From the Cover: Arginine ADP-ribosylation mechanism based on structural snapshots of iota-toxin and actin complex

Clostridium perfringens iota-toxin (Ia) mono-ADP ribosylates Arg177 of actin, leading to cytoskeletal disorganization and cell death. To fully understand the reaction mechanism of arginine-specific mono-ADP ribosyl transferase, the structure of the toxin-substrate protein complex must be characterized. Recently, we solved the crystal structure of Ia in complex with actin and the nonhydrolyzable NAD⁺ analog βTAD (thiazole-4-carboxamide adenine dinucleotide); however, the structures of the NAD⁺-bound form (NAD⁺-Ia-actin) and the ADP ribosylated form [Ia-ADP ribosylated (ADPR)-actin] remain unclear. Accidentally, we found that ethylene glycol as cryo-protectant inhibits ADP ribosylation and crystallized the NAD⁺-Ia-actin complex. Here we report high-resolution structures of NAD⁺-Ia-actin and Ia-ADPR-actin obtained by soaking apo-Ia-actin crystal with NAD⁺ under different conditions. The structures of NAD⁺-Ia-actin and Ia-ADPR-actin represent the pre- and postreaction states, respectively. By assigning the βTAD-Ia-actin structure to the transition state, the strain-alleviation model of ADP ribosylation, which we proposed previously, is experimentally confirmed and improved. Moreover, this reaction mechanism appears to be applicable not only to Ia but also to other ADP ribosyltransferases.     

Angiotensin-(1–7) stimulates hematopoietic progenitor cells in vitro and in vivo

Effects of angiotensin (Ang)-(1–7), an AngII metabolite, on bone marrow-derived hematopoietic cells were studied. We identified Ang-(1–7) to stimulate proliferation of human CD34⁺ and mononuclear cells in vitro. Under in vivo conditions, we monitored proliferation and differentiation of human cord blood mononuclear cells in NOD/SCID mice. Ang-(1–7) stimulated differentially human cells in bone marrow and accumulated them in the spleen. The number of HLA-I⁺ and CD34⁺ cells in the bone marrow was increased 42-fold and 600-fold, respectively. These results indicate a decisive impact of Ang-(1–7) on hematopoiesis and its promising therapeutic potential in diseases requiring progenitor stimulation.     

NAD+ and SIRT3 control microtubule dynamics and reduce susceptibility to antimicrotubule agents

Nicotinamide adenine dinucleotide (NAD⁺) is an endogenous enzyme cofactor and cosubstrate that has effects on diverse cellular and physiologic processes, including reactive oxygen species generation, mitochondrial function, apoptosis, and axonal degeneration. A major goal is to identify the NAD⁺-regulated cellular pathways that may mediate these effects. Here we show that the dynamic assembly and disassembly of microtubules is markedly altered by NAD⁺. Furthermore, we show that the disassembly of microtubule polymers elicited by microtubule depolymerizing agents is blocked by increasing intracellular NAD⁺ levels. We find that these effects of NAD⁺ are mediated by the activation of the mitochondrial sirtuin sirtuin-3 (SIRT3). Overexpression of SIRT3 prevents microtubule disassembly and apoptosis elicited by antimicrotubule agents and knockdown of SIRT3 prevents the protective effects of NAD⁺ on microtubule polymers. Taken together, these data demonstrate that NAD⁺ and SIRT3 regulate microtubule polymerization and the efficacy of antimicrotubule agents.Nicotinamide adenine dinucleotide (NAD⁺) is an endogenous dinucleotide that is present in the cytosol, nucleus, and mitochondria. Athough it serves an important role as a redox cofactor in metabolism, NAD⁺ is also a substrate for several families of enzymes, including the poly(ADP ribose) polymerases and the sirtuin deacetylase enzymes (reviewed in refs. 1 and 2). The level of intracellular NAD⁺ is regulated by many factors, including diet and energy status (3), axonal injury (4), DNA damage (5), and certain disease states (6), suggesting that NAD⁺-dependent signaling is dynamically modulated in diverse contexts.NAD⁺-dependent signaling can be induced by treatment of cells with exogenous NAD⁺, which increases intracellular NAD⁺ levels and results in diverse effects in cells and animals. These effects include enhanced oxygen consumption and ATP production (7), as well as protection from genotoxic stress and apoptosis (3). Mice treated with nicotinamide riboside, a NAD⁺ precursor that is metabolized into NAD⁺, have enhanced oxidative metabolism, increased insulin sensitivity, and protection from high-fat diet-induced obesity (8). These results demonstrate that NAD⁺-dependent pathways can enhance metabolic function and improve a variety of disease phenotypes.An NAD⁺-regulated pathway also inhibits axonal degeneration elicited by axonal transection (4). Treatment of axons with 5–20 mM NAD⁺ markedly delays the axon degenerative process (9). Additionally, animals that express the Wallerian degeneration slow (Wld^S) protein, a fusion of the NAD⁺ biosynthetic enzyme Nicotinamide mononucleotide adenylyl transferase 1 and Ube4a, exhibit markedly delayed degeneration of the distal axonal fragment after axonal transection (10), and expression of Wld^S mitigates disease phenotypes in several neurodegenerative disease models (11–14). Thus, understanding the intracellular pathways regulated by NAD⁺ may be important for understanding the pathogenesis of numerous disorders.Despite the diverse beneficial effects of genetically and pharmacologically augmenting NAD⁺ levels, the cellular processes that are affected by NAD⁺ treatment are incompletely understood. In this study, we show that microtubule dynamics and polymer stability are markedly influenced by NAD⁺ levels. We show that elevation of intracellular NAD⁺ levels markedly alters the stability of microtubule polymers in cells, and renders these polymers resistant to depolymerization elicited by antimicrotubule agents, such as vinblastine, nocodazole, and colchicine. We find that these effects are mediated by sirtuin-3 (SIRT3), a mitochondrial NAD⁺-dependent deacetylase, and that elevated SIRT3 levels also blocks the effects of antimicrotubule agents on the cytoskeleton. Furthermore, we find that both NAD⁺ and SIRT3 reduce the sensitivity of cells to the cytotoxic effects of vinblastine. Taken together, these data identify a new role for NAD⁺ and SIRT3 in regulating the effects of antimicrotubule agents, and link the actions of NAD⁺ to microtubule stabilization in cells.     

