Stearoylation cycle regulates the cell surface distribution of the PCP protein Vangl2.

Defects in planar cell polarity (PCP) have been implicated in diverse human pathologies. Vangl2 is one of the core PCP components crucial for PCP signaling. Dysregulation of Vangl2 has been associated with severe neural tube defects and cancers. However, how Vangl2 protein is regulated at the posttranslational level has not been well understood. Using chemical reporters of fatty acylation and biochemical validation, here we present that Vangl2 subcellular localization is regulated by a reversible S-stearoylation cycle. The dynamic process is mainly regulated by acyltransferase ZDHHC9 and deacylase acyl-protein thioesterase 1 (APT1). The stearoylation-deficient mutant of Vangl2 shows decreased plasma membrane localization, resulting in disruption of PCP establishment during cell migration. Genetically or pharmacologically inhibiting ZDHHC9 phenocopies the effects of the stearoylation loss of Vangl2. In addition, loss of Vangl2 stearoylation enhances the activation of oncogenic Yes-associated protein 1 (YAP), serine-threonine kinase AKT, and extracellular signal-regulated protein kinase (ERK) signaling and promotes breast cancer cell growth and HRas G12V mutant (HRasV12)-induced oncogenic transformation. Our results reveal a regulation mechanism of Vangl2, and provide mechanistic insight into how fatty acid metabolism and protein fatty acylation regulate PCP signaling and tumorigenesis by core PCP protein lipidation.

Assaying Lysosomal Enzyme Activity in Dictyostelium discoideum.

Lysosomes are membrane-enclosed organelles that digest intracellular material. They contain more than 50 different enzymes that can degrade a variety of macromolecules including nucleic acids, proteins, polysaccharides, and lipids. In addition to functioning within lysosomes, lysosomal enzymes are also secreted. Alterations in the levels and activities of lysosomal enzymes dysregulates lysosomes, which can lead to the intralysosomal accumulation of biological material and the development of lysosomal storage diseases (LSDs) in humans. Dictyostelium discoideum has a long history of being used to study the trafficking and functions of lysosomal enzymes. More recently, it has been used as a model system to study several LSDs. In this chapter, we outline the methods for assessing the activity of several lysosomal enzymes in D. discoideum (α-galactosidase, ß-galactosidase, α-glucosidase, ß-glucosidase, ß-N-acetylglucosaminidase, α-mannosidase, cathepsin B, cathepsin D, cathepsin F, palmitoyl protein thioesterase 1, and tripeptidyl peptidase 1).

Overview of carboxyl­terminal modulator protein 1 and its importance in various metabolic regulations (Review).

Acyl­coenzyme A thioesterases (ACOTs) are crucial in mediating lipid metabolic functions, including energy expenditure, hepatic gluconeogenesis and neuronal function. The two distinct types are type I and II ACOTs, the latter of which are 'hotdog' fold superfamily members. Type II ACOTs include carboxyl­terminal modulator protein 1 (CTMP1), also termed thioesterase superfamily member 4 (THEM4), and CTMP2, also termed THEM5. Due to their similar structural features and distinct sequence homology, CTMP1 and CTMP2 stand out from other type II ACOTs. CTMP1 was initially known as a protein kinase B (PKB) inhibitor that attenuates PKB phosphorylation. PKB is the central regulator of various cellular functions, including survival, proliferation, growth and metabolism. Therefore, by inhibiting PKB, CTMP1 can affect various cellular processes. Various other functions of CTMP1 have been revealed, including functions in cancer, brain injury, mitochondrial function and lipid metabolism. CTMP2 is a paralog of CTMP1 and was first identified as a cardiolipin remodeling factor involved in the development of fatty liver. As the functions of CTMP1 and CTMP2 were discovered separately, a review to summarize and connect these findings is essential. The current review delineates the intricate complexity of CTMP regulation across different metabolic pathways and encapsulates the principal discoveries concerning CTMP until the present day.

USP11 promotes prostate cancer progression by up-regulating AR and c-Myc activity.

