Deficiency of Cullin 3, a Protein Encoded by a Schizophrenia and Autism Risk Gene, Impairs Behaviors by Enhancing the Excitability of Ventral Tegmental Area (VTA) DA Neurons.

The dopaminergic neuromodulator system is fundamental to brain functions. Abnormal dopamine (DA) pathway is implicated in psychiatric disorders, including schizophrenia (SZ) and autism spectrum disorder (ASD). Mutations in Cullin 3 (CUL3), a core component of the Cullin-RING ubiquitin E3 ligase complex, have been associated with SZ and ASD. However, little is known about the function and mechanism of CUL3 in the DA system. Here, we show that CUL3 is critical for the function of DA neurons and DA-relevant behaviors in male mice. CUL3-deficient mice exhibited hyperactive locomotion, deficits in working memory and sensorimotor gating, and increased sensitivity to psychostimulants. In addition, enhanced DA signaling and elevated excitability of the VTA DA neurons were observed in CUL3-deficient animals. Behavioral impairments were attenuated by dopamine D2 receptor antagonist haloperidol and chemogenetic inhibition of DA neurons. Furthermore, we identified HCN2, a hyperpolarization-activated and cyclic nucleotide-gated channel, as a potential target of CUL3 in DA neurons. Our study indicates that CUL3 controls DA neuronal activity by maintaining ion channel homeostasis and provides insight into the role of CUL3 in the pathogenesis of psychiatric disorders.SIGNIFICANCE STATEMENT This study provides evidence that Cullin 3 (CUL3), a core component of the Cullin-RING ubiquitin E3 ligase complex that has been associated with autism spectrum disorder and schizophrenia, controls the excitability of dopamine (DA) neurons in mice. Its DA-specific heterozygous deficiency increased spontaneous locomotion, impaired working memory and sensorimotor gating, and elevated response to psychostimulants. We showed that CUL3 deficiency increased the excitability of VTA DA neurons, and inhibiting D2 receptor or DA neuronal activity attenuated behavioral deficits of CUL3-deficient mice. We found HCN2, a hyperpolarization-activated channel, as a target of CUL3 in DA neurons. Our findings reveal CUL3's role in DA neurons and offer insights into the pathogenic mechanisms of autism spectrum disorder and schizophrenia.

Alox15/15-HpETE Aggravates Myocardial Ischemia-Reperfusion Injury by Promoting Cardiomyocyte Ferroptosis.

BACKGROUND: Myocardial ischemia-reperfusion (I/R) injury causes cardiac dysfunction to myocardial cell loss and fibrosis. Prevention of cell death is important to protect cardiac function after I/R injury. The process of reperfusion can lead to multiple types of cardiomyocyte death, including necrosis, apoptosis, autophagy, and ferroptosis. However, the time point at which the various modes of cell death occur after reperfusion injury and the mechanisms underlying ferroptosis regulation in cardiomyocytes are still unclear. METHODS: Using a left anterior descending coronary artery ligation mouse model, we sought to investigate the time point at which the various modes of cell death occur after reperfusion injury. To discover the key molecules involved in cardiomyocyte ferroptosis, we performed a metabolomics study. Loss/gain-of-function approaches were used to understand the role of 15-lipoxygenase (Alox15) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc1α) in myocardial I/R injury. RESULTS: We found that apoptosis and necrosis occurred in the early phase of I/R injury, and that ferroptosis was the predominant form of cell death during the prolonged reperfusion. Metabolomic profiling of eicosanoids revealed that Alox15 metabolites accumulated in ferroptotic cardiomyocytes. We demonstrated that Alox15 expression was specifically increased in the injured area of the left ventricle below the suture and colocalized with cardiomyocytes. Furthermore, myocardial-specific knockout of Alox15 in mice alleviated I/R injury and restored cardiac function. 15-Hydroperoxyeicosatetraenoic acid (15-HpETE), an intermediate metabolite derived from arachidonic acid by Alox15, was identified as a trigger for cardiomyocyte ferroptosis. We explored the mechanism underlying its effects and found that 15-HpETE promoted the binding of Pgc1α to the ubiquitin ligase ring finger protein 34, leading to its ubiquitin-dependent degradation. Consequently, attenuated mitochondrial biogenesis and abnormal mitochondrial morphology were observed. ML351, a specific inhibitor of Alox15, increased the protein level of Pgc1α, inhibited cardiomyocyte ferroptosis, protected the injured myocardium, and caused cardiac function recovery. CONCLUSIONS: Together, our results established that Alox15/15-HpETE-mediated cardiomyocyte ferroptosis plays an important role in prolonged I/R injury.

