Both Forward and Reverse TCA Cycles Operate in Green Sulfur Bacteria

The anoxygenic green sulfur bacteria (GSBs) assimilate CO₂ autotrophically through the reductive (reverse) tricarboxylic acid (RTCA) cycle. Some organic carbon sources, such as acetate and pyruvate, can be assimilated during the phototrophic growth of the GSBs, in the presence of CO₂ or HCO₃⁻. It has not been established why the inorganic carbonis required for incorporating organic carbon for growth and how the organic carbons are assimilated. In this report, we probed carbon flux during autotrophic and mixotrophic growth of the GSB Chlorobaculum tepidum. Our data indicate the following: (a) the RTCA cycle is active during autotrophic and mixotrophic growth; (b) the flux from pyruvate to acetyl-CoA is very low and acetyl-CoA is synthesized through the RTCA cycle and acetate assimilation; (c) pyruvate is largely assimilated through the RTCA cycle; and (d) acetate can be assimilated via both of the RTCA as well as the oxidative (forward) TCA (OTCA) cycle. The OTCA cycle revealed herein may explain better cell growth during mixotrophic growth with acetate, as energy is generated through the OTCA cycle. Furthermore, the genes specific for the OTCA cycle are either absent or down-regulated during phototrophic growth, implying that the OTCA cycle is not complete, and CO₂ is required for the RTCA cycle to produce metabolites in the TCA cycle. Moreover, CO₂ is essential for assimilating acetate and pyruvate through the CO₂-anaplerotic pathway and pyruvate synthesis from acetyl-CoA.     

Reduction of Proteinuria through Podocyte Alkalinization

Podocytes are highly differentiated cells and critical elements for the filtration barrier of the kidney. Loss of their foot process (FP) architecture (FP effacement) results in urinary protein loss. Here we show a novel role for the neutral amino acid glutamine in structural and functional regulation of the kidney filtration barrier. Metabolic flux analysis of cultured podocytes using genetic, toxic, and immunologic injury models identified increased glutamine utilization pathways. We show that glutamine uptake is increased in diseased podocytes to couple nutrient support to increased demand during the disease state of FP effacement. This feature can be utilized to transport increased amounts of glutamine into damaged podocytes. The availability of glutamine determines the regulation of podocyte intracellular pH (pH_i). Podocyte alkalinization reduces cytosolic cathepsin L protease activity and protects the podocyte cytoskeleton. Podocyte glutamine supplementation reduces proteinuria in LPS-treated mice, whereas acidification increases glomerular injury. In summary, our data provide a metabolic opportunity to combat urinary protein loss through modulation of podocyte amino acid utilization and pH_i.     

Functional Insights into Human HMG-CoA Lyase from Structures of Acyl-CoA-containing Ternary Complexes

HMG-CoA lyase (HMGCL) is crucial to ketogenesis, and inherited human mutations are potentially lethal. Detailed understanding of the HMGCL reaction mechanism and the molecular basis for correlating human mutations with enzyme deficiency have been limited by the lack of structural information for enzyme liganded to an acyl-CoA substrate or inhibitor. Crystal structures of ternary complexes of WT HMGCL with the competitive inhibitor 3-hydroxyglutaryl-CoA and of the catalytically deficient HMGCL R41M mutant with substrate HMG-CoA have been determined to 2.4 and 2.2 Å, respectively. Comparison of these β/α-barrel structures with those of unliganded HMGCL and R41M reveals substantial differences for Mg²⁺ coordination and positioning of the flexible loop containing the conserved HMGCL “signature” sequence. In the R41M-Mg²⁺-substrate ternary complex, loop residue Cys²⁶⁶ (implicated in active-site function by mechanistic and mutagenesis observations) is more closely juxtaposed to the catalytic site than in the case of unliganded enzyme or the WT enzyme-Mg²⁺-3-hydroxyglutaryl-CoA inhibitor complex. In both ternary complexes, the S-stereoisomer of substrate or inhibitor is specifically bound, in accord with the observed Mg²⁺ liganding of both C3 hydroxyl and C5 carboxyl oxygens. In addition to His²³³ and His²³⁵ imidazoles, other Mg²⁺ ligands are the Asp⁴² carboxyl oxygen and an ordered water molecule. This water, positioned between Asp⁴² and the C3 hydroxyl of bound substrate/inhibitor, may function as a proton shuttle. The observed interaction of Arg⁴¹ with the acyl-CoA C1 carbonyl oxygen explains the effects of Arg⁴¹ mutation on reaction product enolization and explains why human Arg⁴¹ mutations cause drastic enzyme deficiency.     

