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Aberrant proteins located in the endoplasmic reticulum (ER) undergo rapid ubiquitination by multiple ubiquitin (Ub) E3 ligases and are retrotranslocated to the cytosol as part of the ER-associated degradation (ERAD). Despite several ERAD branches involving different Ub E3 ligases, the molecular machinery responsible for these ERAD branches in mammalian cells remains not fully understood. Through a series of multiplex knockdown/knockout experiments with real-time kinetic measurements, we demonstrate that HERC3 operates independently of the ER-embedded ubiquitin ligases RNF5 and RNF185 (RNF5/185) to mediate the retrotranslocation and ERAD of misfolded CFTR. While RNF5/185 participates in the ERAD process of both misfolded ABCB1 and CFTR, HERC3 uniquely promotes CFTR ERAD. In vitro assay revealed that HERC3 directly interacts with the exposed membrane-spanning domains (MSDs) of CFTR but not with the MSDs embedded in liposomes. Therefore, HERC3 could play a role in the quality control of MSDs in the cytoplasm and might be crucial for the ERAD pathway of select membrane proteins. [ABSTRACT FROM AUTHOR]

Ubiquitin (Ub) is a small, highly conserved protein essential for eukaryotic biology, and is unique in its formation of polyubiquitin chains by conjugation to one of its seven lysine side chains. Here we report that atomic tailoring of Ub side chains – i. e. the insertion, deletion, or replacement of specific atoms – has significant and unexpected consequences on the enzymatic conjugation of Ub oligomers by isopeptide bond formation mediated by E2 conjugating enzymes. These studies employed chemical synthesis and ligation methods to prepare numerous specifically tailored Ub monomers on multi‐milligram scales. While some modifications including N‐terminal acylation and methionine replacement did not affect protein folding or Ub chain formation with Ube2 K, other modifications had a pronounced effect of oligomerization with Ubc13/Mms2. We observed that Ala46Hse mutation obliterates the ability of this Ub monomer to accept another Ub at Lys63 in Ubc13‐mediated conjugations. Exhaustive replacement of all seven lysines with shorter surrogates Orn, Dab, or Dap essentially blocks Ub chain formation, and in the case of Dap, precludes proper folding of the Ub protein. [ABSTRACT FROM AUTHOR]

The spurge Euphorbia characias is known for its latex, which is rich in antioxidant enzymes and anti-phytopathogen molecules. In this study, we identified a novel polyubiquitin protein in the latex and leaves, leading to the first molecular characterization of its coding gene and expressed protein in E. characias. Using consensus-degenerate hybrid oligonucleotide primers (CODEHOP) and rapid amplification of cDNA ends (5′/3′-RACE), we reconstructed the entire open reading frame (ORF) and noncoding regions. Our analysis revealed that the polyubiquitin gene encodes five tandemly repeated sequences, each coding for a ubiquitin monomer with amino acid variations in four of the five repeats. In silico studies have suggested functional differences among monomers. Gene expression peaked during the summer, correlating with high temperatures and suggesting a role in heat stress response. Western blotting confirmed the presence of polyubiquitin in the latex and leaf tissues, indicating active ubiquitination processes. These findings enhance our understanding of polyubiquitin's regulatory mechanisms and functions in E. characias, highlighting its unique structural and functional features. [ABSTRACT FROM AUTHOR]

To explore the effect of K33 only mutant ubiquitin (K33O) on bone marrow‐derived dendritic cells' (BMDCs') maturity, antigen uptake capability, surface molecule expressions and BMDC‐mediated CTL priming, and further investigate the role of PI3K‐Akt engaged in K33O‐increased BMDC maturation, antigen uptake and presentation, surface molecule expressions and BMDC‐based CTL priming. BMDCs were conferred K33O and other ubiquitin mutants (K33R, K48R, K63R‐mutant ubiquitin) incubation or LY294002 and wortmannin pretreatment. PI3K‐Akt phosphorylation, antigen uptake, antigenic presentation and CD86/MHC class I expression in BMDC were determined by western blot or flow cytometry. BMDC‐based CTL proliferation and priming were determined by in vitro mixed lymphocyte reaction (MLR), ex vivo enzyme‐linked immunospot assay (Elispot) and flow cytometry with intracellular staining, respectively. The treatment with K33O effectively augmented PI3K‐Akt phosphorylation, BMDCs' antigen uptake, antigenic presentation, CD86/MHC class I and CD11c expressions. MLR, Elispot and flow cytometry revealed that K33O treatment obviously enhanced CTL proliferation, CTL priming and perforin/granzyme B expression. The pretreatment with PI3K‐Akt inhibitors efficiently abrogated K33O's effects on BMDC. The replenishment of K33 only mutant ubiquitin augments BMDC‐mediated CTL priming in bone marrow‐derived dendritic cells via PI3K‐Akt signalling. [ABSTRACT FROM AUTHOR]

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

Most mature B-cell malignancies originate from the malignant transformation of germinal center (GC) B cells. The GC reaction appears to have a role in malignant transformation, in which a major player of the GC reaction is BCL6, a key regulator of this process. We now demonstrate that BCL6 protein levels were dramatically decreased in Epstein-Barr virus (EBV)-positive lymphoblastoid cell lines and Burkitt’s lymphoma cell lines. Notably, BCL6 degradation was significantly enhanced in the presence of both EBNA3C and FBXO11. Furthermore, the amino-terminal domain of EBNA3C, which contains residues 50–100, interacts directly with FBXO11. The expression of EBNA3C and FBXO11 resulted in a significant induction of cell proliferation. Furthermore, BCL6 protein expression levels were regulated by EBNA3C via the Skp Cullin Fbox (SCF)FBXO11 complex, which mediated its ubiquitylation, and knockdown of FBXO11 suppressed the transformation of lymphoblastoid cell lines. These data provide new insights into the function of EBNA3C in B-cell transformation during GC reaction and raise the possibility of developing new targeted therapies against EBV-associated cancers. IMPORTANCE The novel revelation in our study involves the suppression of BCL6 expression by the essential Epstein-Barr virus (EBV) antigen EBNA3C, shedding new light on our current comprehension of how EBV contributes to lymphomagenesis by impeding the germinal center reaction. It is crucial to note that while several EBV latent proteins are expressed in infected cells, the collaborative mechanisms among these proteins in regulating B-cell development or inducing B-cell lymphoma require additional investigation. Nonetheless, our findings carry significance for the development of emerging strategies aimed at addressing EBV-associated cancers. [ABSTRACT FROM AUTHOR]

