POTENTIAL DIAGNOSTIC BIOMARKERS FOR HUMAN MESENCHYMAL TUMORS, ESPECIALLY LMP2/Β1I AND CYCLIN E1/MIB1 DIFFERENTIAL EXPRESSION: PRUM-IBIO STUDY.

Most mesenchymal tumors found in the uterine corpus are benign tumors; however, uterine leiomyosarcoma is a malignant tumor with unknown risk factors that repeatedly recurs and metastasizes. In some cases, the histopathologic findings of uterine leiomyoma and uterine leiomyosarcoma are similar and surgical pathological diagnosis using excised tissue samples is difficult. It is necessary to analyze the risk factors for human uterine leiomyosarcoma and establish diagnostic biomarkers and treatments. Female mice deficient in the proteasome subunit low molecular mass peptide 2 (LMP2)/ß1i develop uterine leiomyosarcoma spontaneously. MATERIAL AND METHODS: Out of 334 patients with suspected uterine mesenchymal tumors, patients diagnosed with smooth muscle tumors of the uterus were selected from the pathological file. To investigate the expression status of biomarker candidate factors, immunohistochemical staining was performed with antibodies of biomarker candidate factors on thin-cut slides of human uterine leiomyosarcoma, uterine leiomyoma, and other uterine mesenchymal tumors. RESULTS AND DISCUSSION: In human uterine leiomyosarcoma, there was a loss of LMP2/ß1i expression and enhanced cyclin E1 and Ki-67/MIB1 expression. In human uterine leiomyomas and normal uterine smooth muscle layers, enhanced LMP2/ß1i expression and the disappearance of the expression of E1 and Ki-67/MIB1 were noted. The pattern of expression of each factor in other uterine mesenchymal tumors was different from that of uterine leiomyosarcoma. CONCLUSIONS: LMP2/ß1i, cyclin E1, and Ki-67/MIB1 may be candidate factors for biomarkers of human uterine leiomyosarcoma. Further large-cohort clinical trials should be conducted to establish treatments and diagnostics for uterine mesenchymal tumors.

Targeting the immunoproteasome in hypothalamic neurons as a novel therapeutic strategy for high-fat diet-induced obesity and metabolic dysregulation.

OBJECTIVE: Obesity represents a significant global health challenge characterized by chronic low-grade inflammation and metabolic dysregulation. The hypothalamus, a key regulator of energy homeostasis, is particularly susceptible to obesity's deleterious effects. This study investigated the role of the immunoproteasome, a specialized proteasomal complex implicated in inflammation and cellular homeostasis, during metabolic diseases. METHODS: The levels of the immunoproteasome ß5i subunit were analyzed by immunostaining, western blotting, and proteasome activity assay in mice fed with either a high-fat diet (HFD) or a regular diet (CHOW). We also characterized the impact of autophagy inhibition on the levels of the immunoproteasome ß5i subunit and the activation of the AKT pathway. Finally, through confocal microscopy, we analyzed the contribution of ß5i subunit inhibition on mitochondrial function by flow cytometry and mitophagy assay. RESULTS: Using an HFD-fed obese mouse model, we found increased immunoproteasome levels in hypothalamic POMC neurons. Furthermore, we observed that palmitic acid (PA), a major component of saturated fats found in HFD, increased the levels of the ß5i subunit of the immunoproteasome in hypothalamic neuronal cells. Notably, the increase in immunoproteasome expression was associated with decreased autophagy, a critical cellular process in maintaining homeostasis and suppressing inflammation. Functionally, PA disrupted the insulin-glucose axis, leading to reduced AKT phosphorylation and increased intracellular glucose levels in response to insulin due to the upregulation of the immunoproteasome. Mechanistically, we identified that the protein PTEN, a key regulator of insulin signaling, was reduced in an immunoproteasome-dependent manner. To further investigate the potential therapeutic implications of these findings, we used ONX-0914, a specific immunoproteasome inhibitor. We demonstrated that this inhibitor prevents PA-induced insulin-glucose axis imbalance. Given the interplay between mitochondrial dysfunction and metabolic disturbances, we explored the impact of ONX-0914 on mitochondrial function. Notably, ONX-0914 preserved mitochondrial membrane potential and attenuated mitochondrial ROS production in the presence of PA. Moreover, we found that ONX-0914 reduced mitophagy in the presence of PA. CONCLUSIONS: Our findings strongly support the pathogenic involvement of the immunoproteasome in hypothalamic neurons in the context of HFD-induced obesity and metabolic disturbances. Targeting the immunoproteasome highlights a promising therapeutic strategy to mitigate the detrimental effects of obesity on the insulin-glucose axis and cellular homeostasis. This study provides valuable insights into the mechanisms driving obesity-related metabolic diseases and offers potential avenues for developing novel therapeutic interventions.

