RESUMEN
INTRODUCTION: B-domain modification can improve production of recombinant factor VIII (rFVIII) proteins. However, the engineered junction results in non-native peptide sequences with the potential to elicit immune responses via major histocompatibility complex class-II (MHC-II)-binding and CD4+ T cell activation. AIM: Assess the immunogenic potential of B-domain junction peptides of turoctocog alfa and other B-domain-modified rFVIII proteins using in silico and in vitro immunogenicity assessment techniques. METHODS: Peptides with amino acid sequences identical to the B-domain junction of turoctocog alfa, simoctocog alfa and moroctocog alfa were evaluated by in silico peptide-MHC-II binding prediction, in vitro peptide-MHC-II-binding measurement and in vitro T cell-activation assays. Moreover, turoctocog alfa was assessed for peptide presentation on dendritic cells (DCs) using MHC-associated peptide proteomics. RESULTS: In silico analysis predicted virtually no neo-epitopes in the B-domain junction for turoctocog alfa, whereas some were predicted for simoctocog alfa and moroctocog alfa. Turoctocog alfa and moroctocog alfa peptides showed minimal capacity to bind high-frequency MHC-II molecules in vitro, whereas simoctocog alfa peptide demonstrated some degree of binding to approximately half of the MHC-II molecules tested. In line with this, no B-domain peptides from turoctocog alfa were found to be presented on MHC-II complexes on DCs. B-domain junction peptides from all 3 compounds induced T cell responses in only a few percentages of donors. CONCLUSION: All 3 junction peptides were found to have a low immunogenicity potential, suggesting that modification of the B-domain does not constitute an increased immunogenicity risk for any of the products examined.
Asunto(s)
Simulación por Computador , Factor VIII/metabolismo , Hemofilia A/genética , Antígenos de Histocompatibilidad Clase II/metabolismo , Animales , Modelos Animales de Enfermedad , Hemofilia A/metabolismo , Humanos , RatonesRESUMEN
Cofilins are actin binding proteins and regulate actin assembly in vivo. Numerous cofilin homologues have been characterized in various organisms including mammals. In mice, a ubiquitously expressed cofilin (CFL1) and a skeletal muscle specific cofilin (CFL2) have been described. In the present study, we identified and characterized a human CFL2 gene localized on chromosome 14, with high homology to murine CFL2. Furthermore, we provide evidence for differentially spliced CFL2 transcripts (CFL2a and CFL2b). CFL2b is expressed predominantly in human skeletal muscle and heart, while CFL2a is expressed in various tissues. Genetic defects of CFL2 were excluded for one human muscle disorder, the chromosome 14 linked distal myopathy MPD1, and shown to be only possible to be a rare cause of another, nemaline myopathy. In a mouse model of mechanically induced muscle damage the changes of cofilin expression were monitored during the first 10 days of regeneration, with dephosphorylated CFL2 being the major isoform at later stages of muscle regeneration. A similar predominance of dephosphorylated CFL2 was observed in chronically regenerating dystrophin-deficient muscles of Duchenne muscular dystrophy patients. Therefore, the CFL2 isoform may play an important role in normal muscle function and muscle regeneration.
Asunto(s)
Proteínas de Microfilamentos/metabolismo , Músculo Esquelético/metabolismo , Regeneración , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , Mapeo Cromosómico , Cromosomas Humanos Par 14 , Cofilina 2 , Cartilla de ADN , Humanos , Células Híbridas/efectos de la radiación , Sueros Inmunes , Ratones , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/genética , Datos de Secuencia Molecular , Músculo Esquelético/citología , Músculo Esquelético/fisiología , ARN Mensajero/genéticaRESUMEN
MHC class I molecules assemble with peptides in the endoplasmic reticulum (ER). To ensure that only peptide-loaded MHC molecules leave the ER, empty molecules are retained by ER-resident chaperones, most notably the MHC-specific tapasin. ER exit of class I MHC is also controlled by viruses, but for the opposite purpose of preventing peptide presentation to T cells. Interestingly, some viral proteins are able to retain MHC class I molecules in the ER despite being transported. By contrast, other viral proteins exit the ER only upon binding to class I MHC, thereby rerouting newly synthesized class I molecules to intracellular sites of proteolysis. Thus, immune escape can be achieved by reversing, inhibiting or redirecting the chaperone-assisted MHC class I folding, assembly and intracellular transport.
