Cutting Edge: Phagosome-associated Autophagosomes Containing Antigens and Proteasomes Drive TAP-Independent Cross-Presentation.

Activation of naive CD8-positive T lymphocytes is mediated by dendritic cells that cross-present MHC class I (MHC-I)-associated peptides derived from exogenous Ags. The most accepted mechanism involves the translocation of Ags from phagosomes or endolysosomes into the cytosol, where antigenic peptides generated by cytosolic proteasomes are delivered by the transporter associated with Ag processing (TAP) to the endoplasmic reticulum, or an endocytic Ag-loading compartment, where binding to MHC-I occurs. We have described an alternative pathway where cross-presentation is independent of TAP but remains dependent on proteasomes.

mosGILT controls innate immunity and germ cell development in Anopheles gambiae.

Gene-edited mosquitoes lacking a gamma-interferon-inducible lysosomal thiol reductase-like protein, namely (mosGILT) have lower Plasmodium infection, which is linked to impaired ovarian development and immune activation. The transcriptome of mosGILT Anopheles gambiae was therefore compared to wild type (WT) mosquitoes by RNA-sequencing to delineate mosGILT-dependent pathways. Compared to WT mosquitoes, mosGILT A.

mosGILT regulates innate immune genes and expression.

Gene-edited mosquitoes lacking a g amma-interferon-inducible lysosomal thiol reductase-like protein, namely ( ) have lower infection, which is linked to impaired ovarian development and immune activation. The transcriptome of was therefore compared to wild type (WT) by RNA-sequencing to delineate mosGILT-dependent pathways. Compared to WT mosquitoes, demonstrated altered expression of genes related to oogenesis, 20-hydroxyecdysone synthesis, as well as immune-related genes.

A membrane-associated MHC-I inhibitory axis for cancer immune evasion.

Immune-checkpoint blockade has revolutionized cancer treatment, but some cancers, such as acute myeloid leukemia (AML), do not respond or develop resistance. A potential mode of resistance is immune evasion of T cell immunity involving aberrant major histocompatibility complex class I (MHC-I) antigen presentation (AP). To map such mechanisms of resistance, we identified key MHC-I regulators using specific peptide-MHC-I-guided CRISPR-Cas9 screens in AML.

CMPK2 restricts Zika virus replication by inhibiting viral translation.

Flaviviruses continue to emerge as global health threats. There are currently no Food and Drug Administration (FDA) approved antiviral treatments for flaviviral infections. Therefore, there is a pressing need to identify host and viral factors that can be targeted for effective therapeutic intervention.

SARS-CoV-2 accessory proteins ORF7a and ORF3a use distinct mechanisms to down-regulate MHC-I surface expression.

Major histocompatibility complex class I (MHC-I) molecules, which are dimers of a glycosylated polymorphic transmembrane heavy chain and the small-protein β-microglobulin (βm), bind peptides in the endoplasmic reticulum that are generated by the cytosolic turnover of cellular proteins. In virus-infected cells, these peptides may include those derived from viral proteins. Peptide-MHC-I complexes then traffic through the secretory pathway and are displayed at the cell surface where those containing viral peptides can be detected by CD8 T lymphocytes that kill infected cells.

Structural mechanism of tapasin-mediated MHC-I peptide loading in antigen presentation.

Loading of MHC-I molecules with peptide by the catalytic chaperone tapasin in the peptide loading complex plays a critical role in antigen presentation and immune recognition. Mechanistic insight has been hampered by the lack of detailed structural information concerning tapasin-MHC-I. We present here crystal structures of human tapasin complexed with the MHC-I molecule HLA-B*44:05, and with each of two anti-tapasin antibodies.

Protocol for assessing translational regulation in mammalian cell lines by OP-Puro labeling.

Translational regulation is a fundamental step in gene expression with critical roles in biological processes within a cell. Here, we describe a protocol to assess translation activity in mammalian cells by incorporation of O-propargyl-puromycin (OP-Puro). OP-Puro is a puromycin analog that is incorporated into newly synthesized proteins and is detected by click chemistry reaction.

SARS-CoV-2 accessory proteins ORF7a and ORF3a use distinct mechanisms to downregulate MHC-I surface expression.

Unlabelled: Major histocompatibility complex class I (MHC-I) molecules, which are dimers of a glycosylated polymorphic transmembrane heavy chain and the small protein β -microglobulin (β m), bind peptides in the endoplasmic reticulum that are generated by the cytosolic turnover of cellular proteins. In virus-infected cells these peptides may include those derived from viral proteins. Peptide-MHC-I complexes then traffic through the secretory pathway and are displayed at the cell surface where those containing viral peptides can be detected by CD8 T lymphocytes that kill infected cells.

Viperin triggers ribosome collision-dependent translation inhibition to restrict viral replication.

Innate immune responses induce hundreds of interferon-stimulated genes (ISGs). Viperin, a member of the radical S-adenosyl methionine (SAM) superfamily of enzymes, is the product of one such ISG that restricts the replication of a broad spectrum of viruses. Here, we report a previously unknown antiviral mechanism in which viperin activates a ribosome collision-dependent pathway that inhibits both cellular and viral RNA translation.

Impact of Calreticulin and Its Mutants on Endoplasmic Reticulum Function in Health and Disease.

The endoplasmic reticulum (ER) performs key cellular functions including protein synthesis, lipid metabolism and signaling. While these functions are spatially isolated in structurally distinct regions of the ER, there is cross-talk between the pathways. One vital player that is involved in ER function is the ER-resident protein calreticulin (CALR).

