Littérature scientifique sur le sujet « Granzima K »

Abstract Granzymes are highly specific proteases stored in secretory granule in T-cells and NK-cells. Five different granzymes are known in human (A, B, H, K and M). A and B are the best studied and both can induce apotosis when delivered to target cells. The role for granzyme H, K and M are principally unknown. We have studied the expression of granzymes in subsets of T-cells and NK-cells both in fresh circulating cells and cultured cells stimulated with IL-2, IL-15 and OKT3/antiCD28. Protein expression (biosynthesis/immunoprecipitation) and mRNA level (RT-PCR) was studied in parallel. This report focus on Granzyme A and K which are close relatives, both tryptases located 70 kb apart on chromosome 5. Granzyme A have the highest expression in NK-cells were granzyme K are barely detected. Granzyme K on the other hand have a particular high expression in CD8+ T cells. Stimulation with IL-2 or IL-15 give a strong upregulation of both enzymes in NK-cells. Stimulation of CD8+ T cells gives mainly upregulation of granzyme A. Fresh CD4+ T cells show very weak synthesis of granzymes but treatment with IL-2 or IL-15 give a clear upregulation of both granzyme A and K. T-cells (both CD4+ and CD8+) are highly responsive to stimulation with OKT3/anti CD28 producing granzyme B but not A and K. This rather discordant pattern of expression for granzyme A and K talks in favour of different modes of regulation and most likely different functions. A striking difference between granzyme A and K concern the propensity for granzyme A to be secreted into the culture medium after stimulation while this is not the case for granzyme K. The secreted form of granzyme A has a molecular weight slightly higher than the intracellular form indicating the presence of a proform. Secretion of granzymes to the extracellular medium is probably not only an in vitro phenomena since both granzyme A and B can be detected in serum from healthy individuals in picomolar concentrations. Moreover, significant elevation of granzyme levels in serum can bee seen in different diseases with activation of cellular immunity and in T- and NK-cells malignancies. We have previously reported that proforms of certain serine proteases including the five human granzymes have a downregulating activity on myeloid proliferation, a mechanism probably implicated in certain forms of neutropenia. We propose that secreted granzymes (especially A, B and H) constitute a part of T regulatory function working both locally at the site of inflammation and distantly via the circulation.

Abstract Objective. Testosterone depletion induces increased germ cell apoptosis in testes. However, limited studies exist on genes that regulate the germ cell apoptosis. Granzymes (GZM) are serine proteases that induce apoptosis in various tissues. Multiple granzymes, including GZMA, GZMB and GZMN, are present in testes. Th us, we investigated which granzyme may be testosterone responsive and possibly may have a role in germ cell apoptosis aft er testosterone depletion. Methods. Ethylene dimethane sulfonate (EDS), a toxicant that selectively ablates the Leydig cells, was injected into rats to withdraw the testosterone. The testosterone depletion effects after 7 days post-EDS were verified by replacing the testosterone exogenously into EDS-treated rats. Serum or testicular testosterone was measured by radioimmunoassay. Using qPCR, mRNAs of granzyme variants in testes were quantified. The germ cell apoptosis was identified by TUNEL assay and the localization of GZMK was by immunohistochemistry. Results. EDS treatment eliminated the Leydig cells and depleted serum and testicular testosterone. At 7 days post-EDS, testis weights were reduced 18% with increased germ cell apoptosis plus elevation GZMK expression. GZMK was not associated with TUNEL-positive cells, but was localized to stripped cytoplasm of spermatids. In addition, apoptotic round spermatids were observed in the caput epididymis. Conclusions. GZMK expression in testes is testosterone dependent. GZMK is located adjacent to germ cells in seminiferous tubules and the presence of apoptotic round spermatids in the epididymis suggest its role in the degradation of microtubules in ectoplasmic specializations. Thus, overexpression of GZMK may indirectly regulate germ cell apoptosis by premature release of round spermatids from seminiferous tubule lumen.