Asunto(s)
Retículo Endoplásmico/metabolismo , Antígenos de Histocompatibilidad Clase I/química , Chaperonas Moleculares , Péptidos/metabolismo , Proteínas Precoces de Adenovirus/metabolismo , Animales , Antiportadores/metabolismo , Proteínas Portadoras/metabolismo , Glicoproteínas/metabolismo , Aparato de Golgi/metabolismo , Humanos , Proteínas Inmediatas-Precoces/metabolismo , Inmunoglobulinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana , Proteínas de Transporte de Membrana , Modelos Biológicos , Transporte de Proteínas , Proteínas de Unión al ARN/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Proteínas Virales/metabolismo , Virus/metabolismoRESUMEN
Human cytomegalovirus (HCMV) interferes with major histocompatibility complex (MHC) class I antigen presentation by a sequential multistep process to escape T cell surveillance. During the immediate early phase of infection, the glycoprotein US3 prevents intracellular transport of MHC class I molecules. Interestingly, US3 displays a significantly shorter half-life than US3-retained MHC class I molecules. Here we show that US3 associates only transiently with MHC class I molecules, exits the ER, and is inefficiently retrieved from the Golgi. US3 was degraded in a post-Golgi compartment, most likely lysosomes, because: i) Brefeldin A treatment prolonged the half-life of US3; and ii) US3 co-localized with the lysosomal marker protein LAMP in chloroquine-treated cells. In contrast, MHC class I molecules remained stable in the ER. Upon inhibition of protein synthesis MHC class I molecules were released suggesting that a continuous supply of newly synthesized US3 molecules is required for inhibition of transport. Thus, US3 does not seem to retain MHC class I molecules by a retrieval mechanism. Instead, our observations are consistent with US3 preventing MHC class I trafficking by blocking forward transport.
Asunto(s)
Antígenos de Histocompatibilidad Clase I/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Antibacterianos/farmacología , Presentación de Antígeno , Antimaláricos/farmacología , Brefeldino A/farmacología , Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Línea Celular , Separación Celular , Cloroquina/farmacología , Citoplasma/metabolismo , Citosol/metabolismo , Retículo Endoplásmico/metabolismo , Epítopos , Citometría de Flujo , Proteínas Ligadas a GPI , Glicoproteínas , Aparato de Golgi/metabolismo , Células HeLa , Hexosaminidasas/metabolismo , Humanos , Lisosomas/metabolismo , Macrólidos , Proteínas de la Membrana , Microscopía Confocal , Pruebas de Precipitina , Transporte de Proteínas , Puromicina/farmacología , Factores de Tiempo , TransfecciónRESUMEN
Peptide fragments from proteins of intracellular pathogens such as viruses are displayed at the cell surface by MHC class I molecules thus enabling surveillance by cytotoxic T cells. Peptides are produced in the cytosol by proteasomal degradation and translocated into the endoplasmic reticulum by the peptide transporter TAP. Empty MHC class I molecules associate with TAP prior to their acquisition of peptides, a process which is assisted and controlled by a series of chaperones. The first part of this review summarizes our current knowledge of this assembly pathway and describes recent observations that tapasin functions as an endoplasmic reticulum retention molecule for empty MHC class I molecules. To defeat the presentation of virus-derived peptides, several DNA viruses have devised strategies to interfere with MHC class I assembly. Although these evasion strategies have evolved independently and differ mechanistically they often target the same step in this pathway. We compare escape mechanisms of different viruses with particular emphasis on the retention of newly synthesized MHC class I molecules in the endoplasmic reticulum and the inhibition of peptide transport by viral proteins.