Apprehending the NAD+–ADPr-Dependent Systems in the Virus World

NAD⁺ and ADP-ribose (ADPr)-containing molecules are at the interface of virus–host conflicts across life encompassing RNA processing, restriction, lysogeny/dormancy and functional hijacking. We objectively defined the central components of the NAD⁺–ADPr networks involved in these conflicts and systematically surveyed 21,191 completely sequenced viral proteomes representative of all publicly available branches of the viral world to reconstruct a comprehensive picture of the viral NAD⁺–ADPr systems. These systems have been widely and repeatedly exploited by positive-strand RNA and DNA viruses, especially those with larger genomes and more intricate life-history strategies. We present evidence that ADP-ribosyltransferases (ARTs), ADPr-targeting Macro, NADAR and Nudix proteins are frequently packaged into virions, particularly in phages with contractile tails (Myoviruses), and deployed during infection to modify host macromolecules and counter NAD⁺-derived signals involved in viral restriction. Genes encoding NAD⁺–ADPr-utilizing domains were repeatedly exchanged between distantly related viruses, hosts and endo-parasites/symbionts, suggesting selection for them across the virus world. Contextual analysis indicates that the bacteriophage versions of ADPr-targeting domains are more likely to counter soluble ADPr derivatives, while the eukaryotic RNA viral versions might prefer macromolecular ADPr adducts. Finally, we also use comparative genomics to predict host systems involved in countering viral ADP ribosylation of host molecules.     

Cross-dressed CD8α+/CD103+ dendritic cells prime CD8+ T cells following vaccination

