RESUMO
The transporter associated with antigen processing (TAP) is a key player in the major histocompatibility class I-restricted antigen presentation and an attractive target for immune evasion by viruses. Bovine herpesvirus 1 impairs TAP-dependent antigenic peptide transport through a two-pronged mechanism in which binding of the UL49.5 gene product to TAP both inhibits peptide transport and triggers its proteasomal degradation. How UL49.5 promotes TAP degradation has, so far, remained unknown. Here, we use high-content siRNA and genome-wide CRISPR-Cas9 screening to identify CLR2KLHDC3 as the E3 ligase responsible for UL49.5-triggered TAP disposal. We propose that the C terminus of UL49.5 mimics a C-end rule degron that recruits the E3 to TAP and engages the cullin-RING E3 ligase in endoplasmic reticulum-associated degradation.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Degrons , Herpesviridae , Apresentação de Antígeno , Citomegalovirus , Degradação Associada com o Retículo Endoplasmático , Proteínas de Membrana Transportadoras , Peptídeos , Ubiquitina-Proteína Ligases/genética , Herpesviridae/fisiologiaRESUMO
Due to unique features, proline residues may control protein structure and function. Here, we investigated the role of 52PPQ54 residues, indicated by the recently established experimental 3D structure of bovine herpesvirus 1-encoded UL49.5 protein as forming a characteristic proline hinge motif in its N-terminal domain. UL49.5 acts as a potent inhibitor of the transporter associated with antigen processing (TAP), which alters the antiviral immune response. Mechanisms employed by UL49.5 to affect TAP remain undetermined on a molecular level. We found that mutations in the 52PPQ54 region had a vast impact on its immunomodulatory function, increasing cell surface MHC class I expression, TAP levels, and peptide transport efficiency. This inhibitory effect was specific for UL49.5 activity towards TAP but not towards the viral glycoprotein M. To get an insight into the impact of proline hinge modifications on structure and dynamics, we performed all-atom and coarse-grained molecular dynamics studies on the native protein and PPQ mutants. The results demonstrated that the proline hinge sequence with its highly rigid conformation served as an anchor into the membrane. This anchor was responsible for the structural and dynamical behavior of the whole protein, constraining the mobility of the C-terminus, increasing the mobility of the transmembrane region, and controlling the accessibility of the C-terminal residues to the cytoplasmic environment. Those features appear crucial for TAP binding and inhibition. Our findings significantly advance the structural understanding of the UL49.5 protein and its functional regions and support the importance of proline motifs for the protein structure.
Assuntos
Apresentação de Antígeno , Herpesvirus Bovino 1 , Prolina , Herpesvirus Bovino 1/imunologia , Proteínas de Membrana Transportadoras/metabolismo , Prolina/química , Prolina/genética , Motivos de Aminoácidos , Transporte ProteicoRESUMO
Herpes simplex virus 1 (HSV-1) is a well-studied herpesvirus that causes various human diseases. Like other herpesviruses, HSV-1 produces the transmembrane glycoprotein N (gN/UL49.5 protein), which has been extensively studied, but its function in HSV-1 remains largely unknown. The amino-acid sequences and lengths of UL49.5 proteins differ between herpesvirus species. It is, therefore, crucial to determine whether and to what extent the spatial structure of UL49.5 orthologs that are transporter associated with antigen processing (TAP) inhibitors (i.e., of bovine herpesvirus 1; BoHV-1) differ from that of non-TAP inhibitors (i.e., of HSV-1). Our study aimed to examine the 3D structure of the HSV-1-encoded UL49.5 protein in an advanced model of the endoplasmic reticulum (ER) membrane using circular dichroism, 2D nuclear magnetic resonance, and multiple-microsecond all-atom molecular dynamics simulations in an ER membrane mimetic environment. According to our findings, the N-terminus of the HSV-1-encoded UL49.5 adopts a highly flexible, unordered structure in the extracellular part due to the presence of a large number of proline and glycine residues. In contrast to the BoHV-1-encoded homolog, the transmembrane region of the HSV-1-encoded UL49.5 is formed by a single long transmembrane α-helix, rather than two helices oriented perpendicularly, while the cytoplasmic part of the protein (C-terminus) has a short unordered structure. Our findings provide valuable experimental structural information on the HSV-1-encoded UL49.5 protein and offer, based on the obtained structure, insight into its lack of biological activity in inhibiting the TAP-dependent antigen presentation pathway.
