REVIEW ARTICLE SERIES: CELL BIOLOGY AND DISEASE

Unconventional protein secretion – new insights into thepathogenesis and therapeutic targets of human diseasesJiyoon Kim, Heon Yung Gee and Min Goo Lee*

ABSTRACTMost secretory proteins travel through a well-documentedconventional secretion pathway involving the endoplasmic reticulum(ER) and the Golgi complex. However, recently, it has been shownthat a significant number of proteins reach the plasma membrane orextracellular space via unconventional routes. Unconventionalprotein secretion (UPS) can be divided into two types: (i) theextracellular secretion of cytosolic proteins that do not bear a signalpeptide (i.e. leaderless proteins) and (ii) the cell-surface trafficking ofsignal-peptide-containing transmembrane proteins via a route thatbypasses the Golgi. Understanding the UPS pathways is not onlyimportant for elucidating the mechanisms of intracellular traffickingpathways but also has important ramifications for human health,because many of the proteins that are unconventionally secreted bymammalian cells and microorganisms are associated with humandiseases, ranging from common inflammatory diseases to the lethalgenetic disease of cystic fibrosis. Therefore, it is timely andappropriate to summarize and analyze the mechanisms of UPSinvolvement in disease pathogenesis, as they may be of use for thedevelopment of new therapeutic approaches. In this Review, wediscuss the intracellular trafficking pathways of UPS cargos,particularly those related to human diseases. We also outline thediseasemechanisms and the therapeutic potentials of new strategiesfor treating UPS-associated diseases.

KEY WORDS: Unconventional secretion, Human disease,Pathogenesis, Therapeutic target, Leaderless protein

IntroductionAccording to the classic principle of protein secretion, cargoproteins travel by using the conventional pathway from theendoplasmic reticulum (ER) to the Golgi complex, from whichthey subsequently move to the trans-Golgi network (TGN) andfinally to the plasma membrane (Lee et al., 2004). This process isinitiated by recognition of a signal peptide (also known as ‘leadersequence’) at the N-terminus or transmembrane domain of cargoproteins, followed by sequential budding and fusion of vesicularcarriers (Bonifacino and Glick, 2004). Each step of the secretionpathway is under the control of a number of regulatory proteins.Correct regulation of the classic secretory pathway is imperative forthe life and health of the organism (Viotti, 2016). Since thediscovery of vesicular exocytic mechanisms, this classic proteinsecretion pathway involving ER-to-Golgi transport has beenconsidered the only standard mechanism to move proteins out of

the cell. However, discoveries over the last two decades have shownthat an increasing number of proteins use alternative secretorypathways that do not involve the ER-to-Golgi transport (Malhotra,2013; Ponpuak et al., 2015; Rabouille, 2017). These alternativepathways include the extracellular secretion of cytosolic proteinsthat do not bear a signal peptide (i.e. leaderless proteins) (Rubartelli,1997), and cell-surface trafficking of transmembrane proteins via aGolgi-bypassing route. These pathways are collectively referred toas unconventional protein secretion (UPS) (see Box 1).

With a few exceptions, most UPS pathways are induced byvarious cellular stresses, such as nutrient starvation (Cruz-Garciaet al., 2014), mechanical stress (Schotman et al., 2008),inflammation (Schroder and Tschopp, 2010) and ER stress (Geeet al., 2011; Jung et al., 2016). Notably, many disease conditions areassociated with various stresses at the cellular or organismal level(Fulda et al., 2010), indicating the potential of UPS as a promisingemerging target for the development of novel therapeutics to treatassociated human disease.

The number of defined UPS-related diseases continues to expand.For example, sterile inflammation related to Alzheimer’s disease,allergic and autoimmune diseases, and diabetes can be a trigger toinduce unconventional protein secretion (Agosta et al., 2014; Chenet al., 2015; Freigang et al., 2013; Gardella et al., 2002; Schroderand Tschopp, 2010). Heat shock proteins (HSPs) that are secretedunconventionally play a pivotal role in the immunomodulation,proliferation, angiogenesis and invasiveness of cancer (Rodriguezet al., 2009; Sarikonda et al., 2015; Zhang et al., 2012). A number ofautophagy components, the mutations of which are involved invarious diseases (Jiang andMizushima, 2014), participate in UPS ofnumerous cargo proteins (Duran et al., 2010; Kinseth et al., 2007;Manjithaya et al., 2010). Several mutant transmembrane proteins,whose associated trafficking defects to the cell surface causeinherited genetic disorders, such as cystic fibrosis and congenitalhearing loss can, alternatively, reach the plasma membrane byGolgi-bypassing UPS (Gee et al., 2011; Jung et al., 2016). Inaddition, UPS has even been shown to be essential formicroorganisms to mediate their extracellular release and exertbiotrophic pathogenicity (Shoji et al., 2014).

Recent developments in the field have contributed to significantadvances in the understanding of the molecular processes involvedin UPS (Daniels and Brough, 2017; Pompa et al., 2017; Ponpuaket al., 2015; Rabouille, 2017; Santos et al., 2017). Nevertheless, acomprehensive description that adequately explains the role in UPSin disease pathogenesis is currently lacking. In this Review, we willdefine emerging roles for UPS in disease pathogenesis and highlightthe possibility of novel therapeutics that target UPS.

Disease-associated unconventional secretion pathwaysOverview and classificationWith the increased scientific understanding of the alternativesecretion pathways, several researchers have attempted to

Department of Pharmacology, Brain Korea 21 PLUS Project for Medical Sciences,Severance Biomedical Science Institute, Yonsei University College of Medicine,Seoul 120-752, Korea.