Activation of naïve cluster of differentiation (CD)8⁺ cytotoxic T lymphocytes (CTLs) is a tightly regulated process, and specific dendritic cell (DC) subsets are typically required to activate naive CTLs. Potential pathways for antigen presentation leading to CD8⁺ T-cell priming include direct presentation, cross-presentation, and cross-dressing. To distinguish between these pathways, we designed single-chain trimer (SCT) peptide–MHC class I complexes that can be recognized as intact molecules but cannot deliver antigen to MHC through conventional antigen processing. We demonstrate that cross-dressing is a robust pathway of antigen presentation following vaccination, capable of efficiently activating both naïve and memory CD8⁺ T cells and requires CD8α⁺/CD103⁺ DCs. Significantly, immune responses induced exclusively by cross-dressing were as strong as those induced exclusively through cross-presentation. Thus, cross-dressing is an important pathway of antigen presentation, with important implications for the study of CD8⁺ T-cell responses to viral infection, tumors, and vaccines.Professional antigen-presenting cells (APCs) are typically required to activate naïve cluster of differentiation (CD)8⁺ T cells, either by direct priming or cross-priming. In direct priming, infected (viral infection) or directly transfected (DNA vaccination) APCs synthesize the foreign antigen and use endogenous MHC class I pathways of antigen presentation to present antigen and prime CD8⁺ T cells. In cross-priming, APCs are able to capture, process, and present exogenous antigen onto MHC class I molecules through a process known as cross-presentation (1). Cross-priming has been shown to be an essential pathway for immunity to many viral infections and tumors. Although the pathways that lead to cross-presentation remain incompletely understood, increasing evidence suggests that only certain dendritic cell (DC) subsets are efficient in this process.Cross-dressing involves the transfer of intact MHC class I/peptide complexes between cells without the requirement for further processing, representing an alternative pathway of indirect antigen presentation (2, 3). Although cross-dressed DCs can activate memory CD8⁺ T cells following viral infection in vivo (4), it remains unclear whether cross-dressing can prime naïve CD8⁺ T-cell responses, what DC subtypes are required to prime CD8⁺ T cells by cross-dressing, and how robust this pathway is compared with traditional pathways of indirect antigen presentation. These questions must be addressed before the physiologic relevance of cross-dressing can be evaluated in context.To address these questions, we have taken advantage of Batf3-deficient mice and engineered MHC class I single chain trimer (SCT) constructs. Batf3^−/− mice have a selective loss of CD8α⁺ and CD103⁺ DCs, without abnormalities in other hematopoietic cell types or architecture (5). DCs from Batf3^−/− mice are deficient in cross-presentation, and cytotoxic T lymphocyte (CTL) responses to viral infection and syngeneic tumors are impaired in Batf3^−/− mice. Thus, Batf3^−/− mice represent a valuable model system to study cross-presentation, cross-dressing, and the role of CD8α⁺/CD103⁺ DCs following DNA or cellular vaccination. We have previously engineered completely assembled MHC class I SCT whereby all three components of the complex (heavy chain, β₂m, and peptide) are attached by flexible linkers (6). Through progressive molecular engineering, even peptides with low binding affinities can be successfully anchored in the peptide binding groove by a disulfide trap between the first linker and the heavy chain (7–9). Using these experimental tools, we demonstrate that cross-dressing is a robust pathway of antigen presentation following DNA and cellular vaccination, capable of priming naïve and memory CD8⁺ T cells. In addition, we demonstrate that CD8α⁺/CD103⁺ DCs are required to prime CTLs by cross-dressing.     

In vivo NAD assay reveals the intracellular NAD contents and redox state in healthy human brain and their age dependences

NAD is an essential metabolite that exists in NAD⁺ or NADH form in all living cells. Despite its critical roles in regulating mitochondrial energy production through the NAD⁺/NADH redox state and modulating cellular signaling processes through the activity of the NAD⁺-dependent enzymes, the method for quantifying intracellular NAD contents and redox state is limited to a few in vitro or ex vivo assays, which are not suitable for studying a living brain or organ. Here, we present a magnetic resonance (MR) -based in vivo NAD assay that uses the high-field MR scanner and is capable of noninvasively assessing NAD⁺ and NADH contents and the NAD⁺/NADH redox state in intact human brain. The results of this study provide the first insight, to our knowledge, into the cellular NAD concentrations and redox state in the brains of healthy volunteers. Furthermore, an age-dependent increase of intracellular NADH and age-dependent reductions in NAD⁺, total NAD contents, and NAD⁺/NADH redox potential of the healthy human brain were revealed in this study. The overall findings not only provide direct evidence of declined mitochondrial functions and altered NAD homeostasis that accompany the normal aging process but also, elucidate the merits and potentials of this new NAD assay for noninvasively studying the intracellular NAD metabolism and redox state in normal and diseased human brain or other organs in situ.NAD, a multifunctional metabolite found in all living cells, has been the interest of many scientific investigations since its discovery in the early 20th century (1). NAD is known to convert between its oxidized NAD⁺ and reduced NADH forms during the breakdown of nutrients; hence, the intracellular NAD⁺/NADH redox state reflects the metabolic balance of the cell in generating ATP energy through oxidative phosphorylation in mitochondria and/or glycolysis in cytosol (2). More recently, after several protein families associated with cell survival were found to use NAD⁺ as their main substrate with activities also regulated by the availability of the NAD⁺, the full extent of the NAD’s function as a metabolic regulator began to unfold (3–5). A growing number of studies have indicated that NAD⁺ can modulate metabolic signaling pathways and mediate important cellular processes, including calcium homeostasis, gene expression, aging, degeneration, and cell death; therefore, the cellular NAD could serve as a therapeutic target for treating various metabolic or age-related diseases and promoting longevity (6–12).Despite the critical relevance of the intracellular NAD metabolism to human health and diseases, assessment of NAD contents and NAD⁺/NADH redox state is extremely challenging. Only a few invasive techniques based on biochemical assays or autofluorescence methods have been used to analyze tissue samples or cell extracts (13, 14). However, during the preparation of such ex vivo sample, the NAD⁺ and NADH contents are likely altered, because they are highly sensitive to pH, temperature, light, and chemical agent or buffer solution. Thus, accurate quantification and extrapolation to in vivo conditions are difficult, even with the well-established biochemical assays (15). In addition, NAD⁺ does not fluoresce and thus, cannot be detected by fluorometry (16). Clearly, a nondestructive detection and quantification method is highly desired to investigate the NAD contents and redox state in the human body or intact animal models.Recently, we have developed a novel magnetic resonance (MR) -based quantification approach that uses a high-field MR scanner to obtain the endogenous ³¹P MR signals of the NAD molecules in intact animal brains (17). Distinct from earlier ³¹P MR spectroscopy (MRS) studies that reported total NAD contents (18–20), our approach is able to resolve the MR signal of NADH from that of NAD⁺ by taking advantage of their specific spectroscopic characteristics at a given magnetic field strength that can be precisely predicted based on a theoretical model (17). Thus, both NAD⁺ and NADH can be quantified simultaneously by matching the in vivo NAD spectra with the model-simulated spectra. It has been shown that this approach works well in animal brains at ultrahigh fields of 9.4 and 16.4 T (17). In this study, we further exploit the feasibility and potential of this novel approach for noninvasive assessment of intracellular NAD⁺ and NADH contents and NAD⁺/NADH redox state in healthy human brains using a 7-T human MR scanner and ultimately, studying their roles in human aging.     

