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Glaucoma is chronic optic neuropathy whose pathogenesis has been associated with the altered metabolism of Trabecular Meshwork Cells, which is a cell type involved in the synthesis and remodeling of the trabecular meshwork, the main drainage pathway of the aqueous humor. Starting from previous findings supporting altered ubiquitin signaling, in this study, we investigated the ubiquitin-mediated turnover of myocilin (MYOC/TIGR gene), which is a glycoprotein with a recognized role in glaucoma pathogenesis, in a human Trabecular Meshwork strain cultivated in vitro in the presence of dexamethasone. This is a validated experimental model of steroid-induced glaucoma, and myocilin upregulation by glucocorticoids is a phenotypic marker of Trabecular Meshwork strains. Western blotting and native-gel electrophoresis first uncovered that, in the presence of dexamethasone, myocilin turnover by proteasome particles was slower than in the absence of the drug. Thereafter, co-immunoprecipitation, RT-PCR and gene-silencing studies identified STUB1/CHIP as a candidate E3-ligase of myocilin. In this regard, dexamethasone treatment was found to downregulate STUB1/CHIP levels by likely promoting its proteasome-mediated turnover. Hence, to strengthen the working hypothesis about global alterations of ubiquitin-signaling, the first profiling of TMCs ubiquitylome, in the presence and absence of dexamethasone, was here undertaken by diGLY proteomics. Application of this workflow effectively highlighted a robust dysregulation of key pathways (e.g., phospholipid signaling, β-catenin, cell cycle regulation) in dexamethasone-treated Trabecular Meshwork Cells, providing an ubiquitin-centered perspective around the effect of glucocorticoids on metabolism and glaucoma pathogenesis.

Ubiquitination is an evolutionary, ancient system of post-translational modification of proteins that occurs through a cascade involving ubiquitin activation, transfer, and conjugation. The maturation of this system has followed two main pathways. The first is the conservation of a universal structural fold of ubiquitin and ubiquitin-like proteins, which are present in both Archaea and Bacteria, as well as in multicellular Eukaryotes. The second is the rise of the complexity of the superfamily of ligases, which conjugate ubiquitin-like proteins to substrates, in terms of an increase in the number of enzyme variants, greater variation in structural organization, and the diversification of their catalytic domains. Here, we examine the diversity of the ubiquitination system among different organisms, assessing the variety and conservation of the key domains of the ubiquitination enzymes and ubiquitin itself. Our data show that E2 ubiquitin-conjugating enzymes of metazoan phyla are highly conservative, whereas the homology of E3 ubiquitin ligases with human orthologues gradually decreases depending on “molecular clock” timing and evolutionary distance. Surprisingly, Chordata and Echinodermata, which diverged over 0.5 billion years ago during the Cambrian explosion, share almost the same homology with humans in the amino acid sequences of E3 ligases but not in their adaptor proteins. These observations may suggest that, firstly, the E2 superfamily already existed in its current form in the last common metazoan ancestor and was generally not affected by purifying selection in metazoans. Secondly, it may indicate convergent evolution of the ubiquitination system and highlight E3 adaptor proteins as the “upper deck” of the ubiquitination system, which plays a crucial role in chordate evolution.

The application of artificial intelligence (AI) in the analysis of molecular biology data is becoming increasingly widespread. The Western Blot (WB) technique, a cornerstone in proteomic research, facilitates the identification and analysis of proteins, such as the frameshift mutant of ubiquitin B (UBB+1). In our study, we attempted to assess the potential of four different AI models—Gemini, Gemini Advanced, Microsoft Copilot, and ChatGPT 4—in the analysis of WB imagery containing UBB+1, derived from peripheral blood studies of patients suffering from schizophrenia. Participants, all male and diagnosed with schizophrenia, were recruited from the Specialist Psychiatric Care Team of Babinski Hospital in Lodz. After obtaining their informed consent, blood samples were collected and transported to the laboratory of the Department of Medical Biochemistry at the Medical University of Lodz. The samples were processed, synthesis of Ub-48UBB+1 dimers was performed, and the WB technique was applied. The result of the WB analysis, in the form of a photograph with basic labels but without a legend (JPG format), was implemented into ChatGPT 4, Microsoft Copilot, Gemini and Gemini Advanced. Following the implementation of the image, the command ‘Could you analyze the attached photo?’ was added, along with the protocol from Sample Preparation and Synthesis of Ub-48UBB+1 Dimers. The AI models effectively analyzed and interpreted WB images, with variations in their approaches and depth. Gemini excelled in detailing the WB process and biological significance of bands, while Gemini Advanced focused on specific band identification, especially Ub-48UBB+1 dimers. Microsoft Copilot provided a basic overview with less technicality, and ChatGPT 4 offered comprehensive band interpretations, linking them to patient samples and standards, thus confirming the hypothesis about the differing capabilities of these models. This discovery demonstrates the advanced capabilities of ChatGPT 4 and highlights the growing role of AI in scientific research, including the interpretation of results.

Protein ubiquitination is involved in nearly all biological processes in Eukaryotes. To gain precise insights into the function of ubiquitination in these processes, researchers frequently employ ubiquitinated protein probes with well-defined structures. While chemical protein synthesis has afforded a variety of ubiquitinated protein probes, there remains a demand for efficient synthesis methods for complex probes, such as ubiquitinated glycoproteins and ubiquitinated cysteine-containing proteins. In this study, we introduce a new method to obtain ubiquitinated proteins through isopeptide bond formation mediated by δ-selenolysine residues. We synthesized δ-selenolysine derivatives in both L- and D-forms starting from DL-δ-hydroxy-DL-lysine, accomplished by substituting the δ-mesylate with KSeCN and by enzymatic optical resolution with L- and D-aminoacylase. We synthesized ubiquitin (46–76)-α-hydrazide with a δ-seleno-L-lysine residue at position 48, as well as ubiquitin (46–76)-α-thioester, using solid-phase peptide synthesis. Subsequently, the δ-selenolysine-mediated ligation of these peptides, followed by one-pot deselenization, provided the desired isopeptide-linked ubiquitin peptide. The new δ-selenolysine-mediated isopeptide bond formation offers an alternative method to obtain complex ubiquitin- and ubiquitin-like probes with multiple post-translational modifications. These probes hold promise for advancing our understanding of ubiquitin biology.

