The arms race between bacteria and phages led to the development of sophisticated antiphage defense systems, including CRISPR-Cas and restriction-modification systems. Evidence suggests that known ...and unknown defense systems are located in "defense islands" in microbial genomes. Here, we comprehensively characterized the bacterial defensive arsenal by examining gene families that are clustered next to known defense genes in prokaryotic genomes. Candidate defense systems were systematically engineered and validated in model bacteria for their antiphage activities. We report nine previously unknown antiphage systems and one antiplasmid system that are widespread in microbes and strongly protect against foreign invaders. These include systems that adopted components of the bacterial flagella and condensin complexes. Our data also suggest a common, ancient ancestry of innate immunity components shared between animals, plants, and bacteria.
Cyclic-oligonucleotide-based anti-phage signalling systems (CBASS) are a family of defence systems against bacteriophages (hereafter phages) that share ancestry with the cGAS-STING innate immune ...pathway in animals. CBASS systems are composed of an oligonucleotide cyclase, which generates signalling cyclic oligonucleotides in response to phage infection, and an effector that is activated by the cyclic oligonucleotides and promotes cell death. Cell death occurs before phage replication is completed, therefore preventing the spread of phages to nearby cells. Here, we analysed 38,000 bacterial and archaeal genomes and identified more than 5,000 CBASS systems, which have diverse architectures with multiple signalling molecules, effectors and ancillary genes. We propose a classification system for CBASS that groups systems according to their operon organization, signalling molecules and effector function. Four major CBASS types were identified, sharing at least six effector subtypes that promote cell death by membrane impairment, DNA degradation or other means. We observed evidence of extensive gain and loss of CBASS systems, as well as shuffling of effector genes between systems. We expect that our classification and nomenclature scheme will guide future research in the developing CBASS field.
Gasdermin proteins form large membrane pores in human cells that release immune cytokines and induce lytic cell death. Gasdermin pore formation is triggered by caspase-mediated cleavage during ...inflammasome signaling and is critical for defense against pathogens and cancer. We discovered gasdermin homologs encoded in bacteria that defended against phages and executed cell death. Structures of bacterial gasdermins revealed a conserved pore-forming domain that was stabilized in the inactive state with a buried lipid modification. Bacterial gasdermins were activated by dedicated caspase-like proteases that catalyzed site-specific cleavage and the removal of an inhibitory C-terminal peptide. Release of autoinhibition induced the assembly of large and heterogeneous pores that disrupted membrane integrity. Thus, pyroptosis is an ancient form of regulated cell death shared between bacteria and animals.
The recently discovered prokaryotic immune system known as CRISPR (clustered regularly interspaced short palindromic repeats) is based on small RNAs (‘spacers’) that restrict phage and plasmid ...infection. It has been hypothesized that CRISPRs can also regulate self gene expression by utilizing spacers that target self genes. By analyzing CRISPRs from 330 organisms we found that one in every 250 spacers is self-targeting, and that such self-targeting occurs in 18% of all CRISPR-bearing organisms. However, complete lack of conservation across species, combined with abundance of degraded repeats near self-targeting spacers, suggests that self-targeting is a form of autoimmunity rather than a regulatory mechanism. We propose that accidental incorporation of self nucleic acids by CRISPR can incur an autoimmune fitness cost, and this could explain the abundance of degraded CRISPR systems across prokaryotes.
Since the first demonstration that CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against phages and plasmids, numerous studies have yielded key insights into the molecular ...mechanisms governing how these systems attack and degrade foreign DNA. However, the molecular mechanisms underlying the adaptation stage, in which new immunological memory is formed, have until recently represented a major unresolved question. In this Progress article, we discuss recent discoveries that have shown both how foreign DNA is identified by the CRISPR-Cas adaptation machinery and the molecular basis for its integration into the chromosome to form an immunological memory. Furthermore, we describe the roles of each of the specific CRISPR-Cas components that are involved in memory formation, and consider current models for their evolutionary origin.
