Whole exome sequencing in an Italian family with isolated maxillary canine agenesis and canine eruption anomalies

Objective: The aim of this study was the clinical and molecular characterization of a family segregating a trait consisting of a phenotype specifically involving the maxillary canines, including agenesis, impaction and ectopic eruption, characterized by incomplete penetrance and variable expressivity. Design: Clinical standardized assessment of 14 family members and a whole-exome sequencing (WES) of three affected subjects were performed. WES data analyses (sequence alignment, variant calling, annotation and prioritization) were carried out using an in-house implemented pipeline. Variant filtering retained coding and splice-site high quality private and rare variants. Variant prioritization was performed taking into account both the disruptive impact and the biological relevance of individual variants and genes. Sanger sequencing was performed to validate the variants of interest and to carry out segregation analysis. Results: Prioritization of variants “by function” allowed the identification of multiple variants contributing to the trait, including two concomitant heterozygous variants in EDARADD (c.308C>T, p.Ser103Phe) and COL5A1 (c.1588G>A, p.Gly530Ser), specifically associated with a more severe phenotype (i.e. canine agenesis). Differently, heterozygous variants in genes encoding proteins with a role in the WNT pathway were shared by subjects showing a phenotype of impacted/ectopic erupted canines. Conclusions: This study characterized the genetic contribution underlying a complex trait consisting of isolated canine anomalies in a medium-sized family, highlighting the role of WNT and EDA cell signaling pathways in tooth development

SCFβTrCP-mediated degradation of SHARP1 in triple-negative breast cancer

: Triple-negative breast cancer (TNBC) is a subtype of breast cancer associated with metastasis, high recurrence rate, and poor survival. The basic helix-loop-helix transcription factor SHARP1 (Split and Hairy-related Protein 1) has been identified as a suppressor of the metastatic behavior of TNBC. SHARP1 blocks the invasive phenotype of TNBC by inhibiting hypoxia-inducible factors and its loss correlates with poor survival of breast cancer patients. Here, we show that SHARP1 is an unstable protein that is targeted for proteasomal degradation by the E3 ubiquitin ligase complex SCFβTrCP. SHARP1 recruits βTrCP via a phosphodegron encompassing Ser240 and Glu245 which are required for SHARP1 ubiquitylation and degradation. Furthermore, mice injected with TNBC cells expressing the non-degradable SHARP1(S240A/E245A) mutant display reduced tumor growth and increased tumor-free survival. Our study suggests that targeting the βTrCP-dependent degradation of SHARP1 represents a therapeutic strategy in TNBC

Neonatal corticosterone mitigates autoimmune neuropsychiatric disorders associated with streptococcus in mice

Increased glucocorticoid concentrations have been shown to favor resilience towards autoimmune phenomena. Here, we addressed whether experimentally induced elevations in circulating glucocorticoids mitigate the abnormalities exhibited by an experimental model of Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus (PANDAS). This is a pathogenic hypothesis linking repeated exposures to Group-A-beta-hemolytic streptococcus (GAS), autoantibodies targeting selected brain nuclei and neurobehavioral abnormalities. To persistently elevate glucocorticoid concentrations, we supplemented lactating SJL/J mice with corticosterone (CORT; 80\u2009mg/L) in the drinking water. Starting in adolescence (postnatal day 28), developing offspring were exposed to four injections - at bi-weekly intervals - of a GAS homogenate and tested for behavioral, immunological, neurochemical and molecular alterations. GAS mice showed increased perseverative behavior, impaired sensorimotor gating, reduced reactivity to a serotonergic agonist and inflammatory infiltrates in the anterior diencephalon. Neonatal CORT persistently increased circulating glucocorticoids concentrations and counteracted these alterations. Additionally, neonatal CORT increased peripheral and CNS concentrations of the anti-inflammatory cytokine IL-9. Further, upstream regulator analysis of differentially expressed genes in the striatum showed that the regulatory effect of estradiol is inhibited in GAS-treated mice and activated in GAS-treated mice exposed to CORT. These data support the hypothesis that elevations in glucocorticoids may promote central immunomodulatory processes

