Mutations in the mitochondrial protein kinase PINK1 are associated with autosomal recessive Parkinson disease (PD). We and other groups have reported that PINK1 activates Parkin E3 ligase activity both directly via phosphorylation of Parkin serine 65 (Ser65)—which lies within its ubiquitin-like domain (Ubl)—and indirectly through phosphorylation of ubiquitin at Ser65. How Ser65-phosphorylated ubiquitin (ubiquitinPhospho-Ser65) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitinPhospho-Ser65 binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser65 by PINK1 in vitro. Analysis of the Parkin structure, corroborated by site-directed mutagenesis, shows that the conserved His302 and Lys151 residues play a critical role in binding of ubiquitinPhospho-Ser65, thereby promoting Parkin Ser65 phosphorylation and activation of its E3 ligase activity in vitro. Mutation of His302 markedly inhibits Parkin Ser65 phosphorylation at the mitochondria, which is associated with a marked reduction in its E3 ligase activity following mitochondrial depolarisation. We show that the binding of ubiquitinPhospho-Ser65 to Parkin disrupts the interaction between the Ubl domain and C-terminal region, thereby increasing the accessibility of Parkin Ser65. Finally, purified Parkin maximally phosphorylated at Ser65 in vitro cannot be further activated by the addition of ubiquitinPhospho-Ser65. Our results thus suggest that a major role of ubiquitinPhospho-Ser65 is to promote PINK1-mediated phosphorylation of Parkin at Ser65, leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho-Ser65-binding pocket on the surface of Parkin that is critical for the ubiquitinPhospho-Ser65 interaction. This study provides new mechanistic insights into Parkin activation by ubiquitinPhospho-Ser65, which could aid in the development of Parkin activators that mimic the effect of ubiquitinPhospho-Ser65.

No abstract

A human recombinant homo‐oligomeric 5‐HT3 receptor (h5‐HT3A) expressed in a human embryonic kidney cell line (HEK 293) was characterized using the whole‐cell recording configuration of the patch clamp technique. 5‐HT evoked transient inward currents (EC50= 3.4 μM; Hill coefficient = 1.8) that were blocked by the 5‐HT3 receptor antagonist ondansetron (IC50= 103 pM) and by the non‐selective agents metoclopramide (IC50= 69 nM), cocaine (IC50= 459 nM) and (+)‐tubocurarine (IC50= 2.8 μM). 5‐HT‐induced currents rectified inwardly and reversed in sign (E5‐HT) at a potential of −2.2 mV. N‐Methyl‐D‐glucamine was finitely permeant. Permeability ratios PNa/PCs and PNMDG/PCs were 0.90 and 0.083, respectively. Permeability towards divalent cations was assessed from measurements of E5‐HT in media where Ca2+ and Mg2+ replaced Na+. PCa/PCs and PMg/PCs were calculated to be 1.00 and 0.61, respectively. Single channel chord conductance (γ) estimated from fluctuation analysis of macroscopic currents increased with membrane hyperpolarization from 243 fS at −40 mV to 742 fS at −100 mV. Reducing [Ca2+]o from 2 to 0.1 mM caused an increase in the whole‐cell current evoked by 5‐HT. A concomitant reduction in [Mg2+]o produced further potentiation. Fluctuation analysis indicates that a voltage‐independent augmentation of γ contributes to this phenomenon. The data indicate that homo‐oligomeric receptors composed of h5‐HT3A subunits form inwardly rectifying cation‐selective ion channels of low conductance that are permeable to Ca2+ and Mg2+.

No abstract

Matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometry has become a promising alternative for high-throughput drug discovery as new instruments offer high speed, flexibility and sensitivity, and the ability to measure physiological substrates label free. Here we developed and applied high-throughput MALDI TOF mass spectrometry to identify inhibitors of the salt-inducible kinase (SIK) family, which are interesting drug targets in the field of inflammatory disease as they control production of the anti-inflammatory cytokine interleukin-10 (IL-10) in macrophages. Using peptide substrates in in vitro kinase assays, we can show that hit identification of the MALDI TOF kinase assay correlates with indirect ADP-Hunter kinase assays. Moreover, we can show that both techniques generate comparable IC50 data for a number of hit compounds and known inhibitors of SIK kinases. We further take these inhibitors to a fluorescence-based cellular assay using the SIK activity-dependent translocation of CRTC3 into the nucleus, thereby providing a complete assay pipeline for the identification of SIK kinase inhibitors in vitro and in cells. Our data demonstrate that MALDI TOF mass spectrometry is fully applicable to high-throughput kinase screening, providing label-free data comparable to that of current high-throughput fluorescence assays.

