Polyphosphate attachment to lysine repeats is a non-covalent protein modification.

Polyphosphate (polyP) is a chain of inorganic phosphate that is present in all domains of life and affects diverse cellular phenomena, ranging from blood clotting to cancer. A study by Azevedo et al. described a protein modification whereby polyP is attached to lysine residues within polyacidic serine and lysine (PASK) motifs via what the authors claimed to be covalent phosphoramidate bonding. This was based largely on the remarkable ability of the modification to survive extreme denaturing conditions. Our study demonstrates that lysine polyphosphorylation is non-covalent, based on its sensitivity to ionic strength and lysine protonation and absence of phosphoramidate bond formation, as analyzed via 31P NMR. Ionic interaction with lysine residues alone is sufficient for polyP modification, and we present a new list of non-PASK lysine repeat proteins that undergo polyP modification. This work clarifies the biochemistry of polyP-lysine modification, with important implications for both studying and modulating this phenomenon. This Matters Arising paper is in response to Azevedo et al. (2015), published in Molecular Cell. See also the Matters Arising Response by Azevedo et al. (2024), published in this issue.

Modification of histidine repeat proteins by inorganic polyphosphate.

Inorganic polyphosphate (polyP) is a linear polymer of orthophosphate that is present in nearly all organisms studied to date. A remarkable function of polyP involves its attachment to lysine residues via non-enzymatic post-translational modification (PTM), which is presumed to be covalent. Here, we show that proteins containing tracts of consecutive histidine residues exhibit a similar modification by polyP, which confers an electrophoretic mobility shift on NuPAGE gels. Our screen uncovers 30 human and yeast histidine repeat proteins that undergo histidine polyphosphate modification (HPM). This polyP modification is histidine dependent and non-covalent in nature, although remarkably it withstands harsh denaturing conditions-a hallmark of covalent PTMs. Importantly, we show that HPM disrupts phase separation and the phosphorylation activity of the human protein kinase DYRK1A, and inhibits the activity of the transcription factor MafB, highlighting HPM as a potential protein regulatory mechanism.

eEF2K Inhibitor Design: The Progression of Exemplary Structure-Based Drug Design.

The α-kinase, eEF2K, phosphorylates the threonine 56 residue of eEF2 to inhibit global peptide elongation (protein translation). As a master regulator of protein synthesis, in combination with its unique atypical kinase active site, investigations into the targeting of eEF2K represents a case of intense structure-based drug design that includes the use of modern computational techniques. The role of eEF2K is incredibly diverse and has been scrutinized in several different diseases including cancer and neurological disorders-with numerous studies inhibiting eEF2K as a potential treatment option, as described in this paper. Using available crystal structures of related α-kinases, particularly MHCKA, we report how homology modeling has been used to improve inhibitor design and efficacy. This review presents an overview of eEF2K related drug discovery efforts predating from the 1990's, to more recent in vivo studies in rat models. We also provide the reader with a basic introduction to several approaches and software programs used to undertake such drug discovery campaigns. With the recent exciting publication of an eEF2K crystal structure, we present our view regarding the future of eEF2K drug discovery.

Unusual ß1-4-galactosidase activity of an α1-6-mannosidase from Xanthomonas manihotis in the processing of branched hybrid and complex glycans.

Mannosidases are a diverse group of glycoside hydrolases that play crucial roles in mannose trimming of oligomannose glycans, glycoconjugates, and glycoproteins involved in numerous cellular processes, such as glycan biosynthesis and metabolism, structure regulation, cellular recognition, and cell-pathogen interactions. Exomannosidases and endomannosidases cleave specific glycosidic bonds of mannoside linkages in glycans and can be used in enzyme-based methods for sequencing of isomeric glycan structures. α1-6-mannosidase from Xanthomonas manihotis is known as a highly specific exoglycosidase that removes unbranched α1-6 linked mannose residues from oligosaccharides. However, we discovered that this α1-6-mannosidase also possesses an unexpected ß1-4-galactosidase activity in the processing of branched hybrid and complex glycans through our use of enzymatic reactions, high performance anion-exchange chromatography, and liquid chromatography mass spectrometric sequencing. Our docking simulation of the α1-6-mannosidase with glycan substrates reveals potential interacting residues in a relatively shallow pocket slightly differing from its homologous enzymes in the glycoside hydrolase 125 family, which may be responsible for the observed higher promiscuity in substrate binding and subsequent terminal glycan hydrolysis. This observation of novel ß1-4-galactosidase activity of the α1-6-mannosidase provides unique insights into its bifunctional activity on the substrate structure-dependent processing of terminal α1-6-mannose of unbranched glycans and terminal ß1-4-galactose of hybrid and complex glycans. The finding thus suggests the dual glycosidase specificity of this α1-6-mannosidase and the need for careful consideration when used for the structural elucidation of glycan isomers.

Careful conversations: an educational video to support parents in communicating about weight with their children.

BACKGROUND: Parents may struggle to initiate healthy weight-related conversations with their children. Educational videos may be an effective tool for improving parents' knowledge and self-efficacy on this topic. The aim of this pilot study was to develop an educational video to assist parents in weight-related conversations with their child, and to assess changes in parents' self-efficacy on this topic. METHODS: Video development was based on a scoping review and semi-structured interviews with parents. Respondent demographics and user satisfaction were assessed at pre- and post- video, and 4-6 months later. Self-efficacy scores were compared between parent groups based on weight concerns over time. RESULTS: Fifty-seven parents participated in the video questionnaires, and 40 repeated measures 4-6 months later. Significant improvements in self-efficacy in "raising the issue of weight" and "answering questions or concerns" were found after watching the video (p ≤ 0.002) compared to baseline, and scores 4-6 months post baseline remained slightly elevated, but non-significant. Parents with concerns about their child being overweight had significantly lower perceived self-efficacy scores compared to parents with no concerns about their child's weight (p = 0.031). The video was found to be positively received and of relevance to parents across a number of different domains. CONCLUSION(S): Preliminary findings suggest an educational video about initiating weight-related conversations may be an effective tool for increasing parents' perceived self-efficacy in the short term. Further work is needed to validate findings in a randomized controlled trial, and with diverse parent populations. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03664492 . Registered 10 September 2018 - Retrospectively registered.