INTRODUCTION: Frontotemporal lobar degeneration (FTLD) encompasses behavioral variant frontotemporal dementia (bvFTD), progressive supranuclear palsy, corticobasal syndrome/degeneration, and primary progressive aphasias (PPAs). We cross‐validated fluid biomarkers and neuroimaging. METHODS: Seven fluid biomarkers from cerebrospinal fluid and serum were related to atrophy in 428 participants including these FTLD subtypes, logopenic variant PPA (lvPPA), Alzheimer's disease (AD), and healthy subjects. Atrophy was assessed by structural magnetic resonance imaging and atlas‐based volumetry. RESULTS: FTLD subtypes, lvPPA, and AD showed specific profiles for neurofilament light chain, phosphorylated heavy chain, tau, phospho‐tau, amyloid beta1‐42 from serum/cerebrospinal fluid, and brain atrophy. Neurofilaments related to regional atrophy in bvFTD, whereas progranulin was associated with atrophy in semantic variant PPA. Ubiquitin showed no effects. DISCUSSION: Results specify biomarker and atrophy patterns in FTLD and AD supporting differential diagnosis. They identify neurofilaments and progranulin in interaction with structural imaging as promising candidates for monitoring disease progression and therapy. Highlights: Study cross‐validated neuroimaging and fluid biomarkers in dementia.Five kinds of frontotemporal lobar degeneration and two variants of Alzheimer's disease.Study identifies disease‐specific fluid biomarker and atrophy profiles.Fluid biomarkers and atrophy interact in a disease‐specific way.Neurofilaments and progranulin are proposed as biomarkers for diagnosis and therapy. [ABSTRACT FROM AUTHOR]

The E3 ligase-degron interaction determines the specificity of the ubiquitin‒proteasome system. We recently discovered that FEM1B, a substrate receptor of Cullin 2-RING ligase (CRL2), recognizes C-degrons containing a C-terminal proline. By solving several cryo-EM structures of CRL2FEM1B bound to different C-degrons, we elucidate the dimeric assembly of the complex. Furthermore, we reveal distinct dimerization states of unmodified and neddylated CRL2FEM1B to uncover the NEDD8-mediated activation mechanism of CRL2FEM1B. Our research also indicates that, FEM1B utilizes a bipartite mechanism to recognize both the C-terminal proline and an upstream aromatic residue within the substrate. These structural findings, complemented by in vitro ubiquitination and in vivo cell-based assays, demonstrate that CRL2FEM1B-mediated polyubiquitination and subsequent protein turnover depend on both FEM1B-degron interactions and the dimerization state of the E3 ligase complex. Overall, this study deepens our molecular understanding of how Cullin-RING E3 ligase substrate selection mediates protein turnover. A key question in the ubiquitin-proteasome system is how E3 ligases select their substrates. Here, the authors reveal that CRL2FEM1B E3 ligase functions as a dimer and employs a bipartite mode of substrate recognition, requiring a C-terminal proline and an upstream aromatic residue in the target. [ABSTRACT FROM AUTHOR]

Introduction: The Anaphase Promoting Complex (APC/C), an E3 ubiquitin ligase, plays a key role in cell cycle control, but it is also thought to operate in postmitotic neurons. Most studies linking APC/C function to neuron biology employed perturbations of the APC/C activators, cell division cycle protein 20 (Cdc20) and Cdc20 homologue 1 (Cdh1). However, multiple lines of evidence indicate that Cdh1 and Cdc20 can function in APC/C-independent contexts, so that the effects of their perturbation cannot strictly be linked to APC/C function. Methods: We therefore deleted the gene encoding Anaphase Promoting Complex 4 (APC4), a core APC/C component, in neurons cultured from conditional knockout (cKO) mice. Results: Our data indicate that several previously published substrates are actually not APC/C substrates, whereas ubiquitin specific peptidase 1 (USP1) protein levels are altered in APC4 knockout (KO) neurons. We propose a model where the APC/C ubiquitylates USP1 early in development, but later ubiquitylates a substrate that directly or indirectly stabilizes USP1. We further discovered a novel role of the APC/C in regulating the number of neurites exiting somata, but we were unable to confirm prior data indicating that the APC/C regulates neurite length, neurite complexity, and synaptogenesis. Finally, we show that APC4 SUMOylation does not impact the ability of the APC/C to control the number of primary neurites or USP1 protein levels. Discussion: Our data indicate that perturbation studies aimed at dissecting APC/C biology must focus on core APC/C components rather than the APC/C activators, Cdh20 and Cdh1. [ABSTRACT FROM AUTHOR]

Background: Histone ubiquitination modification is emerging as a critical epigenetic mechanism involved in a range of biological processes. In vitro reconstitution of ubiquitinated nucleosomes is pivotal for elucidating the influence of histone ubiquitination on chromatin dynamics. Results: In this study, we introduce a Non-Denatured Histone Octamer Ubiquitylation (NDHOU) approach for generating ubiquitin or ubiquitin-like modified histone octamers. The method entails the co-expression and purification of histone octamers, followed by their chemical cross-linking to ubiquitin using 1,3-dibromoacetone. We demonstrate that nucleosomes reconstituted with these octamers display a high degree of homogeneity, rendering them highly compatible with in vitro biochemical assays. These ubiquitinated nucleosomes mimic physiological substrates in function and structure. Additionally, we have extended this method to cross-linking various histone octamers and three types of ubiquitin-like proteins. Conclusions: Overall, our findings offer an efficient strategy for producing ubiquitinated nucleosomes, advancing biochemical and biophysical studies in the field of chromatin biology. [ABSTRACT FROM AUTHOR]

Background: The proliferation of porcine ovarian granulosa cells (GCs) is essential to follicular development and the ubiquitin–proteasome system is necessary for maintaining cell cycle homeostasis. Previous studies found that the deubiquitinase ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) regulates female reproduction, especially in ovarian development. However, the mechanism by which UCHL1 regulates porcine GC proliferation remains unclear. Results: UCHL1 overexpression promoted GC proliferation, and knockdown had the opposite effect. UCHL1 is directly bound to cyclin B1 (CCNB1), prolonging the half-life of CCNB1 and inhibiting its degradation, thereby promoting GC proliferation. What's more, a flavonoid compound-isovitexin improved the enzyme activity of UCHL1 and promoted the proliferation of porcine GCs. Conclusions: UCHL1 promoted the proliferation of porcine GCs by stabilizing CCNB1, and isovitexin enhanced the enzyme activity of UCHL1. These findings reveal the role of UCHL1 and the potential of isovitexin in regulating proliferation and provide insights into identifying molecular markers and nutrients that affect follicle development. [ABSTRACT FROM AUTHOR]

