RESUMO
Programmed cell death 1 ligand 2 (PD-L2), a member of the B7 immune checkpoint protein family, emerges as a crucial player in immune modulation. Despite its functional overlap with programmed cell death 1 ligand 1 (PD-L1) in binding to the programmed cell death protein 1 (PD-1) on T cells, PD-L2 exhibits a divergent expression pattern and a higher affinity for PD-1. However, the regulatory mechanisms of PD-L2 remain under-explored. Here, our investigations illustrate the pivotal role of cholesterol in modulating PD-L2 stability. Using advanced nuclear magnetic resonance (NMR) and biochemical analyses, we demonstrate a direct and specific binding between cholesterol and PD-L2, mediated by an F-xxx-V-xx-LR motif in its transmembrane domain, distinct from that in PD-L1. This interaction stabilizes PD-L2 and prevents its downstream degradation. Disruption of this binding motif compromises PD-L2's cellular stability, underscoring its potential significance in cancer biology. These findings not only deepen our understanding of PD-L2 regulation in the context of tumors, but also open avenues for potential therapeutic interventions.
Assuntos
Colesterol , Proteína 2 Ligante de Morte Celular Programada 1 , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Receptor de Morte Celular Programada 1 , Domínios Proteicos , Linfócitos T/metabolismo , Células HEK293 , Humanos , Estabilidade Proteica , Proteína 2 Ligante de Morte Celular Programada 1/química , Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , Colesterol/química , Colesterol/metabolismoRESUMO
Programmed Cell Death 1 (PD-1) receptor and its ligands (PD-Ls) are essential to maintain peripheral immune tolerance and to avoid tissue damage. Consequently, altered gene or protein expression of this system of co-inhibitory molecules has been involved in the development of cancer and autoimmunity. Substantial progress has been achieved in the study of the PD-1/PD-Ls system in terms of regulatory mechanisms and therapy. However, the role of the PD-1/PD-Ls pathway in neuroinflammation has been less explored despite being a potential target of treatment for neurodegenerative diseases. Multiple Sclerosis (MS) is the most prevalent, chronic, inflammatory, and autoimmune disease of the central nervous system that leads to demyelination and axonal damage in young adults. Recent studies have highlighted the key role of the PD-1/PD-Ls pathway in inducing a neuroprotective response and restraining T cell activation and neurodegeneration in MS. In this review, we outline the molecular and cellular mechanisms regulating gene expression, protein synthesis and traffic of PD-1/PD-Ls as well as relevant processes that control PD-1/PD-Ls engagement in the immunological synapse between antigen-presenting cells and T cells. Also, we highlight the most recent findings regarding the role of the PD-1/PD-Ls pathway in MS and its murine model, experimental autoimmune encephalomyelitis (EAE), including the contribution of PD-1 expressing follicular helper T (TFH) cells in the pathogenesis of these diseases. In addition, we compare and contrast results found in MS and EAE with evidence reported in other autoimmune diseases and their experimental models, and review PD-1/PD-Ls-targeting therapeutic approaches.
Assuntos
Antígeno B7-H1/fisiologia , Esclerose Múltipla/imunologia , Proteína 2 Ligante de Morte Celular Programada 1/fisiologia , Receptor de Morte Celular Programada 1/fisiologia , Animais , Antígeno B7-H1/química , Antígeno B7-H1/genética , Encéfalo/patologia , Encefalomielite Autoimune Experimental/etiologia , Encefalomielite Autoimune Experimental/imunologia , Regulação da Expressão Gênica , Humanos , Sinapses Imunológicas , Camundongos , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/etiologia , Proteína 2 Ligante de Morte Celular Programada 1/química , Proteína 2 Ligante de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/química , Receptor de Morte Celular Programada 1/genética , Transdução de Sinais/fisiologia , Células T Auxiliares Foliculares/imunologiaRESUMO
BACKGROUND: The programmed cell death protein 1 (PD-1) and its ligand 1 and 2 (PD-L1/PD-L2) regulate the immune system, and the checkpoint pathway can be exploited by malignant cells to evade anti-tumor immune response. Soluble forms (sPD-1/sPD-L1/sPD-L2) exist in the peripheral blood, but their biological and clinical significance is unclear. METHOD: Time-resolved immunofluorometric assay (TRIFMA) and enzyme-linked immunosorbent assay (ELISA) were used to measure sPD-1, sPD-L1, and sPD-L2 levels in serum from 131 lymphoma patients and 22 healthy individuals. RESULTS: Patients had higher sPD-1 and sPD-L2 levels than healthy individuals. In diffuse large B-cell lymphoma, patients with high International Prognostic Index score had higher sPD-1 levels and sPD-L2 levels correlated with subtype according to cell of origin. Compared to other lymphoma types, follicular lymphoma displayed higher sPD-1 and lower sPD-L1 levels along with lower ligand/receptor ratios. CONCLUSION: This is the first study to simultaneously characterize pretherapeutic sPD-1, sPD-L1, and sPD-L2 in a variety of lymphoma subtypes. The relation between higher sPD-1 levels and adverse prognostic factors suggests a possible biological role and potential clinical usefulness of sPD-1. Moreover, the reverse expression pattern in follicular lymphoma and T-cell lymphoma/leukemia may reflect biological information relevant for immunotherapy targeting the PD-1 pathway.
