Academic Edi o : Ke mi L. Ca away
Recei ed: 29 No embe 2024
Re ised: 20 Decembe 2024
Accep ed: 31 Decembe 2024
Published: 7 Janua y 2025
Ci a ion: Conesa-Bakkali, R.;
Mo illo-Huesca, M.; Ma ínez-Fáb egas,
J.. Non-Canonical, Ex alysosomal
Ac i i ies o Lysosomal Pep idases in
Physiological and Pa hological
Condi ions: New Clinical
Oppo uni ies o Cance The apy.
Cells 2025,14, 68. h ps://doi.o g/
10.3390/cells14020068
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Re iew
Non-Canonical, Ex alysosomal Ac i i ies o Lysosomal
Pep idases in Physiological and Pa hological Condi ions: New
Clinical Oppo uni ies o Cance The apy
Ryan Conesa-Bakkali 1, Maca ena Mo illo-Huesca 1and Jona han Ma ínez-Fáb egas 1,2,*
1
Cen o Andaluz de Biología Molecula y Medicina Regene a i a—CABIMER, Uni e sidad de Se illa, Consejo
Supe io de In es igaciones Cien í icas (CSIC), Uni e sidad Pablo de Ola ide, Amé ico Vespucio 24,
41092 Se illa, Spain; yan.conesa@cabime .es (R.C.-B.); maca ena.mo illo@cabime .es (M.M.-H.)
2Depa amen o de Bioquímica Vege al y Biología Molecula , Facul ad de Biología, Uni e sidad de Se illa,
A enida Reina Me cedes, 41012 Se illa, Spain
*Co espondence: jona han.ma inez@cabime .es
Abs ac : Lysosomes a e subcellula compa men s cha ac e ised by an acidic pH, con ain-
ing an ample a ie y o acid hyd olases in ol ed in he ecycling o biopolyme s. Among
hese hyd olases, lysosomal p o eases ha e me ely been conside ed as end-des ina ion
p o eases esponsible o he diges ion o was e p o eins, a icked o he lysosomal com-
pa men h ough au ophagy and endocy osis. Howe e , ecen epo s ha e s a ed o
un a el speci ic oles o hese p o eases in he egula ion o ini ially unexpec ed bio-
logical p ocesses, bo h unde physiological and pa hological condi ions. Fu he mo e,
some lysosomal p o eases a e no longe es ic ed o he lysosomal compa men , as mo e
no el non-canonical, ex alysosomal a ge s a e being iden i ied. Cu en ly, lysosomal
p o eases a e accep ed o play key unc ions in he ex acellula milieu, a ached o he
plasma memb ane and e en in he cy osolic and nuclea compa men s o he cell. Un-
de physiological condi ions, lysosomal p o eases, h ough non-canonical, ex alysosomal
ac i i ies, ha e been linked o cell di e en ia ion, egula ion o gene exp ession, and cell
di ision. Unde pa hological condi ions, hese p o eases ha e been linked o cance , mos ly
h ough hei ex alysosomal ac i i ies in he cy osol and nuclei o cells. In his e iew,
we aim o p o ide a comp ehensi e summa y o ou cu en knowledge abou he ex-
alysosomal, non-canonical unc ions o lysosomal p o eases, bo h unde physiological
and pa hological condi ions, wi h a pa icula in e es in cance , ha could po en ially o e
new oppo uni ies o clinical in e en ion.
Keywo ds: lysosomes; p o eases; nucleus; cy osol; ex alysosomal ac i i ies; cance
1. In oduc ion
Lysosomes, subcellula o ganelles ound in mos euka yo ic cells, a e esponsible
o he deg ada ion and ecycling o ex acellula ma e ial, p e iously in e nalised by
endocy osis [
1
] and/o phagocy osis [
2
], and in acellula componen s seques e ed by
au ophagy [
3
]. Since hei disco e y by Ch is ian de Du e in 1955 [
4
], lysosomes ha e
been he subjec o nume ous impo an s udies and disco e ies, allowing o a be e
unde s anding o hei ole in he cell (Figu e 1). These s udies ha e allowed us o change
ou iew o lysosomes om simple ecycling cen es o complex signalling hubs in ol ed
in he egula ion o mul iple physiological p ocesses.
Cells 2025,14, 68 h ps://doi.o g/10.3390/cells14020068
Cells 2025,14, 68 2 o 34
Lysosomes a e desc ibed
(De Du e e al., 1955)
1955
Lysosomes as ecycling plan s in cells
(Conh, 1963)
1963
Lysosome-phagosome usion
(S aus, 1964)
1964
Fi s desc ip ion o lysosomal s o age
diseases (He s, 1965)
1965
Apop osis e m is coined and
connec ed wi h lysosomes
(Ke e al, 1972)
1972
De Du e ecei es he Nobel P ize o
he disco e y and desc ip ion o
lysosomes
1974
P esence o speci ic ecogni ion si es
in lysosomal enzymes con olling i s
a icking is p oposed
(Hickman & Neu eld, 1972)
Desc ip ion o he key ole o he -
ATPase in he in aluminal acidic pH o
lysosomes
1978 - 1983
Fi s desc ip ion o au ophagy in
yeas s (1988) and la e alida ion
(Takeshige e al., 1992)
1988 - 1992
Recogni ion o he key ole o
lysosomes in an igen gene a ion and
p esen a ion
1990s
Role o Beclin1 in au ophagy induc ion
and umo igenesis inhibi ion
(Liang e al., 1999)
1999
Au ophagy limi s cance p og ession
by limi ing ch omosome ins abili y
(Ma hew e al., 2007)
2007
Role o he mTOR-TFEB-CLEAR
pa hway in lysosomal biogenesis
(Sa diello e al., 2009) Lysosomal-media ed p og ammed cell
dea h is ecognised as a physiological
way o cell dea h
(Galluzzi e al., 2018)
2018
Lysosomal p o eases con ol T eg
di e en ia ion h ough he egula ion o
FoxP3 (S a hopoulo e al., 2018)
Nuclea and cy osolic a ge s o
lysosomal p o eases in cance
Physiological ole o lysosomal
p o eases in cell di ision h ough
con olled lysosomal leakage
(Hamalis o e al., 2020)
Role o lysosomal p o eases in he
ac i a ion o TLRs
(Ewald e al, 2009; Sepul eda e al.,
2009)
2009
2000’s - now
Fi s desc ip ion o lysosomal
media ed cell dea h
(Fi es one e al., 1979)
1979
His one 3 N- e minal clea age by C sL
con ols cell di e en ia ion
(Duncan e al., 2008)
2008
2020
Nuclea and cy osolic a ge s o
lysosomal p o eases in cance
2000’s - now
Se e al g oups add ess how
lysosomal p o eases a e a icked
ou side he lysosomal compa men
2000´s
Di e en s udies demons a e
lysosomal p o eases emain ac i e a
neu al pH
2000’s
Nuclea C sL con ol cell cycle h ough
CDP/Cux
(Goule e al., 2006)
2006
Figu e 1. Timeline showing he main disco e ies in lysosomal biology. In ed boxes, some o he
non-canonical, ex alysosomal unc ions o lysosomal p o eases a e iden i ied. In blue boxes, some o
he main miles ones in lysosome esea ch a e p esen ed [4–21].