African swine fever (ASF) is an acute, hemorrhagic, highly contagious disease in pigs caused by African swine fever virus (ASFV). Our previous study identified that the ASFV MGF300-2R protein functions as a virulence factor and found that MGF300-2R degrades IKKβ via selective autophagy. However, the E3 ubiquitin ligase responsible for IKKβ ubiquitination during autophagic degradation still remains unknown. In order to solve this problem, we first pulled down 328 proteins interacting with MGF300-2R through immunoprecipitation-mass spectrometry. Next, we analyzed and confirmed the interaction between the E3 ubiquitin ligase TRIM21 and MGF300-2R and demonstrated the catalytic role of TRIM21 in IKKβ ubiquitination. Finally, we indicated that the degradation of IKKβ by MGF300-2R was dependent on TRIM21. In summary, our results indicate TRIM21 is the E3 ubiquitin ligase involved in the degradation of IKKβ by MGF300-2R, thereby augmenting our understanding of the functions of MGF300-2R and offering insights into the rational design of live attenuated vaccines and antiviral strategies against ASF. [ABSTRACT FROM AUTHOR]

Host cell protein (HCP) characterization is a crucial quality parameter for biotherapeutic drug safety and stability. With a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach, we identified ubiquitin in ultrafiltration/diafiltration (UF/DF) pools of one of our monoclonal antibody (mAb) products. Since ubiquitin occurs physiologically as a post-translational modification (PTM) involved in many cellular functions, we suspected the possibility that if identified as an HCP, it may occur as a covalent modification on the mAb. In fact, in this study we characterized and quantified the ubiquitin modification on the Fc domain of mAbX by data dependent acquisition (DDA) and data independent acquisition (DIA) – MS workflows. Covalent binding and site localization were confirmed by identifying a characteristic diglycine motif on the modified peptide. Initially observed reduced detectability of ubiquitin in samples prepared with native digestion was attributed to impaired digestion and subsequent removal along with the mAb in the precipitation step. Our work has contributed to a better understanding of ubiquitin as an HCP considering its specific features such as occurrence in different topologies and provided insight into how covalent binding to a drug product can affect its identification by MS when native digestion conditions are used. [ABSTRACT FROM AUTHOR]

Purpose: Despite lung cancer is one of the leading causes of cancer-related deaths, it remains hard to discover effective diagnostic and therapeutic approaches. Moreover, the five-year survival rate is relatively lower than other tumors. So urgent needs for finding a new theranostic target to treat lung cancer effectively. This study aims to present SOCS3 and NOD2 proteins as novel targets for diagnosis and therapy. Methods: We first confirmed SOCS3 expression level in patients' tissues. Then, we applied knockdown and overexpression of SOCS3 on lung cancer cell lines and performed proliferation, migration, and invasion assay. After that, we found NOD2 is a target of SOCS3 and introduced overexpression of NOD2 to A549 for verifying reduced tumorigenicity of lung cancer cells. Results: We identified protein expression level of SOCS3 was frequently higher in tumor tissues than adjacent normal tissues. Truly, overexpression of SOCS3 promoted proliferation, migration, and invasion capacity of lung cancer cells. We found that SOCS3 interacts with NOD2 and SOCS3 ubiquitinates NOD2 directly. Furthermore, lung cancer tissues with higher SOCS3 expression showed lower NOD2 expression. We confirmed overexpression of NOD2 leads to suppressed tumorigenicity of lung cancer cells, and these effects occurred through MAPK pathway. Conclusion: Collectively, our work reveals novel roles of SOCS3 in lung tumorigenesis and proposes SOCS3 as a promising biomarker candidate for therapeutic and diagnostic target for lung cancer. [ABSTRACT FROM AUTHOR]

The process of degrading unwanted or damaged mitochondria by autophagy, called mitophagy, is essential for mitochondrial quality control together with mitochondrial apoptosis. In mammalian cells, pan-Bcl-2 family members including conical Bcl-2 members and non-conical ones are involved in and govern the two processes. We have illustrated recently the BH3 receptor Hsp70 interacts with Bim to mediate both apoptosis and mitophagy. However, whether similar pathways exist in lower eukaryotes where conical Bcl-2 members are absent remained unclear. Here, a specific inhibitor of the Hsp70-Bim PPI, S1g-10 and its analogs were used as chemical tools to explore the role of yeast Bxi1/Ybh3 in regulating mitophagy and apoptosis. Using Om45-GFP processing assay, we illustrated that yeast Ybh3 mediates a ubiquitin-related mitophagy pathway in both yeast and mammalian cells through association with Hsp70, which is in the same manner with Bim. Moreover, by using Bax/Bak double knockout MEF cells, Ybh3 was identified to induce apoptosis through forming oligomerization to trigger mitochondrial outer membrane permeabilization (MOMP) like Bax. We not only illustrated a conserved ubiquitin-related mitophagy pathway in yeast but also revealed the multi-function of Ybh3 which combines the function of BH3-only protein and multi-domain Bax protein as one. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors (GPCRs) are ubiquitously expressed cell surface receptors that mediate numerous physiological responses and are highly druggable. Upon activation, GPCRs rapidly couple to heterotrimeric G proteins and are then phosphorylated and internalized from the cell surface. Recent studies indicate that GPCRs not only localize at the plasma membrane but also exist in intracellular compartments where they are competent to signal. Intracellular signaling by GPCRs is best described to occur at endosomes. Several studies have elegantly documented endosomal GPCR-G protein and GPCR-β-arrestin signaling. Besides phosphorylation, GPCRs are also posttranslationally modified with ubiquitin. GPCR ubiquitination has been studied mainly in the context of receptor endosomal-lysosomal trafficking. However, new studies indicate that ubiquitination of endogenous GPCRs expressed in endothelial cells initiates the assembly of an intracellular p38 mitogen-activated kinase signaling complex that promotes inflammatory responses from endosomes. In this mini-review, we discuss emerging discoveries that provide critical insights into the function of ubiquitination in regulating GPCR inflammatory signaling at endosomes. [ABSTRACT FROM AUTHOR]

