Pyruvate Dehydrogenase Subunit Research Articles

A hub for regulation of mitochondrial metabolism: Fatty acid and lipoic acid biosynthesis.

Having evolved from a prokaryotic origin, mitochondria retain pathways required for the catabolism of energy-rich molecules and for the biosynthesis of molecules that aid catabolism and/or participate in other cellular processes essential for life of the cell. Reviewed here are details of the mitochondrial fatty acid biosynthetic pathway (FAS II) and its role in building both the octanoic acid precursor for lipoic acid biosynthesis (LAS) and longer-chain fatty acids functioning in chaperoning the assembly of mitochondrial multisubunit complexes. Also covered are the details of mitochondrial lipoic acid biosynthesis, which is distinct from that of prokaryotes, and the attachment of lipoic acid to subunits of pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and glycine cleavage system complexes. Special emphasis has been placed on presenting what is currently known about the interconnected paths and loops linking the FAS II-LAS pathway and two other mitochondrial realms, the organellar translation machinery and Fe-S cluster biosynthesis and function.

Formalin-Fixed Paraffin-Embedded Proteomics of Malignant Mesothelioma and New Candidate Biomarkers Thioredoxin and Superoxide Dismutase 2 for Immunohistochemistry

The pathogenesis of malignant mesothelioma (MM) has been extensively investigated, focusing on stress derived from reactive oxygen species. We aimed to identify diagnostic biomarkers of MM by analyzing proteins in formalin-fixed paraffin-embedded specimens using liquid chromatography-mass spectrometry. We extracted proteins from formalin-fixed paraffin-embedded sections of MM tissues (n = 7) and compared their profiles with those of benign mesothelial tissues (n = 4) and alveolar tissue (n = 1). Proteomic data were statistically assessed and profiled using principal component analysis. We were successful in the classification of MM and healthy tissue. The levels of superoxide dismutase 2 (SOD2), an enzyme that converts superoxide anion into oxygen and hydrogen peroxide, and thioredoxin (TXN), which plays a crucial role in reducing disulfide bonds in proteins, primarily contributed to the classification. Other redox-related proteins, such as pyruvate dehydrogenase subunit X, and ceruloplasmin also contributed to the classification. Protein-protein interaction analysis demonstrated that these proteins play essential roles in MM pathogenesis. Immunohistochemistry revealed that TXN levels were significantly lower, whereas SOD2 levels were significantly higher in MM and lung cancer tissues than in controls. Proteomic profiling suggested that MM tissues experienced increased exposure to hydrogen peroxide and other reactive oxygen species. Combining immunohistochemistry for TXN and SOD2 allows for differentiation among MM, lung cancer, and control tissues; hence, TXN and SOD2 may be promising MM biomarkers and therapeutic targets.

Salmonella enterica relies on carbon metabolism to adapt to agricultural environments.

Salmonella enterica, a foodborne and human pathogen, is a constant threat to human health. Agricultural environments, for example, soil and plants, can be ecological niches and vectors for Salmonella transmission. Salmonella persistence in such environments increases the risk for consumers. Therefore, it is necessary to investigate the mechanisms used by Salmonella to adapt to agricultural environments. We assessed the adaptation strategy of S. enterica serovar Typhimurium strain 14028s to agricultural-relevant situations by analyzing the abundance of intermediates in glycolysis and the tricarboxylic acid pathway in tested environments (diluvial sand soil suspension and leaf-based media from tomato and lettuce), as well as in bacterial cells grown in such conditions. By reanalyzing the transcriptome data of Salmonella grown in those environments and using an independent RT-qPCR approach for verification, several genes were identified as important for persistence in root or leaf tissues, including the pyruvate dehydrogenase subunit E1 encoding gene aceE. In vivo persistence assay in tomato leaves confirmed the crucial role of aceE. A mutant in another tomato leaf persistence-related gene, aceB, encoding malate synthase A, displayed opposite persistence features. By comparing the metabolites and gene expression of the wild-type strain and its aceB mutant, fumarate accumulation was discovered as a potential way to replenish the effects of the aceB mutation. Our research interprets the mechanism of S. enterica adaptation to agriculture by adapting its carbon metabolism to the carbon sources available in the environment. These insights may assist in the development of strategies aimed at diminishing Salmonella persistence in food production systems.

