Targeting NOX2 via p47/phox-p22/phox Inhibition with Novel Triproline Mimetics

On the basis of the knowledge that the proline-rich hot spot PPPRPP region of P(151)PSNPPPRPP(160), an oligopeptide derived from the cytosolic portion of p22phox (p22), binds to the single functional bis-SH3 domain of the regulatory protein p47phox (p47), we designed a mimetic of the tripeptide PPP based on NMR and X-ray crystallographic data for the p22(151-161) peptide PPSNPPPRPPA with a peptide construct. Incorporation of the synthetic pseudo-triproline mimetic Pro-Pro-Cyp in a molecule derived from molecular modeling studies led to only a 7-fold diminution in activity in a surface plasmon resonance assay relative to the same molecule containing the natural Pro-Pro-Pro tripeptide. The alternative sequence corresponding to a Pro-Cyp-Pro insertion was inactive. This is a first example of the use of a triproline mimetic to interfere with the formation of the p47-p22 complex, which is critical for the activation of NOX, leading to the production of reactive oxygen species as superoxide anions

Activity-Dependent Neuroplasticity Induced by an Enriched Environment Reverses Cognitive Deficits in Scribble Deficient Mouse

International audiencePlanar cell polarity (PCP) signaling is well known to play a critical role during prenatal brain development; whether it plays specific roles at postnatal stages remains rather unknown. Here, we investigated the role of a key PCP-associated gene scrib in CA1 hippocampal structure and function at postnatal stages. We found that Scrib is required for learning and memory consolidation in the Morris water maze as well as synaptic maturation and NMDAR-dependent bidirectional plasticity. Furthermore, we unveiled a direct molecular interaction between Scrib and PP1/PP2A phosphatases whose levels were decreased in postsynaptic density of conditional knockout mice. Remarkably, exposure to enriched environment (EE) preserved memory formation in CaMK-Scrib −/− mice by recovering synaptic plasticity and maturation. Thus, Scrib is required for synaptic function involved in memory formation and EE has beneficiary therapeutic effects. Our results demonstrate a distinct new role for a PCP-associated protein, beyond embryonic development, in cognitive functions during adulthood

Discovery of neutralizing SARS-CoV-2 antibodies enriched in a unique antigen specific B cell cluster.

Despite development of effective SARS-CoV-2 vaccines, a sub-group of vaccine non-responders depends on therapeutic antibodies or small-molecule drugs in cases of severe disease. However, perpetual viral evolution has required continuous efficacy monitoring as well as exploration of new therapeutic antibodies, to circumvent resistance mutations arising in the viral population. We performed SARS-CoV-2-specific B cell sorting and subsequent single-cell sequencing on material from 15 SARS-CoV-2 convalescent participants. Through screening of 455 monoclonal antibodies for SARS-CoV-2 variant binding and virus neutralization, we identified a cluster of activated B cells highly enriched for SARS-CoV-2 neutralizing antibodies. Epitope binning and Cryo-EM structure analysis identified the majority of neutralizing antibodies having epitopes overlapping with the ACE2 receptor binding motif (class 1 binders). Extensive functional antibody characterization identified two potent neutralizing antibodies, one retaining SARS-CoV-1 neutralizing capability, while both bind major common variants of concern and display prophylactic efficacy in vivo. The transcriptomic signature of activated B cells harboring broadly binding neutralizing antibodies with therapeutic potential identified here, may be a guide in future efforts of rapid therapeutic antibody discovery

Scribble1/AP2 Complex Coordinates NMDA Receptor Endocytic Recycling

The appropriate trafficking of glutamate receptors to synapses is crucial for basic synaptic function and synaptic plasticity. It is now accepted that NMDA receptors (NMDARs) internalize and are recycled at the plasma membrane but also exchange between synaptic and extrasynaptic pools; these NMDAR properties are also key to governing synaptic plasticity. Scribble1 is a large PDZ protein required for synaptogenesis and synaptic plasticity. Herein, we show that the level of Scribble1 is regulated in an activity-dependent manner and that Scribble1 controls the number of NMDARs at the plasma membrane. Notably, Scribble1 prevents GluN2A subunits from undergoing lysosomal trafficking and degradation by increasing their recycling to the plasma membrane following NMDAR activation. Finally, we show that a specific YxxR motif on Scribble1 controls these mechanisms through a direct interaction with AP2. Altogether, our findings define a molecular mechanism to control the levels of synaptic NMDARs via Scribble1 complex signaling

FACS gating strategy.

