Selection and validation of reference genes for RT-qPCR in ophiocordyceps sinensis under different experimental conditions.

The Chinese caterpillar mushroom, Ophiocordyceps sinensis (O. sinensis), is a rarely medicinal fungus in traditional chinese herbal medicine due to its unique medicinal values, and the expression stability of reference genes is essential to normalize its gene expression analysis. In this study, BestKeeper, NormFinder and geNorm, three authoritative statistical arithmetics, were applied to evaluate the expression stability of sixteen candidate reference genes (CRGs) in O. sinensis under different stress [low temperature (4°C), light treatment (300 lx), NaCl (3.8%)] and different development stages (mycelia, primordia and fruit bodies) and formation of morphologic mycelium (aeriasubstrate, hyphae knot mycelium). The paired variation values indicated that two genes could be enough to accurate standardization exposed to different conditions of O.sinensis. Among these sixteen CRGs, 18S ribosomal RNA (18S rRNA) and beta-Tubulin (ß-TUB) showed the topmost expression stability in O.sinensis exposed to all conditions, while glutathione hydrolase proenzym (GGT) and Phosphoglucose isomerase (PGI) showed the least expression stability. The optimal reference gene in different conditions was various. ß-TUB and Ubiquitin (UBQ) were identified as the two most stable genes in different primordia developmental stage, while phosphoglucomutase (PGM) with elongation factor 1-alpha (EF1-α) and 18S rRNA with UBQ were the most stably expressed for differentially morphologic mycelium stages and different stresses, respectively. These results will contribute to more accurate evaluation of the gene relative expression levels in O.sinensis under different conditions using the optimal reference gene in real-time quantitative PCR (RT-qPCR) analysis.

Phosphorylation-dependent deubiquitinase OTUD3 regulates YY1 stability and promotes colorectal cancer progression.

Yin Yang 1 (YY1) is a key transcription factor that has been implicated in the development of several malignancies. The stability of YY1 is regulated by the ubiquitin-proteasome system. The role of deubiquitinases (DUBs) and their impact on YY1 remain to be fully elucidated. In this study, we screened for ubiquitin-specific proteases that interact with YY1, and identified OTUD3 as a DUB for YY1. Over-expressed OTUD3 inhibited YY1 degradation, thereby increasing YY1 protein levels, whereas OTUD3 knockdown or knockout promoted YY1 degradation, thereby decreasing the proliferation of colorectal cancer (CRC). Furthermore, PLK1 mediates OTUD3 S326 phosphorylation, which further enhances OTUD3 binding and deubiquitination of YY1. In CRC tissues, elevated the expression level of OTUD3 and YY1 were significantly associated with poor prognostic outcomes. These findings suggest that the OTUD3-YY1 pathway has therapeutic potential in CRC, and OTUD3 plays a critical role in regulating YY1.

Carpaine alleviates tendinopathy in mice by promoting the ubiquitin-proteasomal degradation of p65 via targeting the E3 ubiquitin ligase LRSAM1.

BACKGROUND: Currently, there are no specific drugs or targets available for the treatment of tendinopathy. However, inflammation has recently been found to play a pivotal role in tendinopathy progression, thereby identifying it as a potential therapeutic target. Carpaine (CA) exhibits potential anti-inflammatory pharmacological properties and may offer a therapeutic option for tendinopathy. PURPOSE: This study aimed to investigate the effectiveness of CA in addressing tendinopathy and uncovering its underlying mechanisms. METHODS: Herein, the efficacy of CA by local administration in vivo in comparison to the first-line drug indomethacin was evaluated in a mouse collagenase-induced tendinopathy (CIT) model. Furthermore, IL-1ß induced a simulated pathological inflammatory microenvironment in tenocytes to investigate its underlying mechanisms in vitro. Further confirmation experiments were performed by overexpressing or knocking down the selective targets of CA in vivo. RESULTS: The findings demonstrated that CA was dose-dependent in treating tendinopathy and that the high-dose group outperformed the first-line drug indomethacin. Mechanistically, CA selectively bound to and enhanced the activity of the E3 ubiquitin ligase LRSAM1 in tendinopathy. This effect mediated the ubiquitination of p65 at lysine 93, subsequently promoting its proteasomal degradation. As a result, the NF-κB pathway was inactivated, leading to a reduction in inflammation of tendinopathy. Consequently, CA effectively mitigated the progression of tendinopathy. Moreover, the LRSAM1 overexpression demonstrated effectiveness in mitigating the tendinopathy progression and its knockdown abolished the therapeutic effects of CA. CONCLUSION: CA attenuates the progression of tendinopathy by promoting the ubiquitin-proteasomal degradation of p65 via increasing the enzyme activity of LRSAM1. The exploration of LRSAM1 has also unveiled a new potential target for treating tendinopathy based on the ubiquitin-proteasomal pathway.