Effects of 2-deoxyglucose and dehydroepiandrosterone on intracellular NAD<Superscript>+</Superscript> level,SIRT1 activity and replicative lifespan of human Hs68 cells

2-Deoxy-d-glucose (2-DG) and dehydroepiandrosterone (DHEA) have been hypothesized to extend lifespan via mimicking calorie restriction (CR). Activation of sirtuins has been proposed to contribute to life extension of CR by increasing intercellular levels of NAD⁺ in several organisms. However, it is unclear whether 2-DG and DHEA may affect intracellular NAD⁺ levels and human sirtuin 1 (SIRT1) activities. Here, using human fibroblast Hs68 cells we showed that 2-DG increased intracellular NAD⁺ levels in both time- and concentration-dependent manners. 2-DG also dose-dependently increased SIRT1 activities and the lifespan (measured as the cumulated growth curve of population doubling levels) of Hs68 cells. In contrast, DHEA at non-cytotoxic concentrations (≤50 μM) did not significantly affect NAD⁺ levels, SIRT1 activities or the lifespan of Hs68 cells. These results suggest that 2-DG extends the lifespan of Hs68 cells by increased NAD⁺ levels and SIRT1 activities, and that 2-DG has a potential as a CR mimetic.     

NAD+ supplementation reduces neuroinflammation and cell senescence in a transgenic mouse model of Alzheimer’s disease via cGAS–STING

Alzheimer''s disease (AD) is a progressive and fatal neurodegenerative disorder. Impaired neuronal bioenergetics and neuroinflammation are thought to play key roles in the progression of AD, but their interplay is not clear. Nicotinamide adenine dinucleotide (NAD⁺) is an important metabolite in all human cells in which it is pivotal for multiple processes including DNA repair and mitophagy, both of which are impaired in AD neurons. Here, we report that levels of NAD⁺ are reduced and markers of inflammation increased in the brains of APP/PS1 mutant transgenic mice with beta-amyloid pathology. Treatment of APP/PS1 mutant mice with the NAD⁺ precursor nicotinamide riboside (NR) for 5 mo increased brain NAD⁺ levels, reduced expression of proinflammatory cytokines, and decreased activation of microglia and astrocytes. NR treatment also reduced NLRP3 inflammasome expression, DNA damage, apoptosis, and cellular senescence in the AD mouse brains. Activation of cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) are associated with DNA damage and senescence. cGAS–STING elevation was observed in the AD mice and normalized by NR treatment. Cell culture experiments using microglia suggested that the beneficial effects of NR are, in part, through a cGAS–STING-dependent pathway. Levels of ectopic (cytoplasmic) DNA were increased in APP/PS1 mutant mice and human AD fibroblasts and down-regulated by NR. NR treatment induced mitophagy and improved cognitive and synaptic functions in APP/PS1 mutant mice. Our findings suggest a role for NAD⁺ depletion-mediated activation of cGAS–STING in neuroinflammation and cellular senescence in AD.

