If you had to swallow a pouch of toxins, you’d like to be certain the pouch was indestructible. Similarly, every eukaryotic cell seeks to ensure that the membranes of its many miniscule sacs called lysosomes that contain damaging digestive enzymes, are in good repair. Hydrolytic enzymes in lysosomes recycle cellular trash and maintain a cell’s normal lifespan.
Leaky lysosomal membranes, technically known as “lysosomal membrane permeabilization” (LMP), are associated with a slew of age-related diseases. For example, the leakage of tau fibrils from compromised lysosomes is a key step in the progression of Alzheimer’s disease.
Healthy cells must therefore be on constant vigil to immediately repair breaches in lysosomal integrity. Yet, how cells detect and address LMP remains unclear.
In a new study published in the journal Nature (“A Phosphoinositide Signaling Pathway Mediates Rapid Lysosomal Repair”), scientists at the University of Pittsburgh, have identified a new signaling pathway that rapidly repairs leaks in lysosomal membranes, ensuring the health and survival of the cell. The authors call it the PITT pathway for “phosphoinositide-initiated membrane tethering and lipid transport,” and as a nod to their institution.
“Our hope was that if we could understand how lysosomes are protected from damage, we could devise new strategies to maintain health-span for patients,” said Toren Finkel, MD, PhD, director of the Aging Institute, professor of medicine at Pitt’s School of Medicine, and senior author of the study.
First author of the study, Jay Xiaojun Tan, PhD, assistant professor of cell biology at Pitt’s School of Medicine and a member of the Aging Institute, said, “Lysosome damage is a hallmark of aging and many diseases, particularly neurodegenerative disorders such as Alzheimer’s. Our study identifies a series of steps that we believe is a universal mechanism for lysosomal repair.”
Tan and Finkel used an unbiased proteomics-based approach and a promiscuous biotinylation enzyme to tag proteins at the lysosomal surface under normal conditions and after damage.
“This novel tool allowed us to identify what proteins are recruited to damaged lysosomes and how they contribute to rapid lysosomal membrane repair,” said Tan.
Finkel said, “This unbiased strategy was critical for discovering the PITT pathway and could be useful for finding quality control pathways for other organelles such as mitochondria.”
They found when lysosomes in cultured cells were experimentally damaged, an enzyme called PI4K2A (type II alpha phosphatidylinositol-4 kinase) rapidly appeared on lysosomes and attracted high levels of a lipid signaling molecule called PtdIns4P (phosphatidylinositol-4-phosphate).
“PtdIns4P is like a red flag. It tells the cell, ‘Hey, we have a problem here,’” said Tan. “This alert system then recruits another group of proteins called ORPs.”
ORP proteins (multiple oxysterol-binding proteins) work like tethers that bind PtdIns4P at one end and the endoplasmic reticulum (ER) at the other, orchestrating extensive new membrane contact sites (MCSs) between damaged lysosomes and the ER. The ER is a labyrinth of channels extending from the cell membrane to the nucleus. It facilitates the biosynthesis of proteins and lipids.
“The endoplasmic reticulum wraps around the lysosome like a blanket,” added Finkel. “Normally, the endoplasmic reticulum and lysosomes barely touch each other, but once the lysosome was damaged, we found they were embracing.”
Lipids such as cholesterol and phosphatidylserine are shuttled to the lysosome from the ER through MCSs to promptly patch up tears in lysosomal membranes. Phosphatidylserine also activates a lipid transfer protein called ATG2, which acts like a bridge to transfer additional lipids to lysosomes to seal tears.
“What’s beautiful about this system is that all components of the PITT pathway were known to exist, but they weren’t known to interact in this sequence or for the function of lysosome repair,” said Finkel. “I believe these findings are going to have many implications for normal aging and for age-related diseases.”
When Tan deleted the gene encoding PI4K2A in a cell line (U2OS) derived from epithelial cells, he found that the spread of tau fibrils and the accumulation of lipofuschin—a signature of aging—increased dramatically. This indicated defects in the PITT pathway could contribute to Alzheimer’s disease.
Earlier studies had shown that loss PI4K2A in humans and mice causes severe neurodegeneration and premature aging, but the molecular underpinnings were not known. The current study provides mechanistic clarification for these observations.
“The PITT pathway is essential for rapid lysosomal repair,” said Tan. “It is activated by multiple disease-related lysosomal perturbing conditions, suggesting that it is a universal lysosomal quality control mechanism.”
In future work, the team plans to investigate the in vivo impact of blocking or triggering the PITT pathway in mouse model studies and to conduct chemical screens to identify small molecules that boost the PITT pathway.
The work was funded by grants from the National Institutes of Health and the UPMC Competitive Medical Research Fund.
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