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1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 Fermentation and Alternative Respiration Compensate for NADH Dehydrogenase Deficiency in a Prokaryotic Model of DJ-1 Associated Parkinsonism. Nadia Messaoudi1, Valérie Gautier1, Julien Dairou3, Mouhad Mihoub1, Gaëlle Lelandais1, Philippe Bouloc2, Ahmed Landoulsi4 and Gilbert Richarme1* 1Stress molecules, Institut Jacques Monod, Université Paris 7, 2 place Jussieu, 75005 Paris, France. de Signalisation et Réseaux de Régulations bactériens, UMR8621, Institut de Génétique et Microbiologie, Orsay, France. 3Université Paris Diderot, Sorbonne Paris Cité, Unité de Biologie Fonctionnelle et Adaptative (BFA) UMR 8251 CNRS, Bioprofiler facility, F-75205, Paris, France. 4Laboratoire de Biochimie et Biologie Moléculaire 03/UR/0902, Faculté des Sciences de Bizerte, Zarzouna 7021, Bizerte, Tunisia. 2Laboratoire Tel: 33 01 57 27 80 57; Fax: 33 01 44 27 57 16; Email: [email protected] *Corresponding author The two first authors contributed equally to the work. Running title : Respirofermentative metabolism in yajL mutant. Keywords : YajL / NADH dehydrogenase 1/ alternative dehydrogenases/ pyruvate oxidase/ NADH/NAD ratio. Abbreviations: MPTP: 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine, RMSD: root mean square deviation. SUMMARY YajL is the closest prokaryotic homolog of Parkinsonism-associated DJ-1, a protein of undefined function involved in the oxidative stress response. We reported recently that YajL and DJ-1 protect cells against oxidative stress-induced protein aggregation by acting as covalent chaperones for the thiol proteome, including the NuoG subunit of NADH dehydrogenase 1, and that NADH dehydrogenase 1 activity is negligible in the yajL mutant. We report here that this mutant compensates for low NADH dehydrogenase activity by utilizing NADH-independent alternative dehydrogenases, including pyruvate oxidase PoxB and D-aminoacid dehydrogenase DadA, and mixed acid aerobic fermentations characterized by acetate, lactate, succinate and ethanol excretion. The yajL mutant has a low adenylate energy charge favouring glycolytic flux, and a high NADH/NAD ratio favouring fermentations over pyruvate dehydrogenase and the Krebs cycle. DNA array analysis showed upregulation of genes involved in glycolytic and pentose phosphate pathways and alternative respiratory pathways. Moreover, the yajL mutant preferentially catabolized pyruvate-forming amino acids over Krebs cycle-related ones, and thus the yajL mutant utilizes pyruvate-centred respiro-fermentative metabolism to compensate for the NADH dehydrogenase 1 defect, and constitutes an interesting model for studying eukaryotic respiratory complex I deficiencies, especially those associated with Alzheimer and Parkinson’s diseases. INTRODUCTION YajL belongs to the PfpI/Hsp31/DJ-1 superfamily which includes chaperones (Quigley et al., 2003 ; Sastry et al., 2002), peptidases (Malki et al., 2005) and the Parkinson’s disease-associated protein DJ-1 (Canet-Aviles et al., 2004 ; Cookson, 2005). The crystal structures of YajL and DJ-1 are strikingly similar (Wilson et al., 2003 ; Wilson et al., 2005), and their backbone structures are essentially identical (0.9 A C RMSD), suggesting that they have similar functions. DJ-1 is involved in the cellular response to oxidative stress, and has been suggested to function as a weak protease (Lee et al., 2003), an oxidative stress activated chaperone (Le et al., 2012 ; Shendelman et al., 2004 ; Zhou et al., 2006), an atypical peroxiredoxin-like peroxidase (AndresMateos et al., 2007 ; Canet-Aviles et al., 2004), a stabilizer of the antioxidant transcriptional regulator Nrf2 (Clements et al., 2006), an apoptosis inhibitor via interaction with Daxx (Junn et al., 2005), a transcriptional or translational regulator of gene expression (Cookson, 2005 ; van der Brug et al., 2008) and a regulator of uncoupling protein expression affecting the production of reactive oxygen species (Guzman et al., 2010). 2 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 Like DJ-1, YajL protects cells against oxidative stress and oxidative stress-induced protein aggregation, possibly through its chaperone function and control of gene expression (Kthiri et al., 2010a ; Kthiri et al., 2010b). We reported recently that YajL and DJ-1 function as covalent chaperones that, upon oxidative stress, form mixed disulfides with proteins containing essential cysteines for catalysis, such as glyceraldehyde-3-phosphate dehydrogenase and peroxidases, or FeS cluster binding, such as aconitases and the NuoG subunit of NADH dehydrogenase 1 (Le et al., 2012). Moreover, YajL and DJ-1 preferentially form mixed disulfides with sulfenylated proteins and protect cells against protein sulfenylation (Gautier et al., 2012). The specific interaction of YajL with the NuoG subunit of NADH dehydrogenase is likely responsible for the NADH dehydrogenase 1 defect of the yajL mutant, which can be rescued by YajL and DJ-1 overproducing plasmids (Le et al., 2012). These results suggest that DJ-1 may be involved in biogenesis and stress protection of mitochondrial complex I (Danielson & Andersen, 2008). Respiratory chain abnormalities in cells of patients with Parkinson’s disease have been reported (Bindoff et al., 1989), and complex I inhibitors such as rotenone and MPTP produce a Parkinson-like syndrome in both human and primate, identifying complex I as a major target in Parkinson’s disease. Moreover, the genetic causes of Parkinson’s disease highlight the importance of mitochondrial dysfunction: parkin, PINK1, DJ-1, LRRK2 and HTRA2 localize to the mitochondria (Schapira & Gegg, 2011) and their deficiency induces mitochondrial defects that contribute to oxidative stress-induced cell death (Irrcher et al., 2010). The importance of complex I deficiencies in the etiology of Parkinson’s disease, the specific involvement of YajL and DJ-1 in its biogenesis, and the scarcity of results describing the metabolic characteristics of DJ-1-deficient cells led us to investigate the metabolism of the yajL mutant to understand how cells compensate for NADH dehydrogenase 1 deficiency. METHODS Bacterial strains and plasmids. The yajL-deficient strain was constructed by Dr. H. Mori (Nara Institute of Sciences and technology, Japan). It contains yajL disrupted by λRed in the E. coli strain BW25113 (lacIq rrnB3 ∆lacZ4787 hsdR514 ∆(araBAD)567 ∆(rhaBAD)568 rph-1), leading to strain JW5067 (Baba et al., 2006). We transduced the yajL mutation into strain MG1655 as described in (Messaoudi et al., 2013). For complementation experiments, the yajL mutant was transformed by plasmid pCA24N-yajL as described in (Le et al., 2012). Cells were grown aerobically in Luria-Bertani medium at 37°C. NAD, NADH, NADP, NADPH, ATP, ADP and AMP pools. Pools of metabolites were measured as described in (Richarme, 1987), and calculations were done by assuming 130 µg of cell protein/ml bacteria at an A600 of 1, and 5 µl of cell volume / mg of protein. The results are the mean values of three experiments (S.E. was less than 15%). NADH dehydrogenase 1 and 2 activities. NADH dehydrogenases 1 and 2 activities in membrane extracts (prepared from exponential phase bacteria harvested at OD 600= 0.4) were assayed by measuring the oxidation of 2,6 dichlorophenolindophenol at 600 nm, with NADH (NADH dehydrogenase 1 + NADH dehydrogenase 2) or deamino-NADH (NADH dehydrogenase 1) as substrates (Cahloum & Gennis, 1993). The assay mixture contained the following in a final volume of 1 ml: 0.1 M Tris, pH 7.5, 100 µM NADH or deamino-NADH, 30 mM KCN and 10 µg membranes prepared by the French Press procedure. An activity of 100 represents 0.18 µmol of NADH/min/mg protein The results are the mean values of three experiments. NADH, succinate, pyruvate, D-alanine, -D-glycerolphosphate and formate oxidase activities. Oxidases present in membrane extracts were estimated by measuring the rate of oxygen uptake using a Clarke-type oxygen electrode. The reaction mixtures contained membranes (3 mg protein) and substrates (0.6 mM NADH, 20 mM succinate, 4 mM lactate, 20 mM a-glycerophosphate) in 100 mM Tris pH 7.5, 20 mM magnesium acetate and 0.25 M sucrose (Wallace & Young, 1977). Oxygen uptake due to the presence of endogenous substrates was allowed to reach a steady state level before substrates were added, except for the estimations for succinate oxidase in which case succinate was added to the reaction mixture before the membranes (Wallace & Young, 1977). The concentration of oxygen dissolved in the reaction buffer at 30°C was taken to be 225 nmol per ml. The results are the mean values of three experiments. 3 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 Analysis of culture supernatants. Bacteria were grown in LB medium (100 ml bacteria in a 1 l Fernbach) to exponential phase (OD600= 0.8)(both strains displayed exponential growth with identical doubling time until OD600= 1.6), and culture supernatants were analyzed enzymatically for the determination of acetate, lactate, ethanol, succinate and formate. These determinations were made by using the corresponding Megazyme assay kits for acetic acid, lactic acid, ethanol, acetic acid and formic acid, following instructions of the manufacturer (Megazyme International Ireland Limited). The results are the mean values of three experiments. DNA microarray measurements. The yajL mutant and its parental strain MG1655 were grown under aeration to exponential phase (OD600= 0.3) in LB rich medium (Kthiri et al., 2010). Total RNAs were extracted and treated twice with Dnase I (Brumaghim et al., 2003; Collier et al., 2004). RNA quality was monitored with a 2100 Bioanalyzer (Agilent, Santa Claran CA, USA). Transcriptome experiments were performed using E. coli Affimetrix DNA chips (Santa Clara, CA, USA) by Cogenics (Newton, MA, USA) according to the standard manufacturer’s instructions. Hybrized arrays were stained using Affimetrix protocol. Samples were duplicated biologically, and we calculated the average of gene expression ratios from