Ketone Supplementation Dampens Subjective and Objective Responses to Alcohol: Evidence From a Preclinical Rat Study and a Randomized, Cross-Over Trial in Healthy Volunteers.

BACKGROUND: Previous preclinical and human studies have shown that a high-fat ketogenic diet and ketone supplements (KS) are efficacious in reducing alcohol craving, alcohol consumption, and signs of alcohol withdrawal. However, the effects of KS on alcohol sensitivity are unknown. METHODS: In this single-blind, cross-over study, 10 healthy participants (3 females) were administered a single, oral dose of a KS (25 g of ketones from D-ß-hydroxybutyric acid and R-1,3-butanediol) or placebo 30 minutes before an oral alcohol dose (0.25 g/kg for women; 0.31 g/kg for men). Assessments of breath alcohol concentration and blood alcohol levels (BAL) and responses on the Drug Effect Questionnaire were repeatedly obtained over 180 minutes after alcohol consumption. In a parallel preclinical study, 8 Wistar rats (4 females) received an oral gavage of KS (0.42 g ketones/kg), water, or the sweetener allulose (0.58 g/kg) followed 15 minutes later by an oral alcohol dose (0.8 g/kg). BAL was monitored for 240 minutes after alcohol exposure. RESULTS: In humans, the intake of KS before alcohol significantly blunted breath alcohol concentration and BAL, reduced ratings of alcohol liking and wanting more, and increased disliking for alcohol. In rats, KS reduced BAL more than either allulose or water. CONCLUSION: KS altered physiological and subjective responses to alcohol in both humans and rats, and the effects were likely not mediated by the sweetener allulose present in the KS drink. Therefore, KS could potentially reduce the intoxicating effects of alcohol.

Hippocampal volume loss in individuals with a history of non-fatal opioid overdose.

Incidence of opioid-related overdoses in the United States has increased dramatically over the past two decades. Despite public emphasis on overdose fatalities, most overdose cases are not fatal. Although there are case reports of amnestic syndromes and acute injury to the hippocampus following non-fatal opioid overdose, the effects of such overdoses on brain structure are poorly understood. Here, we investigated the neuroanatomical correlates of non-fatal opioid overdoses by comparing hippocampal volume in opioid use disorder (OUD) patients who had experienced an opioid overdose (OD; N = 17) with those who had not (NOD; N = 32). Voxel-based morphometry showed lower hippocampal volume in the OD group than in the NOD group, which on post hoc analysis was evident in the left but not the right hippocampus. These findings strengthen the evidence that hippocampal injury is associated with non-fatal opioid overdose, which is hypothesized to underlie overdose-related amnestic syndrome.

Oligomerization of the HECT ubiquitin ligase NEDD4-2/NEDD4L is essential for polyubiquitin chain assembly.

The NEDD4-2 (neural precursor cell-expressed developmentally down-regulated 4-2) HECT ligase catalyzes polyubiquitin chain assembly by an ordered two-step mechanism requiring two functionally distinct E2∼ubiquitin-binding sites, analogous to the trimeric E6AP/UBE3A HECT ligase. This conserved catalytic mechanism suggests that NEDD4-2, and presumably all HECT ligases, requires oligomerization to catalyze polyubiquitin chain assembly. To explore this hypothesis, we examined the catalytic mechanism of NEDD4-2 through the use of biochemically defined kinetic assays examining rates of 125I-labeled polyubiquitin chain assembly and biophysical techniques. The results from gel filtration chromatography and dynamic light-scattering analyses demonstrate for the first time that active NEDD4-2 is a trimer. Homology modeling to E6AP revealed that the predicted intersubunit interface has an absolutely conserved Phe-823, substitution of which destabilized the trimer and resulted in a ≥104-fold decrease in kcat for polyubiquitin chain assembly. The small-molecule Phe-823 mimic, N-acetylphenylalanyl-amide, acted as a noncompetitive inhibitor (Ki = 8 ± 1.2 mm) of polyubiquitin chain elongation by destabilizing the active trimer, suggesting a mechanism for therapeutically targeting HECT ligases. Additional kinetic experiments indicated that monomeric NEDD4-2 catalyzes only HECT∼ubiquitin thioester formation and monoubiquitination, whereas polyubiquitin chain assembly requires NEDD4-2 oligomerization. These results provide evidence that the previously identified sites 1 and 2 of NEDD4-2 function in trans to support chain elongation, explicating the requirement for oligomerization. Finally, we identified a conserved catalytic ensemble comprising Glu-646 and Arg-604 that supports HECT-ubiquitin thioester exchange and isopeptide bond formation at the active-site Cys-922 of NEDD4-2.

The mechanism of neural precursor cell expressed developmentally down-regulated 4-2 (Nedd4-2)/NEDD4L-catalyzed polyubiquitin chain assembly.

