Rana faza razvoja kompleksa za stvaranje viriona tijekom citomegalovirusne infekcije

Projekt Hrvatske zaklade za znanost IP-2014-09-9564

Voditelj projekta: Prof. dr. sc. Pero Lučin
Zavod za fiziologiju i imunologiju

Medicinski fakultet Sveučilišta u Rijeci

 

Istraživački tim

Prof. dr. sc. Pero Lučin, dr. med. • UniRi portfelj • Google Scholar profil
Prof. dr. sc. Hana Mahmutefendić, dipl. ing. mol. biol. • UniRi portfelj • Google Scholar profil
Doc. dr. sc. Gordana Blagojević Zagorac, dr. med. • UniRi portfelj • Google Scholar profil
Doc. dr. sc. Kristina Grabušić • UniRi portfelj • Google Scholar profil
Doktorandi
Ljerka Karleuša, dipl. ing. bioteh. • UniRi portfelj • Google Scholar profil
Valentino Pavišić, dr. med. • UniRi portfelj • Google Scholar profil
Natalia Jug, mag. pharm. inv. • UniRi portfelj • Google Scholar profil
Silvija Lukanović Jurić, dr. med. • UniRi portfelj • Google Scholar profil
Marina Marcelić, mag. pharm. inv • UniRi portfelj • Google Scholar profil
Laboranti
Jelene Đirlić
Ksenija Tulić
Igor Štimac

 

Sažetak istraživanja

Infekcija beta-herpesvirusima izaziva opsežnu reorganiziraju unutarstaničnih odjeljka u svrhu stvaranja okruženja za stvaranje virusa, poznatog kao kompleks za stvaranje virusa (AC, engl. assembly complex). Ova struktura razvija se postupno, počevši od najranijih stadija infekcije, i uključuje stupnjevitu reorganizaciju membranskih organela. Iako je sastav i organizacija AC relativno dobro karakterizirana tijekom infekcije humanim citomegalovirusom (CMV), dio događanja koji vodi razvoju AC-a je slabo poznat. Cilj ovoga projekta je karakterizirati procese koji prethode stvaranju AC-a u stanicama inficiranim mišjim CMV-om (MCMV). MCMV započinje remodeliranje endosomalnog sustava vrlo rano tijekom infekcije, što smo karakterizirali kao razvoj endosomalnog retencijskog odjeljka rane faze (EPERC). Postoji jaka indikacija da je upravo EPERC prethodnih AC-a. Općenito je prihvaćeno da je remodeliranje endosomalnog sustava posredovano malim GTPazama iz Rab i Arf obitelji te fosfoinozitidima.

U predloženom projektu cilj nam je razotkriti procese reorganizacije membranskih organela tijekom rane faze infekcije, koji dovode do stvaranja AC-a. U tu svrhu utvrditi ćemo ulogu malih ATPaza iz obitelji Rab i Arf te njihovih efektorskih proteina tijekom stvaranja EPERC. Naposljetku, karakterizirati ćemo membranske domene EPERC-a tijekom rane faze infekcije obzirom na sastav fosfoinozitida. To će uključivati proučavanje endosomalnih efektorskih proteina koji se vezuju na fosfoinozitide i glavnih endosomalnih enzima koji nadziru konverziju fosfoinozitida i tako mijenjaju osobine membrana. Kako bismo postigli navedene ciljeve koristiti ćemo trostruko imunofluorescencijsko bojanje EPERC-a, fluorescentne boje stvorene genetičkim inženjeringom, utišavanje RNA i farmakološke inhibitore. Konačni ishod predloženog istraživanja biti će rekonstrukcija mehanizma razvoja EPERC-a i identifikacija staničnih proteina koji predstavljaju mjesto djelovanja MCMV-a tijekom rane faze infekcije.

AsCo hipoteza
Radna hipoteza – model razvoja EPERC-a. MCMV inicira razvoj EPERC-a putem (lijevo) prodora ranih endosomalnih vezikula u jukstanuklearno područje i premiještanjem Golgi-a prema periferiji, te putem(desno dolje) inhibicije reciklirajuće rute od ranih endosome prema staničnoj membrane, transportne rute od ranih endosome prema trans-Glogi-u i rute od ranih endosome prema kasnim endosomima (isprekidane crte).