both experiments; genes were considered to be clearly induced if the absolute value of the expression ratio was higher than 2, and genes displaying a too low signal intensity were discarded from the analysis. Microarray analysis and data processing. After image quantification and global signal correction (following the manufacturer’s instructions), the ratios between intensity values measured in yajL mutant and its parent MG1655 were calculated and normalized using the standard Lowess procedure (Messaoudi et al., 2013). This statistical correction has the advantage to remove intensity-dependent effects in the observed log2(ratio) values and is thus well-adapted to correct systematic bias related to low-intensity measures. As an additional filter, probes with low intensity signals in the yajL mutant (<250) compared to background distribution were excluded from subsequent analyses. Reproducibility between results obtained with different microarray replicates was verified and used to eliminate genes with artifactual signals. Finally, to assess whether any of the cellular functions associated to the genes identified as up-regulated in the yajL mutant were observed at a frequency greater than that expected by chance, p-values were calculated as described (Messaoudi et al., 2013) (hypergeometric distribution). Note that this approach was previously successfully applied (Messaoudi et al., 2013). The microarray data were deposited at Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/), Accession No GSE42702. Materials. All chemicals were reagent grade and were obtained from Sigma. RESULTS Increased NADH/NAD ratio. NADH is produced primarily by catabolic reactions, including glycolysis and the Krebs cycle, and is reoxidized by the respiratory chains and/or fermentations, whereas NADPH, which is produced primarily by the pentose phosphate pathway and isocitrate dehydrogenase, drives anabolic reactions (Brumaghim et al., 2003 ; Holm et al.,2010 ; Lin et al., 2004) and acts as a major reductant during oxidative stress (Holm et al., 2010). We measured the nucleotide pools of aerobic bacteria grown in LB medium during the exponential phase and found that the NADH/NAD ratio of the parental strain (NADH/NAD ratio=0.22) was similar to that reported by others (Auriol et al., 2011)), whereas that of the yajL mutant (NADH/NAD ratio=0.72) was similar to ratios observed in NADH dehydrogenase I mutants (Pruss et al., 1994) and anaerobic bacteria (de Graef et al., 1999) (Figure 1A). We also measured the NADH/NAD ratio of the yajL mutant transformed with the YajL-overproducing plasmid pCA24N-yajL, and found that it displayed a ratio of 0.28, close to that of the wild-type strain (not shown). The high NADH/NAD ratio of the yajL mutant likely results from its NADH dehydrogenase defect (see below) and is probably responsible for respiro-fermentative metabolism described below. Increased NADPH/NADP ratio The yajL mutant displayed a higher NADPH/NADP ratio (NADPH/NADP ratio=2.0) than the parental strain (NADPH/NADP ratio=1.4) and other wild-type E. coli strains grown under similar conditions (Andersen & von Meyenburg, 1977) (Figure 1B). This high NADPH/NADP ratio may be important for oxidative stress resistance. Decreased adenylate energy charge. Intracellular ATP, ADP and AMP concentrations were 1.2 ± 0.16, 0.92 ± 0.12 and 0.42 ± 0.06 mM, respectively, in the yajL mutant, versus 2.2 ± 0.28 mM, 0.75 4 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 ± 0.09 mM and 0.21 ± 0.04 mM, respectively, in the parental strain (Figure 1C). Because the mutant displayed higher ADP/ATP and AMP/ATP ratios than the parental strain, its adenylate energy charge (0.58) was lower than that of the parental strain (0.78). This low adenylate energy charge likely favours glycolytic flux (Koebmann et al., 2002), since adenyl nucleotides are allosteric regulators (with AMP and ADP as activators and ATP an inhibitor) of phosphofructokinase and pyruvate kinase. NADH dehydrogenase defect. E. coli possesses two NADH dehydrogenases, namely, NADH dehydrogenase 1, composed of the nuoA-N gene products (a homolog of mitochondrial respiratory complex I) and NADH dehydrogenase 2, the ndh gene product. We measured the oxidation of dichlorophenolindophenol with NADH (NADH dehydrogenase 1 + NADH dehydrogenase 2) or deamino-NADH (NADH dehydrogenase 1). The wild-type strain and the yajL mutant oxidized NADH at relative rates of 100 and 29, respectively, and deamino-NADH at relative rates of 41 and 5.3, respectively (Figure 1D); thus NADH dehydrogenase I was almost inactive in the mutant (13% of the parental strain activity, as reported previously (Le et al., 2012) (we reported previously that the YajL-overproducing plasmid efficiently rescued (up to 86%) the NADH dehydrogenase defect of the yajL mutant (Le et al., 2012)), whereas NADH dehydrogenase 2 was 41% active. The weak global NADH dehydrogenase activity of the yajL mutant may be responsible for the high NADH/NAD ratio, fermentative metabolism and the induction of alternative