Identification and Characterization of an Extramitochondrial Human 3-Hydroxy-3-methylglutaryl-CoA Lyase

3-Hydroxy-3-methylglutaryl-CoA lyase-like protein (HMGCLL1) has been annotated in the Mammalian Genome Collection as a previously unidentified human HMG-CoA lyase (HMGCL). To test the validity of this annotation and evaluate the physiological role of the protein, plasmids were constructed for protein expression in Escherichia coli and Pichia pastoris. Protein expression in E. coli produced insoluble material. In contrast, active HMGCLL1 could be recovered upon expression in P. pastoris. Antibodies were prepared against a unique peptide sequence found in the N terminus of the protein. In immunodetection experiments, the antibodies discriminated between HMGCLL1 and mitochondrial HMGCL. Purified enzyme was characterized and demonstrated to cleave HMG-CoA to acetoacetate and acetyl-CoA with catalytic and affinity properties comparable with human mitochondrial HMGCL. The deduced HMGCLL1 sequence contains an N-terminal myristoylation motif; the putative modification site was eliminated by construction of a G2A HMGCLL1. Modification of both proteins was attempted using human N-myristoyltransferase and [³H]myristoyl-CoA. Wild-type protein was clearly modified, whereas G2A protein was not labeled. Myristoylation of HMGCLL1 affects its cellular localization. Upon transfection of appropriate expression plasmids into COS1 cells, immunofluorescence detection indicates that G2A HMGCLL1 exhibits a diffuse pattern, suggesting a cytosolic location. In contrast, wild-type HMGCLL1 exhibits a punctate as well as a perinuclear immunostaining pattern, indicating myristoylation dependent association with nonmitochondrial membrane compartments. In control experiments with the HMGCL expression plasmid, protein is localized in the mitochondria, as anticipated. The available results for COS1 cell expression, as well as endogenous expression in U87 cells, indicate that HMGCLL1 is an extramitochondrial hydroxymethylglutaryl-CoA lyase.     

Inhibition of Neuronal Cell Mitochondrial Complex I with Rotenone Increases Lipid β-Oxidation,Supporting Acetyl-Coenzyme A Levels

Rotenone is a naturally occurring mitochondrial complex I inhibitor with a known association with parkinsonian phenotypes in both human populations and rodent models. Despite these findings, a clear mechanistic link between rotenone exposure and neuronal damage remains to be determined. Here, we report alterations to lipid metabolism in SH-SY5Y neuroblastoma cells exposed to rotenone. The absolute levels of acetyl-CoA were found to be maintained despite a significant decrease in glucose-derived acetyl-CoA. Furthermore, palmitoyl-CoA levels were maintained, whereas the levels of many of the medium-chain acyl-CoA species were significantly reduced. Additionally, using isotopologue analysis, we found that β-oxidation of fatty acids with varying chain lengths helped maintain acetyl-CoA levels. Rotenone also induced increased glutamine utilization for lipogenesis, in part through reductive carboxylation, as has been found previously in other cell types. Finally, palmitoylcarnitine levels were increased in response to rotenone, indicating an increase in fatty acid import. Taken together, these findings show that alterations to lipid and glutamine metabolism play an important compensatory role in response to complex I inhibition by rotenone.     