Insulin Suppresses Ubiquitination via the Deubiquitinating Enzyme Ubiquitin-Specific Protease 14, Independent of Proteasome Activity in H4IIEC3 Hepatocytes.

Ubiquitin-proteasome dysfunction contributes to obesity-related metabolic disorders, such as diabetes and fatty liver disease. However, the regulation of ubiquitin-proteasome activity by insulin remains to be elucidated. Here, we show that prolonged insulin stimulation activates proteasome function even though it reduces the ubiquitinated proteins in H4IIEC3 hepatocytes. Looking for a pathway by which insulin inhibits ubiquitination, we found that hepatic expression of ubiquitin-specific protease 14 (USP14) was upregulated in the liver of patients with insulin resistance. Indeed, the USP14-specific inhibitor IU1 canceled the insulin-mediated reduction of ubiquitinated proteins. Furthermore, insulin-induced endoplasmic reticulum (ER) stress, which was canceled by IU1, suggesting that USP14 activity is involved in insulin-induced ER stress. Co-stimulation with insulin and IU1 for 2 hours upregulated the nuclear translocation of the lipogenic transcription factor, sterol regulatory element binding protein-1c (SREBP-1c), upregulated the expression of the lipogenic gene, fatty acid synthase (Fasn), and repressed the gluconeogenic genes. In conclusion, insulin activates proteasome function even though it inhibits protein ubiquitination by activating USP14 in hepatocytes. USP14 activation by insulin inhibits mature SREBP-1c while upregulating ER stress and the expression of genes involved in gluconeogenesis. Further understanding mechanisms underlying the USP14 activation and its pleiotropic effects may lead to therapeutic development for obesity-associated metabolic disorders, such as diabetes and fatty liver disease. SIGNIFICANCE STATEMENT: This study shows that insulin stimulation inhibits ubiquitination by activating USP14, independent of its effect on proteasome activity in hepatocytes. USP14 also downregulates the nuclear translocation of the lipogenic transcription factor SREBP-1c and upregulates the expression of genes involved in gluconeogenesis. Since USP14 is upregulated in the liver of insulin-resistant patients, understanding mechanisms underlying the USP14 activation and its pleiotropic effects will help develop treatments for metabolic disorders such as diabetes and fatty liver.

ASPM promotes migration and invasion of anaplastic thyroid carcinoma by stabilizing KIF11.

Abnormal spindle-like microcephaly-associated (ASPM) protein is crucial to the mitotic spindle function during cell replication and tumor progression in multiple tumor types. However, the effect of ASPM in anaplastic thyroid carcinoma (ATC) has not yet been understood. The present study is to elucidate the function of ASPM in the migration and invasion of ATC. ASPM expression is incrementally upregulated in ATC tissues and cell lines. Knockout (KO) of ASPM pronouncedly attenuates the migration and invasion of ATC cells. ASPM KO significantly reduces the transcript levels of Vimentin, N-cadherin, and Snail and increases E-cadherin and Occludin, thereby inhibiting epithelial-to-mesenchymal transition (EMT). Mechanistically, ASPM regulates the movement of ATC cells by inhibiting the ubiquitin degradation of KIF11 and thus stabilizing it via direct binding to it. Moreover, xenograft tumors in nude mice proved that KO of ASPM could ameliorate tumorigenesis and tumor growth accompanied by a decreased protein expression of KIF11 and an inhibition of EMT. In conclusion, ASPM is a potentially useful therapeutic target for ATC. Our results also reveal a novel mechanism by which ASPM inhibits the ubiquitin process in KIF11.

Overexpression of a C3HC4-type E3-ubiquitin ligase contributes to salinity tolerance by modulating Na+ homeostasis in rice.

Soil salinity has a negative effect on crop yield. Therefore, plants have evolved many strategies to overcome decreases in yield under saline conditions. Among these, E3-ubiquitin ligase regulates salt tolerance. We characterized Oryza sativa Really Interesting New Gene (RING) Finger C3HC4-type E3 ligase (OsRFPHC-4), which plays a positive role in improving salt tolerance. The expression of OsRFPHC-4 was downregulated by high NaCl concentrations and induced by abscisic acid (ABA) treatment. GFP-fused OsRFPHC-4 was localized to the plasma membrane of rice protoplasts. OsRFPHC-4 encodes a cellular protein with a C3HC4-RING domain with E3 ligase activity. However, its variant OsRFPHC-4C161A does not possess this activity. OsRFPHC-4-overexpressing plants showed enhanced salt tolerance due to low accumulation of Na+ in both roots and leaves, low Na+ transport in the xylem sap, high accumulation of proline and soluble sugars, high activity of reactive oxygen species (ROS) scavenging enzymes, and differential regulation of Na+ /K+ transporter expression compared to wild-type (WT) and osrfphc-4 plants. In addition, OsRFPHC-4-overexpressing plants showed higher ABA sensitivity under exogenous ABA treatment than WT and osrfphc-4 plants. Overall, these results suggest that OsRFPHC-4 contributes to the improvement of salt tolerance and Na+ /K+ homeostasis via the regulation of changes in Na+ /K+ transporters.

