Unconventional Protein Secretion – New Insights Into the Pathogenesis and Therapeutic Targets of Human Diseases Jiyoon Kim, Heon Yung Gee and Min Goo Lee*

REVIEW ARTICLE SERIES: CELL BIOLOGY AND DISEASE Unconventional protein secretion – new insights into the pathogenesis and therapeutic targets of human diseases Jiyoon Kim, Heon Yung Gee and Min Goo Lee*

ABSTRACT the cell. However, discoveries over the last two decades have shown Most secretory proteins travel through a well-documented that an increasing number of proteins use alternative secretory conventional secretion pathway involving the endoplasmic reticulum pathways that do not involve the ER-to-Golgi transport (Malhotra, (ER) and the Golgi complex. However, recently, it has been shown 2013; Ponpuak et al., 2015; Rabouille, 2017). These alternative that a significant number of proteins reach the plasma membrane or pathways include the extracellular secretion of cytosolic proteins extracellular space via unconventional routes. Unconventional that do not bear a signal peptide (i.e. leaderless proteins) (Rubartelli, protein secretion (UPS) can be divided into two types: (i) the 1997), and cell-surface trafficking of transmembrane proteins via a extracellular secretion of cytosolic proteins that do not bear a signal Golgi-bypassing route. These pathways are collectively referred to peptide (i.e. leaderless proteins) and (ii) the cell-surface trafficking of as unconventional protein secretion (UPS) (see Box 1). signal-peptide-containing transmembrane proteins via a route that With a few exceptions, most UPS pathways are induced by bypasses the Golgi. Understanding the UPS pathways is not only various cellular stresses, such as nutrient starvation (Cruz-Garcia important for elucidating the mechanisms of intracellular trafficking et al., 2014), mechanical stress (Schotman et al., 2008), pathways but also has important ramifications for human health, inflammation (Schroder and Tschopp, 2010) and ER stress (Gee because many of the proteins that are unconventionally secreted by et al., 2011; Jung et al., 2016). Notably, many disease conditions are mammalian cells and microorganisms are associated with human associated with various stresses at the cellular or organismal level diseases, ranging from common inflammatory diseases to the lethal (Fulda et al., 2010), indicating the potential of UPS as a promising genetic disease of cystic fibrosis. Therefore, it is timely and emerging target for the development of novel therapeutics to treat appropriate to summarize and analyze the mechanisms of UPS associated human disease. involvement in disease pathogenesis, as they may be of use for the The number of defined UPS-related diseases continues to expand. ’ development of new therapeutic approaches. In this Review, we For example, sterile inflammation related to Alzheimer s disease, discuss the intracellular trafficking pathways of UPS cargos, allergic and autoimmune diseases, and diabetes can be a trigger to particularly those related to human diseases. We also outline the induce unconventional protein secretion (Agosta et al., 2014; Chen disease mechanisms and the therapeutic potentials of new strategies et al., 2015; Freigang et al., 2013; Gardella et al., 2002; Schroder for treating UPS-associated diseases. and Tschopp, 2010). Heat shock proteins (HSPs) that are secreted unconventionally play a pivotal role in the immunomodulation, KEY WORDS: Unconventional secretion, Human disease, proliferation, angiogenesis and invasiveness of cancer (Rodriguez Pathogenesis, Therapeutic target, Leaderless protein et al., 2009; Sarikonda et al., 2015; Zhang et al., 2012). A number of autophagy components, the mutations of which are involved in Introduction various diseases (Jiang and Mizushima, 2014), participate in UPS of According to the classic principle of protein secretion, cargo numerous cargo proteins (Duran et al., 2010; Kinseth et al., 2007; proteins travel by using the conventional pathway from the Manjithaya et al., 2010). Several mutant transmembrane proteins, endoplasmic reticulum (ER) to the Golgi complex, from which whose associated trafficking defects to the cell surface cause they subsequently move to the trans-Golgi network (TGN) and inherited genetic disorders, such as cystic fibrosis and congenital finally to the plasma membrane (Lee et al., 2004). This process is hearing loss can, alternatively, reach the plasma membrane by initiated by recognition of a signal peptide (also known as ‘leader Golgi-bypassing UPS (Gee et al., 2011; Jung et al., 2016). In sequence’) at the N-terminus or transmembrane domain of cargo addition, UPS has even been shown to be essential for proteins, followed by sequential budding and fusion of vesicular microorganisms to mediate their extracellular release and exert carriers (Bonifacino and Glick, 2004). Each step of the secretion biotrophic pathogenicity (Shoji et al., 2014). pathway is under the control of a number of regulatory proteins. Recent developments in the field have contributed to significant Correct regulation of the classic secretory pathway is imperative for advances in the understanding of the molecular processes involved the life and health of the organism (Viotti, 2016). Since the in UPS (Daniels and Brough, 2017; Pompa et al., 2017; Ponpuak discovery of vesicular exocytic mechanisms, this classic protein et al., 2015; Rabouille, 2017; Santos et al., 2017). Nevertheless, a secretion pathway involving ER-to-Golgi transport has been comprehensive description that adequately explains the role in UPS considered the only standard mechanism to move proteins out of in disease pathogenesis is currently lacking. In this Review, we will define emerging roles for UPS in disease pathogenesis and highlight the possibility of novel therapeutics that target UPS. Department of Pharmacology, Brain Korea 21 PLUS Project for Medical Sciences, Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul 120-752, Korea. Disease-associated unconventional secretion pathways Overview and classification *Author for correspondence ([email protected]) With the increased scientific understanding of the alternative

M.G.L., 0000-0001-7436-012X secretion pathways, several researchers have attempted to Journal of Cell Science

