Biology subjects

Valverde, D.

Publications and source records attributed to Valverde, D..

7 recordsLinked to original sources

bioRxiv2025

alms1 regulates the immune response and brain ageing in zebrafish

The ALMS1 gene plays a crucial role in maintaining cellular homeostasis through its involvement in primary cilium assembly, cytoskeletal regulation, and signalling pathways such as NOTCH and TGF-{beta}. Pathogenic variants in ALMS1 are associated with Alstrom Syndrome (ALMS), a multi-systemic ciliopathy characterised by neurosensory deficits, metabolic disorders, and multi-organ fibrosis. To better understand the tissue-dependent role of ALMS1, we utilised CRISPR/Cas9 technology to develop a zebrafish model with alms1 depletion. Multi-tissue transcriptomic profiling revealed that alms1 depletion has pleiotropic effects on gene expression, with the brain and eyes displaying the most pronounced transcriptomic alterations, including disrupted ciliary function and immune dysregulation. Inflammatory and innate immune pathways along with glutamatergic synapse-related processes were significantly affected in the brain and eyes but with different gene expression signatures. The analysis further highlights tissue-specific processes, primarily associated with organ dysfunction. Additionally, our findings underscore the role of alms1 in regulating age-associated gene expression profiles in the brain, suggesting a link between ciliary dysfunction and accelerated brain ageing. Comparative analyses with Bardet-Biedl Syndrome iPSC models revealed shared pathways, reinforcing the potential of ciliopathies as models for ageing-related disorders. This study provides novel insights into the tissue-specific functions of alms1 and the molecular mechanisms underlying ALMS, paving the way for the development of targeted therapeutic strategies.

genomics↗

bioRxiv2024

Depletion of BBSome Subunits Alters Receptor Endocytosis and Promotes EMT via TGF-β Signaling

Background: Ciliopathies are genetic disorders caused by defects in the structure or function of the cilia and their related structures. Bardet-Biedl syndrome (BBS) is a complex ciliopathy with varied symptoms, probably due to altered membrane receptor signalling pathways. Methods: We investigated the role of BBS1 and BBS4 gene deficiencies in retinal epithelial cells using surface membrane proteomics, co-immunoprecipitation, AlphaFold structural modelling, endocytic trafficking colocalization assays, autophagy flux analysis, and epithelial-to-mesenchymal transition (EMT) functional assays including migration and wound healing. Results: Deficiencies in these BBSome components led to shorter cilia and disrupted receptor endocytosis, with accumulation of TGF-{beta}-pathway and EMT-associated proteins on the plasma membrane of BBS1 KO cells. Co-immunoprecipitation and AlphaFold structural modelling indicated that neither BBS1 nor BBS4 forms stable physical complexes with core endocytic components (EEA1, RAB11, RAB7, LAMP2), suggesting an indirect mechanism. BBS1 KO cells showed sustained TGFBR1 recycling, low basal autophagic flux that increases upon TGF-{beta} stimulation, and the most pronounced mesenchymal phenotype. BBS4 KO cells showed reduced receptor recycling, elevated basal autophagic flux that declines upon stimulation, and the strongest canonical TGF-{beta}/SMAD activation with reduced non-canonical ERK1/2 signalling. Conclusions: Increased EMT markers and enhanced migration in BBS1 KO cells highlight the role of BBSome-dependent receptor trafficking as a potential therapeutic target for retinal degeneration in BBS. The divergent autophagic and signalling phenotypes between BBS1 and BBS4 KO reveal subunit-specific contributions to TGF-{beta} pathway dysregulation.

molecular biology↗

bioRxiv2024

The human ciliopathy protein RSG1 links the CPLANE complex to transition zone architecture

Cilia are essential organelles and variants in genes governing ciliary function result in ciliopathic diseases. The Ciliogenesis and PLANar polarity Effectors (CPLANE) protein complex is essential for ciliogenesis in animals models but remains poorly defined. Notably, all but one subunit of the CPLANE complex have been implicated in human ciliopathy. Here, we identify three families in which variants in the remaining CPLANE subunit CPLANE2/RSG1 also cause ciliopathy. These patients display cleft palate, tongue lobulations and polydactyly, phenotypes characteristic of Oral-Facial-Digital Syndrome. We further show that these alleles disrupt two vital steps of ciliogenesis, basal body docking and recruitment of intraflagellar transport proteins. Moreover, APMS reveals that Rsg1 binds the CPLANE and also the transition zone protein Fam92 in a GTP-dependent manner. Finally, we show that CPLANE is generally required for normal transition zone architecture. Our work demonstrates that CPLANE2/RSG1 is a causative gene for human ciliopathy and also sheds new light on the mechanisms of ciliary transition zone assembly.

