scholarly journals Cyclic Tetra-Adenylate (cA4) Recognition by Csa3; Implications for an Integrated Class 1 CRISPR-Cas Immune Response in Saccharolobus solfataricus

Biomolecules ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1852
Author(s):  
Alexander A. Charbonneau ◽  
Debra M. Eckert ◽  
Colin C. Gauvin ◽  
Nathanael G. Lintner ◽  
C. Martin Lawrence
Keyword(s):  
Immune Response ◽  
Transcription Factors ◽  
Conformational Changes ◽  
Second Messengers ◽  
Nuclease Activity ◽  
Type I ◽  
Independent Manner ◽  
Type Iii ◽  
Class 1 ◽  
Associated Proteins

Csa3 family transcription factors are ancillary CRISPR-associated proteins composed of N-terminal CARF domains and C-terminal winged helix-turn-helix domains. The activity of Csa3 transcription factors is thought to be controlled by cyclic oligoadenyate (cOA) second messengers produced by type III CRISPR-Cas surveillance complexes. Here we show that Saccharolobus solfataricus Csa3a recognizes cyclic tetra-adenylate (cA4) and that Csa3a lacks self-regulating “ring nuclease” activity present in some other CARF domain proteins. The crystal structure of the Csa3a/cA4 complex was also determined and the structural and thermodynamic basis for cA4 recognition are described, as are conformational changes in Csa3a associated with cA4 binding. We also characterized the effect of cA4 on recognition of putative DNA binding sites. Csa3a binds to putative promoter sequences in a nonspecific, cooperative and cA4-independent manner, suggesting a more complex mode of transcriptional regulation. We conclude the Csa3a/cA4 interaction represents a nexus between the type I and type III CRISPR-Cas systems present in S. solfataricus, and discuss the role of the Csa3/cA4 interaction in coordinating different arms of this integrated class 1 immune system to mount a synergistic, highly orchestrated immune response.

scholarly journals Csx3 is a cyclic oligonucleotide phosphodiesterase associated with type III CRISPR–Cas that degrades the second messenger cA4

2020 ◽  
Vol 295 (44) ◽  
pp. 14963-14972
Author(s):  
Sharidan Brown ◽  
Colin C. Gauvin ◽  
Alexander A. Charbonneau ◽  
Nathaniel Burman ◽  
C. Martin Lawrence
Keyword(s):  
Second Messengers ◽  
Second Messenger ◽  
Nuclease Activity ◽  
Type I ◽  
Type Iii ◽  
Cell Death Pathway ◽  
Cyclase Domain ◽  
Protospacer Adjacent Motif ◽  
Core Elements ◽  
Mechanistic Basis

Cas10 is the signature gene for type III CRISPR–Cas surveillance complexes. Unlike type I and type II systems, type III systems do not require a protospacer adjacent motif and target nascent RNA associated with transcriptionally active DNA. Further, target RNA recognition activates the cyclase domain of Cas10, resulting in the synthesis of cyclic oligoadenylate second messengers. These second messengers are recognized by ancillary Cas proteins harboring CRISPR-associated Rossmann fold (CARF) domains and regulate the activities of these proteins in response to invading nucleic acid. Csx3 is a distant member of the CARF domain superfamily previously characterized as a Mn2+-dependent deadenylation exoribonuclease. However, its specific role in CRISPR–Cas defense remains to be determined. Here we show that Csx3 is strongly associated with type III systems and that Csx3 binds cyclic tetra-adenylate (cA4) second messenger with high affinity. Further, Csx3 harbors cyclic oligonucleotide phosphodiesterase activity that quickly degrades this cA4 signal. In addition, structural analysis identifies core elements that define the CARF domain fold, and the mechanistic basis for ring nuclease activity is discussed. Overall, the work suggests that Csx3 functions within CRISPR–Cas as a counterbalance to Cas10 to regulate the duration and amplitude of the cA4 signal, providing an off ramp from the programmed cell death pathway in cells that successfully cure viral infection.

