Intrinsically disordered substrates dictate SPOP subnuclear localization and ubiquitination activity
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Usher, Emery T.↗
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Panel of Engineered Ubiquitin Variants Targeting the Family of Human Ubiquitin Interacting Motifs
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Veggiani, Gianluca↗
Chloroplast Autophagy and Ubiquitination Combine to Manage Oxidative Damage and Starvation Responses
Not provided.
Plant Sciences↗
Differential Gene Expression in Host Ubiquitination Processes in Childhood Malarial Anemia
Background: Malaria remains one of the leading global causes of childhood morbidity and mortality. In holoendemic Plasmodium falciparum transmission regions, such as western Kenya, severe malarial anemia [SMA, hemoglobin (Hb) < 6.0 g/dl] is the primary form of severe disease. Ubiquitination is essential for regulating intracellular processes involved in innate and adaptive immunity. Although dysregulation in ubiquitin molecular processes is central to the pathogenesis of multiple human diseases, the expression patterns of ubiquitination genes in SMA remain unexplored. Methods: To examine the role of the ubiquitination processes in pathogenesis of SMA, differential gene expression profiles were determined in Kenyan children ( n = 44, aged <48 mos) with either mild malarial anemia (M l MA; Hb ≥9.0 g/dl; n = 23) or SMA (Hb <6.0 g/dl; n = 21) using the Qiagen Human Ubiquitination Pathway RT 2 Profiler PCR Array containing a set of 84 human ubiquitination genes. Results: In children with SMA, 10 genes were down-regulated ( BRCC3 , FBXO3 , MARCH5 , RFWD2 , SMURF2 , UBA6 , UBE2A , UBE2D1 , UBE2L3 , UBR1 ), and five genes were up-regulated ( MDM2 , PARK2 , STUB1 , UBE2E3 , UBE2M ). Enrichment analyses revealed Ubiquitin-Proteasomal Proteolysis as the top disrupted process, along with altered sub-networks involved in proteasomal, protein, and ubiquitin-dependent catabolic processes. Conclusion: Collectively, these novel results show that protein coding genes of the ubiquitination processes are involved in the pathogenesis of SMA.
60 APPLIED LIFE SCIENCES↗
A deubiquitylase with an unusually high-affinity ubiquitin-binding domain from the scrub typhus pathogen Orientia tsutsugamushi
Ubiquitin mediated signaling contributes critically to host cell defenses during pathogen infection. Many pathogens manipulate the ubiquitin system to evade these defenses. Here we characterize a likely effector protein bearing a deubiquitylase (DUB) domain from the obligate intracellular bacterium Orientia tsutsugamushi, the causative agent of scrub typhus. The Ulp1-like DUB prefers ubiquitin substrates over ubiquitin-like proteins and efficiently cleaves polyubiquitin chains of three or more ubiquitins. The co-crystal structure of the DUB (OtDUB) domain with ubiquitin revealed three bound ubiquitins: one engages the S1 site, the second binds an S2 site contributing to chain specificity and the third binds a unique ubiquitin-binding domain (UBD). The UBD modulates OtDUB activity, undergoes a pronounced structural transition upon binding ubiquitin, and binds monoubiquitin with an unprecedented ~5 nM dissociation constant. The characterization and high-resolution structure determination of this enzyme should aid in its development as a drug target to counter Orientia infections.
59 BASIC BIOLOGICAL SCIENCES↗
SIRT3 mediates mitofusin 2 ubiquitination and degradation to suppress ischemia reperfusion-induced acute kidney injury
Ischemia reperfusion-induced acute kidney injury (IR-induced AKI) is a life-threatening disease with many complications. Mitofusin 2 (Mfn2) ubiquitination is related to AKI. But the underlying molecular mechanisms remain unknown. This study aimed to probe the mechanism of Mfn2 ubiquitination in IR-induced AKI development. In IR-induced AKI mouse models, orbital blood and urine were collected for assessing kidney function. The kidney injury, ultrastructure of mitochondria, and histopathology in mice were evaluated after injection of G5, an ubiquitination inhibitor. Oxygen glucose deprivation/reoxygenation (OGD/R) models were established in HK-2 cells, and the mitochondria were extracted. Cell viability, apoptosis, oxidative stress, inflammatory reaction, mitochondrial membrane potential, and ATP production were measured. Mfn2 ubiquitination in mouse and cell models was evaluated. si-SIRT3 and pcDNA3.1-SIRT3 were transfected into cell models. Consequently, kidney function in mice was impaired by IR-induced AKI. Mfn2 ubiquitination and degradation promoted IR-induced AKI. OGD/R induced renal tubular epithelial cell injury and disrupted mitochondrial dynamics and functions through promoting Mfn2 ubiquitination. SIRT3 knockdown led to Mfn2 ubiquitination by binding to UBC; while its overexpression alleviated tubular epithelial cell injury. Briefly, SIRT3 mediates Mfn2 ubiquitination to relieve IR-induced AKI. This investigation may offer new insights for the treatment of IR-induced AKI injury.