Alzheimer’s disease (AD) is the most feared neurodegenerative disease and is characterized by progressive cognitive impairment associated with extensive accumulation of amyloid β-peptide (Aβ) plaques and tau neurofibrillary tangles in vulnerable brain regions (1, 2). There are no available treatments. Neuroinflammation, mitochondrial dysfunction, and cellular senescence have been recognized as key drivers of AD (3–6). Microglia are the primary innate immune cells in the brain. Although accumulating evidence challenges the simplified M1-M2 phenotypes of microglia, the classification is still widely in use as microglia can be protective (M2) and detrimental (M1) under different circumstances (7, 8). The detrimental microglia are activated by Aβ and produce interleukin (IL)-1β, TNF-α, and other proinflammatory molecules. The protective microglia secrete the anti-inflammatory cytokines IL-4 and IL-10 (8).The inflammatory response is one of the hallmarks of cellular senescence (9). The senescence-associated secretory phenotype (SASP) includes cessation of cell division and the production of proinflammatory cytokines (10). The SASP is highly correlated with neuroinflammation and has been documented in the brains of AD mouse models (11). During normal aging, the number of senescent cells in tissues increases significantly (10), and evidence suggests that microglia, astrocytes, and oligodendrocyte progenitor cells can become senescent in AD (5, 11, 12). Moreover, senolytic treatments can preserve cognitive function in AD mice (11). However, the mechanisms that result in neuroinflammation and cell senescence in AD remain unclear.Nicotinamide adenine dinucleotide (NAD⁺) plays a central role in cellular metabolism and is also critical for maintaining mitochondrial homeostasis and genome integrity (13). Emerging evidence has identified lower levels of NAD⁺ in affected tissues in many neurodegenerative diseases, including AD (13–16). Supplementation with the NAD⁺ precursor nicotinamide riboside (NR) efficiently increases NAD⁺ and can have beneficial effects on many AD features in mouse models (14). The cyclic GMP-AMP synthase (cGAS)–STING (stimulator of interferon genes) DNA-sensing pathway detects the presence of cytosolic DNA and triggers expression of inflammatory genes that lead to senescence or to the activation of defense mechanisms (17–20). Here, we explore the mechanisms by which NAD⁺ supplementation reduces neuroinflammation and cell senescence in AD. Our results suggest that the cGAS–STING pathway is a therapeutic target for AD.     

Activation of plant quinate:NAD 3-oxidoreductase by Ca and calmodulin

Quinate:NAD⁺ 3-oxidoreductase (EC 1.1.1.24) from carrot cell suspension cultures has previously been shown to be activated by phosphorylation and inactivated by dephosphorylation. Here it is shown that the reactivation of the inactivated quinate:NAD⁺ oxidoreductase is an enzyme-mediated process that requires ATP and protein kinase activity. The reactivation is completely inhibited by EGTA and can be restored by the addition of Ca²⁺. Cyclic AMP at concentrations up to 5 μM did not have any effect on the reactivation either with or without EGTA in the medium. Calmodulin-depleted fractions containing quinate:NAD⁺ oxidoreductase were obtained by passage of the crude extracts through an affinity column of 2-chloro-10-(3-aminopropyl)phenothiazine coupled to Sepharose 4B. The enzyme in this calmodulin-deficient fraction could be inactivated but not reactivated even in the presence of ATP and Ca²⁺. However, addition of bovine brain calmodulin completely restored the activity of the enzyme. Half-maximal activation occurred at 130 nM calmodulin. We conclude from these data that the quinate:NAD⁺ oxidoreductase is activated by a Ca²⁺ - and calmodulin-dependent plant protein kinase.     

Synthesis and structural characterization of isolable phosphine coinage metal ��-complexes

The chemical community has recently witnessed a dramatic increase in the application of cationic gold(I)-phosphine complexes as homogeneous catalysts for organic synthesis. The majority of gold(I)-catalyzed reactions rely on nucleophilic additions to carbon–carbon multiple bonds, which have been activated by coordination to a cationic gold(I) catalyst. However, structural evidence for coordination of cationic gold(I) complexes to alkynes has been limited. Here, we report the crystal structure of a gold(I)-phosphine η²-coordinated alkyne. Related Ag(I) and Cu(I) complexes have been synthesized for comparison. The crystallization of these complexes was enabled by tethering a labile alkyne ligand to a strongly coordinating triarylphosphine. This approach also proved applicable to crystallization of the first gold(I)-phosphine η²-coordinated alkene.     

