cistein peptidasas · estructura de la papaina - papain is a single-chain nonglycosylated...
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CISTEIN PEPTIDASAS
Intermediario
tetraédrico
Intermediario
acil-enzima
Cys25His159
Cys25His159
Sustrato
Cys25His159
Cys25His159
Cys25His159
Cys25His159
ME
CA
NIS
MO
CA
TA
LIT
ICO
PAPAINA: PERSPECTIVA
HISTÓRICA
The importance of papaya latex as a source of enzymes appears to have
been first recognized by G. C. Roy in 1873.
The name 'papaine' was first used by Wurtz & Bouchet (1879) who
partially purified the product.
The suggestion that papain might contain more than one proteinase came
from Vines (1905) and later (Vines, 1909) he reported a partial separation
of two proteinases in papain by NaCl precipitation.
In summary, papaya latex contains the following major cysteine
proteinase components:
- papain (papaya peptidase I)
- chymopapain A
- chymopapains B1-B3
- papaya peptidase II
Distinguishing Features:
Overall, papain can be considered to possess a fairly broad specificity.
The enzyme is stable and active under a wide range of conditions: from
pH 4 to 10 and at temperatures up to 80°C (Glazer & Smith, 1971). It also
retains activity in 8 M urea.
Brocklehurst K, Salih E. “A re-evaluation of the nomenclature of the cysteine proteinases of Carica papaya and a rational basis for
their identification”. Biochem J. 1983 Aug 1;213(2):559-60.
ESTRUCTURA DE LA PAPAINA
- Papain is a single-chain nonglycosylated polypeptide of 212 amino acids (23.429 Da) containing 3 disulfide bonds.
- The initial 2.8 Å resolution structure (Drenth et al., 1968) has been refined to 1.65 Å (Kamphuis et al., 1984)
A number of structures are also available for papain complexes with ligands and inhibitors.
- The polypeptide chain is folded to form a globular protein with two interacting domains delimiting a cleft at the
surface of the enzyme where substrates can bind.
L12-112
208-212
R1-11
113-207Asn175
His159
Cys25
Catepsinas (Clan CA, Familia C1)
Cathepsin: derived from the Greek kathepsein (to digest), proposed for
the protease that was active in a slightly acidic environment.
Serine proteases: cathepsins A and G
Aspartic proteases cathepsins D and E
Lysosomal Cysteine cathepsins (11 in humans): cathepsins B, C, F, H, K, L,
O, S, V, X and W.
Lysosomal cathepsins require a reducing, slightly acidic environment,
such as found in the lysosomes, in order to be optimally active. Therefore,
cysteine cathepsins were initially considered as intracellular enzymes,
responsible for the non-specific, bulk proteolysis in the acidic
environment of the endosomal/lysosomal compartment, where they
degrade intracellular and extracellular proteins.
The majority of cathepsins are ubiquitously expressed in human tissues
such as cathepsins B, H, L, C, X, F, O and V. Their expression profile
indicates that these enzymes are involved in a normal cellular protein
degradation and turnover. In contrast, cathepsins K, W and S show a
restricted cell or tissue-specific distribution, indicating their more
specific roles. For example, cathepsin K is highly expressed in osteoclasts.
Among the matrix-degrading enzymes, cathepsin K is the only enzyme for
which an essential role in bone resorption has been unambiguously
documented in mice and humans.
CRUZIPAIN: MAJOR CYSTEINE PROTEINASE OF
TRYPANOSOMA CRUZI .
1. Present (at different levels) in the four major stages of the
parasite.
2. Reagents able to inhibit the enzyme activity in situ block
the differentiation steps in the life cycle of the parasite.
3. Located in the lysosomes (reservosomes in
epimastigotes), with a secondary localization at the cell
surface.
4. Digests proteins at acidic pH values and short blocked
peptides at variable pH values. Broad specificity.
5. High mannose glycoprotein, monomeric, molecular
weight around 40 kDa.
6. Encoded by a high number of genes (approximately
130 in the Tul 2 strain), arrayed in tandems located in 2 to
4 chromosomes. Differentially expressed in the parasite
stages.
7. Microheterogeneous in ion exchange chromatography
(Mono Q), IEF, RP-HPLC and SDS-PAGE in substrate-
containing minigels.
