USP30 sets a trigger threshold for PINK1–PARKIN amplification of mitochondrial ubiquitylation

The mitochondrial deubiquitylase USP30 negatively regulates the selective autophagy of damaged mitochondria. We present the characterisation of an N-cyano pyrrolidine compound, FT3967385, with high selectivity for USP30. We demonstrate that ubiquitylation of TOM20, a component of the outer mitochondrial membrane import machinery, represents a robust biomarker for both USP30 loss and inhibition. A proteomics analysis, on a SHSY5Y neuroblastoma cell line model, directly compares the effects of genetic loss of USP30 with chemical inhibition. We have thereby identified a subset of ubiquitylation events consequent to mitochondrial depolarisation that are USP30 sensitive. Within responsive elements of the ubiquitylome, several components of the outer mitochondrial membrane transport (TOM) complex are prominent. Thus, our data support a model whereby USP30 can regulate the availability of ubiquitin at the specific site of mitochondrial PINK1 accumulation following membrane depolarisation. USP30 deubiquitylation of TOM complex components dampens the trigger for the Parkin-dependent amplification of mitochondrial ubiquitylation leading to mitophagy. Accordingly, PINK1 generation of phospho-Ser65 ubiquitin proceeds more rapidly in cells either lacking USP30 or subject to USP30 inhibition

A batch correction method for liquid chromatography–mass spectrometry data that does not depend on quality control samples

The need for reproducible and comparable results is of increasing importance in non-targeted metabolomic studies, especially when differences between experimental groups are small. Liquid chromatography– mass spectrometry spectra are often acquired batch-wise so that necessary calibrations and cleaning of the instrument can take place. However this may introduce further sources of variation, such as differences in the conditions under which the acquisition of individual batches is performed. Quality control (QC) samples are frequently employed as a means of both judging and correcting this variation. Here we show that the use of QC samples can lead to problems. The non-linearity of the response can result in substantial differences between the recorded intensities of the QCs and experimental samples, making the required adjustment difficult to predict. Furthermore, changes in the response profile between one QC interspersion and the next cannot be accounted for and QC based correction can actually exacerbate the problems by introducing artificial differences. ‘‘Background correction’’ methods utilise all experimental samples to estimate the variation over time rather than relying on the QC samples alone. We compare non-QC correction methods with standard QC correction and demonstrate their success in reducing differences between replicate samples and their potential to highlight differences between experimental groups previously hidden by instrumental variation

From waste to food : optimising the breakdown of oil palm waste to provide substrate for insects farmed as animal feed

Waste biomass from the palm oil industry is currently burned as a means of disposal and solutions are required to reduce the environmental impact. Whilst some waste biomass can be recycled to provide green energy such as biogas, this investigation aimed to optimise experimental conditions for recycling palm waste into substrate for insects, farmed as a sustainable high-protein animal feed. NMR spectroscopy and LC-HRMS were used to analyse the composition of palm empty fruit bunches (EFB) under experimental conditions optimised to produce nutritious substrate rather than biogas. Statistical pattern recognition techniques were used to investigate differences in composition for various combinations of pre-processing and anaerobic digestion (AD) methods. Pre-processing methods included steaming, pressure cooking, composting, microwaving, and breaking down the EFB using ionic liquids. AD conditions which were modified in combination with pre-processing methods were ratios of EFB:digestate and pH. Results show that the selection of pre-processing method affects the breakdown of the palm waste and subsequently the substrate composition and biogas production. Although large-scale insect feeding trials will be required to determine nutritional content, we found that conditions can be optimised to recycle palm waste for the production of substrate for insect rearing. Pre-processing EFB using ionic liquid before AD at pH6 with a 2:1 digestate:EFB ratio were found to be the best combination of experimental conditions

Integrating transcriptomic techniques and k-means clustering in metabolomics to identify markers of abiotic and biotic stress in Medicago truncatula

Nitrogen-fixing legumes are invaluable crops, but are sensitive to physical and biological stresses. Whilst drought and infection from the soil-borne pathogen Fusarium oxysporum have been studied individually, their combined effects have not been widely investigated

AlacatDesigner-Computational Design of Peptide Concatamers for Protein Quantitation

Protein quantitation via mass spectrometry relies on peptide proxies for the parent protein from which abundances are estimated. Owing to the variability in signal from individual peptides, accurate absolute quantitation usually relies on the addition of an external standard. Typically, this involves stable isotope-labeled peptides, delivered singly or as a concatenated recombinant protein. Consequently, the selection of the most appropriate surrogate peptides and the attendant design in recombinant proteins termed QconCATs are challenges for proteome science. QconCATs can now be built in a "a-la-carte" assembly method using synthetic biology: ALACATs. To assist their design, we present "AlacatDesigner", a tool that supports the peptide selection for recombinant protein standards based on the user's target protein. The user-customizable tool considers existing databases, occurrence in the literature, potential post-translational modifications, predicted miscleavage, predicted divergence of the peptide and protein quantifications, and ionization potential within the mass spectrometer. We show that peptide selections are enriched for good proteotypic and quantotypic candidates compared to empirical data. The software is freely available to use either via a web interface AlacatDesigner, downloaded as a Desktop application or imported as a Python package for the command line interface or in scripts

The deubiquitylase USP15 regulates topoisomerase II alpha to maintain genome integrity

Ubiquitin-specific protease 15 (USP15) is a widely expressed deubiquitylase that has been implicated in diverse cellular processes in cancer. Here we identify topoisomerase II (TOP2A) as a novel protein that is regulated by USP15. TOP2A accumulates during G2 and functions to decatenate intertwined sister chromatids at prophase, ensuring the replicated genome can be accurately divided into daughter cells at anaphase. We show that USP15 is required for TOP2A accumulation, and that USP15 depletion leads to the formation of anaphase chromosome bridges. These bridges fail to decatenate, and at mitotic exit form micronuclei that are indicative of genome instability. We also describe the cell cycle-dependent behaviour for two major isoforms of USP15, which differ by a short serine-rich insertion that is retained in isoform-1 but not in isoform-2. Although USP15 is predominantly cytoplasmic in interphase, we show that both isoforms move into the nucleus at prophase, but that isoform-1 is phosphorylated on its unique S229 residue at mitotic entry. The micronuclei phenotype we observe on USP15 depletion can be rescued by either USP15 isoform and requires USP15 catalytic activity. Importantly, however, an S229D phospho-mimetic mutant of USP15 isoform-1 cannot rescue either the micronuclei phenotype, or accumulation of TOP2A. Thus, S229 phosphorylation selectively abrogates this role of USP15 in maintaining genome integrity in an isoform-specific manner. Finally, we show that USP15 isoform-1 is preferentially upregulated in a panel of non-small cell lung cancer cell lines, and propose that isoform imbalance may contribute to genome instability in cancer. Our data provide the first example of isoform-specific deubiquitylase phospho-regulation and reveal a novel role for USP15 in guarding genome integrity