The molecular determinants of classical pathway complement inhibition by OspEF-related proteins of Borrelia burgdorferi.

The complement system serves as the first line of defense against invading pathogens by promoting opsonophagocytosis and bacteriolysis. Antibody-dependent activation of complement occurs through the classical pathway and relies on the activity of initiating complement proteases of the C1 complex, C1r and C1s. The causative agent of Lyme disease, Borrelia burgdorferi, expresses two paralogous outer surface lipoproteins of the OspEF-related protein family, ElpB and ElpQ, that act as specific inhibitors of classical pathway activation. We have previously shown that ElpB and ElpQ bind directly to C1r and C1s with high affinity and specifically inhibit C2 and C4 cleavage by C1s. To further understand how these novel protease inhibitors function, we carried out a series of hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments using ElpQ and full-length activated C1s as a model of Elp-protease interaction. Comparison of HDX-MS profiles between unbound ElpQ and the ElpQ/C1s complex revealed a putative C1s-binding site on ElpQ. HDX-MS-guided, site-directed ElpQ mutants were generated and tested for direct binding to C1r and C1s using surface plasmon resonance. Several residues within the C-terminal region of ElpQ were identified as important for protease binding, including a single conserved tyrosine residue that was required for ElpQ- and ElpB-mediated complement inhibition. Collectively, our study identifies key molecular determinants for classical pathway protease recognition by Elp proteins. This investigation improves our understanding of the unique complement inhibitory mechanism employed by Elp proteins which serve as part of a sophisticated complement evasion system present in Lyme disease spirochetes.

What is allosteric regulation? Exploring the exceptions that prove the rule!

"Allosteric" was first introduced to mean the other site (i.e., a site distinct from the active or orthosteric site), an adjective for "regulation" to imply a regulatory outcome resulting from ligand binding at another site. That original idea outlines a system with two ligand-binding events at two distinct locations on a macromolecule (originally a protein system), which defines a four-state energy cycle. An allosteric energy cycle provides a quantifiable allosteric coupling constant and focuses our attention on the unique properties of the four equilibrated protein complexes that constitute the energy cycle. Because many observed phenomena have been referenced as "allosteric regulation" in the literature, the goal of this work is to use literature examples to explore which systems are and are not consistent with the two-ligand thermodynamic energy cycle-based definition of allosteric regulation. We emphasize the need for consistent language so comparisons can be made among the ever-increasing number of allosteric systems. Building on the mutually exclusive natures of an energy cycle definition of allosteric regulation versus classic two-state models, we conclude our discussion by outlining how the often-proposed Rube-Goldberg-like mechanisms are likely inconsistent with an energy cycle definition of allosteric regulation.

Oligomeric Changes Regulate Flavin Transfer in Two-Component FMN Reductases Involved in Sulfur Metabolism.

The FMN reductases (SsuE and MsuE of the alkanesulfonate monooxygenase systems) supply reduced flavin to their partner monooxygenases for the desulfonation of alkanesulfonates. Flavin reductases that comprise two-component systems must be able to regulate both flavin reduction and transfer. One mechanism to control these distinct processes is through changes in the oligomeric state of the enzymes. Despite their similar overall structures, SsuE and MsuE showed clear differences in their oligomeric states in the presence of substrates. The oligomeric state of SsuE was converted from a tetramer to a dimer/tetramer equilibrium in the presence of FMN or NADPH in analytical ultracentrifugation studies. Conversely, MsuE shifted from a dimer to a single tetrameric state with FMN, and the NADPH substrate did not induce a similar oligomeric shift. There was a fast tetramer to dimer equilibrium shift occurring at the dimer/dimer interface in H/D-X investigations with apo SsuE. Formation of the SsuE/FMN complex slowed the tetramer/dimer conversion, leading to a slower exchange along the dimer/dimer interface. The oligomeric shift of the MsuE/FMN complex from a dimer to a distinct tetramer showed a decrease in H/D-X in the region around the π-helices at the dimer/dimer interface. Both SsuE and MsuE showed a comparable and significant increase in the melting temperature with the addition of FMN, indicating the conformers formed by each FMN-bound enzyme had increased stability. A mechanism that supports the different structural shifts is rationalized by the different roles these enzymes play in providing reduced flavin to single or multiple monooxygenase enzymes.

