Ubl Conjugation Research Articles

60 Molecular Characterization of Boar Semen in Relation to Sperm Quality

Abstract Artificial insemination (AI) with fresh semen is the primary breeding tool in the swine industry. However, the heterogenous semen doses create challenges in boar studs due to the lack of consistent assessment criteria. Seminal plasma (SP) surrounding spermatozoa is a great source of reliable non-invasive biomarkers of sperm quality. It contains a wide array of bioactive molecules, whose dynamic compositions in response to extrinsic and intrinsic factors influence sperm motility and morphology. Therefore, we hypothesize that the miRNA content of extracellular vesicles (EVs) found in SP may reflect semen quality. Our study aimed to characterize poor vs. good quality semen and compare miRNA profiles of SP-derived EVs. Raw semen was collected (n = 75) from sexually mature Duroc boars at a commercial stud (Prestage Farms, MS) for 8 weeks. Semen samples were subjected to sperm motility and morphology analyses and subsequently classified as poor (n = 38) or good (n = 37) quality based on assessment criteria cut-offs (< 70% and ≥70%, respectively). Thereafter, raw semen samples were subjected to serial centrifugation to collect spermatozoa and SP. Samples were stored at -80oC until analyses. A subset of each frozen-thawed SP was used for NIRS absorbance analysis (ASD FieldSpec3 portable spectrometer – 1mm quartz cuvettes; 750-2500 nm). Other subsets of frozen-thawed SP were ultra-centrifugated to isolate EVs (SP-EV), followed by confirmations through transmission electron microscopy (TEM), dynamic light scattering (DLS), and immuno-western blotting. Five pools of three individual SP-EV samples, corresponding to extremely poor and extremely good quality semen, were constituted for total exosome RNA isolation, small RNA sequencing, miRNA detection, and differential miRNA expression. Messenger RNA targets of miRNA were searched and subjected to functional bioinformatic analyses using various software algorithms. Significant differences were set for P < 0.05. Poor and good quality samples had different spectrum profiles following NIRS analyses. Changes were explicitly observed in water structures at coordinates C1, C5, and C12 (nm), corresponding to water associations with chaotropic solutes (water asymmetrical stretching vibrations-ν3), free water, and kosmotropic solutes (increased H-bonding numbers and strength), respectively. The NIRS-LDA data contrasted two distinctive poor and good quality SP samples. All boar SP-EV were confirmed: round spherical structure; 30 to 400nm of diameter; and CD9+, CD63+, and CD81+ membrane markers. Totals of 281 and 271 miRNAs were identified in poor and good groups, respectively, and 15 were differentially expressed (DE). Only three DE miRNAs (ssc-miR-205, ssc-miR-493-5p, and ssc-miR-378b-3p) permitted targeting of 109 mRNA, which functional analyses indicated associations with nuclear and cytosol localization, as well as with functions, i.e., acetylation, Ubl conjugation, protein kinase binding, potentially altering sperm function. In conclusion, the study indicates that the NIRS tool can distinguish between good and poor semen quality, and the miRNA SP-EV content may reflect semen quality.

Characterization of Extracellular Vesicle-Coupled miRNA Profiles in Seminal Plasma of Boars with Divergent Semen Quality Status.

Sperm heterogeneity creates challenges for successful artificial insemination. Seminal plasma (SP) surrounding sperm is an excellent source for detecting reliable non-invasive biomarkers of sperm quality. Here, we isolated microRNAs (miRNAs) from SP-derived extracellular vesicles (SP-EV) of boars with divergent sperm quality statuses. Raw semen from sexually mature boars was collected for eight weeks. Sperm motility and normal morphology were analyzed, and the sperm was classified as poor- or good-quality based on standard cutoffs of 70% for the parameters measured. SP-EVs were isolated by ultracentrifugation and confirmed by electron microscopy, dynamic light scattering, and Western immunoblotting. The SP-EVs were subjected to total exosome RNA isolation, miRNA sequencing, and bioinformatics analysis. The isolated SP-EVs were round spherical structures approximately 30-400 nm in diameter expressing specific molecular markers. miRNAs were detected in both poor- (n = 281) and good (n = 271)-quality sperm, with fifteen being differentially expressed. Only three (ssc-miR-205, ssc-miR-493-5p, and ssc-miR-378b-3p) allowed gene targeting associated with cellular localization (nuclear and cytosol) and molecular functions (acetylation, Ubl conjugation, and protein kinase binding), potentially impairing sperm quality. PTEN and YWHAZ emerged as essential proteins for protein kinase binding. We conclude that SP-EV-derived miRNAs reflect boar sperm quality to enable therapeutic strategies to improve fertility.