Androgen receptor (AR) is a main driver for castration-resistant prostate cancer (CRPC). c-Myc is an oncogene underlying prostate tumorigenesis. Here, we find that the deubiquitinase USP11 targets both AR and c-Myc in prostate cancer (PCa). USP11 expression was up-regulated in metastatic PCa and CRPC. USP11 knockdown (KD) significantly inhibited PCa cell growth. Our RNA-seq studies revealed AR and c-Myc as the top transcription factors altered after USP11 KD. ChIP-seq analysis showed that either USP11 KD or replacement of endogenous USP11 with a catalytic-inactive USP11 mutant significantly decreased chromatin binding by AR and c-Myc. We find that USP11 employs two mechanisms to up-regulate AR and c-Myc levels: namely, deubiquitination of AR and c-Myc proteins to increase their stability and deubiquitination of H2A-K119Ub, a repressive histone mark, on promoters of AR and c-Myc genes to increase their transcription. AR and c-Myc reexpression in USP11-KD PCa cells partly rescued cell growth defects. Thus, our studies reveal a tumor-promoting role for USP11 in aggressive PCa through upregulation of AR and c-Myc activities and support USP11 as a potential target against PCa.

Methylglyoxal detoxifying gene families in tomato: Genome-wide identification, evolution, functional prediction, and transcript profiling.

Methylglyoxal (MG) is a highly cytotoxic molecule produced in all biological systems, which could be converted into non-toxic D-lactate by an evolutionarily conserved glyoxalase pathway. Glutathione-dependent glyoxalase I (GLYI) and glyoxalase II (GLYII) are responsible for the detoxification of MG into D-lactate in sequential reactions, while DJ-1 domain containing glyoxalase III (GLYIII) catalyzes the same reaction in a single step without glutathione dependency. Afterwards, D-lactate dehydrogenase (D-LDH) converts D-lactate into pyruvate, a metabolically usable intermediate. In the study, a comprehensive genome-wide investigation has been performed in one of the important vegetable plants, tomato to identify 13 putative GLYI, 4 GLYII, 3 GLYIII (DJ-1), and 4 D-LDH genes. Expression pattern analysis using microarray data confirmed their ubiquitous presence in different tissues and developmental stages. Moreover, stress treatment of tomato seedlings and subsequent qRT-PCR demonstrated upregulation of SlGLYI-2, SlGLYI-3, SlGLYI-6A, SlGLYII-1A, SlGLYII-3B, SlDJ-1A, SlDLDH-1 and SlDLDH-4 in response to different abiotic stresses, whereas SlGLYI-6B, SlGLYII-1B, SlGLYII-3A, SlDJ-1D and SlDLDH-2 were downregulated. Expression data also revealed SlGLYII-1B, SlGLYI-1A, SlGLYI-2, SlDJ-1D, and SlDLDH-4 were upregulated in response to various pathogenic infections, indicating the role of MG detoxifying enzymes in both plant defence and stress modulation. The functional characterization of each of these members could lay the foundation for the development of stress and disease-resistant plants promoting sustainable agriculture and production.

USP11 modulates mitotic progression and senescence by regulating the p53-p21 axis through MDM2 deubiquitination.

USP11 is overexpressed in colorectal cancer (CRC) and breast cancer tissues compared to normal tissues, suggesting a role in promoting cell proliferation and inhibiting cell death. In this study, we observed that depleting USP11 inhibits cell proliferation and delays cell cycle progression. This depletion leads to increased p53 protein levels due to an extended half-life, resulting in elevated p21 mRNA levels in a p53-dependent manner. The rise in p53 protein upon USP11 depletion is linked to a reduced half-life of MDM2, a known E3 ligase for p53, via enhanced polyubiquitination of MDM2. These findings indicate that USP11 might act as a deubiquitinase for MDM2, regulating the MDM2-p53-p21 axis. Additionally, USP11 depletion promotes the induction of senescent cells in a manner dependent on its deubiquitinase activity. Our findings provide insights into the physiological significance of high USP11 expression in primary tumors and its reduction in senescent cells, highlighting its potential as a therapeutic target.

Identification of a novel target of sulforaphane: Sulforaphane binds to acyl-protein thioesterase 2 (APT2) and attenuates its palmitoylation.

Sulforaphane (SFaN) is a food-derived compound with several bioactive properties, including atherosclerosis, diabetes, and obesity treatment. However, the mechanisms by which SFaN exerts its various effects are still unclear. To elucidate the mechanisms of the various effects of SFaN, we explored novel SFaN-binding proteins using SFaN beads and identified acyl protein thioesterase 2 (APT2). We also found that SFaN binds to the APT2 via C56 residue and attenuates the palmitoylation of APT2, thereby reducing plasma membrane localization of APT2. This study reveals a novel bioactivity of SFaN as a regulator of APT2 protein palmitoylation.