FBXW7 in breast cancer: mechanism of action and therapeutic potential.

Breast cancer is one of the frequent tumors that seriously endanger the physical and mental well-being in women. F-box and WD repeat domain-containing 7 (FBXW7) is a neoplastic repressor. Serving as a substrate recognition element for ubiquitin ligase, FBXW7 participates in the ubiquitin-proteasome system and is typically in charge of the ubiquitination and destruction of crucial oncogenic proteins, further performing a paramount role in cell differentiation, apoptosis and metabolic processes. Low levels of FBXW7 cause abnormal stability of pertinent substrates, mutations and/or deletions in the FBXW7 gene have been reported to correlate with breast cancer malignant progression and chemoresistance. Given the lack of an effective solution to breast cancer's clinical drug resistance dilemma, elucidating FBXW7's mechanism of action could provide a theoretical basis for targeted drug exploration. Therefore, in this review, we focused on FBXW7's role in a range of breast cancer malignant behaviors and summarized the pertinent cellular targets, signaling pathways, as well as the mechanisms regulating FBXW7 expression. We also proposed novel perspectives for the exploitation of alternative therapies and specific tumor markers for breast cancer by therapeutic strategies aiming at FBXW7.

RNF216 affects the stability of STAU2 in the hypothalamus.

Idiopathic hypogonadotropic hypogonadism (IHH) is a rare disease characterized by gonadal failure due to deficiency in gonadotropin-releasing hormone (GnRH) synthesis, secretion, or action. RNF216 variants have been recently identified in patients with IHH. Ring finger protein 216 (RNF216), as a ubiquitin E3 ligase, catalyzes the ubiquitination of target proteins with high specificity, which consequently modulates the stability, localization, and interaction of the target protein. In this study, we found that RNF216 interacted with Staufen2 (STAU2) and affected the stability of STAU2 through the ubiquitin-proteasome pathway. STAU2, as a double-stranded RNA-binding protein enriched in the nervous system, plays a role in RNA transport, RNA stability, translation, anchoring, and synaptic plasticity. Further, we revealed that STAU2 levels in the hypothalamus of RNF216-/- mice were increased compared with wild-type (WT) mice. The change in STAU2 protein homeostasis may affect a series of RNA cargoes. Therefore, we analyzed the changes in RNA levels in the hypothalamus of RNF216-/- mice and WT mice by RNA sequencing. We found that deletion of RNF216 led to decreased activities of the prolactin signaling pathway, neuroactive ligand-receptor interaction, GnRH signaling pathway, and ovarian steroidogenesis. The weakening of these signal pathways is likely to affect the secretion of GnRH, thereby affecting the development of gonads. Therefore, our study suggests that STAU2 may be a potential therapeutic target for IHH. Further experiments are needed to demonstrate the association between the weakening of these signaling pathways and the RNA-binding protein STAU2.

The Males Absent on the First (MOF) Mediated Acetylation Alters the Protein Stability and Transcriptional Activity of YY1 in HCT116 Cells.

