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Bladder cancer (BC) is the second most prevalent genitourinary malignancy worldwide. Despite recent approvals of immune checkpoint inhibitors and targeted therapy for muscle invasive or recurrent BC, options remain limited for patients with non-muscle invasive BC (NMIBC) refractory to Bacillus Calmette-Guérin (BCG) and chemotherapy. NMIBC is more frequently classified as a luminal subtype, in which increased PPARγ activity is a key feature in promoting tumor growth and evasion of immunosurveillance. Cinobufotalin is one of the major compound of bufadienolides, the primary active components of toad venom that has been utilized in the clinical treatment of cancer. We herein focused on cinobufotalin, examining its anticancer activity and molecular mechanisms in luminal-type NMIBC. Our results newly reveal that cinobufotalin strongly suppresses the viability and proliferation of luminal BC cells with minimal cytotoxic effects on normal uroepithelial cells, and exhibits significant antitumor activity in a RT112 xenograft BC model. Mechanistically, our sub-G1-phase cell accumulation, Annexin V staining, caspase-3/8/9 activation, and PARP activation analyses show that cinobufotalin induces apoptosis in luminal-type BC cells. Cinobufotalin significantly inhibited the levels of PPARγ and its downstream targets, as well as lipid droplet formation and free fatty acid levels in RT112 cells. PPARγ overexpression rescued RT112 cells from cinobufotalin-induced apoptosis and mitigated the downregulation of FASN and PLIN4. Finally, we show seemingly for the first time that cinobufotalin promotes SIAH1/2-mediated proteasomal degradation of PPARγ in luminal BC cells. Together, these findings compellingly support the idea that cinobufotalin could be developed as a promising therapeutic agent for treating luminal-type NMIBC., Competing Interests: Competing interests: The authors declare no competing interests. Ethical approval: All animal experiments followed the ethical guidelines, and the protocols have been reviewed and approved by the institutional Animal Care and Use Committee of Taipei Medical University (IACUC approval no: LAC-2021-0471)., (© 2024. The Author(s).)

Poly (ADP-ribose) polymerase 1 (PARP1) catalyzes poly (ADP) ribosylation reaction, one of the essential post-translational modifications of proteins in eukaryotic cells. Given that PARP1 inhibition can lead to synthetic lethality in cells with compromised homologous recombination, this enzyme has been identified as a potent target for anti-cancer therapeutics. However, the clinical application of existing PARP1 inhibitors is restrained by side effects associated with DNA trapping and off-target effects, highlighting the need for improved therapeutic strategies. By integrating protein degradation technology, we synthesized a PROTAC molecule 180055 based on the Rucaparib junction and VHL ligand, which efficiently and selectively degraded PARP1 and inhibited PARP1 enzyme activity without a noticeable DNA trapping effect. Furthermore, 180055 kills tumor cells carrying BRCA mutations with a minor impact on the growth of normal cells both in vitro and in vivo. This suggests that 180055 is a PARP1-degrading compound with excellent pharmacological efficacy and extremely high biological safety that deserves further exploration and validation in clinical trials., Competing Interests: Competing interests: The authors declare no potential conflicts of interest. Ethics approval and consent to participate: The authors confirm that all methods were performed in accordance with the relevant guidelines and regulations. Protocols involving mice were approved by the Institutional Animal Care and Use Committee of ShanghaiTech University (No. 20220216001) and conducted in compliance with the governmental regulations of China for the care and use of animals., (© 2024. The Author(s).)

Mitochondrial dysfunction is a central aspect of Parkinson's disease (PD) pathology, yet the underlying mechanisms are not fully understood. This study investigates the link between α-Synuclein (α-Syn) pathology and the loss of translocase of the outer mitochondrial membrane 40 (TOM40), unraveling its implications for mitochondrial dysfunctions in neurons. We discovered that TOM40 protein depletion occurs in the brains of patients with Guam Parkinsonism-Dementia (Guam PD) and cultured neurons expressing α-Syn proteinopathy, notably, without corresponding changes in TOM40 mRNA levels. Cultured neurons expressing α-Syn mutants, with or without a mitochondria-targeting signal (MTS) underscores the role of α-Syn's mitochondrial localization in inducing TOM40 degradation. PDe-related etiological factors, such as 6-hydroxydopamine or ROS/metal ions stress, which promotes α-Syn oligomerization, exacerbate TOM40 depletion in PD patient-derived cells with SNCA gene triplication. Although α-Syn interacts with both TOM40 and TOM20 in the outer mitochondrial membrane, degradation is selective for TOM40, which occurs via the ubiquitin-proteasome system (UPS) pathway. Our comprehensive analyses using Seahorse technology, mitochondrial DNA sequencing, and damage assessments, demonstrate that mutant α-Syn-induced TOM40 loss results in mitochondrial dysfunction, characterized by reduced membrane potential, accumulation of mtDNA damage, deletion/insertion mutations, and altered oxygen consumption rates. Notably, ectopic supplementation of TOM40 or reducing pathological forms of α-Syn using ADP-ribosylation inhibitors ameliorate these mitochondrial defects, suggesting potential therapeutic avenues. In conclusion, our findings provide crucial mechanistic insights into how α-Syn accumulation leads to TOM40 degradation and mitochondrial dysfunction, offering insights for targeted interventions to alleviate mitochondrial defects in PD., Competing Interests: Competing interests: The authors declare no competing interests. Ethics approval and consent to participate: Human postmortem brain tissue from Guam PD, Guam ALS, and age-matched Guam non-neurological controls were sourced from the Binghamton Biorepository Archive (ALS-Guam). These samples were obtained as de-identified frozen specimens. The studies involving human samples were conducted in accordance with the ethics board standards at the Binghamton Biorepository Archive (ALS-Guam) and the institutional review boards at the Houston Methodist Research Institute (protocol IBC00001560). Additionally, experiments involving non-neurological control and PD patient-derived neural progenitor cells (hNPSCs) were approved by the institutional review boards at the Houston Methodist Research Institute (protocol IBC00001560)., (© 2024. The Author(s).)

Breast cancer is one of the most prevalent and diverse malignancies, and, with global cases increasing, the need for biomarkers to inform individual sensitivity to chemotherapeutics has never been greater. Our retrospective clinical analysis predicted that the expression of the fragile site-associated tumor suppressor (FATS) gene was associated with the sensitivity of breast cancer to neoadjuvant chemotherapy with paclitaxel. In vitro experiments subsequently demonstrated that FATS significantly increased the inhibitory effects of paclitaxel on breast cancer cells' migration, growth, and survival. An interaction screen revealed that FATS interacted with MYH9 and promoted its degradation via the ubiquitin-proteasome pathway, thereby downregulating Wnt signaling. By overexpressing FATS and MYH9, we demonstrated that FATS enhanced paclitaxel-induced apoptosis in breast cancer cells by degrading MYH9 to downregulate the Wnt pathway. We also demonstrated in a mouse xenograft model that FATS significantly increased the chemosensitivity of breast cancer cells to paclitaxel in vivo. This study presents a new mechanism by which FATS interacts with MYH9 to suppress the Wnt/β-catenin signaling pathway and induce apoptosis, thus enhancing the sensitivity of breast cancer cells to paclitaxel chemotherapy. The results also propose novel biomarkers for predicting breast cancer sensitivity to neoadjuvant chemotherapy with paclitaxel. Finally, we provide in vivo evidence that the combination of paclitaxel with IWR-1, a novel Wnt pathway inhibitor, synergistically suppresses breast cancer growth, laying the foundation for future trials with this drug combination. These results therefore provide a number of potential solutions for more precise treatment of patients with breast cancer in the future., Competing Interests: Competing interests The authors declare no competing interests. Ethics approval and consent to participate The Medical Ethics Committee of Tianjin Cancer Hospital approved the relevant retrospective clinical study protocol (approval number: bc2020105). The experiments were conducted with the fully informed consent of the subjects. The study was conducted in accordance with the principles of the Declaration of Helsinki. The experimental animal protocol for this study was approved by the Animal Ethical and Welfare Committee of Tianjin Medical University Cancer Institute and Hospital (approval number: 2023088)., (© 2024. The Author(s).)

