Biology subjects

Johnson, A. G.

Publications and source records attributed to Johnson, A. G..

15 recordsLinked to original sources

bioRxiv2026

Pyroptolysins are versatile, sterol-sensing bacterial toxins

Gasdermin pores mediate cellular self-sacrifice for the immune defense pyroptosis. Here we describe how the gasdermin fold was repurposed during evolution into a membrane targeting bacterial toxin. These proteins, which we call pyroptolysins, are chimeras of gasdermins and cholesterol-dependent cytolysins. Like the cytolysins, pyroptolysins are active on cholesterol-rich membranes, but unlike cytolysins, they are poorly active on mammalian cells. Structural snapshots of the pyroptolysins confirm their dual homology and reveal pre-organized complexes primed for membrane insertion. Unexpectedly, we found the pyroptolysins to be active in vitro and in vivo on membranes with remarkably low sterol content, displaying insecticidal activity. Our results establish the gasdermin fold as a versatile pore-forming scaffold, capable of inducing cell death from the inside and the outside of cells.

microbiology↗

bioRxiv2026

Structure and Mechanism of a Two-component Lanthipeptide Toxin

The enterococcal cytolysin is a two-component lanthipeptide natural product that co-operatively kills mammalian cells and bacteria. It is a virulence factor in typically commensal strains of gut-dwelling Enterococcus faecalis and causes a fatal form of alcoholic hepatitis. Despite its clinical significance, little is known about the mechanism of action. We present here the molecular details of cytolysins remarkable bioactivity. A high-resolution cryo-electron microscopy structure revealed highly ordered tubular assemblies comprising the two subunits. We further demonstrate these structures disrupt the membranes of eukaryotic and bacterial cells. These results provide the first high-resolution structure of two distinct lanthipeptides interacting with one another and offer an explanation for the unique bioactivity of the cytolysin toxin.

biochemistry↗

bioRxiv2023

CARD-like domains mediate anti-phage defense in bacterial gasdermin systems

Caspase recruitment domains (CARDs) and pyrin domains are important facilitators of inflammasome activity and pyroptosis. Upon pathogen recognition by NLR proteins, CARDs recruit and activate caspases, which, in turn, activate gasdermin pore forming proteins to and induce pyroptotic cell death. Here we show that CARD-like domains are present in defense systems that protect bacteria against phage. The bacterial CARD is essential for protease-mediated activation of certain bacterial gasdermins, which promote cell death once phage infection is recognized. We further show that multiple anti-phage defense systems utilize CARD-like domains to activate a variety of cell death effectors. We find that these systems are triggered by a conserved immune evasion protein that phages use to overcome the bacterial defense system RexAB, demonstrating that phage proteins inhibiting one defense system can activate another. We also detect a phage protein with a predicted CARD-like structure that can inhibit the CARD-containing bacterial gasdermin system. Our results suggest that CARD domains represent an ancient component of innate immune systems conserved from bacteria to humans, and that CARD-dependent activation of gasdermins is conserved in organisms across the tree of life.

microbiology↗

bioRxiv2023

Structural basis of Gabija anti-phage defense and viral immune evasion

Bacteria encode hundreds of diverse defense systems that protect from viral infection and inhibit phage propagation1-5. Gabija is one of the most prevalent anti-phage defense systems, occurring in >15% of all sequenced bacterial and archaeal genomes1,6,7, but the molecular basis of how Gabija defends cells from viral infection remains poorly understood. Here we use X-ray crystallography and cryo-EM to define how Gabija proteins assemble into an [~]500 kDa supramolecular complex that degrades phage DNA. Gabija protein A (GajA) is a DNA endonuclease that tetramerizes to form the core of the anti-phage defense complex. Two sets of Gabija protein B (GajB) dimers dock at opposite sides of the complex and create a 4:4 GajAB assembly that is essential for phage resistance in vivo. We show that a phage-encoded protein Gabija anti-defense 1 (Gad1) directly binds the Gabija GajAB complex and inactivates defense. A cryo-EM structure of the virally inhibited state reveals that Gad1 forms an octameric web that encases the GajAB complex and inhibits DNA recognition and cleavage. Our results reveal the structural basis of assembly of the Gabija anti-phage defense complex and define a unique mechanism of viral immune evasion.

microbiology↗

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

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↗

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

Improving the efficacy and accessibility of intracranial viral vector delivery in non-human primates

