Biology subjects

Schwede, F.

Publications and source records attributed to Schwede, F..

9 recordsLinked to original sources

bioRxiv2026

tRNA-derived second messengers confer bacterial immunity

Bacterial small-molecule messengers that function in diverse immune signaling pathways have been thought to derive strictly from biosynthesis using nucleotide and cofactor substrates. Here we report a nuclease-driven immune signaling pathway in which tRNA cleavage generates a specific small molecule that triggers cell death in response to RNA-guided transcript recognition in prokaryotes. We identify a class of type VI-B CRISPR systems in which the RNA-guided nuclease Cas13 and its accessory transmembrane protein, 2TM{beta}, are co-encoded in an operon and are jointly required for bacteriophage defense. Upon target RNA recognition, Cas13 activates 2TM{beta} by producing mono- and dinucleotide messengers bearing 2',3'-cyclic uridine phosphate ends by tRNA cleavage. Activated 2TM{beta} destroys infected cells by forming a multimeric complex that disrupts cell membranes to mediate cell death. These findings demonstrate RNA-programmed tRNA cleavage for second-messenger biogenesis, revealing a new logic for prokaryotic immune signaling.

microbiology↗

bioRxiv2025

Activating and inhibiting nucleotide signals coordinate bacterial anti-phage defense

The cellular nucleotide pool is a major focal point of the host immune response to viral infection. Immune effector proteins that disrupt the nucleotide pool allow animal and bacterial cells to broadly restrict diverse viruses, but reduced nucleotide availability induces cellular toxicity and can limit host fitness(Ahmad et al., 1998; Goldstone et al., 2011; Hsueh et al., 2022; Itsko & Schaaper, 2014; Tal et al., 2022). Here we discover a bacterial anti-phage defense system named Clover that overcomes this tradeoff by encoding a deoxynucleoside triphosphohydrolase enzyme (CloA) that dynamically responds to both an activating phage cue and an inhibitory nucleotide immune signal produced by a partnering regulatory enzyme (CloB). Analysis of Clover phage restriction in cells and reconstitution of enzymatic function in vitro demonstrate that CloA is a dGTPase that responds to viral enzymes that increase cellular levels of dTTP. To restrain CloA activation in the absence of infection, we show that CloB synthesizes a dTTP-related inhibitory nucleotide signal p3diT (5'-triphosphothymidyl-3'5'-thymidine) that binds to CloA and suppresses activation. Cryo-EM structures of CloA in activated and suppressed states reveal how dTTP and p3diT control distinct allosteric sites and regulate effector function. Our results define how nucleotide signals coordinate both activation and inhibition of antiviral immunity and explain how cells balance defense and immune-mediated toxicity.

microbiology↗

bioRxiv2023

Purine nucleosides replace cAMP in allosteric regulation of PKA in trypanosomatid pathogens

Cyclic nucleotide binding domains (CNB) confer allosteric regulation by cAMP or cGMP to many signalling proteins, including PKA and PKG. PKA of phylogenetically distant Trypanosoma is the first exception as it is cyclic nucleotide-independent and responsive to nucleoside analogues (Bachmaier et al. 2019). Here we show that natural nucleosides inosine, guanosine and adenosine are nanomolar affinity CNB ligands and activators of PKA orthologs of the important tropical pathogens T. brucei, T. cruzi and Leishmania. The sequence and structural determinants of binding affinity, -specificity and kinase activation of PKAR were established by structure-activity relationship (SAR) analysis, co-crystal structures and mutagenesis. Substitution of 2-3 amino acids in the binding sites is sufficient for conversion of CNB domains from nucleoside to cyclic nucleotide specificity. In addition, a trypanosomatid-specific C-terminal helix (D) is required for high affinity binding to CNB-B. The D helix functions as a lid of the binding site that shields ligands from solvent. Selectivity of guanosine for CNB-B and of adenosine for CNB-A results in synergistic kinase activation at low nanomolar concentration. PKA pulldown from rapid lysis establishes guanosine as the predominant ligand in vivo in T. brucei bloodstream forms, whereas guanosine and adenosine seem to synergize in the procyclic developmental stage in the insect vector. We discuss the versatile use of CNB domains in evolution and recruitment of PKA for novel nucleoside-mediated signalling.

biochemistry↗

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

Using cGMP analogues to modulate photoreceptor light sensitivity: Perspectives for the treatment of retinal degeneration.

