Študija z gvanini bogatega zaporedja promotorske regije gena sost, povezanega z metabolizmom kosti

Moškrič, Anja (author), Plavec, Janez (mentor)

Wnt/β-kateninska signalna pot je pomembna za nastajanje kostnega tkiva. Proces inhibira glikoprotein sklerostin. Deluje kot antagonist, saj onemogoča vezavo proteinov Wnt na receptor FZD in koreceptor LRP5/6 na osteoblastih. Posledično se β-katenin v citosolu razgradi in ne more sodelovati pri aktivaciji transkripcije genov, povezanih z nastajanjem kosti. V promotorski regiji gena sost, ki kodira omenjeni glikoprotein, smo identificirali 26 nukleotidov dolgo zaporedje bogato z gvanini (SOST26), za katerega smo pokazali, da lahko tvori od B-DNA različno strukturo, imenovano G-kvadrupleks. G-kvadrupleksi so štiri-vijačne strukture, sestavljene iz dveh ali več G-kvartetov, povezanih z različnimi zankami. Pogosto se nahajajo v telomerah in regulatornih regijah genov, kjer odločilno vplivajo na delovanje telomeraz in na transkripcijo genov. Z uporabo NMR in CD spektroskopije smo določili, da se SOST26 zvije v dvokvartetni G-kvadrupleks z antiparalelno usmerjenostjo verig, kjer sta prva in tretja zanka robni, vmesna zanka pa je diagonalna (t.i. zvitje v obliki košare, ang. basket-type G-quadruplex). Preučili smo tudi vpliv ostankov iz 12 nukleotidov dolge diagonalne zanke na nastanek G-kvadrupleksa SOST26. Opazili smo, da se v zanki tvorita dva G–C bazna para (G8–C17 in C9–G16), ki sta ključna za stabilizacijo osrednje strukture. Na zvitje vplivata tudi T7 in A18, za katera predvidevamo, da igrata pomembno vlogo pri interakciji s 3'-končnim G26, ki se naloži pod spodnji G-kvartet. Poleg G-kvadrupleksa se SOST26 v manjšem deležu zvije tudi v lasnico, vendar je ravnotežje pri temperaturah blizu fiziološki skoraj v celoti pomaknjeno v smer nastanka G-kvadrupleksa. Ugotovili smo, da je G-kvadrupleks SOST26 stabilen še vsaj 10 °C nad fiziološko (T1/2 = 47,8 °C), kar nakazuje, da bi se struktura lahko tvorila tudi v celicah in vplivala na izražanje gena sost.

• Language: Slovenian
• Keywords: Wnt, sklerostin, NMR spektroskopija, G-kvadrupleks
• Work type: Bachelor thesis/paper
• Typology: 2.11 - Undergraduate Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2023
• PID: 20.500.12556/RUL-155006
• COBISS.SI-ID: 162901763
• Publication date in RUL: 13.03.2024

Secondary language

• Language: English
• Title: Study of G-rich sequence from the promoter region of the sost gene, related to bone metabolism

Abstract

The Wnt/β-catenin signaling pathway is important for bone tissue formation. The process is inhibited by the glycoprotein sclerostin, which acts as an antagonist by preventing the binding of Wnt proteins to the FZD receptor and the co-receptor LRP5/6 on osteoblasts. As a result, β-catenin in cytosol is degraded and cannot participate in the activation of bone-related gene transcription. In the promoter region of the sost gene, which encodes the mentioned glycoprotein, we identified a 26-nucleotide-long guanine-rich sequence, hereafter designated as SOST26. We have shown that SOST26 can form a non-B-DNA structure, a so-called G-quadruplex. G-quadruplexes are four-stranded structures composed of two or more stacked G-quartets connected by various loops. They are often found in telomeres and regulatory regions of genes, where they have a crucial impact on regulation of telomerase activity and gene transcription, respectively. Using NMR and CD spectroscopy, we determined that SOST26 folds into a two-quartet G-quadruplex with antiparallel alignment of the G-strands, where the first and third loops are edgewise, while the middle loop is diagonal (i.e. basket-type G-quadruplex). We also examined the influence of residues in the 12-nucleotide-long diagonal loop on the formation of the SOST26 G-quadruplex. We observed that two G–C pairs (G8–C17 and C9–G16) form within the loop and are crucial for stabilization of the central structure. T7 and A18 also affect the folding and are predicted to play an important role in the interaction with the 3'-terminal G26, which stacks below the lower G-quartet. In addition to the G-quadruplex, a minor portion of SOST26 folds into a hairpin structure, but the equilibrium is almost completely shifted towards G-quadruplex formation at temperatures close to physiological conditions. We found that the SOST26 G-quadruplex remains stable at least 10 °C above physiological temperature (T1/2 = 47.8 °C), suggesting that the structure could also form in cells and influence the expression of the sost gene.

• Keywords: Wnt, sclerostin, NMR spectroscopy, G-quadruplex