PD-L1-deficient mice show that PD-L1 on T cells, antigen-presenting cells, and host tissues negatively regulates T cells

Both positive and negative regulatory roles have been suggested for the B7 family member PD-L1(B7-H1). PD-L1 is expressed on antigen-presenting cells (APCs), activated T cells, and a variety of tissues, but the functional significance of PD-L1 on each cell type is not yet clear. To dissect the functions of PD-L1 in vivo, we generated PD-L1-deficient (PD-L1^–/–) mice. CD4⁺ and CD8⁺ T cell responses were markedly enhanced in PD-L1^–/– mice compared with wild-type mice in vitro and in vivo. PD-L1^–/– dendritic cells stimulated greater wild-type CD4⁺ T cell responses than wild-type dendritic cells, and PD-L1^–/– CD4⁺ T cells produced more cytokines than wild-type CD4⁺ T cells in vitro, demonstrating an inhibitory role for PD-L1 on APCs and T cells. In vivo CD8⁺ T cell responses also were significantly enhanced, indicating that PD-L1 has a role in limiting the expansion or survival of CD8⁺ T cells. Studies using the myelin oligodendrocyte model of experimental autoimmune encephalomyelitis showed that PD-L1 on T cells and in host tissues limits responses of self-reactive CD4⁺ T cells in vivo. PD-L1 deficiency converted the 129S4/SvJae strain from a resistant to experimental autoimmune encephalomyelitis-susceptible strain. Transfer of encephalitogenic T cells from wild-type mice into PD-L1^–/– recipients led to exacerbated disease. Disease was even more severe in PD-L1^–/– recipients of PD-L1^–/– T cells. These results demonstrate that PD-L1 on T cells, APCs, and host tissue inhibits naïve and effector T cell responses and plays a critical role in T cell tolerance.PD-L1 (B7-H1) is a ligand for programmed cell death-1 (PD-1) and does not bind to other CD28 family members (1). PD-1 is expressed on activated but not resting CD4⁺ and CD8⁺ T, B, and myeloid cells (2, 3). PD-1^–/– mice develop an autoimmune-like phenotype, which is delayed in onset as compared with CTLA-4^–/– mice (4, 5). This phenotype demonstrates an important negative regulatory role for PD-1 and suggests a role for PD-1 in regulating B and/or T cell tolerance.PD-L1 is expressed on resting and up-regulated on activated B, T, myeloid, and dendritic cells (DCs) (1, 6–9). In contrast to B7–1 and B7–2, PD-L1 also is expressed in nonhematopoietic cells (e.g., microvascular endothelial cells), in nonlymphoid organs (e.g., heart and placenta), and in a variety of tumors (6, 8, 10–12). The expression of PD-L1 within nonlymphoid tissues suggests that PD-L1 may regulate self-reactive T or B cells in peripheral tissues and/or may regulate inflammatory responses in the target organs. However, the roles of PD-L1 on T cells, antigen-presenting cells (APCs), and host tissues are not yet clear. Many potential bidirectional interactions occur between PD-L1 and PD-1 because of the broad expression of PD-L1 and the expression of PD-1 on T cells, B cells, and macrophages. Recent studies using anti-PD-L1 mAbs in vivo have suggested a role for PD-L1 in regulating autoimmune diseases (13, 14). However, these studies could not distinguish the importance of PD-L1 expression on T cells, APCs, and host cells. The function of PD-L1 is also unclear because of conflicting results, with some studies suggesting a stimulatory role and others an inhibitory role (1, 6, 11). To determine the obligatory functions of PD-L1 in vivo, we generated PD-L1-deficient (PD-L1^–/–) mice. Our results indicate that PD-L1 in the T cell, APC, and host tissue plays a critical role in negatively regulating T cell responses.     

Can a single subunit yeast NADH dehydrogenase (Ndi1) remedy diseases caused by respiratory complex I defects?

The proton-translocating NADH-quinone oxidoreductase (complex I) is one of five enzyme complexes in the oxidative phosphorylation system in mammalian mitochondria. Complex I is composed of 46 different subunits, 7 of which are encoded by mitochondrial DNA. Defects of complex I are involved in many human mitochondrial diseases; therefore, the authors proposed to use the NDI1 gene encoding a single subunit NADH dehydrogenase of Saccharomyces cerevisiae for repair of respiratory activity. The yeast NDI1 gene was successfully introduced into 10 mammalian cell lines (two of which were complex I-deficient mutants). The expressed Ndi1 protein was correctly targeted to the matrix side of the inner mitochondrial membranes, was fully functional, and restored the NADH oxidase activity to the complex I-deficient cells. The NDI1-transduced cells were more resistant to complex I inhibitors and diminished production of reactive oxygen species. It was further shown that the Ndi1 protein can be functionally expressed in tissues such as skeletal muscles and brain of rodents. The Ndi1 expression scarcely induced an inflammatory response as assessed by hematoxylin and eosin (H&E) staining. The Ndi1 protein expressed in the substantia nigra (SN) elicited protective effects against neurodegeneration caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment. The Ndi1 protein has a great potential as a molecular remedy for complex I deficiencies.     

