Protein Degradation Research Articles

Design and Discovery of Preclinical Candidate LYG-409 as a Highly Potent and Selective GSPT1 Molecular Glue Degraders.

Molecular glue degraders induce "undruggable" protein degradation by a proximity-induced effect. Inspired by the clinical success of immunomodulatory drugs, we aimed to design novel molecular glue degraders targeting GSPT1. Here, we report the design of a series of GSPT1 molecular glue degraders. LYG-409, a 2H-chromene derivative, was identified as a potent, selective, and orally bioavailable GSPT1 degrader with excellent antitumor activity in vivo (anti-Acute Myeloid Leukemia MV4-11 xenograft model: TGI = 94.34% at 30 mg/kg; prostate cancer 22Rv1 xenograft model: TGI = 104.49% at 60 mg/kg) and in vitro (KG-1 cells: IC50 = 9.50 ± 0.71 nM, DC50 = 7.87 nM) mediated by the degradation of GSPT1. In conclusion, LYG-409 exhibits potent GSPT1 degradation activity, demonstrating promising therapeutic efficacy and favorable safety profile. However, its potential drug resistance profile needs to be thoroughly evaluated in comparison with existing treatments. We hope LYG-409 can provide a valuable direction for the development of GSPT1 degraders.

Development of compounds for targeted degradation of mammalian cryptochrome proteins.

The mammalian cryptochrome proteins (CRY1 and CRY2) are transcriptional repressors most notable for their role in circadian transcriptional feedback. Not all circadian rhythms depend on CRY proteins, however, and the CRY proteins are promiscuous interactors that also regulate many other processes. In cells with chronic CRY deficiency, protein homeostasis is highly perturbed, with a basal increase in cellular stress and activation of key inflammatory signalling pathways. Here, we developed tools to delineate the specific effects of CRY reduction, rather than chronic deficiency, to better understand the direct functions of CRY proteins. Performing a bioluminescence screen and immunoblot validation, we identified compounds that resulted in CRY reduction. Using these compounds, we found that circadian PERIOD2 (PER2) protein rhythms persisted under CRY-depleted conditions. By quantitative mass spectrometry, we found that CRY-depleted cells partially phenocopied the proteomic dysregulation of CRY-deficient cells, but showed minimal circadian phenotypes. We did, however, also observe substantial off-target effects of these compounds on luciferase activity and could not ascertain a specific mechanism of action. This work therefore highlights both the utility and the challenges of targeted protein degradation and bioluminescence reporter approaches in disentangling the contribution of CRY proteins to circadian rhythmicity, homeostasis and innate immune regulation.This article is part of the Theo Murphy meeting issue 'Circadian rhythms in infection and immunity'.

NEDD4-Mediated GSNOR Degradation Aggravates Cardiac Hypertrophy and Dysfunction.

The decrease in S-nitrosoglutathione reductase (GSNOR) leads to an elevation of S-nitrosylation, thereby exacerbating the progression of cardiomyopathy in response to hemodynamic stress. However, the mechanisms under GSNOR decrease remain unclear. Here, we identify NEDD4 (neuronal precursor cell expressed developmentally downregulated 4) as a novel molecule that plays a crucial role in the pathogenesis of pressure overload-induced cardiac hypertrophy, by modulating GSNOR levels, thereby demonstrating significant therapeutic potential. Protein synthesis and degradation inhibitors were used to verify the reasons for the decrease in GSNOR. Mass spectrometry and database filtering were used to uncover NEDD4, the E3 Ub (ubiquitin) ligase, involved in GSNOR decrease. NEDD4 cardiomyocyte-specific deficiency mice were used to evaluate the role of NEDD4 and NEDD4-induced ubiquitination of GSNOR in cardiac hypertrophy in vivo. Both IBM, a highly specific NEDD4 inhibitor, and indole-3-carbinol, a NEDD4 inhibitor currently undergoing phase 2 clinical trial, were used to effectively suppress the NEDD4/GSNOR axis. GSNOR protein levels were reduced, while mRNA levels remained unchanged in myocardium samples from hypertrophic patients and transverse aortic constriction-induced mice, indicating GSNOR is regulated by ubiquitination. NEDD4, an E3 Ub ligase, was associated with GSNOR ubiquitination, which exhibited significantly higher expression levels in hypertrophic myocardial samples. Moreover, either the NEDD4 enzyme-dead mutant or GSNOR nonubiquitylated mutant decreased GSNOR ubiquitination and inhibited cardiac hypertrophic growth. Cardiomyocyte-specific NEDD4 deficiency inhibited cardiac hypertrophy in vitro and in vivo. NEDD4 inhibitor IBM effectively suppressed GSNOR ubiquitination and cardiac hypertrophy. Clinically, indole-3-carbinol, a NEDD4 inhibitor in phase II clinical trials used as an antitumor drug, demonstrated comparable efficacy. Our findings showed that upregulated NEDD4 leads to GSNOR ubiquitination and subsequent degradation, thereby facilitating the progression of cardiac hypertrophy. NEDD4 inhibitors may serve as a potential therapeutic strategy for the treatment of cardiac hypertrophy and heart failure.

