Nuclear speckle rejuvenation alleviates proteinopathies at the expense of YAP1.

Current treatments targeting individual protein quality control have limited efficacy in alleviating proteinopathies, highlighting the prerequisite for a common upstream druggable target capable of global proteostasis modulation. Building on our prior research establishing nuclear speckles as pivotal organelles responsible for global proteostasis transcriptional control, we aim to alleviate proteinopathies through nuclear speckle rejuvenation. We identified pyrvinium pamoate as a small-molecule nuclear speckle rejuvenator that enhances protein quality control while suppressing YAP1 signaling via decreasing the surface tension of nuclear speckle condensates through interaction with the intrinsically disordered region of nuclear speckle scaffold protein SON. In pre-clinical models, pyrvinium pamoate reduced tauopathy and alleviated retina degeneration by promoting autophagy and ubiquitin-proteasome system. Aberrant nuclear speckle morphology, reduced protein quality control and increased YAP1 activity were also observed in human tauopathies. Our study uncovers novel therapeutic targets for tackling protein misfolding disorders within an expanded proteostasis framework encompassing nuclear speckles and YAP1.

E3 ubiquitin ligase ZBTB25 suppresses beta coronavirus infection through ubiquitination of the main viral protease MPro.

The main protease of severe acute respiratory syndrome coronavirus 2, Mpro, is a key viral protein essential for viral infection and replication. Mpro has been the target of many pharmacological efforts; however, the host-specific regulation of Mpro protein remains unclear. Here, we report the ubiquitin-proteasome-dependent degradation of Mpro protein in human cells, facilitated by the human E3 ubiquitin ligase ZBTB25. We demonstrate that Mpro has a short half-life that is prolonged via proteasomal inhibition, with its Lys-100 residue serving as a potential ubiquitin acceptor. Using in vitro binding assays, we observed ZBTB25 and Mpro bind to each other in vitro, and using progressive deletional mapping, we further uncovered the required domains for this interaction. Finally, we used an orthologous beta-coronavirus infection model and observed that genetic ablation of ZBTB25 resulted in a more highly infective virus, an effect lost upon reconstitution of ZBTB25 to deleted cells. In conclusion, these data suggest a new mechanism of Mpro protein regulation as well as identify ZBTB25 as an anticoronaviral E3 ubiquitin ligase.

KIAA0317 regulates SOCS1 stability to ameliorate colonic inflammation.

Dysregulated cytokine signalling is a hallmark of inflammatory bowel diseases. Inflammatory responses of the colon are regulated by the suppressor of cytokine signalling (SOCS) proteins. SOCS1 is a key member of this family, and its function is critical in maintaining an appropriate inflammatory response through the JAK/STAT signalling pathway. Dysregulation of SOCS1 protein has been identified as a causal element in colonic inflammatory diseases. Despite this, it remains unclear how SOCS1 protein is regulated. Here, we identify that SOCS1 protein is targeted for degradation by the ubiquitin proteasome system, mediated by the E3 ubiquitin ligase KIAA0317 during experimental colonic inflammation. We characterize the mechanism of protein-protein interaction and ubiquitin conjugation to SOCS1 and demonstrate that the modulation of SOCS1 protein level leads to stark effects on JAK/STAT inflammatory signalling. Together, these results provide insight into the regulation of colonic inflammation through a new mechanism of ubiquitin-based control of SOCS1 protein.

Cross-Regulation of F-Box Protein FBXL2 with T-bet and TNF-α during Acute and Chronic Lung Allograft Rejection.