Inhibition of TCA cycle improves the anti-PD-1 immunotherapy efficacy in melanoma cells via ATF3-mediated PD-L1 expression and glycolysis

Backgroundanti-Programmed Death-1 (anti-PD-1) immunotherapy has shown promising manifestation in improving the survival rate of patients with advanced melanoma, with its efficacy closely linked to Programmed cell death-Ligand 1 (PD-L1) expression....

Pyruvate dehydrogenase kinase 1–dependent metabolic reprogramming: A promising target for postmenopausal osteoporosis treatment

Postmenopausal osteoporosis (PMOP) is a classic endocrine disorder characterized by an imbalance between bone formation and bone resorption. Abnormal glucose metabolic reprogramming has been widely explored in endocrine disorders, but its role in PMOP has not been elucidated. Icariin (ICA), as a bioactive component of the traditional Chinese medicine Epimedium grandiflorum, has been testified to be effective for PMOP treatment, but its effects on glucose metabolism are poorly studied. In this study, pyruvate dehydrogenase was proved to be a key regulator of the metabolic switch in PMOP and the efficacy of ICA. The augmentation of pyruvate dehydrogenase subunit alpha (PDHA) pSer232, which was excited in osteoclasts; the decreased expression of citrate synthase; elevated expression of lactate dehydrogenase; and changes in the pyruvate, lactate, and citrate pools all indicated the preference for aerobic glycolysis in PMOP. Further study showed that the expression and activity of pyruvate dehydrogenase kinase 1 (PDK1) which regulated the phosphorylation status of PDHA Ser232 were both increased in ovariectomized rats. Furthermore, the genetic knockdown of PDK1 and pharmaceutical inhibition of PDK1 markedly suppressed osteoclastogenesis. Meanwhile, ICA might reverse metabolic reprogramming by inhibiting PDK1. In conclusion, this study revealed that PDK1-dependent metabolic reprogramming is a potential therapeutic target for PMOP treatment. ICA alleviates PMOP by inhibiting PDK1 in osteoclasts, indicating its potential for clinical use.

Externalization of Mitochondrial PDCE2 on Irradiated Endothelium as a Target for Radiation-Guided Drug Delivery and Precision Thrombosis of Pathological Vasculature.

Endothelial cells are highly sensitive to ionizing radiation, and exposure leads to multiple adaptive changes. Remarkably, part of this response is the translocation of normally intracellular proteins to the cell surface. It is unclear whether this ectopic expression has a protective or deleterious function, but, regardless, these surface-exposed proteins may provide unique discriminatory targets for radiation-guided drug delivery to vascular malformations or tumor vasculature. We investigated the ability of an antibody–thrombin conjugate targeting mitochondrial PDCE2 (E2 subunit of pyruvate dehydrogenase) to induce precision thrombosis on irradiated endothelial cells in a parallel-plate flow system. Click-chemistry was used to create antibody–thrombin conjugates targeting PDCE2 as the vascular targeting agent (VTA). VTAs were injected into the parallel-plate flow system with whole human blood circulating over irradiated cells. The efficacy and specificity of fibrin-thrombus formation was assessed relative to non-irradiated controls. The PDCE2-targeting VTA dose-dependently increased thrombus formation: minimal thrombosis was induced in response to 5 Gy radiation; doses of 15 and 25 Gy induced significant thrombosis with equivalent efficacy. Negligible VTA binding or thrombosis was demonstrated in the absence of radiation or with non-targeted thrombin. PDCE2 represents a unique discriminatory target for radiation-guided drug delivery and precision thrombosis in pathological vasculature.

GLUTAMINE DEPRIVATION AFFECTS THE EXPRESSION OF GENES WHICH CONTROL PYRUVATE DEHYDROGENASE ACTIVITY: THE IMPACT OF ERN1 KNOCKDOWN