Despite development of effective SARS-CoV-2 vaccines, a sub-group of vaccine non-responders depends on therapeutic antibodies or small-molecule drugs in cases of severe disease. However, perpetual viral evolution has required continuous efficacy monitoring as well as exploration of new therapeutic antibodies, to circumvent resistance mutations arising in the viral population. We performed SARS-CoV-2-specific B cell sorting and subsequent single-cell sequencing on material from 15 SARS-CoV-2 convalescent participants. Through screening of 455 monoclonal antibodies for SARS-CoV-2 variant binding and virus neutralization, we identified a cluster of activated B cells highly enriched for SARS-CoV-2 neutralizing antibodies. Epitope binning and Cryo-EM structure analysis identified the majority of neutralizing antibodies having epitopes overlapping with the ACE2 receptor binding motif (class 1 binders). Extensive functional antibody characterization identified two potent neutralizing antibodies, one retaining SARS-CoV-1 neutralizing capability, while both bind major common variants of concern and display prophylactic efficacy in vivo. The transcriptomic signature of activated B cells harboring broadly binding neutralizing antibodies with therapeutic potential identified here, may be a guide in future efforts of rapid therapeutic antibody discovery.</div

Cluster distribution of drug development liabilities.

Cluster distribution of drug development liabilities.</p

Single-cell profiling of 7176 B cells from three donor groups by scRNA-seq and V(D)J-seq.

(A) UMAP projection and unsupervised clustering revealing 9 transcriptomic clusters annotated according to selected marker genes. (B) Somatic hyper-mutation percentage compared to inferred naïve germline stratified by isotype and transcriptomic cluster, highlighting one cluster of naïve B cells associated with particularly low mutation rate. The dotted line indicates the threshold used for the selection of mAbs for validation. The number of cells within each cluster or isotype is displayed below. (C) Fraction of cells with each isotype stratified by transcriptomic cluster highlighting the naïve cluster and three IgG-rich clusters. (D) mRNA expression levels given by the log of the normalized UMI counts of selected markers in each transcriptomic cluster. (E) Scaled average expression in each cluster indicating memory B cell, naïve B cell and activated B cell markers highlighting unique transcriptomic profiles of the clusters. The size of the dots indicates the percentage of cells expressing the given marker gene within the cluster.</p

Monoclonal antibody screening.

(A) Neutralization percentage of SARS-CoV-2 pseudovirus shown for each individual mAb supernatant analyzed at 20 μg/ml, shown on y-axis. MAbs are ordered along the x-axis from best (left) to poorest (right) neutralizers. n = 455. Screening was performed once in duplicate determinations. (B) Visualization of the expressed mAbs B cell cluster origin and distribution within all isolated B cells. Neutralizers shown in green ( neutralization, n = 9). Top neutralizers shown in red (<95% neutralization, n = 24). Non-neutralizers shown in blue (<80% neutralization). Background shown in grey (cells not expressed for screening).(C) Distribution of successful neutralizing mAbs, between clusters 6, 8 and remaining clusters (cluster 7 excluded). Hit rate cut-off for defining successful neutralizations was set at 80% pseudovirus neutralization. Hit rates were calculated within each cluster group. n = 455 (D) Predictive performance of Ag scores used for SARS-CoV-2 specific B cell sorting towards neutralization capability in cluster 8. n = 108. Red = SARS-CoV-2 D614G mutant trimer, Blue = SARS-CoV-2 RBD, Orange = SARS-CoV-1 RBD, Green = SARS-CoV-2 trimer. The p-value is based on a Kruskal-Wallis test of the receiver-operator characteristics curve.</p