Biology subjects

Azoitei, M.

Publications and source records attributed to Azoitei, M..

3 recordsLinked to original sources

bioRxiv2026

Control of Guanine Exchange Factor Activity Using De Novo Designed Protein Inhibitors

Guanine Exchange Factors (GEF) of the Dbl family are the main activators of RhoA GTPases. GEF and GTPase activity is tightly regulated at the subcellular level with fast kinetics. Therefore, to fully understand the function of Dbl GEFs requires their study in living cells. Towards developing molecular tools that reversibly and rapidly modulate the activity of endogenous GEFs in living cells, here we developed a general platform for engineering inhibitors against members of the Dbl family of GEFs using generative protein design. Engineered proteins showed high affinity and remarkable specificity for the target GEFs and modulated GEF activity both in vitro and in cells. In a proof-of-principle example, a GEF inhibitor was coupled to a light-activated module, enabling the optogenetic control of its activity in cells. These findings show that generative protein design can create modulators of intracellular signaling and broaden the range of tools available for biological research.

cell biology↗

bioRxiv2021

SARS-CoV-2 vaccination induces neutralizing antibodies against pandemic and pre-emergent SARS-related coronaviruses in monkeys

Betacoronaviruses (betaCoVs) caused the severe acute respiratory syndrome (SARS) and Middle East Respiratory Syndrome (MERS) outbreaks, and now the SARS-CoV-2 pandemic. Vaccines that elicit protective immune responses against SARS-CoV-2 and betaCoVs circulating in animals have the potential to prevent future betaCoV pandemics. Here, we show that immunization of macaques with a multimeric SARS-CoV-2 receptor binding domain (RBD) nanoparticle adjuvanted with 3M-052-Alum elicited cross-neutralizing antibody responses against SARS-CoV-1, SARS-CoV-2, batCoVs and the UK B.1.1.7 SARS-CoV-2 mutant virus. Nanoparticle vaccination resulted in a SARS-CoV-2 reciprocal geometric mean neutralization titer of 47,216, and robust protection against SARS-CoV-2 in macaque upper and lower respiratory tracts. Importantly, nucleoside-modified mRNA encoding a stabilized transmembrane spike or monomeric RBD protein also induced SARS-CoV-1 and batCoV cross-neutralizing antibodies, albeit at lower titers. These results demonstrate current mRNA vaccines may provide some protection from future zoonotic betaCoV outbreaks, and provide a platform for further development of pan-betaCoV nanoparticle vaccines.

immunology↗

bioRxiv2019

Connectivity analysis of GEF/GTPase networks in living cells

The cytoskeleton is regulated by dynamic, multi-layered signaling networks that interconnect Rho family small GTPases with exquisite spatiotemporal precision1. Understanding the organization of these networks is challenging, as protein activation and interaction occur transiently and with precise subcellular localization. We and others have used fluorescent biosensors in living cells to map the activation patterns of the Rho family small GTPases relative to the changes in cell edge dynamics that they produce2-4. These GTPases are controlled by the localized activity of numerous Rho guanine nucleotide exchange factors (RhoGEFs) with overlapping GTPase specificity5,6. Here we extend this analysis to determine functional relationships between GEFs and GTPases in controlling cell edge movements. First, biosensors for GEF activation are produced, and their activity correlated with edge dynamics. We then shift the wavelengths of GTPase biosensors to image and correlate the activation of GEFs and GTPases in the same cell. Using partial correlation analysis, we can parse out from such multiplexed data the contribution of each GEF - GTPase interaction to edge dynamics, i.e. we identify when and where specific GEF activation events regulate specific downstream GTPases to affect motility. We describe biosensors for eight Dbl family RhoGEFs based on a broadly applicable new design strategy (Asef, Tiam1, Vav isoforms, Tim, LARG, and {beta}-Pix), and red shifted biosensors for RhoA, Rac1 and Cdc42. In the context of motility, functional interactions were identified for Asef regulation of Cdc42 and Rac1. This approach exemplifies a powerful means to elucidate the real-time connectivity of signal transduction networks.

cell biology↗