8. Possible causes for the microheterogeneities:
simultaneous expression of several genes; different post-
translational modifications.
COMPARACIÓN DE LAS SECUENCIAS DE CRUZIPAÍNA 1 Y 2.
THE C-TERMINAL DOMAIN OF CRUZIPAIN.
1. Obtained by self-proteolysis of the mature enzyme.
2. Microheterogeneous in SDS-PAGE, IEF and RP- HPLC.
3. Responsible for the high antigenicity of mature cruzipain.
4. Structure: 130 amino acid residues. Among the first 21 there are
7 modified Thr and 7 Pro. In the central part there are 8 Cys,
probably forming 4 disulfide bridges. The C-terminus consists of 27
residues, 18 of which are hydrophilic, 11 from them charged. Only
one Met residue and only one N-glycosilation site, which may be
occupied by high-mannose, hybrid monoantennary or complex
biantennary oligosaccharide in different molecules. There are sialic
acid and sulfate groups linked to some of these oligosaccharides. O-
glycosilation: N-acetyl glucosamine residues.
5. Highly resistant to proteolysis if the disulfide bridges are intact.
PURIFICATION OF THE C-TERMINAL DOMAIN
Al 30 de Mayo de 2017, hay 26 estructuras en la base de datos MEROPS, la
mayoría de ellas en complejo con inhibidores.
FUNCIONES DE LA CRUZIPAINA
1) Proteinasa lisosomal principal del parásito: digestión de
proteínas exógenas.
2) Probable protección del parásito contra la respuesta inmune
del huésped, a través de la degradación de inmunoglobulinas en
la bisagra (mecanismos de “fabulación”).
3) Producción de bradikinina a partir del kininógeno.
Considerada un factor de virulencia del parásito por esta razón.
4) Participación en la penetración del parásito en la célula
huésped.
5) Participación en las etapas de diferenciación en el ciclo
biológico del parásito. Presente en los reservosomas y esencial
para la metaciclogénesis.
EFECTO DE LOS INHIBIDORES-EPIMASTIGOTES
Effects of proteinase inhibitors on the growth and differentiation of Trypanosoma cruzi.
Franke de Cazzulo BM, Martínez J, North MJ, Coombs GH, Cazzulo JJ.
FEMS Microbiol Lett. 1994 Nov 15;124(1):81-6.
EFECTO DE LOS INHIBIDORES SOBRE LA
DIFERENCIACIÓN EPIS-METAS
Effects of proteinase inhibitors on the growth and differentiation of Trypanosoma cruzi.
Franke de Cazzulo BM, Martínez J, North MJ, Coombs GH, Cazzulo JJ.
FEMS Microbiol Lett. 1994 Nov 15;124(1):81-6.
EFECTO DE LOS INHIBIDORES EN LA
INFECCIÓN
tripomastigotes
amastigotes
Effects of proteinase inhibitors on the growth and differentiation of Trypanosoma cruzi.
Franke de Cazzulo BM, Martínez J, North MJ, Coombs GH, Cazzulo JJ.
FEMS Microbiol Lett. 1994 Nov 15;124(1):81-6.
EFECTO DE LOS INHIBIDORES EN LA
INFECCIÓN
Mol Biochem Parasitol. 1993 Mar;58(1):17-24.
“Peptide-fluoromethyl ketones arrest intracellular replication and intercellular transmission of Trypanosoma cruzi”Harth G, Andrews N, Mills AA, Engel JC, Smith R, McKerrow JH.
Inhibidores proteicos de CPs
Stefins (type 1 cystatins)
- single-chain proteins of ~100 amino acid residues. Mr approx. 11,000.
- synthesized without signal peptide, lack carbohydrates, primarily intracellular
proteins but can also be detected in body fluids.
- Stefins do not posses disulphide bonds.
- Stefins belong to the subfamily I25A of the cystatin protein family.
- Present in most major eukaryotic subgroups. Originally, two representatives of
this group, stefins A and B, were found in various mammals, including humans.
In addition, bovine stefin C has been identified as the first Trp-containing stefin
with a prolonged N-terminus.
Cystatins (type 2 cystatins)
- single-chain proteins of ~115 amino acid residues. Mr approx. 13,000.