Cells 2025,14, 68 3 o 34
Lysosomes a e memb ane-limi ed, subcellula componen s cha ac e ised by an acidic
in aluminal pH, illed wi h a ple ho a o acid hyd oly ic enzymes, including lipases, nucle-
ases, glycosidases, sulpha ases, p o eases, e c, ha a e undamen al o he ole o lysosomes
in he deg ada ion and ecycling o biopolyme s. In his ega d, lysosomes a e packed wi h
mo e han i y di e en acid hyd olases, wi h lysosomal p o eases ep esen ing he la ges
g oup [
22
], ha allow lysosomes o play hei ole as he ecycling plan o he cell. These
p o eases can be o ganised in o h ee well-de ined, s uc u ally un ela ed amilies: pepsin-
like aspa yl ca hepsins (ca hepsins (C s) D and C sE), papain-like cys eine ca hepsins (C sB,
C sC, C sF, C sH, C sK, C sL, C sO, C sS, C sV, C sW, and C sX/Z), chymo ypsin-like
se ine ca hepsins (C sA and C sG) and inally, a s uc u ally un ela ed cys eine p o ease
known as legumain o aspa agine endopep idase (AEP), which is closely ela ed o caspases
and sepa ase [
23
–
25
]. Howe e , lysosomes a e nowadays ecognised as much mo e han
me e ecycling cen es, being key playe s in he egula ion o he immune sys em [
15
,
16
,
26
–
29
]
and he no mal physiology o he cell [
24
,
30
–
32
]. This enewed pe cep ion o lysosomes
has allowed us o a ionalise hei key ole in he onse and p og ession o a ple ho a o
human diseases, such as lysosomal s o age diseases (LSDs) [
33
], neu odegene a i e dis-
eases [
34
], au oimmune diso de s [
35
], and cance [
36
,
37
]. Impo an ly, un il qui e ecen ly,
hese p ocesses we e hough o ely on p o eoly ic ac i i ies cons ained o he lysosomal
lumen due o ea lie wo ks epo ing hei in i o dena u a ion a neu al pH [38]. These
unc ions aking place wi hin he lysosomal compa men cons i u e he so-called canonical,
in alysosomal unc ions o lysosomal p o eases. Howe e , as highligh ed in Figu e 1,
ecen , g owing e idence has demons a ed, a leas in some ins ances, ha lysosomal
p o eases emain ac i e a neu al pH, albei in some cases wi h modi ied enzyme kine ics
and subs a e speci ici y [
39
–
42
]. Mo eo e , a g owing body o li e a u e has con i med
hei nuclea and cy osolic localisa ion, demons a ing ha hey emain ac i e in hese
ex alysosomal loca ions, whe e hey play c i ical oles in ini ially unexpec ed p ocesses,
bo h unde physiological and pa hological condi ions (Figu e 1). In his ega d, lysosomal
p o eases, h ough hei ex alysosomal ac i i y—so-called non-canonical ac i i ies— ha e
been linked o cell di ision [
21
,
43
,
44
], p og ammed cell dea h [
43
,
44
], neu o oxici y [
45
–
47
],
immune cell di e en ia ion [
20
,
27
], gene exp ession [
17
] and cance [
48
–
51
]. Howe e , one
o he mos exci ing ques ions in he ield emains o be add essed: How a e lysosomal
p o eases a icked o he cy osol o nuclea compa men s o he cell? In his con ex ,
some seminal wo ks, which will be discussed la e , ha e s a ed o un eil he molecula
mechanisms con olling his key e en [
21
,
52
–
54
] (Figu e 1). In e es ingly, non-canonical,
ex alysosomal unc ions ha e also been epo ed o o he lysosomal hyd olases, such as
glucosylce amidase be a 1 (Gba1), u he ein o cing he non-canonical, ex alysosomal
ole o hese hyd olases, including lysosomal p o eases, in he egula ion o biological
p ocesses, bo h unde physiological and pa hological condi ions [55–57].
The iden i ica ion o he biologically ele an a ge s o some o hese lysosomal p o-
eases, con i ming hei ole in he egula ion o ini ially unexpec ed biological p ocesses,
bo h unde physiological and pa hological condi ions, o e s new he apeu ical oppo -
uni ies o he ea men o a ple ho a human diseases. In his con ex , his manusc ip
aims o p o ide an upda ed, comp ehensi e e iew o he non-canonical, ex alysosomal
biological a ge s and p ocesses egula ed by hese p o eases, bo h unde physiological and
pa hological condi ions, wi h special emphasis in cance .
2. Canonical, In alysosomal Func ions o Lysosomal P o eases
and Disease
Toge he , all hese lysosomal p o eases con ibu e o he unc ion o lysosomes as ecy-
cling plan s, esponsible o he bulk deg ada ion and u no e o was e and endogenous
Cells 2025,14, 68 4 o 34
p o eins [
58
]. Fu he mo e, wi hin he lysosomal compa men , hey a e in ol ed in a
my iad o p ocesses, including he clea ance o in e nalised pa hogens [
59
–
61
], pa hogen
de ec ion and signalling [
15
,
16
,
62
], p ocessing and p esen a ion o endogenous and o -
eign an igens [
61
,
63
], ac i a ion o chemokines and cy okines [
62
,
64
], egula ion o cell
signalling h ough he p o eoly ic deg ada ion o cell su ace ecep o s [
65
,
66
], egula ion
o lysosomal homeos asis [24], and cellula me abolism [67,68] (Figu e 2).
Non-canonical, ex alysosomal unc ions Canonical, in alysosomal unc ions
P og amed
cell dea h
Cance
Cell mig a ion
Cell adhesion
and mo ili y
Cell mi osis
Cell di e en ia ion
DNA epai
Gene egula ion
Me abolism
Cy o oxic killing
An igen gene a ion
Killing o
In acellula pa hogens
Lysosomal
homeos asis
Recycling
Au ophagy
Gene exp ession
FIGURE 2
Figu e 2. Scheme showing he main canonical, in alysosomal ( igh , blue box) and non-canonical,
ex alysosomal (le , ed box) unc ions desc ibed h oughou his e iew.
All hese ac i i ies occu ing wi hin he lysosomal compa men cons i u e he canonical
unc ions o lysosomal p o eases. Impo an ly, dys egula ion o hese cellula p ocesses is
linked o he onse and p og ession o a wide a ie y o human diseases. Mo eo e , dele ion
o indi idual mu ine lysosomal p o eases esul s in clea issue-speci ic pheno ypes/diseases,
hus s eng hening he idea ha hey ha e non- edundan unc ions [
53
,
69
–
71
]. In his con ex ,
he lack o speci ic lysosomal p o eoly ic ac i i ies has been associa ed o di e en o ms o
lysosomal s o age diseases [
72
–
74
]; meanwhile, dys egula ion o he lysosomal ac i i y is
di ec ly linked o he appea ance and p og ession o di e en diseases, such as au oimmune
diseases and cance [75–81].
2.1. Bulk P o ein Deg ada ion and Lysosomal S o age Diseases
As p e iously desc ibed, lysosomes a e packed wi h a comple e se o acid hyd oly ic
enzymes. Unneeded and damaged biomolecules (such as p o eins, nucleic acids, ca bo-
hyd a es, lipids, e c.) as well as damaged subcellula compa men s a e con inuously
a ge ed o he endolysosomal compa men o hei deg ada ion and ecycling (Figu e 2).
