SQSTM1 downregulates avian metapneumovirus subgroup C replication via mediating selective autophagic degradation of viral M2-2 protein.

Avian metapneumovirus subgroup C (aMPV/C), an important pathogen causing acute respiratory infection in chickens and turkeys, contributes to substantial economic losses in the poultry industry worldwide. aMPV/C has been reported to induce autophagy, which is beneficial to virus replication. Sequestosome 1 (SQSTM1/P62), a selective autophagic receptor, plays a crucial role in viral replication by clearing ubiquitinated proteins. However, the relationship between SQSTM1-mediated selective autophagy and aMPV/C replication is unclear. In this study, we found that the expression of SQSTM1 negatively regulates aMPV/C replication by reducing viral protein expression and viral titers. Further studies revealed that the interaction between SQSTM1 and aMPV/C M2-2 protein is mediated via the Phox and Bem1 (PB1) domain of the former, which recognizes a ubiquitinated lysine at position 67 of the M2-2 protein, and finally degrades M2-2 via SQSTM1-mediated selective autophagy. Collectively, our results reveal that SQSTM1 degrades M2-2 via a process of selective autophagy to suppress aMPV/C replication, thereby providing novel insights for the prevention and control of aMPV/C infection.IMPORTANCEThe selective autophagy plays an important role in virus replication. As an emerging pathogen of avian respiratory virus, clarification of the effect of SQSTM1, a selective autophagic receptor, on aMPV/C replication in host cells enables us to better understand the viral pathogenesis. Previous study showed that aMPV/C infection reduced the SQSTM1 expression accompanied by virus proliferation, but the specific regulatory mechanism between them was still unclear. In this study, we demonstrated for the first time that SQSTM1 recognizes the 67th amino acid of M2-2 protein by the interaction between them, followed by M2-2 degradation via the SQSTM1-mediated selective autophagy, and finally inhibits aMPV/C replication. This information supplies the mechanism by which SQSTM1 negatively regulates viral replication, and provides new insights for preventing and controlling aMPV/C infection.

Pepstatin-Based Probes for Photoaffinity Labeling of Aspartic Proteases and Application to Target Identification.

Aspartic proteases are a small class of proteases implicated in a wide variety of human diseases. Covalent chemical probes for photoaffinity labeling (PAL) of these proteases are underdeveloped. We here report a full on-resin synthesis of clickable PAL probes based on the natural product inhibitor pepstatin incorporating a minimal diazirine reactive group. The position of this group in the inhibitor determines the labeling efficiency. The most effective probes sensitively detect cathepsin D, a biomarker for breast cancer, in cell lysates. Moreover, through chemical proteomics experiments and deep learning algorithms, we identified sequestosome-1, an important player in autophagy, as a direct interaction partner and substrate of cathepsin D.

In-Cell Chemical Crosslinking Identifies Hotspots for SQSTM-1/p62-IκBα Interaction That Underscore a Critical Role of p62 in Limiting NF-κB Activation Through IκBα Stabilization.

We have previously documented that in liver cells, the multifunctional protein scaffold p62/SQSTM1 is closely associated with IκBα, an inhibitor of the transcriptional activator NF-κB. Such an intimate p62-IκBα association we now document leads to a marked 18-fold proteolytic IκBα-stabilization, enabling its nuclear entry and termination of the NF-κB-activation cycle. In p62-/--cells, such termination is abrogated resulting in the nuclear persistence and prolonged activation of NF-κB following inflammatory stimuli. Utilizing various approaches both classic (structural deletion, site-directed mutagenesis) as well as novel (in-cell chemical crosslinking), coupled with proteomic analyses, we have defined the precise structural hotspots of p62-IκBα association. Accordingly, we have identified such IκBα hotspots to reside around N-terminal (K38, K47, and K67) and C-terminal (K238/C239) residues in its fifth ankyrin repeat domain. These sites interact with two hotspots in p62: One in its PB-1 subdomain around K13, and the other comprised of a positively charged patch (R183/R186/K187/K189) between its ZZ- and TB-subdomains. APEX proximity analyses upon IκBα-cotransfection of cells with and without p62 have enabled the characterization of the p62 influence on IκBα-protein-protein interactions. Interestingly, consistent with p62's capacity to proteolytically stabilize IκBα, its presence greatly impaired IκBα's interactions with various 20S/26S proteasomal subunits. Furthermore, consistent with p62 interaction with IκBα on an interface opposite to that of its NF-κB-interacting interface, p62 failed to significantly affect IκBα-NF-κB interactions. These collective findings together with the known dynamic p62 nucleocytoplasmic shuttling leads us to speculate that it may be involved in "piggy-back" nuclear transport of IκBα following its NF-κB-elicited transcriptional activation and de novo synthesis, required for termination of the NF-κB-activation cycle. Consequently, mice carrying a liver-specific deletion of p62-residues 68 to 252 reveal age-dependent-enhanced liver inflammation. Our findings reveal yet another mode of p62-mediated pathophysiologically relevant regulation of NF-κB.