Chronic lung allograft dysfunction is the major barrier to long-term survival in lung transplant recipients. Evidence supports type 1 alloimmunity as the predominant response in acute/chronic lung rejection, but the immunoregulatory mechanisms remain incompletely understood. We studied the combinatorial F-box E3 ligase system: F-box protein 3 (FBXO3; proinflammatory) and F-box and leucine-rich repeat protein 2 (FBXL2; anti-inflammatory and regulates TNFR-associated factor [TRAF] protein). Using the mouse orthotopic lung transplant model, we evaluated allografts from BALB/c → C57BL/6 (acute rejection; day 10) and found significant induction of FBXO3 and diminished FBXL2 protein along with elevated T-bet, IFN-γ, and TRAF proteins 1-5 compared with isografts. In the acute model, treatment with costimulation blockade (MR1/CTLA4-Ig) resulted in attenuated FBXO3, preserved FBXL2, and substantially reduced T-bet, IFN-γ, and TRAFs 1-5, consistent with a key role for type 1 alloimmunity. Immunohistochemistry revealed significant changes in the FBXO3/FBXL2 balance in airway epithelia and infiltrating mononuclear cells during rejection compared with isografts or costimulation blockade-treated allografts. In the chronic lung rejection model, DBA/2J/C57BL/6F1 > DBA/2J (day 28), we observed persistently elevated FBXO3/FBXL2 balance and T-bet/IFN-γ protein and similar findings from lung transplant recipient lungs with chronic lung allograft dysfunction versus controls. We hypothesized that FBXL2 regulated T-bet and found FBXL2 was sufficient to polyubiquitinate T-bet and coimmunoprecipitated with T-bet on pulldown experiments and vice versa in Jurkat cells. Transfection with FBXL2 diminished T-bet protein in a dose-dependent manner in mouse lung epithelial cells. In testing type 1 cytokines, TNF-α was found to negatively regulate FBXL2 protein and mRNA levels. Together, our findings show the combinatorial E3 ligase FBXO3/FBXL2 system plays a role in the regulation of T-bet through FBXL2, with negative cross-regulation of TNF-α on FBXL2 during lung allograft rejection.

CD4+ T-Cell Dysfunction in Severe COVID-19 Disease Is Tumor Necrosis Factor-α/Tumor Necrosis Factor Receptor 1-Dependent.

Rationale: Lymphopenia is common in severe coronavirus disease (COVID-19), yet the immune mechanisms are poorly understood. As inflammatory cytokines are increased in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, we hypothesized a role in contributing to reduced T-cell numbers. Objectives: We sought to characterize the functional SARS-CoV-2 T-cell responses in patients with severe versus recovered, mild COVID-19 to determine whether differences were detectable. Methods: Using flow cytometry and single-cell RNA sequence analyses, we assessed SARS-CoV-2-specific responses in our cohort. Measurements and Main Results: In 148 patients with severe COVID-19, we found lymphopenia was associated with worse survival. CD4+ lymphopenia predominated, with lower CD4+/CD8+ ratios in severe COVID-19 compared with patients with mild disease (P < 0.0001). In severe disease, immunodominant CD4+ T-cell responses to Spike-1 (S1) produced increased in vitro TNF-α (tumor necrosis factor-α) but demonstrated impaired S1-specific proliferation and increased susceptibility to activation-induced cell death after antigen exposure. CD4+TNF-α+ T-cell responses inversely correlated with absolute CD4+ counts from patients with severe COVID-19 (n = 76; R = -0.797; P < 0.0001). In vitro TNF-α blockade, including infliximab or anti-TNF receptor 1 antibodies, strikingly rescued S1-specific CD4+ T-cell proliferation and abrogated S1-specific activation-induced cell death in peripheral blood mononuclear cells from patients with severe COVID-19 (P < 0.001). Single-cell RNA sequencing demonstrated marked downregulation of type-1 cytokines and NFκB signaling in S1-stimulated CD4+ cells with infliximab treatment. We also evaluated BAL and lung explant CD4+ T cells recovered from patients with severe COVID-19 and observed that lung T cells produced higher TNF-α compared with peripheral blood mononuclear cells. Conclusions: Together, our findings show CD4+ dysfunction in severe COVID-19 is TNF-α/TNF receptor 1-dependent through immune mechanisms that may contribute to lymphopenia. TNF-α blockade may be beneficial in severe COVID-19.

A Repurposed Drug Screen for Compounds Regulating Aquaporin 5 Stability in Lung Epithelial Cells.

