RIPK4 regulates cell-cell adhesion in epidermal development and homeostasis.

Epidermal development and maintenance are finely regulated events requiring a strict balance between proliferation and differentiation. Alterations in these processes give rise to human disorders such as cancer or syndromes with skin and annexes defects, known as ectodermal dysplasias (EDs). Here, we studied the functional effects of two novel receptor-interacting protein kinase 4 (RIPK4) missense mutations identified in siblings with an autosomal recessive ED with cutaneous syndactyly, palmoplantar hyperkeratosis and orofacial synechiae. Clinical overlap with distinct EDs caused by mutations in transcription factors (i.e. p63 and interferon regulatory factor 6, IRF6) or nectin adhesion molecules was noticed. Impaired activity of the RIPK4 kinase resulted both in altered epithelial differentiation and defective cell adhesion. We showed that mutant RIPK4 resulted in loss of PVRL4/nectin-4 expression in patient epidermis and primary keratinocytes, and demonstrated that PVRL4 is transcriptionally regulated by IRF6, a RIPK4 phosphorylation target. In addition, defective RIPK4 altered desmosome morphology through modulation of plakophilin-1 and desmoplakin. In conclusion, this work implicates RIPK4 kinase function in the p63-IRF6 regulatory loop that controls the proliferation/differentiation switch and cell adhesion, with implications in ectodermal development and cancer.

Advanced Raman Spectroscopy Detection of Oxidative Damage in Nucleic Acid Bases: Probing Chemical Changes and Intermolecular Interactions in Guanosine at Ultralow Concentration.

DNA/RNA synthesis precursors are especially vulnerable to damage induced by reactive oxygen species occurring following oxidative stress. Guanosine triphosphates are the prevalent oxidized nucleotides, which can be misincorporated during replication, leading to mutations and cell death. Here, we present a novel method based on micro-Raman spectroscopy, combined with ab initio calculations, for the identification, detection, and quantification of oxidized nucleotides at low concentration. We also show that the Raman signature in the terahertz spectral range (<100 cm-1) contains information on the intermolecular assembly of guanine in tetrads, which allows us to further boost the oxidative damage detection limit. Eventually, we provide evidence that similar analyses can be carried out on samples in very small volumes at very low concentrations by exploiting the high sensitivity of surface-enhanced Raman scattering combined with properly designed superhydrophobic substrates. These results pave the way for employing such advanced spectroscopic methods for quantitatively sensing the oxidative damage of nucleotides in the cell.

Developmental expression of dysbindin in Muller cells of rat retina.

Dysbindin, the product of the DTNBP1 gene, was identified by yeast two hybrid assay as a binding partner of dystrobrevin, a cytosolic component of the dystrophin protein complex. Although its functional role has not yet been completely elucidated, the finding that dysbindin assembles into the biogenesis of lysosome related organelles complex 1 (BLOC-1) suggests that it participates in intracellular trafficking and biogenesis of organelles and vesicles. Dysbindin is ubiquitous and in brain is expressed primarily in neurons. Variations at the dysbindin gene have been associated with increased risk for schizophrenia. As anomalies in retinal function have been reported in patients suffering from neuropsychiatric disorders, we investigated the expression of dysbindin in the retina. Our results show that differentially regulated dysbindin isoforms are expressed in rat retina during postnatal maturation. Interestingly, we found that dysbindin is mainly localized in Müller cells. The identification of dysbindin in glial cells may open new perspectives for a better understanding of the functional involvement of this protein in visual alterations associated to neuropsychiatric disorders.

The CaMKII/MLC1 Axis Confers Ca2+-Dependence to Volume-Regulated Anion Channels (VRAC) in Astrocytes.