cell biology↗

bioRxiv2022

Phosphoproteomic profiling highlights CDC42 and CDK2 as key players in the regulation of the TGF-β pathway in ALMS1 and BBS1 knockout models

BACKGROUNDThe primary cilium is a sensory organelle that extends from the plasma membrane. It plays a vital role in physiological and developmental processes by controlling different signalling pathways such as WNT, Sonic hedgehog (SHh), and transforming growth factor {beta} (TGF-{beta}). Ciliary dysfunction has been related to different pathologies such as Alstrom (ALMS) or Bardet-Biedl (BBS) syndrome. The leading cause of death in adults with these syndromes is chronic kidney disease (CKD), which is characterised by fibrotic and inflammatory processes often involving the TGF-{beta} pathway. METHODSUsing genomic editing with CRISPR-CAS9 and phosphoproteomics we have studied the TGF-{beta} signalling pathway in knockout (KO) models for ALMS1 and BBS1 genes. We have developed a network diffusion-based analysis pipeline to expand the data initially obtained and to be able to determine which processes were deregulated in TGF-{beta} pathway. Finally, we have analysed protein-protein and kinase-substrate interactions to prioritise candidate genes in the regulation of the TGF-{beta} pathway in ALMS and BBS. RESULTSAnalysis of differentially phosphorylated proteins identified 10 candidate proteins in the ALMS1 KO model and 41 in the BBS1 KO model. After network expansion using a random walk with a restart algorithm, we were able to identify the TGF-{beta} signalling pathway together with other related processes such as endocytosis in the case of ALMS1 or the regulation of the extracellular matrix in BBS1. Protein interaction analyses demonstrated the involvement of CDC42 as a central protein in the interactome in ALMS1 and CDK2 in the case of BBS1. CONCLUSIONIn conclusion, the depletion of ALMS1 and BBS1 affects the TGF-{beta} signalling pathway, conditioning the phosphorylation and activation of several proteins, including CDC42 in the case of ALMS1 and CDK2 in the case of BBS1.

cell biology↗

bioRxiv2022

Loss of the centrosomal protein ALMS1 alters lipid metabolism and the regulation of extracellular matrix-related processes

BackgroundAlstrom syndrome (ALMS) is a rare autosomal recessive disease that is associated with mutations in ALMS1 gene. The main clinical manifestations of ALMS are retinal dystrophy, obesity, type 2 diabetes mellitus, dilated cardiomyopathy and multi-organ fibrosis, characteristic in kidneys and liver. Depletion of the protein encoded by ALMS1 has been associated with the alteration of different processes regulated via the primary cilium, such as the NOTCH or TGF-{beta} signalling pathways. However, the cellular impact of these deregulated pathways in the absence of ALMS1 remains unknown. MethodsIn this study, we integrated RNA-seq and proteomic analysis to determine the gene expression profile of hTERT-BJ-5ta ALMS1 knockout fibroblasts after TGF-{beta} stimulation. In addition, we studied alterations in cross-signalling between the TGF-{beta} pathway and the AKT pathway in this cell line. ResultsWe found that ALMS1 depletion affects the TGF-{beta} pathway and its cross-signalling with other pathways such as PI3K/AKT, EGFR1 or p53. In addition, alterations associated with ALMS1 depletion clustered around the processes of extracellular matrix regulation and lipid metabolism in both the transcriptome and proteome. By studying the enriched pathways of common genes differentially expressed in the transcriptome and proteome, collagen fibril organisation, {beta}-oxidation of fatty acids and eicosanoid metabolism emerged as key processes altered by the absence of ALMS1. Finally, an overactivation of the AKT pathway was determined in the absence of ALMS1 that could be explained by a decrease in PTEN gene expression. ConclusionALMS1 deficiency disrupts cross-signalling between the TGF-{beta} pathway and other dependent pathways in hTERT-BJ-5ta cells. Furthermore, altered cross-signalling impacts the regulation of extracellular matrix-related processes and fatty acid metabolism, and leads to over-activation of the AKT pathway.

cell biology↗

bioRxiv2021

Depletion of ALMS1 inhibits TGF-β/SMAD3/SNAI1 axis altering normal cell migration capacity and epithelial-mesenchyme transition.

