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Mitophagy is a form of autophagy specialized to selectively remove mitochondria. Although the PINK1/Parkin pathway is the best described mitophagy of damaged mitochondria, receptor/mediated mitophagy seems to have a pivotal role in cellular development and specialization. The most studied mitophagy receptor BCL2/adenovirus E1B 19‐kDa‐interacting protein 3‐like (BNIP3L/NIX) is shown to be important for the programmed removal of healthy mitochondria during terminal differentiation of erythrocytes, but its role has been proven in various cell types. Despite recent advances in our understanding of its regulation by phosphorylation and dimerization, there remain numerous questions on how BNIP3L/NIX tightly balances between cellular life and death decisions. This brief review intends to summarize ongoing dilemmas related to BNIP3L/NIX., The inactive form of the BCL2/adenovirus E1B 19‐kDa‐interacting protein 3‐like (BNIP3L/NIX) receptor is found as a monomer phosphorylated on C‐terminal serine 212. Upon mitophagy induction, the receptor is dephosphorylated and this allows BNIP3L/NIX monomers to form the active form of the receptor, BNIP3L/NIX dimer, which is also additionally activated with double phosphorylation near the LIR domain. Dimerization enables stronger recruitment of autophagosomes on selected mitochondria and more efficient mitophagy.

Mitophagy is a conserved intracellular catabolic process responsible for the selective removal of dysfunctional or superfluous mitochondria to maintain mitochondrial quality and need in cells. Here, we examine the mechanisms of receptor-mediated mitophagy activation, with the focus on BNIP3L/NIX mitophagy receptor, proven to be indispensable for selective removal of mitochondria during the terminal differentiation of reticulocytes. The molecular mechanisms of selecting damaged mitochondria from healthy ones are still very obscure. We investigated BNIP3L dimerization as a potentially novel molecular mechanism underlying BNIP3L-dependent mitophagy. Forming stable homodimers, BNIP3L recruits autophagosomes more robustly than its monomeric form. Amino acid substitutions of key transmembrane residues of BNIP3L, BNIP3L(G204A) or BNIP3L(G208V), led to the abolishment of dimer formation, resulting in the lower LC3A-BNIP3L recognition and subsequently lower mitophagy induction. Moreover, we identified the serine 212 as the main amino acid residue at the C-terminal of BNIP3L, which extends to the intermembrane space, responsible for dimerization. In accordance, the phosphomimetic mutation BNIP3L(S212E) leads to a complete loss of BNIP3L dimerization. Thus, the interplay between BNIP3L phosphorylation and dimerization indicates that the combined mechanism of LIR phosphorylation and receptor dimerization is needed for proper BNIP3L-dependent mitophagy initiation and progression. Abbreviations: AMBRA1: autophagy and beclin 1 regulator 1; Baf A1: bafilomycin A(1); BH3: BCL2 homology 3; BNIP3: BCL2 interacting protein 3; BNIP3L/NIX: BCL2 interacting protein 3 like; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CoCl(2): cobalt (II) chloride; FKBP8: FKBP prolyl isomerase 8; FUNDC1: FUN14 domain containing 1; GABARAP: GABA type A receptor-associated protein; GST: glutathione S-transferase; IMM: inner mitochondrial membrane; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; OMM: outer mitochondrial membrane; PHB2: prohibitin 2; PI: propidium iodide; PINK1: PTEN induced kinase 1; TM: transmembrane domain; TOMM20: translocase of outer mitochondrial membrane 20

In the last two decades, accumulating evidence pointed to the importance of autophagy in various human diseases. As an essential evolutionary catabolic process of cytoplasmatic component digestion, it is generally believed that modulating autophagic activity, through targeting specific regulatory actors in the core autophagy machinery, may impact disease processes. Both autophagy upregulation and downregulation have been found in cancers, suggesting its dual oncogenic and tumor suppressor properties during malignant transformation. Identification of the key autophagy targets is essential for the development of new therapeutic agents. Despite this great potential, no therapies are currently available that specifically focus on autophagy modulation. Although drugs like rapamycin, chloroquine, hydroxychloroquine, and others act as autophagy modulators, they were not originally developed for this purpose. Thus, autophagy may represent a new and promising pharmacologic target for future drug development and therapeutic applications in human diseases. Here, we summarize our current knowledge in regard to the interplay between autophagy and malignancy in the most significant tumor types: pancreatic, breast, hepatocellular, colorectal, and lung cancer, which have been studied in respect to autophagy manipulation as a promising therapeutic strategy. Finally, we present an overview of the most recent advances in therapeutic strategies involving autophagy modulators in cancer.