Yin Yang 1 (YY1) is a well-known transcription factor that controls the expression of many genes and plays an important role in the occurrence and development of various cancers. We previously found that the human males absent on the first (MOF)-containing histone acetyltransferase (HAT) complex may be involved in regulating YY1 transcriptional activity; however, the precise interaction between MOF-HAT and YY1, as well as whether the acetylation activity of MOF impacts the function of YY1, has not been reported. Here, we present evidence that the MOF-containing male-specific lethal (MSL) HAT complex regulates YY1 stability and transcriptional activity in an acetylation-dependent manner. First, the MOF/MSL HAT complex was bound to and acetylated YY1, and this acetylation further promoted the ubiquitin-proteasome degradation pathway of YY1. The MOF-mediated degradation of YY1 was mainly related to the 146-270 amino acid residues of YY1. Further research clarified that acetylation-mediated ubiquitin degradation of YY1 mainly occurred through lysine 183. A mutation at the YY1K183 site was sufficient to alter the expression level of p53-mediated downstream target genes, such as CDKN1A (encoding p21), and it also suppressed the transactivation of YY1 on CDC6. Furthermore, a YY1K183R mutant and MOF remarkably antagonized the clone-forming ability of HCT116 and SW480 cells facilitated by YY1, suggesting that the acetylation-ubiquitin mode of YY1 plays an important role in tumor cell proliferation. These data may provide new strategies for the development of therapeutic drugs for tumors with high expression of YY1.

Aloe-emodin targets multiple signaling pathways by blocking ubiquitin-mediated degradation of DUSP1 in nasopharyngeal carcinoma cells.

Aloe-emodin (AE) has been shown to inhibit the proliferation of several cancer cell lines, including human nasopharyngeal carcinoma (NPC) cell lines. In this study, we confirmed that AE inhibited malignant biological behaviors, including cell viability, abnormal proliferation, apoptosis, and migration of NPC cells. Western blotting analysis revealed that AE upregulated the expression of DUSP1, an endogenous inhibitor of multiple cancer-associated signaling pathways, resulting in blockage of the extracellular signal-regulated kinase (ERK)-1/2, protein kinase B (AKT), and p38-mitogen activated protein kinase（p38-MAPK） signaling pathways in NPC cell lines. Moreover, the selective inhibitor of DUSP1, BCI-hydrochloride, partially reversed the AE-induced cytotoxicity and blocked the aforementioned signaling pathways in NPC cells. In addition, the binding between AE and DUSP1 was predicted via molecular docking analysis using AutoDock-Vina software and further verified via a microscale thermophoresis assay. The binding amino acid residues were adjacent to the predicted ubiquitination site (Lys192) of DUSP1. Immunoprecipitation with the ubiquitin antibody, ubiquitinated DUSP1 was shown to be upregulated by AE. Our findings revealed that AE can stabilize DUSP1 by blocking its ubiquitin-proteasome-mediated degradation and proposed an underlying mechanism by which AE-upregulated DUSP1 may potentially target multiple pathways in NPC cells.

Functional Characterization of Potato UBC13-UEV1s Genes Required for Ubiquitin Lys63 Chain to Polyubiquitination.

Ubiquitin-conjugating enzymes (E2s/UBC) are components of the ubiquitin proteasome system (UPS), and the ubiquitin-conjugating enzyme variant (UEV) is one of E2s (ubiquitin-conjugating enzymes, UBC) subfamily. The UEVs and UBC13 play an auxiliary role in mediating Lys63-linked polyUb chain assembly, which is correlated with target protein non-proteolytic functions, such as DNA repair or response to stress. However, the collaborative mechanism of StUBC13 (homologue of AtUBC13) and StUEVs (the UEVs in potato) involved in potato are not fully understood understood. Here, we identified two StUBC13 and seven StUEVs from potato genome. We analyzed protein motif and conserved domain, gene structure, phylogenetic features, cis-acting elements of StUBC13 and StUEVs. Subsequently, we screened StUBC13 partners protein and verified interaction between StUBC13 and StUEVs using yeast two-hybrid, split luciferase complementation (SLC) and bimolecular fluorescence complementation (BiFC) approach. The expression profile and qRT-PCR analysis suggested that StUBC13 and StUEVs gene exhibited a tissue-specific expression and were induced by different stress. Overall, this investigative study provides a comprehensive reference and view for further functional research on StUBC13 and StUEV1s in potato.

