Biology subjects

Kranzusch, P. J.

Publications and source records attributed to Kranzusch, P. J..

30 records · Page 2Linked to original sources

bioRxiv2023

Structure and assembly of a bacterial gasdermin pore

In response to pathogen infection, gasdermin (GSDM) proteins form membrane pores that induce a host cell death process called pyroptosis1-3. Studies of human and mouse GSDM pores reveal the functions and architectures of 24-33 protomers assemblies4-9, but the mechanism and evolutionary origin of membrane targeting and GSDM pore formation remain unknown. Here we determine a structure of a bacterial GSDM (bGSDM) pore and define a conserved mechanism of pore assembly. Engineering a panel of bGSDMs for site-specific proteolytic activation, we demonstrate that diverse bGSDMs form distinct pore sizes that range from smaller mammalian-like assemblies to exceptionally large pores containing >50 protomers. We determine a 3.3 [A] cryo-EM structure of a Vitiosangium bGSDM in an active slinky-like oligomeric conformation and analyze bGSDM pores in a native lipid environment to create an atomic-level model of a full 52-mer bGSDM pore. Combining our structural analysis with molecular dynamics simulations and cellular assays, our results support a stepwise model of GSDM pore assembly and suggest that a covalently bound palmitoyl can leave a hydrophobic sheath and insert into the membrane before formation of the membrane-spanning {beta}-strand regions. These results reveal the diversity of GSDM pores found in nature and explain the function of an ancient post-translational modification in enabling programmed host cell death.

microbiology↗

bioRxiv2023

RNase L activating 2'-5' oligoadenlyates bind ABCF1, -3 and Decr-1

A notable signaling mechanism employed by mammalian innate immune signaling pathways uses nucleotide based second messengers such as 2'-3'-cGAMP and 2'-5'-oligoadenylates (2'-5' OA), which bind and activate STING and RNase L, respectively. Interestingly, the involvement of nucleotide second messengers to activate antiviral responses is evolutionary conserved, evidenced by the identification of an antiviral cGAMP-dependent pathway in Drosophila. Using a mass spectrometry approach, we identified several members of the ABCF family in human, mouse, and Drosophila cell lysates as 2'-5' OA binding proteins, suggesting an evolutionary conserved function. Biochemical characterization of these interactions demonstrates high-affinity binding of 2'-5' OA to ABCF1, which depended on phosphorylated 2'-5' OA and an intact Walker A/B motif of the ABC cassette of ABCF1. As further support for species-specific interactions with 2'-5' OA, we additionally identified that the metabolism enzyme Decr1 from mouse, but not human or Drosophila cells forms a high-affinity complex with 2'-5' OA. A 1.4 [A] co-crystal structure of the mouse Decr1-2'-5' OA complex explains high-affinity recognition of 2'-5' OA and the mechanism of species-specificity. Despite clear evidence of physical interactions, we could not identify profound antiviral functions of ABCF1, ABCF3 or Decr1 or 2'-5' OA-dependent regulation of cellular translation rates as suggested by the engagement of ABCF proteins. Thus, although the biological consequences of the here identified interactions need to be identified, our data suggests that 2'-5' OA can serve as signaling hub to distribute a signal to different recipient proteins.

microbiology↗

bioRxiv2023

Structural homology screens reveal poxvirus-encoded proteins impacting inflammasome-mediated defenses

Viruses acquire host genes via horizontal gene transfer and can express them to manipulate host biology during infections. Some viral and host homologs retain sequence identity, but evolutionary divergence can obscure host origins. We used structural modeling to compare vaccinia virus proteins with metazoan proteomes. We identified vaccinia A47L as a homolog of gasdermins, the executioners of pyroptosis. An X-ray crystal structure of A47 confirmed this homology and cell-based assays revealed that A47 inhibits pyroptosis. We also identified vaccinia C1L as the product of a cryptic gene fusion event coupling a Bcl-2 related fold with a pyrin domain. C1 associates with components of the inflammasome, a cytosolic innate immune sensor involved in pyroptosis, yet paradoxically enhances inflammasome activity, suggesting a benefit to poxvirus replication in some circumstances. Our findings demonstrate the potential of structural homology screens to reveal genes that viruses capture from hosts and repurpose to benefit viral fitness.