RNA Methyltransferase METTL16's Protein Domains Have Differential Functional Effects on Cell Processes.

METTL16, a human m6A RNA methyltransferase, is currently known for its modification of U6 and MAT2A RNAs. Several studies have identified additional RNAs to which METTL16 binds, however whether METTL16 modifies these RNAs is still in question. Moreover, a recent study determined that METTL16 contains more than one RNA-binding domain, leaving the importance of each individual RNA-binding domain unknown. Here we examined the effects of mutating the METTL16 protein in certain domains on overall cell processes. We chose to mutate the N-terminal RNA-binding domain, the methyltransferase domain, and the C-terminal RNA-binding domain. With these mutants, we identified changes in RNA-binding ability, protein and RNA expression, cell cycle phase occupancy, and proliferation. From the resulting changes in RNA and protein expression, we saw effects on cell cycle, metabolism, intracellular transport, and RNA processing pathways, which varied between the METTL16 mutant lines. We also saw significant effects on the G1 and S phase occupancy times and proliferative ability with some but not all the mutants. We have therefore concluded that while METTL16 may or may not m6A-modify all RNAs it binds, its binding (or lack of) has a significant outcome on a variety of cell processes.

2-Aminoimidazole Analogs Target PhoP Altering DNA Binding Activity and Affect Outer Membrane Stability in Gram-Negative Bacteria.

Multidrug-resistant bacteria cause immense public health concerns as once effective antibiotics no longer work against even common infections. Concomitantly, there has been a decline in the discovery of new antibiotics, and the current global clinical pipeline is woefully inadequate, especially against resistant Gram-negative bacteria. One major contribution to Gram-negative resistance is the presence of a protective outer membrane. Consequently, an appealing option for tackling resistance is to adversely affect that outer membrane. With that in mind, we define the response regulator PhoP as a target for new 2-aminoimidazole compounds and show that they affect the integrity of the outer membrane in resistant strains of Escherichia coli and Klebsiella pneumoniae. We also provide empirical evidence for the 2-aminoimidazole mechanism of action.

Racial differences in the limb skeletal muscle transcriptional programs of patients with critical limb ischemia.

Critical limb ischemia (CLI) is the most severe manifestation of peripheral artery disease (PAD) and is characterized by high rates of morbidity and mortality. As with most severe cardiovascular disease manifestations, Black individuals disproportionately present with CLI. Accordingly, there remains a clear need to better understand the reasons for this discrepancy and to facilitate personalized therapeutic options specific for this population. Gastrocnemius muscle was obtained from White and Black healthy adult volunteers and patients with CLI for whole transcriptome shotgun sequencing (WTSS) and enrichment analysis was performed to identify alterations in specific Reactome pathways. When compared to their race-matched healthy controls, both White and Black patients with CLI demonstrated similar reductions in nuclear and mitochondrial encoded genes and mitochondrial oxygen consumption across multiple substrates, indicating a common bioenergetic paradigm associated with amputation outcomes regardless of race. Direct comparisons between tissues of White and Black patients with CLI revealed hemostasis, extracellular matrix organization, platelet regulation, and vascular wall interactions to be uniquely altered in limb muscles of Black individuals. Among traditional vascular growth factor signaling targets, WTSS revealed only Tie1 to be significantly altered from White levels in Black limb muscle tissues. Quantitative reverse transcription polymerase chain reaction validation of select identified targets verified WTSS directional changes and supports reductions in MMP9 and increases in NUDT4P1 and GRIK2 as unique to limb muscles of Black patients with CLI. This represents a critical first step in better understanding the transcriptional program similarities and differences between Black and White patients in the setting of amputations related to CLI and provides a promising start for therapeutic development in this population.

Impact of 17ß-estradiol on complex I kinetics and H2O2 production in liver and skeletal muscle mitochondria.

