Biology subjects

Platani, M.

Publications and source records attributed to Platani, M..

6 recordsLinked to original sources

bioRxiv2026

CysNet: Theorem constrained inference of cysteine redox proteoform states from bottom-up mass spectrometry data

Here, we present CysNet, a theorem-constrained method designed to infer cysteine redox proteoforms, i.e.,oxiforms, from bottom-up, mass spectrometry (MS)-based proteomic data. This overcomes limitations with previous MS redox proteomic approaches, which can quantify residue-resolved cysteine redox states, but leave distinct oxiforms unresolved. CysNet treats each residue-resolved oxidation value as a binary redox-coordinate marginal, enabling theorem-constrained inference of the oxiforms that are necessary, impossible or bounded within the compatible protein-group ensemble. This collapses the vast theoretically possible set of oxiform states to a finite set of allowed values by extracting existence and exclusion constraints from the data, despite the incomplete proteome coverage typical for bottom-up MS datasets. Using CysNet to analyse human induced pluripotent stem cell lines (~6,300 cysteine-containing protein groups, ~22% cysteine coverage), resolved 519 exact oxiforms, inferring 7,000 oxiforms per line. Quantitatively, CysNet bounded the oxiform content to 6.36-8.24 x 1012 protein copies, corresponding to 14-19% of the measured cysteine proteome. These data define the deepest oxiform survey recorded. CysNet revealed a latent structural layer in redox variation between the cell lines, distinguishing changes in oxiform identity (composition) from changes in oxiform weighting (intensity). Hence, CysNet moves bottom-up redox proteomics beyond isolated site-level cataloguing by reconstructing copy-number-weighted oxiform maps, providing a scalable route to deep oxiform information from peptide-level data.

biochemistry↗

bioRxiv2026

ReCap enables deep, copy number-scaled cysteine redox proteomics with minimal exogenous oxidation

Cysteine oxidation analyses require the preservation of the redox state present at harvest and quantitative scaling to relate oxidation to protein copy numbers, rather than only providing fractional oxidation data. Here, we present ReCap, a Redox Capture workflow combining Oxi-DIA, an enrichment-free isotope-encoded DIA workflow, with Oxi-Stop, a simple oxygen-exclusion strategy for cryopreserved tissue. In mouse brains, Oxi-DIA quantified 17,809 cysteine sites belonging to 6,085 protein groups in every sample, enabling matched measurements of residue-resolved oxidation and protein abundance. Atmospheric oxygen exposure during 14 days of cryopreservation distorted the measured cysteine redox state. The resultant increase of an estimated 5.3176 x 1011 {micro}g-1 oxidised cysteine molecules was mitigated by Oxi-Stop, which minimised exogenous oxidation during cryopreservation. Copy-number scaling altered the interpretation of cysteine oxidation values. Although cysteine oxidation was detected across 2,371 sites and 1,439 proteins, 20 sites on abundant proteins accounted for 44% of the oxidised signal. ReCap advances redox proteomics from providing a site catalogue into a biologically weighted map of redox information, revealing cysteine oxidation as a sparse, ordered and quantitatively concentrated signal.

biochemistry↗

bioRxiv2025

PHD1-dependent hydroxylation of RepoMan (CDCA2) on P604 modulates the control of mitotic progression

Prolyl-hydroxylases (PHDs) are oxygen sensing enzymes that mediate the hydroxylation of proline residues. In mammals, three PHD isoforms (PHD1-3) are responsible for proline hydroxylation of Hypoxia Inducible Factor (HIF) alpha, a key regulator of the hypoxia response. In the accompanying paper (Jiang et. al., 2025) we report development of a mass spectrometry-based method to reliably identify proline hydroxylation (OH-Pro) sites on proteins and use this to identify a PHD-dependent OH-Pro modification at Pro604 on the protein RepoMan (CDCA2), a regulatory subunit for protein phosphatase PP1{gamma}, with important roles in mitotic progression and cell viability. Here, we investigate the functional significance of hydroxylation of RepoMan at P604. During M phase, the PP1-RepoMan complex dephosphorylates Thr3 of Histone H3 (H3T3) on chromosomes arms to ensure the correct localisation of the chromosomal passenger complex (CPC) at centromeres. We show that siRNA depletion of PHD1, but not PHD2, increases H3T3 phosphorylation in prometaphase-arrested cells. In cells depleted of endogenous RepoMan, exogenous expression of wild type RepoMan, but not a RepoMan P604A mutant, restored normal H3T3 phosphorylation localisation in prometaphase arrested cells. RepoMan P604 is located proximal to the Short Linear Motifs (SLiMs) that function as binding sites for the serine/threonine Protein Phosphatase 2A (PP2A). The interaction of RepoMan and PP2A-B56{gamma} is reduced in cells expressing RepoMan P604A. Moreover, analyses in both fixed and live cells released from a prometaphase arrest, show that expression of the RepoMan P604A mutant delays completion of mitosis, results in defects in chromosome alignment and segregation and increases levels of cell death. These data support a role for PHD1-mediated prolyl hydroxylation in controlling progression through mitosis, acting, at least in part, via hydroxylation of RepoMan at P604 regulating the interaction of RepoMan with PP2A during chromosome alignment and thereby controlling the levels of Histone H3 phosphorylation at Thr3.

