Papel de la proteína CLMP en la interacción entre epitelio intestinal y Campylobacter jejuni

Premio extraordinario de Trabajo Fin de Máster curso 2012-2013.Biotecnología Molecular, Celular y Genétic

Characterization of the catalytic flexible loop in the dihydroorotase domain of the human multi-enzymatic protein CAD

The dihydroorotase (DHOase) domain of the multifunctional protein carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase (CAD) catalyzes the third step in the de novo biosynthesis of pyrimidine nucleotides in animals. The crystal structure of the DHOase domain of human CAD (huDHOase) revealed that, despite evolutionary divergence, its active site components are highly conserved with those in bacterial DHOases, encoded as monofunctional enzymes. An important element for catalysis, conserved from Escherichia coli to humans, is a flexible loop that closes as a lid over the active site. Here, we combined mutagenic, structural, biochemical, and molecular dynamics analyses to characterize the function of the flexible loop in the activity of CAD's DHOase domain. A huDHOase chimera bearing the E. coli DHOase flexible loop was inactive, suggesting the presence of distinctive elements in the flexible loop of huDHOase that cannot be replaced by the bacterial sequence. We pinpointed Phe-1563, a residue absolutely conserved at the tip of the flexible loop in CAD's DHOase domain, as a critical element for the conformational equilibrium between the two catalytic states of the protein. Substitutions of Phe-1563 with Ala, Leu, or Thr prevented the closure of the flexible loop and inactivated the protein, whereas substitution with Tyr enhanced the interactions of the loop in the closed position and reduced fluctuations and the reaction rate. Our results confirm the importance of the flexible loop in CAD's DHOase domain and explain the key role of Phe-1563 in configuring the active site and in promoting substrate strain and catalysi

Functional and structural deficiencies of Gemin5 variants associated with neurological disorders

Dysfunction of RNA-binding proteins is often linked to a wide range of human disease, particularly with neurological conditions. Gemin5 is a member of the survival of the motor neurons (SMN) complex, a ribosome-binding protein and a translation reprogramming factor. Recently, pathogenic mutations in Gemin5 have been reported, but the functional consequences of these variants remain elusive. Here, we report functional and structural deficiencies associated with compound heterozygosity variants within the Gemin5 gene found in patients with neurodevelopmental disorders. These clinical variants are located in key domains of Gemin5, the tetratricopeptide repeat (TPR)-like dimerization module and the noncanonical RNA-binding site 1 (RBS1). We show that the TPR-like variants disrupt protein dimerization, whereas the RBS1 variant confers protein instability. All mutants are defective in the interaction with protein networks involved in translation and RNA-driven pathways. Importantly, the TPR-like variants fail to associate with native ribosomes, hampering its involvement in translation control and establishing a functional difference with the wild-type protein. Our study provides insights into the molecular basis of disease associated with malfunction of the Gemin5 protei

The GATA3 X308_Splice breast cancer mutation is a hormone context-dependent oncogenic driver

As the catalog of oncogenic driver mutations is expanding, it becomes clear that alterations in a given gene might have different functions and should not be lumped into one class. The transcription factor GATA3 is a paradigm of this. We investigated the functions of the most common GATA3 mutation (X308_Splice) and five additional mutations, which converge into a neoprotein that we called “neoGATA3,” associated with excellent prognosis in patients. Analysis of available molecular data from >3000 breast cancer patients revealed a dysregulation of the ER-dependent transcriptional response in tumors carrying neoGATA3-generating mutations. Mechanistic studies in vitro showed that neoGATA3 interferes with the transcriptional programs controlled by estrogen and progesterone receptors, without fully abrogating them. ChIP-Seq analysis indicated that ER binding is reduced in neoGATA3-expressing cells, especially at distal regions, suggesting that neoGATA3 interferes with the fine tuning of ER-dependent gene expression. This has opposite outputs in distinct hormonal context, having pro- or anti-proliferative effects, depending on the estrogen/progesterone ratio. Our data call for functional analyses of putative cancer drivers to guide clinical application.Institute of Cancer Research of the Medical University Vienna and by the grant P27361-B23 from the Austrian Science Grant (FWF), FXR was supported by SAF2011-29530 and SAF2015-70553-R grants from Ministerio de Economía y Competitividad (Madrid, Spain) (co-funded by the ERDF-EU), Fundación Científica de la Asociación Española Contra el Cáncer. CNIO is supported by Ministerio de Ciencia, Innovación y Universidades as a Centro de Excelencia Severo Ochoa SEV-2015-051

