Biology subjects

Suppanz, I.

Publications and source records attributed to Suppanz, I..

5 recordsLinked to original sources

bioRxiv2026

Sample preparation for direct nanopore based protein identification via tryptic peptide recognition

Rapid, cost-efficient and miniaturized protein analysis is essential for point-of-care clinical diagnostics. Nanopores offer a promising alternative to mass spectrometry, offering high-resolution, single-molecule sensing with a minimal footprint. However, peptide-level protein identification remains challenging due to difficulties in assigning nanopore signals to peptide sequences and the incompatibility of conventional proteolysis workflows with nanopore sensing. Here we integrate a single-pot, solid-phase-enhanced sample preparation (SP3) workflow with nanopore sensing. The SP3 protocol decouples proteolysis from interfering components via magnetic bead cleanup, yielding a digest compatible with nanopore sensing. As a proof of concept, tryptic digests of myelin basic protein measured with an engineered aerolysin variant (AeL-R220S) generated a reproducible five-peak peptide fingerprint. Cross-validation against a synthetic peptide library and LC-MS/MS enabled reference-based assignment of blockade signatures to four MBP-derived peptides. This approach establishes SP3 as a sample-preparation workflow for nanopore sensing, paving the way for label-free identification and characterization of protein targets.

biochemistry↗

bioRxiv2023

Analysis of ageing-dependent thiol oxidation reveals early oxidation of proteins involved in core proteostasis functions

Oxidants have a profound impact on biological systems in physiology and under pathological conditions. Oxidative post-translational modifications of protein thiols are well-recognized as a readily occurring alteration of proteins. Changes in protein thiol redox state can modify the function of proteins and thus can control cellular processes. However, chronic oxidative stress causes oxidative damage to proteins with detrimental consequences for cellular function and organismal health. The development of techniques enabling the site-specific and quantitative assessment of protein thiol oxidation on a proteome-wide scale significantly expanded the number of known oxidation-sensitive protein thiols. However, lacking behind are large-scale data on the redox state of proteins during ageing, a physiological process accompanied by increased levels of endogenous oxidants. Here, we present the landscape of protein thiol oxidation in chronologically aged wild-type Saccharomyces cerevisiae in a time-dependent manner. Our data determine early oxidation targets in key biological processes governing the de novo production of proteins, folding, and protein degradation. Comparison to existing datasets reveals evolutionary conservation of early oxidation targets. To facilitate accessibility and cross-species comparison of the experimental data obtained, we created the OxiAge Database, a free online tool for the research community that integrates current datasets on thiol redoxomes in aged yeast, nematode Caenorhabditis elegans, fruit fly Drosophila melanogaster, and mouse Mus musculus. The database can be accessed through an interactive web application at http://oxiage.ibb.waw.pl.

molecular biology↗

bioRxiv2023

A Msp1-containing complex removes orphaned proteins in the mitochondrial outer membrane of trypanosomes

The AAA-ATPase Msp1 extracts mislocalized outer membrane proteins and thus contributes to mitochondrial proteostasis. Using pull down experiments we show that trypanosomal Msp1 localizes to both glycosomes and the mitochondrial outer membrane, where it forms a stable complex with four outer membrane proteins. The trypanosome-specific pATOM36 mediates complex assembly of a-helically anchored mitochondrial outer membrane proteins such as protein translocase subunits. Inhibition of their assembly triggers a pathway that results in the proteasomal digestion of unassembled substrates. Using inducible single, double and triple RNAi cell lines combined with proteomic analyses we demonstrate that not only Msp1 but also the trypanosomal homolog of the AAA-ATPase VCP are implicated in this quality control pathway. Moreover, in the absence of VCP three out of the four Msp1-interacting mitochondrial proteins are required for efficient proteasomal digestion of pATOM36 substrates suggesting they act in concert with Msp1. pATOM36 is a functional analogue of the yeast MIM complex and possibly of human MTCH2 suggesting that similar mitochondrial quality control pathways linked to Msp1 might also exist in yeast and humans.

biochemistry↗

bioRxiv2021

Protein homeostasis is maintained by proteasomes containing PSMB9 induced by EEF1A2 upon mitochondrial stress

Perturbed proteostasis and mitochondrial dysfunction are often associated with age-related diseases such as Alzheimers and Parkinsons diseases. However, the link between them remains incompletely understood. Mitochondrial dysfunction causes proteostasis imbalance, and cells respond to restore proteostasis by increasing proteasome activity and molecular chaperons in yeast and C. elegans. Here, we demonstrate the presence of similar responses in humans. Mitochondrial dysfunction upregulates a small heat shock protein HSPB1 and an immunoproteasome subunit PSMB9 leading to an increase in proteasome activity. HSPB1 and PSMB9 are required to prevent protein aggregation upon mitochondrial dysfunction. Moreover, PSMB9 expression is dependent on a translation elongation factor EEF1A2, and PSMB9-containing proteasomes are located near mitochondria, enabling fast local degradation of aberrant proteins. Our findings put a step forward in understanding the stress response triggered by mitochondrial dysfunction, and may be useful for therapeutic strategies to prevent or delay the onset of age-related diseases and attenuate their progression.

cell biology↗

bioRxiv2020

Pex14p phosphorylation modulates import of citrate synthase 2 into peroxisomes in Saccharomyces cerevisiae

The peroxisomal biogenesis factor Pex14p is an essential component of the peroxisomal matrix protein import machinery. Together with Pex13p and Pex17p, it is part of the membrane-associated peroxisomal docking complex in yeast, facilitating the binding of cargo-loaded receptor proteins for translocation of cargo proteins into the peroxisome. Furthermore, Pex14p is part of peroxisomal import pores. The central role of Pex14p in peroxisomal matrix protein import processes renders it an obvious target for regulatory mechanisms such as protein phosphorylation. To explore this possibility, we examined the state of Pex14p phosphorylation in Saccharomyces cerevisiae. Phos-tag-SDS-PAGE of Pex14p affinity-purified from solubilized membranes revealed Pex14p as multi-phosphorylated protein. Using mass spectrometry, we identified 16 phosphorylation sites, with phosphorylation hot spots located in the N- and C-terminal regions of Pex14p. Analysis of phosphomimicking and nonphosphorylatable variants of Pex14p revealed a decreased import of GFP carrying a peroxisomal targeting signal type 1, indicating a functional relevance of Pex14p phosphorylation in peroxisomal matrix protein import. We show that this effect can be ascribed to the phosphomimicking mutation at serine 266 of Pex14p (Pex14p-S266D). We further screened the subcellular distribution of 23 native GFP-tagged peroxisomal matrix proteins by high-content fluorescence microscopy. Only Cit2p, the peroxisomal isoform of citrate synthase, was affected in the Pex14p-S266D mutant, showing increased cytosolic localization. Cit2p is part of the glyoxylate cycle, which is required for the production of essential carbohydrates when yeast is grown on non-fermentable carbon sources. Pex14p-S266 phosphosite mutants showed reversed growth phenotypes on oleic acid and ethanol with acetyl-CoA formed in peroxisomes and the cytosol, respectively. Our data point to the control of the peroxisomal import of Cit2p via the state of Pex14p phosphorylation at S266, which may help S. cerevisiae cells to rapidly adjust their carbohydrate metabolism according to the nutritional conditions.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsPTSperoxisomal targeting signal;LC-MSliquid chromatography-mass spectrometry;TEVtobacco etch virus;TPATEV protease cleavage site-Protein AView Full Text

cell biology↗