The mitochondrial carrier Citrin plays a role in regulating cellular energy during carcinogenesis.

Citrin, encoded by SLC25A13 gene, is an inner mitochondrial transporter that is part of the malate-aspartate shuttle, which regulates the NAD+/NADH ratio between the cytosol and mitochondria. Citrullinemia type II (CTLN-II) is an inherited disorder caused by germline mutations in SLC25A13, manifesting clinically in growth failure that can be alleviated by dietary restriction of carbohydrates. The association of citrin with glycolysis and NAD+/NADH ratio led us to hypothesize that it may play a role in carcinogenesis. Indeed, we find that citrin is upregulated in multiple cancer types and is essential for supplementing NAD+ for glycolysis and NADH for oxidative phosphorylation. Consequently, citrin deficiency associates with autophagy, whereas its overexpression in cancer cells increases energy production and cancer invasion. Furthermore, based on the human deleterious mutations in citrin, we found a potential inhibitor of citrin that restricts cancerous phenotypes in cells. Collectively, our findings suggest that targeting citrin may be of benefit for cancer therapy.

Cooperative Substrate-Cofactor Interactions and Membrane Localization of the Bacterial Phospholipase A2 (PLA2) Enzyme, ExoU.

The ExoU type III secretion enzyme is a potent phospholipase A2 secreted by the Gram-negative opportunistic pathogen, Pseudomonas aeruginosa Activation of phospholipase activity is induced by protein-protein interactions with ubiquitin in the cytosol of a targeted eukaryotic cell, leading to destruction of host cell membranes. Previous work in our laboratory suggested that conformational changes within a C-terminal domain of the toxin might be involved in the activation mechanism. In this study, we use site-directed spin-labeling electron paramagnetic resonance spectroscopy to investigate conformational changes in a C-terminal four-helical bundle region of ExoU as it interacts with lipid substrates and ubiquitin, and to examine the localization of this domain with respect to the lipid bilayer. In the absence of ubiquitin or substrate liposomes, the overall structure of the C-terminal domain is in good agreement with crystallographic models derived from ExoU in complex with its chaperone, SpcU. Significant conformational changes are observed throughout the domain in the presence of ubiquitin and liposomes combined that are not observed with either liposomes or ubiquitin alone. In the presence of ubiquitin, two interhelical loops of the C-terminal four-helix bundle appear to penetrate the membrane bilayer, stabilizing ExoU-membrane association. Thus, ubiquitin and the substrate lipid bilayer act synergistically to induce a conformational rearrangement in the C-terminal domain of ExoU.

Efficacy of an Automated Multiple Emitter Whole-Room Ultraviolet-C Disinfection System Against Coronaviruses MHV and MERS-CoV.

Efficient and automated methods of disinfecting surfaces contaminated with the Middle Eastern respiratory syndrome coronavirus (MERS-CoV) may prevent the spread of the virus. Here we report the efficacy and use of an automated triple-emitter whole room UV-C disinfection system to inactivate mouse hepatitis virus, strain A59 (MHV-A59) and MERS-CoV viruses on surfaces with a >5 log10 reduction.

Reduced mitochondrial DNA content and heterozygous nuclear gene mutations in patients with acute liver failure.

OBJECTIVES: Historically, mitochondrial disorders have been associated with predominantly multisystem or neurological symptoms. If present, hepatic complications were thought to be a late feature. Recently, mutations in at least 4 nuclear genes have been identified in infants presenting with rapidly progressive hepatic failure, which may be precipitated by infection or drugs. We aimed to determine whether hepatic mitochondrial DNA (mtDNA) depletion is associated with apparently isolated hepatic failure in individuals with acute liver failure (ALF) of known or unknown etiologies undergoing liver transplant (LT). In addition, we wished to establish whether there was an excess of mutations in gene known to cause hepatic mtDNA depletion. METHODS: Using previously established methods, we demonstrated that end-stage liver disease from known causes did not lead to hepatic mtDNA depletion. RESULTS: Using thresholds derived from receiver-operator curve analysis, 66% of cases with ALF had probable or definite mtDNA depletion, including 34% with definite mtDNA depletion. There was a small but significant increase in the proportion of patients undergoing LT for ALF with heterozygous mutations known to lead to mtDNA depletion and hepatic failure compared with controls (P = 0.001). CONCLUSIONS: Liver disease severe enough to require LT does not cause secondary mtDNA depletion; however, the majority of patients undergoing LT for ALF had reduced mtDNA content, which fell within the range seen in patients with classic mtDNA depletion. A subset of patients with ALF has mutations in genes known to lead to mtDNA depletion and hepatic failure. Together, these results suggest defective mtDNA maintenance is associated with ALF.

