Ubiquitin-proteasome System Pathway Research Articles

Design, synthesis and evaluation of the first DYRK1A degrader for promoting the proliferation of pancreatic β-cells

Dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) is the most promising target for diabetes treatment by promoting β-cell proliferation. The desmethylbellidifolin (DMB) as a DYRK1A inhibitor could facilitate β-cell proliferation in vivo and in vitro. However, DMB has the problem of weak binding affinity to DYRK1A, which means that continuous high concentration administration of DMB is effective for the diabetes. In order to solve this problem, we designed and synthesized a series of DMB-based proteolysis targeting chimeras (PROTACs) by taking advantage of the property of PROTAC that induce protein degradation in a cycle-catalytic manner. MDM2-based PROTAC X1–4P-MDM2 was identified as the most active PROTAC molecule. Mechanism research showed that X1–4P-MDM2 formed a ternary complex with DYRK1A and murine double minute 2 (MDM2), and induced the degradation of DYRK1A through the ubiquitin-proteasome system pathway. At a dose much lower than that of DMB, X1–4P-MDM2 still significantly enhanced β-cell proliferation by inhibiting transforming growth factor beta (TGF-β) and promoting the mitogen-activated protein kinases/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway, which may provide a new strategy for the application of DMB in diabetes.

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.

Estimating the causal effects of genetically predicted plasma proteome on heart failure.

Heart Failure (HF) is the end-stage cardiovascular syndrome with poor prognosis. Proteomics holds great promise in the discovery of novel biomarkers and therapeutic targets for HF. The aim of this study is to investigate the causal effects of genetically predicted plasma proteome on HF using the Mendelian randomization (MR) approach. Summary-level data for the plasma proteome (3,301 healthy individuals) and HF (47,309 cases; 930,014 controls) were extracted from genome-wide association studies (GWASs) of European descent. MR associations were obtained using the inverse variance-weighted (IVW) method, sensitivity analyses, and multivariable MR analyses. Using single-nucleotide polymorphisms as instrumental variables, 1-SD increase in MET level was associated with an approximately 10% decreased risk of HF (odds ratio [OR]: 0.92; 95% confidence interval [CI]: 0.89 to 0.95; p = 1.42 × 10-6), whereas increases in the levels of CD209 (OR: 1.04; 95% CI: 1.02-1.06; p = 6.67 × 10-6) and USP25 (OR: 1.06; 95% CI: 1.03-1.08; p = 7.83 × 10-6) were associated with an increased risk of HF. The causal associations were robust in sensitivity analyses, and no evidence of pleiotropy was observed. The study findings suggest that the hepatocyte growth factor/c-MET signaling pathway, dendritic cells-mediated immune processes, and ubiquitin-proteasome system pathway are involved in the pathogenesis of HF. Moreover, the identified proteins have potential to uncover novel therapies for cardiovascular diseases.

Fowl adenovirus serotype 4 52/55k protein triggers PKR degradation by ubiquitin–proteasome system to evade effective innate immunity

The pro- and inflammatory cytokines fail to effectively inhibit FAdV-4, which has always puzzled us. In the current study, the data determined that the mRNA levels of interferons were significantly enhanced in the livers and LMH cells from 24 h to 72 h post FAdV-4 infection. But the viral load of FAdV-4 was still significantly increased, which meant that FAdV-4 evaded innate immune response. We additionally revealed that the protein levels not mRNA levels of PKR were degraded in host cell at 48 h post FAdV-4 infection. Moreover, the results of over expression and silent expression of PKR revealed that PKR could inhibit FAdV-4 proliferation. These results indicated that FAdV-4 degraded the protein levels of PKR to evade innate immune response. We also found that the protein degradation levels of PKR induced by FAdV-4 were recovery in LHM cells after treatment with proteasome inhibitor MG132, and ubiquitin-specific proteases inhibitor DUB-IN-1. Furthermore, our current data presented that FAdV-4 52/55 K protein directly interacted with PKR and degraded it determined by Co-immunoprecipitation and immunofluorescence. We also determined that 52/55 K protein triggered PKR degradation, and the degradation of PKR could be recovery in LHM cells after treatment with MG132, or DUB-IN-1, respectively. Finally, our data demonstrated that 52/55 K protein was a ubiquitylase that could directly degrade PKR protein in host cells via the ubiquitin-proteasome pathway. Therefore, the current study firstly revealed that FAdV-4 52/55 K protein played the key role in triggering PKR degradation by ubiquitin-proteasome system pathway to escape from innate immunity response.

