Ubiquitin Chain Topology Research Articles

Ubiquitin chain topology in plant cell signaling: a new facet to an evergreen story.

Ubiquitin is a peptide modifier able to form polymers of varying length and linkage as part of a powerful signaling system. Perhaps the best-known aspect of this protein's function is as the driver of targeted protein degradation through the Ubiquitin Proteasome System (UPS). Through the formation of lysine 48-linked polyubiquitin chains, it is able to direct the degradation of tagged proteins by the 26S proteasome, indirectly controlling many processes within the cell. However, recent research has indicated that ubiquitin performs a multitude of other roles within the cell beyond protein degradation. It is able to form 6 other “atypical” linkages though lysine residues at positions 6, 11, 27, 29, 33, and 63. These atypical chains perform a range of diverse functions, including the regulation of iron uptake in response to perceived deficiency, repair of double stranded breaks in the DNA, and regulation of the auxin response through the non-proteasomal degradation of auxin efflux carrier protein PIN1. This review explores the role ubiquitin chain topology plays in plant cellular function. We aim to highlight the importance of these varying functions and the future challenges to be encountered within this field.

Deubiquitinating Enzyme Specificity for Ubiquitin Chain Topology Profiled by Di-Ubiquitin Activity Probes

SummaryPosttranslational modification with ubiquitin (Ub) controls many cellular processes, and aberrant ubiquitination can contribute to cancer, immunopathology, and neurodegeneration. The versatility arises from the ability of Ub to form polymer chains with eight distinct linkages via lysine side chains and the N terminus. In this study, we engineered Di-Ub probes mimicking all eight different poly-Ub linkages and profiled the deubiquitinating enzyme (DUB) selectivity for recognizing Di-Ub moieties in cellular extracts. Mass spectrometric profiling revealed that most DUBs examined have broad selectivity, whereas a subset displays a clear preference for recognizing noncanonical over K48/K63 Ub linkages. Our results expand knowledge of Ub processing enzyme functions in cellular contexts that currently depends largely on using recombinant enzymes and substrates.

Advances in characterizing ubiquitylation sites by mass spectrometry

The attachment of one or more ubiquitin moieties to proteins plays a central regulatory mechanism in eukaryotic cells. Protein ubiquitylation regulates numerous cellular processes, including protein degradation, signal transduction, DNA repair and cell division. The characterization of ubiquitylation is a two-fold challenge that involves the mapping of ubiquitylation sites and the determination of ubiquitin chain topology. This review focuses on the technical advances in the mass spectrometry-based characterization of ubiquitylation sites, which have recently involved the large-scale identification of ubiquitylation sites by peptide-level enrichment strategies. The discovery that ubiquitylation is a widespread modification similar to phosphorylation and acetylation suggests cross-talk may also occur at the post translational modification level.

Deubiquitinases Regulate the Activity of Caspase-1 and Interleukin-1β Secretion via Assembly of the Inflammasome

IL-1β is a potent pro-inflammatory cytokine produced in response to infection or injury. It is synthesized as an inactive precursor that is activated by the protease caspase-1 within a cytosolic molecular complex called the inflammasome. Assembly of this complex is triggered by a range of structurally diverse damage or pathogen associated stimuli, and the signaling pathways through which these act are poorly understood. Ubiquitination is a post-translational modification essential for maintaining cellular homeostasis. It can be reversed by deubiquitinase enzymes (DUBs) that remove ubiquitin moieties from the protein thus modifying its fate. DUBs present specificity toward different ubiquitin chain topologies and are crucial for recycling ubiquitin molecules before protein degradation as well as regulating key cellular processes such as protein trafficking, gene transcription, and signaling. We report here that small molecule inhibitors of DUB activity inhibit inflammasome activation. Inhibition of DUBs blocked the processing and release of IL-1β in both mouse and human macrophages. DUB activity was necessary for inflammasome association as DUB inhibition also impaired ASC oligomerization and caspase-1 activation without directly blocking caspase-1 activity. These data reveal the requirement for DUB activity in a key reaction of the innate immune response and highlight the therapeutic potential of DUB inhibitors for chronic auto-inflammatory diseases.

