Iordan Iordanov

Abolition of mitochondrial substrate-level phosphorylation by itaconic acid produced by LPS-induced Irg1 expression in cells of murine macrophage lineage

Itaconate is a nonamino organic acid exhibiting antimicrobial effects. It has been recently identified in cells of macrophage lineage as a product of an enzyme encoded by immunoresponsive gene 1 (Irg1), acting on the citric acid cycle intermediate cis‐aconitate. In mitochondria, itaconate can be converted by succinate‐coenzyme A (CoA) ligase to itaconyl‐CoA at the expense of ATP (or GTP), and is also a weak competitive inhibitor of complex II. Here, we investigated specific bioenergetic effects of increased itaconate production mediated by LPS‐induced stimulation of Irg1 in murine bone marrow‐derived macrophages (BMDM) and RAW‐264.7 cells. In rotenone‐treated macrophage cells, stimulation by LPS led to impairment in substrate‐level phosphorylation (SLP) of in situ mitochondria, deduced by a reversal in the directionality of the adenine nucleotide translocase operation. In RAW‐264.7 cells, the LPS‐induced impairment in SLP was reversed by short‐interfering RNA(siRNA)—but not scrambled siRNA—treatment directed against Irg1. LPS dose‐dependently inhibited oxygen consumption rates (61‐91%) and elevated glycolysis rates (>21%) in BMDM but not RAW‐264.7 cells, studied under various metabolic conditions. In isolated mouse liver mitochondria treated with rotenone, itaconate dose‐dependently (0.5‐2 mM) reversed the operation of adenine nucleotide translocase, implying impairment in SLP, an effect that was partially mimicked by malonate. However, malonate yielded greater ADP‐induced depolarizations (3‐19%) than itaconate. We postulate that itaconate abolishes SLP due to 1) a “CoA trap” in the form of itaconyl‐CoA that negatively affects the upstream supply of succinyl‐CoA from the α‐ketoglutarate dehydrogenase complex; 2) depletion of ATP (or GTP), which are required for the thioesterification by succinate‐CoA ligase; and 3) inhibition of complex II leading to a buildup of succinate which shifts succinate‐CoA ligase equilibrium toward ATP (or GTP) utilization. Our results support the notion that Irg1‐expressing cells of macrophage lineage lose the capacity of mitochondrial SLP for producing itaconate during mounting of an immune defense.—Németh, B., Doczi, J., Csete, D., Kacso, G., Ravasz, D., Adams, D., Kiss, G., Nagy, A. M., Horvath, G., Tretter, L., Mócsai, A., Csépányi‐Kömi, R., Iordanov, I., Adam‐Vizi, V., Chinopoulos, C. Abolition of mitochondrial substrate‐level phosphorylation by itaconic acid produced by LPS‐induced Irg1 expression in cells of murine macrophage lineage. FASEB J. 30, 286‐300 (2016). www.fasebj.org

Ruling out pyridine dinucleotides as true TRPM2 channel activators reveals novel direct agonist ADP-ribose-2′-phosphate

ADP-ribose-2′-phosphate acts as a direct agonist of TRPM2, whereas NAD, NAAD, and NAADP do not.

The Transmembrane Protein KpOmpA Anchoring the Outer Membrane of Klebsiella pneumoniae Unfolds and Refolds in Response to Tensile Load

In Klebsiella pneumoniae the transmembrane β-barrel forming outer membrane protein KpOmpA mediates adhesion to a wide range of immune effector cells, thereby promoting respiratory tract and urinary infections. As major transmembrane protein OmpA stabilizes Gram-negative bacteria by anchoring their outer membrane to the peptidoglycan layer. Adhesion, osmotic pressure, hydrodynamic flow, and structural deformation apply mechanical stress to the bacterium. This stress can generate tensile load to the peptidoglycan-binding domain (PGBD) of KpOmpA. To investigate how KpOmpA reacts to mechanical stress, we applied a tensile load to the PGBD and observed a detailed unfolding pathway of the transmembrane β-barrel. Each step of the unfolding pathway extended the polypeptide connecting the bacterial outer membrane to the peptidoglycan layer and absorbed mechanical energy. After relieving the tensile load, KpOmpA reversibly refolded back into the membrane. These results suggest that bacteria may reversibly unfold transmembrane proteins in response to mechanical stress.

