Proteostasis Research Articles

Proteostasis perturbation of N-Myc leveraging HSP70 mediated protein turnover improves treatment of neuroendocrine prostate cancer

N-Myc is a key driver of neuroblastoma and neuroendocrine prostate cancer (NEPC). One potential way to circumvent the challenge of undruggable N-Myc is to target the protein homeostasis (proteostasis) system that maintains N-Myc levels. Here, we identify heat shock protein 70 (HSP70) as a top partner of N-Myc, which binds a conserved “SELILKR” motif and prevents the access of E3 ubiquitin ligase, STIP1 homology and U-box containing protein 1 (STUB1), possibly through steric hindrance. When HSP70’s dwell time on N-Myc is increased by treatment with the HSP70 allosteric inhibitor, STUB1 is in close proximity with N-Myc and becomes functional to promote N-Myc ubiquitination on the K416 and K419 sites and forms polyubiquitination chains linked by the K11 and K63 sites. Notably, HSP70 inhibition significantly suppressed NEPC tumor growth, increased the efficacy of aurora kinase A (AURKA) inhibitors, and limited the expression of neuroendocrine-related pathways.

Time-resolved interactome profiling deconvolutes secretory protein quality control dynamics

Many cellular processes are governed by protein–protein interactions that require tight spatial and temporal regulation. Accordingly, it is necessary to understand the dynamics of these interactions to fully comprehend and elucidate cellular processes and pathological disease states. To map de novo protein–protein interactions with time resolution at an organelle-wide scale, we developed a quantitative mass spectrometry method, time-resolved interactome profiling (TRIP). We apply TRIP to elucidate aberrant protein interaction dynamics that lead to the protein misfolding disease congenital hypothyroidism. We deconvolute altered temporal interactions of the thyroid hormone precursor thyroglobulin with pathways implicated in hypothyroidism pathophysiology, such as Hsp70-/90-assisted folding, disulfide/redox processing, and N-glycosylation. Functional siRNA screening identified VCP and TEX264 as key protein degradation components whose inhibition selectively rescues mutant prohormone secretion. Ultimately, our results provide novel insight into the temporal coordination of protein homeostasis, and our TRIP method should find broad applications in investigating protein-folding diseases and cellular processes.

NatB Protects Procaspase-8 from UBR4-Mediated Degradation and Is Required for Full Induction of the Extrinsic Apoptosis Pathway.

N-terminal acetyltransferase B (NatB) is a major contributor to the N-terminal acetylome and is implicated in several key cellular processes including apoptosis and proteostasis. However, the molecular mechanisms linking NatB-mediated N-terminal acetylation to apoptosis and its relationship with protein homeostasis remain elusive. In this study, we generated mouse embryonic fibroblasts (MEFs) with an inactivated catalytic subunit of NatB (Naa20-/-) to investigate the impact of NatB deficiency on apoptosis regulation. Through quantitative N-terminomics, label-free quantification, and targeted proteomics, we demonstrated that NatB does not influence the proteostasis of all its substrates. Instead, our focus on putative NatB-dependent apoptotic factors revealed that NatB serves as a protective shield against UBR4 and UBR1 Arg/N-recognin-mediated degradation. Notably, Naa20-/- MEFs exhibited reduced responsiveness to an extrinsic pro-apoptotic stimulus, a phenotype that was partially reversible upon UBR4 Arg/N-recognin silencing and consequent inhibition of procaspase-8 degradation. Collectively, our results shed light on how the interplay between NatB-mediated acetylation and the Arg/N-degron pathway appears to impact apoptosis regulation, providing new perspectives in the field including in therapeutic interventions.

