Role of NLRP3 inflammasome in nanoparticle adjuvant-mediated immune response.

The nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) inflammasome is pivotal in orchestrating the immune response induced by nanoparticle adjuvants. Understanding the intricate mechanisms underlying the activation of NLRP3 inflammasome by these adjuvants is crucial for deciphering their immunomodulatory properties. This review explores the involvement of the NLRP3 inflammasome in mediating immune responses triggered by nanoparticle adjuvants. It delves into the signaling pathways and cellular mechanisms involved in NLRP3 activation, highlighting its significance in modulating the efficacy and safety of nanoparticle-based adjuvants. A comprehensive grasp of the interplay between NLRP3 inflammasome and nanoparticle adjuvants holds promise for optimizing vaccine design and advancing immunotherapeutic strategies.

SUMO-activated target traps (SATTs) enable the identification of a comprehensive E3-specific SUMO proteome.

Ubiquitin and ubiquitin-like conjugation cascades consist of dedicated E1, E2, and E3 enzymes with E3s providing substrate specificity. Mass spectrometry-based approaches have enabled the identification of more than 6500 SUMO2/3 target proteins. The limited number of SUMO E3s provides the unique opportunity to systematically study E3 substrate wiring. We developed SUMO-activated target traps (SATTs) and systematically identified substrates for eight different SUMO E3s, PIAS1, PIAS2, PIAS3, PIAS4, NSMCE2, ZNF451, LAZSUL (ZNF451-3), and ZMIZ2. SATTs enabled us to identify 427 SUMO1 and 961 SUMO2/3 targets in an E3-specific manner. We found pronounced E3 substrate preference. Quantitative proteomics enabled us to measure substrate specificity of E3s, quantified using the SATT index. Furthermore, we developed the Polar SATTs web-based tool to browse the dataset in an interactive manner. Overall, we uncover E3-to-target wiring of 1388 SUMO substrates, highlighting unique and overlapping sets of substrates for eight different SUMO E3 ligases.

Divalent Ion-Induced Switch in DNA Cleavage of KpnI Endonuclease Probed through Surface-Enhanced Raman Spectroscopy.

We demonstrate the remarkable ability of surface-enhanced Raman spectroscopy (SERS) to track the allosteric changes in restriction endonuclease KpnI (R.KpnI) caused by metal ions. R.KpnI binds and promiscuously cleaves DNA upon activation by Mg2+ ions. However, the divalent ion Ca2+ induces high fidelity cleavage, which can be overcome by higher concentrations of Mg2+ ions. In the absence of any 3D crystal structure, for the first time, we have elucidated the structural underpinnings of such a differential effect of divalent ions on the endonuclease activity. A combined SERS and molecular dynamics (MD) approach showed that Ca2+ ion activates an enzymatic switch in the active site, which is responsible for the high fidelity activity of the enzyme. Thus, SERS in combination with MD simulations provides a powerful tool for probing the link between the structure and activity of enzyme molecules that play vital roles in DNA transactions.

Biochemical characterization of SUMO-conjugating enzymes by in vitro sumoylation assays.

The small ubiquitin-related modifier (SUMO) is a protein of ~10kDa that is covalently conjugated to its substrate proteins in an enzymatic process called sumoylation. This posttranslational modification is an essential regulatory mechanism that plays crucial roles in many cellular pathways. It allows rapid adaptation to environmental changes by switching protein functions due to alternate complex assemblies, changes in intracellular localization, enzymatic activity, or stability. SUMO conjugation is executed by the hierarchical action of E1, E2, and E3 enzymes. Both E2 and E3 enzymes contribute to substrate specificity but with E3 ligases being the more important for this. E1 and E2 activities are essential for all sumoylation reactions but usually-with a few exceptions-modify substrates only inefficiently. Hence, most substrates require the additional action of an E3 ligase or a cofactor. Here, we describe methods to distinguish a bona fide E3 ligase from a cofactor activity by using in vitro sumoylation assays.

Restriction endonuclease triggered bacterial apoptosis as a mechanism for long time survival.