Thioester and Oxyester Linkages in the Ubiquitin System.

The traditional textbook describes ubiquitylation as the conjugation of ubiquitin to a target by forming a covalent bond connecting ubiquitin's carboxy-terminal glycine residue with an acceptor amino acid like lysine or amino-terminal methionine in the substrate protein. While this adequately depicts a significant fraction of cellular ubiquitylation processes, a growing number of ubiquitin modifications do not follow this rule. Recent data demonstrate that ubiquitin can also be efficiently attached to other amino acids, such as cysteine, serine, and threonine, via ester bonding. Initially observed for a virus-encoded ubiquitin ligase, which targets a cysteine residue in a host protein to initiate its degradation, ester-linked ubiquitylation is now shown to also drive regular cellular processes. These ubiquitylation events expand the complexity and diversity of ubiquitin signaling and broaden the capability of cellular messages in the so-called ubiquitin code. Still, questions on the prevalence, relevance, and involvement in physiological and cellular functions await clearing. In this review, we aim to summarize our knowledge on ester-linked ubiquitylation and introduce experimental strategies to circumvent technical issues that complicate analysis of this uncommon posttranslational modification.

Therapeutic potential of metal ions for COVID-19: insights from the papain-like protease of SARS-CoV-2.

Coronaviruses have been responsible for multiple challenging global pandemics, including coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Papain-like protease (PLpro), one of two cysteine proteases responsible for the maturation and infectivity of SARS-CoV-2, processes and liberates functional proteins from the viral polyproteins and cleaves ubiquitin and ISG15 modifications to inhibit innate immune sensing. Consequently, PLpro is an attractive target for developing COVID-19 therapies. PLpro contains a zinc-finger domain important for substrate binding and structural stability. However, the impact of metal ions on the activity and biophysical properties of SARS-CoV-2 PLpro has not been comprehensively studied. Here, we assessed the impacts of metal ions on the catalytic activity of PLpro. Zinc had the largest inhibitory effect on PLpro, followed by manganese. Calcium, magnesium, and iron had smaller or no effects on PLpro activity. EDTA at a concentration of 0.5 mM was essential for PLpro activity, likely by chelating trace metals that inhibit PLpro. IC50 values for ZnCl2, ZnSO4, and MnCl2 of 0.42 ± 0.02 mM, 0.35 ± 0.01 mM, and 2.6 ± 0.3 mM were obtained in the presence of 0.5 mM EDTA; in the absence of EDTA, the estimated IC50 of ZnCl2 was 14 µM. Tryptophan intrinsic fluorescence analysis confirmed the binding of zinc and manganese to PLpro, and differential scanning calorimetry revealed that zinc but not manganese reduced ΔHcal of PLpro. The results of this study provide a reference for further work targeting PLpro to prevent and treat COVID-19.

RNAi and CRISPR-Cas silencing E3-RING ubiquitin ligase AIP2 enhances soybean seed protein content.

The majority of plant protein in the world's food supply is derived from soybean (Glycine max). Soybean is a key protein source for global animal feed and is incorporated into plant-based foods for people, including meat alternatives. Soybean protein content is genetically variable and is usually inversely related to seed oil content. ABI3-interacting protein 2 (AIP2) is an E3-RING ubiquitin ligase that targets the seed-specific transcription factor ABI3. Silencing both soybean AIP2 genes (AIP2a and AIP2b) by RNAi enhanced seed protein content by up to seven percentage points, with no significant decrease in seed oil content. The protein content enhancement did not alter the composition of the seed storage proteins. Inactivation of either AIP2a or AIP2b by a CRISPR-Cas9-mediated mutation increased seed protein content, and this effect was greater when both genes were inactivated. Transactivation assays in transfected soybean hypocotyl protoplasts indicated that ABI3 changes the expression of glycinin, conglycinin, 2S albumin, and oleosin genes, indicating that AIP2 depletion increased seed protein content by regulating activity of the ABI3 transcription factor protein. These results provide an example of a gene-editing prototype directed to improve global food security and protein availability in soybean that may also be applicable to other protein-source crops.

