Proteasome Activity Research Articles

Disrupted Nitric Oxide Homeostasis Impacts Fertility through Multiple Processes Including Protein Quality Control.

Plant fertility is fundamental to plant survival and requires the coordinated interaction of developmental pathways and signaling molecules. Nitric oxide (NO) is a small, gaseous signaling molecule that plays crucial roles in plant fertility as well as other developmental processes and stress responses. NO influences biological processes through S-nitrosation, the posttranslational modification of protein cysteines to S-nitrosocysteine (R-SNO). NO homeostasis is controlled by S-nitrosoglutathione reductase (GSNOR), which reduces S-nitrosoglutathione (GSNO), the major form of NO in cells. GSNOR mutants (hot5-2/gsnor1) have defects in female gametophyte development along with elevated levels of reactive nitrogen species and R-SNOs. To better understand the fertility defects in hot5-2, we investigated the in vivo nitrosoproteome of Arabidopsis (Arabidopsis thaliana) floral tissues coupled with quantitative proteomics of pistils. To identify protein-SNOs, we used an organomercury-based method that involves direct reaction with S-nitrosocysteine, enabling specific identification of S-nitrosocysteine-containing peptides and S-nitrosated proteins. We identified 1102 endogenously S-nitrosated proteins in floral tissues, of which 1049 were unique to hot5-2. Among the identified proteins, 728 were novel S-nitrosation targets. Notably, specific UDP-glycosyltransferases and argonaute proteins are S-nitrosated in floral tissues and differentially regulated in pistils. We also discovered S-nitrosation of subunits of the 26S proteasome together with increased abundance of proteasomal components and enhanced trypsin-like proteasomal activity in hot5-2 pistils. Our data establish a method for nitrosoprotein detection in plants, expand knowledge of the plant S-nitrosoproteome, and suggest that nitro-oxidative modification and NO homeostasis are critical to protein quality control in reproductive tissues.

Plasma Proteome Alterations of Laying Hens Subjected to Heat Stress and Fed a Diet Supplemented with Pequi Oil (Caryocar brasiliense Camb.): New Insights in the Identification of Heat Stress Biomarkers

Heat stress can disrupt the balance between the heat poultry release into the environment and the heat they generate. Pequi oil has antioxidant properties, which may mitigate the heat stress effects. This study aimed to investigate the response of laying hens to pequi oil supplementation under heat stress using a proteomic approach. A total of 96 Lohmann White laying hens with 26 weeks old were housed in a completely randomized design with a 2 × 2 factorial arrangement. They were housed in two climate chambers, thermal comfort temperature ± 24.04 °C with the relative humidity ± 66.35 and heat stress (HS) ± 31.26 °C with the relative humidity ± 60.62. They were fed two diets: a control diet (CON), basal diet (BD) without additives, and with Pequi oil (PO), BD + 0.6% PO. After 84 days, plasma samples were analyzed using Shotgun and LC-MS/MS. Proteins related to anti-inflammation, transport, and the immune system were differentially expressed in hens fed PO and CON under heat stress compared to those in thermoneutral environments. This helps protect against oxidative stress and may support the body’s ability to manage heat-induced damage, stabilizing protein expression under stress conditions. The ovotransferrin proteins, fibrinogen isoforms, apolipoprotein A-I, Proteasome activator subunit 4, Transthyretin, and the enzyme serine Peptidase Inhibitor_Kazal Type 5, which presented Upregulated (Up) equal to 1, present characteristics that may be crucial for enhancing the adaptive responses of hens to thermal stress, thereby increasing their tolerance and minimizing the negative effects of heat on egg production. The data presented in this manuscript provides new insights into the plasma proteome alterations of laying hens fed a diet supplemented with pequi oil during heat stress challenges.

Augmenting Protein Degradation Capacity of PROTAC through Energy Metabolism Regulation and Targeted Drug Delivery.

