A pulse-chasable reporter processing assay for mammalian autophagic flux with HaloTag

eLife, Journal Year: 2022, Volume and Issue: 11

Published: Aug. 8, 2022

Monitoring autophagic flux is necessary for most autophagy studies. The assays currently available mammalian cells are generally complicated and do not yield highly quantitative results. Yeast routinely monitored with the green fluorescence protein (GFP)-based processing assay, whereby amount of GFP proteolytically released from GFP-containing reporters (e.g. GFP-Atg8), detected by immunoblotting, reflects flux. However, this simple effective assay typically inapplicable to because efficiently degraded in lysosomes while more resistant red fluorescent (RFP) accumulates under basal conditions. Here, we report a HaloTag (Halo)-based reporter monitor We found that Halo sensitive lysosomal proteolysis but becomes upon ligand binding. When delivered into autophagy, pulse-labeled Halo-based Halo-LC3 Halo-GFP) processed generate when autophagy. Hence, free immunoblotting or in-gel imaging demonstrate applications monitoring pathways, macroautophagy, selective even bulk nonselective With lysosome-mediated degradation can be easily precisely.

Language: Английский

---

Targeting immunogenic cell stress and death for cancer therapy

Nature Reviews Drug Discovery, Journal Year: 2024, Volume and Issue: 23(6), P. 445 - 460

Published: April 15, 2024

Language: Английский

---

Crosstalk between mitochondrial biogenesis and mitophagy to maintain mitochondrial homeostasis

Journal of Biomedical Science, Journal Year: 2023, Volume and Issue: 30(1)

Published: Oct. 12, 2023

Mitochondrial mass and quality are tightly regulated by two essential opposing mechanisms, mitochondrial biogenesis (mitobiogenesis) mitophagy, in response to cellular energy needs other environmental cues. Great strides have been made uncover key regulators of these complex processes. Emerging evidence has shown that there exists a tight coordination between mitophagy mitobiogenesis, their defects may cause many human diseases. In this review, we will first summarize the recent advances discovery molecular regulations mitobiogenesis then focus on mechanism signaling pathways involved simultaneous regulation tissue or cultured cells needs, stress, pathophysiological conditions. Further studies crosstalk processes at level provide better understanding how cell maintains optimal fitness function under physiological conditions, which holds promise for fighting aging aging-related

Language: Английский

---

Cellular Senescence in Intervertebral Disc Aging and Degeneration: Molecular Mechanisms and Potential Therapeutic Opportunities

Biomolecules, Journal Year: 2023, Volume and Issue: 13(4), P. 686 - 686

Published: April 18, 2023

Closely associated with aging and age-related disorders, cellular senescence (CS) is the inability of cells to proliferate due accumulated unrepaired damage irreversible cell cycle arrest. Senescent are characterized by their senescence-associated secretory phenotype that overproduces inflammatory catabolic factors hamper normal tissue homeostasis. Chronic accumulation senescent thought be intervertebral disc degeneration (IDD) in an population. This IDD one largest age-dependent chronic often neurological dysfunctions such as, low back pain, radiculopathy, myelopathy. (SnCs) increase number aged, degenerated discs, have a causative role driving IDD. review summarizes current evidence supporting CS on onset progression The discussion includes molecular pathways involved as p53-p21CIP1, p16INK4a, NF-κB, MAPK, potential therapeutic value targeting these pathways. We propose several mechanisms including mechanical stress, oxidative genotoxic nutritional deprivation, stress. There still large knowledge gaps research, understanding which will provide opportunities develop interventions treat

Language: Английский

---

ULK1-mediated metabolic reprogramming regulates Vps34 lipid kinase activity by its lactylation

Science Advances, Journal Year: 2023, Volume and Issue: 9(22)

Published: June 2, 2023

Autophagy and glycolysis are highly conserved biological processes involved in both physiological pathological cellular programs, but the interplay between these is poorly understood. Here, we show that glycolytic enzyme lactate dehydrogenase A (LDHA) activated upon UNC-51-like kinase 1 (ULK1) activation under nutrient deprivation. Specifically, ULK1 directly interacts with LDHA, phosphorylates serine-196 when nutrients scarce promotes production. Lactate connects autophagy through Vps34 lactylation (at lysine-356 lysine-781), which mediated by acyltransferase KAT5/TIP60. enhances association of Beclin1, Atg14L, UVRAG, then increases lipid activity. autophagic flux endolysosomal trafficking. skeletal muscle during intense exercise maintains cell homeostasis correlates cancer progress inducing autophagy. Together, our findings describe regulation mechanism integrate glycolysis.

Language: Английский

---