During cell division, the activation of glycolysis is tightly regulated by the action of two ubiquitin ligases, anaphase-promoting complex/ cyclosome-Cdh1 (APC/C-Cdh1) and SKP1/CUL-1/F-box protein-β-transducin repeat-containing protein (SCF-β-TrCP), which control the transient appearance and metabolic activity of the glycolysispromoting enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, isoform 3 (PFKFB3). We now demonstrate that the breakdown of PFKFB3 during S phase occurs specifically via a distinct residue (S 273 ) within the conserved recognition site for SCF-β-TrCP. Glutaminase 1 (GLS1), the first enzyme in glutaminolysis, is also targeted for destruction by APC/C-Cdh1 and, like PFKFB3, accumulates after the activity of this ubiquitin ligase decreases in mid-to-late G1. However, our results show that GLS1 differs from PFKFB3 in that its recognition by APC/C-Cdh1 requires the presence of both a Lys-GluAsn box (KEN box) and a destruction box (D box) rather than a KEN box alone. Furthermore, GLS1 is not a substrate for SCF-β-TrCP and is not degraded until cells progress from S to G2/M. The presence of PFKFB3 and GLS1 coincides with increases in generation of lactate and in utilization of glutamine, respectively. The contrasting posttranslational regulation of PFKFB3 and GLS1, which we have verified by studies of ubiquitination and protein stability, suggests the different roles of glucose and glutamine at distinct stages in the cell cycle. Indeed, experiments in which synchronized cells were deprived of either of these substrates show that both glucose and glutamine are required for progression through the restriction point in mid-tolate G1, whereas glutamine is the only substrate essential for the progression through S phase into cell division.

For S-nitrosothiols and peroxynitrite to interfere with the activity of mitochondrial complex I, prior transition of the enzyme from its active (A) to its deactive, dormant (D) state is necessary. We now demonstrate accumulation of the D-form of complex I in human epithelial kidney cells after prolonged hypoxia. Upon reoxygenation after hypoxia there was an initial delay in the return of the respiration rate to normal. This was due to the accumulation of the D-form and its slow, substratedependent reconversion to the A-form. Reconversion to the A-form could be prevented by prolonged incubation with endogenously generated NO. We propose that the hypoxic transition from the A-form to the D-form of complex I may be protective, because it would act to reduce the electron burst and the formation of free radicals during reoxygenation. However, this may become an early pathophysiological event when NO-dependent formation of S-nitrosothiols or peroxynitrite structurally modifies complex I in its D-form and impedes its return to the active state. These observations provide a mechanism to account for the severe cell injury that follows hypoxia and reoxygenation when accompanied by NO generation.The mechanisms underlying the cellular response to hypoxia and their consequences are not completely understood. Because the mitochondrial respiratory chain is the major consumer of oxygen, mitochondria are likely to play a significant role in regulating its distribution in cells and tissues (1). Cells have the ability to decrease oxygen demand at low [O 2 ] (2, 3), and the affinity of cytochrome c oxidase for oxygen is considered to be the most important factor in the decrease of mitochondrial oxygen consumption during hypoxia (4, 5). The interaction of nitric oxide (NO) 2 with cytochrome c oxidase has been shown to be a significant determinant of the affinity of this enzyme for oxygen and is responsible for reducing cellular consumption of oxygen at low [O 2 ]. Nitric oxide also plays a role in the early reduction of mitochondrial cytochromes that occurs as the [O 2 ] decreases (6, 7). A direct consequence of such reduction is a backlog of electrons at all the redox centers of the respiratory chain, including cytochromes and ubiquinone, as well as the intramitochondrial pool of NAD(P)H.Mitochondrial complex I (EC 1.6.5.3, proton-translocating NADH:ubiquinone oxidoreductase) is responsible for oxidation of matrix NADH by membrane-bound ubiquinone and is the major entry point for electrons to the respiratory chain (8). It is also a major source of mitochondrial reactive oxygen species (ROS) (9 -11). Two catalytically and structurally distinct forms of complex I have been identified in partially purified preparations in vitro: one is a fully competent, "active" A-form, and the other is a dormant, silent, "deactivated" D-form (12). In such systems a so-called pseudo-reversible A/D transition has been described in mammalian and other vertebrate complex I (13). The turnover number of active complex I in submitochondrial particles (SM...

