Highlights d aS impacts lipid homeostasis, triggering excess oleic acid (OA) and diglycerides (DG) d Triglycerides and lipid droplets protect against toxicity by sequestering OA and DG d Stearoyl-CoA desaturase (SCD) inhibition rescues aS toxicity and neuron degeneration d SCD inhibition decreases aS inclusions and increases aS multimerization and solubility

PSD-95, a principal scaffolding component of the post-synaptic density, is targeted to synapses by palmitoylation where it couples NMDA receptor stimulation to production of nitric oxide (NO) by neuronal nitric oxide synthase (nNOS). Here, we show that PSD-95 is physiologically S-nitrosylated. We identify cysteines 3 and 5, which are palmitoylated, as sites of nitrosylation, suggesting a competition between these two modifications. In support of this hypothesis, physiologically produced NO inhibits PSD-95 palmitoylation in granule cells of the cerebellum, decreasing the number of PSD-95 clusters at synaptic sites. Further, decreased palmitoylation, as seen in heterologous cells treated with 2-bromopalmitate or in ZDHHC8 knockout mice deficient in a PSD-95 palmitoyltransferase, results in increased PSD-95 nitrosylation. These data support a model in which NMDA mediated production of NO regulates targeting of PSD-95 to synapses via mutually competitive cysteine modifications. Thus, differential modification of cysteines may represent a general paradigm in signal transduction.

The Fanconi anemia (FA) pathway is a DNA damage-activated signaling pathway which regulates cellular resistance to DNA cross-linking agents. Cloned FA genes and proteins cooperate in this pathway, and monoubiquitination of FANCD2 is a critical downstream event. The cell cycle checkpoint kinase ATR is required for the efficient monoubiquitination of FANCD2, while another checkpoint kinase, ATM, directly phosphorylates FANCD2 and controls the ionizing radiation (IR)-inducible intra-S-phase checkpoint. In the present study, we identify two novel DNA damage-inducible phosphorylation sites on FANCD2, threonine 691 and serine 717. ATR phosphorylates FANCD2 on these two sites, thereby promoting FANCD2 monoubiquitination and enhancing cellular resistance to DNA cross-linking agents. Phosphorylation of the sites is required for establishment of the intra-S-phase checkpoint response. IR-inducible phosphorylation of threonine 691 and serine 717 is also dependent on ATM and is more strongly impaired when both ATM and ATR are knocked down. Threonine 691 is phosphorylated during normal S-phase progression in an ATM-dependent manner. These findings further support the functional connection of ATM/ATR kinases and FANCD2 in the DNA damage response and support a role for the FA pathway in the coordination of the S phase of the cell cycle.Fanconi anemia (FA) is an autosomal recessive, or X-linked, cancer susceptibility disorder characterized by cellular hypersensitivity to DNA cross-linking agents, such as mitomycin C (MMC) (11, 12). The 11 cloned FA genes (A, B, C, D1, D2, E, F, G, L, M, and J) cooperate in a novel DNA damage response pathway, the FA pathway (6). Eight of the FA proteins (A, B, C, E, F, G, L, and M) are subunits of a nuclear E3 ubiquitin ligase required for monoubiquitination of the downstream FANCD2 protein on lysine 561, which is a critical step for the function of the FA pathway (8). The FANCL subunit is the putative catalytic E3 ligase subunit of the complex (19). Monoubiquitinated D2 interacts with FANCD1/BRCA2 (36). The recently cloned FANCJ protein is a helicase (3,15,16,18) which may work in concert with FANCD2-Ub and BRCA2 or may function independently of the FA pathway. Disruption of any of the proteins in the pathway leads to MMC hypersensitivity and chromosome instability.Increasing evidence supports a role for the FA pathway during normal S-phase progression in mammalian cells. First, the FA complex is associated with chromatin during S phase and interacts with the helicase BLM, suggesting that the complex plays a "sensor" role at the advancing replication fork (21, 22) Second, the newly cloned FANCM subunit of the FA complex appears to promote its chromatin association during S phase (20,26). Third, the FA complex activates FANCD2 monoubiquitination during S phase, leading to the association of monoubiquitinated FANCD2 with BRCA1 and RAD51 in replication foci (34). Fourth, the FA pathway is required for normal homologous recombination repair, a mode of DNA repair executed in S phase (28). Finally, t...