*Author for correspondence (mlee@yuhs.ac)

M.G.L., 0000-0001-7436-012X

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© 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs213686. doi:10.1242/jcs.213686

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systematically classify the UPS pathways. For example, Deretic andco-workers have classified autophagy-associated protein secretionpathways (Ponpuak et al., 2015). However, because their reviewalso addresses the overlap between autophagy and conventionalprotein secretion, we will adopt here the UPS classificationproposed by Rabouille and colleagues (Rabouille, 2017;Rabouille et al., 2012). According to this, UPS pathways can bedivided into four types with the UPS of leaderless proteins beingsub-divided into three. They are: (Type I) direct secretion or pore-mediated translocation across the plasma membrane (Ding et al.,2016; Zacherl et al., 2015), (Type II) ATP-binding cassette (ABC)transporter-based secretion (McGrath and Varshavsky, 1989) and,(Type III) membrane-bound organelle (autophagosome/endosome)-based secretion (Duran et al., 2010; Kinseth et al.,2007). The fourth type is the Type IV UPS of leader-sequence-containing transmembrane proteins, which are synthesized in theER and reach the plasma membrane by bypassing the Golgi (Geeet al., 2011). Because the nature of ABC-transporter-mediated UPS(Type II) is not fully characterized, we will consider Type I andType II as a single category of leaderless non-vesicular UPS(Fig. 1). We will discuss these three categories of UPS with a focuson their link to human disease (summarized in Table 1).In addition to the pathways mentioned above, there are other cell

processes that can be considered to be UPS. For example, transportthrough the intercellular channels that function as pathways for thecellular spreading of macromolecules, including pathogens such asviruses and prion-like proteins, could be viewed a specialized formof UPS (see Box 2). Additionally, the senescence-associatedsecretory phenotype (SASP) is characterized by the secretion ofpro-inflammatory and matrix-degrading molecules from senescentcells and is associated with a number of human diseases, includingatherosclerosis, cancer and inflammatory diseases (Coppé et al.,2008). Because many SASP factors are leaderless proteins, UPSmechanisms are thought to be involved in their extracellularsecretion (see Box 3).

Leaderless non-vesicular UPS – Types I and IIThe leaderless non-vesicular class of UPS includes cytoplasmicleaderless proteins that are secreted directly out of the cell eitherthrough plasma membrane pores (Type I) (Steringer et al., 2012) orthrough ABC transporters (Type II) (McGrath and Varshavsky,1989). One of several typical triggers for this type of UPS isinflammation, which leads to the extracellular release of diversecytokines that do not possess a signal peptide (Schroder andTschopp, 2010). A well-known example of a protein that utilizesthis UPS is interleukin (IL)-1β, which is mainly expressed inmyeloid cells, such as macrophages and monocytes. Initially, IL-1βis produced as a 31-kDa inactive form that is cleaved by caspase-1into the 17-kDa mature form. The latter is then recruited by theintracellular NACHT-domain-, LRR-motif- and PYD-containingprotein3 (NLRP3) component of the inflammasome, thus engagingthe immune response (Schroder and Tschopp, 2010). It appears thatmultiple UPS pathways can mediate IL-1β secretion (see below,‘Leaderless vesicular UPS - Type III’), depending on inflammatoryconditions and cell type. Secretion of IL-1β from macrophagesfollowing inflammation is mediated by a type of UPS that requireshyper-permeabilization of the plasma membrane (Bergsbaken et al.,2009; Martín-Sánchez et al., 2016). The precise mechanism of thismembrane hyper-permeabilization is not yet fully understood(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 beenproposed to be involved in the formation of the membrane pore(Ding et al., 2016). The innate immune response evoked bycytokines, such as IL-1βwas originally thought to be the first line ofdefense against non-self (e.g. microorganisms) and to serve as asophisticated system to sense danger signals (Pitanga et al., 2016).However, elevated local or systemic levels of IL-1β have beenassociated with a number of hereditary or acquired human diseases,such as cryopyrin-associated periodic syndrome (Schroder andTschopp, 2010). Therefore, preventing the overt secretion of IL-1βby modulating UPS pathways would be a potential new therapeuticstrategy to overcome these inflammatory diseases (Table 1).

Another important example of Type I UPS is the translocation of acargo through the plasma membrane via self-made lipidic pores.Examples are fibroblast growth factor 2 (FGF2) and the HIV-TATprotein, both of which are recruited to the cytoplasmic leaflet of theplasma membrane by interaction with phosphatidylinositol (4,5)-bisphosphate (PIP2) and then undergo self-oligomerization; this,sequentially, induces membrane insertion, pore formation, andextracellular translocation of FGF2 and HIV-TAT (Debaisieuxet al., 2012; Rabouille, 2017; Zeitler et al., 2015). Phosphorylationof FGF2 by Tec kinases has been shown to be essential for PIP2-mediated translocation of FGF2 (Ebert et al., 2010). However, manyquestions persist concerning this process, including the source of theenergy for self-oligomerization, formation of themembrane pore, andtranslocation across the membrane. Recently, the Na+/K+-ATPasesubunit α1 (ATP1A1), an α-chain of the Na+/K+-ATPaseheterotetramer (Shull et al., 1986), was identified as a regulatoryfactor for FGF2 secretion that is involved in recruitment of FGF2 tothe plasma membrane leaflet (Zacherl et al., 2015). Interestingly, thesecretion of FGF2 is inhibited by the lack of extracellular heparansulfate proteoglycans, which may function as extracellular traps forFGF2 (Nickel, 2007). FGF2 has been shown to be crucial for thedevelopment of the central nervous system and adult neurogenesis,and proposed as a therapeutic target for various neurodegenerativediseases, including Alzheimer’s disease, Parkinson’s disease,multiple sclerosis and traumatic brain injury (Woodbury and Ikezu,