60 APPLIED LIFE SCIENCES↗
Quantitative proteomics reveals extensive lysine ubiquitination and transcription factor stability states in Arabidopsis
Abstract Protein activity, abundance, and stability can be regulated by post-translational modification including ubiquitination. Ubiquitination is conserved among eukaryotes and plays a central role in modulating cellular function; yet, we lack comprehensive catalogs of proteins that are modified by ubiquitin in plants. In this study, we describe an antibody-based approach to enrich ubiquitinated peptides coupled with isobaric labeling to enable quantification of up to 18-multiplexed samples. This approach identified 17,940 ubiquitinated lysine sites arising from 6,453 proteins from Arabidopsis (Arabidopsis thaliana) primary roots, seedlings, and rosette leaves. Gene ontology analysis indicated that ubiquitinated proteins are associated with numerous biological processes including hormone signaling, plant defense, protein homeostasis, and metabolism. We determined ubiquitinated lysine residues that directly regulate the stability of three transcription factors, CRYPTOCHROME-INTERACTING BASIC-HELIX-LOOP-HELIX 1 (CIB1), CIB1 LIKE PROTEIN 2 (CIL2), and SENSITIVE TO PROTON RHIZOTOXICITY1 (STOP1) using in vivo degradation assays. Furthermore, codon mutation of CIB1 to create a K166R conversion to prevent ubiquitination, via CRISPR/Cas9-derived adenosine base editing, led to an early flowering phenotype and increased expression of FLOWERING LOCUS T (FT). These comprehensive site-level ubiquitinome profiles provide a wealth of data for future functional studies related to modulation of biological processes mediated by this post-translational modification in plants.
Biochemistry & Molecular Biology↗
Protein ubiquitination in plant peroxisomes
Protein ubiquitination regulates diverse cellular processes in eukaryotic organisms, from growth and development to stress response. Proteins subjected to ubiquitination can be found in virtually all subcellular locations and organelles, including peroxisomes, single-membrane and highly dynamic organelles ubiquitous in eukaryotes. Peroxisomes contain metabolic functions essential to plants and animals such as lipid catabolism, detoxification of reactive oxygen species (ROS), biosynthesis of vital hormones and cofactors, and photorespiration. Plant peroxisomes possess a complex proteome with functions varying among different tissue types and developmental stages, and during plant response to distinct environmental cues. However, how these diverse functions are regulated at the post-translational level is poorly understood, especially in plants. Here, in this review, we summarized current knowledge of the involvement of protein ubiquitination in peroxisome protein import, remodeling, pexophagy, and metabolism, focusing on plants, and referencing discoveries from other eukaryotic systems when relevant. Based on previous ubiquitinomics studies, we compiled a list of 56 ubiquitinated Arabidopsis peroxisomal proteins whose functions are associated with all the major plant peroxisomal metabolic pathways. This discovery suggests a broad impact of protein ubiquitination on plant peroxisome functions, therefore substantiating the need to investigate this significant regulatory mechanism in peroxisomes at more depths.