Cyclic nucleotide-gated (CNG) channels play an essential role within the phototransduction cascade in vertebrates. Although rod and cone light responses are mediated through similar pathways, each photoreceptor type relies on a specific CNG-channel isoform. In many forms of retinal degeneration, increased cGMP levels initiate a pathophysiological rollercoaster, which starts with an enhanced CNG-channel activation, often in rod photoreceptors. This causes cell death of both rods and cones, and eventually leads to complete blindness. Unfortunately, the targeting of the desired channel isoform still constitutes the bottleneck for many therapeutic schemes. Here, we present a novel strategy, based on cGMP analogues with opposite types of action, which allows for the selective modulation of either rod or cone photoreceptors. A combined treatment with a weak rod-selective CNG-channel inhibitor (Rp-8-Br-PET-cGMPS) and a cone-selective CNG-channel activator (8-pCPT-cGMP) preserved the normal CNG-channel function at physiological and pathological cGMP levels. The effectiveness of this approach was tested and confirmed in explanted mouse retina. Under physiological conditions, the inhibitor silenced the rods selectively and decreased the dependency of cone responses on light intensity. Remarkably, the activator, when applied together with the inhibitor, reinstated only the light responsiveness of cones. Yet, when applied alone, the activator dampened rod responses more strongly than those of cones. Hence, combinations of cGMP analogues with desired properties may elegantly address the isoform-specificity problem in future pharmacological therapies. Beyond therapies for retinal degeneration diseases, treatments based on this strategy may allow modulation of visual performance in certain light environments or disease conditions. One Sentence SummaryA combination of cGMP analogues with opposite types of action can selectively modulate either rod or cone photoreceptor function.

physiology↗

bioRxiv2021

Retinal degeneration: Multilevel protection of photoreceptor and ganglion cell viability and function with the novel PKG inhibitor CN238.

Hereditary retinal degeneration (RD) is often associated with excessive cGMP-signaling in photoreceptors. Previous research has shown that inhibition of cGMP-dependent protein kinase G (PKG) can slow down the loss of photoreceptors in different RD animal models. In this study, we identified a novel PKG inhibitor, the cGMP analogue CN238, with strong protective effects on photoreceptors in retinal degeneration rd1 and rd10 mutant mice. In long-term organotypic retinal explants, CN238 preserved rd1 and rd10 photoreceptor viability and function. Surprisingly, in explanted retinae CN238 also protected retinal ganglion cells from axotomy induced retrograde degeneration and preserved their functionality. Together, these results confirm the strong neuroprotective capacity of PKG inhibitors for both photoreceptors and retinal ganglion cells, thereby significantly broadening their potential applications for the treatment of retinal diseases and possibly neurodegenerative diseases in general.

neuroscience↗

bioRxiv2020

Redefining the role of Ca2+-permeable channels in hereditary photoreceptor degeneration using the D- and L-cis enantiomers of diltiazem

Hereditary degeneration of photoreceptors has been linked to over-activation of Ca2+-permeable channels, excessive Ca2+-influx, and downstream activation of Ca2+-dependent calpain-type proteases. Unfortunately, after more than 20 years of pertinent research, unequivocal evidence proving significant and reproducible photoreceptor protection with Ca2+-channel blockers is still lacking. Here, we show that both D- and L-cis enantiomers of the anti-hypertensive drug diltiazem were very effective at blocking photoreceptor Ca2+-influx, most probably by blocking the pore of Ca2+-permeable channels. Yet, unexpectedly, this block neither reduced the activity of calpain-type proteases, nor did it result in photoreceptor protection. Remarkably, application of the L-cis enantiomer of diltiazem even led to a strong increase in photoreceptor cell death. These findings shed doubt on the previously proposed links between Ca2+ and retinal degeneration and are highly relevant for future therapy development as they may serve to refocus research efforts towards alternative, Ca2+-independent degenerative mechanisms.

neuroscience↗

bioRxiv2020

Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6

Upon target RNA recognition, type III CRISPR-Cas systems produce cyclic oligoadenylate second messengers to activate downstream effectors including Csm6-family ribonucleases via their CARF domains. Here we show that Enteroccocus italicus Csm6 (EiCsm6) degrades its cognate cyclic hexa-AMP (cA6) activator and report the crystal structure of EiCsm6 bound to a cA6 mimic. The structure, combined with biochemical and in vivo functional assays, reveal how cA6 recognition by the CARF domain activates the Csm6 HEPN domains for collateral RNA degradation, and how CARF domain-mediated cA6 cleavage provides an intrinsic off-switch to limit Csm6 activity in the absence of ring nucleases. These mechanisms facilitate rapid invader clearance and ensure termination of CRISPR interference to limit self-toxicity.

biochemistry↗

bioRxiv2017

Type III CRISPR-Cas systems generate cyclic oligoadenylate second messengers to activate Csm6 RNases

In many prokaryotes, type III CRISPR-Cas systems detect and degrade invasive genetic elements by an RNA-guided, RNA-targeting multisubunit interference complex that possesses dual RNase and DNase activities. The CRISPR-associated protein Csm6 additionally contributes to interference by functioning as a standalone ribonuclease that degrades invader RNA transcripts, but the mechanism linking invader sensing to Csm6 activity is not understood. Here we show that Csm6 proteins are activated through a second messenger generated by the type III interference complex. Upon target RNA binding by the type III interference complex, the Cas10 subunit converts ATP into a cyclic oligoadenylate product, which allosterically activates Csm6 by binding to its CARF domain. CARF domain mutations that abolish allosteric activation inhibit Csm6 activity in vivo, and mutations in the Cas10 Palm domain phenocopy loss of Csm6. Together, these results point to a hitherto unprecedented mechanism for regulation of CRISPR interference that bears striking conceptual similarity to oligoadenylate signalling in mammalian innate immunity.

biochemistry↗