The mechanism of Nedd4-2 has been quantitatively explored for the first time using biochemically defined kinetic assays examining rates of I-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 Lys-linked polyubiquitin chains. Full-length Nedd4-2 catalyzes free I-polyubiquitin chain assembly by hyperbolic Michaelis-Menten kinetics with respect to Ubc5B∼ubiquitin thioester concentration ( = 44 ± 6 nm; = 0.020 ± 0.007 s) and substrate inhibition above 0.5 μm ( = 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 ( = 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 ( = 720 ± 340 nm) and non-essential activation at the lower-affinity Site 2 ( = 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 Ca-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.
Background: IpaH bacterial ubiquitin ligases show no homology with eukaryotic ligases, and their mechanism is speculative. Results: IpaH9.8 functions as a cooperative allosteric dimer with two Ubc5ϳubiquitin binding sites per subunit. Conclusion: Kinetic parallels between IpaH and eukaryotic HECT ligases suggest convergent catalytic cycle evolution. Significance: These are the first mechanistic details of the IpaH enzyme catalytic mechanism.
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 >25 μM) 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 ≤25 μM) 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.
Edited by George N. DeMartino 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 E2ϳubiquitin-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 125 I-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 >10 4-fold decrease in k cat for polyubiquitin chain assembly. The small-molecule Phe-823 mimic, N-acetylphenylalanyl-amide, acted as a noncompetitive inhibitor (K i ؍ 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 HECTϳubiquitin 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.
SummarySea anemones discharge cnidae (‘stinging capsules’ including nematocysts) to capture prey and to defend themselves. In the present study, we tested the relationship between the force of test probes striking feeding tentacles and discharge of microbasic p-mastigophore nematocysts into the test probes. In seawater alone, the response curve is bimodal with maximal discharge observed at 0.33 and 1.10 millinewtons (mN) and with minimal discharge at 1.50 mN. Upon activating chemoreceptors for N-acetylated sugars, maximal discharge is observed across a broad range of smaller forces from 0.16 to 0.9 mN before decreasing to a minimum at 1.50 mN. Likewise, in the presence of nearby vibrations at key frequencies, maximal discharge is observed over a broad range of smaller forces before decreasing to a minimum at 1.50 mN. It appears that sensory input indicating proximity of potential prey expands the range of small forces of impact that stimulate maximal discharge (i.e. to less than 1.10 mN) but not at larger forces of impact (i.e. at approximately 1.50 mN). Thus, contact by small prey would stimulate maximal discharge, and all the more so if such contact is accompanied by specific odorants or by vibrations at specific frequencies. Nevertheless, anemones would not maximally discharge nematocysts into large animals that blunder into contact with their tentacles.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.