Cell proliferation is accompanied by an increase in the utilization of glucose and glutamine. The proliferative response is dependent on a decrease in the activity of the ubiquitin ligase anaphasepromoting complex/cyclosome (APC/C)-Cdh1 which controls G1-to-S-phase transition by targeting degradation motifs, notably the KEN box. This occurs not only in cell cycle proteins but also in the glycolysis-promoting enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3 (PFKFB3), as we have recently demonstrated in cells in culture. We now show that APC/C-Cdh1 controls the proliferative response of human T lymphocytes. Moreover, we have found that glutaminase 1 is a substrate for this ubiquitin ligase and appears at the same time as PFKFB3 in proliferating T lymphocytes. Glutaminase 1 is the first enzyme in glutaminolysis, which converts glutamine to lactate, yielding intermediates for cell proliferation. Thus APC/C-Cdh1 is responsible for the provision not only of glucose but also of glutamine and, as such, accounts for the critical step that links the cell cycle with the metabolic substrates essential for its progression.cell cycle | glutaminase | 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3 | proliferation H uman blood T lymphocytes have been used for many years in studies of cell proliferation (1, 2). These cells can be obtained directly from the circulation and therefore avoid the pitfalls associated with the use of cells in culture, which acquire confounding characteristics as a result of their in vitro environment (3). Interest has recently been rekindled in the metabolic changes that underpin cell proliferation in cancer to identify potential targets for chemotherapy (4,5). This has highlighted the need to carry out comparative studies on proliferating normal and tumor cells to ascertain whether an antimetabolic approach to cancer is possible without side effects related to the mechanism of action.The E3 ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C) attached to the activator protein Cdh1 plays a crucial role in controlling G1-to S-phase transition, and therefore proliferation, through the breakdown of cell cycle proteins (6, 7). APC/C-Cdh1 substrates are targeted for degradation through specific recognition motifs, including one known as the KEN box (8). Inactivation of APC/C-Cdh1 in G1 of the cell cycle is necessary for initiation of S phase, in which DNA is replicated and chromosomes are duplicated. We have recently found that APC/CCdh1 links cell cycle activity with that of the enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3 (PFKFB3) (9, 10). PFKFB3-a key regulator of glycolysis (11)-contains a KEN box motif and is thus also broken down by APC/C-Cdh1. Inactivation of APC/C-Cdh1 enables PFKFB3 to up-regulate glycolysis, thus providing the cell with the glucose essential for the subsequent biosynthesis of macromolecules. Our previous studies (9) were carried out in two cell lines; we therefore decided to investigate whether the same mechanis...

