IntroductionIdiopathic pulmonary fibrosis (IPF) is a fatal disease of lung parenchymal scarring triggered by alveolar epithelial cell dysfunction. Inherited forms of pulmonary fibrosis, including ...those caused by mutant forms of surfactant protein C (SFTPC), offer an opportunity to study early pathogenic events which remain poorly understood.ObjectivesWe wished to interrogate specific factors involved in SFTPC trafficking to understand the pathological mistrafficking phenotype caused by the commonest pathogenic mutant, SFTPC-I73T, which aberrantly localises to the plasma membrane.MethodsCRISPR-Cas9 forward genetic screens were employed in an immortalised cell model system to interrogate SFTPC processing and trafficking and a FACS-based readout used to identify altered SFTPC localisation. Alveolar organoids derived from human embryonic lung tissue were transduced with an inducible CRISPRi system for the validation of screen hits.ResultsWe identified several novel targets including candidate proteases and ubiquitin ligases in addition to validating core complexes suspected to be involved in SFTPC trafficking. Of particular note is the E3 ligase ITCH which, when depleted, altered SFTPC localisation in a manner that phenocopies the pathogenic I73T mutant. Further validation is underway using genetic manipulation of a novel human embryonic alveolar organoid system.Abstract S108 Figure 1ITCH depletion results in redistribution of SFTPC to the plasma membrane. (A) Subcellular localisation of GFP-SFTPC WT and I73T expressed in HeLa cells. (B) GFP-SFTPC localises at the plasma membrane in ITCH knockout HeLa cellsConclusionsA cell line model of SFTPC trafficking has allowed us to identify important factors in SFTPC trafficking using forward genetic screens which we can test in a physiological system to understand SFTPC handling in health and disease.
Human cytomegalovirus (HCMV) persists as a subclinical, lifelong infection in the normal human host, but reactivation from latency in immunocompromised subjects results in serious disease. Latency ...and reactivation are defining characteristics of the herpesviruses and are key to understanding their biology; however, the precise cellular sites in which HCMV is carried and the mechanisms regulating its latency and reactivation during natural infection remain poorly understood. Here we present evidence, based entirely on direct analysis of material isolated from healthy virus carriers, to show that myeloid dendritic cell (DC) progenitors are sites of HCMV latency and that their ex vivo differentiation to a mature DC phenotype is linked with reactivation of infectious virus resulting from differentiation-dependent chromatin remodeling of the viral major immediate-early promoter. Thus, myeloid DC progenitors are a site of HCMV latency during natural persistence, and there is a critical linkage between their differentiation to DC and transcriptional reactivation of latent virus, which is likely to play an important role in the pathogenesis of HCMV infection.
Dysfunction of receptors for IgG (FcγRs) has been thought to be involved in the pathogenesis of systemic lupus erythematosus (SLE). We show that a recently described SLE-associated polymorphism of ...FcγRIIb (FcγRIIbT232), encoding a single transmembrane amino acid substitution, is functionally impaired. FcγRIIbT232 is unable to inhibit activatory receptors because it is excluded from sphingolipid rafts, resulting in the unopposed proinflammatory signaling thought to promote SLE.
The mK3, K3, and K5 gene products from the γ2 group of γ‐herpesviruses are the founding members of a family of membrane‐associated ubiquitin E3 ligases. As part of the viral immunoevasion strategy, ...expression of these proteins results in a decrease in cell‐surface major histocompatibility complex class I molecules and other immunoreceptors including intercellular adhesion molecule‐1, CD86, and CD1d. These viral gene products all possess a characteristic cytosolic N‐terminal RING‐CH domain, responsible for ubiquitination of the target protein, and two membrane‐spanning segments required for substrate specificity. For the majority of substrates, ubiquitination at the cell surface leads to rapid internalization and endolysosomal degradation, while mK3 ubiquitinates class I molecules associated with the peptide‐loading complex resulting in proteasome‐mediated degradation. Related viral genes with similar functions have been found in poxviruses, suggesting appropriation of these genes from the eukaryotic host. Ten membrane‐associated RING‐CH (MARCH) human genes with a similar organization have now been identified, and their overexpression leads to ubiquitination and downregulation of a variety of cell‐surface immunoreceptors. While all the MARCH proteins are predicted to act as ubiquitin E3 ligases, their physiological role and substrates remain to be defined.
Models for protein sorting at multivesicular bodies in the endocytic pathway of mammalian cells have relied largely on data obtained from yeast. These data suggest the essential role of four ESCRT ...complexes in multivesicular body protein sorting. However, the putative mammalian ESCRTII complex (hVps25p, hVps22p, and hVps36p) has no proven functional role in endosomal transport. We have characterized the human ESCRTII complex and investigated its function in endosomal trafficking. The human ESCRTII proteins interact with one another, with hVps20p (a component of ESCRTIII), and with their yeast homologues. Our interaction data from yeast two-hybrid studies along with experiments with purified proteins suggest an essential role for the N-terminal domain of hVps22p in the formation of a heterotetrameric ESCRTII complex. Although human ESCRTII is found in the cytoplasm and in the nucleus, it can be recruited to endosomes upon overexpression of dominant-negative hVps4Bp. Interestingly, we find that small interference RNA depletion of mammalian ESCRTII does not affect degradation of epidermal growth factor, a known cargo of the multivesicular body protein sorting pathway. We also show that depletion of the deubiquitinating enzymes AMSH (associated molecule with the SH3 domain of STAM (signal transducing adaptor molecule)) and UBPY (ubiquitin isopeptidase Y) have opposite effects on epidermal growth factor degradation, with UBPY depletion causing dramatic swelling of endosomes. Down-regulation of another cargo, the major histocompatibility complex class I in cells expressing the Kaposi sarcoma-associated herpesvirus protein K3, is unaffected in ESCRTII-depleted cells. Our data suggest that mammalian ESCRTII may be redundant, cargo-specific, or not required for protein sorting at the multivesicular body.
