RESUMEN

In general, apoptotic stimuli lead to activation of caspases. Once activated, a caspase can induce intracellular signaling pathways involving proteolytic activation of other caspase family members. We report the in vitro processing of eight murine procaspases by their enzymatically active counterparts. Caspase-8 processed all procaspases examined. Caspase-1 and -11 processed the effector caspases procaspase-3 and -7, and to a lesser extent procaspase-6. However, vice versa, none of the caspase-1-like procaspases was activated by the effector caspases. This suggests that the caspase-1 subfamily members either act upstream of the apoptosis effector caspases or else are part of a totally separate activation pathway. Procaspase-2 was maturated by caspase-8 and -3, and to a lesser extent by caspase-7, while the active caspase-2 did not process any of the procaspases examined, except its own precursor. Hence, caspase-2 might not be able to initiate a wide proteolytic signaling cascade. Additionally, cleavage data reveal not only proteolytic amplification between caspase-3 and -8, caspase-6 and -3, and caspase-6 and -7, but also positive feedback loops involving multiple activated caspases. Our results suggest the existence of a hierarchic proteolytic procaspase activation network, which would lead to a dramatic increase in multiple caspase activities once key caspases are activated. The proteolytic procaspase activation network might allow that different apoptotic stimuli result in specific cleavage of substrates responsible for typical processes at the cell membrane, the cytosol, the organelles, and the nucleus, which characterize a cell dying by apoptosis.

Asunto(s)

Caspasas/metabolismo , Animales , Caspasas/genética , Caspasas/inmunología , Caspasas/aislamiento & purificación , Activación Enzimática , Escherichia coli , Expresión Génica , Ratones , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo

RESUMEN

Cleavage of structural proteins by caspases has been associated with the severe morphological changes occurring during the apoptotic process. One of the proteins regulating the connection of the actin filament with cadherins in a cell-cell adhesion complex is beta-catenin. During apoptosis, both an N-terminal and a small C-terminal part are removed from beta-catenin. Removal of the N-terminal part may result in a disconnection of the actin filament from a cadherin cell-cell adhesion complex. We demonstrate that caspase-8, -3 and -6 directly proteolyse beta-catenin in vitro. However, the beta-catenin cleavage products generated by caspase-8 were different from those generated by caspase-3 or caspase-6. Caspase-1, -2, -4/11 and -7 did not or only very inefficiently cleave beta-catenin. These data suggest that activation of procaspase-3, -6 or -8 by different stimuli in the cell might result in a differential proteolysis of beta-catenin.

Asunto(s)

Caspasas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Transactivadores , Animales , Cadherinas/metabolismo , Caspasa 3 , Caspasa 6 , Caspasa 8 , Caspasa 9 , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/aislamiento & purificación , Humanos , Hidrólisis , Ratones , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Biosíntesis de Proteínas , Transcripción Genética , beta Catenina

RESUMEN

Mutations in the presenilin (PS) genes PSI and PS2 are involved in Alzheimer's disease (AD). Recently, apoptosis-associated cleavage of PS proteins was identified. Here we demonstrate that PS1 as well as PS2 are substrates for different members of the caspase protein family. Remarkably, the caspases acting on PS1 could be subdivided in two groups. One group, containing caspase-8, -6 and -11, cleaved PSI after residues ENDD329 and to a lesser extent after residues AQRD341. A second group consisting of caspase-3, -7 and -1 acted uniquely on AQRD341. Importantly, these two cleavage sites were also recognized by caspases in the C-terminal PS1 fragment produced by constitutive proteolysis. In decreasing order of activity, caspase-8, -3, -1, -6 and -7 proteolysed PS2 at the recognition site D326SYD329. Caspase-8 and -3 exhibited the highest proteolytic activity on both PS1 and PS2. PS1 and PS2 were not hydrolyzed by caspase-2 and PS2 also not by caspase-11. None of five missense mutations affected the sensitivity of PSI to caspase-mediated cleavage. This suggests that AD pathogenesis associated with PS1 missense mutations cannot be explained by a change in caspase-dependent processing.