59 BASIC BIOLOGICAL SCIENCES↗
The Structure and Immune Regulatory Implications of the Ubiquitin-Like Tandem Domain Within an Avian 2’-5’ Oligoadenylate Synthetase-Like Protein
Post-translational modification of host and viral proteins by ubiquitin and ubiquitin-like proteins plays a key role in a host’s ability to mount an effective immune response. Avian species lack a ubiquitin-like protein found in mammals and other non-avian reptiles; interferon stimulated gene product 15 (ISG15). ISG15 serves as a messenger molecule and can be conjugated to both host and viral proteins leading them to be stabilized, degraded, or sequestered. Structurally, ISG15 is comprised of a tandem ubiquitin-like domain (Ubl), which serves as the motif for post-translational modification. The 2’-5’ oligoadenylate synthetase-like proteins (OASL) also encode two Ubl domains in series near its C-terminus which binds OASL to retinoic acid inducible gene-I (RIG-I). This protein-protein interaction increases the sensitivity of RIG-I and results in an enhanced production of type 1 interferons and a robust immune response. Unlike human and other mammalian OASL homologues, avian OASLs terminate their tandem Ubl domains with the same LRLRGG motif found in ubiquitin and ISG15, a motif required for their conjugation to proteins. Chickens, however, lack RIG-I, raising the question of structural and functional characteristics of chicken OASL (chOASL). By investigating chOASL, the evolutionary history of viruses with deubiquitinases can be explored and drivers of species specificity for these viruses may be uncovered. Here we show that the chOASL tandem Ubl domains shares structural characteristics with mammalian ISG15, and that chOASL can oligomerize and conjugate to itself. In addition, the ISG15-like features of avian OASLs and how they impact interactions with viral deubiquitinases and deISGylases are explored.
59 BASIC BIOLOGICAL SCIENCES↗
Repetitive diacetyl vapor exposure promotes ubiquitin proteasome stress and precedes bronchiolitis obliterans pathology
Bronchiolitis obliterans (BO) is a devastating lung disease seen commonly after lung transplant but also seen following severe respiratory tract infection or chemical inhalation exposure. Diacetyl (DA; 2,3-butanedione), highly reactive alpha-diketone is known to cause BO when inhaled, however, the mechanisms of how inhalation exposure leads to BO development remains poorly understood. In the current work, we combined two clinically relevant models for studying the pathogenesis of DA-induced BO: (1) an in vivo rat model of repetitive DA vapor exposures with recovery and (2) an in vitro model of primary human airway epithelial cells exposed to pure DA vapors. Rats exposed to 5 consecutive days 200 parts-per-million DA 6 hrs per day had worsening survival, persistent hypoxemia, poor weight gain, and histologic evidence of BO 14 days after DA exposure cessation. At the end of exposure, increased expression of the ubiquitin stress protein, ubiquitin-C, accumulated within DA-exposed rat lung homogenates, localized primarily to the airway epithelium, the primary site of BO development, and did not occur in air-exposed controls. Lung proteasome activity increased concurrently after DA exposure. In primary human airway cultures, global proteomics analysis identified 519 significantly modified proteins in DA-exposed samples relative to controls with the ubiquitin proteasome system, endosomal reticulum transport, and the response to unfolded protein pathways being upregulated while cell-cell adhesion and oxidation-reduction pathways being downregulated in DA-exposed samples. Altogether, repetitive DA vapor exposure resulted in abundant protein damage, accumulation of polyubiquinated proteins, and ubiquitin proteasome stress prior to the development of chemical-induced BO pathology.
59 BASIC BIOLOGICAL SCIENCES↗
Structural basis for substrate recognition and chemical inhibition of oncogenic MAGE ubiquitin ligases
Testis-restricted melanoma antigen (MAGE) proteins are frequently hijacked in cancer and play a critical role in tumorigenesis. MAGEs assemble with E3 ubiquitin ligases and function as substrate adaptors that direct the ubiquitination of novel targets, including key tumor suppressors. However, how MAGEs recognize their targets is unknown and has impeded the development of MAGE-directed therapeutics. Here, we report the structural basis for substrate recognition by MAGE ubiquitin ligases. Biochemical analysis of the degron motif recognized by MAGE-A11 and the crystal structure of MAGE-A11 bound to the PCF11 substrate uncovered a conserved substrate binding cleft (SBC) in MAGEs. Mutation of the SBC disrupted substrate recognition by MAGEs and blocked MAGE-A11 oncogenic activity. A chemical screen for inhibitors of MAGE-A11:substrate interaction identified 4-Aminoquinolines as potent inhibitors of MAGE-A11 that show selective cytotoxicity. These findings provide important insights into the large family of MAGE ubiquitin ligases and identify approaches for developing cancer-specific therapeutics.
