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Amyotrophic lateral sclerosis (ALS) is a fatal and incurable neurodegenerative disease affecting motor neurons and characterized by microglia-mediated neurotoxic inflammation whose underlying mechanisms remain incompletely understood. In this work, we reveal that MAPK/MAK/MRK overlapping kinase (MOK), with an unknown physiological substrate, displays an immune function by controlling inflammatory and type-I interferon (IFN) responses in microglia which are detrimental to primary motor neurons. Moreover, we uncover the epigenetic reader bromodomain-containing protein 4 (Brd4) as an effector protein regulated by MOK, by promoting Ser-phospho-Brd4 levels. We further demonstrate that MOK regulates Brd4 functions by supporting its binding to cytokine gene promoters, therefore enabling innate immune responses. Remarkably, we show that MOK levels are increased in the ALS spinal cord, particularly in microglial cells, and that administration of a chemical MOK inhibitor to ALS model mice can modulate Ser-phospho-Brd4 levels, suppress microglial activation, and modify the disease course, indicating a pathophysiological role of MOK kinase in ALS and neuroinflammation.

Compounds that mimic the biological properties of glycosaminoglycans (GAGs) and can be more easily prepared than the native GAG oligosaccharides are highly demanded. Here, we present the synthesis of sulfated oligosaccharides displaying a perfluorinated aliphatic tag at the reducing end as GAG mimetics. The preparation of these molecules was greatly facilitated by the presence of the fluorinated tail since the reaction intermediates were isolated by simple fluorous solid-phase extraction. Fluorescence polarization competition assays indicated that the synthesized oligosaccharides interacted with two heparin-binding growth factors, midkine (MK) and FGF-2, showing higher binding affinities than the natural oligosaccharides, and can be therefore considered as useful GAG mimetics. Moreover, NMR experiments showed that the 3D structure of these compounds is similar to that of the native sequences, in terms of sugar ring and glycosidic linkage conformations. Finally, we also demonstrated that these derivatives are able to block the MK-stimulating effect on NIH3T3 cells growth.

The recent pandemic we are experiencing caused by the coronavirus disease 2019 (COVID-19) has put the world’s population on the rack, with more than 191 million cases and more than 4.1 million deaths confirmed to date. This disease is caused by a new type of coronavirus, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A massive proteomic analysis has revealed that one of the structural proteins of the virus, the E protein, interacts with BRD2 and BRD4 proteins of the Bromodomain and Extra Terminal domain (BET) family of proteins. BETs are essential to cell cycle progression, inflammation and immune response and have also been strongly associated with infection by different types of viruses. The fundamental role BET proteins play in transcription makes them appropriate targets for the propagation strategies of some viruses. Recognition of histone acetylation by BET bromodomains is essential for transcription control. The development of drugs mimicking acetyl groups, and thereby able to displace BET proteins from chromatin, has boosted interest on BETs as attractive targets for therapeutic intervention. The success of these drugs against a variety of diseases in cellular and animal models has been recently enlarged with promising results from SARS-CoV-2 infection studies.

In the last decades, the post-translational modification system by covalent attachment of the SUMO polypeptide to proteins has emerged as an essential mechanism controlling virtually all the physiological processes in the eukaryotic cell. This includes vertebrate development. In the nervous system, SUMO plays crucial roles in synapse establishment and it has also been linked to a variety of neurodegenerative diseases. However, to date, the involvement of the modification of specific targets in key aspects of nervous system development, like patterning and differentiation, has remained largely elusive. A number of recent works confirm the participation of target-specific SUMO modification in critical aspects of nervous system development. Here, we review pioneering and new findings demonstrating the essential role SUMO plays in neurogenesis and other facets of neurodevelopment, which will help to precisely understand the variety of mechanisms SUMO utilizes to control most fundamental processes in the cell.

Neurogenesis is a complex process involving the orchestration of many transcription factors and other proteins. Fine regulation of their activities is crucial for proper progression of neurogenesis. A few decades ago, covalent attachment of Small Ubiquitin-like MOdifier (SUMO) to other proteins was revealed as a major regulator of protein activities, constituting an essential posttranslational modification system in vertebrates. Since then, hundreds of proteins have been shown to be targets of SUMO, which is implicated in controlling many relevant processes in eukaryotic cells. These include the development and function of the nervous system, with SUMO tightly linked to synapsis and to neurodegenerative diseases (Yau et al., 2020). The SUMO protease SENP7 has been involved in neurogenesis (Juárez-Vicente et al., 2016), while sumoylation of BRAF35, a subunit of the LSD1 histone demethylase complex, assures the undifferentiated state of neural progenitors (Ceballos-Chávez et al., 2012). In addition, increased sumoylation in mouse brain-derived neural stem cells results in enhanced neuronal differentiation (Bernstock et al., 2019). However, in general, the implication of SUMO in neuronal differentiation and nervous system development has been poorly studied. We have recently developed a proteomic study aimed at identifying SUMO targets associated either with proliferation or with neuronal differentiation conditions, which has highlighted the relevance of SUMO modification for proper progression of neurogenesis. Here, we discuss our recent results, present specific SUMO targets that are key to the process, and indicate future research directions to uncover the molecular mechanisms underlying SUMO modification of relevant neurogenesis-associated factors.

