Analiza delovanja inhibitorja kinaze Hog1 na aktivnost HwHog1 pri glivi Hortaea werneckii : diplomska naloga

Kejžar, Anja (author), Kos, Janko (mentor), Lenassi, Metka (comentor)

Kljub številnim raziskavam odziva na povišano okoljsko osmolarnost pri ekstremno halotolerantni črni kvasovki Hortaea werneckii, ostaja vprašanje ključne in nenadomestljive vloge MAP kinaze HwHog1 v odgovoru nerešeno. Aktivacija HwHog1 je uravnavana s signalno potjo HOG, ki preko senzorjev v membrani prenese signal na MAP kinazno kaskado, ki sproţi fosforilacijo HwHog1, ta pa nadalje uravnava izraţanje tarčnih genov in delovanje določenih proteinov. Pri reševanju tega problema smo se odločili za kemijsko-genetski pristop, pri čemer ţelimo za inhibicijo aktivnosti HwHog1 uporabiti prvi znan inhibitor aktivnosti nativnega Hog1, homologa proteina HwHog1 pri Saccharomyces cerevisiae. V ta namen smo najprej pripravili kvasni vektor z vstavljenim brezintronskim genom HwHOG1, ga s transformacijo prenesli v sev S. cerevisiae z mutiranim lastnim genom HOG1 in z analizo odtisa western nakazali na prisotnost proteina HwHog1 v celicah S. cerevisiae, transformiranih z omenjenim plazmidom. Pripravljen sistem je omogočil preizkus delovanja inhibitorja na HwHog1 v enakem genetskem ozadju, kot je bilo uporabljeno za testiranje delovanja inhibitorja na Hog1. S testi funkcionalne komplementacije ter detekcijo s protitelesi proti fosfo(p38) smo pokazali, da HwHog1 uspešno komplementira funkcijo MAP kinaze Hog1 v celicah S. cerevisiae, torej lahko sprejme signal od MAPK kinaze Pbs2, se translocira v jedro in tam regulira izraţanje genov, kar omogoči prilagoditev S. cerevisiae na povišano osmolarnost v okolju. Z inhibitorskimi testi na ploščah smo pokazali, da inhibitor PD447, ki dokazano deluje na MAP kinazo Hog1 v S. cerevisiae, učinkovito inhibira tudi HwHog1, homologni protein iz H. werneckii, kadar je ta izraţen v S. cerevisiae. Inhibitor je posledično primeren za nadaljnjo uporabo pri kemijsko-genetskih študijah HOG poti pri H. werneckii. V prihodnjih raziskavah ţelimo preveriti, ali inhibitor PD447 učinkovito deluje tudi na aktivnost HwHog1, kadar se ta izraţa v črni kvasovki H. werneckii in tako prvič pokazati edinstvenost vloge HOG poti in HwHog1 v odzivu na povišano osmolarnost v okolju pri črni kvasovki H. werneckii.

• Language: Slovenian
• Keywords: Hortaea werneckii Hog1 inhibitorji Hog1 MAP kinaza signalna pot Hog
• Work type: Undergraduate thesis
• Typology: 2.11 - Undergraduate Thesis
• Organization: FFA - Faculty of Pharmacy
• Place of publishing: Ljubljana
• Publisher: [A. Kejžar]
• Year: 2011
• Number of pages: VII, 62 f.
• PID: 20.500.12556/RUL-121159
• UDC: 542:577
• COBISS.SI-ID: 3082097
• Publication date in RUL: 30.09.2020

Secondary language

• Language: English
• Title: Analysis of the Hog1 kinase inhibitor activity on the HwHog1 in fungus Hortaea werneckii

Abstract

Despite several previous studies focusing on osmostress response in the extremely halotolerant black yeast Hortaea werneckii, the key role of the MAP kinase HwHog1 remains unsolved. HwHog1 activation is regulated by the HOG signaling pathway, through which the signal is transferred from the membrane osmosensors to the MAP kinase cascade inside the cell. This triggers HwHog1 phosphorylation, which leads to its activation and regulation of expression of target genes and activity of target proteins. To resolve this question, we wanted to employ a chemical-genetic approach using a novel inhibitor of the native Hog1 kinase, a Saccharomyces cerevisiae homologue of the HwHog1 kinase. To this end, we constructed a yeast plasmid with inserted intronless HwHOG1 gene and transformed it into the S. cerevisiae strain with deleted HOG1 gene. Western blotting confirmed the presence of the HwHog1 in the transformed cells. The described system enabled us to study the effect of the inhibitor in the same genetic background as was used for the Hog1 inhibitor testing. Functional complementation assays and western blotting using antibodies against phospho(p38) showed, that HwHog1 was able to complement the MAP kinase Hog1 function in S. cerevisiae Δhog mutant and rescue the osmosensitive phenotype. Evidently, HwHog1 is able to accept the signal from the MAPK kinase Pbs2 and translocate to the nucleus where it regulates the expression of several genes, enabling S. cerevisiae to adapt to the increased osmolarity in the environment. Moreover, the inhibitor plate tests showed that PD447, which is an effective in vivo inhibitor of the Hog1 MAP kinase, efficiently inhibits the HwHog1 kinase when expressed in S. cerevisiae. Altogether we showed the suitability of the use of inhibitor PD447 in the chemical-genetic approach for studying the role of HOG response pathway in H. werneckii. In the future we want to examine the effect of the inhibitor on the HwHog1 kinase activity in H. werneckii and hopefully demonstrate the essential role of the HOG pathway in osmostress response in the black yeast H. werneckii.