Penicillin-binding protein-type thioesterases: An emerging family of non-ribosomal peptide cyclases with biocatalytic potentials.

Macrocyclization of peptides reduces conformational flexibilities, potentially leading to improved drug-like properties, such as target specificities and metabolic stabilities. As chemical methodologies often allow side reactions like epimerization and oligomerization, keen attention has been directed toward enzymatic peptide cyclization using peptide cyclases from specialized metabolic pathways. Penicillin-binding protein-type thioesterases (PBP-type TEs) are a recently identified family of peptide cyclases involved in the biosynthesis of non-ribosomal peptides in actinobacteria. PBP-type TEs have undergone intensive investigation due to their outstanding potential as biocatalysts. This review summarizes the rapidly growing knowledge on PBP-type TEs, with special emphasis on their functions, scopes, and structures, and efforts towards their biocatalytic applications.

Melatonin induced reversibility of vanadium toxicity in muskmelon by regulating antioxidant defense and glyoxalase systems.

Agricultural lands with vanadium (V), pose a significant and widespread threat to crop production worldwide. The study was designed to explore the melatonin (ME) treatment in reducing the V-induced phytotoxicity in muskmelon. The muskmelon seedlings were grown hydroponically and subjected to V (40 mg L-1) stress and exogenously treated with ME (100 µmol L-1) to mitigate the V-induced toxicity. The results showed that V toxicity displayed a remarkably adverse effect on seedling growth and biomass, primarily by impeding root development, the photosynthesis system and the activities of antioxidants. Contrarily, the application of ME mitigated the V-induced growth damage and significantly improved root attributes, photosynthetic efficiency, leaf gas exchange parameters and mineral homeostasis by reducing V accumulation in leaves and roots. Additionally, a significant reduction in the accumulation of reactive oxygen species (ROS), malondialdehyde (MDA) along with a decrease in electrolyte leakage was observed in muskmelon seedlings treated with ME under V-stress. This reduction was attributed to the enhancement in the activities of antioxidants in leaves/roots such as ascorbate (AsA), superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), glutathione peroxidase (GPX), glutathione S-transferase (GST) as compared to the V stressed plants. Moreover, ME also upregulated the chlorophyll biosynthesis and antioxidants genes expression in muskmelon. Given these findings, ME treatment exhibited a significant improvement in growth attributes, photosynthesis efficiency and the activities of antioxidants (enzymatic and non-enzymatic) by regulating their expression of genes against V-stress with considerable reduction in oxidative damage.

USP11 regulates proliferation and apoptosis of human spermatogonial stem cells via HOXC5-mediated canonical WNT/ß-catenin signaling pathway.

Spermatogonial stem cells (SSCs) are capable of transmitting genetic information to the next generations and they are the initial cells for spermatogenesis. Nevertheless, it remains largely unknown about key genes and signaling pathways that regulate fate determinations of human SSCs and male infertility. In this study, we explored the expression, function, and mechanism of USP11 in controlling the proliferation and apoptosis of human SSCs as well as the association between its abnormality and azoospermia. We found that USP11 was predominantly expressed in human SSCs as shown by database analysis and immunohistochemistry. USP11 silencing led to decreases in proliferation and DNA synthesis and an enhancement in apoptosis of human SSCs. RNA-sequencing identified HOXC5 as a target of USP11 in human SSCs. Double immunofluorescence, Co-immunoprecipitation (Co-IP), and molecular docking demonstrated an interaction between USP11 and HOXC5 in human SSCs. HOXC5 knockdown suppressed the growth of human SSCs and increased apoptosis via the classical WNT/ß-catenin pathway. In contrast, HOXC5 overexpression reversed the effect of proliferation and apoptosis induced by USP11 silencing. Significantly, lower levels of USP11 expression were observed in the testicular tissues of patients with spermatogenic disorders. Collectively, these results implicate that USP11 regulates the fate decisions of human SSCs through the HOXC5/WNT/ß-catenin pathway. This study thus provides novel insights into understanding molecular mechanisms underlying human spermatogenesis and the etiology of azoospermia and it offers new targets for gene therapy of male infertility.

Effect of BvAgNP on growth, development, and glyoxalase gene expression analysis in Mammillaria bombycina and Selenicereus undatus.