Assuntos
Antígeno B7-H1/sangue , Biomarcadores Tumorais/sangue , Regulação Leucêmica da Expressão Gênica , Leucemia/sangue , Linfoma de Células B/sangue , Linfoma Difuso de Grandes Células B/sangue , Linfoma de Células T/sangue , Proteína 2 Ligante de Morte Celular Programada 1/sangue , Receptor de Morte Celular Programada 1/sangue , Adulto , Apoptose , Antígeno B7-H1/química , Doadores de Sangue , Estudos de Casos e Controles , Contagem de Células , Testes Diagnósticos de Rotina , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoterapia , Ligantes , Linfoma de Células B/imunologia , Linfoma de Células B/metabolismo , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteína 2 Ligante de Morte Celular Programada 1/química , Receptor de Morte Celular Programada 1/químicaRESUMO
BACKGROUND: Programmed cell death-ligand 1 (PD-L1) is overexpressed in tumor cells, which causes tumor cells to escape T cell killing, and promotes tumor cell survival, cell proliferation, migration, invasion, and angiogenesis. Britannin is a natural product with anticancer pharmacological effects. PURPOSE: In this work, we studied the anticancer potential of britannin and explored whether britannin mediated its effect by inhibiting the expression of PD-L1 in tumor cells. METHODS: In vitro, the mechanisms underlying the inhibition of PD-L1 expression by britannin were investigated by MTT assay, homology modeling and molecular docking, RT-PCR, western blotting, co-immunoprecipitation, and immunofluorescence. The changes in tumor killing activity, cell proliferation, cell cycle, migration, invasion, and angiogenesis were analyzed by T cell killing assays, EdU labeling, colony formation, flow cytometry, wound healing, matrigel transwell invasion, and tube formation, respectively. In vivo, the antitumor activity of britannin was evaluated in the HCT116 cell xenograft model. RESULTS: Britannin reduced the expression of PD-L1 in tumor cells by inhibiting the synthesis of the PD-L1 protein but did not affect the degradation of the PD-L1 protein. Britannin also inhibited HIF-1α expression through the mTOR/P70S6K/4EBP1 pathway and Myc activation through the Ras/RAF/MEK/ERK pathway. Mechanistically, britannin inhibited the expression of PD-L1 by blocking the interaction between HIF-1α and Myc. In addition, britannin could enhance the activity of cytotoxic T lymphocytes and inhibit tumor cell proliferation and angiogenesis by inhibiting PD-L1. Finally, in vivo observations were confirmed by demonstrating the antitumor activity of britannin in a murine xenograft model. CONCLUSION: Britannin inhibits the expression of PD-L1 by blocking the interaction between HIF-1α and Myc. Moreover, britannin stabilizes T cell activity and inhibits proliferation and angiogenesis by inhibiting PD-L1 in cancer. The current work highlights the anti-tumor effect of britannin, providing insights into the development of cancer therapeutics via PD-L1 inhibition.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Lactonas/farmacologia , Neovascularização Patológica/tratamento farmacológico , Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , Sesquiterpenos/farmacologia , Linfócitos T/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Antineoplásicos Fitogênicos/farmacologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Células HCT116 , Humanos , Lactonas/química , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Camundongos Nus , Simulação de Acoplamento Molecular , Neovascularização Patológica/metabolismo , Proteína 2 Ligante de Morte Celular Programada 1/química , Proteína 2 Ligante de Morte Celular Programada 1/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Sesquiterpenos/química , Linfócitos T/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Recent clinical data has shown that some cancers choose to express PDL2 compared to PDL1. Therefore, a detailed and comparative study of the dynamic binding mechanism between PD1/PDL1 and PD1/PDL2 can guide drug design towards PD1. Herein, long time-scale classical molecular dynamics simulation, binding free energy calculation, energy decomposition and homology modeling for PD1/PDL2 were used to shed light on the differences in the binding mechanisms of the PD1/PDL1 and PD1/PDL2 complexes. On one hand, our results reveal a different binding mechanism of PD1 binding to PDL1 and PDL2, which is mainly attributed to the induced-fit from different proteins, that is, the C'D loop of PD1 is essential for PD1/PDL1, while the CD loop of PDL2 is critical for PD1/PDL2. Particularly, the "enclosed" conformation of PDL2 leads to a higher affinity between PD1-PDL2 in comparison to the affinity between PD1-PDL1. For PD1/PDL1, the key residues of N66, Y68, Q75, T76, K78, D85, I126, L128, A132, I134 and E136 are the dominant residues for stabilizing the protein-protein interaction (PPI). For PD1/PDL2, the key residues are mainly concentrated in the FG loop, including N33/Q75/L128/A132/Q133/I134/K135. These findings provide a comprehensive understanding of the distinctive binding kinetics and thermodynamic features, which will contribute meaningfully for the design of peptides and small molecule inhibitors to selectively break the PPI interfaces of PD1/PDL1 and PD1/PDL2.
