Biology subjects

Di Fraia, D.

Publications and source records attributed to Di Fraia, D..

4 recordsLinked to original sources

bioRxiv2023

Automated workflow for BioID improves reproducibility and identification of protein-protein interactions

Proximity dependent biotinylation is an important method to study protein-protein interactions in cells, for which an expanding number of applications has been proposed. The laborious and time consuming sample processing has limited project sizes so far. Here, we introduce an automated workflow on a liquid handler to process up to 96 samples at a time. The automation does not only allow higher sample numbers to be processed in parallel, but also improves reproducibility and lowers the minimal sample input. Furthermore, we combined automated sample processing with shorter liquid chromatography gradients and data-independent acquisition to increase analysis throughput and enable reproducible protein quantitation across a large number of samples. We successfully applied this workflow to optimise the detection of proteasome substrates by proximity-dependent labelling.

biochemistry↗

bioRxiv2023

A comprehensive atlas of the aging vertebrate brain reveals signatures of progressive proteostasis dysfunction

Aging and neurodegeneration entail diverse cellular and molecular hallmarks. Here, we studied the effects of aging on the transcriptome, translatome, and multiple layers of the proteome in the brain of a short-lived killifish. We reveal that aging causes widespread reduction of proteins enriched in basic amino acids that is independent of mRNA regulation, and it is not due to impaired proteasome activity. Instead, we identify a cascade of events where aberrant translation pausing leads to reduced ribosome availability resulting in proteome remodeling independently of transcriptional regulation. Our research uncovers a vulnerable point in the aging brains biology - the biogenesis of basic DNA/RNA binding proteins. This vulnerability may represent a unifying principle that connects various aging hallmarks, encompassing genome integrity and the biosynthesis of macromolecules. One-Sentence SummaryTranslation pausing reshapes the aging brain proteome, revealing vulnerabilities in the biogenesis of nucleic-acid protein.

systems biology↗

bioRxiv2022

ProteasomeID: quantitative mapping of proteasome interactomes and substrates for in vitro and in vivo studies

Proteasomes are essential molecular machines responsible for the degradation of proteins in eukaryotic cells. Altered proteasome activity has been linked to neurodegeneration, auto-immune disorders and cancer. Despite the relevance for human disease and drug development, no method currently exists to monitor proteasome composition and interactions in vivo in animal models. To fill this gap, we developed a strategy based on tagging of proteasomes with promiscuous biotin ligases and generated a new mouse model enabling the quantification of proteasome interactions by mass spectrometry. We show that biotin ligases can be incorporated in fully assembled proteasomes without negative impact on their activity. We demonstrate the utility of our method by identifying novel proteasome-interacting proteins, charting interactomes across mouse organs, and showing that proximity-labeling enables the identification of both endogenous and small molecule-induced proteasome substrates.

biochemistry↗

bioRxiv2021

Conserved exchange of paralog proteins during neuronal differentiation

Gene duplication enables the emergence of new functions by lowering the general evolutionary pressure. Previous studies have highlighted the role of specific paralog genes during cell differentiation, e.g., in chromatin remodeling complexes. It remains unexplored whether similar mechanisms extend to other biological functions and whether the regulation of paralog genes is conserved across species. Here, we analyze the expression of paralogs across human tissues, during development and neuronal differentiation in fish, rodents and humans. While ~80% of paralog genes are co-regulated, a subset of paralogs shows divergent expression profiles, contributing to variability of protein complexes. We identify 78 substitutions of paralog pairs that occur during neuronal differentiation and are conserved across species. Among these, we highlight a substitution between the paralogs SEC23A and SEC23B subunits of the COPII complex. Altering the ratio between these two proteins via RNAi-mediated knockdown is sufficient to influence neuron differentiation. We propose that remodeling of the vesicular transport system via paralog substitutions is an evolutionary conserved mechanism enabling neuronal differentiation.

systems biology↗