The aim of the current investigation was to study the expression of genes encoded pyruvate dehydrogenase subunits (PDHA1, PDHB, PDHX, DLAT, and DLD) in U87 glioma cells in response to glutamine deprivation in U87 glioma cells in relation to knockdown of ERN1 for evaluation of a possible dependence of the expression of these important regulatory genes from glutamine supply and ERN1 signaling. Methods. The expression of PDHA1, PDHB, PDHX, DLAT, and DLD genes was studied by real-time qPCR in control U87 glioma cells (transfected by vector) and cells with knockdown of ERN1 (transfected by dnERN1) after exposure to glutamine deprivation condition. Total RNA was extracted from glioma cells using TRIZOL reagent. An RNA quantity as well as spectral characteristics was measured using NanoDrop One. For reverse transcription of mRNAs we used Thermo Scientific Verso cDNA Synthesis Kit (Germany). The values of mRNA expressions were normalized to the level of ACTB mRNA and represented as percent of control (100 %). Results. It was shown that the expression level of PDH1, PDHB, DLAT, and DLD genes was down-regulated in control glioma cells treated by glutamine deprivation. At the same time, ERN1 knockdown is suppressed the effect of glutamine deprivation on PDHB and DLD gene expressions in glioma cells, but did not change significantly the impact of glutamine deprivation on the expression of PDHA1, DLAT, and PDHX genes. Conclusions. The results of this investigation demonstrated that the expression of PDH1, PDHB, PDHX, DLAT, and DLD genes was significantly affected by exposure of U87 glioma cells under glutamine deprivation condition and that the effect of glutamine deprivation on the expression of most these genes was modified in cells with knockdown of ERN1, a major signaling pathway of the endoplasmic reticulum stress.

Reversing mitochondrial defects in aged hearts: role of mitochondrial calpain activation.

Aging chronically increases endoplasmic reticulum (ER) stress that contributes to mitochondrial dysfunction. Activation of calpain 1 (CPN1) impairs mitochondrial function during acute ER stress. We proposed that aging-induced ER stress led to mitochondrial dysfunction by activating CPN1. We posit that attenuation of the ER stress or direct inhibition of CPN1 in aged hearts can decrease cardiac injury during ischemia-reperfusion by improving mitochondrial function. Male young (3 mo) and aged mice (24 mo) were used in the present study, and 4-phenylbutyrate (4-PBA) was used to decrease the ER stress in aged mice. Subsarcolemmal (SSM) and interfibrillar mitochondria (IFM) were isolated. Chronic 4-PBA treatment for 2 wk decreased CPN1 activation as shown by the decreased cleavage of spectrin in cytosol and apoptosis inducing factor (AIF) and the α1 subunit of pyruvate dehydrogenase (PDH) in mitochondria. Treatment improved oxidative phosphorylation in 24-mo-old SSM and IFM at baseline compared with vehicle. When 4-PBA-treated 24-mo-old hearts were subjected to ischemia-reperfusion, infarct size was decreased. These results support that attenuation of the ER stress decreased cardiac injury in aged hearts by improving mitochondrial function before ischemia. To challenge the role of CPN1 as an effector of the ER stress, aged mice were treated with MDL-28170 (MDL, an inhibitor of calpain 1). MDL treatment improved mitochondrial function in aged SSM and IFM. MDL-treated 24-mo-old hearts sustained less cardiac injury following ischemia-reperfusion. These results support that age-induced ER stress augments cardiac injury during ischemia-reperfusion by impairing mitochondrial function through activation of CPN1.

Abstract 14216: Mitochondrial Dysfunction in Aged Hearts: Role of Mitochondrial Calpain Activation

Introduction: Mitochondrial function is impaired in aged hearts. Calpain 1 (CPN1) is a calcium-dependent cysteine protease located in cytosol (cCPN1) and mitochondria (mCPN1). Activation of CPN1 contributes to mitochondrial dysfunction during ischemia-reperfusion. Induction of acute ER (endoplasm reticulum) stress causes CPN1 activation in adult hearts. We find that aging leads to increased ER stress that may favor CPN1 activation. Hypothesis: ER stress induced CPN1 activation leads to mitochondrial dysfunction during aging. Methods: Male adult (3 mo.) and aged (24 mo.) mice were provided by NIA. 4-PBA (1g/kg/day) was dissolved in drinking water with sucrose (0.2 g/100 ml) as sweetener. Sucrose water was used as vehicle control. Mice were treated for two weeks with 4-PBA or vehicle followed by the isolation of cardiac cytosol, SSM (subsarcolemmal mitochondria) and IFM (interfibrillar mitochondria). Results: Oxidative phosphorylation was decreased in IFM from 24 mo. compared to 3 mo. when glutamate + malate was used as complex I substrate (Fig A). The 4-PBA treatment improved oxidative phosphorylation in aged IFM (Figure A). The cCPN1 was activated in 24 mo. as shown by increased spectrin cleavage (Figure B). The α1 subunit of pyruvate dehydrogenase (PDH) is a substrate of mCPN1. The content of the PDH α1 subunit was decreased in 24 mo. compared to 3 mo., indicating that the mCPN1 was activated during aging (Figure C). The 4-PBA treatment decreased the activation of cCPN1 and mCPN1 in aged mice. SSM function is also impaired during aging (data not shown). Conclusions: Aging increased the activation of both cCPN1 and mCPN1. Attenuation of ER stress in 24 mo. mice using 4-PBA decreased cCPN1 and mCPN1 activation. Decreased CPN1 activation was accompanied by improved mitochondrial function . The results support that activation of calpain 1, most likely mCPN1, is a likely cause of increased mitochondrial dysfunction during aging.