Naturally or surgically induced postmenopausal women are widely prescribed estrogen therapies to alleviate symptoms associated with estrogen loss and to lower the subsequent risk of developing metabolic diseases, including diabetes and nonalcoholic fatty liver disease. However, the molecular mechanisms by which estrogens modulate metabolism across tissues remain ill-defined. We have previously reported that 17ß-estradiol (E2) exerts antidiabetogenic effects in ovariectomized (OVX) mice by protecting mitochondrial and cellular redox function in skeletal muscle. The liver is another key tissue for glucose homeostasis and a target of E2 therapy. Thus, in the present study we determined the effects of acute loss of ovarian E2 and E2 administration on liver mitochondria. In contrast to skeletal muscle mitochondria, E2 depletion via OVX did not alter liver mitochondrial respiratory function or complex I (CI) specific activities (NADH oxidation, quinone reduction, and H2O2 production). Surprisingly, in vivo E2 replacement therapy and in vitro E2 exposure induced tissue-specific effects on both CI activity and on the rate and topology of CI H2O2 production. Overall, E2 therapy protected and restored the OVX-induced reduction in CI activity in skeletal muscle, whereas in liver mitochondria E2 increased CI H2O2 production and decreased ADP-stimulated respiratory capacity. These results offer novel insights into the tissue-specific effects of E2 on mitochondrial function.

Proteolipid domains form in biomimetic and cardiac mitochondrial vesicles and are regulated by cardiolipin concentration but not monolyso-cardiolipin.

Cardiolipin (CL) is an anionic phospholipid mainly located in the inner mitochondrial membrane, where it helps regulate bioenergetics, membrane structure, and apoptosis. Localized, phase-segregated domains of CL are hypothesized to control mitochondrial inner membrane organization. However, the existence and underlying mechanisms regulating these mitochondrial domains are unclear. Here, we first isolated detergent-resistant cardiac mitochondrial membranes that have been reported to be CL-enriched domains. Experiments with different detergents yielded only nonspecific solubilization of mitochondrial phospholipids, suggesting that CL domains are not recoverable with detergents. Next, domain formation was investigated in biomimetic giant unilamellar vesicles (GUVs) and newly synthesized giant mitochondrial vesicles (GMVs) from mouse hearts. Confocal fluorescent imaging revealed that introduction of cytochrome c into membranes promotes macroscopic proteolipid domain formation associated with membrane morphological changes in both GUVs and GMVs. Domain organization was also investigated after lowering tetralinoleoyl-CL concentration and substitution with monolyso-CL, two common modifications observed in cardiac pathologies. Loss of tetralinoleoyl-CL decreased proteolipid domain formation in GUVs, because of a favorable Gibbs-free energy of lipid mixing, whereas addition of monolyso-CL had no effect on lipid mixing. Moreover, murine GMVs generated from cardiac acyl-CoA synthetase-1 knockouts, which have remodeled CL acyl chains, did not perturb proteolipid domains. Finally, lowering the tetralinoleoyl-CL content had a stronger influence on the oxidation status of cytochrome c than did incorporation of monolyso-CL. These results indicate that proteolipid domain formation in the cardiac mitochondrial inner membrane depends on tetralinoleoyl-CL concentration, driven by underlying lipid-mixing properties, but not the presence of monolyso-CL.

Docosahexaenoic acid lowers cardiac mitochondrial enzyme activity by replacing linoleic acid in the phospholipidome.