scholarly journals Fuse to defuse: a self-limiting ribonuclease-ring nuclease fusion for type III CRISPR defence

2020 ◽  
Author(s):  
Aleksei Samolygo ◽  
Januka S. Athukoralage ◽  
Shirley Graham ◽  
Malcolm F. White
Keyword(s):  
Immune Response ◽  
Fusion Protein ◽  
Binding Sites ◽  
Second Messengers ◽  
Nuclease Activity ◽  
Ribonuclease Activity ◽  
Type Iii ◽  
Archaeal Viruses ◽  
Archaeal Species ◽  
Cell Dormancy

AbstractType III CRISPR systems synthesise cyclic oligoadenylate (cOA) second messengers in response to viral infection of bacteria and archaea, potentiating an immune response by binding and activating ancillary effector nucleases such as Csx1. As these effectors are not specific for invading nucleic acids, a prolonged activation can result in cell dormancy or death. To avoid this fate, some archaeal species encode a specialised ring nuclease enzyme (Crn1) to degrade cyclic tetra-adenylate (cA4) and deactivate the ancillary nucleases. Some archaeal viruses and bacteriophage encode a potent ring nuclease anti-CRISPR, AcrIII-1, to rapidly degrade cA4 and neutralise immunity. Homologues of this enzyme (named Crn2) exist in type III CRISPR systems but are uncharacterised. Here we describe an unusual fusion between cA4-activated CRISPR ribonuclease (Csx1) and a cA4-degrading ring nuclease (Crn2) from Marinitoga piezophila. The protein has two binding sites that compete for the cA4 ligand, a canonical cA4-activated ribonuclease activity in the Csx1 domain and a potent cA4 ring nuclease activity in the C-terminal Crn2 domain. The activities of the two constituent enzymes in the fusion protein cooperate to ensure a robust but time-limited cOA-activated ribonuclease activity that is finely tuned to cA4 levels as a second messenger of infection.

scholarly journals Fuse to defuse: a self-limiting ribonuclease-ring nuclease fusion for type III CRISPR defence

2020 ◽  
Vol 48 (11) ◽  
pp. 6149-6156 ◽  
Author(s):  
Aleksei Samolygo ◽  
Januka S Athukoralage ◽  
Shirley Graham ◽  
Malcolm F White
Keyword(s):  
Immune Response ◽  
Binding Sites ◽  
Second Messengers ◽  
Nuclease Activity ◽  
Ribonuclease Activity ◽  
Binding Affinities ◽  
Type Iii ◽  
Archaeal Viruses ◽  
Archaeal Species ◽  
Cell Dormancy

Abstract Type III CRISPR systems synthesise cyclic oligoadenylate (cOA) second messengers in response to viral infection of bacteria and archaea, potentiating an immune response by binding and activating ancillary effector nucleases such as Csx1. As these effectors are not specific for invading nucleic acids, a prolonged activation can result in cell dormancy or death. Some archaeal species encode a specialised ring nuclease enzyme (Crn1) to degrade cyclic tetra-adenylate (cA4) and deactivate the ancillary nucleases. Some archaeal viruses and bacteriophage encode a potent ring nuclease anti-CRISPR, AcrIII-1, to rapidly degrade cA4 and neutralise immunity. Homologues of this enzyme (named Crn2) exist in type III CRISPR systems but are uncharacterised. Here we describe an unusual fusion between cA4-activated CRISPR ribonuclease (Csx1) and a cA4-degrading ring nuclease (Crn2) from Marinitoga piezophila. The protein has two binding sites that compete for the cA4 ligand, a canonical cA4-activated ribonuclease activity in the Csx1 domain and a potent cA4 ring nuclease activity in the C-terminal Crn2 domain. The cA4 binding affinities and activities of the two constituent enzymes in the fusion protein may have evolved to ensure a robust but time-limited cOA-activated ribonuclease activity that is finely tuned to cA4 levels as a second messenger of infection.