60 APPLIED LIFE SCIENCES↗
SPOP mutation induces replication over-firing by impairing Geminin ubiquitination and triggers replication catastrophe upon ATR inhibition
Geminin and its binding partner Cdt1 are essential for the regulation of DNA replication. Here we show that the CULLIN3 E3 ubiquitin ligase adaptor protein SPOP binds Geminin at endogenous level and regulates DNA replication. SPOP promotes K27-linked non-degradative poly-ubiquitination of Geminin at lysine residues 100 and 127. This poly-ubiquitination of Geminin prevents DNA replication over-firing by indirectly blocking the association of Cdt1 with the MCM protein complex, an interaction required for DNA unwinding and replication. SPOP is frequently mutated in certain human cancer types and implicated in tumorigenesis. We show that cancer-associated SPOP mutations impair Geminin K27-linked poly-ubiquitination and induce replication origin over-firing and re-replication. The replication stress caused by SPOP mutations triggers replication catastrophe and cell death upon ATR inhibition. Our results reveal a tumor suppressor role of SPOP in preventing DNA replication over-firing and genome instability and suggest that SPOP-mutated tumors may be susceptible to ATR inhibitor therapy.
59 BASIC BIOLOGICAL SCIENCES↗
Legionella metaeffector MavL reverses ubiquitin ADP-ribosylation via a conserved arginine-specific macrodomain
ADP-ribosylation is a reversible post-translational modification involved in various cellular activities. Removal of ADP-ribosylation requires (ADP-ribosyl)hydrolases, with macrodomain enzymes being a major family in this category. The pathogen Legionella pneumophila mediates atypical ubiquitination of host targets using the SidE effector family in a process that involves ubiquitin ADP-ribosylation on arginine 42 as an obligatory step. Here, we show that the Legionella macrodomain effector MavL regulates this pathway by reversing the arginine ADP-ribosylation, likely to minimize potential detrimental effects caused by the modified ubiquitin. We determine the crystal structure of ADP-ribose-bound MavL, providing structural insights into recognition of the ADP-ribosyl group and catalytic mechanism of its removal. Further analyses reveal DUF4804 as a class of MavL-like macrodomain enzymes whose representative members show unique selectivity for mono-ADP-ribosylated arginine residue in synthetic substrates. We find such enzymes are also present in eukaryotes, as exemplified by two previously uncharacterized (ADP-ribosyl)hydrolases in Drosophila melanogaster . Crystal structures of several proteins in this class provide insights into arginine specificity and a shared mode of ADP-ribose interaction distinct from previously characterized macrodomains. Collectively, our study reveals a new regulatory layer of SidE-catalyzed ubiquitination and expands the current understanding of macrodomain enzymes.
59 BASIC BIOLOGICAL SCIENCES↗
NUB1 traps unfolded FAT10 for ubiquitin-independent degradation by the 26S proteasome
The ubiquitin-like modifier FAT10 targets hundreds of proteins in the mammalian immune system to the 26S proteasome for degradation. This degradation pathway requires the cofactor NUB1, yet the underlying mechanisms remain unknown. Here, we reconstituted a minimal in vitro system with human components and revealed that NUB1 uses the intrinsic instability of FAT10 to trap its N-terminal ubiquitin-like domain in an unfolded state and deliver it to the 26S proteasome for engagement, allowing the degradation of FAT10-ylated substrates in a ubiquitin-independent and p97-independent manner. Using hydrogen–deuterium exchange, structural modeling and site-directed mutagenesis, we identified the formation of an intricate complex with FAT10 that activates NUB1 for docking to the 26S proteasome, and our cryo-EM studies visualized the highly dynamic NUB1 complex bound to the proteasomal Rpn1 subunit during FAT10 delivery and the early stages of ATP-dependent degradation. These findings identified a previously unknown mode of cofactor-mediated, ubiquitin-independent substrate delivery to the 26S proteasome that relies on trapping partially unfolded states for engagement by the proteasomal ATPase motor.
Arkinson, Connor↗
Unlocking the unfolded structure of ubiquitin: Combining time-resolved x-ray solution scattering and molecular dynamics to generate unfolded ensembles
The unfolding dynamics of ubiquitin were studied using a combination of x-ray solution scattering (XSS) and molecular dynamics (MD) simulations. The kinetic analysis of the XSS ubiquitin signals showed that the protein unfolds through a two-state process, independent of the presence of destabilizing salts. In order to characterize the ensemble of unfolded states in atomic detail, the experimental XSS results were used as a constraint in the MD simulations through the incorporation of x-ray scattering derived potential to drive the folded ubiquitin structure toward sampling unfolded states consistent with the XSS signals. We detail how biased MD simulations provide insight into unfolded states that are otherwise difficult to resolve and underscore how experimental XSS data can be combined with MD to efficiently sample structures away from the native state. Our results indicate that ubiquitin samples unfolded in states with a high degree of loss in secondary structure yet without a collapse to a molten globule or fully solvated extended chain. Finally, we propose how using biased-MD can significantly decrease the computational time and resources required to sample experimentally relevant nonequilibrium states.
