[
J Biol Chem,
1995]
The Caenorhabditis elegans Unc-13 protein is a novel member of the phorbol ester receptor family having a single cysteine-rich region with high homology to those present in protein kinase C (PKC) isozymes and the chimaerins, We expressed the cysteine-rich region of Unc-13 in Escherichia coli and quantitatively analyzed its interactions with phorbol esters and related analogs, its phospholipid requirements, and its inhibitor sensitivity, [H-3]Phorbol 12,13-dibutyrate [H-3]PDBu bound with high affinity to the cysteine-rich region of Unc-13 (K-d = 1.3 +/- 0.2 nM). This affinity is similar to that of other single cysteine-rich regions from PKC isozymes as well as n-chimaerin, As also described for PKC isozymes and n-chimaerin, Unc-13 bound diacylglycerol with an affinity about 2 orders of magnitude weaker than [H-3]PDBu. Structure activity analysis revealed significant but modest differences between recombinant cysteine-rich regions of Unc-13 and PKC delta. In addition, Unc-13 required slightly higher concentrations of phospholipid for reconstitution of [H-3]PDBu binding. Calphostin C, a compound described as a selective inhibitor of PKC, was also able to inhibit [H-3]PDBu binding to Unc-13, suggesting that this inhibitor is not able to distinguish between different classes of phorbol ester receptors, In conclusion, although our results revealed some differences in ligand and lipid cofactor sensitivities, Unc-13 represents a high affinity cellular target for the phorbol esters as well as for the lipid second messenger diacylglycerol, at least in C. elegans, The use of phorbol esters or some ''specific'' antagonists of PKC does not distinguish between cellular pathways involving different PKC isozymes or novel phorbol ester receptors such as n-chimaerin or Unc-13.
Relini A, Tortora P, Gatta E, De Gioia L, Airoldi C, Natalello A, Vertemara J, Visentin C, Penco A, Pellistri F, Regonesi ME, Bonanomi M
[
Hum Mol Genet,
2017]
The protein ataxin-3 (ATX3) triggers an amyloid-related neurodegenerative disease when its polyglutamine stretch is expanded beyond a critical threshold. We formerly demonstrated that the polyphenol epigallocatechin-3-gallate (EGCG) could redirect amyloid aggregation of a full-length, expanded ATX3 (ATX3-Q55) towards non-toxic, soluble, SDS-resistant aggregates. Here, we have characterized other related phenol compounds, although smaller in size, i.e., (-)-epigallocatechin gallate (EGC), and gallic acid (GA). We analyzed the aggregation pattern of ATX3-Q55 and of the N-terminal globular Josephin domain (JD) by assessing the time course of the soluble protein, as well its structural features by FTIR and AFM, in the presence and the absence of the mentioned compounds. All of them redirected the aggregation pattern towards soluble, SDS-resistant aggregates. They also prevented the appearance of ordered side-chain hydrogen bonding in ATX3-Q55, which is the hallmark of polyQ-related amyloids. Molecular docking analyses on the JD highlighted three interacting regions, including the central, aggregation-prone one. All three compounds bound to each of them, although with different patterns. This might account for their capability to prevent amyloidogenesis. Saturation transfer difference NMR experiments also confirmed EGCG and EGC binding to monomeric JD. ATX3-Q55 pre-incubation with any of the three compound prevented its calcium-influx-mediated cytotoxicity towards neural cells. Finally, all the phenols significantly reduced toxicity in a transgenic Caenorhabditis elegans strain expressing an expanded ATX3. Overall, our results show that the three polyphenols act in a substantially similar manner. GA, however, might be more suitable for antiamyloid treatments due to its simpler structure and higher chemical stability.