MicroRNAs, including the tumor-suppressor miR-126 and the oncogene miR-221, regulate tumor formation and growth in colitis-associated cancer (CAC) and colorectal cancer (CRC). This study explores the impact of the epithelial cytokine heparin-binding epidermal growth factor (HB-EGF) and its receptor epidermal growth factor receptor (EGFR) on the pathogenesis of CAC and CRC, particularly in the regulation of microRNA-driven tumor growth and protease expression. In murine models of CRC and CAC, lack of miR-126 and elevated miR-221 expression in colonic tissues enhanced tumor formation and growth. MiR-126 downregulation in colon cells established a pro-tumorigenic proteolytic niche by targeting HB-EGF-active metalloproteinase-7, -9 (MMP7/MMP9), disintegrin, and metalloproteinase domain-containing protein 9, and modulating chemokine-mediated recruitment of HB-EGF-loaded inflammatory cells. Mechanistically, downregulation of HB-EGF and EGFR in the colon suppressed miR-221 and enhanced miR-126 expression via activating enhancer-binding protein 2 alpha. Reintroducing miR-126 reduced tumor development and HB-EGF expression. Combining miR-126 reintroduction, which targets specific HB-EGF-active proteases but not ADAM17, with MMP inhibitors like Batimastat or Marimastat effectively suppressed tumor growth. This combination normalized protease expression and balanced miR-126 and miR-221 levels in developing and growing tumors. These findings demonstrate that suppressing HB-EGF and EGFR1 shifts the balance from oncogenic miR-221 to tumor-suppressive miR-126 action. Consequently, normalizing miR-126 expression could open new avenues for treating patients with CAC and CRC, and this normalization is intertwined with the anticancer efficacy of MMP inhibitors., (© 2024. The Author(s).)

Clear cell renal cell carcinoma (ccRCC) is characterized by Von Hippel Lindau (VHL) gene loss of function mutation, which leads to the accumulation of hypoxia-inducible factor 2α (HIF2α). HIF2α has been well-established as one of the major oncogenic drivers of ccRCC, however, its therapeutic targeting remains a challenge. Through an analysis of proteomic data from ccRCCs and adjacent non-tumor tissues, we herein revealed that Ubiquitin-Specific Peptidase 7 (USP7) was upregulated in tumor tissues, and its depletion by inhibitors or shRNAs caused significant suppression of tumor progression in vitro and in vivo. Mechanistically, USP7 expression is activated by the transcription factors FUBP1 and FUBP3, and it promotes tumor progression mainly by deubiquitinating and stabilizing HIF2α. Moreover, the combination of USP7 inhibitors and afatinib (an ERBB family inhibitor) coordinately induce cell death and tumor suppression. In mechanism, afatinib indirectly inhibits USP7 transcription and accelerates the degradation of HIF2α protein, and the combination of them caused a more profound suppression of HIF2α abundance. These findings reveal a FUBPs-USP7-HIF2α regulatory axis that underlies the progression of ccRCC and provides a rationale for therapeutic targeting of oncogenic HIF2α via combinational treatment of USP7 inhibitor and afatinib., (© 2024. The Author(s).)

The current selection of ligands for both proteins of interest (POI) and E3 ubiquitin ligase significantly restricts the scope of targeted protein degradation (TPD) technologies. This study introduces cell-penetrating peptide-induced chimera conjugates (cp-PCCs) targeting the DHHC3 enzyme involved in PD-L1 palmitoylation. This approach disrupts PD-L1's immunosuppressive function, enhancing anti-tumor immunity. We developed cp-PCCs to degrade DHHC3, directly linking DHHC3-mediated PD-L1 palmitoylation to PD-L1 stability on tumor cells. Our research utilized both in vitro assays and in vivo experiments in immune checkpoint blockade-resistant mouse models. We focused on a CRBN-based cp-PCC named PCC16, which demonstrated a DC50 of 102 nmol for DHHC3 degradation and significantly reduced PD-L1 levels. In resistant models, PCC16 not only robustly downregulated PD-L1 but also exhibited substantial anti-tumor activity in vivo without significant toxicity. This outperformed traditional inhibitors, showcasing the potential of cp-PCC technology to bypass current PROTAC limitations. Our findings suggest that cp-PCCs offer a promising method for targeting PD-L1 through DHHC3 inhibition and support their continued exploration as a versatile tool in cancer immunotherapy, especially for tumors resistant to standard treatments., (© 2024. The Author(s).)

Glioma, a malignant and infiltrative neoplasm of the central nervous system, poses a significant threat due to its high mortality rates. Branched-chain amino acid transaminase 1 (BCAT1), a key enzyme in branched-chain amino acid (BCAA) catabolism, exhibits elevated expression in gliomas and correlates strongly with poor prognosis. Nonetheless, the regulatory mechanisms underlying this increased BCAT1 expression remains incompletely understood. In this study, we reveal that ubiquitination at Lys360 facilitates BCAT1 degradation, with low ubiquitination levels contributing to high BCAT1 expression in glioma cells. The Carboxyl terminus of Hsc70-interacting protein (CHIP), an E3 ubiquitin ligase, interacts with BCAT1 via its coiled-coil (CC) domain, promoting its K48-linkage ubiquitin degradation through proteasomal pathway. Moreover, CHIP-mediated BCAT1 degradation induces metabolic reprogramming, and impedes glioma cell proliferation and tumor growth both in vitro and in vivo. Furthermore, a positive correlation is observed between low CHIP expression, elevated BCAT1 levels, and unfavorable prognosis among glioma patients. Additionally, we show that the CHIP/BCAT1 axis enhances glioma sensitivity to temozolomide by reducing glutathione (GSH) synthesis and increasing oxidative stress. These findings underscore the critical role of CHIP/BCAT1 axis in glioma cell proliferation and temozolomide sensitivity, highlighting its potential as a diagnostic marker and therapeutic target in glioma treatment., (© 2024. The Author(s).)

Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related deaths worldwide, necessitating the identification of novel therapeutic targets. Lysosome Associated Protein Transmembrane 4B (LAPTM4B) is involved in biological processes critical to cancer progression, such as regulation of solute carrier transporter proteins and metabolic pathways, including mTORC1. However, the metabolic processes governed by LAPTM4B and its role in oncogenesis remain unknown. In this study, we conducted unbiased metabolomic screens to uncover the metabolic landscape regulated by LAPTM4B. We observed common metabolic changes in several knockout cell models suggesting of a role for LAPTM4B in suppressing ferroptosis. Through a series of cell-based assays and animal experiments, we demonstrate that LAPTM4B protects tumor cells from erastin-induced ferroptosis both in vitro and in vivo. Mechanistically, LAPTM4B suppresses ferroptosis by inhibiting NEDD4L/ZRANB1 mediated ubiquitination and subsequent proteasomal degradation of the cystine-glutamate antiporter SLC7A11. Furthermore, metabolomic profiling of cancer cells revealed that LAPTM4B knockout leads to a significant enrichment of ferroptosis and associated metabolic alterations. By integrating results from cellular assays, patient tissue samples, an animal model, and cancer databases, this study highlights the clinical relevance of the LAPTM4B-SLC7A11-ferroptosis signaling axis in NSCLC progression and identifies it as a potential target for the development of cancer therapeutics., (© 2024. The Author(s).)

The targeted elimination of radio- or chemotherapy-induced senescent cells by so-called senolytic substances represents a promising approach to reduce tumor relapse as well as therapeutic side effects such as fibrosis. We screened an in-house library of 178 substances derived from marine sponges, endophytic fungi, and higher plants, and determined their senolytic activities towards DNA damage-induced senescent HCT116 colon carcinoma cells. The Pan-PI3K-inhibitor wortmannin and its clinical derivative, PX-866, were identified to act as senolytics. PX-866 potently induced apoptotic cell death in senescent HCT116, MCF-7 mammary carcinoma, and A549 lung carcinoma cells, independently of whether senescence was induced by ionizing radiation or by chemotherapeutics, but not in proliferating cells. Other Pan-PI3K inhibitors, such as the FDA-approved drug BAY80-6946 (Copanlisib, Aliqopa®), also efficiently and specifically eliminated senescent cells. Interestingly, only the simultaneous inhibition of both PI3K class I alpha (with BYL-719 (Alpelisib, Piqray®)) and delta (with CAL-101 (Idelalisib, Zydelig®)) isoforms was sufficient to induce senolysis, whereas single application of these inhibitors had no effect. On the molecular level, inhibition of PI3Ks resulted in an increased proteasomal degradation of the CDK inhibitor p21 WAF1/CIP1 in all tumor cell lines analyzed. This led to a timely induction of apoptosis in senescent tumor cells. Taken together, the senolytic properties of PI3K-inhibitors reveal a novel dimension of these promising compounds, which holds particular potential when employed alongside DNA damaging agents in combination tumor therapies., (© 2024. The Author(s).)