Abstract 2333: EMT programs ovarian cancer cells to survive the adipocyte-rich microenvironment

Abstract Background: Metabolic pathways are non-canonical targets of the EMT program. The relevance of re-programming metabolism during the metastasis cascade is underscored in ovarian cancer cells, which primarily metastasizes to the adipocyte-rich omentum. Ovarian cancer-adipocyte interaction has been shown to promote fatty acid (FA) uptake into the cancer cells. These FA are then catabolized via β-oxidation thus providing the energy required for rapid tumor growth. β-oxidation of FA requires efficient mitochondria and oxygen processing. The objective of this study is to determine if E/M status determine adaptation and survival in adipocyte-rich microenvironment. Materials and Methods: RNA seq was performed in Ck18+/Twist- epithelial and Ck18-/Twist+ mesenchymal human ovarian cancer cells. Pathway and Gene Ontology analyses were performed on differentially expressed (DEX) genes (FC>2 and p<0.05) using iPathway Guide. Protein levels were determined by western blot. Oxygen consumption rate was measured using Seahorse XF Analyzer. Cell viability was quantified using CelltoxTM Green viability dye. Results: Pathway analysis of DEX genes with focus on metabolic pathways showed the following DEX pathways: glycolysis/gluconeogenesis (p=0.008); pyruvate metabolism (0.019); fatty acid degradation (p=0.01); fatty acid elongation (0.008). Gene ontology analysis showed significant difference in GO:0006979: response to oxidative stress (p=0.007). Further analysis of DEX genes within these pathways showed upregulation in mesenchymal cancer cells of both pyruvate dehydrogenase subunits, PDHA1 (p = 0.011) and PDHB (p=0.014) and upregulation of carnitine palmitoyltransferase (CPT1; p=0.011), the rate limiting enzyme in FA β-oxidation. The upregulation of these genes was validated at the protein level by western blot analysis, which also showed increased levels of COX-IV in mesenchymal cancer cells compared to epithelial cancer cells. Moreover, mesenchymal cells exhibited enhanced maximal respiration and enhanced spare respiratory capacity. Finally, viability studies conclusively demonstrate the metabolic flexibility in mesenchymal cancer cells, which maintained viability upon nutrient deprivation suggesting metabolic plasticity. In contrast, cell death was observed epithelial cancer cells upon nutrient deprivation, which was rescued by the addition of glucose but not the FA, oleic acid. Conclusion: Ovarian cancer cells acquire enhanced pyruvate shuttling to the TCA cycle, a more efficient FA β-oxidation machinery, as well as more efficient oxygen processing during EMT. These phenotypes are relevant in supporting survival in the adipocyte microenvironment. Further elucidation of EMT related pathways that re-program lipid metabolism may lead to better control of carcinomatosis in ovarian cancer patients. Citation Format: Hussein Chehade, Alexandra Fox, Roslyn Tedja, Radhika Gogoi, Gil Mor, Ayesha B. Alvero. EMT programs ovarian cancer cells to survive the adipocyte-rich microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2333.

