Biology subjects

Valverde, J. M.

Publications and source records attributed to Valverde, J. M..

5 recordsLinked to original sources

bioRxiv2024

A chemical-genetic system to rapidly inhibit the PP2A-B56 phosphatase

AO_SCPLOWBSTRACTC_SCPLOWSerine-threonine phosphatases have been challenging to study because of the lack of specific inhibitors. Their catalytic domains are druggable, but these are shared or very similar between individual phosphatase complexes, precluding their specific inhibition. Instead, phosphatase complexes achieve specificity by interacting with short-linear motifs (SLiMs) in substrates or their binding partners. We develop here a chemical-genetic system to rapidly inhibit these interactions within the PP2A-B56 family. Drug-inducible recruitment of ectopic SLiMs ("directSLiMs") is used to rapidly block the SLiM-binding pocket on the B56 regulatory subunit, thereby displacing endogenous interactors and inhibiting PP2A-B56 activity within seconds. We use this system to characterise PP2A-B56 substrates during mitosis and to identify a novel role for PP2A-B56 in maintaining kinetochore-microtubule attachments at metaphase. The directSLiMs approach can be used to inhibit any other phosphatase, enzyme or protein that uses a critical SLiM-binding interface, providing a powerful strategy to inhibit and characterise proteins once considered ."undruggable".

cell biology↗

bioRxiv2022

Capturing the signalling dynamics of the MAPK-AKT-mTOR pathway in a single targeted phosphoproteomics assay

The MAPK-AKT-mTOR protein network integrates extra- and intracellular signals to determine cellular fate, regulating pivotal biological processes such as cell growth and metabolism. Due to this crucial role, pathway dysregulation has been implicated in multiple diseases, such as metabolic disorders and cancer. The MAPK-AKT-mTOR pathway consists of dozens of proteins and signal transduction is primarily driven by protein phosphorylation. Here, we present a targeted phosphoproteomics assay to study the phosphorylation dynamics of the MAPK-AKT-mTOR pathway in detail with high sensitivity and in a high throughput manner. By using a multi-protease approach, we increased the pathway coverage with phosphosites that were previously inaccessible. This novel approach yields the most comprehensive method for the detailed study of mTOR signalling to date (covering 150 phosphopeptides on more than 70 phosphoproteins), which can be applied to in vitro and in vivo systems and has the sensitivity to be compatible with small sample amounts. We demonstrate the feasibility of this assay to monitor the plasticity of MAPK-AKT-mTOR phosphorylation dynamics in response to cellular stimuli with high temporal resolution and amino acid residue specificity. We found highly dynamic phosphorylation events upon treatment with growth factors, revealing the sequential nature of phosphosites in this signalling pathway. Furthermore, starvation of glucose and amino acids showed upregulation of AKT-targets PRAS40T246 and FOXO3T32, highlighting the role of AKT in cellular response to starvation. These findings illustrate the potential of this assay to obtain new biological insight when monitoring dynamics of functional phosphosites. HighlightsO_LIRobust targeted MS assay to study the phosphorylation dynamics of the MAPK-AKT-mTOR network C_LIO_LIExtended pathway coverage by application of multiple proteases for protein digestion C_LIO_LIHighly sensitive, high throughput and readily applicable assay for in vivo and in vitro systems C_LIO_LIPhosphorylation patterns of MAPK-AKT-mTOR network are highly dynamic and change upon stimulation with growth factors, amino acids and glucose C_LI MotivationThe MAPK-AKT-mTOR protein network integrates extra- and intracellular signals to determine cellular fate, regulating pivotal biological processes such as cell growth and metabolism. Due to this crucial role, pathway dysregulation has been implicated in multiple diseases, such as metabolic disorders and cancer. Our understanding of the complex regulation of this intricate signalling network is incomplete and is hampered by the lack of analytical methods to study its phosphorylation dynamics in detail. In this study, we present a targeted phosphoproteomics assay to monitor the phosphorylation events of the MAPK-AKT-mTOR pathway with amino acid residue specificity and in a high throughput manner. We increased the pathway coverage with phosphosites that were previously inaccessible by the use of multiple proteases for protein digestion. This novel approach yields the most comprehensive method for the detailed study of MAPK-AKT-mTOR signalling to date, which can be applied to in vitro and in vivo human samples and has the sensitivity to be compatible with small amounts of starting material. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=167 SRC="FIGDIR/small/476555v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@50a6a2org.highwire.dtl.DTLVardef@ec125dorg.highwire.dtl.DTLVardef@a9aca8org.highwire.dtl.DTLVardef@1865b5f_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

