The role of conserved BPM1 protein domains MATH, BTB and SPOP in interaction with DMS3, RDM1 and HB6 proteins

Članovi proteinske porodice MATH-BTB sudjeluju u ubikvitinskom putu degradacije proteina kao adaptori E3 ligaze ovisne o kulinu 3 i odgovorni su za specifičnost prema ciljanom supstratu. U uročnjaku (Arabidopsis thaliana L.) postoji šest gena ove obitelji (BPM 1-6). Protein BPM1 uz domene MATH i BTB sadrži i konzerviranu domenu SPOP koja zajedno s domenom BTB sudjeluje u stvaranju proteinskih interakcija s kulinom 3. Domena MATH sudjeluje u selektivnim interakcijama s proteinima ciljanim za proteasomsku razgradnju. Takve interakcije utvrđene su između proteina BPM1 i nekih transkripcijskih faktora poput HB6 koji ima ulogu u odgovorima reguliranim apscizinskom kiselinom u biljnim stanicama. Preliminarna istraživanja su pokazala interakciju proteina BPM1 s proteinima DMS3 i RDM1 koji sudjeluju u metilaciji DNA posredovanoj molekulama RNA. U ovom radu interakcije proteinskih domena BPM1 s transkripcijskim faktorom HB6 i proteinima DMS3 odnosno RDM1 su analizirane metodom kopurifikacije „jedan na jedan“. Konstruirani su odgovarajući ekspresijski plazmidi, optimizirani su postupci poticanja ekspresije i pročišćavanja za svaki pojedini protein ili varijantu proteina te su eksprimirani i pročišćeni rekombinantni proteini iz kemijski transformiranih bakterijskih stanica. Interakcija proteina je provjerena hibridizacijom po Westernu i metodom SDS-PAGE. Potvrđena je interakcija proteina BPM1 i DMS3 posredovana domenom MATH te interakcija BPM1 s RDM1 koja nije specifično posredovana niti jednom pojedinom domenom. Interakcija proteina BPM1 i HB6 nije dokazana uslijed degradacije proteina HB6 .Members of MATH-BTB protein family partake as adaptors of cullin 3 dependant E3 ligase in the ubiquitin proteasome pathway and are responsible for specificity towards a substrate. In Arabidopsis thaliana there are 6 genes from this family (BPM 1-6). BPM1 protein along MATH and BTB domains, contains conserved SPOP domain which together with BTB domain partakes in forming of protein interactions with cullin 3. MATH domain has a role in selective interactions with proteins which are targeted for proteasomal degradation. This kind of interactions are established between BPM1 protein and some transcriptional factors like HB6 which has an important role in abscisic acid dependant response in plant cells. Preliminary research have shown an interaction between BPM1 protein and DMS3 and RDM1 proteins which partake in a DNA methylation directed by RNA molecules. Here interactions between BPM1 protein, HB6 transcription factor and proteins DMS3 and RDM1 were analized with „one to one“ pull down method. Adequate plasmid constructs were made, conditions for induced expression and purification for each protein and protein variant were optimised and recombinant proteins were expressed and purified from chemically transformed bacterial strains. Protein-protein interactions were proven by Western blot and SDS-PAGE. Interaction between BPM1 and DMS3 was confirmed and it is primarily mediated by MATH domain. Positive interaction between BPM1 and RDM1 didn't show preference towards any BPM1 domain. BPM1 and HB6 interaction wasn't confirmed due to HB6 degradation