Nelli Mnatsakanyan

The roles of hydrogenases 3 and 4, and the F0F1-ATPase, in H2 production by Escherichia coli at alkaline and acidic pH.

The hyc operon of Escherichia coli encodes the H2‐evolving hydrogenase 3 (Hyd‐3) complex that, in conjunction with formate dehydrogenase H (Fdh‐H), constitutes a membrane‐associated formate hydrogenlyase (FHL) catalyzing the disproportionation of formate to CO2 and H2 during fermentative growth at low pH. Recently, an operon (hyf) encoding a potential second H2‐evolving hydrogenase (Hyd‐4) was identified in E. coli. In this study the roles of the hyc‐ and hyf‐encoded systems in formate‐dependent H2 production and Fdh‐H activity have been investigated. In cells grown on glucose under fermentative conditions at slightly acidic pH the production of H2 was mostly Hyd‐3‐ and Fdh‐H‐dependent, and Fdh‐H activity was also mainly Hyd‐3‐dependent. However, at slightly alkaline pH, H2 production was found to be largely Hyd‐4, Fdh‐H and F0F1‐ATPase‐dependent, and Fdh‐H activity was partially dependent on Hyd‐4 and F0F1‐ATPase. These results suggest that, at slightly alkaline pH, H2 production and Fdh‐H activity are dependent on both the F0F1‐ATPase and a novel FHL, designated FHL‐2, which is composed of Hyd‐4 and Fdh‐H, and is driven by a proton gradient established by the F0F1‐ATPase.

Hydrogenase 3 But Not Hydrogenase 4 is Major in Hydrogen Gas Production by Escherichia coli Formate Hydrogenlyase at Acidic pH and in the Presence of External Formate

Fermenting Escherichia coli is able to produce formate and molecular hydrogen (H2) when grown on glucose. H2 formation is possessed by two hydrogenases, 3 (Hyd-3) and 4 (Hyd-4), those, in conjunction with formate dehydrogenase H (Fdh-H), constitute distinct membrane-associated formate hydrogenylases. At slightly alkaline pH (pH 7.5), the production of H2 was found to be dependent on Hyd-4 and the F0F1-adenosine triphosphate (ATPase), whereas external formate increased the activity of Hyd-3. In this study with cells grown without and with external formate H2 production dependent on pH was investigated. In both types of cells, H2 production was increased after lowering of pH. At acidic pH (pH 5.5), this production became insensitive either to N,N′-dicyclohexylcarbodiimide or to osmotic shock and it became largely dependent on Fdh-H and Hyd-3 but not Hyd-4 and the F0F1-ATPase. The results indicate that Hyd-3 has a major role in H2 production at acidic pH independently on the F0F1-ATPase.

Cell death disguised: The mitochondrial permeability transition pore as the c-subunit of the F1FO ATP synthase

Ion transport across the mitochondrial inner and outer membranes is central to mitochondrial function, including regulation of oxidative phosphorylation and cell death. Although essential for ATP production by mitochondria, recent findings have confirmed that the c-subunit of the ATP synthase also houses a large conductance uncoupling channel, the mitochondrial permeability transition pore (mPTP), the persistent opening of which produces osmotic dysregulation of the inner mitochondrial membrane and cell death. This review will discuss recent advances in understanding the molecular components of mPTP, its regulatory mechanisms and how these contribute directly to its physiological as well as pathological roles.

Regulation of Escherichia coli Formate Hydrogenlyase Activity by Formate at Alkaline pH

Escherichia coli possesses two hydrogenases, Hyd-3 and Hyd-4. These, in conjunction with formate dehydrogenase H (Fdh-H), constitute distinct membrane-associated formate hydrogenlyases, FHL-1 and FHL-2, both catalyzing the decomposition of formate to H2 and CO2 during fermentative growth. FHL-1 is the major pathway at acidic pH whereas FHL-2 is proposed for slightly alkaline pH. In this study, regulation of activity of these pathways by formate has been investigated. In cells grown under fermentative conditions on glucose in the presence of 30 mM formate at pH 7.5, intracellular pH was decreased to 7.1, the activity of Fdh-H raised 3.5-fold, and the production of H2 became mostly Hyd-3 dependent. These results suggest that at alkaline pH formate increases an activity of Fdh-H and of Hyd-3 both but not of Hyd-4.