Mice repeatedly exposed to Group-A \u3b2-Haemolytic Streptococcus show perseverative behaviors, impaired sensorimotor gating, and immune activation in rostral diencephalon

Repeated exposure to Group-A \u3b2-Haemolytic Streptococcus (GAS) may constitute a vulnerability factor in the onset and course of pediatric motor disturbances. GAS infections/colonization can stimulate the production of antibodies, which may cross the blood brain barrier, target selected brain areas (e.g. basal ganglia), and exacerbate motor alterations. Here, we exposed developing SJL male mice to four injections with a GAS homogenate and evaluated the following domains: motor coordination; general locomotion; repetitive behaviors; perseverative responses; and sensorimotor gating (pre-pulse inhibition, PPI). To demonstrate that behavioral changes were associated with immune-mediated brain alterations, we analyzed, in selected brain areas, the presence of infiltrates and microglial activation (immunohistochemistry), monoamines (HPLC), and brain metabolites (in vivo Magnetic Resonance Spectroscopy). GAS-exposed mice showed increased repetitive and perseverative behaviors, impaired PPI, and reduced concentrations of serotonin in prefrontal cortex, a brain area linked to the behavioral domains investigated, wherein they also showed remarkable elevations in lactate. Active inflammatory processes were substantiated by the observation of infiltrates and microglial activation in the white matter of the anterior diencephalon. These data support the hypothesis that repeated GAS exposure may elicit inflammatory responses in brain areas involved in motor control and perseverative behavior, and result in phenotypic abnormalities

Degradation of Tiam1 by casein kinase 1 and the SCFβTrCP ubiquitin ligase controls the duration of mTOR-S6K signaling

Tiam1 (T-cell lymphoma invasion and metastasis 1) is a guanine nucleotide exchange factor that specifically controls the activity of the small GTPase Rac, a key regulator of cell adhesion, proliferation, and survival. Here, we report that in response to mitogens, Tiam1 is degraded by the ubiquitin-proteasome system via the SCF(βTrCP) ubiquitin ligase. Mitogenic stimulation triggers the binding of Tiam1 to the F-box protein βTrCP via its degron sequence and subsequent Tiam1 ubiquitylation and proteasomal degradation. The proteolysis of Tiam1 is prevented by βTrCP silencing, inhibition of CK1 and MEK, or mutation of the Tiam1 degron site. Expression of a stable Tiam1 mutant that is unable to interact with βTrCP results in sustained activation of the mTOR/S6K signaling and increased apoptotic cell death. We propose that the SCF(βTrCP)-mediated degradation of Tiam1 controls the duration of the mTOR-S6K signaling pathway in response to mitogenic stimuli

A systems-wide screen identifies substrates of the SCFβTrCP ubiquitin ligase

Cellular proteins are degraded by the ubiquitin-proteasome system (UPS) in a precise and timely fashion. Such precision is conferred by the high substrate specificity of ubiquitin ligases. Identification of substrates of ubiquitin ligases is crucial not only to unravel the molecular mechanisms by which the UPS controls protein degradation but also for drug discovery purposes because many established UPS substrates are implicated in disease. We developed a combined bioinformatics and affinity purification-mass spectrometry (AP-MS) workflow for the system-wide identification of substrates of SCF(βTrCP), a member of the SCF family of ubiquitin ligases. These ubiquitin ligases are characterized by a multisubunit architecture typically consisting of the invariable subunits Rbx1, Cul1, and Skp1 and one of 69 F-box proteins. The F-box protein of this member of the family is βTrCP. SCF(βTrCP) binds, through the WD40 repeats of βTrCP, to the DpSGXX(X)pS diphosphorylated motif in its substrates. We recovered 27 previously reported SCF(βTrCP) substrates, of which 22 were verified by two independent statistical protocols, thereby confirming the reliability of this approach. In addition to known substrates, we identified 221 proteins that contained the DpSGXX(X)pS motif and also interacted specifically with the WD40 repeats of βTrCP. Thus, with SCF(βTrCP), as the example, we showed that integration of structural information, AP-MS, and degron motif mining constitutes an effective method to screen for substrates of ubiquitin ligases