FOXK2 is a crucial transcription factor implicated in a wide array of biological activities and yet understanding of its molecular regulation at the level of protein turnover is limited. Here, we identify that FOXK2 undergoes degradation in lung epithelia in the presence of the virulent pathogens Pseudomonas aeruginosa and Klebsiella pneumoniae through ubiquitin-proteasomal processing. FOXK2 through its carboxyl terminus (aa 428–478) binds the Skp-Cullin-F-box ubiquitin E3 ligase subunit FBXO24 that mediates multisite polyubiquitylation of the transcription factor resulting in its nuclear degradation. FOXK2 was detected within the mitochondria and targeted depletion of the transcription factor or cellular expression of FOXK2 mutants devoid of key carboxy terminal domains significantly impaired mitochondrial function. In experimental bacterial pneumonia, Fbxo24 heterozygous mice exhibited preserved mitochondrial function and Foxk2 protein levels compared to WT littermates. The results suggest a new mode of regulatory control of mitochondrial energetics through modulation of FOXK2 cellular abundance. [ABSTRACT FROM AUTHOR]

Background: Activation of Mas-related G protein-coupled receptor C (MrgC) receptors relieves pain, but also leads to ubiquitination of MrgC receptors. Ubiquitination mediates MrgC receptor endocytosis and degradation. However, MrgC degradation pathways and ubiquitin-linked chain types are not known. Methods: N2a cells were treated with cycloheximide (CHX, protein synthesis inhibitor), Mg132 (proteasome inhibitor), 3-Methyladenine (3MA, autophagy lysosome inhibitor) and Chloroquine (CQ, autophagy lysosome inhibitor) to observe the half-life and degradation pathway of MrgC. The location of internalized MrgC receptors and lysosomes (Lyso-Tracker) was observed by immunofluorescence staining. N2a cells were transfected with Myc-MrgC and a series of HA-tagged ubiquitin mutants to study the ubiquitin-linked chain type of MrgC. Results: The amount of MrgC protein decreased with time after CHX treatment of N2a cells. Autophagy lysosome inhibitors can inhibit the degradation of MrgC. The amount of MrgC protein decreased with time after CHX treatment of N2a cells. 3-MA and CQ inhibited the degradation of MrgC protein, whereas Mg-132 did not inhibit it. Partially internalized MrgC receptors were co-labeled with lysosomes. MrgC proteins have multiple topologies of ubiquitin-modified chains. Conclusion: As a member of the G protein-coupled receptor family, MrgC receptors can be degraded over time. The complex topology of the ubiquitin-linked chain mediates the lysosomal degradation of MrgC proteins. [ABSTRACT FROM AUTHOR]

In plants, the ubiquitin (Ub)-26S proteasome system (UPS) regulates numerous biological functions by selectively targeting proteins for ubiquitylation and degradation. However, the regulation of Ub itself on plant growth and development remains unclear. To demonstrate a possible impact of Ub supply, as seen in animals and flies, we carefully analyzed the growth and developmental phenotypes of two different poly-Ub (UBQ) gene overexpression plants of Arabidopsis thaliana. One is transformed with hexa-6His-UBQ (designated 6HU), driven by the cauliflower mosaic virus 35S promoter, while the other expresses hexa-6His-TEV-UBQ (designated 6HTU), driven by the endogenous promoter of UBQ10. We discovered that 6HU and 6HTU had contrasting seed yields. Compared to wildtype (WT), the former exhibited a reduced seed yield, while the latter showed an increased seed production that was attributed to enhanced growth vigor and an elevated silique number per plant. However, reduced seed sizes were common in both 6HU and 6HTU. Differences in the activity and size of the 26S proteasome assemblies in the two transgenic plants were also notable in comparison with WT, suggestive of a contributory role of UBQ expression in proteasome assembly and function. Collectively, our findings demonstrated that exogenous expression of recombinant Ub may optimize plant growth and development by influencing the UPS activities via structural variance, expression patterns, and abundance of free Ub supply. [ABSTRACT FROM AUTHOR]

The climate-driven challenges facing Earth necessitate a comprehensive understanding of the mechanisms facilitating plant resilience to environmental stressors. This review delves into the crucial role of ubiquitin-like modifiers, particularly focusing on ATG8-mediated autophagy, in bolstering plant tolerance to salt stress. Synthesising recent research, we unveil the multifaceted contributions of ATG8 to plant adaptation mechanisms amidst salt stress conditions, including stomatal regulation, photosynthetic efficiency, osmotic adjustment, and antioxidant defence. Furthermore, we elucidate the interconnectedness of autophagy with key phytohormone signalling pathways, advocating for further exploration into their molecular mechanisms. Our findings underscore the significance of understanding molecular mechanisms underlying ubiquitin-based protein degradation systems and autophagy in salt stress tolerance, offering valuable insights for designing innovative strategies to improve crop productivity and ensure global food security amidst increasing soil salinisation. By harnessing the potential of autophagy and other molecular mechanisms, we can foster sustainable agricultural practices and develop stress-tolerant crops resilient to salt stress. [ABSTRACT FROM AUTHOR]

Tumor cell metastasis is the key cause of death in patients with nasopharyngeal carcinoma (NPC). MiR‐2110 was cloned and identified in Epstein–Barr virus (EBV)‐positive NPC, but its role is unclear in NPC. In this study, we investigated the effect of miR‐2110 on NPC metastasis and its related molecular basis. In addition, we also explored whether miR‐2110 can be regulated by cinobufotalin (CB) and participate in the inhibition of CB on NPC metastasis. Bioinformatics, RT‐PCR, and in situ hybridization were used to observe the expression of miR‐2110 in NPC tissues and cells. Scratch, Boyden, and tail vein metastasis model of nude mouse were used to detect the effect of miR‐2110 on NPC metastasis. Western blot, Co‐IP, luciferase activity, colocalization of micro confocal and ubiquitination assays were used to identify the molecular mechanism of miR‐2110 affecting NPC metastasis. Finally, miR‐2110 induced by CB participates in CB‐stimulated inhibition of NPC metastasis was explored. The data showed that increased miR‐2110 significantly suppresses NPC cell migration, invasion, and metastasis. Suppressing miR‐2110 markedly restored NPC cell migration and invasion. Mechanistically, miR‐2110 directly targeted FGFR1 and reduced its protein expression. Decreased FGFR1 attenuated its recruitment of NEDD4, which downregulated NEDD4‐induced phosphatase and tensin homolog (PTEN) ubiquitination and degradation and further increased PTEN protein stability, thereby inactivating PI3K/AKT‐stimulated epithelial–mesenchymal transition signaling and ultimately suppressing NPC metastasis. Interestingly, CB, a potential new inhibitory drug for NPC metastasis, significantly induced miR‐2110 expression by suppressing PI3K/AKT/c‐Jun‐mediated transcription inhibition. Suppression of miR‐2110 significantly restored cell migration and invasion in CB‐treated NPC cells. Finally, a clinical sample assay indicated that reduced miR‐2110 was negatively correlated with NPC lymph node metastasis and positively related to NPC patient survival prognosis. In summary, miR‐2110 is a metastatic suppressor involving in CB‐induced suppression of NPC metastasis. [ABSTRACT FROM AUTHOR]

