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Reorganizacija sučelja između ranih endosoma i endosomalnog reciklirajućeg odjeljka u citomegalovirusnoj infekciji

Projekt Hrvatske zaklade za znanost IP-2020-02-2916

Voditelj projekta: Prof. dr. sc. Hana Mahmutefendić Lučin

Istraživački tim

Prof. dr. sc. Pero Lučin, Sveučilište u Rijeci, Medicinski fakultet

Prof. Dr. Martin Messerle, Medicinski fakultet Hannover

Prof. Dr. Zsolt Ruzsics, Sveučilišna klinika Freiburg Institut za virologiju

Dr. sc. Ljerka Karleuša, Sveučilište u Rijeci, Medicinski fakultet

Igor Štimac, Sveučilište u Rijeci, Medicinski fakultet

Marina Marcelić, Sveučilište u Rijeci, Medicinski fakultet

Barbara Radić, Sveučilište u Rijeci, Medicinski fakultet

Sažetak istraživanja

Biogeneza cVAC (engl. cytoplasmic virion assembly compartment) predstavlja važan, i još nerazjašnjen problem za razumijevanje biologije herpesvirusa. U ovom projektnom prijedlogu ćemo proučavati najranije faze reorganizacije mambranskih organela koje nazivamo preAC (engl. pre-assembly compartment). Ovaj istraživački projekt predstavlja nastavak i produbljenje našeg prethodnoga rada i fokusira se na stanične mehanizme koje koristi mišji citomegalovirus (MCMV) na sučelju između ranih endosoma i endosomalnog reciklirajućeg odjeljaka (EE-ERC), odnosno mjestu za koje pretpostavljamo da predstavlja inicijacijski korak cVAC biogeneze. U tu svrhu, MCMV može ciljati (1) domaćinske efektore koji reguliraju procese sortiranja i recikliranja na EE-ERC sučelju, (2) Rab10 i njegove partnere, regulatorne proteine koji kontroliraju EE-ERC prometovanje, i (3) procese ubikvitinilacije koji kontroliraju endosomalno recikliranje, a djeluju prema principu reostata. Ovi potencijalni učinci MCMV djelovanja predstavljaju tri cilja ovog projektnog prijedloga. Kako bismo ih ispunili testirat ćemo skoro 50 staničnih efektora i regulatora na EE-ERC sučelju i njihovu ulogu u nastanku preAC. Stanice će biti inficirane divljim tipom i različitim MCMV rekombinantama, a domaćinski proteini će biti analizirani knock-down (siRNA, inhibitori) i knock-out eksperimentima (CRISPR-Cas9). Nastanak preAC će se pratiti konfokalnom mikroskopijom i rekonstrukcijom mape interakcija na EE-ERC sučelju pomoću BioID testa, imunoprecipitacije, Western blot analize i subcelularnog frakcioniranja. MCMV učinak na kompleksnu mrežu regulatornih proteina i efektora na EE-ERC sučelju će predstavljati značajan znanstveni doprinos ovog projektnog prijedloga. On će razjasniti ne samo najranije stadije patogeneze beta-herpesvirusne infekcije, već i prostorno-vremenske odnose u neinficiranim stanicama, dva nedovoljno razjašnjena fundamentalna biološka procesa. 

 Hana 1Slika 1: Sustav endosoma. (A) Glavne komponente staničnog endosomalnog sustava su pre-rani endosomi (preEE), rani endosomi (EE), sortirajući endosomi (SE), endosomalni reciklirajući odjeljak (ERC), kasni endosomi (LE) i trans-Golgi mreža (TGN). (B) EE-ERC sučelje je kompleksno područje koje je organizirano putem nekoliko Rab GTPaza, ali njihova je uloga još uvijek slabo karakterizirana. 

