RESUMEN
Hypogonadism is defined as the inadequate gonadal production of testosterone. Low serum testosterone leads to infertility by impairing spermatogenesis and reducing sperm count, however, the impact of hypogonadism in epididymal sperm maturation is poorly understood. From the testis, spermatozoa are transported into the epididymis where they find a specific microenvironment composed of a complex mixture of proteins that facilitate sperm storage and maturation. Inside the epididymal ductule, spermatozoa undergo several changes, resulting in their becoming capable of fertilizing eggs. Protein disulfide isomerases (PDIs) are known to participate in the folding and assembly of secreted proteins in the endoplasmic reticulum. However, little is known about the control and function of PDIs in the testis and epididymis, particularly during male development. The aim of this work was to compare the expression and distribution of PDI and PDIA3 (ERp57) in the testis and epididymis of healthy and GnRH-immunized boars. We detected higher amounts of PDIA3 and PDI in sperm preparations and fluid from the proximal regions of the epididymis of healthy boars. However, we observed an increase in PDIA3 expression in the testis and cauda epididymis in the immunocastrated group. GnRH-immunized boars showed a marked increase in PDI content in cauda spermatozoa and fluid, indicating a possible endocrine dysregulation of PDI. The results of our study suggest that PDIs are associated with epididymal sperm maturation and may be attractive candidates for monitoring male fertility.
Asunto(s)
Epidídimo/metabolismo , Hormona Liberadora de Gonadotropina/inmunología , Proteína Disulfuro Isomerasas/metabolismo , Testículo/metabolismo , Animales , Inmunización , Masculino , Espermatozoides/metabolismo , PorcinosRESUMEN
The ER (endoplasmic reticulum) is the site of protein folding for all eukaryotic secreted and plasma membrane proteins. Disulphide bonds are formed in many of these proteins through a dithiol-disulphide exchange chain comprising two types of protein catalysts: PDI (protein disulphide-isomerase) and ERO (ER oxidoreductase) proteins. This review will examine what we know about ERO function, and will then consider ERO interactions and their implications for mammalian oxidative protein folding.
Asunto(s)
Retículo Endoplásmico/enzimología , Isoenzimas/metabolismo , Oxidorreductasas/metabolismo , Animales , Dimerización , Disulfuros/química , Humanos , Isoenzimas/química , Oxidorreductasas/química , Compuestos de Sulfhidrilo/química , Distribución TisularRESUMEN
The presence of correctly formed disulfide bonds is crucial to the structure and function of proteins that are synthesized in the endoplasmic reticulum (ER). Disulfide bond formation occurs in the ER owing to the presence of several specialized catalysts and a suitable redox potential. Work in yeast has indicated that the ER resident glycoprotein Ero1p provides oxidizing equivalents to newly synthesized proteins via protein disulfide isomerase (PDI). Here we show that Ero1-Lalpha, the human homolog of Ero1p, exists as a collection of oxidized and reduced forms and covalently binds PDI. We analyzed Ero1-Lalpha cysteine mutants in the presumed active site C(391)VGCFKC(397). Our results demonstrate that this motif is important for protein folding, structural integrity, protein half-life and the stability of the Ero1-Lalpha-PDI complex.
Asunto(s)
Glicoproteínas de Membrana/química , Secuencia de Aminoácidos , Animales , Células CHO , Cricetinae , Disulfuros/química , Retículo Endoplásmico/química , Glicosilación , Proteína gp120 de Envoltorio del VIH/química , Células HeLa , Humanos , Oxidación-Reducción , Oxidorreductasas , Conformación Proteica , Pliegue de ProteínaRESUMEN
Our understanding of eukaryotic protein folding in the endoplasmic reticulum has increased enormously over the last 5 years. In this review, we summarize some of the major research themes that have captivated researchers in this field during the last years of the 20th century. We follow the path of a typical protein as it emerges from the ribosome and enters the reticular environment. While many of these events are shared between different polypeptide chains, we highlight some of the numerous differences between proteins, between cell types, and between the chaperones utilized by different ER glycoproteins. Finally, we consider the likely advances in this field as the new century unfolds and we address the prospect of a unified understanding of how protein folding, degradation, and translation are coordinated within a cell.