Immune pathogenesis of primary biliary cholangitis

Primary biliary cholangitis (PBC) is an autoimmune liver disease, mainly characterized by chronic progressive cholestasis. The root cause of PBC is the loss of immune tolerance to autoantigen E2 subunit of pyruvate dehydrogenase (PDC-E2). The unique immunobiological characteristics of intrahepatic bile duct epithelial cells make it an active participant in the pathogenesis of PBC. In recent years, the detection rate of PBC has been increasing year by year, but the clinical situation of ursodeoxycholic acid monotherapy has not changed. Therefore, an in-depth understanding of the immune pathogenesis of PBC will help clinicians better prevent and treat diseases.

The (pro)renin receptor ((P)RR) and soluble (pro)renin receptor (s(P)RR) in pregnancy.

The (pro)renin receptor ((P)RR) is a multi-functional protein that can be proteolytically cleaved and released in a soluble form (s(P)RR). Recently, the (P)RR and s(P)RR have become of interest in pregnancy and its associated pathologies. This is because the (P)RR not only activates tissue renin angiotensin systems, but it is also an integral component of vacuolar-ATPase, activates the wingless/integrated (Wnt)/β-catenin and extracellular signal regulated kinases 1 and 2/mitogen-activated protein kinase signalling pathways, and stabilises the β subunit of pyruvate dehydrogenase. Additionally, s(P)RR is detected in plasma and urine, and maternal plasma levels are elevated in pregnancy complications including fetal growth restriction, preeclampsia and gestational diabetes mellitus. Therefore, s(P)RR has potential as a biomarker for these pregnancy pathologies. Preliminary functional findings suggest that s(P)RR may be important for regulating fluid balance, inflammation and blood pressure, all of which contribute to a successful pregnancy. The (P)RR and s(P)RR regulate pathways that are known to be important in maintaining pregnancy, however their role in the physiological context of pregnancy is poorly characterised. This review summarises the known and potential functions of the (P)RR and s(P)RR in pregnancy, and how their dysregulation may contribute to pregnancy complications. It also highlights the need for further research into the source and function of s(P)RR in pregnancy. Soluble (P)RR levels could be indicative of placental, kidney or liver dysfunction and therefore be a novel clinical biomarker, or therapeutic target, to improve the detection and treatment of pregnancy pathologies.

Nitric Oxide Mediates Direct Restriction of Pyruvate Dehydrogenase Complex via Generation of Nitroxyl During Macrophage Polarization

M1 macrophages “commit” to a metabolic state where increased aerobic glycolysis sustains fast ATP production, independently of Oxidative Phosphorylation (OXPHOS). This is associated with a “broken” mitochondrial TCA cycle and accumulation of metabolites like citrate, succinate and itaconate, important for cellular functions. We have shown that induced Nitric Oxide (NO) levels in Bone Marrow Derived Macrophages (BMDMs) from Wild Type (WT) mice activated with lipopolysaccharide and interferon gamma (LPS + IFNγ) are necessary and sufficient for the repression of OXPHOS and for metabolic reprogramming at mitochondrial Aconitase (ACO2) and Pyruvate Dehydrogenase (PDH). We hypothesize that NOdirectly targets the PDH complex via its critical cofactor lipoate, interacting with its thiol motifs. High-sensitivity proteomics on PDH complex shows that stimulated BMDM and cells exposed to NO have stalled enzymatic machinery, and this correlates with NO-associated lipoate on E2 subunit of PDH. Moreover, through native in gel activity assays we found decreased enzymatic activity of PDH-E3 and shifts in molecular weight of both PDH-E3 and E3 binding protein (E3Bp); in addition we found these positive for nitrogen specie-mediated modifications, which were not easily reversible with common reducing agents. Using chemical approaches we show that interaction of lipoate with NO can generate a reduced NO radical, nitroxyl (HNO), which can interact with thiols to form unstable intermediates that spontaneously rearrange to a sulfinamide; this represents a nearly irreversible thiol modification. Consistently, mass spectrometry analysis of recombinant protein reveals HNO-dependent post translational modification (PTM) of PDHE3. Structurally, HNO-specific modifications on E3 strongly impair homodimer formation, essential for activity. Our work is the first to demonstrate that HNO is produced physiologically in proinflammatory macrophages, and it is locally released within the interaction with lipoate moiety at the level of PDH-E2 and E3Bp, facilitating irreversible modification of distal E3. In turn this leads to inability for deacetylation of lipoate and global impairment of enzyme function. Finally, since the same E3 subunit is shared with two other key cellular metabolic enzymes, Oxoglutarate and Branched-chain alpha-keto acid Dehydrogenases (OGDH and BCKDH) we predict that NO could be responsible for orchestrating macrophage energy metabolism during inflammation also by limiting the activity of these dehydrogenases through irreversible modifications, opening the possibility for new therapeutic manipulations.