A Microbead-Based Flow Cytometry Assay to Assess the Activity of Ubiquitin and Ubiquitin-Like Conjugating Enzymes.

The peptidic posttranslational modifiers of the ubiquitin (Ub) family (ubiquitin-like, UbLs) are conjugated to thousands of proteins to modify their function and fate. Dysregulation of their conjugation/deconjugation pathways is associated with a variety of pathological disorders. However, the techniques currently available to monitor the levels of target modification by UbLs as well as the activity of UbL-conjugating enzymes are limited and generally not quantitative. Here, we describe a microbead-based flow cytometry assay to accurately quantify UbL conjugation activity. It measures the capacity of UbL-conjugating enzymes, either purified or present in cell extracts, to transfer their respective UbL onto target substrates immobilized on color-coded microbeads. Although this protocol describes its use to study protein modification by Ub, SUMO-1 to SUMO-3, and NEDD8, this assay may be applicable to investigating conjugation of any other UbLs. It should therefore prove a precious tool for both screening UbL-conjugating enzymes inhibitors and following UbL pathway dysregulations in both physiological and pathological settings.

E1 Enzymes as Therapeutic Targets in Cancer.

Post-translational modifications of cellular substrates with ubiquitin and ubiquitin-like proteins (UBLs), including ubiquitin, SUMOs, and neural precursor cell-expressed developmentally downregulated protein 8, play a central role in regulating many aspects of cell biology. The UBL conjugation cascade is initiated by a family of ATP-dependent enzymes termed E1 activating enzymes and executed by the downstream E2-conjugating enzymes and E3 ligases. Despite their druggability and their key position at the apex of the cascade, pharmacologic modulation of E1s with potent and selective drugs has remained elusive until 2009. Among the eight E1 enzymes identified so far, those initiating ubiquitylation (UBA1), SUMOylation (SAE), and neddylation (NAE) are the most characterized and are implicated in various aspects of cancer biology. To date, over 40 inhibitors have been reported to target UBA1, SAE, and NAE, including the NAE inhibitor pevonedistat, evaluated in more than 30 clinical trials. In this Review, we discuss E1 enzymes, the rationale for their therapeutic targeting in cancer, and their different inhibitors, with emphasis on the pharmacologic properties of adenosine sulfamates and their unique mechanism of action, termed substrate-assisted inhibition. Moreover, we highlight other less-characterized E1s-UBA6, UBA7, UBA4, UBA5, and autophagy-related protein 7-and the opportunities for targeting these enzymes in cancer. SIGNIFICANCE STATEMENT: The clinical successes of proteasome inhibitors in cancer therapy and the emerging resistance to these agents have prompted the exploration of other signaling nodes in the ubiquitin-proteasome system including E1 enzymes. Therefore, it is crucial to understand the biology of different E1 enzymes, their roles in cancer, and how to translate this knowledge into novel therapeutic strategies with potential implications in cancer treatment.

Ubiquitin-Like Modifiers: Emerging Regulators of Protozoan Parasites.