respiratory pathways (see below). Overexpression of alternative respiratory dehydrogenases. The major respiratory dehydrogenases of E. coli are NADH dehydrogenase 1, NADH dehydrogenase 2 and succinate dehydrogenase. Other aerobic dehydrogenases include pyruvate oxidase PoxB, D-amino acid dehydrogenase DadA, glycerol-3-phosphate dehydrogenase GlpD, formate dehydrogenase FdoGHI, acyl-CoA dehydrogenase FadE, and proline oxidase PutA, all of which transfer electrons from substrates to quinones without using NAD/NADH (Figure 2). The expression levels of mRNAs coding for respiratory dehydrogenases were determined by DNA array experiments (Figure 3A). Mutant/parental strain gene expression ratios were between 0.4 and 0.8 for nuoA-N, and were 0.75 for ndh and 1.3 for sdhABCD. By contrast, the yajL mutant overexpressed several aerobic alternative dehydrogenases, including pyruvate oxidase PoxB (9fold), D-alanine dehydrogenase DadA (counterpart of eukaryotic D-aminoacid dehydrogenases; 4fold), glycerophosphate dehydrogenase GlpD (prokaryotic counterpart of eukaryotic glycerophosphate dehydrogenase GPD2; 12-fold), formate dehydrogenase FdoGHI (3-fold)), acylCoA dehydrogenase FadE (counterpart of eukaryotic acyl-CoA dehydrogenases; 2-fold) and proline dehydrogenase PutA (the counterpart of eukaryotic proline oxidase; 2-fold). Using RT-PCR, we measured the expression of pyruvate oxidase and alanine dehydrogenase (which play a major role in respiration in the yajL mutant, as described below) and glycerol-phosphate dehydrogenase and found that expression of pyruvate oxidase and D-alanine dehydrogenase was increased by 3-fold in the mutant, whereas glycerol-phosphate dehydrogenase was not significantly overexpressed (not shown). As expected for aerobically grown cells, anaerobic dehydrogenases, such as glycerophosphate dehydrogenase GlpABC, formate dehydrogenase FdnGHI, and hydrogenases A and B, were expresssed at low levels in both the yajL mutant and the parental strain (not shown); thus, whereas NADH dehydrogenases and succinate dehydrogenase constitute the major dehydrogenases active in wild-type cells, alternative dehydrogenases are overexpressed in the yajL mutant to compensate for NADH dehydrogenase deficiency. Moderate changes in the expression of quinol oxidases. The major E. coli quinol oxidases comprise CyoABCD, which transfers electrons from quinol to oxygen, and thus performs the functions of mitochondrial complexes 3 and 4, and CydAB, which has a higher affinity for oxygen, and is usually expressed in microaerobiosis (Unden, 1997)). The genes cyoABCD were expressed at similar levels in both strains, whereas expression of cydAB was increased by 1.9-fold in the yajL mutant, up to a level approaching that of cyoABCD (Figure 3B) (the expression of genes cbdABX coding for the second bd-type oxidase was negligible in both strains (not shown)). As expected for aerobic cells, genes coding for anaerobic nitrate, nitrite, DMSO, TMNAO reductases and hydrogenase C were not significantly expressed in either of the two strains (Figure 3B, not shown). However, expression of frdABCD, the genes coding for fumarate reductase, which transfers electrons from quinols to fumarate and is usually repressed in aerobiosis was increased by 1.8-fold in the mutant, suggesting that oxaloacetate-succinate interconversion occurs in the oxaloacetate-succinate direction (in accordance with its high 5 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 NADH/NAD ratio and succinate excretion, as described below). Therefore, in contrast to its impressive overexpression of multiple alternative dehydrogenases, the yajL mutant expressed quinol oxidase CyoABCD at normal levels, and displayed only a 2-fold overexpression of microaerobic quinol oxidase CydAB (Noda et al., 2006) and fumarate reductase. Increased activities of alternative respiratory chains. To confirm the increased expression of alternative dehydrogenases in the yajL mutant, we investigated its dehydrogenase activities. We prepared membranes from the mutant and parental strains by the French Press procedure, incubated them in an oxygraph cell in the presence of respiratory substrates, and measured oxygen consumptions as a function of time (Figure 4) (Wallace & Young, 1977). The mutant/wild-type ratios of NADH and succinate oxidase activities were 0.24 and 0.8, respectively; thus, the yajL mutant displayed low NADH oxidase activity (in accordance with its low NADH dehydrogenase activity), and a succinate oxidase activity slightly lower than that of the parental strain. Pyruvate oxidase and D-alanine oxidase activities were 3-fold higher in the yajL mutant than in the parental strain, suggesting that they compensate for the NADH dehydrogenase deficiency. Replacement of pyruvate dehydrogenase by pyruvate oxidase constitutes a major metabolic shift known as overflow metabolism (see below), in which pyruvate is directly converted into acetate in a reaction that is not limited by high NADH levels, bypassing pyruvate dehydrogenase, the Krebs cycle