The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A.

The neuronal calcium sensor 1 (NCS-1), an EF-hand Ca2+ binding protein, and Ric-8A coregulate synapse number and probability of neurotransmitter release. Recently, the structures of Ric-8A bound to Gα have revealed how Ric-8A phosphorylation promotes Gα recognition and activity as a chaperone and guanine nucleotide exchange factor. However, the molecular mechanism by which NCS-1 regulates Ric-8A activity and its interaction with Gα subunits is not well understood. Given the interest in the NCS-1/Ric-8A complex as a therapeutic target in nervous system disorders, it is necessary to shed light on this molecular mechanism of action at atomic level. We have reconstituted NCS-1/Ric-8A complexes to conduct a multimodal approach and determine the sequence of Ca2+ signals and phosphorylation events that promote the interaction of Ric-8A with Gα. Our data show that the binding of NCS-1 and Gα to Ric-8A are mutually exclusive. Importantly, NCS-1 induces a structural rearrangement in Ric-8A that traps the protein in a conformational state that is inaccessible to casein kinase II-mediated phosphorylation, demonstrating one aspect of its negative regulation of Ric-8A-mediated G-protein signaling. Functional experiments indicate a loss of Ric-8A guanine nucleotide exchange factor (GEF) activity toward Gα when complexed with NCS-1, and restoration of nucleotide exchange activity upon increasing Ca2+ concentration. Finally, the high-resolution crystallographic data reported here define the NCS-1/Ric-8A interface and will allow the development of therapeutic synapse function regulators with improved activity and selectivity.

The inhibition of NCS-1 binding to Ric8a rescues fragile X syndrome mice model phenotypes.

Fragile X syndrome (FXS) is caused by the loss of function of Fragile X mental retardation protein (FMRP). FXS is one of the leading monogenic causes of intellectual disability (ID) and autism. Although it is caused by the failure of a single gene, FMRP that functions as an RNA binding protein affects a large number of genes secondarily. All these genes represent hundreds of potential targets and different mechanisms that account for multiple pathological features, thereby hampering the search for effective treatments. In this scenario, it seems desirable to reorient therapies toward more general approaches. Neuronal calcium sensor 1 (NCS-1), through its interaction with the guanine-exchange factor Ric8a, regulates the number of synapses and the probability of the release of a neurotransmitter, the two neuronal features that are altered in FXS and other neurodevelopmental disorders. Inhibitors of the NCS-1/Ric8a complex have been shown to be effective in restoring abnormally high synapse numbers as well as improving associative learning in FMRP mutant flies. Here, we demonstrate that phenothiazine FD44, an NCS-1/Ric8a inhibitor, has strong inhibition ability in situ and sufficient bioavailability in the mouse brain. More importantly, administration of FD44 to two different FXS mouse models restores well-known FXS phenotypes, such as hyperactivity, associative learning, aggressive behavior, stereotype, or impaired social approach. It has been suggested that dopamine (DA) may play a relevant role in the behavior and in neurodevelopmental disorders in general. We have measured DA and its metabolites in different brain regions, finding a higher metabolic rate in the limbic area, which is also restored with FD44 treatment. Therefore, in addition to confirming that the NCS-1/Ric8a complex is an excellent therapeutic target, we demonstrate the rescue effect of its inhibitor on the behavior of cognitive and autistic FXS mice and show DA metabolism as a FXS biochemical disease marker.

Tools and Biomarkers for the Study of Retinal Ganglion Cell Degeneration.

The retina is part of the central nervous system, its analysis may provide an idea of the health and functionality, not only of the retina, but also of the entire central nervous system, as has been shown in Alzheimer's or Parkinson's diseases. Within the retina, the ganglion cells (RGC) are the neurons in charge of processing and sending light information to higher brain centers. Diverse insults and pathological states cause degeneration of RGC, leading to irreversible blindness or impaired vision. RGCs are the measurable endpoints in current research into experimental therapies and diagnosis in multiple ocular pathologies, like glaucoma. RGC subtype classifications are based on morphological, functional, genetical, and immunohistochemical aspects. Although great efforts are being made, there is still no classification accepted by consensus. Moreover, it has been observed that each RGC subtype has a different susceptibility to injury. Characterizing these subtypes together with cell death pathway identification will help to understand the degenerative process in the different injury and pathological models, and therefore prevent it. Here we review the known RGC subtypes, as well as the diagnostic techniques, probes, and biomarkers for programmed and unprogrammed cell death in RGC.