Branched-chain amino acids regulate intracellular protein turnover in porcine mammary epithelial cells.

Dietary supplementation with branched-chain amino acids (BCAAs) to lactating sows has been reported to enhance their milk production, but the underlying mechanisms remain largely unknown. This study was conducted with porcine mammary epithelial cells (PMECs) to test the hypothesis that individual BCAAs or their mixture stimulates protein synthesis and inhibit proteolysis in PMECs. Cells were cultured at 37 °C in customized Dulbecco's modified Eagle medium containing 5 mmol/L D-glucose, 1 mmol/L L-phenylalanine, L-[ring-2,4-3H]phenylalanine, 0.1 (control), 0.25, 0.5, 1, or 2 mmol/L L-leucine, L-isoleucine or L-valine or an equimolar mixture of the three BCAAs. The culture medium also contained physiological concentrations of other amino acids found in the plasma of lactating sows. Proliferation, protein synthesis, proteolysis, ß-casein production, the mechanistic target of rapamycin (mTOR) signaling, and the ubiquitin-proteasome pathway were determined for PMECs. Cell proliferation and abundances of phosphorylated mTOR, eukaryotic translation initiation factor 4E-binding protein 1, and ribosomal protein S6 kinase ß-1 proteins increased (P < 0.05), but abundances of ubiquitinated protein and 20S proteasome decreased (P < 0.05) when extracellular concentrations of L-leucine, L-isoleucine, L-valine, or an equimolar mixture of BCAAs were increased from 0.1 to 2 mmol/L. Compared with the control, 0.25, 0.5, 1 or 2 mmol/L BCAAs enhanced (P < 0.01) protein (including ß-casein) synthesis, while decreasing (P < 0.05) proteolysis in PMECs in a dose-dependent manner. Collectively, our results indicate that physiological concentrations of BCAAs regulate protein turnover in mammary epithelial cells to favor net protein synthesis through stimulating the mTOR signaling pathway and inhibiting the ubiquitin-proteasome pathway.

Discovery of the oncogenic MDM2, a direct binding target of berberine and a potential therapeutic, in multiple myeloma.

Recent studies have suggested the potency of berberine (BBR) for multiple cancer treatments, including multiple myeloma (MM). However, the direct target and underlying mechanism of BBR remain largely understood in MM. Here, we demonstrated that BBR inhibited cell proliferation and acted synergistically with bortezomib in MM.1S cells. BBR treatment induced MM cell cycle arrest by downregulating several cell cycle-related proteins. Murine double minute 2 (MDM2) as a BBR-binding protein was identified by surface plasmon resonance image (SPRi) analysis and molecular docking. Overexpression of MDM2 is associated with MM progression and a poor prognosis. Knockdown MDM2 by siRNA transfection can repress MM malignant progression and attenuate the BBR sensitivity to MM.1S cells. BBR treatment induced the degradation of MDM2 through the ubiquitin-proteasome system and reactivated P53/P21 in MM cells. Overall, our data has illustrated that MDM2, as a binding protein of BBR for the first time, may serve as a potential therapeutic option for MM.

Laurel Attenuates Dexamethasone-Induced Skeletal Muscle Atrophy In Vitro and in a Rat Model.