Recessive deoxyguanosine kinase deficiency causes juvenile onset mitochondrial myopathy.

Deoxyguanosine kinase (DGUOK) (MIM#601465) deficiency was originally described as the cause of an infantile onset hepatocerebral mitochondrial disease [1]. The classic features of this disorder include significant hepatic failure with nystagmus and hypotonia. Mitochondrial DNA studies reveal significant mitochondrial DNA depletion in the affected tissues. Subsequently it has been shown that the same mutations in this gene may present with isolated acute liver failure without cerebral involvement. In this paper we studied the mitochondrial DNA depletion in cells from a patient presenting with mitochondrial myopathy caused by a novel mutation in DGUOK. Subsequently we developed the method to diagnose this condition using MyoD induced fibroblasts to study the muscle specific phenotype. In addition, supplementation of MyoD induced fibroblasts with dAMP and dGMP resulted in a restoration of mtDNA quantity.

Functionally important ATP binding and hydrolysis sites in Escherichia coli MsbA.

ATP-binding cassette (ABC) transporters make up one of the largest classes of proteins found in nature, and their ability to move a variety of substrates across the membrane using energy from the binding or hydrolysis of ATP is essential to an array of human pathologies and to bacterial viability. MsbA is an essential ABC transporter that specifically transports lipid A across the inner membranes of Gram-negative organisms such as Escherichia coli. The exact mechanisms of function during the binding and hydrolysis of ATP at the molecular level remain unclear. The studies presented and summarized in this work directly address the role and local dynamics of specific residues within the conserved ABC motifs in E. coli MsbA using in vivo growth and biochemical activity assays coupled with site-directed spin labeling electron paramagnetic resonance (EPR) spectroscopy motional and accessibility analysis. This first comprehensive analysis of the specific residues in these motifs within MsbA indicates that closure of the dimer interface does not occur upon ATP binding in this transporter.

Spin-labeled lipid A.

Lipopolysaccharide (LPS), a major component of the outer membranes of gram-negative bacteria, is composed of a polysaccharide chain attached to a lipid A base that contains a disaccharide headgroup with two negative phosphate groups and at least four acyl chains. Lipid A is an essential component of the membranes of a large number of bacteria and is also a substrate for a wide variety of proteins. Here we report the synthesis of a nitroxide spin-labeled lipid A, characterize its localization at the membrane bilayer surface, and demonstrate that it remains a viable substrate for the Escherichia coli lipid flippase MsbA.

Characterization of the LSGGQ and H motifs from the Escherichia coli lipid A transporter MsbA.

ATP-binding cassette (ABC) transporters make up one of the largest superfamilies of proteins known and have been shown to transport substrates ranging from lipids and antibiotics to sugars and amino acids. The dysfunction of ABC transporters has been linked to human pathologies such as cystic fibrosis, hyperinsulinemia, and macular dystrophy. Several bacterial ABC transporters are also necessary for bacterial survival and transport of virulence factors in an infected host. MsbA is a 65 kDa protein that forms a functional homodimer consisting of two six-helix transmembrane domains and two approximately 250 amino acid nucleotide-binding domains (NBD). The NBDs contain several conserved regions such as the Walker A, LSGGQ, and H motif that bind directly to ATP and align it for hydrolysis. MsbA transports lipid A, its native substrate, across the inner membrane of Gram-negative bacteria. The loss or dysfunction of MsbA results in a toxic accumulation of lipid A inside the cell, leading to cell-membrane instability and cell death. Using site-directed spin labeling electron paramagnetic resonance spectroscopy, conserved motifs within the MsbA NBD have been evaluated for structure and dynamics upon substrate binding. It has been determined that the LSGGQ NBD consensus sequence is consistent with an alpha-helical conformation and that these residues maintain extensive tertiary contacts throughout hydrolysis. The dynamics of the LSGGQ and the H-motif region have been studied in the presence of ATP, ADP, and ATP plus vanadate to identify the residues that are directly affected by interactions with the substrate before, after, and during hydrolysis, respectively.