and NADH dehydrogenases (pyruvate oxidase transfers electrons directly from pyruvate to quinones, and acetate is excreted). Glycerol-phosphate dehydrogenase activities were low in both the yajL mutant and the parental strain. Degradation of D-alanine by D-alanine oxidase produces pyruvate, the pyruvate oxidase substrate, so alanine likely constitutes an important metabolite for the yajL mutant. L-alanine is also metabolized after racemisation by alanine racemase DadX, mRNA levels of which are increased by 10-fold in the yajL mutant (not shown). The increased activities of pyruvate and alanine oxidases in the yajL mutant are consistent with the increased excretion of acetate (see below); thus, the yajL mutant displays increased pyruvate and alanine oxidase activities to overcome NADH dehydrogenase deficiency. Alternative oxidases constitute an energetically favourable alternative to fermentations, because, although PoxB and DadA do not expel protons by themselves (Unden, 1998), a proton motive force is generated during electron transfer through cytochrome oxidases. Fermentative metabolism. Compared to the parental strain, the yajL mutant expressed 4-fold more fermentative lactate dehydrogenase LdhA, 3-fold more acetaldehyde/alcohol dehydrogenase AdhE and 2-fold more pyruvate decarboxylase FrsA (which degrades pyruvate to acetaldehyde) (Lee et al., 2012) (Figure 5A). Accordingly, the yajL mutant excreted (as early as in the exponential phase) higher amounts of acetate (2-fold), lactate (8-fold), ethanol (2.6-fold) and succinate (2-fold) than the parental strain (Figure 5B). Therefore, the yajL mutant exhibits active fermentative metabolism in aerobiosis, a characteristic of cells displaying high NADH/NAD ratios (Berrios-Rivera et al., 2004 ; Holm et al., 2010). The observed increase in excretion of acetate, succinate, lactate and ethanol is in accordance with overexpression of pyruvate oxidase, fumarate reductase, lactate dehydrogenase and alcohol dehydrogenase, respectively. Overexpression of glycolytic and pentose phosphate pathway enzymes, and of PEP carboxykinase and malic enzymes. Bacteria grown in LB medium metabolize both carbohydrates and peptides (Baev et al., 2006 ; Yohannes et al., 2004). Glycolytic enzymes were overexpressed in the yajL mutant, especially those acting downstream of aldolase (i.e., after convergence of the glycolytic and pentose phosphate pathways), such as triose phosphate isomerase (tpiA gene product; 2.8-fold), glyceraldehyde-3-phosphate dehydrogenase (gap gene product; 2.1-fold), 3phosphoglycerate kinase (pgk gene product; 2.4-fold), enolase (eno gene product; 1.8-fold) and pyruvate kinase (pyk gene product; 1.7-fold). The expression of pentose phosphate pathway enzymes was also increased, including glucose-6-phosphate dehydrogenase (zwf gene product; 1.8 fold), transaldolase B (aldB gene product; 5 fold) and transketolase (tkt gene product; 1.7-fold), suggesting that this pathway constitutes a privileged route for conversion of hexose to triose and is responsible for the high NADPH/NADP ratio of the mutant (Figure 6A). PEP carboxykinase and malic enzymes were also overexpressed, and may be involved in converting metabolites derived from aspartate and asparagine (fumarate, malate and oxaloacetate) into PEP and pyruvate, thereby feeding the pyruvate oxidase and fermentative pathways. By contrast, PEP carboxylase (ppc gene product), which catalyzes the conversion of PEP into oxaloacetate for 6 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 replenishment of the Krebs cycle, was underexpressed in the yajL mutant (not shown), in accordance with depressed Krebs cycle activity, as discussed below)). These results suggest that metabolism is increased in the yajL mutant through glycolytic and pentose phosphate pathways, in accordance with the observed lower adenylate energy charge, pyruvatebased overflow metabolism, fermentative metabolism and NADPH requirement for combating oxidative stress (Koebmann et al., 2002). The expression levels of PEP carboxykinase (pck gene product) and of a malic enzyme (maeA gene product), which replenish PEP and pyruvate pools from oxaloacetate and malate, are also increased (Figure 6A). Overflow metabolism. In aerobic bacteria, the major route for pyruvate degradation is via pyruvate dehydrogenase, the Krebs cycle and respiratory chains. In E. coli, pyruvate is also metabolized into acetate via either the sequential action of pyruvate dehydrogenase, phosphotransacetylase and acetate kinase, or via the activity of pyruvate oxidase (Eiteman & Altman, 2006). Pyruvate oxidase was overexpressed by several-fold in the yajL mutant, which suggests that the mutant preferentially uses pyruvate oxidase for acetate production. Moreover, pyruvate dehydrogenase is likely inhibited in the mutant by the high NADH/NAD ratio. In the mutant, pyruvate is also degraded into lactate and ethanol, and the pyruvate-alanine interconversion likely functions in the alanine-pyruvate direction via the alanine dehydrogenase DadA and thus