Molecular participants in regulation of the meiotic cell cycle in mammalian oocytes

Meiosis in oocytes consists of two consecutive asymmetric cell divisions, each completed by the extrusion of one set of chromosomes into a small polar body. First polar body (PBI) extrusion is triggered by the inactivation of cyclin-dependent kinase 1 (CDK1), following the degradation of its regulatory subunit cyclin B1 by the ubiquitin proteasome pathway. The present review covers the sequence of events leading to PBI extrusion, and compares them to the corresponding events in mitotic cell division. The latest findings regarding the contribution of ubiquitin chain topology, separase, securin, cyclin B1, CDK1, Polo-like kinase 1 and mitogen-activated protein kinase kinase 1/2 to the regulation of meiosis are discussed.

Cellular functions of the DUBs

Ubiquitylation is a reversible post-translational modification that has emerged as a key regulator of most complex cellular processes. It may rival phosphorylation in scope and exceed it in complexity. The dynamic nature of ubiquitylation events is important for governing protein stability, maintaining ubiquitin homeostasis and controlling ubiquitin-dependent signalling pathways. The human genome encodes ~80 active deubiquitylating enzymes (DUBs, also referred to as deubiquitinases), which exhibit distinct specificity profiles towards the various ubiquitin chain topologies. As a result of their ability to reverse ubiquitylation, these enzymes control a broad range of key cellular processes. In this Commentary we discuss the cellular functions of DUBs, such as their role in governing membrane traffic and protein quality control. We highlight two key signalling pathways--the Wnt and transforming growth factor β (TGF-β) pathways, for which dynamic ubiquitylation has emerged as a key regulator. We also discuss the roles of DUBs in the nucleus, where they govern transcriptional activity and DNA repair pathways.

The Deubiquitinating Enzyme AMSH3 Is Required for Intracellular Trafficking and Vacuole Biogenesis inArabidopsis thaliana

Ubiquitination, deubiquitination, and the formation of specific ubiquitin chain topologies have been implicated in various cellular processes. Little is known, however, about the role of ubiquitin in the development of cellular organelles. Here, we identify and characterize the deubiquitinating enzyme AMSH3 from Arabidopsis thaliana. AMSH3 hydrolyzes K48- and K63-linked ubiquitin chains in vitro and accumulates both ubiquitin chain types in vivo. amsh3 mutants fail to form a central lytic vacuole, accumulate autophagosomes, and mis-sort vacuolar protein cargo to the intercellular space. Furthermore, AMSH3 is required for efficient endocytosis of the styryl dye FM4-64 and the auxin efflux facilitator PIN2. We thus present evidence for a role of deubiquitination in intracellular trafficking and vacuole biogenesis.

Quantitative Analysis of global Ubiquitination in HeLa Cells by Mass Spectrometry

Ubiquitination regulates a host of cellular processes by labeling proteins for degradation, but also by functioning as a regulatory, nonproteolytic posttranslational modification. Proteome-wide strategies to monitor changes in ubiquitination profiles are important to obtain insight into the various cellular functions of ubiquitination. Here we describe generation of stable cell lines expressing a tandem hexahistidine-biotin tag (HB-tag) fused to ubiquitin for two-step purification of the ubiquitinated proteome under fully denaturing conditions. Using this approach we identified 669 ubiquitinated proteins from HeLa cells, including 44 precise ubiquitin attachment sites on substrates and all seven possible ubiquitin chain-linkage types. To probe the dynamics of ubiquitination in response to perturbation of the ubiquitin/proteasome pathway, we combined ubiquitin profiling with quantitative mass spectrometry using the stable isotope labeling with amino acids in cell culture (SILAC) strategy. We compared untreated cells and cells treated with the proteasome inhibitor MG132 to identify ubiquitinated proteins that are targeted to the proteasome for degradation. A number of proteasome substrates were identified. In addition, the quantitative approach allowed us to compare proteasome targeting by different ubiquitin chain topologies in vivo. The tools and strategies described here can be applied to detect changes in ubiquitination dynamics in response to various changes in growth conditions and cellular stress and will contribute to our understanding of the ubiquitin/proteasome system.