Raquel Villanueva1, Silvia Romero-Tamayo1, Ruben Laplaza1,2, Juan Martínez-Olivan1, Adrián Velázquez-Campoy1,3,4,5, Javier Sancho1,4, Patricia Ferreira1, Milagros Medina1. 1. 1 Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Instituto de Biocomputación y Física de Sistemas Complejos (GBsC-CSIC and BIFI-IQFR Joint Units), Universidad de Zaragoza, Zaragoza, Spain. 2. 2 Departamento de Química Física, Universidad de Santiago de Compostela, Santiago de Compostela, Spain. 3. 3 Fundación ARAID, Diputación General de Aragón, Zaragoza, Spain. 4. 4 Aragon Institute for Health Research (IIS Aragon), Zaragoza, Spain. 5. 5 Biomedical Research Networking Centre for Liver and Digestive Diseases (CIBERehd), Madrid, Spain.
Abstract
Aims: The human apoptosis-inducing factor (hAIF) supports OXPHOS biogenesis and programmed cell death, with missense mutations producing neurodegenerative phenotypes. hAIF senses the redox environment of cellular compartments, stabilizing a charge transfer complex (CTC) dimer that modulates the protein interaction network. In this context, we aimed to evaluate the subcellular pH, CTC formation, and pathogenic mutations effects on hAIF stability, and a thermal denaturation high-throughput screening (HTS) assay to discover AIF binders. Results: Apoptotic hAIFΔ1-101 is not stable at intermembrane mitochondrial space (IMS) pH, but the 77-101 residues confer stability to the mitochondrial isoform. hAIF and its CTC populate different conformational ensembles with redox switch to the CTC producing a less stable and compact protein. The pathogenic G308E, ΔR201, and E493V mutations modulate hAIF stability; particularly, ΔR201 causes a population shift to a less stable conformation that remodels active site structure and dynamics. We have identified new molecules that modulate the hAIF reduced nicotinamide adenine dinucleotide (NADH)/oxidized nicotinamide adenine dinucleotide (NAD+) association/dissociation equilibrium and regulate its catalytic efficiency. Innovation: Biophysical methods allow evaluating the regulation of hAIF functional ensembles and to develop an HTS assay to discover small molecules that might modulate hAIF stability and activities. Conclusions: The mitochondrial soluble 54-77 portion stabilizes hAIF at the IMS pH. NADH-redox-linked conformation changes course with strong NAD+ binding and protein dimerization, but they produce a negative impact in overall hAIF stability. Loss of functionality in the R201 deletion is due to distortion of the active site architecture. We report molecules that may serve as leads in the development of hAIF bioactive compounds.
Aims: The human apoptosis-inducing factor (hAIF) supports OXPHOS biogenesis and programmed cell death, with missense mutations producing neurodegenerative phenotypes. hAIF senses the redox environment of cellular compartments, stabilizing a charge transfer complex (CTC) dimer that modulates the protein interaction network. In this context, we aimed to evaluate the subcellular pH, CTC formation, and pathogenic mutations effects on hAIF stability, and a thermal denaturation high-throughput screening (HTS) assay to discover AIF binders. Results: Apoptotic hAIFΔ1-101 is not stable at intermembrane mitochondrial space (IMS) pH, but the 77-101 residues confer stability to the mitochondrial isoform. hAIF and its CTC populate different conformational ensembles with redox switch to the CTC producing a less stable and compact protein. The pathogenic G308E, ΔR201, and E493V mutations modulate hAIF stability; particularly, ΔR201 causes a population shift to a less stable conformation that remodels active site structure and dynamics. We have identified new molecules that modulate the hAIF reduced nicotinamide adenine dinucleotide (NADH)/oxidized nicotinamide adenine dinucleotide (NAD+) association/dissociation equilibrium and regulate its catalytic efficiency. Innovation: Biophysical methods allow evaluating the regulation of hAIF functional ensembles and to develop an HTS assay to discover small molecules that might modulate hAIF stability and activities. Conclusions: The mitochondrial soluble 54-77 portion stabilizes hAIF at the IMS pH. NADH-redox-linked conformation changes course with strong NAD+ binding and protein dimerization, but they produce a negative impact in overall hAIF stability. Loss of functionality in the R201 deletion is due to distortion of the active site architecture. We report molecules that may serve as leads in the development of hAIF bioactive compounds.
Authors: Olga Abian; David Ortega-Alarcon; Ana Jimenez-Alesanco; Laura Ceballos-Laita; Sonia Vega; Hugh T Reyburn; Bruno Rizzuti; Adrian Velazquez-Campoy Journal: Int J Biol Macromol Date: 2020-08-01 Impact factor: 6.953