Postignuća

Znanstvene publikacije

  1. Štimac, I.; Jug Vučko, N.; Blagojević Zagorac, G.; Marcelić, M.; Mahmutefendić Lučin, H.; Lučin, P. (2021). Dynamin Inhibitors Prevent the Establishment of the Cytomegalovirus Assembly Compartment in the Early Phase of Infection. Life 2021, 11, 876. https://doi.org/10.3390/life11090876.
  2. Marcelić M, Mahmutefendić Lučin H, Begonja AJ, Zagorac GB, Lisnić VJ, Lučin P. (2021). Endosomal Phosphatidylinositol-3-Phosphate-Associated Functions Are Dispensable for Establishment of the Cytomegalovirus Pre-Assembly Compartment but Essential for the Virus Growth. Life. 2021; 11(8):859. https://doi.org/10.3390/life11080859.
  3. Mahmutefendić Lučin H, Blagojević Zagorac G, Marcelić M, Lučin P. (2022). Host Cell Signatures of the Envelopment Site within Beta-Herpes Virions. International Journal of Molecular Sciences. 23(17):9994. https://doi.org/10.3390/ijms23179994.
  4. Mahmutefendić Lučin H, Lukanović Jurić S, Marcelić M, Štimac I, Viduka I, Blagojević Zagorac G, , Lučin P. Membraneless Compartmentalization of Nuclear Assembly Sites during Murine Cytomegalovirus Infection. Viruses 2023. 15, 766. https://doi.org/10.3390/v15030766.
  5. Štimac I, Marcelić M, Radić B, Viduka I, Blagojević Zagorac G, Lukanović Jurić S, Rožmanić C, Messerle M, Brizić I, Lučin P, Mahmutefendić Lučin H. SNX27:Retromer:ESCPE-1-mediated early endosomal tubulation impacts cytomegalovirus replication. Front Cell Infect Microbiol. 2024 Sep 18;14:1399761. https://doi.org/10.3389/fcimb.2024.1399761.
  6. Lučin P, Mahmutefendić Lučin H, Blagojević Zagorac G. Cytomegaloviruses reorganize endomembrane system to intersect endosomal and amphisome-like egress pathway. Front Cell Dev Biol. 12/2023 19;11:1328751. https://doi.org/10.3389/fcell.2023.1328751.
  7. Mahmutefendić Lučin H, Štimac I, Marcelić M, Skočaj M, Lisnić B, Omerović A, Vidika I, Radić B, Karleuša Lj, Blagojević Zagorac G, Deželjin M, Jurak Begonja A, Lučin P. Rab10-associated tubulation as an early marker for biogenesis of the assembly compartment in cytomegalovirus-infected cells. Front Cell Dev Biol. 12/2024 https://doi.org/10.3389/fcell.2024.1517236. 