Mitochondrial Encephalomyopathy Associated with Pyruvate Dehydrogenase Complex Deficiency: Eight Clinical Cases

Background. Defects in pyruvate dehydrogenase complex (PDC), involved in the glycolysis products integration into the cells' energy metabolism, are one of the reasons of mitochondrial pathology development. The diagnosis of this condition can be pretty complicated also due to the lack of description of such patients with encephalomyopathy associated with PDC deficiency in Russian population. Clinical Cases Description. We have performed the analysis of clinical manifestations polymorphism of progressive mitochondrial encephalomyopathy caused by pathogenic variants in nuclear X linked gene, PDHA1 (encodes alpha subunit of pyruvate dehydrogenase), in 8 boys aged from 1 to 8 years. The adverse perinatal period was mentioned in all cases. The major features of symptom complex by the time of hospital examination were psychomotor retardation, ataxy, myopathic manifestations. Dystonic attacks were observed in 2 sibs. All patients had changes on brain magnetic resonance imaging: in basal ganglia in 6 children and ventriculomegaly in 2 children. All children had lactic acidosis. Clinical examination has shown that 4 patients had severe damage of nervous system, other 4 patients had moderate damage. Missense mutations in the PDHA1 gene were revealed in 6 children, insertions and duplications including 6 and 16 base pairs — in 2 children. The moderate positive dynamics was noticed as a result of complex treatment of children: stabilization of the overall condition, no metabolic crises, decrease in frequency of dystonic attacks. Conclusion. The clinical polymorphism of mitochondrial encephalomyopathy associated with PDC deficiency is described. The differences in manifestations of severe and moderate forms of disease are shown. The presented description may be useful for medico-genetic counseling and providing medico-genetic care for families.

Genetic dissection of the mitochondrial lipoylation pathway in yeast.

BackgroundLipoylation of 2-ketoacid dehydrogenases is essential for mitochondrial function in eukaryotes. While the basic principles of the lipoylation processes have been worked out, we still lack a thorough understanding of the details of this important post-translational modification pathway. Here we used yeast as a model organism to characterize substrate usage by the highly conserved eukaryotic octanoyl/lipoyl transferases in vivo and queried how amenable the lipoylation system is to supplementation with exogenous substrate.ResultsWe show that the requirement for mitochondrial fatty acid synthesis to provide substrates for lipoylation of the 2-ketoacid dehydrogenases can be bypassed by supplying the cells with free lipoic acid (LA) or octanoic acid (C8) and a mitochondrially targeted fatty acyl/lipoyl activating enzyme. We also provide evidence that the S. cerevisiae lipoyl transferase Lip3, in addition to transferring LA from the glycine cleavage system H protein to the pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase (KGD) E2 subunits, can transfer this cofactor from the PDH complex to the KGD complex. In support of yeast as a model system for human metabolism, we demonstrate that the human octanoyl/lipoyl transferases can substitute for their counterparts in yeast to support respiratory growth and protein lipoylation. Like the wild-type yeast enzyme, the human lipoyl transferase LIPT1 responds to LA supplementation in the presence of the activating enzyme LplA.ConclusionsIn the yeast model system, the eukaryotic lipoylation pathway can use free LA and C8 as substrates when fatty/lipoic acid activating enzymes are targeted to mitochondria. Lip3 LA transferase has a wider substrate specificity than previously recognized. We show that these features of the lipoylation mechanism in yeast are conserved in mammalian mitochondria. Our findings have important implications for the development of effective therapies for the treatment of LA or mtFAS deficiency-related disorders.