Structures of RNA 3'-phosphate cyclase bound to ATP reveal the mechanism of nucleotidyl transfer and metal-assisted catalysis

RNA 3′-phosphate cyclase (RtcA) synthesizes RNA 2′,3′ cyclic phosphate ends via three steps: reaction with ATP to form a covalent RtcA-(histidinyl-Nϵ)-AMP intermediate; transfer of adenylate to an RNA 3′-phosphate to form RNA(3′)pp(5′)A; and attack of the vicinal O2′ on the 3′-phosphorus to form a 2′,3′ cyclic phosphate and release AMP. Here we report the crystal structures of RtcA•ATP, RtcA•ATP•Mn²⁺, and RtcA•ATP•Co²⁺ substrate complexes and an RtcA•AMP product complex. Together with the structures of RtcA apoenzyme and the covalent RtcA–AMP intermediate, they illuminate the mechanism of nucleotidyl transfer, especially the stereochemical transitions at the AMP phosphate, the critical role of the metal in orienting the PP_i leaving group of ATP during step 1, and the protein conformational switches that accompany substrate binding and product release. The octahedral metal complex of RtcA•ATP•Mn²⁺ includes nonbridging oxygens from each of the ATP phosphates, two waters, and Glu14 as the sole RtcA component. Whereas the RtcA adenylylation step is metal-catalyzed, the subsequent steps in the cyclization pathway are metal-independent.     

Sirtuin 1 in rat orthotopic liver transplantation:An I GL-1 preservation solution approach

AIM:To investigate the possible involvement of Sirtuin1(SIRT1)in rat orthotopic liver transplantation(OLT),when Institute Georges Lopez 1(IGL-1)preservation solution is enriched with trimetazidine(TMZ).METHODS:Male Sprague-Dawley rats were used as donors and recipients.Livers were stored in IGL-1 preservation solution for 8h at 4℃,and then underwent OLT according to Kamada’s cuff technique without arterialization.In another group,livers were stored in IGL-1 preservation solution supplemented with TMZ,at10-6 mol/L,for 8 h at 4℃and then underwent OLT.Rats were sacrificed 24 h after reperfusion,and liver and plasma samples were collected.Liver injury(transaminase levels),mitochondrial damage(glutamate dehydrogenase activity)oxidative stress(malondialdehyde levels),and nicotinamide adenine dinucleotide(NAD+),the cofactor necessary for SIRT1 activity,were determined by biochemical methods.SIRT1 and its substrates(acFox O1,ac-p53),the precursor of NAD+,nicotinamide phosphoribosyltransferase(NAMPT),as well as the phosphorylation of adenosine monophosphate activated protein kinase(AMPK),p-m TOR,p-p70S6K(direct substrate of m TOR),autophagy parameters(beclin-1,LC3B)and MAP kinases(p-p38 and p-ERK)were determined by Western blot.RESULTS:Liver grafts preserved in IGL-1 solution enriched with TMZ presented reduced liver injury and mitochondrial damage compared with those preservedin IGL-1 solution alone.In addition,livers preserved in IGL-1+TMZ presented reduced levels of oxidative stress.This was consistent with enhanced SIRT1 protein expression and elevated SIRT1 activity,as indicated by decreased acetylation of p53 and Fox O1.The elevated SIRT1 activity in presence of TMZ can be attributed to the enhanced NAMPT protein and NAD+/NADH levels.Up-regulation of SIRT1 was consistent with activation of AMPK and inhibition of phosphorylation of m TOR and its direct substrate(p-p70S6K).As a consequence,autophagy mediators(beclin-1 and LC3B)were overexpressed.Furthermore,MAP kinases were regulated in livers preserved with IGL-1+TMZ,as they were characterized by enhanced p-ERK and decreased p-p38protein expression.CONCLUSION:Our study shows that IGL-1 preservation solution enriched with TMZ protects liver grafts from the IRI associated with OLT,through SIRT1 up-regulation.     

