Sphingosine kinase 1 regulates HMGB1 translocation by directly interacting with calcium/calmodulin protein kinase II-δ in sepsis-associated liver injury

Previously, we confirmed that sphingosine kinase 1 (SphK1) inhibition improves sepsis-associated liver injury. High-mobility group box 1 (HMGB1) translocation participates in the development of acute liver failure. However, little information is available on the association between SphK1 and HMGB1 translocation during sepsis-associated liver injury. In the present study, we aimed to explore the effect of SphK1 inhibition on HMGB1 translocation and the underlying mechanism during sepsis-associated liver injury. Primary Kupffer cells and hepatocytes were isolated from SD rats. The rat model of sepsis-associated liver damage was induced by intraperitoneal injection with lipopolysaccharide (LPS). We confirmed that Kupffer cells were the cells primarily secreting HMGB1 in the liver after LPS stimulation. LPS-mediated HMGB1 expression, intracellular translocation, and acetylation were dramatically decreased by SphK1 inhibition. Nuclear histone deacetyltransferase 4 (HDAC4) translocation and E1A-associated protein p300 (p300) expression regulating the acetylation of HMGB1 were also suppressed by SphK1 inhibition. HDAC4 intracellular translocation has been reported to be controlled by the phosphorylation of HDAC4. The phosphorylation of HDAC4 is modulated by CaMKII-δ. However, these changes were completely blocked by SphK1 inhibition. Additionally, by performing coimmunoprecipitation and pull-down assays, we revealed that SphK1 can directly interact with CaMKII-δ. The colocalization of SphK1 and CaMKII-δ was verified in human liver tissues with sepsis-associated liver injury. In conclusion, SphK1 inhibition diminishes HMGB1 intracellular translocation in sepsis-associated liver injury. The mechanism is associated with the direct interaction of SphK1 and CaMKII-δ.Subject terms: Hepatotoxicity, Sepsis     

A cross-talk between epithelium and endothelium mediates human alveolar–capillary injury during SARS-CoV-2 infection

COVID-19, caused by SARS-CoV-2, is an acute and rapidly developing pandemic, which leads to a global health crisis. SARS-CoV-2 primarily attacks human alveoli and causes severe lung infection and damage. To better understand the molecular basis of this disease, we sought to characterize the responses of alveolar epithelium and its adjacent microvascular endothelium to viral infection under a co-culture system. SARS-CoV-2 infection caused massive virus replication and dramatic organelles remodeling in alveolar epithelial cells, alone. While, viral infection affected endothelial cells in an indirect manner, which was mediated by infected alveolar epithelium. Proteomics analysis and TEM examinations showed viral infection caused global proteomic modulations and marked ultrastructural changes in both epithelial cells and endothelial cells under the co-culture system. In particular, viral infection elicited global protein changes and structural reorganizations across many sub-cellular compartments in epithelial cells. Among the affected organelles, mitochondrion seems to be a primary target organelle. Besides, according to EM and proteomic results, we identified Daurisoline, a potent autophagy inhibitor, could inhibit virus replication effectively in host cells. Collectively, our study revealed an unrecognized cross-talk between epithelium and endothelium, which contributed to alveolar–capillary injury during SARS-CoV-2 infection. These new findings will expand our understanding of COVID-19 and may also be helpful for targeted drug development.Subject terms: Mechanisms of disease, Viral infection     

Molecular Dynamics Simulations of PIP2 and PIP3 in Lipid Bilayers: Determination of Ring Orientation, and the Effects of Surface Roughness on a Poisson-Boltzmann Description