One of the many routes proposed for the cellular inactivation of endogenous nitric oxide (NO) is by the cytochrome c oxidase of the mitochondrial respiratory chain. We have studied this possibility in human embryonic kidney cells engineered to generate controlled amounts of NO. We have used visible light spectroscopy to monitor continuously the redox state of cytochrome c oxidase in an oxygentight chamber, at the same time as which we measure cell respiration and the concentrations of oxygen and NO. Pharmacological manipulation of cytochrome c oxidase indicates that this enzyme, when it is in turnover and in its oxidized state, inactivates physiological amounts of NO, thus regulating its intra-and extracellular concentrations. This inactivation is prevented by blocking the enzyme with inhibitors, including NO. Furthermore, when cells generating low concentrations of NO respire toward hypoxia, the redox state of cytochrome c oxidase changes from oxidized to reduced, leading to a decrease in NO inactivation. The resultant increase in NO concentration could explain hypoxic vasodilation.hypoxia ͉ mitochondrial respiration ͉ nitric oxide inactivation ͉ nitric oxide synthase D espite much research on its metabolic fate, the way in which the concentration of nitric oxide (NO) is regulated in cells and tissues is at present unresolved. Many routes for its inactivation have been discussed, including interaction with superoxide ions (1), hemoglobin (2-4) or myoglobin (5), accelerated autoxidation favored by partition within cell membranes (6), and interactions with free radicals derived from eicosanoid lipoxygenase (7), cyclo-oxygenase (8), different peroxidases (9), or catalase (10). Interactions with a flavohemoglobin-like NO dioxygenase (11-13) or with an unknown protein (14), and simple partitioning within mitochondrial membranes (15), have also been suggested.Before the discovery of NO as a biological mediator (16) it had been shown that isolated cytochrome c oxidase, the terminal enzyme in the mitochondrial electron transport chain, catalyzes both the oxidation and reduction of NO (17). More recent evidence has suggested that cytochrome c oxidase may provide a metabolic route for NO, either by interaction of NO with the reduced enzyme, leading to the formation of N 2 O, (18,19) or by interaction of NO with the oxidized enzyme, forming nitrite (NO 2 Ϫ ; 20, 21). Although the NO reductase activity of the enzyme is too slow to constitute a physiological mechanism for the removal of NO (22), there is strong evidence in favor of the oxidation of NO to NO 2 Ϫ both by the purified enzyme (23, 24) and by cells (25). Nevertheless, the possibility that cytochrome c oxidase constitutes a significant metabolic route for NO remains controversial (26, 27) and has been directly challenged (28).Clarifying the route(s) of the cellular inactivation of NO will be important for a fuller understanding of its biological functions. We have therefore investigated the role of cytochrome c oxidase in the metabolic fate of NO by using our met...

Nitric oxide (NO), generated endogenously in NO-synthase-transfected cells, increases the reduction of mitochondrial cytochrome c oxidase (CcO) at O2 concentrations ([O2]) above those at which it inhibits cell respiration. Thus, in cells respiring to anoxia, the addition of 2.5 μM L-arginine at 70 μM O2 resulted in reduction of CcO and inhibition of respiration at [O2] of 64.0±0.8 and 24.8±0.8 μM, respectively. This separation of the two effects of NO is related to electron turnover of the enzyme, because the addition of electron donors resulted in inhibition of respiration at progressively higher [O2], and to their eventual convergence. Our results indicate that partial inhibition of CcO by NO leads to an accumulation of reduced cytochrome c and, consequently, to an increase in electron flux through the enzyme population not inhibited by NO. Thus, respiration is maintained without compromising the bioenergetic status of the cell. We suggest that this is a physiological mechanism regulated by the flux of electrons in the mitochondria and by the changing ratio of O2:NO, either during hypoxia or, as a consequence of increases in NO, as a result of cell stress.

