPP2 also moonlights in a complex with the MRPP1 (also known as TRMT10C) protein for N1-methylation of purines at position 9 of mitochondrial tRNA, and in a complex with MRPP1 and MRPP3 (also known as PRORP) proteins for 5'-end processing of mitochondrial precursor tRNA. Inherited mutations in the HSD17B10 gene encoding MRPP2 protein lead to a childhood disorder characterised by progressive neurodegeneration, cardiomyopathy or both. Here we report two patients with novel missense mutations in the HSD17B10 gene (c.34G>C and c.526G>A), resulting in the p.V12L and p.V176M substitutions. Val12 and Val176 are highly conserved residues located at different regions of the MRPP2 structure. Recombinant mutant proteins were expressed and characterised biochemically to investigate their effects towards the functions of MRPP2 and associated complexes in vitro. Both mutant proteins showed significant reduction in the dehydrogenase, methyltransferase and tRNA processing activities compared to wildtype, associated with reduced stability for protein with p.V12L, whereas the protein carrying p.V176M showed impaired kinetics and complex formation. This study therefore identified two distinctive molecular mechanisms to explain the biochemical defects for the novel missense patient mutations., (Copyright © 2017. Published by Elsevier B.V.)

Mitochondrial polycistronic transcripts are extensively processed to give rise to functional mRNAs, rRNAs and tRNAs; starting with the release of tRNA elements through 5'-processing by RNase P (MRPP1/2/3-complex) and 3'-processing by RNase Z (ELAC2). Here, we show using in vitro experiments that MRPP1/2 is not only a component of the mitochondrial RNase P but that it retains the tRNA product from the 5'-processing step and significantly enhances the efficiency of ELAC2-catalyzed 3'-processing for 17 of the 22 tRNAs encoded in the human mitochondrial genome. Furthermore, MRPP1/2 retains the tRNA product after ELAC2 processing and presents the nascent tRNA to the mitochondrial CCA-adding enzyme. Thus, in addition to being an essential component of the RNase P reaction, MRPP1/2 serves as a processing platform for several down-stream tRNA maturation steps in human mitochondria. These findings are of fundamental importance for our molecular understanding of disease-related mutations in MRPP1/2, ELAC2 and mitochondrial tRNA genes., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Pathological cardiac hypertrophy may be associated with reduced expression of glucose transporter 4 (GLUT4) in contrast to exercise-induced cardiac hypertrophy, where GLUT4 levels are increased. However, mice with cardiac-specific deletion of GLUT4 (G4H -/- ) have normal cardiac function in the unstressed state. This study tested the hypothesis that cardiac GLUT4 is required for myocardial adaptations to hemodynamic demands. G4H -/- and control littermates were subjected to either a pathological model of left ventricular pressure overload [transverse aortic constriction (TAC)] or a physiological model of endurance exercise (swim training). As predicted after TAC, G4H -/- mice developed significantly greater hypertrophy and more severe contractile dysfunction. Somewhat surprisingly, after exercise training, G4H -/- mice developed increased fibrosis and apoptosis that was associated with dephosphorylation of the prosurvival kinase Akt in concert with an increase in protein levels of the upstream phosphatase protein phosphatase 2A (PP2A). Exercise has been shown to decrease levels of ceramide; G4H -/- hearts failed to decrease myocardial ceramide in response to exercise. Furthermore, G4H -/- hearts have reduced levels of the transcriptional coactivator peroxisome proliferator-activated receptor-γ coactivator-1, lower carnitine palmitoyl-transferase activity, and reduced hydroxyacyl-CoA dehydrogenase activity. These basal changes may also contribute to the impaired ability of G4H -/- hearts to adapt to hemodynamic stresses. In conclusion, GLUT4 is required for the maintenance of cardiac structure and function in response to physiological or pathological processes that increase energy demands, in part through secondary changes in mitochondrial metabolism and cellular stress survival pathways such as Akt. NEW & NOTEWORTHY Glucose transporter 4 (GLUT4) is required for myocardial adaptations to exercise, and its absence accelerates heart dysfunction after pressure overload. The requirement for GLUT4 may extend beyond glucose uptake to include defects in mitochondrial metabolism and survival signaling pathways that develop in its absence. Therefore, GLUT4 is critical for responses to hemodynamic stresses., (Copyright © 2017 the American Physiological Society.)

