Adenoviral Vector Design for Mitophagy Research

Overview of Mitophagy

Mitochondria are crucial organelles that control cellular energy levels and cell death. The term 'mitophagy' refers to the selective autophagic process for mitochondria. Mitophagy is mediated by NIX and BNIP3 in mammalian cells and induced via multiple routes. The PINK1-parkin pathways have been the most thoroughly explored. This mechanism begins by determining the difference between healthy and damaged mitochondria, whose difference is that PTEN-induced PINK1 accumulates after depolarizing damaged mitochondria's mitochondrial membrane. As for Fig.1, when mitochondria are injured and lose membrane potential, the kinase PTEN-induced PINK1 accumulates and recruits the E3 ubiquitin ligase parkin from the cytosol to the damaged mitochondrion. Parkin ubiquitylates mitochondrial proteins, causing them to be absorbed by isolation membranes, which later combine with lysosomes. Mitophagy receptors on the outer mitochondrial membrane surface are another route for mitophagy. The receptors are NIX1, BNIP3, and FUNDC1. These receptors have LIR consensus sequences that bind LC3/GABARAP, possibly leading to mitochondrial breakdown. Adenovirus is utilized to investigate mitophagy owing to its high efficiency, significant genomic capacity, and limited immunogenicity.

Fig. 1 PiNK1-parkin pathways of mitophagy.¹

Creative Biolabs has launched several adenovirus packaging services for mitophagy research, in which fluorescent tags are fused at the C-termini of the autophagosome marker LC3, and autophagy pathway markers such as Parkin, Bnip3, Nix, and FUNDC1, allowing to detection the formation and fusion of mitophagy.

Ads for Mitophagy Phenotype Research

We labeled autophagosomes with the LC3-GFP and mitochondria with the Mito-Dsred. They co-infect target cells with HBAD-Mito-dsred, a mitochondria-specific localization fluorescence probe that can detect the role of the LC3 protein in autophagy initiation.

• Ad-GFP-LC3

Ad-GFP-LC3 is an adenovirus vector that contains the structural domains of green fluorescent protein (GFP) and microtubule-associated protein 1 light chain 3 (LC3). LC3 is a protein associated with autophagy, and its presence is observed in membrane structures during the autophagic process. By expressing Ad-GFP-LC3, researchers can track the formation and fusion of autophagosomes during the autophagic process.

• Ad-Dsred-Mito

Ad-Dsred-Mito is an adenovirus vector carrying red fluorescent protein (Dsred) and a mitochondrial targeting sequence. This vector is used to label mitochondria, aiding in the study of the localization and removal of mitochondria during the process of mitophagy.

Ads for Mitophagy Pathways Research

We employed the mitophagy pathway marker Parkin-EGFP/Bnip3-EGFP+Nix-EGFP/FUNDC1-EGFP to mark essential mitophagy pathway signaling components and mitochondria with Mito- Dsred. They co-infected target cells with HBAD-Mito-dsred to locate mitochondria. Confocal was used to detect co-localization and identify the mitochondrial translocation of relevant signaling molecules.

• Ad-Parkin-EGFP

Parkin is a protein involved in the regulation of mitophagy. The Ad-Parkin-EGFP vector can be used to investigate the localization and activity of Parkin within cells, providing insights into the mechanisms of mitophagy.

• Ad-Bnip3-EGFP+Ad-Nix-EGFP

BNIP3 and NIX are the mitochondrial outer membrane proteins related to the BH3-only family, which induce mitophagy. The Ad-Bnip3-EGFP+Ad-Nix-EGFP can be used to study how BNIP3 and NIX induce mitophagy.

• Ad-FUNDC1-EGFP

FUNDC1 is a protein associated with mitophagy, binding to the mitochondrial membrane. The Ad-FUNDC1-EGFP vector can be employed to study the function and mechanism of FUNDC1 in the regulation of mitophagy.

Creative Biolabs provides high-quality Ads that titer can reach more than 1x10¹⁰ PFU/mL for mitophagy. We not only have various types of packaged Ads for consumers, but we also have extensive customizing experience to provide customized Ads services for customers. Please contact us for more information.

Reference

1. Sekine, Shiori, and Richard J. Youle. "PINK1 import regulation; a fine system to convey mitochondrial stress to the cytosol." BMC biology 16 (2018): 1-12.