The 26 S proteasome in Entamoeba histolytica: divergence of the substrate binding pockets from host proteasomes.

OBJECTIVE: Proteasomes are conserved proteases crucial for proteostasis in eukaryotes and are promising drug targets for protozoan parasites. Yet, the proteasomes of Entamoeba histolytica remain understudied. The study's objective was to analyse the differences in the substrate binding pockets of amoeba proteasomes from those of host, and computational modelling of ß5 catalytic subunit, with the goal of finding selective inhibitors. RESULTS: Comparative sequence analysis revealed differences in substrate binding sites of E. histolytica proteasomes, especially in the S1 and S3 pockets of the catalytic beta subunits, implying differences in substrate preference and susceptibility to inhibitors from host proteasomes. This was strongly supported by significantly lower sensitivity to MG132 mediated inhibition of amoebic proteasome ß5 subunit's chymotryptic activity compared to human proteasomes, also reflected in lower sensitivity of E. histolytica to MG132 for inhibition of proliferation. Computational models of ß4 and ß5 subunits, and a docked ß4-ß5 model revealed a binding pocket between ß4-ß5, similar to that of Leishmania tarentolae. Selective inhibitors for visceral leishmaniasis, LXE408 and compound 8, docked well to this pocket. This functional and sequence-based analysis predicts differences between amoebic and host proteasomes that can be utilized to develop rationally designed, selective inhibitors against E. histolytica.

Ubiquitination and deubiquitination in cancer: from mechanisms to novel therapeutic approaches.

Ubiquitination, a pivotal posttranslational modification of proteins, plays a fundamental role in regulating protein stability. The dysregulation of ubiquitinating and deubiquitinating enzymes is a common feature in various cancers, underscoring the imperative to investigate ubiquitin ligases and deubiquitinases (DUBs) for insights into oncogenic processes and the development of therapeutic interventions. In this review, we discuss the contributions of the ubiquitin-proteasome system (UPS) in all hallmarks of cancer and progress in drug discovery. We delve into the multiple functions of the UPS in oncology, including its regulation of multiple cancer-associated pathways, its role in metabolic reprogramming, its engagement with tumor immune responses, its function in phenotypic plasticity and polymorphic microbiomes, and other essential cellular functions. Furthermore, we provide a comprehensive overview of novel anticancer strategies that leverage the UPS, including the development and application of proteolysis targeting chimeras (PROTACs) and molecular glues.

Exploring the therapeutic potential of Aloin: unraveling neuroprotective and anticancer mechanisms, and strategies for enhanced stability and delivery.

We investigate the therapeutic potential of Aloin A and Aloin B, two natural compounds derived from Aloe vera leaves, focusing on their neuroprotective and anticancer properties. The structural differences between these two epimers suggest that they may exhibit distinct pharmacological properties. Our investigations revealed that both epimers are not stable in aqueous solution and tend to degrade rapidly, with their concentration decreasing by over 50% within approximately 12 h. These results underscore the importance of addressing issues such as the need for encapsulation into effective drug delivery systems to enhance stability. ThT fluorescence experiments showed that neither compound was able to inhibit Aß amyloid aggregation, indicating that other mechanisms may be responsible for their neuroprotective effects. Next, an equimolar mixture of Aloin A and Aloin B demonstrated an ability to inhibit proteasome in tube tests, which is suggestive of potential anticancer properties, in accordance with antiproliferative effects observed in neuroblastoma SH-SY5Y and HeLa cell lines. Higher water stability and increased antiproliferative activity were observed by encapsulation in carbon dot nanoparticles, suggesting a promising potential for further in vivo studies.

