Parkinson's disease (PD) is a neurodegenerative disorder that commonly occurs in older individuals and clinically manifests as resting tremors, bradykinesia, muscle stiffness, and impaired postural balance. From a genetic perspective, animal models using gene-editing technologies offer distinct advantages in replicating the pathophysiological traits of PD, while also functionally exploring potential treatment targets. In this review, we highlight the available gene- modified animal models related to various mechanisms of PD, including abnormal expression of alpha-synuclein protein, dysfunction of the autophagy-lysosome system, abnormalities in the ubiquitin-proteasome system, and mitochondrial dysfunction. We further discuss their respective strengths, limitations, and prospects, aiming to provide the most up to date information for the application of PD animal models and the advancement of anti-PD drugs.

Ubiquitination is one of the most well-known post-translational modifications in eukaryotes. UBC13 is an E2 ubiquitin coupling enzyme, which interacts with different E3 ligases and exerts ubiquitination activity to assemble and synthesize lysine-63-linked (Lys63) ubiquitin strands, thus playing an important role in cell homeostasis, various diseases caused by inflammation, and the occurrence and development of cancer. In this paper, we review the structure and function of UBC13, summarize the diverse pathways it mediates, and discuss its involvement in bacterial and non-bacterial inflammatory diseases. Additionally, we explore UBC13's role in physiological damage repair mechanisms, cancer development, DNA damage repair, immune cell maturation, and function. Furthermore, We also elucidate the progress of the discovery of small molecule inhibitors targeting UBC13 and summarize their structure, which suggests that targeting UBC13 may be a potential disease treatment strategy.

The proteostasis network involves complex protein signaling cascades. The tagging of proteins with ubiquitin is central to the degradation of cellular proteins, but understanding its exact role in processing proteins is complicated by the complexity and extent of its utilization within cells. Here, we describe the application of a traceless protein delivery strategy to effect the uptake of exogenous ubiquitin into the cytosol of human cells. We find that coadministration of the endosomolytic peptides L17E and, especially, L17ER4 provides not only cytosolic access to ubiquitin but also its functional incorporation into endogenous proteins. By enabling the study of semisynthetic ubiquitin variants in the human cytosol, this strategy could advance the field of ubiquitin biology.

CXXC5, as a member of the zinc-finger CXXC family proteins, interacts with unmodified CpG dinucleotides to modulate the expression of genes involved in cellular proliferation, differentiation, and death in physiology and pathophysiology. Various signaling pathways, including mitogenic 17β-estradiol (E2), contribute to the expression and synthesis of CXXC5. However, how signaling pathways modulate protein levels of CXXC5 in cells is largely unknown. We previously reported that some key regulators, including retinoblastoma 1 and E74-like ETS transcription factor 1, of the G1 to S phase transitions are involved in the expression of CXXC5 in estrogen-responsive MCF-7 cells, derived from a breast adenocarcinoma. We, therefore, predict that the synthesis of CXXC5 is regulated in a cell cycle-dependent manner. We report here that although E2 in synchronized MCF-7 cells augments both transcription and synthesis of CXXC5 in the G1 phase, CXXC5 protein levels are primarily mediated by ubiquitination independently of cell cycle phases. Utilizing the bioUbiquitination approach, which is based on cellular biotinylation of ubiquitin, in HEK293FT cells derived from immortalized human embryonic kidney cells, followed by sequential immunoprecipitation coupled mass spectrometry analyses, we identified ubiquitinated lysine residues of CXXC5. We show in both MCF-7 and HEK293FT cells that the ubiquitinated lysine residues contribute to the degradation of CXXC5 through the ubiquitin-proteasome pathway.

In 2020, sepsis has been defined a worldwide health major issue (World Health Organization). Lung, urinary tract and abdominal cavity are the preferred sites of sepsis-linked infection. Research has highlighted that the advancement of sepsis is not only related to the presence of inflammation or microbial or host pattern recognition. Clinicians and researchers now recognized that a severe immunosuppression is also a common feature found in patients with sepsis, increasing the susceptibility to secondary infections. Lipopolysaccharides (LPS) are expressed on the cell surface of Gram-negative, whereas Gram-positive bacteria express peptidoglycan (PGN) and lipoteichoic acid (LTA). The main mechanism by which LPS trigger host innate immune responses is binding to TLR4-MD2 (toll-like receptor4-myeloid differentiation factor 2), whereas, PGN and LTA are exogenous ligands of TLR2. Nucleotide-binding oligomerization domain (NOD)-like receptors are the most well-characterized cytosolic pattern recognition receptors, which bind microbial molecules, endogenous by-products and environmental triggers. It has been demonstrated that high-density lipoproteins (HDL), besides their major role in promoting cholesterol efflux, possess diverse pleiotropic properties, ranging from a modulation of the immune system to anti-inflammatory, anti-apoptotic, and anti-oxidant functions. In addition, HDL are able at i) binding LPS, preventing the activating of TLR4, and ii) inducing the expression of ATF3 (Activating transcription factor 3), a negative regulator of the TLR signalling pathways, contributing at justifying their capacity to hamper infection-based illnesses. Therefore, reconstituted HDL (rHDL), constituted by apolipoprotein A-I/apolipoprotein A-IMilano complexed with phospholipids, may be considered as a new therapeutic tool for the management of sepsis.

