Inhibicija signaliziranja MyD88 s kratkima različicama MyD88swt in MyD88sL265P

Medved, Ines (avtor), Manček Keber, Mateja (mentor), Župunski, Vera (komentor)

MyD88 (mieloidni diferenciacijski primarni odgovor 88) je najpomembnejša molekula pri naravni imunosti. Deluje kot adapter in povezuje receptorje (TLR, IL-1R), ki sprejemajo zunajcelične signale, s proteini, ki prenašajo signale znotraj celice. Sestavljen je iz domene smrti (DD) na N-koncu, osrednje intermediarne domene (IND) in Toll-interlevkin-1 receptorske domene (TIR) na C-koncu. Po aktivaciji receptorjev MyD88 skupaj z IRAK4 in IRAK1/2 tvori večje agregate midosome, ki so nujni za prenos signala do transkripcijskih faktorjev NF-κB in AP-1. MyD88L265P je oblika MyD88, ki ima v domeni TIR na mestu 265 zamenjan levcin s prolinom. Je konstitutivno aktiven, zato poveča aktivnost NF-κB, signalizacijo JAK-STAT3 in prepisovanje provnetnih citokinov. MyD88L265P je povezan tudi z nastankom bolezni, kot so B celični limfom, Waldenstroemova makroglobulinemija (WM) in kronična limfocitna levkemija. Stimulacija celic z bakterijskimi produkti ali provnetnimi citokini sproži tvorbo kratke oblike MyD88 z alternativnim izrezovanjem. Ob alternativnem izrezovanju se iz MyD88 popolnoma izreže ekson 2, ki vsebuje zapis za celotno domeno IND. Odsotnost domene IND onemogoči aktivacijo NF-κB, ne prepreči pa fosforilacje JNK in izražanje od AP-1 odvisnih genov. MyD88s torej deluje inhibitorno na aktivacijo NF-κB in ima terapevtski potencial pri zdravljenju bolezni, ki jih povzroči prekomerna aktivacija z MyD88 posredovanih poti. V magistrski nalogi smo pokazali, da kratki obliki MyD88swt in MyD88sL265P v celici reagirata in kolokalizirata z dolgo obliko MyD88. Kratki obliki MyD88swt in MyD88sL265P inhibirata aktivacijo NF-κB ob koekspresiji dolgih oblik MyD88wt in MyD88L265P. Opazili smo tudi inhibicijo MyD88 signalne poti po stimulaciji celic s citokinom IL-1β, ni pa prišlo do inhibicije signaliziranja s TNFα, ki je od MyD88 neodvisno. Močno povečano izražanje kratkih oblik MyD88s je obrnilo trend inhibicije v povečanje aktivacije. Ta pojav razlagamo s tem, da povečane količine MyD88swt in MyD88sL265P verjetno pripomorejo k tvorbi midosomskega kompleksa z endogenim MyD88, ki povzroči aktivacijo NF-κB. Čeprav kratke oblike MyD88s kot take zaradi tega niso primerne za zdravljenje, kaže, da bi alternativno izrezovanje eksona 2 z uporabo kratkih protismiselnih oligonukleotidov pri bolnikih z WM lahko imelo terapevtski potencial.

• Jezik: Slovenski jezik
• Ključne besede: MyD88, MyD88L265P, MyD88s, NF-κB
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2022
• PID: 20.500.12556/RUL-136267
• COBISS.SI-ID: 106670339
• Datum objave v RUL: 21.04.2022

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Inhibition of MyD88 signaling using short variants of MyD88swt and MyD88sL265P

Izvleček

MyD88 (myeloid differentiation primary response 88) is the most important molecule in innate immunity. It acts as an adapter and connects receptors (TLR, IL-1R), which receive extracellular signals, with proteins that transmit signals within the cell. It consists of the death domain (DD) at the N-terminus, the central intermediate domain (IND), and the Toll-interleukin-1 receptor domain (TIR) at the C-terminus. Upon activation of MyD88 receptors, together with IRAK4 and IRAK1/2, it forms larger aggregates myddosomes, which are necessary for signal transduction to the transcription factors NF-κB and AP-1. MyD88L265P is a form of MyD88 that has leucine replaced by proline at position 265 in the TIR domain. It is constitutively active and therefore increases NF-κB activity, JAK-STAT3 signaling, and transcription of pro-inflammatory cytokines. MyD88L265P has also been associated with the development of diseases such as B cell lymphoma, Waldenstrom's macroglobulinemia (WM), and chronic lymphocytic leukemia. Stimulation of cells with bacterial products or pro-inflammatory cytokines triggers the formation of a short form of MyD88 by alternative splicing. In alternative splicing, exon 2, which contains the entire IND domain, is completely excised from MyD88. The absence of the IND domain disables NF-κB activation but does not prevent JNK phosphorylation and expression of AP-1 dependent genes. MyD88s, therefore has inhibitory effect on NF-κB activation and has therapeutic potential in the treatment of diseases caused by the overactivation of MyD88-mediated pathways. In the master's thesis, we showed that MyD88swt and MyD88sL265P short forms in the cell react and colocalize with the long-form MyD88. The short forms MyD88swt and MyD88sL265P inhibit NF-κB activation upon coexpression of the long forms MyD88wt and MyD88L265P. Inhibition of MyD88 signaling pathway after stimulation of cells with the cytokine IL-1β was also observed, but there was no inhibition of signaling by TNFα, which is MyD88-independent. Increased expression of short forms reversed the inhibition trend in increasing NF-κB activation. This phenomenon is explained by the fact that increased amounts of MyD88swt and MyD88sL265P are likely to contribute to the formation of a myddosomal complex with endogenous MyD88 that causes NF-κB activation. Although short forms of MyD88s as such are therefore not suitable for treatment, it suggests that alternative splicing of exon 2 using short antisense oligonucleotides in patients with WM could have therapeutic potential.

• Ključne besede: MyD88, MyD88L265P, MyD88s, NF-κB