Single-embryo phosphoproteomics reveals the importance of intrinsic disorder in cell cycle dynamics

Switch-like cyclin-dependent kinase (CDK)-1 activation is thought to underlie the abruptness of mitotic onset, but how CDKs can simultaneously phosphorylate many diverse substrates is unknown, and direct evidence for such phosphorylation dynamics in vivo is lacking. Here, we analysed protein phosphorylation states in single Xenopus embryos throughout synchronous cell cycles. Over a thousand phosphosites were dynamic in vivo, and assignment of cell cycle phases using egg extracts revealed hundreds of S-phase phosphorylations. Targeted phosphoproteomics in single embryos showed switch-like mitotic phosphorylation of diverse protein complexes. The majority of cell cycle-regulated phosphosites occurred in CDK consensus motifs, and 72% located to intrinsically disordered regions. Dynamically phosphorylated proteins, and documented substrates of cell cycle kinases, are significantly more disordered than phosphoproteins in general. Furthermore, 30-50% are components of membraneless organelles. Our results suggest that phosphorylation of intrinsically disordered proteins by cell cycle kinases, particularly CDKs, allows switch-like mitotic cellular reorganisation.

cell biology↗

bioRxiv2021

Automatic segmentation of the rat brain hippocampus in MRI after traumatic brain injury

AO_SCPLOWBSTRACTC_SCPLOWRegistration-based methods are commonly used in the anatomical segmentation of magnetic resonance (MR) brain images. However, they are sensitive to the presence of deforming brain pathologies that may interfere with the alignment of the atlas image with the target image. Our goal was to develop an algorithm for automated segmentation of the normal and injured rat hippocampus. We implemented automated segmentation using a U-Net-like Convolutional Neural Network (CNN). of sham-operated experimental controls and rats with lateral-fluid-percussion induced traumatic brain injury (TBI) on MR images and trained ensembles of CNNs. Their performance was compared to three registration-based methods: single-atlas, multi-atlas based on majority voting and Similarity and Truth Estimation for Propagated Segmentations (STEPS). Then, the automatic segmentations were quantitatively evaluated using six metrics: Dice score, Hausdorff distance, precision, recall, volume similarity and compactness using cross-validation. Our CNN and multi-atlas -based segmentations provided excellent results (Dice scores > 0.90) despite the presence of brain lesions, atrophy and ventricular enlargement. In contrast, the performance of singe-atlas registration was poor (Dice scores < 0.85). Unlike registration-based methods, which performed better in segmenting the contralateral than the ipsilateral hippocampus, our CNN-based method performed equally well bilaterally. Finally, we assessed the progression of hippocampal damage after TBI by applying our automated segmentation tool. Our data show that the presence of TBI, time after TBI, and whether the location of the hippocampus was ipsilateral or contralateral to the injury explained hippocampal volume (p = 0.029, p < 0.001, and p < 0.001 respectively).

bioinformatics↗

bioRxiv2020

Automated skull-stripping and segmentation with Multi-Task U-Net in large mouse brain MRI databases

Skull-stripping and region segmentation are fundamental steps in preclinical magnetic resonance imaging (MRI) studies, and these common procedures are usually performed manually. We present Multi-task U-Net (MU-Net), a convolutional neural network designed to accomplish both tasks simultaneously. MU-Net achieved higher segmentation accuracy than state-of-the-art multi-atlas segmentation methods with an inference time of 0.35 seconds and no pre-processing requirements. We evaluated the performance of our network in the presence of skip connections and recently proposed framing connections, finding the simplest network to be the most effective. We tested MU-Net with an unusually large dataset combining several independent studies consisting of 1,782 mouse brain MRI volumes of both healthy and Huntington animals, and measured average Dice scores of 0.906 (striati), 0.937 (cortex), and 0.978 (brain mask). These high evaluation scores demonstrate that MU-Net is a powerful tool for segmentation and skull-stripping, decreasing inter and intra-rater variability of manual segmentation. The MU-Net code and the trained model are publicly available at https://github.com/Hierakonpolis/MU-Net.

bioinformatics↗