In combina ion, all hese hyd olases a e equi ed o he comple e deg ada ion o his
ca go wi hin he lysosomal compa men [
82
–
87
]. Rema kably, his lysosomal unc ion is
well conse ed h oughou e olu ion, hus highligh ing i s key ole in he main enance o
cellula homeos asis [88–91].
Cells 2025,14, 68 5 o 34
In his con ex , he lack o speci ic lysosomal hyd olases d i es he accumula ion o
speci ic, undiges ed molecules, hence leading o he onse and p og ession o di e en LSDs.
LSDs ep esen a g oup o mo e han 70 di e en , a e inbo n me abolic al e a ions, gene i-
cally un ela ed, ha a e associa ed o mu a ions in p o eins in ol ed in he deg ada ion o
anspo o mac omolecules o in modula o s o he lysosomal mic oen i onmen [
33
,
92
].
E en hough, indi idually, LSDs a ec a low numbe o pa ien s, as a g oup, hei incidence
inc eases up o 1:5000 [
93
–
95
], hus highligh ing i s clinical ele ance. Some examples o
hese diseases a e shown in Table 1, indica ing he gene mu a ion associa ed o i s onse .
Table 1. Some examples o lysosomal s o age diseases indica ing he lysosomal gene mu a ed.
Diseases in which mu a ions in lysosomal p o eases ha e been iden i ied a e highligh ed in bold.
G oup Disease Gene
Glycogen s o age disease Pompe disease GAA
Danon disease LAMP2
Lipidoses Niemann-Pick disease ype C NPC1,NPC2,CTSB,CTSL
Neu onal ce oid lipo uscinoses PPT1,TRP1,CTSD,CTSF,CTSB,CTSL
Lysosomal anspo disease Cys inosis CTNS
Pycnodysos osis CTSK
Mucolipidosis Type I NEU1
Type II GNPTAB
Mucopolysaccha idoses Type I (Hu le synd ome) IDUA
Type II (Hun e synd ome) IDS
Type III (San ilippo synd ome) SGSH,NAGLU,HGSNAT,GNS
Glycop o einoses Galac osialidosis CTSA
Sphingolipidosis
Niemann-Pick disease SMPD1
Fab y disease GLA
Schindle disease NAGA
Tay-Sachs HEXA
Gauche disease GBA
In mos cases, LSDs a e linked o mu a ions in hyd olases and lysosomal anspo e s,
a he han lysosomal p o eases [
33
]. Howe e , he e a e some cases in which he mu a ion
o speci ic lysosomal p o eases has been shown o d i e he onse and p og ession o
LSDs. Speci ically, in mouse models, he loss o some lysosomal p o eases (e.g., CTSA [
72
],
CTSB [
96
], CTSD [
97
,
98
], CTSF [
74
], CTSK [
99
], CTSL [
96
] and CTSS [
97
]) esul s in he
onse and p og ession o LSDs, while in humans, mu a ions in some lysosomal p o eases
ha e been linked o LSDs.
In his ega d, speci ic mu a ions in some lysosomal p o eases in human pa ien s
ha e been linked o he onse and p og ession o di e en o ms o lipidoses. In humans,
se e al pa hogenic mu a ions in he gene encoding C sD a e linked o he congeni al, la e
in an ile o ju enile onse o ype 10 Neu onal Ce oid Lipo uscinosis (NCL) [
100
–
106
],
a se e e neu odegene a i e LSD cha ac e ised by he accumula ion o au o luo escen
lipopigmen s [
107
]. These mu a ions igge di e en deg ees o neu opa hogenesis, de-
pending on he deg ee o C sD inac i a ion ( e iewed in de ail in [
108
]). Simila ly, se e al
mu a ions in he CTSF gene leading o he onse o ype 13 NCL ha e been iden i ied in
humans [
74
,
109
–
111
]. Mo eo e , despi e he lack o iden i ied mu a ions in CTSB and CTSL
leading o LSDs in human pa ien s, mouse models wi h bo h CTSB and CTSL de iciencies
and he double knock-ou de elop a neu opa hology ha esembles human NCL [
112
].
Fu he mo e, a de iciency in CTSB and CTSL in human neu oblas oma cells esul s in he
Cells 2025,14, 68 6 o 34
accumula ion o choles e ol in la e endosomes/lysosomes, leading o a neu opa hology
ha esembles Niemann–Pick disease ype C, also included among lipidoses [
96
] (Table 1).
Pycnodysos osis is a a e, au osomal ecessi e LSD cha ac e ised by he abno mal
ha dening o he bones and educed s a u e [
99
] (Table 1). Wi h espec o his, nonsense,
missense, and s op codon mu a ions in he CTSK gene, leading o C sK de iciency, ha e
been iden i ied in human pa ien s.
Finally, in humans, CTSA mu a ions leading o loss o o educed C sA ac i i y a e
linked o he onse o Galac osialidosis, also known as neu aminidase de iciency wi h
β
-
galac osidase de iciency, which is included among glycop o einoses [
72
] (Table 1). In no mal
condi ions, C sA o ms a complex wi h be a-galac osidase (GLB1) and neu aminidase 1
(NEU1) o p ope ly deg ade glycop o eins [
113
]. Howe e , mu a ions in he CTSA gene,
a ec ing i s in e ac ion wi h GLB1 and NEU1, o i s de iciency, lead o he des abilisa ion
and deg ada ion o hese enzymes, esul ing in he accumula ion o undiges ed ma e ial
and he onse o his LSD.
2.2. Inna e and Adap i e Immuni y and Au oimmune Diseases
Lysosomal ac i i y di ec ly in luences he egula ion o he inna e and adap i e im-
mune esponses [
63
,
114
]. In his con ex , lysosomes play a key ole in he de ec ion and
signalling o pa hogens h ough he ac i a ion o Toll-like ecep o s (TLRs) [
115
]. Fu he -
mo e, lysosomes a e essen ial in he p ocessing and p esen a ion o an igens de i ed om
bo h pa hogens and endogenous p o eins [
116
] (Figu e 2). Fo his eason, lysosomes, and
mo e speci ically lysosomal p o eases, h ough hei canonical, in alysosomal ac i i y, a e
essen ial o he ac i a ion o p ope immune esponses, bu also o he acquisi ion o sel -
ole ance. The e o e, i should no come as a su p ise ha lysosomal dys unc ion has been
linked o he onse and p og ession o a ple ho a o au oimmune diseases [35,64,117,118].
2.2.1. Inna e Immune Response
Du ing he inna e immune esponse, pa hogens can be in e nalised h ough phagocy-
osis and a ge ed o he lysosomes o be diges ed by he lysosomal p o eases. Fu he mo e,
in he case o cy osolic pa hogens and hose ha manage o escape he endolysosomal
sys em gaining access o he cy osol, hey can be cap u ed h ough au ophagy and a ge ed
o he lysosomal compa men o be elimina ed. In his con ex , he lysosomal compa men ,
and mo e speci ically, he lysosomal p o eases, se e as an in acellula de ence sys em,
elimina ing pa hogens and p o ec ing he cells om in ec ion [59] (Figu e 2).