Box 1. Conventional and unconventional proteinsecretion pathwaysMost secretory proteins reach their destination via the ER–Golgi-targetorganelle route, which is referred to as the ‘classic’ or ‘conventional’ proteinsecretion pathway. These secretory proteins contain a signal peptide(the ‘leader sequence’) that directs their translocation into the lumen or tothe ERmembrane. The newly synthesized proteins then exit the ER at anER-exit site (ERES) through coat protein complex II (COPII)-coatedvesicles and, so, reach the Golgi network before being dispatched to theplasma membrane, lysosomes, endosomes or peroxisomes (Gee et al.,2018; Viotti, 2016). Fusion of vesicular intermediates and organelles ismediated by soluble N-ethylmaleimide-sensitive factor (NSF) attachmentprotein (SNAP) receptor proteins (SNAREs), Rab proteins and theirregulators (Mellman and Warren, 2000).In addition to the above-mentioned conventional pathway, eukaryotic

cells also utilize unconventional protein secretion (UPS) for protein sortingand delivery. Initially, the term ‘unconventional secretion’ was used for therelease of cytoplasmic proteins that lack a signal peptide, i.e. leaderlessproteins, into the extracellular medium. Later, it was found that sometransmembrane proteins that are synthesized in the ER, reach the plasmamembrane via a route that bypasses the Golgi complex (Nickel andRabouille, 2009). As described in the text, Rabouille colleagues dividedUPS into four types, i.e. Type I, II and III UPS of leaderless proteins, andType IVUPSofGolgi-bypassing transmembrane proteins (Rabouille et al.,2012). However, the ABC transporter-mediated Type II pathway is not wellstudied and needs additional validation (Rabouille, 2017).

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2014). Therefore, a modulation of the regulatory proteins that areinvolved in the UPS of FGF2, such as ATP1A1 or proteoglycans,might have therapeutic potential for neurodegenerative diseases.Accumulating evidence suggests that extracellular HIV-TAT, whichis also secreted through the Type I UPS occurring through self-madepores, acts as a viral toxin and plays a key role in the progression ofacquired immune-deficiency syndrome (AIDS) (Debaisieux et al.,2012). Therefore, inhibition of UPS regarding HIV-TAT could bebeneficial to the treatment of AIDS.It has been suggested that some leaderless cargos can be secreted

out of the cell by ABC transporters. Since the initial discovery in1989 that a-factor, the mating pheromone of Saccharomycescerevisiae, is secreted from the cell by the ABC transporter Ste6p(McGrath and Varshavsky, 1989), several other proteins, includingthe m-factor of Schizosaccharomyces pombe (Christensen et al.,1997) and hydrophilic acylated surface protein B (HASPB) ofLeishmania species (Denny et al., 2000; Maclean et al., 2012), havebeen found to be exported by ABC transporter-mediatedtranslocation. Leishmania HASPB is a leaderless protein that islocalized to the extracellular face of its plasma membrane.Leishmaniasis is caused by protozoan parasites of Leishmaniaspecies and spreads through the bites of certain types of sandfly.During Leishmaniasis pathogenesis, HASPB has been suggested tobe required for either initial parasite transmission to the host or theestablishment of parasites within host macrophages (Maclean et al.,2012). β-COP and δ-COP, subunits of the COPI coatomer complexthat participates in the conventional secretion pathway, are known to

be involved in the unconventional secretion of HASPB (Ritzerfeldet al., 2011). However, further studies are needed to elucidate theprecise molecular mechanisms of UPS of HASPB, and to answerthe question of the link between these COPI subunits and ABCtransporters. Interestingly, HASPB is modified by lipidation,including N-myristoylation and palmitoylation, which is requiredfor trafficking to the cell surface (Denny et al., 2000). Therefore, thestudy of how the lipidation event is involved in the secretion andfunction of HASPB might contribute to the future development oftherapeutics.

Leaderless vesicular UPS – Type IIIThe leaderless vesicular class (Type III) of UPS involves leaderlesscytoplasmic proteins that are packaged into membrane-boundorganelles, including autophagosomes, lysosomes, endosomes andexosomes. Thus far, numerous studies have examined the substratecargo proteins of leaderless vesicular UPS pathways and theirtransport mechanisms (Dupont et al., 2011; Ejlerskov et al., 2013;Lotze and Tracey, 2005). Although the transport process ofindividual cargo proteins varies greatly, autophagy-relatedvesicular structures appear to be an important transport carrier inType III UPS (Manjithaya and Subramani, 2010). The best-knownrole of autophagy is that of a degradative canonical pathway thatcontributes to nutrient recycling and cellular defense by digestingcytoplasmic components during starvation (Galluzzi et al., 2014), aswell as aggregated proteins (Rogov et al., 2014), damagedorganelles (Yamano et al., 2014) and invading pathogens (Deretic

trans-Golgi

Cis-Golgicis-Golgi

Pore formation

Plasma membrane

ABC transporterLeaderless vesicular UPS

Golgi-bypassingUPS

ER

Nucleus

Leaderless non-vesicular UPS

Conventional

traffic

king

Leaderless proteins

Unconventional trafficking

Key Leaderless cytosolic proteinsER luminal proteins Transmembrane proteins(unconventional)

Transmembrane proteins(conventional)