evolutionary biology↗

bioRxiv2023

cGLRs are a diverse family of pattern recognition receptors in animal innate immunity

cGAS (cyclic GMP-AMP synthase) is an enzyme in human cells that controls an immune response to cytosolic DNA. Upon binding DNA, cGAS synthesizes a nucleotide signal 2'3'-cGAMP that activates the protein STING and downstream immunity. Here we discover cGAS-like receptors (cGLRs) constitute a major family of pattern recognition receptors in animal innate immunity. Building on recent analysis in Drosophila, we use a bioinformatic approach to identify >3,000 cGLRs present in nearly all metazoan phyla. A forward biochemical screen of 140 animal cGLRs reveals a conserved mechanism of signaling including response to dsDNA and dsRNA ligands and synthesis of alternative nucleotide signals including isomers of cGAMP and cUMP-AMP. Using structural biology, we explain how synthesis of distinct nucleotide signals enables cells to control discrete cGLR-STING signaling pathways. Together our results reveal cGLRs as a widespread family of pattern recognition receptors and establish molecular rules that govern nucleotide signaling in animal immunity.

biochemistry↗

bioRxiv2022

Cryo-EM structure of the RADAR supramolecular anti-phage defense complex

RADAR is a two-protein bacterial defense system which was reported to defend against phage by editing messenger RNA. Here we determine cryo-EM structures of the RADAR defense complex, revealing RdrA as a heptameric, two-layered AAA+ ATPase and RdrB as a dodecameric, hollow complex with twelve surface-exposed deaminase active sites. RdrA and RdrB join to form a giant assembly up to 10 MDa, with RdrA docked as a funnel over the RdrB active site. Surprisingly, our structures reveal a RdrB active site that targets mononucleotides, not RNA. We show that RdrB catalyzes ATP-to-ITP conversion in vitro and induces the accumulation of inosine mononucleotides during phage infection in vivo, limiting phage replication. Our results define ATP mononucleotide deamination as a determinant of RADAR immunity and reveal supramolecular assembly of a nucleotide-modifying machine as a novel mechanism of anti-phage defense.

biochemistry↗

bioRxiv2022

Viruses inhibit TIR gcADPR signaling to overcome bacterial defense

The Toll/interleukin-1 receptor (TIR) domain is a key component of immune receptors that identify pathogen invasion in bacteria, plants, and animals. In the bacterial antiphage system Thoeris, as well as in plants, recognition of infection stimulates TIR domains to produce an immune signaling molecule whose molecular structure remained elusive. This molecule binds and activates the Thoeris immune effector, which then executes the immune function. We identified a large family of phage-encoded proteins, denoted here Thoeris anti-defense 1 (Tad1), that inhibit Thoeris immunity. We found that Tad1 proteins are "sponges" that bind and sequester the immune signaling molecule produced by TIR-domain proteins, thus decoupling phage sensing from immune effector activation and rendering Thoeris inactive. A high-resolution crystal structure of Tad1 bound to the signaling molecule revealed that its chemical structure is 1'-2' glycocyclic ADPR (gcADPR), a unique molecule not previously described in other biological systems. Our results define the chemical structure of a central immune signaling molecule, and reveal a new mode of action by which pathogens can suppress host immunity.

microbiology↗

bioRxiv2022

Three functionally distinct classes of cGAS proteins in nature revealed by self-DNA-induced interferon responses

Innate immune pattern recognition receptors (PRRs) emerged early in evolution. It is generally assumed that structurally homologous proteins in distinct species will operate via similar mechanisms. We tested this prediction through the study of interferon responses to self-DNA by the enzymatic PRR cyclic GMP-AMP synthase (cGAS). Contrary to expectations, we identified three functional classes of this PRR in mammals. Class 1 proteins (including human) contained a catalytic domain that was intrinsically self-DNA reactive and stimulated interferon responses in diverse cell types. This reactivity was prevented by an upstream N-terminal domain. Class 2 and 3 proteins were either not self-DNA reactive (including chimpanzee) or included proteins whose N-terminal domain promoted self-DNA reactivity (mouse). While self-DNA reactivity of Class 1 cGAS was linked to an ability to access intra-mitochondrial DNA, mitochondrial localization was not associated with other classes. These studies reveal unexpected diversity in the mechanisms of self-DNA reactivity of a PRR. One Sentence SummaryThe regulation of self-DNA reactivity of cGAS is evolutionarily diverse in mammals.

immunology↗

bioRxiv2022

Sequential action of a tRNA base editor in conversion of cytidine to pseudouridine

Post-transcriptional RNA editing modulates gene expression in a condition-dependent fashion. We recently discovered C-to-{Psi} editing in Vibrio cholerae tRNA. Here, we characterize the biogenesis, regulation, and functions of this previously undescribed RNA editing process. We show that an enzyme, TrcP, mediates the editing of C-to-U followed by the conversion of U to {Psi}, consecutively. AlphaFold-2 predicts that TrcP consists of two globular domains, including a novel cytidine deaminase and a pseudouridylase, along with a long helical domain. The latter domain tethers tRNA substrates during both the C-to-U editing and pseudouridylation, likely enabling a substrate channeling mechanism for efficient catalysis all the way to the terminal product. C-to-{Psi} editing both requires and suppresses other modifications, creating an interdependent network of modifications in the tRNA anticodon loop that facilitates coupling of tRNA modification states to iron availability. Our findings provide mechanistic insights into an RNA editing process that likely promotes environmental adaptation.