La sclerosi multipla (SM) è una malattia degenerativa autoimmune del Sistema Nervoso Centrale (SNC), in cui cellule T CD4 autoreattive attaccano la mielina dei neuroni causando danni al SNC. La SM è anche associata all’infezione del virus Epstein-Barr (EBV), ma il ruolo dei virus nella progressione della SM è dibattuto. Le risposte di cellule T autoreattive e anti-virali sono controllate da popolazioni diverse di cellule T regolatorie, quali le Treg FOXP3+ e le cellule T regolatorie di tipo 1 (Tr1). Entrambe le polazioni sono coinvolte nella SM, ma il ruolo delle cellule Tr1 nella SM in vivo è poco chiaro. L'eomesodermina (Eomes), un fattore di trascrizione che definisce il lineage delle cellule Tr1 e controlla l'espressione del granzima (Gzm)K, ci permette la loro identificazione ex vivo. La soppressione delle risposte immunitarie è mediata da queste cellule dopo attivazione antigenica tantochè la loro specificità antigenica ne è un fattore chiave. Terapie cellulari con cellule regolatorie sono attualmente già in uso clinico per altre malattie immunomediate ma per la SM è ancora necessario determinare quale subset cellulare sia in grado di sopprimere la risposta immunitaria. Lo scopo di questa tesi è capire il ruolo delle cellule Tr1 nella SM, in particolare, analizzare le loro capacità migratorie verso SNC e la loro specificità per antigeni autologhi o virali, al fine di identificare una popolazione adatta per una eventuale terapia cellulare. Pertanto, in questo progetto ho monitorato una coorte di pazienti MS recidivanti-remittenti trattati o meno con Natalizumab ‒ anticorpo che blocca l’ingresso dei linfoci T nel SNC ‒ mediante tecniche di citofluorimetrica multidimensionale. Ho mostrato che nel liquor di pazienti MS attivi le cellule Tr1 GzmK+ Eomes+ sono fortemente e selettivamente arricchite suggerendo un loro ruolo nelle ricadute. Inoltre, le Tr1 risultano ridotte nel sangue dei pazienti MS e con un’alta proliferanzione in vivo, suggerendo che le Tr1 siano reclutate e attivate nel SNC di questi pazienti. Coerentemente, nei pazienti SM trattati con Natalizumab, le Tr1 hanno frequenze e tassi di proliferazione normali. Al contrario, i pazienti SM hanno un numero maggiore di Treg che però sono meno proliferanti in vivo, mentre le CTL restano inalterate. Nei pazienti MS le cellule Tr1 rispondono fortemente e selettivamente all'antigene EBNA-1 associato alla latenza di EBV mentre la risposta è debole negli individui sani. Non si rileva invece alcuna risposta agli antigeni della mielina o del John Cunningham Virus. Inoltre i pazienti trattati con Natalizumab hanno livelli significativamente più elevati di Tr1 specifiche per EBV/EBNA1, suggerendo che le Tr1 EBV-specifiche siano reclutate e/o generate da precursori nel SNC. Le Tr1 mostrano maggiori proprietà antinfiammatorie nei pazienti SM secernendo livelli più alti di IL-10 in risposta a stimolazione policlonale. Infine abbiamo rilevato che nel liquor le Tr1 producono notevoli quantità di IL-10, come avviene anche nel sangue periferico di pazienti SM in risposta a EBV/ EBNA1 . Nel complesso, i risultati supportano l'ipotesi di una risposta immunitaria anti-EBV alterata nel SNC dei pazienti SM, e suggeriscono un doppio ruolo per le cellule Tr1 Eomes: esse possono avere un ruolo benefico nelle ricadute in quanto sono presenti nel SNC e producono la citochina anti-infiammatoria IL-10 ma allo stesso tempo, la specificità per EBV nella fase latente potrebbe essere alla base di una risposta inefficiente al virus e quindi della progressione della MS. Ulteriori indagini sulle cellule Tr1 sono necessarie per comprendere le loro capacità soppressive, i geni coinvolti e il loro ruolo nella SM progressiva.
