Določevanje interakcij med proteinoma PMS2 in novo varianto MLH1

Zavodnik, Tina (author), Petrovič, Uroš (mentor)

Človeška proteina MLH1 in PMS2 sestavljata heterodimer MutLα, ki ima ključno vlogo pri popravljanju neujemanja molekule DNA. Patogene mutacije v genih, ki zapisujejo za oba proteina, povezujemo z Lynchevim sindromom; to je dedna predispozicija za razvoj rakavih obolenj, predvsem raka debelega črevesa in danke. Odkrivanje in dokazovanje patogenih mutacij v genih za MLH1 in PMS2 pripomore k bolj zanesljivi in hitrejši diagnozi Lynchevega sindroma. V diplomskem delu smo se osredotočili na novo delecijsko varianto proteina MLH1 – LRG_216t1:c.2236_2247delCTGCCTGATCTA p.(Leu746_Leu749del) (MLH1_del746-749). Delecijska varianta MLH1_del746-749 je bila najdena v slovenski družini z znano zgodovino rakavih obolenj in je klasificirana kot verjetno patogena. Pri genetskih analizah osebe, ki nosi zapis za delecijsko varianto MLH1_del746-749, so zaznali visoko stopnjo mikrosatelitnih nestabilnosti in nenavadno izgubo izražanja proteina PMS2 pri imunohistokemičnem barvanju tumorskih vzorcev. Na podlagi vseh znanih podatkov smo predpostavili, da delecija štirih aminokislin na skrajnem C-končnem delu proteina MLH1 onemogoči interakcijo s PMS2 in s tem oblikovanje stabilnega heterodimera MutLα. V bioinformatskem delu diplomske naloge smo naredili modele struktur C-končnih delov proteinov PMS2, MLH1_del746-749 in dodatnih štirih patogenih variant proteina MLH1. Poleg tega smo naredili tudi model strukture C-končnega dela heterodimera MutLα, ki do danes še ni eksperimentalno določen. Na podlagi analize struktur smo predpostavili, da MLH1_del746-749 s PMS2 ne more interagirati. Svojo predpostavko smo preverili s tehniko dvohibridnega sistema kvasovke. Metoda dvohibridnega sistema kvasovke se je izkazala za uspešno pri dokazovanju interakcij, saj smo potrdili interakcijo med MLH1 in PMS2, ki je znana iz literature. Poleg tega niti MLH1 niti PMS2 nista sposobna samoaktivacije reporterskega sistema, s čimer smo potrdili, da so rezultati testa interakcij z dvohibridnim sistemom kvasovke med obema proteinoma zanesljivi. Test interakcije med MLH1_del746-749 in PMS2 je pokazal, da proteina med seboj interagirata in tvorita dovolj stabilen kompleks, da se aktivira reporterski sistem in omogoča rast kvasovk na selekcijskem gojišču. Delecija Leu746_Leu749 torej ne prepreči interakcije proteina MLH1 s PMS2 v celicah kvasovke, vpliva pa na stabilnost in funkcijo heterodimera MutLα v človeških celicah.

• Language: Slovenian
• Keywords: MLH1, PMS2, MutLα, Lynchev sindrom, dvohibridni sistem kvasovke
• Work type: Bachelor thesis/paper
• Typology: 2.11 - Undergraduate Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2021
• PID: 20.500.12556/RUL-129751
• COBISS.SI-ID: 79543811
• Publication date in RUL: 07.09.2021

Secondary language

• Language: English
• Title: Determination of protein interactions between PMS2 and a new variant of MLH1

Abstract

Human proteins MLH1 and PMS2 form a heterodimer called MutLα, which plays a key role in DNA mis-match repair system. Pathogenic mutations in MLH1 and PMS2 genes are linked to Lynch syndrome, a hereditary predisposition for developing cancer, particularly colorectal cancer. Identification of pathogenic mutations in MLH1 contributes to a more reliable Lynch syndrome diagnosis. In this work we focused on a new in-frame deletion in MLH1 LRG_216t1:c.2236_2247delCTGCCTGATCTA p.(Leu746_Leu749del) (MLH1_del746-749). MLH1_del746-749 was found in a Slovenian family with a known history of cancers and is classified as probably pathogenic. Genetic test showed high levels of microsatellite instability and an unusual loss of PMS2 expression in immunohistochemical staining of tumour samples. Based on all known data, we hypothesized that the deletion of four amino acids at the C-terminal part of MLH1 precludes its interaction with PMS2 and thus the formation of MutLα heterodimer. In the bioinformatic part of the work we constructed models of the structures of C-terminal parts of proteins PMS2, MLH1_del746-749 and four additional pathogenic variants of MLH1 protein. In addition, we made a model of the C-terminal part of human MutLα, which has not been experimentally determined yet. Based on the analysis of the structures we hypothesized that MLH1_del746-749 could not interact with PMS2. We tested our assumption with the yeast two-hybrid system. The yeast two-hybrid system has been shown to be successful in determining interactions between MLH1 and PMS2, which are known to interact from literature. In addition, neither MLH1 nor PMS2 are capable of self-activation of the yeast two hybrid system, thus confirming the results with the yeast two-hybrid are reliable. The test showed that MLH1_del746-749 interacts with PMS2 which activated reporter genes and enabled yeast growth on selection medium. Deletion Leu746_Leu749 does not prevent the interaction of MLH1 with PMS2 in yeast, but probably affects the stability and function of the MutLα in human cells.

• Keywords: MLH1, PMS2, MutLα, Lynch syndrome, yeast two-hybrid