RESUMEN
Vaccines work largely by generating long-lived plasma cells (LLPCs), but knowledge of how such cells are recruited is sparse. Although it is clear that LLPCs preferentially originate in germinal centers (GCs) and relocate to survival niches in bone marrow where they can persist for decades, the issues of the timing of LLPC recruitment and the basis of their retention remain uncertain. Here, using a genetic timestamping system in mice, we show that persistent PCs accrue in bone marrow at an approximately constant rate of one cell per hour over a period spanning several weeks after a single immunization with a model antigen. Affinity-based selection was evident in persisting PCs, reflecting a relative and dynamic rather than absolute affinity threshold as evidenced by the changing pattern of VH gene somatic mutations conveying increased affinity for antigen. We conclude that the life span of persistent, antigen-specific PCs is in part intrinsic, preprogrammed, and varied and that their final number is related to the duration of the response in a predictable way. This implies that modulating vaccines to extend the duration of the GC reaction will enhance antibody-mediated protective immunity.
Asunto(s)
Médula Ósea , Células Plasmáticas , Animales , Ratones , Centro Germinal , Anticuerpos , InmunidadRESUMEN
Over a decade ago Polymerase δ interacting protein of 38 kDa (PDIP38) was proposed to play a role in DNA repair. Since this time, both the physiological function and subcellular location of PDIP38 has remained ambiguous and our present understanding of PDIP38 function has been hampered by a lack of detailed biochemical and structural studies. Here we show, that human PDIP38 is directed to the mitochondrion in a membrane potential dependent manner, where it resides in the matrix compartment, together with its partner protein CLPX. Our structural analysis revealed that PDIP38 is composed of two conserved domains separated by an α/ß linker region. The N-terminal (YccV-like) domain of PDIP38 forms an SH3-like ß-barrel, which interacts specifically with CLPX, via the adaptor docking loop within the N-terminal Zinc binding domain of CLPX. In contrast, the C-terminal (DUF525) domain forms an immunoglobin-like ß-sandwich fold, which contains a highly conserved putative substrate binding pocket. Importantly, PDIP38 modulates the substrate specificity of CLPX and protects CLPX from LONM-mediated degradation, which stabilises the cellular levels of CLPX. Collectively, our findings shed new light on the mechanism and function of mitochondrial PDIP38, demonstrating that PDIP38 is a bona fide adaptor protein for the mitochondrial protease, CLPXP.
Asunto(s)
Endopeptidasa Clp/metabolismo , Mitocondrias/metabolismo , Proteínas Nucleares/metabolismo , Endopeptidasa Clp/genética , Regulación de la Expresión Génica , Células HeLa , Humanos , Proteínas Nucleares/genética , Proteínas RecombinantesRESUMEN
The transcription factor interferon regulatory factor 4 (IRF4) is critical for the development, maintenance, and function of plasma cells. The mechanism by which IRF4 exerts its action in mature plasma cells has been elusive due to the death of all such cells upon IRF4 loss. While we identify apoptosis as a critical pathway for the death of plasma cells caused by IRF4 loss, we also determine that IRF4 did not regulate the intrinsic apoptotic pathway directly. By using an inducible IRF4 deletion system in the presence of the overexpression of anti-apoptotic BCL2, we identify genes whose expression is coordinated by IRF4 and that in turn specify plasma cell identity and mitochondrial homeostasis.
Asunto(s)
Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Células Plasmáticas/fisiología , Animales , Línea Celular Tumoral , Homeostasis , Humanos , Ratones , Mieloma Múltiple/genética , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Células Plasmáticas/metabolismo , Células Plasmáticas/patología , Plasmacitoma/genética , Plasmacitoma/metabolismo , Plasmacitoma/patología , Transcripción GenéticaRESUMEN
A B cell culture system using BAFF, IL-4 and IL-21 was recently developed that generates B cells with phenotypic and functional characteristics of in vivo-generated germinal center (GC) B cells. Here, we observe discrete influences of each exogenous signal on the expansion and differentiation of a CD40L-activated B cell pool. IL-4 was expressly necessary, but neither BAFF nor IL-21 was required for B cell acquisition of the GC B cell phenotypes of peanut agglutinin binding and loss of CD38 and IgD expression. Both IL-4 and IL-21 enhanced cell cycle entry upon initial activation dose-dependently, and did so additively. Importantly, while both cytokines acted in concert to increase overall BCL6 expression amounts, IL-21 exposure uniquely caused a small proportion of cells to attain a higher level of BCL6 expression, reminiscent of in vivo GC B cells. In contrast, BAFF supported survival of a fraction of memory-like B cells in extended cultures after removal of surrogate T cell-help signals. Thus, by separably programming proliferation, survival and GC phenotype acquisition, IL-4, BAFF and IL-21 drive distinct components of activated B cell fate.
