⚗️ biochemistry

Intralysosomal Amyloidogenesis and Proximity Labeling by Cathepsin C

This study reveals that Cathepsin C converts the lysosomal damage agent LLOMe into amyloid fibrils and, upon membrane rupture, utilizes its endogenous dipeptidyl ligase activity to perform the first fully endogenous, non-engineered proximity labeling within the lysosome, thereby uncovering new mechanisms of lysosomal perturbation and a novel tool for studying lysosomal biology.

Original authors: Elias, R. D., Allen, S., Demiralp, I. I., O'Neill, R. T., Shäfer, J.-H., Siems, H., Montabana, E. A., Ermel, U. H., Ash, C., Abdurrob, F., Yacoubian, D. A., Lederberg, O. L., Serwas, D., Agard, D. A.

Published 2026-07-07

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CC BY 4.0

Original authors: Elias, R. D., Allen, S., Demiralp, I. I., O'Neill, R. T., Shäfer, J.-H., Siems, H., Montabana, E. A., Ermel, U. H., Ash, C., Abdurrob, F., Yacoubian, D. A., Lederberg, O. L., Serwas, D., Agard, D. A., Cravatt, B. F., Kelly, J. W.

Original paper licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). ⚕️ This is an AI-generated explanation of a preprint that has not been peer-reviewed. It is not medical advice. Do not make health decisions based on this content. Read full disclaimer

Imagine your cell as a bustling city, and inside that city, there is a specialized recycling center called the lysosome. Its main job is to break down trash and old parts so the city can reuse the materials. Usually, this recycling center is safe and contained, but sometimes, things go wrong.

Scientists have long used a specific chemical, let's call it LLOMe, to intentionally break the walls of this recycling center in the lab. They knew it caused damage, but they didn't fully understand how it happened. It was like knowing a bomb went off in the recycling plant, but not knowing if it was a fire, a gas leak, or a structural collapse.

This new study acts like a high-tech detective story that solves the mystery in two surprising ways:

1. The "Self-Assembling Trap"

The researchers discovered that when LLOMe enters the recycling center, a specific worker enzyme inside called Cathepsin C chops it up. Instead of just breaking it down into harmless dust, this enzyme accidentally turns the pieces into tiny, rigid building blocks.

Think of it like a construction crew that is supposed to demolish a building but instead starts stacking the bricks into a giant, jagged wall. These blocks snap together to form long, stiff chains called amyloid fibrils. These chains grow right inside the recycling center, acting like a swarm of microscopic spears that eventually poke holes in the walls, causing the center to leak and break.

2. The "Emergency Sticker"

Here is the second, more clever discovery. When the walls of the recycling center finally crack and the damage is done, that same enzyme, Cathepsin C, switches jobs. It stops just chopping things up and starts acting like a glue gun.

Normally, this enzyme is very picky about what it sticks together. But in this damaged, chaotic environment, it becomes "broadly nonspecific." It starts grabbing any nearby protein floating in the mess and sticking a special, invisible "click" tag onto it.

Think of it like a security guard in a burning building who suddenly starts slapping bright neon stickers on everyone standing near the fire. The scientists can then use these stickers to find out exactly which proteins were hanging out in the damaged area.

Why This Matters

The most exciting part is that the scientists didn't have to build a new machine or engineer a special enzyme to do this tagging. They used Cathepsin C, a worker that already exists naturally in every human cell, without any modifications.

It's like discovering that the janitor in your office building has a hidden talent for painting murals on the walls, and you can use that talent to map out the building's layout. This study shows us two things:

How a common chemical breaks the recycling center (by turning into sticky, spear-like chains).
How the cell's own natural tools can be used as a "proximity label" to tag and study what happens right where the damage occurs.

In short, the paper reveals that LLOMe doesn't just break the lysosome; it triggers a chain reaction where the cell's own enzymes build damaging structures and then accidentally start tagging the wreckage, giving scientists a new way to look inside the cell's recycling center.

Technical Summary: Intralysosomal Amyloidogenesis and Proximity Labeling by Cathepsin C

Problem Statement
The lysosome serves as a primary catabolic organelle for degrading intra- and extracellular substrates. A critical area of recent investigation involves lysosomal membrane damage induced by pathologic amyloid fibrils. To study this phenomenon, researchers have frequently utilized the dipeptide ester LLOMe (leucyl-lysine methyl ester) as a model agent to induce lysosomal membrane damage. However, the precise molecular mechanism by which LLOMe causes membranolysis has remained incompletely understood. Furthermore, while the lysosomal protease Cathepsin C is known to process LLOMe, the downstream consequences of this processing within the lysosomal lumen and the potential for utilizing endogenous enzymes for proximity labeling in damaged lysosomes had not been fully characterized.

Methodology
The study employed a multi-faceted approach combining in vitro biochemical assays, cell-based analyses, and advanced structural biology techniques.

Structural Analysis: Cryo-electron microscopy (cryo-EM) and cryo-electron tomography were utilized to visualize the structural organization of LLOMe-derived products within the lysosome.
Biochemical and Cellular Assays: The researchers investigated the enzymatic activities of Cathepsin C in response to LLOMe treatment and lysosomal membrane damage.
Proximity Labeling Strategy: The study explored the ability of Cathepsin C to facilitate the tagging of proteins within the damaged lysosome lumen using a click chemistry handle, leveraging the enzyme's ligase activity.

Key Findings and Results

Intralysosomal Amyloidogenesis: The study reveals that LLOMe is processed by Cathepsin C into oligopeptides. Contrary to previous assumptions, these oligopeptides do not merely accumulate but assemble into cross- $\beta$ -sheet amyloid fibrils specifically within the lysosome. Cryo-EM and tomography confirm that these fibrils are the direct agents of lysosomal membrane damage.
Cathepsin C Ligase Activity: The research demonstrates that lysosomal membrane damage triggers a broadly nonspecific dipeptidyl ligase activity in Cathepsin C. This activity allows the enzyme to attach a click chemistry handle to proximal proteins located within the damaged lysosome lumen.
Endogenous Proximity Labeling: This mechanism represents the first reported instance of a localized proximity labeling approach that exploits a fully endogenous, non-engineered enzyme. While Cathepsin C ligase activity has been previously demonstrated in vitro, this study confirms its occurrence on proximal proteins within living cells.

Significance and Claims
The paper claims to unveil two distinct mechanisms by which dipeptide esters, specifically LLOMe, perturb lysosomal homeostasis:

The generation of amyloid fibrils that physically damage the lysosomal membrane.
The activation of a ligase function in Cathepsin C that enables the labeling of the local proteome in damaged lysosomes.

The authors posit that these findings provide a roadmap for utilizing dipeptide esters in targeted studies of the lysosome. Furthermore, the observation of Cathepsin C's role in dipeptidyl ligation suggests an unexplored biological function for this enzyme in lysosomal biology. Broadly, the work exemplifies how other endogenous enzymes might be similarly exploited for proximity labeling, offering a new paradigm for studying organelle-specific proteomes without the need for engineered tools.

1. The "Self-Assembling Trap"

2. The "Emergency Sticker"

Why This Matters

Technical Summary: Intralysosomal Amyloidogenesis and Proximity Labeling by Cathepsin C

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