Emamectin Benzoate and Microplastics Led to Skeletal Muscle Atrophy in Common Carp via Induced Oxidative Stress, Mitochondrial Dysfunction, and Protein Synthesis and Degradation Imbalance.

Pesticides and plastics have brought convenience to agricultural production and daily life, but they have also led to environmental pollution through residual chemicals. Emamectin benzoate (EMB) is among the most widely used insecticides, which can cause environmental pollution and harm the health of organisms. Additionally, microplastics (MPs), a relatively new type of pollutant, not only are increasing in residual amounts within water bodies and aquatic organisms but also exacerbate pollution by adsorbing other pollutants, leading to a mixed pollution scenario. Nevertheless, the toxicity and mechanism of EMB and MPs on common carp skeletal muscle have not been elucidated. Therefore, we established exposure models for EMB and MPs, and methods such as hematoxylin and eosin staining, immunofluorescence staining, JC-1 staining, and western blotting were employed to investigate the underlying mechanisms of skeletal muscle damage. The results of in vivo and in vitro experiments indicated that exposure to EMB or MPs led to oxidative stress, which in turn caused mitochondrial fusion/fission imbalance (with decreased Mfn1, Mfn2, and OPA1 and increased DRP1), reduced mitochondrial membrane potential, decreased ATP content, reduced protein synthesis, and increased degradation, ultimately resulting in skeletal muscle atrophy. Joint exposure caused more severe damage than single exposure, and the addition of NAC can effectively alleviate skeletal muscle atrophy. In summary, exposure to EMB and/or MPs induced excessive reactive oxygen species (ROS) production, giving rise to mitochondrial dysfunction and an imbalance in skeletal muscle protein synthesis and degradation, ultimately resulting in skeletal muscle atrophy in common carp.

Comparative metagenomics of tropical reef fishes show conserved core gut functions across hosts and diets with diet-related functional gene enrichments.

The benefits of healthy microbiomes for vertebrate hosts include the breakdown of food into more readily usable forms and production of essential vitamins from their host's diet. Compositions of microbial communities in the guts of fish in response to diet have been studied, but there is a lack of a comprehensive understanding of the genome-based metabolic capabilities of specific microbes and how they support their hosts. Therefore, we assembled genomes of several gut microbes collected from the feces of three fish species that were being fed different diets to illustrate how individual microbes can carry out specific steps in the degradation and energy utilization of various food inputs and support their host. We found evidence that fish gut microbial communities share several core functions despite differences in microbial taxonomy. Herbivorous fish harbored a functionally diverse microbial community with plant matter degraders, while the piscivorous and invertivorous fish had microbiomes more specialized in protein degradation.