Insights into real-time chemical processes in a calcium sensor protein-directed dynamic library.

Dynamic combinatorial chemistry (DCC) has proven its potential in drug discovery speeding the identification of modulators of biological targets. However, the exchange chemistries typically take place under specific reaction conditions, with limited tools capable of operating under physiological parameters. Here we report a catalyzed protein-directed DCC working at low temperatures that allows the calcium sensor NCS-1 to find the best ligands in situ. Ultrafast NMR identifies the reaction intermediates of the acylhydrazone exchange, tracing the molecular assemblies and getting a real-time insight into the essence of DCC processes at physiological pH. Additionally, NMR, X-ray crystallography and computational methods are employed to elucidate structural and mechanistic aspects of the molecular recognition event. The DCC approach leads us to the identification of a compound stabilizing the NCS-1/Ric8a complex and whose therapeutic potential is proven in a Drosophila model of disease with synaptic alterations.

Molecular mechanisms that change synapse number.

Synapses are the functional units of the nervous system, and their number and protein composition undergo changes over a wide time scale. These synaptic changes manifest into differential behavioural outputs and, in turn, changes in the external conditions to the individual may elicit changes in synapses. We review here publications appeared during the last 10 years in which advances on molecular and cellular mechanisms for synapse changes have been reported. We focus on synaptic changes occurring in the time range of minutes to hours, mainly.

Deciphering the Inhibition of the Neuronal Calcium Sensor 1 and the Guanine Exchange Factor Ric8a with a Small Phenothiazine Molecule for the Rational Generation of Therapeutic Synapse Function Regulators.

Protein-protein interactions (PPIs) are known to play an essential role between the neuronal calcium sensor 1 (NCS-1) and the guanine exchange factor Ric8a to regulate synapse function, emerging as a druggable interface for synaptopathies such as the fragile X syndrome (FXS). Recently, the phenothiazine FD44 has been identified as an inhibitor of this PPI, decreasing the abnormally high synapse number and enhancing associative learning in a FXS animal model. Here, we have integrated advanced experimental and computational studies to obtain important structural insights into Drosophila NCS-1/FD44 recognition to understand the basis of its affinity and specificity and generate improved PPI regulators. This has allowed the identification of a new small drug-like molecule, IGS-1.76, which efficiently inhibits the human NCS-1/Ric8a complex with improved binding potency. The crystal structure of the Drosophila NCS-1/IGS-1.76 complex demonstrates that the new inhibitor, although chemically different from FD44, shares the same mechanism of action and constitutes a new hit candidate for FXS.

Interference of the complex between NCS-1 and Ric8a with phenothiazines regulates synaptic function and is an approach for fragile X syndrome.

The protein complex formed by the Ca2+ sensor neuronal calcium sensor 1 (NCS-1) and the guanine exchange factor protein Ric8a coregulates synapse number and probability of neurotransmitter release, emerging as a potential therapeutic target for diseases affecting synapses, such as fragile X syndrome (FXS), the most common heritable autism disorder. Using crystallographic data and the virtual screening of a chemical library, we identified a set of heterocyclic small molecules as potential inhibitors of the NCS-1/Ric8a interaction. The aminophenothiazine FD44 interferes with NCS-1/Ric8a binding, and it restores normal synapse number and associative learning in a Drosophila FXS model. The synaptic effects elicited by FD44 feeding are consistent with the genetic manipulation of NCS-1. The crystal structure of NCS-1 bound to FD44 and the structure-function studies performed with structurally close analogs explain the FD44 specificity and the mechanism of inhibition, in which the small molecule stabilizes a mobile C-terminal helix inside a hydrophobic crevice of NCS-1 to impede Ric8a interaction. Our study shows the drugability of the NCS-1/Ric8a interface and uncovers a suitable region in NCS-1 for development of additional drugs of potential use on FXS and related synaptic disorders.

A new overgrowth syndrome is due to mutations in RNF125.