Datasets from an interaction proteomics screen for substrates of the SCFβTrCP ubiquitin ligase

An affinity purification-mass spectrometry (AP-MS) method was employed to identify novel substrates of the SCFβTrCP ubiquitin ligase. A FLAG-HA tagged version of the F-box protein βTrCP2, the substrate recognition subunit of SCFβTrCP, was used as bait. βTrCP2 wild type and the two mutants βTrCP2-R447A and βTrCP2-ΔF were expressed and purified from HEK293T cells to be able to discriminate between potential substrates of SCFβTrCP and unspecific binders. Affinity-purified samples were analyzed by mass spectrometry-based proteomics, applying ultra-high performance liquid chromatography (UHPLC) coupled to high-resolution tandem mass spectrometry. The raw mass spectrometry data have been deposited to the PRIDE partner repository with the identifiers PXD001088 and PXD001224. The present dataset is associated with a research resource published in T.Y. Low, M. Peng, R. Magliozzi, S. Mohammed, D. Guardavaccaro, A.J.R. Heck, A systems-wide screen identifies substrates of the SCFβTrCP ubiquitin ligase

Datasets from an interaction proteomics screen for substrates of the SCF(βTrCP) ubiquitin ligase

An affinity purification-mass spectrometry (AP-MS) method was employed to identify novel substrates of the SCF(βTrCP) ubiquitin ligase. A FLAG-HA tagged version of the F-box protein βTrCP2, the substrate recognition subunit of SCF(βTrCP), was used as bait. βTrCP2 wild type and the two mutants βTrCP2-R447A and βTrCP2-ΔF were expressed and purified from HEK293T cells to be able to discriminate between potential substrates of SCF(βTrCP) and unspecific binders. Affinity-purified samples were analyzed by mass spectrometry-based proteomics, applying ultra-high performance liquid chromatography (UHPLC) coupled to high-resolution tandem mass spectrometry. The raw mass spectrometry data have been deposited to the PRIDE partner repository with the identifiers PXD001088 and PXD001224. The present dataset is associated with a research resource published in T.Y. Low, M. Peng, R. Magliozzi, S. Mohammed, D. Guardavaccaro, A.J.R. Heck, A systems-wide screen identifies substrates of the SCF(βTrCP) ubiquitin ligase. Sci. Signal. 7 (2014) rs8-rs8, 10.1126/scisignal.2005882

Neonatal corticosterone mitigates autoimmune neuropsychiatric disorders associated with streptococcus in mice

Contains fulltext : 201375.pdf (publisher's version ) (Open Access)12 p

Nodal Signaling Range Is Regulated by Proprotein Convertase-Mediated Maturation

Tissue patterning is established by extracellular growth factors or morphogens. Although different theoretical models explaining specific patterns have been proposed, our understanding of tissue pattern establishment invivo remains limited. In many animal species, left-right patterning is governed by a reaction-diffusion system relying on the different diffusivity of an activator, Nodal, and an inhibitor, Lefty. In a genetic screen, we identified a zebrafish loss-of-function mutant for the proprotein convertase FurinA. Embryological and biochemical experiments demonstrate that cleavage of the Nodal-related Spaw proprotein into a mature form by FurinA is required for Spaw gradient formation and activation of Nodal signaling. We demonstrate that FurinA is required cell-autonomously for the long-range signaling activity of Spaw and no other Nodal-related factors. Combined insilico and invivo approaches support a model in which FurinA controls the signaling range of Spaw by cleaving its proprotein into a mature, extracellular form, consequently regulating left-right patterning