Fig. 1. Protein secretion pathways. Theconventional secretion pathway involvesthe ER-to-Golgi transport of cargo (blueand yellow circles). However, a number ofproteins use alternative secretorypathways, known as unconventionalprotein secretion (UPS) that do notinvolve ER-to-Golgi transport.Leaderless cytosolic proteins (orangecircles) can be secreted by the cell vianon-vesicular routes, such as through amembrane pore and the ABC transporter(Leaderless non-vesicular UPS). Forexample, UPS of FGF2 involves poreformation within the membrane, Teckinase, ATP1A1 and extracellularheparan sulfate proteoglycan. Inaddition, a-factor of Saccharomycescerevisiae requires the ABC transporterSte6P. Another subset of leaderlesscytosolic proteins, including IL-18, IL-33,α-synuclein, amyloid-β and IDE, can besecreted through autophagy-associatedvesicles, such as lysosomes and lateendosomes (Leaderless vesicular UPS).In addition, transmembrane proteins (redcircles) can reach the plasma membraneby bypassing the Golgi (Golgi-bypassingUPS). Examples for this are αPS1integrin, Mpl, and ΔF508-CFTR, whichcan be transported to the plasmamembrane by GRASP-dependent UPS.

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et al., 2015). In addition to this classic role in maintaining cellhealth, mounting evidence suggests that autophagy also plays a rolein UPS (Duran et al., 2010; Manjithaya and Subramani, 2010). Thistype of secretory autophagy has been shown to deliver several typesof cytoplasmic (Type III UPS) and transmembrane proteins (TypeIV UPS) to the cell surface for their secretion (Ponpuak et al., 2015).

Indeed, several cytokines and inflammatory mediators useautophagy components as a means of their unconventionalsecretion, suggesting a close link between inflammation andautophagy (see Table 1) (Manjithaya and Subramani, 2010). Recentstudies have shown that the secretion of IL-1β, particularly that fromnon-immune cells, is mediated by vesicular structures, including those

Table 1. Overview of UPS pathways associated with diseases and disorders

Cargo Classificiationa Involved proteins/processesRelated diseases/disorders/inabilities/injuries

Therapeuticpotentialb References

IL-1β A NLRP3 inflammasome, gasdermin-N, Caspase-1, endolysosomalvesicle, exosome,hyperpermeabilization

Hemorrhagic disorders,Alzheimer’s disease, gout,Diabetes, atherosclerosis,CAPS

Inhibition A: (Andrei et al., 1999; Ding et al., 2016;Duewell et al., 2010; Dutra et al.,2014; Gardella et al., 2002; Henekaet al., 2013; Kuemmerle-Deschner,2015; Martinon et al., 2006; Masterset al., 2010; Qu et al., 2007)

B: (Lopez-Castejon and Brough, 2011;Schroder and Tschopp, 2010;Zhang et al., 2015)

B ATG5, ATG7, RAB8, MVB, HSP90,GRASP55, GRASP65

Inhibition

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, hemorrhagicdisorders, cancer,atherosclerosis, SASP

Inhibition (Freigang et al., 2013; Greenhalghet al., 2012; Laberge et al., 2015;Luheshi et al., 2011; Singer et al.,2006)

FGF2 A PIP2, Tec kinase, heparan sulfateproteoglycans, ATP1A1

Alzheimer’s disease,Parkinson’s disease, MS,TBI

Activation (Nickel, 2010; Woodbury and Ikezu,2014; Zacherl et al., 2015)

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,

PRTN3AMD, asthma Activation (Doyle et al., 2014; Gu et al., 1997;

Lopez-Castejon and Brough, 2011;Murai et al., 2015; Sugawara et al.,2001)

HMGB1 B ATG5, secretory lysosome Sepsis, lung disease, arthritis,stroke, hemorrhagic shock,cancer

Inhibition (Abraham et al., 2000; Kokkola et al.,2003; Liu et al., 2007; Qin et al.,2006; Thorburn et al., 2009; Yanget 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,DKK3

B ATG7 Cancer Inhibition (Kraya et al., 2015)

Mpl C ATG5, GRASP55 Myeloproliferative cancer Inhibition (Cleyrat et al., 2014)CFTR C GRASP55, GRASP65, ATG1, ATG5,

ATG7, ATG8, SEC16, IRE1αCystic fibrosis, pancreatitis,bronchiectasis, infertility

Activation (Gee et al., 2011; Kim et al., 2016;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; Mochizukiet al., 1996)

M2 mutant ofsmoothened

Cd RAB8A Cancer Inhibition (Hoffmeister et al., 2011; Rubin and deSauvage, 2006; Wang et al., 2009)

Peripherin-2 Cd COPII Vitelliform macular dystrophy,ADRP

Activation (Tian et al., 2014; Wells et al., 1993)

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 Cmember 14, FAM3C, family with sequence similarity 3member C; FGF2, fibroblast growth factor2; 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.

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of the autophagy and endosome pathways (Dupont et al., 2011; Zhanget al., 2015). However, the general role of autophagy in IL-1βsecretion remains ill-defined. For example, autophagy has beensuggested to inhibit both the cleavage of pro-IL-1β by caspase-1 andthe activity of the NLRP3 inflammasome, which may lead to theinhibition of IL-1β secretion (Harris and Rubinsztein, 2011). Withregards to the clinical significance of unconventional IL-1β secretionduring inflammation, the plasticity of IL-1β secretion under differentconditions and cell types should be considered. Because, as discussedabove, a significant portion of IL-1β secretion is mediated by non-vesicular UPS in macrophages, it remains to be determined whetherinhibiting vesicular UPS of IL-1β in non-macrophage cells hastherapeutic potential for human inflammatory diseases.Another distinct example of Type III UPS is the secretion of high-

mobility group box 1 (HMGB1) protein, a leaderless nuclear proteinthat is secreted into the extracellular space in an unconventionalmanner (Lotze and Tracey, 2005). It has been shown that autophagycomponents, such as ATG5 (Dupont et al., 2011) and secretory