Dysregulated proteasome activity and steroid hormone biosynthesis are associated with mortality among patients with acute COVID-19.

The persistence of coronavirus disease 2019 (COVID-19)-related hospitalization severely threatens medical systems worldwide and has increased the need for reliable detection of acute status and prediction of mortality. We applied a systems biology approach to discover acute-stage biomarkers that could predict mortality. A total 247 plasma samples were collected from 103 COVID-19 (52 surviving COVID-19 patients and 51 COVID-19 patients with mortality), 51 patients with other infectious diseases (IDCs) and 41 healthy controls (HCs). Paired plasma samples were obtained from survival COVID-19 patients within 1 day after hospital admission and 1-3 days before discharge. There were clear differences between COVID-19 patients and controls, as well as substantial differences between the acute and recovery phases of COVID-19. Samples from patients in the acute phase showed suppressed immunity and decreased steroid hormone biosynthesis, as well as elevated inflammation and proteasome activation. These findings were validated by enzyme-linked immunosorbent assays and metabolomic analyses in a larger cohort. Moreover, excessive proteasome activity was a prominent signature in the acute phase among patients with mortality, indicating that it may be a key cause of poor prognosis. Based on these features, we constructed a machine learning panel, including four proteins [C-reactive protein (CRP), proteasome subunit alpha type (PSMA)1, PSMA7, and proteasome subunit beta type (PSMB)1)] and one metabolite (urocortisone), to predict mortality among COVID-19 patients (area under the receiver operating characteristic curve: 0.976) on the first day of hospitalization. Our systematic analysis provides a novel method for the early prediction of mortality in hospitalized COVID-19 patients.

In Silico Design, Synthesis, and Evaluation of PROTAC Against Hematopoietic Prostaglandin D Synthase.

Chemical protein knockdown technology using proteolysis-targeting chimeras (PROTACs) to hijack the endogenous ubiquitin-proteasome system is a powerful strategy to degrade disease-related proteins. This chapter describes in silico design of a hematopoietic prostaglandin D synthase (H-PGDS) degrader, PROTAC(H-PGDS), using a docking simulation of the ternary complex of H-PGDS/PROTAC/E3 ligase as well as the synthesis of the designed PROTAC(H-PGDS)s and evaluation of their H-PGDS degradation activity.

Synergistic induction of mitotic pyroptosis and tumor remission by inhibiting proteasome and WEE family kinases.

Mitotic catastrophe (MC), which occurs under dysregulated mitosis, represents a fascinating tactic to specifically eradicate tumor cells. Whether pyroptosis can be a death form of MC remains unknown. Proteasome-mediated protein degradation is crucial for M-phase. Bortezomib (BTZ), which inhibits the 20S catalytic particle of proteasome, is approved to treat multiple myeloma and mantle cell lymphoma, but not solid tumors due to primary resistance. To date, whether and how proteasome inhibitor affected the fates of cells in M-phase remains unexplored. Here, we show that BTZ treatment, or silencing of PSMC5, a subunit of 19S regulatory particle of proteasome, causes G2- and M-phase arrest, multi-polar spindle formation, and consequent caspase-3/GSDME-mediated pyroptosis in M-phase (designated as mitotic pyroptosis). Further investigations reveal that inhibitor of WEE1/PKMYT1 (PD0166285), but not inhibitor of ATR, CHK1 or CHK2, abrogates the BTZ-induced G2-phase arrest, thus exacerbates the BTZ-induced mitotic arrest and pyroptosis. Combined BTZ and PD0166285 treatment (named BP-Combo) selectively kills various types of solid tumor cells, and significantly lessens the IC50 of both BTZ and PD0166285 compared to BTZ or PD0166285 monotreatment. Studies using various mouse models show that BP-Combo has much stronger inhibition on tumor growth and metastasis than BTZ or PD0166285 monotreatment, and no obvious toxicity is observed in BP-Combo-treated mice. These findings disclose the effect of proteasome inhibitors in inducing pyroptosis in M-phase, characterize pyroptosis as a new death form of mitotic catastrophe, and identify dual inhibition of proteasome and WEE family kinases as a promising anti-cancer strategy to selectively kill solid tumor cells.