Regulation of a Dynamic Interaction between Two Microtubule-binding Proteins,EB1 and TIP150, by the Mitotic p300/CBP-associated Factor (PCAF) Orchestrates Kinetochore Microtubule Plasticity and Chromosome Stability during Mitosis

The microtubule cytoskeleton network orchestrates cellular dynamics and chromosome stability in mitosis. Although tubulin acetylation is essential for cellular plasticity, it has remained elusive how kinetochore microtubule plus-end dynamics are regulated by p300/CBP-associated factor (PCAF) acetylation in mitosis. Here, we demonstrate that the plus-end tracking protein, TIP150, regulates dynamic kinetochore-microtubule attachments by promoting the stability of spindle microtubule plus-ends. Suppression of TIP150 by siRNA results in metaphase alignment delays and perturbations in chromosome biorientation. TIP150 is a tetramer that binds an end-binding protein (EB1) dimer through the C-terminal domains, and overexpression of the C-terminal TIP150 or disruption of the TIP150-EB1 interface by a membrane-permeable peptide perturbs chromosome segregation. Acetylation of EB1-PCAF regulates the TIP150 interaction, and persistent acetylation perturbs EB1-TIP150 interaction and accurate metaphase alignment, resulting in spindle checkpoint activation. Suppression of the mitotic checkpoint serine/threonine protein kinase, BubR1, overrides mitotic arrest induced by impaired EB1-TIP150 interaction, but cells exhibit whole chromosome aneuploidy. Thus, the results identify a mechanism by which the TIP150-EB1 interaction governs kinetochore microtubule plus-end plasticity and establish that the temporal control of the TIP150-EB1 interaction by PCAF acetylation ensures chromosome stability in mitosis.     

Insulin resistance and metabolic derangements in obese mice are ameliorated by a novel peroxisome proliferator-activated receptor γ-sparing thiazolidinedione

Currently approved thiazolidinediones (TZDs) are effective insulin-sensitizing drugs that may have efficacy for treatment of a variety of metabolic and inflammatory diseases, but their use is limited by side effects that are mediated through ectopic activation of the peroxisome proliferator-activated receptor γ (PPARγ). Emerging evidence suggests that the potent anti-diabetic efficacy of TZDs can be separated from the ability to serve as ligands for PPARγ. A novel TZD analog (MSDC-0602) with very low affinity for binding and activation of PPARγ was evaluated for its effects on insulin resistance in obese mice. MSDC-0602 treatment markedly improved several measures of multiorgan insulin sensitivity, adipose tissue inflammation, and hepatic metabolic derangements, including suppressing hepatic lipogenesis and gluconeogenesis. These beneficial effects were mediated at least in part via direct actions on hepatocytes and were preserved in hepatocytes from liver-specific PPARγ(-/-) mice, indicating that PPARγ was not required to suppress these pathways. In conclusion, the beneficial pharmacology exhibited by MSDC-0602 on insulin sensitivity suggests that PPARγ-sparing TZDs are effective for treatment of type 2 diabetes with reduced risk of PPARγ-mediated side effects.     

Widespread and Enzyme-independent N?-Acetylation and N?-Succinylation of Proteins in the Chemical Conditions of the Mitochondrial Matrix

Alterations in mitochondrial protein acetylation are implicated in the pathophysiology of diabetes, the metabolic syndrome, mitochondrial disorders, and cancer. However, a viable mechanism responsible for the widespread acetylation in mitochondria remains unknown. Here, we demonstrate that the physiologic pH and acyl-CoA concentrations of the mitochondrial matrix are sufficient to cause dose- and time-dependent, but enzyme-independent acetylation and succinylation of mitochondrial and nonmitochondrial proteins in vitro. These data suggest that protein acylation in mitochondria may be a chemical event facilitated by the alkaline pH and high concentrations of reactive acyl-CoAs present in the mitochondrial matrix. Although these results do not exclude the possibility of enzyme-mediated protein acylation in mitochondria, they demonstrate that such a mechanism may not be required in its unique chemical environment. These findings may have implications for the evolutionary roles that the mitochondria-localized SIRT3 deacetylase and SIRT5 desuccinylase have in the maintenance of metabolic health.     