lysosomes (Gardella et al., 2002), are involved in the activesecretion of HMGB1, although HMGB1 can also be passivelysecreted from necrotic cells (Scaffidi et al., 2002) or apoptotic cells(Qin et al., 2006) through permeabilized membranes after itsdissociation from chromosomes (Falciola et al., 1997). HMGB1 is amain mediator of endotoxin shock (Wang et al., 1999) and acts onseveral immune cells to trigger inflammatory responses in the formof a damage-associated molecular pattern (DAMP), which theninitiates and perpetuates a noninfectious inflammatory responsethrough the activation of its receptors, which include the Toll-likereceptors (TLRs) TLR2 and TLR4, and the receptor for advancedglycation and end product (RAGE, also known as AGER) (Scaffidiet al., 2002). Other functions of extracellular HMGB1 are in thematuration of dendritic cells, the production of pro-inflammatorycytokines in myeloid cells, the induction of cell adhesion moleculesin endothelial cells, and the progression of cancer (Sims et al.,2010). Therefore, the modulation of HMGB1 secretion is a potentialway to treat its associated diseases.

In addition to IL-1β and HMGB1, several other inflammatorymediators, such as IL-18 and IL-33, have been shown to be secretedby UPS, particularly by autophagy-associated mechanisms (Dereticet al., 2012; Murai et al., 2015). Potential therapeutic approachesthat might alleviate excessive inflammatory responses bymodulating UPS include the following strategies: (i) inhibition ofcaspase-1-mediated cleavage of IL-1β to treat diseases, such asgout, atherosclerosis and diabetes (Burns et al., 2003; Schroder andTschopp, 2010); (ii) inhibition of cathepsin G and elastase-mediatedcleavage of IL-33 to prevent inflammatory diseases, for instance,arthritis and chronic obstructive pulmonary disease (COPD) (Cayroland Girard, 2009; Lefrancais et al., 2012); (iii) inhibition of calpain-mediated cleavage of IL-1α to provide therapy for diseasesincluding stroke and atherosclerosis (Zheng et al., 2013) and; (iv)inhibition of caspase-1 and proteinase-mediated cleavage of IL-18to provide therapy for diseases such as age-related maculardegeneration (Gu et al., 1997; Sugawara et al., 2001).

Secretory autophagy may play a role in the extracellular transportof aggregation-prone proteins, such as α-synuclein and amyloid-β(Ejlerskovet al., 2013;Nilsson et al., 2013).α-Synuclein, particularlyin its aggregated forms, has been implicated in the pathogenesis ofParkinson’s disease and other related neurological disorders (Kahle,2008; Polymeropoulos et al., 1997). The extracellular secretion of α-synucleinmay result in cell-to-cell transmission of protein aggregatesthat occur in many neurodegenerative disorders (Lee et al., 2005,2010). Therefore, reducing of the unconventional secretion of α-synuclein by inhibiting autophagy possibly has therapeutic potentialfor Parkinson’s disease (Ejlerskov et al., 2013). However, the role ofautophagy in α-synuclein secretion was later challenged by otherresearchers who showed that autophagy inhibition promotes α-synuclein secretion (Lee et al., 2013; Lee and Lee, 2016). In the caseof amyloid-β, the main component of the amyloid plaques found inthe brains of Alzheimer patients, it is unclear whether reducingextracellular amyloid-β secretion would be beneficial for thetreatment of Alzheimer’s disease. For example, inhibitingautophagy through neuron-specific deletion of Atg7 in miceaggravated the neurotoxic phenotype due to the accumulation ofintracellular amyloid-β aggregates (Nilsson et al., 2013).Interestingly, the secretion of insulin-degrading enzyme (IDE), amain endogenous amyloid-β-degrading enzyme that is released fromastrocytes, has been shown to be mediated by autophagy-based UPS(Son et al., 2016). Therefore, stimulation of IDE secretion fromastrocytes by modulating the UPS pathway constitutes a potentialstrategy to cope with Alzheimer’s disease (Son et al., 2016).

Box 2. Intercellular channels – plasmodesmata andtunneling nanotubesMacromolecules, such as proteins and RNA, can be also transporteddirectly to other cells through the intercellular channels. A well-knownexample are plasmodesmata, intercellular channels in plants and somealgae (Knox and Benitez-Alfonso, 2014). The viral replication complexesof the Tobacco mosaic virus exhibit cell-to-cell movement throughplasmodesmata, which can be regarded as a kind of UPS (Heinlein,2015). In animals, tunneling nanotubes (TNTs) have been suggested toserve as the main pathway for such macromolecular transport (Gerdeset al., 2013; Rustom et al., 2004). Of note, the tunneling transport throughTNTs can be used as a pathway for the cellular spreading of pathogens,such as viruses and prion-like proteins. For example, influenza A virushas been recently reported to spread through TNTs (Kumar et al., 2017).Additionally, TNT-meditated spreading of α-synuclein, a prion-likeaggregated protein, uses lysosomal vesicles and has been associatedwith Parkinson’s disease (Abounit et al., 2016; Gousset et al., 2009).However, further research is needed to evaluate the precise nature ofTNTs and determine whether this type of protein transport can bespecifically modulated to develop therapeutic strategies to treat humandiseases.