Astrocytes, the main glial cells of the central nervous system, play a key role in brain volume control due to their intimate contacts with cerebral blood vessels and the expression of a distinctive equipment of proteins involved in solute/water transport. Among these is MLC1, a protein highly expressed in perivascular astrocytes and whose mutations cause megalencephalic leukoencephalopathy with subcortical cysts (MLC), an incurable leukodystrophy characterized by macrocephaly, chronic brain edema, cysts, myelin vacuolation, and astrocyte swelling. Although, in astrocytes, MLC1 mutations are known to affect the swelling-activated chloride currents (ICl,swell) mediated by the volume-regulated anion channel (VRAC), and the regulatory volume decrease, MLC1's proper function is still unknown. By combining molecular, biochemical, proteomic, electrophysiological, and imaging techniques, we here show that MLC1 is a Ca2+/Calmodulin-dependent protein kinase II (CaMKII) target protein, whose phosphorylation, occurring in response to intracellular Ca2+ release, potentiates VRAC-mediated ICl,swell. Overall, these findings reveal that MLC1 is a Ca2+-regulated protein, linking volume regulation to Ca2+ signaling in astrocytes. This knowledge provides new insight into the MLC1 protein function and into the mechanisms controlling ion/water exchanges in the brain, which may help identify possible molecular targets for the treatment of MLC and other pathological conditions caused by astrocyte swelling and brain edema.

The interaction with HMG20a/b proteins suggests a potential role for beta-dystrobrevin in neuronal differentiation.

alpha and beta dystrobrevins are cytoplasmic components of the dystrophin-associated protein complex that are thought to play a role as scaffold proteins in signal transduction and intracellular transport. In the search of new insights into the functions of beta-dystrobrevin, the isoform restricted to non-muscle tissues, we performed a two-hybrid screen of a mouse cDNA library to look for interacting proteins. Among the positive clones, one encodes iBRAF/HMG20a, a high mobility group (HMG)-domain protein that activates REST (RE-1 silencing transcription factor)-responsive genes, playing a key role in the initiation of neuronal differentiation. We characterized the beta-dystrobrevin-iBRAF interaction by in vitro and in vivo association assays, localized the binding region of one protein to the other, and assessed the kinetics of the interaction as one of high affinity. We also found that beta-dystrobrevin directly binds to BRAF35/HMG20b, a close homologue of iBRAF and a member of a co-repressor complex required for the repression of neural specific genes in neuronal progenitors. In vitro assays indicated that beta-dystrobrevin binds to RE-1 and represses the promoter activity of synapsin I, a REST-responsive gene that is a marker for neuronal differentiation. Altogether, our data demonstrate a direct interaction of beta-dystrobrevin with the HMG20 proteins iBRAF and BRAF35 and suggest that beta-dystrobrevin may be involved in regulating chromatin dynamics, possibly playing a role in neuronal differentiation.

Exploiting SERS sensitivity to monitor DNA aggregation properties.

In the last decades, DNA has been considered far more than the system carrying the essential genetic instructions. Indeed, because of the remarkable properties of the base-pairing specificity and thermoreversibility of the interactions, DNA plays a central role in the design of innovative architectures at the nanoscale. Here, combining complementary DNA strands with a custom-made solution of silver nanoparticles, we realize plasmonic aggregates to exploit the sensitivity of Surface Enhanced Raman Spectroscopy (SERS) for the identification/detection of the distinctive features of DNA hybridization, both in solution and on dried samples. Moreover, SERS allows monitoring the DNA aggregation process by following the temperature variation of a specific spectroscopic marker associated with the Watson-Crick hydrogen bond formation. This temperature-dependent behavior enables us to precisely reconstruct the melting profile of the selected DNA sequences by spectroscopic measurements only.

Diverse driving forces underlie the invariant occurrence of the T42A, E139D, I282V and T468M SHP2 amino acid substitutions causing Noonan and LEOPARD syndromes.

