A trimeric Rab7 GEF controls NPC1-dependent lysosomal cholesterol export

Abstract: Cholesterol import in mammalian cells is mediated by the LDL receptor pathway. Here, we perform a genome-wide CRISPR screen using an endogenous cholesterol reporter and identify >100 genes involved in LDL-cholesterol import. We characterise C18orf8 as a core subunit of the mammalian Mon1-Ccz1 guanidine exchange factor (GEF) for Rab7, required for complex stability and function. C18orf8-deficient cells lack Rab7 activation and show severe defects in late endosome morphology and endosomal LDL trafficking, resulting in cellular cholesterol deficiency. Unexpectedly, free cholesterol accumulates within swollen lysosomes, suggesting a critical defect in lysosomal cholesterol export. We find that active Rab7 interacts with the NPC1 cholesterol transporter and licenses lysosomal cholesterol export. This process is abolished in C18orf8-, Ccz1- and Mon1A/B-deficient cells and restored by a constitutively active Rab7. The trimeric Mon1-Ccz1-C18orf8 (MCC) GEF therefore plays a central role in cellular cholesterol homeostasis coordinating Rab7 activation, endosomal LDL trafficking and NPC1-dependent lysosomal cholesterol export

Varicellovirus UL 49.5 proteins differentially affect the function of the transporter associated with antigen processing, TAP

Cytotoxic T-lymphocytes play an important role in the protection against viral infections, which they detect through the recognition of virus-derived peptides, presented in the context of MHC class I molecules at the surface of the infected cell. The transporter associated with antigen processing (TAP) plays an essential role in MHC class I–restricted antigen presentation, as TAP imports peptides into the ER, where peptide loading of MHC class I molecules takes place. In this study, the UL49.5 proteins of the varicelloviruses bovine herpesvirus 1 (BHV-1), pseudorabies virus (PRV), and equine herpesvirus 1 and 4 (EHV-1 and EHV-4) are characterized as members of a novel class of viral immune evasion proteins. These UL49.5 proteins interfere with MHC class I antigen presentation by blocking the supply of antigenic peptides through inhibition of TAP. BHV-1, PRV, and EHV-1 recombinant viruses lacking UL49.5 no longer interfere with peptide transport. Combined with the observation that the individually expressed UL49.5 proteins block TAP as well, these data indicate that UL49.5 is the viral factor that is both necessary and sufficient to abolish TAP function during productive infection by these viruses. The mechanisms through which the UL49.5 proteins of BHV-1, PRV, EHV-1, and EHV-4 block TAP exhibit surprising diversity. BHV-1 UL49.5 targets TAP for proteasomal degradation, whereas EHV-1 and EHV-4 UL49.5 interfere with the binding of ATP to TAP. In contrast, TAP stability and ATP recruitment are not affected by PRV UL49.5, although it has the capacity to arrest the peptide transporter in a translocation-incompetent state, a property shared with the BHV-1 and EHV-1 UL49.5. Taken together, these results classify the UL49.5 gene products of BHV-1, PRV, EHV-1, and EHV-4 as members of a novel family of viral immune evasion proteins, inhibiting TAP through a variety of mechanisms

A brief report on combination chemotherapy and anti–programmed death (ligand) 1 treatment in small-cell lung cancer: Did we choose the optimal chemotherapy backbone?

Extensive-stage small-cell lung cancer (ES-SCLC) is an aggressive cancer that remains very hard to treat. The life expectancy of a patient diagnosed with this disease has not changed over the past three decades. Recently, three large clinical studies showed a survival benefit by adding an anti–programmed death (ligand) 1 (PD-(L)1 antibody to the current chemotherap

A CD8+ T cell immune evasion protein specific to Epstein-Barr virus and its close relatives in Old World primates

γ1-Herpesviruses such as Epstein-Barr virus (EBV) have a unique ability to amplify virus loads in vivo through latent growth-transforming infection. Whether they, like α- and β-herpesviruses, have been driven to actively evade immune detection of replicative (lytic) infection remains a moot point. We were prompted to readdress this question by recent work (Pudney, V.A., A.M. Leese, A.B. Rickinson, and A.D. Hislop. 2005. J. Exp. Med. 201:349–360; Ressing, M.E., S.E. Keating, D. van Leeuwen, D. Koppers-Lalic, I.Y. Pappworth, E.J.H.J. Wiertz, and M. Rowe. 2005. J. Immunol. 174:6829–6838) showing that, as EBV-infected cells move through the lytic cycle, their susceptibility to EBV-specific CD8+ T cell recognition falls dramatically, concomitant with a reductions in transporter associated with antigen processing (TAP) function and surface human histocompatibility leukocyte antigen (HLA) class I expression. Screening of genes that are unique to EBV and closely related γ1-herpesviruses of Old World primates identified an early EBV lytic cycle gene, BNLF2a, which efficiently blocks antigen-specific CD8+ T cell recognition through HLA-A–, HLA-B–, and HLA-C–restricting alleles when expressed in target cells in vitro. The small (60–amino acid) BNLF2a protein mediated its effects through interacting with the TAP complex and inhibiting both its peptide- and ATP-binding functions. Furthermore, this targeting of the major histocompatibility complex class I pathway appears to be conserved among the BNLF2a homologues of Old World primate γ1-herpesviruses. Thus, even the acquisition of latent cycle genes endowing unique growth-transforming ability has not liberated these agents from evolutionary pressure to evade CD8+ T cell control over virus replicative foci