Aquaporin 5 (AQP5) is expressed in several cell types in the lung and regulates water transport, which contributes to barrier function during injury and the composition of glandular secretions. Reduced AQP5 expression is associated with barrier dysfunction during acute lung injury, and strategies to enhance its expression are associated with favorable phenotypes. Thus, pharmacologically enhancing AQP5 expression could be beneficial. Here, we optimized a high-throughput assay designed to detect AQP5 abundance using a cell line stably expressing bioluminescent-tagged AQP5. We then screened a library of 1153 compounds composed of FDA-approved drugs for their effects on AQP5 abundance. We show compounds Niclosamide, Panobinostat, and Candesartan Celexitil increased AQP5 abundance, and show that Niclosamide has favorable cellular toxicity profiles. We determine that AQP5 levels are regulated in part by ubiquitination and proteasomal degradation in lung epithelial cells, and mechanistically Niclosamide increases AQP5 levels by reducing AQP5 ubiquitination and proteasomal degradation. Functionally, Niclosamide stabilized AQP5 levels in response to hypotonic stress, a stimulus known to reduce AQP5 levels. In complementary assays, Niclosamide increased endogenous AQP5 in both A549 cells and in primary, polarized human bronchial epithelial cells compared to control-treated cells. Further, we measured rapid cell volume changes in A549 cells in response to osmotic stress, an effect controlled by aquaporin channels. Niclosamide-treated A549 cell volume changes occurred more rapidly compared to control-treated cells, suggesting that increased Niclosamide-mediated increases in AQP5 expression affects functional water transport. Taken together, we describe a strategy to identify repurposed compounds for their effect on AQP5 protein abundance. We validated the effects of Niclosamide on endogenous AQP5 levels and in regulating cell-volume changes in response to tonicity changes. Our findings highlight a unique approach to screen for drug effects on protein abundance, and our workflow can be applied broadly to study compound effects on protein abundance in lung epithelial cells.

Discovery of bactericides as an acute mitochondrial membrane damage inducer.

Mitochondria evolved from endosymbiotic bacteria to become essential organelles of eukaryotic cells. The unique lipid composition and structure of mitochondrial membranes are critical for the proper functioning of mitochondria. However, stress responses that help maintain the mitochondrial membrane integrity are not well understood. One reason for this lack of insight is the absence of efficient tools to specifically damage mitochondrial membranes. Here, through a compound screen, we found that two bis-biguanide compounds, chlorhexidine and alexidine, modified the activity of the inner mitochondrial membrane (IMM)-resident protease OMA1 by altering the integrity of the IMM. These compounds are well-known bactericides whose mechanism of action has centered on their damage-inducing activity on bacterial membranes. We found alexidine binds to the IMM likely through the electrostatic interaction driven by the membrane potential as well as an affinity for anionic phospholipids. Electron microscopic analysis revealed that alexidine severely perturbated the cristae structure. Notably, alexidine evoked a specific transcriptional/proteostasis signature that was not induced by other typical mitochondrial stressors, highlighting the unique property of alexidine as a novel mitochondrial membrane stressor. Our findings provide a chemical-biological tool that should enable the delineation of mitochondrial stress-signaling pathways required to maintain the mitochondrial membrane homeostasis.

A high-throughput screen for TMPRSS2 expression identifies FDA-approved compounds that can limit SARS-CoV-2 entry.

SARS-CoV-2 (2019-nCoV) is the pathogenic coronavirus responsible for the global pandemic of COVID-19 disease. The Spike (S) protein of SARS-CoV-2 attaches to host lung epithelial cells through the cell surface receptor ACE2, a process dependent on host proteases including TMPRSS2. Here, we identify small molecules that reduce surface expression of TMPRSS2 using a library of 2,560 FDA-approved or current clinical trial compounds. We identify homoharringtonine and halofuginone as the most attractive agents, reducing endogenous TMPRSS2 expression at sub-micromolar concentrations. These effects appear to be mediated by a drug-induced alteration in TMPRSS2 protein stability. We further demonstrate that halofuginone modulates TMPRSS2 levels through proteasomal-mediated degradation that involves the E3 ubiquitin ligase component DDB1- and CUL4-associated factor 1 (DCAF1). Finally, cells exposed to homoharringtonine and halofuginone, at concentrations of drug known to be achievable in human plasma, demonstrate marked resistance to SARS-CoV-2 infection in both live and pseudoviral in vitro models. Given the safety and pharmacokinetic data already available for the compounds identified in our screen, these results should help expedite the rational design of human clinical trials designed to combat active COVID-19 infection.

A Fbxo48 inhibitor prevents pAMPKα degradation and ameliorates insulin resistance.