biochemistry↗

bioRxiv2021

Bacterial gasdermins reveal an ancient mechanism of cell death

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. Here we discover gasdermin homologs encoded in bacteria that execute prokaryotic cell death. Structures of bacterial gasdermins reveal a conserved pore-forming domain that is stabilized in the inactive state with a buried lipid modification. We demonstrate that bacterial gasdermins are activated by dedicated caspase-like proteases that catalyze site-specific cleavage and removal of an inhibitory C-terminal peptide. Release of autoinhibition induces the assembly of >200 [A] pores that form a mesh-like structure and disrupt membrane integrity. These results demonstrate that caspase-mediated activation of gasdermins is an ancient form of regulated cell death shared between bacteria and animals.

microbiology↗

bioRxiv2019

Structure and mechanism of a cyclic trinucleotide-activated bacterial endonuclease mediating bacteriophage immunity

Bacteria possess an array of defenses against foreign invaders, including diverse nucleases that target and destroy the genome of an invading bacteriophage or foreign DNA element. A recently-described bacteriophage immunity pathway employs a cGAS/DncV-like nucleotidyltransferase to produce a cyclic tri-AMP second messenger, which activates the DNA endonuclease effector NucC. Here, we show that NucC is related to restriction enzymes but uniquely assembles into a homotrimer in solution. cAAA binding in a conserved allosteric pocket promotes assembly of two NucC trimers into a homohexamer competent for double-strand DNA cleavage. We propose that NucC mediates bacteriophage immunity either through global activation, causing altruistic cell death and abortive infection, or through local activation and targeted phage genome destruction. Finally, we identify NucC homologs in type III CRISPR-Cas systems, where they likely function as accessory nucleases activated by cyclic oligoadenylate second messengers synthesized by these systems effector complexes.

biochemistry↗

bioRxiv2018

cGAMP signaling controls a transient surface-associated lifestyle

A newfound signaling pathway employs a GGDEF enzyme with unique activity compared to the majority of homologs associated with bacterial cyclic di-GMP signaling. This system provides a rare opportunity to study how signaling proteins natively gain distinct function. Using genetic knockouts, riboswitch reporters, and RNA-Seq, we show that GacA, the Hypr GGDEF in Geobacter sulfurreducens, specifically regulates cyclic GMP-AMP (3,3-cGAMP) levels in vivo to stimulate gene expression associated with metal reduction separate from electricity production. To reconcile these in vivo findings with prior in vitro results that showed GacA was promiscuous, we developed a full kinetic model combining experimental data and mathematical modeling to reveal mechanisms that contribute to in vivo specificity. A 1.4 [A]-resolution crystal structure of the Geobacter Hypr GGDEF domain was determined to understand the molecular basis for those mechanisms, including key cross-dimer interactions. Together these results demonstrate that specific signaling can result from a promiscuous enzyme.

biochemistry↗

bioRxiv2018

Recurrent loss-of-function mutations reveal costs to OAS1 antiviral activity in primates

Immune responses counteract infections and can also cause collateral damage to hosts. We investigated functional outcomes of variation in the rapidly evolving antiviral double-stranded RNA (dsRNA) sensing factor Oligoadenylate Synthetase 1 (OAS1) in primates as a model for understanding how individual immune pathways evolve to minimize deleterious effects on host fitness. Upon binding of dsRNAs, OAS1 polymerizes ATP into 2'-5' linked oligoadenylate (2-5A), which in turn activates Latent Ribonuclease (RNase L) to kill virus infected cells. OAS1 can undergo auto-activation by host encoded RNAs, raising the question of how it might evolve to mitigate RNase L-mediated cytotoxicity. Using a new yeast-based growth assay, we observed a pattern of frequent loss of 2-5A synthesis by OAS1 from several species. In gorillas, we identified a polymorphism in a conserved substrate binding residue that severely decreases catalytic function. In contrast, lowered 2-5A generation previously associated with variation in humans results from production of unstable OAS1 isoforms. Examination of OAS1 function in monkeys revealed a spectrum of activities, including the complete loss of 2-5A synthesis in tamarins. Frequent loss of catalytic activity in primates suggests that costs associated with OAS1 activation can be so detrimental to host fitness that its pathogen-protective effects are repeatedly forfeited.

genetics↗