Multiple sclerosis (MS) is a degenerative autoimmune disease of the Central Nervous System (CNS), where autoreactive CD4+T-cells are believed to attack the myelin sheath of neurons causing CNS damage. MS is also associated with viral infections, in particular with Epstein–Barr Virus (EBV), but the role of viruses in MS progression is debated. Auto-reactive and overshooting anti-viral T-cell responses are controlled by regulatory T-cell subsets, namely FOXP3+Treg and IL-10-producing type 1 regulatory cells (Tr1) cells. Both subsets were proposed to be involved in MS, but the role of Tr1 cells in vivo in MS remains unclear. Eomesodermin (Eomes), a putative lineage-defining transcription factor of Tr1 cells that controls directly the expression of Granzyme (Gzm)K, allows their analysis ex vivo. Notably, in order to suppress immune responses efficiently, regulatory T-cells have to be activated by antigens, and their antigen specificity is a key feature. Cell-therapy with regulatory T-cells was established in other immune-mediated diseases, but the subset that efficiently suppresses pathogenic T-cells in MS needs first to be identified. The aim of this thesis is to understand the role of Tr1-cells in MS, in particular, to analyze their CNS-homing capacities and their specificity for self- or viral-antigens, in order to identify subsets that are suited for MS cell-therapy. Therefore, in this project I monitored a cohort of relapsing-remitting MS patient that were either untreated or treated with Natalizumab ‒ the anti-α4 integrin antibody that block the CNS-homing of lymphocytes ‒ by multidimensional cytometric analysis. I found that GzmK+Tr1 cells ‒ and not FOXP3+Treg or GzmB+CTL (cytotoxic lymphocytes) ‒ are strongly and selectively enriched in the cerebrospinal fluid (CSF) of active MS patients, suggesting a role in relapses. Moreover, Tr1 cells were reduced in the blood of MS patients and were highly proliferating in vivo, suggesting that Tr1 cells are recruited and activated in the CNS of MS patients. Consistently, Natalizumab-treated MS patients showed normal Tr1 frequencies and proliferation rates. Conversely, MS patients had strikingly higher frequencies of Tregs and a reduced in vivo turnover, while CTL were unaltered. To assess ex vivo the antigen specificity, a new assay was successfully established. Tr1 and their putative precursors cells responded strongly and selectively to the EBV latency-associated antigen EBNA1 in MS patients, and not with lytic ones, but responded only weakly in healthy individuals. They also failed to respond to myelin antigens or to the John Cunningham Virus. Interestingly, Natalizumab-treated patients had significantly higher levels of EBV-specific Tr1 cells, suggesting that these cells are recruited to and/or generated from precursors in the CNS. Tr1 cells have enhanced anti-inflammatory properties in MS patients, secreting higher levels of IL-10 in response to polyclonal stimulation. Moreover, we have preliminary evidences that Tr1 cells produce also considerable amounts of IL-10 in the CSF and even in response to EBV/EBNA1 in the blood of MS patients. Overall, our results are consistent with the notion that there is a dysregulated immune response against EBV in the CNS of MS patients, and suggest a dual role for Eomes+Tr1 cells regulating EBV-specific and not myelin-reactive T-cells. A key finding for this project is that Tr1 cells may have a beneficial role in relapses since they are present in the CNS and produce the anti-inflammatory cytokine IL-10. But at the same time, the specificity for EBV in the latent phase could be at the basis of the inefficient response to the virus and therefore of MS progression. In the future a better understanding of Tr1 cell role in MS could lead to novel therapeutic approaches, although further investigations on Tr1 cells are needed to understand their suppressive abilities, the genes involved and their role in progressive MS.

Granzymes are a family of serine proteases that were once thought to function exclusively as mediators of cytotoxic lymphocyte-induced target cell death. However, non-lethal roles for granzymes, including Granzyme K (GzK), have been recently proposed. As recent studies have observed elevated levels of GzK in plasma of patients diagnosed with sepsis, we hypothesized that extracellular GzK induces a pro-inflammatory response in endothelial cells. In the present study, extracellular GzK proteolytically activated Protease Activated Receptor-1 (PAR-1) leading to increased IL-6 and MCP-1 production in Human Umbilical Venous Endothelial Cells (HUVEC). Enhanced expression of ICAM-1 along with an increased capacity for adherence of THP-1 cells was also observed. Characterization of downstream pathways implicated the MAPK p38 pathway for ICAM-1 expression, and both the p38 and the ERK1/2 pathways in cytokine production. GzK also increased TNFα–induced inflammatory adhesion molecule expression. Furthermore, the physiological inhibitor of GzK, IαIp, significantly inhibited GzK activity in vitro. In summary, extracellular GzK is not cytotoxic but promotes a pro-inflammatory response in endothelial cells.
Medicine, Faculty of
Pathology and Laboratory Medicine, Department of
Graduate

Estructura Tridimensional de Granzima B humana
Granzima B es la proteina prototípica de la familia de serina proteasas que se encuentran en células NK y CTLs. GzmB induce apoptosis mediante activación de las caspasas, y está implicada en la etiología de la artritis reumatoidea. Hemos cristalizado y resuelto la estructura 3D de la GzmB humana a una resolución de 3.1Å. GzmB muestra un plegamiento similar al de catepsina G y quimasa humanas. GzmB exhibe una especificidad de substrato muy inusual ya que corta tras Asp, debido a la presencia de Arg226 en bolsillo de especificidad S1. El sitio de unión del substrato está diseñado para acomodar y cortar hexapéptidos, i.e. IETD_SG, secuencia presente en el lugar de activación de la caspasa-3 y de Bid, sus substratos fisiológicos. Esta estructura ayudará en el diseño de inhibidores que podrian ser usados en la cura de enfermedades inflamatorias crónicas.