Numerous bacterial pathogens have evolved tactics to interfere with the host ubiquitination network to evade clearance by the innate immune system. Nevertheless, the subtle antagonism between a bacterial ubiquitinase and a host deubiquitinase, through which they modify their respective targets within a multifaceted network, has yet to be characterized. BRCC3 isopeptidase complex (BRISC) is a newly identified K63-specific deubiquitinase complex that plays a crucial role in cellular signaling pathways such as inflammation. NleG, a type III secretion system (T3SS) effector, contains a conserved RING E3 ubiquitin ligase domain that interacts with host ubiquitination machinery, along with a distinct substrate-recognition domain that targets host proteins. Here, one particular variant, NleG6, was identified as mediating K27- and K29-linked polyubiquitination at residues K89 and K114 of ABRAXAS2/FAM175B, a scaffolding protein within the BRISC complex, leading to its degradation through TOLLIP (toll interacting protein)-mediated selective autophagy. Further investigations elucidated that ABRAXAS2 degradation triggered the subsequent degradation of adjacent BRCC3, which in turn, hindered TNIP1/ABIN1 degradation, ultimately inhibiting NFKB/NF-κB (nuclear factor kappa B)-mediated inflammatory responses. This chain of events offers valuable insights into the NFKB activation by the K63-specific deubiquitinating role of BRISC, unveiling how bacteria manipulate ubiquitin regulation and selective autophagy within the BRISC network to inhibit the host's inflammatory response and thus dominate a pathogen-host tug-of-war.Abbreviations: 3-MA: 3-methyladenine; A/E: attaching and effacing; ATG7: autophagy related 7; BafA1: bafilomycin A1; BNIP3L/Nix: BCL2 interacting protein 3 like; BRISC: BRCC3 isopeptidase complex; Cas9: CRISPR-associated system 9; co-IP: co-immunoprecipitation; CQ: chloroquine; CRISPR: clustered regulatory interspaced short palindromic repeat; DAPI: 4',6-diamidino2-phenylindole; DMSO: dimethyl sulfoxide; DUB: deubiquitinating enzyme; E. coli: Escherichia coli; EHEC: enterohemorrhagic Escherichia coli; EPEC: enteropathogenic Escherichia coli; GFP: green fluorescent protein; LEE: locus of enterocyte effacement; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MG132: cbz-leu-leu-leucinal; MOI: multiplicity of infection; NBR1: NBR1 autophagy cargo receptor; NC: negative control; NFKB/NF-κB: nuclear factor kappa B; NH4Cl: ammonium chloride; OPTN: optineurin; SQSTM1/p62: sequestosome 1; sgRNAs: small guide RNAs; T3SS: type III secretion system; TNF: tumor necrosis factor; TOLLIP: toll interacting protein; TRAF: TNF receptor associated factor; TUBB: tubulin beta class I; WCL: whole cell lysate; WT: wide type.

NUMB is a tumor suppressor gene that functions by inhibiting the action of the NOTCH proto-oncogene and enhancing the levels and activity of the tumor suppressor protein p53. In breast cancer (BC), NUMB loss of function (LOF), mediated by various molecular mechanisms, is a frequent and causal event. Herein, it is established that loss of NUMB protein, resulting from protein hyper-degradation, is the prevalent mechanism of NUMB LOF in BC. Through an RNAi-based screening, the CRL7FBXW8 complex is identified as the E3 ligase complex responsible for NUMB hyper-degradation in BC. Genetic and pharmacological inhibition of CRL7FBXW8 rescues the transformation-related phenotypes induced by NUMB LOF in BC cell lines and in patient-derived xenografts. These effects are directly dependent on the restoration of NUMB protein levels. Thus, enhanced CRL7FBXW8 activity, through its interference with the tumor suppressor activity of NUMB, is a causal alteration in BC, suggesting it as a potential therapeutic target for precision medicine.

AIDS, caused by HIV-1, is a devastating condition that severely compromises the human immune system, often resulting in fatal consequences. The primary therapeutic approach for AIDS involves a combination of multiple agents, known as "cocktail therapy," aimed at maximizing and sustainably suppressing viral replication within patients. The ongoing discovery of novel compounds and the establishment of innovative research strategies have become the mandatory path to provide increasingly effective treatment options for AIDS. Peptide-based fusion inhibitors, exemplified as enfuvirtide, are able to target the six-helix bundle fusion core in HIV-1 envelope protein and function during the early stage of viral invasion. However, the prolonged and intensive use of enfuvirtide in clinical settings has posed significant challenges, including the emergence of drug resistance. N-peptide fusion inhibitors, whose sequences are different from enfuvirtide, exhibit potential anti-HIV-1 activity and inhibition of drug-resistant strains through the advanced coiled-coil conformation and are expected to serve as novel peptide inhibitors in the iteration of enfuvirtide. This paper provides a comprehensive summary of N-peptide fusion inhibitor research and development (R&D) to date, with the aim of providing investigators with prospective ideas for exploring antivirals.

Homologous to E6AP C terminus (HECT) ubiquitin ligases play key roles in essential pathways such as DNA repair, cell cycle control, or protein quality control. Tom1 is one of five HECT ubiquitin E3 ligases in budding yeast S. cerevisiae and is prototypical for a ligase with pleiotropic functions such as ubiquitin chain amplification, orphan quality control, and DNA damage response. Structures of full-length HECT ligases, including the Tom1 ortholog HUWE1, have been reported, but how domains beyond the conserved catalytic module contribute to catalysis remains largely elusive. Here, through cryoelectron microscopy (cryo-EM) snapshots of Tom1 during an active ubiquitination cycle, we demonstrate that the extended domain architecture directly contributes to activity. We identify a Tom1-ubiquitin architecture during ubiquitination involving a non-canonical ubiquitin-binding site in the solenoid shape of Tom1. We demonstrate that this ubiquitin-binding site coordinates a structural ubiquitin contributing to the fidelity of K48 poly-ubiquitin chain assembly.