Overgrowth syndromes (OGS) are a group of disorders in which all parameters of growth and physical development are above the mean for age and sex. We evaluated a series of 270 families from the Spanish Overgrowth Syndrome Registry with no known OGS. We identified one de novo deletion and three missense mutations in RNF125 in six patients from four families with overgrowth, macrocephaly, intellectual disability, mild hydrocephaly, hypoglycemia, and inflammatory diseases resembling Sjögren syndrome. RNF125 encodes an E3 ubiquitin ligase and is a novel gene of OGS. Our studies of the RNF125 pathway point to upregulation of RIG-I-IPS1-MDA5 and/or disruption of the PI3K-AKT and interferon signaling pathways as the putative final effectors.

The guanine-exchange factor Ric8a binds to the Ca²âº sensor NCS-1 to regulate synapse number and neurotransmitter release.

The conserved Ca(2+)-binding protein Frequenin (homolog of the mammalian NCS-1, neural calcium sensor) is involved in pathologies that result from abnormal synapse number and probability of neurotransmitter release per synapse. Both synaptic features are likely to be co-regulated but the intervening mechanisms remain poorly understood. We show here that Drosophila Ric8a (a homolog of mammalian synembryn, which is also known as Ric8a), a receptor-independent activator of G protein complexes, binds to Frq2 but not to the virtually identical homolog Frq1. Based on crystallographic data on Frq2 and site-directed mutagenesis on Frq1, the differential amino acids R94 and T138 account for this specificity. Human NCS-1 and Ric8a reproduce the binding and maintain the structural requirements at these key positions. Drosophila Ric8a and Gαs regulate synapse number and neurotransmitter release, and both are functionally linked to Frq2. Frq2 negatively regulates Ric8a to control synapse number. However, the regulation of neurotransmitter release by Ric8a is independent of Frq2 binding. Thus, the antagonistic regulation of these two synaptic properties shares a common pathway, Frq2-Ric8a-Gαs, which diverges downstream. These mechanisms expose the Frq2-Ric8a interacting surface as a potential pharmacological target for NCS-1-related diseases and provide key data towards the corresponding drug design.

Cerebrospinal fluid level of YKL-40 protein in preclinical and prodromal Alzheimer's disease.

BACKGROUND: An increase in YKL-40 levels seems to correlate with disease severity and poor prognosis in many diseases, including several neurodegenerative diseases such as multiple sclerosis, amyotrophic lateral sclerosis, and Alzheimer's disease (AD). Specifically, YKL-40 protein is increased in mild AD with respect to controls, both in cerebrospinal fluid (CSF) and plasma. OBJECTIVE: We hypothesize that subjects in the preclinical (Pre-AD) and prodromal (Prod-AD) stage of AD could already present an increase in CSF YKL-40 levels with respect to healthy controls and idiopathic REM sleep behavior disorder (iRBD) subjects, included as a control group of a distinct neurological disease. METHODS: We measured CSF YKL-40 levels using a commercial ELISA kit in a cohort of 95 subjects, consisting of controls (n = 43), Pre-AD (n = 18), Prod-AD (n = 22), and iRBD (n = 12) subjects. We explored for possible correlations of YKL-40 levels with demographic characteristics, a wide battery of neuropsychological tests, and the AD CSF biomarkers: amyloid-ß42 (Aß42), total-tau protein (t-tau), and phosphorylated-tau protein (p-tau). RESULTS: We detected statistically significant differences between Prod-AD patients and controls. YKL-40 levels showed a significant correlation with t-tau and p-tau levels in the predementia AD continuum and the Pre-AD group. We also observed significant correlations with the MMSE, FCSRT, and M@T tests within the AD continuum, but not in iRBD subjects. CONCLUSION: Our data suggest that CSF YKL-40 levels, although not useful as a diagnostic marker for Prod-AD, may be a valuable marker to detect early physiopathological changes potentially linked with the neurodegenerative process.

Frq2 from Drosophila melanogaster: cloning, expression, purification, crystallization and preliminary X-ray analysis.

Drosophila melanogaster contains two calcium-binding proteins, Frq1 and Frq2, in the nervous system that control the number of synapses and the probability of release. To understand the differential function of the two proteins, whose sequence is only 5% dissimilar, the crystal structures of Frq1 and Frq2 are needed. Here, the cloning, expression, purification, crystallization and preliminary crystallographic analysis of Frq2 are presented. The full-length protein was purified using a two-step chromatographic procedure. Two different diffracting crystal forms were obtained using a progressive streak-seeding method and detergents.

Isoform-specific regulation of a steroid hormone nuclear receptor by an E3 ubiquitin ligase in Drosophila melanogaster.