 

Postignuća

 

Publikacije

  1. Mahmutefendić H., Blagojević Zagorac G., Grabušić K., Karleuša Lj., Maćešić S., Momburg F., Lučin P. (2017). Late Endosomal Recycling of Open MHC-I Conformers. Journal of Cellular Physiology, 232(4), 872-887. doi: 10.1002/jcp.25495.

  2. Gordana Blagojević Zagorac, Hana Mahmutefendić, Senka Maćešić, Ljerka Karleuša, Pero Lučin (2017). Quantitative analysis of endocytic recycling of membrane proteins by monoclonal antibody-based recycling assays. Journal of Cellular Physiology, 232(3), 463-476. doi: 10.1002/jcp.2550.

  3. Karleuša Lj., Mahmutefendić H., Ilić Tomaš M., Blagojević Zagorac G., Lučin P. (2018). Landmarks of endosomal remodeling in the early phase of cytomegalovirus infection. Virology, 515, 108-122. (DOI: 10.1016/j.virol.2017. 12.001).

  4. Mahmutefendić H., Blagojević Zagorac G., Maćešić S., Lučin P. (2018). Chapter 6: Rapid Endosomal Recycling. Open access peer-reviewed Edited volume: Peripheral Membrane Proteins. Edited by Shihori Tanabe. Published: July 25th 2018 by IntechOpen (DOI: 10.5772/intechopen.71809; ISBN: 978-1-78923-513-5; Print ISBN: 978-1-78923-512-8), pp83-104. DOI: 10.5772/intechopen.75685

  5. Kuharić J., Grabušić K., Sotošek Tokmadžić V., Štifter S., Tulić K., Shevchuk O., Lučin P., Šustić A. (2018). Severe traumatic brain injury induces early changes in the physical properties and protein composition of intracranial extracellular vesicles. Journal of Neurotrauma, (https://doi.org/10.1089/neu.2017.5515).

Prezentacije

  1. Karleuša Lj., Grabušić K., Mahmutefendić H., Blagojević Zagorac G., Lučin P. Endosomal remodeling during early phase of Murine Cytomegalovirus infection (Robinskon College), Biochemical Society/FEBS Focused Meeting, Signalling 2015, Cellular Functions of Phosphoinositides and Inositol Phosphates. Cambridge, 1-4 Sepetember 2015. Abstract book, page 29.
  2. Karleuša, Lj., Grabušić, K.,  Mahmutefendić, H., Blagojević Zagorac, G., Ilić Tomaš, M., Lučin, P. Remodelling of endosomal system during murine cytomegalovirus infection. Annual Meeting of Croatian Phyiological Society with international Participation, September 18th - 20th2015. Abstract book pages 37-38
  3. Blagojević Zagorac G., Mahmutefendić H., Mačešić S., Karleuša Lj., Lučin P.: Chasing recycled MHC class I molecules. 4th European Congress of Immunology - ECI 2015, Vienna, Austria, September 6-9, 2015. Abstract book, p246.
  4. Mahmutefendić H., Blagojević Zagorac G., Grabušić K., Karleuša Lj., Momburg F., Lučin P. Late endosomal trafficking and endosecretory recycling route of open MHC-I conformers. 4th European Congress of Immunology - ECI 2015 - Vienna, Vienna, Austria, September 6-9, 2015. Abstract book, pp246-247.

  5. Lučin P., Mahmutefendić H., Blagojević Zagorac G. Pathways of endosomal recycling of MHC-I proteins. Congress of the Croatian Society for Biochemistry and Molecular Biology. Split, June 1-4, 2016. Book of abstracts, p. 37.

  6. Lukanović Jurić S., Jug N., Pavišić V., Karleuša Lj., Mahmutefendić H., Blagojević Zagorac G., Volarević S., Lučin P. Autophagy in MCMV infected cells. 11. Godišnji simpozij Hrvatskog društva fiziologa s međunarodnim sudjelovanjem „Homeostaza-od stanice do organizma“ Zagreb, 17.-18. rujna 2016.