Asunto(s)

Caspasas/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Sitios de Unión , Humanos , Proteínas de la Membrana/genética , Presenilina-1 , Presenilina-2 , Conejos

RESUMEN

Several caspases are mediators of apoptotic cell death. We describe a novel murine member of this growing protein family. Based on homology and especially on the substrate specificity, this new procaspase is identified as the murine counterpart of human procaspase-8. The protein exhibits a rather low similarity (76%) and identity (70%) to human procaspase-8. Procaspase-8 mRNA is expressed in all adult mouse tissues examined, the highest levels being reached in kidney, liver and lung. Procaspase-8 mRNA expression is highest in seven-day old embryos, but also during later stages of development the expression was fairly high. Both human and murine procaspase-8 are very weak substrates for granzyme B as compared to procaspase-3. Murine procaspases-1, 2, 3, 6, 7, 8, 11/4 and 12 are processed by recombinant murine caspase-8, suggesting a key role in the procaspase activation cascade. In addition, murine caspase-8 induced cell death that was inhibited both by cytokine response modifier A and p35. In vitro experiments demonstrated that p35 inhibits caspase-8 directly.

Asunto(s)

Caspasas/genética , Secuencia de Aminoácidos , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Apoptosis/fisiología , Secuencia de Bases , Caspasa 8 , Caspasa 9 , Inhibidores de Caspasas , Caspasas/metabolismo , Línea Celular , Clonación Molecular , Cartilla de ADN/genética , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Granzimas , Humanos , Técnicas In Vitro , Proteínas Inhibidoras de la Apoptosis , Ratones , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/metabolismo , Serpinas/genética , Serpinas/farmacología , Especificidad por Sustrato , Distribución Tisular , Transfección , Proteínas Virales/genética , Proteínas Virales/farmacología

RESUMEN

Caspases are cysteinyl aspartate-specific proteinases, many of which play a central role in apoptosis. Here, we report the identification of a new murine caspase homologue, viz. caspase-14. It is most related to human/murine caspase-2 and human caspase-9, possesses all the typical amino acid residues of the caspases involved in catalysis, including the QACRG box, and contains no or only a very short prodomain. Murine caspase-14 shows 83% similarity to human caspase-14. Human caspase-14 is assigned to chromosome 19p13.1. Northern blot analysis revealed that mRNA expression of caspase-14 is undetectable in all mouse adult tissues examined except for skin, while it is abundantly expressed in mouse embryos. In contrast to many other caspase family members, murine caspase-14 is not cleaved by granzyme B, caspase-1, caspase-2, caspase-3, caspase-6, caspase-7 or caspase-11, but is weakly processed into p18 and p11 subunits by murine caspase-8. No aspartase activity of murine caspase-14 could be generated by bacterial or yeast expression. Transient overexpression of murine caspase-14 in mammalian cells did not elicit cell death and did not interfere with caspase-8-induced apoptosis. In conclusion, caspase-14 is a member of the caspase family but no proteolytic or biological activities have been identified so far. The high constitutive expression levels in embryos and specific expression in adult skin suggest a role in ontogenesis and skin physiology.

Asunto(s)

Caspasas/genética , Secuencia de Aminoácidos , Animales , Caspasa 1/química , Caspasa 14 , Caspasas/biosíntesis , Caspasas/química , Línea Celular , Clonación Molecular , Células HeLa , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transcripción Genética , Transfección

RESUMEN

The aspartase granzyme B is one of the major components of the granules involved in cell killing by cytotoxic T lymphocytes. Granzyme B has been shown to activate the apoptotic death pathway in the target cell, and this involves activation of members of the caspase (CASP) protein family. Therefore, activational cleavage of mouse (m) CASP proforms by granzyme B was examined in vitro. CASP can be subdivided in the CASP-1 (interleukin-1 beta-converting enzyme; ICE) subfamily, the CASP-2 (Ich1) subfamily, and the CASP-3 (CPP32) subfamily. Our results reveal that the proforms of the CASP-3 subfamily members mCASP-3 and mCASP-7 are hydrolyzed by granzyme B, while proforms of CASP-2 and CASP-1 subfamily members are not directly cleaved. Only one CASP-3 subfamily member, pro-mCASP-6, was not proteolytically cleaved by granzyme B. These results indicate that two members of the CASP-3 subfamily, but no others, become activated by granzyme B.

Asunto(s)

Caspasas , Serina Endopeptidasas/metabolismo , Animales , Caspasa 1 , Caspasa 2 , Caspasa 3 , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Granzimas , Hidrólisis , Ratones , Biosíntesis de Proteínas , Proteínas/genética , Proteínas/metabolismo , Transcripción Genética