Missense PTPN11 mutations cause Noonan and LEOPARD syndromes (NS and LS), two developmental disorders with pleiomorphic phenotypes. PTPN11 encodes SHP2, an SH2 domain-containing protein tyrosine phosphatase functioning as a signal transducer. Generally, different substitutions of a particular amino acid residue are observed in these diseases, indicating that the crucial factor is the residue being replaced. For a few codons, only one substitution is observed, suggesting the possibility of specific roles for the residue introduced. We analyzed the biochemical behavior and ligand-binding properties of all possible substitutions arising from single-base changes affecting codons 42, 139, 279, 282 and 468 to investigate the mechanisms underlying the invariant occurrence of the T42A, E139D and I282V substitutions in NS and the Y279C and T468M changes in LS. Our data demonstrate that the isoleucine-to-valine change at codon 282 is the only substitution at that position perturbing the stability of SHP2's closed conformation without impairing catalysis, while the threonine-to-alanine change at codon 42, but not other substitutions of that residue, promotes increased phosphopeptide-binding affinity. The recognition specificity of the C-SH2 domain bearing the E139D substitution differed substantially from its wild-type counterpart acquiring binding properties similar to those observed for the N-SH2 domain, revealing a novel mechanism of SHP2's functional dysregulation. Finally, while functional selection does not seem to occur for the substitutions at codons 279 and 468, we point to deamination of the methylated cytosine at nucleotide 1403 as the driving factor leading to the high prevalence of the T468M change in LS.

Peroxynitrite induces tyrosine residue modifications in synaptophysin C-terminal domain, affecting its interaction with src.

Peroxynitrite is a potent oxidant that contributes to tissue damage in neurodegenerative disorders. We have previously reported that treatment of rat brain synaptosomes with peroxynitrite induced post-translational modifications in pre- and post-synaptic proteins and stimulated soluble N-ethylmaleimide sensitive fusion proteins attachment receptor complex formation and endogenous glutamate release. In this study we show that, following peroxynitrite treatment, the synaptic vesicle protein synaptophysin (SYP) can be both phosphorylated and nitrated in a dose-dependent manner. We found that tyrosine-phosphorylated, but not tyrosine-nitrated, SYP bound to the src tyrosine kinase and enhanced its catalytic activity. These effects were mediated by direct and specific binding of the SYP cytoplasmic C-terminal tail with the src homology 2 domain. Using mass spectrometry analysis, we mapped the SYP C-terminal tail tyrosine residues modified by peroxynitrite and found one nitration site at Tyr250 and two phosphorylation sites at Tyr263 and Tyr273. We suggest that peroxynitrite-mediated modifications of SYP may be relevant in modulating src signalling of synaptic terminal in pathophysiological conditions.

Association of dystrobrevin and regulatory subunit of protein kinase A: a new role for dystrobrevin as a scaffold for signaling proteins.

The dystrophin-related and -associated protein dystrobrevin is a component of the dystrophin-associated protein complex, which directly links the cytoskeleton to the extracellular matrix. It is now thought that this complex also serves as a dynamic scaffold for signaling proteins, and dystrobrevin may play a role in this context. Since dystrobrevin involvement in signaling pathways seems to be dependent on its interaction with other proteins, we sought new insights and performed a two-hybrid screen of a mouse brain cDNA library using beta-dystrobrevin, the isoform expressed in non-muscle tissues, as bait. Among the positive clones characterized after the screen, one encodes the regulatory subunit RIalpha of the cAMP-dependent protein kinase A (PKA). We confirmed the interaction by in vitro and in vivo association assays, and mapped the binding site of beta-dystrobrevin on RIalpha to the amino-terminal region encompassing the dimerization/docking domain of PKA regulatory subunit. We also found that the domain of interaction for RIalpha is contained in the amino-terminal region of beta-dystrobrevin. We obtained evidence that beta-dystrobrevin also interacts directly with RIIbeta, and that not only beta-dystrobrevin but also alpha-dystrobrevin interacts with PKA regulatory subunits. We show that both alpha and beta-dystrobrevin are specific phosphorylation substrates for PKA and that protein phosphatase 2A (PP2A) is associated with dystrobrevins. Our results suggest a new role for dystrobrevin as a scaffold protein that may play a role in different cellular processes involving PKA signaling.

Molecular basis of dystrobrevin interaction with kinesin heavy chain: structural determinants of their binding.