BACKGROUND: Silver nanoparticles (AgNPs) have been used in plant tissue culture as growth stimulants, promoting bud initiation, germination, and rooting. In prior studies, AgNPs were synthesized and characterized by green synthesis using extracts from Beta vulgaris var. cicla (BvAgNP), and their functionality as seed disinfectant and antimicrobial was verified. In this study, we evaluated the effect of BvAgNP on the growth and development of Mammillaria bombycina and Selenicereus undatus in vitro, as well as the expression of glyoxalase genes. METHODS: Explants from M. bombycina and S. undatus in vitro were treated with 25, 50, and 100 mg/L of BvAgNP. After 90 days, morphological characteristics were evaluated, and the expression of glyoxalase genes was analyzed by qPCR. RESULTS: All treatments inhibited rooting for M. bombycina and no bud initiation was observed. S. undatus, showed a maximum response in rooting and bud generation at 25 mg/L of BvAgNP. Scanning electron microscopy (SEM) results exhibited a higher number of vacuoles in stem cells treated with BvAgNP compared to the control for both species. Expression of glyoxalase genes in M. bombycina increased in all treatments, whereas it decreased for S. undatus, however, increasing in roots. CONCLUSIONS: This study presents the effects of BvAgNP on the growth and development of M. bombycina and S. undatus, with the aim of proposing treatments that promote in vitro rooting and bud initiation.

A protein sequence-based deep transfer learning framework for identifying human proteome-wide deubiquitinase-substrate interactions.

Protein ubiquitination regulates a wide range of cellular processes. The degree of protein ubiquitination is determined by the delicate balance between ubiquitin ligase (E3)-mediated ubiquitination and deubiquitinase (DUB)-mediated deubiquitination. In comparison to the E3-substrate interactions, the DUB-substrate interactions (DSIs) remain insufficiently investigated. To address this challenge, we introduce a protein sequence-based ab initio method, TransDSI, which transfers proteome-scale evolutionary information to predict unknown DSIs despite inadequate training datasets. An explainable module is integrated to suggest the critical protein regions for DSIs while predicting DSIs. TransDSI outperforms multiple machine learning strategies against both cross-validation and independent test. Two predicted DUBs (USP11 and USP20) for FOXP3 are validated by "wet lab" experiments, along with two predicted substrates (AR and p53) for USP22. TransDSI provides new functional perspective on proteins by identifying regulatory DSIs, and offers clues for potential tumor drug target discovery and precision drug application.

OTUB2 silencing promotes ovarian cancer via mitochondrial metabolic reprogramming and can be synthetically targeted by CA9 inhibition.

Ovarian cancer is an aggressive gynecological tumor characterized by a high relapse rate and chemoresistance. Ovarian cancer exhibits the cancer hallmark of elevated glycolysis, yet effective strategies targeting cancer cell metabolic reprogramming to overcome therapeutic resistance in ovarian cancer remain elusive. Here, we revealed that epigenetic silencing of Otubain 2 (OTUB2) is a driving force for mitochondrial metabolic reprogramming in ovarian cancer, which promotes tumorigenesis and chemoresistance. Mechanistically, OTUB2 silencing destabilizes sorting nexin 29 pseudogene 2 (SNX29P2), which subsequently prevents hypoxia-inducible factor-1 alpha (HIF-1α) from von Hippel-Lindau tumor suppressor-mediated degradation. Elevated HIF-1α activates the transcription of carbonic anhydrase 9 (CA9) and drives ovarian cancer progression and chemoresistance by promoting glycolysis. Importantly, pharmacological inhibition of CA9 substantially suppressed tumor growth and synergized with carboplatin in the treatment of OTUB2-silenced ovarian cancer. Thus, our study highlights the pivotal role of OTUB2/SNX29P2 in suppressing ovarian cancer development and proposes that targeting CA9-mediated glycolysis is an encouraging strategy for the treatment of ovarian cancer.

Challenging Aromaticity: Revealing a Thioesterase Domain in a Fungal Nonreducing Polyketide Synthase Governing the Production of 3-Methylene Isochromanone.