P62/SQSTM1 upregulates NQO1 transcription via Nrf2/Keap1a signaling pathway to resist microcystins-induced oxidative stress in freshwater mussel Cristaria plicata.

Microcystins (MCs) are the most frequent and widely distributed type of cyanotoxin in aquatic systems, and they cause an imbalance of the body's oxidative system. In a previous experiment, we demonstrated that the mollusk Cristaria plicata can protect against MC-induced oxidative damage through the nuclear factor erythroid 2-related factor 2(Nrf2)/Kelch-like epichlorohydrin-related protein-1 (Keap1) pathway. Here, we evaluated whether selective autophagy affects the Nrf2/Keap1a anti-oxidative stress pathway in C. plicata. Full-length cDNA sequences of p62/SQSTM1 from C. plicata (Cpp62) were divided into 2484 bp fragments. From N-terminal to C-terminal, the amino acid sequence of Cpp62 contained PB1 (Phox and Bem1p domain), ZNF (zinc finger domain) chain, LIR (LC3 interacting region) and UBA (ubiquitin-associated domain) domains, but not the KIR (Keap1 interacting region) domain. We confirmed that Cpp62 did not bind to CpKeap1a in vitro, and the relative level of Cpp62 was the highest in the hepatopancreas. Moreover, MCs significantly upregulated the mRNA and protein levels of Cpp62 in the hepatopancreas after CpKeap1a knockdown, whereas Nrf2 upregulated the transcription levels of Cpp62, suggesting that MCs increased Cpp62 expression via the Nrf2/Keap1a signaling pathway. Moreover, Cpp62 and CpNrf2 proteins have a strong affinity for the NQO1 promoter, but MCs inhibited the ability of CpNrf2 and Cpp62 to upregulate luciferase activity. The results show that Nrf2 and the p62 protein induced p62 expression by binding to ARE (antioxidant response element) sequences in the p62 promoter of C. plicata, thereby promoting p62 to resist MC-induced oxidative stress. Therefore, we speculate that MCs induce p62-dependent autophagy in C. plicata, resulting in the inhibition of Nrf2 transcription and Cpp62 promoter activity. These findings help to reveal the mechanism by which the p62-Nrf2/Keap1 pathway mitigates MC-induced oxidative damage in mussels.

SQSTM1/p62 promotes miR-198 loading into extracellular vesicles and its autophagy-related secretion.

MicroRNA dysregulation is a hallmark of hepatocellular carcinoma (HCC), leading to tumor growth and metastasis. Previous screening on patient specimens identified miR-198 as the most downregulated miRNA in HCC. Here, we show that miR-198 compensation leads to self-release into extracellular vesicles (EVs). Importantly, the vesicular secretion is mediated by autophagy-related pathway, initiated by sequestration of p62/miR-198 complexes in autophagosome-associated vesicle fractions. miR-198 is selectively recognized and loaded by p62 into autophagosomal fractions, whereas mutated miR-198 forms neither induce autophagy and nor interact with p62. Gain and loss of function experiments, using a CRIPR/Cas knockout (KO) and transgenic site-specific p62 mutants, identified p62 as an essential repressor of cellular miR-198 abundancy. Notably, EVs, harboring miR-198/p62 protein complexes, can be uptaken by cells in the close vicinity, leading to change of gene expression in recipient cells. In conclusion, miR-198 enhances autophagy; conversely autophagic protein p62 reduces the miR-198 levels by sorting into extracellular space. miR-198 is at first transcribed as primary miRNA, after being processed into single stranded mature miR-198 form, it is transported into cytoplasm ①. By interaction with p62 protein, miR-198 conglomerates and forms a binding complex ②. Since LC3 protein is an interaction partner of p62 protein, hence miR-198 is included into autophagosomes ③. By fusion with multivesicular bodies (MVB), miR-198-binding complex was recruited into amphisomes ④, the latter of which quickly turns into secretory MVB containing intraluminal vesicles⑤. By fusion with cell membrane, intraluminal vesicles were released into extracellular space as EVs ⑥.