Ferroptosis is a form of regulated cell death that emerges to be relevant for therapy-resistant and dedifferentiating cancers. Although several lines of evidence suggest that ferroptosis is a type of autophagy-dependent cell death, the underlying molecular mechanisms remain unclear. Fin56, a type 3 ferroptosis inducer, triggers ferroptosis by promoting glutathione peroxidase 4 (GPX4) protein degradation via a not fully understood pathway. Here, we determined that Fin56 induces ferroptosis and autophagy in bladder cancer cells and that Fin56-triggered ferroptosis mechanistically depends on the autophagic machinery. Furthermore, we found that autophagy inhibition at different stages attenuates Fin56-induced oxidative stress and GPX4 degradation. Moreover, we investigated the effects of Fin56 in combination with Torin 2, a potent mTOR inhibitor used to activate autophagy, on cell viability. We found that Fin56 synergizes with Torin 2 in cytotoxicity against bladder cancer cells. Collectively, our findings not only support the concept that ferroptosis is a type of autophagy-dependent cell death but imply that the combined application of ferroptosis inducers and mTOR inhibitors is a promising approach to improve therapeutic options in the treatment of bladder cancer., (© 2021. The Author(s).)

Metformin, the first-line drug for type II diabetes, has recently been considered an anticancer agent. However, the molecular target and underlying mechanism of metformin's anti-cancer effects remain largely unclear. Herein, we report that metformin treatment increases the sensitivity of hepatocarcinoma cells to methotrexate (MTX) by suppressing the expression of the one-carbon metabolism enzyme DHFR. We show that the combination of metformin and MTX blocks nucleotide metabolism and thus effectively inhibits cell cycle progression and tumorigenesis. Mechanistically, metformin not only transcriptionally represses DHFR via E2F4 but also promotes lysosomal degradation of the DHFR protein. Notably, metformin dramatically increases the response of patient-derived hepatocarcinoma organoids to MTX without obvious toxicity to organoids derived from normal liver tissue. Taken together, our findings identify an important role for DHFR in the suppressive effects of metformin on therapeutic resistance, thus revealing a therapeutically targetable potential vulnerability in hepatocarcinoma., (© 2021. The Author(s).)

Androgen receptor splice variant 7 (AR-V7), a form of ligand-independent and constitutively activating variant of androgen receptor (AR), is considered as the key driver to initiate castration-resistant prostate cancer (CRPC). Because AR-V7 lacks ligand-binding domain, the AR-targeted therapies that aim to inactivate AR signaling through disrupting the interaction between AR and androgen are limited in CRPC. Thus, the emergence of AR-V7 has become the greatest challenge for treating CRPC. Targeting protein degradation is a recently proposed novel avenue for cancer treatment. Our previous studies have been shown that the oncoprotein AR-V7 is a substrate of the proteasome. Identifying novel drugs that can trigger the degradation of AR-V7 is therefore critical to cure CRPC. Here we show that nobiletin, a polymethoxylated flavonoid derived from the peel of Citrus fruits, exerts a potent anticancer activity via inducing G0/G1 phase arrest and enhancing the sensitivity of cells to enzalutamide in AR-V7 positive PC cells. Mechanically, we unravel that nobiletin selectively induces proteasomal degradation of AR-V7 (but not AR). This effect relies on its selective inhibition of the interactions between AR-V7 and two deubiquitinases USP14 and USP22. These findings not only enrich our understanding on the mechanism of AR-V7 degradation, but also provide an efficient and druggable target for overcoming CRPC through interfering the stability of AR-V7 mediated by the interaction between AR-V7 and deubiquitinase., (© 2021. The Author(s).)

Lung epithelial cell death is a prominent feature of acute lung injury and acute respiratory distress syndrome (ALI/ARDS), which results from severe pulmonary infection leading to respiratory failure. Multiple mechanisms are believed to contribute to the death of epithelia; however, limited data propose a role for epigenetic modifiers. In this study, we report that a chromatin modulator protein arginine N-methyltransferase 4/coactivator-associated arginine methyltransferase 1 (PRMT4/CARM1) is elevated in human lung tissues with pneumonia and in experimental lung injury models. Here PRMT4 is normally targeted for its degradation by an E3 ubiquitin ligase, SCF FBXO9 , that interacts with PRMT4 via a phosphodegron to ubiquitinate the chromatin modulator at K228 leading to its proteasomal degradation. Bacterial-derived endotoxin reduced levels of SCF FBXO9 thus increasing PRMT4 cellular concentrations linked to epithelial cell death. Elevated PRMT4 protein caused substantial epithelial cell death via caspase 3-mediated cell death signaling, and depletion of PRMT4 abolished LPS-mediated epithelial cell death both in cellular and murine injury models. These findings implicate a unique molecular interaction between SCF FBXO9 and PRMT4 and its regulation by endotoxin that impacts the life span of lung epithelia, which may play a key role in the pathobiology of tissue injury observed during critical respiratory illness., (© 2021. The Author(s).)

Pediatric gliomas comprise a broad range of brain tumors derived from glial cells. While high-grade gliomas are often resistant to therapy and associated with a poor outcome, children with low-grade gliomas face a better prognosis. However, the treatment of low-grade gliomas is often associated with severe long-term adverse effects. This shows that there is a strong need for improved treatment approaches. Here, we highlight the potential for repurposing disulfiram to treat pediatric gliomas. Disulfiram is a drug used to support the treatment of chronic alcoholism and was found to be effective against diverse cancer types in preclinical studies. Our results show that disulfiram efficiently kills pediatric glioma cell lines as well as patient-derived glioma stem cells. We propose a novel mechanism of action to explain disulfiram's anti-oncogenic activities by providing evidence that disulfiram induces the degradation of the oncoprotein MLL. Our results further reveal that disulfiram treatment and MLL downregulation induce similar responses at the level of histone modifications and gene expression, further strengthening that MLL is a key target of the drug and explaining its anti-oncogenic properties., (© 2021. The Author(s).)

Human immunodeficiency type 1 (HIV)-infected macrophages (HIV-Mφ) are a reservoir for latent HIV infection and a barrier to HIV eradication. In contrast to CD4+ T cells, HIV-Mφ are resistant to the cytopathic effects of acute HIV infection and have increased expression of cell survival factors, including X-linked inhibitor of apoptosis (XIAP), baculoviral IAP repeat containing (BIRC) 2/cIAP1, beclin-1, BCL2, BCL-xl, triggering receptor expressed on myeloid cells 1, mitofusin (MFN) 1, and MFN2. DIABLO/SMAC mimetics are therapeutic agents that affect cancer cell survival and induce cell death. We found that DIABLO/SMAC mimetics (LCL-161, AT-406 (also known as SM-406 or Debio 1143), and birinapant) selectively kill HIV-Mφ without increasing bystander cell death. DIABLO/SMAC mimetic treatment of HIV-Mφ-induced XIAP and BIRC2 degradation, leading to the induction of autophagy and the formation of a death-inducing signaling complex on phagophore membranes that includes both pro-apoptotic or necroptotic (FADD, receptor-interacting protein kinase (RIPK) 1, RIPK3, caspase 8, and MLKL) and autophagy (ATG5, ATG7, and SQSTM1) proteins. Genetic or pharmacologic inhibition of early stages of autophagy, but not late stages of autophagy, ablated this interaction and inhibited apoptosis. Furthermore, DIABLO/SMAC mimetic-mediated apoptosis of HIV-Mφ is dependent upon tumor necrosis factor signaling. Our findings thus demonstrate that DIABLO/SMAC mimetics selectively induce autophagy-dependent apoptosis in HIV-Mφ.

APOE4 is a major risk factor for sporadic Alzheimer's disease; however, it is unclear how it exerts its pathological effects. Others and we have previously shown that autophagy is impaired in APOE4 compared to APOE3 astrocytes, and demonstrated differences in the expression of mitochondrial dynamics proteins in brains of APOE3 and APOE4 transgenic mice. Here, we investigated the effect of APOE4 expression on several aspects of mitochondrial function and network dynamics, including fusion, fission, and mitophagy, specifically in astrocytes. We found that APOE3 and APOE4 astrocytes differ in their mitochondrial dynamics, suggesting that the mitochondria of APOE4 astrocytes exhibit reduced fission and mitophagy. APOE4 astrocytes also show impaired mitochondrial function. Importantly, the autophagy inducer rapamycin enhanced mitophagy and improved mitochondrial functioning in APOE4 astrocytes. Collectively, the results demonstrate that APOE4 expression is associated with altered mitochondrial dynamics, which might lead to impaired mitochondrial function in astrocytes. This, in turn, may contribute to the pathological effects of APOE4 in Alzheimer's disease.

