Project description:Axoplasmic proteomics from sciatic or centrally projecting branches of sciatic DRG identifies unique protein enrichment and signalling pathways, including prior and subsequent to a spinal regeneration-incompetent versus sciatic regeneration-competent axonal injury.
Project description:The dorsoventral gradient of BMP signaling plays an essential role in embryonic patterning, with high BMP signal activating ventral-lateral mesoderm markers directly, and low BMP signal inducing neural tissues. The Zinc finger SWIM domain-containing protein 4 (zswim4) is expressed in the dorsal blastopore lip at the onset of Xenopus gastrula and then enriched at the forming neuroectoderm at mid-gastrula stages. Overexpression of zswim4 in Xenopus embryos causes inhibition of the anterior axis and shortened, curved body, and knockdown or knockout of zswim4 disturbed embryonic body axis formation and head development. The expression of ventral-lateral mesoderm marker genes was reduced after zswim4 overexpression and increased in embryos with zswim4 knockdown. Neural marker genes were repressed in zswim4 morphant. Mechanistically Zswim4 attenuates BMP signal through reducing protein stability of Smad1 in both Xenopus embryos and HEK293T cells. Zswim4 interacts with Smad1 and promotes ubiquitination of Smad1 in HEK293T cells. To identify the interaction partner of Zswim4 in regulating Smad1 stability, we performed SILAC based IP in HEK293T cells, and the precipitates were analyzed by Mass Spectrometry.
Project description:Developmental and epileptic encephalopathies (DEEs) are a group of rare childhood disorders characterized by severe epilepsy and cognitive deficits. Numerous DEE genes have been discovered thanks to advances in genomic diagnosis, yet putative molecular links between these disorders are unknown. CDKL5 deficiency disorder (CDD, DEE2), one of the most common genetic epilepsies, is caused by loss-of-function mutations in the brain-enriched kinase CDKL5. To elucidate CDKL5 function, we looked for CDKL5 substrates using a SILAC-based phosphoproteomic screen. We identified the voltage-gated Ca2+ channel Cav2.3 (encoded by CACNA1E) as a novel physiological target of CDKL5 in mice and humans. Recombinant channel electrophysiology and interdisciplinary characterization of Cav2.3 phosphomutant mice revealed that loss of Cav2.3 phosphorylation leads to channel gain-of-function via slower inactivation and enhanced cholinergic stimulation, resulting in increased neuronal excitability. Our results thus show that CDD is partly a channelopathy. The properties of unphosphorylated Cav2.3 closely resemble those described for CACNA1E gain-of-function mutations causing DEE69, a disorder sharing clinical features with CDD. We show that these two single-gene diseases are mechanistically related and could be ameliorated with Cav2.3 inhibitors.
Project description:DNA/RNA helicase Upf1 seems to interact with axonal transcripts in response to nerve growth factor (NGF, A. Ludanyi, M. Gaspari and A. Riccio, unpublished data). In order to shed light on Upf1 mechanism of action and to identify potential associations with molecules having cleavage activity, we performed an AP-MS experiment comprising Upf1 immunoprecipitation, on-beads digestion, isotopic labelling (18O) and quantitative LC-MS/MS analysis.
Project description:BRCA2 maintains genome stability by facilitating DNA repair via homologous recombination and replication fork stability. Loss of BRCA2 is deleterious for survival of normal cells, but is paradoxically tolerated in cancer cells. Using quantitative mass-spectrometry, differences in protein expression were identified that might shed light on how breast cancer cells (HCC38) survive in the absence of BRCA2.
Project description:Intracellular signaling nodes can sample multiple inputs and induce different cellular outputs. High combinatorial between their numerous potential conformational intermediaries having distinct enzymatic activities and/or repertoire of partners makes it challenging to determine their dynamic coding molecularly. We have combined protein engineering with optogenetic to investigate coding functions of specific intermediaries of the kinase Src, a representative example of versatile signaling node. Optogenetic is used to generate local flux of specific Src intermediaries, as Src dimers, into adhesion sites. Modulations of this molecular flux are sufficient to code different Src signaling pathways and Src-dependent cellular outputs implicated in migration or invasion. This response selectivity is based on the ability for each flux of Src conformational intermediary to generate distinct Src signaling waves in protein-protein interaction networks revealed by time-resolved analysis of phosphotyrosine-proteome. Thus, the pleiotropy of a signaling node is molecularly coded by modulation of its local fluxes of specific conformational intermediaries.
