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Proximity labelling identification of ULK1 wild-type (WT) and ULK1-K46I (kinase dead) interactors upon mitochondria tethering in U2OS WT cells

ABSTRACT: This project involved generating three stable cell lines using a Flp-In system, each containing specific integrated constructs: "FKBPx2-myc-APEX2-ULK1", "FKBPx2-myc-APEX2-ULK1-K46I" (kinase-dead mutant), and "FKBPx2-myc-APEX2" (control). All cell lines additionally express an outer mitochondrial membrane-targeted construct consisting of truncated FIS193–152 fused to FRB (FRB-FIS193–152). Upon treatment with a heterodimerizer (rapalog), the cytosolic FKBP-tagged constructs are recruited to mitochondria via FRB-rapalog-FKBP interactions, enabling controlled mitochondrial tethering. The constructs contain APEX2 peroxidase, which catalyzes biotin-phenol biotinylation of nearby proteins upon H₂O₂ treatment, allowing proximity-dependent protein identification. The primary objective was to investigate the interactomes of wild-type ULK1 versus the kinase-dead ULK1-K46I mutant when localized to mitochondria. By comparing the biotinylated protein profiles between these conditions and the APEX2-only control, we aimed to identify ULK1-specific interacting partners and determine how kinase activity affects these interactions in the mitochondrial environment. This approach provides insights into ULK1's role in mitophagy and autophagy regulation.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Flp-in-t-rex Cell

SUBMITTER: David Hollenstein

LAB HEAD: Prof. Dr. Claudine Kraft

PROVIDER: PXD068381 | Pride | 2026-04-24

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	2023.03_UP000005640_9606_Homo_sapiens_1protein_per_gene.fasta	Fasta
	20230810_E1_NEO3_Babic_Kraft_MPLGmbH_Academic_MPLID23159_s10_41_23p.raw	Raw
	20230810_E1_NEO3_Babic_Kraft_MPLGmbH_Academic_MPLID23159_s11_42_19p.raw	Raw
	20230810_E1_NEO3_Babic_Kraft_MPLGmbH_Academic_MPLID23159_s12_43_18p.raw	Raw
	20230810_E1_NEO3_Babic_Kraft_MPLGmbH_Academic_MPLID23159_s13_51_24p.raw	Raw

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Project description:Why age is the primary driver of the most common dementia-Alzheimer’s disease (AD)? Autophagy plays fundamental roles in cellular homeostasis, metabolic energetics, and in maintaining cellular resilience such as via eliminating damaged mitochondria. It is proposed that impaired autophagy leads to compromised catabolic degradation of amyloid beta (Aβ) and Tau pathologies, the disease-defining pathologies of AD; however, molecular mechanisms on whether and if yes, why reduced autophagy during ageing and its contribution to AD, especially in human samples, are largely elusive. By using serum and CSF samples from a healthy ageing cohort (n = 22 with 4-year follow up samples) and an AD cohort (n = 391), we show that the abundance of ULK1 protein in serum and CSF decreases with increasing age and is extremely lower in AD patients than in age-matched controls. In a large number of postmortem human brain samples (n = 72), expression of ULK1 in entorhinal cortical and hippocampal neurons in AD patients is significantly lower than in neurons from cognitive normal controls. To investigate whether ULK1 reduction is a bystander or cause/risk of AD, we generated a ubiquitously ULK1-ov mouse model and an AAV-based CA1 neuronal ULK1-ov mouse model in Aβ-bearing background; ULK1 overexpression stimulates autophagic and phagocytic degradation of Aβ, improves mitochondrial quality, thereby delaying progressive cognitive loss. Similarly, in hTau.P301S mice, overexpression of ULK1 inhibits Tau pathology and inhibits memory loss; mechanistically, ULK1 upregulation increases autophagy which increases cellular NAD+, leading to the inhibition of acetylated Tau174 (a cause of Tau pathology) through the NAD+/SIRT1 axis. ULK1 is likely a druggable target for AD as small molecule activators of ULK1 antagonize AD progression which is dependent on PINK1- and FUNDC1-related mitophagy. We propose age-dependent autophagy impairment risks AD due to ULK1 reduction, opening a new window for anti-AD therapeutics.

Dataset Information

Proximity labelling identification of ULK1 wild-type (WT) and ULK1-K46I (kinase dead) interactors upon mitochondria tethering in U2OS WT cells

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