Tight regulation of gene expression is achieved through the coordinated action of transcription factors and cofactors that often can act as both repressors and activators in response to regulatory signals, with their activity modulated by context-specific signal transduction pathways that also impinge on their transient and cyclical recruitment to chromatin. However, the mechanisms underlying the intricate interplay between the regulatory strategies controlling cofactors' activity and localization across subcellar domains remain poorly understood. Here, we investigated the role of G-Protein Pathway Suppressor 2 (GPS2), a transcriptional cofactor critical for maintaining cellular homeostasis via regulation of mitochondrial biogenesis, stress response, lipid metabolism, insulin signaling, and inflammation, in MCF-7 breast cancer cells. By integration of biochemical assays with genome-wide RNA sequencing and Chromatin immunoprecipitation-Seq analyses, we show that nuclear GPS2 is required for licensing histone deacetylase 3 recruitment to chromatin via restricted ubiquitination by tumor necrosis factor receptor-associated factor 6 (TRAF6), an E3 ubiquitin ligase previously shown to regulate the switch from repressive to activating functions of the nuclear receptor corepressor (NCoR)/silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) complex and here unexpectedly found to translocate to the nucleus in response to IL-1β stimulation. Nuclear TRAF6 is recruited to chromatin via direct interaction with the corepressors NCoR/SMRT, and TRAF6-mediated ubiquitination of TGF-beta activated kinase 1 (MAP3K7) binding protein 2 (TAB2), a facultative component of the NCoR/SMRT complex, contributes to corepressor clearance from target regulatory regions. Together, these results reveal an exquisite mechanism for coordinating the local regulation of cofactor activity with proinflammatory signaling pathways.

Liver cancer is one of the most common malignant tumours in humans, and a large proportion of patients are diagnosed at an advanced stage due to rapid growth and distant metastasis; thus, there is an urgent need to identify critical genes involved in the development of liver cancer. In this study, we investigated the role of RING finger protein 157 (RNF157) in liver cancer proliferation and the related mechanisms. First, we used bioinformatics counting for database mining of RNF157 expression in hepatocellular carcinoma and paracancerous tissues and its relationship with prognosis. We detected the expression level of RNF157 in human samples and different liver cancer cell lines via fluorescence quantitative polymerase chain reaction (Q-PCR), Western blot, and immunohistochemistry (IHC). Subsequently, virus transfection was used to construct knockdown and overexpressed stably transfected cell lines to test the effect of RNF157 on the proliferation of liver cancer. Immunoprecipitation (Co-IP) was used to verify the binding and regulatory relationship between RNF157 and RIG-I/DDX58. We found that RNF157 expression was significantly upregulated in liver cancer tissues and correlated with poor prognosis. In addition, knockdown of RNF157 expression inhibited liver cancer cell proliferation and overexpression of RNF157 promoted liver cancer cell proliferation. Co-IP results revealed that RNF157 binds to and downregulates the expression level of RIG-I/DDX58, further suggesting that RNF157 binds to and ubiquitinates RIG-I/DDX58 at residue 48 of the lysine, which leads to a significant increase in the expression level of RIG-I/DDX58. In this study, we found that RNF157 is a tumour-promoting ubiquitin ligase that promotes liver cancer growth by targeting RIG-I/DDX58 for degradation. Thus, RNF157 has the potential to be a therapeutic target for liver cancer.

Powdery mildew caused by Leveillula taurica adversely affects the development and growth of pepper plants. However, there have been few reports on the fine mapping and quantitative trait locus (QTLs) gene cloning of resistance genes to powdery mildew in pepper. Herein, an F2 segregating population was constructed using the high resistance material "NuMex Suave Red" and the extremely susceptible material "c89" for bulked segregant analysis and DNA re-sequencing (BSA-seq). Molecular markers were used to achieve fine mapping, followed by expression verification. A major QTL located on chromosome 5 (Chr5, 7.20-11.75 Mb) that is associated with resistance to powdery mildew in pepper was mapped using BSA-seq. A narrow interval of 64.86 kb encompassing five genes was refined using InDel and KSAP molecular markers developed from the QTL region. Among them, the expression of the ubiquitin-conjugating enzyme E2 gene, Capana05g000392, was significantly upregulated in multiple resistant materials. In addition, there was a single nucleotide polymorphism (SNP) of A/G in the 241st position of the CDS sequence of Capana05g000392, which in turn leads to an amino acid polymorphism of M/V between susceptible parent and resistant parent. Overall, these results indicate that the Capana05g000392 gene may serve as a robust potential factor against powdery mildew in pepper. These findings further elucidate the genetic mechanism of resistance to powdery mildew in pepper and facilitate molecular marker-assisted breeding.

Jasmonic acid (JA) and gibberellin (GA) coordinate many aspects of plant growth and development, including anthocyanin biosynthesis. However, the crossover points of JA and GA signals and the pathways through which they interact to regulate anthocyanin biosynthesis are poorly understood. Here, we investigated the molecular mechanism by which the zinc finger protein (ZFP) transcription factor Malus domestica ZFP7 (MdZFP7) regulates anthocyanin biosynthesis by integrating JA and GA signals at the transcriptional and post-translational levels. MdZFP7 is a positive regulator of anthocyanin biosynthesis, which fulfills its role by directly activating the expression of MdMYB1 and enhancing the transcriptional activation of MdWRKY6 on the target genes MdDFR and MdUF3GT. MdZFP7 integrates JA and GA signals by interacting with the JA repressor apple JASMONATE ZIM-DOMAIN2 (MdJAZ2) and the GA repressor apple REPRESSOR-of-ga1-3-like 3a (MdRGL3a). MdJAZ2 weakens the transcriptional activation of MdMYB1 by MdZFP7 and disrupts the MdZFP7-MdWRKY6 interaction, thereby reducing the anthocyanin biosynthesis promoted by MdZFP7. MdRGL3a contributes to the stimulation of anthocyanin biosynthesis by MdZFP7 by sequestering MdJAZ2 from the MdJAZ2-MdZFP7 complex. The E3 ubiquitin ligase apple BOI-related E3 ubiquitin-protein ligase 3 (MdBRG3), which is antagonistically regulated by JA and GA, targets the ubiquitination degradation of MdZFP7. The MdBRG3-MdZFP7 module moves the crosstalk of JA and GA signals from the realm of transcriptional regulation and into the protein post-translational modification. In conclusion, this study not only elucidates the node-role of MdZFP7 in the integration of JA and GA signals, but also describes the transcriptional and post-translational regulatory network of anthocyanin biosynthesis with MdZFP7 as the hub.