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Leaderless non-vesicular UPS – Types I and II Box 1. Conventional and unconventional protein The leaderless non-vesicular class of UPS includes cytoplasmic secretion pathways leaderless proteins that are secreted directly out of the cell either Most secretory proteins reach their destination via the ER–Golgi-target through plasma membrane pores (Type I) (Steringer et al., 2012) or organelle route, which is referred to as the ‘classic’ or ‘conventional’ protein through ABC transporters (Type II) (McGrath and Varshavsky, secretion pathway. These secretory proteins contain a signal peptide 1989). One of several typical triggers for this type of UPS is ‘ ’ (the leader sequence ) that directs their translocation into the lumen or to inflammation, which leads to the extracellular release of diverse the ER membrane. The newly synthesized proteins then exit the ER at an ER-exit site (ERES) through coat protein complex II (COPII)-coated cytokines that do not possess a signal peptide (Schroder and vesicles and, so, reach the Golgi network before being dispatched to the Tschopp, 2010). A well-known example of a protein that utilizes plasma membrane, lysosomes, endosomes or peroxisomes (Gee et al., this UPS is interleukin (IL)-1β, which is mainly expressed in 2018; Viotti, 2016). Fusion of vesicular intermediates and organelles is myeloid cells, such as macrophages and monocytes. Initially, IL-1β mediated by soluble N-ethylmaleimide-sensitive factor (NSF) attachment is produced as a 31-kDa inactive form that is cleaved by caspase-1 protein (SNAP) receptor proteins (SNAREs), Rab proteins and their into the 17-kDa mature form. The latter is then recruited by the regulators (Mellman and Warren, 2000). In addition to the above-mentioned conventional pathway, eukaryotic intracellular NACHT-domain-, LRR-motif- and PYD-containing cells also utilize unconventional protein secretion (UPS) for protein sorting protein3 (NLRP3) component of the inflammasome, thus engaging and delivery. Initially, the term ‘unconventional secretion’ was used for the the immune response (Schroder and Tschopp, 2010). It appears that release of cytoplasmic proteins that lack a signal peptide, i.e. leaderless multiple UPS pathways can mediate IL-1β secretion (see below, proteins, into the extracellular medium. Later, it was found that some ‘Leaderless vesicular UPS - Type III’), depending on inflammatory transmembrane proteins that are synthesized in the ER, reach the plasma conditions and cell type. Secretion of IL-1β from macrophages membrane via a route that bypasses the Golgi complex (Nickel and Rabouille, 2009). As described in the text, Rabouille colleagues divided following inflammation is mediated by a type of UPS that requires UPS into four types, i.e. Type I, II and III UPS of leaderless proteins, and hyper-permeabilization of the plasma membrane (Bergsbaken et al., Type IV UPS of Golgi-bypassing transmembrane proteins (Rabouille et al., 2009; Martín-Sánchez et al., 2016). The precise mechanism of this 2012). However, the ABC transporter-mediated Type II pathway is not well membrane hyper-permeabilization is not yet fully understood studied and needs additional validation (Rabouille, 2017). (Rabouille, 2017), but the N-terminal domain of gasdermin-D, one of the regulators of pyroptosis that is also produced by caspase- 1-mediated cleavage following initiation of inflammation, has been proposed to be involved in the formation of the membrane pore systematically classify the UPS pathways. For example, Deretic and (Ding et al., 2016). The innate immune response evoked by co-workers have classified autophagy-associated protein secretion cytokines, such as IL-1β was originally thought to be the first line of pathways (Ponpuak et al., 2015). However, because their review defense against non-self (e.g. microorganisms) and to serve as a also addresses the overlap between autophagy and conventional sophisticated system to sense danger signals (Pitanga et al., 2016). protein secretion, we will adopt here the UPS classification However, elevated local or systemic levels of IL-1β have been proposed by Rabouille and colleagues (Rabouille, 2017; associated with a number of hereditary or acquired human diseases, Rabouille et al., 2012). According to this, UPS pathways can be such as cryopyrin-associated periodic syndrome (Schroder and divided into four types with the UPS of leaderless proteins being Tschopp, 2010). Therefore, preventing the overt secretion of IL-1β sub-divided into three. They are: (Type I) direct secretion or pore- by modulating UPS pathways would be a potential new therapeutic mediated translocation across the plasma membrane (Ding et al., strategy to overcome these inflammatory diseases (Table 1). 2016; Zacherl et al., 2015), (Type II) ATP-binding cassette (ABC) Another important example of Type I UPS is the translocation of a transporter-based secretion (McGrath and Varshavsky, 1989) and, cargo through the plasma membrane via self-made lipidic pores. (Type III) membrane-bound organelle (autophagosome/ Examples are fibroblast growth factor 2 (FGF2) and the HIV-TAT endosome)-based secretion (Duran et al., 2010; Kinseth et al., protein, both of which are recruited to the cytoplasmic leaflet of the 2007). The fourth type is the Type IV UPS of leader-sequence- plasma membrane by interaction with phosphatidylinositol (4,5)- containing transmembrane proteins, which are synthesized in the bisphosphate (PIP2) and then undergo self-oligomerization; this, ER and reach the plasma membrane by bypassing the Golgi (Gee sequentially, induces membrane insertion, pore formation, and et al., 2011). Because the nature of ABC-transporter-mediated UPS extracellular translocation of FGF2 and HIV-TAT (Debaisieux (Type II) is not fully characterized, we will consider Type I and et al., 2012; Rabouille, 2017; Zeitler et al., 2015). Phosphorylation Type II as a single category of leaderless non-vesicular UPS of FGF2 by Tec kinases has been shown to be essential for PIP2- (Fig. 1). We will discuss these three categories of UPS with a focus mediated translocation of FGF2 (Ebert et al., 2010). However, many on their link to human disease (summarized in Table 1). questions persist concerning this process, including the source of the In addition to the pathways mentioned above, there are other cell energy for self-oligomerization, formation of the membrane pore, and processes that can be considered to be UPS. For example, transport translocation across the membrane. Recently, the Na+/K+-ATPase through the intercellular channels that function as pathways for the subunit α1(ATP1A1),anα-chain of the Na+/K+-ATPase cellular spreading of macromolecules, including pathogens such as heterotetramer (Shull et al., 1986), was identified as a regulatory viruses and prion-like proteins, could be viewed a specialized form factor for FGF2 secretion that is involved in recruitment of FGF2 to of UPS (see Box 2). Additionally, the senescence-associated the plasma membrane leaflet (Zacherl et al., 2015). Interestingly, the secretory phenotype (SASP) is characterized by the secretion of secretion of FGF2 is inhibited by the lack of extracellular heparan pro-inflammatory and matrix-degrading molecules from senescent sulfate proteoglycans, which may function as extracellular traps for cells and is associated with a number of human diseases, including FGF2 (Nickel, 2007). FGF2 has been shown to be crucial for the atherosclerosis, cancer and inflammatory diseases (Coppé et al., development of the central nervous system and adult neurogenesis, 2008). Because many SASP factors are leaderless proteins, UPS and proposed as a therapeutic target for various neurodegenerative mechanisms are thought to be involved in their extracellular diseases, including Alzheimer’s disease, Parkinson’s disease, secretion (see Box 3). multiple sclerosis and traumatic brain injury (Woodbury and Ikezu, Journal of Cell Science

