Stabilization of cytokine mRNAs in iNKT cells requires the serine-threonine kinase IRE1alpha

Activated invariant natural killer T (iNKT) cells rapidly produce large amounts of cytokines, but how cytokine mRNAs are induced, stabilized and mobilized following iNKT activation is still unclear. Here we show that an endoplasmic reticulum stress sensor, inositol-requiring enzyme 1α (IRE1α), links key cellular processes required for iNKT cell effector functions in specific iNKT subsets, in which TCR-dependent activation of IRE1α is associated with downstream activation of p38 MAPK and the stabilization of preformed cytokine mRNAs. Importantly, genetic deletion of IRE1α in iNKT cells reduces cytokine production and protects mice from oxazolone colitis. We therefore propose that an IRE1α-dependent signaling cascade couples constitutive cytokine mRNA expression to the rapid induction of cytokine secretion and effector functions in activated iNKT cells

Hydrophobic surface patches on LoIA of Pseudomonas aeruginosa are essential for lipoprotein binding.

Many lipoproteins reside in the outer membrane (OM) of Gram-negative bacteria, and their biogenesis is dependent on the Lol (localization of lipoproteins) system. The periplasmic chaperone LolA accepts OM-destined lipoproteins that are released from the inner membrane by the LolCDE complex and transfers them to the OM receptor LolB. The exact nature of the LolA-lipoprotein complex is still unknown. The crystal structure of Escherichia coli LolA features an open β-barrel covered by α helices that together constitute a hydrophobic cavity, which would allow the binding of one acyl chain. However, OM lipoproteins contain three acyl chains, and the stoichiometry of the LolA-lipoprotein complex is 1:1. Here we present the crystal structure of Pseudomonas aeruginosa LolA that projects clear hydrophobic surface patches. Since these patches are large enough to accommodate acyl chains, their role in lipoprotein binding was investigated. Several LolA mutant proteins were created, and their functionality was assessed by studying their capacity to release lipoproteins produced in sphaeroplasts. Interruption of the largest hydrophobic patch completely destroyed the lipoprotein-releasing capacity of LolA, while interruption of smaller patches apparently reduced efficiency. Thus, the results show a new lipoprotein transport model that places (some of) the acyl chains on the hydrophobic surface patches. © 2010 Elsevier Ltd

Structure-aided optimization of non-nucleoside M. tuberculosis thymidylate kinase inhibitors

Mycobacterium tuberculosis thymidylate kinase (MtTMPK) has emerged as an attractive target for rational drug design. We recently investigated new families of non-nucleoside MtTMPK inhibitors in an effort to diversify MtTMPK inhibitor chemical space. We here report a new series of MtTMPK inhibitors by combining the Topliss scheme with rational drug design approaches, fueled by two co-crystal structures of MtTMPK in complex with developed inhibitors. These efforts furnished the most potent MtTMPK inhibitors in our assay, with two analogues displaying low micromolar MIC values against H37Rv Mtb. Prepared inhibitors address new sub-sites in the MtTMPK nucleotide binding pocket, thereby offering new insights into its druggability. We studied the role of efflux pumps as well as the impact of cell wall permeabilizers for selected compounds to potentially provide an explanation for the lack of correlation between potent enzyme inhibition and whole-cell activity. © 2021 Elsevier Masson SA

Disruption of endocytosis through chemical inhibition of clathrin heavy chain function

Clathrin mediated endocytosis CME is a highly conserved and essential cellular process in eukaryotic cells, but its dynamic and vital nature makes it challenging to study using classical genetics tools. In contrast, although small molecules can acutely and reversibly perturb CME, the few chemical CME inhibitors that have been applied to plants are either ineffective or show undesirable side effects. Here, we identify the previously described endosidin9 ES9 as an inhibitor of clathrin heavy chain CHC function in both Arabidopsis and human cells through affinity based target isolation, in vitro binding studies and X ray crystallography. Moreover, we present a chemically improved ES9 analog, ES9 17, which lacks the undesirable side effects of ES9 while retaining the ability to target CHC. ES9 and ES9 17 have expanded the chemical toolbox used to probe CHC function, and present chemical scaffolds for further design of more specific and potent CHC inhibitors across different system

Study of the active site residues of a glycoside hydrolase family 8 xylanase

Site-directed mutagenesis and a comparative characterisation of the kinetic parameters, pH dependency of activity and thermal stability of mutant and wild-type enzymes have been used in association with crystallographic analysis to delineate the functions of several active site residues in a novel glycoside hydrolase family 8 xylanase. Each of the residues investigated plays an essential role in this enzyme: E78 as the general acid, D281 as the general base and in orientating the nucleophilic water molecule, Y203 in maintaining the position of the nucleophilic water molecule and in structural integrity and D144 in sugar ring distortion and transition state stabilization. Interestingly, although crystal structure analyses and the pH-activity profiles clearly identify the functions of E78 and D281, substitution of these residues with their amide derivatives results in only a 250-fold and 700-fold reduction in their apparent k(cat) values, respectively. This, in addition to the observation that the proposed general base is not conserved in all glycoside hydrolase family 8 enzymes, indicates that the mechanistic architecture in this family of inverting enzymes is more complex than is conventionally believed and points to a diversity in the identity of the mechanistically important residues as well as in the arrangement of the intricate microenvironment of the active site among members of this family. (c) 2005 Elsevier Ltd. All rights reserved