The adenosine monophosphate (AMP)-activated protein kinase (Ampk) is a central regulator of metabolic pathways, and increasing Ampk activity has been considered to be an attractive therapeutic target. Here, we have identified an orphan ubiquitin E3 ligase subunit protein, Fbxo48, that targets the active, phosphorylated Ampkα (pAmpkα) for polyubiquitylation and proteasomal degradation. We have generated a novel Fbxo48 inhibitory compound, BC1618, whose potency in stimulating Ampk-dependent signaling greatly exceeds 5-aminoimidazole-4-carboxamide-1-ß-ribofuranoside (AICAR) or metformin. This compound increases the biological activity of Ampk not by stimulating the activation of Ampk, but rather by preventing activated pAmpkα from Fbxo48-mediated degradation. We demonstrate that, consistent with augmenting Ampk activity, BC1618 promotes mitochondrial fission, facilitates autophagy and improves hepatic insulin sensitivity in high-fat-diet-induced obese mice. Hence, we provide a unique bioactive compound that inhibits pAmpkα disposal. Together, these results define a new pathway regulating Ampk biological activity and demonstrate the potential utility of modulating this pathway for therapeutic benefit.

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.

A high throughput screen for TMPRSS2 expression identifies FDA-approved and clinically advanced compounds that can limit SARS-CoV-2 entry.

SARS-CoV-2 (2019-nCoV) is the pathogenic coronavirus responsible for the global pandemic of COVID-19 disease. The Spike (S) protein of SARS-CoV-2 attaches to host lung epithelial cells through the cell surface receptor ACE2, a process dependent on host proteases including TMPRSS2. Here, we identified small molecules that can reduce surface expression of TMPRSS2 using a 2,700 FDA-approved or current clinical trial compounds. Among these, homoharringtonine and halofuginone appear the most potent agents, reducing endogenous TMPRSS2 expression at sub-micromolar concentrations. These effects appear to be mediated by a drug-induced alteration in TMPRSS2 protein stability. We further demonstrate that halofuginone modulates TMPRSS2 levels through proteasomal-mediated degradation that involves the E3 ubiquitin ligase component DDB1- and CUL4-associated factor 1 (DCAF1). Finally, cells exposed to homoharringtonine and halofuginone, at concentrations of drug known to be achievable in human plasma, demonstrated marked resistance to SARS-CoV-2 pseudoviral infection. Given the safety and pharmacokinetic data already available for the compounds identified in our screen, these results should help expedite the rational design of human clinical trials designed to combat COVID-19 infection.

Chemical inhibition of FBXO7 reduces inflammation and confers neuroprotection by stabilizing the mitochondrial kinase PINK1.

Mitochondrial quality control is mediated by the PTEN-induced kinase 1 (PINK1), a cytoprotective protein that is dysregulated in inflammatory lung injury and neurodegenerative diseases. Here, we show that a ubiquitin E3 ligase receptor component, FBXO7, targets PINK1 for its cellular disposal. FBXO7, by mediating PINK1 ubiquitylation and degradation, was sufficient to induce mitochondrial injury and inflammation in experimental pneumonia. A computational simulation-based screen led to the identification of a small molecule, BC1464, which abrogated FBXO7 and PINK1 association, leading to increased cellular PINK1 concentrations and activities, and limiting mitochondrial damage. BC1464 exerted antiinflammatory activity in human tissue explants and murine lung inflammation models. Furthermore, BC1464 conferred neuroprotection in primary cortical neurons, human neuroblastoma cells, and patient-derived cells in several culture models of Parkinson's disease. The data highlight a unique opportunity to use small molecule antagonists that disrupt PINK1 interaction with the ubiquitin apparatus to enhance mitochondrial quality, limit inflammatory injury, and maintain neuronal viability.

A small molecule NRF2 activator BC-1901S ameliorates inflammation through DCAF1/NRF2 axis.

