Site-specific ubiquitination of MLKL targets it to endosomes and targets Listeria and Yersinia to the lysosomes

Phosphorylation of the pseudokinase mixed lineage kinase domain-like protein (MLKL) by the protein kinase RIPK3 targets MLKL to the cell membrane, where it triggers necroptotic cell death. We report that conjugation of K63-linked polyubiquitin chains to distinct lysine residues in the N-terminal HeLo domain of phosphorylated MLKL (facilitated by the ubiquitin ligase ITCH that binds MLKL via a WW domain) targets MLKL instead to endosomes. This results in the release of phosphorylated MLKL within extracellular vesicles. It also prompts enhanced endosomal trafficking of intracellular bacteria such as Listeria monocytogenes and Yersinia enterocolitica to the lysosomes, resulting in decreased bacterial yield. Thus, MLKL can be directed by specific covalent modifications to differing subcellular sites, whence it signals either for cell death or for non-deadly defense mechanisms.

Plasma RIPK3 And HMGB1 Predict Severe COVID-19 Progression In ICU Patients: A Single-Center Cohort Study

Background: Severe progression of coronavirus disease 2019 (COVID‑19) causes respiratory failure and critical illness. Recently, these pathologies have been associated with necroptosis, a receptor‑interacting serine/threonine‑protein kinase 3 (RIPK3) dependent regulated form of inflammatory cell death. Investigations of indicator necroptosis proteins like RIPK3, mixed lineage kinase domain‑like pseudokinase (MLKL), receptor‑interacting serine/threonine‑protein kinases 1 (RIPK1), and high‑mobility group box 1 (HMGB1) in clinical COVID‑19 manifestations are lacking.Methods: A prospective prolonged cohort study including 46 intensive care unit (ICU) patients classified with moderate and severe COVID‑19 was conducted with daily measured plasma levels of indicator necroptosis proteins like RIPK3, MLKL, RIPK1, and HMGB1 by enzyme‑linked immunosorbent assay (ELISA). On this basis, a multiple logistic (regression) classification for the prediction of severe COVID‑19 progression was performed. Results: We found significantly elevated RIPK3, MLKL, HMGB1, and RIPK1 levels in COVID‑19 patients admitted to the ICU compared to healthy controls throughout the ongoing disease, indicating necroptotic processes. Above all, with combined measurements of RIPK3 and HMGB1 plasma levels, we were able to time‑independently predict COVID‑19 severity with 84% accuracy, 90% sensitivity, and 76% specificity.Conclusion: We suggest that HMGB1 and RIPK3 are potential biomarkers to identify high‑risk COVID‑19 patients and developed a classifier for COVID‑19 severity.

MLKL D144K mutation activates the necroptotic activity of the N-terminal MLKL domain

Mixed-lineage kinase domain-like protein (MLKL) is an essential effector protein of necroptotic cell death. The four-helix bundle domain (4HB) presented by the first 125 amino acids of the N terminal domain is sufficient for its necroptotic activity. However, it has been proposed that the subsequent helix H6 of the brace region has a regulatory effect on its necroptotic activity. How the brace region restrains the necroptotic activity of the N-terminal domain of MLKL is currently unknown. Here, we demonstrate the importance of helix H6 to constrain the necroptotic activity. A single amino acid mutation D144K was able to activate the necroptotic activity of the N terminal domain of MLKL by removing helix H6 away from 4HB domain. This enabled protein oligomerization and membrane translocation. Moreover, a biophysical comparison revealed that helix H6 becomes partially unstructured due to D144K mutation, leading to a lower overall thermodynamic stability of the mutant protein compared to the wild type.

Ras-Related C3 Botulinum Toxin Substrate 1 Combining With the Mixed Lineage Kinase 3- Mitogen-Activated Protein Kinase 7- c-Jun N-Terminal Kinase Signaling Module Accelerates Diabetic Nephropathy

Ras-related C3 botulinum toxin substrate 1 (RAC1) activation plays a vital role in diabetic nephropathy (DN), but the exact mechanism remains unclear. In this study, we attempted to elucidate the precise mechanism of how RAC1 aggravates DN through cellular and animal experiments. In this study, DN was induced in mice by intraperitoneal injection of streptozotocin (STZ, 150mg/kg), and the RAC1 inhibitor NSC23766 was administered by tail vein injection. Biochemical indicators, cell proliferation and apoptosis, and morphological changes in the kidney were detected. The expression of phosphorylated c-Jun N-terminal kinase (p-JNK), nuclear factor-κB (NF-κB), and cleaved caspase-3 and the interaction between RAC1 and the mixed lineage kinase 3 (MLK3)-mitogen-activated protein kinase 7 (MKK7)-JNK signaling module were determined. Furthermore, the colocalization and direct co-interaction of RAC1 and MLK3 were confirmed. Our results showed that RAC1 accelerates renal damage and increases the expression of p-JNK, NF-κB, and cleaved caspase-3. However, inhibition of RAC1 ameliorated DN by downregulating p-JNK, NF-κB, and cleaved caspase-3. Also, RAC1 promoted the assembly of MLK3-MKK7-JNK, and NSC23766 blocked the interaction between RAC1 and MLK3-MKK7-JNK and inhibited the assembly of the MLK3-MKK7-JNK signaling module. Furthermore, RAC1 was combined with MLK3 directly, but the RAC1 Y40C mutant inhibited the interaction between RAC1 and MLK3. We demonstrated that RAC1 combining with MLK3 activates the MLK3-MKK7-JNK signaling module, accelerating DN occurrence and development, and RAC1 Y40 is an important site for binding of RAC1 to MLK3. This study illustrates the cellular and molecular mechanisms of how RAC1 accelerates DN and provides evidence of DN-targeted therapy.