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Fig. 1. Protein secretion pathways. The Unconvent ional t conventional secretion pathway involves raffic king the ER-to-Golgi transport of cargo (blue and yellow circles). However, a number of Leaderless proteins use alternative secretory vesicular UPS pathways, known as unconventional Leaderless non-vesicular UPS ABC transporter protein secretion (UPS) that do not involve ER-to-Golgi transport. Pore formation Leaderless cytosolic proteins (orange Golgi-bypassing circles) can be secreted by the cell via non-vesicular routes, such as through a UPS membrane pore and the ABC transporter Conventionaltrafficking (Leaderless non-vesicular UPS). For example, UPS of FGF2 involves pore formation within the membrane, Tec kinase, ATP1A1 and extracellular heparan sulfate proteoglycan. In addition, a-factor of Saccharomyces cerevisiae requires the ABC transporter trans -Golgi Plasma membrane Ste6P. Another subset of leaderless cytosolic proteins, including IL-18, IL-33, α-synuclein, amyloid-β and IDE, can be secreted through autophagy-associated cis-GolgiCis-Golgi vesicles, such as lysosomes and late endosomes (Leaderless vesicular UPS). Leaderless proteins In addition, transmembrane proteins (red circles) can reach the plasma membrane by bypassing the Golgi (Golgi-bypassing UPS). Examples for this are αPS1 integrin, Mpl, and ΔF508-CFTR, which ER can be transported to the plasma membrane by GRASP-dependent UPS.

Nucleus

Key Transmembrane proteins ER luminal proteins Leaderless cytosolic proteins Transmembrane proteins (conventional) (unconventional)

2014). Therefore, a modulation of the regulatory proteins that are be involved in the unconventional secretion of HASPB (Ritzerfeld involved in the UPS of FGF2, such as ATP1A1 or proteoglycans, et al., 2011). However, further studies are needed to elucidate the might have therapeutic potential for neurodegenerative diseases. precise molecular mechanisms of UPS of HASPB, and to answer Accumulating evidence suggests that extracellular HIV-TAT, which the question of the link between these COPI subunits and ABC is also secreted through the Type I UPS occurring through self-made transporters. Interestingly, HASPB is modified by lipidation, pores, acts as a viral toxin and plays a key role in the progression of including N-myristoylation and palmitoylation, which is required acquired immune-deficiency syndrome (AIDS) (Debaisieux et al., for trafficking to the cell surface (Denny et al., 2000). Therefore, the 2012). Therefore, inhibition of UPS regarding HIV-TAT could be study of how the lipidation event is involved in the secretion and beneficial to the treatment of AIDS. function of HASPB might contribute to the future development of It has been suggested that some leaderless cargos can be secreted therapeutics. out of the cell by ABC transporters. Since the initial discovery in 1989 that a-factor, the mating pheromone of Saccharomyces Leaderless vesicular UPS – Type III cerevisiae, is secreted from the cell by the ABC transporter Ste6p The leaderless vesicular class (Type III) of UPS involves leaderless (McGrath and Varshavsky, 1989), several other proteins, including cytoplasmic proteins that are packaged into membrane-bound the m-factor of Schizosaccharomyces pombe (Christensen et al., organelles, including autophagosomes, lysosomes, endosomes and 1997) and hydrophilic acylated surface protein B (HASPB) of exosomes. Thus far, numerous studies have examined the substrate Leishmania species (Denny et al., 2000; Maclean et al., 2012), have cargo proteins of leaderless vesicular UPS pathways and their been found to be exported by ABC transporter-mediated transport mechanisms (Dupont et al., 2011; Ejlerskov et al., 2013; translocation. Leishmania HASPB is a leaderless protein that is Lotze and Tracey, 2005). Although the transport process of localized to the extracellular face of its plasma membrane. individual cargo proteins varies greatly, autophagy-related Leishmaniasis is caused by protozoan parasites of Leishmania vesicular structures appear to be an important transport carrier in species and spreads through the bites of certain types of sandfly. Type III UPS (Manjithaya and Subramani, 2010). The best-known During Leishmaniasis pathogenesis, HASPB has been suggested to role of autophagy is that of a degradative canonical pathway that be required for either initial parasite transmission to the host or the contributes to nutrient recycling and cellular defense by digesting establishment of parasites within host macrophages (Maclean et al., cytoplasmic components during starvation (Galluzzi et al., 2014), as 2012). β-COP and δ-COP, subunits of the COPI coatomer complex well as aggregated proteins (Rogov et al., 2014), damaged that participates in the conventional secretion pathway, are known to organelles (Yamano et al., 2014) and invading pathogens (Deretic Journal of Cell Science