Cardiac mitochondrial phospholipid acyl chains regulate respiratory enzymatic activity. In several diseases, the rodent cardiac phospholipidome is extensively rearranged; however, whether specific acyl chains impair respiratory enzyme function is unknown. One unique remodeling event in the myocardium of obese and diabetic rodents is an increase in docosahexaenoic acid (DHA) levels. Here, we first confirmed that cardiac DHA levels are elevated in diabetic humans relative to controls. We then used dietary supplementation of a Western diet with DHA as a tool to promote cardiac acyl chain remodeling and to study its influence on respiratory enzyme function. DHA extensively remodeled the acyl chains of cardiolipin (CL), mono-lyso CL, phosphatidylcholine, and phosphatidylethanolamine. Moreover, DHA lowered enzyme activities of respiratory complexes I, IV, V, and I+III. Mechanistically, the reduction in enzymatic activities were not driven by a dramatic reduction in the abundance of supercomplexes. Instead, replacement of tetralinoleoyl-CL with tetradocosahexaenoyl-CL in biomimetic membranes prevented formation of phospholipid domains that regulate enzyme activity. Tetradocosahexaenoyl-CL inhibited domain organization due to favorable Gibbs free energy of phospholipid mixing. Furthermore, in vitro substitution of tetralinoleoyl-CL with tetradocosahexaenoyl-CL blocked complex-IV binding. Finally, reintroduction of linoleic acid, via fusion of phospholipid vesicles to mitochondria isolated from DHA-fed mice, rescued the major losses in the mitochondrial phospholipidome and complexes I, IV, and V activities. Altogether, our results show that replacing linoleic acid with DHA lowers select cardiac enzyme activities by potentially targeting domain organization and phospholipid-protein binding, which has implications for the ongoing debate about polyunsaturated fatty acids and cardiac health.

The Manganese-Dependent Pyruvate Kinase PykM Is Required for Wild-Type Glucose Utilization by Brucella abortus 2308 and Its Virulence in C57BL/6 Mice.

Pyruvate kinase plays a central role in glucose catabolism in bacteria, and efficient utilization of this hexose has been linked to the virulence of Brucella strains in mice. The brucellae produce a single pyruvate kinase which is an ortholog of the Bradyrhizobium manganese (Mn)-dependent pyruvate kinase PykM. A biochemical analysis of the Brucella pyruvate kinase and phenotypic analysis of a Brucella abortus mutant defective in high-affinity Mn import indicate that this enzyme is an authentic PykM ortholog which functions as a Mn-dependent enzyme in vivo The loss of PykM has a negative impact on the capacity of the parental 2308 strain to utilize glucose, fructose, and galactose but not on its ability to utilize ribose, xylose, arabinose, or erythritol, and a pykM mutant displays significant attenuation in C57BL/6 mice. Although the enzyme pyruvate phosphate dikinase (PpdK) can substitute for the loss of pyruvate kinase in some bacteria and is also an important virulence determinant in Brucella, a phenotypic analysis of B. abortus 2308 and isogenic pykM, ppdK, and pykM ppdK mutants indicates that PykM and PpdK make distinctly different contributions to carbon metabolism and virulence in these bacteria.IMPORTANCE Mn plays a critical role in the physiology and virulence of Brucella strains, and the results presented here suggest that one of the important roles that the high-affinity Mn importer MntH plays in the pathogenesis of these strains is supporting the function of the Mn-dependent kinase PykM. A better understanding of how the brucellae adapt their physiology and metabolism to sustain their intracellular persistence in host macrophages will provide knowledge that can be used to design improved strategies for preventing and treating brucellosis, a disease that has a significant impact on both the veterinary and public health communities worldwide.

15(V/K) kinetic isotope effect and steady-state kinetic analysis for the transglutaminase 2 catalyzed deamidation and transamidation reactions.

The Ca2+-dependent deamidation and transamidation activities of transglutaminase 2 (TG2) are important to numerous physiological and pathological processes. Herein, we have examined the steady-state kinetics and 15(V/K) kinetic isotope effects (KIEs) for the TG2-catalyzed deamidation and transamidation of N-Benzyloxycarbonyl-l-Glutaminylglycine (Z-Gln-Gly) using putrescine as the acyl acceptor substrate. Kinetic parameters determined from initial velocity plots are consistent with previously proposed mechanisms. Significant differences in the 15(V/K) KIEs on NH3 release determined for the deamidation (0.2%) and the transamidation (2.3%) of Z-Gln-Gly suggest the rate-limiting steps of TG2 active site acylation are dependent on the presence of the acyl acceptor. We propose a plausible mechanistic explanation where substrate-induced conformational changes may play a role in promoting catalysis.

Multiple non-catalytic ADAMs are novel integrin α4 ligands.

