Regulation of receptor-mediated mitophagy in erythroid lineage cells - MitoReg (IP-2020-02-3883)

(CSF Research project)

Project leader: prof. dr. sc. Ivana Novak Nakir

Project timeframe: 13.09.2021.- 12.09.2025.

Prof. Jörn Dengjel, PhD, University of Fribourg, Switzerland
Dr. sc. Vladimir Rogov, Goethe University, Frankfurt am Main, Germany
Prof. dr. sc. Irena Drmić Hofman, University of Split, School of Medicine and University department for health studies, Split, Croatia

Postdoctoral fellow:
dr. sc. Mija Marinković

PhD student:
mag. chem. Ana Rožić (employed through CSF grant "Young Researchers' Career Development Project" DOK-2021-02-4248)

Project summary:
Quality control maintenance of mitochondria and homeostasis in the cell is dependent on mitophagy, a specialized form of the autophagy that selectively removes mitochondria. Two key steps are indispensable for this basic cellular process of damaged or unnecessary mitochondria removal: induction of the autophagy machinery and priming of mitochondria destined for degradation. Mitochondrial priming is mediated by PINK1-PARKIN pathway or through specialized mitochondrial receptors. Receptor-mediated mitophagy is extensively studied in reticulocytes where mitophagy is indispensable for their maturation into erythrocytes, as well as in retina. For both differentiation moments, it is necessary to establish a link between the NIX and LC3/GABARAP adaptors, which are an integral part of the autophagosomal vesicle needed for degradation. The focus of this project proposal is to study mitophagy mechanisms, w ith emphasis on receptor-mediated mitophagy, in erythroid cell line to better understand terminal erythrocyte differentiation. Our previous results show that phosphorylation and dimerization of NIX are key for receptor activation, and therefore the initiation of mitophagy. This project will in detail analyze the protein interactions of NIX with its kinases and phosphatases identified in the lab and clarify their activity in cells using biochemical, molecular and cellular biology methods. Furthermore, we will investigate new interaction partners of NIX and other mitophagy receptors with emphasis on mitochondrial proteins by mass spectrometry methods to unveil signaling pathways that trigger mitophagy. Finally, by analyzing the status of autophagy and mitophagy in bone marrow cells of myeloproliferative disease patients using quantitative PCR, Western blot and flow cytometry, we will tackle the possible role of these processes in the onset of the disease and open the door to grasp the role of autophagy in abnormal differentiation of erythroid cells.

-  Mija Marinković is the winner of the Science Award for 2021 in the category of young scientists awarded by the University of Split. Congratulations.

Project publications:

A brief overview of BNIP3L/NIX receptor-mediated mitophagy. Marinković M. and Novak I. 2021. FEBS Open Bio, Oct 1, Online ahead of print.

Finished project:

The role of autophagy receptors in selective removal of mitochondria - AutoMito

(CSF Installation research project)


Project leader: prof. dr. sc. Ivana Novak Nakir

Project timeframe: 01.03.2015.- 30.09.2018.

prof. dr. sc. Ivan Đikić, Goethe University School of Medicine, Frankfurt am Main, Germany
dr. sc. Nathan Brady, German Cancer Research Center (DKFZ) and Bioquant, Heidelberg, Germany

PhD student:
mag. mol. biol. Mija Marinković (employed through CSF grant "Young Researchers' Career Development Project" DOK-2014-06-9538) 

Postdoctoral fellow: Maja Buljubašić (from July 2015 till Dec 2017)

Project summary:
Autophagy is cellular degradation pathway essential for cell survival, development and differentiation. Its digestive property has been shown important in different pathologies including cancer, neurodegeneration and pathogen removal. Discoveries of specific autophagy receptors have opened a new chapter in the autophagy field. Of great interest is the removal of damaged or excess mitochondria through specialized autophagy- mitophagy. Together with colleagues, I have identified and characterized mitochondrial proteins Nix/Bnip3L and Bnip3 as a mitophagy receptors for recruiting the autophagic machinery to damaged mitochondria and help their elimination (Novak et al, 2010; Zhu et al, 2013). We demonstrated that receptors mediate mitochondrial clearance during reticulocyte differentiation. Our goal is to further study autophagy receptors and proteins associated with mitochondrial clearance to better understand the role of mitophagy and its mechanism. We will study: (1) phosphorylation and dimerization of receptors and the consequences of this events on mitophagy progression; (2)factors that regulate tight connection of apoptosis and mitophagy; (3)potential interplay between mitophagy receptors and other mitophagy-related proteins, like  E3-ligase Parkin and kinase PINK1; (4)the effects of mitophagy on cancer development. To reach our goals we will use established methodology in our laboratories including protein identification and interaction techniques (Western blot, IP, GST-pull down, RNAi), immunofluorescent microscopy, mass spectrometry, ITC and NMR. All experiments will be performed using cultured cells. This project proposal has a high capacity to generate very interesting data with high possibility for publications in top scientific journals. Fundamentally, generated data will help to understand the physiological and pathological conditions, such as neurodegeneration and cancer and allow us to manipulate mitophagy to fight diseases.

