Biology subjects

Impens, F.

Publications and source records attributed to Impens, F..

28 records · Page 2Linked to original sources

bioRxiv2022

Single-cell profiling identifies a spectrum of human unconventional intraepithelial T lineage cells

In the human thymus, a CD10+ PD-1+ TCR{beta}+ differentiation pathway diverges from the conventional single positive T cell lineages at the early double positive stage. These cells are phenotypically and functionally similar to murine unconventional intraepithelial lymphocyte (uIEL) precursors. Here, the progeny of the human uIEL lineage was identified in antigen-inexperienced blood. The uIELs in thymus and peripheral blood share a transcriptomic profile, characterized by hallmark transcription factors (i.e. ZNF683 and IKZF2), and polyclonal TCR repertoire with autoreactive features, exhibiting a bias towards early TCR alpha chain rearrangements. Single-cell RNA sequencing confirmed a common developmental trajectory between the thymic and peripheral uIELs, and clearly delineated this unconventional lineage in peripheral blood. This population is phenotypically defined as CD3+ TCR{beta}+ CD4- CCR7- CD26-. It contains CD10+ recent thymic emigrants, Helios+ KIR+ CD8+ Tregs and CD8+ T cells. Thus, the uIEL lineage represents a well-defined but heterogeneous, unconventional TCR{beta}+ lineage mostly confined in human within the CD8 single positive T cells. SummaryBilliet et al. identify the postthymic progeny of the intraepithelial lymphocyte precursors in human based on shared characteristics of the T cell receptor repertoire and the transcriptome. This lineage represents a well-defined but heterogeneous, unconventional TCR{beta}+ lineage mostly confined within the CD8 single positive T cells.

immunology↗

bioRxiv2021

Proteome-wide cellular thermal shift assay reveals novel crosstalk between brassinosteroid and auxin signaling

Despite the growing interest in using chemical genetics in plant research, small-molecule target identification remains a major challenge. The cellular thermal shift assay coupled with high-resolution mass-spectrometry (CETSA MS) that monitors changes in the thermal stability of proteins caused by their interactions with small molecules, other proteins, or post-translational modifications allows the identification of drug targets, or the study of protein-metabolite and protein-protein interactions mainly in mammalian cells. To showcase the applicability of this method in plants, we applied CETSA MS to intact Arabidopsis thaliana cells and identified the thermal proteome of the plant-specific glycogen synthase kinase 3 (GSK3) inhibitor, bikinin. A comparison between the thermal- and the phospho-proteomes of bikinin revealed the auxin efflux carrier PIN-FORMED1 (PIN1) as a novel substrate of the Arabidopsis GSK3s that negatively regulate the brassinosteroid signaling. We established that PIN1 phosphorylation by the GSK3s is essential for maintaining its intracellular polarity that is required for auxin-mediated regulation of vascular patterning in the leaf thus, revealing a novel crosstalk between brassinosteroid and auxin signaling. Significance StatementChemical genetics, which investigates the biological processes using small molecules, is gaining interest in plant research. However, a major challenge is to uncover the mode of action of the small molecule. Here, we applied the cellular thermal shift assay coupled with mass spectrometry (CETSA MS) to intact Arabidopsis cells and showed that bikinin, the plant-specific glycogen synthase kinase 3 (GSK3) inhibitor, changed the thermal stability of some of its direct targets and putative GSK3 interacting proteins. In combination with phosphoproteomics, we also revealed that GSK3s phosphorylate the auxin carrier PIN-FORMED1 (PIN1) and regulated its polarity that is required for the vascular patterning in the leaf.

plant biology↗

bioRxiv2021

Optineurin links Hace1-dependent Rac ubiquitylation to integrin-mediated mechanotransduction to control bacterial invasion and cell division

Extracellular matrix (ECM) elasticity is perceived by cells via focal adhesion structures, which transduce mechanical cues into chemical signalling to conform cell behaviour. Although the contribution of ECM compliance to the control of cell migration or division has been extensively studied, little has been reported regarding infectious processes. We have studied how mechanical properties of the ECM impact invasion of cells by the extraintestinal Escherichia coli pathogen UTI89. We show that UTI89 takes advantage, via its CNF1 toxin, of integrin mechanoactivation to trigger its invasion into cells. We identified OPTN as a protein regulated by ECM stiffness whose function is required for bacterial invasion and integrin mechanical coupling and for stimulation of HACE1 E3 ligase activity towards the Rac1 GTPase. We showed that OPTN knockdown cells display enhanced Rac1 activation, strong mechanochemical adhesion signalling and increased cyclin D1 translation, together with enhanced cell proliferation independent of ECM stiffness. Despite such features, OPTN knockdown cells displayed defective traction force buildup associated with limited cellular invasion by UTI89. Together, our data indicate that OPTN, through a new role in mechanobiology, supports CNF1-producing uropathogenic E. coli invasion and links HACE1-mediated ubiquitylation of Rac1 to ECM mechanical properties and integrin mechanotransduction.