F0 Cysteine, bCys21, in the Escherichia coli ATP Synthase Is Involved in Regulation of Potassium Uptake and Molecular Hydrogen Production in Anaerobic Conditions

The single cysteine in the b subunit of the membranous F0 sector and the 19 cysteines in extramembranous F1 sector of the Escherichia coli ATP synthase were replaced by alanine. When cells were grown under anaerobic conditions on glucose, the kcat for ATP hydrolysis of membrane vesicles containing the bCys21Ala mutant enzyme, but not enzymes with other cysteine replacements, was lower, while ATP-driven H+ pumping was unchanged. However, the ATP-dependent increase in the number of accessible thiol groups in membrane vesicles was negated. Furthermore, K+ uptake and molecular hydrogen production by whole cells and protoplasts was greatly decreased. These results indicate a role for the F0 subunit bCys21 in the functionality of F0F1 and coupling to other membranous activities under fermentative conditions.

Experimental determination of the vertical alignment between the second and third transmembrane segments of muscle nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChR) are members of the Cys‐loop ligand‐gated ion channel superfamily. Muscle nAChR are heteropentamers that assemble from two α, and one each of β, γ, and δ subunits. Each subunit is composed of three domains, extracellular, transmembrane and intracellular. The transmembrane domain consists of four α‐helical segments (M1–M4). Pioneering structural information was obtained using electronmicroscopy of Torpedo nAChR. The recently solved X‐ray structure of the first eukaryotic Cys‐loop receptor, a truncated (intracellular domain missing) glutamate‐gated chloride channel α (GluClα) showed the same overall architecture. However, a significant difference with regard to the vertical alignment between the channel‐lining segment M2 and segment M3 was observed. Here, we used functional studies utilizing disulfide trapping experiments in muscle nAChR to determine the spatial orientation between M2 and M3. Our results are in agreement with the vertical alignment as obtained when using the GluClα structure as a template to homology model muscle nAChR, however, they cannot be reconciled with the current Torpedo nAChR model. The vertical M2–M3 alignments as observed in X‐ray structures of prokaryotic Gloeobacter violaceus ligand‐gated ion channel and GluClα are in agreement. Our results further confirm that this alignment in Cys‐loop receptors is conserved between prokaryotes and eukaryotes.

Physiological roles of the mitochondrial permeability transition pore.

Neurons experience high metabolic demand during such processes as synaptic vesicle recycling, membrane potential maintenance and Ca2+ exchange/extrusion. The energy needs of these events are met in large part by mitochondrial production of ATP through the process of oxidative phosphorylation. The job of ATP production by the mitochondria is performed by the F1FO ATP synthase, a multi-protein enzyme that contains a membrane-inserted portion, an extra-membranous enzymatic portion and an extensive regulatory complex. Although required for ATP production by mitochondria, recent findings have confirmed that the membrane-confined portion of the c-subunit of the ATP synthase also houses a large conductance uncoupling channel, the mitochondrial permeability transition pore (mPTP), the persistent opening of which produces osmotic dysregulation of the inner mitochondrial membrane, uncoupling of oxidative phosphorylation and cell death. Recent advances in understanding the molecular components of mPTP and its regulatory mechanisms have determined that decreased uncoupling occurs in states of enhanced mitochondrial efficiency; relative closure of mPTP therefore contributes to cellular functions as diverse as cardiac development and synaptic efficacy.

Functional Chimeras of GLIC Obtained by Adding the Intracellular Domain of Anion- and Cation-Conducting Cys-Loop Receptors

Pentameric ligand-gated ion channels (pLGICs), also called Cys-loop receptors in eukaryotic superfamily members, play diverse roles in neurotransmission and serve as primary targets for many therapeutic drugs. Structural studies of full-length eukaryotic pLGICs have been challenging because of glycosylation, large size, pentameric assembly, and hydrophobicity. X-ray structures of prokaryotic pLGICs, including the Gloeobacter violaceus LGIC (GLIC) and the Erwinia chrysanthemi LGIC (ELIC), and truncated eukaryotic pLGICs have significantly improved and complemented the understanding of structural details previously obtained with acetylcholine-binding protein and Torpedo nicotinic acetylcholine receptors. Prokaryotic pLGICs share their overall structural features with eukaryotic pLGICs for the ligand-binding extracellular and channel-lining transmembrane domains. The large intracellular domain (ICD) is present only in eukaryotic members and is characterized by a low level of sequence conservation and significant variability in length (50-250 amino acids), making the ICD a potential target for the modulation of specific pLGIC subunits. None of the structures includes a complete ICD. Here, we created chimeras by adding the ICD of cation-conducting (nAChR-α7) and anion-conducting (GABAρ1, Glyα1) eukaryotic homopentamer-forming pLGICs to GLIC. GLIC-ICD chimeras assemble into pentamers to form proton-gated channels, as does the parent GLIC. Additionally, the sensitivity of the chimeras toward modulation of functional maturation by chaperone protein RIC-3 is preserved as in those of the parent eukaryotic channels. For a previously described GLIC-5HT3A-ICD chimera, we now provide evidence of its successful large-scale expression and purification to homogeneity. Overall, the chimeras provide valuable tools for functional and structural studies of eukaryotic pLGIC ICDs.