Asunto(s)
Retículo Endoplásmico/metabolismo , Glicoproteínas/metabolismo , Pliegue de Proteína , Procesamiento Proteico-Postraduccional , Animales , HumanosRESUMEN
MHC class I molecules are cell surface glycoproteins that play a pivotal role in the response to intracellular pathogens. The loading of MHC class I molecules with antigenic substrates takes place in the endoplasmic reticulum. This requires a functional TAP transporter, which translocates peptides into the endoplasmic reticulum from the cytosol. The generation of antigenic peptides from polypeptide precursors is thought to be mediated in the cytosol by the proteasome. Previously, we have demonstrated that inhibiting the proteasome with the specific covalent inhibitor lactacystin results in a direct reduction of peptide-loaded MHC class I molecules. This indicates that the proteasome is the limiting step in the MHC class I pathway. In this study we use isoelectric focusing to demonstrate that two related MHC class I alleles, HLA-A3 and HLA-A11, as well as HLA-B35 do not follow this behavior. In contrast to other class I alleles expressed by the same cells, these alleles are loaded with peptides and mature normally when proteasome activity is severely inhibited. Our observations highlight a new level of diversity in the MHC class I system and indicate that there are allele-specific differences in the linkage between proteasome activity and MHC class I peptide loading.
Asunto(s)
Alelos , Cisteína Endopeptidasas/metabolismo , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Complejos Multienzimáticos/metabolismo , Péptidos/metabolismo , Línea Celular , Cisteína Endopeptidasas/efectos de los fármacos , Cisteína Endopeptidasas/inmunología , Inhibidores de Cisteína Proteinasa/farmacología , Antígenos HLA-A/genética , Antígenos HLA-A/inmunología , Antígenos HLA-A/metabolismo , Antígeno HLA-A11 , Antígeno HLA-A3/genética , Antígeno HLA-A3/inmunología , Células HeLa , Humanos , Focalización Isoeléctrica , Complejos Multienzimáticos/efectos de los fármacos , Complejos Multienzimáticos/inmunología , Péptidos/inmunología , Complejo de la Endopetidasa ProteasomalRESUMEN
Proteasomes are proteolytic complexes involved in non-lysosomal degradation which are localized in both the cytoplasm and the nucleus. The dynamics of proteasomes in living cells is unclear, as is their targeting to proteins destined for degradation. To investigate the intracellular distribution and mobility of proteasomes in vivo, we generated a fusion protein of the proteasome subunit LMP2 and the green fluorescent protein (GFP). The LMP2-GFP chimera was quantitatively incorporated into catalytically active proteasomes. The GFP-tagged proteasomes were located within both the cytoplasm and the nucleus. Within these two compartments, proteasomes diffused rapidly, and bleaching experiments demonstrated that proteasomes were transported slowly and unidirectionally from the cytoplasm into the nucleus. During mitosis, when the nuclear envelope has disintegrated, proteasomes diffused rapidly throughout the dividing cell without encountering a selective barrier. Immediately after cell division, the restored nuclear envelope formed a new barrier for the diffusing proteasomes. Thus, proteasomes can be transported unidirectionally over the nuclear membrane, but can also enter the nucleus upon reassembly during cell division. Since proteasomes diffuse rapidly in the cytoplasm and nucleus, they may perform quality control by continuous collision with intracellular proteins, and degrading those proteins that are properly tagged or misfolded.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Complejos Multienzimáticos/metabolismo , Proteínas/metabolismo , Presentación de Antígeno , Transporte Biológico , Compartimento Celular , División Celular , Núcleo Celular/metabolismo , Cisteína Endopeptidasas/genética , Citoplasma/metabolismo , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Movimiento , Complejos Multienzimáticos/genética , Complejo de la Endopetidasa Proteasomal , Proteínas/genética , Proteínas Recombinantes de Fusión/metabolismo , Células Tumorales CultivadasAsunto(s)
Linfocitos B/inmunología , Células Dendríticas/inmunología , Trasplante de Riñón/inmunología , Trasplante de Piel/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Trasplante Homólogo/inmunología , Animales , Formación de Anticuerpos , Antígenos de Histocompatibilidad Clase I/inmunología , Complejo Mayor de Histocompatibilidad , Modelos Biológicos , Ratas , Ratas Endogámicas Lew , Ratas EndogámicasRESUMEN