cell biology↗

bioRxiv2021

Ring Finger Protein 213 Assembles into a Sensor for ISGylated Proteins with Antimicrobial Activity

ISG15 is an interferon-stimulated, ubiquitin-like protein that can conjugate to substrate proteins (ISGylation) to counteract microbial infection, but the underlying mechanisms remain elusive. Here, we used a viral-like particle trapping technology to identify ISG15-binding proteins and discovered Ring Finger Protein 213 (RNF213) as an ISG15 interactor and cellular sensor of ISGylated proteins. RNF213 is a poorly-characterized, interferon-induced megaprotein that is frequently mutated in Moyamoya disease, a rare cerebrovascular disorder. We found that interferon induces ISGylation and oligomerization of RNF213 on lipid droplets, where it acts as a sensor for ISGylated proteins. We showed that RNF213 has broad antimicrobial activity in vitro and in vivo, counteracting infection with Listeria monocytogenes, herpes simplex virus 1 (HSV-1), human respiratory syncytial virus (RSV) and coxsackievirus B3 (CVB3), and we observed a striking co-localization of RNF213 with intracellular bacteria. Together, our findings provide novel molecular insights into the ISGylation pathway and reveal RNF213 as a key antimicrobial effector.

cell biology↗

bioRxiv2019

A simple approach for accurate peptide quantification in MS-based proteomics

Despite its growing popularity and use, bottom-up proteomics remains a complex analytical methodology. Its general workflow consists of three main steps: sample preparation, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and computational data analysis. Quality assessment of the different steps and components of this workflow is instrumental to identify technical flaws and to avoid loss of precious measurement time and sample material. However, assessment of the extent of sample losses along the sample preparation protocol, in particular after proteolytic digestion, is not yet routinely implemented because of the lack of an accurate and straightforward method to quantify peptides. Here, we report on the use of a microfluidic UV/visible spectrophotometer to quantify MS-ready peptides directly in MS loading solvent, consuming only 2 l of sample. We determined the optimal peptide amount for LC-MS/MS analysis on a Q Exactive HF mass spectrometer using a dilution series of a commercial K562 cell digest. Careful evaluation of selected LC and MS parameters allowed us to define 3 g as an optimal peptide amount to be injected on this particular LC-MS/MS system. Finally, using tryptic digests from human HEK293T cells, we showed that injecting equal peptide amounts, rather than approximated ones, results into less variable LC-MS/MS and protein quantification data. The obtained quality improvement together with easy implementation of the approach makes it possible to routinely quantify MS-ready peptides as a next step in daily proteomics quality control.

biochemistry↗

bioRxiv2019

Establishment of Proximity-dependent Biotinylation Approaches in Different Plant Model Systems

Proximity-dependent biotin labelling (PDL) uses a promiscuous biotin ligase (PBL) or a peroxidase fused to a protein of interest. This enables covalent biotin labelling of proteins and allows subsequent capture and identification of interacting and neighbouring proteins without the need for the protein complex to remain intact. To date, only few papers report on the use of PDL in plants. Here we present the results of a systematic study applying a variety of PDL approaches in several plant systems using various conditions and bait proteins. We show that TurboID is the most promiscuous variant in several plant model systems and establish protocols which combine Mass Spectrometry-based analysis with harsh extraction and washing conditions. We demonstrate the applicability of TurboID in capturing membrane-associated protein interactomes using Lotus japonicus symbiotically active receptor kinases as test-case. We further benchmark the efficiency of various PBLs in comparison with one-step affinity purification approaches. We identified both known as well as novel interactors of the endocytic TPLATE complex. We furthermore present a straightforward strategy to identify both non-biotinylated as well as biotinylated peptides in a single experimental setup. Finally, we provide initial evidence that our approach has the potential to infer structural information of protein complexes.

