RESUMEN
The cystine/glutamate antiporter system xc- has been identified as the major source of extracellular glutamate in several brain regions as well as a modulator of neuroinflammation, and genetic deletion of its specific subunit xCT (xCT-/-) is protective in mouse models for age-related neurological disorders. However, the previously observed oxidative shift in the plasma cystine/cysteine ratio of adult xCT-/- mice led to the hypothesis that system xc- deletion would negatively affect life- and healthspan. Still, till now the role of system xc- in physiological aging remains unexplored. We therefore studied the effect of xCT deletion on the aging process of mice, with a particular focus on the immune system, hippocampal function, and cognitive aging. We observed that male xCT-/- mice have an extended lifespan, despite an even more increased plasma cystine/cysteine ratio in aged compared to adult mice. This oxidative shift does not negatively impact the general health status of the mice. On the contrary, the age-related priming of the innate immune system, that manifested as increased LPS-induced cytokine levels and hypothermia in xCT+/+ mice, was attenuated in xCT-/- mice. While this was associated with only a very moderate shift towards a more anti-inflammatory state of the aged hippocampus, we observed changes in the hippocampal metabolome that were associated with a preserved hippocampal function and the retention of hippocampus-dependent memory in male aged xCT-/- mice. Targeting system xc- is thus not only a promising strategy to prevent cognitive decline, but also to promote healthy aging.
Asunto(s)
Sistema de Transporte de Aminoácidos y+ , Cistina , Sistema de Transporte de Aminoácidos y+/genética , Sistema de Transporte de Aminoácidos y+/metabolismo , Animales , Cisteína , Cistina/metabolismo , Ácido Glutámico , Hipocampo/metabolismo , Longevidad , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
We recently described two proteasome subtypes that are intermediate between the standard proteasome and the immunoproteasome. They contain only one (ß5i) or two (ß1i and ß5i) of the three inducible catalytic subunits of the immunoproteasome. They are present in tumor cells and abundant in normal human tissues. We described two tumor antigenic peptides that are uniquely produced by these intermediate proteasomes. In this work, we studied the production by intermediate proteasomes of tumor antigenic peptides known to be produced exclusively by the immunoproteasome (MAGE-A3(114-122), MAGE-C2(42-50), MAGE-C2(336-344)) or the standard proteasome (Melan-A(26-35), tyrosinase(369-377), gp100(209-217)). We observed that intermediate proteasomes efficiently produced the former peptides, but not the latter. Two peptides from the first group were equally produced by both intermediate proteasomes, whereas MAGE-C2(336-344) was only produced by intermediate proteasome ß1i-ß5i. Those results explain the recognition of tumor cells devoid of immunoproteasome by CTL recognizing peptides not produced by the standard proteasome. We also describe a third antigenic peptide that is produced exclusively by an intermediate proteasome: peptide MAGE-C2(191-200) is produced only by intermediate proteasome ß1i-ß5i. Analyzing in vitro digests, we observed that the lack of production by a given proteasome usually results from destruction of the antigenic peptide by internal cleavage. Interestingly, we observed that the immunoproteasome and the intermediate proteasomes fail to cleave between hydrophobic residues, despite a higher chymotrypsin-like activity measured on fluorogenic substrates. Altogether, our results indicate that the repertoire of peptides produced by intermediate proteasomes largely matches the repertoire produced by the immunoproteasome, but also contains additional peptides.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Antígeno MART-1/metabolismo , Monofenol Monooxigenasa/metabolismo , Proteínas de Neoplasias/metabolismo , Fragmentos de Péptidos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Procesamiento Proteico-Postraduccional/inmunología , Antígeno gp100 del Melanoma/metabolismo , Secuencia de Aminoácidos , Presentación de Antígeno/inmunología , Antígenos de Neoplasias/biosíntesis , Antígenos de Neoplasias/genética , Línea Celular Tumoral , Células Clonales , Epítopos de Linfocito T/biosíntesis , Epítopos de Linfocito T/metabolismo , Células HEK293 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Antígeno MART-1/biosíntesis , Melanoma/enzimología , Melanoma/genética , Melanoma/inmunología , Monofenol Monooxigenasa/biosíntesis , Proteínas de Neoplasias/biosíntesis , Fragmentos de Péptidos/biosíntesis , Complejo de la Endopetidasa Proteasomal/genética , Antígeno gp100 del Melanoma/biosíntesisRESUMEN
Most antigenic peptides presented by MHC class I molecules result from the degradation of intracellular proteins by the proteasome. In lymphoid tissues and cells exposed to IFNγ, the standard proteasome is replaced by the immunoproteasome, in which all of the standard catalytic subunits ß1, ß2, and ß5 are replaced by their inducible counterparts ß1i, ß2i, and ß5i, which have different cleavage specificities. The immunoproteasome thereby shapes the repertoire of antigenic peptides. The existence of additional forms of proteasomes bearing a mixed assortment of standard and inducible catalytic subunits has been suggested. Using a new set of unique subunit-specific antibodies, we have now isolated, quantified, and characterized human proteasomes that are intermediate between the standard proteasome and the immunoproteasome. They contain only one (ß5i) or two (ß1i and ß5i) of the three inducible catalytic subunits of the immunoproteasome. These intermediate proteasomes represent between one-third and one-half of the proteasome content of human liver, colon, small intestine, and kidney. They are also present in human tumor cells and dendritic cells. We identified two tumor antigens of clinical interest that are processed exclusively either by intermediate proteasomes ß5i (MAGE-A3(271-279)) or by intermediate proteasomes ß1i-ß5i (MAGE-A10(254-262)). The existence of these intermediate proteasomes broadens the repertoire of antigens presented to CD8 T cells and implies that the antigens presented by a given cell depend on their proteasome content.