The mechanism of Nedd4-2 has been quantitatively explored for the first time using biochemically defined kinetic assays examining rates of 125I-polyubiquitin chain assembly as a functional readout. We demonstrate that Nedd4-2 exhibits broad specificity for E2 paralogs of the Ubc4/5 clade to assemble Lys63-linked polyubiquitin chains. Full-length Nedd4-2 catalyzes free 125I-polyubiquitin chain assembly by hyperbolic Michaelis-Menten kinetics with respect to Ubc5B∼ubiquitin thioester concentration (Km = 44 ± 6 nm; kcat = 0.020 ± 0.007 s-1) and substrate inhibition above 0.5 µm (Ki = 2.5 ± 1.3 µm) that tends to zero velocity, requiring ordered binding at two functionally distinct E2∼ubiquitin-binding sites. The Ubc5BC85A product analog non-competitively inhibits Nedd4-2 (Ki = 2.0 ± 0.5 µm), consistent with the presence of the second E2-binding site. In contrast, the isosteric Ubc5BC85S-ubiquitin oxyester substrate analog exhibits competitive inhibition at the high-affinity Site 1 (Ki = 720 ± 340 nm) and non-essential activation at the lower-affinity Site 2 (Kact = 750 ± 260 nm). Additional studies utilizing Ubc5BF62A, defective in binding the canonical E2 site, demonstrate that the cryptic Site 1 is associated with thioester formation, whereas binding at the canonical site (Site 2) is associated with polyubiquitin chain elongation. Finally, previously described Ca2+-dependent C2 domain-mediated autoinhibition of Nedd4-2 is not observed under our reported experimental conditions. These studies collectively demonstrate that Nedd4-2 catalyzes polyubiquitin chain assembly by an ordered two-step mechanism requiring two dynamically linked E2∼ubiquitin-binding sites analogous to that recently reported for E6AP, the founding member of the Hect ligase family.

Neurotoxic mechanisms by which the USP14 inhibitor IU1 depletes ubiquitinated proteins and Tau in rat cerebral cortical neurons: Relevance to Alzheimer's disease.

In Alzheimer's disease proteasome activity is reportedly downregulated, thus increasing it could be therapeutically beneficial. The proteasome-associated deubiquitinase USP14 disassembles polyubiquitin-chains, potentially delaying proteasome-dependent protein degradation. We assessed the protective efficacy of inhibiting or downregulating USP14 in rat and mouse (Usp14axJ) neuronal cultures treated with prostaglandin J2 (PGJ2). IU1 concentrations (HIU1>25µM) reported by others to inhibit USP14 and be protective in non-neuronal cells, reduced PGJ2-induced Ub-protein accumulation in neurons. However, HIU1 alone or with PGJ2 is neurotoxic, induces calpain-dependent Tau cleavage, and decreases E1~Ub thioester levels and 26S proteasome assembly, which are energy-dependent processes. We attribute the two latter HIU1 effects to ATP-deficits and mitochondrial Complex I inhibition, as shown herein. These HIU1 effects mimic those of mitochondrial inhibitors in general, thus supporting that ATP-depletion is a major mediator of HIU1-actions. In contrast, low IU1 concentrations (LIU1≤25µM) or USP14 knockdown by siRNA in rat cortical cultures or loss of USP14 in cortical cultures from ataxia (Usp14axJ) mice, failed to prevent PGJ2-induced Ub-protein accumulation. PGJ2 alone induces Ub-protein accumulation and decreases E1~Ub thioester levels. This seemingly paradoxical result may be attributed to PGJ2 inhibiting some deubiquitinases (such as UCH-L1 but not USP14), thus triggering Ub-protein stabilization. Overall, IU1-concentrations that reduce PGJ2-induced accumulation of Ub-proteins are neurotoxic, trigger calpain-mediated Tau cleavage, lower ATP, E1~Ub thioester and E1 protein levels, and reduce proteasome activity. In conclusion, pharmacologically inhibiting (with low or high IU1 concentrations) or genetically down-regulating USP14 fail to enhance proteasomal degradation of Ub-proteins or Tau in neurons.

Convergent evolution in the assembly of polyubiquitin degradation signals by the Shigella flexneri IpaH9.8 ligase.

The human pathogen Shigella flexneri subverts host function and defenses by deploying a cohort of effector proteins via a type III secretion system. The IpaH family of 10 such effectors mimics ubiquitin ligases but bears no sequence or structural homology to their eukaryotic counterpoints. Using rates of (125)I-polyubiquitin chain formation as a functional read out, IpaH9.8 displays V-type positive cooperativity with respect to varying concentrations of its Ubc5B∼(125)I-ubiquitin thioester co-substrate in the nanomolar range ([S]½ = 140 ± 32 nm; n = 1.8 ± 0.1) and cooperative substrate inhibition at micromolar concentrations ([S]½ = 740 ± 240 nm; n = 1.7 ± 0.2), requiring ordered binding to two functionally distinct sites per subunit. The isosteric substrate analog Ubc5BC85S-ubiquitin oxyester acts as a competitive inhibitor of wild-type Ubc5B∼(125)I-ubiquitin thioester (Ki = 117 ± 29 nm), whereas a Ubc5BC85A product analog shows noncompetitive inhibition (Ki = 2.2 ± 0.5 µm), consistent with the two-site model. Re-evaluation of a related IpaH3 crystal structure (PDB entry 3CVR) identifies a symmetric dimer consistent with the observed cooperativity. Genetic disruption of the predicted IpaH9.8 dimer interface reduces the solution molecular weight and significantly ablates the kcat but not [S]½ for polyubiquitin chain formation. Other studies demonstrate that cooperativity requires the N-terminal leucine-rich repeat-targeting domain and is transduced through Phe(395). Additionally, these mechanistic features are conserved in a distantly related SspH2 Salmonella enterica ligase. Kinetic parallels between IpaH9.8 and the recently revised mechanism for E6AP/UBE3A (Ronchi, V. P., Klein, J. M., and Haas, A. L. (2013) E6AP/UBE3A ubiquitin ligase harbors two E2∼ubiquitin binding sites. J. Biol. Chem. 288, 10349-10360) suggest convergent evolution of the catalytic mechanisms for prokaryotic and eukaryotic ligases.