7 research outputs found
The Phosphoinositide Kinase PIKfyve Promotes Cathepsin-S-Mediated Major Histocompatibility Complex Class II Antigen Presentation
Contains fulltext :
201259.pdf (publisher's version ) (Open Access
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
Pioglitazone and bladder cancer risk: a multipopulation pooled, cumulative exposure analysis
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