Isorhamnetin and anti-PD-L1 antibody dual-functional mesoporous silica nanoparticles improve tumor immune microenvironment and inhibit YY1-mediated tumor progression.

BACKGROUND: The immune checkpoint inhibitor (ICI) anti-PD-L1 monoclonal antibody can inhibit the progress of hepatocellular carcinoma (HCC). Epithelial-mesenchymal transformation (EMT) can promote tumor migration and the formation of immune-suppression microenvironment, which affects the therapeutic effect of ICI. Yin-yang-1 (YY1) is an important transcription factor regulating proliferation, migration and EMT of tumor cells. This work proposed a drug-development strategy that combined the regulation of YY1-mediated tumor progression with ICIs for the treatment of HCC. METHODS: We first studied the proteins that regulated YY1 expression by using pull-down, co-immunoprecipitation, and duo-link assay. The active compound regulating YY1 content was screened by virtual screening and cell-function assay. Isorhamnetin (ISO) and anti-PD-L1 antibody dual-functional mesoporous silica nanoparticles (HMSN-ISO@ProA-PD-L1 Ab) were prepared as an antitumor drug to play a synergistic anti-tumor role. RESULTS: YY1 can specifically bind with the deubiquitination enzyme USP7. USP7 can prevent YY1 from ubiquitin-dependent degradation and stabilize YY1 expression, which can promote the proliferation, migration and EMT of HCC cells. Isorhamnetin (ISO) were screened out, which can target USP7 and promote YY1 ubiquitin-dependent degradation. The cell experiments revealed that the HMSN-ISO@ProA-PD-L1 Ab nanoparticles can specifically target tumor cells and play a role in the controlled release of ISO. HMSN-ISO@ProA-PD-L1 Ab nanoparticles inhibited the growth of Hepa1-6 transplanted tumors and the effect was better than that of PD-L1 Ab treatment group and ISO treatment group. HMSN-ISO@ProA-PD-L1 Ab nanoparticles also exerted a promising effect on reducing MDSC content in the tumor microenvironment and promoting T-cell infiltration in tumors. CONCLUSIONS: The isorhamnetin and anti-PD-L1 antibody dual-functional nanoparticles can improve tumor immune microenvironment and inhibit YY1-mediated tumor progression. This study demonstrated the possibility of HCC treatment strategies based on inhibiting USP7-mediated YY1 deubiquitination combined with anti-PD-L1 monoclonal Ab.

Simplified Synthesis of Renieramycin T Derivatives to Target Cancer Stem Cells via ß-Catenin Proteasomal Degradation in Human Lung Cancer.

Cancer stem cells (CSCs) found within cancer tissue play a pivotal role in its resistance to therapy and its potential to metastasize, contributing to elevated mortality rates among patients. Significant strides in understanding the molecular foundations of CSCs have led to preclinical investigations and clinical trials focused on CSC regulator ß-catenin signaling targeted interventions in malignancies. As part of the ongoing advancements in marine-organism-derived compound development, it was observed that among the six analogs of Renieramycin T (RT), a potential lead alkaloid from the blue sponge Xestospongia sp., the compound DH_32, displayed the most robust anti-cancer activity in lung cancer A549, H23, and H292 cells. In various lung cancer cell lines, DH_32 exhibited the highest efficacy, with IC50 values of 4.06 ± 0.24 µM, 2.07 ± 0.11 µM, and 1.46 ± 0.06 µM in A549, H23, and H292 cells, respectively. In contrast, parental RT compounds had IC50 values of 5.76 ± 0.23 µM, 2.93 ± 0.07 µM, and 1.52 ± 0.05 µM in the same order. Furthermore, at a dosage of 25 nM, DH_32 showed a stronger ability to inhibit colony formation compared to the lead compound, RT. DH_32 was capable of inducing apoptosis in lung cancer cells, as demonstrated by increased PARP cleavage and reduced levels of the proapoptotic protein Bcl2. Our discovery confirms that DH_32 treatment of lung cancer cells led to a reduced level of CD133, which is associated with the suppression of stem-cell-related transcription factors like OCT4. Moreover, DH_32 significantly suppressed the ability of tumor spheroids to form compared to the original RT compound. Additionally, DH_32 inhibited CSCs by promoting the degradation of ß-catenin through ubiquitin-proteasomal pathways. In computational molecular docking, a high-affinity interaction was observed between DH_32 (grid score = -35.559 kcal/mol) and ß-catenin, indicating a stronger binding interaction compared to the reference compound R9Q (grid score = -29.044 kcal/mol). In summary, DH_32, a newly developed derivative of the right-half analog of RT, effectively inhibited the initiation of lung cancer spheroids and the self-renewal of lung cancer cells through the upstream process of ß-catenin ubiquitin-proteasomal degradation.