The steroid hormone 20-hydroxyecdysone (20E) regulates gene transcription through the heterodimeric nuclear receptor composed of ecdysone receptor (EcR) and Ultraspiracle (USP). The EcR gene encodes three protein isoforms--A, B1, and B2--with variant N-terminal domains that mediate tissue and developmental stage-specific responses to 20E. Ariadne-1a is a conserved member of the RING finger family of ubiquitin ligases first identified in Drosophila melanogaster. Loss-of-function mutations at key cysteines in either of the two RING finger motifs, as well as general overexpression of this enzyme, cause lethality in pupae, which suggests a requirement in metamorphosis. Here, we show that Ariadne-1a binds specifically the isoform A of EcR and ubiquitylates it. Co-immunoprecipitation experiments indicate that the full sequence of EcRA is required for this binding. Protein levels of EcRA and USP change in opposite directions when those of ARI-1a are genetically altered. This is an isoform-specific, E3-dependent regulatory mechanism for a steroid nuclear receptor. Further, qRT-PCR experiments show that the ARI-1a levels lead to the transcriptional regulation of Eip78C, Eip74EF, Eip75B, and Br-C, as well as that of EcR and usp genes. Thus, the activity of this enzyme results in the regulation of dimerizing receptors at the protein and gene transcription levels. This fine-tuned orchestration by a conserved ubiquitin ligase is required during insect metamorphosis and, likely, in other steroid hormone-controlled processes across species.

Troponin I and Tropomyosin regulate chromosomal stability and cell polarity.

The Troponin-Tropomyosin (Tn-Tm) complex regulates muscle contraction through a series of Ca(2+)-dependent conformational changes that control actin-myosin interactions. Members of this complex in Drosophila include the actin-binding protein Troponin I (TnI), and two Tropomyosins (Tm1 and Tm2), which are thought to form heterodimers. We show here that pre-cellular embryos of TnI, Tm1 and Tm2 mutants exhibit abnormal nuclear divisions with frequent loss of chromosome fragments. During cellularization, apico-basal polarity is also disrupted as revealed by the defective location of Discs large (Dlg) and its ligand Rapsynoid (Raps; also known as Partner of Inscuteable, Pins). In agreement with these phenotypes in early development, on the basis of RT-PCR assays of unfertilized eggs and germ line mosaics of TnI mutants, we also show that TnI is part of the maternal deposit during oogenesis. In cultures of the S2 cell line, native TnI is immunodetected within the nucleus and immunoprecipitated from nuclear extracts. SUMOylation at an identified site is required for the nuclear translocation. These data illustrate, for the first time, a role for TnI in the nucleus and/or the cytoskeleton of non-muscle cells. We propose that the Tn-Tm complex plays a novel function as regulator of motor systems required to maintain nuclear integrity and apico-basal polarity during early Drosophila embryogenesis.

Autophagy inhibition compromises degradation of ubiquitin-proteasome pathway substrates.

The two main routes that cells use for degrading intracellular proteins are the ubiquitin-proteasome and autophagy-lysosome pathways, which have been thought to have largely distinct clients. Here, we show that autophagy inhibition increases levels of proteasome substrates. This is largely due to p62 (also called A170/SQSTM1) accumulation after autophagy inhibition. Excess p62 inhibits the clearance of ubiquitinated proteins destined for proteasomal degradation by delaying their delivery to the proteasome's proteases. Our data show that autophagy inhibition, which was previously believed to only affect long-lived proteins, will also compromise the ubiquitin-proteasome system. This will lead to increased levels of short-lived regulatory proteins, like p53, as well as the accumulation of aggregation-prone proteins, with predicted deleterious consequences.

Evolution of the insulin receptor family and receptor isoform expression in vertebrates.