Post-translational protein regulation allows for fine-tuning of cellular functions and involves a wide range of modifications, including ubiquitin and ubiquitin-like modifiers (Ubls). The dynamic balance of Ubl conjugation and removal shapes the fates of target substrates, in turn modulating various cellular processes. The mechanistic aspects of Ubl pathways and their biological roles have been largely established in yeast, plants, and mammalian cells. However, these modifiers may be utilised differently in highly specialised and divergent organisms, such as parasitic protozoa. In this review, we explore how these parasites employ Ubls, in particular SUMO, NEDD8, ATG8, ATG12, URM1, and UFM1, to regulate their unconventional cellular physiology. We discuss emerging data that provide evidence of Ubl-mediated regulation of unique parasite-specific processes, as well as the distinctive features of Ubl pathways in parasitic protozoa. We also highlight the potential to leverage these essential regulators and their cognate enzymatic machinery for development of therapeutics to protect against the diseases caused by protozoan parasites.

Discovery and optimization of pyrazolopyrimidine sulfamates as ATG7 inhibitors

Autophagy is postulated to be required by cancer cells to survive periods of metabolic and/or hypoxic stress. ATG7 is the E1 enzyme that is required for activation of Ubl conjugation pathways involved in autophagosome formation. This article describes the design and optimization of pyrazolopyrimidine sulfamate compounds as potent and selective inhibitors of ATG7. Cellular levels of the autophagy markers, LC3B and NBR1, are regulated following treatment with these compounds.

Correction: Sensitive detection of protein ubiquitylation using a protein fragment complementation assay.

There was an error in J. Cell Sci. (2020) 133, jcs240093 (doi:10.1242/jcs.240093).In their article, the authors omitted to mention ubiquitin-mediated fluorescence complementation (UbFC), a method previously designed to probe the conjugation of ubiquitin or ubiquitin-like proteins (Ubls) to their substrates (Fang and Kerppola, 2004; Sung et al, 2013). UbFC and NUbiCA are similar in that both methods rely on a protein-fragment complementation assay to detect Ubl conjugation, but the reporters (fluorescent proteins for UbFC, the NanoLuc luciferase for NUbiCA) and assay conditions (live or fixed cells for UbFC, purified proteins for NUbiCA) are different. UbFC is appealing since it enables direct visualization of the subcellular localization of Ubl conjugates in living cells, which is not possible with NUbiCA. Yet, the irreversible assembly and slow maturation of fluorescent proteins (Hu et al., 2002; Kodama and Hu, 2012) precludes real-time monitoring of Ubl conjugation and deconjugation events, which may complicate the interpretation of UbFC results, especially for short-lived or dynamically modified conjugates. Other approaches based on FRET and BRET have also been devised to probe ubiquitylation of proteins in living cells, but their sensitivity has not yet been carefully assessed (Ganesan et al., 2006; Batters et al., 2010; Riching et al., 2018).The authors apologise to readers for this omission, which does not impact the results or conclusions of the paper.

Formation of the Alarmones Diadenosine Triphosphate and Tetraphosphate by Ubiquitin- and Ubiquitin-like-Activating Enzymes

Diadenosine polyphosphates (ApnAs) such as diadenosine tri- and tetraphosphates are formed in prokaryotic as well as eukaryotic cells. Since upon stress intracellular ApnA concentrations increase, it was postulated that ApnAs are alarmones triggering stress-adaptive processes. The major synthesis pathway of ApnAs is assumed to be a side reaction of amino acid activation. How this process is linked to stress adaptation remains enigmatic. The first step of one of the most prominent eukaryotic post-translational modification systems-the conjugation of ubiquitin (Ub) and ubiquitin-like proteins (Ubl) to target proteins-involves the formation of an adenylate as intermediate. Like ApnA formation, Ub and Ubl conjugation is significantly enhanced during stress conditions. Here, we demonstrate that diadenosine tri- and tetraphosphates are indeed synthesized during activation of Ub and Ubls. This links one of the most prevalent eukaryotic protein-modification systems to ApnA formation for the first time.

CDK5RAP3, a UFL1 substrate adaptor, is crucial for liver development.

