The deubiquitinase USP40 preserves endothelial integrity by targeting the heat shock protein HSP90ß.

Endothelial cell (EC) barrier disruption and inflammation are the pathological hallmarks of vascular disorders and acute infectious diseases and related conditions, including the coronavirus disease 2019 (COVID-19) and sepsis. Ubiquitination plays a critical role in regulating the stability, intracellular trafficking, and enzymatic activity of proteins and is reversed by deubiquitinating enzymes (DUBs). The role of DUBs in endothelial biology is largely unknown. In this study, we report that USP40, a poorly characterized DUB, prevents EC barrier disruption through reductions in the activation of RhoA and phosphorylation of myosin light chain (MLC) and cofilin. Furthermore, USP40 reduces EC inflammation through the attenuation of NF-ĸB activation, ICAM1 expression, and leukocyte-EC adhesion. We further show that USP40 activity and expression are reduced in response to endotoxin challenge. Global depletion of USP40 and EC-targeted USP40 depletion in mice exacerbated experimental lung injury, whereas lentiviral gene transfer of USP40 protected against endotoxin-induced lung injury. Using an unbiased approach, we discovered that the protective effect of USP40 occurs through the targeting of heat shock protein 90ß (HSP90ß) for its deubiquitination and inactivation. Together, these data reveal a critical protective role of USP40 in vascular injury, identifying a unique mechanistic pathway that profoundly impacts endothelial function via DUBs.

Non-Lethal Doses of RSL3 Impair Microvascular Endothelial Barrier through Degradation of Sphingosie-1-Phosphate Receptor 1 and Cytoskeletal Arrangement in A Ferroptosis-Independent Manner.

The excess microvascular endothelial permeability is a hallmark of acute inflammatory diseases. Maintenance of microvascular integrity is critical to preventing leakage of vascular components into the surrounding tissues. Sphingosine-1-phosphate (S1P) is an active lysophospholipid that enhances the endothelial cell (EC) barrier via activation of its receptor S1PR1. Here, we delineate the effect of non-lethal doses of RSL3, an inhibitor of glutathione peroxidase 4 (GPX4), on EC barrier function. Low doses of RSL3 (50-100 nM) attenuated S1P-induced human lung microvascular barrier enhancement and the phosphorylation of AKT. To investigate the molecular mechanisms by which RSL3 attenuates S1P's effect, we examined the S1PR1 levels. RSL3 treatment reduced S1PR1 levels in 1 h, whereas the effect was attenuated by the proteasome and lysosome inhibitors as well as a lipid raft inhibitor. Immunofluorescence staining showed that RSL3 induced S1PR1 internalization from the plasma membrane into the cytoplasm. Furthermore, we found that RSL3 (100 and 200 nM) increased EC barrier permeability and cytoskeletal rearrangement without altering cell viability. Taken together, our data delineates that non-lethal doses of RSL3 impair EC barrier function via two mechanisms. RSL3 attenuates S1P1-induced EC barrier enhancement and disrupts EC barrier integrity through the generation of 4-hydroxynonena (4HNE). All these effects are independent of ferroptosis.

ISGylation of NF-κBp65 by SCFFBXL19 E3 Ligase Diminishes Endothelial Inflammation.

BACKGROUND: NF-κB (nuclear factor kappa B) plays a pivotal role in endothelial cell (EC) inflammation. Protein ISGylation is regulated by E3 ISG15 (interferon-stimulated gene 15) ligases; however, ISGylation of NF-κBp65 and its role in EC functions have not been investigated. Here, we investigate whether p65 is ISGylated and the role of its ISGylation in endothelial functions. METHODS: In vitro ISGylation assay and EC inflammation were performed. EC-specific transgenic mice were utilized in a murine model of acute lung injury. RESULTS: We find that NF-κBp65 is ISGylated in resting ECs and that the posttranslational modification is reversible. TNFα (tumor necrosis factor alpha) and endotoxin stimulation of EC reduce p65 ISGylation, promoting its serine phosphorylation through reducing its association with a phosphatase WIP1 (wild-type p53-induced phosphatase 1). Mechanistically, an SCF (Skp1-Cul1-F-box) protein E3 ligase SCFFBXL19 is identified as a new ISG15 E3 ligase that targets and catalyzes ISGylation of p65. Depletion of FBXL19 (F-box and leucine-rich repeat protein 19) increases p65 phosphorylation and EC inflammation, suggesting a negative correlation between p65 ISGylation and phosphorylation. Moreover, EC-specific FBXL19 overexpressing humanized transgenic mice exhibit reduced lung inflammation and severity of experimental acute lung injury. CONCLUSIONS: Together, our data reveal a new posttranslational modification of p65 catalyzed by a previously unrecognized role of SCFFBXL19 as an ISG15 E3 ligase that modulates EC inflammation.