The ubiquitin-proteasome system (UPS) is responsible for degrading over 70-80% of cellular proteins. Consequently, proteolysis-targeting chimeras (PROTACs) are developed to induce the ubiquitination and subsequent degradation of proteins of interest (POIs) by the UPS. To amplify the therapeutic efficacy of PROTACs, energy metabolism regulation is first harnessed to boost UPS function in tumor cells. Proteomic and ubiquitinome analyzes reveal that total ubiquitinated proteins and proteasome activity are significantly increased in 143B and MDA-MB-231 tumor cells following fasting-mimicking diet (FMD) treatment. As a result, the degradation efficiency of PROTACs targeting focal adhesion kinase (FAK-P) or bromodomain-containing protein 4 (BRD4-P) is significantly enhanced in FMD-treated 143B and MDA-MB-231 tumor cells. Then, silica-coated iron oxide nanoparticles are developed modified with tumor cell membranes for targeted delivery of PROTACs. Magnetic resonance imaging (MRI) and fluorescence imaging confirm that nanocarriers significantly improve the delivery efficiency of PROTACs in FMD-treated 143B or MDA-MB-231 tumors. In vivo studies demonstrate that the antitumor efficacy of FAK-P and BRD4-P is greatly augmented when combined with targeted delivery and FMD treatment. Overall, this study presents a strategy to enhance the efficacy of PROTACs in cancer therapy.

Azobenzene-Tagged Photopeptides Exhibiting Excellent Selectivity and Light-Induced Cytotoxicity in MCF-7 Cells over HeLa and A549.

The precise regulation of proteasome activity has become a focal point in current research, particularly its implications in cancer treatment. Bortezomib is used for treating multiple myeloma and is found to be ineffective against solid tumors. A spatiotemporal control over the proteasome is one of the solutions to resolve these issues using external stimuli, such as light. Thus, we designed and synthesized azobenzene-containing tripeptide vinyl sulfones 1, 2, 3, and 4, as the azobenzene moiety can impart E↔Z isomerism upon exposure to UV light. Further, the hydrophobicity of these peptides was fine-tuned by systematically varying the size of hydrophobic amino acids at the P1, P2, and P3 positions. The light-induced Z isomers of these photopeptides showed excellent cellular potency in HeLa, MCF-7, and A549 cell lines. Photopeptide 4 with valine at the proximal position, phenylalanine at P2, and leucine at the P1 positions exhibited 19.3- and 6.6-fold cellular potency in MCF-7 and A549 cells, respectively.

Osteoprotegerin secretion and its inhibition by RANKL in osteoblastic cells visualized using bioluminescence imaging

Bone remodeling is regulated by the interaction between receptor activator of nuclear factor kappa-B ligand (RANKL) and its receptor RANK on osteoblasts and osteoclasts, respectively. Osteoprotegerin (OPG) is secreted from osteoblasts and inhibits osteoclast differentiation by acting as a decoy receptor for RANKL. Despite its importance, the mechanism underlying the secretion of OPG remains poorly understood. Here, we applied a method of video-rate bioluminescence imaging using a fusion protein with Gaussia luciferase (GLase) and visualized the secretion of OPG from living mouse osteoblastic MC3T3-E1 cells. The bioluminescence imaging revealed that the secretion of OPG fused to GLase (OPG-GLase) occurred frequently and widely across the cell surface. Notably, co-expression of RANKL significantly reduced the secretion of OPG-GLase, indicating an inhibitory role of RANKL on OPG secretion within cells. Further imaging and biochemical analyses using deletion mutants of OPG and RANKL, as well as RANKL mutants that cause autosomal recessive osteopetrosis, demonstrated the essential role of protein-protein interaction between OPG and RANKL in the inhibition of OPG secretion. Treatment with proteasome inhibitors resulted in increased levels of OPG in both culture medium and cell lysates. However, the fold-increase of OPG was similar regardless of the presence or absence of RANKL, suggesting that the regulation of OPG secretion by RANKL is independent of proteasome activity. This report visualized the secretion of OPG from living cells and provided evidence for a novel intracellular inhibitory effect of RANKL on OPG secretion.