Many human cancers over-express B cell lymphoma 2 (Bcl-2) or X-linked inhibitor of apoptosis (IAP) proteins to evade cell death. The pro-apoptotic ARTS (Sept4_i2) protein binds directly to both Bcl-2 and XIAP and promotes apoptosis by stimulating their degradation via the ubiquitin-proteasome system (UPS). Here we describe a small molecule, A4, that mimics the function of ARTS. Microscale thermophoresis assays showed that A4 binds XIAP, but not cellular inhibitor of apoptosis protein 1 (cIAP1). A4 binds to a distinct ARTS binding pocket in the XIAP-BIR3 (baculoviral IAP repeat 3) domain. Like ARTS, A4 stimulated poly-ubiquitylation and UPS-mediated degradation of XIAP and Bcl-2, but not cIAP1, resulting in caspase-9 and -3 activation and apoptosis. In addition, over-expression of XIAP rescued HeLa cells from A4-induced apoptosis, consistent with the idea that A4 kills by antagonizing XIAP. On the other hand, treatment with the SMAC-mimetic Birinapant induced secretion of tumour necrosis factor-α (TNFα) and killed ~50% of SKOV-3 cells, and addition of A4 to Birinapant-treated cells significantly reduced secretion of TNFα and blocked Birinapant-induced apoptosis. This suggests that A4 acts by specifically targeting XIAP. The effect of A4 was selective as peripheral blood mononuclear cells and normal human breast epithelial cells were unaffected. Furthermore, proteome analysis revealed that cancer cell lines with high levels of XIAP were particularly sensitive to the killing effect of A4. These results provide proof of concept that the ARTS binding site in XIAP is "druggable". A4 represents a novel class of dual-targeting compounds stimulating apoptosis by UPS-mediated degradation of important anti-apoptotic oncogenes.

Spinal muscular atrophy (SMA) is a severe neuromuscular disorder caused by loss of the survival motor neuron 1 (SMN1) gene. SMA is characterized by the degeneration of spinal cord motoneurons (MNs), progressive skeletal muscle atrophy, and weakness. The cellular and molecular mechanisms causing MN loss of function are only partially known. Recent advances in SMA research postulate the role of calpain protease regulating survival motor neuron (SMN) protein and the positive effect on SMA phenotype of treatment with calpain inhibitors. We analyzed the level of calpain pathway members in mice and human cellular SMA models. Results indicate an increase of calpain activity in SMN-reduced MNs. Spinal cord analysis of SMA mice treated with calpeptin, a calpain inhibitor, showed an increase of SMN, calpain, and its endogenous inhibitor calpastatin in MNs. Finally, in vitro calpeptin treatment prevented microtubule-associated protein 1A/1B-light chain 3 (LC3) increase in MNs neurites, indicating that calpain inhibition may reduce autophagosome accumulation in neuron prolongations, but not in soma. Thus, our results show that calpain activity is increased in SMA MNs and its inhibition may have a beneficial effect on SMA phenotype through the increase of SMN in spinal cord MNs.

Hydrogen sulfide (H 2 S) is now widely considered the third endogenous gasotransmitter and plays critical roles in cancer biological processes. In this study, we demonstrate that 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADT-OH), the most widely used moiety for synthesising slow-releasing H 2 S donors, induces melanoma cell death in vitro and in vivo. Consistent with previous reports, ADT-OH inhibited IκBɑ degradation, resulting in reduced NF-κB activation and subsequent downregulation of the NF-κB-targeted anti-apoptotic proteins XIAP and Bcl-2. More importantly, we found that ADT-OH suppressed the ubiquitin-induced degradation of FADD by downregulating the expression of MKRN1, an E3 ubiquitin ligase of FADD. In addition, ADT-OH had no significant therapeutic effect on FADD-knockout B16F0 cells or FADD-knockdown A375 cells. Based on these findings, we evaluated the combined effects of ADT-OH treatment and FADD overexpression on melanoma cell death in vivo using a mouse xenograft model. As expected, tumour-specific delivery of FADD through a recombinant Salmonella strain, VNP-FADD, combined with low-dose ADT-OH treatment significantly inhibited tumour growth and induced cancer cell apoptosis. Taken together, our data suggest that ADT-OH is a promising cancer therapeutic drug that warrants further investigation into its potential clinical applications.

Progressive degeneration of retinal ganglion cells (RGCs) will cause a blinding disease. Most of the study is focusing on the RGCs itself. In this study, we demonstrate a decline of the presynaptic rod bipolar cells (RBCs) response precedes RGCs loss and a decrease of protein kinase Cα (PKCα) protein expression in RBCs dendrites, using whole-cell voltage-clamp, electroretinography (ERG) measurements, immunostaining and co-immunoprecipitation. We present evidence showing that N-methyl D-aspartate receptor subtype 2B (NR2B)/protein interacting with C kinase 1 (PICK1)-dependent degradation of PKCα protein in RBCs contributes to RBCs functional loss. Mechanistically, NR2B forms a complex with PKCα and PICK1 to promote the degradation of PKCα in a phosphorylation- and proteasome-dependent manner. Similar deficits in PKCα expression and response sensitivity were observed in acute ocular hypertension and optic never crush models. In conclusion, we find that three separate experimental models of neurodegeneration, often used to specifically target RGCs, disrupt RBCs function prior to the loss of RGCs. Our findings provide useful information for developing new diagnostic tools and treatments for retinal ganglion cells degeneration disease.

As pathogenic Parkin mutations result in the defective clearance of damaged mitochondria, Parkin-dependent mitophagy is thought to be protective against the dopaminergic neurodegeneration observed in Parkinson's disease. Recent studies, however, have demonstrated that Parkin can promote cell death in the context of severe mitochondrial damage by degrading the pro-survival Bcl-2 family member, Mcl-1. Therefore, Parkin may act as a 'switch' that can shift the balance between protective or pro-death pathways depending on the degree of mitochondrial damage. Here, we report that the Parkin interacting protein, Bcl-2-associated athanogene 5 (BAG5), impairs mitophagy by suppressing Parkin recruitment to damaged mitochondria and reducing the movement of damaged mitochondria into the lysosomes. BAG5 also enhanced Parkin-mediated Mcl-1 degradation and cell death following severe mitochondrial insult. These results suggest that BAG5 may regulate the bi-modal activity of Parkin, promoting cell death by suppressing Parkin-dependent mitophagy and enhancing Parkin-mediated Mcl-1 degradation.

Targeting oncogenic proteins for degradation using proteolysis-targeting chimera (PROTAC) recently has drawn increasing attention in the field of cancer research. Bromodomain and extra-terminal (BET) family proteins are newly identified cancer-related epigenetic regulators, which have a role in the pathogenesis and progression of osteosarcoma. In this study, we investigated the in vitro and in vivo anti-osteosarcoma activity by targeting BET with a PROTAC molecule BETd-260. The results showed that BETd-260 completely depletes BET proteins and potently suppresses cell viability in MNNG/HOS, Saos-2, MG-63, and SJSA-1 osteosarcoma cell lines. Compared with BET inhibitors HJB-97 and JQ1, the activity of BETd-260 increased over 1000 times. Moreover, BETd-260 substantially inhibited the expression of anti-apoptotic Mcl-1, Bcl-xl while increased the expression of pro-apoptotic Noxa, which resulted in massive apoptosis in osteosarcoma cells within hours. In addition, pro-oncogenic protein c-Myc also was substantially inhibited by BETd-260 in the OS cells. Of note, BETd-260 induced degradation of BET proteins, triggered apoptosis in xenograft osteosarcoma tumor tissue, and profoundly inhibited the growth of cell-derived and patient-derived osteosarcoma xenografts in mice. Our findings indicate that BET PROTACs represent a promising therapeutic agent for human osteosarcoma.

Clusterin (CLU) is induced in many organs after tissue injury or remodeling. Recently, we show that CLU levels are increased in plasma and left ventricle (LV) after MI, however, the mechanisms involved are not yet elucidated. On the other hand, it has been shown that the activity of the protein degradation systems (PDS) is affected after MI with a decrease in ubiquitin proteasome system (UPS) and an increase in macroautophagy. The aim of this study was to decipher if the increased CLU levels after MI are in part due to the alteration of PDS activity. Rat neonate cardiomyocytes (NCM) were treated with different modulators of UPS and macroautophagy in order to decipher their role in CLU expression, secretion, and degradation. We observed that inhibition of UPS activity in NCM increased CLU mRNA levels, its intracellular protein levels (p-CLU and m-CLU) and its secreted form (s-CLU). Macroautophagy was also induced after MG132 treatment but is not active. The inhibition of macroautophagy induction in MG132-treated NCM increased CLU mRNA and m-CLU levels, but not s-CLU compared to NCM only treated by MG132. We also demonstrate that CLU can be degraded in NCM through proteasome and lysosome by a macroautophagy independent pathway. In another hand, CLU silencing in NCM has no effect either on macroautophagy or apoptosis induced by MG132. However, the overexpression of CLU secreted isoform in H9c2 cells, but not in NCM decreased apoptosis after MG132 treatment. Finally, we observed that increased CLU levels in hypertrophied NCM and in failing human hearts are associated with proteasome inhibition and macroautophagy alteration. All these data suggest that increased CLU expression and secretion after MI is, in part, due to a defect of UPS and macroautophagy activities in the heart and may have a protective effect by decreasing apoptosis induced by proteasome inhibition.