Revisiting graph neural networks from hybrid regularized graph signal reconstruction.

Graph neural networks (GNNs) have shown strong graph-structured data processing capabilities. However, most of them are generated based on the message-passing mechanism and lack of the systematic approach to guide their developments. Meanwhile, a unified point of view is hard to explain the design concepts of different GNN models. This paper presents a unified optimization framework from hybrid regularized graph signal reconstruction to establish the connection between the aggregation operations of different GNNs, showing that exploring the optimal solution is the process of GNN information aggregation. We use this new framework to mathematically explain several classic GNN models and summarizes their commonalities and differences from a macro perspective. The proposed framework not only provides convenience to understand GNNs, but also has a guiding significance for the proposal of new GNNs. Moreover, we design a model-driven fixed-point iteration method and a data-driven dictionary learning network according to the corresponding optimization objective and sparse representation. Then the new model, GNN based on model-driven and data-driven (GNN-MD), is established by using alternating iteration methods. We also theoretically analyze its convergence. Numerous node classification experiments on multiple datasets illustrate that the proposed GNN-MD has excellent performance and outperforms all baselines on high-feature-dimension datasets.

IL-37-induced activation of glycogen synthase kinase 3ß promotes IL-1R8/Sigirr phosphorylation, internalization, and degradation in lung epithelial cells.

Interleukin (IL)-37 diminishes a variety of inflammatory responses through ligation to its receptor IL-1R8/Sigirr. Sigirr is a Toll like receptor/IL-1R family member. We have shown that Sigirr is not stable in response to IL-37 treatment. IL-37-induced Sigirr degradation is mediated by the ubiquitin-proteasome system, and the process is reversed by a deubiquitinase, USP13. However, the molecular mechanisms by which USP13 regulates Sigirr stability have not been revealed. In this study, we investigate the roles of glycogen synthesis kinase 3ß (GSK3ß) in Sigirr phosphorylation and stability. IL-37 stimulation induced Sigirr phosphorylation and degradation, as well as activation of GSK3ß. Inhibition of GSK3ß attenuated IL-37-induced Sigirr phosphorylation, while exogenous expressed GSK3ß promoted Sigirr phosphorylation at threonine (T)372 residue. Sigirr association with GSK3ß was detected. Amino acid residues 51-101 in GSK3ß were identified as the Sigirr binding domain. These data indicate that GSK3ß mediates IL-37-induced threonine phosphorylation of Sigirr. Further, we investigated the role of GSK3ß-mediated phosphorylation of Sigirr in Sigirr degradation. Inhibition of GSK3ß attenuated IL-37-induced Sigirr degradation, while T372 mutant of Sigirr was resistant to IL-37-mediated degradation. Furthermore, inhibition of Sigirr phosphorylation prevented Sigirr internalization and association with USP13, suggesting GSK3ß promotes Sigirr degradation through disrupting Sigirr association with USP13.

SCF FBXW17 E3 ubiquitin ligase regulates FBXL19 stability and cell migration.

The Skp1-Cul1-F-box protein (SCF) E3 ligase complex is one of the largest ubiquitin E3 ligase families. FBXL19, a F-box protein in SCFFBXL19 E3 ligase complex, regulates a variety of cellular responses including cell migration. We have shown that FBXL19 is not stable and its degradation is mediated by the ubiquitin-proteasome system, while the ubiquitin E3 ligase for FBXL19 ubiquitination and degradation has not been identified. In the study, we discovered that a new ubiquitin E3 ligase, SCFFBXW17 , ubiquitinates and induces FBXL19 degradation. Exogenous FBXW17 targets FBXL19 for its ubiquitination and degradation. Lysine 114 in FBXL19 is a potential ubiquitin acceptor site. Acetylation of FBXL19 attenuated SCFFBXW17 -mediated FBXL19 degradation. SCFFBXL19 E3 ligase reduced Rac1 levels and cell migration, while the effects were attenuated by exogenous FBXW17. Downregulation of FBXW17 attenuated lysophosphatidic acid-induced lamellipodia formation and Rac1 accumulation at migration leading edge. Taken together with our previous studies, FBXL19 is degraded by the ubiquitin-proteasome system and its site-specific ubiquitination is mediated by SCFFBXW17 E3 ligase, which promotes cell migration.

