The ubiquitin system and jasmonate signaling

The ubiquitin (Ub) system is involved in most, if not all, biological processes in eukaryotes. The major specificity determinants of this system are the E3 ligases, which bind and ubiquitinate specific sets of proteins and are thereby responsible for target recruitment to the proteasome or other cellular processing machineries. The Ub system contributes to the regulation of the production, perception and signal transduction of plant hormones. Jasmonic acid (JA) and its derivatives, known as jasmonates (JAs), act as signaling compounds regulating plant development and plant responses to various biotic and abiotic stress conditions. We provide here an overview of the current understanding of the Ub system involved in JA signaling

What Has the Study of the K3 and K5 Viral Ubiquitin E3 Ligases Taught Us about Ubiquitin-Mediated Receptor Regulation?

Cells communicate with each other and the outside world through surface receptors, which need to be tightly regulated to prevent both overstimulation and receptor desensitization. Understanding the processes involved in the homeostatic control of cell surface receptors is essential, but we are not alone in trying to regulate these receptors. Viruses, as the ultimate host pathogens, have co-evolved over millions of years and have both pirated and adapted host genes to enable viral pathogenesis. K3 and K5 (also known as MIR1 and MIR2) are viral ubiquitin E3 ligases from Kaposi’s Sarcoma Associated Herpesvirus (KSHV) which decrease expression of a number of cell surface receptors and have been used to interrogate cellular processes and improve our understanding of ubiquitin-mediated receptor endocytosis and degradation. In this review, we summarize what has been learned from the study of these viral genes and emphasize their role in elucidating the complexity of ubiquitin in receptor regulation

Proteomic techniques to probe the ubiquitin landscape

Protein ubiquitination is a powerful modulator of cellular functions. Classically linked to the degradation of proteins, it also plays a role in intracellular localization, DNA damage response, vesicle fusion events, and the immune and transcriptional responses. Ubiquitin is versatile and can code for several distinct signals, either by adding a single ubiquitin or forming a chain of ubiquitins on the target protein. The enzymatic cascade associated with the cellular process determines the nature of the modification. Numerous efforts have been made for the identification of ubiquitin acceptor sites in the target proteins using genetic, biochemical or mass-spectrometry based proteomic methods, such as affinity-based enrichment of ubiquitinated proteins, and antibody-based enrichment of modified peptides. Modern instrumentation enables quantitative mass-spectrometry strategies to identify and characterize hundreds of ubiquitin substrates in a single analysis making it the dominant method for ubiquitin site detection. Characterization of the inter-ubiquitin connectivity in ubiquitin polymers has also moved into focus, with the field of targeted proteomics techniques proving invaluable for identifying and quantifying linkage types found in such polyubiquitin chains. This review seeks to provide an overview of the many mass-spectrometry based proteomics techniques available for exploring this dynamic field

Sumo paralogs: redundancy and divergencies.

Although sharing a common conjugation pathway, SUMO1, SUMO2/3 and SUMO4 seem to play preferential roles in the cell. Recently, many regulatory mechanisms contributing to SUMO paralogs specific modification have emerged. SUMO enzymes can discriminate between SUMO paralogs at both conjugation and deconjugation levels. Moreover, many substrates possess characteristics that promote their preference for different SUMO family members. A better knowledge of the mechanisms promoting SUMO specific modification will improve our understanding of the functions of SUMO paralogs in distinct cellular pathways

Co-Chaperones in Targeting and Delivery of Misfolded Proteins to the 26S Proteasome

Protein homeostasis (proteostasis) is essential for the cell and is maintained by a highly conserved protein quality control (PQC) system, which triages newly synthesized, mislocalized and misfolded proteins. The ubiquitin-proteasome system (UPS), molecular chaperones, and co-chaperones are vital PQC elements that work together to facilitate degradation of misfolded and toxic protein species through the 26S proteasome. However, the underlying mechanisms are complex and remain partly unclear. Here, we provide an overview of the current knowledge on the co-chaperones that directly take part in targeting and delivery of PQC substrates for degradation. While J-domain proteins (JDPs) target substrates for the heat shock protein 70 (HSP70) chaperones, nucleotide-exchange factors (NEFs) deliver HSP70-bound substrates to the proteasome. So far, three NEFs have been established in proteasomal delivery: HSP110 and the ubiquitin-like (UBL) domain proteins BAG-1 and BAG-6, the latter acting as a chaperone itself and carrying its substrates directly to the proteasome. A better understanding of the individual delivery pathways will improve our ability to regulate the triage, and thus regulate the fate of aberrant proteins involved in cell stress and disease, examples of which are given throughout the review