contributes to pyruvate formation. Therefore, overflow metabolism is active in the yajL mutant, with a significant fraction of pyruvate being metabolized into acetate via pyruvate oxidase and into lactate and ethanol by lactate and alcohol dehydrogenases. Depressed Krebs cycle. In aerobic wild-type bacteria, substrates are completely oxidized via the Krebs cycle, and the NADH that is produced is oxidized by respiratory chains. Several findings suggest that the Krebs cycle is weakly active in the yajL mutant, as follows: i) a high NADH/NAD ratio is known to inhibit pyruvate dehydrogenase, citrate synthase, ketoglutarate and malate dehydrogenases; ii) NADH dehydrogenases, which oxidize Krebs cycle NADH are weakly active; iii) alternative dehydrogenases utilize Krebs cycle-independent substrates (mainly pyruvate and alanine; iv) the mutant overexpresses fumarate dehydrogenase and excretes succinate, which suggests that the oxaloacetate/succinate interconversion occurs in the oxaloacetate-succinate direction, and vi) the ketoglutarate pool is 10-fold smaller than that of the parental strain (11 µM versus 110 µM; not shown), suggesting that ketoglutarate dehydrogenase activity is negligible (Kmketoglutarate=140 µM). Therefore, the Krebs cycle is likely depressed in the yajL mutant, and pyruvate-dependent metabolism is characterized by fermentation and pyruvate-related respiration involving D-alanine dehydrogenase and pyruvate oxidase. Defective aminoacid metabolism. Because the yajL mutant displays reduced Krebs cycle activity and contains low ketoglutarate levels, growth on mixed amino acids is affected, and degradation of pyruvate-forming, transaminase-independent amino acids is favoured, over that of Krebs cyclerelated, transaminase-dependent amino acids (aminoacid degradation via transaminases requires alpha-ketoglutarate as a substrate). We cultured the yajL mutant and the parental strain in tryptone broth (a casein hydrolysate lacking carbohydrates), monitored growth by optical density, and removed aliquots of the medium for amino acid analysis at appropriate intervals. The yajL mutant grew more slowly than the parental strain in tryptone broth, whereas both strains grew at similar rates in LB medium (which contains sugars and peptides) (not shown), suggesting that it harbours an amino acid utilization defect. Both strains consumed alanine, serine, glycine, aspartate, asparagine and tryptophan rapidly, as reported by others for wild-type strains (Pruss et al., 1994) (Figure 6B). The efficient consumption of alanine, serine, glycine and tryptophan by the mutant can be explained by their degradation into pyruvate without transamination. The yajL mutant also actively consumed asparagine and aspartate, which are mainly degraded into fumarate by aspartase (AspA), with fumarate being converted either (via oxaloacetate) into PEP by PEP carboxykinase, or (via malate) into pyruvate by malic enzymes. By contrast, the yajL mutant consumed glutamate, glutamine, arginine, phenylalanine, tyrosine, methionine, valine, isoleucine and leucine less efficiently than the parental strain. These amino acids are degraded via Krebs cycle intermediates (oxoglutarate, succinyl CoA and fumarate) and require a transamination, so their decreased utilization may be explained by the depressed Krebs cycle activity of the mutant and its low ketoglutarate pools; thus, the yajL mutant preferentially degrades pyruvateforming amino acids, that do not require transamination for their degradation. DISCUSSION 7 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 We show in this study that a mutant defective in YajL, the prokaryotic homolog of the Parkinsonism-associated protein DJ-1, displays impressive metabolic changes, including NADH dehydrogenase deficiency, a high NADH/NAD ratio, decreased adenylate energy charge, and pyruvate-centred metabolism involving alternative dehydrogenases and fermentation (Figure 7). High NADH/NAD and NADPH/NADP ratios. The high NADH/NAD ratio of the yajL mutant is similar to those of NADH dehydrogenase I mutants (Husain et al., 2008) and anaerobic bacteria (de Graef et al., 1999). High NADH/NAD ratios reduce the flow of material through pyruvate dehydrogenase and the Krebs cycle, and favour fermentation and overflow metabolism. The NADPH/NADP ratio in the yajL mutant is higher than in the parental strain. NADPH acts as a reductant for peroxides and protein disulfides during oxidative stress, and might be protective against endogenous oxidative stress in the mutant (Husain et al., 2008). Decreased adenylate energy charge. The low adenylate energy charge of the yajL mutant likely triggers high glycolytic flux (Koebmann et al., 2002), since ADP and AMP are allosteric activators of phosphofructokinase and pyruvate kinase. The low adenylate energy charge probably results from fermentative metabolism (Unden, 1998) and alternative respiratory pathways which are less efficient than NADH dehydrogenase in generating a protonmotive force (Unden & Bongaerts, 1997). Less tightly coupled