Vault RNA1-1 riboregulates the autophagic function of p62 by binding to lysine 7 and arginine 21, both of which are critical for p62 oligomerization.

Cellular processes can be regulated at multiple levels, including transcriptional, post-transcriptional, and post-translational mechanisms. We have recently shown that the small, noncoding vault RNA1-1 negatively riboregulates p62 oligomerization in selective autophagy through direct interaction with the autophagic receptor. This function is highly specific for this Pol III transcript, but the determinants of this specificity and a mechanistic explanation of how vault RNA1-1 inhibits p62 oligomerization are lacking. Here, we combine biochemical and functional experiments to answer these questions. We show that the PB1 domain and adjacent linker region of p62 (aa 1-122) are necessary and sufficient for specific vault RNA1-1 binding, and we identify lysine 7 and arginine 21 as key hinges for p62 riboregulation. Chemical structure probing of vault RNA1-1 further reveals a central flexible loop within vault RNA1-1 that is required for the specific interaction with p62. Overall, our data provide molecular insight into how a small RNA riboregulates protein-protein interactions critical to the activation of specific autophagy.

Protocol to identify drug-binding sites in proteins using solution NMR spectroscopy.

Dusquetide is a next-generation IDR (innate defense regulator) targeting the major autophagy receptor protein SQSTM1/p62 and modulating the innate immune response. Here, we describe a protocol for determining dusquetide-binding sites of p62 by solution NMR spectroscopy. Step-by-step technique details were provided, including sample preparation, NMR experiment setup, data processing, and binding site analysis. This protocol could be applied to characterize other small molecules targeting the ZZ domain of p62 (9 kDa) or other proteins containing ZZ domains. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2022).

Sequestosome 1 Is Part of the Interaction Network of VAPB.

VAPB (Vesicle-Associated-membrane Protein-associated protein B) is a tail-anchored membrane protein of the endoplasmic reticulum that can also be detected at the inner nuclear membrane. As a component of many contact sites between the endoplasmic reticulum and other organelles, VAPB is engaged in multiple protein interactions with a plethora of binding partners. A mutant version of VAPB, P56S-VAPB, which results from a single point mutation, is involved in a familial form of amyotrophic lateral sclerosis (ALS8). We performed RAPIDS (rapamycin- and APEX-dependent identification of proteins by SILAC) to identify proteins that interact with or are in close proximity to P56S-VAPB. The mutation abrogates the interaction of VAPB with many known binding partners. Here, we identify Sequestosome 1 (SQSTM1), a well-known autophagic adapter protein, as a major interaction/proximity partner of P56S-VAPB. Remarkably, not only the mutant protein, but also wild-type VAPB interacts with SQSTM1, as shown by proximity ligation assays and co-immunoprecipiation experiments.

Effects of Weak Nonspecific Interactions with ATP on Proteins.

Adenosine triphosphate (ATP) is an immensely well-studied metabolite serving multiple key biochemical roles as the major chemical energy currency in living systems, a building block of ribonucleic acids, and a phosphoryl group donor in kinase-mediated signaling. Intriguingly, ATP has been recently proposed to act as a hydrotrope that inhibits aggregation of amyloidogenic proteins; however, the underlying mechanism and the general physicochemical effect that coexistence with ATP exerts on proteins remain unclear. By combining NMR spectroscopy and MD simulations, here we observed weak but unambiguously measurable and concentration-dependent noncovalent interactions between ATP and various proteins. The interactions were most pronounced for an intrinsically disordered protein (α-synuclein) and for residues in flexible regions (e.g., loops or termini) of two representative folded proteins (ubiquitin and the dimeric ubiquitin-binding domain of p62). As shown by solution NMR, a consequence of the ATP-protein interaction was altered hydration of solvent-exposed residues in the protein. The observation that ATP interacted with all three proteins suggests that ATP is a general nonspecific binder of proteins. Several complementary biophysical methods further confirmed that, at physiological concentrations of ∼5-10 mM, ATP starts to form oligomeric states via magnesium-chelating and chelation-independent mechanisms, in agreement with previous studies. Although the observed ATP-protein interaction was relatively weak overall, the high ratio of ATP (monomeric free ATP, mono- and divalent ion-bound ATP, oligomeric and chelated ATP) to proteins in cells suggests that most proteins are likely to encounter transient interactions with ATP (and chemically similar metabolites) that confer metabolite-mediated protein surface protection.