Colorectal cancer (CRC) is a common malignant gastrointestinal tumor with high mortality worldwide. Drug resistance and cytotoxicity to normal cells are the main causes of chemotherapeutic treatment failure in CRC. Therefore, extracting the bioactive compounds from natural products with anti-carcinogenic activity and minimal side-effects is a promising strategy against CRC. The present study aims to evaluate the anti-carcinogenic properties of avenanthramides (AVNs) extracted from oats bran and clarify the underlying molecular mechanisms. We demonstrated that AVNs treatment suppressed mitochondrial bioenergetic generation, resulting in mitochondrial swelling and increased reactive oxygen species (ROS) production. Further study indicated that AVNs treatment significantly reduced DDX3 expression, an oncogenic RNA helicase highly expressed in human CRC tissues. DDX3 overexpression reversed the ROS-mediated CRC apoptosis induced by AVNs. Of note, we identified Avenanthramide A (AVN A) as the effective ingredient in AVNs extracts. AVN A blocked the ATPase activity of DDX3 and induced its degradation by directly binding to the Arg287 and Arg294 residues in DDX3. In conclusion, these innovative findings highlight that AVNs extracts, in particular its bioactive compound AVN A may crack the current hurdles in the way of CRC treatment.

Acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) has been a major obstacle in the treatment of non-small cell lung cancer (NSCLC) patients. AXL has been reported to mediate EGFR-TKIs. Recently, third generation EGFR-TKI osimertinib has been approved and yet its acquired resistance mechanism is not clearly understood. We found that AXL is involved in both gefitinib and osimertinib resistance using in vitro and in vivo model. In addition, AXL overexpression was correlated with extended protein degradation rate. We demonstrate targeting AXL degradation is an alternative route to restore EGFR-TKIs sensitivity. We confirmed that the combination effect of YD, an AXL degrader, and EGFR-TKIs can delay or overcome EGFR-TKIs-driven resistance in EGFR-mutant NSCLC cells, xenograft tumors, and patient-derived xenograft (PDX) models. Therefore, combination of EGFR-TKI and AXL degrader is a potentially effective treatment strategy for overcoming and delaying acquired resistance in NSCLC.

Autophagy is a vital process that involves degradation of long-lived proteins and dysfunctional organelles and contributes to cellular metabolism. Glioma-initiating cells (GICs) have the ability to self-renew, differentiate into heterogeneous types of tumor cells, and sustain tumorigenicity; thus, GICs lead to tumor recurrence. Accumulating evidence indicates that autophagy can induce stem cell differentiation and increase the lethality of temozolomide against GICs. However, the mechanism underlying the regulation of GIC self-renewal by autophagy remains uncharacterized. In the present study, autophagy induced by AZD8055 and rapamycin treatment suppressed GIC self-renewal in vitro. We found that autophagy inhibited Notch1 pathway activation. Moreover, autophagy activated Notch1 degradation, which is associated with maintenance of the self-renewal ability of GICs. Furthermore, autophagy abolished the tumorigenicity of CD133 + U87-MG neurosphere cells in an intracranial model. These findings suggest that autophagy regulating GICs self-renewal and tumorigenicity is probably bound up with Notch1 degradation. The results of this study could aid in the design of autophagy-based clinical trials for glioma treatments, which may be of great value.

Dynamin-related protein 1 (DRP1) is a key molecule to regulate mitochondrial fission. DRP1 activity is modulated by phosphorylation and S-nitrosylation on serine and cysteine residues, respectively. However, it is still unexplored whether S-nitrosylation of DRP1 affects its phosphorylation. In the present study, we found that N ω -nitro-L-arginine methyl ester hydrochloride (L-NAME, a NOS inhibitor) abolished S-nitrosylated (SNO-DRP1) and DRP1-serine (S) 616 phosphorylation levels in CA1 neurons under physiological condition. L-NAME led to mitochondrial elongation. In spite of the sustained NO synthesis, status epilepticus (a prolonged seizure activity, SE) diminished SNO-DRP1 and DRP1-S616 levels in CA1 neurons, accompanied by the reduced protein disulfide isomerase (PDI) expression and mitochondrial elongation. SE did not influence thioredoxin 1 (Trx1, a denitrosylating enzyme) activity, which was unaffected by L-NAME under physiological and post-SE condition. PDI knockdown decreased SNO-DRP1 and DRP1-S616 levels concomitant with mitochondrial elongation in CA1 neurons without altered NO synthesis under physiological condition. These findings indicate that PDI may be a NO donor of DRP1 to regulate DRP1-S616 phosphorylation, independent of Trx1 activity. Therefore, we suggest that PDI-mediated S-nitrosylation of DRP1 may be one of the major regulatory modifications for mitochondrial dynamics.

Epstein-Barr virus (EBV) can infect cells in latent and lytic period and cause serious disease. Epstein-Barr virus nuclear antigen 1 (EBNA1) is essential for the maintenance of the EBV DNA episome, replication and transcription. 2-phenylethynesulfonamide (PES) is a small molecular inhibitor of Heat shock protein 70 (Hsp70), which can interact with Hsp70 and disrupts its association with co-chaperones and substrate proteins of Hsp70. In our study, we found that PES could decrease the expression of EBNA1, which is independent of effects on EBNA1 transcription or proteasomal degradation pathway. The central glycine-alanine repeats domain was not required for inhibition of EBNA1 expression by PES. Also, PES could reduce the amount of intracellular EBV genomic DNA. PES inhibited proliferation and migration but induced cell cycle arrest and apoptosis of EBV positive cells. In addition, silencing of Hsp70 decreased expression of EBNA1 and the amounts of intracellular EBV genomic DNA, and PES increased this effect on a dose-dependent manner. On the contrast, over-expression of Hsp70 enhanced the expression of EBNA1 and the amounts of intracellular EBV genomic DNA, but PES inhibited this effect on a dose-dependent manner. Furthermore, Hsp70 interacted with EBNA1 but PES interfered this interaction. Our results indicate that PES suppresses replication and carcinogenicity of Epstein-Barr virus via inhibiting the molecular chaperone function of Hsp70.

Atherosclerosis was considered to induce many vascular-related complications, such as acute myocardial infarction and stroke. Abnormal lipid metabolism and its peroxidation inducing blood-brain barrier (BBB) leakage were associated with the pre-clinical stage of stroke. Dapsone (DDS), an anti-inflammation and anti-oxidation drug, has been found to have protective effects on vascular. However, whether DDS has a protective role on brain microvessels during lipid oxidation had yet to be elucidated. We investigated brain microvascular integrity in a high-fat diet (HFD) mouse model. We designed this study to explore whether DDS had protective effects on brain microvessels under lipid oxidation and tried to explain the underlying mechanism. In our live optical study, we found that DDS significantly attenuated brain microvascular leakage through reducing serum oxidized low-density lipoprotein (oxLDL) in HFD mice (p < 0.001), and DDS significantly inhibited LDL oxidation in vitro (p < 0.001). Our study showed that DDS protected tight junction proteins: ZO-1 (p < 0.001), occludin (p < 0.01), claudin-5 (p < 0.05) of microvascular endothelial cells in vivo and in vitro. DDS reversed LAMP1 aggregation in cytoplasm, and decreased the destruction of tight junction protein: ZO-1 in vitro. We first revealed that DDS had a protective role on cerebral microvessels through preventing tight junction ZO-1 from abnormal degradation by autophagy and reducing lysosome accumulation. Our findings suggested the significance of DDS in protecting brain microvessels under lipid metabolic disorders, which revealed a novel potential therapeutic strategy in brain microvascular-related diseases.