Structural Basis for Regulation of the Human Acetyl-CoA Thioesterase 12 and Interactions with the Steroidogenic Acute Regulatory Protein-related Lipid Transfer (START) Domain

Acetyl-CoA plays a fundamental role in cell signaling and metabolic pathways, with its cellular levels tightly controlled through reciprocal regulation of enzymes that mediate its synthesis and catabolism. ACOT12, the primary acetyl-CoA thioesterase in the liver of human, mouse, and rat, is responsible for cleavage of the thioester bond within acetyl-CoA, producing acetate and coenzyme A for a range of cellular processes. The enzyme is regulated by ADP and ATP, which is believed to be mediated through the ligand-induced oligomerization of the thioesterase domains, whereby ATP induces active dimers and tetramers, whereas apo- and ADP-bound ACOT12 are monomeric and inactive. Here, using a range of structural and biophysical techniques, it is demonstrated that ACOT12 is a trimer rather than a tetramer and that neither ADP nor ATP exert their regulatory effects by altering the oligomeric status of the enzyme. Rather, the binding site and mechanism of ADP regulation have been determined to occur through two novel regulatory regions, one involving a large loop that links the thioesterase domains (Phe¹⁵⁴-Thr¹⁷⁸), defined here as RegLoop1, and a second region involving the C terminus of thioesterase domain 2 (Gln³⁰⁴-Gly³²⁶), designated RegLoop2. Mutagenesis confirmed that Arg³¹² and Arg³¹³ are crucial for this mode of regulation, and novel interactions with the START domain are presented together with insights into domain swapping within eukaryotic thioesterases for substrate recognition. In summary, these experiments provide the first structural insights into the regulation of this enzyme family, revealing an alternate hypothesis likely to be conserved throughout evolution.     

The Endoplasmic Reticulum-based Acetyltransferases,ATase1 and ATase2, Associate with the Oligosaccharyltransferase to Acetylate Correctly Folded Polypeptides

The endoplasmic reticulum (ER) has two membrane-bound acetyltransferases responsible for the endoluminal N^ϵ-lysine acetylation of ER-transiting and -resident proteins. Mutations that impair the ER-based acetylation machinery are associated with developmental defects and a familial form of spastic paraplegia. Deficient ER acetylation in the mouse leads to defects of the immune and nervous system. Here, we report that both ATase1 and ATase2 form homo- and heterodimers and associate with members of the oligosaccharyltransferase (OST) complex. In contrast to the OST, the ATases only modify correctly folded polypetides. Collectively, our studies suggest that one of the functions of the ATases is to work in concert with the OST and “select” correctly folded from unfolded/misfolded transiting polypeptides.     

Impact of Peripheral Ketolytic Deficiency on Hepatic Ketogenesis and Gluconeogenesis during the Transition to Birth

Preservation of bioenergetic homeostasis during the transition from the carbohydrate-laden fetal diet to the high fat, low carbohydrate neonatal diet requires inductions of hepatic fatty acid oxidation, gluconeogenesis, and ketogenesis. Mice with loss-of-function mutation in the extrahepatic mitochondrial enzyme CoA transferase (succinyl-CoA:3-oxoacid CoA transferase, SCOT, encoded by nuclear Oxct1) cannot terminally oxidize ketone bodies and develop lethal hyperketonemic hypoglycemia within 48 h of birth. Here we use this model to demonstrate that loss of ketone body oxidation, an exclusively extrahepatic process, disrupts hepatic intermediary metabolic homeostasis after high fat mother''s milk is ingested. Livers of SCOT-knock-out (SCOT-KO) neonates induce the expression of the genes encoding peroxisome proliferator-activated receptor γ co-activator-1a (PGC-1α), phosphoenolpyruvate carboxykinase (PEPCK), pyruvate carboxylase, and glucose-6-phosphatase, and the neonate''s pools of gluconeogenic alanine and lactate are each diminished by 50%. NMR-based quantitative fate mapping of ¹³C-labeled substrates revealed that livers of SCOT-KO newborn mice synthesize glucose from exogenously administered pyruvate. However, the contribution of exogenous pyruvate to the tricarboxylic acid cycle as acetyl-CoA is increased in SCOT-KO livers and is associated with diminished terminal oxidation of fatty acids. After mother''s milk provokes hyperketonemia, livers of SCOT-KO mice diminish de novo hepatic β-hydroxybutyrate synthesis by 90%. Disruption of β-hydroxybutyrate production increases hepatic NAD⁺/NADH ratios 3-fold, oxidizing redox potential in liver but not skeletal muscle. Together, these results indicate that peripheral ketone body oxidation prevents hypoglycemia and supports hepatic metabolic homeostasis, which is critical for the maintenance of glycemia during the adaptation to birth.     