The ADAM (a disintegrin and metalloprotease) protein family uniquely exhibits both catalytic and adhesive properties. In the well-defined process of ectodomain shedding, ADAMs transform latent, cell-bound substrates into soluble, biologically active derivatives to regulate a spectrum of normal and pathological processes. In contrast, the integrin ligand properties of ADAMs are not fully understood. Emerging models posit that ADAM-integrin interactions regulate shedding activity by localizing or sequestering the ADAM sheddase. Interestingly, 8 of the 21 human ADAMs are predicted to be catalytically inactive. Unlike their catalytically active counterparts, integrin recognition of these "dead" enzymes has not been largely reported. The present study delineates the integrin ligand properties of a group of non-catalytic ADAMs. Here we report that human ADAM11, ADAM23, and ADAM29 selectively support integrin α4-dependent cell adhesion. This is the first demonstration that the disintegrin-like domains of multiple catalytically inactive ADAMs are ligands for a select subset of integrin receptors that also recognize catalytically active ADAMs.

Kinetic and Thermodynamic Analysis of Acetyl-CoA Activation of Staphylococcus aureus Pyruvate Carboxylase.

Allosteric regulation of pyruvate carboxylase (PC) activity is pivotal to maintaining metabolic homeostasis. In contrast, dysregulated PC activity contributes to the pathogenesis of numerous diseases, rendering PC a possible target for allosteric therapeutic development. Recent research efforts have focused on demarcating the role of acetyl-CoA, one of the most potent activators of PC, in coordinating catalytic events within the multifunctional enzyme. Herein, we report a kinetic and thermodynamic analysis of acetyl-CoA activation of the Staphylococcus aureus PC (SaPC)-catalyzed carboxylation of pyruvate to identify novel means by which acetyl-CoA synchronizes catalytic events within the PC tetramer. Kinetic and linked-function analysis, or thermodynamic linkage analysis, indicates that the substrates of the biotin carboxylase and carboxyl transferase domain are energetically coupled in the presence of acetyl-CoA. In contrast, both kinetic and energetic coupling between the two domains is lost in the absence of acetyl-CoA, suggesting a functional role for acetyl-CoA in facilitating the long-range transmission of substrate-induced conformational changes within the PC tetramer. Interestingly, thermodynamic activation parameters for the SaPC-catalyzed carboxylation of pyruvate are largely independent of acetyl-CoA. Our results also reveal the possibility that global conformational changes give rise to observed species-specific thermodynamic activation parameters. Taken together, our kinetic and thermodynamic results provide a possible allosteric mechanism by which acetyl-CoA coordinates catalysis within the PC tetramer.

Pyruvate Occupancy in the Carboxyl Transferase Domain of Pyruvate Carboxylase Facilitates Product Release from the Biotin Carboxylase Domain through an Intermolecular Mechanism.

Protein structure, ligand binding, and catalytic turnover contributes to the governance of catalytic events occurring at spatially distinct domains in multifunctional enzymes. Coordination of these catalytic events partially rests on the ability of spatially discrete active sites to communicate with other allosteric and active sites on the same polypeptide chain (intramolecular) or on different polypeptide chains (intermolecular) within the holoenzyme. Often, communication results in long-range effects on substrate binding or product release. For example, pyruvate binding to the carboxyl transferase (CT) domain of pyruvate carboxylase (PC) increases the rate of product release in the biotin carboxylase (BC) domain. In order to address how CT domain ligand occupancy is "sensed" by other domains, we generated functional, mixed hybrid tetramers using the E218A (inactive BC domain) and T882S (low pyruvate binding, low activity) mutant forms of PC. The apparent Ka pyruvate for the pyruvate-stimulated release of Pi catalyzed by the T882S:E218A[1:1] hybrid tetramer was comparable to the wild-type enzyme and nearly 10-fold lower than that for the T882S homotetramer. In addition, the ratio of the rates of oxaloacetate formation to Pi release for the WT:T882S[1:1] and E218A:T882S[1:1] hybrid tetramer-catalyzed reactions was 0.5 and 0.6, respectively, while the T882S homotetramer exhibited a near 1:1 coupling of the two domains, suggesting that the mechanisms coordinating catalytic events is more complicated that we initially assumed. The results presented here are consistent with an intermolecular communication mechanism, where pyruvate binding to the CT domain is "sensed" by domains on a different polypeptide chain within the tetramer.