Asunto(s)
Factor Activador de Células B/metabolismo , Linfocitos B/citología , Ligando de CD40/metabolismo , Centro Germinal/metabolismo , Interleucina-4/metabolismo , Interleucinas/metabolismo , Activación de Linfocitos , Células 3T3 , Animales , Proliferación Celular , Supervivencia Celular , Regulación de la Expresión Génica , Cambio de Clase de Inmunoglobulina , Memoria Inmunológica , Ratones , Ratones Endogámicos C57BL , Fenotipo , Proteínas Proto-Oncogénicas c-bcl-6/metabolismoRESUMEN
The maintenance of mitochondrial protein homeostasis (proteostasis) is crucial for correct cellular function. Recently, several mutations in the mitochondrial protease CLPP have been identified in patients with Perrault syndrome 3 (PRLTS3). These mutations can be arranged into two groups, those that cluster near the docking site (hydrophobic pocket, Hp) for the cognate unfoldase CLPX (i.e. T145P and C147S) and those that are adjacent to the active site of the peptidase (i.e. Y229D). Here we report the biochemical consequence of mutations in both regions. The Y229D mutant not only inhibited CLPP-peptidase activity, but unexpectedly also prevented CLPX-docking, thereby blocking the turnover of both peptide and protein substrates. In contrast, Hp mutations cause a range of biochemical defects in CLPP, from no observable change to CLPP activity for the C147S mutant, to dramatic disruption of most activities for the "gain-of-function" mutant T145P - including loss of oligomeric assembly and enhanced peptidase activity.
Asunto(s)
Endopeptidasa Clp/genética , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Variación Genética , Disgenesia Gonadal 46 XX/diagnóstico , Disgenesia Gonadal 46 XX/genética , Pérdida Auditiva Sensorineural/diagnóstico , Pérdida Auditiva Sensorineural/genética , Endopeptidasa Clp/química , Endopeptidasa Clp/metabolismo , Disgenesia Gonadal 46 XX/metabolismo , Pérdida Auditiva Sensorineural/metabolismo , Humanos , Mitocondrias/genética , Mitocondrias/metabolismo , Modelos Moleculares , Mutación , Conformación ProteicaRESUMEN
Systemic lupus erythematosus (SLE) is a progressive autoimmune disease characterized by increased sensitivity to self-antigens, auto-antibody production, and systemic inflammation. B cells have been implicated in disease progression and as such represent an attractive therapeutic target. Lyn is a Src family tyrosine kinase that plays a major role in regulating signaling pathways within B cells as well as other hematopoietic cells. Its role in initiating negative signaling cascades is especially critical as exemplified by Lyn-/- mice developing an SLE-like disease with plasma cell hyperplasia, underscoring the importance of tightly regulating signaling within B cells. This review highlights recent advances in our understanding of the function of the Src family tyrosine kinase Lyn in B lymphocytes and its contribution to positive and negative signaling pathways that are dysregulated in autoimmunity.
Asunto(s)
Linfocitos B/fisiología , Lupus Eritematoso Sistémico/inmunología , Familia-src Quinasas/genética , Animales , Autoinmunidad/genética , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Noqueados , Transducción de Señal/genética , Familia-src Quinasas/metabolismoRESUMEN
Maintenance of mitochondrial protein homeostasis is critical for proper cellular function. Under normal conditions resident molecular chaperones and proteases maintain protein homeostasis within the organelle. Under conditions of stress however, misfolded proteins accumulate leading to the activation of the mitochondrial unfolded protein response (UPR(mt)). While molecular chaperone assisted refolding of proteins in mammalian mitochondria has been well documented, the contribution of AAA+ proteases to the maintenance of protein homeostasis in this organelle remains unclear. To address this gap in knowledge we examined the contribution of human mitochondrial matrix proteases, LONM and CLPXP, to the turnover of OTC-∆, a folding incompetent mutant of ornithine transcarbamylase, known to activate UPR(mt). Contrary to a model whereby CLPXP is believed to degrade misfolded proteins, we found that LONM, and not CLPXP is responsible for the turnover of OTC-∆ in human mitochondria. To analyse the conformational state of proteins that are recognised by LONM, we examined the turnover of unfolded and aggregated forms of malate dehydrogenase (MDH) and OTC. This analysis revealed that LONM specifically recognises and degrades unfolded, but not aggregated proteins. Since LONM is not upregulated by UPR(mt), this pathway may preferentially act to promote chaperone mediated refolding of proteins.