Apoptosis Inducing Factor (AIF), a highly conserved mitochondrial flavoprotein, is generally known as a caspase-independent effector in the intrinsic apoptosis pathway. Beside this apoptotic function, recent studies demonstrate that AIF is also able to regulate the cell energy homeostasis by promoting the biogenesis and the function of multi-subunit respiratory complexes. Although the underlying molecular mechanisms have not been yet elucidated, it is clear that this role is played thanks to the interaction of AIF with CHCHD4, a soluble inter membrane space (IMS) protein which promotes the entrance in the IMS and the oxidative folding of substrates belonging to the respiratory complexes’ subunits. Given the interest in deeply understanding the AIF vital role in mitochondria, we decided to investigate the AIF-CHCHD4 interaction from both the functional and the structural point of view. We focused on the study of the possible impact of the 27-residues N-terminal portion of CHCHD4, which effectively mimics the protein binding site with AIF, on the catalytic activity and NAD+-binding ability of AIF. The peptide turned out to stimulate the DCIP-NADH reductase activity of AIF, allowing the estimation of the apparent Kd of the AIF-peptide complex in the sub-micromolar range. Another interesting finding was the ability of AIF to bind NAD+ only in the presence of peptide. The experiments indicate that NAD+ complexation is strongly stimulated by lowering the temperature and that such effect is secondary to the AIF-peptide interaction. The same effect was obtained even with the full length form of CHCHD4. Another important feature observed is that CHCHD4 or its peptide binding also modulates the AIF reactivity towards thiol-containing compounds revealing a possible role of AIF in assisting the oxidative folding of disulfide-containing polypeptides in mitochondria. The interaction between AIF and CHCHD4 was then further investigated through different biophysical techniques such as analytic Size Exclusion Chromatography (SEC), ThermoFAD, Microscale Thermophoresis and Isothermal Titration Calorimetry. In particular, we found out that AIF and CHCHD4 form a heterodimeric complex, whose stability is temperature-dependent, with a binding affinity in the low micromolar range for oxidised AIF and in the low nanomolar range for reduced AIF. For the determination of relevant structural features of the complex, we used AlphaFold software to build up a model, which was then experimentally confirmed by mass-spectrometry analyses on the cross-linked AIF-CHCHD4 complex. Since peptide and NAD+ bindings display strong positive cooperativity, we used this information to set up the optimal conditions for X-ray diffraction studies, through which we determined the crystal structure of the complex. The binding site of AIF for CHCHD4 was further validated by mutagenesis experiments on AIF.
Il fattore di induzione dell’apoptosi (AIF), una flavoproteina mitocondriale altamente conservata, è generalmente noto per il suo ruolo nell’attivazione del processo di apoptosi intrinseca in maniera indipendente dalle caspasi. Oltre a questa funzione pro-apoptotica, studi recenti hanno dimostrato che AIF è anche in grado di regolare l’omeostasi energetica cellulare promuovendo la biogenesi e la funzione dei complessi respiratori multi-subunità. Sebbene i meccanismi molecolari alla base non siano stati ad oggi chiariti, è noto che questo ruolo è svolto grazie all’interazione di AIF con CHCHD4, una proteina solubile dello spazio inter-membrana dei mitocondri (IMS), che promuove l’ingresso nell’IMS e il folding ossidativo dei substrati appartenenti alle subunità dei complessi respiratori. Dato l'interesse nel chiarire il ruolo vitale di AIF nei mitocondri, abbiamo deciso di indagare l'interazione AIF-CHCHD4 sia dal punto di vista funzionale che strutturale. Ci siamo concentrati sullo studio del possibile impatto della porzione N-terminale di CHCHD4 (27 residui), sufficiente per legare AIF, sull'attività catalitica di AIF e sulla sua capacità di legare il NAD+. Si è scoperto che il peptide stimola l'attività DCIP-NADH reduttasica di AIF ed è stata stimata una Kd apparente del complesso AIF-peptide nel basso micromolare. Un’altra interessante scoperta è stata la capacità di AIF di legare NAD+ solo in presenza del peptide. Gli esperimenti indicano che l’interazione con NAD+ è fortemente stimolata dall'abbassamento della temperatura e che tale effetto è secondario all'interazione AIF-peptide. Lo stesso effetto è stato ottenuto anche con la forma intera di CHCHD4. Inoltre, è stato osservato che il legame di CHCHD4 o del suo peptide modula anche la reattività di AIF verso composti contenenti gruppi tiolici, suggerendo un possibile ruolo di AIF nell'assistere il folding ossidativo dei polipeptidi contenenti disolfuri all’interno dei mitocondri. L'interazione tra AIF e CHCHD4 è stata poi ulteriormente studiata attraverso diverse tecniche biofisiche come la cromatografia analitica ad esclusione molecolare, ThermoFAD, termoforesi e calorimetria. In particolare, abbiamo scoperto che AIF e CHCHD4 formano un complesso eterodimerico, la cui stabilità dipende dalla temperatura, con un'affinità di legame nel basso micromolare per AIF ossidata e nel basso nanomolare per AIF ridotta. Per la determinazione delle caratteristiche strutturali del complesso, abbiamo utilizzato il software AlphaFold per costruire un modello, che è stato poi confermato sperimentalmente da analisi di spettrometria di massa sul complesso AIF-CHCHD4 covalentemente legato. Poiché il legame del peptide e di NAD+ mostrano una forte cooperatività positiva, abbiamo utilizzato queste informazioni per determinare le condizioni ottimali per gli studi di diffrazione a raggi X, attraverso i quali abbiamo determinato la struttura cristallografica del complesso. Il sito di legame di AIF per CHCHD4 è stato ulteriormente validato tramite esperimenti di mutagenesi su AIF.