Granzyme B y el Receptor Manosa-6-Fosfato Independiente de Cationes
GzmB cristalizó como dímero, mediante la interdigitación de las cadenas de azúcares unidas a Asn65 en los dos monómeros. GzmB es captada por las células mediante el Receptor Manosa-6-Fosfato Independiente de Cationes. Sugerimos que la GzmB dimérica sea la forma internalizada por las células diana. Hemos modelado cómo posiblemente GzmB se une a su receptor celular.
Estructura Tridimensional de pro-Granzyme K humana
Hemos determinado la estructura 3D de la pGzmK a una resolución de 2.2Å. pGzmK se parece más a una serina proteasa activa, a pesar de ser un zimógeno. Esta proteina carece de triada zimogénica y utiliza un nuevo mecanismo de estabilización de la forma inactiva.
Estructura Tridimensional de una Procarboxipeptidasa de Helicoverpa armigera. H. armigera es un insecto cuya plaga afecta a un gran número de países. Hemos determinado la estructura 3D de una nueva carboxipeptidasa (PCPAHa) de larvas de H. armigera, la primera resuelta de un insecto hasta la fecha. El zimógeno de PCPAHa muestra una estructura similar a las ya resueltas de mamífero. Su sitio de activación presenta el motivo (Ala)5Lys. Es curioso apreciar similar motivo ((Ala)6Lys) cerca del extremo N-terminal del enzima activo. Ser255 agranda el bolsillo S1´de especificidad y influencia las preferencias de substrato de ésta enzima. Hemos modelado el extremo C-terminal del LCI dentro del sitio activo de PCPAHa.
Crystal Structure of Human Granzyme B
Granzyme B is the prototypic member of the granzymes, trypsin-like serine proteinases localized in activated NK cells and CTLs. GzmB triggers apoptosis by activating the caspases, and is implicated in the etiology of rheumatoid arthritis. Human GzmB has been crystallized and its structure has been determined to 3.1 Å resolution. GzmB overall fold is similar to that found in cathepsin G and human chymase. GzmB exhibits an unusual substrate specificity as it cleaves after Asp residues due to the presence of Arg226 at the back of the S1-specificity pocket. GzmB substrate binding site is designed to fit and cleave hexapeptides, i.e. IETD_SG, sequence present in the activation site of caspase-3 and Bid, physiological substrates of GzmB. This structure would help in the design of inhibitors for a treatment of chronic inflammatory disorders.
Granzyme B and the Cation-Independent Mannose-6-Phosphate Receptor
Our crystal structure of GzmB unexpectedly revealed a dimer, mediated by the interdigitation of the sugar chains attached to Asn65 in the two monomers. The uptake of GzmB is effected by the cation-independent mannose-6-phosphate (M6P) receptor. We suggest that the GzmB dimer would be the form preferentially recognized by its receptor. To investigate the probable binding mode of GzmB to its cell receptor we have modeled the binding of the GzmB dimer to the M6P-receptor.
Crystal Structure of Human Pro-Granzyme K
We have determined the crystal structure of human pGzmK at 2.2 resolution. The overall fold of pGzmK is most similar to that found in active serine proteinases rather than in zymogens. An unusual feature of pGzmK is that the residues Ser32, His40 and Asp194 do not form a zymogen triad, while pGzmK uses a novel mechanism for zymogen stabilization.
Crystal Structure of a Procarboxypeptidase from Helicoverpa armigera
H. armigera is one of the most serious insect pests worldwide. We present the 2.5 Å crystal structure from this novel procarboxypeptidase (PCPAHa) from H. armigera larvae, the first one reported for an insect. PCPAHa zymogen has a 3D structure similar to the corresponding mammalian digestive carboxypeptidases. The activation site contains the motif (Ala)5Lys. It is noteworthy the occurrence of the same (Ala)6Lys near the C-terminus of the active enzyme. Ser255 enlarges the S1' specificity pocket and influences the substrate preferences of the enzyme. The C-terminal tail of LCI was modeled into PCPAHa active site.