Bisecting GlcNAc modification of vesicular GAS6 regulates CAFs activation and breast cancer metastasis

BackgroundCancer-associated fibroblasts (CAFs) are a pivotal component of the tumor microenvironment (TME), playing key roles in tumor initiation, metastasis, and chemoresistance. While glycosylation is known to regulate various cellular processes, its impact on CAFs activation remains insufficiently explored.MethodsWe assessed the correlation between bisecting GlcNAc levels and CAFs markers (α-SMA, PDGFRA, PDGFRB) in breast cancer tissues. The effects of small extracellular vesicles (sEVs) derived from MDA-MB-231/OEvec and MDA-MB-231/OEMGAT3 cells on CAFs activation were examined using western blotting, transwell, and collagen contraction assays. Proteomic analysis was performed to identify dysregulated proteins in sEVs from different cell lines. The role of GAS6 in CAFs activation was validated through in vitro and in vivo experiments. The impact of bisecting GlcNAc modification on GAS6 expression and function was analyzed through protein degradation and N-glycosylation site mutation. The effect of activated CAFs on breast cancer metastasis was evaluated using western blotting and transwell assays.ResultsWe found that low bisecting GlcNAc levels were associated with CAFs activation within the TME of breast cancer. Breast cancer-derived sEVs stimulated the conversion of normal fibroblasts to CAFs, with GAS6 in sEVs playing a key role by interacting with AXL receptors on fibroblasts. Introducing GAS6 into normal fibroblasts induced their conversion into CAFs, which enhanced breast cancer cell metastasis. Notably, GAS6 was decorated with bisecting GlcNAc, which promoted its degradation in donor cells, leading to reduced GAS6 levels in sEVs and attenuating GAS6-mediated CAFs activation.ConclusionTaken together, our findings provide new insights into the functional role of bisecting GlcNAc on GAS6-mediated CAFs activation in breast cancer.

A Rapid and Reversible Molecular "Switch" Regulating Protein Expression in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii, a prominent chassis in synthetic biology, faces limitations in regulating the expression of exogenous genes. A destabilization domain (DD)/Shield-1 system, originally derived from mammals, offers a ligand-dependent control of stability, making it a valuable tool. This system utilises the destabilization domain to induce rapid degradation of target protein unless stabilised by Shield-1, a synthetic ligand. Upon the addition of Shield-1,the degradation is halted, leading to the accumulation and stabilisation of the target protein. This system has demonstrated successful regulation of foreign protein expression in mammals, parasites, and plants. In this study, the DD/Shield-1 system was harnessed to regulate the expression of the paromomycin resistance gene and luciferase encoding gene in Chlamydomonas, revealing its capability for rapid, stable, and reversible protein expression regulation in microalgae, serving as a molecular switch. Furthermore, this regulation exhibits reagent dependency, enhancing its applicability in practical production. A strain with induced expression of the gene-editing protein, LbCas12a, was successfully constructed and then tested for gene editing. The findings not only enrich the toolkit for Chlamydomonas molecular studies but offer a promising technique for regulating the expression and validating the functionality of exogenous proteins in microalgae.

The Kinesin Kar3 is Required for Endoplasmic Reticulum-Associated Degradation.

Degradation of aberrant, excess, and regulatory proteins at the endoplasmic reticulum (ER) is a conserved feature of eukaryotic cells, disruption of which contributes to disease. While remarkable progress has been made in recent years, mechanisms and genetic requirements for ER-Associated Degradation (ERAD) remain incompletely understood. We recently conducted a screen for genes required for turnover of a model ER translocon-associated substrate of the Hrd1 ubiquitin ligase in Saccharomyces cerevisiae. This screen revealed loss of Kar3 impedes degradation of Deg1*-Sec62, which persistently and aberrantly engages the translocon. Kar3 is a microtubule-associated kinesin 14 family member that impacts multiple aspects of microtubule dynamics during cell division and karyogamy. We investigated involvement of Kar3 and its cofactors in ERAD. Loss of Kar3 hindered ERAD mediated by three ubiquitin ligases but did not impair turnover of a soluble nuclear protein. Further, KAR3 deletion caused hypersensitivity to conditions associated with proteotoxic stress. Kar3's cytoplasmic cofactor Vik1 was also required for efficient degradation of Deg1*-Sec62. Our results reveal a profound and underappreciated role for microtubule-associated proteins in ERAD.