Non-human primates (NHPs) are precious resources for cutting edge neuroscientific research, including large-scale viral vector-based experimentation such as optogenetics. We propose to improve surgical outcomes by enhancing surgical preparation practices of convection-enhanced delivery (CED), which is an efficient viral vector infusion technique for large brains such as NHPs. Here we present both real-time and next-day MRI data of CED in the brains of ten NHPs, and we present a quantitative, inexpensive, and practical bench-side model of the in vivo CED data. Our bench-side model is composed of food coloring infused into a transparent agar phantom, and the spread of infusion is optically monitored over time. Our proposed method approximates CED infusions into the cortex, thalamus, medial temporal lobe and caudate nucleus of NHPs, confirmed by MRI data acquired with either gadolinium-based or manganese-based contrast agents co-infused with optogenetic viral vectors. These methods and data serve to guide researchers and surgical team members in key surgical preparations for intracranial viral delivery using CED in NHPs, and thus improve expression targeting and efficacy and, as a result, reduce surgical risks.

neuroscience↗

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↗

bioRxiv2020

Dynamic competition between SARS-CoV-2 NSP1 and mRNA on the human ribosome inhibits translation initiation

SARS-CoV-2 recently emerged as a human pathogen and is the causative agent of the COVID-19 pandemic. A molecular framework of how the virus manipulates host cellular machinery to facilitate infection remains unclear. Here, we focus on SARS-CoV-2 NSP1, which is proposed to be a virulence factor that inhibits protein synthesis by directly binding the human ribosome. Using extract-based and reconstitution experiments, we demonstrate that NSP1 inhibits translation initiation on model human and SARS-CoV-2 mRNAs. NSP1 also specifically binds to the small (40S) ribosomal subunit, which is required for translation inhibition. Using single-molecule fluorescence assays to monitor NSP1-40S subunit binding in real time, we demonstrate that eukaryotic translation initiation factors (eIFs) modulate the interaction: NSP1 rapidly and stably associates with most ribosomal pre-initiation complexes in the absence of mRNA, with particular enhancement and inhibition by eIF1 and eIF3j, respectively. Using model mRNAs and an inter-ribosomal-subunit FRET signal, we elucidate that NSP1 competes with RNA segments downstream of the start codon to bind the 40S subunit and that the protein is unable to associate rapidly with 80S ribosomes assembled on an mRNA. Collectively, our findings support a model where NSP1 associates with the open head conformation of the 40S subunit to inhibit an early step of translation, by preventing accommodation of mRNA within the entry channel. SIGNIFICANCE STATEMENTSARS-CoV-2 is the causative agent of the COVID-19 pandemic. A molecular framework for how SARS-CoV-2 manipulates host cellular machinery to facilitate infection is needed. Here, we integrate biochemical and single-molecule strategies to reveal molecular insight into how NSP1 from SARS-CoV-2 inhibits translation initiation. NSP1 directly binds to the small (40S) subunit of the human ribosome, which is modulated by human initiation factors. Further, NSP1 and mRNA compete with each other to bind the ribosome. Our findings suggest that the presence of NSP1 on the small ribosomal subunit prevents proper accommodation of the mRNA. How this competition disrupts the many steps of translation initiation is an important target for future studies.

biophysics↗

bioRxiv2019

A memory of RPS25 loss drives resistance phenotypes

In order to maintain cellular protein homeostasis, ribosomes are safeguarded against dysregulation by myriad processes. Many cell types can nonetheless withstand genetic lesions of certain ribosomal protein genes, some of which are linked to diverse cellular phenotypes and human disease. However, the direct and indirect consequences from sustained alterations in ribosomal protein levels are poorly understood. To address this knowledge gap, we studied in vitro and cellular consequences that follow genetic knockout of the ribosomal proteins RPS25 or RACK1 in a human cell line, as both proteins are implicated in direct translational control. Prompted by the unexpected detection of an off-target ribosome alteration in the RPS25 knockout, we closely interrogated cellular phenotypes. We found that multiple RPS25 knockout clones display viral- and toxin-resistance phenotypes that cannot be rescued by functional cDNA expression, suggesting that RPS25 loss elicits a cell state transition. We characterized this state and found that it underlies pleiotropic phenotypes and has a common rewiring of gene expression. Rescuing RPS25 expression by genomic locus repair failed to correct for the phenotypic and expression hysteresis. Our findings illustrate how the elasticity of cells to a ribosome perturbation can drive specific phenotypic outcomes that are indirectly linked to translation.