Various hormones, kinases, and stressors (fasting, heat shock) stimulate 26S proteasome activity. To understand how its capacity to degrade ubiquitylated proteins can increase, we studied mouse ZFAND5, which promotes protein degradation during muscle atrophy. Cryo-electron microscopy showed that ZFAND5 induces large conformational changes in the 19S regulatory particle. ZFAND5s AN1 Zn-finger domain interacts with the Rpt5 ATPase and its C terminus with Rpt1 ATPase and Rpn1, a ubiquitin-binding subunit. Upon proteasome binding, ZFAND5 widens the entrance of the substrate translocation channel, yet it associates only transiently with the proteasome. Dissociation of ZFAND5 then stimulates opening of the 20S proteasome gate. Using single-molecule microscopy, we showed that ZFAND5 binds ubiquitylated substrates, prolongs their association with proteasomes, and increases the likelihood that bound substrates undergo degradation, even though ZFAND5 dissociates before substrate deubiquitylation. These changes in proteasome conformation and reaction cycle can explain the accelerated degradation and suggest how other proteasome activators may stimulate proteolysis.

Ubiquitin phosphorylation at Ser65 increases the population of a rare C-terminally retracted (CR) conformation. Transition between the Major and CR ubiquitin conformations is critical for promoting mitochondrial degradation. The mechanisms by which the Major and CR conformations of Ser65-phosphorylated (pSer65) ubiquitin interconvert, however, remain unresolved. Here, we perform all-atom molecular dynamics simulations using the string method with swarms of trajectories to calculate the lowest free-energy path between these two conformers. Our analysis reveals the existence of a Bent intermediate in which the C-terminal residues of the β5 strand shift to resemble the CR conformation, while pSer65 retains contacts resembling the Major conformation. This stable intermediate was reproduced in well-tempered metadynamics calculations but was less stable for a Gln2Ala mutant that disrupts contacts with pSer65. Lastly, dynamical network modeling reveals that the transition from the Major to CR conformations involves a decoupling of residues near pSer65 from the adjacent β1 strand.

Synucleinopathies are neurodegenerative disorders characterized by the accumulation of α-synuclein containing Lewy bodies. Ubiquitination, a key post-translational modification, has been recognized as a pivotal regulator of α-synuclein’s cellular dynamics, influencing its degradation, aggregation, and associated neurotoxicity. This review examines comprehensively the current understanding of α-synuclein ubiquitination and its role in the pathogenesis of synucleinopathies, particularly in the context of Parkinson’s disease. We explore the molecular mechanisms responsible for α-synuclein ubiquitination, with a focus on the roles of E3 ligases and deubiquitinases implicated in the degradation process which occurs primarily through the endosomal lysosomal pathway. The review further discusses how the dysregulation of these mechanisms contributes to α-synuclein aggregation and LB formation and offers suggestions for future investigations into the role of α-synuclein ubiquitination. Understanding these processes may shed light on potential therapeutic avenues that can modulate α-synuclein ubiquitination to alleviate its pathological impact in synucleinopathies.

Abstract Ubiquitination is an enzymatic process characterized by the covalent attachment of ubiquitin to target proteins, thereby modulating their degradation, transportation, and signal transduction. By precisely regulating protein quality and quantity, ubiquitination is essential for maintaining protein homeostasis, DNA repair, cell cycle regulation, and immune responses. Nevertheless, the diversity of ubiquitin enzymes and their extensive involvement in numerous biological processes contribute to the complexity and variety of diseases resulting from their dysregulation. The ubiquitination process relies on a sophisticated enzymatic system, ubiquitin domains, and ubiquitin receptors, which collectively impart versatility to the ubiquitination pathway. The widespread presence of ubiquitin highlights its potential to induce pathological conditions. Ubiquitinated proteins are predominantly degraded through the proteasomal system, which also plays a key role in regulating protein localization and transport, as well as involvement in inflammatory pathways. This review systematically delineates the roles of ubiquitination in maintaining protein homeostasis, DNA repair, genomic stability, cell cycle regulation, cellular proliferation, and immune and inflammatory responses. Furthermore, the mechanisms by which ubiquitination is implicated in various pathologies, alongside current modulators of ubiquitination are discussed. Enhancing our comprehension of ubiquitination aims to provide novel insights into diseases involving ubiquitination and to propose innovative therapeutic strategies for clinical conditions.

Ubiquitination typically involves covalent linking of ubiquitin (Ub) to a lysine residue on a protein substrate. Recently, new facets of this process have emerged, including Ub modification of non-proteinaceous substrates like ADP-ribose by the DELTEX E3 ligase family. Here, we show that the DELTEX family member DTX3L expands this non-proteinaceous substrate repertoire to include single-stranded DNA and RNA. Although the N-terminal region of DTX3L contains single-stranded nucleic acid binding domains and motifs, the minimal catalytically competent fragment comprises the C-terminal RING and DTC domains (RD). DTX3L-RD catalyses ubiquitination of the 3’-end of single-stranded DNA and RNA, as well as double-stranded DNA with a 3’ overhang of two or more nucleotides. This modification is reversibly cleaved by deubiquitinases. NMR and biochemical analyses reveal that the DTC domain binds single-stranded DNA and facilitates the catalysis of Ub transfer from RING-bound E2-conjugated Ub. Our study unveils the direct ubiquitination of nucleic acids by DTX3L, laying the groundwork for understanding its functional implications.