scholarly journals Cryo-EM structure of phosphodiesterase 6 reveals insights into the allosteric regulation of type I phosphodiesterases

2019 ◽  
Vol 5 (2) ◽  
pp. eaav4322 ◽  
Author(s):  
Sahil Gulati ◽  
Krzysztof Palczewski ◽  
Andreas Engel ◽  
Henning Stahlberg ◽  
Lubomir Kovacik
Keyword(s):  
Conformational Changes ◽  
Second Messengers ◽  
Structural Features ◽  
Full Length ◽  
Type I ◽  
Regulatory Domains ◽  
Catalytic Domains ◽  
Cryo Electron Microscopy ◽  
Density Map ◽  
G Protein Coupled

Cyclic nucleotide phosphodiesterases (PDEs) work in conjunction with adenylate/guanylate cyclases to regulate the key second messengers of G protein–coupled receptor signaling. Previous attempts to determine the full-length structure of PDE family members at high-resolution have been hindered by structural flexibility, especially in their linker regions and N- and C-terminal ends. Therefore, most structure-activity relationship studies have so far focused on truncated and conserved catalytic domains rather than the regulatory domains that allosterically govern the activity of most PDEs. Here, we used single-particle cryo–electron microscopy to determine the structure of the full-length PDE6αβ2γ complex. The final density map resolved at 3.4 Å reveals several previously unseen structural features, including a coiled N-terminal domain and the interface of PDE6γ subunits with the PDE6αβ heterodimer. Comparison of the PDE6αβ2γ complex with the closed state of PDE2A sheds light on the conformational changes associated with the allosteric activation of type I PDEs.

scholarly journals STING: a master regulator in the cancer-immunity cycle

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Yuanyuan Zhu ◽  
Xiang An ◽  
Xiao Zhang ◽  
Yu Qiao ◽  
Tongsen Zheng ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Immune Responses ◽  
Type I Interferons ◽  
Type I ◽  
Type I Ifn ◽  
Negative Effects ◽  
Independent Manner ◽  
Adaptive Immune ◽  
Cancer Immunity

Abstract The aberrant appearance of DNA in the cytoplasm triggers the activation of cGAS-cGAMP-STING signaling and induces the production of type I interferons, which play critical roles in activating both innate and adaptive immune responses. Recently, numerous studies have shown that the activation of STING and the stimulation of type I IFN production are critical for the anticancer immune response. However, emerging evidence suggests that STING also regulates anticancer immunity in a type I IFN-independent manner. For instance, STING has been shown to induce cell death and facilitate the release of cancer cell antigens. Moreover, STING activation has been demonstrated to enhance cancer antigen presentation, contribute to the priming and activation of T cells, facilitate the trafficking and infiltration of T cells into tumors and promote the recognition and killing of cancer cells by T cells. In this review, we focus on STING and the cancer immune response, with particular attention to the roles of STING activation in the cancer-immunity cycle. Additionally, the negative effects of STING activation on the cancer immune response and non-immune roles of STING in cancer have also been discussed.

scholarly journals Type I and type III interferon-induced immune response: It's a matter of kinetics and magnitude

Hepatology ◽  
2014 ◽  
Vol 59 (4) ◽  
pp. 1225-1228 ◽  
Author(s):  
David Olagnier ◽  
John Hiscott
Keyword(s):  
Immune Response ◽  
Type I ◽  
Type Iii ◽  
Type Iii Interferon

scholarly journals Synergistic activation of type III protein kinase C by cis-fatty acid and diacylglycerol

1992 ◽  
Vol 282 (1) ◽  
pp. 33-39 ◽  
Author(s):  
S G Chen ◽  
K Murakami
Keyword(s):  
Protein Kinase C ◽  
Fatty Acid ◽  
Protein Kinase ◽  
Second Messengers ◽  
Type I ◽  
Physiological Concentration ◽  
Type Iii ◽  
Kinase C ◽  
Synergistic Activation ◽  
Protein Kinase C Activation

