Biology subjects

Luescher, B.

Publications and source records attributed to Luescher, B..

5 recordsLinked to original sources

bioRxiv2025

Deciphering cytokine-driven ADP-ribosylation signaling networks via Af1521-based mass spectrometry analysis of labile Glu/Asp-linkages

ADP-ribosylation (ADPr) is a regulatory post-translational modification targeting nine amino acid residues. Glutamate- and aspartate-linked ADPr (Glu/Asp-ADPr) is chemically unstable during sample preparation for conventional mass spectrometry (MS)-based proteomics workflows, limiting its detection. Here, we systematically assessed the stability of ADPr linkages using synthetic peptides and confirmed that ester-linked Glu/Asp-ADPr is lost under alkaline conditions, elevated temperatures, or through hydrolysis by wildtype Af1521. We established an acidic enrichment workflow encompassing an Af1521 mutant, robustly preserving Glu/Asp-ADPr enabling their site-specific and systems-wide MS analysis. Applying this strategy to cytokine-stimulated A549 and HeLa cells, we identified >600 Glu/Asp-ADPr and >200 Cys-ADPr sites. Our analysis uncovered that Glu/Asp-ADPr marks distinct cytoplasmic protein networks enriched in immune functions, contrasting with Ser-ADPr typically observed on nuclear and chromatin-associated proteins. Quantitative profiling revealed reproducible ADPr patterns specific to cell type and treatment. Notably, PARP10 promoted Glu/Asp modification of ubiquitin, highlighting crosstalk between ADPr and ubiquitin signaling. Across interferon treatments, we identified a conserved network of antiviral PARPs and associated cofactors extensively modified on Glu/Asp residues, emphasizing residue-specific ADPr as a regulator of innate immune signaling. Together, our work establishes an MS-based proteomics workflow for identification of Glu/Asp-ADPr, provides a resource of site-specific modification events, and reveals residue-specific ADPr dynamics in immune signaling.

cell biology↗

bioRxiv2022

Four decades of phenology in an alpine amphibian: trends, stasis and climatic drivers

O_LIStrong phenological shifts in response to changes in climatic conditions have been reported for many species, including amphibians, which are expected to breed earlier. Phenological shifts in breeding are observed in a wide number of amphibian populations, but less is known about populations living at high elevations, which are predicted to be more sensitive to climate change than lowland populations. C_LIO_LIThe goal of this study is to assess the main factors determining the timing of breeding in an alpine population of the common toad (Bufo bufo) and to describe the observed shifts in its breeding phenology. C_LIO_LIWe modelled the effect of environmental variables on the start and peak dates of the breeding season using 39 years of individual-based data. In addition, we investigated the effect of the lunar cycle, as well as the individual variation in breeding phenology. Finally, to assess the individual heterogeneity in the timing of breeding, we calculated the repeatability of the timing of arrival at the breeding site. C_LIO_LIBreeding advanced to earlier dates in the first years of the study but the trend continued only until the mid 1990s, and stabilised afterwards. Overall, toads are now breeding on average around 30 days earlier than at the start of the study period. High temperatures and low snow cover in winter and spring, as well as reduced spring precipitation were all associated with earlier breeding. Additionally, we found evidence of males arriving on average before females at the breeding site but no clear and strong effect of the lunar cycle. We only found weak evidence of among-individual variation in shifts in the breeding phenology, as well as a low repeatability of arrival timing. C_LIO_LIOur findings show that the observed changes in breeding phenology are strongly associated with the environmental conditions. These results contribute to filling a knowledge gap on the effects ssof climate change on alpine amphibian populations. Moreover, we show that changes in phenology, especially in the mountains, can be hard to predict as local microclimatic conditions do not necessarily reflect the observed global climatic trends. C_LI

ecology↗

bioRxiv2022

Systematic analysis of ADP-ribose detection reagents and optimisation of sample preparation to detect ADP-ribosylation in vitro and in cells

Recent evidence suggests that modification of substrates with a single ADP-ribose (ADPr) is important in for example antiviral immunity and cancer. However, the endogenous substrates and the extent of mono-ADP-ribosylation are still largely unclear. Several reagents were developed to detect ADP-ribosylation but it is unknown whether they recognise only ADPr, amino acid-ADPr linkages or a combination of ADPr with a protein backbone. We screened the affinity of selected reagents for enzymatically, chemically and in cell generated ADP-ribosylation on glutamate, cysteine, serine, arginine, threonine and RNA by blotting, as well as analysed the subcellular sites of ADP-ribosylation using immunofluorescence confocal microscopy. We furthermore observed that the modification is heat-labile and optimised sample preparation procedures. Our comparison of the available reagents, as well as optimisation of sample preparation, will allow future work further dissecting the function of ADP-ribosylation in cells, both on protein and on RNA substrates.