Ovarian cancer (OC) is one of the most common malignancies and a leading cause of death among women worldwide. The ubiquitin pathway plays an important role in OC development. Using the single nucleotide polymorphism data obtained using the prevalence and dominance strategies, four ubiquitin marker genes were identified according to their expression levels: BARD1, BRCA2, FANCA, and BRCA1. Based on these four genes, a consensus clustering of OC expression data was performed. The significant differences in the survival analysis, ESTIMATE, and CIBERSORT results among the clusters indicated the pivotal role of these four genes in OC development. Of the ubiquitin-representative genes in each cluster, two ubiquitin genes, TOP2A and MYLIP, were identified in the survival risk model after univariate survival, Least Absolute Shrinkage and Selection Operator regression, and multivariate survival analyses. The reliability and robustness of both the training and validation data were confirmed by comparing the significant survival difference between high- and low-risk patients. We further explored the association between our risk model and clinical outcomes as well as predicted potentially interacting drugs. The co-expression network showed clear interactions among the four marker genes and two model genes and between high- and low-risk differentially expressed genes. Significantly enriched genes were found in pathways associated with ion channels, channel activity, and neuroactive ligand–receptor interactions. Therefore, suggesting the involvement of ubiquitin genes in the survival and development of OC through neurohumoral regulation. Our results will provide valuable reference or supplementary information for studies investigating OC diagnosis and therapies.

Van Gogh-like 2 (Vangl2), a core planar cell polarity component, plays an important role in polarized cellular and tissue morphology induction, growth development, and cancer. However, its role in regulating inflammatory responses remains elusive. Here, we report that Vangl2 is upregulated in patients with sepsis and identify Vangl2 as a negative regulator of The nuclear factor-kappaB (NF-κB) signaling by regulating the protein stability and activation of the core transcription component p65. Mice with myeloid-specific deletion of Vangl2 (Vangl2ΔM) are hypersusceptible to lipopolysaccharide (LPS)-induced septic shock. Vangl2-deficient myeloid cells exhibit enhanced phosphorylation and expression of p65, therefore, promoting the secretion of proinflammatory cytokines after LPS stimulation. Mechanistically, NF-κB signaling-induced-Vangl2 recruits E3 ubiquitin ligase PDLIM2 to catalyze K63-linked ubiquitination on p65, which serves as a recognition signal for cargo receptor NDP52-mediated selective autophagic degradation. Taken together, these findings demonstrate Vangl2 as a suppressor of NF-κB-mediated inflammation and provide insights into the crosstalk between autophagy and inflammatory diseases.

Ubiquitination is one of the most conserved post-translational modifications and together with mRNA translation contributes to cellular protein homeostasis (proteostasis). Temporal and spatial regulation of proteostasis is particularly important during synaptic plasticity, when translation of specific mRNAs requires tight regulation. Mutations in genes encoding regulators of mRNA translation and in ubiquitin ligases have been associated with several neurodevelopmental disorders. RNA metabolism and translation are regulated by RNA-binding proteins, critical for the spatial and temporal control of translation in neurons. Several ubiquitin ligases also regulate RNA-dependent mechanisms in neurons, with numerous ubiquitination events described in splicing factors and ribosomal proteins. Here we will explore how ubiquitination regulates translation in neurons, from RNA biogenesis to alternative splicing and how dysregulation of ubiquitin signaling can be the underlying cause of pathology in neurodevelopmental disorders, such as Fragile X syndrome. Finally we propose that targeting ubiquitin signaling is an attractive novel therapeutic strategy for neurodevelopmental disorders where mRNA translation and ubiquitin signaling are disrupted.

Loss-of-function Parkin mutations lead to early-onset of Parkinson’s disease. Parkin is an auto-inhibited ubiquitin E3 ligase activated by dual phosphorylation of its ubiquitin-like (Ubl) domain and ubiquitin by the PINK1 kinase. Herein, we demonstrate a competitive binding of the phospho-Ubl and RING2 domains towards the RING0 domain, which regulates Parkin activity. We show that phosphorylated Parkin can complex with native Parkin, leading to the activation of autoinhibited native Parkin in trans. Furthermore, we show that the activator element (ACT) of Parkin is required to maintain the enzyme kinetics, and the removal of ACT slows the enzyme catalysis. We also demonstrate that ACT can activate Parkin in trans but less efficiently than when present in the cis molecule. Furthermore, the crystal structure reveals a donor ubiquitin binding pocket in the linker connecting REP and RING2, which plays a crucial role in Parkin activity.

G-Protein Pathway Suppressor 2 (GPS2) is an inhibitor of non-proteolytic K63 ubiquitination mediated by the E2 ubiquitin-conjugating enzyme Ubc13. Previous studies have associated GPS2-mediated restriction of ubiquitination with the regulation of insulin signaling, inflammatory responses and mitochondria-nuclear communication across different tissues and cell types. However, a detailed understanding of the targets of GPS2/Ubc13 activity is lacking. Here, we have dissected the GPS2-regulated K63 ubiquitome in mouse embryonic fibroblasts and human breast cancer cells, unexpectedly finding an enrichment for proteins involved in RNA binding and translation on the outer mitochondrial membrane. Validation of selected targets of GPS2-mediated regulation, including the RNA-binding protein PABPC1 and translation factors RPS1, RACK1 and eIF3M, revealed a mitochondrial-specific strategy for regulating the translation of nuclear-encoded mitochondrial proteins via non-proteolytic ubiquitination. Removal of GPS2-mediated inhibition, either via genetic deletion or stress-induced nuclear translocation, promotes the import-coupled translation of selected mRNAs leading to the increased expression of an adaptive antioxidant program. In light of GPS2 role in nuclear-mitochondria communication, these findings reveal an exquisite regulatory network for modulating mitochondrial gene expression through spatially coordinated transcription and translation.

The ubiquitin-proteasome system (UPS) controls the majority of protein degradation in cells and dysregulation of the UPS has been implicated in the pathophysiology of numerous neurodegenerative disorders, including Alzheimer’s disease. Further, strong evidence supports a critical role for the UPS in synaptic plasticity and memory formation. However, while proteasome function is known to decrease broadly in the brain across the lifespan, whether it changes in the hippocampus, a region critical for memory storage and among the first impacted in Alzheimer’s disease, at rest and following learning in the aged brain remains unknown. Further, which proteins have altered targeting for protein degradation in the aged hippocampus has yet to be explored and whether learning in advanced age interacts with changes in ubiquitin-proteasome function across the lifespan remains unknown. Here, using proteasome activity assays and unbiased proteomic analyses, we report age-dependent changes in proteasome activity and degradation-specific K48 polyubiquitin protein targeting in the hippocampus and retrosplenial cortex of male and female rats across the lifespan. In the hippocampus, the targets of altered protein degradation were involved in transcription and astrocyte structure or G-protein and Interferon signaling in males and females, respectively. Importantly, we found that contextual fear conditioning led to an increase in proteasome activity and K48 polyubiquitin protein targeting in the hippocampus of aged male rats, a result in direct contrast to what was previously reported in young adult animals. Together, these data suggest that changes in protein degradation in the hippocampus across the lifespan may be contributing to age-related memory loss.

