Determining the molecular mehanisms regulating lysosome damage and repair pathways

master's thesis

• Title (croatian): Određivanje molekularnih mehanizama u regulaciji oštećenja i popravka lizosoma
• Author: Mihaela Božić
• Mentor: David McEwan (mentor)
• Mentor: Inga Marijanović (mentor)
• Committee member: Inga Marijanović (predsjednik povjerenstva)
• Committee member: Nada Oršolić (član povjerenstva)
• Committee member: Maria Špoljar (član povjerenstva)
• Committee member: Sofia Ana Blažević (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2018-09-28, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Lysosomes are organelles surrounded by a single lipid layer, encapsulating hydrolytic enzymes,
and serve as cell’s primary catabolic compartments. Lysosome membranes are easily damaged
by the intake of lyososomotropic agents such as L-Leucyl-L-Leucine methyl ester (LLOMe).
This event can be recognized by recruitment of Galectin3 to the damaged membranes. Damaged
lysosomes can be further repaired, or cleared by autophagy. Autophagic degradation of the
lysosomes themselves is termed lysophagy. Little is known of how the cell senses that damage,
how it is repaired, or how LLOMe mediated lysosome damage impacts autophagy flux. By
overexpressing fluorophore tagged Galectin3 damaged lysosomes can be counted and LLOMe
influence characterized. siRNA knock down of specific genes will result in higher number of
Galectin3 signal when silenced genes are involved in lysosome damage recognition and
repair/clearance. Here, we provide 7 genes identified as possible mediators of the lysosomal
biogenesis pathways that can serve as base for future studies and further validation. Identifying
those pathways could serve a therapeutic potential in tumours with high levels of cathepsins,
inflammations, infections, and neurodegenerative diseases. We also characterize influence of
LLOMe intake on autophagy flux, being simultaneously a potent autophagy inducer, and
inhibitor of autophagy progression by autophagosome-lysosome fusion.

Abstract (croatian)

Lizosomi su organeli omeđeni jednostukom membranom, sadržavaju hidrolitičke enzime i
služe kao primarni katabolički organeli eukariotskih stanica. Lizosomi su vrlo podložni
oštećenju pri ulasku lizozomotropnih spojeva, kao što je detergent L-leucil-L-leucin metil ester
(LLOMe), u stanicu. Takav događaj može se prepoznati prema regrutaciji proteina Galektin3
na oštećenu membranu lizosoma. Oštećeni lizosomi posljedično bivaju popravljeni ili
razgrađeni putem autofagije. Do sada je vrlo malo poznat poces prepoznavanja i popravlja takva
oštećenja, te kako oštećenje lizosoma izazvano detergentom LLOMe utječe na process
autofagije. Ekspresijom fuzijskog proteina GFP-Galektin3 oštećeni lizosoma mogu se
vizualizirati i izbrojati. Utišavanje pojedinih gena pomoću siRNA rezultira u povećanom broju
Galektin3 u stanici ako su ti geni uključeni u prepoznavanje oštećenih lizosoma ili njihov
popravak/degradaciju. U ovom radu predlažemo 7 gena prepoznatih kao medijatore puta
biogeneze lizosoma koji mogu poslužiti kao baza za daljnja istraživanja i detaljnu
karakterizaciju staničnih signalnih puteva. Potpuno razumijevanje mehanizma prepoznavanja
oštećenih lizosoma i njihovog popravka može poslužiti pri identifikaciji terapeutske mete kod
tumora s visokom razinom katepsina, upala, infekcija i neurodegenerativnih bolesti. Također
pružamo uvid u utjecaj LLOMe na proces autofagije i označavamo LLOMe kao snažan
induktor autofagije, te istovremeno inhibitor napretka progresije autofagije pri koraku fuzije
autofagosoma s lizosomom.

• Keywords: autophagy; lysophagy; LMP; LLOMe; Galectin3; siRNA
• Keywords (croatian): autofagija; lizofagija; LMP; LLOMe; galectin3; siRNA
• Language: english
• URN:NBN: urn:nbn:hr:217:147253
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2020-09-28
• Created on: 2018-10-29 12:00:16