The Y498T499-SARS-CoV-2 spike (S) protein interacts poorly with rat ACE2 and does not affect the rat lung.

The rat is a useful laboratory model for respiratory diseases. SARS-CoV-2 proteins, such as the spike (S) protein, can induce inflammation. This study has investigated the ability of the Q498Y, P499T (QP-YT) amino acid change, described in the S-protein of the mouse-adapted laboratory SARS-CoV-2 MA strain, to interact with rat angiotensin converting enzyme-2 (ACE2) and stimulate responses in rat lungs. A real-time S-ACE2 quantitative fusion assay shows that ancestral and L452R S-proteins fuse with human but not rat ACE2 expressed on HEK293 (human embryonic kidney-293) cells. The QP-YT S-protein retains the ability to fuse with human ACE2 and increases the binding to rat ACE2. Although lower lung of the rat contains both ACE2 and TMPRSS2 (transmembrane serine protease 2) target cells, intratracheal delivery of ancestral or QP-YT S-protein pseudotyped lentivirus did not induce measurable respiratory changes, inflammatory infiltration or innate mRNA responses. Isolation of primary cells from rat alveoli demonstrated the presence of cells expressing ACE2 and TMPRSS2. Infection of these cells, however, with ancestral or QP-YT S-protein pseudotyped lentivirus was not observed, and the QP-YT S-protein pseudotyped lentivirus poorly infected HEK293 cells expressing rat ACE2. Analysis of the amino acid changes across the S-ACE2 interface highlights not only the Y498 interaction with H353 as a likely facilitator of binding to rat ACE2 but also other amino acids that could improve this interaction. Thus, rat lungs contain cells expressing receptors for SARS-CoV-2, and the QP-YT S-protein variant can bind to rat ACE2, but this does not result in infection or stimulate responses in the lung. Further, amino acid changes in S-protein may enhance this interaction to improve the utility of the rat model for defining the role of the S-protein in driving lung inflammation.

Phosphorylation by Aurora B kinase regulates caspase-2 activity and function.

Mitotic catastrophe (MC) is an important oncosuppressive mechanism that serves to eliminate cells that become polyploid or aneuploid due to aberrant mitosis. Previous studies have demonstrated that the activation and catalytic function of caspase-2 are key steps in MC to trigger apoptosis and/or cell cycle arrest of mitotically defective cells. However, the molecular mechanisms that regulate caspase-2 activation and its function are unclear. Here, we identify six new phosphorylation sites in caspase-2 and show that a key mitotic kinase, Aurora B kinase (AURKB), phosphorylates caspase-2 at the highly conserved residue S384. We demonstrate that phosphorylation at S384 blocks caspase-2 catalytic activity and apoptosis function in response to mitotic insults, without affecting caspase-2 dimerisation. Moreover, molecular modelling suggests that phosphorylation at S384 may affect substrate binding by caspase-2. We propose that caspase-2 S384 phosphorylation by AURKB is a key mechanism that controls caspase-2 activation during mitosis.

Fluid-induced lung injury-role of TRPV4 channels.

Administration of bolus intravenous fluid is associated with respiratory dysfunction and increased mortality, findings with no clear mechanistic explanation. The objective of this study was to examine whether bolus intravenous (i.v.) fluid administration results in acute lung injury in a rat model and further, to examine whether this injury is associated with transient receptor potential vallinoid (TRPV)4 channel function and endothelial inflammatory response. Healthy male Sprague-Dawley rats were administered 60 ml/kg 0.9% saline i.v. over 30 min. Manifestation of acute lung injury was assessed by lung physiology, morphology, and markers of inflammation. The role of TRPV4 channels in fluid-induced lung injury was subsequently examined by the administration of ruthenium red (RR) in this established rat model and again in TRPV4 KO mice. In endothelial cell culture, permeability and P-selectin expression were measured following TRPV4 agonist with and without antagonist; 0.9% saline resulted in an increase in lung water, lavage protein and phospholipase A2, and plasma angiopoietin-2, with worsening in arterial blood oxygen (PaO2), lung elastance, surfactant activity, and lung histological injury score. These effects were ameliorated following i.v. fluid in rats receiving RR. TRPV4 KO mice did not develop lung edema. Expression of P-selectin increased in endothelial cells following administration of a TRPV4 agonist, which was ameliorated by simultaneous addition of RR. Bolus i.v. 0.9% saline resulted in permeability pulmonary edema. Data from ruthenium red, TRPV4 KO mice, and endothelial cell culture suggest activation of TRPV4 and release of angiopoietin 2 and P-selectin as the central mechanism.