Muscle proteolysis via ubiquitin-proteasome system (UPS) is activated by BthTx-I Lys49 PLA2 but not by BthTx-II Asp49 PLA2 and Bothrops jararacussu venom

Bites by viperid snakes belonging to Bothrops genus produce fast and intense local edema, inflammation, bleeding and myonecrosis. In this study, we investigated the role of Myogenic Regulatory Factors (MRFs: MyoD; Myog), negatively regulated by GDF-8 (Myostatin), and ubiquitin-proteasome system pathway (UPS: MuRF-1; Fbx-32) in gastrocnemius muscle regeneration after Bothrops jararacussu snake venom (Bjussu) or its isolated phospholipase A2 myotoxins, BthTx-I (Lys-49 PLA2) and BthTx-II (Asp-49 PLA2) injection. Male Swiss mice received a single intra-gastrocnemius injection of crude Bjussu, at a dose/volume of 0.83 mg/kg/20 μl, and BthTx-I or BthTx-II, at a dose/volume of 2.5 mg/kg/20 μl. Control mice (Sham) received an injection of sterile saline solution (NaCl 0.9%; 20 μl). At 24, 48, 72 and 96 h post injection, right gastrocnemius was collected for protein expression analyses. Based on the temporal expressional dynamics of MyoD, Myog and GDF-8/Myostatin, it was possible to propose that the myogenesis pathway was impacted most badly by BthTx-II followed by BthTx-I and lastly by B. jararacussu venom, thus suggesting that catalytic activity has likely inhibitory role on the satellite cells-mediated reparative myogenesis pathway. Inversely, the catalytic activity seems to be not a determinant for the activation of proteins ubiquitination by MuRF-1 and Fbx-32/Atrogin-1 E3 proteasome ligases, given proteolysis pathway through UPS was activated neither after Bjussu, nor after BthTx-II, but just after the catalytically-inactive BthTx-I Lys-49 PLA2-homologue exposure. The findings of this study disclose interesting perspective for further mechanistic studies about pathways that take part in the atrophy and repair after permanent damage induced by bothropic snakebites.

407. The Effect of Streptococcus pneumoniae Pneumonia on Atherosclerosis

BackgroundClinical studies consistently find an increase in the risk of acute coronary syndrome (ACS) in the weeks following pneumonia, although the mechanisms underlying this finding are unknown. ACS most commonly occurs as a result of thrombosis at the site of ruptured atherosclerotic plaques. We hypothesized that the systemic inflammatory response to pneumococcal pneumonia leads to acute localized inflammatory changes within established atherosclerotic plaques, favoring plaque instability and rupture, thereby resulting in ACS.MethodsMale ApoE-/- mice, a well-established model of atherosclerosis, were fed an atherogenic diet for 7–8 weeks before intranasal infection with Streptococcus pneumoniae or mock infection. Mice were sacrificed 2 or 8 weeks post-infection. Formalin-fixed, paraffin-embedded aortic sinus plaque sections were analyzed to assess markers of plaque vulnerability to rupture. To characterise post-pneumonic plaque macrophage phenotype, aortic sinus plaque cryosections 2 weeks post pneumonia/mock infection were immunostained for MAC-3 to identify macrophage-rich areas. These plaque regions were collected using laser capture microdissection and RNA extracted for microarray analysis.Results S. pneumoniae infection was associated with increased aortic sinus atherosclerotic plaque macrophage content (18.1 vs. 8.0%; P < 0.05) at 2 weeks post infection, but no significant difference in aortic sinus plaque burden, plaque smooth muscle or collagen content. There was no significant difference in any of these plaque vulnerability markers at 8 weeks post infection. Microarray analysis of laser capture micro-dissected plaque macrophages identified downregulation of the expression of three genes coding for specific E3 ubiquitin ligases following pneumonia. Pathway analysis identified a significant perturbation in the ubiquitin proteasome system pathway as a result.ConclusionIn this murine model, pneumococcal pneumonia resulted in increased atherosclerotic plaque macrophage content, a marker of plaque instability, at 2 weeks post infection. Pneumonia may therefore lead to an increased propensity for atherosclerotic plaques to rupture soon after pneumonia, due to infiltration of macrophages into the plaque. Disclosures All authors: No reported disclosures.

