RESUMO
In the current issue of Molecular Cell, Liu et al. (2020) show that the secretion of cancer-linked forms of mutant calreticulin allow cancer cells to escape protective immune responses induced by chemotherapeutic and immunotherapeutic drugs, thereby promoting tumor growth.
Assuntos
Calreticulina , Neoplasias , Humanos , FagocitoseRESUMO
The extreme polymorphisms of HLA class I proteins result in structural variations in their peptide binding sites to achieve diversity in Ag presentation. External factors could independently constrict or alter HLA class I peptide repertoires. Such effects of the assembly factor tapasin were assessed for HLA-B*44:05 (Y116) and a close variant, HLA-B*44:02 (D116), which have low and high tapasin dependence, respectively, for their cell surface expression. Analyses of the HLA-B*44:05 peptidomes in the presence and absence of tapasin reveal that peptides with C-terminal tryptophans and higher predicted affinities are preferentially selected by tapasin, coincident with reduced frequencies of peptides with other C-terminal amino acids, including leucine. Comparisons of the HLA-B*44:05 and HLA-B*44:02 peptidomes indicate the expected structure-based alterations near the peptide C termini, but also C-terminal amino acid frequency and predicted affinity changes among the unique and shared peptide groups for B*44:02 and B*44:05. Overall, these findings indicate that the presence of tapasin and the tapasin dependence of assembly alter HLA class I peptide-binding preferences at the peptide C terminus. The particular C-terminal amino acid preferences that are altered by tapasin are expected to be determined by the intrinsic peptide-binding specificities of HLA class I allotypes. Additionally, the findings suggest that tapasin deficiency and reduced tapasin dependence expand the permissive affinities of HLA class I-bound peptides, consistent with prior findings that HLA class I allotypes with low tapasin dependence have increased breadth of CD8+ T cell epitope presentation and are more protective in HIV infections.
Assuntos
Infecções por HIV , Triptofano , Humanos , Antígeno HLA-B44/metabolismo , Triptofano/metabolismo , Peptídeos/metabolismo , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Imunoglobulinas/metabolismo , Ligação Proteica , Antígenos HLA-B/genética , Antígenos HLA-B/metabolismoRESUMO
Myeloproliferative neoplasms (MPNs) are cancers involving dysregulated production and function of myeloid lineage hematopoietic cells. Among MPNs, Essential thrombocythemia (ET), Polycythemia Vera (PV) and Myelofibrosis (MF), are driven by mutations that activate the JAK-STAT signalling pathway. Somatic mutations of calreticulin (CRT), an endoplasmic reticulum (ER)-localized lectin chaperone, are driver mutations in approximately 25% of ET and 35% of MF patients. The MPN-linked mutant CRT proteins have novel frameshifted carboxy-domain sequences and lack an ER retention motif, resulting in their secretion. Wild type CRT is a regulator of ER calcium homeostasis and plays a key role in the assembly of major histocompatibility complex (MHC) class I molecules, which are the ligands for antigen receptors of CD8+ T cells. Mutant CRT-linked oncogenesis results from the dysregulation of calcium signalling in cells and the formation of stable complexes of mutant CRT with myeloproliferative leukemia (MPL) protein, followed by downstream activation of the JAK-STAT signalling pathway. The intricate participation of CRT in ER protein folding, calcium homeostasis and immunity suggests the involvement of multiple mechanisms of mutant CRT-linked oncogenesis. In this review, we highlight recent findings related to the role of MPN-linked CRT mutations in the dysregulation of calcium homeostasis, MPL activation and immunity.