For an effective CD8+ cytotoxic T cell response to occur during infection, MHC class I molecules must be loaded with antigenic peptides in the endoplasmic reticulum. The cytosolic factor responsible for peptide generation is believed to be the proteasome, with the TAP heterodimer mediating peptide transport into the endoplasmic reticulum. However, the rate-determining step(s) in this intracellular pathway of Ag presentation is currently unresolved. The availability of a specific and irreversible proteasome inhibitor called lactacystin has enabled us to determine the amount of proteasomes required for the peptide loading of MHC class I molecules in four cell types. In the absence of the IFN-gamma-inducible proteasome subunits LMP2 and LMP7, the trypsin-like (but not the chymotrypsin-like) activity of the proteasome is directly related to MHC class I peptide loading. However, IFN-gamma stimulation or assimilation of catalytic LMP2 and LMP7 subunits into proteasomes causes both chymotrypsin- and trypsin-like activities of the proteasome to become limiting for the loading of class I molecules. Our data suggest that upon full IFN-gamma stimulation, peptide supply by the proteasome is the limiting step in the assembly of MHC class I polypeptides. This mechanism may enable the cell to prevent competition between novel Ags and the pool of endogenous proteins for binding to MHC class I molecules.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Interferón gamma/farmacología , Activación de Linfocitos , Complejos Multienzimáticos/metabolismo , Linfocitos T Citotóxicos/enzimología , Línea Celular Transformada , Quimotripsina/metabolismo , Quimotripsina/fisiología , Cisteína Endopeptidasas/fisiología , Humanos , Activación de Linfocitos/efectos de los fármacos , Melanoma/enzimología , Melanoma/inmunología , Complejos Multienzimáticos/fisiología , Complejo de la Endopetidasa Proteasomal , Proteínas/metabolismo , Linfocitos T Citotóxicos/inmunología , Tripsina/metabolismo , Tripsina/fisiología , Células Tumorales Cultivadas , Proteínas de la Matriz Viral/deficienciaRESUMEN
LEW rats with long-surviving (> 100 days) (DA x LEW)F1 kidney allografts were generated by treating the recipients with cyclosporine for 14 days after grafting. All rats were monitored after transplantation for the development of antibodies to intact donor class I MHC molecules. Cyclosporine completely suppressed the early antibody response to intact DA class I MHC molecules in all 19 LEW rats. However, 17 of the 19 rats developed antibodies between four and six weeks after grafting-i.e., between two and four weeks after the cessation of cyclosporine therapy, and maintained high levels of antibody to the donor class I molecules in spite of the long-term presence of the allograft. The 2 rats that did not produce antibodies to donor class I MHC molecules, along with one of the 17 that did produce antibodies, were immunized with a synthetic peptide corresponding to a region of the DA class I MHC molecule known to be recognized by LEW CD4+ T cells via the indirect recognition pathway. All 3 long survivors developed self APC-dependent CD4+ T cell proliferation to the immunizing donor peptides, and strong antibody responses to these peptides. However, none of these long survivors suffered rejection episodes as a consequence of the peptide immunization. In one of the two long-surviving rats without antibodies to intact donor class I MHC molecules at the time of peptide priming, the peptide priming resulted in the prompt development of strong antibodies to intact donor class I molecules. However, the other of these 2 rats did not produce such antibodies after peptide priming. Thus in this model of kidney allograft tolerance, with long-term exposure of the recipient's immune system to donor antigens without evidence of rejection, none of the animals develops tolerance for the indirect T cell recognition of donor class I MHC antigens. In occasional animals, B cells potentially reactive to intact donor class I molecules are present and are adequately exposed to antigen but are quiescent because of the absence of T cell help, perhaps as a consequence of reversible T cell suppression or anergy. In other occasional animals, B cell nonreactivity (anergy or tolerance) to intact donor class I molecules appears to develop.