SUMOylation is a post-translational modification that has emerged in recent decades as a mechanism involved in controlling diverse physiological processes and that is essential in vertebrates. The SUMO pathway is regulated by several enzymes, proteases and ligases being the main actors involved in the control of sumoylation of specific targets. Dysregulation of the expression, localization and function of these enzymes produces physiological changes that can lead to the appearance of different types of cancer, depending on the enzymes and target proteins involved. Among the most studied proteases and ligases, those of the SENP and PIAS families stand out, respectively. While the proteases involved in this pathway have specific SUMO activity, the ligases may have additional functions unrelated to sumoylation, which makes it more difficult to study their SUMO-associated role in cancer process. In this review we update the knowledge and advances in relation to the impact of dysregulation of SUMO proteases and ligases in cancer initiation and progression.

La neurogénesis es un proceso esencial que implica un gran número de eventos celulares en los que intervienen numerosos factores de transcripción sometidos a una estricta regulación. La modificación post-traduccional por unión covalente del polipéptido SUMO a otras proteínas es esencial en vertebrados y regula multitud de procesos celulares, pero su implicación en diferenciación neuronal no ha sido estudiada en profundidad. En este sentido, el principal objetivo de esta tesis ha sido la identificación de factores de transcripción relevantes cuya actividad durante el proceso de neurogénesis esté modulada por sumoilación. Para ello se llevaron a cabo estudios funcionales de proteínas diferencialmente sumoiladas en condiciones de proliferación y de diferenciación neuronal, entre las que se encontró Utf1, identificándose como una nueva diana de SUMO. Experimentos de ganancia de función demostraron diferencias marcadas entre los efectos sobre la neurogénesis de las versiones silvestre y mutantes de sumoilación de una selección de proteínas. Mientras que la sumoilación de Prox1, Sall4a, Trim24, Utf1 y Etv6 se asoció con un efecto positivo sobre la neurogénesis, la sumoilación de Kctd15 se asoció con un efecto negativo. Hemos determinado dos mecanismos por los que SUMO regula la función previamente descrita de Utf1 de controlar la expresión de genes bivalentes. Por un lado la sumoilación modula su afinidad por la cromatina y por otro lado facilita su unión a la enzima Dcp1 (“mRNA-decapping enzyme”) a través de una secuencia SIM (“SUMO interacting motif”) de interacción no covalente con SUMO conservada en proteínas Dcp1 de distintos organismos. La proteína etv6, se ha descrito previamente que se modifica por SUMO en su extremo amino-terminal, existiendo una isoforma alternativa truncada que carece de este sitio de sumoilación. Mediante el sistema CRISPR/Cas9 hemos generado: a) líneas celulares carentes de la isoforma larga de Etv6, que fenotípicamente presentan un cierto gr

Post-translational modification by covalent attachment of the Small ubiquitin-like modifier (Sumo) polypeptide regulates a multitude of processes in vertebrates. Despite demonstrated roles of Sumo in the development and function of the nervous system, the identification of key factors displaying a sumoylation-dependent activity during neurogenesis remains elusive. Through a SILAC (stable isotope labeling by/with amino acids in cell culture)-based proteomic approach, we have identified the Sumo proteome of the model cell line P19 under proliferation and neuronal differentiation conditions. More than 300 proteins were identified as putative Sumo targets differentially associated with one or the other condition. A group of proteins of interest were validated and investigated in functional studies. Among these, Utf1 was revealed as a new Sumo target. Gain-of-function experiments demonstrated marked differences between the effects on neurogenesis of overexpressing wild-type and sumoylation mutant versions of the selected proteins. While sumoylation of Prox1, Sall4a, Trim24, and Utf1 was associated with a positive effect on neurogenesis in P19 cells, sumoylation of Kctd15 was associated with a negative effect. Prox1, Sall4a, and Kctd15 were further analyzed in the vertebrate neural tube of living embryos, with similar results. Finally, a detailed analysis of Utf1 showed the sumoylation dependence of Utf1 function in controlling the expression of bivalent genes. Interestingly, this effect seems to rely on two mechanisms: sumoylation modulates binding of Utf1 to the chromatin and mediates recruitment of the messenger RNA-decapping enzyme Dcp1a through a conserved SIM (Sumo-interacting motif). Altogether, our results indicate that the combined sumoylation status of key proteins determines the proper progress of neurogenesis.

