Isolation and Characterization of Monomeric Human RAD51: A Novel Tool for Investigating Homologous Recombination in Cancer

RAD51 is a key player in the homologous recombination pathway. Upon DNA damage, RAD51 is transported into the nucleus by BRCA2, where it can repair DNA double-strand breaks. Due to the structural complexity and dynamics, researchers have not yet clarified the mechanistic details of every step of RAD51 recruitment and DNA repair. RAD51 possesses an intrinsic tendency to form oligomeric structures, which make it challenging to conduct biochemical and biophysical investigations. Here, for the first time, we report on the isolation and characterization of a human monomeric RAD51 recombinant form, obtained through a double mutation, which preserves the protein’s integrity and functionality. We investigated different buffers to identify the most suitable condition needed to definitively stabilize the monomer. The monomer of human RAD51 provides the community with a unique biological tool for investigating RAD51-mediated homologous recombination, and paves the way for more reliable structural, mechanistic, and drug discovery studies

A Numb-Mdm2 fuzzy complex reveals an isoformspecific involvement of Numb in breast cancer

Numb functions as an oncosuppressor by inhibiting Notch signaling and stabilizing p53. This latter effect depends on the interaction of Numb with Mdm2, the E3 ligase that ubiquitinates p53 and commits it to degradation. In breast cancer (BC), loss of Numb results in a reduction of p53-mediated responses including sensitivity to genotoxic drugs and maintenance of homeostasis in the stem cell compartment. In this study, we show that the Numb-Mdm2 interaction represents a fuzzy complex mediated by a short Numb sequence encompassing its alternatively spliced exon 3 (Ex3), which is necessary and sufficient to inhibit Mdm2 and prevent p53 degradation. Alterations in the Numb splicing pattern are critical in BC as shown by increased chemoresistance of tumors displaying reduced levels of Ex3-containing isoforms, an effect that could be mechanistically linked to diminished p53 levels. A reduced level of Ex3-less Numb isoforms independently predicts poor outcome in BCs harboring wild-type p53. Thus, we have uncovered an important mechanism of chemoresistance and progression in p53-competent BCs

A tetracationic porphyrin with dual anti-prion activity

Prions are deadly infectious agents made of PrPSc, a misfolded variant of the cellular prion protein (PrPC) which self-propagates by inducing misfolding of native PrPC. PrPSc can adopt different pathogenic conformations (prion strains), which can be resistant to potential drugs, or acquire drug resistance, hampering the development of effective therapies. We identified Zn(II)-BnPyP, a tetracationic porphyrin that binds to distinct domains of native PrPC, eliciting a dual anti-prion effect. Zn(II)-BnPyP binding to a C-terminal pocket destabilizes the native PrPC fold, hindering conversion to PrPSc; Zn(II)-BnPyP binding to the flexible N-terminal tail disrupts N- to C-terminal interactions, triggering PrPC endocytosis and lysosomal degradation, thus reducing the substrate for PrPSc generation. Zn(II)-BnPyP inhibits propagation of different prion strains in vitro, in neuronal cells and organotypic brain cultures. These results identify a PrPC-targeting compound with an unprecedented dual mechanism of action which might be exploited to achieve anti-prion effects without engendering drug resistance

Persistence of Anti-SARS-CoV-2 Antibodies in Non-Hospitalized COVID-19 Convalescent Health Care Workers

Although antibody response to SARS-CoV-2 can be detected early during the infection, several outstanding questions remain to be addressed regarding the magnitude and persistence of antibody titer against different viral proteins and their correlation with the strength of the immune response. An ELISA assay has been developed by expressing and purifying the recombinant SARS-CoV-2 Spike Receptor Binding Domain (RBD), Soluble Ectodomain (Spike), and full length Nucleocapsid protein (N). Sera from healthcare workers affected by non-severe COVID-19 were longitudinally collected over four weeks, and compared to sera from patients hospitalized in Intensive Care Units (ICU) and SARS-CoV-2-negative subjects for the presence of IgM, IgG and IgA antibodies as well as soluble pro-inflammatory mediators in the sera. Non-hospitalized subjects showed lower antibody titers and blood pro-inflammatory cytokine profiles as compared to patients in Intensive Care Units (ICU), irrespective of the antibodies tested. Noteworthy, in non-severe COVID-19 infections, antibody titers against RBD and Spike, but not against the N protein, as well as pro-inflammatory cytokines decreased within a month after viral clearance. Thus, rapid decline in antibody titers and in pro-inflammatory cytokines may be a common feature of non-severe SARS-CoV-2 infection, suggesting that antibody-mediated protection against re-infection with SARS-CoV-2 is of short duration. These results suggest caution in using serological testing to estimate the prevalence of SARS-CoV-2 infection in the general population

Fast native-SAD phasing for routine macromolecular structure determination

We describe a data collection method that uses a single crystal to solve X-ray structures by native SAD (single-wavelength anomalous diffraction). We solved the structures of 11 real-life examples, including a human membrane protein, a protein-DNA complex and a 266-kDa multiprotein-ligand complex, using this method. The data collection strategy is suitable for routine structure determination and can be implemented at most macromolecular crystallography synchrotron beamlines