Neuro-intestinal acetylcholine signalling regulates the mitochondrial stress response in Caenorhabditis elegans

Neurons coordinate inter-tissue protein homeostasis to systemically manage cytotoxic stress. In response to neuronal mitochondrial stress, specific neuronal signals coordinate the systemic mitochondrial unfolded protein response (UPRmt) to promote organismal survival. Yet, whether chemical neurotransmitters are sufficient to control the UPRmt in physiological conditions is not well understood. Here, we show that gamma-aminobutyric acid (GABA) inhibits, and acetylcholine (ACh) promotes the UPRmt in the Caenorhabditis elegans intestine. GABA controls the UPRmt by regulating extra-synaptic ACh release through metabotropic GABAB receptors GBB-1/2. We find that elevated ACh levels in animals that are GABA-deficient or lack ACh-degradative enzymes induce the UPRmt through ACR-11, an intestinal nicotinic α7 receptor. This neuro-intestinal circuit is critical for non-autonomously regulating organismal survival of oxidative stress. These findings establish chemical neurotransmission as a crucial regulatory layer for nervous system control of systemic protein homeostasis and stress responses.

Abstract Mo123: Post-transcriptional Mechanisms of BAG3 Regulation in Ischemia-Reperfusion Injury

The co-chaperone BAG3 is critical for protein quality control at the cardiac sarcomere. BAG3 binds to Hsp70 and coordinates the assembly of the CASA (chaperone-assisted selective autophagy) complex, thus supporting protein homeostasis and cardiomyocyte contractility. Decreased BAG3 levels are associated with heart disease, whereas BAG3 overexpression rescues ventricular function in animal models of heart failure (HF). Despite BAG3’s potential as a therapeutic target, the mechanisms underlying BAG3 regulation are largely unresolved. Here, we investigate the mechanisms of BAG3 downregulation after stress. We found that BAG3 protein is reduced in human cardiomyopathies compared to non-failing hearts, yet there is an increase in bag3 mRNA transcript, suggesting BAG3’s downregulation in heart disease may be controlled post-transcriptionally. To identify these post-transcriptional pathways, we subjected neonatal rat ventricular myocytes (NRVMs) to prolonged hypoxia-reoxygenation (H/R) stress, which recapitulated the decrease in BAG3 levels observed in human heart disease. Notably, disrupting Hsp70 binding to BAG3 in NRVMs via the drug JG-98 decreased BAG3’s half-life by ~90%, suggesting that Hsp70 protects BAG3 from degradation. Loss of Hsp70-mediated protection could contribute to declining BAG3 levels, so we quantified Hsp70 abundance after H/R stress in NRVMs, finding no significant change. We also found that overexpressing inducible Hsp70 did not rescue BAG3 levels. This data suggests that BAG3 downregulation in H/R stress is not caused by loss of Hsp70 binding/protection. To examine BAG3 regulation in vivo, we subjected wildtype mice to ischemia-reperfusion injury. After 24 hours, we observed a decrease in BAG3 levels in the left ventricle and no significant change in Hsp70 abundance. Interestingly, the decline in full-length BAG3 (85 kDa) was accompanied by an increase in a BAG3 cleavage product at 74 kDa. We analyzed this product via mass spectrometry, discovering that it lacks a third of the WW domain, which is involved in autophagy. In future experiments, BAG3 cleavage will be explored as a potential mechanism of BAG3 loss. Such mechanisms will provide insight into how to maintain BAG3 levels, and thus cardiac function, during stress.

Scenedesmus acuminatus as a potential phycoremediator: Enrichment and detoxification of cadmium/lead