Assuntos
Calreticulina , Transtornos Mieloproliferativos , Humanos , Cálcio/metabolismo , Calreticulina/genética , Calreticulina/metabolismo , Carcinogênese , Linfócitos T CD8-Positivos/metabolismo , Transformação Celular Neoplásica , Janus Quinase 2/metabolismo , Mutação , Transtornos Mieloproliferativos/genéticaRESUMO
Homodimeric class 1 cytokine receptors include the erythropoietin (EPOR), thrombopoietin (TPOR), granulocyte colony-stimulating factor 3 (CSF3R), growth hormone (GHR), and prolactin receptors (PRLR). These cell-surface single-pass transmembrane (TM) glycoproteins regulate cell growth, proliferation, and differentiation and induce oncogenesis. An active TM signaling complex consists of a receptor homodimer, one or two ligands bound to the receptor extracellular domains, and two molecules of Janus Kinase 2 (JAK2) constitutively associated with the receptor intracellular domains. Although crystal structures of soluble extracellular domains with ligands have been obtained for all of the receptors except TPOR, little is known about the structure and dynamics of the complete TM complexes that activate the downstream JAK-STAT signaling pathway. Three-dimensional models of five human receptor complexes with cytokines and JAK2 were generated here by using AlphaFold Multimer. Given the large size of the complexes (from 3220 to 4074 residues), the modeling required a stepwise assembly from smaller parts, with selection and validation of the models through comparisons with published experimental data. The modeling of active and inactive complexes supports a general activation mechanism that involves ligand binding to a monomeric receptor followed by receptor dimerization and rotational movement of the receptor TM α-helices, causing proximity, dimerization, and activation of associated JAK2 subunits. The binding mode of two eltrombopag molecules to the TM α-helices of the active TPOR dimer was proposed. The models also help elucidate the molecular basis of oncogenic mutations that may involve a noncanonical activation route. Models equilibrated in explicit lipids of the plasma membrane are publicly available.
Assuntos
Citocinas , Receptores de Citocinas , Humanos , Janus Quinase 2 , Ligantes , Transdução de SinaisRESUMO
Human leukocyte antigen (HLA) class I allotypes vary in their ability to present peptides in the absence of tapasin, an essential component of the peptide loading complex. We quantified tapasin dependence of all allotypes that are common in European and African Americans (n = 97), which revealed a broad continuum of values. Ex vivo examination of cytotoxic T cell responses to the entire HIV-1 proteome from infected subjects indicates that tapasin-dependent allotypes present a more limited set of distinct peptides than do tapasin-independent allotypes, data supported by computational predictions. This suggests that variation in tapasin dependence may impact the strength of the immune responses by altering peptide repertoire size. In support of this model, we observed that individuals carrying HLA class I genotypes characterized by greater tapasin independence progress more slowly to AIDS and maintain lower viral loads, presumably due to increased breadth of peptide presentation. Thus, tapasin dependence level, like HLA zygosity, may serve as a means to restrict or expand breadth of the HLA-I peptide repertoire across humans, ultimately influencing immune responses to pathogens and vaccines.
Assuntos
Apresentação de Antígeno/genética , Infecções por HIV , Antígenos de Histocompatibilidade Classe I , Proteínas de Membrana Transportadoras , Infecções por HIV/genética , Infecções por HIV/imunologia , HIV-1/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Proteínas do Vírus da Imunodeficiência Humana/imunologia , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/imunologia , Proteínas de Membrana Transportadoras/metabolismo , Peptídeos/imunologia , Peptídeos/metabolismo , Linfócitos T Citotóxicos/imunologia , Carga Viral/genética , Carga Viral/imunologiaRESUMO
Nef is an HIV-encoded accessory protein that enhances pathogenicity by down-regulating major histocompatibility class I (MHC-I) expression to evade killing by cytotoxic T lymphocytes (CTLs). A potent Nef inhibitor that restores MHC-I is needed to promote immune-mediated clearance of HIV-infected cells. We discovered that the plecomacrolide family of natural products restored MHC-I to the surface of Nef-expressing primary cells with variable potency. Concanamycin A (CMA) counteracted Nef at subnanomolar concentrations that did not interfere with lysosomal acidification or degradation and were nontoxic in primary cell cultures. CMA specifically reversed Nef-mediated down-regulation of MHC-I, but not CD4, and cells treated with CMA showed reduced formation of the Nef:MHC-I:AP-1 complex required for MHC-I down-regulation. CMA restored expression of diverse allotypes of MHC-I in Nef-expressing cells and inhibited Nef alleles from divergent clades of HIV and simian immunodeficiency virus, including from primary patient isolates. Lastly, we found that restoration of MHC-I in HIV-infected cells was accompanied by enhanced CTL-mediated clearance of infected cells comparable to genetic deletion of Nef. Thus, we propose CMA as a lead compound for therapeutic inhibition of Nef to enhance immune-mediated clearance of HIV-infected cells.