Two Classes of Cholesterol Binding Sites for the β2AR Revealed by Thermostability and NMR

Cholesterol binding to G protein-coupled receptors (GPCRs) and modulation of their activities in membranes is a fundamental issue for understanding their function. Despite the identification of cholesterol binding sites in high-resolution x-ray structures of the ?2 adrenergic receptor (β2AR) and other GPCRs, the binding affinity of cholesterol for this receptor and exchange rates between the free and bound cholesterol remain unknown. In this study we report the existence of two classes of cholesterol binding sites in β2AR. By analyzing the β2AR unfolding temperature in lipidic cubic phase (LCP) as a function of cholesterol concentration we observed high-affinity cooperative binding of cholesterol with sub-nM affinity constant. In contrast, saturation transfer difference (STD) NMR experiments revealed the existence of a second class of cholesterol binding sites, in fast exchange on the STD NMR timescale. Titration of the STD signal as a function of cholesterol concentration provided a lower limit of 100 mM for their dissociation constant. However, these binding sites are specific for both cholesterol and β2AR, as shown with control experiments using ergosterol and a control membrane protein (KpOmpA). We postulate that this specificity is mediated by the high-affinity bound cholesterol molecules and propose the formation of transient cholesterol clusters around the high-affinity binding sites.

Local and Global Dynamics in Klebsiella pneumoniae Outer Membrane Protein a in Lipid Bilayers Probed at Atomic Resolution

The role of membrane proteins in cellular mechanism strongly depends on their dynamics, and solid-state magic-angle spinning (MAS) nuclear magnetic resonance (NMR) is a unique method to exhaustively characterize motions of proteins in a lipid environment. Herein, we make use of advances in 1H-detected MAS NMR to describe the dynamics of the membrane domain of the Outer membrane protein A of Klebsiella pneumoniae (KpOmpA). By measuring 1H-15N dipolar-coupling as well as 15N R1 and R1ρ relaxation rates at fast (60 kHz) MAS and high magnetic field (1 GHz), we were able to describe the motions of the residues of the β-barrel as a collective rocking of low amplitude and of hundreds of nanoseconds time scale. Residual local motions at the edges of the strands, underscored by enhanced 15N R1ρ relaxation rates, report on the mobility of the connected loops. In agreement with MAS NMR data, proteolysis experiments performed on the full length KpOmpA as well as on its membrane domain, reconstituted in liposomes or in detergent micelles, revealed in all cases the existence of a unique trypsin cleavage site within the membrane domain (out of 16 potential Lys and Arg sites). This site is located in the extracellular loop L3, showing that it is highly accessible to protein-protein interactions. KpOmpA is involved in cell-cell recognition, for adhesion and immune response mechanisms. The L3 region may therefore play a key role in pathogenicity.

Putative chanzyme activity of TRPM2 cation channel is unrelated to pore gating

Significance Ion channels are protein pores that allow passive transmembrane ion flow. These pores are opened and closed (gated) by various cellular signals. Typically, the mechanism of gating conformational changes is an equilibrium process, but for some channels, gating is an irreversible cycle, for example, linked to an enzymatic activity. For equilibrium mechanisms, channel activity is readily modulated by energetic stabilization of closed or open states, whereas for cyclic gating, alteration of transition-state stabilities most effectively modulates activity. Transient receptor potential melastatin 2 (TRPM2), a cation channel involved in multiple physiologic and pathophysiologic processes, possesses enzymatic activity, which cleaves its activating ligand. This work addresses, and rules out, a suggested link between that catalysis and pore gating in TRPM2, classifying it among the channels that gate at equilibrium. Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable cation channel expressed in immune cells of phagocytic lineage, pancreatic β cells, and brain neurons and is activated under oxidative stress. TRPM2 activity is required for immune cell activation and insulin secretion and is responsible for postischemic neuronal cell death. TRPM2 is opened by binding of ADP ribose (ADPR) to its C-terminal cytosolic nudix-type motif 9 (NUDT9)-homology (NUDT9-H) domain, which, when expressed in isolation, cleaves ADPR into AMP and ribose-5-phosphate. A suggested coupling of this enzymatic activity to channel gating implied a potentially irreversible gating cycle, which is a unique feature of a small group of channel enzymes known to date. The significance of such a coupling lies in the conceptually distinct pharmacologic strategies for modulating the open probability of channels obeying equilibrium versus nonequilibrium gating mechanisms. Here we examine the potential coupling of TRPM2 enzymatic activity to pore gating. Mutation of several residues proposed to enhance or eliminate NUDT9-H catalytic activity all failed to affect channel gating kinetics. An ADPR analog, α-β-methylene-ADPR (AMPCPR), was shown to be entirely resistant to hydrolysis by NUDT9, but nevertheless supported TRPM2 channel gating, albeit with reduced apparent affinity. The rate of channel deactivation was not slowed but, rather, accelerated in AMPCPR. These findings, as well as detailed analyses of steady-state gating kinetics of single channels recorded in the presence of a range of concentrations of ADPR or AMPCPR, identify TRPM2 as a simple ligand-gated channel that obeys an equilibrium gating mechanism uncoupled from its enzymatic activity.