Heavy metal pollution in water can cause severe environmental problems, harm the ecosystem, and ultimately threaten human health. The traditional methods used to remove heavy metals from water are expensive, inefficient, and can cause secondary pollution. However, microalgae have potential applications in the removal of toxic heavy metal pollutants from water. In this study, six green algae were screened in terms of their ability to adsorb cadmium (Cd) and lead (Pb) from a liquid medium. Ion content analyses confirmed that Scenedesmus acuminatus had the strongest Cd- and Pb-absorption ability. S. acuminatus was subjected to Cd and Pb treatments, and then transcriptome sequencing analysis was conducted. The results of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses indicated that the “proteasome” pathway was enriched with differentially expressed genes (DEGs) under Cd treatment, and the “photosynthesis” pathway was enriched with DEGs under Pb treatment. These results imply that the proteasome pathway maintained protein homeostasis and cellular activity by degrading damaged proteins under Cd toxicity, and that photosynthesis was affected by Pb toxicity in S. acuminatus. In conclusion, S. acuminatus showed a strong ability to accumulate Cd/Pb and might alleviate heavy metal toxicity and reduce heavy metal efflux using the ubiquitin-proteasome system. The results of this study identify a novel microalgal strain with potential applications in phytoextraction and phytoremediation, and provide information about the molecular mechanisms of Cd/Pb accumulation in this alga. These data provide a theoretical basis for reducing Cd/Pb toxicity and will be useful for devising strategies to protect aquatic animals and plants.

Arginyltransferase 1 (ATE1)-mediated proteasomal degradation of viral haemagglutinin protein: a unique host defence mechanism.

The extensive protein production in virus-infected cells can disrupt protein homeostasis and activate various proteolytic pathways. These pathways utilize post-translational modifications (PTMs) to drive the ubiquitin-mediated proteasomal degradation of surplus proteins. Protein arginylation is the least explored PTM facilitated by arginyltransferase 1 (ATE1) enzyme. Several studies have provided evidence supporting its importance in multiple physiological processes, including ageing, stress, nerve regeneration, actin formation and embryo development. However, its function in viral pathogenesis is still unexplored. The present work utilizes Newcastle disease virus (NDV) as a model to establish the role of the ATE1 enzyme and its activity in pathogenesis. Our data indicate a rise in levels of N-arginylated cellular proteins in the infected cells. Here, we also explore the haemagglutinin-neuraminidase (HN) protein of NDV as a presumable target for arginylation. The data indicate that the administration of Arg amplifies the arginylation process, resulting in reduced stability of the HN protein. ATE1 enzyme activity inhibition and gene expression knockdown studies were also conducted to analyse modulation in HN protein levels, which further substantiated the findings. Moreover, we also observed Arg addition and probable ubiquitin modification to the HN protein, indicating engagement of the proteasomal degradation machinery. Lastly, we concluded that the enhanced levels of the ATE1 enzyme could transfer the Arg residue to the N-terminus of the HN protein, ultimately driving its proteasomal degradation.

Potential use of HSP70 as an indicator of heat stress in dairy cows – a review

Heat stress (HS) poses significant challenges to the dairy industry, resulting in reduced milk production, impaired reproductive performance, and compromised animal welfare. Therefore, understanding the molecular mechanisms underlying cellular responses to HS is crucial for developing effective strategies to mitigate its adverse effects. Heat shock protein 70 (HSP70) has emerged as a potential player involved in cellular thermotolerance in dairy cows. This review provides a comprehensive overview of the role of HSP70 as a molecular chaperone in cellular thermotolerance in dairy cows under HS. HSP70 facilitates proper protein folding and prevents the aggregation of denatured proteins. By binding to misfolded proteins, it helps maintain protein homeostasis and prevents the accumulation of damaged proteins during HS. Additionally, HSP70 interacts with various regulatory proteins and signaling pathways, contributing to the cellular adaptive response to HS. The upregulation of HSP70 expression in response to HS is regulated by a complex network involving heat-shock factors (HSFs), heat-shock element-binding proteins, and HSF co-chaperones. Therefore, HSP70 holds the potential to be a useful indicator of tissue stress due to its role in maintaining cellular balance, and as it is released both inside and outside cells in response to stress. Traditional methods of measuring HSP70 in blood samples are labor-intensive, and with the process being potentially stressful for the animals and may subsequently affect the results. Therefore, measuring HSP expression in cow's milk has shown promise as an easy, non-invasive, and accurate way to detect HS in dairy cows. Monitoring HSP70 levels in milk can be applied as a supplementary approach to identify HS or HS resistance of individual cows, selection of suitable animals and to guide targeted management strategies. However, despite the potential advantages of using HSP70 as a biomarker for monitoring HS on dairy cows, challenges remain in standardizing measurement protocols, establishing species-specific reference ranges, addressing inter-individual variations, and determining the specificity of changes in HSP70 due to HS. Future research should focus on developing non-invasive techniques for HSP70 detection, with consideration of climatic conditions, and unravelling the molecular interactions and regulatory networks involving HSP70.