Cystic fibrosis (CF) is a genetic disorder caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a selective anion channel expressed in the epithelium of various organs. The most frequent mutation is F508del. This mutation leads to a misfolded CFTR protein quickly degraded via ubiquitination in the endoplasmic reticulum. Although preventing ubiquitination stabilizes the protein, functionality is not restored due to impaired plasma membrane transport. However, inhibiting the ubiquitination process can improve the effectiveness of correctors which act as chemical chaperones, facilitating F508del CFTR trafficking to the plasma membrane. Previous studies indicate a crosstalk between SUMOylation and ubiquitination in the regulation of CFTR. In this study, we investigated the potential of inhibiting SUMOylation to increase the effects of correctors and enhance the rescue of the F508del mutant across various cell models. In the widely used CFBE41o-cell line expressing F508del-CFTR, inhibiting SUMOylation substantially boosted F508del expression, thereby increasing the efficacy of correctors. Interestingly, this outcome did not result from enhanced stability of the mutant channel, but rather from augmented cytomegalovirus (CMV) promoter-mediated gene expression of F508del-CFTR. Notably, CFTR regulated by endogenous promoters in multiple cell lines or patient cells was not influenced by SUMOylation inhibitors.

Ubiquitin chains are flexible multidomain proteins that have important biological functions in cellular signalling. Computational studies with all-atom molecular dynamics simulations of the conformational spaces of polyubiquitins can be challenging due to the system size and a multitude of long-lived meta-stable states. Coarse graining is an efficient approach to overcome this problem—at the cost of losing high-resolution details. Recently, we proposed the back-mapping based sampling (BMBS) approach that reintroduces atomistic information into a given coarse grained (CG) sampling based on a two-dimensional (2D) projection of the conformational landscape, produces an atomistic ensemble and allows to systematically compare the ensembles at the two levels of resolution. Here, we apply BMBS to K48-linked tri-ubiquitin, showing its applicability to larger systems than those it was originally introduced on and demonstrating that the algorithm scales very well with system size. In an extension of the original BMBS we test three different seeding strategies, i.e. different approaches from where in the CG landscape atomistic trajectories are initiated. Furthermore, we apply a recently introduced conformational clustering algorithm to the back-mapped atomistic ensemble. Thus, we obtain insight into the structural composition of the 2D landscape and illustrate that the dimensionality reduction algorithm separates different conformational characteristics very well into different regions of the map. This cluster analysis allows us to show how atomistic trajectories sample conformational states, move through the projection space and in sum converge to an atomistic conformational landscape that slightly differs from the original CG map, indicating a correction of flaws in the CG template.

The overactivity of keratinocyte cytoplasmic signaling contributes to several cutaneous inflammatory and immune pathologies. An important emerging complement to proteins responsible for this overactivity is signal repression brought about by several proteins and protein complexes with the native role of limiting inflammation. The signaling repression by these proteins distinguishes them from transmembrane receptors, kinases, and inflammasomes, which drive inflammation. For these proteins, defects or deficiencies, whether naturally arising or in experimentally engineered skin inflammation models, have clearly linked them to maintaining keratinocytes in a non-activated state or returning cells to a post-inflamed state after a signaling event. Thus, together, these proteins help to resolve acute inflammatory responses or limit the development of chronic cutaneous inflammatory disease. We present here an integrated set of demonstrated or potentially inflammation-repressive proteins or protein complexes (linear ubiquitin chain assembly complex [LUBAC], cylindromatosis lysine 63 deubiquitinase [CYLD], tumor necrosis factor alpha-induced protein 3-interacting protein 1 [TNIP1], A20, and OTULIN) for a comprehensive view of cytoplasmic signaling highlighting protein players repressing inflammation as the needed counterpoints to signal activators and amplifiers. Ebb and flow of players on both sides of this inflammation equation would be of physiological advantage to allow acute response to damage or pathogens and yet guard against chronic inflammatory disease. Further investigation of the players responsible for repressing cytoplasmic signaling would be foundational to developing new chemical-entity pharmacologics to stabilize or enhance their function when clinical intervention is needed to restore balance.

The size of seeds is particularly important for agricultural development, as it is a key trait that determines yield. It is controlled by the coordinated development of the integument, endosperm, and embryo. Large seeds are an important way of improving the ultimate “sink strength” of crops, providing more nutrients for early plant growth and showing certain tolerance to abiotic stresses. There are several pathways for regulating plant seed size, including the HAIKU (IKU) pathway, ubiquitin–proteasome pathway, G (Guanosine triphosphate) protein regulatory pathway, mitogen-activated protein kinase (MAPK) pathway, transcriptional regulators pathway, and phytohormone regulatory pathways including the auxin, brassinosteroid (BR), gibberellin (GA), jasmonic acid (JA), cytokinin (CK), Abscisic acid (ABA), and microRNA (miRNA) regulatory pathways. This article summarizes the seed size regulatory network and prospective ways of improving yield. We expect that it will provide a valuable reference to researchers in related fields.