Resveratrol directly suppresses proteolysis possibly via PKA/CREB signaling in denervated rat skeletal muscle.

Although there are reports that polyphenol resveratrol (Rsv) may cause muscle hypertrophy in basal conditions and attenuate muscle wasting in catabolic situations, its mechanism of action is still unclear. Our study evaluated the ex vivo effects of Rsv on protein metabolism and intracellular signaling in innervated (sham-operated; Sham) and 3-day sciatic denervated (Den) rat skeletal muscles. Rsv (10-4 M) reduced total proteolysis (40%) in sham muscles. Den increased total proteolysis (~40%) in muscle, which was accompanied by an increase in the activities of ubiquitin-proteasome (~3-fold) and lysosomal (100%) proteolytic systems. Rsv reduced total proteolysis (59%) in Den muscles by inhibiting the hyperactivation of ubiquitin-proteasome (50%) and lysosomal (~70%) systems. Neither Rsv nor Den altered calcium-dependent proteolysis in muscles. Mechanistically, Rsv stimulated PKA/CREB signaling in Den muscles, and PKA blockage by H89 (50µM) abolished the antiproteolytic action of the polyphenol. Rsv reduced FoxO4 phosphorylation (~60%) in both Sham and Den muscles and Akt phosphorylation (36%) in Den muscles. Rsv also caused a homeostatic effect in Den muscles by returning their protein synthesis rates to levels similar to Sham muscles. These data indicate that Rsv directly inhibits the proteolytic activity of lysosomal and ubiquitin-proteasome systems, mainly in Den muscles through, at least in part, the activation of PKA/CREB signaling.

[Effect and mechanism of ubiquitin-like protein FAT10 on Angâ ¡ induced endothelial cell inflammation].

Objective: To investigate the role and related mechanism of ubiquitin-like protein FAT10 in the angiotensin Ⅱ (AngⅡ)-induced endothelial cell inflammatory responses. Methods: The Western blot was used to detect the protein expression of FAT10 in 16-weeks old WKY rat carotid artery, thoracic aorta artery, renal artery and vascular smooth muscle cells (VSMC), human umbilical vein endothelial cells (HUVEC) and human breast cancer cells (MDA-MB-231). The optimal concentration and stimulation time of AngⅡ on inducing the highest FAT10 in HUVEC were determined. The following plasmids were constructed: control plasmid, overexpression FAT10 plasmid (Flag-FAT10), invalid interference plasmid, and interference FAT10 plasmid (sh-FAT10). These plasmids were then transfected into HUVEC cells and divided into following groups: control group, Flag-FAT10 group, invalid interference group, and sh-FAT10 group. After culturing with 100 nmol/L AngⅡ for 36 h, the control group and the Flag-FAT10 group were treated with reactive oxygen species scavenger N-acetyl-L-cysteine ​​(NAC), the protein expression levels of the inflammatory factor monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α) were measured. Laser confocal microscopy was used to detect the generation levels of reactive oxygen species in the cells of vrious groups. Results: FAT10 was expressed in carotid artery, thoracic aorta, and renal artery of normal blood pressure rats and expressed in HUVEC, VSMC, MDA-MB-231. The expression level of FAT10 gradually increased in proportion to the increase of the time and concentration of AngⅡ stimulation in HUVEC, and the expression level of FAT10 was the highest when the HUVEC was treated with 100 nmol/L AngⅡ for 36 h (P<0.01). The protein expression level of MCP-1 (P<0.001) and TNF-α (P<0.01) was higher in AngⅡ treated HUVEC with FAT10 overexpression, while the expression level of MCP-1 and TNF-α protein was lower in AngⅡ treated HUVEC with FAT10 knockdown (all P<0.01). The level of intracellular reactive oxygen species (ROS) production was significantly increased with FAT10 overexpression (P<0.001), and the level of ROS was decreased when the expression of FAT10 was interfered (P<0.05). The increased level of MCP-1 and TNF-α proteins in FAT10 overexpressed HUVEC was reversed by NAC (all P<0.05). Conclusion: FAT10 promotes the release of inflammatory factors induced by AngⅡ in endothelial cells by increasing the level of intracellular ROS production.