Chemistry↗
The Legionella Effector SdjA Is a Bifunctional Enzyme That Distinctly Regulates Phosphoribosyl Ubiquitination
Legionella pneumophila promotes its survival and replication in phagocytes by actively modulating cellular processes using effectors injected into host cells by its Dot/Icm type IV secretion system. Many of these effectors function to manipulate the ubiquitin network of infected cells, thus contributing to the biogenesis of the Legionella-containing vacuole (LCV), which is permissive for bacterial replication. Among these, members of the SidE effector family (SidEs) catalyze ubiquitination of functionally diverse host proteins by a mechanism that is chemically distinct from the canonical three-enzyme cascade. The activity of SidEs is regulated by two mechanisms: reversal of the phosphoribosyl ubiquitination by DupA and DupB and direct inactivation by SidJ, which is a calmodulin-dependent glutamylase. In many L. pneumophila strains, SidJ belongs to a two-member protein family. Its homolog SdjA appears to function differently from SidJ despite the high-level similarity in their primary sequences. Here, we found that SdjA is a bifunctional enzyme that exhibits distinct activities toward members of the SidE family. It inhibits the activity of SdeB and SdeC by glutamylation. Unexpectedly, it also functions as a deglutamylase that reverses SidJ-induced glutamylation on SdeA. Our results reveal that an enzyme can catalyze two completely opposite biochemical reactions, which highlights the distinct regulation of phosphoribosyl ubiquitination by the SidJ effector family.
59 BASIC BIOLOGICAL SCIENCES↗
Bacterial effectors mimicking ubiquitin-proteasome pathway tweak plant immunity
Plant pathogenic Gram-negative bacteria evade the host plant immune system by secreting Type III (T3E) and Type IV effector (T4E) proteins into the plant cytoplasm. Mostly T3Es are secreted into the plant cells to establish pathogenicity by affecting the vital plant process viz. metabolic pathways, signal transduction and hormonal regulation. Ubiquitin-26S proteasome system (UPS) exists as one of the important pathways in plants to control plant immunity and various cellular processes by employing several enzymes and enzyme components. Pathogenic and non-pathogenic bacteria are found to secrete effectors into plants with structural and/or functional similarity to UPS pathway components like ubiquitin E3 ligases, F-box domains, cysteine proteases, inhibitor of host UPS or its components, etc. The bacterial effectors mimic UPS components and target plant resistance proteins for degradation by proteasomes, thereby taking control over the host cellular activities as a strategy to exert virulence. Thus, the bacterial effectors circumvent plant cellular pathways leading to infection and disease development. Finally, this review highlights known bacterial T3E and T4E proteins that function and interfere with the ubiquitination pathway to regulate the immune system of plants.
59 BASIC BIOLOGICAL SCIENCES↗
Gene commander in the trash heap: Transcriptional regulation and ubiquitination modification mediated by RNF6 in carcinogenesis
Highlights: • Reveal the dysregulation of RNF6 in cancer. • Describe the molecular function of RNF6. • Summarize the molecular mechanism mediated by RNF6. RING finger protein 6 (RNF6), a RING finger protein, has been identified as a potential tumor promoter in several cancers. However, the exact mechanism of RNF6 in cancer remains elusive. As in various diseases, RNF6 may be involved in regulating cell growth, cell proliferation, invasion, cell cycle progression, apoptosis and cell adhesion through E3 ligase-mediated ubiquitination. Thus, the research on RNF6 is mainly focused on the ubiquitination of RNF6 in recent years. This article summarizes the role of RNF6 ubiquitination in various physiological and pathological mechanisms, such as Akt/mTOR signaling pathway, Wnt/β-catenin pathway, RNF6/ERα/Bcl-xL axis, and provides knowledge and understanding for the treatment of diseases.
60 APPLIED LIFE SCIENCES↗