Microscopy of Lewy bodies in Parkinson’s disease (PD) suggests they are not solely filamentous deposits of α-synuclein (αS) but also contain vesicles and other membranous material. We previously reported the existence of native αS tetramers/multimers and described engineered mutations of the αS KTKEGV repeat motifs that abrogate the multimers. The resultant excess monomers accumulate in lipid membrane-rich inclusions associated with neurotoxicity exceeding that of natural familial PD mutants, such as E46K. Here, we use the αS “3K” (E35K+E46K+E61K) engineered mutation to probe the mechanisms of reported small-molecule modifiers of αS biochemistry and then identify compounds via a medium-throughput automated screen. αS 3K, which forms round, vesicle-rich inclusions in cultured neurons and causes a PD-like, l-DOPA–responsive motor phenotype in transgenic mice, was fused to YFP, and fluorescent inclusions were quantified. Live-cell microscopy revealed the highly dynamic nature of the αS inclusions: for example, their rapid clearance by certain known modulators of αS toxicity, including tacrolimus (FK506), isradipine, nilotinib, nortriptyline, and trifluoperazine. Our automated 3K cellular screen identified inhibitors of stearoyl-CoA desaturase (SCD) that robustly prevent the αS inclusions, reduce αS 3K neurotoxicity, and prevent abnormal phosphorylation and insolubility of αS E46K. SCD inhibition restores the E46K αS multimer:monomer ratio in human neurons, and it actually increases this ratio for overexpressed wild-type αS. In accord, conditioning 3K cells in saturated fatty acids rescued, whereas unsaturated fatty acids worsened, the αS phenotypes. Our cellular screen allows probing the mechanisms of synucleinopathy and refining drug candidates, including SCD inhibitors and other lipid modulators.

Serine racemase (SR) generates D-serine, a coagonist with glutamate at NMDA receptors. We show that SR is physiologically S-nitrosylated leading to marked inhibition of enzyme activity. Inhibition involves interactions with the cofactor ATP reflecting juxtaposition of the ATP-binding site and cysteine-113 (C113), the site for physiological S-nitrosylation. NMDA receptor physiologically enhances SR S-nitrosylation by activating neuronal nitricoxide synthase (nNOS) . These findings support a model whereby postsynaptic stimulation of nitric-oxide (NO) formation feeds back to presynaptic cells to S-nitrosylate SR and decrease D-serine availability to postsynaptic NMDA receptors.neuronal nitric-oxide synthase ͉ NMDA receptor ͉ S-nitrosylation G lutamate neurotransmission through NMDA receptors requires a coagonist originally thought to be glycine. Recent studies indicate that in most portions of the brain, D-serine is the physiological coagonist because selective degradation of D-serine but not glycine markedly reduces NMDA transmission (1, 2), whereas retraction of D-serine-producing glia in the hypothalamus of lactating rats also diminishes NMDA transmission (2). D-serine is formed from L-serine by serine racemase (SR), which, like D-serine, is selectively enriched in glia (2, 3), although recent studies indicate some neuronal localization (4). SR, a pyridoxal phosphate-requiring enzyme, also displays an absolute requirement for ATP, which is not hydrolyzed during SR activation (5). SR binds the glutamate receptor interacting protein, which also binds to AMPA subtypes of glutamate receptors with glutamate receptor interacting protein markedly activating SR and providing a means whereby glutamate stimulation of SR-containing cells augments D-serine formation (6).In postsynaptic cells, NMDA signaling is mediated in part by neuronal nitric-oxide synthase (nNOS) because calcium entering through NMDA receptor channels binds to calmodulin associated with nNOS (7,8). Extensive studies have documented a feedback from postsynaptic to presynaptic glutamatergic nerve terminals, which modulates NMDA neurotransmission, especially in long-term potentiation (9). Nitric oxide (NO) may be a retrograde messenger of long-term potentiation (10-12), although the area is controversial (12). Because SR is a component of the NMDA synaptic complex, we wondered whether it is influenced by NO. In the present study, we demonstrate that SR is physiologically S-nitrosylated leading to inhibition of enzyme activity mediated by interactions with ATP. NMDA transmission stimulates SR S-nitrosylation suggesting a feedback mechanism to diminish presynaptic formation of D-serine. ResultsWe demonstrated S-nitrosylation of SR in multiple ways. Incubation of the NO donor S-nitroso-glutathione (GSNO) with SR in vitro leads to robust nitrosylation (Fig. 1A). In HEK293 cells, treatment with the NO donor sodium nitroprusside also provides S-nitrosylation (Fig. 1B). NO produced by nNOS S-nitrosylates SR, as is evident in HEK293 cells transfected with nNOS (Fi...