Box 3. Senescence-associated secretory phenotypesCellular senescence is thought to be a program of arrested proliferationand altered gene expression that can be triggered by many stresses(Kang et al., 2015). Compared to the culture medium of quiescent cells,that of senescent cells is enriched with secreted proteins. Thesecharacterize the so-called senescence-associated secretory phenotype[SASP; also known as the senescence messaging secretome (SMS)],and include interleukins (i.e. IL-1α, IL-1β and IL-6), chemokines [i.e. IL-8and growth-regulated alpha protein (CXCL1)], growth factors [i.e. basicfibroblast growth factor (bFGF) and hepatocyte growth factor (HGF)] andextracellular proteases [i.e. matrix metalloproteinase (MMP)-1, MMP-3and MMP-13]. SASP is associated with several human diseases,including atherosclerosis, chronic kidney disease, Crohn’s disease andvarious cancers (He and Sharpless, 2017). Although recent studies onSASP have identified several specific transcriptional regulators,including GATA4 (Kang et al., 2015), the intracellular secretorymechanisms of the underlying factors are poorly understood. Becausemany SASP factors, such as IL-1β and IL-8, are leaderless proteins, UPSmechanisms are thought to play a role in SASP. Characterizing the UPSroute of SASP factors and identifying their regulatory components will beof great importance to future research of ageing.

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Golgi-bypassing UPS – Type IVProteins undergoing Golgi-bypassing UPS have a signal peptidethat is recognized by the signal recognition particle (SRP), whichrecruits the protein to the ER while it is being synthesized on theribosome (Blobel and Dobberstein, 1975; Walter et al., 1981). Incontrast to conventional secretion cargos that travel through theentire Golgi, these UPS cargos bypass at least part of the Golgicomplex on their way to the cell surface (Gee et al., 2018). Cargosfor Golgi-bypassing UPS include position-specific antigen subunitalpha 1 (αPS1) integrin in Drosophila (Schotman et al., 2008,2009), myeloproliferative leukemia virus oncogene (Mpl) (Cleyratet al., 2014), the ion channel cystic fibrosis transmembraneconductance regulator (CFTR) and the ion-transporting membraneprotein pendrin (Gee et al., 2011; Jung et al., 2016), as well as theciliary membrane proteins polycystin-2, the M2 mutant ofSmoothened (Hoffmeister et al., 2011) and peripherin 2 (Tianet al., 2014). In addition, CD45 (PTPRC), connexin 26, connexin30, pannexin 1, pannexin 3, serglycin and scramblase 1 have alsobeen shown to reach the cell surface by unconventional secretion(Baldwin and Ostergaard, 2002; Martin et al., 2001; Merregaertet al., 2010; Penuela et al., 2007; Qu et al., 2009; Scully et al., 2012).Although all Golgi-bypassing plasma-membrane proteins aretypically referred to as Golgi-bypassing UPS cargos, theindividual trafficking routes for the different proteins are notidentical (Fig. 2). For example, peripherin 2 reaches cilia throughCOPII-dependent exit from the ER, distinguishing it from otherUPS cargos that rely on COPII-independent routes, such as Mpl(Cleyrat et al., 2014) and CFTR (Gee et al., 2011).

From a therapeutic perspective, it is of great interest that somedisease-causing membrane proteins that have defects in proteinfolding or cell-surface trafficking, such as CFTR and pendrinmutants (Gee et al., 2011; Jung et al., 2016), can be transported tothe plasma membrane by UPS pathways. CFTR is an epithelialanion channel, and its loss of function as a result from geneticmutations causes cystic fibrosis (MIM 219700) and several otherepithelial diseases, such as bronchiectasis and chronic pancreatitis(LaRusch et al., 2014; Lee et al., 2003). The most common disease-causing mutation of CFTR is the deletion of Phe at position 508(ΔF508). Pendrin, a protein encoded by SLC26A4, is atransmembrane protein that exhibits Cl− to HCO3

− or Cl− to I−

exchange activity in the inner ear, thyroid follicles and renal corticalcollecting ducts (Mount and Romero, 2004). Mutations in SLC26A4cause non-syndromic recessive deafness with an enlarged vestibularaqueduct (deafness autosomal recessive 4, DFNB4, [MIM 600791])and Pendred syndrome (PDS, [MIM 274600]) (Everett et al., 1997;Li et al., 1998), a common cause of hereditary hearing loss inhumans. Specifically, p.H723R (His723Arg) is one of the mostprevalent pathological mutations (Dossena et al., 2009; Lee et al.,2014). Both the lack of Phe508 in CFTR (ΔF508-CFTR) andsubstitution of His723 for Arg in pendrin (H723R-Pendrin) result inprotein misfolding, retention in the ER and subsequent degradationby the ER-associated degradation (ERAD) pathway (Ward et al.,1995; Yoon et al., 2008). Consequently, only negligible amounts ofΔF508-CFTR and H723R-Pendrin reach the plasma membrane, andmost of the ion-transporting activity at the cell surface is lost(Amaral, 2004; Yoon et al., 2008). Although the mutant proteins

MplMye

lopro

lifera

tive

canc

er

Cystic f

ibrosis, p

ancreatitis

,

bronchiectasis

, infertili

ty

DFNB4, Pendred syndrome Polycystickidney disease

Cancer

ΔF508-CFTR

H723R-Pendrin Polycystin-2

M2 mutant of

Smoothened

Plasmamembrane

ER

Peripherin-2

vitelliform m

acular

dystrophy, ADRP

GRASP HSP70 RAB8AATG IRE1αDNAJC14 RAB18COPIISEC16

Nucleus

Key

Fig. 2. Known molecular arrangements involved in UPS of transmembrane proteins bypassing the Golgi. Immature Mpl involved in myeloproliferativecancer 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 Smoothenedwhich can induce cancer, is transported to the plasma membrane by RAB8A-mediated UPS. Peripherin 2, whose mutations cause ophthalmic diseases, can betransported to the plasma membrane by COPII-mediated UPS.

