The emerging roles of autophagy in homeostasis of Lysosome-Related Organelles

Xinran Wang^1,2Luan Zhang^1,2Xueqin Cao²Yuting Zhao^1*

• ¹Institute of Future Agriculture, Northwest A&F University, Yangling, China
• ²Northwest A&F University College of Life Sciences, Yangling, China

Introduction Canonical autophagy mediates the quality control of damaged organelles selectively, such as the clearance of mitochondria (mitophagy) and lysosomes (lysophagy) (Picca et al., 2023;Vargas et al., 2023). Selective autophagy receptors recognize organelle cargos, some of which depend on cargo ubiquitination (Vargas et al., 2023). Lysosome-Related Organelles (LROs) refer to a variety of secretory compartments, including melanosomes in pigment cells, Weibel-Palade bodies (WPBs) in endothelial cells, lamellar bodies (LBs) in type II alveolar epithelial cells, Histocompatibility Complex (MHC) class II compartments in antigen-presenting cells (APCs), secretory granules (SGs) in mast cells, and so on (Delevoye et al., 2019). However, it remains unclear if autophagy regulates the clearance of LROs, and if known or novel autophagy receptors are involved. Two recent studies uncover the mechanisms underlying the selective autophagy of melanosomes (melanophagy) – the first LRO selective autophagy (Lee et al., 2024;Park et al., 2024), which shares common strategy with other types of selective autophagy. Noncanonical autophagy has been implicated in the formation, maturation and secretion of various LROs (Ushio et al., 2011;Torisu et al., 2013;Ramkumar et al., 2017;Morishita et al., 2020;Li et al., 2022;Sarango et al., 2022;Omari et al., 2024), suggesting the complex roles of autophagy in LRO regulation and demanding in-depth research on autophagy-LROs. In this opinion piece, we compare the molecular mechanisms reported by recent studies on melanophagy. We also discuss the current understanding of the roles of autophagy – mostly noncanonical, in regulating LRO biogenesis and secretion, and propose future studies to investigate the roles of autophagy in LRO homeostasis. Selective autophagy of melanosomes (melanophagy) Selective autophagy of damaged organelles has been extensively studied (Vargas et al., 2023). In general, after cellular organelles get damaged by stresses, specific E3 ligases mediate the polyubiquitination of specific cargo substrates on or in the organelles which become accessible upon damage; selective autophagy receptors interact with both polyubiquitinated cargo substrates and lipidated ATG8/LC3/GABARAP family proteins, to recruit isolation membrane to the cargo - damaged LROs and autophagy organelles; with isolation membrane growth and closure, autophagosome enclosing cargo is formed, then fuses with lysosome leading to the degradation of the cargo inside (Figure 1A). The latter process shares the same machinery as bulk or non-selective autophagy, including ULK1 complex, ATG9, class III phosphatidylinositol 3-kinase (PI3K) complex, and ATG8-conjugation system. Some selective autophagy receptors are resident proteins in the cargo organelles (i.e., ER-phagy) thus polyubiquitination is not required (Vargas et al., 2023). Phosphorylation on E3 ubiquitin ligases or selective autophagy receptors by certain protein kinases can further regulate the process of selective autophagy (Vargas et al., 2023). It is surprising that little is known if selective autophagy of LROs occurs. Melanosomes are LROs in pigment cells like skin melanocytes and retinal pigment epithelial cells. Recently, two groups have reported that autophagy plays a canonical role in melanosome degradation (Park et al., 2020;Lee et al., 2024;Park et al., 2024), demonstrating the first example of LRO selective autophagy. β-Mangostin reduces intracellular and extracellular amounts of melanosomes in α-melanocyte stimulating hormone (MSH)-stimulated melanocytes, and such effect is reverted by autophagy inhibition via ATG5 (a component of ATG8-conjugation system) knockdown, FIP200 (a subunit of ULK1 complex) knockdown, or 3-Methyladenine treatment (Class III PI3K inhibitor) (Lee et al., 2024). Similarly, 3,4,5-trimethoxycinnamate thymol ester (TCTE) inhibits skin pigmentation in an autophagy-dependent manner, as TCTE reduces α-MSH-stimulated pigmentation and such reduction is restored by ATG5 knockdown (Park et al., 2020). Furthermore, β-mangostin induces degradation of melanosomes but not mitochondria, endoplasmic reticulum (ER), or peroxisomes, indicating that the process is selective (Lee et al., 2024). If β-mangostin and TCTE induce selective autophagy of melanosome (melanophagy), what selective autophagy receptors and possible E3 ubiquitin ligases participate the process? As summarized in Table 1, OPTN/optineurin is identified as the melanophagy receptor, screened from several known selective autophagy receptors including NBR1, SQSTM1/p62, FUNDC1, NDP52, NIX, and TAX1BP1 (Lee et al., 2024;Park et al., 2024). K63-linked polyubiquitination on total melanosome proteins is enhanced upon β-mangostin treatment, while polyubiquitination on melanosome marker MLANA/Melan-A is increased upon TCTE stimulation. OPTN colocalizes with melanosomes via ubiquitin-binding domain (UBD) or interacts with MLANA in response to stresses, completing the step of cargo recognition. OTPN also binds to lipidated LC3B to recruit isolation membrane to melanosome. E3 ubiquitin ligases RCHY1 and ITCH are identified to regulate the polyubiquitination of melanosome cargo substrates. TBK1 phosphorylates OPTN (on mouse Ser 187, corresponding to human Ser 177) to activate OPTN during β-mangostin-induced melanophagy, and OPTN is required for TBK1 activation on melanosome as well. PTK2 phosphorylates ITCH to promote polyubiquitination of MLANA during TCTE-induced melanophagy. Taken together, the molecular mechanism of melanophagy follow the paradigm of selective autophagy, with two pathways β-mangostin-RCHY1-TBK1-OPTN and TCTE-PTK2-ITCH-MLANA-OPTN characterized (Figure 1B). It is uncertain whether selective autophagy of other types of LROs exists. Since LROs are usually too big for efficient proteasomal degradation, we think it is highly likely that autophagic/lysosomal degradation is employed to clear unwanted or damaged LROs. Future studies can utilize similar strategies of the abovementioned melanophagy studies to identify selective autophagy receptors and regulators of other LROs. Roles of noncanonical autophagy in regulating biogenesis and secretion of LROs The canonical role of autophagy is to recognize cargos, pack them in double-membraned autophagosomes and target them for lysosomal degradation, which is crucial for quality control of organelles upon stresses, as in the processes of melanophagy. During the past decade, noncanonical (non-degradative, or autophagosome-independent) roles of autophagy have been extensively LROs and autophagy investigated (Piletic et al., 2023;Deretic et al., 2024) and implicated in regulating the internalization of extracellular components, the secretion of soluble or membrane-enclosed cargos, and noncanonical functions of autophagy proteins. Earlier studies suggest that autophagy plays distinct roles on LRO biogenesis as well as maturation and motility, and secretion, most of which are noncanonical (Figure 1C). During the biogenesis of melanosomes (melanogenesis), UVRAG, a subunit of Class III PI3K complex 2 which mediates autophagosome maturation to autolysosome, is required for cell pigmentation independent of Class III PI3K complex 2 activity; instead, UVRAG interacts with BLOC-1 complex and regulates BLOC-1 stability and distribution, leading to proper melanogenic cargo-sorting (Yang et al., 2018). LC3B, a major ATG8 family protein on autophagosomes, localizes to melanosomes to facilitate transport on microtubules, while ATG4B which regulates LC3B lipidation and delipidation mediates melanosome translocation to actin filaments and transport (Ramkumar et al., 2017). Beclin 1, another subunit of Class III PI3K complex, as well as LC3B and ATG7 have been suggested to activate MITF, the major transcription factor for melanogenic gene expression; however, the underlying molecular mechanisms remain elusive (Lee et al., 2022). MHC class II, mainly expressed in B cells, monocytes, macrophages, dendritic cells and so on, present antigenic peptides on the cell surface to CD4+ T cells (Rock et al., 2016;Pishesha et al., 2022). The antigenic peptides presented by MHC class II are processed in specialized endolysosomal compartments - MHC class II compartments, and loaded onto MHC class II, involving both canonical and noncanonical autophagy (Münz, 2022). Autophagy receptor TAX1BP1 not only facilitates autophagic degradation of intracellular antigens and delivery to MHC class II compartments, but also stabilizes invariant chain CD74/MHC class II complex to ensure the proper presentation of high affinity peptides (Sarango et al., 2022). LC3-associated phagocytosis (LAP) is a type of noncanonical autophagy (degradative, autophagosome-independent) and functions in immune responses, where ATG8/LC3 is conjugated to single-membraned phagosomes, replying on a subset of canonical autophagy machinery such as UVRAG/Beclin 1-containing Class III PI3K complex and ATG8-conjugation system but not ULK1 complex (Peña-Martinez et al., 2022). LAP accelerates extracellular antigen internalization and processing for MHC class II, under the regulation of ATG4B oxidation (Ligeon et al., 2021;Münz, 2022). Secretory autophagy or autophagy-dependent secretion (New and Thomas, 2019;Piletic et al., 2023), another type of noncanonical autophagy (non-degradative, most likely autophagosome-dependent), regulates the content release of several LROs, including Weibel-Palade bodies (WPBs) in endothelial cells, lamellar bodies (LBs) in type II alveolar epithelial cells and secretory granules (SGs) in mast cells. The secretion of von Willebrand factor (VWF) from WPBs requires autophagy machinery, as autophagy inhibition (ATG5 or ATG7 knockdown, inhibitors of lysosomes/autolysosomes chloroquine or Bafilomycin A1) blocks VWF secretion, and WPBs are close to or within LC3-positive autophagosomes (Torisu et al., 2013). The secretion of surfactant from lung LBs relies on autophagy, as autophagy inhibition (FIP200 or ATG7 knockout, 3-Methyladenine) impairs lung LB maturation and surfactant protein secretion, and lung LBs fuse with LC3B-positive autophagosomes (Morishita et al., 2020;Li et al., 2022). The degranulation process of mast cells, during which mast cells release inflammatory mediators like histamine and β-hexosaminidase from SGs upon antigen stimulation, is autophagy(ATG7)-dependent as well (Ushio et al., 2011). CD63-positive SGs fuse with LC3-positive late endosomes (amphisomes) and release exosomes upon stimulation (Omari et al., 2024). Investigating roles of autophagy in regulating homeostasis of LROs As discussed above, autophagy and autophagy proteins participate in different aspects of LROs homeostasis, from the formation and content sorting to LROs, to the content release and breakdown of LROs and autophagy LROs (Figure 1). We think crucial questions shall be addressed in the field, like whether selective autophagy of LROs other than melanosomes take place, what molecular mechanisms underlying noncanonical autophagy of LROs are, and how canonical and noncanonical autophagy are coordinated on the same type of LROs. In addition to screening known autophagy receptors as in melanophagy studies, we propose that LRO cargos and autophagy receptors can be investigated using proteomic approach. In autophagy-deficient cells (like FIP200 or ATG5 deletion), cargos and autophagy receptors shall decrease in lysosomes and increase in whole cells. Therefore, proteomic analyses on purified lysosomes (i.e., through LysoIP (Abu-Remaileh et al., 2017)) and whole cell lysate from autophagy-sufficient and autophagy-deficient cells can narrow down the list of cargos and autophagy receptors (Herhaus et al., 2024). Candidate LRO cargos and autophagy receptors can then be found by overlapping with LRO proteomes. Alternatively, ATG8 family proteins can be used as baits in search of autophagy receptors from purified LROs. To validate if a candidate autophagy receptor is selective for certain LROs, the following criteria shall be met: (1) deletion of the candidate autophagy receptor increases the level of LRO cargos; (2) the candidate autophagy receptor localizes to the LROs and the colocalization is likely enhanced upon stress; (3) the candidate autophagy receptor interacts with ATG8 family proteins; (4) the candidate autophagy receptor is a resident protein of the LROs, or interacts with LRO cargos in a polyubiquitination-dependent manner. As for MHC class II compartments, known lysophagy receptors TAX1BP1 and p62 shall be the on the shortlist. Autophagy machinery can regulate internalization of extracellular components, during the processes of LC3-associated phagocytosis (LAP), micropinocytosis (LAM), or endocytosis (LANDO) (Magné and Green, 2022;Deretic et al., 2024). It will be interesting to test if LAM or LANDO impacts MHC class II compartments in addition to LAP. Secretory autophagy also consists of different processes, such as LC3-dependent extracellular vesicle loading and secretion (LDELS), secretory autophagy during lysosome inhibition (SALI), and so on (Debnath and Leidal, 2022;Deretic et al., 2024). It will be important to elucidate the molecular details of the secretory autophagy