In addi ion, lysosomal p o eases a e esponsible o he p o eoly ic p ocessing nec-
essa y o he comple e ac i a ion o TLRs, which a e esponsible o he de ec ion o
Pa hogen-Associa ed Molecula Pa e ns (PAMPs), pa hogen-speci ic molecules whose
ecogni ion by ecep o s o he inna e immune sys em igge s a apid and gene alised
immune esponse [
119
–
121
]. This esponse includes he p oduc ion o p o-in lamma o y
cy okines, such as umou nec osis ac o (TNF) and in e leukin-1 (IL-1), which p omo e
in lamma ion and he ec ui men o immune cells o he si e o in ec ion, and he induc-
ion o phagocy osis, a p ocess by which phagocy ic cells engul and des oy in ading
mic oo ganisms [15,16,115,117–119,122].
Finally, lysosomes a e also in ol ed in he inna e immune esponse agains i us-
in ec ed cells o cance cells h ough sec e o y lysosomes. These specialised subcellula
compa men s a e lysosome- ela ed o ganelles cha ac e ised by he ca abolic unc ions
o lysosomes, while p esen ing inducible sec e o y capabili ies [
29
,
123
,
124
]. Sec e o y
lysosomes a e p esen in bo h cy o oxic CD8
+
T lymphocy es and Na u al Kille (NK)
cells, and hey a e packed wi h a ull se o acid lysosomal hyd olases as well as le hal
p o eins such as pe o ins and g anzymes [
120
,
121
] (Figu e 2). When hese cells encoun e
Cells 2025,14, 68 7 o 34
i us-in ec ed hos cells and cance cells, he sec e o y lysosomes use wi h hei plasma
memb ane, eleasing hei con en o he ex acellula milieu. Upon elease, pe o in o ms
a po e in he plasma memb ane o he a ge cell, allowing g anzymes and lysosomal
p o eases o igge he ac i a ion o apop osis [121,125,126].
2.2.2. Adap i e Immune Response
The ole o lysosomes in he egula ion o he immune sys em also includes impo an
unc ions in he adap i e immune esponse. In his ega d, lysosomal p o eases a e essen ial
playe s in he p ocessing and p esen a ion o an igens. In specialised an igen-p esen ing
cells (APCs)—such as mac ophages, dend i ic cells, and B lymphocy es—bo h endogenous
and pa hogen-de i ed p o eins a e a ge ed o he lysosomes, whe e hey a e diges ed
in o smalle agmen s o gene a e an igens. These an igens a e subsequen ly loaded in o
Majo His ocompa ibili y Complex II (MHC-II) molecules o be p esen ed o CD4
+
T cells.
Fo hese easons, lysosomal p o eases no only play an essen ial ole in he ac i a ion o
p ope immune esponses agains speci ic pa hogens, bu also gua an ee he ecogni ion
o sel -an igens and he acquisi ion and de elopmen o sel - ole ance [
65
,
117
,
122
,
127
]
(Figu e 2).
2.2.3. Role o Lysosomal P o eases in Au oimmune Diseases
In au oimmune diseases, he immune sys em mis akenly a acks i s own cells and
issues, leading o dys unc ions in a ious sys ems and o gans. The ole played by lyso-
somes in he cen al pa hways o he immune sys em (including an igen p ocessing and
p esen a ion, cy okine p ocessing, ac i a ion o TLR-media ed signalling, e c.) jus i ies
i s con ibu ion o he onse and p og ession o hese diseases (Figu e 2). Mo eo e , lyso-
somes play a mul i ace ed ole in he onse and p og ession o hese ypes o diso de s, as
inc eased le els o au ophagy, high exp ession le els o speci ic lysosomal enzymes, and
ele a ed luminal pH o cells ha e been con i med in pa ien s su e ing om hese ypes o
diso de s [
64
,
66
,
84
,
128
]. In his ega d, he a o emen ioned al e a ions ega ding he p ope
unc ioning o lysosomes can lead o he gene a ion and p esen a ion o new sel -an igens,
hus igge ing abe an immune esponses agains i s own cells and issues. Fu he mo e,
lysosomes a e also key in he ac i a ion o cy okines and chemokines in immune cells [
61
];
he e o e, changes in he lysosomal compa men a ec ing his p ocessing can a ec he
immune unc ion o cells, hus leading o au oimmune diseases [61,129].
Fo all hese easons, lysosomes ha e been associa ed o he onse and p og ession o
a wide a ie y o au oimmune diseases [
123
]. In e es ingly, ele a ed exp ession le els o
di e en lysosomal p o eases ha e been linked o some o hese pa hologies, such as Sys emic
Lupus E y hema osus (SLE) [
80
,
124
,
129
–
131
], Rheuma oid A h i is (RA) [
127
,
128
,
132
–
139
]
and Amyo ophic La e al Scle osis (ALS) [127,128,132–139].
SLE is an au oimmune diso de cha ac e ised by he p oduc ion o au oan ibodies,
abe an in lamma ion, and mul iple o gan damage. The abno mal p ocessing and p esen a-
ion o an igens is conside ed one o he i s e en s in ol ed in he onse o he disease [
140
].
In his ega d, inc eased le els o exp ession and ac i i y o lysosomal p o eases (C sB,
C sD, C sL, and C sS, among o he s), known o po ay c i ical oles in an igen p ocessing
and p esen a ion, ha e been epo ed o be al e ed in SLE [
83
,
134
,
136
,
137
]. Fu he mo e, in
a mouse model, C sK was shown o be linked o SLE h ough he p o eoly ic p ocessing
and ac i a ion o TLR7 [131].
RA is an au oimmune disease in which he immune sys em w ongly a acks he join s,
leading o in lamma ion, join des uc ion, and bone damage [
141
]. In RA pa ien s, lysosomes
a e o e ac i e in in lamma o y cells, showing inc eased le els o C sB, C sD, C sG, C sK, C sL,
and C sS ha con ibu e o mos o he clinical mani es a ions o RA [127,128,132–139].
Cells 2025,14, 68 8 o 34
The causes o ALS emain as ly unknown; howe e , inc easing e idence suppo s
he p esence o a dys egula ed immune esponse con ibu ing o he pa hogenesis. Wi h
espec o his, inc eased exp ession le els o a ious lysosomal p o eases, such as C sB,
C sD, C sX, and C sZ, ha e been de ec ed in pa ien s wi h ALS and/o mouse models,
hus e ealing a po en ial ole o hese p o eases in he onse and/o p og ession o his
pa hology [
142
–
147
], al hough he con ibu ion o hese inc eased le els o he onse and
p og ession o ALS a he molecula le el s ill emains o be de e mined.
2.3. Cance
Beyond hei classical ole in p o ein u no e and an igen gene a ion and p esen-
a ion, lysosomes a e known o play key unc ions in ene gy homeos asis, gene a ion o
building blocks o cell g ow h, and immune escape h ough hei canonical, in alysosomal
unc ions ( e iewed in [
36
]) (Figu e 2). In his con ex , lysosomal p o eases, h ough hei
canonical, in alysosomal unc ions, ha e been shown o play cen al oles in he onse and
p og ession o cance .
Lysosomal p o eases a e esponsible o he deg ada ion o unneeded p o eins wi hin
he lysosome, p o iding cells wi h building blocks o he syn hesis o new p o eins, hus
p omo ing cell g ow h. In e es ingly, cance cells a e cha ac e ised by accele a ed a es
o g ow h and inc eased demand o ene gy supply. This explains why cance cells show
inc eased lysosomal ac i i y and au ophagy, as hey could allow hem o sus ain ele a ed
p oli e a ion a es in condi ions when nu ien s become limi ing [148].