Assuntos
Herpes Simples , Herpesviridae , Herpesvirus Humano 1 , Humanos , Apresentação de Antígeno , Herpesvirus Humano 1/metabolismo , Proteínas do Envelope Viral/química , Proteínas de Membrana Transportadoras/metabolismo , Herpesviridae/metabolismoRESUMO
Biotherapeutics, and antimicrobial proteins in particular, are of increasing interest for human medicine. An important challenge in the development of such therapeutics is their potential immunogenicity, which can induce production of anti-drug-antibodies, resulting in altered pharmacokinetics, reduced efficacy, and potentially severe anaphylactic or hypersensitivity reactions. For this reason, the development and application of effective deimmunization methods for protein drugs is of utmost importance. Deimmunization may be achieved by unspecific shielding approaches, which include PEGylation, fusion to polypeptides (e.g., XTEN or PAS), reductive methylation, glycosylation, and polysialylation. Alternatively, the identification of epitopes for T cells or B cells and their subsequent deletion through site-directed mutagenesis represent promising deimmunization strategies and can be accomplished through either experimental or computational approaches. This review highlights the most recent advances and current challenges in the deimmunization of protein therapeutics, with a special focus on computational epitope prediction and deletion tools.
RESUMO
Tumor immunity represents a new avenue for cancer therapy. Immune checkpoint inhibitors have successfully improved outcomes in several tumor types. In addition, currently, immune cell-based therapy is also attracting significant attention. However, the clinical efficacy of these treatments requires further improvement. The mechanisms through which cancer cells escape the immune response must be identified and clarified. Cancer stem cells (CSCs) play a central role in multiple aspects of malignant tumors. CSCs can initiate tumors in partially immunocompromised mice, whereas non-CSCs fail to form tumors, suggesting that tumor initiation is a definitive function of CSCs. However, the fact that non-CSCs also initiate tumors in more highly immunocompromised mice suggests that the immune evasion property may be a more fundamental feature of CSCs rather than a tumor-initiating property. In this review, we summarize studies that have elucidated how CSCs evade tumor immunity and create an immunosuppressive milieu with a focus on CSC-specific characteristics and functions. These profound mechanisms provide important clues for the development of novel tumor immunotherapies.
RESUMO
Human leukocyte antigen (HLA) class I molecules are ligands for antigen receptors of cytotoxic T cells (CTL) and inhibitory receptors of natural killer (NK) cells. The high degree of HLA class I polymorphism allows for the selection of distinct and diverse sets of antigenic peptide ligands for presentation to CTL. The extensive polymorphisms of the HLA class I genes also result in large variations in their intracellular folding and assembly characteristics. Recent findings indicate that North American HLA-B variants differ significantly in the stabilities of their peptide-deficient forms and in the requirements for the endoplasmic reticulum (ER)-resident factor tapasin for proper assembly. In HIV-infected individuals, the presence of tapasin-independent HLA-B allotypes links to more rapid progression to death. Further studies are important to better understand how the intrinsic structural characteristics of HLA class I folding intermediates affect immune responses mediated by CTL and NK cells.
Assuntos
Apresentação de Antígeno/genética , Células Apresentadoras de Antígenos/imunologia , Antígenos HLA-B/imunologia , Polimorfismo Genético , Animais , Células Apresentadoras de Antígenos/citologia , Citosol/imunologia , Citosol/metabolismo , Antígenos HLA-B/genética , Humanos , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/imunologia , Modelos Moleculares , Peptídeos/imunologia , Peptídeos/metabolismo , Receptores de Células Matadoras Naturais/genética , Receptores de Células Matadoras Naturais/imunologia , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologiaRESUMO
Signal peptide (SP) domains have a common motif but also sequence specific features. This knowledge was mainly ignored by immunologists who considered SP as generic, short-lived, targeting sequences. Consequently, while SP-derived MHC class I, class II and HLA-E epitopes have been isolated, their use as antigen-specific vaccine candidates (VCs) was mostly neglected. Recently we demonstrated the rational of selecting entire SP domains as multi-epitope long peptide VCs based on their high T and B-cell epitope densities. This review summarizes preclinical and clinical results demonstrating the various advantages of human SP domain VCs derived from both bacterial and tumor antigens. Such vaccine design provides for a straightforward, yet unique immunotherapeutic means of generating robust, non-toxic, diversified, combined antigen-specific CD4+/CD8+ T/B-cell immunity, irrespective of patient HLA repertoire also in disease associated transporter-associated with antigen processing (TAP) deficiencies. Subsequent clinical trials will further assess the full potential of this approach.
Assuntos
Vacinas Bacterianas/imunologia , Vacinas Anticâncer/imunologia , Sinais Direcionadores de Proteínas , Vacinação/métodos , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Ensaios Clínicos como Assunto , Avaliação Pré-Clínica de Medicamentos , Epitopos/imunologia , HumanosRESUMO
The durable long term clinical benefits seen for certain patients treated with immunotherapy agents has suggested there is significant therapeutic potential to be derived from these agents, as shown by the increasing prominence of this treatment strategy in upcoming clinical trials. There has been a renewed interest and focus on the drivers of tumoral antigen recognition, and the pathways by which various cells of the immune system can stimulate, propagate and execute an effective anti-tumor response. Various challenges lie ahead in the further development of these treatments, including induction of an endogenous anti-tumor response, tumor microenvironment modulation, and T-cell response amplification. Novel treatment combinations may prove of significant added benefit by immunogenic intensification.