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have some defects in protein folding, they retain a certain level offunctional activity if they reach the cell surface (Gee et al., 2011;Jung et al., 2016). Therefore, many research efforts are invested inapproaches that facilitate the membrane targeting of ΔF508-CFTRand H723R-Pendrin.Notably, the blockade of conventional ER-to-Golgi trafficking,

which induces ER stress and the unfolded protein response (UPR),has been shown to also evoke unconventional cell-surfacetrafficking of CFTR and pendrin (Gee et al., 2011; Jung et al.,2016). It is, however, difficult to adopt a direct activation of ERstress as a therapeutic strategy because it is likely to give rise tomany unfavorable side effects (Yoshida, 2007). The basicmechanisms through which CFTR and pendrin reach the cellmembrane appear to be similar. For example, expression of bothΔF508-CFTR and H723R-Pendrin at the plasma membrane wasabolished by the knockdown of IRE1. This finding indicates thatIRE1 plays a major role in the UPS of CFTR and pendrin mutants,when considering the three UPR signaling arms consisting of IRE1,PERK and ATF6 (Gee et al., 2011; Jung et al., 2016). The molecularmechanism of how IRE1 facilitates Type IV UPS is not clearlyunderstood; however, a recent report has shown that IRE1 augmentsthe expression and function of SEC16A, which forms the ER exitsites for the UPS of ΔF508-CFTR (Piao et al., 2017), suggesting thatSEC16A is a downstream target of IRE1-mediated upregulation ofUPS. In contrast to the finding that the same ER stress signals canactivate UPS of both CFTR and pendrin, some of the key molecularfactors that are involved in the UPS of these two membrane proteinsare different. For example, Golgi reassembly stacking proteins(GRASPs) are required for the UPS of CFTR (Gee et al., 2011),whereas the HSP70 co-chaperone DNAJC14 is involved in the UPSof pendrin (Jung et al., 2016) (see below, ‘Potential therapeutictargets’). Theoretically, an activation of the cargo-specific pathwaywould be more desirable to develop therapeutics for each disease asit might help to minimize the adverse events caused by theactivation of common pathways that might also induce UPS ofuntoward cargos.In addition to CFTR and pendrin, several other transmembrane

proteins that are associated with human diseases can arrive at theplasma membrane by UPS. Polycystin-2, mutations of which areassociated with autosomal-dominant polycystic kidney disease(Mochizuki et al., 1996), is transported to cilia by UPS; this ismediated by RAB8A, a small GTPase, which plays a role invesicular traffic from the trans-Golgi network to the plasmamembrane (Hoffmeister et al., 2011). Activating mutations ofSmoothened can induce medulloblastoma, basal-cell carcinoma,pancreatic cancer and prostate cancer by unregulated activation ofthe hedgehog pathway (Rubin and de Sauvage, 2006), and thisprotein is also transported to the cell surface via UPS (Hoffmeisteret al., 2011). Patients with myeloproliferative neoplasms (MPNs)often carry either an activating form of JAK2 or mutations in the ERresident protein calreticulin (Klampfl et al., 2013; Nangalia et al.,2013), which leads to accumulation of immature Mpl in the ER(Kralovics et al., 2003; Moliterno et al., 1998). It has been reportedthat immature Mpl utilizes an unconventional autophagic secretorypathway to reach the cell surface (Cleyrat et al., 2014). Therefore,either the activation or inhibition of their unconventional secretioncould have therapeutic potential for any associated diseases(Table 1).

Potential therapeutic targetsAlthough the precise nature and mechanisms of UPS are not yetfully understood, the accumulated knowledge thus far suggests that

research into the following three areas would be especially helpfulin identifying druggable targets for human diseases, as well as tofurther elucidate the underlying mechanism of UPS.

First, a promising research area of UPS is the identification of thevesicular carrier involved in leaderless vesicular and Golgi-bypassing UPS. Results from previous studies have provided a listof candidates for vesicular carriers that include COPII-coatedvesicles (Tian et al., 2014), autophagy vesicles (Cleyrat et al., 2014;Gee et al., 2011), lipid droplets (Jung et al., 2016) and endosomes(Zhang et al., 2015). It is also possible that multiple vesicularsystems are involved in a single UPS event. For example, it has beensuggested that both autophagosome and endosome/multivesicularbody (MVB) components are sequentially involved in the vesicularUPS of IL-1β (Zhang et al., 2015). Our recent results also indicatethat early autophagosomal components, MVBs and RAB8A-dependent recycling vesicles sequentially mediate the ER stress-induced UPS of CFTR (our unpublished observation). To be able tomodulate the UPS pathway with a clear therapeutic potential fordiverse human diseases, the characterization of the vesicular systeminvolved in the UPS of each cargo of interest and their regulatoryprocesses are of paramount importance.

Second, GRASPs have emerged as an interesting regulator of theUPS pathway of various cargo proteins. GRASPs were initiallyidentified as factors required for the stacking of Golgi cisternae byusing in vitro assays (Barr et al., 1997). Two isoforms, GRASP55and GRASP65, exist in vertebrates (Barr et al., 1997; Shorter et al.,1999). Interestingly, several studies have demonstrated thatGRASPs are involved in Golgi-bypassing UPS in bothinvertebrate and vertebrate models, although they were initiallydescribed as Golgi-associated proteins (Cleyrat et al., 2014; Kinsethet al., 2007; Schotman et al., 2008). For example, the GRASPhomologs in Dictyostelium and Drosophila, respectively, mediatethe transport of acyl-CoA-binding protein (AcbA) and α-integrin atspecific developmental stages through an unconventional Golgi-independent route (Gee et al., 2011; Kinseth et al., 2007; Schotmanet al., 2008). Furthermore, as discussed above, GRASPs participatein the UPS of the mammalian transmembrane proteins CFTR andMpl (Cleyrat et al., 2014; Gee et al., 2011). However, the preciseroles of GRASPs in UPS are largely unknown. Interestingly,monomerization and ER re-localization of GRASP55 byphosphorylation on its serine 441 residue appear to be critical forthe UPS of CFTR (Kim et al., 2016). Therefore, in order to be ableto develop therapeutics for cystic fibrosis, a feasible approach mightbe to screen for small-molecule compounds that can inducephosphorylation and, thus, activation of GRASP55.