Distinct TORC1 signalling branches regulate Adc17 proteasome assembly chaperone expression.

When stressed, cells need to adapt their proteome to maintain protein homeostasis. This requires increased proteasome assembly. Increased proteasome assembly is dependent on increased production of proteasome assembly chaperones. In Saccharomyces cerevisiae, inhibition of the growth-promoting kinase complex TORC1 causes increased proteasome assembly chaperone translation, including that of Adc17. This is dependent upon activation of the mitogen-activated protein kinase (MAPK) Mpk1 and relocalisation of assembly chaperone mRNA to patches of dense actin. We show here that TORC1 inhibition alters cell wall properties to induce these changes by activating the cell wall integrity pathway through the Wsc1, Wsc3 and Wsc4 sensor proteins. We demonstrate that, in isolation, these signals are insufficient to drive protein expression. We identify that the TORC1-activated S6 kinase Sch9 must be inhibited as well. This work expands our knowledge on the signalling pathways that regulate proteasome assembly chaperone production.

Quantitative Measurement of Rate of Targeted Protein Degradation.

Targeted protein degradation (TPD) is a therapeutic approach that leverages the cell's natural machinery to degrade targets instead of inhibiting them. This is accomplished by using mono- or bifunctional small molecules designed to induce the proximity of target proteins and E3 ubiquitin ligases, leading to ubiquitination and subsequent proteasome-dependent degradation of the target. One of the most significant attributes of the TPD approach is its proposed catalytic mechanism of action, which permits substoichiometric exposure to achieve the desired pharmacological effects. However, apart from one in vitro study, studies supporting the catalytic mechanism of degraders are largely inferred based on potency. A more comprehensive understanding of the degrader catalytic mechanism of action can help aspects of compound development. To address this knowledge gap, we developed a workflow for the quantitative measurement of the catalytic rate of degraders in cells. Comparing a selective and promiscuous BTK degrader, we demonstrate that both compounds function as efficient catalysts of BTK degradation, with the promiscuous degrader exhibiting faster rates due to its ability to induce more favorable ternary complexes. By leveraging computational modeling, we show that the catalytic rate is highly dynamic as the target is depleted from cells. Further investigation of the promiscuous kinase degrader revealed that the catalytic rate is a better predictor of optimal degrader activity toward a specific target compared to degradation magnitude alone. In summary, we present a versatile method for mapping the catalytic activity of any degrader for TPD in cells.

Targeting Microglial Immunoproteasome: A Novel Approach in Neuroinflammatory-Related Disorders.

It is widely acknowledged that the aging process is linked to the accumulation of damaged and misfolded proteins. This phenomenon is accompanied by a decrease in proteasome (c20S) activity, concomitant with an increase in immunoproteasome (i20S) activity. These changes can be attributed, in part, to the chronic neuroinflammation that occurs in brain tissues. Neuroinflammation is a complex process characterized by the activation of immune cells in the central nervous system (CNS) in response to injury, infection, and other pathological stimuli. In certain cases, this immune response becomes chronic, contributing to the pathogenesis of various neurological disorders, including chronic pain, Alzheimer's disease, Parkinson's disease, brain traumatic injury, and others. Microglia, the resident immune cells in the brain, play a crucial role in the neuroinflammatory response. Recent research has highlighted the involvement of i20S in promoting neuroinflammation, increased activity of which may lead to the presentation of self-antigens, triggering an autoimmune response against the CNS, exacerbating inflammation, and contributing to neurodegeneration. Furthermore, since i20S plays a role in breaking down accumulated proteins during inflammation within the cell body, any disruption in its activity could lead to a prolonged state of inflammation and subsequent cell death. Given the pivotal role of i20S in neuroinflammation, targeting this proteasome subtype has emerged as a potential therapeutic approach for managing neuroinflammatory diseases. This review delves into the mechanisms of neuroinflammation and microglia activation, exploring the potential of i20S inhibitors as a promising therapeutic strategy for managing neuroinflammatory disorders.