Asunto(s)
Presentación de Antígeno/inmunología , Antiportadores/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Inmunoglobulinas/inmunología , Virus/inmunología , Animales , Retículo Endoplásmico/inmunología , Humanos , Proteínas de Transporte de Membrana , Péptidos/inmunología , Proteínas Virales/inmunologíaRESUMEN
The requirement of mitochondrial hsp70 (mt-hsp70) for the import of a series of preproteins containing hydrophobic sorting signals into isolated yeast mitochondria was investigated. Here we demonstrate that the presence of such a sorting signal in proximity to the N-terminal matrix-targeting sequence of a preprotein can secure a translocating polypeptide chain in the import channel in a manner that does not require mt-hsp70 activity. Trapping the translocating chain in this fashion leads to efficient processing by the mitochondrial processing peptidase and to complete translocation across the outer mitochondrial membrane into the intermembrane space. These mt-hsp70-independent effects appear to be exerted at the level of the inner membrane through an interaction of the hydrophobic core of the sorting signal with component(s) of the translocase of the inner membrane. Hydrophobic sorting signals of inner membrane proteins inserted into the membrane from the matrix, as well as those of intermembrane space proteins, are capable of causing this mt-hsp70-independent stabilization, demonstrating that this phenomenon is not unique to those preproteins normally sorted to the intermembrane space.
Asunto(s)
Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Transporte de Membrana , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial , Precursores de Proteínas/metabolismo , Señales de Clasificación de Proteína/metabolismo , Proteínas de Saccharomyces cerevisiae , Adenosina Trifosfato/metabolismo , Transporte Biológico , Proteínas Portadoras/metabolismo , División Celular/efectos de los fármacos , Proteínas Fúngicas/metabolismo , Proteínas de la Membrana/metabolismo , Metaloendopeptidasas/metabolismo , Metotrexato/farmacología , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Modelos Moleculares , Saccharomyces cerevisiae , Tetrahidrofolato Deshidrogenasa/metabolismo , Peptidasa de Procesamiento MitocondrialRESUMEN
Human cytomegalovirus (HCMV) inhibits MHC class I antigen presentation by a sequential multistep process involving a family of unique short (US) region-encoded glycoproteins. US3 retains class I molecules, whereas US2 and US11 mediate the cytosolic degradation of heavy chains by the proteosomes. In US6-transfected cells, however, intracellular transport of class I molecules is impaired because of defective peptide translocation by transporters associated with antigen processing (TAP). Peptide transport is restored in HCMV mutants lacking US6. In contrast to the cytosolic herpes simplex virus protein ICP47, US6 interacts with TAP inside the endoplasmic reticulum lumen, as shown by US6 derivatives lacking the transmembrane and cytoplasmic domains and by the observation that US6 does not prevent peptides from binding to TAP. Thus, HCMV targets TAP for immune escape by a molecular mechanism different from that of herpes simplex virus.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/farmacología , Citomegalovirus/química , Retículo Endoplásmico/fisiología , Péptidos/metabolismo , Proteínas de Unión al ARN/farmacología , Proteínas del Envoltorio Viral/farmacología , Proteínas Virales/farmacología , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Secuencia de Aminoácidos , Unión Competitiva , Transporte Biológico/inmunología , Regulación hacia Abajo/inmunología , Retículo Endoplásmico/química , Retículo Endoplásmico/virología , Células HeLa , Antígenos de Histocompatibilidad Clase I/biosíntesis , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Datos de Secuencia Molecular , Péptidos/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Precytochrome b2 is targeted to the mitochondrial intermembrane space by a dual targeting sequence comprising 80 amino acids. A kinetic analysis of intramitochondrial sorting was performed. The intermediate-size form accumulated transiently in the matrix. When import was performed in the presence of metal chelators to prevent the first processing by the matrix processing peptidase, > 40% of the imported precursor was localized in the matrix. A deletion of 13 amino acids in the intermembrane space sorting sequence caused partial inhibition of the first processing, and a transient accumulation of the precursor form in the matrix was also observed. The decrease in this matrix-localized precursor form paralleled an increase in the mature-size form in the intermembrane space. A point mutation in the mitochondrial targeting sequence (N-terminal to the sorting sequence) resulted in missorting to the matrix space. Furthermore, a chimeric protein consisting of the initial 85 residues of cytochrome b2 fused to dihydrofolate reductase was partially targeted to the matrix at 15 degrees C, but not at 25 degrees C. Together, the results presented here indicate that cytochrome b2 passes through the matrix on its sorting pathway to the intermembrane space.
