A Generic Platform for Cellular Screening Against Ubiquitin Ligases

Ubiquitin signalling regulates most aspects of cellular life, thus deregulation of ubiquitylation has been linked with a number of diseases. E3 ubiquitin ligases provide substrate selectivity in ubiquitylation cascades and are therefore considered to be attractive targets for developing therapeutic molecules. In contrast to established drug target classes, such as protein kinases, GPCRs, hormone receptors and ion channels, ubiquitin drug discovery is in its early stages. This is, in part, due to the complexity of the ubiquitylation pathways and the lack of robust quantitative technologies that allow high-throughput screening of inhibitors. Here we report the development of a Ubiquitin Ligase Profiling system, which is a novel and generic cellular technology designed to facilitate identification of selective inhibitors against RING type E3 ubiquitin ligases. Utilization of this system requires a single co-transfection of cells with assay vectors, thereby enabling readout of E3 ubiquitin ligase catalytic activity within the cellular environment. Therefore, our robust high-throughput screening platform offers novel opportunities for the development of inhibitors against this difficult-to-target E3 ligase enzyme class

A methodology for detecting the orthology signal in a PPI network at a functional complex level

Contains fulltext : 93730.pdf (preprint version ) (Open Access)13 p

Structural and non-coding variants increase the diagnostic yield of clinical whole genome sequencing for rare diseases

BACKGROUND: Whole genome sequencing is increasingly being used for the diagnosis of patients with rare diseases. However, the diagnostic yields of many studies, particularly those conducted in a healthcare setting, are often disappointingly low, at 25–30%. This is in part because although entire genomes are sequenced, analysis is often confined to in silico gene panels or coding regions of the genome. METHODS: We undertook WGS on a cohort of 122 unrelated rare disease patients and their relatives (300 genomes) who had been pre-screened by gene panels or arrays. Patients were recruited from a broad spectrum of clinical specialties. We applied a bioinformatics pipeline that would allow comprehensive analysis of all variant types. We combined established bioinformatics tools for phenotypic and genomic analysis with our novel algorithms (SVRare, ALTSPLICE and GREEN-DB) to detect and annotate structural, splice site and non-coding variants. RESULTS: Our diagnostic yield was 43/122 cases (35%), although 47/122 cases (39%) were considered solved when considering novel candidate genes with supporting functional data into account. Structural, splice site and deep intronic variants contributed to 20/47 (43%) of our solved cases. Five genes that are novel, or were novel at the time of discovery, were identified, whilst a further three genes are putative novel disease genes with evidence of causality. We identified variants of uncertain significance in a further fourteen candidate genes. The phenotypic spectrum associated with RMND1 was expanded to include polymicrogyria. Two patients with secondary findings in FBN1 and KCNQ1 were confirmed to have previously unidentified Marfan and long QT syndromes, respectively, and were referred for further clinical interventions. Clinical diagnoses were changed in six patients and treatment adjustments made for eight individuals, which for five patients was considered life-saving. CONCLUSIONS: Genome sequencing is increasingly being considered as a first-line genetic test in routine clinical settings and can make a substantial contribution to rapidly identifying a causal aetiology for many patients, shortening their diagnostic odyssey. We have demonstrated that structural, splice site and intronic variants make a significant contribution to diagnostic yield and that comprehensive analysis of the entire genome is essential to maximise the value of clinical genome sequencing

Self-oligomerization regulates stability of survival motor neuron protein isoforms by sequestering an SCF^Slmb degron

Spinal muscular atrophy (SMA) is caused by homozygous mutations in human SMN1. Expression of a duplicate gene (SMN2) primarily results in skipping of exon 7 and production of an unstable protein isoform, SMNΔ7. Although SMN2 exon skipping is the principal contributor to SMA severity, mechanisms governing stability of survival motor neuron (SMN) isoforms are poorly understood. We used a Drosophila model system and label-free proteomics to identify the SCFSlmb ubiquitin E3 ligase complex as a novel SMN binding partner. SCFSlmb interacts with a phosphor degron embedded within the human and fruitfly SMN YG-box oligomerization domains. Substitution of a conserved serine (S270A) interferes with SCFSlmb binding and stabilizes SMNΔ7. SMA-causing missense mutations that block multimerization of full-length SMN are also stabilized in the degron mutant background. Overexpression of SMNΔ7S270A, but not wild-type (WT) SMNΔ7, provides a protective effect in SMA model mice and human motor neuron cell culture systems. Our findings support a model wherein the degron is exposed when SMN is monomeric and sequestered when SMN forms higher-order multimers

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Deubiquitinase-based analysis of ubiquitin chain architecture using Ubiquitin Chain Restriction (UbiCRest)

Protein ubiquitination is a versatile protein modification that regulates virtually all cellular processes. This versatility originates from polyubiquitin chains, which can be linked in eight distinct ways. The combinatorial complexity of eight linkage types in homotypic (one chain type per polymer) and heterotypic (multiple linkage types per polymer) chains poses significant problems for biochemical analysis. Here we describe UbiCRest, in which substrates (ubiquitinated proteins or polyubiquitin chains) are treated with a panel of linkage-specific deubiquitinating enzymes (DUBs) in parallel reactions, followed by gel-based analysis. UbiCRest can be used to show that a protein is ubiquitinated, to identify which linkage type(s) are present on polyubiquitinated proteins and to assess the architecture of heterotypic polyubiquitin chains. DUBs used in UbiCRest can be obtained commercially; however, we include details for generating a toolkit of purified DUBs and for profiling their linkage preferences in vitro. UbiCRest is a qualitative method that yields insights into ubiquitin chain linkage types and architecture within hours, and it can be performed on western blotting quantities of endogenously ubiquitinated proteins