Protein modification by ubiquitin and ubiquitin-like proteins (UBLs) regulates numerous biological functions. The UFM1 system, a novel UBL conjugation system, is implicated in mouse development and hematopoiesis. However, its broad biological functions and working mechanisms remain largely elusive. CDK5RAP3, a possible ufmylation substrate, is essential for epiboly and gastrulation in zebrafish. Herein, we report a crucial role of CDK5RAP3 in liver development and hepatic functions. Cdk5rap3 knockout mice displayed prenatal lethality with severe liver hypoplasia, as characterized by delayed proliferation and compromised differentiation. Hepatocyte-specific Cdk5rap3 knockout mice suffered post-weaning lethality, owing to serious hypoglycemia and impaired lipid metabolism. Depletion of CDK5RAP3 triggered endoplasmic reticulum stress and activated unfolded protein responses in hepatocytes. We detected the in vivo interaction of CDK5RAP3 with UFL1, the defined E3 ligase in ufmylation. Notably, loss of CDK5RAP3 altered the ufmylation profile in liver cells, suggesting that CDK5RAP3 serves as a novel substrate adaptor for this UBL modification. Collectively, our study identifies CDK5RAP3 as an important regulator of ufmylation and suggests the involvement of ufmylation in mammalian development.

Whole-genome methylation profiling from PBMCs in acute-exacerbation COPD patients with good and poor responses to corticosteroid treatment

Identifying heterogeneity in chronic obstructive pulmonary disease (COPD) phenotypes is important for the development of personalized medicine. Genome-wide analysis was used to compare the methylation levels of peripheral blood mononuclear cell (PBMC) samples from 24 acute-exacerbation (AE) COPD patients with good/poor response to corticosteroid therapy and 12 non-COPD controls. Pyrosequencing was employed to validate the genome-wide analysis. In the dataset specific to COPD patients with a good response, enrichment was identified for the following: genes in the Ubl conjugation pathway, nicotinamide nucleotide metabolism, the alkaloid metabolic process, and regulation of the glucose metabolic process. Validation results confirmed CpG sites in PRKAG2 with different methylation levels in COPD patients and normal subjects. The CpG sites of ALOX5AP were specifically associated with a good response. The results suggested that a good response to corticosteroid treatment for AE-COPD should be considered a distinct subtype according to the putative epigenetic mechanism.

Dissecting Distinct Roles of NEDDylation E1 Ligase Heterodimer APPBP1 and UBA3 Reveals Potential Evolution Process for Activation of Ubiquitin-related Pathways

Despite the similar enzyme cascade in the Ubiquitin and Ubiquitin-like peptide(Ubl) conjugation, the involvement of single or heterodimer E1 activating enzyme has been a mystery. Here, by using a quantitative Förster Resonance Energy Transfer (FRET) technology, aided with Analysis of Electrostatic Similarities Of Proteins (AESOP) computational framework, we elucidate in detail the functional properties of each subunit of the E1 heterodimer activating-enzyme for NEDD8, UBA3 and APPBP1. In contrast to SUMO activation, which requires both subunits of its E1 heterodimer AOS1-Uba2 for its activation, NEDD8 activation requires only one of two E1 subunits, UBA3. The other subunit, APPBP1, only contributes by accelerating the activation reaction rate. This discovery implies that APPBP1 functions mainly as a scaffold protein to enhance molecular interactions and facilitate catalytic reaction. These findings for the first time reveal critical new mechanisms and a potential evolutionary pathway for Ubl activations. Furthermore, this quantitative FRET approach can be used for other general biochemical pathway analysis in a dynamic mode.

Abstract 1310: Identification of pleiotropic cancer susceptibility variants from genome-wide association studies reveals functional characteristics

