In vitro reconstitution of the initiation of mitophagosome formation

Informations projet

Mitophagy

N° de convention de subvention: 101062916

DOI

10.3030/101062916

Projet clôturé

Date de signature de la CE 13 Mai 2022

Date de début 1 Juin 2022

Date de fin 31 Mai 2024

Financé au titre de

• Marie Skłodowska-Curie Actions (MSCA)

Coût total

Aucune donnée

Contribution de l’UE

€ 199 440,96

Coordonné par

UNIVERSITAT WIEN
Austria

Periodic Reporting for period 1 - Mitophagy (In vitro reconstitution of the initiation of mitophagosome formation)

Période du rapport: 2022-06-01 au 2024-05-31

Résumé du contexte et des objectifs généraux du projet

Mitochondrial quality control is essential for cellular health, as it selectively targets damaged mitochondria for degradation through a process known as mitophagy. The primary regulatory mechanisms for PINK1/Parkin-dependent mitophagy, including the prevention of overactivation and ensuring swift progression, are not fully understood. Our research focuses on elucidating the roles of TBK1 adaptors, NAP1 and SINTBAD, in regulating mitophagy. To address this, we combined a bottom-up biochemical reconstitution approach with cellular genome engineering to characterise individual steps in presence/absence of NAP1 and SINTBAD.

The project's objectives were therefore following:

(i) Determine how NAP1 and SINTBAD modulate the initiation and progression of mitophagy.
(ii) Understand the competitive dynamics between NAP1/SINTBAD and OPTN in TBK1 recruitment.
(iii) Investigate the supportive role of NAP1/SINTBAD in NDP52-mediated mitophagy.

By addressing these objectives, the project aims to provide insights into cellular strategies that balance mitophagy activation and progression, preventing pathway hyperactivity while ensuring efficient clearance of damaged mitochondria. This understanding could have significant implications for diseases associated with mitochondrial dysfunction, such as neurodegenerative diseases and mitochondrial encephalopathies.

Travail effectué depuis le début du projet jusqu’à la fin de la période considérée dans le rapport et principaux résultats atteints jusqu’à présent

The research resulted in the establishment of following protocols:

(i) Recruitment and Co-degradation Analysis: We analyzed how NAP1/SINTBAD are recruited to and co-degraded with damaged mitochondria during mitophagy.
(ii) Functional Assays: Using cellular and in vitro reconstitutions, we assessed the inhibitory and supportive roles of NAP1/SINTBAD in mitophagy.
(iii) Interaction Studies: Detailed investigations were conducted to understand the competitive binding dynamics between NAP1/SINTBAD and OPTN for TBK1.
(iv) Mitophagy Induction Experiments: We evaluated the sufficiency of NAP1/SINTBAD recruitment in inducing mitophagy.

The main achievements include:

(i) Demonstrating that NAP1/SINTBAD are key regulators of mitophagy initiation by restricting OPTN-driven TBK1 recruitment.
(ii) Showing that NAP1/SINTBAD support NDP52-mediated mitophagy by stabilizing the interaction between NDP52, TBK1, and FIP200.
(iii) Establishing a model where NAP1/SINTBAD act as rheostats, increasing the threshold for mitophagy initiation but promoting pathway progression once initiated.

Progrès au-delà de l’état des connaissances et impact potentiel prévu (y compris l’impact socio-économique et les conséquences sociétales plus larges du projet jusqu’à présent)

The results indicate that NAP1 and SINTBAD play dual roles in mitophagy:

(I) They act as inhibitors by competing with OPTN for TBK1 binding, thus controlling the initiation phase.
(ii) They promote NDP52-mediated mitophagy, ensuring effective progression of the process once initiated.The potential impacts of these findings are significant:

Further Research:
(i) These insights open new avenues for research into selective autophagy pathways and their regulation.
(ii) Therapeutic Development: Understanding these regulatory mechanisms can inform the development of therapeutic strategies targeting mitochondrial dysfunction in neurodegenerative diseases and other conditions.
(iii) Biotechnological Applications: The ability to modulate mitophagy could lead to advances in biotechnology, particularly in areas requiring precise control of cellular health and function.