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Table 1. Overview of UPS pathways associated with diseases and disorders Related diseases/disorders/ Therapeutic Cargo Classificiationa Involved proteins/processes inabilities/injuries potentialb References IL-1β A NLRP3 inflammasome, gasdermin- Hemorrhagic disorders, Inhibition A: (Andrei et al., 1999; Ding et al., 2016; N, Caspase-1, endolysosomal Alzheimer’s disease, gout, Duewell et al., 2010; Dutra et al., vesicle, exosome, Diabetes, atherosclerosis, 2014; Gardella et al., 2002; Heneka hyperpermeabilization CAPS et al., 2013; Kuemmerle-Deschner, B ATG5, ATG7, RAB8, MVB, HSP90, Inhibition 2015; Martinon et al., 2006; Masters GRASP55, GRASP65 et al., 2010; Qu et al., 2007) B:(Lopez-Castejon and Brough, 2011; Schroder and Tschopp, 2010; Zhang et al., 2015) IL-33 Nc Caspase-1 Asthma, COPD, arthritis Inhibition (Kearley et al., 2015; Palmer et al., 2009; Prefontaine et al., 2009) IL-1α Nc Calpains Stroke, hemorrhagic Inhibition (Freigang et al., 2013; Greenhalgh disorders, cancer, et al., 2012; Laberge et al., 2015; atherosclerosis, SASP Luheshi et al., 2011; Singer et al., 2006)

FGF2 A PIP2, Tec kinase, heparan sulfate Alzheimer’s disease, Activation (Nickel, 2010; Woodbury and Ikezu, proteoglycans, ATP1A1 Parkinson’s disease, MS, 2014; Zacherl et al., 2015) TBI

TAT A PIP2 AIDS Inhibition (Debaisieux et al., 2012; Zeitler et al., 2015) HASPB A Acylation, β-COP, δ-COP Leishmaniasis Inhibition (Denny et al., 2000; Maclean et al., 2012) PfCDPK1 A Acylation Inhibition (Möskes et al., 2004) IL-18 B ATG5, GRASP55, caspase-1, AMD, asthma Activation (Doyle et al., 2014; Gu et al., 1997; PRTN3 Lopez-Castejon and Brough, 2011; Murai et al., 2015; Sugawara et al., 2001) HMGB1 B ATG5, secretory lysosome Sepsis, lung disease, arthritis, Inhibition (Abraham et al., 2000; Kokkola et al., stroke, hemorrhagic shock, 2003; Liu et al., 2007; Qin et al., cancer 2006; Thorburn et al., 2009; Yang et al., 2006; Zhang et al., 2011) Galectin-3 B Beclin1 Cancer, heart disease, stroke Inhibition (Ohman et al., 2014) α-Synuclein B ATG5, RAB8 Parkinson’s disease Inhibition (Ejlerskov et al., 2013) IDE B Autophagosome, RAB8A, GRASP55 Alzheimer’s disease Activation (Son et al., 2016) LIF, FAM3C, B ATG7 Cancer Inhibition (Kraya et al., 2015) DKK3 Mpl C ATG5, GRASP55 Myeloproliferative cancer Inhibition (Cleyrat et al., 2014) CFTR C GRASP55, GRASP65, ATG1, ATG5, Cystic fibrosis, pancreatitis, Activation (Gee et al., 2011; Kim et al., 2016; ATG7, ATG8, SEC16, IRE1α bronchiectasis, infertility LaRusch et al., 2014; Piao et al., 2017) Pendrin C HSP70, DNAJC14, RAB18, IRE1α DFNB4, Pendred syndrome Activation (Dossena et al., 2009; Jung et al., 2016) Polycystin-2 Cd RAB8A PKD Activation (Hoffmeister et al., 2011; Mochizuki et al., 1996) M2 mutant of Cd RAB8A Cancer Inhibition (Hoffmeister et al., 2011; Rubin and de smoothened Sauvage, 2006; Wang et al., 2009) Peripherin-2 Cd COPII Vitelliform macular dystrophy, Activation (Tian et al., 2014; Wells et al., 1993) ADRP aClassification: A, leaderless non-vesicular UPS; B, leaderless vesicular UPS; C, Golgi-bypassing UPS; N, not determined. bTherapeutic potential: Activation (activation of UPS is expected to have a therapeutic effect); Inhibition (inhibition of UPS is expected to have a therapeutic effect). cUnclassified (cytoplasmic leaderless protein; a specific UPS route has not been established), dBypass trans-Golgi and move directly from cis-Golgi to cilia. ADRP, autosomal-dominant retinitis pigmentosa; AMD, age-related macular degeneration; ATG, autophagy-related gene product; ATP1A1, ATPase Na+/K+ transporting subunit alpha 1; CAPS, cryopyrin-associated autoinflammatory syndrome; CFTR, cystic fibrosis transmembrane conductance regulator, COPD, chronic obstructive pulmonary disease; COPII, coat protein complex II; DFNB4, deafness autosomal recessive 4 with enlarged vestibular aqueduct; DKK3, dickkopf-related protein 3; DNAJC14, DnaJ homolog subfamily C member 14, FAM3C, family with sequence similarity 3 member C; FGF2, fibroblast growth factor 2; GRASP, Golgi reassembly stacking protein; HMGB1, high-mobility group box 1; IDE, insulin-degrading enzyme; IRE1α, inositol-requiring enzyme 1α; LIF, leukemia inhibitory factor; Mpl, myeloproliferative leukemia virus oncogene; MS, multiple sclerosis; MVB, multivesicular body; NLRP3, NLR family pyrin-domain- containing 3; PIP2, phosphatidylinositol (4,5)-bisphosphate; PKD, polycystic kidney disease; PRTN3, proteinase 3; RAB, Ras-associated binding protein; SASP, senescence-associated secretory phenotype; TBI, traumatic brain injury. et al., 2015). In addition to this classic role in maintaining cell Indeed, several cytokines and inflammatory mediators use health, mounting evidence suggests that autophagy also plays a role autophagy components as a means of their unconventional in UPS (Duran et al., 2010; Manjithaya and Subramani, 2010). This secretion, suggesting a close link between inflammation and type of secretory autophagy has been shown to deliver several types autophagy (see Table 1) (Manjithaya and Subramani, 2010). Recent of cytoplasmic (Type III UPS) and transmembrane proteins (Type studies have shown that the secretion of IL-1β, particularly that from