A three-dimensional valve-on-chip microphysiological system implicates cell cycle progression, cholesterol metabolism and protein homeostasis in early calcific aortic valve disease progression

BackgroundCalcific aortic valve disease (CAVD) is one of the most common forms of valvulopathy, with a 50 % elevated risk of a fatal cardiovascular event, and greater than 15,000 annual deaths in North America alone. The treatment standard is valve replacement as early diagnostic, mitigation, and drug strategies remain underdeveloped. The development of early diagnostic and therapeutic strategies requires the fabrication of effective in vitro valve mimetic models to elucidate early CAVD mechanisms. MethodsIn this study, we developed a multilayered physiologically relevant 3D valve-on-chip (VOC) system that incorporated aortic valve mimetic extracellular matrix (ECM), porcine aortic valve interstitial cell (VIC) and endothelial cell (VEC) co-culture and dynamic mechanical stimuli. Collagen and glycosaminoglycan (GAG) based hydrogels were assembled in a bilayer to mimic healthy or diseased compositions of the native fibrosa and spongiosa. Multiphoton imaging and proteomic analysis of healthy and diseased VOCs were performed. ResultsCollagen-based bilayered hydrogel maintained the phenotype of the VICs. Proteins related to cellular processes like cell cycle progression, cholesterol biosynthesis, and protein homeostasis were found to be significantly altered and correlated with changes in cell metabolism in diseased VOCs. This study suggested that diseased VOCs may represent an early, adaptive disease initiation stage, which was corroborated by human aortic valve proteomic assessment. ConclusionsIn this study, we developed a collagen-based bilayered hydrogel to mimic healthy or diseased compositions of the native fibrosa and spongiosa layers. When the gels were assembled in a VOC with VECs and VICs, the diseased VOCs revealed key insights about the CAVD initiation process. Statement of significanceCalcific aortic valve disease (CAVD) elevates the risk of death due to cardiovascular pathophysiology by 50 %, however, prevention and mitigation strategies are lacking, clinically. Developing tools to assess early disease would significantly aid in the prevention of disease and in the development of therapeutics. Previously, studies have utilized collagen and glycosaminoglycan-based hydrogels for valve cell co-cultures, valve cell co-cultures in dynamic environments, and inorganic polymer-based multilayered hydrogels; however, these approaches have not been combined to make a physiologically relevant model for CAVD studies. We fabricated a bi-layered hydrogel that closely mimics the aortic valve and used it for valve cell co-culture in a dynamic platform to gain mechanistic insights into the CAVD initiation process using proteomic and multiphoton imaging assessment.

Deciphering the Intracellular Action of the Antimicrobial Peptide A11 via an In-Depth Analysis of Its Effect on the Global Proteome of Acinetobacter baumannii.