A bacterial type III effector hijacks plant ubiquitin proteases to evade degradation.

Gram-negative bacterial pathogens inject effector proteins inside plant cells using a type III secretion system. These effectors manipulate plant cellular functions and suppress the plant immune system in order to promote bacterial proliferation. Despite the fact that bacterial effectors are exogenous threatening proteins potentially exposed to the protein degradation systems inside plant cells, effectors are relative stable and able to perform their virulence functions. In this work, we found that RipE1, an effector protein secreted by the bacterial wilt pathogen, Ralstonia solanacearum, undergoes phosphorylation of specific residues inside plant cells, and this promotes its stability. Moreover, RipE1 associates with plant ubiquitin proteases, which contribute to RipE1 deubiquitination and stabilization. The absence of those specific phosphorylation sites or specific host ubiquitin proteases leads to a substantial decrease in RipE1 protein accumulation, indicating that RipE1 hijacks plant post-translational modification regulators in order to promote its own stability. These results suggest that effector stability or degradation in plant cells constitute another molecular event subject to co-evolution between plants and pathogens.

Locating Polyubiquitin Receptors on the 19S Regulatory Proteasome of S. cerevisiae by Cross-Linking Mass Spectrometry.

The effectiveness of state-of-the-art cross-linking strategies and mass spectrometry (MS) detection was explored in an important biological context, namely, the ubiquitin-proteasome system, which is responsible for most of the regulated protein degradation in eukaryotic cells. The locations of possible binding sites on the S. cerevisiae 19S proteasome regulatory particle for Lys48 linked polyubiquitin chains were examined using cross-linking strategies and MS based detection by comparing two types of cross-linkers: a (bis)-sulfosuccinimidyl suberate (BS3) and diethyl suberothioimidate (DEST). The well-established BS3-based strategy produced 328 cross-linked peptides; however, no ubiquitin-19S cross-links were observed. The recently developed DEST-based approach produced fewer (146) linkages overall, but these included six ubiquitin-19S cross-links. Some of these cross-links are predicted by the canonical view of ubiquitin recognition, but others suggest novel insights into how the proteasome recognizes its substrates. A discussion of these strategies and structural implications for polyubiquitin-proteasome binding is provided.

Upregulation of FAM129B protects against glucocorticoid-induced skeletal muscle atrophy via regulating long non-coding RNA NEAT1.

Skeletal muscle atrophy, manifested by a reduction in muscle size and quantity, is primarily attributed to excessive protein catabolism. FAM129B, an antioxidant protein, has been previously implicated in muscle growth and development in cattle. Aim of this study is to elucidate the role of FAM129B in muscle atrophy. FAM129B was consistently down-regulation in muscle atrophy models in vitro and in vivo and in human steroid-treated gluteus muscles. FAM129B depletion resulted in myotubes atrophy with reduced diameter, increased MuRF-1 and Atrogin-1. Conversely, FAM129B overexpression ameliorated muscle atrophy by increasing myotube diameter and reducing Atrogin-1 and MuRF-1. Mice overexpressing FAM129B exhibited resistance to muscle atrophy, evidenced by increased grip strength, increased tibial anterior weight, increased myofiber cross-sectional area and decreased MuRF-1 and Atrogin-1. RNA sequencing revealed NEAT1 as a downstream gene of FAM129B. Mechanistically, FAM129B was found to influence the stability of NEAT1 by directly binding to it. The enhanced stability of NEAT1 subsequently led to increased FoxO1 expression and subsequent protein degradation. Our study has provided evidence that the upregulation of FAM129B rescues the Dex-induced skeletal muscle atrophy, suggesting that FAM129B may be a potential target for alleviating skeletal muscle atrophy.

Macrocyclic Peptide-Based Dual-Sensor Platform for Linkage-Specific Visualization of Ubiquitin Chain Assembling in Live Cells.