- Cystatins contain a signal peptide responsible for secretion through the cell
membrane to the extracellular milieu, recognized as extracellular proteins.
- All type 2 cystatins contain two highly conserved intra-molecular disulphide
bridges, with the exception of human cystatin F, which possesses an additional
disulphide bridge, thus stabilizing the N-terminal part of the protein.
- The human type 2 cystatins are grouped into subfamily I25B of the cystatin
family I25.
- Cystatins are more widely distributed proteins than stefins. Currently, seven
members of this type of inhibitor have been identified: cystatin C, salivary
cystatins (cystatins S, SA and SN), cystatin D, cystatin E/M and cystatin F
(leukocystatin).
INHIBIDORES PROTEICOS DE CPS
Kininogens (type 3 cystatins)
- Kininogens: precursor molecules of the kinins, can be released through either
the plasma kallikrein-kinin system or tissue kallikrein. An alternative route
may involve cysteine cathepsins
- Kininogens are multifunctional and multidomain glycoproteins comprising 3
distinct types:
- high-molecular-weight kininogen (HK)
- low-molecular-weight kininogen (LK)
- T-kininogen (TK), an acute phase protein only found in rats.
Both human HK and LK are products of the same gene, resulting from the
alternative mRNA splicing, while TK is encoded by the TK-gene.
- The mature kininogens are single-chain that after cleavage by kallikreins,
release the kinin segment and are converted into two-chain proteins: a heavy-
and a light-chain. The heavy chains of HK and LK have an identical amino acid
sequence, whereas the light-chain of HK is much longer than that of LK.
- The heavy chains of HK and LK are composed of three tandemly repeated type
2 cystatin-like domains (domains 1, 2, 3) containing eight disulphide bridges.
Only the second and third domains inhibit the papain-like proteases. The
inhibitory domains 2 and 3 are more closely related than domain 1. Both HK
and LK bind two molecules of various cysteine proteases including cathepsins
and cruzipain with high affinity.
- Like type 2 cystatins, the kininogens are grouped into subfamily I25B of the
cystatin family I25.
HMW: 88 - 114 kDa LMW: 50 - 66 kDa
D6: Unión a pre-kalikreína y Factor IX D5: His-rich, unión a
endotelios
Kininógenos: Gen único, 27 kpb, 11 exones.
The molecule consists mainly of a straight five turn -helix, a five stranded
antiparallel β-pleated sheet which is twisted and wrapped around this -helix
Estructura de la estefina B
“Trunk”
1st loop 2nd loop
Turk V, Bode W. “The cystatins: protein inhibitors of cysteine proteinases.”FEBS Jul 22;285(2):213-9.
Interacción entre la cistatina de clara de huevo y la
papaína
- Both hairpin loops and the N-
terminus form a hydrophobic wedge
shaped ‘edge’ which is highly
complementary to the active site cleft
of papain.
-The first and the second hairpin loops
have precisely the appropriate shape
and size to fill the more open part of
the active site cleft of papain which
represents the S1’-S2’ subsites.
- The amino-terminal segment of
cystatin (Gly9-Ala10) is directed
towards the substrate subsite S2, but
in an inappropriate conformation and
too far away to be attacked by the
reactive site Cys-25. Due to the rigid
positioning of Ala10 in cystatin away
from Cys25 in papain, a cleavage of the
Gly9-Ala10 bond is impossible.
- The proposed model was recently
verified by the crystallized
stoichiometric complex of human
stefin B and papain.
M T Stubbs, B Laber, W Bode, R Huber, R Jerala, B Lenarcic, and V Turk. “The refined 2.4 A X-ray crystal structure of
recombinant human stefin B in complex with the cysteine proteinase papain: a novel type of proteinase inhibitor interaction.”EMBO J. Jun 1990; 9(6): 1939–1947.
Estructura 3D del complejo Stefina B humana-
Papaína
- The conserved residues form a
tripartite wedge, which penetrates
into the papain active site.
- The main interactions are provided
by the amino-terminal ‘trunk’ and the
first hairpin loop containing the
highly conserved QVVAG region, with
minor contributions coming from the
second hairpin loop.