Abstract Background: There exists compelling evidence that some genetic variants are associated with the risk of multiple cancers (i.e., pleiotropy). However, the biological mechanisms of the pleiotropic effects are unclear. Thus, we investigated the functional effects for genetic variants associated with the risk of multiple cancers. Methods: The National Human Genome Research Institute-European Bioinformatics Institute (NHGRI-EBI) GWAS Catalog contains 28,643 variant-trait associations with p < 10-5. We utilized the Experimental Factor Ontology (EFO) to classify cancer traits and obtained all associations between variants and cancer risk. Based on pairwise linkage disequilibrium (LD) determined from the European (EUR) population of Phase 3 of the 1000 Genomes Project in LDlink, correlated variants (R-squared ≥ 0.8) were clustered into groups. Variant groups associated with the risk of multiple cancers were annotated using the Ensembl Variant Effect Predictor (VEP), and tested for functional enrichment using the DAVID Functional Annotation Tool. Results: We identified 1,456 variant-cancer risk associations. The majority (57.1%) of the associations were discovered in European ancestry populations, 19.0% in East Asians, 7.9% in an African Americans or Afro-Caribbeans, and 7.1% in Hispanics or Latin Americans. Removing duplicates, we found 1,034 unique variant-cancer risk associations for 1,005 unique variants and 27 unique cancer sites. After clustering correlated variants, we identified 29 pleiotropic variant groups, of which 2 were associated with risk of five different cancer sites. Variant group rs10936599 and rs12696304 within the MYNN gene (7.8 kb from the TERC gene) was associated with leukemia, multiple myeloma, and colorectal, skin, and bladder cancer. Variant group rs31489, rs31490, rs401681, and rs4975616 within the CLPTM1L gene (22.8 kb from the TERT gene) was associated with leukemia, and lung, pancreatic, skin, and bladder cancer. Among the 42 unique variants that composed the 29 pleiotropic variant groups, 41 variants were within a known gene, and only 1 variant was in an intergenic region. The 41 variants mapped to 26 genes, which were enriched in cellular response to hypoxia (p= 0.0071), establishment of protein localization to telomere (p= 0.0071), and ubl conjugation (p= 0.0072). Conclusion: This study identified and functionally characterized genetic variants showing pleiotropic effects on cancer risk. Our findings improve the understanding of shared biological mechanisms common to different cancers. Clinical implications could include the classification of cancers based on etiology, genetic testing for multiple cancers, and repurposing cancer treatments. Citation Format: Yi-Hsuan Wu, Rebecca E. Graff, Michael N. Passarelli, Thomas J. Hoffmann, Elad Ziv, John S. Witte. Identification of pleiotropic cancer susceptibility variants from genome-wide association studies reveals functional characteristics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1310. doi:10.1158/1538-7445.AM2017-1310

Ubiquitin-like Protein Conjugation: Structures, Chemistry, and Mechanism.

Ubiquitin-like proteins (Ubl’s) are conjugated to target proteins or lipids to regulate their activity, stability, subcellular localization, or macromolecular interactions. Similar to ubiquitin, conjugation is achieved through a cascade of activities that are catalyzed by E1 activating enzymes, E2 conjugating enzymes, and E3 ligases. In this review, we will summarize structural and mechanistic details of enzymes and protein cofactors that participate in Ubl conjugation cascades. Precisely, we will focus on conjugation machinery in the SUMO, NEDD8, ATG8, ATG12, URM1, UFM1, FAT10, and ISG15 pathways while referring to the ubiquitin pathway to highlight common or contrasting themes. We will also review various strategies used to trap intermediates during Ubl activation and conjugation.

A comprehensive platform for the analysis of ubiquitin-like protein modifications using in vivo biotinylation

Post-translational modification by ubiquitin and ubiquitin-like proteins (UbLs) is fundamental for maintaining protein homeostasis. Efficient isolation of UbL conjugates is hampered by multiple factors, including cost and specificity of reagents, removal of UbLs by proteases, distinguishing UbL conjugates from interactors, and low quantities of modified substrates. Here we describe bioUbLs, a comprehensive set of tools for studying modifications in Drosophila and mammals, based on multicistronic expression and in vivo biotinylation using the E. coli biotin protein ligase BirA. While the bioUbLs allow rapid validation of UbL conjugation for exogenous or endogenous proteins, the single vector approach can facilitate biotinylation of most proteins of interest. Purification under denaturing conditions inactivates deconjugating enzymes and stringent washes remove UbL interactors and non-specific background. We demonstrate the utility of the method in Drosophila cells and transgenic flies, identifying an extensive set of putative SUMOylated proteins in both cases. For mammalian cells, we show conjugation and localization for many different UbLs, with the identification of novel potential substrates for UFM1. Ease of use and the flexibility to modify existing vectors will make the bioUbL system a powerful complement to existing strategies for studying this important mode of protein regulation.