Lipopolysaccharide reduces USP13 stability through c-Jun N-terminal kinase activation in Kupffer cells.

Protein ubiquitination regulates protein stability, cellular localization, and enzyme activity. Deubiquitinases catalyze the removal of ubiquitin from target proteins and reverse ubiquitination. USP13, a deubiquitinase, has been shown to regulate a variety of cellular responses including inflammation; however, the molecular regulation of USP13 has not been demonstrated. In this study, we revealed that USP13 is degraded in response to lipopolysaccharide (LPS) in Kupffer cells. USP13 levels are significantly decreased in inflamed organs, including liver tissues from septic mice. LPS reduces USP13 protein stability, not transcription, in Kupffer cells. Furthermore, LPS increases USP13 polyubiquitination. Inhibition of proteasome, but not lysosome or immunoproteasome, attenuates LPS-induced USP13 degradation, suggesting USP13 degradation is mediated by the ubiquitin-proteasome system. A catalytically inactive form of USP13 exhibits similar degree of degradation compared with USP13 wild-type, suggesting that USP13 degradation is not dependent on its activity. Furthermore, USP13 degradation is dependent on new protein synthesis. Inhibition of c-Jun N-terminal kinase (JNK) attenuates USP13 degradation, indicating that JNK-dependent new protein synthesis is necessary for USP13 degradation. This study reveals a molecular mechanism of regulation of USP13 degradation in Kupffer cells in response to bacterial endotoxin.

Two distinct E3 ligases, SCFFBXL19 and HECW1, degrade thyroid transcription factor 1 in normal thyroid epithelial and follicular thyroid carcinoma cells, respectively.

Thyroid transcription factor 1 (TTF1) regulates the tissue-specific expression of genes. However, the molecular regulation of TTF1 in thyroid normal and carcinoma cells has not been revealed. Here we identify 2 distinct ubiquitin E3 ligases that are responsible for TTF1 degradation in normal thyroid cells and carcinoma cells, respectively. Phorbol myristate acetate induced TTF1 protein degradation in the ubiquitin-proteasome system in both HTori3 thyroid follicular epithelial cells and follicular thyroid carcinoma 133 (FTC133) cells. Lysine 151 residue was identified as a ubiquitin acceptor site within TTF1 in both cell types. Overexpression of E3 ubiquitin protein ligase 1 containing HECT, C2, and WW domain (HECW1) induced TTF1 degradation and ubiquitination in Htori3 cells but not in FTC133 cells. Overexpression of ubiquitin E3 ligase subunit FBXL19 increased TTF1 ubiquitination and degradation in FTC133 cells, but it had no effect on TTF1 levels in Htori3 cells. Overexpression of TTF1 increased thyroglobulin and sodium/iodide symporter mRNA levels, cell migration, and proliferation in HTori3 cells, whereas the effects were reversed by the overexpression of HECW1. This study reveals an undiscovered molecular mechanism by which TTF1 ubiquitination and degradation is regulated by different E3 ligases in thyroid normal and tumor cells.-Liu, J., Dong, S., Wang, H., Li, L., Ye, Q., Li, Y., Miao, J., Jhiang, S., Zhao, J., Zhao, Y. Two distinct E3 ligases, SCFFBXL19 and HECW1, degrade thyroid transcription factor 1 in normal thyroid epithelial and follicular thyroid carcinoma cells, respectively.

Albumin Nanovectors in Cancer Therapy and Imaging.

Albumin nanovectors represent one of the most promising carriers recently generated because of the cost-effectiveness of their fabrication, biocompatibility, safety, and versatility in delivering hydrophilic and hydrophobic therapeutics and diagnostic agents. In this review, we describe and discuss the recent advances in how this technology has been harnessed for drug delivery in cancer, evaluating the commonly used synthesis protocols and considering the key factors that determine the biological transport and the effectiveness of such technology. With this in mind, we highlight how clinical and experimental albumin-based delivery nanoplatforms may be designed for tackling tumor progression or improving the currently established diagnostic procedures.