Alzheimer's disease (AD) is a dreadful neurodegenerative disease that leads to severe impairment of cognitive function, leading to a drastic decline in the quality of life. The primary pathological features of AD include senile plaques (SPs) and intracellular neurofibrillary tangles (NFTs), comprising aggregated amyloid β (Aβ) and hyperphosphorylated tau protein, respectively, in the hippocampus of AD patients. Histone deacetylase 6 (HDAC6) is a key enzyme in this neurodegenerative disease, in particular, as it relates to tau hyperphosphorylation. This study aimed to investigate the protective effects and mechanism of the novel HDAC6 inhibitor, MPT0G211, using an AD model. Our results indicated that MPT0G211 significantly reduced tau phosphorylation and aggregation, the processes highly correlated with the formation of NFTs. This HDAC6 inhibitory activity resulted in an increase in acetylated Hsp90, which decreased Hsp90 and HDAC6 binding, causing ubiquitination of phosphorylated tau proteins. In addition, a significant increase of phospho-glycogen synthase kinase-3β (phospho-GSK3β) on Ser9 (the inactive form) through Akt phosphorylation was associated with the inhibition of phospho-tau Ser396 in response to MPT0G211 treatment. In AD in vivo models, MPT0G211 appeared to ameliorate learning and memory impairment in animals. Furthermore, MPT0G211 treatment reduced the amount of phosphorylated tau in the hippocampal CA1 region. In summary, MPT0G211 treatment appears to be a promising strategy for improving the AD phenotypes, including tau hyperphosphorylation and aggregation, neurodegeneration, and learning and memory impairment, making it a valuable agent for further investigation.

Despite success of ERBB2-targeted therapies such as lapatinib, resistance remains a major clinical concern. Multiple compensatory receptor tyrosine kinase (RTK) pathways are known to contribute to lapatinib resistance. The heterogeneity of these adaptive responses is a significant hurdle for finding most effective combinatorial treatments. The goal of this study was to identify a unifying molecular mechanism whose targeting could help prevent and/or overcome lapatinib resistance. Using the MMTV-ERBB2;mutant p53 (R175H) in vivo mouse model of ERBB2-positive breast cancer, together with mouse and human cell lines, we compared lapatinib-resistant vs. lapatinib-sensitive tumor cells biochemically and by kinome arrays and evaluated their viability in response to a variety of compounds affecting heat shock response. We found that multiple adaptive RTKs are activated in lapatinib-resistant cells in vivo, some of which have been previously described (Axl, MET) and some were novel (PDGFRα, PDGFRβ, VEGFR1, MUSK, NFGR). Strikingly, all lapatinib-resistant cells show chronically activated HSF1 and its transcriptional targets, heat shock proteins (HSPs), and, as a result, superior tolerance to proteotoxic stress. Importantly, lapatinib-resistant tumors and cells retained sensitivity to Hsp90 and HSF1 inhibitors, both in vitro and in vivo, thus providing a unifying and actionable therapeutic node. Indeed, HSF1 inhibition simultaneously downregulated ERBB2, adaptive RTKs and mutant p53, and its combination with lapatinib prevented development of lapatinib resistance in vitro. Thus, the kinome adaptation in lapatinib-resistant ERBB2-positive breast cancer cells is governed, at least in part, by HSF1-mediated heat shock pathway, providing a novel potential intervention strategy to combat resistance.

Proton pump inhibitors (PPIs) play a role in antitumor activity, with studies showing specialized impacts of PPIs on cancer cell apoptosis, metastasis, and autophagy. In this study, we demonstrated that pantoprazole (PPI) increased autophagosomes formation and affected autophagic flux depending on the pH conditions. PPI specifically elevated SQSTM1 protein levels by increasing SQSTM1 transcription via NFE2L2 activation independent of the specific effect of PPI on autophagic flux. Via decreasing proteasome subunits expression, PPI significantly impaired the function of the proteasome, accompanied by the accumulation of undegraded poly-ubiquitinated proteins. Notably, PPI-induced autophagy functioned as a downstream response of proteasome inhibition by PPI, while suppressing protein synthesis abrogated autophagy. Blocking autophagic flux in neutral pH condition or further impairing proteasome function with proteasome inhibitors, significantly aggravated PPI cytotoxicity by worsening protein degradation ability. Interestingly, under conditions of mitochondrial stress, PPI showed significant synergism when combined with Bcl-2 inhibitors. Taken together, these findings provide a new understanding of the impact of PPIs on cancer cells' biological processes and highlight the potential to develop more efficient and effective combination therapies.

Aberrant activation of Wnt/β-catenin signalling is critical in the progression of human cancers, especially colorectal cancer (CRC). Therefore, inhibition of Wnt/β-catenin signalling is a significant potential target for CRC therapy. Here, we identified for the first time that Physalin F (PF), a steroid derivative isolated from Physalis angulate, acts as an antagonist of Wnt/β-catenin signalling. In vitro, PF decreased Wnt3a-induced TOPFlash reporter activity in HEK293T cells and promoted the formation of the β-catenin destruction complex. Importantly, PF also inhibited Wnt/β-catenin signalling and accelerated the degradation of β-catenin in CRC cells. However, PF did not affect the stabilization of Axin or the interaction of β-catenin with E-cadherin. Interestingly, we further found that PF promoted YAP binding to the β-catenin destruction complex, which facilitated the ubiquitination and degradation of β-catenin. Silencing and pharmacological inhibition of YAP reversed the formation of the β-catenin destruction complex induced by PF, implying that YAP binding to the β-catenin destruction complex was responsible for PF-mediated inhibition of Wnt/β-catenin signalling. Furthermore, PF observably inhibited tumour growth by down-regulating β-catenin in tumour-bearing mice. Collectively, our findings indicated that PF inhibited Wnt/β-catenin signalling by accelerating the ubiquitination and degradation of β-catenin in a YAP-dependent manner and therefore PF could be a novel potential candidate for CRC therapy.

In type 2 diabetes, amyloid oligomers, chronic hyperglycemia, lipotoxicity, and pro-inflammatory cytokines are detrimental to beta-cells, causing apoptosis and impaired insulin secretion. The histone acetyl transferase p300, involved in remodeling of chromatin structure by epigenetic mechanisms, is a key ubiquitous activator of the transcriptional machinery. In this study, we report that loss of p300 acetyl transferase activity and expression leads to beta-cell apoptosis, and most importantly, that stress situations known to be associated with diabetes alter p300 levels and functional integrity. We found that proteasomal degradation is the mechanism subserving p300 loss in beta-cells exposed to hyperglycemia or pro-inflammatory cytokines. We also report that melatonin, a hormone produced in the pineal gland and known to play key roles in beta-cell health, preserves p300 levels altered by these toxic conditions. Collectively, these data imply an important role for p300 in the pathophysiology of diabetes.

Accumulating evidence revealed that mesenchymal stem cells (MSCs) confer cardioprotection against myocardial infarction (MI). However, the poor survival and engraftment rate of the transplanted cells limited their therapeutic efficacy in the heart. The enhanced leptin production associated with hypoxia preconditioning contributed to the improved MSCs survival. Mitochondrial integrity determines the cellular fate. Thus, we aimed to investigate whether leptin can enhance mitochondrial integrity of human MSCs (hMSCs) to protect against various stress. In vivo, transplantation of leptin-overexpressing hMSCs into the infarcted heart resulted in improved cell viability, leading to enhanced angiogenesis and cardiac function. In vitro, pretreatment of hMSCs with recombinant leptin (hMSCs-Lep pre ) displayed improved cell survival against severe ischemic condition (glucose and serum deprivation under hypoxia), which was associated with increased mitochondrial fusion. Subsequently, Optic atrophy 1 (OPA1), a mitochondrial inner membrane protein that regulates fusion and cristae structure, was significantly elevated in the hMSCs-Lep pre group, and the protection of leptin was abrogated by targeting OPA1 with a selective siRNA. Furthermore, OMA1, a mitochondrial protease that cleaves OPA1, decreased in a leptin-dependent manner. Pretreatment of cells with an inhibitor of the proteasome (MG132), prevented leptin-induced OMA1 degradation, implicating the ubiquitination/proteasome system as a part of the protective leptin pathway. In addition, GSK3 inhibitor (SB216763) was also involved in the degradation of OMA1. In conclusion, in the hostile microenvironment caused by MI, (a) leptin can maintain the mitochondrial integrity and prolong the survival of hMSCs; (b) leptin-mediated mitochondrial integrity requires phosphorylation of GSK3 as a prerequisite for ubiquitination-depended degradation of OMA1 and attenuation of long-OPA1 cleavage. Thus, leptin targeting the GSK3/OMA1/OPA1 signaling pathway can optimize hMSCs therapy for cardiovascular diseases such as MI.

HDAC3 is involved in deacetylation of histone and non-histone proteins, having a key role in the regulation of gene transcription and also in the process of tumorgenesis. However, how HDAC3 is regulated in cancer remains largely unclear. Here, we showed that PIWIL2 can interact with HDAC3, leading to stabilization of HDAC3 from ubiquitin-mediated degradation by competitive association with E3 ubiquitin ligase Siah2. Furthermore, we found that expression of PIWIL2 enhanced HDAC3 activity via CK2α. PIWIL2 facilitated the interaction between HDAC3 and CK2α, thus exhibiting a promotion on the HDAC3 phosphorylation by CK2α. Further work showed that PIWIL2 could promote cell proliferation and suppress cell apoptosis via regulating HDAC3. Our present study firstly revealed that PIWIL2 can play a role in HDAC3-mediated epigenetic regulation on cancer cell proliferation and apoptosis. These findings provide a novel insight into the roles of PIWIL2 in tumorigenesis.