NRF2 is a master regulator of cellular anti-oxidant and anti-inflammatory responses, and strategies to augment NRF2-dependent responses may beneficial in many diseases. Basal NRF2 protein level is constrained by constitutive KEAP1-mediated degradation, but in the presence of electrophiles, NRF2 ubiquitination is inhibited. Impeded NRF2 degradation increases NRF2 protein, resulting in up-regulation of anti-oxidant gene transcription, and decreased inflammation. KEAP1-independent mechanisms regulating NRF2 stability have also been reported. Here we employed an HTS approach and identified a small molecule, BC-1901S, that stabilized NRF2 and increased its activity. BC-1901S activated NRF2 by inhibiting NRF2 ubiquitination in a KEAP1-independent manner. It further increased NRF2-dependent anti-oxidant gene transcription, and exhibited anti-inflammatory effects in vitro and in vivo. Further, we identified a new NRF2-interacting partner, DDB1 and CUL4 Associated Factor 1 (DCAF1), an E3 ligase that targeted NRF2 for proteasomal degradation. Mechanistically, BC-1901S directly bound to DCAF1 and disrupted NRF2/DCAF1 interaction, thus activating NRF2. These findings provide new insights in NRF2 biology and NRF2 based anti-inflammatory therapy.

Kelch-like protein 42 is a profibrotic ubiquitin E3 ligase involved in systemic sclerosis.

Systemic scleroderma (SSc) is an autoimmune disease that affects over 2.5 million people globally. SSc results in dysfunctional connective tissues with excessive profibrotic signaling, affecting skin, cardiovascular, and particularly lung tissue. Over three-quarters of individuals with SSc develop pulmonary fibrosis within 5 years, the main cause of SSc mortality. No approved medicines to manage lung SSc currently exist. Recent research suggests that profibrotic signaling by transforming growth factor ß (TGF-ß) is directly tied to SSc. Previous studies have also shown that ubiquitin E3 ligases potently control TGF-ß signaling through targeted degradation of key regulatory proteins; however, the roles of these ligases in SSc-TGF-ß signaling remain unclear. Here we utilized primary SSc patient lung cells for high-throughput screening of TGF-ß signaling via high-content imaging of nuclear translocation of the profibrotic transcription factor SMAD family member 2/3 (SMAD2/3). We screened an RNAi library targeting ubiquitin E3 ligases and observed that knockdown of the E3 ligase Kelch-like protein 42 (KLHL42) impairs TGF-ß-dependent profibrotic signaling. KLHL42 knockdown reduced fibrotic tissue production and decreased TGF-ß-mediated SMAD activation. Using unbiased ubiquitin proteomics, we identified phosphatase 2 regulatory subunit B'ϵ (PPP2R5ϵ) as a KLHL42 substrate. Mechanistic experiments validated ubiquitin-mediated control of PPP2R5ϵ stability through KLHL42. PPP2R5ϵ knockdown exacerbated TGF-ß-mediated profibrotic signaling, indicating a role of PPP2R5ϵ in SSc. Our findings indicate that the KLHL42-PPP2R5ϵ axis controls profibrotic signaling in SSc lung fibroblasts. We propose that future studies could investigate whether chemical inhibition of KLHL42 may ameliorate profibrotic signaling in SSc.

The RNFT2/IL-3Rα axis regulates IL-3 signaling and innate immunity.

Interleukin-3 (IL-3) receptor α (IL-3Rα) is the α subunit of the ligand-specific IL-3R and initiates intracellular signaling in response to IL-3. IL-3 amplifies proinflammatory signaling and cytokine storm in murine sepsis models. Here we found that RNFT2 (RING finger transmembrane-domain containing protein 2, also TMEM118), a previously uncharacterized RING finger ubiquitin E3 ligase, negatively regulated IL-3-dependent cellular responses through IL-3Rα ubiquitination and degradation in the proteasome. In vitro, IL-3 stimulation promoted IL-3Rα proteasomal degradation dependent on RNFT2, and we identified IL-3Rα lysine 357 as a ubiquitin acceptor site. We determined that LPS priming reduces RNFT2 abundance, extends IL-3Rα half-life, and sensitizes cells to the effects of IL-3, acting synergistically to increase proinflammatory signaling. In vivo, IL-3 synergized with LPS to exacerbate lung inflammation in LPS and Pseudomonas aeruginosa-challenged mice; conversely, IL-3 neutralization reduced LPS-induced lung injury. Further, RNFT2 overexpression reduced lung inflammation and injury, whereas Rnft2 knockdown exacerbated inflammatory responses in LPS-induced murine lung injury. Last, we examined RNFT2 and IL-3Rα in human lung explants from patients with cystic fibrosis and also showed that IL-3 is elevated in mechanically ventilated critically ill humans at risk for acute respiratory distress syndrome. These results identify RNFT2 as a negative regulator of IL-3Rα and show a potential role for the RNFT2/IL-3Rα/IL-3 axis in regulating innate immune responses in the lung.