Thioesterase (TE) domain exerts a great influence over the structure of the final product and TE-released nonreduced polyketides (nrPKs) retain aromaticity. 3-Methylene isochromanones are lactones with a unique olefin at C3 that disrupts the aromaticity, whose biosynthetic details are speculative. Our study unveils the complete biosynthesis of ascochin, in which the construction of the 3-methylene isochromanone backbone is achieved by a nonreducing polyketide synthase (nrPKS) alone and two subsequent oxidations are involved. Intriguingly, the TEAscD serves as a gatekeeper to direct the product release toward formation of nonaromatic 3-methylene isochromanone, rather than the typical aromatic product.

Caveolin-2 palmitoylation turnover facilitates insulin receptor substrate-1-directed lipid metabolism by insulin receptor tyrosine kinase.

Here, we show that insulin induces palmitoylation turnover of Caveolin-2 (Cav-2) in adipocytes. Acyl protein thioesterases-1 (APT1) catalyzes Cav-2 depalmitoylation, and zinc finger DHHC domain-containing protein palmitoyltransferase 21 (ZDHHC21) repalmitoylation of the depalmitoylated Cav-2 for the turnover, thereby controlling insulin receptor (IR)-Cav-2-insulin receptor substrate-1 (IRS-1)-Akt-driven signaling. Insulin-induced palmitoylation turnover of Cav-2 facilitated glucose uptake and fat storage through induction of lipogenic genes. Cav-2-, APT1-, and ZDHHC21-deficient adipocytes, however, showed increased induction of lipolytic genes and glycerol release. In addition, white adipose tissues from insulin sensitive and resistant obese patients exhibited augmented expression of LYPLA1 (APT1) and ZDHHC20 (ZDHHC20). Our study identifies the specific enzymes regulating Cav-2 palmitoylation turnover, and reveals a new mechanism by which insulin-mediated lipid metabolism is controlled in adipocytes.

Inhibitors of the Thioesterase Activity of Mycobacterium tuberculosis Pks13 Discovered Using DNA-Encoded Chemical Library Screening.

DNA-encoded chemical library (DEL) technology provides a time- and cost-efficient method to simultaneously screen billions of compounds for their affinity to a protein target of interest. Here we report its use to identify a novel chemical series of inhibitors of the thioesterase activity of polyketide synthase 13 (Pks13) from Mycobacterium tuberculosis (Mtb). We present three chemically distinct series of inhibitors along with their enzymatic and Mtb whole cell potency, the measure of on-target activity in cells, and the crystal structures of inhibitor-enzyme complexes illuminating their interactions with the active site of the enzyme. One of these inhibitors showed a favorable pharmacokinetic profile and demonstrated efficacy in an acute mouse model of tuberculosis (TB) infection. These findings and assay developments will aid in the advancement of TB drug discovery.

Thioesterases as tools for chemoenzymatic synthesis of macrolactones.

Macrocycles are a key functional group that can impart unique properties into molecules. Their synthesis has led to the development of many outstanding chemical methodologies and yet still remains challenging. Thioesterase (TE) domains are frequently responsible for macrocyclization in natural product biosynthesis and provide unique strengths for the enzymatic synthesis of macrocycles. In this feature article, we describe our work to characterize the substrate selectivity of TEs and to use these enzymes as biocatalysts. Our efforts have shown that the linear thioester activated substrates are loaded on TEs with limited substrate selectivity to generate acyl-enzyme intermediates. We show that cyclization of the acyl-enzyme intermediates can be highly selective, with competing hydrolysis of the acyl-enzyme intermediates. The mechanisms controlling TE-mediated macrocyclization versus hydrolysis are a significant unsolved problem in TE biochemistry. The potential of TEs as biocatalysts was demonstrated by using them in the chemoenzymatic total synthesis of macrocyclic depsipeptide natural products. This article highlights the strengths and potential of TEs as biocatalysts as well as their limitations, opening exciting research opportunities including TE engineering to optimize these powerful biocatalysts.

A Versatile Thioesterase Involved in Dimerization during Cinnamoyl Lipid Biosynthesis.

