- Calcium/calmodulin-dependent protein kinase II δ (CaMKIIδ) has a pivotal role in cardiac signaling. Constitutive and deleterious CaMKII “autonomous” activation is induced by oxidative stress, and the previously reported mechanism involves oxidation of methionine residues in the regulatory domain. Here, we demonstrate that covalent oxidation leads to a disulfide bond with Cys273 in the regulatory domain causing autonomous activity. Autonomous activation was induced by treating CaMKII with diamide or histamine chloramine, two thiol-oxidizing agents.
- The evolutionarily conserved bacterial proteins MnmE and MnmG collectively install a carboxymethylaminomethyl (cmnm) group at the fifth position of wobble uridines of several tRNA species. While the reaction catalyzed by MnmEG is one of the central steps in the biosynthesis of the methylaminomethyl (mnm) posttranscriptional tRNA modification, details of the reaction remain elusive. Glycine is known to be the source of the carboxy methylamino moiety of cmnm, and a tetrahydrofolate (THF) analog is thought to supply the one carbon that is appended to the fifth position of U.
- The MqsRA toxin-antitoxin system is a component of the Escherichia coli stress response. Free MqsR, a ribonuclease, cleaves mRNAs containing a 5′-GC-3′ sequence causing a global shutdown of translation and the cell to enter a state of dormancy. Despite a general understanding of MqsR function, the molecular mechanism(s) by which MqsR binds and cleaves RNA and how one or more of these activities is inhibited by its cognate antitoxin MqsA is still poorly understood. Here, we used NMR spectroscopy coupled with mRNA cleavage assays to identify the molecular mechanism of MqsR substrate recognition and the MqsR residues that are essential for its catalytic activity.
- The pyrrolysyl-tRNA synthetase (PylRS) facilitates the cotranslational installation of the 22nd amino acid pyrrolysine. Owing to its tolerance for diverse amino acid substrates, and its orthogonality in multiple organisms, PylRS has emerged as a major route to install noncanonical amino acids into proteins in living cells. Recently, a novel class of PylRS enzymes was identified in a subset of methanogenic archaea. Enzymes within this class (ΔPylSn) lack the N-terminal tRNA-binding domain that is widely conserved amongst PylRS enzymes, yet remain active and orthogonal in bacteria and eukaryotes.
- The ChaC family of γ-glutamyl cyclotransferases is conserved throughout all Kingdoms and catalyzes the degradation of GSH. So far, the ChaC family proteins in trypanosomal parasites are missing in the literature. Here, we report two members of the ChaC family of γ-glutamyl cyclotransferases (LmChaC2a and LmChaC2b) in the unicellular pathogen Leishmania. Activity measurements suggest that these proteins catalyze degradation of GSH but no other γ-glutamyl peptides. Recombinant LmChaC2a protein shows ∼17-fold lower catalytic efficiency (kcat ∼ 0.9 s−1) than LmChaC2b (kcat ∼ 15 s−1), although they showed comparable Km values (∼1.75 mM for LmChaC2a and ∼2.0 mM for LmChaC2b) toward GSH.
- Hepatitis C virus (HCV) is a major cause of liver-related diseases and hepatocellular carcinoma. The helicase domain of one of the nonstructural proteins of HCV, NS3 (nonstructural protein 3), is essential for viral replication; however, its specific biological role is still under investigation. Here, we set out to determine the interaction between a purified recombinant full length NS3 and synthetic guanine-rich substrates that represent the conserved G-quadruplex (G4)-forming sequences in the HCV-positive and HCV-negative strands.
- Mitochondria harbor the bacteria-inherited iron–sulfur cluster assembly (ISC) machinery to generate [2Fe–2S; iron–sulfur (Fe–S)] and [4Fe–4S] proteins. In yeast, assembly of [4Fe–4S] proteins specifically involves the ISC proteins Isa1, Isa2, Iba57, Bol3, and Nfu1. Functional defects in their human equivalents cause the multiple mitochondrial dysfunction syndromes, severe disorders with a broad clinical spectrum. The bacterial Iba57 ancestor YgfZ was described to require tetrahydrofolate (THF) for its function in the maturation of selected [4Fe–4S] proteins.
- Specific DNA methylation at CpG and non-CpG sites is essential for chromatin regulation. The DNA methyltransferase DNMT3A interacts with target sites surrounded by variable DNA sequences with its TRD and RD loops, but the functional necessity of these interactions is unclear. We investigated CpG and non-CpG methylation in a randomized sequence context using WT DNMT3A and several DNMT3A variants containing mutations at DNA-interacting residues. Our data revealed that the flanking sequence of target sites between the −2 and up to the +8 position modulates methylation rates >100-fold.
- Sonic hedgehog (Shh) signaling is a key component of embryonic development and is a driving force in several cancers. Hedgehog acyltransferase (Hhat), a member of the membrane-bound O-acyltransferase family of enzymes, catalyzes the attachment of palmitate to the N-terminal cysteine of Shh, a posttranslation modification critical for Shh signaling. The activity of Hhat has been assayed in cells and in vitro, and cryo-EM structures of Hhat have been reported, yet several unanswered questions remain regarding the enzyme’s reaction mechanism, substrate specificity, and the impact of the latter on Shh signaling.