Asunto(s)
Linfocitos B/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Trasplante de Riñón/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Formación de Anticuerpos , Tolerancia Inmunológica , Isoanticuerpos/biosíntesis , Activación de Linfocitos , Datos de Secuencia Molecular , Péptidos/química , Péptidos/inmunología , Ratas , Ratas Endogámicas Lew , Ratas EndogámicasRESUMEN
In this article, we propose that T cell help for B cells can occur via an unusual three-cell cluster, with recipient CD4+ T helper cells interacting via direct allorecognition with donor dendritic cell class II MHC antigens, recipient B cells interacting with MHC class I (or any other) antigen on the donor dendritic cell surface, and noncognate (i.e., antigen nonspecific) T-B collaboration. In this noncognate pathway, antigen processing by B cells is not required and T cell help is potent because of the high precursor T cell frequency for direct recognition of allogeneic class II MHC molecules. The data supporting this hypothesis are: 1. LEW rat strain recipients of interstitial dendritic cell-free (DAxLEW)F1 kidney allografts were shown to have no detectable antibody to donor class I MHC antigens at day 7 after grafting. By contrast, LEW recipients of normal (DAxLEW)F1 kidneys had strong antibody responses. 2. Consistent wih important role for donor dendritic cells in the early antibody response to donor class I MHC antigens was the finding that it was dependent on donor class II MHC antigens. PVG recipients, previously immunized with pure DA RT1.B class II MHC antigens, had virtually no antibody response to the class I MHC antigens of DA kidney allografts. 3. We confirmed the low and high responder status of PVG and LEW rats, respectively, to DA class I antigens by studying antibody responses to pure DA class I antigens. However, PVG and LEW recipients of DA kidney allografts did not differ in their antibody response to the donor DA class I MHC antigens. This is consistent with this response not requiring the processing and presentation of DA class I antigen by PVG recipients. 4. LEW recipients of interstitial dendritic cell-free (DAxLEW)F1 kidney allografts did eventually develop a strong antibody response to DA class I antigens, but this was delayed by several weeks. That this delayed antibody response was probably mediated by conventional T-B collaboration and that T help was rate limiting in this situation, was demonstrated by immunizing LEW recipients with a DA class I peptide. This markedly accelerated the kinetics of the antibody response to the dendritic cell-free (DAxLEW)F1 kidneys.
Asunto(s)
Células Dendríticas/fisiología , Cooperación Linfocítica , Linfocitos T/fisiología , Secuencia de Aminoácidos , Animales , Formación de Anticuerpos , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase II/fisiología , Trasplante de Riñón/inmunología , Masculino , Ratones , Datos de Secuencia Molecular , Conejos , Ratas , Ratas Endogámicas Lew , Trasplante HomólogoAsunto(s)
Presentación de Antígeno/fisiología , Antígenos de Histocompatibilidad Clase I/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Miembro 3 de la Subfamilia B de Transportadores de Casetes de Unión a ATP , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico Activo , Cisteína Endopeptidasas/inmunología , Cisteína Endopeptidasas/metabolismo , Retículo Endoplásmico/inmunología , Retículo Endoplásmico/metabolismo , Humanos , Datos de Secuencia Molecular , Complejos Multienzimáticos/inmunología , Complejos Multienzimáticos/metabolismo , Oligopéptidos/genética , Oligopéptidos/inmunología , Oligopéptidos/metabolismo , Complejo de la Endopetidasa ProteasomalRESUMEN
This report demonstrates for the first time that indirect T cell allorecognition of donor antigens can contribute to the effector mechanism of rejection of vascularized organ allografts. LEW (RT1(1)) rats were primed for indirect T cell allorecognition of DA (RT1av1) classical class I MHC molecules by immunization with synthetic 22-24 amino acid peptides corresponding to the alpha-helices of the RT1-A class I molecule. These rats received (DA x LEW) F1 kidney grafts that had been depleted of donor interstitial dendritic cells to minimize the direct T cell allorecognition response to the graft. The peptide-immunized rats rejected their grafts more rapidly than did control immunized rats, in terms of both graft function and survival. Moreover, the kinetics of antibody production to intact donor class I molecules after kidney transplantation was much more rapid in the peptide-immunized rats, suggesting that T cell help is the rate-limiting factor for antibody production to donor antigens in this model. It was of interest that we could not detect an antibody response to donor peptides after kidney graft rejection.