Biology subjects

Salido, E.

Publications and source records attributed to Salido, E..

7 recordsLinked to original sources

bioRxiv2026

Allelic Variation at 9p21.3 Orchestrates Widespread RNA Splicing Shifts Governing Vascular Smooth Muscle Cell Plasticity

Genetic risk for coronary artery disease (CAD) has been linked to variants across more than 300 genomic loci. Whether and how these loci interface with RNA processing to drive disease-relevant cellular phenotypes remains unknown. Here, we applied haplotype-biased genome editing in induced pluripotent stem cells (iPSCs), followed by differentiation into vascular smooth muscle cells (VSMCs), to address how genetic variation at the strongest CAD locus - the 9p21.3 CAD risk locus - affects RNA processing and alternative splicing genome-wide. Using long-read RNA sequencing, we identified distinct allele-specific transcriptional programs driven by the two major haplotypes at 9p21.3, risk and non-risk. We unravel extensive reprogramming of mRNA splicing across the transcriptome, which leads to VSMC aberrant phenotypic modulation. The 9p21.3 risk haplotype disrupts transcript isoform expression and usage across multiple genomic loci implicated in diverse stages of atherosclerotic plaque development. We prioritized DDX5, previously implicated in CAD through GWAS. Isoform-specific modulation of DDX5 in VSMCs was sufficient to mitigate the 9p21.3 risk-associated molecular signature. Together, this work provides the first comprehensive isoform-level transcriptomic comparison of the two major haplotypes at the 9p21.3 locus and identifies a 9p21.3-DDX5 axis as a key regulator of VSMC phenotypic plasticity. These findings uncover allele-specific reprogramming of RNA splicing as a previously unrecognized mechanism underlying cardiovascular disease susceptibility and present a resource of targetable transcripts with potential relevance across vascular pathologies.

genetics↗

bioRxiv2025

Phosphorylation of Ser81 in human AGT reversibly inactivate enzyme function and mimics catalytic defects of certain PH1-causing mutations

Phosphorylation is fundamental to modulate protein function and stabilty. There have detected about 300000 site-specic Phosphorylation sites in over 20000 human proteins. However, only 5% of the sites have been experimenrally characterized. In this work, we investigated a phosphorylation event in AGT, an important enzyme due to its detoxifying role of glyoxylate and hundreds of mutations cause a rare disease (primary hyperoxaluria type I or PH1). We analyzed the effect of phosphomimetic mutations Ser81 on the WT proten, the common polymorpshim LM, and the most common disease-associted variants (LM-G170R and LM-I244T). Using biochemical, biophysical and cell biology approaches, we show that phosphorylation at S81 dramatically affects PLP/PMP binding pose and disrupts enzyme activity, without pertubing its subcellular location to peroxisomes. This reversible phenotype is similar to the irreversible effects of some PH1-causing mutaions. Thus, we provide evidence for a novel regulatory mechanism for PH1, in health and disease.

biophysics↗

bioRxiv2025

Nanobodies as therapies for loss-of-function misfolding diseases.

Misfolding diseases that result in loss of function represent a considerable burden for both individuals and society. Primary hyperoxaluria type 1 (PH1) is a rare genetic disorder caused by mutations in the alanine:glyoxylate aminotransferase 1 (AGT) enzyme. The underlying molecular mechanisms causing PH1 are associated with protein misfolding (enhanced aggregation and mitochondrial mistargeting). The main therapeutic approach to increase patients lifespan and quality of life is a double kidney and liver transplantation. Alternative treatments such as gene and enzyme replacement and pharmacological chaperones are currently being introduced, but other alternatives are necessary. In this work, we developed and characterized a novel biotechnological approach using six single-domain nanobodies (NB-AGT-1 to -6) as potential therapeutics for PH1 misfolding. We show that NB-AGTs are very stable proteins and bind to pathogenic and non-pathogenic variants of AGT with extreme affinities (with Kd values from low nM to low pM). Structural studies showed that NB-AGTs bind to different epitopes of AGT with selectivity for different AGT variants. Experiments in cellular PH1 models showed that internalization of engineered NB-AGT-3 enhanced the specific activity of disease-associated variants. Overall, we show that NBs are a novel and promising approach to treat PH1 and other loss-of-function misfolding diseases.

biophysics↗

bioRxiv2024

Thermodynamic versus kinetic basis for the high conformational stability of nanobodies for therapeutic applications

Single domain nanobodies (NB) are powerful tools for biotechnological and therapeutic applications. They strongly bind to their targets and are very stable. Early studies showed that NB unfolding is reversible and can be analyzed by equilibrium thermodynamics whereas more recent studies focused on their kinetic stability in very harsh conditions, far from storage or physiological temperatures (4-37{degrees}C). Here we reinforce the thermodynamic view in which a simple two-state denaturation model is applicable. We found that thermal stability of NB actually reflect thermodynamic stabilities in wide range of temperatures (18-100{degrees}C). We also modeled their structure observing subtle differences. We expect that our approach will be helpful to improve our capacity to enhance structure-function-stability relationships of NB.

biophysics↗

bioRxiv2024

The 9p21.3 coronary artery disease risk locus drives vascular smooth muscle cells to osteo-chondrogenic state