RESUMEN

Seven members of the murine caspase (mCASP) family were cloned and functionally characterized by transient overexpression: mCASP-1 (mICE), mCASP-2 (Ich1), mCASP-3 (CPP32), mCASP-6 (Mch2), mCASP-7 (Mch3), mCASP-11 (TX) and mCASP-12. mCASP-11 is presumably the murine homolog of human CASP-4. Although mCASP-12 is related to human CASP-5 (ICErel-III), it is most probably a new CASP-1 family member. On the basis of sequence homology, the caspases can be divided into three subfamilies: first, mCASP-1, mCASP-11 and mCASP-12; second, mCASP-2; third, mCASP-3, mCASP-6 and mCASP-7. The tissue distribution of the CASP-1 subfamily transcripts is more restricted than that of the CASP-3 subfamily transcripts, suggesting that the transcriptional regulation of the CASP members within one subfamily is related, but is quite different between the CASP-1 and the CASP-3 subfamilies. Transient overexpression of each of the seven CASPs induced apoptosis in mammalian cells. Only two, mCASP-1 as well as mCASP-3, were able to process precursor interleukin (IL)-1beta to biologically active IL-1beta. In addition, mCASP-3 is the predominant PARP-cleaving enzyme in vivo.

Asunto(s)

Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Secuencia de Aminoácidos , Animales , Apoptosis/genética , Caspasa 1 , Clonación Molecular , Humanos , Interleucina-1/genética , Interleucina-1/metabolismo , Ratones , Datos de Secuencia Molecular , Poli(ADP-Ribosa) Polimerasas/metabolismo , Biosíntesis de Proteínas , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Distribución Tisular , Transcripción Genética

RESUMEN

We describe a novel phage display system that affords the surface expression and hence affinity selection of cDNAs. The strategy is based on a new approach to functionally display proteins on filamentous phage through the attachment to the C-terminus of the minor coat protein VI. The utility of the method was evaluated using a cDNA library derived from the parasite Ancylostoma caninum. cDNA sequences were fused in each of the three reading frames to the 3'-end of the M13 gene VI expressed by a phagemid vector. Phages rescued from this cDNA expression library were subjected to biopanning against two serine proteases, trypsin and the human coagulation factor Xa. This led to the identification of cDNAs encoding novel members of two different families of serine protease inhibitors. The authenticity of the cDNA selected with trypsin as the target was demonstrated by purifying the encoded potent Kunitz-type inhibitor from an Ancylostoma caninum extract. The rapid isolation of specific cDNAs with the protein VI monovalent display system should facilitate the search for novel biologically important ligands.

Asunto(s)

Cápside/genética , ADN Complementario/genética , Secuencia de Aminoácidos , Ancylostoma/enzimología , Ancylostoma/genética , Animales , Secuencia de Bases , Biotinilación , Western Blotting , Factor Xa/metabolismo , Biblioteca de Genes , Vectores Genéticos , Humanos , Datos de Secuencia Molecular , Proteínas de Plantas/genética , Serina Endopeptidasas/genética , Inhibidores de Serina Proteinasa/genética , Tripsina/metabolismo , Inhibidores de Tripsina , alfa-Amilasas/antagonistas & inhibidores

RESUMEN

Site-specific substitutions of arginine for lysine in the thermostable D-xylose isomerase (XI) from Actinoplanes missouriensis are shown to impart significant heat stability enhancement in the presence of sugar substrates most probably by interfering with nonenzymatic glycation. The same substitutions are also found to increase heat stability in the absence of any sugar derivatives, where a mechanism based on prevention of glycation can no longer be invoked. This rather conservative substitution is moreover shown to improve thermostability in two other structurally unrelated proteins, human copper, zinc-superoxide dismutase (CuZnSOD) and D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus subtilis. The stabilizing effect of Lys----Arg substitutions is rationalized on the basis of a detailed analysis of the crystal structures of wild-type XI and of engineered variants with Lys----Arg substitution at four distinct locations, residues 253, 309, 319, and 323. Molecular model building analysis of the structures of wild-type and mutant CuZnSOD (K9R) and GAPDH (G281K and G281R) is used to explain the observed stability enhancement in these proteins. In addition to demonstrating that even thermostable proteins can lend themselves to further stability improvement, our findings provide direct evidence that arginine residues are important stabilizing elements in proteins. Moreover, the stabilizing role of electrostatic interactions, particularly between subunits in oligomeric proteins, is documented.

Asunto(s)

Isomerasas Aldosa-Cetosa , Arginina/química , Estabilidad de Enzimas , Arginina/genética , Carbohidrato Epimerasas/química , Carbohidrato Epimerasas/genética , Clonación Molecular , Activación Enzimática , Gliceraldehído-3-Fosfato Deshidrogenasas/química , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Glicosilación , Calor , Humanos , Lisina/química , Lisina/genética , Mutagénesis Sitio-Dirigida , Conformación Proteica , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Superóxido Dismutasa/química , Superóxido Dismutasa/genética , Difracción de Rayos X