Programmed cell death (PCD) under certain conditions is one of the features of bacterial altruism. Given the bacterial diversity and varied life style, different PCD mechanisms must be operational that remain largely unexplored. We describe restriction endonuclease (REase) mediated cell death by an apoptotic pathway, beneficial for isogenic bacterial communities. Cell death is pronounced in stationary phase and when the enzyme exhibits promiscuous DNA cleavage activity. We have elucidated the molecular mechanism of REase mediated cell killing and demonstrate that released nutrients from dying cells support the growth of the remaining cells in the population. These findings illustrate a new intracellular moonlighting role for REases which are otherwise established host defence arsenals. REase induced PCD appears to be a cellular design to replenish nutrients for cells undergoing starvation stress and the phenomenon could be wide spread in bacteria, given the abundance of restriction-modification (R-M) systems in the microbial population.

Draft Genome Sequence of Klebsiella pneumoniae OK8, a Multidrug-Resistant Mouse and Human Pathogen.

We report here the draft genome sequence of Klebsiella pneumoniae OK8, a multidrug-resistant strain which was isolated in 1976 from a human and is known to be a mouse pathogen.

Increasing cleavage specificity and activity of restriction endonuclease KpnI.

Restriction enzyme KpnI is a HNH superfamily endonuclease requiring divalent metal ions for DNA cleavage but not for binding. The active site of KpnI can accommodate metal ions of different atomic radii for DNA cleavage. Although Mg(2+) ion higher than 500 µM mediates promiscuous activity, Ca(2+) suppresses the promiscuity and induces high cleavage fidelity. Here, we report that a conservative mutation of the metal-coordinating residue D148 to Glu results in the elimination of the Ca(2+)-mediated cleavage but imparting high cleavage fidelity with Mg(2+). High cleavage fidelity of the mutant D148E is achieved through better discrimination of the target site at the binding and cleavage steps. Biochemical experiments and molecular dynamics simulations suggest that the mutation inhibits Ca(2+)-mediated cleavage activity by altering the geometry of the Ca(2+)-bound HNH active site. Although the D148E mutant reduces the specific activity of the enzyme, we identified a suppressor mutation that increases the turnover rate to restore the specific activity of the high fidelity mutant to the wild-type level. Our results show that active site plasticity in coordinating different metal ions is related to KpnI promiscuous activity, and tinkering the metal ion coordination is a plausible way to reduce promiscuous activity of metalloenzymes.

Ca(2+) binding to the ExDxD motif regulates the DNA cleavage specificity of a promiscuous endonuclease.

Most of the restriction endonucleases (REases) are dependent on Mg(2+) for DNA cleavage, and in general, Ca(2+) inhibits their activity. R.KpnI, an HNH active site containing ßßα-Me finger nuclease, is an exception. In presence of Ca(2+), the enzyme exhibits high-fidelity DNA cleavage and complete suppression of Mg(2+)-induced promiscuous activity. To elucidate the mechanism of unusual Ca(2+)-mediated activity, we generated alanine variants in the putative Ca(2+) binding motif, E(132)xD(134)xD(136), of the enzyme. Mutants showed decreased levels of DNA cleavage in the presence of Ca(2+). We demonstrate that ExDxD residues are involved in Ca(2+) coordination; however, the invariant His of the catalytic HNH motif acts as a general base for nucleophile activation, and the other two active site residues, D148 and Q175, also participate in Ca(2+)-mediated cleavage. Insertion of a 10-amino acid linker to disrupt the spatial organization of the ExDxD and HNH motifs impairs Ca(2+) binding and affects DNA cleavage by the enzyme. Although ExDxD mutant enzymes retained efficient cleavage at the canonical sites in the presence of Mg(2+), the promiscuous activity was greatly reduced, indicating that the carboxyl residues of the acidic triad play an important role in sequence recognition by the enzyme. Thus, the distinct Ca(2+) binding motif that confers site specific cleavage upon Ca(2+) binding is also critical for the promiscuous activity of the Mg(2+)-bound enzyme, revealing its role in metal ion-mediated modulation of DNA cleavage.

Promiscuous restriction is a cellular defense strategy that confers fitness advantage to bacteria.

Most bacterial genomes harbor restriction-modification systems, encoding a REase and its cognate MTase. On attack by a foreign DNA, the REase recognizes it as nonself and subjects it to restriction. Should REases be highly specific for targeting the invading foreign DNA? It is often considered to be the case. However, when bacteria harboring a promiscuous or high-fidelity variant of the REase were challenged with bacteriophages, fitness was maximal under conditions of catalytic promiscuity. We also delineate possible mechanisms by which the REase recognizes the chromosome as self at the noncanonical sites, thereby preventing lethal dsDNA breaks. This study provides a fundamental understanding of how bacteria exploit an existing defense system to gain fitness advantage during a host-parasite coevolutionary "arms race."