A Viral Ubiquitin Ligase Has Substrate Preferential SUMO Targeted Ubiquitin Ligase Activity that Counteracts Intrinsic Antiviral Defence

Intrinsic antiviral resistance represents the first line of intracellular defence against virus infection. During herpes simplex virus type-1 (HSV-1) infection this response can lead to the repression of viral gene expression but is counteracted by the viral ubiquitin ligase ICP0. Here we address the mechanisms by which ICP0 overcomes this antiviral response. We report that ICP0 induces the widespread proteasome-dependent degradation of SUMO-conjugated proteins during infection and has properties related to those of cellular SUMO-targeted ubiquitin ligases (STUbLs). Mutation of putative SUMO interaction motifs within ICP0 not only affects its ability to degrade SUMO conjugates, but also its capacity to stimulate HSV-1 lytic infection and reactivation from quiescence. We demonstrate that in the absence of this viral countermeasure the SUMO conjugation pathway plays an important role in mediating intrinsic antiviral resistance and the repression of HSV-1 infection. Using PML as a model substrate, we found that whilst ICP0 preferentially targets SUMO-modified isoforms of PML for degradation, it also induces the degradation of PML isoform I in a SUMO modification-independent manner. PML was degraded by ICP0 more rapidly than the bulk of SUMO-modified proteins in general, implying that the identity of a SUMO-modified protein, as well as the presence of SUMO modification, is involved in ICP0 targeting. We conclude that ICP0 has dual targeting mechanisms involving both SUMO- and substrate-dependent targeting specificities in order to counteract intrinsic antiviral resistance to HSV-1 infection

A conserved ubiquitin ligase of the nuclear envelope/endoplasmic reticulum that functions in both ER-associated and Mat a2 repressor degradation

Substrate discrimination in the ubiquitin–proteasome system is believed to be dictated by specific combinations of ubiquitin–protein ligases (E3s) and ubiquitin-conjugating enzymes (E2s). Here we identify Doa10/Ssm4 as a yeast E3 that is embedded in the endoplasmic reticulum (ER)/nuclear envelope yet can target the soluble transcription factor Mat2. Doa10 contains an unusual RING finger, which has ubiquitin-ligase activity in vitro and is essential in vivo for degradation of 2 via its Deg1 degradation signal. Doa10 functions with two E2s, Ubc6 and Ubc7, to ubiquitinate Deg1-bearing substrates, and it is also required for the degradation of at least one ER membrane protein. Interestingly, different short-lived ER proteins show distinct requirements for Doa10 and another ER-localized E3, Hrd1. Nevertheless, the two E3s overlap in function: A doa10 hrd1 mutant is far more sensitive to cadmium relative to either single mutant and displays strong constitutive induction of the unfolded protein response; this suggests a role for both E3s in eliminating aberrant ER proteins. The likely human ortholog of DOA10 is in the cri-du-chat syndrome critical region on chromosome 5p, suggesting that defective ubiquitin ligation might contribute to this common genetic disorder

How ubiquitination regulates the TGF-β signalling pathway: New insights and new players

Ubiquitination of protein species in regulating signal transduction pathways is universally accepted as of fundamental importance for normal development, and defects in this process have been implicated in the progression of many human diseases. One pathway that has received much attention in this context is transforming growth factor-beta (TGF-ß) signalling, particularly during the regulation of epithelial-mesenchymal transition (EMT) and tumour progression. While E3-ubiquitin ligases offer themselves as potential therapeutic targets, much remains to be unveiled regarding mechanisms that culminate in their regulation. With this in mind, the focus of this review highlights the regulation of the ubiquitination pathway and the significance of a recently described group of NEDD4 E3-ubiquitin ligase isoforms in the context of TGF-ß pathway regulation. Moreover, we now broaden these observations to incorporate a growing number of protein isoforms within the ubiquitin ligase superfamily as a whole, and discuss their relevance in defining a new ‘iso-ubiquitinome’