Manganese inhibits HBV transcription and promotes HBsAg degradation at non-toxic levels

Chronic hepatitis B virus (HBV) infection continues to pose a significant global health challenge. However, therapeutic measures for a cure are lacking in clinical practice. Manganese, an essential trace element, has garnered attention due to its potential to activate innate immune pathways and its significant role in antiviral and antitumor immunity. Yet, the specific impact of manganese on chronic hepatitis B has been largely unexplored. Our research reveals that manganese substantially inhibits HBV replication in hepatocellular carcinoma cells at non-toxic levels. This suppression occurs independently of well-known anti-HBV innate immune pathways, such as the cGAS-STING pathway. Mechanistically, manganese decreases HBV transcription by diminishing the levels of liver-specific transcription factors. Furthermore, it activates the mTOR pathway, enhancing HBsAg ubiquitination through the upregulation of the ubiquitin ligase β-TrCP and increasing proteasome activity via the augmentation of its subunits, leading to a ubiquitin-dependent degradation of HBsAg. Significantly, our study also uncovers a notable clinical correlation between manganese levels and chronic hepatitis B infection. These findings position manganese as a critical element in diminishing HBV replication, offering a new direction in the management of chronic hepatitis B.

Lymphocyte Antigen 6 Family Member D (LY6D) Affects Stem Cell Phenotype and Progression of Pancreatic Adenocarcinoma.

The membrane-bound protein lymphocyte antigen 6 family member D (LY6D), a marker of early B cell lineage is reportedly expressed in several human malignancies and has been implicated in cancer stemness. However, its expression and role in cancer stemness remain largely unexplored in pancreatic ductal adenocarcinoma (PDAC). The aim of this study was to clarify the role of LY6D in PDAC. We conducted functional analysis of LY6D to evaluate its impact on the malignant features of PDAC cells in vitro. Using our in-house developed stem cell separation technique, which isolates cells with low proteasome activity and CD44 v9 cell surface marker for cancer stem cells, we performed sphere formation and chemosensitivity tests and tumor formation assay in mice, through knockdown of LY6D expression. Immuno-histopathological analysis was also conducted to reveal the clinical significance of LY6D in PDAC. In vitro functional assays demonstrated that LY6D was critically involved in promoting the cancer malignant phenotype, including increased invasive ability, drug resistance, migration capacity, and cancer stemness. Immunohistopathological analysis revealed that high LY6D expression levels were associated with high recurrence rates and poorer prognosis in PDAC. Our study showed that LY6D is a novel prognostic indicator and plays a key role in regulation of cancer stemness in PDAC.

Functional 20S Proteasomes in Retroviruses: Evidence in Favor.

Proteasomes are barrel-like cellular protein complexes responsible for the degradation of most intracellular proteins. Earlier, it has been shown that during assembly, hundreds of different cellular proteins are incorporated into retro-and herpes viruses. Among detected cellular proteins, there were different proteasome subunits (PS). Previous reports postulated the incorporation of 20S proteasome subunits and subunits of proteasome regulator complexes inside retroviruses. Here, we demonstrated the association of functional 20S proteasome with gammaretroviruses, betaretroviruses, and lentiviruses. Cleaved proteasome subunits β1, β2 and β5 were detected in tested viruses. Using fluorescent peptides and a cell-permeable proteasome activity probe, proteasome activity was detected in endogenous and exogenous retroviruses, including recombinant HIV-1. Taken together, our data favors the insertion of functional proteasomes into the retroviruses during assembly. The possible role of proteasomes in retroviruses is discussed.

Exploration of novel 20S proteasome activators featuring anthraquinone structures and their application in hypoxic cardiomyocyte protection

Under hypoxic conditions, the accumulation of misfolded proteins primarily relies on the autonomous activity of 20S proteasome for degradation. The buildup of toxic proteins in cardiomyocyte contribute to various cardiovascular diseases. Therefore, enhancing the 20S proteasome degradation capacity and restoring protein homeostasis in myocardial cells with small molecule activators represent a promising therapeutic strategy for the treatment of ischemic cardiomyopathy. In this study, the lead compound 8016–8398 was identified through virtual screening, and subsequent structure optimization resulted in a series of highly potent 20S proteasome activators. Intracellular protein degradation assessment revealed that these compounds possessed abilities to alleviate endoplasmic reticulum stress, as demonstrated by the luciferase reporter system. Additionally, selected compound B-03 significantly enhanced the survival rate of hypoxic-damaged cardiomyocytes. Mechanistic investigations verified B-03 rescued hypoxic damaged cardiomyocyte through apoptosis inhibition and proliferation promotion.