Electrodelivery of Drugs into Cancer Cells in the Presence of Poloxamer 188

In the present study it is shown that poloxamer 188, added before or immediately after an electrical pulse used for electroporation, decreases the number of dead cells and at the same time does not reduce the number of reversible electropores through which small molecules (cisplatin, bleomycin, or propidium iodide) can pass/diffuse. It was suggested that hydrophobic sections of poloxamer 188 molecules are incorporated into the edges of pores and that their hydrophilic parts act as brushy pore structures. The formation of brushy pores may reduce the expansion of pores and delay the irreversible electropermeability. Tumors were implanted subcutaneously in both flanks of nude mice using HeLa cells, transfected with genes for red fluorescent protein and luciferase. The volume of tumors stopped to grow after electrochemotherapy and the use of poloxamer 188 reduced the edema near the electrode and around the subcutaneously growing tumors.

Two transgenic mouse models for beta subunit components of succinate-CoA ligase yielding pleiotropic metabolic alterations

Succinate-CoA ligase (SUCL) is a heterodimer enzyme composed of Suclg1 α-subunit and a substrate-specific Sucla2 or Suclg2 β-subunit yielding ATP or GTP, respectively. In humans, the deficiency of this enzyme leads to encephalomyopathy with or without methylmalonyl aciduria, in addition to resulting in mitochondrial DNA depletion. We generated mice lacking either one Sucla2 or Suclg2 allele. Sucla2 heterozygote mice exhibited tissue- and age-dependent decreases in Sucla2 expression associated with decreases in ATP-forming activity, but rebound increases in cardiac Suclg2 expression and GTP-forming activity. Bioenergetic parameters including substrate-level phosphorylation (SLP) were not different between wild-type and Sucla2 heterozygote mice unless a submaximal pharmacological inhibition of SUCL was concomitantly present. mtDNA contents were moderately decreased, but blood carnitine esters were significantly elevated. Suclg2 heterozygote mice exhibited decreases in Suclg2 expression but no rebound increases in Sucla2 expression or changes in bioenergetic parameters. Surprisingly, deletion of one Suclg2 allele in Sucla2 heterozygote mice still led to a rebound but protracted increase in Suclg2 expression, yielding double heterozygote mice with no alterations in GTP-forming activity or SLP, but more pronounced changes in mtDNA content and blood carnitine esters, and an increase in succinate dehydrogenase activity. We conclude that a partial reduction in Sucla2 elicits rebound increases in Suclg2 expression, which is sufficiently dominant to overcome even a concomitant deletion of one Suclg2 allele, pleiotropically affecting metabolic pathways associated with SUCL. These results as well as the availability of the transgenic mouse colonies will be of value in understanding SUCL deficiency.

Dynamics of Klebsiella pneumoniae OmpA transmembrane domain: The four extracellular loops display restricted motion behavior in micelles and in lipid bilayers

The transmembrane domain of Klebsiella pneumoniae OmpA (KpOmpA) possesses four long extracellular loops that exhibit substantial sequence variability throughout OmpA homologs in Enterobacteria, in comparison with the highly conserved membrane-embedded β-barrel core. These loops are responsible for the immunological properties of the protein, including cellular and humoral recognition. In addition to key features revealed by structural elucidation of the KpOmpA transmembrane domain in detergent micelles, studies of protein dynamics provide insight into its function and/or mechanism of action. We have investigated the dynamics of KpOmpA in a lipid bilayer, using magic angle spinning solid-state NMR. The dynamics of the β-barrel and loop regions were probed by the spin-lattice relaxation times of the C(α) and C(β) atoms of the serine and threonine residues, and by cross-polarization dynamics. The β-barrel core of the protein is rigid; the C-terminal halves of two of the four extracellular loops (L1 and L3), which are particularly long in KpOmpA, are highly mobile. The other two loops (L2 and L4), which are very similar to their homologs in Escherichia coli OmpA, and the N-terminal halves of L1 and L3 exhibit more restricted motions. We suggest a correlation between the sequence variability and the dynamics of certain loop regions, which accounts for their respective contributions to the structural and immunological properties of the protein.

Cytotoxic activity of platinum(II) and palladium(II) complexes of N-3-pyridinylmethanesulfonamide: the influence of electroporation.

The series of complexes: cis-[Pd(PMSA)2X2], cis-[Pt(PMSA)2X2], trans-[Pt(PMSA)2I2] and [Pt(PMSA)4]Cl2 (PMSA = N-3-pyridinylmethanesulfonamide; X = Cl, Br, I), previously synthesized and characterized by us, as well as the free ligand PMSA, were tested for their cytotoxic activity without electroporation - against murine leukemia F4N and human SKW-3 and MDA-MB-231 tumour cell lines - and with electroporation - against the latter two cell lines. The majority of the complexes exhibited cytotoxic effects (IC50 < 100 μmol/l) under the conditions of electroporation. Both cis- and trans-[Pt(PMSA)2I2] had pronounced cytotoxic effects (29 - 61 μmol/l against MDA-MB-231 cells).