Treadmill exercise promotes E3 ubiquitin ligase to remove amyloid ß and P-tau and improve cognitive ability in APP/PS1 transgenic mice.

BACKGROUND: Moderate physical exercise is conducive to the brains of healthy humans and AD patients. Previous reports have suggested that treadmill exercise plays an anti-AD role and improves cognitive ability by promoting amyloid clearance, inhibiting neuronal apoptosis, reducing oxidative stress level, alleviating brain inflammation, and promoting autophagy-lysosome pathway in AD mice. However, few studies have explored the relationships between the ubiquitin-proteasome system and proper exercise in AD. The current study was intended to investigate the mechanism by which the exercise-regulated E3 ubiquitin ligase improves AD. METHODS: Both wild type and APP/PS1 transgenic mice were divided into sedentary (WTC and ADC) and exercise (WTE and ADE) groups (n = 12 for each group). WTE and ADE mice were subjected to treadmill exercise of 12 weeks in order to assess the effect of treadmill running on learning and memory ability, Aß plaque burden, hyperphosphorylated Tau protein and E3 ubiquitin ligase. RESULTS: The results indicated that exercise restored learning and memory ability, reduced Aß plaque areas, inhibited the hyperphosphorylation of Tau protein activated PI3K/Akt/Hsp70 signaling pathway, and improved the function of the ubiquitin-proteasome system (increased UCHL-1 and CHIP levels, decreased BACE1 levels) in APP/PS1 transgenic mice. CONCLUSIONS: These findings suggest that exercise may promote the E3 ubiquitin ligase to clear ß-amyloid and hyperphosphorylated Tau by activating the PI3K/Akt signaling pathway in the hippocampus of AD mice, which is efficient in ameliorating pathological phenotypes and improving learning and memory ability.

How in vitro maturation changes the proteome of ovine cumulus-oocyte complexes?

The present study evaluated the effects of in vitro maturation (IVM) on the proteome of cumulus-oocyte complexes (COCs) from ewes. Extracted COC proteins were analyzed by LC-MS/MS. Differences in protein abundances (p < 0.05) and functional enrichments in immature versus in vitro-matured COCs were evaluated using bioinformatics tools. There were 2550 proteins identified in the COCs, with 89 and 87 proteins exclusive to immature and mature COCs, respectively. IVM caused downregulation of 84 and upregulation of 34 proteins. Major upregulated proteins in mature COCs were dopey_N domain-containing protein, structural maintenance of chromosomes protein, ubiquitin-like modifier-activating enzyme 2. Main downregulated proteins in mature COCs were immunoglobulin heavy constant mu, inter-alpha-trypsin inhibitor heavy chain 2, alpha-2-macroglobulin. Proteins exclusive to mature COCs and upregulated after IVM related to immune response, complement cascade, vesicle-mediated transport, cell cycle, and extracellular matrix organization. Proteins of immature COCs and downregulated after IVM were linked to metabolic processes, immune response, and complement cascade. KEGG pathways and miRNA-regulated genes attributed to downregulated and mature COC proteins related to complement and coagulation cascades, metabolism, humoral response, and B cell-mediated immunity. Thus, IVM influenced the ovine COC proteome. This knowledge supports the future development of efficient IVM protocols for Ovis aries.

Dihydroartemisinin suppresses renal fibrosis in mice by inhibiting DNA-methyltransferase 1 and increasing Klotho.