Dystrobrevins are a family of widely expressed dystrophin-associated proteins that comprises alpha and beta isoforms and displays significant sequence homology with several protein-binding domains of the dystrophin C-terminal region. The complex distribution of the multiple dystrobrevin isoforms suggests that the variability of their composition may be important in mediating their function. We have recently identified kinesin as a novel dystrobrevin-interacting protein and localized the dystrobrevin-binding site on the cargo-binding domain of neuronal kinesin heavy chain (Kif5A). In the present study, we assessed the kinetics of the dystrobrevin-Kif5A interaction by quantitative pull-down assay and surface plasmon resonance (SPR) analysis and found that beta-dystrobrevin binds to kinesin with high affinity (K(D) approximately 40 nM). Comparison of the sensorgrams obtained with alpha and beta-dystrobrevin at the same concentration of analyte showed a lower affinity of alpha compared to that of beta-dystrobrevin, despite their functional domain homology and about 70% sequence identity. Analysis of the contribution of single dystrobrevin domains to the interaction revealed that the deletion of either the ZZ domain or the coiled-coil region decreased the kinetics of the interaction, suggesting that the tertiary structure of dystrobrevin may play a role in regulating the interaction of dystrobrevin with kinesin. In order to understand if structural changes induced by post-translational modifications could affect dystrobrevin affinity for kinesin, we phosphorylated beta-dystrobrevin in vitro and found that it showed reduced binding capacity towards kinesin. The interaction between the adaptor/scaffolding protein dystrobrevin and the motor protein kinesin may play a role in the transport and targeting of components of the dystrophin-associated protein complex to specific sites in the cell, with the differences in the binding properties of dystrobrevin isoforms reflecting their functional diversity within the same cell type. Phosphorylation events could have a regulatory role in this context.

Identification of ß-Dystrobrevin as a Direct Target of miR-143: Involvement in Early Stages of Neural Differentiation.

Duchenne Muscular Dystrophy, a genetic disorder that results in a gradual breakdown of muscle, is associated to mild to severe cognitive impairment in about one-third of dystrophic patients. The brain dysfunction is independent of the muscular pathology, occurs early, and is most likely due to defects in the assembly of the Dystrophin-associated Protein Complex (DPC) during embryogenesis. We have recently described the interaction of the DPC component ß-dystrobrevin with members of complexes that regulate chromatin dynamics, and suggested that ß-dystrobrevin may play a role in the initiation of neuronal differentiation. Since oxygen concentrations and miRNAs appear as well to be involved in the cellular processes related to neuronal development, we have studied how these factors act on ß-dystrobrevin and investigated the possibility of their functional interplay using the NTera-2 cell line, a well-established model for studying neurogenesis. We followed the pattern of expression and regulation of ß-dystrobrevin during the early stages of neuronal differentiation induced by exposure to retinoic acid (RA) under hypoxia as compared with normoxia, and found that ß-dystrobrevin expression is regulated during RA-induced differentiation of NTera-2 cells. We also found that ß-dystrobrevin pattern is delayed under hypoxic conditions, together with a delay in the differentiation and an increase in the proliferation rate of cells. We identified miRNA-143 as a direct regulator of ß-dystrobrevin expression, demonstrated that ß-dystrobrevin is expressed in the nucleus and showed that, in line with our previous in vitro results, ß-dystrobrevin is a repressor of synapsin I in live cells. Altogether the newly identified regulatory pathway miR-143/ß-dystrobrevin/synapsin I provides novel insights into the functions of ß-dystrobrevin and opens up new perspectives for elucidating the molecular mechanisms underlying the neuronal involvement in muscular dystrophy.

Biomolecular interactions by Surface Plasmon Resonance technology.

The Surface Plasmon Resonance (SPR) technique makes it possible to measure biomolecular interactions in real-time with a high degree of sensitivity and without the need of label. The information obtained is both qualitative and quantitative and it is possible to obtain the kinetic parameters of the interaction. This new technology has been used to study a diverse set of interaction partners of biological interest, such as protein-protein, protein-lipids, protein- nucleic acids or protein and low molecular weight molecules such as drugs, substrates and cofactors. In addition to basic biomedical research, the SPR biosensor has recently been used in food analysis, proteomics, immunogenicity and drug discovery.

Expression of dystrophin-associated proteins during neuronal differentiation of P19 embryonal carcinoma cells.