Kongresna priopćenja

  1. Mahmutefendić Lučin H, Štimac I, Jug Vučko N, Blagojević Zagorac G, Marcelić M, Lučin P. Dynamine inhibitor(s) blocks development of cytomegalovirus assembly compartment in the early phase of infection. 45th Annual International Herpesvirus Workshop. Virtual, August 2-6, 2021. Abstract book, p. 189. 
  2. Štimac I, Blagojević Zagorac G, Lučin P, Mahmutefendić Lučin H.MCMV influence on intracellular membrane localization molecules that regulates endosomal recycling.The 15th Annual Symposium of the Croatian Physiological Society with international participation „Homeostasis – From Cell to Organ“. October 7th – 8th, 2021 Zagreb. (Štimac, poster presentation).
  3. Karleuša  Lj, Jug Vučko N, Pavišić V, Lukanović Jurić S, Mahmutefendić Lučin H, Blagojević Zagorac G, Lisnić B, Lučin P. Expansion of early endosome – endosomal recycling compartment interface initiates the establishment of the cytomegalovirus assembly compartment. The 15th Annual Symposium of the Croatian Physiological Society with international participation „Homeostasis – From Cell to Organ“. October 7th – 8th, 2021 Zagreb.
  4. Štimac I, Marcelić M, Blagojević Zagorac G, Lučin P, Mahmutefendić Lučin H. Dynamin-2 is important for production of infective virions in murine cytomegalovirus (MCMV) infection.6th European Congress of Immunology. September 1-4 on virtualeci2021.com.Eur. J. Immunol. 2021. 51 (Suppl. 1): 301.
  5. Radić B, Ružić T, Lukanović Jurić S, Štimac I, Lisnić B, , Blagojević Zagorac G, Lučin P, Mahmutefendić Lučin H. Murine Cytomegalovirus M48 Protein Deubiquitylation Function Is Dispensable for ViralReplication, but Is Important for Host Cell Membrane Remodeling. 46th International Herpesvirus Workshop, Virtual Conference, July 25th-29th. Poster presentation (8,52). Abstract book p82.
  6. Štimac I, Viduka I, Blagojević Zagorac G, Marcelić M, Lučin P. Sorting Nexin 4 and Sorting Nexin 27 are Important for Generation of Pre-Assembly Compartment in the Early Phase of Murine Cytomegalovirus Infection. 46th International Herpesvirus Workshop, Virtual Conference, July 25th-29th. Poster presentation (7.32). Abstract book p146.
  7. Radić B, Ružić T, Štimac I, Lisnić B, Lukanović Jurić S, Blagojević Zagorac G, Lučin P, Mahmutefendić Lučin H. Deubiquitinilation function of murine cytomegalovirus M48 protein influence remodeling of WASH1 endosomal network in the early phase of infection. 3rd Congress of Physiological Societies and 5th Congress of Croatian Physiological Society. 22nd - 24th September 2022, Plitvička Jezera, Croatia. Abstract book, p143.
  8. Štimac I, Blagojević Zagorac G, Marcelić M, Lučin P, Mahmutefendić Lučin H. Rab10 GTPase is involved in membrane remodeling to form assembly compartment in the early phase of murine cytomegalovirus infection. 3rd Regional  Congress of Physiological Societies and 5th Congress of Croatian Physiological Society. 22nd - 24th September 2022, Plitvička Jezera, Croatia. Abstract book, p51.
  9. Štimac I, Marcelić M, Lisnić B, Radić B, Blagojević Zagorac G, Lučin P, Mahmutefendić Lučin H. Cooperation with Rab10 key interaction partners AS-160, MICAL-L1 and ACAP-2 is enhanced in the early phase of murine cytomegalovirus infection. Poster prezentacija na 18th Congress of the International Union of Microbiological Societies (IUMS 2024), Firenza, Italia, 23-25.10.2024, Frontiers Abstract Book str. 2312, DOI: 10.3389/978-2-8325-5119-6
  10. Radić B, Štimac I, Blagojević Zagorac G, Lučin P, Mahmutefendić Lučin H. Extensive disruption of ubiquitination processes by Pyr41 delays murine cytomegalovirus replication cycle in infected fibroblasts. E-poster prezentacija na 18th Congress of the International Union of Microbiological Societies (IUMS 2024), Firenza, Italia, 23-25.10.2024, Frontiers Abstract Book str. 732, DOI: 10.3389/978-2-8325-5119.
  11. Štimac I, Skočaj M, Marcelić M, Karleuša Lj, Blagojević Zagorac G, Radić B, Viduka I, Sepčić K, Lučin P, Mahmutefendić Lučin H. Rab10 can be used as a reliable marker for the assembly compartment in fibroblasts infected with murine cytomegalovirus. Poster prezentacija na Drugom kongresu doktoranada biomedicine i zdravstva Medicinskog fakulteta „Science and us”. Rijeka, 16.-18.05.2024., abstract book str. 127.
  12. Panevska A, Blagojević Zagorac G, Skočaj M, Viduka I, Štimac I, Mahmutefendić Lučin H, Sepčić K, Lučin P. Modulation of lipid composition of cell membranes during cytomegalovirus infection. Poster prezentacija na Drugom kongresu doktoranada biomedicine i zdravstva Medicinskog fakulteta „Science and us”. Rijeka, 16.-18.05.2024., abstract book str. 131.
  13. Radić B, Štimac I, Marcelić M, Blagojević Zagorac G, Lučin P, Mahmutefendić Lučin H. Murine cytomegalovirus affects the activation of WASH1 protein for regulation of endosomal recycling by hiperubiquitination. Poster prezentacija na Drugom kongresu doktoranada biomedicine i zdravstva Medicinskog fakulteta „Science and us”. Rijeka, 16.-18.05.2024., abstract book str. 120.
  14. Jug Vučko N, Pavišić V, Mahmutefendić Lučin H, Štimac I, Lučin P, Blagojević Zagorac G. Expression and intracellular localization of Arf guanine nucleotide exchange factors in Balb 3T3 fibroblasts infected with murine cytomegalovirus. Poster prezentacija naDrugom kongresu doktoranada biomedicine i zdravstva Medicinskog fakulteta „Science and us”. Rijeka, 16.-18.05.2024., abstract book str. 101.
  15. Blagojević Zagorac G, Pavišić V, Mahmutefendić Lučin H, Jug Vučko N, Štimac I, Karleuša Lj, Viduka I, Radić B, Lučin P. Arf GTPases are required for progression of cytomegalovirus replication cycle. Poster prezentacija na EMBO Workshop “Pathogen immunity and signaling”, Venezia, Italija, 08-12.04.2024., abstract book str. 29.
  16. Radić B, Ružić T, Štimac I, Lisnić B, Blagojević Zagorac G, Lučin P, Mahmutefendić Lučin H. Murine cytomegalovirus M48 protein deubiquitynilates EHD1 and Rab11-FIP1 proteins and inhibits transferrin recycling.  Poster presentacija na 12 th  EFIS-EJI South Eastern European Immunology (SEEIS 2023), Trogir, 20-23.10.2023., abstract book str. 39.
  17. Štimac I, Radić B, Blagojević Zagorac G, Marcelić M, Lisnić B, Lučin P, Mahmutefendić Lučin H. EHBP1 and Rabin8 GTPase recruit Rab10 GTPase in Murine cytomegalovirus assembly compartment. Poster prezentacija na 2023 Annual Meeting of the Croatian Immunological Society, Brijuni, 05 - 07.10.2023., abstract book str. 33.
  18. Radić B, Ružić T, Štimac I, Lisnić B, Blagojević Zagorac G, Lučin P, Mahmutefendić Lučin H. Ubiquitinilation of Rab5 guanine nucleotide exchange factor (GEF) Rabex5 and deubiquitinilation of Rab11FIP1 affects transferrin recycling in MCMV infected cells. Poster prezentacija na 2023 Annual Meeting of the Croatian Immunological Society, Brijuni, 05- 07.10.2023, abstract book str. 33.

  19. Radić B, Štimac I, Marcelić M, Blagojević Zagorac G, Lučin P, Mahmutefendić Lučin H. Murine cytomegalovirus disrupts endosomal recycling by changing the ubiquitination status of EHD1 and MICALL1. Poster prezentacija na Power of viruses, International Virology Symposium, 25.9. -28. 9.  2024; Zadar, abstract book str. 106. 

DOKTORSKA DISERTACIJA

Igor Štimac: Biogeneza citomegalovirusnog odjeljka za sklapanje viriona preuređivanjem sortiranja u endosomalnim reciklirajućim domenama (21.10.2024). Medicinski fakultet Sveučilišta u Rijeci (Mentorica: Prof. dr. sc. Hana Mahmutefendić Lučin).