Radiation modulates the peptide repertoire, enhances MHC class I expression, and induces successful antitumor immunotherapy

Radiotherapy is one of the most successful cancer therapies. Here the effect of irradiation on antigen presentation by MHC class I molecules was studied. Cell surface expression of MHC class I molecules was increased for many days in a radiation dose-dependent manner as a consequence of three responses. Initially, enhanced degradation of existing proteins occurred which resulted in an increased intracellular peptide pool. Subsequently, enhanced translation due to activation of the mammalian target of rapamycin pathway resulted in increased peptide production, antigen presentation, as well as cytotoxic T lymphocyte recognition of irradiated cells. In addition, novel proteins were made in response to γ-irradiation, resulting in new peptides presented by MHC class I molecules, which were recognized by cytotoxic T cells. We show that immunotherapy is successful in eradicating a murine colon adenocarcinoma only when preceded by radiotherapy of the tumor tissue. Our findings indicate that directed radiotherapy can improve the efficacy of tumor immunotherapy

Protein Kinase A-induced tamoxifen resistance is mediated by anchoring protein AKAP13

Background: Estrogen Receptor alpha (ERaα)-positive breast cancer patients receive endocrine therapy, often in the form of tamoxifen. However, resistance to tamoxifen is frequently observed. A signalling cascade that leads to tamoxifen resistance is dictated by activation of the Protein Kinase A (PKA) pathway, which leads to phosphorylation of ERaα on Serine 305 and receptor activation, following tamoxifen binding. Thus far, it remains elusive what protein complexes enable the PKA-ERaα interaction resulting in ERaα Serine 305 phosphorylation. Methods: We performed immunohistochemistry to detect ERaαSerine 305 phosphorylation in a cohort of breast cancer patients who received tamoxifen treatment in the metastatic setting. From the same tumor specimens, Agilent 44 nK gene expression analyses were performed and integrated with clinicopathological data and survival information. In vitro analyses were performed using MCF7 breast cancer cells, which included immunoprecipitations and Fluorescence Resonance Energy Transfer (FRET) analyses to illustrate ERaα complex formation. siRNA mediated knockdown experiments were performed to assess effects on ERaαSerine 305 phosphorylation status, ERaα/PKA interactions and downstream responsive gene activity. Results: Stratifying breast tumors on ERaα Serine 305 phosphorylation status resulted in the identification of a gene network centered upon AKAP13. AKAP13 mRNA expression levels correlate with poor outcome in patients who received tamoxifen treatment in the metastatic setting. In addition, AKAP13 mRNA levels correlate with ERaαSerine 305 phosphorylation in breast tumor samples, suggesting a functional connection between these two events. In a luminal breast cancer cell line, AKAP13 was found to interact with ERaα as well as with a regulatory subunit of PKA. Knocking down of AKAP13 prevented PKA-mediated Serine 305 phosphorylation of ERaα and abrogated PKA-driven tamoxifen resistance, illustrating that AKAP13 is an essential protein in this process. Conclusions: We show that the PKA-anchoring protein AKAP13 is essential for the phosphorylation of ERaαS305, which leads to tamoxifen resistance both in cell lines and tamoxifen-treated breast cancer patients

Varicellovirus UL49.5 Proteins Differentially Affect the Function of the Transporter Associated with Antigen Processing, TAP

Cytotoxic T-lymphocytes play an important role in the protection against viral infections, which they detect through the recognition of virus-derived peptides, presented in the context of MHC class I molecules at the surface of the infected cell. The transporter associated with antigen processing (TAP) plays an essential role in MHC class I–restricted antigen presentation, as TAP imports peptides into the ER, where peptide loading of MHC class I molecules takes place. In this study, the UL49.5 proteins of the varicelloviruses bovine herpesvirus 1 (BHV-1), pseudorabies virus (PRV), and equine herpesvirus 1 and 4 (EHV-1 and EHV-4) are characterized as members of a novel class of viral immune evasion proteins. These UL49.5 proteins interfere with MHC class I antigen presentation by blocking the supply of antigenic peptides through inhibition of TAP. BHV-1, PRV, and EHV-1 recombinant viruses lacking UL49.5 no longer interfere with peptide transport. Combined with the observation that the individually expressed UL49.5 proteins block TAP as well, these data indicate that UL49.5 is the viral factor that is both necessary and sufficient to abolish TAP function during productive infection by these viruses. The mechanisms through which the UL49.5 proteins of BHV-1, PRV, EHV-1, and EHV-4 block TAP exhibit surprising diversity. BHV-1 UL49.5 targets TAP for proteasomal degradation, whereas EHV-1 and EHV-4 UL49.5 interfere with the binding of ATP to TAP. In contrast, TAP stability and ATP recruitment are not affected by PRV UL49.5, although it has the capacity to arrest the peptide transporter in a translocation-incompetent state, a property shared with the BHV-1 and EHV-1 UL49.5. Taken together, these results classify the UL49.5 gene products of BHV-1, PRV, EHV-1, and EHV-4 as members of a novel family of viral immune evasion proteins, inhibiting TAP through a variety of mechanisms