Wild-type p53 acts as a tumor suppressor, but p53 is frequently mutated and inactivated in tumor cells, promoting cancer progression, invasion, and metastasis. Thus, compounds that reactivate p53 may be leveraged for cancer treatment, and the development of drugs targeting p53 reactivation is actively progressing. Notably, natural products exhibit diverse structures and biological activities and are used as therapeutic agents for various diseases worldwide. This review discusses the natural products that inhibit p53 degradation through p53-Mdm2 interaction, promote p53 reactivation by inducing conformational changes, and exhibit p53-dependent growth inhibition.

Transcription factors, pivotal in gene expression regulation, are essential in cancer progression. Their function is meticulously regulated by post-translational modifications, including ubiquitination. This process, which marks proteins for degradation, can either enhance or inhibit the function of transcription factors, contingent on the context. In cancers, dysregulated ubiquitination of transcription factors contributes to the hallmark of uncontrolled growth and survival of tumors. For example, tumor suppressors such as p53 might be degraded prematurely due to abnormal ubiquitination, causing genomic instability. On the other hand, oncogenic transcription factors may gain stability via ubiquitination, thus facilitating tumorigenesis. Targeting the ubiquitin-proteasome system (UPS) therefore could be a viable therapeutic approach in cancer. Emerging treatments aim to block the ubiquitination of oncogenic transcription factors or to stabilize tumor suppressors. This review underscores the critical impact of transcription factor-altered ubiquitination on cancer progression. Additionally, it outlines innovative therapeutic approaches that involve inhibitors or drugs directed at specific ubiquitin E3 ligases and deubiquitinases (DUBs) that regulate transcription factor activity.

Isopeptide bonds (IPBs)─formed between the amine group of a Lys residue and the carboxamide/carboxy group of Asn/Gln or Asp/Glu─play essential roles in many biological processes, ranging from cellular signaling and regulation to blood clotting and bacterial pathogenesis. The formation of IPBs is not a spontaneous process and requires enzymatic machinery that provides a specialized active site environment to enable this challenging catalytic reaction. Here we report the de novo design and characterization of two proteins (dnIPB-1 and dnIPB-2) capable of autocatalytic IPB formation. While these designed proteins preserve the key active-site residues of their structural template (the bacterial pilin protein RrgA), they possess less than 31% sequence identity to RrgA. Extensive structural and Ala-scanning analyses indicate that IPB formation requires a solvent-protected core motif composed of several critical residues, yet there is also a large tolerance to different protein topologies and overall protein sizes in terms of accommodating an IPB-forming motif. Notably, the structural insights gained from the study of dnIPB-1 and dnIPB-2 also guided the redesign of an initially failed construct (dnIPB-3) and enabled it to form an IPB, highlighting the value of de novo design in examining sequence-structure-function relationships not explored in natural evolution. Our study highlights the versatility of IPBs as designable elements which can be used to construct functional proteins or protein-based materials with enhanced chemical, thermal, and mechanical stabilities.

Inducible protein degradation systems are an important but untapped resource for the study of protein function in plant cells. Unlike mutagenesis or transcriptional control, regulated degradation of proteins of interest allows the study of the biological mechanisms of highly dynamic cellular processes involving essential proteins. While systems for targeted protein degradation are available for research and therapeutics in animals, there are currently limited options in plant biology. Targeted protein degradation systems rely on target ubiquitination by E3 ubiquitin ligases. Systems that are available or being developed in plants can be distinguished primarily by the type of E3 ubiquitin ligase involved, including those that utilize Cullin-RING ligases, bacterial novel E3 ligases, and N-end rule pathway E3 ligases, or they can be controlled by proteolysis targeting chimeras. Target protein ubiquitination leads to degradation by the proteasome or targeting to the vacuole, with both pathways being ubiquitous and important for the endogenous control of protein abundance in plants. Targeted proteolysis approaches for plants will likely be an important tool for basic research and to yield novel traits for crop biotechnology.

The E3 ubiquitin ligase RNF187, also known as RING domain AP1 coactivator-1, is a member of the RING finger family. RNF187 is indispensable for the proliferation and migration of GC-1 cells derived from mouse spermatogonia and GC-2 cells derived from spermatocytes. However, it remains unclear whether RNF187 plays a crucial role in the self-renewal and migration of human spermatogonial stem cells (SSCs). In this study, we observed a positive correlation between RNF187 expression and the proliferation and migration of human SSCs. Through co-immunoprecipitation and mass spectrometry analyses, we identified WD repeat-containing protein 77 (WDR77) as an interacting partner of RNF187. Specifically, RNF187 recognises the K118 site of WDR77 through lysine 48-linked polyubiquitination, subsequently mediating its degradation via the ubiquitin-proteasome system (UPS). Further studies have revealed that decreased expression of WDR77 diminishes the symmetric dimethylation at H4R3 (H4R3me2s) catalysed by its interacting protein, the arginine methyltransferase PRMT5. This, in turn, relieves the transcriptional repression of early growth response protein 1 (EGR1), a positive regulator for human SSC maintenance. In conclusion, this study has unveiled a pivotal role for RNF187 in the proliferation and migration of human SSCs. This may provide a promising strategy for addressing non-obstructive azoospermia (NOA) caused by SSC dysfunction.