The potential antimicrobial activity and low propensity to induce the development of bacterial resistance have rendered antimicrobial peptides (AMPs) as novel and ideal candidate therapeutic agents for the treatment of infections caused by drug-resistant pathogenic bacteria. The targeting of bacterial membranes by AMPs has been typically considered their sole mode of action; however, increasing evidence supports the existence of multiple and complementary functions of AMPs that result in bacterial death. An in-depth characterization of their mechanism of action could facilitate further research and development of AMPs with higher potency. The current study employs biophysics and proteomics approaches to unveil the mechanisms underlying the antibacterial activity of A11, a potential candidate AMP, against Acinetobacter baumannii, a leading cause of hospital-acquired infections (HAIs) and consequently, a serious global threat. A11 peptide was found to induce membrane depolarization to a high extent, as revealed by flow cytometry and electron microscopy analyses. The prompt intracellular penetration of A11 peptide, observed using confocal microscopy, was found to occur concomitantly with a very low degree of membrane lysis, suggesting that its mode of action predominantly involves a nonlytic killing mechanism. Quantitative proteomics analysis employed for obtaining insights into the mechanisms underlying the antimicrobial activity of A11 peptide revealed that it disrupted energy metabolism, interfered with protein homeostasis, and inhibited fatty acid synthesis that is essential for cell membrane integrity; all these impacted the cellular functions of A. baumannii. A11 treatment also impacted signal transduction associated with the regulation of biofilm formation, hindered the stress response, and influenced DNA repair processes; these are all crucial survival mechanisms of A. baumannii. Additionally, robust antibacterial activity was exhibited by A11 peptide against multidrug-resistant (MDR) and extensively drug-resistant (XDR) clinical isolates of A. baumannii; moreover, A11 peptide exhibited synergy with levofloxacin and minocycline as well as low propensity for inducing resistance. Taken together, the findings emphasize the therapeutic potential of A11 peptide as an antibacterial agent against drug-resistant A. baumannii and underscore the need for further investigation.

In vivo RNAi screening identifies multiple deubiquitinases required for the maintenance of intestinal homeostasis in Drosophila

Deubiquitinases (DUBs) are essential for the maintenance of protein homeostasis and assembly of proteins into functional complexes. Despite growing interest in DUBs biological functions, the roles of DUBs in regulating intestinal stem cells (ISCs) and gut homeostasis remain largely unknown. Here, we perform an in vivo RNAi screen through induced knock-down of DUBs expression in adult midgut ISCs and enteroblasts (EBs) to identify DUB regulators of intestinal homeostasis in Drosophila. We screen 43 DUBs and identify 8 DUBs that are required for ISCs homeostasis. Knocking-down of usp1, CG7857, usp5, rpn8, usp10 and csn5 decreases the number of ISCs/EBs, while knocking-down of CG4968 and usp8 increases the number of ISCs/EBs. Moreover, knock-down of usp1, CG4968, CG7857, or rpn8 in ISCs/EBs disrupts the intestinal barrier integrity and shortens the lifespan, indicating the requirement of these DUBs for the maintenance of gut homeostasis. Furthermore, we provide evidences that USP1 mediates ISC lineage differentiation via modulating the Notch signaling activity. Our study identifies, for the first time, the deubiquitinases required for the maintenance of intestinal homeostasis in Drosophila, and provide new insights into the functional links between the DUBs and intestinal homeostasis.

Endoplasmic reticulum unfolded protein response transcriptional targets of XBP-1s mediate rescue from tauopathy

Pathological tau disrupts protein homeostasis (proteostasis) within neurons in Alzheimer’s disease (AD) and related disorders. We previously showed constitutive activation of the endoplasmic reticulum unfolded protein response (UPRER) transcription factor XBP-1s rescues tauopathy-related proteostatic disruption in a tau transgenic Caenorhabditis elegans (C. elegans) model of human tauopathy. XBP-1s promotes clearance of pathological tau, and loss of function of the ATF-6 branch of the UPRER prevents XBP-1s rescue of tauopathy in C. elegans. We conducted transcriptomic analysis of tau transgenic and xbp-1s transgenic C. elegans and found 116 putative target genes significantly upregulated by constitutively active XBP-1s. Among these were five candidate XBP-1s target genes with human orthologs and a previously known association with ATF6 (csp-1, dnj-28, hsp-4, ckb-2, and lipl-3). We examined the functional involvement of these targets in XBP-1s-mediated tauopathy suppression and found loss of function in any one of these genes completely disrupts XBP-1s suppression of tauopathy. Further, we demonstrate upregulation of HSP-4, C. elegans BiP, partially rescues tauopathy independent of other changes in the transcriptional network. Understanding how the UPRER modulates pathological tau accumulation will inform neurodegenerative disease mechanisms and direct further study in mammalian systems with the long-term goal of identifying therapeutic targets in human tauopathies.