Toll-like Receptor 8 Stability Is Regulated by Ring Finger 216 in Response to Circulating MicroRNAs.

TLR8 (Toll-like receptor 8) is an intracellular pattern recognition receptor that senses RNA in endosomes to initiate innate immune signaling through NF-κB, and mechanisms regulating TLR8 protein abundance are not completely understood. Protein degradation is a cellular process controlling protein concentrations, accomplished largely through ubiquitin transfer directed by E3 ligase proteins to substrates. In the present study, we show that TLR8 has a short half-life in THP-1 monocytes (∼1 h) and that TLR8 is ubiquitinated and degraded in the proteasome. Treatment with the TLR8 agonist R848 causes rapid depletion of TLR8 concentrations at early time points, an effect blocked by proteasomal inhibition. We show a novel role for RNF216 (ring finger protein 216), an E3 ligase that targets TLR8 for ubiquitination and degradation. RNF216 overexpression reduces TLR8 concentrations, whereas RNF216 knockdown stabilizes TLR8. We describe a potential role for TLR8 activation by circulating RNA ligands in humans with acute respiratory distress syndrome (ARDS): Plasma and extracted RNA fractions from subjects with ARDS activated TLR8 in vitro. MicroRNA (miRNA) expression profiling revealed several circulating miRNAs from subjects with ARDS. miRNA mimics promoted TLR8 proteasomal degradation in THP-1 cells. These data show that TLR8 proteasomal disposal through RNF216 in response to RNA ligands regulates TLR8 cellular concentrations and may have implications for innate immune signaling. In addition, TLR8 activation by circulating RNA ligands may be a previously underrecognized stimulus contributing to excessive innate immune signaling characteristic of ARDS.

KIAA0317 regulates pulmonary inflammation through SOCS2 degradation.

Dysregulated proinflammatory cytokine release has been implicated in the pathogenesis of several life-threatening acute lung illnesses such as pneumonia, sepsis, and acute respiratory distress syndrome. Suppressors of cytokine signaling proteins, particularly SOCS2, have recently been described as antiinflammatory mediators. However, the regulation of SOCS2 protein has not been described. Here we describe a mechanism of SOCS2 regulation by the action of the ubiquitin E3 ligase KIAA0317. KIAA0317-mediated degradation of SOCS2 exacerbated inflammation in vitro, and depletion of KIAA0317 in vivo ameliorated pulmonary inflammation. KIAA0317-knockout mice exhibited resistance to LPS-induced pulmonary inflammation, while KIAA03017 reexpression mitigated this effect. We uncovered a small molecule inhibitor of KIAA0317 protein (BC-1365) that prevented SOCS2 degradation and attenuated LPS- and P. aeruginosa-induced lung inflammation in vivo. These studies show KIAA0317 to be a critical mediator of pulmonary inflammation through its degradation of SOCS2 and a potential candidate target for therapeutic inhibition.

The RING-type E3 ligase RNF186 ubiquitinates Sestrin-2 and thereby controls nutrient sensing.

Nutrient sensing is a critical cellular process controlling metabolism and signaling. mTOR complex 1 (mTORC1) is the primary signaling hub for nutrient sensing and, when activated, stimulates anabolic processes while decreasing autophagic flux. mTORC1 receives nutrient status signals from intracellular amino acid sensors. One of these sensors, Sestrin-2, functions as an intracellular sensor of cytosolic leucine and inhibitor of mTORC1 activity. Genetic studies of Sestrin-2 have confirmed its critical role in regulating mTORC1 activity, especially in the case of leucine starvation. Sestrin-2 is known to be transcriptionally controlled by several mechanisms; however, the post-translational proteolytic regulation of Sestrin-2 remains unclear. Here, we explored how Sestrin-2 is regulated through the ubiquitin proteasome system. Using an unbiased screening approach of an siRNA library targeting ubiquitin E3 ligases, we identified a RING-type E3 ligase, ring finger protein 186 (RNF186), that critically mediates the Sestrin-2 ubiquitination and degradation. We observed that RNF186 and Sestrin-2 bind each other through distinct C-terminal motifs and that Lys-13 in Sestrin-2 is a putative ubiquitin acceptor site. RNF186 knockdown increased Sestrin-2 protein levels and decreased mTORC1 activation. These results reveal a new mechanism of E3 ligase control of mTORC1 activity through the RNF186-Sestrin-2 axis, suggesting that RNF186 inhibition may be a potential strategy to increase levels of the mTORC1 inhibitor Sestrin-2.