Inhibition of Bcl-xL prevents pro-death actions of |[Delta]|N-Bcl-xL at the mitochondrial inner membrane during glutamate excitotoxicity

ABT-737 is a pharmacological inhibitor of the anti-apoptotic activity of B-cell lymphoma-extra large (Bcl-xL) protein; it promotes apoptosis of cancer cells by occupying the BH3-binding pocket. We have shown previously that ABT-737 lowers cell metabolic efficiency by inhibiting ATP synthase activity. However, we also found that ABT-737 protects rodent brain from ischemic injury in vivo by inhibiting formation of the pro-apoptotic, cleaved form of Bcl-xL, ΔN-Bcl-xL. We now report that a high concentration of ABT-737 (1 μM), or a more selective Bcl-xL inhibitor WEHI-539 (5 μM) enhances glutamate-induced neurotoxicity while a low concentration of ABT-737 (10 nM) or WEHI-539 (10 nM) is neuroprotective. High ABT-737 markedly increased ΔN-Bcl-xL formation, aggravated glutamate-induced death and resulted in the loss of mitochondrial membrane potential and decline in ATP production. Although the usual cause of death by ABT-737 is thought to be related to activation of Bax at the outer mitochondrial membrane due to sequestration of Bcl-xL, we now find that low ABT-737 not only prevents Bax activation, but it also inhibits the decline in mitochondrial potential produced by glutamate toxicity or by direct application of ΔN-Bcl-xL to mitochondria. Loss of mitochondrial inner membrane potential is also prevented by cyclosporine A, implicating the mitochondrial permeability transition pore in death aggravated by ΔN-Bcl-xL. In keeping with this, we find that glutamate/ΔN-Bcl-xL-induced neuronal death is attenuated by depletion of the ATP synthase c-subunit. C-subunit depletion prevented depolarization of mitochondrial membranes in ΔN-Bcl-xL expressing cells and substantially prevented the morphological change in neurites associated with glutamate/ΔN-Bcl-xL insult. Our findings suggest that low ABT-737 or WEHI-539 promotes survival during glutamate toxicity by preventing the effect of ΔN-Bcl-xL on mitochondrial inner membrane depolarization, highlighting ΔN-Bcl-xL as an important therapeutic target in injured brain.

Direct interaction of the resistance to inhibitors of cholinesterase type 3 protein with the serotonin receptor type 3A intracellular domain.

Pentameric ligand‐gated ion channels (pLGIC) are expressed in both excitable and non‐excitable cells that are targeted by numerous clinically used drugs. Assembly from five identical or homologous subunits yields homo‐ or heteromeric pentamers, respectively. The protein known as Resistance to Inhibitors of Cholinesterase (RIC‐3) was identified to interfere with assembly and functional maturation of pLGICs. We have shown previously for serotonin type 3A homopentamers (5‐HT3A) that the interaction with RIC‐3 requires the intracellular domain (ICD) of this pLGIC. After expression in Xenopus laevis oocytes RIC‐3 attenuated serotonin‐induced currents in 5‐HT3A wild‐type channels, but not in functional 5‐HT3AglvM3M4 channels that have the 115‐amino acid ICD replaced by a heptapeptide. In complementary experiments we have shown that engineering the Gloeobacter violaceus ligand‐gated ion channel (GLIC) to contain the 5‐HT3A‐ICD confers sensitivity to RIC‐3 in oocytes to otherwise insensitive GLIC. In this study, we identify endogenous RIC‐3 protein expression in X. laevis oocytes. We purified RIC‐3 to homogeneity after expression in Echericia coli. By using heterologously over‐expressed and purified RIC‐3 and the chimera consisting of the 5‐HT3A‐ICD and the extracellular and transmembrane domains of GLIC in pull‐down experiments, we demonstrate a direct and specific interaction between the two proteins. This result further underlines that the domain within 5‐HT3AR that mediates the interaction with RIC‐3 is the ICD. Importantly, this is the first experimental evidence that the interaction between 5‐HT3AR‐ICD and RIC‐3 does not require other proteins. In addition, we demonstrate that the pentameric assembly of the GLIC‐5‐HT3A‐ICD chimera interacts with RIC‐3.