molecular biology↗

bioRxiv2021

An Enhanced Sampling Approach to the Induced Fit Docking Problem in Protein-Ligand Binding: the case of mono-ADP-ribosylation hydrolases inhibitors

A variety of enhanced sampling methods can predict free energy landscapes associated with protein/ligand binding events, characterizing in a precise way the intermolecular interactions involved. Unfortunately, these approaches are challenged by not uncommon induced fit mecchanisms. Here, we present a variant of the recently reported volume-based metadynamics (MetaD) method which describes ligand binding even when it affects protein structure. The validity of the approach is established by applying it to a substrate/enzyme complexes of pharmacological relevance: this is the mono-ADP-ribose (ADPr) in complex with mono-ADP-ribosylation hydrolases (MacroD1 and MacroD2), where induced-fit phenomena are known to be operative. The calculated binding free energies are consistent with experiments, with an absolute error less than 0.5 kcal/mol. Our simulations reveal that in all circumstances the active loops, delimiting the boundaries of the binding site, rearrange from an open to a closed conformation upon ligand binding. The calculations further provide, for the first time, the molecular basis of the experimentally observed affinity changes in ADPr binding on passing from MacroD1 to MacroD2 and all its mutants. Our study paves the way to investigate in a completely general manner ligand binding to proteins and receptors.

biochemistry↗

bioRxiv2020

Mono-ADP-ribosylation by ARTD10 restricts Chikungunya virus replication by interfering with the proteolytic activity of nsP2

A subset of intracellular mono-ADP-ribosyltransferases diphtheria toxin-like (ARTDs, aka mono-PARPs) is induced by type I interferons. Some of these mono-ARTDs feature antiviral activity while certain RNA viruses, including Chikungunya virus (CHIKV), encode mono-ADP-ribosylhydrolases, suggesting a role for mono-ADP-ribosylation (MARylation) in host-virus conflicts. CHIKV expresses four non-structural proteins (nsP1-nsP4), with nsP3 containing a macrodomain that hydrolyzes and thereby reverses protein MARylation in vitro and in cells. This de-MARylation activity is essential as hydrolase inactivating mutations result in replication defective virus. However, the substrates of MARylation during CHIKV infection are unknown and thus it is unclear how the macrodomain contributes to virus replication and how mono-ARTD-dependent MARylation confers antiviral immunity. We identified ARTD10 and ARTD12 as restriction factors for CHIKV replication in a catalytic activity-dependent manner. CHIKV replication requires processing of the non-structural polyprotein nsP1-4 by the nsP2-encoded protease and the assembly of the four individual nsPs into a functional replication complex. Expression of ARTD10 and ARTD12 resulted in a reduction of processed nsPs. Similarly, MAR hydrolase inactive CHIKV replicon mutants revealed a decrease in processed nsPs, comparable to an nsP2 protease defective mutant. This suggested that the macrodomain contributes to nsP2 protease activity. In support, a hydrolase-deficient virus was complemented by a protease-deficient virus. We hypothesized that MARylation regulates the proteolytic function of nsP2. Indeed, we found that nsP2 is MARylated by ARTD10. This inhibited nsP2 protease activity, thereby preventing polyprotein processing and consequently virus replication. This inhibition was antagonized by the MAR hydrolase activity of nsP3. Together, our findings provide a mechanistic explanation for the need of the viral MAR hydrolase for efficient replication of CHIKV. Author SummaryInfectious diseases still pose major health threats. Especially fast evolving viruses find ever new strategies to manipulate the immune response. With climate warming and increased human mobility vector-borne pathogens like Chikungunya virus (CHIKV) spread and cause world-wide epidemics. Beyond the acute phase, CHIKV patients regularly suffer from chronic rheumatism. This entails a decline in life quality and an economic burden. To date no drugs are approved and the mode of pathogenesis remains elusive. Here we describe a mechanistic function of the CHIKV nsP3 macrodomain. We found that the viral nsP2 is mono-ADP-ribosylated interfering with its auto-proteolytic function. The nsP3 macrodomain removes this modification and restores the protease activity that is essential for replication. Because macrodomains are highly conserved they might represent broad antiviral targets.

microbiology↗