Umetne lipidne kapljice s sfingomielinom in holesterolom in njihove interakcije z beljakovinskimi tvorci por

Vezočnik, Valerija (author), Maček, Peter (mentor), Žagar, Ema (comentor)

Naravne lipidne sisteme je zaradi njihove kompleksnosti, težavnosti izolacije ter morebitne neobstojnosti večinoma precej težko raziskovati in zato imajo natančno definirani modelni sistemi ključno vlogo pri razumevanju le-teh. Do sedaj so se za študije naravnih lipidnih kapljic (LK) večinoma uporabljale neobstojne in slabo okarakterizirane umetne lipidne strukture, kjer so dodatno težavo povzročale še slabo opisane tehnike za njihovo pripravo. Tako se je porodila ideja, da bi lahko lipidne nanoemulzije zaradi svoje zgradbe in lastnosti služile kot modelni lipidni sistem ne zgolj za študije naravnih LK, ampak tudi za namene raziskovanja bioloških membran ter njihovih interakcij s proteini. V naši študiji smo se osredotočili na pripravo in karakterizacijo umetnih LK oziroma lipidnih nanoemulzij LK, sestavljenih iz sfingomielina (SM), holesterola (Chol) in trioleoil glicerola (TOG), kjer se SM in Chol na površini urejata v lipidni monosloj, notranjost pa zapolnjuje TOG. O lastnostih umetnih lipidnih veziklov oziroma Langmuirjevega monosloja iz SM/Chol je veliko znanega, nanoemulzije LK v tej sestavi pa v literaturi še niso bile opisane, zato je naša raziskava pripomogla k boljšemu poznavanju lastnosti le-teh. Poleg karakterizacije LK predstavljajo pomemben del naše raziskovalne študije tudi njihove interakcije z izbranimi beljakovinskimi tvorci por, ki v membranah tvorijo transmembranske pore. Čeprav mehanizem nastanka le-teh še ni čisto pojasnjen, velja, da lahko poteka preko nastanka t.i. pre-por. Za namene naše študije smo tako izbrali od sfingomielina-odvisni citolizin ekvinatoksin II (EqTx II), od holesterola odvisna toksina perfringolizin O (PFO) in listeriolizin O ter bikomponentna tvorca por pleurotolizin B in ostreolizin A. S primerjalno študijo njihovih interakcij z nanoemulzijami LK in velikimi unilamelarnimi vezikli smo pokazali, da transmembranske pore v lipidnih monoslojih LK ne morejo nastati. Še več, kot prvi smo tudi uspešno vizualizirali različne stopnje nastanka pre-pore za primer PFO ter strukture (pre)por EqTx II. Dokazali smo, da dobro definirane umetne LK z natančno izdelanim protokolom priprave predstavljajo pomembo orodje na področju bioloških in bio(medicinskih) raziskav. Naša študija prav tako potrjuje, da je moč z interdisciplinarnim raziskovalnim pristopom in uporabo zgolj temeljnih znanj uspešno povezati različna znanstvena področja.

• Language: Slovenian
• Keywords: lipidne kapljice, liposomi, tvorba transmembranske pore, pre-pora, interakcije med lipidi in proteini, modelni lipidni sistem
• Work type: Doctoral dissertation
• Organization: MF - Faculty of Medicine
• Year: 2021
• PID: 20.500.12556/RUL-127423
• COBISS.SI-ID: 130164483
• Publication date in RUL: 06.06.2021

Secondary language

• Language: English
• Title: Artificial lipid droplets with sphingomyelin and cholesterol and their interaction with pore-forming proteins

Abstract

Researching natural lipid systems is often challenging due to their complexity, possible instability and isolation difficulty. Well-defined model lipid systems, therefore, play a key role in their understanding. So far, unstable and not well-characterized artificial lipid structures prepared by poorly designed techniques have been used for studying natural lipid droplets (LDs). As nanoemulsions share many physico-chemical characteristics in common with natural LDs, our study was therefore stimulated by the idea that nanoemulsions of LDs might serve as a model lipid system for studying not only natural LDs, but also biological membranes and their interaction with lipid specific proteins. Our study focused on the preparation and characterization of artificial LDs named nanoemulsions of LDs, composed of a trioleoylglycerol (TOG) core coated with a sphingomyelin (SM) and cholesterol (Chol) monolayer. This lipid combination was chosen as nanoemulsions covered by combined SM and Chol have been poorly characterized in contrast to respective SM/Chol vesicles or Langmuir monolayers. As such, our study contributed to a better understanding of their properties. Besides LDs’ characterization, an important part of our research was also dedicated to studying their interaction with selected pore forming proteins that form transmembrane pores in membranes. Although the exact mechanism of transmembrane pore formation has not been precisely explained yet, it is assumed that pore formation is a multi-step process, involving the formation of structures called pre-pores. For the purpose of our study, sphingomyelin-dependent cytolysin Equinatoxin II (EqTx II), cholesterol-dependent toxins Perfringolysin O (PFO) and Listeriolysin O, and a binary pore-forming protein complex Ostreolysin A (OlyA)/Pleurotolysin B (PlyB) were selected. Herein, we confirmed that transmembrane pores on the LDs monolayers could not be formed. Furthermore, different stages of PFO pre-pore formation were successfully visualized as well as the (pre)pore structures formed by EqTx II. Our study confirmed that well-defined artificial LD with a precisely designed preparation procedure might play an important role in the field of biological and bio(medical) research. Herein, a great advantage of interdisciplinary research approach using the fundamental knowledge was successfully demonstrated.

• Keywords: lipid droplets, liposomes, transmembrane pore formation, pre-pore, protein-lipid interaction, model lipid system