Renal fibrosis is an unavoidable end result of all forms of progressive chronic kidney diseases (CKD). Discovery of efficacious drugs against renal fibrosis is in crucial need. In a preliminary study we found that a derivative of artemisinin, dihydroartemisinin (DHA), exerted strong renoprotection, and reversed renal fibrosis in adenine-induced CKD mouse model. In this study we investigated the anti-fibrotic mechanisms of DHA, particularly its specific target in renal cells. Renal fibrosis was induced in mice by unilateral ureteral obstruction (UUO) or oral administration of adenine (80 mg · kg-1), the mice received DHA (30 mg · kg-1 · d-1, i.g.) for 14 or 21 days, respectively. We showed that DHA administration markedly attenuated the inflammation and fibrotic responses in the kidneys and significantly improved the renal function in both the renal fibrosis mouse models. In adenine-treated mice, DHA was more effective than 5-azacytidine against renal fibrosis. The anti-fibrotic effects of DHA were also observed in TGF-ß1-treated HK-2 cells. In order to determine the target protein of DHA, we conducted pull-down technology coupled with shotgun proteomics using a small-molecule probe based on the structure of DHA (biotin-DHA). As a results, DNA methyltransferase 1 (DNMT1) was identified as the anti-fibrotic target of DHA in 3 different types of renal cell lines (HK-2, HEK293 and 3T3). We demonstrated that DHA directly bound to Asn 1529 and Thr 1528 of DNMT1 with a Kd value of 8.18 µM. In primary mouse renal tubular cells, we showed that DHA (10 µM) promoted DNMT1 degradation via the ubiquitin-proteasome pathway. DHA-reduced DNMT1 expression effectively reversed Klotho promoter hypermethylation, which led to the reversal of Klotho protein loss in the kidney of UUO mice. This subsequently resulted in inhibition of the Wnt/ß-catenin and TGF-ß/Smad signaling pathways and consequently conferred renoprotection in the animals. Knockdown of Klotho abolished the renoprotective effect of DHA in UUO mice. Our study reveals a novel pharmacological activity for DHA, i.e., renoprotection. DHA exhibits this effect by targeting DNMT1 to reverse Klotho repression. This study provides an evidence for the possible clinical application of DHA in the treatment of renal fibrosis.

Gamma radiation induced changes in expression of heat shock proteins (Hsc70 and Hsp83) in the dengue vector Aedes aegypti (L.).

We aimed to assess the effect of gamma radiation on the expression of heat shock proteins Hsc70 and Hsp83 in Aedes aegypti. Adult males were irradiated with 50Gy of gamma radiation, and changes in the expression of proteins in SDS-PAGE gel bands corresponding to molecular weights ~60-75kDa and ~80-95kDa were analyzed at two different time points 6 and 12-hour post-irradiation, using a temporal mass spectrometry based semi-quantitative analysis. A 2-3-fold increase was observed in both proteins Hsc70 and Hsp83, at both time points. In addition, the experiment also revealed the overexpression of several other molecules such as Arginine Kinase - known to be upregulated in certain insects during stress, Esterase B1- implicated in insecticide resistance, and also down-regulation of the 26S proteasome non-ATPase regulatory subunit 1 and ubiquitin-activating enzyme E1 - both known to be involved in ubiquitin-mediated protein degradation. The results taken together with existing data on Hsp83 and Hsc70, indicate that these proteins may enhance the survival of Ae. aegypti following gamma radiation and could serve as molecular markers for the detection of radiation-induced stress.

Downregulation of SHANK-associated RH domain-interacting protein elevates interleukin-33 expression by stimulating the Janus kinase 2/signal transducer and activator of transcription signalling pathway in HaCaT cells.

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease in which T-helper type 2 (Th2) immune responses are dominant. SH3 and multiple ankyrin repeat domains (SHANK)-associated RH domain-interacting protein (SHARPIN) is expressed at low levels in AD, resulting in the upregulation of the signal transducer and activator of transcription (STAT)3 protein and the Th2 cytokine, interleukin (IL)-33. However, the roles of SHARPIN in AD are not yet fully elucidated. AIM: To evaluate the signalling interactions of SHARPIN and IL-33 in order to improve understanding of AD pathogenesis. METHODS: Western blotting was used to detect the Janus kinase (JAK)/STAT signalling proteins and IL-33 protein in HaCaT cells to determine the key proteins mediating the interaction between SHARPIN and IL-33. The findings were validated by immunofluorescence and immunohistochemical staining. Chromatin immunoprecipitation assays were used to evaluate the activity of STAT3 at the IL-33 promoter. RESULTS: We found that phosphorylated (p)JAK2 and pSTAT3 were upregulated in SHARPIN-knockdown HaCaT cells. Subsequent chromatin immunoprecipitation assays revealed that STAT3 binds to the IL-33 promoter to mediate IL-33 expression. Moreover, SHARPIN-mediated expression of IL-33 was reduced after treatment of HaCaT cells with the JAK/STAT inhibitor ruxolitinib. STAT3 and IL-33 expression levels were higher in AD skin lesion tissues than in normal skin tissues. CONCLUSION: These findings suggest that SHARPIN modulates inflammation in HaCaT cells by inhibiting JAK/STAT signalling, supporting the application of SHARPIN as a potential therapeutic target for AD.

Inhibition of SCF ubiquitin ligases by engineered ubiquitin variants that target the Cul1 binding site on the Skp1-F-box interface.

