Biology subjects

Lamond, A. I.

Publications and source records attributed to Lamond, A. I..

26 records · Page 2Linked to original sources

bioRxiv2018

Optimising multi-batch TMT analysis to mitigate inflation of missing values, false positives and diminished inter batch accuracy

Multiplexing strategies for large-scale proteomic analyses have become increasingly prevalent, TMT in particular. Here we used a large iPSC proteomic experiment with twenty-four 10-plex TMT batches to evaluate the effect of integrating multiple TMT batches within a single analysis. We reveal a significant inflation rate of missing protein and peptide values and show that precision decreases as multiple batches are integrated. Additionally, we explore the effect of false positives using Y chromosome specific peptides as an internal control to quantify the effect of co-isolation interference, as well as primary and secondary reporter ion interference. Based on the results we suggest solutions to mitigate these effects. We show using a reference line can increase precision by normalising the quantification across batches and we propose experimental designs that minimise the effect of cross population reporter ion interference.

biochemistry↗

bioRxiv2018

The iPSC proteomic compendium

Induced pluripotent stem cell (iPSC) technology holds great potential for therapeutic and research purposes. The Human Induced Pluripotent Stem Cell Initiative (HipSci) was established to generate a panel of high-quality iPSCs, from healthy and disease cohorts, with accompanying multi-omics and phenotypic data. Here, we present a proteomic analysis of 217 HipSci iPSC lines obtained from 163 donors.\n\nThis dataset provides a comprehensive proteomic map of iPSCs, identifying >16,000 protein groups. We analyse how the expression profiles of proteins involved in cell cycle, metabolism and DNA repair contribute to key features of iPSC biology and we identify potential new regulators of the primed pluripotent state. To facilitate access, all these data have been integrated into the Encyclopedia of Proteome Dynamics (www.peptracker.com/epd), where it can be browsed interactively. Additionally, we generated an iPSC specific spectral library for DIA which we deposited in PRIDE along with the raw and processed mass-spectrometry data.

biochemistry↗

bioRxiv2018

Composition of the Survival Motor Neuron (SMN) complex in Drosophila melanogaster

Spinal Muscular Atrophy (SMA) is caused by homozygous mutations in the human survival motor neuron 1 (SMN1) gene. SMN protein has a well-characterized role in the biogenesis of small nuclear ribonucleoproteins (snRNPs), core components of the spliceosome. SMN is part of an oligomeric complex with core binding partners, collectively called Gemins. Biochemical and cell biological studies demonstrate that certain Gemins are required for proper snRNP assembly and transport. However, the precise functions of most Gemins are unknown. To gain a deeper understanding of the SMN complex in the context of metazoan evolution, we investigated the composition of the SMN complex in Drosophila melanogaster. Using a stable transgenic line that exclusively expresses Flag-tagged SMN from its native promoter, we previously found that Gemin2, Gemin3, Gemin5, and all nine classical Sm proteins, including Lsm10 and Lsm11, co-purify with SMN. Here, we show that CG2941 is also highly enriched in the pulldown. Reciprocal co-immunoprecipitation reveals that epitope-tagged CG2941 interacts with endogenous SMN in Schneider2 cells. Bioinformatic comparisons show that CG2941 shares sequence and structural similarity with metazoan Gemin4. Additional analysis shows that three other genes (CG14164, CG31950 and CG2371) are not orthologous to Gemins 6-7-8, respectively, as previously suggested. In D.melanogaster, CG2941 is located within an evolutionarily recent genomic triplication with two other nearly identical paralogous genes (CG32783 and CG32786). RNAi-mediated knockdown of CG2941 and its two close paralogs reveals that Gemin4 is essential for organismal viability.

genetics↗

bioRxiv2017

FAM192A/PIP30 regulates Cajal body dynamics by antagonizing PA28γ/coilin interaction

PA28{gamma} is a nuclear activator of the 20S proteasome involved in the regulation of several essential cellular processes, such as cell proliferation, apoptosis, nuclear dynamics and cellular stress response. Unlike the 19S regulator of the proteasome, which specifically recognizes ubiquitylated proteins, PA28{gamma} promotes the degradation of several substrates by the proteasome in an ATP- and ubiquitin-independent manner. However its exact mechanisms of action are unclear and likely to involve additional partners that remain to be identified. Here we report the identification of the first cofactor of PA28{gamma}, PIP30/FAM192A. PIP30 binds directly and specifically via its C-terminal end and in an interaction stabilized by casein kinase 2 phosphorylation to both free and 20S proteasome-associated PA28{gamma}. Its recruitment to proteasome-containing complexes depends on PA28{gamma} and its expression increases the association of PA28{gamma} with the 20S proteasome in cells. Further dissection of its possible roles shows that PIP30 alters PA28{gamma}-dependent activation of peptide degradation by the 20S proteasome in vitro and negatively controls in cells the presence of PA28{gamma} in Cajal Bodies by inhibition of its association with the key Cajal body component coilin. Altogether, our data show that PIP30 deeply affects PA28{gamma} interactions with cellular proteins, including 20S proteasome, demonstrating that it is an important regulator of PA28{gamma} in cells and thus a new player in the control of the multiple functions of the proteasome within the nucleus.\n\nSignificance StatementThe 20S proteasome is a key actor of the control of protein levels and integrity in cells. To perform its multiple functions, it works with a series of regulators, among which a nuclear complex called PA28{gamma}. In particular, PA28{gamma} participates in the regulation of cell proliferation and nuclear dynamics. We describe here the characterization of a novel protein, PIP30/FAM192A, which binds tightly to PA28{gamma} and favors its interaction with the 20S proteasome while inhibiting its association with coilin, a central component of nuclear Cajal bodies. Thus PIP30/FAM192A critically controls the interactome and consequently the functions of PA28{gamma}, and appears to be a new player in the fine regulation of intracellular proteostasis in the cell nucleus.