Intracellular monitoring of protein ubiquitination and differentiating polyubiquitin chain topology are crucial for understanding life processes and drug discovery, which is challenged by the high complexity of the ubiquitination process and a lack of molecular tools. Herein, a synthetic dual-sensor platform specific for K48-linked ubiquitin oligomers was tailored for in situ visualization of polyubiquitin chain assembling in live biosystems. This is achieved using macrocyclic peptides as recognition motifs and a tetraphenylethylene derivative as an activatable reporter. The efficient cell penetration, tight binding, and protection of polyubiquitin delivered a "freeze-and-image" approach, allowing fluorescent readout of polyubiquitin linkage type and chain elongation without perturbing the physiological environment. Motivated by these unique features, mapping of K48-ubiquitination dynamics during protein degradation was facilely achieved. Rapid, sensitive, and intracellular assessment of the mechanism of action and potency dependence for proteolysis-targeting chimeras (PROTACs) was demonstrated, presenting the sensors as promising molecular tools for PROTAC drug development.

Unleashing the Power of Covalent Drugs for Protein Degradation.

Targeted protein degradation (TPD) has emerged as a significant therapeutic approach for a variety of diseases, including cancer. Advances in TPD techniques, such as molecular glue (MG) and lysosome-dependent strategies, have shown substantial progress since the inception of the first PROTAC in 2001. The PROTAC methodology represents the forefront of TPD technology, with ongoing evaluation in more than 20 clinical trials for the treatment of diverse medical conditions. Two prominent PROTACs, ARV-471 and ARV-110, are currently undergoing phase III and II clinical trials, respectively. Traditional PROTACs are encountering obstacles such as limited binding affinity and a restricted range of E3 ligase ligands for facilitating the protein of interest (POI) degradation. Covalent medicines offer the potential to enhance PROTAC efficacy by enabling the targeting of previously considered "undruggable" shallow binding sites. Strategic alterations allow PROTAC to establish covalent connections with particular target proteins, including Kirsten rat sarcoma viral oncogene homolog (KRAS), Bruton's tyrosine kinase (BTK), epidermal growth factor receptor (EGFR), as well as E3 ligases such as DDB1 and CUL4 associated factor 16 (DCAF16) and Kelch-like ECH-associated protein 1 (Keap1). The concept of covalent degradation has also been utilized in various new forms of degraders, including covalent molecule glue (MG), in-cell click-formed proteolysis targeting chimera (CLIPTAC), HaloPROTAC, lysosome-targeting chimera (LYTAC) and GlueTAC. This review focuses on recent advancements in covalent degraders beyond covalent PROTACs and examines obstacles and future directions pertinent to this field.

KGF impedes TRIM21-enhanced stabilization of keratin 10 mediating differentiation in hypopharyngeal cancer.

KGF, also known as FGF7, is a member of the fibroblast growth factor (FGF) family that binds with high affinity to the FGF receptor 2b (FGFR2b) and regulates various cellular processes, including cell proliferation and differentiation in a variety of tumors. However, its potential role in hypopharyngeal cancer (HPC) remains largely unknown. In our study, we observed increased expression of FGFR2b in HPC. KGF treatment inhibited the expression of the differentiation marker keratin 10 (K10) protein at the post-transcriptional level in FaDu cells. Furthermore, treatment with the proteasome inhibitor MG132 was found to attenuate KGF-induced K10 reduction, suggesting the involvement of the ubiquitin-proteasome system. Using mass spectrometry and immunoprecipitation analysis, we identified the E3 ubiquitin ligase TRIM21 as a K10-interacting protein. Unexpectedly, instead of causing degradation, TRIM21 enhanced K10 protein stability through K6-linked ubiquitination of K10 at lysine 163 (K163) in the context of KGF exposure. Meanwhile, KGF treatment decreased TRIM21 protein levels, which were regulated by the p38 MAPK pathway, leading to K48-linked ubiquitination-mediated degradation of TRIM21. Notably, TRIM21 knockdown significantly promoted proliferation, inhibited differentiation and migration of FaDu cells, whereas TRIM21 overexpression had opposite effects in vitro and suppressed xenograft tumor growth in vivo. Our study demonstrates that TRIM21 may act as a tumor suppressor in HPC. However, TRIM21 overexpression decreased the sensitivity of FaDu cells to 5-fluorouracil, whereas TRIM21 knockdown or KGF administration significantly increased 5-fluorouracil sensitivity. Taken together, these findings highlight the intricate balance between protein stabilization and degradation orchestrated by KGF. This ubiquitination-mediated non-degradation mechanism of TRIM21 may provide novel therapeutic strategies for HPC and other cancers.