The Role of Protein Quantity Control in Polyglutamine Spinocerebellar Ataxias.

Polyglutamine spinocerebellar ataxias (polyQ SCAs) represent the most prevalent subtype of SCAs. The primary pathogenic mechanism is believed to be the gain-of-function neurotoxicity of polyQ proteins. Strategies such as enhancing the degradation or inhibiting the accumulation of these mutant proteins are pivotal for reducing their toxicity and slowing disease progression. The protein quality control (PQC) system, comprising primarily molecular chaperones and the ubiquitin‒proteasome system (UPS), is essential for maintaining protein homeostasis by regulating protein folding, trafficking, and degradation. Notably, polyQ proteins can disrupt the PQC system by sequestering its critical components and impairing its proteasomal functions. Therefore, restoring the PQC system through genetic or pharmacological interventions could potentially offer beneficial effects and alleviate the symptoms of the disease. Here, we will provide a review on the distribution, expression, and genetic or pharmacological intervention of protein quality control system in cellular or animal models of PolyQ SCAs.

Establishment of a stable THP-1 cell line expressing PSMB9-eGFP-His protein and detection of immunoproteasome activity

The ubiquitin/proteasome system (UPS) plays a crucial role in maintaining cellular protein homeostasis. The catalytic activity of proteasome in the UPS is regulated by β1 (PSMB6), β2 (PSMB7), and β5 (PSMB5) subunits. Interferon (IFN)-γ, tumor necrosis factor (TNF)-α, inflammation, and oxidative stress can induce the replacement of β1, β2, and β5 with their respective immuno-subunits β1i (PSMB9), β2i (PSMB10), and β5i (PSMB8), which can be assembled into the immunoproteasome. Compared with the standard proteasome, the immunoproteasome exerts enhanced regulatory effects on immune responses, such as processing and presenting MHC class Ⅰ antigens, production of pro-inflammatory cytokines, and T cell differentiation and proliferation. Abnormal aggregation of immunoproteasomes can cause neurodegenerative diseases like Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis. To explore the function of PSMB9 after bacterial infection, we constructed a lentivirus plasmid overexpressing PSMB9-eGFP-His and transfected the plasmid into HEK293T cells for packaging by using a triple-plasmid system in this study. After screening with puromycin, we obtained a stable human leukemia monocytic THP-1 cell line expressing the fusion protein of PSMB9. Western blotting (WB) and fluorescence microscopy verified the expression of the fusion protein in the stable THP-1 cells. Quantitative PCR (qPCR) was employed to measure the copies of PSMB9-eGFP in THP-1 cells. Immunofluorescence results found that eGFP-His did not affect the subcellular localization of PSMB9. The purification with nickel affinity chromatography confirmed that the fusion protein could be assembled into the 20S immunoproteasome and exhibited cleaving activity for fluorescent peptide substrates. These results indicated that the PSMB9-eGFP fusion gene was integrated into the chromosome, and could be stably expressed in the constructed THP-1 cell line. This cell line can be utilized for the research on subcellular localization, dynamic expression, and activity of PSMB9 in live cells at different infection conditions and disease stages. It also provides a model for the stable cell lines construction of other immunoproteasome subunits PSMB8 and PSMB10.

Bag-1-mediated HSF1 phosphorylation regulates expression of heat shock proteins in breast cancer cells.

