The Phosphoinositide Kinase PIKfyve Promotes Cathepsin-S-Mediated Major Histocompatibility Complex Class II Antigen Presentation

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Computation-Guided Rational Design of a Peptide Motif That Reacts with Cyanobenzothiazoles via Internal Cysteine–Lysine Relay

Site-selective protein modification based on covalent reactions of peptide tags and small molecules is a key capability for basic research as well as for the development of new therapeutic bioconjugates. Here, we describe the computation-guided rational design of a cysteine- and lysine-containing 11-residue peptide sequence that reacts with 2-cyanobenzothiazole (CBT) derivatives. Our data show that the cysteine residue reversibly reacts with the nitrile group on the CBT moiety to form an intermediate thioimidate, which undergoes irreversible <i>SN</i> transfer to the lysine residue, yielding an amidine-linked product. The concepts outlined herein lay a foundation for future development of peptide tags in the context of site-selective modification of lysine residues within engineered microenvironments

<i>In Vitro</i> and <i>In Vivo</i> Enzyme Activity Screening via RNA-Based Fluorescent Biosensors for <i>S</i>‑Adenosyl‑l‑homocysteine (SAH)

High-throughput enzyme activity screens are essential for target characterization and drug development, but few assays employ techniques or reagents that are applicable to both <i>in vitro</i> and live cell settings. Here, we present a class of selective and sensitive fluorescent biosensors for <i>S</i>-adenosyl-l-homocysteine (SAH) that provide a direct “mix and go” activity assay for methyltransferases (MTases), an enzyme class that includes several cancer therapeutic targets. Our riboswitch-based biosensors required an alternate inverted fusion design strategy, but retained full selectivity for SAH over its close structural analogue, the highly abundant methylation cofactor <i>S</i>-adenosyl-l-methionine (SAM). The level of ligand selectivity for these fluorescent biosensors exceeded that of commercial antibodies for SAH and proved critical to cellular applications, as we employed them to measure methylthioadenosine nucleosidase (MTAN) activity in live Escherichia coli. In particular, we were able to monitor <i>in vivo</i> increase of SAH levels upon chemical inhibition of MTAN using flow cytometry, which demonstrates high-throughput, single cell measurement of an enzyme activity associated with the biosynthesis of quorum sensing signal AI-2. Thus, this study presents RNA-based fluorescent biosensors as promising molecular reagents for high-throughput enzymatic assays that successfully bridge the gap between <i>in vitro</i> and <i>in vivo</i> applications

Evolutionary success of prokaryotes

How can the evolutionary success of prokaryotes be explained ? How did they manage to survive conditions that have fluctuated, with drastic events over 3.5 billion years ? Which significant metabolisms and mechanisms have appeared over the course of evolution that have permitted them to survive the most inhospitable conditions from the physicochemical point of view ? In a 'Red Queen Race', prokaryotes have always run sufficiently fast to adapt to constraints imposed by the environment and the other living species with which they have established interactions. If the criterion retained to define the level of evolution of an organism is its capacity to survive and to yield the largest number of offsprings, prokaryotes must be considered highly evolved organisms