Assuntos
Antígeno B7-H1/metabolismo , Simulação de Dinâmica Molecular , Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , Antígeno B7-H1/química , Proteína 2 Ligante de Morte Celular Programada 1/química , Ligação Proteica , Conformação Proteica , Estabilidade ProteicaRESUMO
Programmed cell death protein 1 (PD-1) is an inhibitory receptor on T lymphocytes that is critical for modulating adaptive immunity. As such, it has been successfully exploited for cancer immunotherapy. Programmed death ligand 1 (PD-L1) and PD-L2 are ligands for PD-1; the former is ubiquitously expressed in inflamed tissues, whereas the latter is restricted to antigen-presenting cells. PD-L2 binds to PD-1 with 3-fold stronger affinity compared with PD-L1. To date, this affinity discrepancy has been attributed to a tryptophan (W110PD-L2) that is unique to PD-L2 and has been assumed to fit snuggly into a pocket on the PD-1 surface. Contrary to this model, using surface plasmon resonance to monitor real-time binding of recombinantly-expressed and -purified proteins, we found that W110PD-L2 acts as an "elbow" that helps shorten PD-L2 engagement with PD-1 and therefore lower affinity. Furthermore, we identified a "latch" between the C and D ß-strands of the binding face as the source of the PD-L2 affinity advantage. We show that the 3-fold affinity advantage of PD-L2 is the consequence of these two opposing features, the W110PD-L2 "elbow" and a C-D region "latch." Interestingly, using phylogenetic analysis, we found that these features evolved simultaneously upon the emergence of placental mammals, suggesting that PD-L2-affinity tuning was part of the alterations to the adaptive immune system required for placental gestation.
Assuntos
Antígeno B7-H1/química , Placenta/metabolismo , Proteína 2 Ligante de Morte Celular Programada 1/química , Sequência de Aminoácidos , Animais , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Proliferação de Células , Feminino , Humanos , Ligantes , Ativação Linfocitária , Camundongos , Mutagênese Sítio-Dirigida , Filogenia , Gravidez , Proteína 2 Ligante de Morte Celular Programada 1/classificação , Proteína 2 Ligante de Morte Celular Programada 1/genética , Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , Ligação Proteica , Domínios Proteicos , Estrutura Terciária de Proteína , Alinhamento de Sequência , Eletricidade EstáticaRESUMO
Immune checkpoint blockade of programmed death-1 (PD-1) by monoclonal antibody drugs has delivered breakthroughs in the treatment of cancer. Nonetheless, small-molecule PD-1 inhibitors could lead to increases in treatment efficacy, safety, and global access. While the ligand-binding surface of apo-PD-1 is relatively flat, it harbors a striking pocket in the murine PD-1/PD-L2 structure. An analogous pocket in human PD-1 may serve as a small-molecule drug target, but the structure of the human complex is unknown. Because the CC' and FG loops in murine PD-1 adopt new conformations upon binding PD-L2, we hypothesized that mutations in these two loops could be coupled to pocket formation and alter PD-1's affinity for PD-L2. Here, we conducted deep mutational scanning in these loops and used yeast surface display to select for enhanced PD-L2 binding. A PD-1 variant with three substitutions binds PD-L2 with an affinity two orders of magnitude higher than that of the wild-type protein, permitting crystallization of the complex. We determined the X-ray crystal structures of the human triple-mutant PD-1/PD-L2 complex and the apo triple-mutant PD-1 variant at 2.0 Å and 1.2 Å resolution, respectively. Binding of PD-L2 is accompanied by formation of a prominent pocket in human PD-1, as well as substantial conformational changes in the CC' and FG loops. The structure of the apo triple-mutant PD-1 shows that the CC' loop adopts the ligand-bound conformation, providing support for allostery between the loop and pocket. This human PD-1/PD-L2 structure provide critical insights for the design and discovery of small-molecule PD-1 inhibitors.