plant biology↗

bioRxiv2019

The future of peptide-centric Data-Independent Acquisition is predicted

Data-Independent Acquisition (DIA) generates comprehensive yet complex mass spectrometric data, which imposes the use of data-dependent acquisition (DDA) libraries for deep peptide-centric detection. We here show that DIA can be redeemed from this dependency by combining predicted fragment intensities and retention times with narrow window DIA. This eliminates variation in library building and omits stochastic sampling, finally making the DIA workflow fully deterministic. Especially for clinical proteomics, this has the potential to facilitate inter-laboratory comparison. Significance of the StudyData-independent acquisition (DIA) is quickly developing into the most comprehensive strategy to analyse a sample on a mass spectrometer. Correspondingly, a wave of data analysis strategies has followed suit, improving the yield from DIA experiments with each iteration. As a result, a worldwide wave of investments in DIA is already taking place in anticipation of clinical applications. Yet, there is considerable confusion about the most useful and efficient way to handle DIA data, given the plethora of possible approaches with little regard for compatibility and complementarity. In our manuscript, we outline the currently available peptide-centric DIA data analysis strategies in a unified graphic called the DIAmond DIAgram. This leads us to an innovative and easily adoptable approach based on predicted spectral information. Most importantly, our contribution removes what is arguably the biggest bottleneck in the field: the current need for Data Dependent Acquisition (DDA) prior to DIA analysis. Fractionation, stochastic data acquisition, processing and identification all introduce bias in the library. By generating libraries through data independent, i.e. deterministic acquisition, stochastic sampling in the DIA workflow is now fully omitted. This is a crucial step towards increased standardization. Additionally, our results demonstrate that a proteome-wide predicted spectral library can surrogate an exhaustive DDA Pan-Human library that was built based on 331 prior DDA runs.

bioinformatics↗

bioRxiv2019

Crippling the bacterial cell wall molecular machinery

Lytic transglycosylases (LT) are redundant enzymes that play a critical role in peptidoglycan (PG) recycling and metabolism. LT(s) role in cell wall-modifying complexes and usefulness as antimicrobial drug targets remain elusive. We determined at high-resolution a structure of the membrane-bound homolog of the soluble LT from Neisseria species with a disordered active site helix (alpha helix 30). Alpha helix 30 is crucial for binding PG during catalysis1. Here we show using an alpha helix 30 deletion strain that LT (LtgA) determines the integrity of the cell wall, participates in cell division and separation, and can be manipulated to impair the fitness of the human pathogen Neisseria meningitidis during infection. Characterization of ltgA helix deleted strain interactome identified glycan chain remodeling enzymes whose function appear to be modulated by LTs. Targeting LTs can disrupt the PG machinery, which is fatal for the bacterium, a new approach for antibiotic development.

biochemistry↗

bioRxiv2018

Digging deeper into the human proteome: A novel nanoflow LCMS setup using micro pillar array columns (μPAC™)

In bottom-up proteomics, capillaries up to 75 cm long with internal diameters of 50 to 100 {micro}m packed with sub-2-{micro}m C18-functionalized particles are routinely used in combination with high-resolution mass spectrometry. Unlike such conventional liquid chromatography (LC) columns, micro pillar array columns ({micro}PAC) are fabricated using micromachining technology, resulting in perfectly ordered chromatographic separation beds, leading to a minimized analyte dispersion while column permeability is increased by one order of magnitude. This allows using very long columns (up to 200 cm) at only a fraction of the pressure needed to operate packed bed columns. To validate {micro}PAC column performances, different amounts of tryptic digests of HEK293T cell lysates were prepared and separated using a 200 cm {micro}PAC column or a 40 cm long conventional column. Using an Orbitrap Elite instrument, on average 25% more proteins were identified with the {micro}PAC column. Moreover, the rate at which the peak width increases with gradient time is much lower on the {micro}PAC column. For a 10-hour long gradient, average peak widths below 0.5 min were observed, resulting in consistent identification of over 5,000 proteins. Combining long solvent gradients and this new type of LC column, substantial improvements in proteome coverage could be obtained. Finally, we demonstrated high reproducibility and durability of the {micro}PAC column. Data are available via ProteomeXchange with identifiers PXD011547 and PXD013235.

biochemistry↗

bioRxiv2018

Parl Deficiency In Mouse Causes Coenzyme Q Depletion, Complex III Defects, And Leigh-Like Syndrome

The mitochondrial intramembrane rhomboid protease Parl has been implicated in diverse functions in vitro, but its physiological role in vivo remains unclear. Here we show that Parl ablation in mouse causes a striking necrotizing encephalomyelopathy similar to Leigh syndrome, a mitochondrial disease characterized by disrupted energy production. Mice with conditional Parl deficiency in the nervous system, but not in muscle, develop a similar phenotype as germline Parl knockouts demonstrating the vital role of Parl in neurological homeostasis. Genetic modification of two major Parl substrates, Pink1 and Pgam5, do not modify this severe neurological phenotype. Parl-/- brain mitochondria are affected by defects in Complex III activity and in coenzyme Q biosynthesis. Parl is necessary for the stable expression of Ttc19, required for Complex III activity, and of Coq4, essential in coenzyme Q biosynthesis. Thus, Parl plays a previously overseen constitutive role in the maintenance of the respiratory chain in the nervous system, and its deficiency causes progressive mitochondrial dysfunction and Leigh-like syndrome.

molecular biology↗