An Experimentally Induced Mutation in the UBA Domain of p62 Changes the Sensitivity of Cisplatin by Up-Regulating HK2 Localisation on the Mitochondria and Increasing Mitophagy in A2780 Ovarian Cancer Cells.

The study of cisplatin sensitivity is the key to the development of ovarian cancer treatment strategies. Mitochondria are one of the main targets of cisplatin, its self-clearing ability plays an important role in determining the fate of ovarian cancer cells. First, we proved that the sensitivity of ovarian cancer cells to cisplatin depends on mitophagy, and p62 acts as a broad autophagy receptor to regulate this process. However, p62's regulation of mitophagy does not depend on its location on the mitochondria. Our research shows that the mutation of the UBA domain of p62 increases the localisation of HK2 on the mitochondria, thereby increasing the phosphorylated ubiquitin form of parkin, then stabilising the process of mitophagy and ultimately cell survival. Collectively, our results showed that a mutation in the UBA domain of p62 regulates the level of apoptosis stimulated by cisplatin in ovarian cancer.

The high stability of the three-helix bundle UBA domain of p62 protein as revealed by molecular dynamics simulations.

The ubiquitin-associated (UBA) domain is an important motif in the modulation of many molecular functionalities. It has been mainly associated with ubiquitin-mediated proteolysis, a multistep mechanism in which undesirable proteins are tagged with polyubiquitin chains for degradation in the proteasome complex. Comparison among UBA domains reveals a quite small structural variability, displaying an overall fold with a tightly packed three-helix bundle, and a common conserved hydrophobic patch on their surface that is important for ubiquitin binding. Mutations in the UBA domain, mainly in the highly conserved hydrophobic patch, induce conformational instabilities, which can be related to weak affinity for ubiquitin. This raises the question whether such hydrophobic patch presents conserved structural arrangement for selective recognition and protein binding. A concern that led us to investigate the stability of the p62-UBA domain as a case study regarding its structural arrangement as a function of temperature and two NaCl concentrations. Our results reveal that the temperature range and ionic strengths considered in this work produced a negligible effect on the three-helix bundle fold of p62-UBA domain.

Structure and function of p62/SQSTM1 in the emerging framework of phase separation.

p62/SQSTM1 is a multiprotein interaction hub forming cellular punctate structures known as p62 bodies. p62 is centrally involved in the degradation of ubiquitinated cargo through autophagy, as well as in a wide range of signaling activities as part of the cellular response to nutrient sensing, oxidative stress, infection, immunity, and inflammation. Structural work has shown that p62 forms flexible filamentous assemblies composed of an N-terminal PB1-domain scaffold and a C-terminal binding platform, including folded recognition domains and structurally disordered binding motifs. In the cell, these filaments are part of cellular p62 bodies that display properties of liquid-liquid-phase separation. Here, we review the accumulated structural and functional work of p62 and integrate them with the emerging framework of filamentous biomolecular condensates.

The Interaction of the Tumor Suppressor FAM46C with p62 and FNDC3 Proteins Integrates Protein and Secretory Homeostasis.

FAM46C is a non-canonical poly(A) polymerase uniquely mutated in up to 20% of multiple myeloma (MM) patients, implying a tissue-specific tumor suppressor function. Here, we report that FAM46C selectively stabilizes mRNAs encoding endoplasmic reticulum (ER)-targeted proteins, thereby concertedly enhancing the expression of proteins that control ER protein import, folding, N-glycosylation, and trafficking and boosting protein secretion. This role requires the interaction with the ER membrane resident proteins FNDC3A and FNDC3B. In MM cells, FAM46C expression raises secretory capacity beyond sustainability, inducing ROS accumulation, ATP shortage, and cell death. FAM46C activity is regulated through rapid proteasomal degradation or the inhibitory interaction with the ZZ domain of the autophagic receptor p62 that hinders its association with FNDC3 proteins via sequestration in p62+ aggregates. Altogether, our data disclose a p62/FAM46C/FNDC3 circuit coordinating sustainable secretory activity and survival, providing an explanation for the MM-specific oncosuppressive role of FAM46C and uncovering potential therapeutic opportunities against cancer.

