RESUMO
RNA-targeting type VI CRISPR-Cas effectors are widely used in RNA applications. Cas13h is a recently identified subtype of Cas13 ribonuclease, with strong RNA cleavage activity and robust in vivo RNA knockdown efficiency. However, little is known regarding its biochemical properties and working mechanisms. Biochemical characterization of Cas13h1 indicated that it lacks in vitro pre-crRNA processing activity and adopts a central seed. The cleavage activity of Cas13h1 is enhanced by a R(G/A) 5'-PFS, and inhibited by tag:anti-tag RNA pairing. We determined the structures of Cas13h1-crRNA binary complex at 3.1 Å and Cas13h1-crRNA-target RNA ternary complex at 3.0 Å. The ternary complex adopts an elongated architecture, and encodes a nucleotide-binding pocket within Helical-2 domain to recognize the guanosine at the 5'-end of the target RNA. Base pairing between crRNA guide and target RNA disrupts Cas13h1-guide interactions, leading to dramatic movement of HEPN domains. Upon target RNA engagement, Cas13h1 adopts a complicated activation mechanism, including separation of HEPN catalytic residues and destabilization of the active site loop and NTD domain, to get activated. Collectively, these insights expand our understanding into Cas13 effectors.
RESUMO
BACKGROUND: Previous studies showed that monocytes from patients with unstable coronary disease exhibit a greatly enhanced production of interleukin-6 (IL-6) in response to lipopolysaccharide and artery injury. Moreover, accumulating evidence suggest that C-reactive protein (CRP) may have direct proinflammatory effects on the cells of vascular wall. Whether this enhanced inflammatory response also exists, however, in cultured monocytes from patients with unstable angina, in response to CRP, has not been investigated. METHODS: Monocytes were isolated from blood of 15 healthy volunteers (normal control), 15 patients with stable angina, and 15 patients with unstable angina by Ficoll density gradient and were stimulated by 20 microg/ml of CRP for 24 h. Measurements of IL-6 and tumor necrosis factor-a (tnf-alpha) were performed from supernatants of cultured medium in duplicate, using a commercial assay kit. RESULTS: the data showed that IL-6 and tnf-alpha concentrations of spontaneous secretion (baseline) were higher in patients with unstable angina than in patients with stable angina and normal control (IL-6: 179+/-19 vs. 87+/-6 and 89+/-8 pg/ml, p<0.05, respectively; tnf-alpha: 69+/-13 vs. 30+/-4 and 27+/-3 pg/ml, p<0.05, respectively). CRP induced the enhanced release of IL-6 and tnf-alpha by 17-fold and 23-fold increase, respectively, in patients with unstable angina, while it did about 11-fold and 19-fold increase in patients with stable angina and normal group (IL-6: 3129+/-333 vs. 991+/-134 and 987+/-102 pg/ml, p<0.01, respectively; tnf-alpha: 1554+/-784 vs. 560+/-135 and 558+/-152 pg/ml, p<0.01, respectively). CONCLUSIONS: Increased baseline concentrations of IL-6 and tnf-alpha can be a marker of the hyperresponsiveness of the inflammatory system in patients with unstable coronary disease. CRP could enhance even further this response, suggesting that a persisted and enhanced inflammatory responsiveness to CRP may be involved in the pathogenesis of unstable coronary disease.