IV UPS) to the cell surface for their secretion (Ponpuak et al., 2015). non-immune cells, is mediated by vesicular structures, including those Journal of Cell Science

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lysosomes (Gardella et al., 2002), are involved in the active Box 2. Intercellular channels – plasmodesmata and secretion of HMGB1, although HMGB1 can also be passively tunneling nanotubes secreted from necrotic cells (Scaffidi et al., 2002) or apoptotic cells Macromolecules, such as proteins and RNA, can be also transported (Qin et al., 2006) through permeabilized membranes after its directly to other cells through the intercellular channels. A well-known dissociation from chromosomes (Falciola et al., 1997). HMGB1 is a example are plasmodesmata, intercellular channels in plants and some main mediator of endotoxin shock (Wang et al., 1999) and acts on algae (Knox and Benitez-Alfonso, 2014). The viral replication complexes several immune cells to trigger inflammatory responses in the form of the Tobacco mosaic virus exhibit cell-to-cell movement through plasmodesmata, which can be regarded as a kind of UPS (Heinlein, of a damage-associated molecular pattern (DAMP), which then 2015). In animals, tunneling nanotubes (TNTs) have been suggested to initiates and perpetuates a noninfectious inflammatory response serve as the main pathway for such macromolecular transport (Gerdes through the activation of its receptors, which include the Toll-like et al., 2013; Rustom et al., 2004). Of note, the tunneling transport through receptors (TLRs) TLR2 and TLR4, and the receptor for advanced TNTs can be used as a pathway for the cellular spreading of pathogens, glycation and end product (RAGE, also known as AGER) (Scaffidi such as viruses and prion-like proteins. For example, influenza A virus et al., 2002). Other functions of extracellular HMGB1 are in the has been recently reported to spread through TNTs (Kumar et al., 2017). Additionally, TNT-meditated spreading of α-synuclein, a prion-like maturation of dendritic cells, the production of pro-inflammatory aggregated protein, uses lysosomal vesicles and has been associated cytokines in myeloid cells, the induction of cell adhesion molecules with Parkinson’s disease (Abounit et al., 2016; Gousset et al., 2009). in endothelial cells, and the progression of cancer (Sims et al., However, further research is needed to evaluate the precise nature of 2010). Therefore, the modulation of HMGB1 secretion is a potential TNTs and determine whether this type of protein transport can be way to treat its associated diseases. specifically modulated to develop therapeutic strategies to treat human In addition to IL-1β and HMGB1, several other inflammatory diseases. mediators, such as IL-18 and IL-33, have been shown to be secreted by UPS, particularly by autophagy-associated mechanisms (Deretic et al., 2012; Murai et al., 2015). Potential therapeutic approaches of the autophagy and endosome pathways (Dupont et al., 2011; Zhang that might alleviate excessive inflammatory responses by et al., 2015). However, the general role of autophagy in IL-1β modulating UPS include the following strategies: (i) inhibition of secretion remains ill-defined. For example, autophagy has been caspase-1-mediated cleavage of IL-1β to treat diseases, such as suggested to inhibit both the cleavage of pro-IL-1β by caspase-1 and gout, atherosclerosis and diabetes (Burns et al., 2003; Schroder and the activity of the NLRP3 inflammasome, which may lead to the Tschopp, 2010); (ii) inhibition of cathepsin G and elastase-mediated inhibition of IL-1β secretion (Harris and Rubinsztein, 2011). With cleavage of IL-33 to prevent inflammatory diseases, for instance, regards to the clinical significance of unconventional IL-1β secretion arthritis and chronic obstructive pulmonary disease (COPD) (Cayrol during inflammation, the plasticity of IL-1β secretion under different and Girard, 2009; Lefrancais et al., 2012); (iii) inhibition of calpain- conditions and cell types should be considered. Because, as discussed mediated cleavage of IL-1α to provide therapy for diseases above, a significant portion of IL-1β secretion is mediated by non- including stroke and atherosclerosis (Zheng et al., 2013) and; (iv) vesicular UPS in macrophages, it remains to be determined whether inhibition of caspase-1 and proteinase-mediated cleavage of IL-18 inhibiting vesicular UPS of IL-1β in non-macrophage cells has to provide therapy for diseases such as age-related macular therapeutic potential for human inflammatory diseases. degeneration (Gu et al., 1997; Sugawara et al., 2001). Another distinct example of Type III UPS is the secretion of high- Secretory autophagy may play a role in the extracellular transport mobility group box 1 (HMGB1) protein, a leaderless nuclear protein of aggregation-prone proteins, such as α-synuclein and amyloid-β that is secreted into the extracellular space in an unconventional (Ejlerskov et al., 2013; Nilsson et al., 2013). α-Synuclein, particularly manner (Lotze and Tracey, 2005). It has been shown that autophagy in its aggregated forms, has been implicated in the pathogenesis of components, such as ATG5 (Dupont et al., 2011) and secretory Parkinson’s disease and other related neurological disorders (Kahle, 2008; Polymeropoulos et al., 1997). The extracellular secretion of α- synuclein may result in cell-to-cell transmission of protein aggregates that occur in many neurodegenerative disorders (Lee et al., 2005, Box 3. Senescence-associated secretory phenotypes 2010). Therefore, reducing of the unconventional secretion of α- Cellular senescence is thought to be a program of arrested proliferation and altered gene expression that can be triggered by many stresses synuclein by inhibiting autophagy possibly has therapeutic potential (Kang et al., 2015). Compared to the culture medium of quiescent cells, for Parkinson’s disease (Ejlerskov et al., 2013). However, the role of that of senescent cells is enriched with secreted proteins. These autophagy in α-synuclein secretion was later challenged by other characterize the so-called senescence-associated secretory phenotype researchers who showed that autophagy inhibition promotes α- [SASP; also known as the senescence messaging secretome (SMS)], synuclein secretion (Lee et al., 2013; Lee and Lee, 2016). In the case α β and include interleukins (i.e. IL-1 , IL-1 and IL-6), chemokines [i.e. IL-8 of amyloid-β, the main component of the amyloid plaques found in and growth-regulated alpha protein (CXCL1)], growth factors [i.e. basic fibroblast growth factor (bFGF) and hepatocyte growth factor (HGF)] and the brains of Alzheimer patients, it is unclear whether reducing extracellular proteases [i.e. matrix metalloproteinase (MMP)-1, MMP-3 extracellular amyloid-β secretion would be beneficial for the and MMP-13]. SASP is associated with several human diseases, treatment of Alzheimer’s disease. For example, inhibiting including atherosclerosis, chronic kidney disease, Crohn’s disease and autophagy through neuron-specific deletion of Atg7 in mice various cancers (He and Sharpless, 2017). Although recent studies on aggravated the neurotoxic phenotype due to the accumulation of SASP have identified several specific transcriptional regulators, intracellular amyloid-β aggregates (Nilsson et al., 2013). including GATA4 (Kang et al., 2015), the intracellular secretory Interestingly, the secretion of insulin-degrading enzyme (IDE), a mechanisms of the underlying factors are poorly understood. Because β many SASP factors, such as IL-1β and IL-8, are leaderless proteins, UPS main endogenous amyloid- -degrading enzyme that is released from mechanisms are thought to play a role in SASP. Characterizing the UPS astrocytes, has been shown to be mediated by autophagy-based UPS route of SASP factors and identifying their regulatory components will be (Son et al., 2016). Therefore, stimulation of IDE secretion from of great importance to future research of ageing. astrocytes by modulating the UPS pathway constitutes a potential