Phosphorylation of ubiquitin and the ubiquitin-like domain of Parkin, mediated by the kinase PINK1, is essential for the liberation of the E3 ligase from its autoinhibited state. In this issue of Structure , Lenka et al. 1 provide the structural basis for the specificity and stronger Parkin activation by phospho-NEDD8 compared to phospho-ubiquitin. Phosphorylation of ubiquitin and the ubiquitin-like domain of Parkin, mediated by the kinase PINK1, is essential for the liberation of the E3 ligase from its autoinhibited state. In this issue of Structure , Lenka et al. provide the structural basis for the specificity and stronger Parkin activation by phospho-NEDD8 compared to phospho-ubiquitin. [ABSTRACT FROM AUTHOR]

In a recent issue of Nature , Chambers et al. 1 combined bioinformatics, biochemistry, and X-ray crystallography to uncover the presence of a ubiquitin-like machinery in bacteria, which was believed to be unique to archaea and eukaryotes. This study highlights the prevalence of a ubiquitin-like system in bacteria that was later adopted by the eukaryotes for various purposes such as protein degradation. In a recent issue of Nature , Chambers et al. combined bioinformatics, biochemistry, and X-ray crystallography to uncover the presence of a ubiquitin-like machinery in bacteria, which was believed to be unique to archaea and eukaryotes. This study highlights the prevalence of ubiquitin-like system in bacteria that was later adopted by the eukaryotes for various purposes such as protein degradation. [ABSTRACT FROM AUTHOR]

Endolysosomal defects in neurons are central to the pathogenesis of prion and other neurodegenerative disorders. In prion disease, prion oligomers traffic through the multivesicular body (MVB) and are routed for degradation in lysosomes or for release in exosomes, yet how prions impact proteostatic pathways is unclear. We found that prion-affected human and mouse brain showed a marked reduction in Hrs and STAM1 (ESCRT-0), which route ubiquitinated membrane proteins from early endosomes into MVBs. To determine how the reduction in ESCRT-0 impacts prion conversion and cellular toxicity in vivo, we prion-challenged conditional knockout mice (male and female) having Hrs deleted from neurons, astrocytes, or microglia. The neuronal, but not astrocytic or microglial, Hrs-depleted mice showed a shortened survival and an acceleration in synaptic derangements, including an accumulation of ubiquitinated proteins, deregulation of phosphorylated AMPA and metabotropic glutamate receptors, and profoundly altered synaptic structure, all of which occurred later in the prion-infected control mice. Finally, we found that neuronal Hrs (nHrs) depletion increased surface levels of the cellular prion protein, PrPC, which may contribute to the rapidly advancing disease through neurotoxic signaling. Taken together, the reduced Hrs in the prion-affected brain hampers ubiquitinated protein clearance at the synapse, exacerbates postsynaptic glutamate receptor deregulation, and accelerates neurodegeneration.SIGNIFICANCE STATEMENT Prion diseases are rapidly progressive neurodegenerative disorders characterized by prion aggregate spread through the central nervous system. Early disease features include ubiquitinated protein accumulation and synapse loss. Here, we investigate how prion aggregates alter ubiquitinated protein clearance pathways (ESCRT) in mouse and human prion-infected brain, discovering a marked reduction in Hrs. Using a prion-infection mouse model with neuronal Hrs (nHrs) depleted, we show that low neuronal Hrs is detrimental and markedly shortens survival time while accelerating synaptic derangements, including ubiquitinated protein accumulation, indicating that Hrs loss exacerbates prion disease progression. Additionally, Hrs depletion increases the surface distribution of prion protein (PrPC), linked to aggregate-induced neurotoxic signaling, suggesting that Hrs loss in prion disease accelerates disease through enhancing PrPC-mediated neurotoxic signaling.

HUWE1 is a large, enigmatic HECT-domain ubiquitin ligase implicated in the regulation of diverse pathways, including DNA repair, apoptosis, and differentiation. How HUWE1 engages its structurally diverse substrates and how HUWE1 activity is regulated are unknown. Using unbiased quantitative proteomics, we find that HUWE1 targets substrates in a largely cell-type-specific manner. However, we identify C16orf72/HAPSTR1 as a robust HUWE1 substrate in multiple cell lines. Previously established physical and genetic interactions between HUWE1 and HAPSTR1 suggest that HAPSTR1 positively regulates HUWE1 function. Here, we show that HAPSTR1 is required for HUWE1 nuclear localization and nuclear substrate targeting. Nuclear HUWE1 is required for both cell proliferation and modulation of stress signaling pathways, including p53 and nuclear factor κB (NF-κB)-mediated signaling. Combined, our results define a role for HAPSTR1 in gating critical nuclear HUWE1 functions.

Many G protein-coupled receptors (GPCRs) reside within cilia of mammalian cells and must undergo regulated exit from cilia for the appropriate transduction of signals such as hedgehog morphogens. Lysine 63-linked ubiquitin (UbK63) chains mark GPCRs for regulated removal from cilia, but the molecular basis of UbK63 recognition inside cilia remains elusive. Here, we show that the BBSome-the trafficking complex in charge of retrieving GPCRs from cilia-engages the ancestral endosomal sorting factor target of Myb1-like 2 (TOM1L2) to recognize UbK63 chains within cilia of human and mouse cells. TOM1L2 directly binds to UbK63 chains and the BBSome, and targeted disruption of the TOM1L2/BBSome interaction results in the accumulation of TOM1L2, ubiquitin, and the GPCRs SSTR3, Smoothened, and GPR161 inside cilia. Furthermore, the single-cell alga Chlamydomonas also requires its TOM1L2 ortholog in order to clear ubiquitinated proteins from cilia. We conclude that TOM1L2 broadly enables the retrieval of UbK63-tagged proteins by the ciliary trafficking machinery.