Asunto(s)
L-Lactato Deshidrogenasa/metabolismo , Mitocondrias/enzimología , Secuencia de Aminoácidos , Precursores Enzimáticos/metabolismo , Cinética , L-Lactato Deshidrogenasa/química , L-Lactato Deshidrogenasa (Citocromo) , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/metabolismo , Temperatura , Tetrahidrofolato Deshidrogenasa/metabolismoRESUMEN
Cytochrome b2 is sorted into the intermembrane space of mitochondria by a bipartite N-terminal targeting and sorting presequence. In an attempt to define the sorting pathway we have identified an as yet unknown import intermediate. Cytochrome b2-dihydrofolate reductase (DHFR) fusion proteins were arrested in the presence of methotrexate (MTX) so that the DHFR domain was at the surface of the outer membrane while the N-terminus reached into the intermembrane space where the sorting signal was removed. This membrane-spanning, mature-sized species was efficiently chased into the mitochondria upon removal of MTX. Thus, an intermediate was generated which was exposed to the intermembrane space but was still associated with the inner membrane. This intermediate was also found upon direct import of cytochrome b2 and derived fusion proteins. These membrane-bound mature-sized cytochrome b2 species loop through the matrix and could be recovered in a complex with mt-Hsp70 and the inner membrane MIM44/ISP45, a component of the inner membrane import apparatus. This novel sorting intermediate can only be explained by a pathway in which cytochrome b2 passes through the matrix. The existence of such an intermediate is inconsistent with a pathway by which entrance of the mature part of cytochrome b2 into the matrix is stopped by the sorting sequence; however, its presence is fully consistent with the conservative sorting pathway.
Asunto(s)
L-Lactato Deshidrogenasa/metabolismo , Mitocondrias/metabolismo , Procesamiento Proteico-Postraduccional , Saccharomyces cerevisiae/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/metabolismo , Cinética , L-Lactato Deshidrogenasa/biosíntesis , L-Lactato Deshidrogenasa/química , L-Lactato Deshidrogenasa (Citocromo) , Metotrexato/farmacología , Modelos Estructurales , Conformación Proteica , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Tetrahidrofolato Deshidrogenasa/biosíntesis , Tetrahidrofolato Deshidrogenasa/metabolismoRESUMEN
The role of ATP in the matrix for the import of precursor proteins into the various mitochondrial subcompartments was investigated by studying protein translocation at experimentally defined ATP levels. Proteins targeted to the matrix were neither imported or processed when matrix ATP was depleted. Import and processing of precytochrome b2 (pb2), a precursor carrying a bipartite presequence, into the intermembrane space was also strongly dependent on matrix ATP. Preproteins, consisting of 220 or more residues of pb2 fused to dihydrofolate reductase, showed the same requirement for matrix ATP, whereas the import of shorter fusion proteins (up to 167 residues of pb2) was largely independent of matrix ATP. For those intermembrane-space-targeted proteins that did need matrix ATP, the dependence could be relieved either by unfolding these proteins prior to import or by introducing a deletion into the mature portion of the protein thereby impairing the tight folding of the cytochrome b2 domain. These results suggest the following: (a) The import of matrix-targeted preproteins, in addition to a membrane potential delta psi, requires matrix ATP [most likely to facilitate reversible binding of mitochondrial heat-shock protein 70 (mt-Hsp70) to incoming precursors], for two steps, securing the presequence on the matrix side of the inner membrane and for the completion of translocation; (b) in the case of intermembrane-space-targeted precursors with bipartite signals, the function of ATP/mt-Hsp70 is not obligatory, as components of the intermembrane-space-sorting pathway may substitute for ATP/mt-Hsp70 function (however, if a tightly folded domain is present in the precursor, ATP/mt-Hsp70 is indispensable); (c) unfolding on the mitochondrial surface of tightly folded segments of preproteins is facilitated by matrix-ATP/mt-Hsp70.