The dystrophin gene that is defective in Duchenne muscular dystrophy shows a complex transcriptional control based on several promoters driving independent cell-type-specific expression of different isoforms. Dystrophin isoforms together with dystroglycan, a transmembrane protein which in turn binds to extracellular matrix, are the core of a complex of proteins, the dystrophin-associated protein (DAP) complex, which also comprises cytoplasmic elements like dystrobrevin. Whereas the molecular organization of DAP complex in muscle is well documented, the composition of a similar complex in the nervous system remains largely unknown. We followed by competitive PCR the expression of DAP complex components during retinoic acid (RA)-induced neuronal differentiation of P19 cells. Transcripts for the full-length dystrophin, Dp427, and the short isoform, Dp71, as well as for alpha-dystrobrevin 2 increased in parallel with days in culture after RA stimulation, while dystroglycan, alpha-dystrobrevin 1 and 3, and beta-dystrobrevin were constitutively expressed. The upregulation of some of the components of the dystrophin complex during neuronal maturation suggests functional flexibility of the complex in the nervous system, where specific associations between different isoforms of DAP complex components could possibly organize distinct DAP complex-like complexes.

Phosphorylation on threonine 11 of ß-dystrobrevin alters its interaction with kinesin heavy chain.

Dystrobrevin family members (α and ß) are cytoplasmic components of the dystrophin-associated glycoprotein complex, a multimeric protein complex first isolated from skeletal muscle, which links the extracellular matrix to the actin cytoskeleton. Dystrobrevin shares high homology with the cysteine-rich and C-terminal domains of dystrophin and a common domain organization. The ß-dystrobrevin isoform is restricted to nonmuscle tissues, serves as a scaffold for signaling complexes, and may participate in intracellular transport through its interaction with kinesin heavy chain. We have previously characterized the molecular determinants affecting the ß-dystrobrevin-kinesin heavy chain interaction, among which is cAMP-dependent protein kinase [protein kinase A (PKA)] phosphorylation of ß-dystrobrevin. In this study, we have identified ß-dystrobrevin residues phosphorylated in vitro by PKA with pull-down assays, surface plasmon resonance measurements, and MS analysis. Among the identified phosphorylated residues, we demonstrated, by site-directed mutagenesis, that Thr11 is the regulatory site for the ß-dystrobrevin-kinesin interaction. As dystrobrevin may function as a signaling scaffold for kinases/phosphatases, we also investigated whether ß-dystrobrevin is phosphorylated in vitro by kinases other than PKA. Thr11 was phosphorylated by protein kinase C, suggesting that this represents a key residue modified by the activation of different signaling pathways.

beta-dystrobrevin, a kinesin-binding receptor, interacts with the extracellular matrix components pancortins.

The dystrobrevins (alpha and beta) are components of the dystrophin-associated protein complex (DPC), which links the cytoskeleton to the extracellular matrix and serves as a scaffold for signaling proteins. The precise functions of the beta-dystrobrevin isoform, which is expressed in nonmuscle tissues, have not yet been determined. To gain further insights into the role of beta-dystrobrevin in brain, we performed a yeast two-hybrid screen and identified pancortin-2 as a novel beta-dystrobrevin-binding partner. Pancortins-1-4 are neuron-specific olfactomedin-related glycoproteins, highly expressed during brain development and widely distributed in the mature cerebral cortex of the mouse. Pancortins are important constituents of the extracellular matrix and are thought to play an essential role in neuronal differentiation. We characterized the interaction between pancortin-2 and beta-dystrobrevin by in vitro and in vivo association assays and mapped the binding site of pancortin-2 on beta-dystrobrevin to amino acids 202-236 of the beta-dystrobrevin molecule. We also found that the domain of interaction for beta-dystrobrevin is contained in the B part of pancortin-2, a central region that is common to all four pancortins. Our results indicate that beta-dystrobrevin could interact with all members of the pancortin family, implying that beta-dystrobrevin may be involved in brain development. We suggest that dystrobrevin, a motor protein receptor that binds kinesin heavy chain, might play a role in intracellular transport of pancortin to specific sites in the cell.