Assuntos
HIV-1 , Interações Hospedeiro-Patógeno , Macrolídeos , Linfócitos T Citotóxicos , Células Cultivadas , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/imunologia , Humanos , Macrolídeos/imunologia , Macrolídeos/farmacologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/virologia , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
Cluster of differentiation 8 (CD8) is a cell surface glycoprotein, which is expressed as 2 forms, αα homodimer or αß heterodimer. Peptide-loaded major histocompatibility complex class I (pMHC-I) molecules are major ligands for both forms of CD8. CD8αß is a coreceptor for the T cell receptor (TCR) and binds to the same cognate pMHC-I as the TCR, thus enabling or augmenting T cell responses. The function of CD8αα homodimers is largely unknown. While CD8αß heterodimer is expressed exclusively on CD8+ T cells, the CD8αα homodimer is present in subsets of T cells and human natural killer (NK) cells. Here, we report that the CD8αα homodimer functions as a coreceptor for KIR3DL1, an inhibitory receptor of NK cells that is specific for certain MHC-I allotypes. CD8αα enhances binding of pMHC-I to KIR3DL1, increases KIR3DL1 clustering at the immunological synapse, and augments KIR3DL1-mediated inhibition of NK cell activation. Additionally, interactions between pMHC-I and CD8αα homodimers regulate KIR3DL1+ NK cell education. Together, these findings reveal another dimension to the modulation of NK cell activity.
Assuntos
Antígenos CD8/química , Antígenos CD8/metabolismo , Multimerização Proteica , Receptores KIR3DL1/metabolismo , Animais , Imunofluorescência , Antígenos HLA-B/química , Antígenos HLA-B/metabolismo , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Camundongos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Receptores KIR3DL1/química , Relação Estrutura-AtividadeRESUMO
Type 2 diabetes mellitus (T2DM) is characterized by impaired glucose-stimulated insulin secretion and increased peripheral insulin resistance. Unremitting endoplasmic reticulum (ER) stress can lead to beta-cell apoptosis and has been linked to type 2 diabetes. Although many studies have attempted to link ER stress and T2DM, the specific effects of ER stress on beta-cell function remain incompletely understood. To determine the interrelationship between ER stress and beta-cell function, here we treated insulin-secreting INS-1(832/13) cells or isolated mouse islets with the ER stress-inducer tunicamycin (TM). TM induced ER stress as expected, as evidenced by activation of the unfolded protein response. Beta cells treated with TM also exhibited concomitant alterations in their electrical activity and cytosolic free Ca2+ oscillations. As ER stress is known to reduce ER Ca2+ levels, we tested the hypothesis that the observed increase in Ca2+ oscillations occurred because of reduced ER Ca2+ levels and, in turn, increased store-operated Ca2+ entry. TM-induced cytosolic Ca2+ and membrane electrical oscillations were acutely inhibited by YM58483, which blocks store-operated Ca2+ channels. Significantly, TM-treated cells secreted increased insulin under conditions normally associated with only minimal release, e.g. 5 mm glucose, and YM58483 blocked this secretion. Taken together, these results support a critical role for ER Ca2+ depletion-activated Ca2+ current in mediating Ca2+-induced insulin secretion in response to ER stress.