The Degradation of Botulinum Neurotoxin Light Chains Using PROTACs.

Botulinum neurotoxins are some of the most potent natural toxins known; they cause flaccid paralysis by inhibiting synaptic vesicle release. Some serotypes, notably serotype A and B, can cause persistent paralysis lasting for several months. Because of their potency and persistence, botulinum neurotoxins are now used to manage several clinical conditions, and there is interest in expanding their clinical applications using engineered toxins with novel substrate specificities. It will also be beneficial to engineer toxins with tunable persistence. We have investigated the potential use of small-molecule proteolysis-targeting chimeras (PROTACs) to vary the persistence of modified recombinant botulinum neurotoxins. We also describe a complementary approach that has potential relevance for botulism treatment. This second approach uses a camelid heavy chain antibody directed against botulinum neurotoxin that is modified to bind the PROTAC. These strategies provide proof of principle for the use of two different approaches to fine tune the persistence of botulinum neurotoxins by selectively targeting their catalytic light chains for proteasomal degradation.

Ubiquitin: Not just a one-way ticket to the proteasome, but a therapeutic dial to fine-tune the molecular landscape of disease.

Recently, there is a rise in studies that recognize the importance of targeting ubiquitin and related molecular machinery in various therapeutic contexts. Here we briefly discuss the history of ubiquitin, its biological roles in protein degradation and beyond, as well as the current state of ubiquitin-targeting therapeutics across diseases. We conclude that targeting ubiquitin machinery is approaching a renaissance, and tapping its full potential will require embracing a wholistic perspective of ubiquitin's multifaceted roles.

Cellular turnover and degradation of the most common missense cystathionine beta-synthase variants causing homocystinuria.

Homocystinuria (HCU) due to cystathionine beta-synthase (CBS) deficiency is the most common inborn error of sulfur amino acid metabolism. Recent work suggests that missense pathogenic mutations-regardless of their topology-cause instability of the C-terminal regulatory domain, which likely translates into CBS misfolding, impaired assembly, and loss of function. However, it is unknown how instability of the regulatory domain translates into cellular CBS turnover and which degradation pathways are involved in CBS proteostasis. Here, we developed a human HEK293-based cellular model lacking intrinsic CBS and stably overexpressing wild-type (WT) CBS or its 10 most common missense HCU mutants. We found that HCU mutants, except the I278T variant, expressed similarly or better than CBS WT, with some of them showing impaired oligomerization, activity and response to allosteric activator S-adenosylmethionine. Cellular stability of all HCU mutants, except P49L and A114V, was significantly lower than the stability of CBS WT, suggesting their increased degradation. Ubiquitination analysis of CBS WT and two representative CBS mutants (T191M and I278T) showed that proteasomal degradation is the major pathway for CBS disposal, with a minor involvement of lysosomal-autophagic and endoplasmic reticulum-associated degradation (ERAD) pathways for HCU mutants. Proteasomal inhibition significantly increased the half-life and activity of T191M and I278T CBS mutants. Lysosomal and ERAD inhibition had only a minor impact on CBS turnover, but ERAD inhibition rescued the activity of T191M and I278T CBS mutants similarly as proteasomal inhibition. In conclusion, the present study provides new insights into proteostasis of CBS in HCU.

Mutations in nucleotide metabolism genes bypass proteasome defects in png-1/NGLY1-deficient Caenorhabditis elegans.

