RESUMO
Amyotrophic lateral sclerosis (ALS) is a debilitating disease characterized by progressive loss of voluntary motor neurons leading to muscle atrophy, weight loss and respiratory failure. Evidence suggests that inflammation, oxidative stress, mitochondrial dysfunction, apoptosis, glutamate excitotoxicity and proteasomal dysfunction are all responsible for ALS pathogenesis. We review neuroprotective agents with the ability to reduce ALS-related bodyweight loss, summarize the various therapies tested on animal models targeting the proposed molecular mechanisms, compare their effects on bodyweight loss, muscle damage, disease onset, duration and survival, and analyze their structure-activity relationships, with the overall goal of creating a screening strategy for further clinical application.
Assuntos
Esclerose Lateral Amiotrófica/tratamento farmacológico , Atrofia Muscular/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Animais , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Humanos , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacologia , Relação Estrutura-Atividade , Redução de Peso/efeitos dos fármacosRESUMO
We report a comparative study in which a single-molecule fluorescence resonance energy transfer approach was used to examine how the binding of two families of HIV-1 viral proteins to viral RNA hairpins locally changes the RNA secondary structures. The single-molecule fluorescence resonance energy transfer results indicate that the zinc finger protein (nucleocapsid) locally melts the TAR RNA and RRE-IIB RNA hairpins, whereas arginine-rich motif proteins (Tat and Rev) may strengthen the hairpin structures through specific binding interactions. Competition experiments show that Tat and Rev can effectively inhibit the nucleocapsid-chaperoned annealing of complementary DNA oligonucleotides to the TAR and RRE-IIB RNA hairpins, respectively. The competition binding data presented here suggest that the specific nucleic acid binding interactions of Tat and Rev can effectively compete with the general nucleic acid binding/chaperone functions of the nucleocapsid protein, and thus may in principle help regulate critical events during the HIV life cycle.
Assuntos
Arginina/metabolismo , HIV-1/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , RNA Viral/metabolismo , Dedos de Zinco , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Ensaio de Desvio de Mobilidade Eletroforética , Transferência Ressonante de Energia de Fluorescência , Repetição Terminal Longa de HIV/genética , Cinética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ligação Proteica , RNA Viral/química , RNA Viral/genética , Elementos de Resposta/genética , Produtos do Gene rev do Vírus da Imunodeficiência Humana/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/químicaRESUMO
Reverse transcription of the HIV-1 genome involves several nucleic acid rearrangement steps that are catalyzed (chaperoned) by the nucleocapsid protein (NC), including the annealing of the transactivation response region (TAR) RNA of the genome to the complementary sequence (TAR DNA) in minus-strand strong-stop DNA. It has been extremely challenging to obtain unambiguous mechanistic details on the annealing process at the molecular level because of the kinetic involvement of a complex and heterogeneous set of nucleic acid/protein complexes of variable structure and variable composition. Here, we investigate the in vitro annealing mechanism using a multistep single-molecule spectroscopy kinetic method. In this approach, an immobilized hairpin is exposed to a multistep programmed concentration sequence of NC, model complementary targeted-oligonucleotides, and buffer-only solutions. The sequence controllably "drags" single immobilized TAR hairpins among the kinetic stable states of the reaction mechanism; i.e., reactants, intermediates, and products. This single-molecule spectroscopy method directly probes kinetic reversibility and the chaperone (catalytic) role of NC at various stages along the reaction sequence, giving access to previously inaccessible kinetic processes and rate constants. By employing target oligonucleotides for specific TAR regions, we kinetically trap and investigate structural models for putative nucleation complexes for the annealing process. The new results lead to a more complete and detailed understanding of the ability of NC to promote nucleic acid/nucleic acid rearrangement processes. This includes information on the ability of NC to chaperone "reverse annealing" in single-strand transfer and the first observation of partially annealed, conformational substates in the annealing mechanism.