Chemical protein synthesis has emerged as a powerful approach for producing ubiquitin (Ub) and ubiquitin-like modifiers (Ubls) in both their free and conjugated forms, particularly when recombinant or enzymatic strategies are challenging. By providing precise control over the assembly of Ub and Ubls, chemical synthesis enables the generation of complex constructs with site-specific modifications that facilitate detailed functional and structural studies. Ub and Ubls are central regulators of protein homeostasis, regulating a wide range of cellular processes such as cell cycle progression, transcription, DNA repair, and apoptosis. Ubls share an evolutionary link with Ub, resembling its structure and following a parallel conjugation pathway that results in a covalent isopeptide bond with their cellular substrates. Despite their structural similarities and sequence homology, Ub and Ubls exhibit distinct functional differences. Understanding Ubl biology is essential for unraveling how cells maintain their regulatory networks and how disruptions in these pathways contribute to various diseases. In this review, we highlight the chemical methodologies and strategies available for studying Ubls and advancing our comprehensive understanding of the Ubl system in health and disease.

Cytoplasmic K63-linked polyubiquitin signals have well-established roles in endocytosis and selective autophagy. However, how these signals help to direct different cargos to different intracellular trafficking routes is unclear. Here we report that, when the K63-polyubiquitin signal is blocked by intracellular expression of a high-affinity sensor (named Vx3), many proteins originating from the plasma membrane are found trapped in clusters of small vesicles that colocalize with ATG9A, a transmembrane protein that plays an essential role in autophagy. Importantly, whereas ATG9A is required for cluster formation, other core autophagy machinery as well as selective autophagy cargo receptors are not required. Although the cargos are sequestered in the vesicular clusters in an ATG9-dependent manner, additional signals are needed to induce LC3 conjugation. Upon removal of the Vx3 block, K63-polyubiquitylated cargos are rapidly delivered to lysosomes. These observations suggest that ATG9A plays an unexpected role in the trafficking of K63-polyubiquitin-modified membrane proteins.

Nitrate is the main source of nitrogen in plants. Nitrate stimulation causes changes in plant secondary metabolites, including anthocyanins. However, the molecular mechanism underlying how nitrate regulates anthocyanin biosynthesis remains unclear. In this study, we identified a nitrate response factor MdLBD36 in apple. This factor positively regulated nitrate deficiency-induced anthocyanin biosynthesis by promoting the transcriptional activity of MdABI5, an important regulator of anthocyanins, and directly activated MdABI5 expression. The E3 ubiquitin ligase MdBRG3 promoted the ubiquitinated degradation of MdLBD36 to reduce anthocyanin biosynthesis under nitrate-sufficient conditions. Nitrate deficiency-activated MdMPK7 maintained the stimulating effect of MdLBD36 on anthocyanin biosynthesis by counteracting the MdBRG3-mediated degradation of MdLBD36. Nitrate coordinated gibberellin (GA) signaling to regulate anthocyanin biosynthesis. The GA signaling repressor MdRGL2a contributed to MdLBD36-promoted anthocyanin biosynthesis by enhancing the MdLBD36-MdABI5 interaction and increasing the MdLBD36 transcriptional activation of MdABI5. In summary, our results elucidate the molecular framework of the coordinated regulation of the nitrate signaling response and anthocyanin biosynthesis by ubiquitination and phosphorylation. This study revealed the cross talk between nitrate and GA signaling in the regulation of anthocyanin biosynthesis and provides references for an in-depth exploration of the nitrate signal transduction pathway and its interactions with hormones.

Rad6 is an E2 ubiquitin-conjugating enzyme that plays critical roles in genome maintenance and proteostasis. Rad6 is frequently overexpressed in many cancers and promotes cancer development, progression, and chemotherapy resistance.

Given its role in cancer development and progression, Rad6 is an underexplored therapeutic target. Previous research identified compound SMI#9 as a small molecule inhibitor of Rad6. Despite its potency, SMI#9 has limited efficacy in vivo due to its limiting water solubility and the presence of a labile ester group.

To address these limitations, we prepared a series of SMI#9 analogs in which the ester group was replaced with a secondary amine, and their effects on Rad6B-mediated ubiquitination of histone H2A were evaluated. In vivo interaction with Rad6 was assessed using cellular thermal shift assays. SMI#9 analog effects on cell survival and migration of triple negative and endocrine-resistant breast cancer, and melanoma cells were measured using MTT and Boyden chamber assays. Autophagy, mitochondrial function, and β-catenin localization were measured using CytoID, Mitotracker, and immunostaining, respectively. Cellular uptakes of analogs were determined by mass spectroscopy.

Analogs #4 and #6 inhibited H2A ubiquitination, induced autophagy and mitochondrial dysfunction, downregulated intracellular β-catenin, and inhibited proliferation. #6 targets Rad6 in vivo. #4 and #6 are chemically related, and #4 undergoes in vivo conversion to #6.

#6 retains all the properties of SMI#9 but with lesser potency. However, its improved water solubility and metabolic stability allows for in vivo studies that were previously precluded due to the poor physicochemical properties of SMI#9.

The tumor microenvironment is a complex environment comprising tumor cells, non-tumor cells, and other critical non-cellular components. Some studies about tumor microenvironment have recently achieved remarkable progress in tumor treatment. As a substantial part of post-translational protein modification, ubiquitination is a crucial player in maintaining protein stability in cell signaling, cell growth, and a series of cellular life activities, which are also essential for regulating tumor cells or other non-tumor cells in the tumor microenvironment. This review focuses on the role and function of ubiquitination and deubiquitination modification in the tumor microenvironment while discussing the prospect of developing inhibitors targeting ubiquity-related enzymes, thereby providing ideas for future research in cancer therapy.