Asunto(s)
Adenosina Trifosfato/metabolismo , Mitocondrias/metabolismo , Precursores de Proteínas/metabolismo , Transporte Biológico Activo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Choque Térmico/metabolismo , Membranas Intracelulares/metabolismo , L-Lactato Deshidrogenasa/metabolismo , L-Lactato Deshidrogenasa (Citocromo) , Neurospora crassa/genética , Neurospora crassa/metabolismo , Pliegue de Proteína , Precursores de Proteínas/química , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/metabolismo , Tetrahidrofolato Deshidrogenasa/metabolismoRESUMEN
We have cloned the yeast PRE2 gene by complementation of pre2 mutants, which are defective in the chymotrypsin-like activity of the 20 S proteasome (multicatalytic-multifunctional proteinase complex). The PRE2 gene, a beta-type member of the proteasomal gene family, is essential for life and codes for a 287-amino acid proteasomal subunit with a predicted molecular mass of 31.6 kDa. Missense mutations in two pre2 mutant alleles were identified. They led to enhanced sensitivity of yeast cells against stress. At the same time, pre2 mutants accumulated ubiquitinated proteins. The Pre2 protein shows striking homology to the human Ring10 protein (60% identity excluding the 70 amino-terminal residues), which is encoded in the major histocompatibility complex class II region. It represents a component of the low molecular mass polypeptide complex, previously shown to be a special type of the 20 S proteasome. The low molecular mass polypeptide complex is assumed to be involved in antigen presentation, generating peptides from cytosolic protein antigens, which are subsequently presented to cytotoxic T-lymphocytes on the cell surface. The high homology of Pre2 to Ring10 implies the hypothesis that Ring10 is a subunit of the low molecular mass polypeptide complex central in its chymotryptic activity. One might further suggest that replacement of constitutive proteasomal components by functionally related major histocompatibility complex-linked low molecular mass polypeptides, as is Ring10, adapts mammalian proteasomes for functions in the immune response.
Asunto(s)
Quimotripsina/metabolismo , Cisteína Endopeptidasas/genética , Proteínas Fúngicas/genética , Complejo Mayor de Histocompatibilidad , Complejos Multienzimáticos/genética , Proteínas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Ubiquitinas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , ADN de Hongos , Proteínas Fúngicas/metabolismo , Humanos , Datos de Secuencia Molecular , Complejo de la Endopetidasa Proteasomal , Saccharomyces cerevisiae/enzimología , Homología de Secuencia de AminoácidoRESUMEN
Proteinase yscE, the yeast proteasome, is a member of the nonlysosomal, high molecular mass (approximately 700 kDa) multifunctional proteinase complexes that are highly conserved from yeast to man. We have isolated mutants defective in one of the three proteolytic activities of the enzyme complex, i.e. in cleavage of peptide bonds after acidic amino acids. Using one of these mutants (pre4-1), we cloned the PRE4 gene and uncovered an open reading frame with 266 amino acids coding for a predicted protein of 29.4 kDa. The protein proved to be a subunit of proteinase yscE. The Pre4 amino acid sequence shows strong homology to the beta-subunit of the Xenopus laevis proteasome. Chromosomal deletion of the PRE4 gene is lethal. The pre4-1 mutant allele was cloned and sequenced. The mutant protein is shortened by 15 amino acids at the carboxyl terminus. Mutations (pre1-1, pre2-2) in the chymotrypsin-like activity of proteinase yscE uncovered the enzyme to be involved in ubiquitin-linked and stress-dependent proteolytic pathways. In contrast to these mutants, pre4-1 mutants did not exhibit any apparent stress-dependent phenotypes. However, pre1-1 pre4-1 double mutants showed enhanced canavanine sensitivity and increased accumulation of ubiquitin protein conjugates, as compared with pre1-1 single mutants.