Assuntos
Cálcio/metabolismo , Estresse do Retículo Endoplasmático , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Animais , Sinalização do Cálcio , Cátions Bivalentes/metabolismo , Linhagem Celular , Células Cultivadas , Diabetes Mellitus Tipo 2/metabolismo , Masculino , Camundongos , RatosRESUMO
α1-antitrypsin (AAT) regulates the activity of multiple proteases in the lungs and liver. A mutant of AAT (E342K) called ATZ forms polymers that are present at only low levels in the serum and induce intracellular protein inclusions, causing lung emphysema and liver cirrhosis. An understanding of factors that can reduce the intracellular accumulation of ATZ is of great interest. We now show that calreticulin (CRT), an endoplasmic reticulum (ER) glycoprotein chaperone, promotes the secretory trafficking of ATZ, enhancing the media:cell ratio. This effect is more pronounced for ATZ than with AAT and is only partially dependent on the glycan-binding site of CRT, which is generally relevant to substrate recruitment and folding by CRT. The CRT-related chaperone calnexin does not enhance ATZ secretory trafficking, despite the higher cellular abundance of calnexin-ATZ complexes. CRT deficiency alters the distributions of ATZ-ER chaperone complexes, increasing ATZ-BiP binding and inclusion body formation and reducing ATZ interactions with components required for ER-Golgi trafficking, coincident with reduced levels of the protein transport protein Sec31A in CRT-deficient cells. These findings indicate a novel role for CRT in promoting the secretory trafficking of a protein that forms polymers and large intracellular inclusions. Inefficient secretory trafficking of ATZ in the absence of CRT is coincident with enhanced accumulation of ER-derived ATZ inclusion bodies. Further understanding of the factors that control the secretory trafficking of ATZ and their regulation by CRT could lead to new therapies for lung and liver diseases linked to AAT deficiency.
Assuntos
Calreticulina/metabolismo , Transporte Proteico/fisiologia , alfa 1-Antitripsina/metabolismo , Animais , Sítios de Ligação , Calnexina/deficiência , Calnexina/genética , Calnexina/metabolismo , Calreticulina/deficiência , Calreticulina/genética , Linhagem Celular , Retículo Endoplasmático/metabolismo , Humanos , Corpos de Inclusão/metabolismo , Camundongos , Mutagênese Sítio-Dirigida , Polissacarídeos/química , Polissacarídeos/metabolismo , Ligação Proteica , Dobramento de Proteína , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , alfa 1-Antitripsina/química , alfa 1-Antitripsina/genéticaRESUMO
The endoplasmic reticulum (ER) is an organelle that mediates the proper folding and assembly of proteins destined for the cell surface, the extracellular space and subcellular compartments such as the lysosomes. The ER contains a wide range of molecular chaperones to handle the folding requirements of a diverse set of proteins that traffic through this compartment. The lectin-like chaperones calreticulin and calnexin are an important class of structurally-related chaperones relevant for the folding and assembly of many N-linked glycoproteins. Despite the conserved mechanism of action of these two chaperones in nascent protein recognition and folding, calreticulin has unique functions in cellular calcium signaling and in the immune response. The ER-related functions of calreticulin in the assembly of major histocompatibility complex (MHC) class I molecules are well-studied and provide many insights into the modes of substrate and co-chaperone recognition by calreticulin. Calreticulin is also detectable on the cell surface under some conditions, where it induces the phagocytosis of apoptotic cells. Furthermore, mutations of calreticulin induce cell transformation in myeloproliferative neoplasms (MPN). Studies of the functions of the mutant calreticulin in cell transformation and immunity have provided many insights into the normal biology of calreticulin, which are discussed.