Ceramide-tamoxifen regimen targets bioenergetic elements in acute myelogenous leukemia.

The objective of our study was to determine the mechanism of action of the short-chain ceramide analog, C6-ceramide, and the breast cancer drug, tamoxifen, which we show coactively depress viability and induce apoptosis in human acute myelogenous leukemia cells. Exposure to the C6-ceramide-tamoxifen combination elicited decreases in mitochondrial membrane potential and complex I respiration, increases in reactive oxygen species (ROS), and release of mitochondrial proapoptotic proteins. Decreases in ATP levels, reduced glycolytic capacity, and reduced expression of inhibitors of apoptosis proteins also resulted. Cytotoxicity of the drug combination was mitigated by exposure to antioxidant. Cells metabolized C6-ceramide by glycosylation and hydrolysis, the latter leading to increases in long-chain ceramides. Tamoxifen potently blocked glycosylation of C6-ceramide and long-chain ceramides. N-desmethyltamoxifen, a poor antiestrogen and the major tamoxifen metabolite in humans, was also effective with C6-ceramide, indicating that traditional antiestrogen pathways are not involved in cellular responses. We conclude that cell death is driven by mitochondrial targeting and ROS generation and that tamoxifen enhances the ceramide effect by blocking its metabolism. As depletion of ATP and targeting the "Warburg effect" represent dynamic metabolic insult, this ceramide-containing combination may be of utility in the treatment of leukemia and other cancers.

Increasing mitochondrial membrane phospholipid content lowers the enzymatic activity of electron transport complexes.

Activities of the enzymes involved in cellular respiration are markedly influenced by the composition of the phospholipid environment of the inner mitochondrial membrane. Contrary to previous suppositions, we show that fusion of mitochondria isolated from healthy cardiac muscle with cardiolipin or dioleoylphosphatidylcholine results in a 2-6-fold reduction in the activity of complexes I, II, and IV. The activity of complex III was unaffected by increased phospholipid levels. Phospholipid content had an indiscriminate yet detrimental effect on the combined activities of complexes I+III and II+III. These results have strong implications for therapeutic lipid replacement strategies, in which phospholipid modification of the mitochondria is proposed to enhance mitochondrial function.

Increasing levels of cardiolipin differentially influence packing of phospholipids found in the mitochondrial inner membrane.

It is essential to understand the role of cardiolipin (CL) in mitochondrial membrane organization given that changes in CL levels contribute to mitochondrial dysfunction in type II diabetes, ischemia-reperfusion injury, heart failure, breast cancer, and aging. Specifically, there are contradictory data on how CL influences the molecular packing of membrane phospholipids. Therefore, we determined how increasing levels of heart CL impacted molecular packing in large unilamellar vesicles, modeling heterogeneous lipid mixtures found within the mitochondrial inner membrane, using merocyanine (MC540) fluorescence. We broadly categorized lipid vesicles of equal mass as loosely packed, intermediate, and highly packed based on peak MC540 fluorescence intensity. CL had opposite effects on loosely versus highly packed vesicles. Exposure of loosely packed vesicles to increasing levels of CL dose-dependently increased membrane packing. In contrast, increasing amounts of CL in highly packed vesicles decreased the packing in a dose-dependent manner. In vesicles that were categorized as intermediate packing, CL had either no effect or decreased packing at select doses in a dose-independent manner. Altogether, the results aid in resolving some of the discrepant data by demonstrating that CL displays differential effects on membrane packing depending on the composition of the lipid environment. This has implications for mitochondrial protein activity in response to changing CL levels in microdomains of varying composition.

Mitochondria inside acute myeloid leukemia cells hydrolyze ATP to resist chemotherapy.

