Naslov
Exploring protein-protein interactions of S- adenosyl homocysteine hydrolase (SAHH) using bi- molecular fluorescence complementation (BiFC)

Autori
Lepur, Adriana ; Kovačević, Lucija ; Belužić, Robert ; Grbeša, Ivana ; Muñoz Torres, Pau Marc ; Trmčić-Cvitaš, Jelena ; Vugrek, Oliver

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
PROCEEDINGS 3rd CROATIAN MICROSCOPY CONGRESS with International Participation / Ambriović Ristov, Andreja ; Gajović, Andreja ; Weber, Igor ; Vidoš, Ana - Zagreb : Institut Ruđer Bošković ; Hrvatsko mikroskopijsko društvo, 2015, 59-59

ISBN
978-953-7941-05-5

Skup
3rd CROATIAN MICROSCOPY CONGRESS with International Participation

Mjesto i datum
Zadar, Hrvatska, 26.04.2015. - 29.04.2015

Vrsta sudjelovanja
Predavanje

Vrsta recenzije
Domaća recenzija

Ključne riječi
S-adenosyl homocysteine hydrolase ; split-Venus fluorescent protein ; cell imaging ; interactome ; Adenosylhomocystein hydrolase-Like 1

Sažetak
S-adenosyl homocysteine hydrolase (SAHH) is the only mammalian enzyme capable of hydrolyzing S- adenosyl homocysteine – a byproduct in cellular methylation reactions. Thus, SAHH is crucial for maintaining the cellular methylation potential, indirectly impacting methylation of DNA, mRNA, tRNA, lipid and protein (1). SAHH deficiency leads to serious developmental defects - muscular hypotonia, brain white matter atrophy and liver inflammation. SAHH interacting partners could help elucidate the regulation of its expression, enzymatic activity and intracellular dynamics. In search for SAHH interactors we used a bi- molecular fluorescence complementation (BiFC) approach. To do so, we cloned proteins of interest into suitable host vectors and fused them to fluorescent Venus protein that is split into two parts. Subsequently, we expressed the fused proteins in either bacteria or in cultured human cells. Detection of fluorescence indicates reassembly of split-Venus, hinting at protein- protein interactions between proteins of interest. Using BiFC we confirmed SAHH self- association in human kidney cell line Hek293T. SAHH mutant that lacks C-terminal region necessary for SAHH multimerisation, served as a negative control. We also observed SAHH interaction with Adenosylhomocystein hydrolase- Like 1 (AHCYL1), a protein closely related to SAHH (2). This interaction plays a role in SAHH shuttling between the cytoplasm and the nucleus, where SAHH contributes to DNA methylation. Also, we found a few unexpected SAHH interactions, e.g. with TLR7. TLR7 mediates a response to viral single-stranded RNA after macrophages internalize it in endosomes. This interaction is less surprising if we know that the elements involved in TLR-mediated RNA responses are controlled by modifications such as methylation. Dysregulation of SAHH can also change DNA methylation pattern - a hallmark of malignant lesions, which are regularly coupled with progressive inflammation (3). To explore this further, we assessed SAHH and/or AHCYL1 levels in lung cancer samples and human monocytes, and compared it to the expression of an inflammatory marker Galectin-3. Immunocytochemistry showed that monocytes down- regulate SAHH after differentiation, but preserve AHCYL1 expression. When we used LPS to induce pro inflammatory macrophage phenotype, the cells stopped expressing AHCYL1 protein completely. Next, we aim to study how SAHH influences the immune function of monocytes and macrophages, while exploring its interactome using bi- molecular fluorescence complementation. Since SAHH dysfunction leads to numerous pathologies including cancer, hepatitis, neurological and vascular disorders, it is crucial to dig deeper into its regulation and intracellular functions.

Izvorni jezik
Engleski

Znanstvena područja
Biologija, Biotehnologija

POVEZANOST RADA