- The carboxyl-terminus provides an
additional interaction with respect to
chicken cystatin.
PURIFICACIÓN DE LA CISTATINA DE CLARA DE
HUEVO
GRUPO INACTIVADOR DE LA PAPAÍNA
Kd (M)
6 x 10-13
7.1 x 10-13
1.4 x 10-11
2.1 x 10-7
1.5 x 10-6
1.0 x 10-5
Calpainas
(Clan CA, Familia C2)
The first reports on calpain came from two different groups in
the 1960s who noted the presence of a calcium-activated
proteolytic activity in soluble extracts from rat brain and
skeletal muscle.
These activities were caused by an intracellular cysteine
protease not associated with the lysosome and having an
optimum activity at neutral pH, which clearly distinguished it
from the cathepsin family of proteases.
Shortly thereafter, the activity was found to be attributable to
two main isoforms, μ-calpain and m-calpain, that differed
primarily in their calcium requirements in vitro. Their names
reflect the fact that they are activated by micro- and nearly
millimolar concentrations of Ca2+, respectively.
These two isoforms were shown to have distinct (although
similar) large (80 kDa) subunits, but a common small (28 kDa)
subunit.
When the sequence of this enzyme became known, it was given
the name “calpain”, to recognize it as a hybrid of two well-
known proteins at the time, the calcium-regulated signalling
protein, calmodulin, and the cysteine protease of papaya,
papain.
GOLL D E et al. Physiol Rev 2003;83:731-801
©2003 by American Physiological Society
CALPAIN DOMAIN STRUCTURE
80 kDa subunit
The 80-kDa subunits of μ- and m-calpain
share 55–65% sequence homology.
I: no sequence homology, 72–86%
similarity between species.
II: peptidase domain, catalytic triad: IIa-
>Cys, IIb->His and Asn. IIa and IIb each
bind one atom of Ca2+ in a 8-9 aa loop.
85-93% simil among different species.
III: no sequence homology, it may bind
phospholipids. The sequence at the
domain II/III boundary does not have an
EF-hand conformation in the
crystallographic structure of rat or
human m-calpain, and this region does
not seem to bind Ca2+ in m-calpain.
IV: calmodulin-like domain, -helical
folds of 170 aa, contains 4 EF-hands
that bind Ca2+ and 1 EF-hand that is
involved in dimerization contacts
between the two subunits.
28 kDa subunit
V: Gly rich domain, binds phospholipids
VI: calmodulin-like domain, -helical
folds of 170 aa, contains 4 EF-hands
that bind Ca2+ and 1 EF-hand that is
involved in dimerization contacts
between the two subunits.
µ- and m-isoforms have distinct large (80 kDa) subunits, but a common small (28 kDa)
subunit.
En ausencia de Ca2+, la Cys del
sitio activo está a 10.5 Å de His y
Asn; el Ca2+ induciría el cambio
conformacional que los acerca a
3.7Å y forma la tríada catalítica.
El clivage autocatalítico entre los
residuos 19 y 20 del dominio I
debe ser por una reacción
bimolecular, pues esos residuos
están a 40 Å del sitio activo (en su
forma sin Ca2+). Esta autólisis se
produce sólo en presencia de Ca2+,
y reduce la concentración del
catión necesaria para su actividad
proteolítica, de 3 – 50 mM a 0.5 -
2.0 mM para la m-calpaína y de
400 – 800 mM a 50 - 150 mM para
la m-calpaína. El PM de la
subunidad de 80 kDa baja a 76 (m)
o 78 (m) kDa, en tanto que la
subunidad de 28 kDa disminuye a
20.5 kDa.
Estructura de dos moléculas de m-calpaína
Se usó una construcción que
tiene 85 % de secuencia de la
m-calpaína; los primeros 48
residuos y los últimos 62 de
la subunidad mayor son los
de la m-calpaína. Esto se
hizo porque la m-calpaína se
expresa muy mal. Es muy
parecida a la m-calpaína,
pero los residuos del sitio
activo están mucho mas
próximos en ausencia de
Ca2+ , y algunas porciones de
los motivos EF-hand están
desordenados y son mas
flexibles. Esto sugiere que la
m-calpaína libre de Ca2+
podría estar ya parcialmente
activada, y por eso requerir
una menor concentración de
Ca2+ .