The conserved SKN-1A/Nrf1 transcription factor regulates the expression of proteasome subunit genes and is essential for maintenance of adequate proteasome function in animal development, aging, and stress responses. Unusual among transcription factors, SKN-1A/Nrf1 is a glycoprotein synthesized in the endoplasmic reticulum (ER). N-glycosylated SKN-1A/Nrf1 exits the ER and is deglycosylated in the cytosol by the PNG-1/NGLY1 peptide:N-glycanase. Deglycosylation edits the protein sequence of SKN-1A/Nrf1 by converting N-glycosylated asparagine residues to aspartate, which is necessary for SKN-1A/Nrf1 transcriptional activation of proteasome subunit genes. Homozygous loss-of-function mutations in the peptide:N-glycanase (NGLY1) gene cause NGLY1 deficiency, a congenital disorder of deglycosylation. There are no effective treatments for NGLY1 deficiency. Since SKN-1A/Nrf1 is a major client of NGLY1, the resulting proteasome deficit contributes to NGLY1 disease. We sought to identify targets for mitigation of proteasome dysfunction in NGLY1 deficiency that might indicate new avenues for treatment. We isolated mutations that suppress the sensitivity to proteasome inhibitors caused by inactivation of the NGLY1 ortholog PNG-1 in Caenorhabditis elegans. We identified multiple suppressor mutations affecting 3 conserved genes: rsks-1, tald-1, and ent-4. We show that the suppressors act through a SKN-1/Nrf-independent mechanism and confer proteostasis benefits consistent with amelioration of proteasome dysfunction. ent-4 encodes an intestinal nucleoside/nucleotide transporter, and we show that restriction of nucleotide availability is beneficial, whereas a nucleotide-rich diet exacerbates proteasome dysfunction in PNG-1/NGLY1-deficient C. elegans. Our findings suggest that dietary or pharmacological interventions altering nucleotide availability have the potential to mitigate proteasome insufficiency in NGLY1 deficiency and other diseases associated with proteasome dysfunction.

Structural modification of the propyl linker of cjoc42 in combination with sulfonate ester and triazole replacements for enhanced gankyrin binding and anti-proliferative activity.

Liver cancer is a complex disease that involves various oncoproteins and the inactivation of tumor suppressor proteins (TSPs). Gankyrin is one such oncoprotein, first identified in human hepatocellular carcinoma, that is known to inactivate multiple TSPs, leading to proliferation and metastasis of tumor cells. Despite this, there has been limited development of small molecule gankyrin binders for the treatment of liver cancer. In this study, we are reporting the structure-based design of gankyrin-binding small molecules which inhibit the proliferation of HuH6 and HepG2 cells while also increasing the levels of certain TSPs, such as Rb and p53. Interestingly the first molecule to exhibit inhibition by 3D structure stabilization is seen. These results suggest a possible mechanism for small-molecule inhibition of gankyrin and demonstrate that gankyrin is a viable therapeutic target for the treatment of liver cancer.

DNA break induces rapid transcription repression mediated by proteasome-dependent RNAPII removal.

A DNA double-strand break (DSB) jeopardizes genome integrity and endangers cell viability. Actively transcribed genes are particularly detrimental if broken and need to be repressed. However, it remains elusive how fast the repression is initiated and how far it influences the neighboring genes on the chromosome. We adopt a recently developed, very fast CRISPR to generate a DSB at a specific genomic locus with precise timing, visualize transcription in live cells, and measure the RNA polymerase II (RNAPII) occupancy near the broken site. We observe that a single DSB represses the transcription of the damaged gene in minutes, which coincides with the recruitment of a damage repair protein. Transcription repression propagates bi-directionally along the chromosome from the DSB for hundreds of kilobases, and proteasome is evoked to remove RNAPII in this process. Our method builds a foundation to measure the rapid kinetic events around a single DSB and elucidate the molecular mechanism.

Structure and Functions of HMGB3 Protein.

HMGB3 protein belongs to the group of HMGB proteins from the superfamily of nuclear proteins with high electrophoretic mobility. HMGB proteins play an active part in almost all cellular processes associated with DNA-repair, replication, recombination, and transcription-and, additionally, can act as cytokines during infectious processes, inflammatory responses, and injuries. Although the structure and functions of HMGB1 and HMGB2 proteins have been intensively studied for decades, very little attention has been paid to HMGB3 until recently. In this review, we summarize the currently available data on the molecular structure, post-translational modifications, and biological functions of HMGB3, as well as the possible role of the ubiquitin-proteasome system-dependent HMGB3 degradation in tumor development.