IMPORTANCEStudies suggest gut worms induce immune responses that can protect against multiple sclerosis (MS). To our knowledge, there are no controlled treatment trials with helminth in MS.OBJECTIVE To determine whether hookworm treatment has effects on magnetic resonance imaging (MRI) activity and T regulatory cells in relapsing MS.DESIGN, SETTING, AND PARTICIPANTS This 9-month double-blind, randomized, placebo-controlled trial was conducted between September 2012 and March 2016 in a modified intention-to-treat population (the data were analyzed June 2018) at the University of Nottingham, Queen's Medical Centre, a single tertiary referral center. Patients aged 18 to 61 years with relapsing MS without disease-modifying treatment were recruited from the MS clinic. Seventy-three patients were screened; of these, 71 were recruited (2 ineligible/ declined).INTERVENTIONS Patients were randomized (1:1) to receive either 25 Necator americanus larvae transcutaneously or placebo. The MRI scans were performed monthly during months 3 to 9 and 3 months posttreatment. MAIN OUTCOMES AND MEASURESThe primary end point was the cumulative number of new/enlarging T2/new enhancing T1 lesions at month 9. The secondary end point was the percentage of cluster of differentiation (CD) 4+CD25 high CD127 neg T regulatory cells in peripheral blood.RESULTS Patients (mean [SD] age, 45 [9.5] years; 50 women [71%]) were randomized to receive hookworm (35 [49.3%]) or placebo (36 [50.7%]). Sixty-six patients (93.0%) completed the trial. The median cumulative numbers of new/enlarging/enhancing lesions were not significantly different between the groups by preplanned Mann-Whitney U tests, which lose power with tied data (high number of zeroactivity MRIs in the hookworm group, 18/35 [51.4%] vs 10/36 [27.8%] in the placebo group). The percentage of CD4+CD25 high CD127 neg T cells increased at month 9 in the hookworm group (hookworm, 32 [4.4%]; placebo, 34 [3.9%]; P = .01). No patients withdrew because of adverse effects. There were no differences in adverse events between groups except more application-site skin discomfort in the hookworm group (82% vs 28%). There were 5 relapses (14.3%) in the hookworm group vs 11 (30.6%) receiving placebo.CONCLUSIONS AND RELEVANCE Treatment with hookworm was safe and well tolerated. The primary outcome did not reach significance, likely because of a low level of disease activity. Hookworm infection increased T regulatory cells, suggesting an immunobiological effect of hookworm. It appears that a living organism can precipitate immunoregulatory changes that may affect MS disease activity.

Multiple sclerosis (MS) is an immune-mediated inflammatory demyelinating disease of the central nervous system. It was previously shown that toll-like receptor (TLR)-2 signaling plays a key role in the murine experimental autoimmune encephalomyelitis (EAE) model of MS, and that TLR2-stimulation of regulatory T cells (Tregs) promotes their conversion to T helper 17 (Th17) cells. Here, we sought potential sources of TLR2 stimulation and evidence of TLR2 activity in MS patient clinical samples. Soluble TLR2 (sTLR2) was found to be significantly elevated in sera of MS patients (n = 21), in both relapse and remission, compared to healthy controls (HC) (n = 24). This was not associated with the acute phase reaction (APR) as measured by serum C-reactive protein (CRP) level, which was similarly increased in MS patients compared to controls. An independent validation cohort from a different ethnic background showed a similar upward trend in mean sTLR2 values in relapsing-remitting MS (RRMS) patients, and significant differences in sTLR2 values between patients and HC were preserved when the data from the two cohorts were pooled together (n = 41 RRMS and 44 HC, P = 0.0006). TLR2-stimulants, measured using a human embryonic kidney (HEK)-293 cells transfectant reporter assay, were significantly higher in urine of MS patients than HC. A screen of several common urinary tract infections (UTI)-related organisms showed strong induction of TLR2-signaling in the same assay. Taken together, these results indicate that two different markers of TLR2-activity—urinary TLR2-stimulants and serum sTLR2 levels—are significantly elevated in MS patients compared to HC.

We have developed a respiration chamber that allows intact cells to be studied under controlled oxygen (O(2)) conditions. The system measures the concentrations of O(2) and nitric oxide (NO) in the cell suspension, while the redox state of cytochrome c oxidase is continuously monitored optically. Using human embryonic kidney cells transfected with a tetracycline-inducible NO synthase we show that the inactivation of NO by cytochrome c oxidase is dependent on both O(2) concentration and electron turnover of the enzyme. At a high O(2) concentration (70 microM), and while the enzyme is in turnover, NO generated by the NO synthase upon addition of a given concentration of l-arginine is partially inactivated by cytochrome c oxidase and does not affect the redox state of the enzyme or consumption of O(2). At low O(2) (15 microM), when the cytochrome c oxidase is more reduced, inactivation of NO is decreased. In addition, the NO that is not inactivated inhibits the cytochrome c oxidase, further reducing the enzyme and lowering O(2) consumption. At both high and low O(2) concentrations the inactivation of NO is decreased when sodium azide is used to inhibit cytochrome c oxidase and decrease electron turnover.