Project publications:

1. Dimerization of mitophagy receptor BNIP3L/NIX is essential for recruitment of autophagic machineryMarinković M, Šprung M and Novak IAutophagy, 2021. April. IF: 16.016

2. Autophagy modulation in cancer: current knowledge on action and therapy. Marinković M, Šprung M, Buljubašić M and Novak I, Oxidative Medicine and Cellular Longevity, 2018. Jan. IF: 4.868 

3. Phosphorylation of the mitochondrial autophagy receptor Nix enhances its interaction with LC3 proteins.
Rogov VV, Suzuki H, Marinković M, Lang V, Kato R, Kawasaki M, Buljubašić M, Šprung M, Rogova N, Wakatsuki S, Hamacher-Brady A, Dötsch V, Dikic I, Brady NR, Novak ISci Rep. April 2017. IF: 4.116

4. Flow Cytometer Monitoring of Bnip3- and Bnip3L/Nix-Dependent Mitophagy.
Šprung M, Dikic I, Novak I. Methods Mol Biol. 2017 Mar

International conferences attended by project leader:

1. Conference "FEBS3+ From molecules to living systems. Siofok, Mađarska. 02.-05.09.2018. Invited speaker.

2. Conference "83rd Harden Conference Autophagy - from Molecules to Disease II". Warwickshire, UK, 03.-07. 06. 2018. Invited speaker.

3. Conference "EMBO Autophagy - From molecular principles to human diseases". Cavtat, Hrvatska. 25.-29. 09. 2017. Poster presentation.

4. Conference "Transautophagy Annual Meeting". Varšava, Poljska. 06.-07.10. 2016. Invited speaker..

5. Conference "Protein Signaling - from pathways to networks", Kopenhagen, Danska. 02.-06.10.2016. Poster prezentation.

6. Conference "HDMBM Congress - On occasion of the 40th Anniversary", Split, Hrvatska. 01.-04.06.2016. Invited speaker.

7. Conference "EMBO - Autophagy signalling and progression in health and disease", Chia, Italija. 09.-12.09.2015. Poster prezentation.

Project in media:
1. Glasnik Medicinskog fakulteta u Splitu (September 2020)
2. Jutarnji list (July 2020)
3. Slobodna dalmacija - o publikaciji u časopisu Autophagy (May 2020)
4. - press release about Autophagy publication (April 2020)
5. Jutarnji list - press release about Autophagy publication (April 2020)
6. Croatian national television, HRT, Treći element - Čišćenje stanice (S4E03)
7. Croatian national radio, HR1, Oko znanosti
8. Croatian national television, HRT, Znanstveni krugovi

Other lectures by project leader:
1. Nobel na Ruđeru - medicina, Institut Ruđer Bošković, Zagreb, 21.10.2016.
2. Prirodoslovno-matematički fakultet u Splitu, Split, 23.11.2016.
3. Croatian society of Biochemistry and Molecular biology Seminar. Split. 06.02.2018.
4. Inter-organelle communication and neurodegeneration, Mini Symposium, Medils, Split. 25.-28.03.2018. 
5. "Praktična znanja za studente" - international student organized conference. School of Medicine in Split. Split. 06.-08.04.2018.
6. HRZZ Tenue Track Pilot programme presentation. HAZU, Zagreb. 15.05.2018.

International conferences and workshops attendance by PhD student Mija Marinković:

1. Conference "HDMBM Congress - On occasion of the 40th Anniversary", Poster. Split, Hrvatska. 01.-04.06.2016.
2. Conference "Protein Signaling - from pathways to networks", Poster.Kopenhagen, Danska. 02.-06.10.2016. 
3. Spetses Summer School an EACR-FEBS advanced lecture course "Molecular Mechanisms in Signal Transduction and Cancer" - poster. Spetses, Grčka. 16.-24.08.2017
4. Flow cytometry intensice course 2017. Dječja bolnica Srebrnjak. Zagreb. 23.-27.10.2017. 
5. Gordon Research Seminars: Autophagy in Stress, Development and Disease - poster. Barga, Italija. 17.-18.03.2018.
6. Gordon Research Conference: Autophagy in Stress, Development and Disease - oral presentation. Barga, Italija. 18.-23.03.2018. 
7. Applications of Cell-Based Luciferase Reporter Assays. PMFST, Split. 26.04.2018.
8. Flow Cytometry Mini Congress and S32TM Cell Sorter workshop, Medils, Split. 09.-10.05.2018.
9. Conference "FEBS3+ From molecules to living systems. - poster. Siofok, Mađarska. 02.-05.09.2018. 


International conferences and workshops attendance by postdoctoral fellow Maja Buljubašić:

1. Conference "HDMBM Congress - On occasion of the 40th Anniversary", Poster prezentacija. Split, Hrvatska. 01.-04.06.2016.
2. Workshop "Protein expression, purification and characterization - PEPC10", EMBL Hamburg, Njemačka. 11.-20-09.2016.
3. Conference "EMBO Autophagy - From molecular principles to human diseases". Cavtat, Hrvatska. 25.-29. 09. 2017.
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