The extracellular ubiquitin–proteasome system is involved in sperm binding to and/or penetration of the vitelline coat (VC), a proteinaceous egg coat, during fertilization of the ascidian (Urochordata) Halocynthia roretzi. It is also known that the sperm receptor on the VC, HrVC70, is ubiquitinated and degraded by the sperm proteasome during the sperm penetration of the VC and that a 700-kDa ubiquitin-conjugating enzyme complex is released upon sperm activation on the VC, which is designated the “sperm reaction”. However, the de novo function of ubiquitin-activating enzyme (UBA/E1) during fertilization is poorly understood. Here, we show that PYR-41, a UBA inhibitor, strongly inhibited the fertilization of H. roretzi. cDNA cloning of UBA1 and UBA6 from H. roretzi gonads was carried out, and their 3D protein structures were predicted to be very similar to those of human UBA1 and UBA6, respectively, based on AlphaFold2. These two genes were transcribed in the ovary and testis and other organs, among which the expression of both was highest in the ovary. Immunocytochemistry showed that these enzymes are localized on the sperm head around a mitochondrial region and the follicle cells surrounding the VC. These results led us to propose that HrUBA1, HrUBA6, or both in the sperm head mitochondrial region and follicle cells may be involved in the ubiquitination of HrVC70, which is responsible for the fertilization of H. roretzi.

The loss of the E3 ligase Parkin, in a familial form of Parkinson’s disease, is thought to cause the failure of both the polyubiquitination of abnormal mitochondria and the consequent induction of mitophagy, resulting in abnormal mitochondrial accumulation. However, this has not been confirmed in patient autopsy cases or animal models. More recently, the function of Parkin as a redox molecule that directly scavenges hydrogen peroxide has attracted much attention. To determine the role of Parkin as a redox molecule in the mitochondria, we overexpressed various combinations of Parkin, along with its substrates FAF1, PINK1, and ubiquitin in cell culture systems. Here, we observed that the E3 Parkin monomer was surprisingly not recruited to abnormal mitochondria but self-aggregated with or without self-ubiquitination into the inner and outer membranes, becoming insoluble. Parkin overexpression alone generated aggregates without self-ubiquitination, but it activated autophagy. These results suggest that for damaged mitochondria, the polyubiquitination of Parkin substrates on the mitochondria is not indispensable for mitophagy.

Protein ubiquitylation acts as a complex cell signaling mechanism since the formation of different mono- and polyubiquitin chains determines the substrate’s fate in the cell. E3 ligases define the specificity of this reaction by catalyzing the attachment of ubiquitin to the substrate protein. Thus, they represent an important regulatory component of this process. Large HERC ubiquitin ligases belong to the HECT E3 protein family and comprise HERC1 and HERC2 proteins. The physiological relevance of the Large HERCs is illustrated by their involvement in different pathologies, with a notable implication in cancer and neurological diseases. Understanding how cell signaling is altered in these different pathologies is important for uncovering novel therapeutic targets. To this end, this review summarizes the recent advances in how the Large HERCs regulate the MAPK signaling pathways. In addition, we emphasize the potential therapeutic strategies that could be followed to ameliorate the alterations in MAPK signaling caused by Large HERC deficiencies, focusing on the use of specific inhibitors and proteolysis-targeting chimeras.

The WD40 repeat-containing F-box proteins (FBXWs) family belongs to three major classes of F-box proteins. Consistent with the function of other F-box proteins, FBXWs are E3 ubiquitin ligases to mediate protease-dependent protein degradation. However, the roles of several FBXWs remain elusive. In the present study, via integrative analysis of transcriptome profiles from The Cancer Genome Atlas (TCGA) datasets, we found that FBXW9 was upregulated in the majority of cancer types, including breast cancer. FBXW expression was correlated with the prognosis of patients with various types of cancers, especially for FBXW4, 5, 9, and 10. Moreover, FBXWs were associated with infiltration of immune cells, and expression of FBXW9 was associated with poor prognosis of patients receiving anti-PD1 therapy. We predicted several substrates of FBXW9, and TP53 was the hub gene in the list. Downregulation of FBXW9 increased the expression of p21, a target of TP53, in breast cancer cells. FBXW9 was also strongly correlated with cancer cell stemness, and genes correlated with FBXW9 were associated with several MYC activities according to gene enrichment analysis in breast cancer. Cell-based assays showed that silencing of FBXW9 inhibited cell proliferation and cell cycle progression in breast cancer cells. Our study highlights the potential role of FBXW9 as a biomarker and promising target for patients with breast cancer.

The ubiquitin-26S proteasome system and autophagy are two major protein degradation machineries encoded in all eukaryotic organisms. While the UPS is responsible for the turnover of short-lived and/or soluble misfolded proteins under normal growth conditions, the autophagy-lysosomal/vacuolar protein degradation machinery is activated under stress conditions to remove long-lived proteins in the forms of aggregates, either soluble or insoluble, in the cytoplasm and damaged organelles. Recent discoveries suggested an integrative function of these two seemly independent systems for maintaining the proteome homeostasis. One such integration is represented by their reciprocal degradation, in which the small 76-amino acid peptide, ubiquitin, plays an important role as the central signaling hub. In this review, we summarized the current knowledge about the activity control of proteasome and autophagosome at their structural organization, biophysical states, and turnover levels from yeast and mammals to plants. Through comprehensive literature studies, we presented puzzling questions that are awaiting to be solved and proposed exciting new research directions that may shed light on the molecular mechanisms underlying the biological function of protein degradation.

The ubiquitin pathway, one of the main actors regulating cell signaling processes and cellular protein homeostasis, is directly involved in the pathophysiology of amyotrophic lateral sclerosis (ALS). We first analyzed, by a next-generation sequencing (NGS) strategy, a series of genes of the ubiquitin pathway in two cohorts of familial and sporadic ALS patients comprising 176 ALS patients. We identified several pathogenic variants in different genes of this ubiquitin pathway already described in ALS, such as FUS, CCNF and UBQLN2. Other variants of interest were discovered in new genes studied in this disease, in particular in the HECW1 gene. We have shown that the HECT E3 ligase called NEDL1, encoded by the HECW1 gene, is expressed in neurons, mainly in their somas. Its overexpression is associated with increased cell death in vitro and, very interestingly, with the cytoplasmic mislocalization of TDP-43, a major protein involved in ALS. These results give new support for the role of the ubiquitin pathway in ALS, and suggest further studies of the HECW1 gene and its protein NEDL1 in the pathophysiology of ALS.