Host-Response Subphenotypes Offer Prognostic Enrichment in Patients With or at Risk for Acute Respiratory Distress Syndrome.

OBJECTIVES: Classification of patients with acute respiratory distress syndrome into hyper- and hypoinflammatory subphenotypes using plasma biomarkers may facilitate more effective targeted therapy. We examined whether established subphenotypes are present not only in patients with acute respiratory distress syndrome but also in patients at risk for acute respiratory distress syndrome (ARFA) and then assessed the prognostic information of baseline subphenotyping on the evolution of host-response biomarkers and clinical outcomes. DESIGN: Prospective, observational cohort study. SETTING: Medical ICU at a tertiary academic medical center. PATIENTS: Mechanically ventilated patients with acute respiratory distress syndrome or ARFA. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We performed longitudinal measurements of 10 plasma biomarkers of host injury and inflammation. We applied unsupervised latent class analysis methods utilizing baseline clinical and biomarker variables and demonstrated that two-class models (hyper- vs hypoinflammatory subphenotypes) offered improved fit compared with one-class models in both patients with acute respiratory distress syndrome and ARFA. Baseline assignment to the hyperinflammatory subphenotype (39/104 [38%] acute respiratory distress syndrome and 30/108 [28%] ARFA patients) was associated with higher severity of illness by Sequential Organ Failure Assessment scores and incidence of acute kidney injury in patients with acute respiratory distress syndrome, as well as higher 30-day mortality and longer duration of mechanical ventilation in ARFA patients (p < 0.0001). Hyperinflammatory patients exhibited persistent elevation of biomarkers of innate immunity for up to 2 weeks postintubation. CONCLUSIONS: Our results suggest that two distinct subphenotypes are present not only in patients with established acute respiratory distress syndrome but also in patients at risk for its development. Hyperinflammatory classification at baseline is associated with higher severity of illness, worse clinical outcomes, and trajectories of persistently elevated biomarkers of host injury and inflammation during acute critical illness compared with hypoinflammatory patients. Our findings provide strong rationale for examining treatment effect modifications by subphenotypes in randomized clinical trials to inform precision therapeutic approaches in critical care.

Ex vivo lung perfusion as a human platform for preclinical small molecule testing.

The acute respiratory distress syndrome (ARDS) causes an estimated 70,000 US deaths annually. Multiple pharmacologic interventions for ARDS have been tested and failed. An unmet need is a suitable laboratory human model to predictively assess emerging therapeutics on organ function in ARDS. We previously demonstrated that the small molecule BC1215 blocks actions of a proinflammatory E3 ligase-associated protein, FBXO3, to suppress NF-κB signaling in animal models of lung injury. Ex vivo lung perfusion (EVLP) is a clinical technique that maintains lung function for possible transplant after organ donation. We used human lungs unacceptable for transplant to model endotoxemic injury with EVLP for 6 hours. LPS infusion induced inflammatory injury with impaired oxygenation of pulmonary venous circulation. BC1215 treatment after LPS rescued oxygenation and decreased inflammatory cytokines in bronchoalveolar lavage. RNA sequencing transcriptomics from biopsies taken during EVLP revealed robust inflammatory gene induction by LPS with a strong signal for NF-κB-associated transcripts. BC1215 treatment reduced the LPS induction of genes associated with inflammatory and host defense gene responses by Gene Ontology (GOterm) and pathways analysis. BC1215 also significantly antagonized LPS-mediated NF-κB activity. EVLP may provide a unique human platform for preclinical study of chemical entities such as FBXO3 inhibitors on tissue physiology.