Effects of different wet distillers’ grains ratios on fermentation quality, nitrogen fractions and bacterial communities of total mixed ration silage

ObjectiveWet distiller’s grains (WDG) are rich in crude protein, yet challenging to preserve. Nevertheless, incorporating WDG into total mixed ration (TMR) silage holds promise for enhancing fermentation quality. This study investigated the effects of varying WDG proportions on nitrogen composition, fermentation quality, and microorganisms in TMR silage.MethodsThree TMR formulations were prepared: (1) 0% WDG (T0), (2) 15% WDG (T15), and (3) 30% WDG (T30) were ensiled for 7, 15, 30 and 60 days.ResultsAfter 7 days of ensiling, butyric acid was detected in T0 and T15 groups, while T30 exhibited significantly lower levels (p < 0.05). Both T15 and T30 treatments led to improved V-scores of TMR silage. Non-protein nitrogen (NPN) production was slower in T30, with significant increases observed in NPN levels for T0 and T15 after 30 days (p < 0.05). However, the abundance of Clostridium was extremely low in the present study. Protein degradation and and butyric acid production may be attributed to Weissella.ConclusionThe fermentation quality of TMR silage is always decreasing during storage, so its storage time should be minimized. Incorporating 30% WDG reduced abundance of Weissella, resulting in less protein degradation and better fermentation quality in TMR silage.

TMT-labelled quantitative proteomics reveals the mechanism of Rhodotorula mucilaginosa on proteolysis of dry-cured ham: Structural protein degradation, amino acid release and taste improvement.

To investigate the mechanism of Rhodotorula mucilaginosa on structural protein degradation and taste development of Jinhua ham, the effects of Rhodotorula mucilaginosa and Pichia kudriavzevii on proteolytic enzyme activities, surface hydrophobicity, myofibril microstructure, protein degradation, free amino acids and sensory attributes were investigated during the dry-ripening of Jinhua ham. The inoculation of Rhodotorula mucilaginosa EIODSF019 (RE) and Rhodotorula mucilaginosa XZY63-3 (RX) consistently exhibited higher proteolytic enzyme activities compared with Pichia kudriavzevii XS-5 (PK). The decrease of α-helix exposing more internal hydrophobic groups of myofibrillar proteins, contributed to higher surface hydrophobicity of RE compared with PK and RX. RE showed the highest proteolysis index among all groups, which could be attributed to more degradation of myosin, actin and troponin; the changes were confirmed by the intense breakdown of myofibrils observed by atomic force microscopy and transmission electron microscopy. 36 down-regulated proteins mainly derived from myofibrils and catalysis-related enzymes were identified in RE by TMT-labeled quantitative proteomics analysis. The degradation of myosin, actin and troponin showed the most intense response to the accumulation of glutamic acid, lysine and alanine. Partial least square regression analysis and correlation analysis revealed that the breakdown of MYH14, MYH3, TNNI1 and TNNTI was highly correlated with improvement of umami, richness and aftertaste.

Metabolic activities are selective modulators for individual segmentation clock processes

Numerous cellular and molecular processes during embryonic development prompt the fundamental question of how their tempos are coordinated and whether a common global modulator exists. While the segmentation clock tempo scales with the kinetics of gene expression and degradation processes of the core clock gene Hes7 across mammals, the coordination of these processes remains unclear. This study examines whether metabolic activities serve as a global modulator for the segmentation clock, finding them to be selective instead. Several metabolic inhibitions extend the clock period but affect key processes differently: glycolysis inhibition slows Hes7 protein degradation and production delay without altering intron delay, while electron transport chain inhibition extends intron delay without influencing the other processes. Combinations of distinct metabolic inhibitions exhibit synergistic effects. We propose that the scaled kinetics of segmentation clock processes across species may result from combined selective modulators shaped by evolutionary constraints, rather than a single global modulator.

