Journal Club

Leucine aminopeptidase LyLAP enables lysosomal degradation of membrane proteins

Science 28 Mar 2025 Vol 387, Issue 6

INTRODUCTION

Transmembrane proteins constitute about 20 to 30% of the human proteome and are crucial for processes such as nutrient transport, signaling, and cell adhesion. These proteins are degraded within lysosomes, where specialized enzymes break them down into single amino acids. Macropinocytosis and phagocytosis may accelerate membrane protein turnover, especially in pathologies such as pancreatic ductal adenocarcinoma PDA, where cells rely on enhanced lysosomal activity for nutrient acquisition. However, the lysosomal degradation of membrane proteins is incompletely understood; specifically, how the hydrophobic, lipid-embedded regions of these proteins are degraded is unclear. Efficient degradation of transmembrane domains is critical because their hydrophobic nature can result in aggregation, while their lipid-intercalating properties can compromise lysosomal membrane integrity.

RATIONALE

Lysosomal proteolysis of hydrophobic α-helical domains is a key but poorly understood step in the life cycle of integral membrane proteins and is likely critical in cell types that engage in high rates of endocytosis, including phagocytic immune cells and certain cancer types. However, no lysosomal enzymes that mediate this activity have been identified.

RESULTS

By combining lysosomal proteomics with functional genomics, we identified PLBD1 (phospholipase B domain–containing 1), renamed LyLAP (lysosomal leucine aminopeptidase), as a top-ranking lysosomal hydrolase in cells that undergo high rates of endocytosis, specifically PDA and phagocytic immune cells. In PDA cells, LyLAP knockdown led to both morphological and functional defects; lysosomes became grossly enlarged and deacidified, accumulating undigested substrates, including proteins and lipids.

On the basis of its structural features, LyLAP was predicted to hydrolyze amide bonds, but its true substrates remained unknown. By reconstituting LyLAP-depleted lysosomes with recombinant LyLAP protein, we identified hydrophobic peptides as its bona fide substrates. LyLAP processively degraded these peptides starting at their amino termini, which most often consist of hydrophobic amino acids, such as leucine. Most LyLAP substrate peptides identified mapped to the membrane-spanning regions of integral membrane proteins, many of which reside at the plasma membrane and are trafficked to the lysosome for degradation.

Consistent with the key role for LyLAP in degrading hydrophobic peptides, these substrates accumulated in LyLAP-depleted lysosomes. This accumulation destabilized the lysosomal limiting membrane and impaired lysosomal function.

LyLAP was the most up-regulated lysosomal hydrolase in PDA cell lines and PDA patient samples. Knockdown of LyLAP was sufficient to arrest proliferation and cause cell death in PDA cell lines.

CONCLUSION

Our discovery of LyLAP addresses a major gap in lysosomal proteolysis. Its processive activity on hydrophobic residues ensures complete degradation of single-pass and multi-pass transmembrane proteins. Questions remain about how LyLAP cooperates with other hydrolases, the sequence of proteolytic events, and alternative mechanisms in LyLAP-deficient cells.

The elevated expression of LyLAP in PDA and their strongly reduced viability upon LyLAP ablation suggest a model where, by delivering large amounts of integral membrane proteins to the lysosome, the elevated macropinocytic activity of PDA cells makes them highly dependent on LyLAP function.