Prevention of muscle atrophy contributes to improved quality of life and life expectancy. In this study, we investigated the effects of laurel, selected from 34 spices and herbs, on dexamethasone (DEX)-induced skeletal muscle atrophy and deciphered the underlying mechanisms. Co-treatment of C2C12 myotubes with laurel for 12 h inhibited the DEX-induced expression of intracellular ubiquitin ligases-muscle atrophy F-box (atrogin-1/MAFbx) and muscle RING finger 1 (MuRF1)-and reduction in myotube diameter. Male Wistar rats were supplemented with 2% laurel for 17 days, with DEX-induced skeletal muscle atrophy occurring in the last 3 days. Laurel supplementation inhibited the mRNA expression of MuRF1, regulated DNA damage and development 1 (Redd1), and forkhead box class O 1 (Foxo1) in the muscles of rats. Mechanistically, we evaluated the effects of laurel on the cellular proteolysis machinery-namely, the ubiquitin/proteasome system and autophagy-and the mTOR signaling pathway, which regulates protein synthesis. These data indicated that the amelioration of DEX-induced skeletal muscle atrophy induced by laurel, is mainly mediated by the transcriptional inhibition of downstream factors of the ubiquitin-proteasome system. Thus, laurel may be a potential food ingredient that prevents muscle atrophy.

α-mangostin derivative 4e as a PDE4 inhibitor promote proteasomal degradation of alpha-synuclein in Parkinson's disease models through PKA activation.

BACKGROUND: Parkinson's disease (PD) is a multi-factorial neurodegenerative disease affecting motor function of patients. The hall markers of PD are dopaminergic neuron loss in the midbrain and the presence of intra-neuronal inclusion bodies mainly composed of aggregation-prone protein alpha-synuclein (α-syn). Ubiquitin-proteasome system (UPS) is a multi-step reaction process responsible for more than 80% intracellular protein degradation. Impairment of UPS function has been observed in the brain tissue of PD patients. PDE4 inhibitors have been shown to activate cAMP-PKA pathway and promote UPS activity in Alzheimer's disease model. α-mangostin is a natural xanthonoid with broad biological activities, such as antioxidant, antimicrobial and antitumour activities. Structure-based optimizations based on α-mangostin produced a potent PDE4 inhibitor, 4e. Herein, we studied whether 4e could promote proteasomal degradation of α-syn in Parkinson's disease models through PKA activation. METHODS: cAMP Assay was conducted to quantify cAMP levels in samples. Model UPS substrates (Ub-G76V-GFP and Ub-R-GFP) were used to monitor UPS-dependent activity. Proteasome activity was investigated by short peptide substrate, Suc-LLVY-AMC, cleavage of which by the proteasome increases fluorescence sensitivity. Tet-on WT, A30P, and A53T α-syn-inducible PC12 cells and primary mouse cortical neurons from A53T transgenic mice were used to evaluate the effect of 4e against α-syn in vitro. Heterozygous A53T transgenic mice were employed to assess the effect of 4e on the clearance of α-syn in vivo, and further validations were applied by western blotting and immunohistochemistry. RESULTS: Taken together, α-mangostin derivative 4e, a PDE4 inhibitor, efficiently activated the cAMP/PKA pathway in neuronal cells, and promoted UPS activity as evidenced by enhanced degradation of UPS substrate Ub-G76V-GFP and Ub-R-GFP, as well as elevated proteasomal enzyme activity. Interestingly, 4e dramatically accelerated degradation of inducibly-expressed WT and mutant α-syn in PC12 cells, in a UPS dependent manner. Besides, 4e consistently activated PKA in primary neuron and A53T mice brain, restored UPS inhibition and alleviated α-syn accumulation in the A53T mice brain. CONCLUSIONS: 4e is a natural compound derived highly potent PDE4 inhibitor. We revealed its potential effect in promoting UPS activity to degrade pathogenic proteins associated with PD.

Studying the ubiquitin code through biotin-based labelling methods.