The class III deacetylase sirtuin 1 (SIRT1), a member of the sirtuin family proteins, plays a key role in many types of cancers including colorectal cancer (CRC). Here we report that SIRT1 suppressed CRC metastasis in vitro and in vivo as a negative regulator for miR-15b-5p transcription. Mechanistically, SIRT1 impaired regulatory effects of activator protein (AP-1) on miR-15b-5p trans-activation through deacetylation of AP-1. Importantly, acyl-CoA oxidase 1 (ACOX1), a key enzyme of the fatty acid oxidation (FAO) pathway, was found as a direct target for miR-15b-5p. SIRT1 expression was positively correlated with ACOX1 expression in CRC cells and in xenografts. Moreover, ACOX1 overexpression attenuated the augmentation of migration and invasion of CRC cells by miR-15b-5p overexpression. In conclusion, our study demonstrated a functional role of the SIRT1/miR-15b-5p/ACOX1 axis in CRC metastasis and suggested a potential target for metastatic CRC therapy., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Ascomycin is a 23-membered polyketide macrolide with high immunosuppressant and antifungal activity. As the lower production in bio-fermentation, global metabolic analysis is required to further explore its biosynthetic network and determine the key limiting steps for rationally engineering. To achieve this goal, an engineering approach guided by a metabolic network model was implemented to better understand ascomycin biosynthesis and improve its production., Results: The metabolic conservation of Streptomyces species was first investigated by comparing the metabolic enzymes of Streptomyces coelicolor A3(2) with those of 31 Streptomyces strains, the results showed that more than 72% of the examined proteins had high sequence similarity with counterparts in every surveyed strain. And it was found that metabolic reactions are more highly conserved than the enzymes themselves because of its lower diversity of metabolic functions than that of genes. The main source of the observed metabolic differences was from the diversity of secondary metabolism. According to the high conservation of primary metabolic reactions in Streptomyces species, the metabolic network model of Streptomyces hygroscopicus var. ascomyceticus was constructed based on the latest reported metabolic model of S. coelicolor A3(2) and validated experimentally. By coupling with flux balance analysis and using minimization of metabolic adjustment algorithm, potential targets for ascomycin overproduction were predicted. Since several of the preferred targets were highly associated with ethylmalonyl-CoA biosynthesis, two target genes hcd (encoding 3-hydroxybutyryl-CoA dehydrogenase) and ccr (encoding crotonyl-CoA carboxylase/reductase) were selected for overexpression in S. hygroscopicus var. ascomyceticus FS35. Both the mutants HA-Hcd and HA-Ccr showed higher ascomycin titer, which was consistent with the model predictions. Furthermore, the combined effects of the two genes were evaluated and the strain HA-Hcd-Ccr with hcd and ccr overexpression exhibited the highest ascomycin production (up to 438.95 mg/L), 1.43-folds improvement than that of the parent strain FS35 (305.56 mg/L)., Conclusions: The successful constructing and experimental validation of the metabolic model of S. hygroscopicus var. ascomyceticus showed that the general metabolic network model of Streptomyces species could be used to analyze the intracellular metabolism and predict the potential key limiting steps for target metabolites overproduction. The corresponding overexpression strains of the two identified genes (hcd and ccr) using the constructed model all displayed higher ascomycin titer. The strategy for yield improvement developed here could also be extended to the improvement of other secondary metabolites in Streptomyces species.