Beyond genetics: Deciphering the impact of missense variants in CAD deficiency

16 páginas, 5 figuras, 1 tablaCAD is a large, 2225 amino acid multienzymatic protein required for de novo pyrimidine biosynthesis. Pathological CAD variants cause a developmental and epileptic encephalopathy which is highly responsive to uridine supplements. CAD deficiency is difficult to diagnose because symptoms are nonspecific, there is no biomarker, and the protein has over 1000 known variants. To improve diagnosis, we assessed the pathogenicity of 20 unreported missense CAD variants using a growth complementation assay that identified 11 pathogenic variants in seven affected individuals; they would benefit from uridine treatment. We also tested nine variants previously reported as pathogenic and confirmed the damaging effect of seven. However, we reclassified two variants as likely benign based on our assay, which is consistent with their long-term follow-up with uridine. We found that several computational methods are unreliable predictors of pathogenic CAD variants, so we extended the functional assay results by studying the impact of pathogenic variants at the protein level. We focused on CAD's dihydroorotase (DHO) domain because it accumulates the largest density of damaging missense changes. The atomic-resolution structures of eight DHO pathogenic variants, combined with functional and molecular dynamics analyses, provided a comprehensive structural and functional understanding of the activity, stability, and oligomerization of CAD's DHO domain. Combining our functional and protein structural analysis can help refine clinical diagnostic workflow for CAD variants in the genomics era.This work was supported by grant RTI2018-098084-B-I00 financed by MCIN/AEI/10.13039/501100011033/ and “FEDER Unamanera de hacer Europa,” by grant PID2021-128468NBI00 financed by MCIN/AEI/10.13039/501100011033 and by a grant from Fundacion Ram on Areces Ciencias de la Vida (XX National Call) to SR-M. FdC-O is a postdoctoral 1182 del CAÑO-OCHOA ET AL. 15732665, 2023, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jimd.12667 by Csic Organización Central Om (Oficialia Mayor) (Urici), Wiley Online Library on [13/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License fellow of the Generalitat Valenciana (APOSTD 2021). AR-d-C is supported by salary from the European Commission–Next Generation EU through the CSIC Global Health Platform (PTI+ Salud Global) established by EU Council Regulation 2020/2094. HHF, BN, and SMP were supported by The Rocket Fund, R01DK099551, and U54 NS115198. SMP is also supported by a training component of U54 NS115198. MPW is supported by an MSCA Individual Fellowship (894669) and an FWO Senior Postdoctoral Fellowship (1289023N). X-ray diffraction experiments at synchrotrons were done through the participation of SR-M in the BAG proposals 2017082302, 2018082950, 2019093709, 2020074406, 2021075216, and 2022075911 at ALBA, and MX-2076, MX-2351, and MX-2452 at European Synchrotron Radiation Facility. The authors thank the ALBA synchrotron staff and Max H. Nanao at beamtime ID23-2 at the ESRF synchrotron for assistance.Peer reviewe

Afatinib Exerts Immunomodulatory Effects by Targeting the Pyrimidine Biosynthesis Enzyme CAD