The Deubiquitinating Enzyme USP20 Regulates the TNFα-Induced NF-κB Signaling Pathway through Stabilization of p62.

p62/sequestosome-1 is a scaffolding protein involved in diverse cellular processes such as autophagy, oxidative stress, cell survival and death. It has been identified to interact with atypical protein kinase Cs (aPKCs), linking these kinases to NF-κB activation by tumor necrosis factor α (TNFα). The diverse functions of p62 are regulated through post-translational modifications of several domains within p62. Among the enzymes that mediate these post-translational modifications, little is known about the deubiquitinating enzymes (DUBs) that remove ubiquitin chains from p62, compared to the E3 ligases involved in p62 ubiquitination. In this study, we first demonstrate a role of ubiquitin-specific protease USP20 in regulating p62 stability in TNFα-mediated NF-κB activation. USP20 specifically binds to p62 and acts as a positive regulator for NF-κB activation by TNFα through deubiquitinating lysine 48 (K48)-linked polyubiquitination, eventually contributing to cell survival. Furthermore, depletion of USP20 disrupts formation of the atypical PKCζ-RIPK1-p62 complex required for TNFα-mediated NF-κB activation and significantly increases the apoptosis induced by TNFα plus cycloheximide or TNFα plus TAK1 inhibitor. These findings strongly suggest that the USP20-p62 axis plays an essential role in NF-κB-mediated cell survival induced by the TNFα-atypical PKCζ signaling pathway.

p62/SQSTM1, a Central but Unexploited Target: Advances in Its Physiological/Pathogenic Functions and Small Molecular Modulators.

p62/SQSTM1, encoded by gene SQSTM1, is widely known as an adaptor protein of selective autophagy to promote aggregate-prone proteins for degradation. It is also a stress-induced scaffold protein involved in Nrf2 activation to resist oxidative stress. Multiple domains of p62 interact with several essential pathways implicated in cell differentiation and proliferation, placing p62 at a significant position to mediate cell survival and apoptosis. The p62 protein has been suggested as a potential target in recent years, since its abnormal expression or SQSTM1 gene mutation is tightly associated with various diseases including cancer such as hepatocellular carcinoma and prostate cancer, neurodegenerative disorders such as Alzheimer's disease and amyotrophic lateral sclerosis, atherosclerosis, and Paget's disease of bone. In this review, we will discuss the relationship between p62 and these diseases, and we attempt to put forward novel methods for current diagnosis or therapy by regulating the p62 expression level.

Pinpoint analysis of a protein in slow exchange using F1F2-selective ZZ-exchange spectroscopy: assignment and kinetic analysis.

ZZ-exchange spectroscopy is widely used to study slow exchange processes in biomolecules, especially determination of exchange rates and assignment of minor peaks. However, if the exchange cross peaks overlap or the populations are skewed, kinetic analysis is hindered. In order to analyze slow exchange protein dynamics under such conditions, here we have developed a new method by combining ZZ-exchange and F1F2-selective NMR spectroscopy. We demonstrate the utility of this method by examining the monomer-dimer transition of the ubiquitin-associated domain of p62, successfully assigning the minor (monomeric) peaks and obtaining the exchange rates, which cannot be achieved by ZZ-exchange alone.

Structural basis of p62/SQSTM1 helical filaments and their role in cellular cargo uptake.

p62/SQSTM1 is an autophagy receptor and signaling adaptor with an N-terminal PB1 domain that forms the scaffold of phase-separated p62 bodies in the cell. The molecular determinants that govern PB1 domain filament formation in vitro remain to be determined and the role of p62 filaments inside the cell is currently unclear. We here determine four high-resolution cryo-EM structures of different human and Arabidopsis PB1 domain assemblies and observed a filamentous ultrastructure of p62/SQSTM1 bodies using correlative cellular EM. We show that oligomerization or polymerization, driven by a double arginine finger in the PB1 domain, is a general requirement for lysosomal targeting of p62. Furthermore, the filamentous assembly state of p62 is required for autophagosomal processing of the p62-specific cargo KEAP1. Our results show that using such mechanisms, p62 filaments can be critical for cargo uptake in autophagy and are an integral part of phase-separated p62 bodies.