Classical molecular-dynamics simulations have been performed to examine the interplay between ubiquitin and its hydration-water sub-layers, chiefly from a vibrational-mode and IR viewpoint—where we analyse individual sub-layers characteristics. The vibrational Density of States (VDOS) revealed that the first solvation sub-shell indicates a confined character therein. For layers of increasing distance from the surface, the adoption of greater bulk-like spectral behaviour was evident, suggesting that vibrational harmonisation to bulk occurs within 6–7 Å of the surface.

RNA-binding proteins (RBPs) and RNAs can form dynamic, liquid droplet-like cytoplasmic condensates, known as stress granules (SGs), in response to a variety of cellular stresses. This process is driven by liquid–liquid phase separation, mediated by multivalent interactions between RBPs and RNAs. The formation of SGs allows a temporary suspension of certain cellular activities such as translation of unnecessary proteins. Meanwhile, non-translating mRNAs may also be sequestered and stalled. Upon stress removal, SGs are disassembled to resume the suspended biological processes and restore the normal cell functions. Prolonged stress and disease-causal mutations in SG-associated RBPs can cause the formation of aberrant SGs and/or impair SG disassembly, consequently raising the risk of pathological protein aggregation. The machinery maintaining protein homeostasis (proteostasis) includes molecular chaperones and co-chaperones, the ubiquitin-proteasome system, autophagy, and other components, and participates in the regulation of SG metabolism. Recently, proteostasis has been identified as a major regulator of SG turnover. Here, we summarize new findings on the specific functions of the proteostasis machinery in regulating SG disassembly and clearance, discuss the pathological and clinical implications of SG turnover in neurodegenerative disorders, and point to the unresolved issues that warrant future exploration.

Endoplasmic reticulum stress (ERS) is closely related to the occurrence and progression of metabolic liver disease. The treatment targeting glucose-regulated protein 94 (GRP94) for liver disease has gotten much attention, but the specific effect of GRP94 on hepatocyte apoptosis is still unclear. So far, all the studies on GRP94 have been conducted in mice or rats, and little study has been reported on pigs, which share more similarities with humans. In this study, we used low-dose (LD) and high-dose (HD) tunicamycin (TM) to establish ERS models on piglet livers and immortalized porcine hepatic stellate cells (HSCs). On the piglet ERS model we found that ERS could significantly (p < 0.01) stimulate the secretion and synthesis of insulin-like growth factor (IGF-1), IGF-1 receptor (IGF-1R), and IGF-binding protein (IGFBP)-1 and IGFBP-3; however, with the increase in ERS degree, the effect of promoting secretion and synthesis significantly (p < 0.01) decreased. In addition, the ubiquitin protein and ubiquitination-related gene were significantly increased (p < 0.05) in the LD group compared with the vehicle group. The protein level of Active-caspase 3 was significantly increased (p < 0.01) in the HD group, however, the TUNEL staining showed there was no significant apoptosis in the piglet liver ERS model. To explore the biofunction of ER chaperone GRP94, we used shRNA to knock down the expression of GRP94 in porcine HSCs. Interestingly, on porcine HSCs, the knockdown of GRP94 significantly (p < 0.05) decreased the secretion of IGF-1, IGFBP-1 and IGFBP-3 under ERS, but had no significant effect on these under normal condition, and knockdown GRP94 had a significant (p < 0.01) effect on the UBE2E gene and ubiquitin protein from the analysis of two-way ANOVA. On porcine HSCs apoptosis, the knockdown of GRP94 increased the cell apoptosis in TUNEL staining, and the two-way ANOVA analysis shows that knockdown GRP94 had a significant (p < 0.01) effect on the protein levels of Bcl-2 and Caspase-3. For CCK-8 assay, ERS had a significant inhibitory(p < 0.05) effect on cell proliferation when treated with ERS for 24 h, and both knockdown GRP94 and ERS had a significant inhibitory(p < 0.05) effect on cell proliferation when treated with ERS for 36 h and 48 h. We concluded that GRP94 can protect the cell from ERS-induced apoptosis by promoting the IGF-1 system and ubiquitin. These results provide valuable information on the adaptive mechanisms of the liver under ERS, and could help identify vital functional genes to be applied as possible diagnostic biomarkers and treatments for diseases induced by ERS in the future.

Injury to the central nervous system (CNS) results in permanent damage and lack of function in most vertebrate animals, due to their limited regenerative capacities. In contrast, echinoderms can fully regenerate their radial nerve cord (RNC) following transection, with little to no scarring. Investigators have associated the regenerative capacity of some organisms to the stress response and inflammation produced by the injury. Here, we explore the gene activation profile of the stressed holothurian CNS. To do this, we performed RNA sequencing on isolated RNC explants submitted to the stress of transection and enzyme dissection and compared them with explants kept in culture for 3 days following dissection. We describe stress-associated genes, including members of heat-shock families, ubiquitin-related pathways, transposons, and apoptosis that were differentially expressed. Surprisingly, the stress response does not induce apoptosis in this system. Other genes associated with stress in other animal models, such as hero proteins and those associated with the integrated stress response, were not found to be differentially expressed either. Our results provide a new viewpoint on the stress response in the nervous system of an organism with amazing regenerative capacities. This is the first step in deciphering the molecular processes that allow echinoderms to undergo fully functional CNS regeneration, and also provides a comparative view of the stress response in other organisms.