The APOBEC3 (A3) proteins are host antiviral cellular proteins that hypermutate the viral genome of diverse viral families. In retroviruses, this process requires A3 packaging into viral particles1-4. The lentiviruses encode a protein, Vif, that antagonizes A3 family members by targeting them for degradation. Diversification of A3 allows host escape from Vif whereas adaptations in Vif enable cross-species transmission of primate lentiviruses. How this 'molecular arms race' plays out at the structural level is unknown. Here, we report the cryogenic electron microscopy structure of human APOBEC3G (A3G) bound to HIV-1 Vif, and the hijacked cellular proteins that promote ubiquitin-mediated proteolysis. A small surface explains the molecular arms race, including a cross-species transmission event that led to the birth of HIV-1. Unexpectedly, we find that RNA is a molecular glue for the Vif-A3G interaction, enabling Vif to repress A3G by ubiquitin-dependent and -independent mechanisms. Our results suggest a model in which Vif antagonizes A3G by intercepting it in its most dangerous form for the virus-when bound to RNA and on the pathway to packaging-to prevent viral restriction. By engaging essential surfaces required for restriction, Vif exploits a vulnerability in A3G, suggesting a general mechanism by which RNA binding helps to position key residues necessary for viral antagonism of a host antiviral gene.

The AAA+ ATPase Cdc48 utilizes the cofactor Ufd1/Npl4 to bind and thread polyubiquitinated substrates for their extraction from complexes or membranes and often for subsequent proteasomal degradation. Previous studies indicated that Cdc48 engages polyubiquitin chains through the Npl4-mediated unfolding of an initiator ubiquitin; yet, the underlying principles remain largely unknown. Using FRET-based assays, we revealed the mechanisms and kinetics of ubiquitin unfolding, insertion into the ATPase, and unfolding of the ubiquitin-attached substrate. We found that Cdc48 uses Ufd1's UT3 domain to bind a K48-linked ubiquitin on the initiator's proximal side of the chain, thereby directing the initiator toward rapid unfolding by Npl4 and engagement by Cdc48. Ubiquitins on the initiator's distal side increase substrate affinity and facilitate unfolding but impede substrate release from Cdc48-Ufd1/Npl4 in the absence of additional cofactors. Our findings explain how Cdc48-UN efficiently processes substrates with K48-linked chains of 4-6 ubiquitins, which represent most cellular polyubiquitinated proteins.

Abstract Mutations in PINK1 and Parkin cause early-onset Parkinson’s Disease (PD). PINK1 is a kinase which functions as a mitochondrial damage sensor and initiates mitochondrial quality control by accumulating on the damaged organelle. There, it phosphorylates ubiquitin, which in turn recruits and activates Parkin, an E3 ubiquitin ligase. Ubiquitylation of mitochondrial proteins leads to the autophagic degradation of the damaged organelle. Pharmacological modulation of PINK1 constitutes an appealing avenue to study its physiological function and develop therapeutics. In this study, we used a thermal shift assay with insect PINK1 to identify small molecules that inhibit ATP hydrolysis and ubiquitin phosphorylation. PRT062607, an SYK inhibitor, is the most potent inhibitor in our screen and inhibits both insect and human PINK1, with an IC50 in the 0.5–3 µM range in HeLa cells and dopaminergic neurons. The crystal structures of insect PINK1 bound to PRT062607 or CYC116 reveal how the compounds interact with the ATP-binding pocket. PRT062607 notably engages with the catalytic aspartate and causes a destabilization of insert-2 at the autophosphorylation dimer interface. While PRT062607 is not selective for PINK1, it provides a scaffold for the development of more selective and potent inhibitors of PINK1 that could be used as chemical probes.

Abstract Liver cancer is the most prevalent fatal malignancy across the globe. The present study aims to explore the molecular mechanism of E3 ligase WWP1 in liver cancer cell proliferation and invasion/migration. RT‐qPCR and Western blot were performed to detect WWP1, KLF14, and VEPH1 expressions in liver cancer cell lines. Furthermore, WWP1 expression was silenced in cells, followed by the detection of cell viability, proliferation, and invasion/migration by CCK‐8, colony formation, and Transwell assays, respectively. ChIP was used to analyze the binding relationship between WWP1 and KLF14. We measured the KLF14 ubiquitination level and KLF14 enrichment on the VEPH1 promoter after MG132 treatment. Dual‐luciferase reporter assay was used to validate the binding relationship between KLF14 and VEPH1. Consequently, WWP1 was highly expressed in liver cancer cells; WWP1 silencing reduced the proliferation and invasion/migration of liver cancer cells. Mechanistically, WWP1 promoted KLF14 ubiquitination degradation; KLF14 was enriched on the VEPH1 promoter to promote its transcription and protein expression. Inhibiting KLF14 or VEPH1 partially minimized the inhibitory effect of WWP1 silencing on liver cancer cell proliferation and invasion/migration. In summary, WWP1 degrades KLF14 through ubiquitination, hence repressing VEPH1 expression and accelerating proliferation and invasion/migration of liver cancer cells.

Background: Preeclampsia (PE) is a serious pregnancy-specific syndrome associated with the inadequate invasion of trophoblast cells and inflammation of the uterus. A previous study found that lncRNA HOXD-AS1 promotes PE. However, its regulatory network requires additional exploration. Methods: HOXD-AS1-targeted miRNAs and genes were predicted by different databases in a bioinformatics analysis. The expression HOXD-AS1 and its potential m6A methylase (METTL3) were detected in placentas from healthy female patients with PE. The targeting relationship and role of the HOXD-AS1/miR-135a/β-TRCP axis in trophoblast cells were demonstrated by overexpression/knockdown assays. The levels of β-TRCP downstream pathway proteins IκBα, NF-κB, and p65 were measured. The levels of inflammatory factors in cell supernatants were detected by ELISA. To verify the molecular mechanism of β-TRCP in PE, IκBα was co-overexpressed in β-TRCP in trophoblast cells. Results: The levels of METTL3, HOXD-AS1, and β-TRCP were elevated in PE placental tissues, while miR-135a levels were reduced. MiR-135a was found to be targeted by HOXD-AS1, and HOXD-AS1 expression was maintained at a high level by METTL3-mediated m6A methylation. Overexpression of METTL3, HOXD-AS1, and β-TRCP, and knockdown of miR-135a in HTR-8/SVneo cells all inhibited cell invasion and migration, and promoted apoptosis and the secretion of inflammatory factors. Knockdown of METTL3, HOXD-AS1, and β-TRCP, and overexpression of miR-135a had the opposite effects. Furthermore, IκBα expression was negatively associated with β-TRCP expression, and the levels of NF-κB, p65, and NLRP3 were positively regulated by β-TRCP. A high level of β-TRCP expression attenuated the effect of HOXD-AS1 knockdown in trophoblast cells. Conclusion: METTL3 functioned to maintain a high level of HOXD-AS1 expression in PE, which influenced inflammation and the migration and invasion of trophoblast cells via the miR-135a/β-TRCP axis and NF-κB pathway.