Biology subjects

Paulo, J.

Publications and source records attributed to Paulo, J..

8 recordsLinked to original sources

bioRxiv2026

Ribosome Quality Control Mitigates Proteotoxic Stress in Aneuploid Cells

Aneuploidy is widespread in tumors, but how cancer cells adapt to aneuploidy-induced cellular stresses remains poorly understood. Here, we focus on the mechanisms employed to cope with proteostasis disruption, a major stress caused by aneuploidy. We show that aneuploid cells exhibit a significant accumulation of ribosomes enclosed within autophagosomes, ultimately degraded through lysosome-mediated processes. Our data also indicate that limited folding capacity of newly synthesized polypeptides leads to lysosome-mediated degradation of ribosomes. We also found that the E3 ligase ZNF598 marks these ribosomes for degradation, thus clearing translationally-impaired ribosomes. Importantly, highly aneuploid tumors display a positive correlation with ZNF598 expression, while being negatively associated with ribosomal signatures. This suggests that ribosome-associated quality control is crucial for cancer cell survival under proteotoxic stress. Our study uncovers molecular events in response to proteotoxic stress in aneuploid cells and suggests that components of ribosome-associated quality control, including ZNF598, could serve as promising targets in cancer therapy.

cell biology↗

bioRxiv2024

A Distributed Computing Solution for Privacy-Preserving Genome-Wide Association Studies

Breakthroughs in sequencing technologies led to an exponential growth of genomic data, providing unprecedented biological in-sights and new therapeutic applications. However, analyzing such large amounts of sensitive data raises key concerns regarding data privacy, specifically when the information is outsourced to third-party infrastructures for data storage and processing (e.g., cloud computing). Current solutions for data privacy protection resort to centralized designs or cryptographic primitives that impose considerable computational overheads, limiting their applicability to large-scale genomic analysis. We introduce GO_SCPLOWYOSAC_SCPLOW, a secure and privacy-preserving distributed genomic analysis solution. Unlike in previous work, GO_SCPLOWYOSAC_SCPLOW follows a distributed processing design that enables handling larger amounts of genomic data in a scalable and efficient fashion. Further, by leveraging trusted execution environments (TEEs), namely Intel SGX, GO_SCPLOWYOSAC_SCPLOW allows users to confidentially delegate their GWAS analysis to untrusted third-party infrastructures. To overcome the memory limitations of SGX, we implement a computation partitioning scheme within GO_SCPLOWYOSAC_SCPLOW. This scheme reduces the number of operations done inside the TEEs while safeguarding the users genomic data privacy. By integrating this security scheme in Glow, GO_SCPLOWYOSAC_SCPLOW provides a secure and distributed environment that facilitates diverse GWAS studies. The experimental evaluation validates the applicability and scalability of GO_SCPLOWYOSAC_SCPLOW, reinforcing its ability to provide enhanced security guarantees. Further, the results show that, by distributing GWASes computations, one can achieve a practical and usable privacy-preserving solution.

bioinformatics↗

bioRxiv2023

ALS-related p97 R155H mutation disrupts lysophagy in iPSC-derived motor neurons

Mutations in the AAA+ ATPase p97 (also known as valosin containing protein, VCP) cause multisystem proteinopathy 1 (MSP-1) which includes amyotrophic-lateral sclerosis (ALS); however, the pathogenic mechanisms that contribute to motor neuron loss in familial ALS caused by p97 mutations remain obscure. Here, we use two distinct induced pluripotent stem cell models differentiated into spinal motor neurons to investigate how p97 mutations perturb the motor neuron proteome. Using multiplexed quantitative proteomics in these cells, we find that motor neurons harboring the p97 R155H mutation have deficits in lysosome quality control and the selective autophagy of lysosomes (lysophagy). p97 R155H motor neurons are unable to efficiently clear damaged lysosomes and have reduced viability. Additionally, lysosomes in mutant motor neurons have increased pH compared to their wildtype counterparts. The endo-lysosomal damage repair (ELDR) complex is required for clearance of damaged lysosomes and involves UBXD1-p97 interaction which is disrupted in mutant motor neurons. Finally, we report that inhibition of the ATPase activity of p97 R155H using the ATP competitive inhibitor CB-5083 rescues lysophagy defects in mutant motor neurons. These results add to the increasing evidence that endo-lysosomal dysfunction is a key aspect of disease pathogenesis in p97-related disorders.

neuroscience↗

bioRxiv2021

The p97-UBXD8 complex modulates ER-Mitochondria contact sites by modulating membrane lipid saturation and composition

The intimate association between the endoplasmic reticulum (ER) and mitochondrial membranes at ER-mitochondria contact sites serves as a platform for several critical cellular processes, in particular lipid synthesis. Enzymes involved in lipid biosynthesis are enriched at contacts and membrane lipid composition at contacts is distinct relative to surrounding membranes. How contacts are remodeled and the subsequent biological consequences of altered contacts such as perturbed lipid metabolism remains poorly understood. Here we show that the p97 AAA-ATPase and its ER-tethered ubiquitin-X domain adaptor 8 (UBXD8) regulate the prevalence of ER-mitochondria contacts. The p97-UBXD8 complex localizes to contacts and loss of this complex increases contacts in a manner that is dependent on p97 catalytic activity. Quantitative proteomics of purified contacts demonstrates alterations in proteins regulating lipid metabolism upon loss of UBXD8. Furthermore, lipidomics studies indicate significant changes in distinct lipid species in UBXD8 knockout cells. We show that loss of p97-UBXD8 results in perturbed contacts due to an increase in membrane lipid saturation via SREBP1 and the lipid desaturase SCD1. Aberrant contacts in p97-UBXD8 loss of function cells can be rescued by supplementation with unsaturated fatty acids or overexpression of SCD1. Perturbation of contacts and inherent lipid synthesis is emerging as a hallmark to a variety of human disorders such as neurodegeneration. Notably, we find that the SREBP1-SCD1 pathway is negatively impacted in the brains of mice with p97 mutations that cause neurodegeneration. Our results suggest that contacts are exquisitely sensitive to alterations to membrane lipid composition and saturation in a manner that is dependent on p97-UBXD8.

cell biology↗

bioRxiv2021

Transcriptomic and proteomic retinal pigment epithelium signatures of age-related macular degeneration.