Third, increasing evidence suggests that molecular chaperones,such as heat shock 70 kDa proteins (HSP70s), HSP90s andmembers of the DNAJ-protein family (also known as HSP40s),play a role in the UPS of diverse cargos (Jung et al., 2016; Zhanget al., 2015). The correct folding of secretory and transmembraneproteins is ensured by ER quality control (ERQC) systems (Vembarand Brodsky, 2008). During endoplasmic-reticulum-associatedprotein degradation (ERAD), misfolded proteins that do not passERQC are retro-translocated to the cytoplasm and degraded in anubiquitin-dependent process by the proteasome. Molecularchaperones have crucial roles in the recognition of misfoldedproteins and the heat shock cognate protein 70 (Hsc70; encoded byHSPA8), a member of the HSP70 family, is one of the importantchaperones involved in this process. However, HSP70 chaperonesdo not function alone. In addition to protein folding anddegradation, HSP70s are involved in a myriad of biologicalprocesses, including protein–protein interaction and intracellular

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trafficking of various proteins (Kampinga and Craig, 2010; Younget al., 2003). Much of the functional diversity of HSP70 is thoughtto be driven by cofactors, including chaperone DNAJ proteins(Kampinga and Craig, 2010). Indeed, as mentioned above, theDNAJ protein DNAJC14 plays a crucial role in the UPS ofmisfolded pendrin (Jung et al., 2016). It appears that the activationof the chaperone machinery stimulates both UPS and the ERADpathway to relieve the protein burden in the ER during ER stress(Ron and Walter, 2007). Among the many co-chaperones,DNAJC14 appears to be specialized in assisting Hsc70 duringUPS, whereas other co-chaperones, such as CHIP, are involved inERAD (Meacham et al., 2001). Although Hsc70 is required for theUPS of ΔF508-CFTR, DNAJC14 is not (Jung et al., 2016),suggesting the presence of unknown co-chaperone that is specificfor UPS of CFTR. In addition, it has been shown that the Hsc70 co-chaperone DNAJC5 participates in Type III UPS of misfoldedcytosolic proteins (Xu et al., 2018). Interestingly, DNAJC14, whichmediates the UPS of transmembrane proteins, has ER-membrane-localizing transmembrane domains, whereas DNAJC5, which isinvolved in the UPS of cytosolic proteins, does not have anytransmembrane domains (Kampinga and Craig, 2010). Identifyingcargo-interacting domains in these proteins and determining theprinciples of how these co-chaperones recognize their UPS substratecargos will be important in order to develop strategies for cargo-specific modulation of UPS for therapeutic applications.

Conclusion and perspectivesUPS mechanisms are found in all organisms, including yeast, fungi,plants, Drosophila and mammals (Rabouille, 2017). It is useful toclassify UPS pathways and define similarities in these complexmechanisms of UPS. However, the current classification systembased on cargo protein category may not be sufficient to encompassthe complexities of UPS. For example, the UPS of IL-1β hascharacteristics of both non-vesicular (Type I) and vesicular (TypeIII) UPS. Additionally, Type III vesicular UPS of IL-1β involvescomponents of the autophagosome and GRASPs (Dupont et al.,2011; Zhang et al., 2015), which are also involved in Type IV UPSof transmembrane proteins Mpl and CFTR (Cleyrat et al., 2014;Gee et al., 2011), pointing to some overlap between the differentpathways. Even within UPS of transmembrane proteins, theprocesses involved exhibit considerable diversity depending onthe particular cargos (Fig. 2). Therefore, the identification of themolecular machinery and the processes that govern the UPS of eachspecific cargo is essential in developing therapeutic strategies forrelated diseases. Broad questions remain about (a) the molecularnature of carriers at the plasma membrane that mediate non-vesicular UPS of cytosolic cargos, (b) types of vesicular carrierinvolved in the vesicular UPS and, (c) characteristics andintramolecular determinants of transmembrane proteins that aretransported by using Golgi-bypassing UPS. More specifically,advancing recent findings related to mechanisms of UPS will haveimmediate therapeutic potential, particularly by identifying thecrucial regulators that divert secretory autophagy from degradativecanonical autophagy, by investigating the molecular switch thatactivates the UPS function of GRASP from its classic Golgimembrane-tethering function, and by identifying the types ofmolecular chaperone involved in UPS and determining how thesechaperones recognize UPS cargos.Insights of the above aspects will bring us closer to understand

why these proteins are transported unconventionally via variousroutes and how exactly the different UPS pathways are involved inthe pathogenesis of human diseases. This continued line of

investigation will, ultimately, open a new era of therapeutics fornumerous human diseases, including cancers, inflammatorydiseases and some genetic disorders, such as cystic fibrosis andPendred syndrome.

AcknowledgementsWe thank Dong Soo Chang for the assistance with illustrations.

Competing interestsThe authors declare no competing or financial interests.

FundingThis work was supported by grant 2013R1A3A2042197 from the National ResearchFoundation, the Ministry of Science, ICT & Future Planning, Republic of Korea, andgrant HI15C1543 of the Korea Health Technology R&D Project through the KoreaHealth Industry Development Institute (KHIDI), funded by the Ministry of Health &Welfare, Republic of Korea.

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