Page 225 - ebook

Isolation of lysosomal activator regulating lysosomal

damages and cell death

Heemin Park , Shin-Hyeon Ryu , Seo-hyun Kim , and Yong-Keun Jung *,1
1
1
1

1 Apoptosis and Molecular Disease Laboratory; School of biological science, Seoul National University, Seoul 08826, Korea

Abstract

Lysosomes contribute to maintaining cellular homeostasis via degradation and recycling of subcellular substances called autophagy, along with a protective action against foreign materials, such

as pathogens, and cellular protein aggregates. Therefore, cells avoid cytotoxicity of the invading pathogen or suppress cell death by degrading cytotoxic proteins and its aggregates through the

lysosomal function. We aimed to isolate small molecules that affect lysosomal damages and activities. In this study, we established a cell-based lysosomal activity assay and screened a bioactive

compound library to isolate new mediators affecting lysosomal damage. We generated HeLa/GFP-Galectin3 stable cell line and observed GFP-Gal3 puncta formation when exposed to

lysosomotropic LLOME. We isolated several compounds that affected the LLOME-induced GFP-Gal3 puncta formation. Among them, we found that mA receptor antagonist (mARA) rescued the

processing of cathepsin D and the lysosomal activity as assayed with DQ-red BSA. In addition, we found that mARA alleviated LLOME-induced apoptosis. Thus, we intend to broaden our

understanding on the lysosomal regulation to lysosomal disease models.

Result 3. mA receptor antagonists increase lysosomal activity

1. Establishment of a cell-based assay for identifying mediators

affecting regulation of intracellular lysosomal damage and activity

(A) HeLa cells were co-treated with 10 μM of

indicated compound and 10 μg/ml of DQ-

red-BSA. for 2 h. Then, 200 μM of LLoMe

was additively treated for 6 h. Images were

captured by fluorescence microscopy.

(B) Quantification of red fluorescence

intensity corrected by area in (A). Bars

represent mean ± S.E.M., compared with

nontreat(DMSO) control, one-way ANOVA.

n=3, cell number = 164-173

(C) Receptor M antagonist T03G06 dose

dependently increases lysosomal activity. 10

ug/ml of DQ-red-BSA was co-treated with

DMSO or 1/5/10 μM of compound T03G06 for

2 h, and then 200 μM of LLoMe was

additively treated for 6 h. Images were

captured by fluorescence microscopy.

(A) Schematic representation of the establishment of GFP-Gal3 expressing HeLa cell line and the LLoMe-

induced lysophagy and GFP-Gal3 puncta formation model. (B) Representative image of LLoMe-inudced GFP-

Gal3 puncta formation. HeLa/GFP-Gal3 cells were treated with 250 μM of LLoMe for 4 h. The images were 4. mA receptor antagonist T03G06(mARA) increases lysosomal activity

captured under fluorescence microscopy. (C) HeLa/GFP-Gal3 cells were plated in 12-well culture plate and

incubeated for 24 h. Cells were treated with or without 250 uM of LLoMe for 6 h. Cells were lysed with RIPA

buffer and whole cell lysate was subjected to western blot analysis using anti-Cathepsin D, anti-LC3, anti-GFP,

anti-β actin antibodies.

2. Screening strategy to isolate bioactive compounds regulating

lysosomal damage and activity

(A) HeLa cells were plated in 12-well culture plate. Cells were treated with indicated concentration of AQ-RA

741. After 2 h, 0, or 500 μM of LLoMe was added. After 6 h, cells were harvested and lysed with RIPA lysis buffer.

Samples were subjected to western blot analysis. (B,C) Quantification of western blot analysis. Relative band

intensity of conversion of LC3-I to LC3-II(B), and process of pro-Cathepsin D to mature-Cathepsin D was

measured.

5. T03G06 decreases LLoMe- 6. T03G06 decreased oAβ-

induced cell death induced cell death in HT22

C e ll d ea th as s a y (P I s ta in in g ) 1 6h

A  -in d u c e d c y to to x ic ity (c e ll d e a th )
*** *
** *
2 0 n .s
*** * 2 5

)
(A) Schematic representation of the screening strategy to isolate bioactive compounds regulating lysosomal t (% 1 5 ) (% 2 0

damage and activity using Tocriscreen mini bioactive compound library. The compound library was prepared s h c M A

at 1 mM. HeLa/GFP-Gal3 cells were plated in 96-well culture plate and incubated for 24 h. Cells were treated e o 1 0 in e 1 5

with 10 μM each of the compounds for 2 h. Then, 200 μM of LLoMe was added. After 4 h, lysosomal damage H I+ lc a 1 0

was assessed based on GFP-Gal3 puncta formation. The compounds showing decreased number of GFP-Gal3 I/P 5 C I+

puncta were isolated. (B) Pharmacological feature of Tocriscreen 2.0 Mini compound library. Various type of P I/P P 5

target classes(left) and its primary actions(right) are shown. 0

S O S O 1  M 5  M 0

M D M 1 0 u M S O S O ) ) ) P F
Discussion C trl_ D T 03G 06 C trl_ D M D M 0 6 (1  M 0 6 (5  M 0 6 (1 0  M

LLO M e (200 M) T 0 3 G T 0 3 G T 0 3 G

⚫ Novel regulators of lysosomal activity were isolated via functional screening on HeLa/GFP-Gal3 cells were plated in 96-well o A  (5  M )

Bioactive compound library culture plate and incubated for 24h. 1% DMSO HT22 cells were plated in 96-well culture plate

⚫ As putative activator of lysosome, T03G06 upregulates lysosomal activity and recovers or indicated concentration of AQ-RA 741 was with full media(10 % FBS). Indicated

lysosomal damage done by lysosomotropic agent, LLOMe. treated for 2 h, and then 0 or 200 uM of LLoMe concentration of compound and 5 μM of
oligomeric Aβ were co-treated with serum-free
⚫ TFEB phosphorylation at Ser211, which is the target residue of mTOR, was decreased was treated. After 16h, cells were stained with DMEM, and then cells were incubated for 48 h.
PI/Hoechst and cell death rate (PI/PI+Hoechst,
upon T03G06. This indicates that T03G06 may upregulate lysosomal activity through cell number) was calculated. Images were Cells were stained with PI+Cacein AM and cell

control of TFEB activity. captured by fluorescence microscopy. death rate was counted.