Induced pluripotent stem cells generated from patients with geographic atrophy as well as healthy individuals were differentiated to retinal pigment epithelium (RPE) cells. By integrating transcriptional profiles of 127,659 RPE cells generated from 43 individuals with geographic atrophy and 36 controls with genotype data, we identified 439 expression Quantitative Trait (eQTL) loci in cis that were associated with disease status and specific to subpopulations of RPE cells. We identified loci linked to two genes with known associations with geographic atrophy - PILRB and PRPH2, in addition to 43 genes with significant genotype x disease interactions that are candidates for novel genetic associations for geographic atrophy. On a transcriptome-only level, we identified molecular pathways significantly upregulated in geographic atrophy-RPE including in extracellular cellular matrix reorganisation, neurodegeneration, and mitochondrial functions. We subsequently implemented a large-scale proteomics analysis, confirming modification in proteins associated with these pathways. We also identified six significant protein (p) QTL that regulate protein expression in the RPE cells and in geographic atrophy - two of which share variants with cis-eQTL. Transcriptome-wide association analysis identified genes at loci previously associated with age-related macular degeneration. Further analysis conditional on disease status, implicated statistically significant RPE-specific eQTL. This study uncovers important differences in RPE homeostasis associated with geographic atrophy.

neuroscience↗

bioRxiv2020

O-GlcNAc regulates gene expression by controlling detained intron splicing

Intron detention in precursor RNAs serves to regulate expression of a substantial fraction of genes in eukaryotic genomes. How detained intron (DI) splicing is controlled is poorly understood. Here we show that a ubiquitous post-translational modification called O-GlcNAc, which is thought to integrate signaling pathways as nutrient conditions fluctuate, controls detained intron splicing. Using specific inhibitors of the enzyme that installs O-GlcNAc (O-GlcNAc transferase, or OGT) and the enzyme that removes O-GlcNAc (O-GlcNAcase, or OGA), we first show that O-GlcNAc regulates splicing of the highly conserved detained introns in OGT and OGA to control mRNA abundance in order to buffer O-GlcNAc changes. We show that OGT and OGA represent two distinct paradigms for how DI splicing can control gene expression. We also show that when DI splicing of the O-GlcNAc-cycling genes fails to restore O-GlcNAc homeostasis, there is a global change in detained intron levels. Strikingly, almost all detained introns are spliced more efficiently when O-GlcNAc levels are low, yet other alternative splicing pathways change minimally. Our results demonstrate that O-GlcNAc controls detained intron splicing to tune system-wide gene expression, providing a means to couple nutrient conditions to the cells transcriptional regime.

cell biology↗

bioRxiv2019

Unmasking functional redundancy of deubiquitylases enables specificity profiling and discovery of novel proteasome substrates

Deubiquitylating enzymes (DUBs) counteract ubiquitylation to control stability or activity of substrates. Identification of DUB substrates is challenging because multiple DUBs act on the same substrates, thwarting genetic approaches. Here, we circumvented redundancy by broadly inhibiting DUBs in Xenopus egg extract. DUB inhibition increases ubiquitylation of hundreds of proteins, depleting free ubiquitin without inducing widespread degradation. Restoring available ubiquitin led to proteasomal degradation of over thirty proteins, indicating that deubiquitylation is essential to maintain their stability. We confirmed their DUB-dependent stability with recombinant human proteins, demonstrating evolutionary conservation. We profiled the ability of DUBs to rescue protein stability, and found that USP7 has a unique ability to broadly antagonize proteasomal degradation. Together, we provide a comprehensive characterization of ubiquitin dynamics in the Xenopus system, identify new DUB substrates, and present a new approach to characterize physiological DUB specificity that overcomes challenges posed by DUB redundancy

biochemistry↗

bioRxiv2018

Identification of endogenous Adenomatous polyposis coli interaction partners and β-catenin-independent targets by proteomics

Adenomatous polyposis coli (APC) is the most frequently mutated gene in colorectal cancer. APC negatively regulates the pro-proliferative Wnt signaling pathway by promoting the degradation of {beta}-catenin, but the extent to which APC exerts Wnt/{beta}-catenin-independent tumor suppressive activity is unclear. To identify interaction partners and {beta}-catenin-independent targets of endogenous, full-length APC, we applied label-free and multiplexed TMT mass spectrometry. Affinity enrichment-mass spectrometry revealed over 150 previously unidentified APC interaction partners. Moreover, our global proteomic analysis revealed that roughly half of the protein expression changes that occur in response to APC loss are independent of {beta}-catenin. By combining these two analyses, we identified Misshapen-like kinase 1 (MINK1) as a putative substrate of an alternative APC-containing destruction complex and provide evidence for the potential contribution of MINK1 to APC mutant phenotypes. Collectively, our results highlight the extent and importance of Wnt-independent APC functions in epithelial biology and disease.

cancer biology↗