Asunto(s)
Presentación de Antígeno , Antígenos de Histocompatibilidad Clase I/metabolismo , Complejo de la Endopetidasa Proteasomal/clasificación , Complejo de la Endopetidasa Proteasomal/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Línea Celular Tumoral , Humanos , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/genética , Subunidades de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Tissue inflammation is accompanied by the cytokine-mediated replacement of constitutive proteasomes by immunoproteasomes that finally leads to an optimized generation of MHC class I restricted epitopes for Ag presentation. The brain is considered an immunoprivileged organ, where both the special anatomy as well as active tolerance mechanisms repress the development of inflammatory responses and help to prevent immunopathological damage. We analyzed the immunoproteasome expression in the brain after an infection with lymphocytic choriomeningitis virus (LCMV) and could show that LCMV-infection of mice leads to the transcriptional induction of inducible proteasome subunits in the brain. However, compared with other organs, i.p. and even intracranial infection with LCMV only led to a faint expression of mature immunoproteasome in the brain and resulted in the accumulation of immunoproteasomal precursors. By immunohistology, we could identify microglia-like cells as the main producers of immunoproteasome, whereas in astrocytes immunoproteasome expression was almost exclusively restricted to nuclei. Neither the immunoproteasome subunits low molecular mass polypeptide 2 nor multicatalytic endopeptidase complex-like-1 were detected in neurons or oligodendrocytes. In vitro studies of IFN-γ-stimulated primary astrocytes suggested that the observed accumulation of immunoproteasomal precursor complexes takes place in this cell population. Functionally, the lack of immunoproteasomes protracted and lowered the severity of LCMV-induced meningitis in LMP7(-/-) mice suggesting a contribution of immunoproteasomes in microglia to exacerbate immunopathological damage. We postulate a posttranslationally regulated mechanism that prevents abundant and inappropriate immunoproteasome assembly in the brain and may contribute to the protection of poorly regenerating cells of the CNS from immunopathological destruction.
Asunto(s)
Encéfalo/inmunología , Cisteína Endopeptidasas/biosíntesis , Coriomeningitis Linfocítica/inmunología , Complejo de la Endopetidasa Proteasomal/biosíntesis , Complejo de la Endopetidasa Proteasomal/inmunología , Animales , Astrocitos/inmunología , Astrocitos/metabolismo , Western Blotting , Encéfalo/metabolismo , Cisteína Endopeptidasas/inmunología , Electroforesis en Gel de Poliacrilamida , Inmunohistoquímica , Coriomeningitis Linfocítica/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/inmunología , Microglía/metabolismo , Neuronas/inmunología , Neuronas/metabolismo , Oligodendroglía/inmunología , Oligodendroglía/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The proteasome is responsible for selective degradation of proteins. It exists in mammalian cells under four main subtypes, which differ by the combination of their catalytic subunits: the standard proteasome (ß1-ß2-ß5), the immunoproteasome (ß1i-ß2i-ß5i) and the two intermediate proteasomes (ß1-ß2-ß5i and ß1i-ß2-ß5i). The efficiency of the four proteasome subtypes to degrade ubiquitinated or oxidized proteins remains unclear. Using cells expressing exclusively one proteasome subtype, we observed that ubiquitinated p21 and c--myc were degraded at similar rates, indicating that the four 26S proteasomes degrade ubiquitinated proteins equally well. Under oxidative stress, we observed a partial dissociation of 26S into 20S proteasomes, which can degrade non-ubiquitinated oxidized proteins. Oxidized calmodulin and hemoglobin were best degraded in vitro by the three ß5i-containing 20S proteasomes, while their native forms were not degraded. Circular dichroism analyses indicated that ubiquitin-independent recognition of oxidized proteins by 20S proteasomes was triggered by the disruption of their structure. Accordingly, ß5i-containing 20S proteasomes degraded unoxidized naturally disordered protein tau, while 26S proteasomes did not. Our results suggest that the three ß5i-containing 20S proteasomes, namely the immunoproteasome and the two intermediate proteasomes, might help cells to eliminate proteins containing disordered domains, including those induced by oxidative stress.
Asunto(s)
Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Ubiquitinación , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Células HEK293 , Humanos , Oxidación-Reducción , Proteínas Proto-Oncogénicas c-myc/metabolismoRESUMEN
The immunoproteasome (IP) is usually viewed as favoring the production of antigenic peptides presented by MHC class I molecules, mainly because of its higher cleavage activity after hydrophobic residues, referred to as the chymotrypsin-like activity. However, some peptides have been found to be better produced by the standard proteasome. The mechanism of this differential processing has not been described. By studying the processing of three tumor antigenic peptides of clinical interest, we demonstrate that their differential processing mainly results from differences in the efficiency of internal cleavages by the two proteasome types. Peptide gp100(209-217) (ITDQVPSFV) and peptide tyrosinase369-377 (YMDGTMSQV) are destroyed by the IP, which cleaves after an internal hydrophobic residue. Conversely, peptide MAGE-C2(336-344) (ALKDVEERV) is destroyed by the standard proteasome by internal cleavage after an acidic residue, in line with its higher postacidic activity. These results indicate that the IP may destroy some antigenic peptides due to its higher chymotrypsin-like activity, rather than favor their production. They also suggest that the sets of peptides produced by the two proteasome types differ more than expected. Considering that mature dendritic cells mainly contain IPs, our results have implications for the design of immunotherapy strategies.