Chloroplasts are ancient organelles responsible for photosynthesis and various biosynthetic functions essential to most life on Earth. Many of these functions require tightly controlled regulatory processes to maintain homeostasis at the protein level. One such regulatory mechanism is the ubiquitin-proteasome system whose fundamental role is increasingly emerging in chloroplasts. In particular, the role of E3 ubiquitin ligases as determinants in the ubiquitination and degradation of specific intra-chloroplast proteins. Here, we highlight recent advances in understanding the roles of plant E3 ubiquitin ligases SP1, COP1, PUB4, CHIP, and TT3.1 as well as the ubiquitin-dependent segregase CDC48 in chloroplast function.

Ubiquitin (ub) is a small, highly conserved protein widely expressed in eukaryotic cells. Ubiquitination is a post-translational modification catalyzed by enzymes that activate, conjugate, and ligate ub to proteins. Substrates can be modified either by addition of a single ubiquitin molecule (monoubiquitination), or by conjugation of several ubs (polyubiquitination). Monoubiquitination acts as a signaling mark to control diverse biological processes. The cellular and spatial distribution of ub is determined by the opposing activities of ub ligase enzymes, and deubiquitinases (DUBs), which remove ub from proteins to generate free ub. In mammalian cells, 1–2% of total histone H2B is monoubiquitinated. The SAGA (Spt Ada Gcn5 Acetyl-transferase) is a transcriptional coactivator and its DUB module removes ub from H2Bub1. The mammalian SAGA DUB module has four subunits, ATXN7, ATXN7L3, USP22, and ENY2. Atxn7l3−/− mouse embryos, lacking DUB activity, have a five-fold increase in H2Bub1 retention, and die at mid-gestation. Interestingly, embryos lacking the ub encoding gene, Ubc, have a similar phenotype. Here we provide a current overview of data suggesting that H2Bub1 retention on the chromatin in Atxn7l3−/− embryos may lead to an imbalance in free ub distribution. Thus, we speculate that ATXN7L3-containing DUBs impact the free cellular ub pool during development.

In all eukaryotes, autophagy is the main pathway for nutrient recycling, which encapsulates parts of the cytoplasm and organelles in double-membrane vesicles, and then fuses with lysosomes/vacuoles to degrade them. Autophagy is a highly dynamic and relatively complex process influenced by multiple factors. Under normal growth conditions, it is maintained at basal levels. However, when plants are subjected to biotic and abiotic stresses, such as pathogens, drought, waterlogging, nutrient deficiencies, etc., autophagy is activated to help cells to survive under stress conditions. At present, the regulation of autophagy is mainly reflected in hormones, second messengers, post-transcriptional regulation, and protein post-translational modification. In recent years, the degradation mechanism of autophagy-related proteins has attracted much attention. In this review, we have summarized how autophagy-related proteins are degraded in yeast, animals, and plants, which will help us to have a more comprehensive and systematic understanding of the regulation mechanisms of autophagy. Moreover, research progress on the degradation of autophagy-related proteins in plants has been discussed.

The potency and selectivity of a small molecule inhibitor are key parameters to assess during the early stages of drug discovery. In particular, it is very informative for characterizing compounds in a relevant cellular context in order to reveal potential off-target effects and drug efficacy. Activity-based probes are valuable tools for that purpose, however, obtaining cellular target engagement data in a high-throughput format has been particularly challenging. Here, we describe a new methodology named ABPP-HT (high-throughput-compatible activity-based protein profiling), implementing a semi-automated proteomic sample preparation workflow that increases the throughput capabilities of the classical ABPP workflow approximately ten times while preserving its enzyme profiling characteristics. Using a panel of deubiquitylating enzyme (DUB) inhibitors, we demonstrate the feasibility of ABPP-HT to provide compound selectivity profiles of endogenous DUBs in a cellular context at a fraction of time as compared to previous methodologies.

Flow cytometry becomes a common method for analysis of spermatozoa quality. Standard sperm characteristics such as viability, acrosome and chromatin integrity, oxidative damage (ROS) etc. can be easily assess in any animal semen samples. Moreover, several fertility-related markers were observed in humans and some other mammals. However, these fertility biomarkers have not been previously studied in ram. The aim of this study was to optimize the flow-cytometric analysis of these standard and novel markers in ram semen. Ram semen samples from Slovak native sheep breeds were analyzed using CASA system for motility and concentration and were subsequently stained with several fluorescent dyes or specific antibodies to evaluate sperm viability (SYBR-14), apoptosis (Annexin V, YO-PRO-1, FLICA, Caspases 3/7), acrosome status (PNA, LCA, GAPDHS), capacitation (merocyanine 540, FLUO-4 AM), mitochondrial activity (MitoTracker Green, rhodamine 123, JC-1), ROS (CM-H2DCFDA, DHE, MitoSOX Red, BODIPY), chromatin (acridine orange), leukocyte content, ubiquitination and aggresome formation, and overexpression of negative biomarkers (MKRN1, SPTRX-3, PAWP, H3K4me2). Analyzed semen samples were divided into two groups according to viability as indicators of semen quality: Group 1 (viability over 60%) and Group 2 (viability under 60%). Significant (p < 0.05) differences were found between these groups in sperm motility and concentration, apoptosis, acrosome integrity (only PNA), mitochondrial activity, ROS production (except for DHE), leukocyte and aggresome content, and high PAWP expression. In conclusion, several standard and novel fluorescent probes have been confirmed to be suitable for multiplex ram semen analysis by flow cytometry as well as several antibodies have been validated for the specific detection of ubiquitin, PAWP and H3K4me2 in ram spermatozoa.

Post-translational modification (PTM) is an essential mechanism for enhancing the functional diversity of proteins and adjusting their signaling networks. The reversible conjugation of ubiquitin (Ub) and ubiquitin-like proteins (Ubls) to cellular proteins is among the most prevalent PTM, which modulates various cellular and physiological processes by altering the activity, stability, localization, trafficking, or interaction networks of its target molecules. The Ub/Ubl modification is tightly regulated as a multi-step enzymatic process by enzymes specific to this family. There is growing evidence that the dysregulation of Ub/Ubl modifications is associated with various diseases, providing new targets for drug development. In this review, we summarize the recent progress in understanding the roles and therapeutic targets of the Ub and Ubl systems in the onset and progression of human diseases, including cancer, neurodegenerative disorders, and heart diseases.