strategy to cope with Alzheimer’s disease (Son et al., 2016). Journal of Cell Science

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Golgi-bypassing UPS – Type IV From a therapeutic perspective, it is of great interest that some Proteins undergoing Golgi-bypassing UPS have a signal peptide disease-causing membrane proteins that have defects in protein that is recognized by the signal recognition particle (SRP), which folding or cell-surface trafficking, such as CFTR and pendrin recruits the protein to the ER while it is being synthesized on the mutants (Gee et al., 2011; Jung et al., 2016), can be transported to ribosome (Blobel and Dobberstein, 1975; Walter et al., 1981). In the plasma membrane by UPS pathways. CFTR is an epithelial contrast to conventional secretion cargos that travel through the anion channel, and its loss of function as a result from genetic entire Golgi, these UPS cargos bypass at least part of the Golgi mutations causes cystic fibrosis (MIM 219700) and several other complex on their way to the cell surface (Gee et al., 2018). Cargos epithelial diseases, such as bronchiectasis and chronic pancreatitis for Golgi-bypassing UPS include position-specific antigen subunit (LaRusch et al., 2014; Lee et al., 2003). The most common disease- alpha 1 (αPS1) integrin in Drosophila (Schotman et al., 2008, causing mutation of CFTR is the deletion of Phe at position 508 2009), myeloproliferative leukemia virus oncogene (Mpl) (Cleyrat (ΔF508). Pendrin, a protein encoded by SLC26A4,isa − − − − et al., 2014), the ion channel cystic fibrosis transmembrane transmembrane protein that exhibits Cl to HCO3 or Cl to I conductance regulator (CFTR) and the ion-transporting membrane exchange activity in the inner ear, thyroid follicles and renal cortical protein pendrin (Gee et al., 2011; Jung et al., 2016), as well as the collecting ducts (Mount and Romero, 2004). Mutations in SLC26A4 ciliary membrane proteins polycystin-2, the M2 mutant of cause non-syndromic recessive deafness with an enlarged vestibular Smoothened (Hoffmeister et al., 2011) and peripherin 2 (Tian aqueduct (deafness autosomal recessive 4, DFNB4, [MIM 600791]) et al., 2014). In addition, CD45 (PTPRC), connexin 26, connexin and Pendred syndrome (PDS, [MIM 274600]) (Everett et al., 1997; 30, pannexin 1, pannexin 3, serglycin and scramblase 1 have also Li et al., 1998), a common cause of hereditary hearing loss in been shown to reach the cell surface by unconventional secretion humans. Specifically, p.H723R (His723Arg) is one of the most (Baldwin and Ostergaard, 2002; Martin et al., 2001; Merregaert prevalent pathological mutations (Dossena et al., 2009; Lee et al., et al., 2010; Penuela et al., 2007; Qu et al., 2009; Scully et al., 2012). 2014). Both the lack of Phe508 in CFTR (ΔF508-CFTR) and Although all Golgi-bypassing plasma-membrane proteins are substitution of His723 for Arg in pendrin (H723R-Pendrin) result in typically referred to as Golgi-bypassing UPS cargos, the protein misfolding, retention in the ER and subsequent degradation individual trafficking routes for the different proteins are not by the ER-associated degradation (ERAD) pathway (Ward et al., identical (Fig. 2). For example, peripherin 2 reaches cilia through 1995; Yoon et al., 2008). Consequently, only negligible amounts of COPII-dependent exit from the ER, distinguishing it from other ΔF508-CFTR and H723R-Pendrin reach the plasma membrane, and UPS cargos that rely on COPII-independent routes, such as Mpl most of the ion-transporting activity at the cell surface is lost (Cleyrat et al., 2014) and CFTR (Gee et al., 2011). (Amaral, 2004; Yoon et al., 2008). Although the mutant proteins