Gastrodin Improves the Activity of the Ubiquitin-Proteasome System and the Autophagy-Lysosome Pathway to Degrade Mutant Huntingtin.

Gastrodin (GAS) is the main chemical component of the traditional Chinese herb Gastrodia elata (called "Tianma" in Chinese), which has been used to treat neurological conditions, including headaches, epilepsy, stroke, and memory loss. To our knowledge, it is unclear whether GAS has a therapeutic effect on Huntington's disease (HD). In the present study, we evaluated the effect of GAS on the degradation of mutant huntingtin protein (mHtt) by using PC12 cells transfected with N-terminal mHtt Q74. We found that 0.1-100 µM GAS had no effect on the survival rate of Q23 and Q74 PC12 cells after 24-48 h of incubation. The ubiquitin-proteasome system (UPS) is the main system that clears misfolded proteins in eukaryotic cells. Mutated Htt significantly upregulated total ubiquitinated protein (Ub) expression, decreased chymotrypsin-like, trypsin-like and caspase-like peptidase activity, and reduced the colocalization of the 20S proteasome with mHtt. GAS (25 µM) attenuated all of the abovementioned pathological changes, and the regulatory effect of GAS on mHtt was found to be abolished by MG132, a proteasome inhibitor. The autophagy-lysosome pathway (ALP) is another system for misfolded protein degradation. Although GAS downregulated the expression of autophagy markers (LC3II and P62), it increased the colocalization of LC3II with lysosomal associated membrane protein 1 (LAMP1), which indicates that ALP was activated. Moreover, GAS prevented mHtt-induced neuronal damage in PC12 cells. GAS has a selective effect on mHtt in Q74 PC12 cells and has no effect on Q23 and proteins encoded by other genes containing long CAGs, such as Rbm33 (10 CAG repeats) and Hcn1 (>30 CAG repeats). Furthermore, oral administration of 100 mg/kg GAS increased grip strength and attenuated mHtt aggregates in B6-hHTT130-N transgenic mice. This is a high dose (100 mg/kg GAS) when compared with experiments on HD mice with other small molecules. We will design more doses to evaluate the dose-response relationship of the inhibition effect of GAS on mHtt in our next study. In summary, GAS can promote the degradation of mHtt by activating the UPS and ALP, making it a potential therapeutic agent for HD.

PARK2 suppresses the proliferation of high-grade serous ovarian carcinoma via inducing the proteasomal degradation of ZNF703.

High-grade serous ovarian cancer (HGSC) is an aggressive disease with poor prognosis. The oncoprotein ZNF703 is implicated in driving HGSC pathogenesis, but factors regulating its abundance remain unclear. In this study, we aim to investigate the potential connection between ZNF703 dysregulation and ubiquitin-mediated protein degradation in HGSC. Bioinformatics prediction was performed using BioGRID database. HGSC representative cell lines were utilized for in vitro and in vivo studies. Results showed that ZNF703 protein was stabilized upon proteasome inhibition, suggesting a regulation via ubiquitination. The ubiquitin E3 ligase PARK2 was found to interact with ZNF703 in a dose-dependent manner, promoting its polyubiquitination and subsequent proteasomal degradation. Re-expression of PARK2 in HGSC cells led to reduced ZNF703 levels together with decreased Cyclin D1/E1 abundance and G1 cell cycle arrest. ZNF703 overexpression alone increased S phase cells, Cyclin D1/E1 levels, and xenograft tumor growth, while co-expression with PARK2 mitigated these oncogenic effects. Collectively, our findings identify ZNF703 as a bona fide substrate of PARK2, reveal a tumor suppressive function for PARK2 in attenuating ZNF703-mediated G1/S transition and HGSC growth through instigating its degradation. This study elucidates a pivotal PARK2-ZNF703 axis with therapeutic implications for targeted intervention in HGSC.