The cinnamoyl lipid compound youssoufene A1 (1), featuring a unique dearomatic carbon-bridged dimeric skeleton, exhibits increased inhibition against multidrug resistant Enterococcus faecalis as compared to monomeric youssoufenes. However, the formation process of this intriguing dearomatization/dimerization remains unknown. In this study, an unusual "gene-within-gene" thioesterase (TE) gene ysfF was functionally characterized. The gene was found to naturally encodes two proteins, an entire YsfF with α/ß-hydrolase and 4-hydroxybenzoyl-CoA thioesterase (4-HBT)-like enzyme domains, and a nested YsfFHBT (4-HBT-like enzyme). Using an intracellular tagged carrier-protein tracking (ITCT) strategy, in vitro reconstitution and in vivo experiments, we found that: i) both domains of YsfF displayed thioesterase activities; ii) YsfF/YsfFHBT could accomplish the 6π-electrocyclic ring closure for benzene ring formation; and iii) YsfF and cyclase YsfX together were responsible for the ACP-tethered dearomatization/dimerization process, possibly through an unprecedented Michael-type addition reaction. Moreover, site-directed mutagenesis experiments demonstrated that N301, E483 and H566 of YsfF are critical residues for both the 6π-electrocyclization and dimerization processes. This study enhances our understanding of the multifunctionality of the TE protein family.

Depalmitoylation and cell physiology: APT1 as a mediator of metabolic signals.

More than half of the global population is obese or overweight, especially in Western countries, and this excess adiposity disrupts normal physiology to cause chronic diseases. Diabetes, an adiposity-associated epidemic disease, affects >500 million people, and cases are projected to exceed 1 billion before 2050. Lipid excess can impact physiology through the posttranslational modification of proteins, including the reversible process of S-palmitoylation. Dynamic palmitoylation cycling requires the S-acylation of proteins by acyltransferases and the depalmitoylation of these proteins mediated in part by acyl-protein thioesterases (APTs) such as APT1. Emerging evidence points to tissue-specific roles for the depalmitoylase APT1 in maintaining homeostasis in the vasculature, pancreatic islets, and liver. These recent findings raise the possibility that APT1 substrates can be therapeutically targeted to treat the complications of metabolic diseases.

USP11 Exacerbates Radiation-Induced Pneumonitis by Activating Endothelial Cell Inflammatory Response via OTUD5-STING Signaling.

PURPOSE: Radiation-induced pneumonitis (RIP) seriously limits the application of radiation therapy in the treatment of thoracic tumors, and its etiology and pathogenesis remain elusive. This study aimed to elucidate the role of ubiquitin-specific peptidase 11 (USP11) in the progression of RIP and the associated underlying mechanisms. METHODS AND MATERIALS: Changes in cytokines and infiltrated immune cells were detected by enzyme-linked immunosorbent assays and immunohistochemistry after exposure to 20 Gy x-ray with whole-thorax irradiation. The effects of USP11 expression on endothelial cell proliferation and apoptosis were analyzed by costaining of CD31/Ki67 and CD31/caspase-3 in vivo, and the production of cytokines and reactive oxygen species was confirmed by reverse-transcription polymerase chain reaction and flow cytometry in vitro. Comprehensive proteome and ubiquitinome analyses were used for USP11 substrate screening after radiation. Results were verified by Western blotting and coimmunoprecipitation experiments. Recombinant adeno-associated virus lung vectors expressing OTUD5 were used for localized overexpression of OTUD5 in mouse pulmonary tissue, and immunohistochemistry was conducted to analyze cytokine expression. RESULTS: The progression of RIP was significantly alleviated by reduced expression of proinflammatory cytokines in both Usp11-knockout (Usp11-/-) mice and in mice treated with the USP11 inhibitor mitoxantrone. Likewise, the absence of USP11 resulted in decreased permeability of pulmonary vessels and neutrophils and macrophage infiltration. The proliferation rates of endothelial cells were prominently increased in the Usp11-/- lung, whereas apoptosis in Usp11-/- lungs decreased after irradiation compared with that observed in Usp11+/+ lungs. Conversely, USP11 overexpression increased proinflammatory cytokine expression and reactive oxygen species production in endothelial cells after radiation. Comprehensive proteome and ubiquitinome analyses indicated that USP11 overexpression upregulates the expression of several deubiquitinating enzymes, including USP22, USP33, and OTUD5. We demonstrate that USP11 deubiquitinates OTUD5 and implicates the OTUD5-STING signaling pathway in the progression of the inflammatory response in endothelial cells. CONCLUSIONS: USP11 exacerbates RIP by triggering an inflammatory response in endothelial cells both in vitro and in vivo, and the OTUD5-STING pathway is involved in the USP11-dependent promotion of RIP. This study provides experimental support for the development of precision intervention strategies targeting USP11 to mitigate RIP.