Structural Analysis of the 20S Proteasome Using Native Mass Spectrometry and Ultraviolet Photodissociation.

Owing to the role of the 20S proteasome in a wide spectrum of pathologies, including neurodegenerative disorders, proteasome-associated autoinflammatory syndromes (PRAAS), and cardiovascular diseases, understanding how its structure and composition contribute to dysfunction is crucial. As a 735 kDa protein assembly, the 20S proteasome facilitates normal cellular proteostasis by degrading oxidized and misfolded proteins. Declined proteasomal activity, which can be attributed to perturbations in the structural integrity of the 20S proteasome, is considered one of the main contributors to multiple proteasome-related diseases. Devising methods to characterize the structures of 20S proteasomes provides necessary insight for the development of drugs and inhibitors that restore proper proteasomal function. Here, native mass spectrometry was combined with multiple dissociation techniques, including ultraviolet photodissociation (UVPD), to identify the protein subunits comprising the 20S proteasome. UVPD, demonstrating an ability to uncover structural features of large (>300 kDa) macromolecular complexes, provided complementary information to conventional collision-based methods. Additionally, variable-temperature electrospray ionization was combined with UV photoactivation to study the influence of solution temperature on the stability of the 20S proteasome.

β-Amyloid impairs Proteasome structure and function. Proteasome activation mitigates amyloid induced toxicity and cognitive deficits.

Alzheimer's Disease (AD) is the leading cause of dementia globally, affecting around 50 million people and marked by cognitive decline and the accumulation of β-amyloid plaques and hyperphosphorylated tau. The limited treatment options and numerous failed clinical trials targeting β-amyloid (Aβ) highlight the need for novel approaches. Lowered proteasome activity is a consistent feature in AD, particularly in the hippocampus. Impaired proteasome function in AD is hypothesized to stem from direct inhibition by β-amyloid or hyperphosphorylated tau, disrupting critical neuronal processes such as memory formation and synaptic plasticity. This study tests the hypothesis that AD related deficits are driven in part by impaired proteasome function as a consequence of inhibition by Aβ. We evaluated how proteasome function is modulated by Aβ and the capacity of two proteasome-activating compounds, TAT1-8,9-TOD and TAT1-DEN to rescue Aβ-induced impairment in vitro, as well as survival deficits in cell culture and Aβ-induced cognitive deficits in Drosophila and mouse models. Our study demonstrates that oligomeric β-amyloid binds to the 20S proteasome and impairs its activity and conformational stability. The oligomers also destabilize the 26S proteasome to release the free 20S proteasome. Treatment with proteasome activators TAT1-8,9TOD and TAT1-DEN rescue the 20S proteasome function and reduces cell death caused by Aβ42 toxicity in SK-N-SH cells. In Drosophila models overexpressing Aβ42, oral administration of proteasome agonists delayed mortality and restored cognitive function. Chronic treatment with TAT1-DEN protected against deficits in working memory caused by Aβ42 in mice and in hAPP(J20) mice with established deficits, acute TAT1-DEN treatment significantly improved spatial learning, with treated mice performing comparably to controls. Aβ has dual impacts on 20S and 26S proteasome function and stability. Proteasome activation using TAT1-8,9TOD and TAT1-DEN shows promise in mitigating AD-like deficits by protecting against amyloid toxicity and enhancing proteasome function. These findings suggest that targeting proteasome activity could be a viable therapeutic approach for AD, warranting further investigation into the broader impacts of proteasome modulation on AD pathology.

Delineating cysteine-reactive compound modulation of cellular proteostasis processes.