Molecular dynamics (MD) simulations of phosphatidylinositol (4,5)-bisphosphate (PIP₂) and phosphatidylinositol (3,4,5)-trisphosphate (PIP₃) in 1-palmitoyl 2-oleoyl phosphatidylcholine (POPC) bilayers indicate that the inositol rings are tilted ∼40° with respect to the bilayer surface, as compared with 17° for the P-N vector of POPC. Multiple minima were obtained for the ring twist (analogous to roll for an airplane). The phosphates at position 1 of PIP₂ and PIP₃ are within an Ångström of the plane formed by the phosphates of POPC; lipids in the surrounding shell are depressed by 0.5-0.8 Å, but otherwise the phosphoinositides do not substantially perturb the bilayer. Finite size artifacts for ion distributions are apparent for systems of ∼26 waters/lipid, but, based on simulations with a fourfold increase of the aqueous phase, the phosphoinositide positions and orientations do not show significant size effects. Electrostatic potentials evaluated from Poisson-Boltzmann (PB) calculations show a strong dependence of potential height and ring orientation, with the maxima on the −25 mV surfaces (17.1 ± 0.1 Å for PIP₂ and 19.4 ± 0.3 Å for PIP₃) occurring near the most populated orientations from MD. These surfaces are well above the background height of 10 Å estimated for negatively charged cell membranes, as would be expected for lipids involved in cellular signaling. PB calculations on microscopically flat bilayers yield similar maxima as the MD-based (microscopically rough) systems, but show less fine structure and do not clearly indicate the most probable regions. Electrostatic free energies of interaction with pentalysine are also similar for the rough and flat systems. These results support the utility of a rigid/flat bilayer model for PB-based studies of PIP₂ and PIP₃ as long as the orientations are judiciously chosen.     

The Functional Repertoire of Survivin's Tails

Survivin is a multitasking protein that can inhibit cell death and that is essential for mitosis. Due to these prosurvival activities and the correlation of its expression with tumor resistance to conventional cancer treatments, survivin has received much attention as a potential oncotherapeutic target. Nevertheless, many questions regarding its exact role at the molecular level remain to be elucidated. In this study we ask whether the extreme C- and NH₂ termini of survivin are required for it to carry out its cytoprotective and mitotic duties. When assayed for their ability to act as a cytoprotectant, both survivin_1–120 and survivin_11–142 were able to protect cells against TRAIL-mediated apoptosis, but when challenged with irradiation cells expressing survivin_11–142 had no survival advantage. During mitosis, however, removing the NH₂ terminal 10 amino acids (survivin_11–142) had no apparent effect but truncating 22 amino acids from the C-terminus (survivin_1–120) prevented survivin from transferring to the midzone microtubules during anaphase. Collectively the data herein presented suggest that the C-terminus is required for cell division, and that the NH₂ terminus is dispensable for apoptosis and mitosis but required for protection from irradiation.     

Chicken riboflavin-binding protein. cDNA sequence and homology with milk folate-binding protein

The Rd gene is expressed in the livers and oviducts of laying hens and codes for the riboflavin-binding protein (RfBP) of egg yolk and egg white. A lambda gt11 cDNA library derived from chicken oviduct poly(A)+ RNA was screened with polyclonal rabbit antiserum to chicken RfBP. Positive clones were isolated and rescreened with a mixed oligonucleotide probe corresponding to residues 20-25 of the mature protein. The largest cDNA clone (969 base pairs) was subcloned into plasmid pIBI21, and the nucleotide sequence was determined by the dideoxynucleotide method. This clone contained the entire coding region for RfBP. The published amino acid sequence of the mature protein was confirmed. In addition, the following 17-residue signal peptide was deduced: Met-Leu-Arg-Phe-Ala-Ile-Thr-Leu-Phe-Ala-Val-Ile-Thr-Ser-Ser-Thr-Cys. Unexpectedly, the nucleotide sequence codes for 2 adjacent arginine residues at the carboxyl terminus that are not observed in the mature protein. The amino acid sequence of RfBP is homologous with bovine milk folate-binding protein. Eight of the nine pairs of cysteines involved in disulfide bonds in RfBP are conserved in folate-binding protein, as are all of the tryptophan residues. Sequence identity between homologous regions of these two vitamin-binding proteins is more than 30%.