Production of Human ATG Proteins for Lipidation Assays.

Humans express several orthologs of yeast Atg8, in the LC3 and GABARAP families, which play crucial roles in autophagy through their covalent ligation to lipids, typically phosphatidylethanolamine (PE), in a process known as lipidation. Lipidation of LC3 and GABARAP regulates numerous facets of the autophagy process, including regulating expansion of the phagophore membrane, recruiting selected cargoes for degradation, and providing an autophagosome membrane-bound platform mediating dynamic interactions with other regulatory proteins. LC3 and GABARAP are families of related ubiquitin-like proteins (UBLs) (referred to here collectively as LC3/GABARAP), and their lipidation involves a divergent UBL conjugation cascade including ATG7, ATG3, and ATG12~ATG5-ATG16L1 acting as E1, E2, and E3 enzymes, respectively. ATG7 initiates LC3/GABARAP conjugation by catalyzing their C-terminal adenylation and conjugation to the catalytic cysteine of ATG3. Ultimately, the ATG12~ATG5-ATG16L1 complex catalyzes LC3/GABARAP ligation to a primary amino group on PE or other acceptor lipids. This chapter describes methods for expressing and purifying human LC3 or GABARAP, ATG7, ATG3, and the ATG12~ATG5-ATG16L1 complex for in vitro studies of LC3/GABARAP lipidation.

Crystallographic Characterization of ATG Proteins and Their Interacting Partners.

Autophagosome formation and specific substrate recruitment during autophagy require ligation of the ubiquitin-like protein (UBL) Atg8 to the head group of the lipid phosphatidylethanolamine. Atg8 lipidation is mediated by distinctive UBL cascades involving autophagy-specific E1, E2, and E3 enzymes that differ substantially in sequence from components of other UBL conjugation cascades. Structural studies are important for elucidating the roles of Atg proteins that regulate multiple steps involved in autophagy. This chapter describes methods to prepare and crystallize selected proteins and complexes involved in autophagy UBL conjugation pathways, as a guide for strategies for structural and biochemical characterization of Atg proteins.

Autophagy protein 12 plays an essential role in Acanthamoeba encystation

Autophagy is a well conserved, catabolic process in eukaryotic cells. Previously, we identified two novel ubiquitin like conjugation systems (Atg12 and Atg8) in the autophagy process of Acanthamoeba castellanii. To obtain more specific information on the Atg12 ubiquitin like conjugation system during encystation of Acanthamoeba, we characterized the function of Atg12. Knockdown of AcAg12 in trophozoites resulted in inhibition of cyst formation. Analysis of subcellular localization showed that AcAtg12 was evenly distributed in the trophozoites during early encystation, started to accumulate partially as dots or fragments, and then co-localized with the vesicle of the autophagic structure. However, the mRNA expression of AcAtg12 was maintained at a constant level during encystation as well as in trophozoites. Ultrastructural analysis with TEM showed that AcAtg12-knockdown cells showed vacuolization, lack of cyst wall formation, and numerical decline of autophagic structures, compared with the control cells. Interestingly, these knockdown cells began to round-up and swell, and then burst at 144 h post encystation. Taken together, our results might provide a better understanding of the Atg12 UBL conjugation system in Acanthamoeba and other cyst forming protozoan parasites.