respiratory chains may allow the yajL mutant to escape to the back pressure from the proton motive force and achieve efficient overflow metabolism (Unden, 1998). Moreover, loosely coupled respiratory chains are advantageous for reducing the formation of reactive oxygen species by respiratory chains. Whereas in bacteria, a decrease in the proton motive force occurs by the use of alternative respiratory chains, in eukaryotic cells this is mediated by uncoupling proteins. Interestingly, underexpression of uncoupling proteins 4 and 5 in DJ-1 deficient cells results in defective uncoupling and the overproduction of reactive oxygen species (Guzman et al., 2010). NADH dehydrogenase 1 deficiency. The NADH dehydrogenase 1 activity of the yajL mutant is 7fold lower than that of the parental strain. Since the NuoG subunit of NADH dehydrogenase 1 is a YajL substrate (Le et al., 2012), the low NADH dehydrogenase 1 activity of the mutant can be explained by its YajL deficiency. The relationship between Parkinson’s disease and complex I inhibition has been extensively studied (Danielson & Andersen, 2008). In the yajL mutant, NADH dehydrogenase 2 is not able to restore a normal NADH/NAD ratio, so the mutant excretes fermentation products and induces NADH-independent alternative dehydrogenases. Human cells do not possess the second NADH dehydrogenase found in bacteria, yeasts and plants, that confers significant flexibility to their respiratory chains. Transkingdom gene therapy, however, is a promising strategy for remedying complex I deficiency in humans (Schiff et al., 2012). Overexpression of alternative respiratory dehydrogenases. Alternative dehydrogenases are overexpressed in the yajL mutant and several display increased activity, including pyruvate oxidase PoxB and D-alanine dehydrogenase DadA. In contrast to NADH dehydrogenase 1 which contains many subunits and several FeS clusters, and is sensitive to dysregulated biogenesis and environnemental stresses, the alternative dehydrogenases PoxB, GlpD and DadA each consist of a single protein devoid of FeS clusters. The overexpression of alternative dehydrogenases in the mutant could be induced by the stress regulator sigma S, which is a known inducer of pyruvate oxidase PoxB and alanine dehydrogenase DadA, and is overexpressed in the mutant in the exponential phase (Messaoudi et al., 2013). Several NADH-independent dehydrogenases described in this study have an eukaryotic counterpart, including succinate dehydrogenase (eukaryotic complex II), glycerophosphate dehydrogenase GlpD (mitochondrial glycerol-3-phosphate dehydrogenase GPD2), D-alanine dehydrogenase DadA (Damino acid oxidases), acyl-CoA dehydrogenase FadE (fatty acyl-CoA dehydrogenases) and proline oxidase PutA (mitochondrial proline oxidase ProDH). Succinate dehydrogenase likely displays reduced activity in complex I-deficient eukaryotic cells because of inhibition of the Krebs cycle by the high NADH/NAD ratio. Glycerol-3-phosphate dehydrogenase GPD2, which is part of the NADH shuttle that transfers the reducing power of cytoplasmic NADH to mitochondrial quinones, is interesting because in contrast to the malate/aspartate shuttle, it circumvents mitochondrial NADH production and may bypass NADH dehydrogenase defects. Eukaryotic D-amino acid oxidases (which are restricted to D-amino acid detoxification) are of limited metabolic interest. Eukaryotic acyl-CoA dehydrogenases are involved in the first step of fatty acyl-CoA degradation, and transfer hydrogens to quinones; however, the next oxidoreduction step of -oxidation and acetyl CoA oxidation in the Krebs cycle produce NADH, so they would be of little use to overcome NADH 8 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 dehydrogenase defects (Abdallah et al., 2007; Bindoff et al., 1989; Cahloun & Gennis, 1993; Koebmann et al., 2002; Kthiri et al., 2012; Schapira & Gegg, 2011). Proline oxidase catalyzes the conversion of proline to 1-pyrroline-5-carboxylate and transfers hydrogens to quinones via FAD, whereas the reverse reaction, catalyzed by 1-pyrroline-5-carboxylate reductase uses NAD(P)H as a cofactor: consequently, both reactions produce a cycle of proline synthesis and degradation that can transfer redox potential between cellular compartments, and might contribute to bypassing NADH dehydrogenase 1 deficiency. Increase in expression of microaerobic quinol oxidase and fumarate reductase. The yajL mutant overexpresses the microaerobic ubiquinol oxidase CydAB (Unden, 1998), and also fumarate reductase which catalyzes the reverse reaction of succinate dehydrogenase, suggesting that the oxaloacetate/succinate branch of the Krebs cycle functions in the reverse direction, as favoured by the high NADH/NAD ratio, and in accordance with increased excretion of succinate. Fermentative metabolism. The yajL mutant displays mixed acid fermentation in accordance with its high NADH/NAD ratio. Human cells utilize lactic fermentations, especially when respiration is unable to cope with high metabolic flux: this occurs when