Covalent modulators and covalent degrader molecules have emerged as drug modalities with tremendous therapeutic potential. Toward realizing this potential, mass spectrometry-based chemoproteomic screens have generated proteome-wide maps of potential druggable cysteine residues. However, beyond these direct cysteine-target maps, the full scope of direct and indirect activities of these molecules on cellular processes and how such activities contribute to reported modes of action, such as degrader activity, remains to be fully understood. Using chemoproteomics, we identified a cysteine-reactive small molecule degrader of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nonstructural protein 14 (nsp14), which effects degradation through direct modification of cysteines in both nsp14 and in host protein disulfide isomerases. This degrader activity was further potentiated by generalized electrophile-induced global protein ubiquitylation, proteasome activation and widespread aggregation and depletion of host proteins, including the formation of stress granules. Collectively, we delineate the wide-ranging impacts of cysteine-reactive electrophilic compounds on cellular proteostasis processes.

The effective multiplicity of infection for HCMV depends on activity of the cellular 20S proteasome.

HCMV infection and reactivation continues to contribute to morbidity and mortality around the world. Antiviral compounds are available but have limitations. Here, we have defined the impact of the proteasome inhibitor bortezomib on HCMV replication. Proteasomal activities play a critical role in temporal changes required for replication. We demonstrate that disrupting these activities inhibits viral replication while likely supporting increased antiviral activity of the anti-HCMV agent, maribavir. Using a combination of live cell imaging and computational tools, we discover that a subset of infected cells progresses to fulminant infection which we define as the effective MOI, and this subset would otherwise be missed when analyzing the average of the population.

Enhanced cell stress response and protein degradation capacity underlie artemisinin resistance in Plasmodium falciparum.

Malaria remains a global health burden, killing over half a million people each year. Decreased therapeutic efficacy to artemisinin, the most efficacious antimalarial, has been detected in sub-Saharan Africa, a worrying fact given that over 90% of deaths occur on this continent. Mutations in Kelch13 are the most well-established molecular marker for artemisinin resistance, but these do not explain all artemisinin-resistant isolates. Understanding the biological underpinnings of drug resistance is key to curbing the emergence and spread of artemisinin resistance. Artemisinin-mediated non-specific alkylation leads to the accumulation of misfolded and damaged proteins and activation of the parasite unfolded protein response (UPR). In addition, the parasite proteasome is vital to artemisinin resistance, as we have previously shown that chemical inhibition of the proteasome or mutations in the β2 proteasome subunit increase parasite susceptibility to dihydroartemisinin (DHA), the active metabolite of artemisinins. Here, we investigate parasites with mutations at the Kelch13 and/or 19S and 20S proteasome subunits with regard to UPR regulation and proteasome activity in the context of artemisinin resistance. Our data show that perturbing parasite proteostasis kills parasites, early parasite UPR signaling dictates DHA survival outcomes, and DHA susceptibility correlates with impairment of proteasome-mediated protein degradation. Importantly, we show that functional proteasomes are required for artemisinin resistance in a Kelch13-independent manner, and compound-selective proteasome inhibition demonstrates why artemisinin-resistant Kelch13 mutants remain susceptible to the related antimalarial peroxide OZ439. These data provide further evidence for targeting the parasite proteasome and UPR to overcome existing artemisinin resistance.IMPORTANCEDecreased therapeutic efficacy represents a major barrier to malaria treatment control strategies. The malaria proteasome and accompanying unfolded protein response are crucial to artemisinin resistance, revealing novel antimalarial therapeutic strategies.

Comparative Study of Cutaneous Squamous Cell Carcinogenesis in Different Hairless Murine Models