La neurogénesis es un proceso esencial que implica un gran número de eventos celulares en los que intervienen numerosos factores de transcripción sometidos a una estricta regulación. La modificación post-traduccional por unión covalente del polipéptido SUMO a otras proteínas es esencial en vertebrados y regula multitud de procesos celulares, pero su implicación en diferenciación neuronal no ha sido estudiada en profundidad. En este sentido, el principal objetivo de esta tesis ha sido la identificación de factores de transcripción relevantes cuya actividad durante el proceso de neurogénesis esté modulada por sumoilación. Para ello se llevaron a cabo estudios funcionales de proteínas diferencialmente sumoiladas en condiciones de proliferación y de diferenciación neuronal, entre las que se encontró Utf1, identificándose como una nueva diana de SUMO. Experimentos de ganancia de función demostraron diferencias marcadas entre los efectos sobre la neurogénesis de las versiones silvestre y mutantes de sumoilación de una selección de proteínas. Mientras que la sumoilación de Prox1, Sall4a, Trim24, Utf1 y Etv6 se asoció con un efecto positivo sobre la neurogénesis, la sumoilación de Kctd15 se asoció con un efecto negativo. Hemos determinado dos mecanismos por los que SUMO regula la función previamente descrita de Utf1 de controlar la expresión de genes bivalentes. Por un lado la sumoilación modula su afinidad por la cromatina y por otro lado facilita su unión a la enzima Dcp1 (“mRNA-decapping enzyme”) a través de una secuencia SIM (“SUMO interacting motif”) de interacción no covalente con SUMO conservada en proteínas Dcp1 de distintos organismos. La proteína etv6, se ha descrito previamente que se modifica por SUMO en su extremo amino-terminal, existiendo una isoforma alternativa truncada que carece de este sitio de sumoilación. Mediante el sistema CRISPR/Cas9 hemos generado: a) líneas celulares carentes de la isoforma larga de Etv6, que fenotípicamente presentan un cierto gr

Despite the growing evidence on impact of chromatin remodeling alterations in several genetic disorders, the comprehension of embryonic consequences remains a scientific challenge. The correct embryonic patterning is finely regulated and epigenetic changes are crucial to modulate the chromatin accessibility and gene expression. The numerous inborn genetic disorders caused by mutations in genes coding chromatin players prove that the consequences of chromatin alterations seriously affect embryonic development. For instance, since the idea of a unique group of syndromes named “Mendelian disorders of the epigenetic machinery” was proposed by Bjornsson in 2015 the number of recognized chromatinopathies has grown from 44 to 82 in 2019 (Bjornsson, 2015; Fahrner and Bjornsson, 2019).

Cornelia de Lange Syndrome (CdLS) is a human developmental syndrome with complex multisystem phenotypic features. It has been traditionally considered a cohesinopathy together with other phenotypically related diseases because of their association with mutations in subunits of the cohesin complex. Despite some overlap, the clinical manifestations of cohesinopathies vary considerably and, although their precise molecular mechanisms are not well defined yet, the potential pathomechanisms underlying these diverse developmental defects have been theoretically linked to alterations of the cohesin complex function. The cohesin complex plays a critical role in sister chromatid cohesion, but this function is not affected in CdLS. In the last decades, a non-cohesion-related function of this complex on transcriptional regulation has been well established and CdLS pathoetiology has been recently associated to gene expression deregulation. Up to 70% of CdLS cases are linked to mutations in the cohesin-loading factor NIPBL, which has been shown to play a prominent function on chromatin architecture and transcriptional regulation. Therefore, it has been suggested that CdLS can be considered a transcriptomopathy. Actually, CdLS-like phenotypes have been associated to mutations in chromatin-associated proteins, as KMT2A, AFF4, EP300, TAF6, SETD5, SMARCB1, MAU2, ZMYND11, MED13L, PHIP, ARID1B, NAA10, BRD4 or ANKRD11, most of which have no known direct association with cohesin. In the case of BRD4, a critical highly investigated transcriptional coregulator, an interaction with NIPBL has been recently revealed, providing evidence on their cooperation in transcriptional regulation of developmentally important genes. This new finding reinforces the notion of an altered gene expression program during development as the major etiological basis for CdLS. In this review, we intend to integrate the recent available evidence on the molecular mechanisms underlying the clinical manifestations of

Midkine (MK) is a neurotrophic factor that participates in the embryonic central nervous system (CNS) development and neural stem cell regulation, interacting with sulfated glycosaminoglycans (GAGs). Chondroitin sulfate (CS) is the natural ligand in the CNS. In this work, we describe the interactions between a library of synthetic models of CS-types and mimics. We did a structural study of this library by NMR and MD (Molecular Dynamics), concluding that the basic shape is controlled by similar geometry of the glycosidic linkages. Their 3D structures are a helix with four residues per turn, almost linear. We have studied the tetrasaccharide-midkine complexes by ligand observed NMR techniques and concluded that the shape of the ligands does not change upon binding. The ligand orientation into the complex is very variable. It is placed inside the central cavity of MK formed by the two structured beta-sheets domains linked by an intrinsically disordered region (IDR). Docking analysis confirmed the participation of aromatics residues from MK completed with electrostatic interactions. Finally, we test the biological activity by increasing the MK expression using CS tetrasaccharides and their capacity in enhancing the growth stimulation effect of MK in NIH3T3 cells.