oxygen availability is low during intense exercise, or in respiratory chain diseases and cancer. In respiratory chain diseases, fermentation increases as a consequence of the respiratory chain defect, whereas in cancer cells, a high rate of glucose metabolism (aerobic glycolysis, known as the Warburg effect) sustains anabolic reactions (DNA and RNA synthesis), and fermentative metabolism protects cells from intermittent hypoxia. Amino acid degradation. The yajL mutant favours degradation of pyruvate-forming, transaminaseindependent amino acids over that of Krebs cycle-related, transaminase-dependent amino acids. Such a metabolic status might occur in many metabolic diseases, including cancer, mitochondrial respiratory chain diseases and Parkinson’s disease, in which respiratory activities are compromised and/or fermentative metabolism is accelerated. In these cases, adequate amino acid supplementation of patients, considering the two sets of amino acids described above, might be of benefit. Finally, the present study exemplifies the consequences of a single gene deficiency on cellular metabolism and constitutes an interesting model for the study of DJ-1 deficiencies and Parkinson’s disease, as well as respiratory chain deficiencies. The remarkable ability of bacteria to cope with NADH dehydrogenase deficiency resulting from YajL deficiency may influence our thinking with regard to the way in which we treat respiratory chain deficiencies in eukaryotic cells by taking into accounts the differences and similarities between prokaryotic and eukaryotic cells. ACKNOWLEDGEMENTS. 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The oxidation state of DJ-1 regulates its chaperone activity toward alpha-synuclein. J Mol Biol 356, 1036-1048. FIGURE LEGENDS. Figure 1. Nucleotide pools and NADH dehydrogenase activities. A) NAD (black bars) and NADH (grey bars) pools, and B) NADP (black bars) and NADPH (grey bars) pools were measured as described in Methods. C) ATP (black bars), ADP (dark grey bars) and AMP (light grey bars) pools were measured as described in Methods. D) The activities of NADH dehydrogenases 1 (black) and 2 (grey) in membrane extracts were assayed by measuring the oxidation of 2,6 dichlorophenol indophenol at 600 nm with NADH (NADH dehydrogenase 1 + NADH dehydrogenase 2) or deamino-NADH (NADH dehydrogenase 1) as substrates. An activity of 100 represents 0.18 µmol of NADH/min/mg protein. Figure 2. Topology and arrangement of aerobic and anaerobic respiration enzymes in E. coli. Topology and membrane insertion was determined from biochemical data or from protein sequence properties such as hydrophobicity or signal sequence. Q and QH 2 stand for quinones and reduced quinones, respectively. The orientation and topology of the subunits were derived from the E. coli database and experimental evidence. This figure is taken from Unden & Bongaerts, 1997 with permission. Figure 3. Expression of respiratory chains. mRNAs expression values are taken from microarray data; A) Expression of respiratory dehydrogenases in the yajL mutant (grey bars) and the parental strain (black bars); B) Expression of ubiquinol oxidases and reductases in the yajL mutant (grey bars) and the parental strain (black bars). Figure 4. NADH, succinate, pyruvate, D-alanine and -D-glycerolphosphate oxidase activities. A) Oxidases present in membrane extracts of the yajL mutant (grey bars) and the parental strain (black bars) were estimated by measuring the rate of oxygen uptake using a Clarke-type oxygen electrode. Reaction mixtures contained membranes (3 mg protein) and substrates (0.6 mM NADH, 20 mM succinate, 4 mM lactate, 20 mM a-glycerophosphate) in a solution of 100 mM Tris (pH 7.5), 20 mM magnesium acetate and 0.25 M sucrose. Figure 5. Fermentation. A) Expression of fermentative enzymes in the yajL mutant (grey bars) and the parental strain (black bars). B) Excretion of acetate, lactate, ethanol, succinate and formate from the yajL mutant (grey bars) and the parental strain (black bars). Bacteria were grown in LB medium at 37°C, and culture supernatants were tested at different times for the presence of fermentation products as described in Methods. “1” represents a concentration of 1 mM in the culture medium for all products, except acetate for which it represents a concentration of 10 mM. Figure 6. Expression of intermediary metabolism enzymes and amino acid catabolism. A) Ratios of mRNA expression values (taken from microarray data) for several metabolic enzymes involved in glycolysis, the HMP pathway and neoglucogenesis. B) Amino acid utilization efficiencies (ratio of the yajL mutant/parental strain) by bacteria during exponential growth on casein hydrolysate medium during the second hour after dilution from overnight cultures. Figure 7. Metabolic scheme proposed for intermediary metabolism in the yajL mutant. The width of the arrows is proportional to the flux through metabolic pathways. Hollow arrows represent amino acid degradation pathways, and elongated grey arrows fermentative pathways.