Assuntos
Sinalização do Cálcio , Sinalização do Cálcio/genética , Calnexina/genética , Calnexina/metabolismo , Calreticulina/genética , Calreticulina/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Sistema Imunitário , Dobramento de ProteínaRESUMO
Major histocompatibility complex class I (MHC-I) molecules present antigenic peptides to CD8+ T cells, and are also important for natural killer (NK) cell immune surveillance against infections and cancers. MHC-I molecules are assembled via a complex assembly pathway in the endoplasmic reticulum (ER) of cells. Peptides present in the cytosol of cells are transported into the ER via the transporter associated with antigen processing (TAP). In the ER, peptides are assembled with MHC-I molecules via the peptide-loading complex (PLC). Components of the MHC-I assembly pathway are frequently targeted by viruses, in order to evade host immunity. Many viruses encode inhibitors of TAP, which is thought to be a central source of peptides for the assembly of MHC-I molecules. However, human MHC-I (HLA-I) genes are highly polymorphic, and it is conceivable that several variants can acquire peptides via TAP-independent pathways, thereby conferring resistance to pathogen-derived inhibitors of TAP. To broadly assess TAP-independent expression within the HLA-B locus, expression levels of 27 frequent HLA-B alleles were tested in cells with deficiencies in TAP. Approximately 15% of tested HLA-B allotypes are expressed at relatively high levels on the surface of TAP1 or TAP2-deficient cells and occur in partially peptide-receptive forms and Endoglycosidase H sensitive forms on the cell surface. Synergy between high peptide loading efficiency, broad specificity for peptides prevalent within unconventional sources and high intrinsic stability of the empty form allows for deviations from the conventional HLA-I assembly pathway for some HLA-B*35, HLA-B*57 and HLA-B*15 alleles. Allotypes that display higher expression in TAP-deficient cells are more resistant to viral TAP inhibitor-induced HLA-I down-modulation, and HLA-I down-modulation-induced NK cell activation. Conversely, the same allotypes are expected to mediate stronger CD8+ T cell responses under TAP-inhibited conditions. Thus, the degree of resistance to TAP inhibition functionally separates specific HLA-B allotypes.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Antígenos HLA-B/genética , Antígenos HLA-B/imunologia , Vigilância Imunológica/imunologia , Humanos , Evasão da Resposta Imune/imunologiaRESUMO
Calreticulin is a calcium-binding chaperone that is normally localized in the endoplasmic reticulum. Calreticulin is detectable on the surface of apoptotic cells under some apoptosis-inducing conditions, where it promotes the phagocytosis and immunogenicity of dying cells. However, the precise mechanism by which calreticulin, a soluble protein, localizes to the outer surface of the plasma membrane of dying cells is unknown, as are the molecular mechanisms that are relevant to calreticulin-induced cellular phagocytosis. Calreticulin comprises three distinct structural domains: a globular domain, an extended arm-like P-domain, and a C-terminal acidic region containing multiple low-affinity calcium binding sites. We show that calreticulin, via its C-terminal acidic region, preferentially interacts with phosphatidylserine (PS) compared with other phospholipids and that this interaction is calcium dependent. Additionally, exogenous calreticulin binds apoptotic cells via a higher-affinity calcium-dependent mode that is acidic region dependent. Exogenous calreticulin also binds live cells, including macrophages, via a second, lower-affinity P-domain and globular domain-dependent, but calcium-independent binding mode that likely involves its generic polypeptide binding site. Truncation constructs lacking the acidic region or arm-like P-domain of calreticulin are impaired in their abilities to induce apoptotic cell phagocytosis by murine peritoneal macrophages. Taken together, the results of this investigation provide the first molecular insights into the phospholipid binding site of calreticulin as a key anchor point for the cell surface expression of calreticulin on apoptotic cells. These findings also support a role for calreticulin as a PS-bridging molecule that cooperates with other PS-binding factors to promote the phagocytosis of apoptotic cells.