No abstract

Of the 16 non-structural proteins (Nsps) encoded by SARS CoV-2, Nsp3 is the largest and plays important roles in the viral life cycle. Being a large, multidomain, transmembrane protein, Nsp3 has been the most challenging Nsp to characterize. Encoded within Nsp3 is the papain-like protease domain (PLpro) that cleaves not only the viral polypeptide but also K48-linked polyubiquitin and the ubiquitin-like modifier, ISG15, from host cell proteins. We here compare the interactors of PLpro and Nsp3 and find a largely overlapping interactome. Intriguingly, we find that near full length Nsp3 is a more active protease compared to the minimal catalytic domain of PLpro. Using a MALDI-TOF based assay, we screen 1971 approved clinical compounds and identify five compounds that inhibit PLpro with IC50s in the low micromolar range but showed cross reactivity with other human deubiquitinases and had no significant antiviral activity in cellular SARS-CoV-2 infection assays. We therefore looked for alternative methods to block PLpro activity and engineered competitive nanobodies that bind to PLpro at the substrate binding site with nanomolar affinity thus inhibiting the enzyme. Our work highlights the importance of studying Nsp3 and provides tools and valuable insights to investigate Nsp3 biology during the viral infection cycle.

1 The g-aminobutyric acid (GABA)-modulatory and GABA-mimetic actions of etomidate at mammalian GABA A receptors are favoured by b 2 -or b 3 -versus b 1 -subunit containing receptors, a selectivity which resides with a single transmembrane amino acid (b 2 N290 , b 3 N289 , b 1 S290 ). Here, we have utilized the Xenopus laevis oocyte expression system in conjunction with the two-point voltage clamp technique to determine the in¯uence of the equivalent amino acid (M314) on the actions of this anaesthetic at an etomidate-insensitive invertebrate GABA receptor (Rdl) of Drosophila melanogaster. 2 Complementary RNA-injected oocytes expressing the wild type Rdl GABA receptor and voltageclamped at 760 mV responded to bath applied GABA with a concentration-dependent inward current response and a calculated EC 50 for GABA of 20+0.4 mM. Receptors in which the transmembrane methionine residue (M314) had been exchanged for an asparagine (Rdl M314N ) or a serine (Rdl M314S ) also exhibited a concentration-dependent inward current response to GABA, but in both cases with a reduced EC 50 of 4.8+0.2 mM. 3 Utilizing the appropriate GABA EC 10 , etomidate (300 mM) had little e ect on the agonist-evoked current of the wild type Rdl receptor. By contrast, at Rdl M314N receptors, etomidate produced a clear concentration-dependent enhancement of GABA-evoked currents with a calculated EC 50 of 64+3 mM and an E max of 68+2% (of the maximum response to GABA). 4 The actions of etomidate at Rdl M314N receptors exhibited an enantioselectivity common to that found for mammalian receptors, with 100 mM R-(+)-etomidate and S- (7)-etomidate enhancing the current induced by GABA (EC 10 ) to 52+6% and 12+1% of the GABA maximum respectively. 5 The e ects of this mutation were selective for etomidate as the GABA-modulatory actions of 1 mM pentobarbitone at wild type Rdl (49+4% of the GABA maximum) and Rdl M314N receptors (53+2% of the GABA maximum) were similar. Additionally, the modest potentiation of GABA produced by the anaesthetic neurosteroid 5a-pregnan-3a-ol-20-one (Rdl=25+4% of the GABA maximum) was not altered by this mutation (Rdl M314N =18+3% of the GABA maximum). 6 Etomidate acting at b 1 (S290)-containing mammalian GABA A receptors is known to produce only a modest GABA-modulatory e ect. Similarly, etomidate acting at Rdl M314S receptors produced an enhancement of GABA but the magnitude of the e ect was reduced compared to Rdl M314N receptors. 7 Etomidate acting at human a 6 b 3 g 2L receptors is known to produce a large enhancement of GABAevoked currents and at higher concentrations this anaesthetic directly activates the GABA A receptor complex. Mutation of the human b 3 subunit asparagine to methionine (b 3 N289M found in the equivalent position in Rdl completely inhibited both the GABA-modulatory and GABA-mimetic action of etomidate (10 ± 300 mM) acting at a 6 b 3 N289M g 2L receptors. 8 It was concluded that, although invertebrate and mammalian proteins exhibit limited sequence homology, allosteric modi®cation of their function by et...