New macular findings in individuals with biallelic KLHL7 gene mutation

ObjectiveThe ubiquitin-proteasome system pathway has been recognised as a crucial cellular mechanism for the proper function of photoreceptor cells. In particular, ubiquitin ligases (E3s) recognise and ubiquitinate specific proteins for...

Integrated analysis of mRNA and miRNA expression profiles reveals muscle growth differences between adult female and male Chinese concave-eared frogs (Odorrana tormota)

The Chinese concave-eared torrent frog (Odorrana tormota) is the first known non-mammalian vertebrate that can communicate using ultrasound. In this species, females are approximately four times as large as males, in which the female growth rate is obviously higher than that of male. Until now, the molecular mechanisms underlying muscle growth development differences between male and female frogs have not been reported. Here, we integrated mRNA and miRNA expression profiles to reveal growth differences in the hindlimb muscles of 2-year-old frogs. Among 569 differentially expressed genes (DEGs), 69 were associated with muscle growth and regeneration. Fifty-one up-regulated genes in females were potentially involved in promoting muscle growth and regeneration, whereas 18 up-regulated genes in males may lead to muscle growth inhibition and fast-twitch muscle fiber contraction. 244 DEGs were enriched in mTOR and other protein synthesis signaling pathways, and protein degradation pathways, including lysosomal protease, calpain, caspase, and ubiquitin–proteasome system pathways. It may interpret why female muscles grow faster than males. Based on expression differences of genes involved in glycolysis and oxidative metabolism, we speculated that the proportion of slow muscle fiber was higher and that of fast muscle fiber was lower in female compared with male muscle. Additionally, 767 miRNAs were identified, including 217 new miRNAs, and 6248 miRNA-negatively regulated mRNAs were predicted. The miRNA target genes were enriched in pathways related to muscle growth, protein synthesis, and degradation. Thus, in addition to the identified mRNA differential expressions, miRNAs may play other important roles in the differential regulation of hindlimb muscle growth between female and male O. tormota.

Chemical characterization and cytotoxic potential of an ellagitannin-enriched fraction from Fragaria vesca leaves

The hepatocellular carcinoma, a primary malignancy of the liver, has a very poor prognosis and a lower survival rate. Moreover, the inefficacy of conventional therapies emphasizes the importance of discovering new bioactive compounds. Several studies clearly state that plant-derived polyphenols, namely ellagitannins, have several health benefits. Fragaria vesca leaves contain high amounts of polyphenols, being especially rich in ellagitannins. Therefore, this study aimed to characterize an ellagitannin-enriched fraction (EEF) from F. vesca leaves and to unveil the anticancer potential of this fraction on human hepatocellular carcinoma cells.The analysis of EEF by HPLC-PDA-ESI/MSn allowed the detection of 12 ellagitannins. The cell viability of both EEF and crude extract was determined after 24h of cells treatment and the half-maximal inhibitory concentration (IC50) was evaluated. The IC50 of the EEF (113μg/mL) was about 6 times lower than the IC50 of the crude extract (690μg/mL). Furthermore, EEF induced cell cycle arrest at G2/M checkpoint and decreased cell proliferation in a dose-dependent way. This fraction also induced an accumulation of LC3-II protein through blockage of autophagic flux, and inhibited chymotrypsin-like activity of 26S proteasome. These results showed, for the first time, that EEF from F. vesca leaves inhibits both, autophagic and ubiquitin-proteasome system pathways, two main intracellular protein degradation systems that are targets for anticancer therapies. Additionally, a proteomic analysis allowed the identification of 914 proteins, among which 133 were modulated after cells treatment with EEF, most of them related to metabolic pathways. Overall, this study shows that the EEF from F. vesca leaves decreased cell proliferation, inhibited the proteolytic mechanisms and modulated the metabolic pathways of the cell. Additionally this study points out F. vesca as a source of valuable molecules with anticancer potential, suggesting that ellagitannins, the polyphenols identified in this fraction, could be useful in the development of new fine-tuned therapeutic strategies against carcinogenesis.