Skp1-Cul1-F-box (SCF) E3 ligases play key roles in multiple cellular processes through ubiquitination and subsequent degradation of substrate proteins. Although Skp1 and Cul1 are invariant components of all SCF complexes, the 69 different human F-box proteins are variable substrate binding modules that determine specificity. SCF E3 ligases are activated in many cancers and inhibitors could have therapeutic potential. Here, we used phage display to develop specific ubiquitin-based inhibitors against two F-box proteins, Fbw7 and Fbw11. Unexpectedly, the ubiquitin variants bind at the interface of Skp1 and F-box proteins and inhibit ligase activity by preventing Cul1 binding to the same surface. Using structure-based design and phage display, we modified the initial inhibitors to generate broad-spectrum inhibitors that targeted many SCF ligases, or conversely, a highly specific inhibitor that discriminated between even the close homologs Fbw11 and Fbw1. We propose that most F-box proteins can be targeted by this approach for basic research and for potential cancer therapies.

Inhibiting heat shock protein 90 and the ubiquitin-proteasome pathway impairs metabolic homeostasis and leads to cell death in human pancreatic cancer cells.

BACKGROUND: Heat shock protein 90 (HSP90) and the ubiquitin-proteasome pathway play crucial roles in the homeostasis of pancreatic cancer cells. This study combined for the first time the HSP90 inhibitor ganetespib (Gan) and the proteasome inhibitor carfilzomib (Carf) to target key mechanisms of homeostasis in pancreatic cancer. It was hypothesized that Gan plus Carf would elicit potent antitumor activity by modulating complementary homeostatic processes. METHODS: In vitro and in vivo effects of this combination on mechanisms of cell growth and viability were evaluated with human pancreatic cancer cell lines (MIA PaCa-2 and HPAC). RESULTS: Combined treatment with Gan and Carf significantly decreased cell viability. The mechanism varied by cell line and involved G2 -M cell-cycle arrest accompanied by a consistent reduction in key cell-cycle regulatory proteins and concomitant upregulation of p27. Further studies revealed increased autophagy markers, including the upregulation of autophagy related 7 and light chain 3 cleavage, and evidence of apoptosis (increased Bax expression and processing of caspase 3). Immunoblot analyses confirmed the modulation of other pathways that influence cell viability, including phosphoinositide 3-kinase/Akt and nuclear factor κB. Finally, the treatment of athymic mice bearing HPAC tumors with Gan and Carf significantly reduced tumor growth in vivo. An immunoblot analysis of freshly isolated tumors from animals at the end of the study confirmed in vivo modulation of key signaling pathways. CONCLUSIONS: The results reveal Gan plus Carf to be a promising combination with synergistic antiproliferative, apoptotic, and pro-autophagy effects in preclinical studies of pancreatic cancer and will further the exploration of the utility of this treatment combination in clinical trials. Cancer 2017;123:4924-33. © 2017 American Cancer Society.

Semisynthetic UbH2A reveals different activities of deubiquitinases and inhibitory effects of H2A K119 ubiquitination on H3K36 methylation in mononucleosomes.

Using a genetically incorporated azidonorleucine for ubiquitin installation, we prepared multi-milligram quantities of H2AK119ub (ubH2A). With a native isopeptide linkage, the synthetic ubH2A was used to study the activity of deubiquitinases and crosstalk between H2A ubiquitination and H3K36 methylation in the context of chemically defined mononucleosomes.

Krüppel-like factor 9 (KLF9) prevents colorectal cancer through inhibition of interferon-related signaling.

UNLABELLED: Expression of the transcription factor Krüppel-like factor 9 (KLF9) is frequently reduced in colorectal cancers, although a tumor suppressive role has not been established. To determine if KLF9 suppresses intestinal adenoma formation, we generated mice of distinct Klf9 genotypes in the background of the Apc (Min/+) mouse and compared their adenoma burdens at 16 weeks of age. While small intestine adenoma burden remained unchanged among Klf9 genotypes, male and female Apc(Min/+)/Klf9(-/-) and Apc(Min/+)/Klf9(+/-) mice exhibited significantly more colon adenomas than their Apc(Min/+)/Klf9(+/+) counterparts. Microarray analysis showed significant increases in the expression of interferon-induced genes in the colon mucosa of female Apc (Min/+)/Klf9(+/-) and Apc(Min/+)/Klf9(-/-) compared to Apc(Min/+)/Klf9(+/+) mice, prior to overt adenoma occurrence. Gene upregulation was confirmed by qPCR of colon mucosa and by siRNA knockdown of KLF9 in human HT29 colorectal cancer cells. Increases in expression of these genes were further augmented by supplementation with Interferon ß1. Circulating levels of the cytokine, interferon-stimulated gene 15 (ISG15) were increased in Apc(Min/+)/Klf9(+/-) and Apc(Min/+)/Klf9(-/-) mice relative to Apc(Min/+)/Klf9(+/+). Additionally, colon mucosal levels of ISG15 were increased in Apc(Min/+)/Klf9(+/-) mice. Chromatin immunoprecipitation demonstrated KLF9 recruitment to the ISG15 promoter. Lastly, treatment with ISG15 suppressed apoptosis in HT29 cells, in the presence and absence of 5-fluorouracil (5FU). Results show KLF9 to be a haploinsufficient suppressor of colon tumorigenesis in Apc(Min/+) mice in part, by repression of ISG15 and the latter's antiapoptotic function. SUMMARY: Krüppel-like factor 9 (KLF9) is a haploinsufficient tumor suppressor in the ApcMin/+ mouse colon by suppressing expression of ISG15, an apoptosis-inhibiting cytokine.