According to the World Health Organization in 2022, 2.3 million women were diagnosed with breast cancer. Investigating the interaction networks between Bcl-2-associated athanogene (Bag)-1 and other chaperone proteins may further the current understanding of the regulation of protein homeostasis in breast cancer cells and contribute to the development of treatment options. The present study aimed to determine the interactions between Bag-1 and heat shock proteins (HSPs); namely, HSP90, HSP70 and HSP27, to elucidate their role in promoting heat shock factor-1 (HSF1)-dependent survival of breast cancer cells. HER2-negative (MCF-7) and HER2-positive (BT-474) cell lines were used to examine the impact of Bag-1 expression on HSF1 and HSPs. We demonstrated that Bag-1 overexpression promoted HER2 expression in breast cancer cells, thereby resulting in the concurrent constitutive activation of the HSF1-HSP axis. The activation of HSP results in the stabilization of several tumor-promoting HSP clients such as AKT, mTOR and HSF1 itself, which substantially accelerates tumor development. Our results suggest that Bag-1 can modulate the chaperone activity of HSPs, such as HSP27, by directly or indirectly regulating the phosphorylation of HSF1. This modulation of chaperone activity can influence the activation of genes involved in cellular homeostasis, thereby protecting cells against stress.

The ISR downstream target ATF4 represses long-term memory in a cell type–specific manner

The integrated stress response (ISR), a pivotal protein homeostasis network, plays a critical role in the formation of long-term memory (LTM). The precise mechanism by which the ISR controls LTM is not well understood. Here, we report insights into how the ISR modulates the mnemonic process by using targeted deletion of the activating transcription factor 4 (ATF4), a key downstream effector of the ISR, in various neuronal and non-neuronal cell types. We found that the removal of ATF4 from forebrain excitatory neurons (but not from inhibitory neurons, cholinergic neurons, or astrocytes) enhances LTM formation. Furthermore, the deletion of ATF4 in excitatory neurons lowers the threshold for the induction of long-term potentiation, a cellular model for LTM. Transcriptomic and proteomic analyses revealed that ATF4 deletion in excitatory neurons leads to upregulation of components of oxidative phosphorylation pathways, which are critical for ATP production. Thus, we conclude that ATF4 functions as a memory repressor selectively within excitatory neurons.

Regenerating Chemotherapeutics through Copper-Based Nanomedicine: Disrupting Protein Homeostasis for Enhanced Tumor Therapy.

The bis-(diethyldithiocarbamate)-copper (CuET), the disulfiram (DSF)-Cu complex, has exhibited noteworthy anti-tumor property. However, its efficacy is compromised due to the inadequate oxidative conditions and the limitation of bioavailable copper. Because CuET can inactivate valosin-containing protein (VCP), a bioinformatic pan-cancer analysis of VCP is first conducted in this study to identify CuET as a promising anticancer drug for diverse cancer types. Then, based on the drug action mechanism, a nanocomposite of CuET and copper oxide (CuO) is designed and fabricated utilizing bovine serum albumin (BSA) as the template (denoted as CuET-CuO@BSA, CCB). CCB manifests peroxidase (POD)-mimicking activity to oxidize the tumor endogenous H2O2 to generate reactive oxygen species (ROS), enhancing the chemotherapy effect of CuET. Furthermore, the cupric ions released after enzymatic reaction can regenerate CuET, which markedly perturbs intracellular protein homeostasis and induces apoptosis of tumor cells. Meanwhile, CCB triggers cuproptosis by inducing the aggregation of lipoylated proteins. The multifaceted action of CCB effectively inhibits tumor progression. Therefore, this study presents an innovative CuET therapeutic strategy that creates an oxidative microenvironment in situ and simultaneously self-supply copper source for CuET regeneration through the combination of CuO nanozyme with CuET, which holds promise for application of CuET for effective tumor therapy.

Actin-bundling protein fimbrin serves as a new auxin biosynthesis orchestrator in Arabidopsis root tips.