Autophagy plays a central role in degrading misfolded proteins such as mutated superoxide dismutase 1 (SOD1), which forms aggregates in motor neurons and is involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). Autophagy is activated when UNC-51-like kinase 1 (ULK1) is phosphorylated at S555 and activated by AMP-activated protein kinase (AMPK). Autophagy is suppressed when ULK1 is phosphorylated at S757 by the mechanistic target of rapamycin (mTOR). Whether p70 S6 kinase 1 (S6K1), a serine/threonine kinase downstream of mTOR, can also regulate autophagy remains uncertain. Here we report that inhibition of S6K1 by A77 1726, the active metabolite of an anti-inflammatory drug leflunomide, induced mTOR feedback activation and ULK1 S757 phosphorylation in NSC34 cells, a hybrid mouse motoneuron cell line. Unexpectedly, A77 1726 did not suppress but rather induced autophagy by increasing AMPK T172 and ULK1 S555 phosphorylation. Similar observations were made with PF-4708671, a specific S6K1 inhibitor, or with S6K1 siRNA. Further studies showed that A77 1726 induced AMPK phosphorylation by activating the TGF-β-activated kinase 1 (TAK1). Functional studies revealed that A77 1726 induced co-localization of mutant SOD1 G93A protein aggregates with autophagosomes and accelerated SOD1 G93A protein degradation, which was blocked by inhibition of autophagy through autophagy-related protein 7 (ATG7) siRNA. Our study suggests that S6K1 inhibition induces autophagy through TAK1-mediated AMPK activation in NSC34 cells, and that blocking S6K1 activity by a small molecule inhibitor such as leflunomide may offer a new strategy for ALS treatment.

Oxidative stress generates reactive oxygen species (ROS) that can promote or inhibit cardiac differentiation of stem cells dependent on the intensity of stimuli as well as cellular context in redox and differentiation status. In the current study, we confirmed that suitable intensity of hydrogen peroxide at the formation stage of embryoid bodies (EBs) effectively favored the formation of spontaneously beating cardiomyocytes from P19 embryonal carcinoma cells. Mechanistic studies implicated that extrinsic ROS enhanced the Caspase-mediated degradation of Oct4 and Nanog, two factors that governing pluripotent property. Further experiments suggested that a cohort of Nanog together with histone deacetylase 4 (Hdac4) played a critical role in establishing and maintaining the silent transcriptional status of Gata4 and Nkx2.5 in undifferentiated cells. Thus, an impulse of hydrogen peroxide depleted Nanog and Hdac4 via a caspase-dependent manner to ameliorate the repression on Gata4 and Nkx2.5 promoters, thereby generating a persistent activation on cardiac differentiation program. Meanwhile, we found that excessive ROS-activated JNK cascade to facilitate the ubiquitination and subsequent degradation of Gata4 protein. Overall, our results indicate that suitable ROS promotes the activation of Gata4 in transcription, while excessive ROS targets Gata4 protein for proteasome-dependent degradation. Gata4 is an important modulator balancing the promoting and inhibitory effects of oxidative stress on differentiation program of cardiomyogenesis.

Proteasome inhibitors have emerged as an effective therapy for the treatment of haematological malignancies; however, their efficacy can be limited by the development of tumour resistance mechanisms. Novel combination strategies including the addition of TLR adjuvants to increase cell death and augment immune responses may help enhance their effectiveness. Although generally thought to inhibit inflammatory responses and NF-κB activation, we found that under specific conditions proteasome inhibitors can promote inflammatory responses by mediating IL-1β maturation and secretion after TLR stimulation. This was dependent on the timing of proteasome inhibition relative to TLR stimulation where reversal of treatment order could alternatively increase or inhibit IL-1β secretion (P < 0.001). TLR stimulation combined with proteasome inhibition enhanced cell death in vitro and delayed tumour development in vivo in NOD SCID mice (P < 0.01). However, unlike IL-1β secretion, cell death occurred similarly regardless of treatment order and was only partially caspase dependent, possessing characteristics of both apoptosis and necrosis as indicated by activation of caspase-1, 3, 8 and RIP3 phosphorylation. Although stimulation of various TLRs was capable of driving IL-1β production, TLR4 stimulation was the most effective at increasing cell death in THP-1 and U937 cells. TLR4 stimulation and proteasome inhibition independently activated the RIP3 necroptotic pathway and ultimately reduced the effectiveness of caspase/necroptosis inhibitors in mitigating overall levels of cell death. This strategy of combining TLR stimulation with proteasome inhibition may improve the ability of proteasome inhibitors to generate immunogenic cell death and increase anti-tumour activity.

Individuals exposed to long-term spaceflight often experience cardiovascular dysfunctions characterized by orthostatic intolerance, disability on physical exercise, and even frank syncope. Recent studies have showed that the alterations of cardiovascular system are closely related to the functional changes of endothelial cells. We have shown previously that autophagy can be induced by simulated microgravity in human umbilical vein endothelial cells (HUVECs). However, the mechanism of enhanced autophagy induced by simulated microgravity and its role in the regulation of endothelial function still remain unclear. We report here that 48 h clinorotation promoted cell migration in HUVECs by induction of autophagy. Furthermore, clinorotation enhanced autophagy by the mechanism of human murine double minute 2 (HDM2)-dependent degradation of cytoplasmic p53 at 26S proteasome, which results in the suppression of mechanistic target of rapamycin (mTOR), but not via activation of AMPK in HUVECs. These results support the key role of HDM2-p53 in direct downregulation of mTOR, but not through AMPK in microgravity-induced autophagy in HUVECs.

The ubiquitin E3 ligase DELTEX1 (DTX1) is specifically downregulated in gastric cancer tissues, and expression of DTX1 is linked to better prognoses and survival in gastric cancer. Cellular FLICE inhibitory protein (c-FLIP) is known for its pivotal role in the resistance of cancer cells to death receptor-induced cell death. Here, we show that DTX1 is an E3 ligase for c-FLIP in gastric cancer cells. DTX1 promoted c-FLIP downregulation. Overexpression of DTX1 sensitized gastric cancer cells to TRAIL-induced apoptosis, whereas DTX1-knockdown attenuated apoptosis induction. DTX1 binds c-FLIP L and directs it into the endosome-lysosomal pathway for proteasome-independent degradation. Moreover, induction of DTX1 in AGS cells by geldanamycin conferred susceptibility of those cells to TRAIL-induced apoptosis. Our results reveal a tumor-suppressive role for DTX1 and suggest a new approach to increasing TRAIL efficacy by raising DTX1 levels in gastric cancer therapy. DTX1 also enhanced c-FLIP degradation and FasL-induced and TRAIL-induced apoptosis in T cells, suggesting that DTX1 constitutes one of the physiological mechanisms regulating c-FLIP stability.

The carboxyl terminus of Hsp70-interacting protein (CHIP) acts as a ubiquitin E3 ligase and a link between the chaperones Hsp70/90 and the proteasome system, playing a vital role in maintaining protein homeostasis. CHIP regulates a number of proteins involved in a myriad of physiological and pathological processes, but the underlying mechanism of action via posttranslational modification has not been extensively explored. In this study, we investigated a novel modulatory mode of CHIP and its effect on CHIP enzymatic activity. ISG15, an ubiquitin-like modifier, is induced by type I interferon (IFN) stimulation and can be conjugated to target proteins (ISGylation). Here we demonstrated that CHIP may be a novel target of ISGylation in HEK293 cells stimulated with type I IFN. We also found that Lys143/144/145 and Lys287 residues in CHIP are important for and target residues of ISGylation. Moreover, ISGylation promotes the E3 ubiquitin ligase activity of CHIP, subsequently causing a decrease in levels of oncogenic c-Myc, one of its many ubiquitination targets, in A549 lung cancer cells and inhibiting A549 cell and tumor growth. In conclusion, the present study demonstrates that covalent ISG15 conjugation produces a novel CHIP regulatory mode that enhances the tumor-suppressive activity of CHIP, thereby contributing to the antitumor effect of type I IFN.