Investigating Synthesis of the MalS Malic Enzyme during Bacillus subtilis Spore Germination and Outgrowth and the Influence of Spore Maturation and Sporulation Conditions

Spore-forming bacteria of the orders Bacillales and Clostridiales play a major role in food spoilage and foodborne diseases. When environmental conditions become favorable, these spores can germinate as the germinant receptors located on the spore's inner membrane are activated via germinant binding. This leads to the formation of vegetative cells via germination and subsequent outgrowth and potential deleterious effects on foods. The present report focuses on analysis of the synthesis of the MalS (malic enzyme) protein during Bacillus subtilis spore germination by investigating the dynamics of the presence and fluorescence level of a MalS-GFP (MalS-green fluorescent protein) fusion protein using time-lapse fluorescence microscopy. Our results show an initial increase in MalS-GFP fluorescence intensity within the first 15 min of germination, followed by a discernible drop and stabilization of the fluorescence throughout spore outgrowth as reported previously (L. Sinai, A. Rosenberg, Y. Smith, E. Segev, and S. Ben-Yehuda, Mol Cell 57:695-707, 2015, https://doi.org/10.1016/j.molcel.2014.12.019). However, in contrast to the earlier report, both Western blotting and SILAC (stable isotopic labeling of amino acids in cell culture) analysis showed there was no increase in MalS-GFP levels during the 15 min after the addition of germinants and that MalS synthesis did not begin until more than 90 min after germinant addition. Thus, the increase in MalS-GFP fluorescence early in germination is not due to new protein synthesis but is perhaps due to a change in the physical environment of the spore cores. Our findings also show that different sporulation conditions and spore maturation times affect expression of MalS-GFP and the germination behavior of the spores, albeit to a minor extent, but still result in no changes in MalS-GFP levels early in spore germination.IMPORTANCE The spores formed by Bacillus subtilis remain in a quiescent state for extended periods due to their dormancy and resistance features. Dormancy is linked to a very low level of core water content and a phase-bright state of spores. The present report, focusing on proteins MalS and PdhD (pyruvate dehydrogenase subunit D) and complementary to our companion report published in this issue, aims to shed light on a major dilemma in the field, i.e., whether protein synthesis, in particular that of MalS, takes place in phase-bright spores. Clustered MalS-GFP in dormant spores diffuses throughout the spore as germination proceeds. However, fluorescence intensity measurements, supported by Western blot analysis and SILAC proteomics, confirm that there is no new MalS protein synthesis in bright-phase dormant spores.

Mitochondrial Proteome of Affected Glutamatergic Neurons in a Mouse Model of Leigh Syndrome.

Defects in mitochondrial function lead to severe neuromuscular orphan pathologies known as mitochondrial disease. Among them, Leigh Syndrome is the most common pediatric presentation, characterized by symmetrical brain lesions, hypotonia, motor and respiratory deficits, and premature death. Mitochondrial diseases are characterized by a marked anatomical and cellular specificity. However, the molecular determinants for this susceptibility are currently unknown, hindering the efforts to find an effective treatment. Due to the complex crosstalk between mitochondria and their supporting cell, strategies to assess the underlying alterations in affected cell types in the context of mitochondrial dysfunction are critical. Here, we developed a novel virus-based tool, the AAV-mitoTag viral vector, to isolate mitochondria from genetically defined cell types. Expression of the AAV-mitoTag in the glutamatergic vestibular neurons of a mouse model of Leigh Syndrome lacking the complex I subunit Ndufs4 allowed us to assess the proteome and acetylome of a subset of susceptible neurons in a well characterized model recapitulating the human disease. Our results show a marked reduction of complex I N-module subunit abundance and an increase in the levels of the assembly factor NDUFA2. Transiently associated non-mitochondrial proteins such as PKCδ, and the complement subcomponent C1Q were also increased in Ndufs4-deficient mitochondria. Furthermore, lack of Ndufs4 induced ATP synthase complex and pyruvate dehydrogenase (PDH) subunit hyperacetylation, leading to decreased PDH activity. We provide novel insight on the pathways involved in mitochondrial disease, which could underlie potential therapeutic approaches for these pathologies.

Metabolic mechanism of colistin resistance and its reverting in Vibrio alginolyticus.