Myostatin (Mstn) deficiency leads to skeletal muscle overgrowth and Mstn inhibition is considered as a promising treatment for muscle-wasting disorders. Mstn gene deletion in mice also causes metabolic changes with decreased mitochondria content, disturbance in mitochondrial respiratory function and increased muscle fatigability. However the impact of MSTN deficiency on these metabolic changes is not fully elucidated. Here, we hypothesized that lack of MSTN will alter skeletal muscle membrane lipid composition in relation with pronounced alterations in muscle function and metabolism. Indeed, phospholipids and in particular cardiolipin mostly present in the inner mitochondrial membrane, play a crucial role in mitochondria function and oxidative phosphorylation process. We observed that Mstn KO muscle had reduced fat membrane transporter levels (FAT/CD36, FABP3, FATP1 and FATP4) associated with decreased lipid oxidative pathway (citrate synthase and β-HAD activities) and impaired lipogenesis (decreased triglyceride and free fatty acid content), indicating a role of mstn in muscle lipid metabolism. We further analyzed phospholipid classes and fatty acid composition by chromatographic methods in muscle and mitochondrial membranes. Mstn KO mice showed increased levels of saturated and polyunsaturated fatty acids at the expense of monounsaturated fatty acids. We also demonstrated, in this phenotype, a reduction in cardiolipin proportion in mitochondrial membrane versus the proportion of others phospholipids, in relation with a decrease in the expression of phosphatidylglycerolphosphate synthase and cardiolipin synthase, enzymes involved in cardiolipin synthesis. These data illustrate the importance of lipids as a link by which MSTN deficiency can impact mitochondrial bioenergetics in skeletal muscle., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Scope: The aim of this work was to study the urinary metabolomics changes of participants that consumed beer, nonalcoholic beer (na-beer), and gin., Methods and Results: Thirty-three males at high cardiovascular risk between 55 and 75 years old participated in an open, randomized, crossover, controlled trial with three nutritional interventions consisting of beer, na-beer, and gin for 4 wk. Diet and physical activity was monitored throughout the study and compliance was assessed by measurement of urinary isoxanthohumol. Metabolomic analysis was performed in urine samples by LC coupled to an LTQ-Orbitrap mass spectrometer combined with univariate and multivariate statistical analysis. Ten metabolites were identified. Eight were exogenous metabolites related to beer, na-beer, or gin consumption, but two of them were related to endogenic changes: hydroxyadipic acid linked to fatty acid oxidation, and 4-guanidinobutanoic acid, which correlated with a decrease in urinary creatinine. Plasmatic acylcarnitines were quantified by targeted MS. A regular and moderate consumption of beer and na-beer decreased stearoylcarnitine concentrations., Conclusion: Humulinone and 2,3-dihydroxy-3-methylvaleric acid showed to be potential biomarkers of beer and na-beer consumption. Moreover, the results of this trial provide new evidence that the nonalcoholic fraction of beer may increase fatty oxidation., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Acute fatty liver of pregnancy (AFLP) is a rare liver disease unique to pregnancy that can lead to acute liver failure. The prognosis, initially often fatal for both mother and child, has been improved by prompt delivery. The diagnosis should be highly suspected if the mother presents epigastric pain, nausea and/or vomiting, or polyuria-polydipsia in the third trimester of pregnancy. AFLP has been found associated with a genetic deficiency of fatty acid beta-oxidation, which may cause sudden death in infancy. Consequently, the mother and her newborn should undergo screening for this deficiency., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Lysine acetylation is a reversible posttranslational modification and is particularly important in the regulation of mitochondrial metabolic enzymes. Acetylation uses acetyl-CoA derived from fuel metabolism as a cofactor, thereby linking nutrition to metabolic activity. In the present study, we investigated how mitochondrial acetylation status in the heart is controlled by food intake and how these changes affect mitochondrial metabolism. We found that there was a significant increase in cardiac mitochondrial protein acetylation in mice fed a long-term high-fat diet and that this change correlated with an increase in the abundance of the mitochondrial acetyltransferase-related protein GCN5L1. We showed that the acetylation status of several mitochondrial fatty acid oxidation enzymes (long-chain acyl-CoA dehydrogenase, short-chain acyl-CoA dehydrogenase, and hydroxyacyl-CoA dehydrogenase) and a pyruvate oxidation enzyme (pyruvate dehydrogenase) was significantly upregulated in high-fat diet-fed mice and that the increase in long-chain and short-chain acyl-CoA dehydrogenase acetylation correlated with increased enzymatic activity. Finally, we demonstrated that the acetylation of mitochondrial fatty acid oxidation proteins was decreased after GCN5L1 knockdown and that the reduced acetylation led to diminished fatty acid oxidation in cultured H9C2 cells. These data indicate that lysine acetylation promotes fatty acid oxidation in the heart and that this modification is regulated in part by the activity of GCN5L1. NEW & NOTEWORTHY Recent research has shown that acetylation of mitochondrial fatty acid oxidation enzymes has greatly contrasting effects on their activity in different tissues. Here, we provide new evidence that acetylation of cardiac mitochondrial fatty acid oxidation enzymes by GCN5L1 significantly upregulates their activity in diet-induced obese mice., (Copyright © 2017 the American Physiological Society.)