Assuntos
Proteína 2 Ligante de Morte Celular Programada 1/química , Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/química , Receptor de Morte Celular Programada 1/metabolismo , Regulação Alostérica , Substituição de Aminoácidos , Antígeno B7-H1/química , Antígeno B7-H1/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Descoberta de Drogas , Humanos , Modelos Moleculares , Complexos Multiproteicos/química , Mutação , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/genética , Conformação Proteica , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Cancer cells can avoid and suppress immune responses through activation of inhibitory immune checkpoint proteins, such as PD-1, PD-L1, and CTLA-4. Blocking the activities of these proteins with monoclonal antibodies, and thus restoring T cell function, has delivered breakthrough therapies against cancer. In this review, we describe the latest work on structural characterization of the checkpoint proteins, their interactions with cognate ligands and with therapeutic antibodies. Structures of the extracellular portions of these proteins reveal that they all have a similar modular structure, composed of small domains similar in topology to the domains found in antibodies. Structural basis for blocking the PD-1/PD-L1 interaction by small molecules is illustrated with the compound BMS-202 that binds to and induces dimerization of PD-L1.
Assuntos
Antineoplásicos Imunológicos/química , Antígeno B7-H1/química , Proteína 2 Ligante de Morte Celular Programada 1/química , Receptor de Morte Celular Programada 1/química , Animais , Antineoplásicos Imunológicos/metabolismo , Antígeno B7-H1/metabolismo , Sítios de Ligação , Humanos , Simulação de Acoplamento Molecular , Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Ligação ProteicaRESUMO
Many eukaryotic regulatory proteins adopt distinct bound and unbound conformations, and use this structural flexibility to bind specifically to multiple partners. However, we lack an understanding of how an interface can select some ligands, but not others. Here, we present a molecular dynamics approach to identify and quantitatively evaluate the interactions responsible for this selective promiscuity. We apply this approach to the anticancer target PD-1 and its ligands PD-L1 and PD-L2. We discover that while unbound PD-1 exhibits a hard-to-drug hydrophilic interface, conserved specific triggers encoded in the cognate ligands activate a promiscuous binding pathway that reveals a flexible hydrophobic binding cavity. Specificity is then established by additional contacts that stabilize the PD-1 cavity into distinct bound-like modes. Collectively, our studies provide insight into the structural basis and evolution of multiple binding partners, and also suggest a biophysical approach to exploit innate binding pathways to drug seemingly undruggable targets.
Assuntos
Antígeno B7-H1/química , Proteína 2 Ligante de Morte Celular Programada 1/química , Receptor de Morte Celular Programada 1/química , Conformação Proteica , Antígeno B7-H1/metabolismo , Simulação de Dinâmica Molecular , Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Ligação ProteicaRESUMO
Programmed death-1 (PD-1) is a co-inhibitory molecule and is seen in CD4+ and CD8+ T cells. Upon binding to its ligands, programmed death ligand-1 (PD-L1) and -2 (PD-L2), PD-1 negatively regulates interleukin 2 (IL-2) production and T cell proliferation. Activated effector T-cells, which kill cancer cells, can be affected by PD-1 signaling in some lymphoid neoplasm that express PD-L1 or PD-L2. PD-L1 expression in tumor cells can be induced by extrinsic signal (i.e. interferon gamma) or intrinsic signals, such as genetic aberrations involving 9p24.1, latent Epstein-Barr virus infection, PD-L1 3'- untranslated region disruptions, and activated Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. Anti-PD-1 therapy improves the overall response rate to treatment in patients with lymphoid neoplasms, particularly relapsed/refractory classical Hodgkin lymphoma. Inspired by their success in treating patients with classical Hodgkin lymphoma, medical practitioners have expanded PD-1 therapy, given as a single therapy or in combination with other drugs, to patients with other types of lymphoma. In this review, current clinical trials with anti-PD-1 or anti-PD-L1 drugs are summarized. The results of numerous clinical trials will broaden our understanding of PD-1 pathway and shall expand the list of patients who will get benefit from these agents including those who suffer from lymphoid neoplasms.