Mo eo e , lysosomes, h ough hei cen al ole in an igen gene a ion and p esen a ion
and he ac i a ion o he immune esponse, can play a cen al ole in immune escape in
cance h ough he deg ada ion o speci ic an igens, hus jus i ying he inc eased lysosomal
p o eoly ic ac i i y obse ed in cance cells [36,149].
3. Non-Canonical, Ex alysosomal Roles o Lysosomal P o eases
in Cance
These canonical, in aluminal ac i i ies o lysosomal p o eases ha e been conside ed,
o a long ime, as he only unc ions execu ed by hese p o eases. Howe e , eme ging
e idence is e ealing a non-canonical ole o hese p o eases, highligh ing hei ole in he
egula ion o key p ocesses, bo h unde physiological and pa hological condi ions, wi h
a special ele ance in cance (Figu e 2). Mo eo e , ex acellula , cy osolic, and nuclea
speci ic a ge s o some o hese lysosomal p o eases ha e been iden i ied, hus e ealing
ini ially unexpec ed unc ions and inc easing he numbe o biological p ocesses egula ed
by hese p o eases (Figu e 2). Howe e , un il ecen ly, wo main ac o s ha e limi ed
he cha ac e isa ion o hese non-canonical, ex alysosomal unc ions. Fi s , he acidic
lysosomal pH, conside ed essen ial o he ac i i y o hese lysosomal p o eases, and
epo s indica ing hei in i o dena u a ion a neu al pH ha e bo h limi ed he s udy o
hei ex alysosomal ac i i ies [
38
]. Second, un il ecen ly, he lack o knowledge on he
molecula mechanisms con olling he ex alysosomal localisa ion o hese p o eases has
u he con ibu ed o he idea ha hese non-canonical, ex alysosomal ac i i ies we e
simple a i ac s, wi h no ele an biological unc ions. In his e iew, we will summa ise
ou cu en knowledge on 1. how lysosomal p o eases main ain hei ac i i y a neu al
pH and how changes in he pH a ec hei ac i i y and speci ici y ou side he lysosomal
compa men , and 2. how hey each hese subcellula compa men s. Fu he mo e, we
will p esen ou cu en knowledge on he non-canonical, ex alysosomal unc ions o hese
p o eases unde physiological and pa hological condi ions, wi h a special ocus in cance .
Cells 2025,14, 68 9 o 34
3.1. E ec o he pH in he Ac i i y o Lysosomal P o eases
Lysosomes a e cha ac e ised by an acidic pH a ound 4.5, in which lysosomal p o-
eases emain olded, achie ing hei maximum ac i i y, wi h some epo s showing ha
lysosomal p o eases become dena u ed and inac i a ed a neu al pH [
38
]. This idea has
impac ed he s udy and unde s anding o he non-canonical, ex alysosomal ac i i ies o
hese p o eases. Howe e , ecen epo s ha e demons a ed ha hese p o eases can be
de ec ed in he ex acellula space, bu also in he nuclea and cy osolic compa men s o
he cell, whe e hey e ain hei ac i i y.
In his ega d, one o he i s examples o hese ex alysosomal ac i i ies was desc ibed
in 1992, when C sB was shown o be in ol ed in he deg ada ion o he ex acellula ma ix
(ECM), bo h unde acidic and neu al pH [
150
]. Howe e , la e wo k demons a ed ha
a la ge a ay o lysosomal p o eases (i.e., C sF, C sK, C sL, C sS and C sV), and no jus
C sB, a e able o deg ade he componen s o he ECM in he ex acellula space, u he
demons a ing ha hey emain ac i e a neu al pH [
150
–
152
] (Figu e 2). Fu he mo e,
some in e ac ions ha e been shown o con ibu e o he s abilisa ion and ac i i y o hese
p o eases a neu al pH. In his con ex , in e ac ions be ween C sB and hepa in, and high
subs a e concen a ions in he case o C sL, ha e been shown o con ibu e o he s abili-
sa ion o bo h p o eases a neu al pH [
39
]. Fu he mo e, ecen epo s ha e con i med
ha a leas some o hese p o eases emain ac i e ou side he lysosomal compa men a
neu al pH, albei showing educed enzyme kine ics and subs a e speci ici y. Consis en ly,
se e al epo s ha e co obo a ed ha AEP, C sB, C sL, and C sS e ain e icien (al hough
subop imal) ac i i y a neu al pH [40,41,54,153,154].
Toge he , all hese epo s demons a e ha lysosomal p o eases e ain hei ac i -
i y ou side he lysosomal compa men a neu al pH, u he ein o cing he biological
ele ance o hei non-canonical, ex alysosomal ac i i ies in he egula ion o ini ially
unexpec ed biological p ocesses.
3.2. Ex alysosomal T a icking o Lysosomal P o eases
Ano he majo limi a ion ega ding he non-canonical, ex alysosomal ac i i ies o
hese p o eases is ela ed o hei a icking. Lysosomal p o eases a e syn hesised as inac i e
p o- o ms ha need o be a icked o he endolysosomal compa men o p ocessing and
ac i a ion a acidic pH [
27
]. In his con ex , lysosomal p o eases can each he ex acellula
space h ough lysosomal exocy osis o h ough al e na i e a icking ou es [
155
–
157
],
whe e hese p o eases ha e been shown o play key oles, bo h unde physiological and
pa hological condi ions ( u he discussed in he upcoming sec ions).
Rema kably, ecen epo s ha e s a ed o expand he non-canonical, ex alysosomal
ac i i ies o hese p o eases h ough he iden i ica ion o bo h cy osolic and nuclea a ge s,
bo h unde physiological and pa hological condi ions. Howe e , he p ecise mechanism
explaining how lysosomal p o eases each hese compa men s emains one o he mos
in iguing ques ions in he ield. Impo an ly, ecen ad ances ha e s a ed o un a el
how hese p o eases can each hese ex alysosomal compa men s. In his ega d, cy oso-
lic exp ession wi hou a icking h ough he endoplasmic e iculum has been epo ed,
e ealing ha some human lysosomal p o eases can be exp essed as ansc ip a ian s lack-
ing he signal pep ide (i.e., C sL) [
55
,
56
,
158
]. Howe e , o he human lysosomal p o eases
(C sD, C sS, and C sV), also linked o non-canonical, ex alysosomal ac i i ies, only show
ansc ip a ian s coding o he ull-leng h p o ein, hus including he signal pep ide [
54
].
E en hough, in some speci ic cases, such as human C sL, he exis ence o ansc ip a i-
an s could explain hei cy osolic/nuclea localisa ion, cu en ly he e is li le e idence
o suppo his as he gene al mechanism con olling he ex alysosomal localisa ion and
ac i i y o hese p o eases.
Cells 2025,14, 68 16 o 34
glioma cells. Fu he mo e, i s inhibi ion sensi ised glioma cells o i adia ion [
222
,
238
]. In-
e es ingly, a ecen epo has also iden i ied C sL as a no el playe in DNA epai , u he
alida ing hese obse a ions [
239
]. Some o he molecula a ge s egula ed by nuclea C sL
ha e been iden i ied, hus p o iding he mechanis ic insigh explaining he ole o nuclea
C sL in DNA damage esponse and adio esis ance (see Ca hepsin L sec ion o u he
de ails). Thus, i would be in e es ing o check whe he C sB a ge s he same p o eins as
C sL, o whe he i s ole in DNA epai and adio esis ance is comple ely un ela ed.