  7. Jug N., Mahmutefendic H., Blagojević Zagorac G., Karleruša Lj., Lučin P. IFNγ influence the endocytic pathways of surface glycoproteins. The 11th Annual CPS Symposium with International Participation. Zagreb, 17-18. 09. 2016.

  8. Pavišić V., Blagojević Zagorac G., Mahmutefendić H., Karleuša Lj., Jug N., Lukanović Jurić S., Lučin P. Role of the ARF proteins during the MCMV infection. The 11th Annual CPS Symposium with International Participation. Zagreb, 17-18. 09. 2016.

  9. Karleuša Lj., Mahmutefendić H., Blagojević Zagorac G., Lučin P. Disruption of proteasomal function, endosomal acidification and actin integration influence IE1 expression in MCMV infected cells. 2016 Annual Meeting of Croatian Immunological Society, Ogulin 14.-15.10.2016.

  10. Blagojević Zagorac G, Mahmutefendić H, Maćešić S, Karleuša Lj, Lučin P. Chasing Recycled Molecules by Monoclonal Antibody-Based Recycling Assays. 2016 Annual Meeting of Croatian Immunological Society, Ogulin 14.-15.10.2016.

  11. Mahmutefendić H, Karleuša L, Ilić Tomaš M, Blagojević Zagorac G, Jug N, Lukanović Jurić S, Pavišić V, Marcelić M, Lučin P. Murine cytomegalovirus restrains endocytic trafficking in sorting endosomes to initiate remodeling of membranous system for its egress. 4th Congress of Croatian Physiological Society and 2nd Regional Congress of the Physiological Societies, September 21st – 24th 2017, Dubrovnik, Croatia.

  12. Marcelić M, Mahmutefendić H, Jurak Begonja A, Blagojević Zagorac G, Karleuša L, Jug N, Lukanović Jurić S, Pavišić V, Lučin P. PI3P is important for early phase endosomal retention compartment (EPERC) formation in MCMV infected fibroblasts. 4th Congress of Croatian Physiological Society and 2nd Regional Congress of the Physiological Societies, September 21st – 24th 2017, Dubrovnik, Croatia.

  13. Jug N, Mahmutefendic H, Lukanović Jurić S, Karleuša L, Blagojević Zagorac G, Pavišić V, Marcelić M, Lučin P. Cellular serine proteases are important for inhibition of transferrin recycling after MCMV infected cells. 4th Congress of Croatian Physiological Society and 2nd Regional Congress of the Physiological Societies, September 21st – 24th 2017, Dubrovnik, Croatia.

  14. Pavišić V, Blagojević Zagorac G, Mahmutefendić H, Karleuša L, Lučin P. ARF5 protein and the MCMV infection. 4th Congress of Croatian Physiological Society and 2nd Regional Congress of the Physiological Societies, September 21st – 24th 2017, Dubrovnik, Croatia.

  15. Blagojevic Zagorac G., Mahmutefendic H, Pavisic V, Jug N, Karleuša L, Lucin P. Influence of different dynamin inhibitors on transferrin/transferrin receptor internalization and recycling. 4th Congress of Croatian Physiological Society and 2nd Regional Congress of the Physiological Societies, September 21st – 24th 2017, Dubrovnik, Croatia.

  16. Lukanović Jurić S, Jug N, Pavišić V, Karleuša L, Blagojević Zagorac G, Marcelić M, Lučin P, Mahmutefendić H. Contribution of cellular proteases in infection process of murine cytomegalovirus. 4th Congress of Croatian Physiological Society and 2nd Regional Congress of the Physiological Societies, September 21st – 24th 2017, Dubrovnik, Croatia.

  17. Lučin P., Mahmutefendić H., Blagojević Zagorac G. Remodeling of endosomal system by viral infection. 4th Congress of Croatian Physiological Society & 2nd Regional Congress of the Physiological Societies. 21-24 September, 2017, Dubrovnik, Croatia.