34 research outputs found
2016 EMBO Chemical Biology Conference
Get PDFFrom August 31st- September 3rd the biennual EMBO Chemical Biology conference, hosted by the European Molecular Biology Laboratory (EMBL) in Heidelberg took place. The conference series has earned a well-deserved reputation as one of the finest meetings representing this discipline of life science research. A feature of this EMBO meeting is the consistency of the venue (EMBL is in a delightful location after all) and the organisers (Maja Kohn, John Overington and Carsten Schultz). This ensures familiarity and high complementarity with prior meetings. Indeed, a policy of the organising committee is that no speaker can be talk more than once so one should choose their opportunity wisely.The typical format was adopted where the conference is divided into sections based on topic and talks were a mixture of 4 keynote speakers and 38 speakers, the latter being invited or selected from abstracts. Almost 200 posters were also displayed throughout the meetin
An atypical ubiquitin ligase at the heart of neural development and programmed axon degeneration
Get PDFThe degeneration of nerve fibres following injury was first described by Augustus Waller over 170 years ago. Initially assumed to be a passive process, it is now evident that axons respond to insult via regulated cellular signaling events resulting in their programmed degeneration. Pro-survival and pro-degenerative factors have been identified and their regulatory mechanisms are beginning to emerge. The ubiquitin system has been implicated in the pro-degenerative process and a key component is the ubiquitin E3 ligase MYCBP2 (also known as PHR1). Ubiquitin E3 ligases are tasked with the transfer of the small protein modifier ubiquitin to substrates and consist of hundreds of members. They can be classified as single subunit systems or as multi-subunit complexes. Their catalytic domains can also be assigned to three general architectures. Hints that MYCBP2 might not conform to these established formats came to light and it is now clear from biochemical and structural studies that MYCBP2 is indeed an outlier in terms of its modus operandi. Furthermore, the unconventional way in which MYCBP2 transfers ubiquitin to substrates has been linked to neurodevelopmental and pro-degenerative function. Herein, we will summarize these research developments relating to the unusual features of MYCBP2 and postulate therapeutic strategies that prevent Wallerian degeneration. These have exciting potential for providing relief from pathological neuropathies and neurodegenerative diseases.</p
Genetically directed production of recombinant, isosteric and non-hydrolyzable ubiquitin conjugates
Get PDFWe describe the genetically directed incorporation of aminooxy functionality into recombinant proteins using a mutant Methanosarcina barkeri pyrrolysyl-tRNA synthetase/tRNACUA pair. This allows the general production of non-hydrolyzable ubiquitin conjugates of recombinant origin via bioorthogonal oxime ligation. This is exemplified by the preparation of non-hydrolyzable versions of diubiquitin, polymeric ubiquitin chains and ubiquitinated SUMO. We demonstrate that the conjugates exhibit unrivalled isostery with the native isopeptide bond through both structural and biophysical characterization. Furthermore, the conjugates function as nanomolar inhibitors of deubiquitinating enzymes and are recognized by linkage–specific antibodies. This technology should provide a versatile platform for the development of powerful research tools for studying deubiquitinating enzymes and for defining the cellular roles of diverse polyubiquitin linkages
Beyond Lysine Ubiquitination
No full textThe covalent attachment of ubiquitin to substrates controls virtually all aspects of the cell. With rare exceptions, ubiquitin was understood to exert its effects by becoming attached to the amino group of lysine residues within protein substrates. Recent discoveries from our lab and others have revealed that dedicated writers (E3 ligases) and erasers (DUBs) of non-lysine ubiquitination are intrinsic to eukaryotes. This highlights that attachment to sites beyond lysine are physiologically and perhaps pathologically important. These E3s tend to be highly divergent from their established counterparts and "activity-based" chemical biology approaches have been instrumental in identifying them. I will present work from my lab on technologies we have used to uncover unusual E3 ligases and the striking nature of their mechanism and substrate specificity. I will also discuss our characterisation of a small DUB family that is highly selective at removing ubiquitin from hydroxy amino acids. These new developments indicate that non-lysine ubiquitination is an integral component of the ubiquitin system that is subject to sophisticated regulation.</p
Semisynthetic Src SH2 domains demonstrate altered phosphopeptide specificity induced by incorporation of unnatural lysine derivatives