Evolutionary genomic approaches provide powerful tools to understand variation in and evolution of physiological processes. Untargeted genomic or transcriptomic screens can identify functionally annotated candidate genes linked to specific physiological processes, in turn suggesting evolutionary roles for these processes. Such studies often aim to inform modeling of the potential of natural populations to adapt to climate change, but these models are most accurate when evolutionary responses are repeatable, and thus predictable. Here, we synthesize the evolutionary genetic and comparative transcriptomic literature on terrestrial and marine invertebrates to assess whether evolutionary responses to temperature are repeatable within populations, across populations and across species. There is compelling evidence for repeatability, sometimes even across species. However, responses to laboratory selection and geographic variation across thermal gradients appear to be highly idiosyncratic. We also survey whether genetic/transcriptomic studies repeatedly identify candidate genes in three functional groups previously associated with the response to thermal stress: heat shock protein (Hsp) genes, proteolysis genes and immunity genes. Multiple studies across terrestrial and marine species identify candidates included in these gene sets. Yet, each of the gene sets are identified in only a minority of studies. Together, these patterns suggest that there is limited predictability of evolutionary responses to natural selection, including across studies within species. We discuss specific patterns for the candidate gene sets, implications for predictive modeling, and other potential applications of evolutionary genetics in elucidating physiology and gene function. Finally, we discuss limitations of inferences from available evolutionary genetic studies and directions for future research.

The occurrence and progression of traumatic brain injury involve a complex process. The pathophysiological mechanisms triggered by neuronal damage include various forms of programmed cell death, including ferroptosis. We observed upregulation of TNFAIP3 in mice after traumatic brain injury. Overexpression of TNFAIP3 inhibits HT-22 proliferation and cell viability through ferroptosis. Mechanistically, TNFAIP3 interacts with the HMOX1 protein and promotes its stability through the deubiquitination pathway. Additionally, TNFAIP3 can enhance lipoperoxidation, mitochondrial damage, and neuronal cell death by promoting ACSL3 degradation via NEDD4-mediated ubiquitination. Mice injected with AAV-shTNFAIP3 exhibited reduced neuronal degeneration and improved motor and cognitive function following cortical impact injury. In conclusion, our findings demonstrate that TNFAIP3 deficiency inhibits neuronal cell ferroptosis and ameliorates cognitive impairment caused by traumatic brain injury and demonstrate its potential applicability in the treatment of traumatic brain injury.

The E2/E3 hybrid enzyme UBE2O plays important roles in key biological events, but its autoubiquitination mechanism remains largely unclear. In this study, we determined the crystal structures of full-length (FL) UBE2O from Trametes pubescens (tp) and its ubiquitin-conjugating (UBC) domain. The dimeric FL-tpUBE2O structure revealed interdomain interactions between the conserved regions (CR1-CR2) and UBC. The dimeric intermolecular and canonical ubiquitin/UBC interactions are mechanistically important for UBE2O functions in catalyzing the formation of free polyubiquitin chains and substrate ubiquitination. Beyond dimerization, autoubiquitination within the CR1-CR2 domain also regulates tpUBE2O activity. Additionally, we show that tpUBE2O catalyzes the formation of all seven types of polyubiquitin chains in vitro. The CR1-CR2/UBC and canonical ubiquitin/UBC interactions are important for the polyubiquitination of AMP-activated protein kinase α2 (AMPKα2) by human UBE2O (hUBE2O), which leads to tumorigenesis. These structural insights lay the groundwork for understanding UBE2O's mechanisms and developing structure-based therapeutics targeting UBE2O.

Tripartite motif-containing proteins (TRIMs), comprising the greatest subfamily of E3 ubiquitin ligases with approximately 80 members of this family, are widely distributed in mammalian cells. TRIMs actively participate in ubiquitination of target proteins, a type of post-translational modification associated with protein degradation and other functions. Tripartite motif-containing protein 29 (TRIM29), a member of the TRIM family, differs from other members of this family in that it lacks the RING finger structural domain containing cysteine and histidine residues that mediates DNA binding, protein-protein interactions, and ubiquitin ligase, at its N-terminus. The expression of TRIM29 was initially found to be associated with cancer and diabetic nephropathy progression, and antiviral immunity which is triggered by virus-derived nucleic acids binding to pattern recognition receptors (PRRs) on immune cells. Recently, TRIM29 has also been explored as a diagnostic biomarker and therapeutic target for some immune-related diseases. Here, we review the functions of TRIM29 in the progression of diseases and the inherent mechanisms, as well as the remaining gaps in the literature. A thorough understanding of the detailed regulatory mechanisms of TRIM29 will ultimately facilitate the development of different therapeutic strategies for various diseases.

Owing to the scarcity of cultivable land in China, the agricultural sector is primarily focused on grain and oil crops. Simultaneously, the cultivation of cotton has gradually shifted towards regions characterized by elevated soil salinity levels. Additionally, the mechanism behind cotton's ability to tolerate salt remains elusive. In this study, we identified the Z9807 genotype as highly tolerant to salt stress, exhibiting superior leaf wilting resistance, antioxidant activity, catalase activity, K+/Na+ ratio, and growth compared to the salt-sensitive ZJ0102. Comparative transcriptome analysis revealed marked differences in salt stress responses between Z9807 and ZJ0102. This study identified a considerable number of differentially expressed genes associated with salt tolerance across multiple time points. By integration of QTL and GWAS mapping data, we successfully identified 621 candidate genes associated with salt tolerance. Weighted gene correlation network analysis exhibited three co-expression modules related to salt-tolerant Z9807 samples, ultimately identifying 15 core salt-tolerant candidate genes. We also conducted in-depth research on the salt tolerance of the stress-associated protein (SAP) GhSAP6 (GhSAP6_At and GhSAP6_Dt homologs). Results revealed that these candidate genes may inhibit salt tolerance through Virus-Induced Gene Silencing (VIGS) and transgenic overexpression assays conducted in Arabidopsis thaliana. Furthermore, we used yeast two-hybrid and luciferase assay experiments to confirm the ubiquitin degradation pathway between selected interacting proteins and verified the interaction with RAD23C. This study will provide new insights into the mechanisms related to salt tolerance in upland cotton.