Assuntos
Calreticulina/metabolismo , Citofagocitose , Retículo Endoplasmático/metabolismo , Macrófagos Peritoneais/imunologia , Fragmentos de Peptídeos/metabolismo , Fosfatidilserinas/metabolismo , Animais , Apoptose , Células Cultivadas , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Ligação ProteicaRESUMO
In 2001, The American Association of Immunologists Committee on the Status of Women conducted a survey examining the percentage of women faculty members within immunology departments or women in immunology graduate programs across 27 institutions in the United States, comparing it to the percentage of women receiving a Ph.D. Here, we examine the representation of women across these same 27 immunology departments and programs to examine changes in gender equity over the last 15 years.
Assuntos
Academias e Institutos/estatística & dados numéricos , Alergia e Imunologia , Educação de Pós-Graduação , Docentes/estatística & dados numéricos , Universidades , Mulheres , Alergia e Imunologia/educação , Feminino , Humanos , Estados Unidos , Recursos HumanosRESUMO
The MHC class I peptide loading complex (PLC) facilitates the assembly of MHC class I molecules with peptides, but factors that regulate the stability and dynamics of the assembly complex are largely uncharacterized. Based on initial findings that ATP, in addition to MHC class I-specific peptide, is able to induce MHC class I dissociation from the PLC, we investigated the interaction of ATP with the chaperone calreticulin, an endoplasmic reticulum (ER) luminal, calcium-binding component of the PLC that is known to bind ATP. We combined computational and experimental measurements to identify residues within the globular domain of calreticulin, in proximity to the high-affinity calcium-binding site, that are important for high-affinity ATP binding and for ATPase activity. High-affinity calcium binding by calreticulin is required for optimal nucleotide binding, but both ATP and ADP destabilize enthalpy-driven high-affinity calcium binding to calreticulin. ATP also selectively destabilizes the interaction of calreticulin with cellular substrates, including MHC class I molecules. Calreticulin mutants that affect ATP or high-affinity calcium binding display prolonged associations with monoglucosylated forms of cellular MHC class I, delaying MHC class I dissociation from the PLC and their transit through the secretory pathway. These studies reveal central roles for ATP and calcium binding as regulators of calreticulin-substrate interactions and as key determinants of PLC dynamics.
Assuntos
Trifosfato de Adenosina/imunologia , Calbindina 2/imunologia , Retículo Endoplasmático/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Trifosfato de Adenosina/genética , Animais , Calbindina 2/genética , Cálcio/imunologia , Retículo Endoplasmático/genética , Antígenos de Histocompatibilidade Classe I/genética , Camundongos , Camundongos Knockout , Ligação Proteica/genética , Ligação Proteica/imunologia , Estrutura Terciária de ProteínaRESUMO
TAP1/TAP2 complexes translocate peptides from the cytosol to the endoplasmic reticulum lumen to enable immune surveillance by CD8(+) T cells. Peptide transport is preceded by peptide binding to a cytosol-accessible surface of TAP1/TAP2 complexes, but the location of the TAP peptide-binding pocket remains unknown. Guided by the known contributions of polymorphic TAP variants to peptide selection, we combined homology modeling of TAP with experimental measurements to identify several TAP residues that interact with peptides. Models for peptide-TAP complexes were generated, which indicate bent conformation for peptides. The peptide binding site of TAP is located at the hydrophobic boundary of the cytosolic membrane leaflet, with striking parallels to the glutathione binding site of NaAtm1, a transporter that functions in bacterial heavy metal detoxification. These studies illustrate the conservation of the ligand recognition modes of bacterial and mammalians transporters involved in peptide-guided cellular surveillance.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Sítios de Ligação/genética , Proteínas de Membrana Transportadoras/genética , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Membro 3 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Sequência de Aminoácidos , Animais , Galinhas , Retículo Endoplasmático/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Complexo Principal de Histocompatibilidade/genética , Modelos Moleculares , Polimorfismo Genético , Ligação Proteica/genética , Estrutura Terciária de Proteína , Transporte Proteico/genética , Ratos , Células Sf9 , SpodopteraRESUMO
Calreticulin is a calcium-binding chaperone that has several functions in the immune response. In the endoplasmic reticulum (ER), calreticulin facilitates the folding of major histocompatibility complex (MHC) class I molecules and their assembly factor tapasin, thereby influencing antigen presentation to cytotoxic T cells. Although calreticulin is normally ER-resident, it is found at the cell surface of living cancer cells and dying cells. Here, calreticulin promotes cellular phagocytic uptake. In tumor vaccine models, drugs that induce cell surface calreticulin confer enhanced tumor protection in an extracellular calreticulin-dependent manner. Much remains to be understood about the roles of calreticulin in these distinct functions. Further investigations are important towards advancing basic knowledge of glycoprotein-folding pathways, and towards developing new cancer therapeutic strategies.