Assuntos
Anticorpos Monoclonais/farmacologia , Linfoma/tratamento farmacológico , Linfoma/patologia , Terapia de Alvo Molecular/métodos , Antígeno B7-H1/química , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Humanos , Proteína 2 Ligante de Morte Celular Programada 1/química , Proteína 2 Ligante de Morte Celular Programada 1/genética , Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/química , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/patologiaRESUMO
Previous reports from this group have indicated that the immunoinhibitory programmed death (PD)-1 receptor and its ligand, PD-L1, are involved in the mechanism of immune evasion of bovine chronic infection. However, no functional analysis of bovine PD-L2 in cattle has been reported. Thus, in this study, the molecular function of bovine PD-L2 was analyzed in vitro. Recombinant PD-L2 (PD-L2-Ig), which comprises an extracellular domain of bovine PD-L2 fused to the Fc portion of rabbit IgG1, was prepared based on the cloned cDNA sequence for bovine PD-L2. Bovine PD-L2-Ig bound to bovine PD-1-expressing cells and addition of soluble bovine PD-1-Ig clearly inhibited the binding of PD-L2-Ig to membrane PD-1 in a dose-dependent manner. Cell proliferation and IFN-γ production were significantly enhanced in the presence of PD-L2-Ig in peripheral blood mononuclear cells (PBMCs) from cattle. Moreover, PD-L2-Ig significantly enhanced IFN-γ production from virus envelope peptides-stimulated PBMCs derived from bovine leukemia virus-infected cattle. Interestingly, PD-L2-Ig-induced IFN-γ production was further enhanced by treatment with anti-bovine PD-1 antibody. These data suggest potential applications of bovine PD-L2-Ig as a therapy for bovine diseases.
Assuntos
Proteína 2 Ligante de Morte Celular Programada 1/genética , Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Sequência de Bases , Bovinos , Clonagem Molecular , Interferon gama/biossíntese , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Dados de Sequência Molecular , Filogenia , Proteína 2 Ligante de Morte Celular Programada 1/química , Proteína 2 Ligante de Morte Celular Programada 1/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Ligação Proteica , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
PD-1, a receptor expressed by T cells, B cells, and monocytes, is a potent regulator of immune responses and a promising therapeutic target. The structure and interactions of human PD-1 are, however, incompletely characterized. We present the solution nuclear magnetic resonance (NMR)-based structure of the human PD-1 extracellular region and detailed analyses of its interactions with its ligands, PD-L1 and PD-L2. PD-1 has typical immunoglobulin superfamily topology but differs at the edge of the GFCC' sheet, which is flexible and completely lacks a C" strand. Changes in PD-1 backbone NMR signals induced by ligand binding suggest that, whereas binding is centered on the GFCC' sheet, PD-1 is engaged by its two ligands differently and in ways incompletely explained by crystal structures of mouse PD-1 · ligand complexes. The affinities of these interactions and that of PD-L1 with the costimulatory protein B7-1, measured using surface plasmon resonance, are significantly weaker than expected. The 3-4-fold greater affinity of PD-L2 versus PD-L1 for human PD-1 is principally due to the 3-fold smaller dissociation rate for PD-L2 binding. Isothermal titration calorimetry revealed that the PD-1/PD-L1 interaction is entropically driven, whereas PD-1/PD-L2 binding has a large enthalpic component. Mathematical simulations based on the biophysical data and quantitative expression data suggest an unexpectedly limited contribution of PD-L2 to PD-1 ligation during interactions of activated T cells with antigen-presenting cells. These findings provide a rigorous structural and biophysical framework for interpreting the important functions of PD-1 and reveal that potent inhibitory signaling can be initiated by weakly interacting receptors.