Mechanistic Study of Uba5 Enzyme and the Ufm1 Conjugation Pathway

E1 enzymes activate ubiquitin or ubiquitin-like proteins (Ubl) via an adenylate intermediate and initiate the enzymatic cascade of Ubl conjugation to target proteins or lipids. Ubiquitin-fold modifier 1 (Ufm1) is activated by the E1 enzyme Uba5, and this pathway is proposed to play an important role in the endoplasmic reticulum (ER) stress response. However, the mechanisms of Ufm1 activation by Uba5 and subsequent transfer to the conjugating enzyme (E2), Ufc1, have not been studied in detail. In this work, we found that Uba5 activated Ufm1 via a two-step mechanism and formed a binary covalent complex of Uba5∼Ufm1 thioester. This feature contrasts with the three-step mechanism and ternary complex formation in ubiquitin-activating enzyme Uba1. Uba5 displayed random ordered binding with Ufm1 and ATP, and its ATP-pyrophosphate (PPi) exchange activity was inhibited by both AMP and PPi. Ufm1 activation and Uba5∼Ufm1 thioester formation were stimulated in the presence of Ufc1. Furthermore, binding of ATP to Uba5∼Ufm1 thioester was required for efficient transfer of Ufm1 from Uba5 to Ufc1 via transthiolation. Consistent with the two-step activation mechanism, the mechanism-based pan-E1 inhibitor, adenosine 5'-sulfamate (ADS), reacted with the Uba5∼Ufm1 thioester and formed a covalent, tight-binding Ufm1-ADS adduct in the active site of Uba5, which prevented further substrate binding or catalysis. ADS was also shown to inhibit the Uba5 conjugation pathway in the HCT116 cells through formation of the Ufm1-ADS adduct. This suggests that further development of more selective Uba5 inhibitors could be useful in interrogating the roles of the Uba5 pathway in cells.

Structural and functional insights to ubiquitin-like protein conjugation.

Attachment of ubiquitin (Ub) and ubiquitin-like proteins (Ubls) to cellular proteins regulates numerous cellular processes including transcription, the cell cycle, stress responses, DNA repair, apoptosis, immune responses, and autophagy, to name a few. The mechanistically parallel but functionally distinct conjugation pathways typically require the concerted activities of three types of protein: E1 Ubl-activating enzymes, E2 Ubl carrier proteins, and E3 Ubl ligases. E1 enzymes initiate pathway specificity for each cascade by recognizing and activating cognate Ubls, followed by catalyzing Ubl transfer to cognate E2 protein(s). Under certain circumstances, the E2 Ubl complex can direct ligation to the target protein, but most often requires the cooperative activity of E3 ligases. Reviewed here are recent structural and functional studies that improve our mechanistic understanding of E1-, E2-, and E3-mediated Ubl conjugation.

Specific substrate recognition and thioester intermediate determinations in ubiquitin and SUMO conjugation cascades revealed by a high-sensitive FRET assay

Ubiquitin and ubiquitin-like proteins (Ubls), such as SUMO, are covalently conjugated to their targets by related, but distinct enzymatic conjugation reactions that involve the dynamic E1-E2-E3 enzyme cascade. E1s activate Ubls by catalyzing Ubl C-terminal adenylation, with the help of ATP, to form a covalent thioester bond. Subsequently, Ubls are transferred to E2 to generate a thioester-linked product. In previous studies, we showed the dynamic processes and thioester intermediates of SUMO with its E1 and E2 conjugating enzymes. Studies of the enzyme specificity of the Ubl conjugation cascade are normally carried out by tedious biochemical processes, and the reaction intermediates are often difficult to capture because they are unstable and have short half-lives. Here, using our recently developed robust quantitative FRET-based technology, we describe systematic investigations of enzymatic specificity and thioester intermediate determination of ubiquitin with its E1-E2 ligases in conjugation with SUMO and its ligases. Our technology easily determined the strong specificity of enzyme-substrate interactions and thioester intermediates in ubiquitination and SUMOylation cascades. The traditional FRET pair ECFP/EYFP lacked adequate signals for these assays. However, in contrast, the highly sensitive FRET pair CyPet/YPet was easily harnessed to define the reaction specificities and intermediates. In addition, the thioester intermediates can be readily monitored by a newly defined FRET index parameter. These results provide an example of a systems biology approach to determine Ubl conjugation specificity and demonstrate that a robust FRET technology can be used to identify enzymes and substrates in other Ubl pathways.