Ca hepsin D
Se e al epo s ha e demons a ed ha C sD is o e exp essed in a a ie y o human
umou s, wi h his o e exp ession posi i ely co ela ing wi h poo p ognosis and educed
o e all su i al [193,226,227,240–244], as illus a ed in Figu e 4.
FIGURE 4
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*****
*
B
Low c sd G oup
High c sd G oup
Log ank p=0.019
HR(high)=1.5
p(HR)=0.021
n(high)=80
n(low)=81
Low c sd G oup
High c sd G oup
Log ank p=0.016
HR(high)=1.3
p(HR)=0.016
n(high)=212
n(low)=212
Figu e 4. CTSD exp ession le els and Kaplan–Meie analyses illus a ing he posi i e co ela ion
be ween high CTSD exp ession le els and poo p ognosis and educed o e all su i al, as p e iously
epo ed [
193
,
226
,
227
,
240
–
244
]. Da a we e ob ained using he GEPIA2 online ool o analyse The
Cance Genome A las (TCGA) da abase. (A) Exp ession le els o CTSD in Glioblas oma (GBM), O a ian
se ous cys adenoca cinoma (OV), Panc ea ic adenoca cinoma (PAAD), Tes icula Ge m Cell Tumou s
(TGCT), Li e Hepa ocellula Ca cinoma (LIHC) and Thy oid Ca cinoma (THCA) pa ien s (T = umou ,
ed boxes, N = no mal, g ey boxes). * p< 0.01. (B) Kaplan–Meie analyses showing he o e all su i al
in pa ien s exp essing low (blue line) s. high ( ed line) CTSD le els in OV and GBM pa ien s.
Cells 2025,14, 68 17 o 34
Nuclea C sD has been epo ed o speci ically a ge he nuclea ep esso T icho-
hino-phalangeal synd ome 1 (TRPS1) and he nuclea chape one Scy he/BAG6 (BAT3),
con olling cell cycle p og ession and ans o ma ion o b eas cance cells. In his case, he
au ho s demons a ed he nuclea localisa ion o C sD, whe e i was speci ically bound o
he ch oma in ac ions, hus ein o cing he nuclea unc ions o C sD. Fu he mo e, he
au ho s wen on o demons a e he nuclea co-localisa ion o C sD wi h bo h TRPS1 and
Scy he/BAG6. In e es ingly, in his case, he ole o C sD seemed o be independen o i s
p o eoly ic ac i i y, hus aising he possibili y ha C sD has nuclea ac i i ies un ela ed o
i s adi ional enzyma ic ac i i y [245].
Ca hepsin L
C sL has been epo ed o be o e exp essed in a as majo i y o human cance s,
wi h i s o e exp ession being a ma ke o poo p ognosis and educed o e all su -
i al [51,222,238,246–254], as illus a ed in Figu e 5.
C sL is one o he bes s udied lysosomal p o eases ega ding i s nuclea ac i i y no
only in cance , bu also unde physiological condi ions. As a ma e o ac , i is among
he i s lysosomal p o eases desc ibed o localise wi hin he nuclea compa men [
255
].
In e es ingly, ecen s udies ha e s a ed o iden i y he speci ic a ge s and he biological
p ocesses egula ed by his p o ease ou side he lysosomal compa men , hus allowing us
o a ionalise i s nucleo-cy osolic localisa ion.
In 2004, a seminal wo k in he ield e ealed an unexpec ed ole o nuclea C sL
by p o eoly ically p ocessing CDP/Cux, p omo ing he ansi ion om G1 o S phase,
being p oposed as a no el mechanism o cell ans o ma ion con ibu ing o umo igene-
sis
[13,176]
. Rema kably, in gas ic cance , i has been demons a ed ha he p o eoly ic
p ocessing o CDP/Cux by nuclea C sL induces angiogenesis by al e ing he gene exp es-
sion pa e n, hus p omo ing cance cell su i al [
256
]. In his s udy, he au ho s u he
demons a ed he nuclea ole o C sL, showing ha pa ien s wi h inc eased nuclea le els
o C sL p esen ed educed o e all su i al compa ed o pa ien s showing low le els o
nuclea C sL.
In 2006, ano he ole o C sL in he nuclei o mouse emb yonic s em cells du ing
di e en ia ion h ough he egula ion o he le els o non-ace yla ed his one 3 was e-
po ed [
17
], hus e ealing a po en ial ole o C sL in he egula ion o gene exp ession
h ough he emo al o epigene ic ma ks in he N- ail o his ones [
257
]. Rema kably, in
colon cance cells, a ole o nuclea C sL in cell p oli e a ion and cell cycle p og ession
was epo ed. The au ho s demons a ed ha nuclea C sL p omo es cell cycle p og ession
and p oli e a ion o colon cance cells by speci ically a ge ing his one 3. In ag eemen
wi h hese obse a ions, colo ec al cance pa ien s exp essing high le els o C sL showed
wo se p ognosis and educed o e all su i al when compa ed o pa ien s exp essing low
C sL le els [
258
]. The au ho s u he con i med he nuclea ole o C sL, showing, bo h
by Wes e n blo ing and con ocal mic oscopy, ha i speci ically accumula ed in he nuclei
o colon cance cells du ing he G1/G0 phase and ha i accumula ed in he lysosomal
compa men du ing he S and G2/M phases. Fu he mo e, he au ho s we e able o
demons a e ha nuclea C sL e ained i s ac i i y, eaching i s maximum a he G1/G0
phase, hus alida ing i s accumula ion du ing his phase o he cell cycle.
In b eas cance cells, i has been epo ed ha he loss o B eas Cance Type 1 Sus-
cep ibili y P o ein (BRCA1) igge s he nuclea deg ada ion o TP53-binding p o ein 1
(TP53BP1). TP53BP1 is a double-s and b eak (DSB) epai p o ein ha , in he absence
o BRCA1, se es as a molecula eplacemen igge ing cell cycle a es and cell dea h
in esponse o DNA damage. Howe e , i s C sL-media ed deg ada ion allows cance
cells o a oid cell g ow h a es and educe cell dea h in esponse o DNA damage. The
Cells 2025,14, 68 18 o 34
au ho s u he con i med he nuclea localisa ion o C sL in iple nega i e b eas cance
pa ien s, con i ming a nega i e co ela ion be ween he le els o nuclea C sL and TP53BP1,
u he ein o cing he nuclea ac i i y o C sL. Fu he mo e, his nuclea ole o C sL in
b eas cance cells has been shown o play an impo an ole in cance esis ance o ea -
men [
49
], hus o e ing he possibili y o designing no el app oaches aimed a sensi ising
cance cells o con en ional chemo he apy and adio he apy app oaches aimed a inducing
DNA damage.
Finally, in b eas cance pa ien s, nuclea C sL has been shown o in e ac wi h he
Cyclin-Dependen Kinase 2-associa ed P o ein 1 (CDK2AP1), an inhibi o o CDK2, hus e-
ealing a po en ial ole o C sL as a egula o o cell cycle in b eas cance cells, con ibu ing
o abe an cance cell p oli e a ion [259].