The ethylmalonyl-CoA pathway is used in place of the glyoxylate cycle by Methylobacterium extorquens AM1 during growth on acetate

Acetyl-CoA assimilation was extensively studied in organisms harboring the glyoxylate cycle. In this study, we analyzed the metabolism of the facultative methylotroph Methylobacterium extorquens AM1, which lacks isocitrate lyase, the key enzyme in the glyoxylate cycle, during growth on acetate. MS/MS-based proteomic analysis revealed that the protein repertoire of M. extorquens AM1 grown on acetate is similar to that of cells grown on methanol and includes enzymes of the ethylmalonyl-CoA (EMC) pathway that were recently shown to operate during growth on methanol. Dynamic 13C labeling experiments indicate the presence of distinct entry points for acetate: the EMC pathway and the TCA cycle. 13C steady-state metabolic flux analysis showed that oxidation of acetyl-CoA occurs predominantly via the TCA cycle and that assimilation occurs via the EMC pathway. Furthermore, acetyl-CoA condenses with the EMC pathway product glyoxylate, resulting in malate formation. The latter, also formed by the TCA cycle, is converted to phosphoglycerate by a reaction sequence that is reversed with respect to the serine cycle. Thus, the results obtained in this study reveal the utilization of common pathways during the growth of M. extorquens AM1 on C1 and C2 compounds, but with a major redirection of flux within the central metabolism. Furthermore, our results indicate that the metabolic flux distribution is highly complex in this model methylotroph during growth on acetate and is fundamentally different from organisms using the glyoxylate cycle.     

LPS impairs phospholipid synthesis by triggering beta-transducin repeat-containing protein (beta-TrCP)-mediated polyubiquitination and degradation of the surfactant enzyme acyl-CoA:lysophosphatidylcholine acyltransferase I (LPCAT1)

Acyl-CoA:lysophosphatidylcholine acyltransferase 1 (LPCAT1) is a relatively newly described and yet indispensable enzyme needed for generation of the bioactive surfactant phospholipid, dipalmitoylphosphatidylcholine (DPPtdCho). Here, we show that lipopolysaccharide (LPS) causes LPCAT1 degradation using the Skp1-Cullin-F-box ubiquitin E3 ligase component, β-transducin repeat-containing protein (β-TrCP), that polyubiquitinates LPCAT1, thereby targeting the enzyme for proteasomal degradation. LPCAT1 was identified as a phosphoenzyme as Ser(178) within a phosphodegron was identified as a putative molecular recognition site for glycogen synthase kinase-3β (GSK-3β) phosphorylation that recruits β-TrCP docking within the enzyme. β-TrCP ubiquitinates LPCAT1 at an acceptor site (Lys(221)), as substitution of Lys(221) with Arg abrogated LPCAT1 polyubiquitination. LPS profoundly reduced immunoreactive LPCAT1 levels and impaired lung surfactant mechanics, effects that were overcome by siRNA to β-TrCP and GSK-3β or LPCAT1 gene transfer, respectively. Thus, LPS appears to destabilize the LPCAT1 protein by GSK-3β-mediated phosphorylation within a canonical phosphodegron for β-TrCP docking and site-specific ubiquitination. LPCAT1 is the first lipogenic substrate for β-TrCP, and the results suggest that modulation of the GSK-3β-SCFβ(TrCP) E3 ligase effector pathway might be a unique strategy to optimize dipalmitoylphosphatidylcholine levels in sepsis.     

Influence of Allosteric Regulators on Individual Steps in the Reaction Catalyzed by Mycobacterium tuberculosis 2-Hydroxy-3-oxoadipate Synthase