Despite early optimism, therapeutics targeting oxidative phosphorylation (OxPhos) have faced clinical setbacks, stemming from their inability to distinguish healthy from cancerous mitochondria. Herein, we describe an actionable bioenergetic mechanism unique to cancerous mitochondria inside acute myeloid leukemia (AML) cells. Unlike healthy cells which couple respiration to the synthesis of ATP, AML mitochondria were discovered to support inner membrane polarization by consuming ATP. Because matrix ATP consumption allows cells to survive bioenergetic stress, we hypothesized that AML cells may resist cell death induced by OxPhos damaging chemotherapy by reversing the ATP synthase reaction. In support of this, targeted inhibition of BCL-2 with venetoclax abolished OxPhos flux without impacting mitochondrial membrane potential. In surviving AML cells, sustained polarization of the mitochondrial inner membrane was dependent on matrix ATP consumption. Mitochondrial ATP consumption was further enhanced in AML cells made refractory to venetoclax, consequential to downregulations in both the proton-pumping respiratory complexes, as well as the endogenous F1-ATPase inhibitor ATP5IF1. In treatment-naive AML, ATP5IF1 knockdown was sufficient to drive venetoclax resistance, while ATP5IF1 overexpression impaired F1-ATPase activity and heightened sensitivity to venetoclax. Collectively, our data identify matrix ATP consumption as a cancer-cell intrinsic bioenergetic vulnerability actionable in the context of mitochondrial damaging chemotherapy.

Oxamate is an alternative substrate for pyruvate carboxylase from Rhizobium etli.

Oxamate, an isosteric and isoelectronic inhibitory analogue of pyruvate, enhances the rate of enzymatic decarboxylation of oxaloacetate in the carboxyl transferase domain of pyruvate carboxylase (PC). It is unclear, though, how oxamate exerts a stimulatory effect on the enzymatic reaction. Herein, we report direct (13)C nuclear magnetic resonance (NMR) evidence that oxamate acts as a carboxyl acceptor, forming a carbamylated oxamate product and thereby accelerating the enzymatic decarboxylation reaction. (13)C NMR was used to monitor the PC-catalyzed formation of [4-(13)C]oxaloacetate and subsequent transfer of (13)CO(2) from oxaloacetate to oxamate. In the presence of oxamate, the apparent K(m) for oxaloacetate is artificially suppressed (from 15 to 4-5 µM). Interestingly, the steady-state kinetic analysis of the initial rates determined at varying concentrations of oxaloacetate and fixed concentrations of oxamate revealed initial velocity patterns inconsistent with a simple ping-pong-type mechanism. Rather, the patterns suggest the existence of an alternate decarboxylation pathway in which an unstable intermediate is formed. The steady-state kinetic analysis coupled with the normal (13)(V/K) kinetic isotope effect observed on C-4 of oxaloacetate [(13)(V/K) = 1.0117 ± 0.0005] indicates that the transfer of CO(2) from carboxybiotin to oxamate is the partially rate-limiting step of the enzymatic reaction. The catalytic mechanism proposed for the carboxylation of oxamate is similar to that proposed for the carboxylation of pyruvate, which occurs via the formation of an enol intermediate.

Pan-tissue mitochondrial phenotyping reveals lower OXPHOS expression and function across cancer types.

Targeting mitochondrial oxidative phosphorylation (OXPHOS) to treat cancer has been hampered due to serious side-effects potentially arising from the inability to discriminate between non-cancerous and cancerous mitochondria. Herein, comprehensive mitochondrial phenotyping was leveraged to define both the composition and function of OXPHOS across various murine cancers and compared to both matched normal tissues and other organs. When compared to both matched normal tissues, as well as high OXPHOS reliant organs like heart, intrinsic expression of the OXPHOS complexes, as well as OXPHOS flux were discovered to be consistently lower across distinct cancer types. Assuming intrinsic OXPHOS expression/function predicts OXPHOS reliance in vivo, these data suggest that pharmacologic blockade of mitochondrial OXPHOS likely compromises bioenergetic homeostasis in healthy oxidative organs prior to impacting tumor mitochondrial flux in a clinically meaningful way. Although these data caution against the use of indiscriminate mitochondrial inhibitors for cancer treatment, considerable heterogeneity was observed across cancer types with respect to both mitochondrial proteome composition and substrate-specific flux, highlighting the possibility for targeting discrete mitochondrial proteins or pathways unique to a given cancer type.