Assuntos
Calreticulina/imunologia , Sistema Imunitário/imunologia , Animais , Calreticulina/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Sistema Imunitário/química , Espaço Intracelular/imunologia , Fagócitos/imunologia , Dobramento de ProteínaRESUMO
MHC class I polymorphisms are known to influence outcomes in a number of infectious diseases, cancers, and inflammatory diseases. Human MHC class I H chains are encoded by the HLA-A, HLA-B, and HLA-C genes. These genes are highly polymorphic, with the HLA-B locus being the most variable. Each HLA class I protein binds to a distinct set of peptide Ags, which are presented to CD8(+) T cells. HLA-disease associations have been shown in some cases to link to the peptide-binding characteristics of individual HLA class I molecules. In this study, we show that polymorphisms at the HLA-B locus profoundly influence the assembly characteristics of HLA-B molecules and the stabilities of their peptide-deficient forms. In particular, dependence on the assembly factor tapasin is highly variable, with frequent occurrence of strongly tapasin-dependent or independent allotypes. Several polymorphic HLA-B residues located near the C-terminal end of the peptide are key determinants of tapasin-independent assembly. In vitro refolded forms of tapasin-independent allotypes assemble more readily with peptides compared to tapasin-dependent allotypes that belong to the same supertype, and, during refolding, reduced aggregation of tapasin-independent allotypes is observed. Paradoxically, in HIV-infected individuals, greater tapasin-independent HLA-B assembly confers more rapid progression to death, consistent with previous findings that some HLA-B allotypes shown to be tapasin independent are associated with rapid progression to multiple AIDS outcomes. Together, these findings demonstrate significant variations in the assembly of HLA-B molecules and indicate influences of HLA-B-folding patterns upon infectious disease outcomes.
Assuntos
Apresentação de Antígeno , Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Loci Gênicos/imunologia , Antígenos HLA-B/imunologia , Peptídeos/imunologia , Síndrome da Imunodeficiência Adquirida/genética , Síndrome da Imunodeficiência Adquirida/imunologia , Antígenos/genética , Linhagem Celular Tumoral , HIV-1/genética , HIV-1/imunologia , Antígenos HLA-B/genética , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/imunologia , Peptídeos/genética , Polimorfismo Genético/genética , Polimorfismo Genético/imunologia , Dobramento de ProteínaRESUMO
The transporter associated with antigen processing (TAP) plays a critical role in the MHC class I antigen presentation pathway. TAP translocates cellular peptides across the endoplasmic reticulum membrane in an ATP hydrolysis-dependent manner. We used FRET spectroscopy in permeabilized cells to delineate different conformational states of TAP in a native subcellular membrane environment. For these studies, we tagged the TAP1 and TAP2 subunits with enhanced cyan fluorescent protein and enhanced yellow fluorescent protein, respectively, C-terminally to their nucleotide binding domains (NBDs), and measured FRET efficiencies under different conditions. Our data indicate that both ATP and ADP enhance the FRET efficiencies but that neither induces a maximally closed NBD conformation. Additionally, peptide binding induces a large and significant increase in NBD proximity with a concentration dependence that is reflective of individual peptide affinities for TAP, revealing the underlying mechanism of peptide-stimulated ATPase activity of TAP. Maximal NBD closure is induced by the combination of peptide and non-hydrolysable ATP analogs. Thus, TAP1-TAP2 NBD dimers are not fully stabilized by nucleotides alone, and substrate binding plays a key role in inducing the transition state conformations of the NBD. Taken together, these findings show that at least three steps are involved in the transport of peptides across the endoplasmic reticulum membrane for antigen presentation, corresponding to three dynamically and structurally distinct conformational states of TAP. Our studies elucidate structural changes in the TAP NBD in response to nucleotides and substrate, providing new insights into the mechanism of ATP-binding cassette transporter function.