Targeting SUMO conjugates for degradation: The human RING finger RNF4 as a specialized ubiquitin ligase

Ubiquitin and the small ubiquitin related modifier (SUMO) belong to a group of small proteins that can be covalently attached to lysine side chains of other proteins, thereby changing their function, localization, interaction partners or stability. The conjugation reactions are mediated by an enzymatic cascade of specific activating, conjugating and ligating enzymes. A ubiquitin chain of at least four K48-linked ubiquitin molecules target substrate proteins for degradation by the proteasome. Several interconnections exist between the ubiquitin and SUMO system, with the latest discoveries made in yeast by identifying E3 ubiquitin ligases that target SUMO conjugates for ubiquitylation and subsequent degradation by the proteasome. These ubiquitin ligases for SUMO conjugates (ULS) recognize especially high molecular weight SUMO conjugates, probably modified with SUMO chains. In mammals, out of the three conjugatable SUMO paralogs, only SUMO-2/3 are able to form chains. Upon stress induction, the free pool of SUMO-2/3 is rapidly conjugated to cellular target proteins. These conjugates are under proteasomal control, implicating that the ULS pathway is conserved in humans. This work identified the RING finger protein RNF4 as a human ULS protein, confirming previous observations in which RNF4 complemented yeast ULS deletion phenotypes. RNF4 comprises a RING domain which is present in many E3 ligases and a stretch of up to four SUMO interaction motifs (SIMs) that confer binding to SUMO. In order to demonstrate ULS activity for RNF4, an in vitro ubiquitylation assay for SUMO conjugates has been developed. For that purpose, SUMOylated proteins were generated and purified as in vitro substrates from E. coli. RNF4 efficiently in vitro ubiquitylated SUMO modified PML while unmodified PML was not recognized as a substrate. This result is in line with recent studies in cells demonstrating that RNF4 targets PML in a SUMO-dependent manner after arsenic trioxide treatment, a drug which is applied in acute promyelocytic leukemia (APL). By investigating the SUMO binding properties of the RNF4 SIM domain, it became apparent that the interaction was especially enhanced by the presence of SUMO chains of more than two SUMOs. In addition, a SIM type specific recognition was noticed for different SUMO paralogs, which emphasizes the idea that there is also a SUMO paralogs specific regulation. Finally, in an attempt to find other ULS regulated cellular proteins, an RNF4 SIM domain construct was used to isolate poly- or multi-SUMOylated proteins from cells subjected to diverse cell stresses

Selectivity of the CUBAN domain in the recognition of ubiquitin and NEDD8

Among the members of the Ubiquitin‐like (Ubl) protein family, Neural precursor cell expressed developmentally down‐regulated protein 8 (NEDD8) is the closest in sequence to ubiquitin (57% identity). The two modification mechanisms and their functions, however, are highly distinct and the two Ubls are not interchangeable. A complex network of interactions between modifying enzymes and adaptors, most of which are specific while others are promiscuous, ensures selectivity. Many domains that bind the ubiquitin hydrophobic patch also bind NEDD8 while no domain that specifically binds NEDD8 has yet been described. Here we report an unbiased selection of domains that bind ubiquitin and/or NEDD8 and we characterize their specificity/promiscuity. Many ubiquitin binding domains bind ubiquitin preferentially and, to a lesser extent, NEDD8. In a few cases, the affinity of these domains for NEDD8 can be increased by substituting the alanine at position 72 with arginine, as in ubiquitin. We have also identified a unique domain, mapping to the carboxyl‐end of the protein KHNYN, which has a starkly preference for NEDD8. Given its ability to bind neddylated cullins we have named this domain CUBAN (Cullin Binding domain Associating with NEDD8). We present here the solution structure of the CUBAN domain both in the isolated form and in complex with NEDD8. The results contribute to the understanding of the discrimination mechanism between ubiquitin and the Ubl. They also provide new insights on the biological role of a ill‐defined protein, whose function is hitherto only predicted