Background: In recent decades, a significant global increase in the incidence of non-melanoma skin cancer has been observed. To explore the pathogenesis of and potential therapeutic approaches for squamous cell carcinoma, various in vivo studies using mouse models have been conducted. However, investigations comparing different hairless mouse models, with or without melanin, as well as models with hypercholesterolemia and immunosuppression, in terms of their ability to induce squamous cell carcinoma have yet to be undertaken. Methods: Four mouse strains, namely SKH-hr1, SKH-hr2, SKH-hr2+ApoE, and immunodeficient Nude (Foxn1 knockout), were exposed to UVA and UVB radiation three times per week, initially to 1 Minimal Erythemal Dose (MED), incrementally increased weekly to a maximum dose of 3 MED. Clinical evaluation, photodocumentation, and biophysical parameters were monitored, along with proteasome protein activity and histopathological assessments. Results: The SKH-hr1 model primarily developed actinic keratosis without significant progression to invasive squamous cell carcinoma (SCC), while the SKH-hr2 and SKH-hr2+ApoE models exhibited a higher likelihood and intensity of papilloma and aggressive SCC formation, with the latter showing upregulated proteasome activity. Histopathological analysis confirmed the presence of poorly differentiated, invasive SCCs in the SKH-hr2 and SKH-hr2+ApoE models, contrasting with the less aggressive SCCs in the Nude mice and the mixed lesions observed in the SKH-hr1 mice. Conclusions: The SKH-hr2+ApoE and SKH-hr2 mice were identified as the most suitable for further exploration of squamous cell carcinogenesis. In contrast, the SKH-hr1 mice were found to be the least suitable, even though they are albino. Notably, proteasome analysis revealed a potential role of proteasome activity in squamous cell carcinogenesis.

Altered Proteasome Composition in Aging Brains, Genetic Proteasome Augmentation Mitigates Age-Related Cognitive Declines, and Acute Proteasome Agonist Treatment Rescues Age-Related Cognitive Deficits in Mice.

The aging brain experiences a significant decline in proteasome function, The proteasome is critical for many key neuronal functions including neuronal plasticity, and memory formation/retention. Treatment with proteasome inhibitors impairs these processes. Our study reveals a marked reduction in 20S and 26S proteasome activities in aged mice brains driven by reduced functionality of aged proteasome. This is matched by a decline in 20S proteasome but an increase in 26S proteasome. Our data suggests this may be a compensatory response to reduced functionality. By overexpressing the proteasome subunit PSMB5 in the neurons of mice, enhancing proteasome function, we slowed age-related declines in spatial learning and memory as well neuromuscular declines. We then showed acute treatment with a proteasome activator to rescue spatial learning and memory deficits in aged mice. These findings highlight the potential of proteasome augmentation as a therapeutic strategy to mitigate age-related cognitive declines.

Localized synthesis of molecular chaperones sustains neuronal proteostasis.

Neurons are challenged to maintain proteostasis in neuronal projections, particularly with the physiological stress at synapses to support intercellular communication underlying important functions such as memory and movement control. Proteostasis is maintained through regulated protein synthesis and degradation and chaperone-assisted protein folding. Using high-resolution fluorescent microscopy, we discovered that neurons localize a subset of chaperone mRNAs to their dendrites, particularly more proximal regions, and increase this asymmetric localization following proteotoxic stress through microtubule-based transport from the soma. The most abundant chaperone mRNA in dendrites encodes the constitutive heat shock protein 70, HSPA8. Proteotoxic stress in cultured neurons, induced by inhibiting proteasome activity or inducing oxidative stress, enhanced transport of Hspa8 mRNAs to dendrites and the percentage of mRNAs engaged in translation on mono and polyribosomes. Knocking down the ALS-related protein Fused in Sarcoma (FUS) and a dominant mutation in the heterogenous nuclear ribonucleoprotein A2/B1 (HNRNPA2B1) impaired stress-mediated localization of Hspa8 mRNA to dendrites in cultured murine motor neurons and human iPSC-derived neurons, respectively, revealing the importance of these RNA-binding proteins in maintaining proteostasis. These results reveal the increased dendritic localization and translation of the constitutive HSP70 Hspa8 mRNA as a crucial neuronal stress response to uphold proteostasis and prevent neurodegeneration.