Colistin is a last-line antibiotic against Gram-negative multidrug-resistant bacteria, but the increased resistance poses a huge challenge to this drug. However, the mechanisms underlying such resistance are largely unexplored. The present study first identified the mutations of two genes encoding AceF subunit of pyruvate dehydrogenase (PDH) and TetR family transcriptional regulator in colistin-resistant Vibrio alginolyticus (VA-RCT ) through genome sequencing. Then, gas chromatography-mass spectroscopy-based metabolomics was adopted to investigate metabolic responses since PDH plays a role in central carbon metabolism. Colistin resistance was associated with the reduction of the central carbon metabolism and energy metabolism, featuring the alteration of the pyruvate cycle, a recently characterized energy-producing cycle. Metabolites in the pyruvate cycle reprogramed colistin-resistant metabolome to colistin-sensitive metabolome, resulting in increased gene expression, enzyme activity or protein abundance of the cycle and sodium-translocating nicotinamide adenine dinucleotide-ubiquinone oxidoreductase. This reprogramming promoted the production of the proton motive force that enhances the binding between colistin and lipid A in lipopolysaccharide. Moreover, this metabolic approach was effective against VA-RCT in vitro and in vivo as well as other clinical isolates. These findings reveal a previously unknown mechanism of colistin resistance and develop a metabolome-reprogramming approach to promote colistin efficiency to combat with colistin-resistant bacteria.

Acquisition of exogenous fatty acids renders apicoplast-based biosynthesis dispensable in tachyzoites of Toxoplasma

Toxoplasma gondii is a common protozoan parasite that infects a wide range of hosts, including livestock and humans. Previous studies have suggested that the type 2 fatty acid synthesis (FAS2) pathway, located in the apicoplast (a nonphotosynthetic plastid relict), is crucial for the parasite's survival. Here we examined the physiological relevance of fatty acid synthesis in T. gondii by focusing on the pyruvate dehydrogenase complex and malonyl-CoA-[acyl carrier protein] transacylase (FabD), which are located in the apicoplast to drive de novo fatty acid biosynthesis. Our results disclosed unexpected metabolic resilience of T. gondii tachyzoites, revealing that they can tolerate CRISPR/Cas9-assisted genetic deletions of three pyruvate dehydrogenase subunits or FabD. All mutants were fully viable in prolonged cultures, albeit with impaired growth and concurrent loss of the apicoplast. Even more surprisingly, these mutants displayed normal virulence in mice, suggesting an expendable role of the FAS2 pathway in vivo Metabolic labeling of the Δpdh-e1α mutant showed reduced incorporation of glucose-derived carbon into fatty acids with medium chain lengths (C14:0 and C16:0), revealing that FAS2 activity was indeed compromised. Moreover, supplementation of exogenous C14:0 or C16:0 significantly reversed the growth defect in the Δpdh-e1α mutant, indicating salvage of these fatty acids. Together, these results demonstrate that the FAS2 pathway is dispensable during the lytic cycle of Toxoplasma because of its remarkable flexibility in acquiring fatty acids. Our findings question the long-held assumption that targeting this pathway has significant therapeutic potential for managing Toxoplasma infections.

Solanum lycopersicum (tomato) possesses mitochondrial and plastidial lipoyl synthases capable of increasing lipoylation levels when expressed in bacteria

Lipoic acid (LA) and its reduced form (dihydrolipoic acid, DHLA) have unique antioxidant properties among such molecules. Moreover, after a process termed lipoylation, LA is an essential prosthetic group covalently-attached to several key multi-subunit enzymatic complexes involved in primary metabolism, including E2 subunits of pyruvate dehydrogenase (PDH). The metabolic pathway of lipoylation has been extensively studied in Escherichia coli and Arabidopsis thaliana in which protein modification occurs via two routes: de novo synthesis and salvage. Common to both pathways, lipoyl synthase (LIP1 in plants, LipA in bacteria, EC 2.8.1.8) inserts sulphur atoms into the molecule in a final, activating step. However, despite the detection of LA and DHLA in other plant species, including tomato (Solanum lycopersicum), no plant LIP1s have been characterised to date from species other than Arabidopsis. In this work, we present the identification and characterisation of two LIPs from tomato, SlLIP1 and SlLIP1p. Consistent with in silico data, both are widely-expressed, particularly in reproductive organs. In line with bioinformatic predictions, we determine that yellow fluorescent protein tagged versions of SlLIP1 and SlLIP1p are mitochondrially- and plastidially-localised, respectively. Both possess the molecular hallmarks and domains of well-characterised bacterial LipAs. When heterologously-expressed in an E. coli lipA mutant, both are capable of complementing specific growth phenotypes and increasing lipoylation levels of E2 subunits of PDH in vivo, demonstrating that they do indeed function as lipoyl synthases.