Poly(ADP-ribose) polymerase-dependent energy depletion occurs through inhibition of glycolysis

Excessive poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) activation kills cells via a cell-death process designated “parthanatos” in which PAR induces the mitochondrial release and nuclear translocation of apoptosis-inducing factor to initiate chromatinolysis and cell death. Accompanying the formation of PAR are the reduction of cellular NAD⁺ and energetic collapse, which have been thought to be caused by the consumption of cellular NAD⁺ by PARP-1. Here we show that the bioenergetic collapse following PARP-1 activation is not dependent on NAD⁺ depletion. Instead PARP-1 activation initiates glycolytic defects via PAR-dependent inhibition of hexokinase, which precedes the NAD⁺ depletion in N-methyl-N-nitroso-N-nitroguanidine (MNNG)-treated cortical neurons. Mitochondrial defects are observed shortly after PARP-1 activation and are mediated largely through defective glycolysis, because supplementation of the mitochondrial substrates pyruvate and glutamine reverse the PARP-1–mediated mitochondrial dysfunction. Depleting neurons of NAD⁺ with FK866, a highly specific noncompetitive inhibitor of nicotinamide phosphoribosyltransferase, does not alter glycolysis or mitochondrial function. Hexokinase, the first regulatory enzyme to initiate glycolysis by converting glucose to glucose-6-phosphate, contains a strong PAR-binding motif. PAR binds to hexokinase and inhibits hexokinase activity in MNNG-treated cortical neurons. Preventing PAR formation with PAR glycohydrolase prevents the PAR-dependent inhibition of hexokinase. These results indicate that bioenergetic collapse induced by overactivation of PARP-1 is caused by PAR-dependent inhibition of glycolysis through inhibition of hexokinase.Pharmacological inhibition or genetic deletion of poly(ADP-ribose) polymerase-1 (PARP-1) is dramatically protective against a variety of toxic insults including ischemia reperfusion injury in the heart, brain, and other organs (1, 2). PARP-1 activation also may play a role in several neurologic disorders such as Parkinson disease (PD), Alzheimer''s disease (AD), autoimmune encephalomyelitis, and multiple sclerosis (3–5). PARP-1 activation plays a prominent role in necrotic cell death; the necrotic cell-death program initiated by PARP-1 activation has been designated “parthanatos” to distinguish it from other forms of cell death (6, 7). Cell death via PARP-1 activation is thought to occur through the formation of poly(ADP-ribose) (PAR), which acts as a death signal to cause the release of apoptosis-inducing factor (AIF) from the mitochondria (8–10). AIF then translocates to the nucleus, causing nuclear condensation, genomic DNA fragmentation, and cell death (9, 10).One of key features of parthanatos is consumption of NAD⁺ caused by parylation, the addition of PAR on PARP-1 itself and ribosylation of PARP-1 substrates (6, 11). Accompanying the NAD⁺-dependent parylation is a drop in cellular ATP levels and metabolic collapse that have been ascribed to the consumption of NAD⁺ and subsequent restoration of NAD⁺ levels, requiring four ATP molecules for each NAD⁺ molecule (12). Early studies suggested that the decrements in NAD⁺ and ATP led to cell death through energy collapse (12–14). However, many studies have challenged this notion, because cell death has been shown to be independent of the loss of cellular energy stores (11, 15, 16) but dependent on PAR signaling (8–11). In cells lacking the PAR-degradative enzyme PAR glycohydrolase (PARG), activation of PARP-1 leads to cell death that does not require NAD⁺ depletion. Instead, cell death occurs through PAR activation of parthanatos (17, 18). In addition, reductions in ATP levels have been shown to precede the reduction in NAD⁺, thus challenging the idea that the reduction in ATP is caused by the consumption of NAD⁺ (15, 16). Thus the initiator of the mechanism underlying the decrements in ATP after PARP-1 activation is not known. Here we show that that the bioenergetic collapse after PARP-1 activation is not dependent on NAD⁺ depletion but instead is caused by the PAR-dependent inhibition of glycolysis that occurs through the inhibition of hexokinase (HK). This study suggests that pharmacological interventions independent of NAD⁺ are potential targets to prevent bioenergetic defects and cell death in diseases that involve PARP-1 activation.     

Restoration of electron transport without proton pumping in mammalian mitochondria

We have restored the CoQ oxidative capacity of mouse mtDNA-less cells (ρ° cells) by transforming them with the alternative oxidase Aox of Emericella nidulans. Cotransforming ρ° cells with the NADH dehydrogenase of Saccharomyces cerevisiae, Ndi1 and Aox recovered the NADH DH/CoQ reductase and the CoQ oxidase activities. CoQ oxidation by AOX reduces the dependence of ρ° cells on pyruvate and uridine. Coexpression of AOX and NDI1 further improves the recycling of NAD⁺. Therefore, 2 single-protein enzymes restore the electron transport in mammalian mitochondria substituting >80 nuclear DNA-encoded and 11 mtDNA-encoded proteins. Because those enzymes do not pump protons, we were able to split electron transport and proton pumping (ATP synthesis) and inquire which of the metabolic deficiencies associated with the loss of oxidative phosphorylation should be attributed to each of the 2 processes.