MiR-326 overexpression inhibits colorectal cancer cell growth and proteasome activity by targeting PNO1: unveiling a novel therapeutic intervention strategy

Proteasome inhibition emerges as a promising strategy for cancer prevention. PNO1, pivotal for colorectal cancer (CRC) progression, is involved in proteasome assembly in Saccharomyces cerevisiae. Hence, we aimed to explore the role of PNO1 in proteasome assembly and its up- and down-streams in CRC. Here, we demonstrated that PNO1 knockdown suppressed CRC cells growth, proteasome activities and assembly, as well as CDKN1B/p27Kip1 (p27) degradation. Moreover, p27 knockdown partially attenuated the inhibition of HCT116 cells growth by PNO1 knockdown. The up-stream studies of PNO1 identified miR-326 as a candidate miRNA directly targeting to CDS-region of PNO1 and its overexpression significantly down-regulated PNO1 protein expression, resulting in suppression of cell growth, decrease of proteasome activities and assembly, as well as increasing the stability of p27 in CRC cells. These findings indicated that miR-326 overexpression can suppress CRC cell growth, acting as an endogenous proteasome inhibitor by targeting PNO1.

Aripiprazole, but Not Olanzapine, Alters the Response to Oxidative Stress in Fao Cells by Reducing the Activation of Mitogen-Activated Protein Kinases (MAPKs) and Promoting Cell Survival.

Prolonged use of atypical antipsychotics (AAPs) is commonly associated with increased cardiovascular disease risk. While weight gain and related health issues are generally considered the primary contributors to this risk, direct interference with mitochondrial bioenergetics, particularly in the liver where these drugs are metabolized, is emerging as an additional contributing factor. Here, we compared the effects of two AAPs with disparate metabolic profiles on the response of Fao hepatoma cells to oxidative stress: olanzapine (OLA), which is obesogenic, and aripiprazole (ARI), which is not. Results showed that cells treated with ARI exhibited resistance to H2O2-induced oxidative stress, while OLA treatment had the opposite effect. Despite enhanced survival, ARI-treated cells exhibited higher apoptotic rates than OLA-treated cells when exposed to H2O2. Gene expression analysis of pro- and anti-apoptotic factors revealed that ARI-treated cells had a generally blunted response to H2O2, contrasting with a heightened response in OLA-treated cells. This was further supported by the reduced activation of MAPKs and STAT3 in ARI-treated cells in response to H2O2, whereas OLA pre-treatment enhanced their activation. The loss of stress response in ARI-treated cells was consistent with the observed increase in the mitochondrial production of O2•-, a known desensitizing factor. The physiological relevance of O2•- in ARI-treated cells was demonstrated by the increase in mitophagy flux, likely related to mitochondrial damage. Notably, OLA treatment protected proteasome activity in Fao cells exposed to H2O2, possibly due to the better preservation of stress signaling and mitochondrial function. In conclusion, this study highlights the underlying changes in cell physiology and mitochondrial function by AAPs. ARI de-sensitizes Fao cells to stress signaling, while OLA has the opposite effect. These findings contribute to our understanding of the metabolic risks associated with prolonged AAP use and may inform future therapeutic strategies.

ProEnd: a comprehensive database for identifying HbYX motif-containing proteins across the tree of life

The proteasome plays a crucial role in cellular homeostasis by degrading misfolded, damaged, or unnecessary proteins. Understanding the regulatory mechanisms of proteasome activity is vital, particularly the interaction with activators containing the hydrophobic-tyrosine-any amino acid (HbYX) motif. Here, we present ProEnd, a comprehensive database designed to identify and catalog HbYX motif-containing proteins across the tree of life. Using a simple bioinformatics pipeline, we analyzed approximately 73 million proteins from 22,000 reference proteomes in the UniProt/SwissProt database. Our findings reveal the widespread presence of HbYX motifs in diverse organisms, highlighting their evolutionary conservation and functional significance. Notably, we observed an interesting prevalence of these motifs in viral proteomes, suggesting strategic interactions with the host proteasome. As validation two novel HbYX proteins found in this database were experimentally tested by pulldowns, confirming that they directly interact with the proteasome, with one of them directly activating it. ProEnd’s extensive dataset and user-friendly interface enable researchers to explore the potential proteasomal regulator landscape, generating new hypotheses to advance proteasome biology. This resource is set to facilitate the discovery of novel therapeutic targets, enhancing our approach to treating diseases such as neurodegenerative disorders and cancer.