cell biology↗

bioRxiv2017

Proteomic analysis of cell cycle progression in asynchronous cultures, including mitotic subphases, using PRIMMUS

The temporal regulation of protein abundance and post-translational modifications is a key feature of cell division. Recently, we analysed gene expression and protein abundance changes during interphase under minimally perturbed conditions (Ly et al. 2014; Ly et al. 2015). Here we show that by using specific intracellular immunolabeling protocols, FACS separation of interphase and mitotic cells, including mitotic subphases, can be combined with proteomic analysis by mass spectrometry. Using this PRIMMUS (PRoteomic analysis of Intracellular iMMUnolabeled cell Subsets) approach, we now compare protein abundance and phosphorylation changes in interphase and mitotic fractions from asynchronously growing human cells. We identify a set of 115 phosphorylation sites increased during G2, which we term early risers. This set includes phosphorylation of S738 on TPX2, which we show is important for TPX2 function and mitotic progression. Further, we use PRIMMUS to provide a proteome-wide analysis of protein abundance remodeling between prophase, prometaphase and anaphase.

biochemistry↗

bioRxiv2017

A protein phosphatase network controls temporal and spatial dynamics of differentiation commitment in human epidermis

Epidermal homeostasis depends on a balance between stem cell renewal and terminal differentiation. The transition between the two cell states, termed commitment, is poorly understood. Here we characterise commitment by integrating transcriptomic and proteomic data from disaggregated primary human keratinocytes held in suspension for up to 12h to induce differentiation. We find that cell detachment induces a network of protein phosphatases. The pro-commitment phosphatases - including DUSP6, PPTC7, PTPN1, PTPN13 and PPP3CA - promote differentiation by negatively regulating ERK MAPK and positively regulating AP1 transcription factors. Their activity is antagonised by concomitant upregulation of DUSP10. Boolean network modelling of phosphatase interactions identifies commitment as an inherently unstable biological switch between the stem and differentiated cell states. Furthermore, phosphatase expression is spatially regulated both in vivo and in vitro. We conclude that an auto-regulatory phosphatase network maintains epidermal homeostasis by controlling the onset and duration of commitment.

developmental biology↗

bioRxiv2017

Characterisation Of The Biflavonoid Hinokiflavone As A Pre-mRNA Splicing Modulator That Inhibits SENP

Here, we identify the plant biflavanoid hinokiflavone as an inhibitor of splicing in vitro and modulater of alternative splicing in multiple human cell lines. Hinokiflavone inhibits splicing in vitro by blocking one or more early steps of spliceosome assembly, leading to accumulation of the A complex. Multiple human cell lines treated with hinokiflavone show changes in the alternative splicing of different pre-mRNA substrates, but little or no change in transcription. They also show altered subnuclear organization, specifically of splicing factors required for A complex formation, which relocalized together with SUMO1 and SUMO2 into enlarged nuclear speckles. While most cell lines treated with hinokiflavone showed cell cycle arrest and eventual cell death, dependent on time and concentration, the promyelocytic NB4 cell line, which expresses the SUMO target PML-RARalpha fusion protein, was exquisitely sensitive to apoptosis following hinokiflavone treatment. Hinokiflavone treatment increased protein SUMOylation levels, both in in vitro splicing reactions and in cells, with little or no effect on levels of ubiquitinylated proteins. Hinokiflavone also inhibited the catalytic activity of purified E. coli expressed SUMO protease, SENP1 in vitro, indicating the increase in SUMOylated proteins results primarily from inhibition of de-SUMOylation. Using a quantitative proteomics assay we identified many SUMO2 sites whose levels increased following hinokiflavone treatment, with the major targets including 6 proteins that are associated with U2 snRNP and required for A complex formation. These data identify hinokiflavone as a SUMO protease inhibitor and indicate SUMOylation of splicing factors may be important for modulating splice site selection.

biochemistry↗

bioRxiv2016

The Aquarius/EMB-4 helicase licenses co-transcriptional gene silencing

Small RNAs (sRNAs) play an ancient role in genome defence against transposable elements. In animals, plants and fungi small RNAs guide Argonaute proteins to nascent RNA transcripts to induce co-transcriptional gene silencing. In animals the link between small RNA pathways and the transcriptional machinery remains unclear. Here we show that the Caenorhabditis elegans germline Argonaute HRDE-1 physically interacts with the conserved RNA helicase Aquarius/EMB-4. We demonstrate that the Aquarius/EMB-4 helicase activity is required to initiate small RNA-induced co-transcriptional gene silencing. HRDE-1 and Aquarius/EMB-4 are required to silence the transcription of overlapping sets of transposable elements. Surprisingly, removal of introns from a small RNA pathway target abolishes the requirement for Aquarius/EMB-4, but not HRDE-1, for gene silencing. We conclude that the Aquarius/EMB-4 helicase activity allows HRDE-1/sRNA complexes to efficiently engage nascent RNA transcripts - in competition with the general RNA processing machinery. We postulate that Aquarius/EMB-4 facilitates the surveillance of the nascent transcriptome to detect and silence transposable elements through small RNA pathways.

molecular biology↗