Mechanistic insight into protein modification and sulfur mobilization activities of noncanonical E1 and associated ubiquitin‐like proteins of Archaea

Abstract

Here we provide the first detailed biochemical study of a noncanonical E1-like enzyme with broad specificity for cognate ubiquitin-like (Ubl) proteins that mediates Ubl protein modification and sulfur mobilization to form molybdopterin and thiolated tRNA. Isothermal titration calorimetry and in vivo analyses proved useful in discovering that environmental conditions, ATP binding and Ubl type controlled the mechanism of association of the Ubl protein with its cognate E1-like enzyme (SAMP and UbaA of the archaeon Haloferax volcanii, respectively). Further analysis revealed ATP hydrolysis triggered the formation of thioester and peptide bonds within the Ubl:E1-like complex. Importantly, the thioester was an apparent precursor to Ubl protein modification but not sulfur mobilization. Comparative modeling to MoeB/ThiF guided the discovery of key residues within the adenylation domain of UbaA that were needed to bind ATP as well as residues that were specifically needed to catalyze the downstream reactions of sulfur mobilization and/or Ubl protein modification. UbaA was also found to be Ubl-automodified at lysine residues required for early (ATP binding) and late (sulfur mobilization) stages of enzyme activity revealing multiple layers of auto-regulation. Cysteine residues, distinct from the canonical E1 ‘active site’ cysteine, were found important in UbaA function supporting a model that this non-canonical E1 is structurally flexible in its active site to allow Ubl~adenylate, Ubl~E1-like thioester and cysteine persulfide(s) intermediates to form.

High expression of ubiquitin-conjugating enzyme E2A predicts poor prognosis in hepatocellular carcinoma

“…However, accurate indicators for the prognosis of HCC are limited. Consequently, there is an urgency to develop a novel biomarker for the prognosis of HCC.The ubiquitin system serves a critical role in numerous cellular events, including the cell cycle regulation, DNA repair, stress responses, metabolic homeostasis, organelle biosynthesis and apoptosis (16,17). A recent study demonstrated that the hepatitis C virus NS3 protein enhanced HCC cell invasion by promoting protein phosphatase magnesium-dependent 1A ubiquitination and degradation (18), which indicated degradation and ubiquitination of proteins were involed in …”

In vitro Analysis of Ubiquitin-like Protein Modification in Archaea

“…Haloferax volcanii LR03 ( Δsamp1 Δsamp2 Δsamp3 ΔmsrA ΔubaA ) carrying plasmid pJAM3010 (a plasmid encoding msrA-strepII under control of the P2 rrnA constitutive promoter) (Fu et al , 2017) or pJAM1209 (a plasmid encoding his 6 -ubaA under control of the P2 rrnA constitutive promoter) (Hepowit et al , 2016) or without carrying any plasmid…”

“…Escherichia coli Rosetta (DE3) (Novagen, Merck, catalog number: 70954-3) carrying plasmid pJAM3200 (a plasmid encoding msrA-strepII under control of T7 promoter) from the Maupin-Furlow lab (Fu et al , 2017) or plasmid pJAM1132 (a plasmid encoding flag-his 6 -samp2 under control of T7 promoter) from the Maupin-Furlow lab (Hepowit et al , 2016)…”

Biosynthesis of Sulfur-Containing tRNA Modifications: A Comparison of Bacterial, Archaeal, and Eukaryotic Pathways

“…This is based on the observations that (i) the ncs6 gene homologs are widespread in archaeal genomes [8]; (ii) the deletion of the ncs6 homolog ( ncsA ) in Haloferax volcanii resulted in only non-thiolated tRNA Lys UUU [71]; (iii) Ncs6 homologs form complexes with the ubiquitin-like small archaeal modifier protein (SAMP), which has high structural homology to Urm1, in H. volcanii [72] and in M. maripaludis [51]; (iv) the H. volcanii E1-like protein UbaA activates SAMP in formation of a thioester intermediate [73]; (v) the deletion of either samp2 or ubaA in H. volcanii eliminated thiolated tRNA Lys UUU [52]; and (vi) the M. maripaludis Ncs6 homolog has a [3Fe-4S] cluster [46]. These findings suggest that both an Fe–S cluster containing Ncs6 homolog and an activated ubiquitin-like protein are required for s 2 U34 formation in Archaea.…”