NIMA-related kinase 9-mediated phosphorylation of the microtubule-associated LC3B protein at Thr-50 suppresses selective autophagy of p62/sequestosome 1.

Human ATG8 family proteins (ATG8s) are active in all steps of the macroautophagy pathway, and their lipidation is essential for autophagosome formation. Lipidated ATG8s anchored to the outer surface of the phagophore serve as scaffolds for binding of other core autophagy proteins and various effector proteins involved in trafficking or fusion events, whereas those at the inner surface are needed for assembly of selective autophagy substrates. Their scaffolding role depends on specific interactions between the LC3-interacting region (LIR) docking site (LDS) in ATG8s and LIR motifs in various interaction partners. LC3B is phosphorylated at Thr-50 within the LDS by serine/threonine kinase (STK) 3 and STK4. Here, we identified LIR motifs in STK3 and atypical protein kinase Cζ (PKCζ) and never in mitosis A (NIMA)-related kinase 9 (NEK9). All three kinases phosphorylated LC3B Thr-50 in vitro A phospho-mimicking substitution of Thr-50 impaired binding of several LIR-containing proteins, such as ATG4B, FYVE, and coiled-coil domain-containing 1 (FYCO1), and autophagy cargo receptors p62/sequestosome 1 (SQSTM1) and neighbor of BRCA1 gene (NBR1). NEK9 knockdown or knockout enhanced degradation of the autophagy receptor and substrate p62. Of note, the suppression of p62 degradation was mediated by NEK9-mediated phosphorylation of LC3B Thr-50. Consistently, reconstitution of LC3B-KO cells with the phospho-mimicking T50E variant inhibited autophagic p62 degradation. PKCζ knockdown did not affect autophagic p62 degradation, whereas STK3/4 knockouts inhibited autophagic p62 degradation independently of LC3B Thr-50 phosphorylation. Our findings suggest that NEK9 suppresses LC3B-mediated autophagy of p62 by phosphorylating Thr-50 within the LDS of LC3B.

Oligomer Model of PB1 Domain of p62/SQSTM1 Based on Crystal Structure of Homo-Dimer and Calculation of Helical Characteristics.

Autophagy is an important process for protein recycling. Oligomerization of p62/SQSTM1 is an essential step in this process and is achieved in two steps. Phox and Bem1p (PB1) domains can oligomerize through both basic and acidic surfaces in each molecule. The ZZ-type zinc finger (ZZ) domain binds to target proteins and promotes higheroligomerization of p62. This mechanism is an important step in routing target proteins to the autophagosome. Here, we determined the crystal structure of the PB1 homo-dimer and modeled the p62 PB1 oligomers. These oligomer models were represented by a cylindrical helix and were compared with the previously determined electron microscopic map of a PB1 oligomer. To accurately compare, we mathematically calculated the lead length and radius of the helical oligomers. Our PB1 oligomer model fits the electron microscopy map and is both bendable and stretchable as a flexible helical filament.

NIPSNAP1 and NIPSNAP2 act as "eat me" signals to allow sustained recruitment of autophagy receptors during mitophagy.

Removal of damaged mitochondria is vital for cellular homeostasis especially in non-dividing cells, like neurons. Damaged mitochondria that cannot be repaired by the ubiquitin-proteasomal system are cleared by a form of selective autophagy known as mitophagy. Following damage, mitochondria become labelled with 'eat-me' signals that selectively determine their degradation. Recently, we identified the mitochondrial matrix proteins, NIPSNAP1 (nipsnap homolog 1) and NIPSNAP2 as 'eat-me' signals for damaged mitochondria. NIPSNAP1 and NIPSNAP2 accumulate on the mitochondrial outer membrane following mitochondrial depolarization, recruiting autophagy receptors and adaptors, as well as human Atg8 (autophagy-related 8)-family proteins to facilitate mitophagy. The NIPSNAPs allow a sustained recruitment of SQSTM1-like receptors (SLRs) to ensure efficient mitophagy. Zebrafish lacking Nipsnap1 show decreased mitophagy in the brain coupled with increased ROS production, loss of dopaminergic neurons and strongly reduced locomotion.