The recent advance of PROTACs targeting BCR-ABL for the treatment of chronic myeloid leukemia.

The chronic myeloid leukemia is a malignant hematopoietic disorder in which the BCR-ABL kinase has been identified as the causative protein. The inhibitors targeting BCR-ABL kinase have been extensively employed in clinical management of chronic myeloid leukemia, significantly enhancing survival rates and prognosis for patients. Despite the extensive utilization of 1st to 4th generation BCR-ABL inhibitors in clinical therapy, the emergence of drug-resistant mutations necessitates an urgent quest for novel therapeutic strategies. The proteolysis targeting chimera technology represents an innovative strategy for protein degradation, directly degrading BCR-ABL fusion proteins while circumventing challenges associated with drug resistance. This review article provides an overview of current research progress on inhibitors and proteolysis targeting chimeras for the treatment of chronic myeloid leukemia through targeting BCR-ABL. We anticipate that this timely and comprehensive review will serve as a source of inspiration and guidance for pharmaceutical chemists in the development of highly potent BCR-ABL inhibitors and proteolysis targeting chimeras.

BTG3 inhibits porcine epidemic diarrhea virus replication by promoting viral S2 protein degradation through the autophagy and proteasome pathways.

BTG3, which belongs to the BTG/Tob gene family, is involved in various physiological processes. Infection with porcine epidemic diarrhea virus (PEDV), an alphacoronavirus, is associated with high mortality rates among piglets, contributing to major economic losses. This study elucidated a novel mechanism through which BTG3 suppresses PEDV replication. Endogenous BTG3 protein expression was upregulated in PEDV-infected host cells. PEDV replication was suppressed upon BTG3 overexpression but enhanced upon BTG3 knockdown. Additionally, BTG3 inhibited viral proliferation by targeting and degrading the S2 subunit of the PEDV spike (S) protein through both autophagy and proteasome pathways. BTG3 interacted and co-localized with the S2 protein, promoting S2 protein degradation through the recruitment of the cargo receptor NDP52 and the E3 ubiquitin ligase MARCHF8. In summary, this study elucidated a novel antiviral mechanism in which the host BTG3 targeted the viral S2 protein to inhibit PEDV proliferation through autophagy and proteasome pathways. These findings indicate that BTG3 is a potential novel target for the prevention and control of PEDV.

The Role of E3 Ubiquitin Ligase Gene FBK in Ubiquitination Modification of Protein and Its Potential Function in Plant Growth, Development, Secondary Metabolism, and Stress Response.

As a crucial post-translational modification (PTM), protein ubiquitination mediates the breakdown of particular proteins, which plays a pivotal role in a large number of biological processes including plant growth, development, and stress response. The ubiquitin-proteasome system (UPS) consists of ubiquitin (Ub), ubiquitinase, deubiquitinating enzyme (DUB), and 26S proteasome mediates more than 80% of protein degradation for protein turnover in plants. For the ubiquitinases, including ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3), the FBK (F-box Kelch repeat protein) is an essential component of multi-subunit E3 ligase SCF (Skp1-Cullin 1-F-box) involved in the specific recognition of target proteins in the UPS. Many FBK genes have been identified in different plant species, which regulates plant growth and development through affecting endogenous phytohormones as well as plant tolerance to various biotic and abiotic stresses associated with changes in secondary metabolites such as phenylpropanoid, phenolic acid, flavonoid, lignin, wax, etc. The review summarizes the significance of the ubiquitination modification of protein, the role of UPS in protein degradation, and the possible function of FBK genes involved in plant growth, development, secondary metabolism, and stress response, which provides a systematic and comprehensive understanding of the mechanism of ubiquitination and potential function of FBKs in plant species.