Sterile alpha and TIR motif-containing 1 (SARM1) is a protein involved in programmed death of injured axons. Following axon injury or a drug-induced insult, the TIR domain of SARM1 degrades the ...essential molecule nicotinamide adenine dinucleotide (NAD+), leading to a form of axonal death called Wallerian degeneration. Degradation of NAD+ by SARM1 is essential for the Wallerian degeneration process, but accumulating evidence suggest that other activities of SARM1, beyond the mere degradation of NAD+, may be necessary for programmed axonal death. In this study we show that the TIR domains of both human and fruit fly SARM1 produce 1''-2' and 1''-3' glycocyclic ADP-ribose (gcADPR) molecules as minor products. As previously reported, we observed that SARM1 TIR domains mostly convert NAD+ to ADPR (for human SARM1) or cADPR (in the case of SARM1 from Drosophila melanogaster). However, we now show that human and Drosophila SARM1 additionally convert ~0.1-0.5% of NAD+ into gcADPR molecules. We find that SARM1 TIR domains produce gcADPR molecules both when purified in vitro and when expressed in bacterial cells. Given that gcADPR is a second messenger involved in programmed cell death in bacteria and likely in plants, we propose that gcADPR may play a role in SARM1-induced programmed axonal death in animals.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Identifying active site residues strictly from protein three-dimensional structure is a difficult task, especially for proteins that have few or no homologues. We transformed protein structures into ...residue interaction graphs (RIGs), where amino acid residues are graph nodes and their interactions with each other are the graph edges. We found that active site, ligand-binding and evolutionary conserved residues, typically have high closeness values. Residues with high closeness values interact directly or by a few intermediates with all other residues of the protein. Combining closeness and surface accessibility identified active site residues in 70% of 178 representative structures. Detailed structural analysis of specific enzymes also located other types of functional residues. These include the substrate binding sites of acetylcholinesterases and subtilisin, and the regions whose structural changes activate MAP kinase and glycogen phosphorylase. Our approach uses single protein structures, and does not rely on sequence conservation, comparison to other similar structures or any prior knowledge. Residue closeness is distinct from various sequence and structure measures and can thus complement them in identifying key protein residues. Closeness integrates the effect of the entire protein on single residues. Such natural structural design may be evolutionary maintained to preserve interaction redundancy and contribute to optimal setting of functional sites.
Inteins comprise a large family of phylogenetically widespread self-splicing protein catalysts that colonize diverse host proteins. The evolutionary and functional relationship between the intein and ...the split-host protein, the exteins, is largely unknown. To probe an association, we developed an in vivo and in vitro intein assay based on FRET. The FRET assay reports cleavage of the intein from its N-terminal extein. Applying this assay to randomized extein libraries, we show that the nature of the extein substrate bordering the intein can profoundly influence intein activity. Residues proximal to the intein-splicing junction in both N- and C-terminal exteins can accelerate the N-terminal cleavage rate by >4-fold or attenuate cleavage by 1,000-fold, both resulting in compromised self-splicing efficiency. The existence and the magnitude of extein effects require consideration for maximizing the utility of inteins in biotechnological applications, and they predict biases in intein integration sites in nature.
Prokaryotic adaptive immune systems are composed of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins. These systems adapt to new threats by ...integrating short nucleic acids, termed spacers, into the CRISPR array. The functional motifs in the repeat and the mechanism by which a constant repeat size is maintained are still elusive. Here, through a series of mutations within the repeat of the CRISPR-Cas type I-E, we identify motifs that are crucial for adaptation and show that they serve as anchor sites for two molecular rulers determining the size of the new repeat. Adaptation products from various repeat mutants support a model in which two motifs in the repeat bind to two different sites in the adaptation complex that are 8 and 16 bp away from the active site. This model significantly extends our understanding of the adaptation process and broadens the scope of its applications.
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•Inverted repeats in the type I-E CRISPR-Cas system are essential for adaptation•Each inverted repeat encodes a motif serving as an anchor site for a molecular ruler•These molecular rulers determine the spacer insertion site regardless of the sequence•The findings support a model considering all known steps in spacer adaptation
Goren et al. map elements that are essential for adaptation in the E. coli CRISPR-Cas type I-E repeat. Two elements were identified as anchor sites for two molecular rulers that maintain a constant repeat size. Their findings support a comprehensive model for spacer adaptation.
The goal of in vitro compartmentalization (IVC) is to divide a large reaction between many microscopic compartments. This technique was first developed to generate artificial cells for the directed ...evolution of proteins. Typically, an aqueous solution of genes and an in vitro transcription-translation system is stirred (or homogenized) into an oil-surfactant mixture to create a water-in-oil (w/o) emulsion with 1010 aqueous droplets per ml of emulsion. The majority of droplets contain no more than a single gene along with all of the molecular machinery needed to express that gene.
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