Allosteric regulation often controls key branch points in metabolic processes. Mycobacterium tuberculosis 2-hydroxy-3-oxoadipate synthase (HOAS), a thiamin diphosphate (ThDP)-dependent enzyme, produces 2-hydroxy-3-oxoadipate using 2-ketoglutarate and glyoxylate. The proposed chemical mechanism in analogy with other ThDP-dependent carboligases involves multiple ThDP-bound covalent intermediates. Acetyl coenzyme A is an activator, and GarA, a forkhead association domain-containing protein known to regulate glutamate metabolism, is an allosteric inhibitor of HOAS. Steady state kinetics using assays to study the first half and the full catalytic cycle suggested that the regulators act at different steps in the overall mechanism. To explore the modes of regulation and to test the effects on individual catalytic steps, we performed circular dichroism (CD) studies using a non-decarboxylatable 2-ketoglutarate analog and determined the distribution of ThDP-bound covalent intermediates during the steady state of the HOAS reaction using one-dimensional ¹H gradient carbon heteronuclear single quantum coherence NMR. The results suggest that acetyl coenzyme A acts as a mixed V and K type activator and predominantly affects the predecarboxylation steps. GarA does not inhibit the formation of the predecarboxylation analog and does not affect the accumulation of the postdecarboxylation covalent intermediate derived from 2-ketoglutarate; however, it decreases the abundance of the product ThDP adduct in the HOAS pathway. Thus, the two regulators act on different halves of the catalytic cycle in an unusual regulatory regime.     

The Structural Basis for Endotoxin-induced Allosteric Regulation of the Toll-like Receptor 4 (TLR4) Innate Immune Receptor

As part of the innate immune system, Toll-like receptor 4 (TLR4) recognizes bacterial cell surface lipopolysaccharide (LPS) by forming a complex with a lipid-binding co-receptor, MD-2. In the presence of agonist, TLR4·MD-2 dimerizes to form an active receptor complex, leading to initiation of intracellular inflammatory signals. TLR4 is of great biomedical interest, but its pharmacological manipulation is complicated because even subtle variations in the structure of LPS can profoundly impact the resultant immunological response. Here, we use atomically detailed molecular simulations to gain insights into the nature of the molecular signaling mechanism. We first demonstrate that MD-2 is extraordinarily flexible. The “clamshell-like” motions of its β-cup fold enable it to sensitively match the volume of its hydrophobic cavity to the size and shape of the bound lipid moiety. We show that MD-2 allosterically transmits this conformational plasticity, in a ligand-dependent manner, to a phenylalanine residue (Phe-126) at the cavity mouth previously implicated in TLR4 activation. Remarkably, within the receptor complex, we observe spontaneous transitions between active and inactive signaling states of Phe-126, and we confirm that Phe-126 is indeed the “molecular switch” in endotoxic signaling.     

Triggering Receptor Expressed on Myeloid Cells 1 (TREM-1)-mediated Bcl-2 Induction Prolongs Macrophage Survival

Triggering receptor expressed on myeloid cells 1 (TREM-1) is a superimmunoglobulin receptor expressed on myeloid cells that plays an important role in the amplification of inflammation. Recent studies suggest a role for TREM-1 in tumor-associated macrophages with relationship to tumor growth and progression. Whether the effects of TREM-1 on inflammation and tumor growth are mediated by an alteration in cell survival signaling is not known. In these studies, we show that TREM-1 knock-out macrophages exhibit an increase in apoptosis of cells in response to lipopolysaccharide (LPS) suggesting a role for TREM-1 in macrophage survival. Specific ligation of TREM-1 with monoclonal TREM-1 (mTREM-1) or overexpression of TREM-1 with adeno-TREM-1 induced B-cell lymphoma-2 (Bcl-2) with depletion of the key executioner caspase-3 prevents the cleavage of poly(ADP-ribose) polymerase. TREM-1 knock-out cells showed lack of induction of Bcl2 with an increase in caspase-3 activation in response to lipopolysaccharide. In addition overexpression of TREM-1 with adeno-TREM-1 led to an increase in mitofusins (MFN1 and MFN2) and knockdown of TREM-1 decreased the expression of mitofusins suggesting that TREM-1 contributes to the maintenance of mitochondrial integrity favoring cell survival. Investigations into potential mechanisms by which TREM-1 alters cell survival showed that TREM-1-induced Bcl-2 in an Egr2-dependent manner. Furthermore, our data shows that expression of Egr2 in response to specific ligation of TREM-1 is ERK mediated. These data for the first time provide novel mechanistic insights into the role of TREM-1 as an anti-apoptotic protein that prolongs macrophage survival.