Transcripts of the KRAS locus are alternatively spliced to generate two proteins, KRAS4A and KRAS4B, which differ in their membrane-targeting sequences. These splice variants have been conserved for more than 450 million years, suggesting non-overlapping functions driven by differential membrane association. Here, we use proximity labeling to map the differential interactomes of the KRAS splice variants. We find 24 and 10 proteins that interact specifically with KRAS4A or KRAS4B, respectively. The KRAS interacting protein most specific to KRAS4A is BIRC6, a large member of the inhibitor of apoptosis protein family unique in possessing E2/E3 ubiquitin ligase activity. We find that this interaction takes place on the Golgi apparatus and results in the mono- and di-ubiquitination of KRAS4A at lysines 128 and 147. Silencing BIRC6 diminishes GTP loading of and growth stimulation by KRAS4A but not KRAS4B. Thus, BIRC6 is a ubiquitin ligase that inhibits apoptosis and also modifies KRAS4A.

Hakai protein (CBLL1 gene) was identified as an E3 ubiquitin ligase of E-cadherin complex, inducing its ubiquitination and degradation, thus inducing epithelial-to-mesenchymal transition. Most of the knowledge about the protein was associated to its E3 ubiquitin ligase canonical role. However, important recent published research has highlighted the noncanonical role of Hakai, independent of its E3 ubiquitin ligase activity, underscoring its involvement in the N6-methyladenosine (m6A) writer complex and its impact on the methylation of RNA. The involvement of Hakai in this mRNA modification process has renewed the relevance of this protein as an important contributor in cancer. Moreover, Hakai potential as a cancer biomarker and its prognostic value in malignant disease also emphasize its untapped potential in precision medicine, which would also be discussed in detail in our review. The development of the first small-molecule inhibitor that targets its atypical substrate binding domain is a promising step that could eventually lead to patient benefit, and we would cover its discovery and ongoing efforts toward its use in clinic.

The modification of proteins and other biomolecules with the small protein ubiquitin has enthralled scientists from many disciplines for decades, creating a broad research field. Ubiquitin research is particularly rich in molecular and mechanistic understanding due to a plethora of (poly)ubiquitin structures alone and in complex with ubiquitin machineries. Furthermore, due to its favorable properties, ubiquitin serves as a model system for many biophysical and computational techniques. Here, we review the current knowledge of ubiquitin signals through a ubiquitin-centric, structural biology lens. We amalgamate the information from 240 structures in the Protein Data Bank (PDB), combined with single-molecule, molecular dynamics, and nuclear magnetic resonance (NMR) studies, to provide a comprehensive picture of ubiquitin and polyubiquitin structures and dynamics. We close with a discussion of the latest frontiers in ubiquitin research, namely the modification of ubiquitin by other post-translational modifications (PTMs) and the notion that ubiquitin is attached to biomolecules beyond proteins.

Diabetes has a substantial impact on public health, highlighting the need for novel treatments. Ubiquitination, an intracellular protein modification process, is emerging as a promising strategy for regulating pathological mechanisms. We hypothesize that ubiquitination plays a critical role in the development and progression of diabetes and its complications, and that understanding these mechanisms can lead to new therapeutic approaches.

To uncover the research trends and advances in diabetes ubiquitination and its complications, we conducted a bibliometric analysis.

Studies on ubiquitination in diabetes mellitus and its complications were retrieved from the Web of Science Core Collection. Visual mapping analysis was conducted using the CiteSpace software.

We gathered 791 articles published over the past 23 years, focusing on ubiquitination in diabetes and its associated complications. These articles originated from 54 countries and 386 institutions, with China as the leading contributor. Shanghai Jiao Tong University has the highest number of publications in this field. The most prominent authors contributing to this research area include Wei-Hua Zhang, with Zhang Y being the most frequently cited author. Additionally, The Journal of Biological Chemistry is noted as the most cited in this field. The predominant keywords included expression, activation, oxidative stress, phosphorylation, ubiquitination, degradation, and insulin resistance.

The role of ubiquitination in diabetes and its complications, such as diabetic nephropathy and cardiomyopathy, is a key research focus. However, these areas require further investigations.

The 26S proteasome degrades the majority of cellular proteins and affects all aspects of cellular life. Therefore, the 26S proteasome abundance, proper assembly, and activity in different life contexts need to be precisely controlled. Impaired proteasome activity is considered a causative factor in several serious disorders. Recent advances in proteasome biology have revealed that the proteasome can be activated by different factors or small molecules. Thus, activated ubiquitin-dependent proteasome degradation has effects such as extending the lifespan in different models, preventing the accumulation of protein aggregates, and reducing their negative impact on cells. Increased 26S proteasome-mediated degradation reduces proteotoxic stress and can potentially improve the efficacy of engineered degraders, such as PROTACs, particularly in situations characterized by proteasome malfunction. Here, emerging ideas and recent insights into the pharmacological activation of the proteasome at the transcriptional and posttranslational levels are summarized.

Tonne-Kalscheuer syndrome (TOKAS; MIM: 300978) is an X-linked recessive disorder with devastating consequences for patients, such as intellectual disability, developmental delay, and multiple congenital abnormalities. TOKAS is associated with hemizygous variants in the RLIM gene, which encodes a RING-type E3 ubiquitin ligase. The current sustained increase in reported RLIM variants of uncertain significance creates an urgent need to develop assays that can screen these variants and experimentally determine their pathogenicity and disease association. Here, we engineered flow cytometry-based RLIM-specific reporters to measure RLIM activity in TOKAS. This paper describes the design and use of RLIM-specific reporters to determine the pathogenicity of a TOKAS RLIM gene variant. Our data demonstrate that RLIM-specific flow cytometry reporters based on either the full length or a degron region of the substrate REX1 measure RLIM activity in cells. Further, we describe the TOKAS variant RLIM p.Asn581Lys and, using reporter assays, determine that it disrupts RLIM catalytic activity. These data reveal how the p.Asn581Lys variant impairs RLIM function and suggests pathogenic mechanisms. The use of RLIM-specific reporters will greatly accelerate the resolution of variants of uncertain significance and disease association in TOKAS.