BackgroundALMS1 is a ubiquitous gene associated with Alstrom syndrome (ALMS). The main symptoms of ALMS affect multiple organs and tissues, generating at last, multi-organic fibrosis in the lungs, kidneys and liver. TGF-{beta} is one of the main pathways implicated in fibrosis, controlling the cell cycle, apoptosis, cell migration, cell adhesion and epithelial-mesenchymal transition (EMT). Nevertheless, the role of ALMS1 gene in fibrosis generation and other implicated processes such as cell migration or cell adhesion via the TGF-{beta} pathway has not been elucidated yet. MethodsInitially, we evaluated how depletion of ALMS1 affects different processes like apoptosis, cell cycle and mitochondrial activity in HeLa cells. Then, we performed proteomic profiling with TGF-{beta} stimuli in HeLa ALMS1 -/- cells and validated the results by examining different EMT biomarkers using qPCR. The expression of these EMT biomarkers were also studied in hTERT-BJ-5ta ALMS1 -/-. Finally, we also evaluated the SMAD3 and SMAD2 phosphorylation and cell migration capacity in both models. ResultsDepletion of ALMS1 generated apoptosis resistance to thapsigargin (THAP) and C2-Ceramide (C2-C), and G2/M cell cycle arrest in HeLa cells. For mitochondrial activity, results did not show significant differences between ALMS1 +/+ and ALMS1 -/-. Proteomic results showed inhibition of downstream pathways regulated by TGF-{beta}. The protein-coding genes (PCG) were associated with processes like focal adhesion or cell-substrate adherens junction in HeLa. SNAI1 showed an opposite pattern to what would be expected when activating the EMT in HeLa and BJ-5ta. Finally, in BJ-5ta model a reduced activation of SMAD3 but not SMAD2 were also observed. In HeLa models no alterations in the canonical TGF-{beta} pathway were observed but both cell lines showed a reduction in migration capacity. ConclusionALMS1 has a role in controlling the cell cycle and the apoptosis processes. Moreover, the depletion of ALMS1 affects the signal transduction through the TGF-{beta} and other processes like the cell migration and adhesion capacity.

cell biology↗

bioRxiv2021

Common variation in EDN1 regulatory regions highlights the role of PPARγ as a key regulator of Endothelin in vitro

Pulmonary Arterial Hypertension (PAH) is a rare disease caused by the obliteration of the pulmonary arterioles, increasing pulmonary vascular resistance and eventually causing right heart failure. Endothelin-1 is a vasoconstrictor peptide whose levels are indicators of disease progression and its pathway is one of the commonest targeted by current treatments. We sequenced the EDN1 untranslated regions of a small subset of PAH patients, predicted the effect in silico, and used a luciferase assay with the different genotypes to analyze its influence on gene expression. Finally, we used siRNAs against the major transcription factors predicted for these regions (PPAR{gamma}, KLF4, and VDR) to assess Endothelin-1 expression in cell culture and validate the binding sites. First, we detected a SNP in the 5UTR (rs397751713) and another in the 3regulatory region (rs2859338) that increased luciferase activity in vitro depending on their genotype. We determined in silico that KLF4/PPAR{gamma} could be binding in the rs397751713 and VDR in rs2859338. By using siRNAs and luciferase, we determined that PPAR{gamma} binds differentially in rs397751713. PPAR{gamma} and VDR Knock-Down, increased EDN1 mRNA levels and Endothelin-1 production in PAECs, while PPAR{gamma} and KLF4 Knock-Down increased the Endothelin-1 production in HeLa. In conclusion, common variants in EDN1 regulatory regions could alter Endothelin-1 levels. We were able to validate that PPAR{gamma} binds in rs397751713 and is key to regulate Endothelin-1. Also, KLF4 and VDR regulate Endothelin-1 production in a cell-dependent manner, but for VDR this interaction does not happen by binding directly to the regions we studied.

cell biology↗