Substrate Specificity
The substrate specificity of calpain is relatively restricted, and most
oligopeptides are not hydrolyzed. Casein is the most popular
substrate for in vitro assays, and it is used either as the natural
protein or modified with various chromophores, fluorescent
reagents or isotopes.
The rules governing the specificity of the calpains remain unclear.
It seems that calpain recognizes the overall three-dimensional
structure of its substrates more than the primary structure. Even so,
hydrophobic (Tyr, Met, Leu, Val) and Arg residues tend to be
preferred in position P2 (Mellgren & Murachi, 1990).
Protein kinases, phosphatases, phospholipases, cytoskeletal
proteins, membrane proteins, cytokines, transcription factors, lens
proteins, calmodulin-binding proteins and others have been
suggested to be in vivo substrates, but clear evidence has not yet
been obtained. Calpain proteolyzes these proteins in a limited
manner rather than digesting them to small peptides, suggesting
that it may modulate the functions of the substrate proteins by
cutting their interdomain regions (Saido et al., 1994).
There is no evidence of any difference in substrate specificity
between μ- and m-calpains.
CALPASTATINAS – Productos de un único gen con 34 exones en ratón, pero
el uso de diferentes promotores o el splicing alternativo origina numerosas isoformas.
Son polipéptidos casi completamente desordenados (determinado por CD y NMR).
Los 4 dominios I, II, III y IV son inhibidores; los 12 residuos en el sub-dominio B son
esenciales para la inhibición. Los sub-dominios A y C se unen a los dominios IV y VI de
la calpaína, respectivamente la unión requiere Ca2+. Inhibidores competitivos con un
Kd < 3 nM. Las calpaínas son inhibidas por leupeptina, E-64, antipaína, cistatinas.
Caspasas
(Clan CD, Familia C14)
Las caspasas son una familia de CPs específicas para cortar proteínas
blanco en uniones peptídicas en las que el Asp aporta el carbonilo. Su
nombre viene de C (CPs), Asp (por el sitio de corte) y asas como
enzimas.
La acción concertada de caspasas es responsable de la apoptosis, una
forma de muerte celular programada esencial para el desarrollo
embrionario, entre otros procesos. Además de la apoptosis, un
subgrupo de la familia C14 está involucrado en la inflamación, como
activadoras de pro-citoquinas.
La actividad no regulada de caspasas sería letal, y para prevenirla la
célula las mantiene en un estado latente como zimógenos, cuya
activación se hace por un mecanismo conservado sujeto a una
regulación estricta.
Las caspasas apoptóticas se clasifican en iniciadoras y ejecutoras,
dependiendo de su punto de entrada en la cascada apoptótica. Las
iniciadoras son las primeras en ser activadas en una vía particular de
muerte celular, y constituyen el primer paso en una mínima cascada de
dos pasos por activar a las caspasas ejecutoras.
Una vez activadas por un estímulo específico, las caspasas ejecutoras
llevan a cabo una proteolisis de ciertos sustratos, lo que gatilla una
cascada de eventos que culmina en una muerte celular ordenada, que,
a diferencia de la necrosis, no causa inflamación.
(A) Caspase organization: a prodomain precedes the catalytic domain,
composed of two covalently linked subunits. Sites for (auto)proteolysis at
Asp residues are indicated. (B) activation mechanisms. Initiators are
monomers that activate by prodomain-mediated dimerization.
Executioners are dimers that activate by cleavage of intersubunit
linkers. Following activation, additional proteolytic events mature the
caspases to more stable forms, prone to regulation.