Both ubiquitination and SUMOylation are dynamic post-translational modifications that regulate thousands of target proteins to control virtually every cellular process. Unfortunately, the detailed mechanisms of how all these cellular processes are regulated by both modifications remain unclear. Target proteins can be modified by one or several moieties, giving rise to polymers of different morphology. The conjugation cascades of both modifications comprise a few activating and conjugating enzymes but close to thousands of ligating enzymes (E3s) in the case of ubiquitination. As a result, these E3s give substrate specificity and can form polymers on a target protein. Polymers can be quickly modified forming branches or cleaving chains leading the target protein to its cellular fate. The recent development of mass spectrometry(MS) -based approaches has increased the understanding of ubiquitination and SUMOylation by finding essential modified targets in particular signaling pathways. Here, we perform a concise overview comprising from the basic mechanisms of both ubiquitination and SUMOylation to recent MS-based approaches aimed to find specific targets for particular E3 enzymes.

The ubiquitin pathway regulates the function of many proteins and controls cellular protein homeostasis. In recent years, it has attracted great interest in neurodevelopmental and neurodegenerative diseases. Here, we have presented the first review on the roles of the 9 proteins of the HECT E3 ligase NEDD4 subfamily in the development and function of neurons in the central nervous system (CNS). We discussed their regulation and their direct or indirect involvement in neurodevelopmental diseases, such as intellectual disability, and neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease or Amyotrophic Lateral Sclerosis. Further studies on the roles of these proteins, their regulation and their targets in neurons will certainly contribute to a better understanding of neuronal function and dysfunction, and will also provide interesting information for the development of therapeutics targeting them.

Many neurodegenerative disorders including Huntington's Disease are hallmarked by intracellular protein aggregates that are decorated by ubiquitin and different ubiquitin ligases and deubiquitinating enzymes. The protein aggregates observed in Huntington's Disease are caused by a polyglutamine expansion in the N-terminus of the huntingtin protein (Htt). Improving the degradation of mutant Htt via the Ubiquitin Proteasome System prior to aggregation would be a therapeutic strategy to delay or prevent the onset of Huntington's Disease for which there is currently no cure. Here we examine the current approaches used to study the ubiquitination of both soluble Htt as well as insolubilized Htt present in aggregates, and we describe what is known about involved (de)ubiquitinating enzymes. Furthermore, we discuss novel methodologies to study the dynamics of Htt ubiquitination in living cells using fluorescent ubiquitin probes, to identify and quantify Htt ubiquitination by mass spectrometry-based approaches, and various approaches to identify involved ubiquitinating enzymes.

Protein ubiquitination is a key post-translational modification regulating a wide range of biological processes. Ubiquitination involves the covalent attachment of the small protein ubiquitin to a lysine of a protein substrate. In addition to its well-established role in protein degradation, protein ubiquitination plays a role in protein-protein interactions, DNA repair, transcriptional regulation, and other cellular functions. Understanding the mechanisms and functional relevance of ubiquitin as a signaling system requires the generation of antibodies or alternative reagents that specifically detect ubiquitin in a site-specific manner. However, in contrast to other post-translational modifications such as acetylation, phosphorylation, and methylation, the instability and size of ubiquitin−76 amino acids–complicate the preparation of suitable antigens and the generation antibodies detecting such site-specific modifications. As a result, the field of ubiquitin research has limited access to specific antibodies. This severely hampers progress in understanding the regulation and function of site-specific ubiquitination in many areas of biology, specifically in epigenetics and cancer. Therefore, there is a high demand for antibodies recognizing site-specific ubiquitin modifications. Here we describe a strategy for the development of site-specific ubiquitin antibodies. Based on a recently developed antibody against site-specific ubiquitination of histone H2B, we provide detailed protocols for chemical synthesis methods for antigen preparation and discuss considerations for screening and quality control experiments.

The interferon (IFN)-stimulated gene product 15 (ISG15) represents an ubiquitin-like protein (Ubl), which in a process termed ISGylation can be covalently linked to target substrates via a cascade of E1, E2, and E3 enzymes. Furthermore, ISG15 exerts functions in its free form both, as an intracellular and as a secreted protein. In agreement with its role as a type I IFN effector, most functions of ISG15 and ISGylation are linked to the anti-pathogenic response. However, also key roles in other cellular processes such as protein translation, cytoskeleton dynamics, exosome secretion, autophagy or genome stability and cancer were described. Ubiquitin-specific protease 18 (USP18) constitutes the major ISG15 specific protease which counteracts ISG15 conjugation. Remarkably, USP18 also functions as a critical negative regulator of the IFN response irrespective of its enzymatic activity. Concordantly, lack of USP18 function causes fatal interferonopathies in humans and mice. The negative regulatory function of USP18 in IFN signaling is regulated by various protein–protein interactions and its stability is controlled via proteasomal degradation. The broad repertoire of physiological functions and regulation of ISG15 and USP18 offers a variety of potential intervention strategies which might be of therapeutic use. Due to the high mutation rates of pathogens which are often species specific and constantly give rise to a variety of immune evasion mechanisms, immune effector systems are under constant evolutionarily pressure. Therefore, it is not surprising that considerable differences in ISG15 with respect to function and sequence exist even among closely related species. Hence, it is essential to thoroughly evaluate the translational potential of results obtained in model organisms especially for therapeutic strategies. This review covers existing and conceptual assay systems to target and identify modulators of ISG15, ISGylation, USP18 function, and protein–protein interactions within this context. Strategies comprise mouse models for translational perspectives, cell-based and biochemical assays as well as chemical probes.