Micromolar concentrations of cis-fatty acid synergistically activate type III protein kinase C with diacylglycerol. This synergistic effect occurs at low concentrations of cis-fatty acid and diacylglycerol, and it is capable of inducing almost full activation of this protein kinase C subtype at a physiologically relevant Ca2+ concentration (2 microM). The synergistic activation mode can be observed even in the absence of Ca2+, but micromolar Ca2+ significantly enhances the type III protein kinase C activation. cis-Fatty acid also augments the diacylglycerol-induced activation of other subtypes (type I and II), although the effect is smaller than that observed in type III. Neither the diacylglycerol- nor the cis-fatty acid-dependent mode of activation can fully activate any of these subtypes at a physiological concentration of Ca2+ (2 microM). Our results suggest that the generation of three second messengers, i.e. the increase in intracellular Ca2+ concentration and the generation of both cis-fatty acid and diacylglycerol in the cell, may be necessary signals for protein kinase C activation, particularly for type III protein kinase C.

scholarly journals Free ISG15 as a dimer generates IL-1β-producing CD8α+ dendritic cells at the site of infection

2017 ◽  
Author(s):  
Anna Napolitano ◽  
Annemarthe G. van der Veen ◽  
Monique Bunyan ◽  
Annabel Borg ◽  
Svend Kjaer ◽  
...  
Keyword(s):  
Immune Response ◽  
Dendritic Cells ◽  
Type I ◽  
Type I Ifn ◽  
Independent Manner ◽  
Molecular Determinants ◽  
Live Parasite ◽  
Ifn Γ ◽  
Covalently Linked ◽  
First Time

AbstractISG15 is strongly induced after type I IFN stimulation producing a protein comprised of two ubiquitin-like domains. Intracellularly, ISG15 can be covalently linked and modify the function of target proteins (ISGylation). In addition, free unconjugated ISG15 can be released from cells. We found that ISG15 is released in the serum of Toxoplasma gondii infected mice early after infection in a type-I IFN independent manner. Once in the extracellular space, free ISG15 forms dimers and enhances the release of key cytokines involved in the immune response to the parasite: IL-12, IFN-γ, and IL-1β. Its action is dependent on an actively invading and replicating live parasite. ISG15 induces an increase of IL-1β later during infection by leading to increased IL-1β producing CD8α+ dendritic cells at the site of infection. Here, we define for the first time the molecular determinants of active free ISG15 and link ISG15 to IL-1β production by CD8α+ dendritic cells. Thus we define ISG15 as a novel secreted modulator of the cytokine response during Toxoplasma infection.

scholarly journals CRISPR-Cas3 induces broad and unidirectional genome editing in human cells

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Hiroyuki Morisaka ◽  
Kazuto Yoshimi ◽  
Yuya Okuzaki ◽  
Peter Gee ◽  
Yayoi Kunihiro ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Cleavage ◽  
Exon Skipping ◽  
Nuclease Activity ◽  
Human Cells ◽  
Eukaryotic Cells ◽  
Type I ◽  
Crispr System ◽  
Class 1 ◽  
Component Class

AbstractAlthough single-component Class 2 CRISPR systems, such as type II Cas9 or type V Cas12a (Cpf1), are widely used for genome editing in eukaryotic cells, the application of multi-component Class 1 CRISPR has been less developed. Here we demonstrate that type I-E CRISPR mediates distinct DNA cleavage activity in human cells. Notably, Cas3, which possesses helicase and nuclease activity, predominantly triggered several thousand base pair deletions upstream of the 5′-ARG protospacer adjacent motif (PAM), without prominent off-target activity. This Cas3-mediated directional and broad DNA degradation can be used to introduce functional gene knockouts and knock-ins. As an example of potential therapeutic applications, we show Cas3-mediated exon-skipping of the Duchenne muscular dystrophy (DMD) gene in patient-induced pluripotent stem cells (iPSCs). These findings broaden our understanding of the Class 1 CRISPR system, which may serve as a unique genome editing tool in eukaryotic cells distinct from the Class 2 CRISPR system.

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