The cellular response to DNA damage is composed of cell cycle checkpoint and DNA repair mechanisms that serve to ensure proper replication of the genome prior to cell division. The function of the DNA damage response during DNA replication in S-phase is critical to this process. Recent evidence has suggested a number of interrelationships of DNA replication and cellular DNA damage responses. These include S-phase checkpoints which suppress replication initiation or elongation in response to DNA damage. Also, many components of the DNA damage response are required either for the stabilization of, or for restarting, stalled replication forks. Further, translesion synthesis permits DNA replication to proceed in the presence of DNA damage and can be coordinated with subsequent repair by homologous recombination (HR). Finally, cohesion of sister chromatids is established coincident with DNA replication and is required for subsequent DNA repair by homologous recombination. Here we review these processes, all of which occur at, or are related to, the advancing replication fork. We speculate that these multiple interdependencies of DNA replication and DNA damage responses integrate the many steps necessary to ensure accurate duplication of the genome.

Adipogenesis, the conversion of precursor cells into adipocytes, is associated with obesity and is mediated by glucocorticoids acting via hitherto poorly characterized mechanisms. Dexras1 is a small G protein of the Ras family discovered on the basis of its marked induction by the synthetic glucocorticoid dexamethasone. We show that Dexras1 mediates adipogenesis and diet-induced obesity. Adipogenic differentiation of 3T3-L1 cells is abolished with Dexras1 depletion, whereas overexpression of Dexras1 elicits adipogenesis. Adipogenesis is markedly reduced in mouse embryonic fibroblasts from Dexras1-deleted mice, whereas adiposity and dietinduced weight gain are diminished in the mutant mice.insulin | cyclic AMP | nitric oxide | Cushing disease

Genetic and biochemical evidence attributes neuronal loss in Parkinson’s disease (PD) and related brain diseases to dyshomeostasis of the 14 kDa protein α-synuclein (αS). There is no consensus on how αS exerts toxicity. Explanations range from disturbed vesicle biology to proteotoxicity caused by fibrillar aggregates. To probe these mechanisms further, robust cellular toxicity models are needed, but their availability is limited. We previously reported that a shift from dynamic multimers to monomers is an early event in αS dyshomeostasis, as caused by familial PD (fPD)-linked mutants such as E46K. Excess monomers accumulate in round, lipid-rich inclusions. Engineered αS ‘3K’ (E35K+E46K+E61K) amplifies E46K, causing a PD-like, L-DOPA-responsive motor phenotype in transgenic mice. Here, we present a cellular model of αS neurotoxicity after transducing human neuroblastoma cells to express yellow fluorescent protein (YFP)-tagged αS 3K in a doxycycline-dependent manner. αS-3K::YFP induction causes pronounced growth defects that accord with cell death. We tested candidate compounds for their ability to restore growth, and stearoyl-CoA desaturase (SCD) inhibitors emerged as a molecule class with growth-restoring capacity, but the therapeutic window varied among compounds. The SCD inhibitor MF-438 fully restored growth while exerting no apparent cytotoxicity. Our αS bioassay will be useful for elucidating compound mechanisms, for pharmacokinetic studies, and for compound/genetic screens.