molecular biology↗

bioRxiv2019

Mammalian Y RNAs are modified at discrete guanosine residues with N-glycans

Glycans modify lipids and proteins to mediate inter- and intramolecular interactions across all domains of life. RNA, another multifaceted biopolymer, is not thought to be a major target of glycosylation. Here, we challenge this view with evidence that mammalian cells use RNA as a third scaffold for glycosylation in the secretory pathway. Using a battery of chemical and biochemical approaches, we find that a select group of small noncoding RNAs including Y RNAs are modified with complex, sialylated N-glycans (glycoRNAs). These glycoRNA are present in multiple cell types and mammalian species, both in cultured cells and in vivo. Finally, we find that RNA glycosylation depends on the canonical N-glycan biosynthetic machinery within the ER/Golgi luminal spaces. Collectively, these findings suggest the existence of a ubiquitous interface of RNA biology and glycobiology suggesting an expanded role for glycosylation beyond canonical lipid and protein scaffolds.

molecular biology↗

bioRxiv2019

Ribosomal protein RACK1 facilitates efficient translation of poliovirus and other viral IRESs

Viruses have evolved various strategies to ensure efficient translation using host cell ribosomes and translation factors. In addition to cleaving translation initiation factors required for host cell translation, poliovirus (PV) uses an internal ribosome entry site (IRES) to bypass the need for these translation initiation factors. Recent studies also suggest that viruses have evolved to exploit specific ribosomal proteins to enhance translation of their viral proteins. The ribosomal protein receptor for activated C kinase 1 (RACK1), a protein of the 40S ribosomal subunit, was previously shown to mediate translation of the 5' cricket paralysis virus and hepatitis C virus IRESs. Here we found that while translation of a PV dual-luciferase reporter shows only a moderate dependence on RACK1, PV translation in the context of a viral infection is drastically reduced. We observed significantly reduced poliovirus plaque size and a delayed host cell translational shut-off suggesting that loss of RACK1 increases the length of the virus life cycle. Our findings further illustrate the involvement of the cellular translational machinery in PV infection and how viruses usurp the function of specific ribosomal proteins.

molecular biology↗

bioRxiv2019

eIF5B gates the transition from translation initiation to elongation

Translation initiation determines both the quantity and identity of the protein product by establishing the reading frame for protein synthesis. In eukaryotic cells, numerous translation initiation factors (eIFs) prepare ribosomes for polypeptide elongation, yet the underlying dynamics of this process remain enigmatic1-4. A central question is how eukaryotic ribosomes transition from translation initiation to elongation. Here, we applied in vitro single-molecule fluorescence microscopy approaches to monitor directly in real time the pathways of late translation initiation and the transition to elongation using a purified yeast Saccharomyces cerevisiae translation system. This transition was remarkably slower in our eukaryotic system than that reported for Escherichia coli5-7. The slow entry to elongation was defined by a long residence time of eIF5B on the 80S ribosome after joining of individual ribosomal subunits, which is catalyzed by this universally conserved initiation factor. Inhibition of eIF5B GTPase activity following subunit joining prevented eIF5B dissociation from the 80S complex, thereby preventing elongation. Our findings illustrate how eIF5B dissociation serves as a kinetic checkpoint for the transition from initiation to elongation, and its release may be governed by a conformation of the ribosome complex that triggers GTP hydrolysis.

biochemistry↗

bioRxiv2019

RACK1 on and off the ribosome

Receptor for activated C kinase 1 (RACK1) is a eukaryote-specific ribosomal protein implicated in diverse biological functions. To engineer ribosomes for specific fluorescent labeling, we selected RACK1 as an target given its location on the small ribosomal subunit and other properties. However, prior results suggested that RACK1 has roles both on and off the ribosome, and such an exchange might be related to its various cellular functions and hinder our ability to use RACK1 as a stable fluorescent tag for the ribosome. In addition, the kinetics of spontaneous exchange of RACK1 or any ribosomal protein from a mature ribosome in vitro remain unclear. To address these issues, we engineered fluorescently-labeled human ribosomes via RACK1, and applied bulk and single-molecule biochemical analyses to track RACK1 on and off the human ribosome. Our results demonstrate that, despite its cellular non-essentiality from yeast to humans, RACK1 readily re-associates with the ribosome, displays limited conformational dynamics, and remains stably bound to the ribosome for hours in vitro. This work sheds insight onto the biochemical basis of ribosomal protein exchange on and off a mature ribosome and provides tools for single-molecule analysis of human translation.

biochemistry↗