A potent GPX4 degrader to induce ferroptosis in HT1080 cells.

Glutathione peroxidase 4 (GPX4) is the most promising target for inducing ferroptosis. GPX4-targeting strategies primarily focus on inhibiting its activity or adjusting its cellular level. However, small inhibitors have limitations due to the covalent reactive alkyl chloride moiety, which could lead to poor selectivity and suboptimal pharmacokinetic properties. Herein, we designed and synthesized a series of proteolysis targeting chimeras (PROTACs) by connecting RSL3, a small molecule inhibitor of GPX4, with six different ubiquitin ligase ligands. As a highly effective degrader, compound 18a is a potent degrader (DC50, 48h = 1.68 µM, Dmax, 48h = 85 %). It also showed an obvious anti-proliferative effect with the IC50 value of 2.37 ± 0.17 µM in HT1080. Mechanism research showed that compound 18a formed a ternary complex with GPX4 and cIAP and induced the degradation of GPX4 through the ubiquitin-proteasome system pathway. Furthermore, compound 18a also induced the accumulation of lipid peroxides and mitochondrial depolarization, subsequently triggering ferroptosis. Our work demonstrated the practicality and efficiency of the PROTAC strategy and offered a promising avenue for designing degraders to induce ferroptosis in cancer cells.

Plasminogen deficiency exacerbates skeletal muscle loss during mechanical unloading in developing mice.

Mechanical-unloading-induced skeletal muscle atrophy results in physical frailty and disability. Elucidating its mechanism is required to establish effective countermeasures for this muscle adaptation. First, we analyzed the proteome profile in the gastrocnemius (Gast) and soleus muscles of space-flown mice raised under microgravity or artificial 1-g for 30 days, and found that the expression levels of fibrinolysis-related proteins were significantly elevated in the mechanical-unloaded muscles. Next, we investigated the roles of the fibrinolytic system in skeletal muscle atrophy induced by mechanical unloading on the ground. Eight-week-old male mice with plasminogen gene deficiency (Plg-/-) and their wild-type littermates were divided into control and hindlimb-suspended groups and were raised for 21 days. Plasminogen deficiency significantly enhanced the decrease in muscle mass at the lower limbs of mice following hindlimb unloading, and the Gast muscle atrophy was more prominent in Plg-/- mice. In addition, plasminogen deficiency significantly increased the expression of autophagy-related markers, beclin1 mRNA and LC3B protein, in the mechanical-unloaded Gast muscles, but did not affect the increase in the gene expression of ubiquitin ligases, atrogin-1 and MuRF1. Neither plasminogen deficiency nor hindlimb unloading affected the Akt/mechanistic target of rapamycin pathway in the Gast muscles. These results suggested that plasminogen deficiency might accelerate protein breakdown via the autophagy-lysosome, but not the ubiquitin-proteasome, system in the mechanical-unloaded Gast muscles. In conclusion, we first showed that plasminogen deficiency exacerbated the Gast muscle atrophy in hindlimb-unloaded mice. Plasminogen and the fibrinolysis system might play some protective roles against muscle atrophy induced by mechanical unloading in developing mice.NEW & NOTEWORTHY The expression levels of fibrinolysis-related proteins, including plasminogen, were significantly elevated in the gastrocnemius (Gast) and soleus muscles of mice following 30-day microgravity exposure. Plasminogen deficiency exacerbated atrophy of the Gast, but not the soleus, muscles in mice following 21-day hindlimb suspension. It was also suggested that protein breakdown via the autophagy-lysosome system was accelerated in the Gast muscles. Plasminogen might play some protective roles against muscle atrophy induced by mechanical unloading in developing mice.