of WPBs, lung LBs and mast cell SGs. These LROs colocalize with LC3 and require ATG7 (a component of ATG8-conjugation system) for secretion, however, little is known whether upstream autophagy proteins like ULK1 complex, ATG9, and Class III PI3K complex are necessary, and whether induction of autophagy can promote the secretion of theses LROs. Damaged mitochondria can be released to the extracellular space in an autophagy-dependent manner, rather than degradation via mitophagy (Nicolás-Avila et al., 2020;Gong et al., 2024). It will be intriguing if damaged LROs can be released out of cells like damaged mitochondria, serving as an alternative path for clearance. With a better understanding of how canonical and noncanonical autophagy regulates different stages of LROs life cycle, we expect key autophagy proteins and modulators will soon be uncovered that coordinate the different roles of autophagy in maintaining homeostasis of LROs. LROs and autophagy FIGURE 1. Roles of autophagy in maintaining homeostasis of Lysosome-Related Organelles (LROs). (A) General scheme of selective autophagy, LC3-associated phagocytosis (LAP) and secretory autophagy. Selective autophagy receptor recognizes s specific cargo substrates which are usually polyubiquinated on cargo organelles, conferring the selectivity. Selective autophagy receptor recruits isolation membrane via interacting with lipidated ATG8/LC3/GABARAP family proteins and the subsequent events (isolation membrane growth and closure, autophagosome formation and fusion with lysosome, autolysosome formation and degradation, dark blue arrows) are the same as canonical, degradative autophagy. During LAP, surface receptor recognizes extracellular pathogen, then LAPosome decorated with lipidated LC3 is formed to internalize pathogen before fusion with lysosome and cargo degradation (light blue arrows). Secretory autophagy consists of various processes, where autophagosome, amphisome (autophagosome fused with late endosome) or autolysosome may fuse with the plasma membrane and release content (dark purple arrows). Cyto refers to cytoplasm while Ex refers to extracellular space. For simplicity, lipidated LC3 is not shown on autophagosome, amphisome or autolysosome. (B) Mechanisms of melanophagy. Two recent studies reveal the molecular mechanisms of selective autophagy of melanosomes - melanophagy. Upon β-mangostin stress, melanosome proteins are polyubiquinated by E3 ubiquitin ligase RCHY1, recognized by selective autophagy receptor OPTN which recruits active protein kinase TBK1 and gets phosphorylated. Upon TCTE stress, melanosome protein MLANA is polyubiquinated by E3 ubiquitin ligase ITCH which gets phosphorylated by active protein kinase PTK2, recognized by OPTN as well. OPTN interacts with lipidated LC3B on isolation membrane to target melanosomes for degradation via canonical autophagy. (C) Different roles of autophagy in maintaining homeostasis of LROs. (1) LAP regulates the loading of antigen peptide to MHC class II in antigen-presenting cell MHC class II compartments. TAX1BP1 plays a noncanonical role in stabilizing CD74/MHC class II for proper presentation (light purple arrow). (2) In mast cell, CD63-positive secretory granules fuse with amphisome and release inflammatory mediators and exosomes. (3) In endothelial cell, secretory autophagy regulates the release of von Willebrand factor (VWF) from Weibel-Palade bodies. (4) In type II alveolar epithelial cells, fusion with autophagosomes leads to maturation of lamellar bodies and secretory autophagy regulates the release of surfactant. (5) In melanocytes, LC3B and ATG4B regulate melanosome transport on microtubules and actin filaments, respectively; this process is considered noncanonical as autophagic degradation in not involved (light purple arrow). LROs and autophagy LROs and autophagy TABLE 1. Comparison of two recent studies on melanophagy. Research Lee et al., 2024 Park et al., 2024 Stress β-Mangostin 3,4,5-Trimethoxycinnamate thymol ester (TCTE) Cell type Melanocyte (B16F10) Melanocyte (B16F10) Cargo for degradation Melanosome Melanosome Selective autophagy receptor OPTN OPTN ATG8 protein LC3B LC3B Polyubiquitinated cargo substrate K63-linked, substrates not specified MLANA E3 ubiquitin ligase RCHY1 ITCH Upstream kinase TBK1 PTK2 Phosphorylated substrate (site) OPTN (mouse S187) ITCH (site not specified) Inhibitor ATG5↓, FIP200↓, 3-Methyladenine (→class III PI3K), BX-795(→TBK1) ATG5↓, Dichlone (→ITCH), Y15 (→PTK2) LROs and autophagy