The molecular phylogeny of the vertebrate insulin receptor (IR) family was reconstructed under maximum likelihood (ML) to establish homologous relationships among its members. A sister group relationship between the orphan insulin-related receptor (IRR) and the insulin-like growth factor 1 receptor (IGF1R) to the exclusion of the IR obtained maximal bootstrap support. Although both IR and IGF1R were identified in all vertebrates, IRR could not be found in any teleost fish. The ancestral character states at each position of the receptor molecule were inferred for IR, IRR + IGF1R, and all 3 paralogous groups based on the recovered phylogeny using ML in order to determine those residues that could be important for the specific function of IR. For 18 residues, ancestral character state of IR was significantly distinct (probability >0.95) with respect to the corresponding inferred ancestral character states both of IRR + IGF1R and of all 3 vertebrate paralogs. Most of these IR distinct (shared derived) residues were located on the extracellular portion of the receptor (because this portion is larger and the rate of generation of IR shared derived sites is uniform along the receptor), suggesting that functional diversification during the evolutionary history of the family was largely generated modifying ligand affinity rather than signal transduction at the tyrosine kinase domain. In addition, 2 residues at positions 436 and 1095 of the human IR sequence were identified as radical cluster-specific sites in IRR + IGF1R. Both Ir and Irr have an extra exon (namely exon 11) with respect to Igf1r. We used the molecular phylogeny to infer the evolution of this additional exon. The Irr exon 11 can be traced back to amphibians, whereas we show that presence and alternative splicing of Ir exon 11 seems to be restricted exclusively to mammals. The highly divergent sequence of both exons and the reconstructed phylogeny of the vertebrate IR family strongly indicate that both exons were acquired independently by each paralog.

The regulated expression of chimeric tyrosine hydroxylase-insulin transcripts during early development.

Biological complexity does not appear to be simply correlated with gene number but rather other mechanisms contribute to the morphological and functional diversity across phyla. Such mechanisms regulate different transcriptional, translational and post-translational processes and include the recently identified transcription induced chimerism (TIC). We have found two novel chimeric transcripts in the chick and quail that result from the fusion of tyrosine hydroxylase (TH) and insulin into a single mature transcript. The th and insulin genes are located in tandem and they are generally transcribed independently. However, it appears that two chimeric transcripts containing exons from both the genes can also be produced in a regulated manner. The TH-INS1 and TH-INS2 chimeras differ in their insulin gene content, and they encode two novel isoforms of the TH protein with markedly reduced functionality when compared with the canonical TH. In addition, the TH-INS1 chimeric mRNA generates a small amount of insulin. We propose that TIC is an additional mechanism that can be employed to further regulate TH and insulin expression according to the specific needs of developing vertebrates.

¿Qué sabemos de la función de la proinsulina en la vida embrionaria? / What do we know about the function of proinsulin in the embryo?

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Developmental regulation of a proinsulin messenger RNA generated by intron retention.

Proinsulin gene expression regulation and function during early embryonic development differ remarkably from those found in postnatal organisms. The embryonic proinsulin protein content decreased from gastrulation to neurulation in contrast with the overall proinsulin messenger RNA increase. This is due to increasing levels of a proinsulin mRNA variant generated by intron 1 retention in the 5' untranslated region. Inclusion of intron 1 inhibited proinsulin translation almost completely without affecting nuclear export or cytoplasmic decay. The novel proinsulin mRNA isoform expression was developmentally regulated and tissue specific. The proportion of intron retention increased from gastrulation to organogenesis, was highest in the heart tube and presomitic region, and could not be detected in the pancreas. Notably, proinsulin addition induced cardiac marker gene expression in the early embryonic stages when the translationally active transcript was expressed. We propose that regulated unproductive splicing and translation is a mechanism that regulates proinsulin expression in accordance with specific requirements in developing vertebrates.

Upstream AUGs in embryonic proinsulin mRNA control its low translation level.

Proinsulin is expressed prior to development of the pancreas and promotes cell survival. Here we study the mechanism affecting the translation efficiency of a specific embryonic proinsulin mRNA. This transcript shares the coding region with the pancreatic form, but presents a 32 nt extended leader region. Translation of proinsulin is markedly reduced by the presence of two upstream AUGs within the 5' extension of the embryonic mRNA. This attenuation is lost when the two upstream AUGs are mutated to AAG, leading to translational efficiency similar to that of the pancreatic mRNA. The upstream AUGs are recognized as initiator codons, because expression of upstream ORF is detectable from the embryonic transcript, but not from the mutated or the pancreatic mRNAs. Strict regulation of proinsulin biosynthesis appears to be necessary, since exogenous proinsulin added to embryos in ovo decreased apoptosis and generated abnormal developmental traits. A novel mechanism for low level proinsulin expression thus relies on upstream AUGs within a specific form of embryonic proinsulin mRNA, emphasizing its importance as a tightly regulated developmental signal.