Elevated tumor suppressor p53 expression has been associated with heart diseases, including the diabetic heart. However, its precise role in the pathogenesis of diabetic cardiomyopathy (DCM) remains unclear. We hypothesized that the development of DCM is attributed to up-regulated p53-mediated both early cardiac cell death and persistent cell senescence, glycolytic and angiogenetic dysfunctions. The present study investigated the effect of p53 inhibition with its specific inhibitor pifithrin-α (PFT-α) on the pathogenesis of DCM and its associated mechanisms. Type 1 diabetes was induced with multiple low doses of streptozotocin. Both hyperglycemic and age-matched control mice were treated with and without PFT-α five times a week for 2 months and then sacrificed at 3 and 6 months post-diabetes. Treatment with PFT-α significantly prevented the progression of diabetes-induced cardiac remodeling and dysfunction (i.e., DCM). Mechanistically, the inhibition of p53 prevented the cardiac apoptosis during early-stage diabetes (0.5 month), attenuated diabetes-induced cell senescence (3 and 6 months), and improved both glycolytic and angiogenic defects by increasing hypoxia-induced factor (HIF)-1α protein stability and upregulating HIF-1α transcription of specific target genes at 3 and 6 months after diabetes. Therefore, the targeted inhibition of p53 in diabetic individuals may provide a novel approach for the prevention of DCM.

Excess production of reactive oxygen species (ROS) is known to cause apoptotic cell death. However, the molecular mechanisms whereby ROS induce apoptosis remain elusive. Here we show that the NHL-repeat-containing protein 2 (NHLRC2) thioredoxin-like domain protein is cleaved by caspase-8 in ROS-induced apoptosis in the HCT116 human colon cancer cell line. Treatment of HCT116 cells with the oxidant tert-butyl hydroperoxide (tBHP) induced apoptosis and reduced NHLRC2 protein levels, whereas pretreatment with the antioxidant N-acetyl-L-cysteine prevented apoptosis and the decrease in NHLRC2 protein levels seen in tBHP-treated cells. Furthermore, the ROS-induced decrease in NHLRC2 protein levels was relieved by the caspase inhibitor z-VAD-fmk. We found that the thioredoxin-like domain of NHLRC2 interacted with a proenzyme form of caspase-8, and that caspase-8 cleaved NHLRC2 protein at Asp580 in vitro. Furthermore, siRNA-mediated knockdown of caspase-8 blocked the ROS-induced decrease in NHLRC2 protein levels. Both shRNA and CRISPR-Cas9-mediated loss of NHLRC2 resulted in an increased susceptibility of HCT116 cells to ROS-induced apoptosis. These results suggest that excess ROS production causes a caspase-8-mediated decrease in NHLRC2 protein levels, leading to apoptotic cell death in colon cancer cells, and indicate an important role of NHLRC2 in the regulation of ROS-induced apoptosis.

Apoptosis of renal tubular epithelial cells is a key feature of the pathogenicity associated with tubulointerstitial fibrosis and other kidney diseases. One factor that regulates important cellular processes like apoptosis and cell proliferation is HRD1, an E3 ubiquitin ligase that acts by promoting ubiquitylation and degradation of its target protein. However, the detailed mechanisms by which HRD1 acts as a regulator of apoptosis in renal tubular epithelial cells have not been established. In our previous liquid chromatography-tandem mass spectrometry (LC-MS/MS) study (Mol Endocrinol. 2016;30:600-613), we demonstrated that one substrate of HRD1 was eIF2α, a critical protein in the PERK-eIF2α-ATF4-CHOP signaling pathway of endoplasmic reticulum (ER) stress. Here, we show that eIF2α expression was increased and HRD1 expression decreased when apoptosis was induced in HKC-8 cells by palmitic acid (PA) or high glucose (HG). HRD1 expression was also lower in kidney tissues from mice with diabetic nephropathy (DN) than in control mice. Forced expression of HRD1 also inhibited apoptosis in HKC-8 cells, while HRD1 overexpression decreased the expression of phosphorylated eIF2α and eIF2α. Further analysis indicated that HRD1 interacted with eIF2α and promoted its ubiquitylation and degradation by the proteasome. Moreover, the HRD1 protection of PA-treated HKC-8 cells was blunted by transfection with Myc-eIF2α. Thus, eIF2α ubiquitylation by HRD1 protects tubular epithelial cells from apoptosis caused by HG and PA, indicating a novel upstream target for therapeutic prevention of renal tubulointerstitial injury.

STAT1, which carries tumor suppressor functions in several models, consists of two isoforms, namely STAT1α and STAT1β. The biological function and significance of STAT1β has never been examined in human cancer. We examined STAT1β function in esophageal squamous cell carcinoma (ESCC) by transfecting a STAT1β gene into various ESCC cell lines. The interaction between STAT1α and STAT1β was examined by using co-immunoprecipitation and confocal microscopy. The prognostic significance of STAT1β expression, detectable by immunohistochemistry and western blot, was evaluated in a large cohort of ESCC patients. Enforced expression of STAT1β induced and prolonged the expression and phosphorylation of STAT1α in ESCC cells, and these effects were amplified by gamma-interferon (IFN-γ). We also found that STAT1β interacts with STAT1α and decreases STAT1α degradation by the proteasome. Moreover, STAT1β substantially increased the DNA binding and transcription activity of STAT1. STAT1β also sensitized ESCC cells to chemotherapeutic agents, including cisplatin and 5-flurouracil. Using western blot and immunohistochemistry, we found that STAT1β was frequently decreased in esophageal cancer, as compared to their adjacent benign esophageal epithelial tissue. Loss of STAT1β significantly correlated with lymph node metastasis, invasion and shorter overall survival in ESCC patients. Therefore, STAT1β plays a key role in enhancing the tumor suppressor function of STAT1α, in ESCC, in a manner that can be amplified by IFN-γ.

Autophagy promotes cancer cell survival and drug resistance by degrading harmful cellular components and maintaining cellular energy levels. Disruption of autophagy may be a promising approach to sensitize cancer cells to anticancer drugs. The combination of autophagic inhibitors, such as chloroquine (CQ) and lucanthone with conventional cancer therapeutics has been investigated in clinical trials, but adverse drug-drug interactions are a high possibility. Here we designed and synthesized a novel, small-molecule library based on an acridine skeleton and the CQ structure with various modifications and substitutions and screened the compounds for effective autophagy inhibition. We found that 9-chloro-2-(3-(dimethylamino)propyl)pyrrolo[2,3,4-kl]acridin-1(2H)-one (LS-1-10) was the most effective from our library at inhibiting autophagic-mediated degradation and could decrease the viability of multiple colon cancer cells. In addition, LS-1-10 induced DNA damage and caspase 8-mediated apoptosis. Overall, this small molecule was more efficient at reducing the viability of cancer cells than other conventional chemotherapeutic agents, such as CQ and amsacrine. The anticancer and autophagy-inhibiting activities of LS-1-10 were confirmed in vivo in a xenograft mouse model. Collectively, this study has identified a new and efficient single compound with both autophagy-inhibiting and anticancer activity, which may provide a novel approach for cancer therapy.

Parkinson's disease (PD) is one of the most common neurodegenerative diseases, characterized by degeneration of dopaminergic neurons in the substantia nigra. α-synuclein (α-syn) and PTEN-induced putative kinase (PINK)1 are two critical proteins associated with the pathogenesis of PD. α-syn induces mitochondrial deficits and apoptosis, PINK1 was found to alleviate α-syn-induced toxicity, but the mechanistic details remain obscure. Here, we show that PINK1 interacts with α-syn mainly in the cytoplasm, where it initiates autophagy. This interaction was dependent on the kinase activity of PINK1 and was abolished by deletion of the kinase domain or a G309D point mutation, an inactivating mutation in the kinase domain. Interaction between PINK1 and α-syn stimulated the removal of excess α-syn, which prevented mitochondrial deficits and apoptosis. Our findings provide evidence for a novel mechanism underlying the protective effects of PINK1 against α-syn-induced neurodegeneration and highlight a novel therapeutic target for PD treatment.

Glioblastoma multiforme (GBM) is the most prevalent form of malignant brain tumor. Amlexanox, a novel compound, has been shown to have anti-cancer potential. In this study, the anti-tumoral effects and the underlying mechanisms of amlexanox were investigated. Amlexanox significantly suppressed proliferation and invasion and induced apoptosis in glioblastoma cells. Furthermore, we found that amlexanox altered the protein expression of the Hippo pathway by downregulating IKBKE. Our data indicates that IKBKE directly targets LATS1/2 and induces degradation of LATS1/2, thereby inhibiting the activity of the Hippo pathway. In vivo results further confirmed the tumor inhibitory effect of amlexanox via the downregulation of IKBKE, and amlexanox induced no apparent toxicity. Collectively, our studies suggest that amlexanox is a promising therapeutic agent for the treatment of GBM.