Translational shutdown and evasion of the innate immune response by SARS-CoV-2 NSP14 protein.

The ongoing COVID-19 pandemic has caused an unprecedented global health crisis. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19. Subversion of host protein synthesis is a common strategy that pathogenic viruses use to replicate and propagate in their host.

HLA tapasin independence: broader peptide repertoire and HIV control.

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 ( = 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.

Quantitating Endosomal Escape of a Library of Polymers for mRNA Delivery.

Endosomal escape is a key step for intracellular drug delivery of nucleic acids, but reliable and sensitive methods for its quantitation remain an unmet need. In order to rationally optimize the mRNA transfection efficiency of a library of polymeric materials, we designed a deactivated Renilla luciferase-derived molecular probe whose activity can be restored only in the cytosol. This probe can be coencapsulated with mRNA in the same delivery vehicle, thereby accurately measuring its endosomal escape efficiency.

Disruption of mosGILT in Anopheles gambiae impairs ovarian development and Plasmodium infection.

Plasmodium infection in Anopheles is influenced by mosquito-derived factors. We previously showed that a protein in saliva from infected Anopheles, mosquito gamma-interferon-inducible lysosomal thiol reductase (mosGILT), inhibits the ability of sporozoites to traverse cells and readily establish infection of the vertebrate host. To determine whether mosGILT influences Plasmodium within the mosquito, we generated Anopheles gambiae mosquitoes carrying mosaic mutations in the mosGILT gene using CRISPR/CRISPR associated protein 9 (Cas9).

Intrinsic expression of viperin regulates thermogenesis in adipose tissues.

Viperin is an interferon (IFN)-inducible multifunctional protein. Recent evidence from high-throughput analyses indicates that most IFN-inducible proteins, including viperin, are intrinsically expressed in specific tissues; however, the respective intrinsic functions are unknown. Here we show that the intrinsic expression of viperin regulates adipose tissue thermogenesis, which is known to counter metabolic disease and contribute to the febrile response to pathogen invasion.

Proteasomal degradation within endocytic organelles mediates antigen cross-presentation.

During MHC-I-restricted antigen processing, peptides generated by cytosolic proteasomes are translocated by the transporter associated with antigen processing (TAP) into the endoplasmic reticulum, where they bind to newly synthesized MHC-I molecules. Dendritic cells and other cell types can also generate MHC-I complexes with peptides derived from internalized proteins, a process called cross-presentation. Here, we show that active proteasomes within cross-presenting cell phagosomes can generate these peptides.

A personal retrospective on the mechanisms of antigen processing.

My intention here is to describe the history of the molecular aspects of the antigen processing field from a personal perspective, beginning with the early identification of the species that we now know as MHC class I and MHC class II molecules, to the recognition that their stable surface expression and detection by T cells depends on peptide association, and to the unraveling of the biochemical and cell biological mechanisms that regulate peptide binding. One goal is to highlight the role that serendipity or, more colloquially, pure blind luck can play in advancing the research enterprise when it is combined with an appropriately receptive mind. This is not intended to be an overarching review, and because of my own work I focus primarily on studies of the human MHC.

miRNA-mediated TUSC3 deficiency enhances UPR and ERAD to promote metastatic potential of NSCLC.

Non-small cell lung carcinoma (NSCLC) is leading cause of cancer-related deaths in the world. The Tumor Suppressor Candidate 3 (TUSC3) at chromosome 8p22 known to be frequently deleted in cancer is often found to be deleted in advanced stage of solid tumors. However, the role of TUSC3 still remains controversial in lung cancer and context-dependent in several cancers.

Cytosolic Processing Governs TAP-Independent Presentation of a Critical Melanoma Antigen.

Cancer immunotherapy has been flourishing in recent years with remarkable clinical success. But as more patients are treated, a shadow is emerging that has haunted other cancer therapies: tumors develop resistance. Resistance is often caused by defects in the MHC class I Ag presentation pathway critical for CD8 T cell-mediated tumor clearance.

A novel probe to assess cytosolic entry of exogenous proteins.

Dendritic cells use a specialized pathway called cross-presentation to activate CD8 T cells by presenting peptides from exogenous protein antigens on major histocompatibility complex class I molecules. Considerable evidence suggests that internalized antigens cross endocytic membranes to access cytosolic proteasomes for processing. The mechanism of protein dislocation represents a major unsolved problem.

A mosquito salivary gland protein partially inhibits Plasmodium sporozoite cell traversal and transmission.

The key step during the initiation of malaria is for motile Plasmodium parasites to exit the host dermis and infect the liver. During transmission, the parasites in the form of sporozoites, are injected together with mosquito saliva into the skin. However, the contribution of vector saliva to sporozoite activity during the establishment of the initial infection of the liver is poorly understood.

Tumor-associated calreticulin variants functionally compromise the peptide loading complex and impair its recruitment of MHC-I.

Major histocompatibility complex-I-βm dimers (MHC-I) bind peptides derived from intracellular proteins, enabling the immune system to distinguish between normal cells and those expressing pathogen-derived or mutant proteins. The peptides bind to MHC-I in the endoplasmic reticulum (ER), and this binding is facilitated by the eptide oading omplex (PLC), which contains calreticulin (CRT). CRT associates with MHC-I via a conserved glycan present on MHC-I and recruits it to the PLC for peptide binding.