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Adenosina Trifosfatases/química , Membrana Celular/química , Peptídeos/química , Multimerização Proteica/fisiologia , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Membro 3 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Animais , Membrana Celular/genética , Membrana Celular/metabolismo , Humanos , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Células Sf9 , SpodopteraRESUMO
Calreticulin is an endoplasmic reticulum chaperone with specificity for monoglucosylated glycoproteins. Calreticulin also inhibits precipitation of nonglycosylated proteins and thus contains generic protein-binding sites, but their location and contributions to substrate folding are unknown. We show that calreticulin binds glycosylated and nonglycosylated proteins with similar affinities but distinct interaction kinetics. Although both interactions involve the glycan-binding site or its vicinity, the arm-like proline-rich (P-) domain of calreticulin contributes to binding non/deglycosylated proteins. Correspondingly, ensemble FRET spectroscopy measurements indicate that glycosylated and nonglycosylated proteins induce "open" and "closed" P-domain conformations, respectively. The co-chaperone ERp57 influences substrate-binding kinetics and induces a closed P-domain conformation. Together with analysis of the interactions of calreticulin with cellular proteins, these findings indicate that the recruitment of monoglucosylated proteins to calreticulin is kinetically driven, whereas the P-domain and co-chaperone contribute to stable substrate binding. Substrate sequestration in the cleft between the glycan-binding site and P-domain is a likely mechanism for calreticulin-assisted protein folding.
Assuntos
Calreticulina/metabolismo , Polissacarídeos/metabolismo , Animais , Sítios de Ligação , Calreticulina/química , Calreticulina/genética , Retículo Endoplasmático/metabolismo , Glicosilação , Humanos , Imunoglobulinas/química , Imunoglobulinas/metabolismo , Cinética , Camundongos , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutagênese Sítio-Dirigida , Polissacarídeos/química , Ligação Proteica , Conformação Proteica , Isomerases de Dissulfetos de Proteínas/metabolismo , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
ABSTRACT: Somatic mutants of calreticulin (CRT) drive myeloproliferative neoplasms (MPNs) via binding to the thrombopoietin receptor (MPL) and aberrant activation of the JAK/STAT pathway. Compared with healthy donors, platelets from mutant CRT-expressing patients with MPN display low cell surface MPL. Additionally, coexpression of MPL with an MPN-linked CRT mutant (CRTDel52) reduces cell surface MPL, suggesting that CRTDel52 may induce MPL degradation. We show that lysosomal degradation is relevant to the turnover of CRTDel52 and MPL. Furthermore, CRTDel52 increases the lysosomal localization and degradation of MPL. Mammalian target of rapamycin (mTOR) inhibitors reduce cellular CRTDel52 and MPL, secreted CRTDel52 levels, and impair CRTDel52-mediated cell proliferation. mTOR inhibition also reduces colony formation and differentiation of CD34+ cells from patients with MPN but not from healthy donors. Together, these findings indicate that low-surface MPL is a biomarker of mutant CRT-mediated MPN and that induced degradation of CRTDel52 and MPL is an avenue for therapeutic intervention.