Project description:Interferon-induced transmembrane protein 3 (IFITM3) is a restriction factor that limits viral pathogenesis and exerts poorly understood immunoregulatory functions. Here, using human and mouse models, we demonstrate that IFITM3 promotes MyD88-dependent, TLR-mediated IL-6 production following exposure to cytomegalovirus (CMV). IFITM3 also restricts IL-6 production in response to influenza and SARS-CoV-2. In dendritic cells, IFITM3 binds to the reticulon 4 isoform Nogo-B and promotes its proteasomal degradation. We reveal that Nogo-B mediates TLR-dependent pro-inflammatory cytokine production and promotes viral pathogenesis in vivo, and in the case of TLR2 responses, this process involves alteration of TLR2 cellular localization. Nogo-B deletion abrogates inflammatory cytokine responses and associated disease in virus-infected IFITM3-deficient mice. Thus, we uncover Nogo-B as a driver of viral pathogenesis and highlight an immunoregulatory pathway in which IFITM3 fine-tunes the responsiveness of myeloid cells to viral stimulation.
Project description:Epithelial to Mesenchymal Transition (EMT) renders epithelial cells to acquire migratory characteristics during development and cancer metastasis. While epigenetic and splicing changes have been implicated in EMT, the mechanisms governing their crosstalk remain poorly understood. Here, we identify C2H2 zinc finger protein, ZNF827, a novel factor, is strongly induced during important EMT mediated processes including in brain development and breast cancer metastasis and is required for the molecular and phenotypic changes underlying EMT in these processes. Mechanistically, ZNF827 mediated these responses by orchestrating a large-scale remodeling of the splicing landscape by recruiting HDAC1 for epigenetic modulation of distinct genomic loci, thereby slowing RNA Pol II progression and altering the splicing of transcripts encoding key EMT regulators in cis. These findings reveal an unprecedented complexity between epigenetic landscape and splicing and identifies ZNF827 as a master regulator coupling these processes during EMT in brain development and breast cancer metastasis.
Project description:Mutations in the human CDKL5 gene have been associated with early onset seizure variant of Rett Syndrome. In order to investigate the potential involvement of CDKL5 in the regulation of gene expression, we compared expression profiles in WT vs CDKL5-mutated IPS clones from two patients (one male and one female) with different mutations using two-colour microarray experiments. For the female patient (CDKL5 mutation p.Gln347X) we compared one clone expressing the mutant CDKL5 allele to another clone from the same patient that expresses the wild-type allele. Maintenance of X chromosome inactivation and the resulting mono-allelic expression of CDKL5 were confirmed by androgen receptor assay and direct sequencing of CDKL5 mRNA. The clone derived from the male patient (CDKL5 mutation p.Thr288Ile) was compared to a clone derived from a normal newborn male. For each mutant/control pair four technical replicates were performed for a total of eight chip hybridizations.
Project description:Zebrafish has been recognized as a robust model organism in biomedical research for the investigation of human genetic disorders. Recently, we proposed the homozygous cdkl5sa21938 mutant zebrafish as a model of CDKL5 deficiency disorder (CDD), a rare developmental epileptic encephalopathy associated with broad range of symptoms. Since neurological phenotypes are the primary focus of investigations, the understanding of CDKL5 function beyond the brain remains limited. Therefore, the aim of this study was to expand the knowledge about Cdkl5-associated molecular mechanisms using the proposed cdkl5 zebrafish model and to evaluate their similarity to findings in mammalian systems. For this purpose, we conducted RNA-sequencing of whole cdkl5-/- mutant zebrafish and compare it to their wild-type siblings at two different stages of development (5 and 35 dpf). Most significant DEGs were related to muscle, neuronal and visual systems which are affected in CDD. Gene Ontology (GO) analysis revealed that at both stages the downregulated DEGs were mainly enriched in muscle development, extracellular matrix and actin cytoskeleton functions while at 35 dpf the upregulated DEGs were mainly enriched in eye development functions. KEGG pathway analysis showed the enrichment of downregulated DEGs in focal adhesion and ECM-receptor interaction pathways at both stages. Additionally, we found that DEGs related to neuronal development were mainly downregulated at both stages while several upregulated DEGs were enriched in synaptic signaling at 35dpf. Moreover, we identified many downregulated key DEGs of cartilage development at both stages and bone development at 35 dpf, potential explaining the impaired cdkl5-/- mutant craniofacial cartilage and bone mineralization defects and the CDD skeletal phenotypes. In conclusion, this study shows that Cdkl5 loss in the cdkl5-/- mutant zebrafish lead to the dysregulation of several genes involved in functions known to be associated with CDKL5 in mammalian systems and provide novel insights into the less studied CDKL5 functions and phenotypes.