DFNB4, Pendred syndrome Polycystic kidney disease

H723R-Pendrin Polycystin-2

Cancer M2 mutant of Smoothened Cystic fibrosis, pancreatitis, bronchiectasis, infertility

ΔF508-CFTR

vitelliform macular dystrophy, ADRP

cancer Myeloproliferative Peri Mpl pherin-2

Plasma ER membrane Nucleus

Key GRASP DNAJC14 HSP70 SEC16 COPII ATG IRE1α RAB18 RAB8A

Fig. 2. Known molecular arrangements involved in UPS of transmembrane proteins bypassing the Golgi. Immature Mpl involved in myeloproliferative cancer reaches the plasma membrane by a GRASP55- and ATG5-mediated pathway. ΔF508-CFTR, which causes cystic fibrosis because of a trafficking defect, can be transported to the plasma membrane by a pathway involving GRASP, ATGs (ATG1, -5, -7 and -8), SEC16A and IRE1α. The substitution mutant H723R- Pendrin, which causes congenital hearing loss, can reach the plasma membrane by a route that is mediated by HSP70, DNAJC14, RAB18 and IRE1α. Polycystin-2, whose mutations cause autosomal-dominant polycystic kidney disease, is transported by RAB8A-mediated UPS. The M2 mutant of Smoothened which can induce cancer, is transported to the plasma membrane by RAB8A-mediated UPS. Peripherin 2, whose mutations cause ophthalmic diseases, can be transported to the plasma membrane by COPII-mediated UPS. Journal of Cell Science

6 REVIEW Journal of Cell Science (2018) 131, jcs213686. doi:10.1242/jcs.213686 have some defects in protein folding, they retain a certain level of research into the following three areas would be especially helpful functional activity if they reach the cell surface (Gee et al., 2011; in identifying druggable targets for human diseases, as well as to Jung et al., 2016). Therefore, many research efforts are invested in further elucidate the underlying mechanism of UPS. approaches that facilitate the membrane targeting of ΔF508-CFTR First, a promising research area of UPS is the identification of the and H723R-Pendrin. vesicular carrier involved in leaderless vesicular and Golgi- Notably, the blockade of conventional ER-to-Golgi trafficking, bypassing UPS. Results from previous studies have provided a list which induces ER stress and the unfolded protein response (UPR), of candidates for vesicular carriers that include COPII-coated has been shown to also evoke unconventional cell-surface vesicles (Tian et al., 2014), autophagy vesicles (Cleyrat et al., 2014; trafficking of CFTR and pendrin (Gee et al., 2011; Jung et al., Gee et al., 2011), lipid droplets (Jung et al., 2016) and endosomes 2016). It is, however, difficult to adopt a direct activation of ER (Zhang et al., 2015). It is also possible that multiple vesicular stress as a therapeutic strategy because it is likely to give rise to systems are involved in a single UPS event. For example, it has been many unfavorable side effects (Yoshida, 2007). The basic suggested that both autophagosome and endosome/multivesicular mechanisms through which CFTR and pendrin reach the cell body (MVB) components are sequentially involved in the vesicular membrane appear to be similar. For example, expression of both UPS of IL-1β (Zhang et al., 2015). Our recent results also indicate ΔF508-CFTR and H723R-Pendrin at the plasma membrane was that early autophagosomal components, MVBs and RAB8A- abolished by the knockdown of IRE1. This finding indicates that dependent recycling vesicles sequentially mediate the ER stress- IRE1 plays a major role in the UPS of CFTR and pendrin mutants, induced UPS of CFTR (our unpublished observation). To be able to when considering the three UPR signaling arms consisting of IRE1, modulate the UPS pathway with a clear therapeutic potential for PERK and ATF6 (Gee et al., 2011; Jung et al., 2016). The molecular diverse human diseases, the characterization of the vesicular system mechanism of how IRE1 facilitates Type IV UPS is not clearly involved in the UPS of each cargo of interest and their regulatory understood; however, a recent report has shown that IRE1 augments processes are of paramount importance. the expression and function of SEC16A, which forms the ER exit Second, GRASPs have emerged as an interesting regulator of the sites for the UPS of ΔF508-CFTR (Piao et al., 2017), suggesting that UPS pathway of various cargo proteins. GRASPs were initially SEC16A is a downstream target of IRE1-mediated upregulation of identified as factors required for the stacking of Golgi cisternae by UPS. In contrast to the finding that the same ER stress signals can using in vitro assays (Barr et al., 1997). Two isoforms, GRASP55 activate UPS of both CFTR and pendrin, some of the key molecular and GRASP65, exist in vertebrates (Barr et al., 1997; Shorter et al., factors that are involved in the UPS of these two membrane proteins 1999). Interestingly, several studies have demonstrated that are different. For example, Golgi reassembly stacking proteins GRASPs are involved in Golgi-bypassing UPS in both (GRASPs) are required for the UPS of CFTR (Gee et al., 2011), invertebrate and vertebrate models, although they were initially whereas the HSP70 co-chaperone DNAJC14 is involved in the UPS described as Golgi-associated proteins (Cleyrat et al., 2014; Kinseth of pendrin (Jung et al., 2016) (see below, ‘Potential therapeutic et al., 2007; Schotman et al., 2008). For example, the GRASP targets’). Theoretically, an activation of the cargo-specific pathway homologs in Dictyostelium and Drosophila, respectively, mediate would be more desirable to develop therapeutics for each disease as the transport of acyl-CoA-binding protein (AcbA) and α-integrin at it might help to minimize the adverse events caused by the specific developmental stages through an unconventional Golgi- activation of common pathways that might also induce UPS of independent route (Gee et al., 2011; Kinseth et al., 2007; Schotman untoward cargos. et al., 2008). Furthermore, as discussed above, GRASPs participate In addition to CFTR and pendrin, several other transmembrane in the UPS of the mammalian transmembrane proteins CFTR and proteins that are associated with human diseases can arrive at the Mpl (Cleyrat et al., 2014; Gee et al., 2011). However, the precise plasma membrane by UPS. Polycystin-2, mutations of which are roles of GRASPs in UPS are largely unknown. Interestingly, associated with autosomal-dominant polycystic kidney disease monomerization and ER re-localization of GRASP55 by (Mochizuki et al., 1996), is transported to cilia by UPS; this is phosphorylation on its serine 441 residue appear to be critical for mediated by RAB8A, a small GTPase, which plays a role in the UPS of CFTR (Kim et al., 2016). Therefore, in order to be able vesicular traffic from the trans-Golgi network to the plasma to develop therapeutics for cystic fibrosis, a feasible approach might membrane (Hoffmeister et al., 2011). Activating mutations of be to screen for small-molecule compounds that can induce Smoothened can induce medulloblastoma, basal-cell carcinoma, phosphorylation and, thus, activation of GRASP55. pancreatic cancer and prostate cancer by unregulated activation of Third, increasing evidence suggests that molecular chaperones, the hedgehog pathway (Rubin and de Sauvage, 2006), and this such as heat shock 70 kDa proteins (HSP70s), HSP90s and protein is also transported to the cell surface via UPS (Hoffmeister members of the DNAJ-protein family (also known as HSP40s), et al., 2011). Patients with myeloproliferative neoplasms (MPNs) play a role in the UPS of diverse cargos (Jung et al., 2016; Zhang often carry either an activating form of JAK2 or mutations in the ER et al., 2015). The correct folding of secretory and transmembrane resident protein calreticulin (Klampfl et al., 2013; Nangalia et al., proteins is ensured by ER quality control (ERQC) systems (Vembar 2013), which leads to accumulation of immature Mpl in the ER and Brodsky, 2008). During endoplasmic-reticulum-associated (Kralovics et al., 2003; Moliterno et al., 1998). It has been reported protein degradation (ERAD), misfolded proteins that do not pass that immature Mpl utilizes an unconventional autophagic secretory ERQC are retro-translocated to the cytoplasm and degraded in an pathway to reach the cell surface (Cleyrat et al., 2014). Therefore, ubiquitin-dependent process by the proteasome. Molecular either the activation or inhibition of their unconventional secretion chaperones have crucial roles in the recognition of misfolded could have therapeutic potential for any associated diseases proteins and the heat shock cognate protein 70 (Hsc70; encoded by (Table 1). HSPA8), a member of the HSP70 family, is one of the important chaperones involved in this process. However, HSP70 chaperones Potential therapeutic targets do not function alone. In addition to protein folding and Although the precise nature and mechanisms of UPS are not yet degradation, HSP70s are involved in a myriad of biological fully understood, the accumulated knowledge thus far suggests that processes, including protein–protein interaction and intracellular Journal of Cell Science