Ubiquitination of proteins is a sophisticated post-translational modification implicated in the regulation of an ever-growing abundance of cellular processes. Recent insights into different layers of complexity have shaped the concept of the ubiquitin code. Key players in determining this code are the number of ubiquitin moieties attached to a substrate, the architecture of polyubiquitin chains, and post-translational modifications of ubiquitin itself. Ubiquitination can induce conformational changes of substrates and alter their interactive profile, resulting in the formation of signaling complexes. Here we focus on a distinct type of ubiquitination that is characterized by an inter-ubiquitin linkage through the N-terminal methionine, called M1-linked or linear ubiquitination. Formation, recognition, and disassembly of linear ubiquitin chains are highly specific processes that are implicated in immune signaling, cell death regulation and protein quality control. Consistent with their role in influencing signaling events, linear ubiquitin chains are formed in a transient and spatially regulated manner, making their detection and quantification challenging.

The regulation of ubiquitination status in the cell is controlled by ubiquitin ligases acting in tandem with deubiquitinating enzymes. Ubiquitination controls many key processes in the cell from division to death making its tight regulation key to optimal cell function. Activity based protein profiling has emerged as a powerful technique to study these important enzymes. With around 100 deubiquitinating enzymes and 600 ubiquitin ligases in the human genome targeting a subclass of these enzymes or even a single enzyme is a compelling strategy to unpick this complex system. In this review we will discuss different approaches adopted, including activity-based probes centered around ubiquitin-protein, ubiquitin-peptide and mutated ubiquitin scaffolds. We examine challenges faced and opportunities presented to increase specificity in activity-based protein profiling of the ubiquitin conjugation/deconjugation machinery.

Virus infection of eukaryotes triggers cellular innate immune response, a major arm of which is the type I interferon (IFN) family of cytokines. Binding of IFN to cell surface receptors triggers a signaling cascade in which the signal transducer and activator of transcription 2 (STAT2) plays a key role, ultimately leading to an antiviral state of the cell. In retaliation, many viruses counteract the immune response, often by the destruction and/or inactivation of STAT2, promoted by specific viral proteins that do not possess protease activities of their own. This review offers a summary of viral mechanisms of STAT2 subversion with emphasis on degradation. Some viruses also destroy STAT1, another major member of the STAT family, but most viruses are selective in targeting either STAT2 or STAT1. Interestingly, degradation of STAT2 by a few viruses requires the presence of both STAT proteins. Available evidence suggests a mechanism in which multiple sites and domains of STAT2 are required for engagement and degradation by a multi-subunit degradative complex, comprising viral and cellular proteins, including the ubiquitin–proteasomal system. However, the exact molecular nature of this complex and the alternative degradation mechanisms remain largely unknown, as critically presented here with prospective directions of future study.

Protein ubiquitination is a highly orchestrated process that controls diverse aspects of human biology. Dysregulation of this process can lead to various disease states including cancer, neurodegeneration, and autoimmunity. It is the correction of these dysregulated pathways, as well as the enticing ability to manipulate protein stability, that have instigated intense research into the therapeutic control of protein ubiquitination. A major bottleneck in the development and validation of small molecule modulators is the availability of a suitable high-throughput assay for enzyme activity. Herein, we present a new assay, which we term UbiReal, that uses fluorescence polarization to monitor all stages of Ub conjugation and deconjugation in real time. We use the assay to validate a chemical inhibitor of the E1 ubiquitin-activating enzyme, as well as to assess the activities and specificities of E2s, E3s, and deubiquitinases. The sensitivity and accessibility of this approach make it an excellent candidate for high-throughput screens of activity modulators, as well as a valuable tool for basic research into the mechanisms of ubiquitin regulation.

Technologies that induce targeted protein degradation by small molecules have been developed recently. Chimeric small molecules such as Proteolysis Targeting Chimeras (PROTACs) and Specific and Non-genetic IAP-dependent Protein Erasers (SNIPERs), and E3 modulators such as thalidomides, hijack the cellular machinery for ubiquitylation, and the ubiquitylated proteins are subjected to proteasomal degradation. This has motivated drug development in industry and academia because “undruggable targets” can now be degraded by targeted protein degradation.

The ubiquitin–proteasome system (UPS) is the major non-lysosomal pathway responsible for regulated degradation of intracellular proteins in eukaryotes. As the principal proteolytic pathway in the cytosol and the nucleus, the UPS serves two main functions: the quality control function (i.e., removal of damaged, misfolded, and functionally incompetent proteins) and a major regulatory function (i.e., targeted degradation of a variety of short-lived regulatory proteins involved in cell cycle control, signal transduction cascades, and regulation of gene expression and metabolic pathways). Aberrations in the UPS are implicated in numerous human pathologies such as cancer, neurodegenerative disorders, autoimmunity, inflammation, or infectious diseases. Therefore, the UPS has become an attractive target for drug discovery and development. For the past two decades, much research has been focused on identifying and developing compounds that target specific components of the UPS. Considerable effort has been devoted to the development of both second-generation proteasome inhibitors and inhibitors of ubiquitinating/deubiquitinating enzymes. With the feature of unique structure and bioactivity, secondary metabolites (natural products) serve as the lead compounds in the development of new therapeutic drugs. This review, for the first time, summarizes fungal secondary metabolites found to act as inhibitors of the UPS components.