cell biology↗

bioRxiv2023

Screening for variable drug responses using human iPSC cohorts

We have developed a laboratory-based drug screening platform that uses a cohort of human induced pluripotent stem cell (hiPSC) lines, derived from different donors, to predict variable drug responses of potential clinical relevance. This builds on recent findings that pluripotent hiPSC lines express a broad repertoire of gene transcripts and proteins, whose expression levels reflect the genetic identity of the donor. We demonstrate that a cohort of hiPSC lines from different donors can be screened efficiently in their pluripotent state, using high-throughput Cell Painting assays. Variable phenotypic responses between hiPSC lines were detected with a wide range of clinically approved drugs, in use across multiple disease areas. Furthermore, information on mechanisms of drug-cell interactions underlying the observed variable responses was derived by using quantitative proteomic analysis to compare sets of hiPSC lines that had been stratified objectively, based upon variable response, Cell Painting data. We propose that information derived from comparative drug screening, using curated libraries of hiPSC lines from different donors, can help to improve the delivery of safe new drugs suitable for a broad range of genetic backgrounds and sexual diversity within human populations.

cell biology↗

bioRxiv2021

A quantitative comparison of human embryonic and induced stem cell proteomes

Human induced pluripotent stem cells (hiPSCs) have great potential to be used as alternatives to embryonic stem cells (hESCs) in regenerative medicine and disease modelling, thereby avoiding many of the ethical issues arising from the use of embryo-derived cells. However, despite clear similarities between the two cell types, it is likely they are not identical. In this study, we characterise the proteomes of multiple hiPSC and hESC lines derived from independent donors. We find that while hESCs and hiPSCs express a near identical set of proteins, they show consistent quantitative differences in the expression levels of a wide subset of proteins. hiPSCs have increased total protein content, while maintaining a comparable cell cycle profile to hESCs. The proteomic data show hiPSCs have significantly increased abundance of vital cytoplasmic and mitochondrial proteins required to sustain high growth rates, including nutrient transporters and metabolic proteins, which correlated with phenotypic differences between hiPSCs and hESCs. Thus, higher levels of glutamine transporters correlated with increased glutamine uptake, while higher levels of proteins involved in lipid synthesis correlated with increased lipid droplet formation. Some of the biggest metabolic changes were seen in proteins involved in mitochondrial metabolism, with corresponding enhanced mitochondrial potential, shown experimentally using high-resolution respirometry. hiPSCs also produced higher levels of secreted proteins, including ECM components and growth factors, some with known tumorigenic properties, as well as proteins involved in the inhibition of the immune system. Our data indicate that reprogramming of human fibroblasts to iPSCs effectively restores protein expression in cell nuclei to a state comparable to hESCs, but does not similarly restore the profile of cytoplasmic and mitochondrial proteins, with consequences for cell phenotypes affecting growth and metabolism. The data improve understanding of the molecular differences between induced and embryonic stem cells, with implications for potential risks and benefits for their use in future disease modelling and therapeutic applications.

biochemistry↗

bioRxiv2017

Rapid degradation and 3D CLEM of condensin reveal chromatin compaction uncoupled from chromosome architecture in mitosis

The requirement for condensin in chromosome formation in somatic cells remains unclear as imperfectly condensed chromosomes do form in conventional condensindepleted cells. Here we have dissected the role of condensin at different stages of vertebrate mitosis by combining auxin-mediated rapid depletion of condensin subunit SMC2 with chemical genetics to obtain near-synchronous mitotic entry of chicken DT40 cells. We analysed the outcomes by live and fixed-cell microscopy methods, including 3D correlative light and serial block face scanning electron microscopy. Following rapid depletion of condensin, chromosomal defects were obvious. The chromatin was compacted normally, but formed a single mass of mitotic chromosomes clustered at one side of a bent mitotic spindle. Cultures arrest at prometaphase, eventually exiting mitosis without segregating chromosomes. Experiments titrating the auxin concentration suggest a previously unsuspected dual role of condensin, as different condensin levels are required for anaphase chromosome segregation and formation of a normal chromosome architecture.

cell biology↗