Caspasa 3 (ejecutora) Caspasa 8 (iniciadora)
En ambos casos después de su activación (proteólisis en 3,
dimerización, que la activa, y proteólisis ulterior en 8)
Intrinsic and extrinsic apoptotic pathways:
The intrinsic pathway
En respuesta a diversos stresses
celulares, como daño al DNA, o stress del
retículo endoplásmico, la vía intrínseca
se transduce por miembros de la familia
Bcl-2. Bcl-2 homology 3 (BH3)-only
activa directamente a otros dos
miembros de la familia Bcl-2, BAX y BAC,
que se homo-oligomerizarían para formar
poros en la membrana externa de la
mitocondria. Esto permitiría que
proteínas pro-apoptóticas, como el
citocromo c y el segundo activador
mitocondrial de caspasas (Smac o
DIABLO), salgan del espacio
intermembrana al citosol. El citocromo c
activará otra proteína adaptadora,
llamada apoptotic protease-activating
factor-I (Apaf-I), que llevará a la
formación del apoptosoma.
a) Apaf-1 functional units: CARD, N-terminal caspase-recruitment domain which is responsible for recruiting caspase-9; NOD or
NB-ARC: a nucleotide-binding and oligomerization domain responsible for (d)ATP-dependent oligomerization, and WD40 region,
responsible for binding of cytochrome c. b) Apoptosome formation: In the absence of an apoptotic signal, Apaf-1 exists in a locked
autoinhibited form (WD40 region binds back to the remainder of the protein). Following an apoptotic signal, the binding of
cytochrome c to the WD40 region releases the lock leading to a semi-open, still autoinhibited, form of Apaf-1. This rearrangement is
concurrent with the hydrolysis of the bound nucleotide to (d)ADP. Neither oligomerization nor caspase-9 binding to the CARD
domain of Apaf-1 is possible in this conformation. (d)ATP nucleotide exchange leads to oligomerization and apoptosome formation.
The apoptosome adopts a wheel-like structure with the cytochrome-c-bound WD40 regions sticking out like spokes, whereas the NB-
ARC regions and CARDs form the central part of the wheel.
CASPASE-9 ACTIVATION: THE APOPTOSOME
EXTRINSIC APOPTOTIC PATHWAY: CASPASE-8
ACTIVATION: DISC (Death-Induced Signaling Complex)
-La vía extrínseca es responsable de la
eliminación de células no deseadas durante
el desarrollo, la educación del sistema
inmune y la eliminación mediada por el
sistema inmune de células de tumores.
-Se inicia por la unión de su ligando a un
receptor de muerte trans-membrana de la
superfamilia del receptor de factor de
necrosis type I; un miembro particular de
esta familia, Fas (también conocido como
CD95 or APO-1), se ha transformado en el
paradigma para el estudio de la vía
extrínseca.
- Al unirse al ligando, el receptor Fas forma
microagregados en la superficie celular,
permitiendo que la molécula adaptadora
FADD (Fas-associated protein with death
domain) sea reclutada por su cola a través
de un mecanismo de varios pasos.
-El FADD recluta los zimógenos de la
caspasa-8 a través de la interacción entre
sus N-terminal death effector domains
(DEDs).
- Es dentro de este death-inducing
signaling complex (DISC) que la caspasa-8
iniciadora se activa.
La proteólisis de la quinasa MST1 de mamífero resulta
en la translocación de un fragmento catalíticamente
activo de esta quinasa al núcleo, donde fosforila a la
histona H2B y provoca la condensación de la cromatina.
-El clivaje de ICAD (inhibitor of caspase-activated
DNase) libera CAD (caspase-activated DNase), la cual
cataliza el clivaje inter-nucleosomal del DNA.
- El clivaje mediado por caspasas de la lamin nuclear
debilita la lamina permitiendo la fragmentación nuclear,
y proteínas de la membrana nuclear son también
proteolizadas.
- La proteolisis de proteínas en sitios focales de adhesión
y sitios de adhesión célula-célula permite la liberación y
retracción celular.
-La actividad de caspasas lleva a la exposición de
fosfatidilserine (PS) y otras señales fagocíticas en la
superficie celular.
- La proteólisis del efector Rho ROCK1 lleva a la
contracción del citoesqueleto de actina y causa blebbing
de la membrana plasmática y fragmentación nuclear.
-Las caspasas también clivan proteínas del Golgi
causando la fragmentación del aparato de Golgi.
-Finalmente, funciones celulares importantes como la
traducción son afectadas a través de la proteólisis
mediada por caspasas de múltiples factores de iniciación
de la traduccción (eIFs).
Las caspasas efectoras desmantelan diversas estructuras celulares a través del clivaje de
sustratos específicos. Colectivamente, estos eventos proteolíticos producen los cambios fenotípicos
de la célula que son característicos de la apoptosis.