13 páginas, 7 figurasCurrent clinical trials of combined EGFR-tyrosine kinase inhibitors (TKI) and immune checkpoint blockade (ICB) therapies show no additional effect. This raises questions regarding whether EGFR-TKIs attenuate ICB-enhanced CD8+ T lymphocyte function. Here we show that the EGFR-TKI afatinib suppresses CD8+ T lymphocyte proliferation, and we identify CAD, a key enzyme of de novo pyrimidine biosynthesis, to be a novel afatinib target. Afatinib reduced tumor-infiltrating lymphocyte numbers in Lewis lung carcinoma (LLC)-bearing mice. Early afatinib treatment inhibited CD8+ T lymphocyte proliferation in patients with non-small cell lung cancer, but their proliferation unexpectedly rebounded following long-term treatment. This suggests a transient immunomodulatory effect of afatinib on CD8+ T lymphocytes. Sequential treatment of afatinib with anti-PD1 immunotherapy substantially enhanced therapeutic efficacy in MC38 and LLC-bearing mice, while simultaneous combination therapy showed only marginal improvement over each single treatment. These results suggest that afatinib can suppress CD8+ T lymphocyte proliferation by targeting CAD, proposing a timing window for combined therapy that may prevent the dampening of ICB efficacy by EGFR-TKIs. SIGNIFICANCE: This study elucidates a mechanism of afatinib-mediated immunosuppression and provides new insights into treatment timing for combined targeted therapy and immunotherapy. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/12/3270/F1.large.jpg.This study was supported by Taiwan Ministry of Science and Technology grants MOST 104-2320-B-002-044-MY3, MOST 106-2320-B-002-046-MY3, and MOST 108-2320-B-002-024-MY3, National Health Research Institutes grants NHRI-EX106-10401BI and NHRI-EX109-10725BI, National Taiwan University grants NTU107L890504 and NTU110L893503 to M.-S. Lee, and National Taiwan University Hospital grants 106-003451, 107-003849, 108-004269, and 109-004720 to C.-C. Ho. This work was also supported by MINECO grants BFU2016-80570-R and RTI2018-098084-B-I00 (AEI/FEDER, UE). The authors would like to thank the Laboratory Animal Core Facility at the College of Medicine, National Taiwan University for their servicesPeer reviewe

Functional characterization of CAD, an antitumoral target controlling the de novo pyrimidine biosynthesis

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 26-06-2019Esta tesis tiene embargado el acceso al texto completo hasta el 26-12-2020Pyrimidine nucleotides are essential compounds for the synthesis of nucleic acids and other key cellular processes. The cells obtain the pyrimidines through two different metabolic pathways depending on their developmental stage. In differentiated cells, pyrimidines are obtained mainly by recycling through salvage pathways, and the de novo synthesis of pyrimidines is low. In contrast, when cells grow and proliferate, the activation of de novo synthesis is necessary to fuel replication and to manufacture of other essential macromolecules. In animals, three of the six enzymatic activities that constitute the de novo synthesis pathway, carbamoyl phosphate synthetase (CPS), aspartate transcarbamoylase (ATC) and dihydroorotase (DHO) are fused into a single multifunctional protein called CAD. This multienzyme protein initiates and controls the de novo synthesis of pyrimidines and is overexpressed in different types of cancer, which makes it a potential target for the development of antitumoral compounds. In recent years, our group has characterized the DHO and ATC enzymatic domains of human CAD, but beyond knowing the atomic structure and kinetic properties, it is necessary to study CAD in a cellular context to better understand its functioning and move towards designing compounds that regulate their activity and may have a therapeutic value. In the course of this thesis, we have addressed the study of the subcellular localization of CAD using fluorescent chimeras and generating, through CRISPR/Cas9 technology, the first human CAD knockout and GFP-CAD-knockin cell lines. Our results show that CAD is a protein present exclusively in the cytosol that, contradicting results published by other groups, is not transported to the nucleus during the cell cycle. Until recently, it was thought that due to the central role of CAD in the synthesis of pyrimidines, mutations that compromised its activity would have a lethal effect, explaining that no diseases were associated with this gene. However, since 2015, it is known that CAD-deficit is a serious metabolic disease in children at an early age who die if they are not diagnosed in time. Until now, patients are diagnosed by exome sequencing, with the associated difficulty of distinguishing between possible pathogenic mutations and undescribed variants of the protein. Thanks to the molecular tools developed in this thesis, we set up a simple cell assay that allows the identification of pathogenic mutations, helping in the correct diagnosis and treatment of patients. In addition, we have studied the effect of the pathogenic mutations on the structure and activity of the isolated CAD domains. These clinical mutations have helped us to discover key elements for the functioning of the protein. This detailed study of the mechanisms of CAD has led us to characterize in detail a flexible loop in the DHO domain of human CAD, and to describe its participation in the catalytic mechanism of the enzyme