FIGURE 5
A
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*
*
*
B
Low c sl G oup
High c sl G oup
Log ank p=0.0093
HR(high)=1.6
p(HR)=0.0099
n(high)=81
n(low)=81
Low c sl G oup
High c sl G oup
Log ank p=0.029
HR(high)=1.7
p(HR)=0.031
n(high)=91
n(low)=91
Figu e 5. CTSL exp ession le els and Kaplan–Meie analyses illus a ing he posi i e co ela ion
be ween high CTSL exp ession le els and poo p ognosis and educed o e all su i al, as p e iously
epo ed [
51
,
222
,
238
,
246
–
254
]. Da a we e ob ained using he GEPIA2 online ool o analyse The
Cance Genome A las (TCGA) da abase. (A) Exp ession le els o CTSL in Glioblas oma (GBM),
Esophageal Ca cinoma (ESCA) and B ain Lowe G ade Glioma (LGG). (T = umou , ed boxes,
N = no mal
, g ey boxes). * p< 0.01. (B) Kaplan–Meie analyses showing he o e all su i al in
pa ien s exp essing low (blue line) s. high ( ed line) CTSL le els in LIHC and GBM pa ien s.
Cells 2025,14, 68 19 o 34
Ca hepsin V
C sV, also known as CTSL2, a p o ein highly ela ed o C sL, has been shown o be
o e exp essed in a a ie y o human umou s, wi h high exp ession co ela ing wi h wo se
p ognosis and educed o e all su i al [260–267], as illus a ed in Figu e 6.
FIGURE 6
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*
**
*
*
*
*
B
Low c s G oup
High c s G oup
Log ank p=2x10-4
HR(high)=4.8
p(HR)=0.00072
n(high)=38
n(low)=38
Low c s G oup
High c s G oup
Log ank p=0.0058
HR(high)=1.5
p(HR)=0.0062
n(high)=201
n(low)=201
Figu e 6. CTSV exp ession le els and Kaplan–Meie analyses illus a ing he posi i e co ela ion
be ween high CTSV exp ession le els and poo p ognosis and educed o e all su i al as p e iously
epo ed [
260
–
267
]. Da a we e ob ained using he GEPIA2 online ool o analyse The Cance Genome
A las (TCGA) da abase. (A) Exp ession le els o CTSV in Ad enoco ical Ca cinoma (ACC), Bladde
U o helial Ca cinoma (BLCA), Head and Neck Squamous Cell Ca cinoma (HNSC), Lung Adeno-
ca cinoma (LUAD), Panc ea ic Adenoca cinoma (PAAD), S omach Adenoca cinoma (STAD), and
U e ine Ca cinosa coma (UCS) pa ien s. (T = umou , ed boxes, N = no mal, g ey boxes). * p< 0.01.
(B) Kaplan–Meie analyses showing he o e all su i al in pa ien s exp essing low (blue line) s.
high ( ed line) CTSV le els in ACC and BLCA pa ien s.
C sV is no exp essed in mouse, hus limi ing ou unde s anding o i s po en ial ole
in he onse and p og ession o human umou s. Howe e , he nuclea localisa ion o
C sV has been epo ed in he case o hy oid ca cinoma cells, con ibu ing o he inc eased
p oli e a ion o cance cells, speci ically accumula ing in he nuclei o hese cells du ing
he S phase [
261
]. Fu he mo e, in he case o b eas cance cells, nuclea C sV has been
epo ed o supp ess he exp ession o he T ans-ac ing T-cell-speci ic ansc ip ion ac o
(GATA3) and p omo ing he s abili y o bo h his one 3 and his one 4 h ough he egula ion
o he chape one sNASP [
260
], hus po en ially con ibu ing o cell p oli e a ion and he
Cells 2025,14, 68 20 o 34
egula ion o gene exp ession. Fu he mo e, he au ho s demons a ed ha nuclea C sV
accumula es speci ically du ing he S and G2/M phases, wi h C sV knock-down esul ing
in G2/M a es .
Aspa aginyl Endopep idase
AEP, a unique lysosomal cys eine p o ease wi h an exquisi e speci ici y owa ds as-
pa agine esidues in he clea age si es and e olu iona ily ela ed o caspases and sepa ase,
is o e exp essed in a as majo i y o human solid umou s, wi h i s o e exp ession co ela -
ing wi h educed o e all su i al in a a ie y o human solid umou s
[48,50,202,268–278]
,
as illus a ed in Figu e 7.
FIGURE 7
A
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***
*
*
B
Low lgmn G oup
High lgmn G oup
Log ank p=0.01
HR(high)=2
p(HR)=0.012
n(high)=77
n(low)=77
Low lgmn G oup
High lgmn G oup
Log ank p=0.062
HR(high)=1.7
p(HR)=0.065
n(high)=535
n(low)=129
Figu e 7. AEP exp ession le els and Kaplan–Meie analyses illus a ing he posi i e co ela ion
be ween high AEP exp ession le els and poo p ognosis and educed o e all su i al, as p e iously
epo ed [
48
,
50
,
202
,
268
–
278
]. Da a we e ob ained using he GEPIA2 online ool o analyse The Cance
Genome A las (TCGA) da abase. (A) Exp ession le els o AEP in B eas In asi e Ca cinoma (BRCA),
Glioblas oma (GBM), S omach Adenoca cinoma (STAD), Tes icula Ge m Cell Tumou s (TGCT) and
Thymoma (THYM) pa ien s. (T = umou , ed boxes, N = no mal, g ey boxes). * p< 0.01. (B) Kaplan–
Meie analyses showing he o e all su i al in pa ien s exp essing low (blue line) s. high ( ed line)
AEP le els in STAD and BRCA pa ien s.
In colo ec al cance cells, AEP shows a nuclea localisa ion, speci ically a ge ing his-
one 3.1, hus po en ially playing a simila ole o C sL in he egula ion o gene exp ession
Cells 2025,14, 68 21 o 34
h ough he egula ion o ch oma in s uc u e [
279
]. The au ho s demons a ed he nuclea
localisa ion o AEP in di e en colo ec al cance cell lines, con i ming ha i e ains i s
p o eoly ic ac i i y a neu al pH and u he alida ing ha AEP can e icien ly clea e
his one 3 a bo h acidic (pH 5.0) and neu al pH (pH 7.0).
Recen ly, a no el ole o AEP has been iden i ied in glioblas oma. In his s udy,
he au ho s demons a ed ha AEP is o e exp essed in umou -associa ed mac ophages
(TAM) in esponse o hypoxia h ough HiIF1
α
egula ion. In his con ex , AEP p omo es
TAM immunosupp essi e pola iza ion ia he GSK-3
β
-STAT3 pa hway, hus p omo ing
cance p og ession.
Finally, ano he ex alysosomal, non-canonical unc ion o AEP has been ecen ly
epo ed in glioblas oma. In his case, Zhang e al. epo ed ha , unde hypoxia and
nu ien dep i a ion condi ions, bo h common ea u es o solid umou s, AEP speci ically
clea es DEAD-box helicase 3 X-linked (DDX3X), an ATP-dependen RNA helicase, in he
cy osolic compa men o cance cells. This unca ed o m o DDX3X ansloca es and
accumula es in he nucleus, igge ing al e na i e RNA splicing ha con ibu es o he
adap a ion o cance cells o ha sh mic oen i onmen s. Thus, his wo k e eals a no el ole
o AEP in p omo ing umou su i al and p oli e a ion h ough he egula ion o nuclea
biological p ocesses, such as al e na i e splicing [280].