Post-translational modifications of cellular substrates by members of the ubiquitin (Ub) and ubiquitin-like (UbL) family are crucial for regulating protein homeostasis in organisms. The term "ubiquitin code" encapsulates how this diverse family of modifications, via adding single UbLs or different types of UbL chains, leads to specific fates for substrates. Cancer, neurodegeneration and other conditions are sometimes linked to underlying errors in this code. Studying these modifications in cells is particularly challenging since they are usually transient, scarce, and compartment-specific. Advances in the use of biotin-based methods to label modified proteins, as well as their proximally-located interactors, facilitate isolation and identification of substrates, modification sites, and the enzymes responsible for writing and erasing these modifications, as well as factors recruited as a consequence of the substrate being modified. In this review, we discuss site-specific and proximity biotinylation approaches being currently applied for studying modifications by UbLs, highlighting the pros and cons, with mention of complementary methods when possible. Future improvements may come from bioengineering and chemical biology but even now, biotin-based technology is uncovering new substrates and regulators, expanding potential therapeutic targets to manipulate the Ub code.

Screening of the ubiquitin-proteasome system activators for anti-Alzheimer's disease by the high-content fluorescence imaging system.

Ubiquitin-proteasome system (UPS) plays an important role in neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD). The discovery of UPS activators for anti-neurodegenerative diseases is becoming increasingly important. In this study, we aimed to identify potential UPS activators using the high-throughput screening method with the high-content fluorescence imaging system and validate the neuroprotective effect in the cell models of AD. At first, stable YFP-CL1 HT22 cells were successfully constructed by transfecting the YFP-CL1 plasmid into HT22 cells, together with G418 screening. The degradation activity of the test compounds via UPS was monitored by detecting the YFP fluorescence intensity reflected by the ubiquitin-proteasome degradation signal CL1. By employing the high-content fluorescence imaging system, together with stable YFP-CL1 HT22 cells, the UPS activators were successfully screened from our established TCM library. The representative images were captured and analyzed, and quantification of the YFP fluorescence intensity was performed by flow cytometry. Then, the neuroprotective effect of the UPS activators was investigated in pEGFP-N1-APP (APP), pRK5-EGFP-Tau P301L (Tau P301L), or pRK5-EGFP-Tau (Tau) transiently transfected HT22 cells using fluorescence imaging, flow cytometry, and Western blot. In conclusion, our study established a high-content fluorescence imaging system coupled with stable YFP-CL1 HT22 cells for the high-throughput screening of the UPS activators. Three compounds, namely salvianolic acid A (SAA), salvianolic acid B (SAB), and ellagic acid (EA), were identified to significantly decrease YFP fluorescence intensity, which suggested that these three compounds are UPS activators. The identified UPS activators were demonstrated to clear AD-related proteins, including APP, Tau, and Tau P301L. Therefore, these findings provide a novel insight into the discovery and development of anti-AD drugs.

Probing Protein-protein Interactions and Druggable Site Identification: Mechanistic Binding Events Between Ubiquitin and Zinc Finger with UFM1-specific Peptidase Domain Protein (ZUFSP).

BACKGROUND: Deubiquitinating enzymes (DUBs) protein family have been implicated in some deregulated pathways involved in carcinogeneses, such as cell cycle, gene expression, and DNA damage response (DDR). Zinc finger with UFM1-specific peptidase domain protein (ZUFSP) is one of the recently discovered members of the DUBs. OBJECTIVES: To identify and cross-validate the ZUFSP binding site using the bioinformatic tools, including SiteMap&Metapocket, respectively. To understand the molecular basis of complementary ZUFSP-Ub interaction and associated structural events using MD Simulation. METHODS: In this study, four binding pockets were predicted, characterized, and cross-validated based on physiochemical features such as site score, druggability score, site volume, and site size. Also, a molecular dynamics simulation technique was employed to determine the impact of ubiquitin-binding on ZUFSP. RESULTS: Site 1 with a site score 1.065, Size 102, D scores 1.00, and size volume 261 was predicted to be the most druggable site. Structural studies revealed that upon ubiquitin-binding, the motional movement of ZUFSP was reduced when compared to the unbound ZUFSP. Also, the ZUFSP helical arm (ZHA) domain orient in such a way that it moves closer to the Ub; this orientation enables the formation of a UBD which is very peculiar to ZUFSP. CONCLUSION: The impact of ubiquitin on ZUFSP movement and the characterization of its predicted druggable site can be targeted in the development of therapeutics.