BackgroundGenome-wide association studies have identified common genetic variants at [~]300 human genomic loci linked to coronary artery disease (CAD) susceptibility. Among these genomic regions, the most impactful is the 9p21.3 CAD risk locus, which spans a 60 kb gene desert and encompasses [~]80 SNPs in high linkage disequilibrium. Despite nearly two decades since its discovery, the role of the 9p21.3 locus in cells of the vasculature remains incompletely resolved. MethodsWe differentiated induced pluripotent stem cells (iPSCs) from risk and non-risk donors at 9p21.3 into vascular smooth muscle cells. We performed single-cell transcriptomic profiling, including co-embedding and comparison with publicly available human arterial datasets. We conducted functional characterization using migration and calcification assays and confirmed our findings on iPSC-VSMCs derived from additional donors. Finally, we used overexpression of ANRIL followed by gene expression analysis. ResultsWe demonstrated that iPSC-VSMCs harboring the 9p21.3 risk haplotype preferentially adopt an osteochondrogenic state and show remarkable similarity to fibrochondrocytes from human artery tissue. The transcriptional profile and functional assessment of migration and calcification capacity across iPSC-VSMCs lines from multiple donors concordantly resemble an osteochondrogenic state. Importantly, we identified numerous transcription factors driving different VSMC state trajectories. Additionally, we prioritized LIMCH1 and CRABP1 as signature genes critical for defining the risk transcriptional program. Finally, overexpression of a short isoform of ANRIL in non-risk cells was sufficient to induce the osteochondrogenic transcriptional signature. ConclusionsOur study provides new insights into the mechanism of the 9p21.3 risk locus and defines its previously undescribed role in driving a disease-prone transcriptional and functional state in VSMCs concordant with an osteochondrogenic-like state. Our data suggest that the 9p21.3 risk haplotype likely promotes arterial calcification, through altered expression of ANRIL, in a cell-type specific and cell-autonomous manner, providing insight into potential risk assessment and treatment for carriers. HighlightsThe 9p21.3 CAD risk locus promotes the transition of iPSC-derived Vascular Smooth Muscle Cells (iPSC-VSMCs) to an osteochondrogenic phenotype, both transcriptionally and functionally. iPSC-VSMCs carrying the risk haplotype at 9p21.3 display a distinct transcriptomic signature, including osteochondrogenic markers SOX9 and COL2A1, along with two novel markers: LIMCH1 and CRABP1. Knockout of the entire haplotype reverts this signature to a non-risk state, demonstrating a clear genotype-to-phenotype causal effect of the risk allele. Short isoform 12 of the lncRNA ANRIL, which partially overlaps the 9p21.3 locus, is sufficient to induce this transcriptional signature. The iPSC-VSMC transcriptional profile strongly resembles that of ex-vivo human arterial VSMCs, providing a human-specific model to study VSMC phenotypic alterations and investigate the effects of large CAD haplotypes on vascular wall cells.

cell biology↗

bioRxiv2022

Effect of naturally-occurring mutations on the stability and function of cancer-associated NQO1: comparison of experiments and computation.

Recent advances in DNA sequencing technologies are revealing a large variability in the human genome. Our capacity to establish genotype-phenotype correlations in such large-scale is, however, limited. This task is particularly challenging due to the multifunctional nature of proteins. Here we describe an extensive analysis of the stability and function of naturally-occurring variants (found in the COSMIC and gnomAD databases) of the cancer-associated human NAD(P)H: quinone oxidoreductase 1 (NQO1). First, we performed in silico saturation mutagenesis studies (>5000 substitutions) aimed to identify regions in NQO1 important for stability and function. We then experimentally characterized twenty-two naturally-occurring variants in terms of protein levels during bacterial expression, solubility, thermal stability and coenzyme binding. These studies showed a good overall correlation between experimental analysis and computational predictions; also the magnitude of the effects of the substitutions are similarly distributed in variants from the COSMIC and gnomAD databases. Outliers in these experimental-computational genotype-phenotype correlations remain, and we discuss these on the grounds and limitations of our approaches. Our work represents a further step to characterize the mutational landscape of NQO1 in the human genome and may help to improve high-throughput in silico tools for genotype-phenotype correlations in multifunctional proteins associated with disease.

biophysics↗

bioRxiv2020

ARID2 deficiency promotes tumor progression and is associated with higher sensitivity to PARP inhibition in lung cancer

The survival rate in lung cancer remains stubbornly low and there is an urgent need for the identification of new therapeutic targets. Last decades research has evidenced a clear role of chromatin structure in cancer development and several members of the SWI/SNF chromatin remodeling complexes have been described altered in different tumor types. Nevertheless, the precise mechanisms of their impact on cancer progression, as well as the application of this knowledge to cancer patient management are largely unknown. In this study, we have performed targeted sequencing of a cohort of lung cancer patients on genes involved in chromatin structure, as well as functional experiments to identify the molecular mechanisms linking alterations of chromatin remodeling genes and tumor development. We have identified ARID2 production loss in 20% of lung cancer patients. Additionally, we have shown that ARID2-deficiency provokes profound chromatin structural changes, alters the transcriptional programme and impairs DNA repair which bolster the proliferative and metastatic potential of the cells both in vitro and in vivo. Moreover, we have demonstrated that ARID2 deficiency significantly affects the sensitivity of the cells to PARP inhibition. All these results support that ARID2 is a bona-fide tumor suppressor gene in lung cancer that might be exploited therapeutically.

cancer biology↗