7 REVIEW Journal of Cell Science (2018) 131, jcs213686. doi:10.1242/jcs.213686 trafficking of various proteins (Kampinga and Craig, 2010; Young investigation will, ultimately, open a new era of therapeutics for et al., 2003). Much of the functional diversity of HSP70 is thought numerous human diseases, including cancers, inflammatory to be driven by cofactors, including chaperone DNAJ proteins diseases and some genetic disorders, such as cystic fibrosis and (Kampinga and Craig, 2010). Indeed, as mentioned above, the Pendred syndrome. DNAJ protein DNAJC14 plays a crucial role in the UPS of misfolded pendrin (Jung et al., 2016). It appears that the activation Acknowledgements of the chaperone machinery stimulates both UPS and the ERAD We thank Dong Soo Chang for the assistance with illustrations. pathway to relieve the protein burden in the ER during ER stress Competing interests (Ron and Walter, 2007). Among the many co-chaperones, The authors declare no competing or financial interests. DNAJC14 appears to be specialized in assisting Hsc70 during UPS, whereas other co-chaperones, such as CHIP, are involved in Funding ERAD (Meacham et al., 2001). Although Hsc70 is required for the This work was supported by grant 2013R1A3A2042197 from the National Research UPS of ΔF508-CFTR, DNAJC14 is not (Jung et al., 2016), Foundation, the Ministry of Science, ICT & Future Planning, Republic of Korea, and grant HI15C1543 of the Korea Health Technology R&D Project through the Korea suggesting the presence of unknown co-chaperone that is specific Health Industry Development Institute (KHIDI), funded by the Ministry of Health & for UPS of CFTR. In addition, it has been shown that the Hsc70 co- Welfare, Republic of Korea. chaperone DNAJC5 participates in Type III UPS of misfolded cytosolic proteins (Xu et al., 2018). Interestingly, DNAJC14, which References mediates the UPS of transmembrane proteins, has ER-membrane- Abounit, S., Bousset, L., Loria, F., Zhu, S., de Chaumont, F., Pieri, L., Olivo- Marin, J. C., Melki, R. and Zurzolo, C. (2016). Tunneling nanotubes spread localizing transmembrane domains, whereas DNAJC5, which is fibrillar alpha-synuclein by intercellular trafficking of lysosomes. EMBO J. 35, involved in the UPS of cytosolic proteins, does not have any 2120-2138. transmembrane domains (Kampinga and Craig, 2010). Identifying Abraham, E., Arcaroli, J., Carmody, A., Wang, H. and Tracey, K. J. (2000). HMG- 1 as a mediator of acute lung inflammation. J. Immunol. 165, 2950-2954. cargo-interacting domains in these proteins and determining the Agosta, F., Dalla Libera, D., Spinelli, E. G., Finardi, A., Canu, E., Bergami, A., principles of how these co-chaperones recognize their UPS substrate Bocchio Chiavetto, L., Baronio, M., Comi, G., Martino, G. et al. (2014). 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