No full textSite-directed mutagenesis to the 20 natural amino acids becomes a limitation when evaluating subtle perturbations of an amino acid side chain within a protein. To further the study of Src homology 2 (SH2) domain ligand binding, we have developed a system allowing its semisynthesis from three fragments by native chemical ligation. We have replaced a key lysine residue with lysyl derivatives possessing progressively shorter aliphatic side chains. Biophysical characterization of these SH2 domain analogs has allowed for the first time a systematic dissection of the side chain length contribution from a lysine residue to ligand binding. We show that the specificity of the SH2 domain of the Src kinase can be altered by incorporation of such lysyl derivatives, thereby demonstrating the potential of the technique for the development of SH2 domain-based research tools and therapeutics
Chemical Ubiquitination for Decrypting a Cellular Code
Get PDFThe modification of proteins with ubiquitin (Ub) is an important regulator of eukaryotic biology and deleterious perturbation of this process is widely linked to the onset of various diseases. The regulatory capacity of the Ub signal is high and, in part, arises from the capability of Ub to be enzymatically polymerised to form polyubiquitin (polyUb) chains of 8 different linkage types. These distinct polyUb topologies can then be site-specifically conjugated to substrate proteins to elicit a number of cellular outcomes. Therefore, to further elucidate the biological significance of substrate ubiquitination, methodologies that allow the production of defined polyUb species and substrate proteins that are site-specifically modified with them are essential to progress our understanding. Many chemically inspired methods have recently emerged which fulfil many of the criteria necessary towards achieving deeper insight into Ub biology. With a view to providing immediate impact to traditional biology research labs, the aim of this review is to provide an overview of the techniques that are available for preparing Ub conjugates and polyUb chains with focus on approaches that use recombinant protein building blocks. These approaches either produce a native isopeptide, or analogue thereof, that can be hydrolyzable or non-hydrolyzable by deubiquitinases. The most significant biological insights that have already been garnered using such approaches will also be summarized
A new dawn beyond lysine ubiquitination
Get PDFThe ubiquitin system has become synonymous with the modification of lysine residues. However, the substrate scope and diversity of the conjugation machinery have been underappreciated, bringing us to an epoch in ubiquitin system research. The striking discoveries of metazoan enzymes dedicated toward serine and threonine ubiquitination have revealed the important role of nonlysine ubiquitination in endoplasmic reticulum-associated degradation, immune signaling and neuronal processes, while reports of nonproteinaceous substrates have extended ubiquitination beyond the proteome. Bacterial effectors that bypass the canonical ubiquitination machinery and form unprecedented linkage chemistry further redefine long-standing dogma. While chemical biology approaches have advanced our understanding of the canonical ubiquitin system, further study of noncanonical ubiquitination has been hampered by a lack of suitable tools. This Perspective aims to consolidate and contextualize recent discoveries and to propose potential applications of chemical biology, which will be instrumental in unraveling this new frontier of ubiquitin research.</p
Photocrosslinking Activity-Based Probes for Ubiquitin RING E3 Ligases
Get PDFActivity-based protein profiling is an invaluable technique for studying enzyme biology and facilitating the development of therapeutics. Ubiquitin E3 ligases (E3s) are one of the largest enzyme families and regulate a host of (patho)physiological processes. The largest subtype are the RING E3s of which there are >600 members. RING E3s have adaptor-like activity that can be subject to diverse regulatory mechanisms and have become attractive drug targets. Activity-based probes (ABPs) for measuring RING E3 activity do not exist. Here we re-engineer ubiquitin-charged E2 conjugating enzymes to produce photocrosslinking ABPs. We demonstrate activity-dependent profiling of two divergent cancer-associated RING E3s, RNF4 and c-Cbl, in response to their native activation signals. We also demonstrate profiling of endogenous RING E3 ligase activation in response to epidermal growth factor (EGF) stimulation. These photocrosslinking ABPs should advance E3 ligase research and the development of selective modulators against this important class of enzymes. Activity-based probes (ABPs) are valuable research tools for studying enzyme function. Ubiquitin E3 ligases are one of the largest enzyme families yet ABPs for this enzyme class do not exist. Mathur et al. developed photocrosslinking ABPs for RING E3s and using activity-based proteomics demonstrate activity-dependent readout of diverse E3 activation.</p
The Role of Water in Computational and Experimental Derivation of Binding Thermodynamics in SH2 Domains
No full text
