CISTEIN PEPTIDASAS

Intermediario

tetraédrico

Intermediario

acil-enzima

Cys25His159

Cys25His159

Sustrato

Cys25His159

Cys25His159

Cys25His159

Cys25His159

ME

CA

NIS

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CA

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LIT

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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.