Plants delicately regulate endogenous auxin levels through the coordination of transport, biosynthesis, and inactivation, which is crucial for growth and development. While it is well-established that the actin cytoskeleton can regulate auxin levels by affecting polar transport, its potential role in auxin biosynthesis has remained largely unexplored. Using LC-MS/MS-based methods combined with fluorescent auxin marker detection, we observed a significant increase in root auxin levels upon deletion of the actin bundling proteins AtFIM4 and AtFIM5. Fluorescent observation, immunoblotting analysis, and biochemical approaches revealed that AtFIM4 and AtFIM5 affect the protein abundance of the key auxin synthesis enzyme YUC8 in roots. AtFIM4 and AtFIM5 regulate the auxin synthesis enzyme YUC8 at the protein level, with its degradation mediated by the 26S proteasome. This regulation modulates auxin synthesis and endogenous auxin levels in roots, consequently impacting root development. Based on these findings, we propose a molecular pathway centered on the 'actin cytoskeleton-26S proteasome-YUC8-auxin' axis that controls auxin levels. Our findings shed light on a new pathway through which plants regulate auxin synthesis. Moreover, this study illuminates a newfound role of the actin cytoskeleton in regulating plant growth and development, particularly through its involvement in maintaining protein homeostasis via the 26S proteasome.

Advances in Ubiquitination and Proteostasis in Retinal Degeneration.

Retinal degeneration (RD) is a group of chronic blinding diseases characterised by progressive retinal cell death. As the disease progresses, vision deteriorates due to retinal cell death and impaired retinal integrity, eventually leading to complete loss of vision. Therefore, the function and environmental homeostasis of the retina have an important impact on the pathogenesis and treatment of RD. Ubiquitination, as a complex post-translational modification process, plays an essential role in maintaining retinal homeostasis and normal function. It covalently combines ubiquitin with protein through a series of enzyme-mediated reactions, and participates in cell processes such as gene transcription, cell cycle process, DNA repair, apoptosis and immune response. At the same time, it plays a central role in protein degradation. There are two major protein degradation systems in eukaryotic cells: the ubiquitin-proteasome system and the autophagy-lysosomal system. The protein degradation pathway maintains retinal protein homeostasis by reducing abnormal protein accumulation in the retina through two modes of degradation. Either dysregulation of ubiquitination or disruption of protein homeostasis may lead to the development of RD. This article aims to comprehensively review recent research progress on ubiquitin-related genes, proteins and protein homeostasis in the pathogenesis of RD, and to summarize the potential targeted therapy strategies for it. The review is expected to provide valuable guidance for further development and application of ubiquitination in RD.

A eukaryotic-like ubiquitination system in bacterial antiviral defence.

Ubiquitination pathways have crucial roles in protein homeostasis, signalling and innate immunity1-3. In these pathways, an enzymatic cascade of E1, E2 and E3 proteins conjugates ubiquitin or a ubiquitin-like protein (Ubl) to target-protein lysine residues4. Bacteria encode ancient relatives of E1 and Ubl proteins involved in sulfur metabolism5,6, but these proteins do not mediate Ubl-target conjugation, leaving open the question of whether bacteria can perform ubiquitination-like protein conjugation. Here we demonstrate that a bacterial operon associated with phage defence islands encodes a complete ubiquitination pathway. Two structures of a bacterial E1-E2-Ubl complex reveal striking architectural parallels with canonical eukaryotic ubiquitination machinery. The bacterial E1 possesses an amino-terminal inactive adenylation domain and a carboxy-terminal active adenylation domain with a mobile α-helical insertion containing the catalytic cysteine (CYS domain). One structure reveals a pre-reaction state with the bacterial Ubl C terminus positioned for adenylation, and a second structure mimics an E1-to-E2 transthioesterification state with the E1 CYS domain adjacent to the bound E2. We show that a deubiquitinase in the same pathway preprocesses the bacterial Ubl, exposing its C-terminal glycine for adenylation. Finally, we show that the bacterial E1 and E2 collaborate to conjugate Ubl to target-protein lysine residues. Together, these data reveal that bacteria possess bona fide ubiquitination systems with strong mechanistic and architectural parallels to canonical eukaryotic ubiquitination pathways, suggesting that these pathways arose first in bacteria.