The mitophagy receptor Nix interacts with LC3/GABARAP proteins, targeting mitochondria into autophagosomes for degradation. Here we present evidence for phosphorylation-driven regulation of the Nix:LC3B interaction. Isothermal titration calorimetry and NMR indicate a ~100 fold enhanced affinity of the serine 34/35-phosphorylated Nix LC3-interacting region (LIR) to LC3B and formation of a very rigid complex compared to the non-phosphorylated sequence. Moreover, the crystal structure of LC3B in complex with the Nix LIR peptide containing glutamic acids as phosphomimetic residues and NMR experiments revealed that LIR phosphorylation stabilizes the Nix:LC3B complex via formation of two additional hydrogen bonds between phosphorylated serines of Nix LIR and Arg11, Lys49 and Lys51 in LC3B. Substitution of Lys51 to Ala in LC3B abrogates binding of a phosphomimetic Nix mutant. Functionally, serine 34/35 phosphorylation enhances autophagosome recruitment to mitochondria in HeLa cells. Together, this study provides cellular, biochemical and biophysical evidence that phosphorylation of the LIR domain of Nix enhances mitophagy receptor engagement.

Recent discoveries of autophagic receptors that recognize specific cellular cargo have opened a new chapter in the autophagy field. Selective removal of damaged organelles or protein aggregates is essential for proper cellular homeostasis and survival. Until today, autophagy receptors and their actions have been described in several aspects of selective autophagy including xenophagy, aggrephagy, pexophagy and mitophagy. Receptors have been proven indispensable for the initiation and finalization of specific cargo removal by autophagy. Mitophagy receptors, scAtg32, BNIP3 and BNIP3L/NIX, in LIR-dependent manner, interact with Atg8/LC3/GABARAP family proteins and recruit autophagy machinery to damaged mitochondria. Moreover, it has been shown that BNIP3L/NIX mediates mitochondrial clearance during reticulocyte differentiation. Recent discovery of its homologue BNIP3 enlightened the role of mitophagy receptors in cellular fate, where either survival by mitophagy or death by apoptosis is chosen. Autophagic function of the receptors is regulated by phosphorylation but what the trigger is for the phosphorylation and activation of mitophagy is still obscure. Fine-tuning of mitophagy regulation (and general autophagy) is the major challenge for researchers of our time and their answers will allow us to better understand the role of receptors and autophagy in disease development.

Nedavna istraživanja ukazuju da autofagija posreduje u selektivnom uklanjanju proteinskih agregata, organela poput mitohondrija i ribosoma ili patogena. Za selektivno uklanjanje mitohondija odgovoran je mitohondrijski protein Nix koji se ponaša kao receptor koji regulira mehanizam autofagije. Cilj ovoga istraživanja je ustanoviti ulogu dimerizacije proteina Nix u aktivaciji mitofagije. Ciljanom mutagenezom napravljena su dva tipa mutanti u proteinu Nix; koji tvore samo monomer i oni koji formiraju monomere i dimere. Za utvrđivanje sposobnosti vezanja autofagosomalnih proteina za divlji tip i mutante proteina Nix korištene su metode GST Pull-down i Western imunodetekcija. Imunofluorescencijska mikroskopija korištena je za određivanje stanične lokalizacije proteina Nix i za određivanje mitofagijske aktivnost u normalnim uvjetima kao i u prisutnosti mitohondrijskog otrova CCCP. Prema očekivanjima, rezultati pokazuju povećanu aktivnost mitofagije u stanicama koje prekomjerno eksprimiraju normalnu varijantu proteina Nix. Nasuprot tome, mutante koje gube sposobnost stvaranja dimera pokazuju smanjenu aktivnost mitofagije. Iz rezultata se zaključuje da je dimerizacija proteina Nix jedan od mogućih mehanizama kojim se povećava aktivnost mitofagije. Recent evidence indicates that autophagy mediates selective removal of protein aggregates, organelles such as mitochondria or ribosomes and microbes in cells. Recently, the role of mitochondrial protein Nix as a receptor for selective removal of mitochondria by autophagy was described. The aim of this research was to determine the role of Nix dimerisation in mitophagy activation. Using site directed mutagenesis, two types of mutants were made, with ability to form only monomers or to form both monomers and dimers. GST Pull-down and Western blot analysis were used to determine binding of different Nix forms to autophagosomal proteins. Immunofluorescence microscopy was applied to define Nix localization in cells and to quantify mitophagic activity in normal conditions and in the presence of mitochondrial poison CCCP. As expected, results showed increased mitophagy activity in the cells overexpressing wild type Nix that form dimers. Nix mutants without the ability to form dimers showed reduced mitophagic activity. In conclusion, Nix dimerisation is a possible additional mechanism how autophagic machinery is recruited to mitoichondria to enhance their removal.