Artificial Zinc-Finger transcription factors libraries: Identification of HIV-1 cellular restriction factors and Therapeutic application in HIV-1 infection.

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Gene-targeted strategies to eliminate HIV latent cells through synthetic activators and suicide lentivectors

Publication . Perdigão, PRL; Gonçalves, João, 1967-; Marta, Mariana Santa, 1978-

Despite the success of antiretroviral therapy, a cure for HIV-1 infection remains elusive. The persistence of cellular reservoirs harboring transcriptionally silent (latent) HIV provirus is responsible for the viremia rebound observed following treatment withdrawal. Stimulation of latent viral expression is considered critical to target HIV reservoirs for elimination through a “shock and kill” approach. Pharmacological drugs have systematically proven ineffective to drastically reduce the reservoir size and may cause severe side effects owing to their indiscriminate mode of action. In the present thesis, gene-targeted strategies were explored to stimulate and eliminate HIV latent cells. To stimulate latent virus expression, we designed synthetic activators based on transcription activator-like effector (TALE) proteins that recognize conserved regions on HIV 5’LTR promoter. Four TALE activators strongly induced HIV transcription, acting in cooperation to specifically enhance viral expression from cell line models of HIV-1 latency. Moreover, we show that histone deacetylase inhibitors can further enhance the effect of TALE-mediated activation in highly repressed latent cells. To further potentiate the elimination of stimulated latent cells, we conjugated an HIV-responsive suicide lentivector to our TALE activator technology. For this purpose, we incorporated a modified 5’LTR promoter into the suicidal lentivector as a safety mechanism to dissociate TALE-driven activation, restricting the responsiveness of this plasmid to the HIV regulatory proteins. The therapeutic plasmid was capable of specifically eliminate latently infected cells stimulated by TALE activators through a Tat/Rev-dependent expression of the diphtheria toxin. Finally, we presented a “gene-free” approach to specifically activate latent HIV expression through protein delivery of cell-penetrating zinc-finger activators (CPP-ZFA). A single activator based on Cys2His2 zinc-finger domains proved effective at inducing viral expression from the primer binding site downstream of 5’LTR promoter. When conjugated with positively charged nuclear localization signal repeats, this synthetic activator efficiently translocated across cell membrane without the need of carriers. Short-term presence of CPP-ZFA following protein delivery was sufficient to stimulate gene expression in HIV-1 latent cells, offering a safer alternative to avoid off-target effects from prolonged exposure to these synthetic activators. In resume, this work provides proof-of-concept that synthetic activators and suicide lentivectors constitute promising candidates for the eradication of HIV-1 reservoirs through gene-targeted strategies.

2017Tese de doutoramento

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Estimation of kinetic parameters related to biochemical interactions between hydrogen peroxide and signal transduction proteins

Publication . Brito, Paula; Antunes, Fernando

The lack of kinetic data concerning the biological effects of reactive oxygen species is slowing down the development of the field of redox signaling. Herein, we deduced and applied equations to estimate kinetic parameters from typical redox signaling experiments. H2O2-sensing mediated by the oxidation of a protein target and the switch-off of this sensor, by being converted back to its reduced form, are the two processes for which kinetic parameters are determined. The experimental data required to apply the equations deduced is the fraction of the H2O2 sensor protein in the reduced or in the oxidized state measured in intact cells or living tissues after exposure to either endogenous or added H2O2. Either non-linear fittings that do not need transformation of the experimental data or linearized plots in which deviations from the equations are easily observed can be used. The equations were shown to be valid by fitting to them virtual time courses simulated with a kinetic model. The good agreement between the kinetic parameters estimated in these fittings and those used to simulate the virtual time courses supported the accuracy of the kinetic equations deduced. Finally, equations were successfully tested with real data taken from published experiments that describe redox signaling mediated by the oxidation of two protein tyrosine phosphatases, PTP1B and SHP-2, which are two of the few H2O2-sensing proteins with known kinetic parameters. Whereas for PTP1B estimated kinetic parameters fitted in general the present knowledge, for SHP-2 results obtained suggest that reactivity toward H2O2 as well as the rate of SHP-2 regeneration back to its reduced form are higher than previously thought. In conclusion, valuable quantitative kinetic data can be estimated from typical redox signaling experiments, thus improving our understanding about the complex processes that underlie the interplay between oxidative stress and redox signaling responses.

2014Artigo científico

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Entidade financiadora

Fundação para a Ciência e a Tecnologia

Programa de financiamento

3599-PPCDT

Número da atribuição

VIH/SAU/0020/2011