Dietary Selenium Deficiency and Excess Accelerate Ubiquitin-Mediated Protein Degradation in the Muscle of Rainbow Trout (Oncorhynchus mykiss) via Akt/FoxO3a and NF-κB Signaling Pathways.

Selenium (Se) deficiency and excess can lead to protein degradation in fish. However, the underlying mechanisms remain unclear. Ubiquitin proteasome system (UPS) is the main pathway of muscle proteolysis. This study aimed to investigate the effect and molecular mechanism of dietary Se on ubiquitin-mediated muscle protein degradation in rainbow trout (Oncorhynchus mykiss). The fish were fed with the Se-deficient diet (0 mg/kg, DSe), Se-adequate diet (4 mg/kg, ASe), and Se-excessive diet (16 mg/kg, ESe), respectively. After a 10-week feeding trial, the growth performance, body composition, antioxidant enzyme activities, and UPS-related gene and protein expressions were detected. Results indicated that DSe and ESe diets significantly decreased the weight gain rate, specific growth rate, feed efficiency, and muscle crude protein content compared with ASe diet. The histological analysis showed that the mean diameter of muscle fibers was significantly decreased in DSe and ESe groups. And DSe and ESe diets significantly increased the contents of malondialdehyde and nitric oxide, but reduced the glutathione peroxidase activity. Additionally, the abundance of muscle ubiquitinated proteins and the expression levels of MuRF1 and Atrogin-1 were significantly increased in DSe and ESe groups. Compared to ASe diet, DSe and ESe diets significantly decreased the phosphorylation level of Akt Ser473 and the ratio of p-FoxO3a/FoxO3a, but significantly increased the phosphorylation level of IκBα and upregulated the expressions of TNF-α, IL-8, and NF-κB. Overall, this study indicated that dietary Se deficiency and excess accelerated the ubiquitin-mediated muscle protein degradation through regulating Akt/FoxO3a and NF-κB signaling pathways in rainbow trout.

Pharmacological characterization of a novel metal-based proteasome inhibitor Na-AuPT for cancer treatment.

The ubiquitin-proteasome system (UPS) is essential for maintaining cell homeostasis by orchestrating the protein degradation, but is impaired in various diseases, including cancers. Several proteasome inhibitors, such as bortezomib, are currently used in cancer treatment, but associated toxicity limits their widespread application. Recently metal complex-based drugs have attracted great attention in tumor therapy; however, their application is hindered by low water-solubility and poor absorbency. Herein, we synthesized a new type of gold (I) complex named Na-AuPT, and further characterized its anticancer activity. Na-AuPT is highly water-soluble (6 mg/mL), and it was able to potently inhibit growth of a panel of 11 cancer cell lines (A549, SMMC7721, H460, HepG2, BEL7402, LNCap, PC3, MGC-803, SGC-7901, U266, and K562). In A549 and SMMC7721 cells, Na-AuPT (in a range of 2.5-20 µM) inhibited the UPS function in a dose-dependent fashion by targeting and inhibiting both 20 S proteasomal proteolytic peptidases and 19 S proteasomal deubiquitinases. Furthermore, Na-AuPT induced caspase-dependent apoptosis in A549 and SMMC7721 cells, which was prevented by the metal chelator EDTA. Administration of Na-AuPT (40 mg · kg-1 · d-1, ip) in nude mice bearing A549 or SMMC7721 xenografts significantly inhibited the tumor growth in vivo, accompanied by increased levels of total ubiquitinated proteins, cleaved caspase 3 and Bax protein in tumor tissue. Moreover, Na-AuPT induced cell death of primary mononuclear cells from 5 patients with acute myeloid leukemia ex vivo with an average IC50 value of 2.46 µM. We conclude that Na-AuPT is a novel metal-based proteasome inhibitor that may hold great potential for cancer therapy.