1 Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Level 5 Box 157, Hills Road, Cambridge CB2 2QQ, UK
2 Cambridge Institute for Medical Research, School of Clinical Medicine, ...University of Cambridge, Addenbrooke's Hospital, Level 5 Box 157, Hills Road, Cambridge CB2 2QQ, UK
Correspondence J. H. Sinclair js{at}mole.bio.cam.ac.uk
Human cytomegalovirus (HCMV) is a frequent cause of major disease following primary infection or reactivation from latency in immunocompromised patients. Infection of non-permissive mononuclear cells is used for analyses of HCMV latency in vitro . Using this approach, it is shown here that repression of lytic gene expression following experimental infection of CD34 + cells, a site of HCMV latency in vivo , correlates with recruitment of repressive chromatin around the major immediate-early promoter (MIEP). Furthermore, long-term culture of CD34 + cells results in carriage of viral genomes in which the MIEP remains associated with transcriptionally repressive chromatin. Finally, specific differentiation of long-term cultures of infected CD34 + cells to mature dendritic cells results in acetylation of histones bound to the MIEP, concomitant loss of heterochromatin protein 1 and the reactivation of HCMV. These data are consistent with ex vivo analyses of latency and may provide a model for further analyses of the mechanisms involved during latency and reactivation.
Published online ahead of print on 31 August 2005 as DOI 10.1099/vir.0.81161-0.
The 70 kDa mycobacterial heat shock protein (Mtb HSP70) stimulates mononuclear cells to release CC-chemokines. We now show that this function of Mtb HSP70, but not human HSP70, is dependent on the ...cell surface expression of CD40. Deletion of the CD40 cytoplasmic tail abolished, and CD40 antibody inhibited, Mtb HSP70 stimulation of CC-chemokine release. Mtb HSP70 stimulated THP1, KG1 cells, and monocyte-derived dendritic cells to produce RANTES. Specific binding of CD40-transfected HEK 293 cells to Mtb HSP70 was demonstrated by surface plasmon resonance. Coimmunoprecipitation of Mtb HSP70 with CD40 indicates a physical association between these molecules. The results suggest that CD40 is critical in microbial HSP70 binding and stimulation of RANTES production.
Assembly of MHC class I–β2 microglobulin (β2m) dimers in the endoplasmic reticulum involves two chaperones. Calnexin has previously been shown to interact with free class I heavy chains. Here, we ...show that the related chaperone, calreticulin, binds human class I–β2m dimers prior to peptide loading. Calreticulin remains associated with at least a subset of class I molecules when they, in turn, bind to TAP. Further evidence suggests that the interaction of class I–β2m dimers with TAP occurs via a novel uncharacterized 48 kDa glycoprotein, tapasin, which can bind independently to TAP and class I–β2m–calreticulin complexes. Tapasin is absent from the mutant cell line .220, in which class I–TAP association and peptide loading is defective.
Defective ribosomal products provide an important supply of endogenous peptides for entry into the classic MHC class I antigen presentation pathway. The recruitment of endoplasmic reticulum membrane ...during phagosome biogenesis allows exogenous antigens to be translocated into the cytosol as well as providing access to the class I peptide transport and loading machinery. This combination of features provides a mechanism for cross-presentation by specialised antigen presenting cells. Recent studies have shed new light on these pathways and have also described the emerging K3 family of viral ubiquitin E3 ligases, which constitutively ubiquitinate and degrade MHC class I molecules and other immunoreceptors.
The Kaposi's sarcoma‐associated herpes virus gene product K3 (KK3) subverts the MHC class I antigen presentation pathway by downregulating MHC class I from the plasma membrane. We now show that KK3 ...associates with MHC class I molecules and promotes ubiquitylation of class I after export from the endoplasmic reticulum. Ubiquitylation requires the KK3 N‐terminal plant homeodomain and provides the signal for class I internalization at the plasma membrane. Once internalized, ubiquitylated MHC class I is targeted to the late endocytic pathway, where it is degraded. Depletion by small interfering RNA of TSG101, a ubiquitin enzyme 2 variant protein involved in late endosomal sorting, prevents class I degradation and preserves cell surface class I expression in KK3‐expressing cells. These results suggest a mechanism by which the KK3‐induced class I ubiquitylation provides a signal for both internalization and sorting to the late endosomal pathway for degradation. KK3 is the first viral gene product that subverts the trafficking of a host protein via the ubiquitin‐dependent endosomal sorting machinery.
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