Wound healing is a complex biological process involving multiple cellular and molecular mechanisms. Ubiquitination, a crucial post-translational modification, plays a vital role in regulating various aspects of wound healing through protein modification and degradation. This review comprehensively examines the molecular mechanisms of ubiquitination in wound healing, focusing on its regulation of inflammatory responses, macrophage polarization, angiogenesis, and the activities of fibroblasts and keratinocytes. We discuss how ubiquitination modifies key signaling pathways, including TGF-β/Smad3, NF-κB, and HIF-α, which are essential for proper wound healing. Understanding these mechanisms provides insights into potential therapeutic strategies for treating impaired wound healing, particularly in conditions such as diabetes. The review highlights recent advances in understanding ubiquitination's role in wound healing and discusses future research directions for developing targeted therapeutic approaches.

Interferon regulatory factor 3 (IRF3) is a central hub transcription factor that controls host antiviral innate immunity. The expression and function of IRF3 are tightly regulated by the post-translational modifications. However, it is unknown whether unanchored ubiquitination and deubiquitination of IRF3 involve modulating antiviral innate immunity against RNA viruses. Here, we find that USP5, a deubiquitinase (DUB) regulating unanchored polyubiquitin, is downregulated during host anti-RNA viral innate immunity in a type I interferon (IFN-I) receptor (IFNAR)-dependent manner. USP5 is further identified to inhibit IRF3-triggered antiviral immune responses through its DUB enzyme activity. K48-linked unanchored ubiquitin promotes IRF3-driven transcription of IFN-β and induction of IFN-stimulated genes (ISGs) in a dose-dependent manner. USP5 simultaneously removes both K48-linked unanchored and K63-linked anchored polyubiquitin chains on IRF3. Our study not only provides evidence that unanchored ubiquitin regulates anti-RNA viral innate immunity but also proposes a novel mechanism for DUB-controlled IRF3 activation, suggesting that USP5 is a potential target for the treatment of RNA viral infectious diseases.

Ubiquitination is one of the most important post-translational modifications (PTMs) and involves the covalent attachment of ubiquitin to a lysine residue on a target protein. Despite ubiquitination playing a crucial role in regulating cellular processes, the ubiquitinated proteome has not been studied extensively in recombinant Chinese hamster ovary (CHO) cells. Moreover, ubiquitination modification in CHO cells is likely to have an impact on protein function related to the efficient productivity of biopharmaceuticals. In this chapter, we describe a comprehensive protocol for ubiquitin di-Glycine (diGly) peptide enrichment using an immunoprecipitation method from recombinant CHO cell proteins followed by Liquid chromatography-Mass spectrometry (LC-MS) analysis of the ubiquitinated proteome. The methods described are also applicable to differential ubiquitinated proteomic studies.

Lung cancer (LC) is a highly malignant and metastatic form of cancer. The global incidence of and mortality from LC is steadily increasing; the mean 5-year overall survival (OS) rate for LC is less than 20%. This frustrating situation may be attributed to the fact that the pathogenesis of LC remains poorly understood and there is still no cure for mid to advanced LC. Methylation at the N6-position of adenosine (N6mA) of RNA (m(6)A) is widely present in human tissues and organs, and has been found to be necessary for cell development and maintenance of homeostasis. However, numerous basic and clinical studies have demonstrated that RNA m(6)A is deregulated in many human malignancies including LC. This can drive LC malignant characteristics such as proliferation, stemness, invasion, epithelial-mesenchymal transition (EMT), metastasis, and therapeutic resistance. Intriguingly, an increasing number of studies have also shown that eliminating RNA m(6)A dysfunction can exert significant anti-cancer effects on LC such as suppression of cell proliferation and viability, induction of cell death, and reversal of treatment insensitivity. The current review comprehensively discusses the therapeutic potential of RNA m(6)A and its underlying molecular mechanisms in LC, providing useful information for the development of novel LC treatment strategies.

The Pro/N-degron recognizing C-terminal to LisH (CTLH) complex is an E3 ligase of emerging interest in the developmental biology field and for targeted protein degradation (TPD) modalities. The human CTLH complex forms distinct supramolecular ring-shaped structures dependent on the multimerization of WDR26 or muskelin β-propeller proteins. Here, we find that, in HeLa cells, CTLH complex E3 ligase activity is dictated by an interplay between WDR26 and muskelin in tandem with muskelin autoregulation. Proteomic experiments revealed that complex-associated muskelin protein turnover is a major ubiquitin-mediated degradation event dependent on the CTLH complex in unstimulated HeLa cells. We observed that muskelin and WDR26 binding to the scaffold of the complex is interchangeable, indicative of the formation of separate WDR26 and muskelin complexes, which correlated with distinct proteomes in WDR26 and muskelin knockout cells. We found that mTOR inhibition-induced degradation of Pro/N-degron containing protein HMGCS1 is distinctly regulated by a muskelin-specific CTLH complex. Finally, we found that mTOR inhibition also activated muskelin degradation, likely as an autoregulatory feedback mechanism to regulate CTLH complex activity. Thus, rather than swapping substrate receptors, the CTLH E3 ligase complex controls substrate selectivity through the differential association of its β-propeller oligomeric subunits WDR26 and muskelin.