UNDERSTANDING THE PRO-SURVIVAL ROLE OF AIF: STRUCTURAL AND FUNCTIONAL STUDY OF AIF-CHCHD4 COMPLEX / E. Fagnani ; tutor: A. Aliverti ; co-tutor: M. Milani ; coordinatore del dottorato: R. Mantovani. Dipartimento di Bioscienze, 2024. 36. ciclo, Anno Accademico 2022/2023.
UNDERSTANDING THE PRO-SURVIVAL ROLE OF AIF: STRUCTURAL AND FUNCTIONAL STUDY OF AIF-CHCHD4 COMPLEX
E. Fagnani
2024
Abstract
Apoptosis Inducing Factor (AIF), a highly conserved mitochondrial flavoprotein, is generally known as a caspase-independent effector in the intrinsic apoptosis pathway. Beside this apoptotic function, recent studies demonstrate that AIF is also able to regulate the cell energy homeostasis by promoting the biogenesis and the function of multi-subunit respiratory complexes. Although the underlying molecular mechanisms have not been yet elucidated, it is clear that this role is played thanks to the interaction of AIF with CHCHD4, a soluble inter membrane space (IMS) protein which promotes the entrance in the IMS and the oxidative folding of substrates belonging to the respiratory complexes’ subunits. Given the interest in deeply understanding the AIF vital role in mitochondria, we decided to investigate the AIF-CHCHD4 interaction from both the functional and the structural point of view. We focused on the study of the possible impact of the 27-residues N-terminal portion of CHCHD4, which effectively mimics the protein binding site with AIF, on the catalytic activity and NAD+-binding ability of AIF. The peptide turned out to stimulate the DCIP-NADH reductase activity of AIF, allowing the estimation of the apparent Kd of the AIF-peptide complex in the sub-micromolar range. Another interesting finding was the ability of AIF to bind NAD+ only in the presence of peptide. The experiments indicate that NAD+ complexation is strongly stimulated by lowering the temperature and that such effect is secondary to the AIF-peptide interaction. The same effect was obtained even with the full length form of CHCHD4. Another important feature observed is that CHCHD4 or its peptide binding also modulates the AIF reactivity towards thiol-containing compounds revealing a possible role of AIF in assisting the oxidative folding of disulfide-containing polypeptides in mitochondria. The interaction between AIF and CHCHD4 was then further investigated through different biophysical techniques such as analytic Size Exclusion Chromatography (SEC), ThermoFAD, Microscale Thermophoresis and Isothermal Titration Calorimetry. In particular, we found out that AIF and CHCHD4 form a heterodimeric complex, whose stability is temperature-dependent, with a binding affinity in the low micromolar range for oxidised AIF and in the low nanomolar range for reduced AIF. For the determination of relevant structural features of the complex, we used AlphaFold software to build up a model, which was then experimentally confirmed by mass-spectrometry analyses on the cross-linked AIF-CHCHD4 complex. Since peptide and NAD+ bindings display strong positive cooperativity, we used this information to set up the optimal conditions for X-ray diffraction studies, through which we determined the crystal structure of the complex. The binding site of AIF for CHCHD4 was further validated by mutagenesis experiments on AIF.File | Dimensione | Formato | |
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