4. Pe spec i es
Lysosomal dys unc ion is linked o se e al human maladies a ec ing millions o peo-
ple a ound he wo ld, including LSDs, au oimmune diseases, neu odegene a i e diso de s,
and cance . Eme ging e idence abou non-canonical, ex alysosomal ac i i ies a e s a ing
o e eal a mo e in ica e ole o lysosomal p o eases, bo h unde physiological and pa ho-
logical condi ions, hus p o iding new clinical oppo uni ies o in e en ion. Impo an ly,
hese ex alysosomal ac i i ies ha e been la gely o e looked due o he
in i o
dena u a-
ion o hese p o eases a neu al pH and hei implica ion in he ac i a ion o cell dea h.
Howe e , ecen epo s ha e demons a ed ha hese p o eases main ain hei ac i i y
in he nucleus and cy osol o he cells, whe e hey con ol key aspec s o cell physiology
bo h unde physiological and pa hological condi ions. The e o e, in o de o unde s and
he ex en o hei ole bo h unde physiological and pa hological condi ions, a numbe o
ques ions s ill need o be add essed.
In his con ex , he iden i ica ion and cha ac e isa ion o sec e o y lysosomes ha e
allowed us o unde s and how hese p o eases loca e o he ex acellula compa men . In
his ex acellula milieu, lysosomal p o eases a e nowadays known o play key oles in ECM
emodelling, angiogenesis, in asion, me as asis, e c. Howe e , al hough ecen ad ances
a e s a ing o shed ligh on he mechanism ha allows hese p o eases o each he nucleo-
cy osolic compa men o cells, a leas unde speci ic condi ions, u he in es iga ion on
his opic is s ill equi ed. On he o he hand, he iden i ica ion and cha ac e isa ion o
nuclea and cy osolic a ge s o some o hese p o eases, bo h unde physiological and
pa hological condi ions, has s a ed o e eal hei non-canonical, ex alysosomal oles.
Howe e , ecen p o eomics s udies ha e iden i ied no el, po en ial ex alysosomal a ge s
o hese p o eases. The e o e, a comple e cha ac e isa ion o he p o eins a ge ed by hese
p o eases in he nuclea and cy osolic compa men s o he cell and he biological p ocesses
hey egula e will p o ide us wi h a deepe unde s anding o hei oles, bu also wi h
no el a ge s wi h he po en ial o be ansla ed in o he clinical se ing o cance ea men .
In a pa hological con ex , he canonical ole o lysosomes in diseases such as LSDs and
au oimmune diseases is well cha ac e ised, as desc ibed abo e. Howe e , he accumula ing
e idence un eiling a non-canonical, ex alysosomal ac i i y o hese p o eases migh
equi e conside ing he possible con ibu ion o hese new ac i i ies o he onse and
Cells 2025,14, 68 22 o 34
de elopmen o hese diseases. Fo example, abe an p ocessing o su ace ma ke s by
ex acellula lysosomal p o eases can lead o he gene a ion o new epi opes ha could
po en ially igge abno mal immune esponses, u he con ibu ing o he onse and
p og ession o au oimmune diseases.
Impo an ly, unexpec ed ex alysosomal a ge s and unc ions o hese lysosomal
p o eases occu ing in he ex acellula milieu, bu also in he cy osolic and nuclea com-
pa men s, whe e hey pa ake a key ole in he onse and p og ession o cance , a e s a ing
o eme ge. This aises wo exci ing ques ions: A e hese non-canonical, ex alysosomal
unc ions exclusi ely happening unde pa hological condi ions, o a e hey s ill uniden i-
ied, exace ba ed physiological unc ions o hese p o eases ou side he lysosome, esul ing
in he onse and p og ession o hese pa hologies? Which o he p ocesses egula ed by
hese p o eases a e we s ill missing, bo h unde physiological and pa hological condi ions?
Accumula ing e idence suppo s an unan icipa ed ole o lysosomal p o eases in he
cy osol and nucleus o cance cells. In his con ex , hese p o eases, by a ge ing speci ic
cy osolic o nuclea a ge s, con ibu e o he abe an cell cycle o cance cells by a ge ing
key egula o s o he cell cycle. Fu he mo e, lysosomal p o eases, h ough non-canonical,
ex alysosomal unc ions, ha e also been shown o a ge key p o eins in ol ed in he
egula ion o gene exp ession, hus con ibu ing o he al e ed gene exp ession pa e ns
obse ed in cance cells. Mo eo e , key p o eins in ol ed in DNA damage esponse ha e
been cha ac e ised as no el a ge s o hese lysosomal p o eases, hus explaining hei
ole in he inc eased esis ance o cance cells o con en ional chemo- and adio he apy
app oaches aimed a inducing DNA damage. The cha ac e isa ion o hese molecula
mechanisms and he iden i ica ion o no el a ge s and biological p ocesses con olled by
hese p o eases has he po en ial o p o ide new oppo uni ies o he design o inno a i e
clinical he apies in he ea men o cance ocused on dis up ing he ole o hese p o eases
in p omo ing cance onse and p og ession.
Au ho Con ibu ions: R.C.-B.—Concep ualiza ion, W i ing— e iew and edi ing; M.M.-H.—
Concep ualiza ion, W i ing— e iew and edi ing; J.M.-F.—Concep ualiza ion, Funding acquisi ion,
W i ing—o iginal d a , W i ing— e iew and edi ing. All au ho s ha e ead and ag eed o he
published e sion o he manusc ip .
Funding: This wo k was suppo ed by he Conseje ía de T ans o mación Económica, Indus ia,
Conocimien o y Uni e sidades unde he EMERGIA p og amme (EMC21_00124) om he Andalu-
sian Regional Go e nmen . J.M.-F. was also unded by he Eu opean Union’s Ho izon 2020 esea ch
and inno a ion p og amme unde he Ma ie Sklodowska-Cu ie p og amme (H2020-MSCA-IF-2020,
101025429), he Spanish Minis y o Science and Inno a ion unde he Ramon y Cajal p og amme
(RYC2021-032389-I) and he VII PPIT o he Uni e si y o Se ille (2023/00000479) g an ed o J.M.-F.
Da a A ailabili y S a emen : No applicable.
Acknowledgmen s: We hank Reyes Sanles-Falagán o he c i ical eading o he manusc ip .
This wo k was unded by he Conseje ía de T ans o mación Económica, Indus ia, Conocimien o
y Uni e sidades unde he EMERGIA p og amme (EMC21_00124) om he Andalusian Regional
Go e nmen g an ed o J.M.-F. J.M.-F. was suppo ed by he Eu opean Union’s Ho izon 2020 esea ch
and inno a ion p og amme unde he Ma ie Sklodowska-Cu ie p og amme (H2020-MSCA-IF-2020,
101025429), he Spanish Minis y o Science and Inno a ion unde he Ramon y Cajal p og amme
(RYC2021-032389-I) and he VII PPIT o he Uni e si y o Se ille (2023/00000479).
Con lic s o In e es : The au ho s decla e no con lic s o in e es . The unde s had no ole in he design
o he s udy; in he collec ion, analyses, o in e p e a ion o da a; in he w i ing o he manusc ip ; o
in he decision o publish he esul s.
Cells 2025,14, 68 23 o 34
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