Abstract LB-257: Discovery and characterization of novel, highly potent and selective USP7 inhibitors

Abstract Over the past decade, protein ubiquitination has emerged as an important post-translational modification with roles in a plethora of cellular processes, and dysregulation in the ubiquitin proteasome system pathway (UPS) has been implicated in multiple human disorders including cancer. Ubiquitin specific proteases (USPs) are cysteine proteases that catalyse the de-ubiquitination of numerous protein substrates including tumor suppressors and oncogenes, hence regulating their levels and/or functions. USPs therefore represent a fast growing and attractive target class for pharmacological intervention. USP7 in particular has attracted considerable attention for its implications in multiple key oncogenic pathways including most notably MDM2/p53, PTEN and DNA damage. As part of a focussed effort towards targeting USPs, fragment screening was performed against a panel of family members, including USP7. Hits were identified by surface plasmon resonance and validated using orthogonal biophysical techniques (NMR, thermophoresis). Subsequent hit expansion identified molecules for which high-resolution co-crystal structures have been solved providing unique opportunities for structure-based design. Medicinal chemistry optimisation has yielded a series of novel, reversible and potent USP7 inhibitors (e.g. IC50 &amp;lt; 10 nM) with excellent selectivity profiles against deubiquitinating (DUBs) and other non-related enzymes. These inhibitors are cell-permeable and also exhibit potent target engagement in cells (e.g. EC50 &amp;lt; 30 nM). In line with the mechanism of action, further cellular profiling has demonstrated effects on p53, p21 and MDM2 levels in a concentration-dependant manner. From a translational viewpoint, initial studies aimed at identifying cell lines sensitive to these inhibitors will also be discussed. In summary, we report the discovery and detailed biochemical and cellular profiling of novel, potent and selective inhibitors of USP7. These molecules have drug-like properties and may provide opportunities for the development of new anticancer therapeutics. Citation Format: Gerald Gavory, Colin O'dowd, Keeva McClelland, Ewa Odrzywol, Alan Brown, Stephanie Burton, Oliver Barker, Frank Burkamp, Matt Helm, Iain James, Jakub Flasz, Elias Arkoudis, Tim Harrison. Discovery and characterization of novel, highly potent and selective USP7 inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-257. doi:10.1158/1538-7445.AM2015-LB-257

Effects of leucine and HMB supplementation under proteasome pathways during different sarcopenic conditions (1163.9)

In this study we have compared the effects of leucine supplementation and its metabolite β‐Hydroxy‐β‐Methyl‐Butyrate (HMB) on the ubiquitin‐proteasome system pathway during two distinct atrophic conditions, hindlimb immobilization and dexamethasone treatment. Leucine supplementation was able to minimize the reduction in rat soleus mass and cross sectional area (CSA) driven by immobilization, but not during dexamethasone treatment. Interestingly, HMB supplementation was unable to provide protection against mass loss or CSA in all treatments. While leucine supplementation was able to minimize increased expression of both Atrogin and MuRF1 in immobilized rats, leucine was only able to minimize Atrogin in dexamethasone treated rats. In contrast, HMB was unable to restrain the increase in those atrogenes in immobilized rats, but in dexamethasone treated rats, HMB minimized gene expression of Atrogin. Leucine supplementation maintained soleus tetanic peak force in immobilized rats at normal level; differently, HMB treatment failed to maintain tetanic peak force. The present data suggested that the anti‐atrophic effects of leucine are not mediated by its metabolite HMB.

Temporal Separation of Aggregation and Ubiquitination during Early Inclusion Formation in Transgenic Mice Carrying the Huntington’s Disease Mutation

Abnormal insoluble ubiqitinated protein aggregates are found in the brains of Huntington’s disease (HD) patients and in mice transgenic for the HTT mutation. Here, we describe the earliest stages of visible NII formation in brains of R6/2 mice killed between 2 and 6 weeks of age. We found that huntingtin-positive aggregates formed rapidly (within 24–48 hours) in a spatiotemporal manner similar to that we described previously for ubiquitinated inclusions. However, in most neurons, aggregates are not ubiquitinated when they first form. It has always been assumed that mutant huntingtin is recognised as ‘foreign’ and consequently ubiquitinated and targeted for degradation by the ubiquitin-proteasome system pathway. Our data, however, suggest that aggregation and ubiquitination are separate processes, and that mutant huntingtin fragment is not recognized as ‘abnormal’ by the ubiquitin-proteasome system before aggregation. Rather, mutant Htt appears to aggregate before it is ubiquitinated, and then either aggregated huntingtin is ubiquitinated or ubiquitinated proteins are recruited into aggregates. Our findings have significant implications for the role of the ubiquitin-proteasome system in the formation of aggregates, as they suggest that this system is not involved until after the first aggregates form.