Characterization of the Walker A motif of MsbA using site-directed spin labeling electron paramagnetic resonance spectroscopy.

MsbA is an ABC transporter that transports lipid A across the inner membrane of Gram-negative bacteria such as Escherichia coli. Without functional MsbA present, bacterial cells accumulate a toxic amount of lipid A within their inner membranes. A crystal structure of MsbA was recently obtained that provides an excellent starting point for functional dynamics studies in membranes [Chang and Roth (2001) Science 293, 1793-1800]. Although a structure of MsbA is now available, several functionally important motifs common to ABC transporters are unresolved in the crystal structure. The Walker A domain, one of the ABC transporter consensus motifs that is directly involved in ATP binding, is located within a large unresolved region of the MsbA ATPase domain. Site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy is a powerful technique for characterizing local areas within a large protein structure in addition to detecting and following changes in local structure due to dynamic interactions. MsbA reconstituted into lipid membranes has been evaluated by EPR spectroscopy, and it has been determined that the Walker A domain forms an alpha-helical structure, which is consistent with the structure of this motif observed in other crystallized ABC transporters. In addition, the interaction of the Walker A residues with ATP before, during, and after hydrolysis was followed using SDSL EPR spectroscopy in order to identify the residues directly involved in substrate binding and hydrolysis.

Resting state conformation of the MsbA homodimer as studied by site-directed spin labeling.

MsbA is the ABC transporter for lipid A and is found in the inner membranes of Gram-negative bacteria such as Escherichia coli. Without MsbA present, bacterial cells accumulate a toxic amount of lipid A within their inner membranes. A crystal structure of MsbA was recently obtained that provides an excellent starting point for functional dynamics studies in membranes [Chang, and Roth (2001) Science 293, 1793-1800]. Although a structure of MsbA is now available, many questions remain concerning its mechanism of transport. Site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy is a powerful approach for characterizing local areas within a large protein structure in addition to detecting and following changes in local structure due to dynamic interactions within a protein. The quaternary structure of the resting state of the MsbA homodimer reconstituted into lipid membranes has been evaluated by SDSL EPR spectroscopy and chemical cross-linking techniques. SDSL and cross-linking results are consistent with the controversial resting state conformation of the MsbA homodimer found in the crystal structure, with the tips of the transmembrane helices forming a dimer interface. The position of MsbA in the membrane bilayer along with the relative orientation of the transmembrane helical bundles with respect to one another has been determined. Characterization of the resting state of the MsbA homodimer is essential for future studies on the functional dynamics of this membrane transporter.

The mechanism of action of the Pseudomonas aeruginosa-encoded type III cytotoxin, ExoU.

Pseudomonas aeruginosa delivers the toxin ExoU to eukaryotic cells via a type III secretion system. Intoxication with ExoU is associated with lung injury, bacterial dissemination and sepsis in animal model and human infections. To search for ExoU targets in a genetically tractable system, we used controlled expression of the toxin in Saccharomyces cerevisiae. ExoU was cytotoxic for yeast and caused a vacuolar fragmentation phenotype. Inhibitors of human calcium-independent (iPLA(2)) and cytosolic phospholipase A(2) (cPLA(2)) lipase activity reduce the cytotoxicity of ExoU. The catalytic domains of patatin, iPLA(2) and cPLA(2) align or are similar to ExoU sequences. Site-specific mutagenesis of predicted catalytic residues (ExoUS142A or ExoUD344A) eliminated toxicity. ExoU expression in yeast resulted in an accumulation of free palmitic acid, changes in the phospholipid profiles and reduction of radiolabeled neutral lipids. ExoUS142A and ExoUD344A expressed in yeast failed to release palmitic acid. Recombinant ExoU demonstrated lipase activity in vitro, but only in the presence of a yeast extract. From these data we conclude that ExoU is a lipase that requires activation or modification by eukaryotic factors.