Triazole‐based deubiquitylase (DUB)‐resistant ubiquitin (Ub) probes have recently emerged as effective tools for the discovery of Ub chain‐specific interactors in proteomic studies, but their structural diversity is limited. A new family of DUB‐resistant Ub probes is reported based on isopeptide‐N‐ethylated dimeric or polymeric Ub chains, which can be efficiently prepared by a one‐pot, ubiquitin‐activating enzyme (E1)‐catalyzed condensation reaction of recombinant Ub precursors to give various homotypic and even branched Ub probes at multi‐milligram scale. Proteomic studies using label‐free quantitative (LFQ) MS indicated that the isopeptide‐N‐ethylated Ub probes may complement the triazole‐based probes in the study of Ub interactome. Our study highlights the utility of modern protein synthetic chemistry to develop structurally and new families of tool molecules needed for proteomic studies. [ABSTRACT FROM AUTHOR]

We use Langevin dynamics simulations to study how the presence of a deep knot affects the time it takes to thread a polypeptide chain through a narrow pore by pulling mechanically at its end. The polypeptide was designed to contain a knotted unstructured segment inserted between two β-hairpins, which prevented the knot from slipping off the chain ends. In the range of forces studied (40–200 pN), the mean translocation time increased with the knot complexity. The type 52 knot, which was recently discovered in the structure of human ubiquitin hydrolase and is the most complex knot found in the protein databank, slows down translocation by about two orders of magnitude, as compared to the unknotted chain. In contrast to the unknotted chain case, the translocation mechanism of knotted chains involves multiple slippage events suggesting that the corresponding free energy landscape is rugged and involves multiple metastable minima. [ABSTRACT FROM AUTHOR]

The ubiquitin system, present in all eukaryotes, contributes to regulating multiple types of cellular protein processes such as cell signaling, cell cycle, and receptor trafficking, and it affects the immune response. In most types of cancer, unusual events in ubiquitin-mediated signaling pathway modulation can lead to a variety of clinical outcomes, including tumor formation and metastasis. Similarly, ubiquitination acts as a core component, which contributes to the alteration of cell signaling activity, dictating biosignal turnover and protein fates. As lung cancer acquires the most commonly mutated proteins, changes in the ubiquitination of the proteins contribute to the development of lung cancer. Various inhibitors targeting the ubiquitin system have been developed for clinical applications in lung cancer treatment. In this review, we summarize the current research advances in therapeutics for lung cancer by targeting the ubiquitin system.

Selective endocytosis followed by degradation is a major mechanism for downregulating plasma membrane transporters in response to specific environmental cues. In Saccharomyces cerevisiae, this endocytosis is promoted by ubiquitylation catalyzed by the Rsp5 ubiquitin-ligase, targeted to transporters via adaptors of the alpha-arrestin family. However, the molecular mechanisms of this targeting and their control according to conditions remain incompletely understood. In this work, we dissect the molecular mechanisms eliciting the endocytosis of Can1, the arginine permease, in response to cycloheximide-induced TORC1 hyperactivation. We show that cycloheximide promotes Rsp5-dependent Can1 ubiquitylation and endocytosis in a manner dependent on the Bul1/2 alpha-arrestins. Also crucial for this downregulation is a short acidic patch sequence in the N-terminus of Can1 likely acting as a binding site for Bul1/2. The previously reported inhibition by cycloheximide of transporter recycling, from the trans-Golgi network to the plasma membrane, seems to additionally contribute to efficient Can1 downregulation. Our results also indicate that, contrary to the previously described substrate-transport elicited Can1 endocytosis mediated by the Art1 alpha-arrestin, Bul1/2-mediated Can1 ubiquitylation occurs independently of the conformation of the transporter. This study provides further insights into how distinct alpha-arrestins control the ubiquitin-dependent downregulation of a specific amino acid transporter under different conditions.

Glycosyltransferase OGT catalyzes the conjugation of O-linked β-D-N-acetylglucosamine (O-GlcNAc) to Ser and Thr residues of the cellular proteins and regulates many key processes in the cell. Here, we report the identification of OGT as a ubiquitination target of HECT-type E3 ubiquitin (UB) ligase E6AP, whose overexpression in HEK293 cells would induce the degradation of OGT. We also found that the expression of E6AP in HeLa cells with the endogenous expression of the E6 protein of the human papillomavirus (HPV) would accelerate OGT degradation by the proteasome and suppress O-GlcNAc modification of OGT substrates in the cell. Overall, our study establishes a new mechanism of OGT regulation by the ubiquitin–proteasome system (UPS) that mediates the crosstalk between protein ubiquitination and O-GlcNAcylation pathways underlying diverse cellular processes.

Post-translational modification of the DNA replication machinery by ubiquitin and SUMO plays key roles in the faithful duplication of the genetic information. Among other functions, ubiquitination and SUMOylation serve as signals for the extraction of factors from chromatin by the AAA ATPase VCP. In addition to the regulation of DNA replication initiation and elongation, we now know that ubiquitination mediates the disassembly of the replisome after DNA replication termination, a process that is essential to preserve genomic stability. Here, we review the recent evidence showing how active DNA replication restricts replisome ubiquitination to prevent the premature disassembly of the DNA replication machinery. Ubiquitination also mediates the removal of the replisome to allow DNA repair. Further, we discuss the interplay between ubiquitin-mediated replisome disassembly and the activation of CDK1 that is required to set up the transition from the S phase to mitosis. We propose the existence of a ubiquitin–CDK1 relay, where the disassembly of terminated replisomes increases CDK1 activity that, in turn, favors the ubiquitination and disassembly of more replisomes. This model has important implications for the mechanism of action of cancer therapies that induce the untimely activation of CDK1, thereby triggering premature replisome disassembly and DNA damage.

Exposure of rodents to 48Ti and their sham counterparts. Unique protein signatures were identified in the hippocampal proteome of: (1) sham rats, (2) Ti-exposed rats, (3) Ti-exposed rats that had sham-like spatial memory performance, and (4) Ti-exposed rats that impaired spatial memory performance. Approximately 14% (159) of the proteins detected in hippocampal proteome of sham rats were not detected in the Ti-exposed rats. We explored the possibility that the loss of the Sham-only proteins may arise as a result of SR-induced changes in protein homeostasis. SR-exposure was associated with a switch towards increased pro-ubiquitination proteins from that seen in Sham. These data suggest that the role of the ubiquitin-proteome system as a determinant of SR-induced neurocognitive deficits needs to be more thoroughly investigated.