Biology subjects

Morales-Sanfrutos, J.

Publications and source records attributed to Morales-Sanfrutos, J..

4 recordsLinked to original sources

bioRxiv2026

A comprehensive HA-tagged PRC1 toolkit enables standardized chromatin profiling and proteomic analysis in Drosophila melanogaster

Polycomb group (PcG) proteins are evolutionarily conserved epigenetic regulators that maintain transcriptional states during development and are frequently misregulated in disease. Here, we generated a comprehensive collection of endogenously HA-tagged alleles for the core components of Polycomb Repressive Complex 1 (PRC1) and additional chromatin regulators in Drosophila melanogaster using CRISPR/Cas9-mediated genome engineering. We show that endogenous HA tagging preserves protein expression, chromatin localization, and genome-wide binding profiles, enabling direct comparison using a common antibody to analyze distinct PcG subunits. Proteomic analyses recovered known components of canonical and non-canonical PRC1 and PRC2 complexes and identified additional PcG-associated factors. Notably, multiple components of the nuclear pore complex (NPC) were detected as PcG interactors, and genetic analyses confirmed a functional interaction between them. This resource provides a platform for standardized analysis of Polycomb function and chromatin regulation that can easily be extended to other chromatin regulatory complexes and applied across different developmental stages. Article SummaryPolycomb group (PcG) proteins are essential regulators of development and genome function. Comparative studies of individual PcG components are often limited by the availability and quality of specific antibodies. Here, we generated a collection of Drosophila melanogaster lines carrying endogenous HA epitope tags in core components of the canonical Polycomb Repressive Complex 1 (PRC1), along with additional chromatin regulators. These tagged proteins faithfully reproduce native chromatin binding and can be used for chromatin mapping and protein interaction studies using a single, highly specific antibody. This standardized resource provides a practical tool for investigating PcG function and chromatin regulation in vivo.

molecular biology↗

bioRxiv2025

Proteome-wide multipoint internal calibration curves for mass spectrometry-based quantitative proteomics

Mass spectrometry (MS)-based proteomics is known for its high accuracy in quantifying peptides and proteins using various calibration strategies, including internal and external calibration curves. While external multi-point calibration curves are created from serial dilutions, they often fail to account for sample-specific matrix effects. In contrast, internal calibration curves account for sample matrix but face scalability and cost challenges for whole proteome analyses. In this manuscript we present a novel TMT-based multipoint internal calibration curve strategy, referred to as TMTCal, which enables the generation of internal calibration curves for all peptides identified within a proteome within a single experiment. We applied this strategy to human ovarian cancer cells to evaluate the linear quantitative responses of all the identified peptides and reveal the significant proteome changes associated with cisplatin treatment.

biochemistry↗

bioRxiv2020

Inhibition of protein N-myristoylation blocks Plasmodium falciparum intraerythrocytic development, egress, and invasion

We have combined chemical biology and genetic modification approaches to investigate the importance of protein myristoylation in the human malaria parasite, Plasmodium falciparum. Parasite treatment during schizogony in the last ten to fifteen hours of the erythrocytic cycle with IMP-1002, an inhibitor of N-myristoyl transferase (NMT), led to a significant blockade in parasite egress from the infected erythrocyte. Two rhoptry proteins were mislocalized in the cell, suggesting that rhoptry function is disrupted. We identified sixteen NMT substrates for which myristoylation was significantly reduced by NMT inhibitor treatment, and of these, six proteins were substantially reduced in abundance. In a viability screen, we showed that for four of these proteins replacement of the N-terminal glycine with alanine to prevent myristoylation had a substantial effect on parasite fitness. In detailed studies of one NMT substrate, glideosome associated protein 45 (GAP45), loss of myristoylation had no impact on protein location or glideosome assembly, in contrast to the disruption caused by GAP45 gene deletion, but GAP45 myristoylation was essential for erythrocyte invasion. Therefore, there are at least three mechanisms by which inhibition of NMT can disrupt parasite development and growth: early in parasite development, leading to the inhibition of schizogony and formation of pseudoschizonts, which has been described previously; at the end of schizogony, with disruption of rhoptry formation, merozoite development and egress from the infected erythrocyte; and at invasion, when impairment of motor complex function prevents invasion of new erythrocytes. These results underline the importance of P. falciparum NMT as a drug target because of the pleiotropic effect of its inhibition.

microbiology↗

bioRxiv2019

SirT7 auto-ADP-ribosylation regulates glucose starvation response through macroH2A1.1

Sirtuins are key players in the response to oxidative, metabolic and genotoxic stress, and are involved in genome stability, metabolic homeostasis and aging. Originally described as NAD+-dependent deacetylases, some sirtuins are also characterized by a poorly understood mono-ADP-ribosyltransferase (mADPRT) activity. Here we report that the deacetylase SirT7 is a dual sirtuin as it also features auto-mADPRT activity. Molecular and structural evidence suggests that this novel activity occurs at a second previously undefined active site that is physically separated in another domain. Specific abrogation of this activity alters SirT7 chromatin distribution, suggesting a role for this modification in SirT7 chromatin binding specificity. We uncover an epigenetic pathway by which ADP-ribosyl-SirT7 is recognized by the ADP-ribose reader macroH2A1.1, a histone variant involved in chromatin organization, metabolism and differentiation. Glucose starvation (GS) boosts this interaction and promotes SirT7 relocalization to intergenic regions in a macroH2A1-dependent manner. Both SirT7 activities are in turn required to promote GS-dependent enrichment of macroH2A1 in a subset of nearby genes, which results in their specific up- or downregulation. Consistently, the expression changes of these genes associated to calorie restriction (CR) or aging are abrogated in SirT7-/- mice, reinforcing the link between Sirtuins, CR and aging. Our work provides a novel perspective about sirtuin duality and suggests a key role for SirT7/macroH2A1.1 axis in mammalian glucose homeostasis, calorie restriction signaling and aging.

molecular biology↗