CAD, A Multienzymatic Protein at the Head of de Novo Pyrimidine Biosynthesis

CAD is a 1.5 MDa particle formed by hexameric association of a 250 kDa protein that carries the enzymatic activities for the first three steps in the de novo biosynthesis of pyrimidine nucleotides: glutamine-dependent Carbamoyl phosphate synthetase, Aspartate transcarbamoylase and Dihydroorotase. This metabolic pathway is essential for cell growth and proliferation and is conserved in all living organisms. However, the fusion of the first three enzymatic activities of the pathway into a single multienzymatic protein only occurs in animals. In prokaryotes, by contrast, these activities are encoded as distinct monofunctional enzymes that function independently or by forming more or less transient complexes. Whereas the structural information about these enzymes in bacteria is abundant, the large size and instability of CAD has only allowed a fragmented characterization of its structure. Here we retrace some of the most significant efforts to decipher the architecture of CAD and to understand its catalytic and regulatory mechanisms

Deciphering CAD: Structure and function of a mega-enzymatic pyrimidine factory in health and disease

14 páginas, 6 figuras, 1 tabla.CAD is a 1.5 MDa particle formed by hexameric association of a 250 kDa protein divided into different enzymatic domains, each catalyzing one of the initial reactions for de novo biosynthesis of pyrimidine nucleotides: glutaminase-dependent Carbamoyl phosphate synthetase, Aspartate transcarbamoylase, and Dihydroorotase. The pathway for de novo pyrimidine synthesis is essential for cell proliferation and is conserved in all living organisms, but the covalent linkage of the first enzymatic activities into a multienzymatic CAD particle is unique to animals. In other organisms, these enzymatic activities are encoded as monofunctional proteins for which there is abundant structural and biochemical information. However, the knowledge about CAD is scarce and fragmented. Understanding CAD requires not only to determine the three-dimensional structures and define the catalytic and regulatory mechanisms of the different enzymatic domains, but also to comprehend how these domains entangle and work in a coordinated and regulated manner. This review summarizes significant progress over the past 10 years toward the characterization of CAD's architecture, function, regulatory mechanisms, and cellular compartmentalization, as well as the recent finding of a new and rare neurometabolic disorder caused by defects in CAD activities.Fundación Ramón Areces, Grant/AwardNumber: XX National Call; area of RareDiseases; Ministerio de Ciencia eInnovación, Grant/Award Number:RTI2018-098084-B-100; AEI/FEDER; UEPeer reviewe

A Tailored Strategy to Crosslink the Aspartate Transcarbamoylase Domain of the Multienzymatic Protein CAD

CAD is a 1.5 MDa hexameric protein with four enzymatic domains responsible for initiating de novo biosynthesis of pyrimidines nucleotides: glutaminase, carbamoyl phosphate synthetase, aspartate transcarbamoylase (ATC), and dihydroorotase. Despite its central metabolic role and implication in cancer and other diseases, our understanding of CAD is poor, and structural characterization has been frustrated by its large size and sensitivity to proteolytic cleavage. Recently, we succeeded in isolating intact CAD-like particles from the fungus Chaetomium thermophilum with high yield and purity, but their study by cryo-electron microscopy is hampered by the dissociation of the complex during sample grid preparation. Here we devised a specific crosslinking strategy to enhance the stability of this mega-enzyme. Based on the structure of the isolated C. thermophilum ATC domain, we inserted by site-directed mutagenesis two cysteines at specific locations that favored the formation of disulfide bridges and covalent oligomers. We further proved that this covalent linkage increases the stability of the ATC domain without damaging the structure or enzymatic activity. Thus, we propose that this cysteine crosslinking is a suitable strategy to strengthen the contacts between subunits in the CAD particle and facilitate its structural characterization