Lidia M. Duncan

CD40 Is a Cellular Receptor Mediating Mycobacterial Heat Shock Protein 70 Stimulation of CC-Chemokines

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.

Lysine‐63‐linked ubiquitination is required for endolysosomal degradation of class I molecules

MHC class I molecules display peptides from endogenous and viral proteins for immunosurveillance by cytotoxic T lymphocytes (CTL). The importance of the class I pathway is emphasised by the remarkable strategies employed by different viruses to downregulate surface class I and avoid CTL recognition. The K3 gene product from Kaposis sarcoma‐associated herpesvirus (KSHV) is a viral ubiquitin E3 ligase which ubiquitinates and degrades cell surface MHC class I molecules. We now show that modification of K3‐associated class I by lysine‐63‐linked polyubiquitin chains is necessary for their efficient endocytosis and endolysosomal degradation and present three lines of evidence that monoubiquitination of class I molecules provides an inefficient internalisation signal. This lysine‐63‐linked polyubiquitination requires both UbcH5b/c and Ubc13‐conjugating enzymes for initiating mono‐ and subsequent polyubiquitination of class I, and the clathrin‐dependent internalisation is mediated by the epsin endocytic adaptor. Our results explain how lysine‐63‐linked polyubiquitination leads to degradation by an endolysosomal pathway and demonstrate a novel mechanism for endocytosis and endolysosomal degradation of class I, which may be applicable to other receptors.

Ubiquitylation of MHC class I by the K3 viral protein signals internalization and TSG101‐dependent degradation

The Kaposis 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.

Downregulation of cell surface receptors by the K3 family of viral and cellular ubiquitin E3 ligases

Summary:  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.

Cloning of JAM-2 and JAM-3: an Emerging Junctional Adhesion Molecular Family

Throughout embryonic and early postnatal development, endothelial cells proliferate and differentiate to form new blood vessels via vasculogenesis and angiogenesis (1, 2). In adult organisms the endothelium defines the blood-tissue barrier and consists of non-cycling quiescent cells. These polarized cells are linked to each other by tight junctions and adherens junctions to form a continuous layer of cells. JAM (hereafter referred as JAM-1) and VE-cadherin were characterized as endothelial adhesion molecules participating in tight and adherens intercellular junctions respectively (3, 4). These molecules were shown to regulate vascular functions such as monocyte transmigration or paracellular permeability, probably as a consequence of their structural contribution to the vessel wall. It is well established that the regulated and coordinated expression of adhesion molecules is necessary to maintain normal vascular functions such as tissue homeostasis, vascular permeability, leukocyte emigration, fibrinolysis, coagulation and vasotonus. Temporary changes in the adhesion properties of vascular endothelium have been observed during inflammation, tumor growth, wounding or angiogenesis (5-7). The presence of a growing tumor increases the local concentration of angiogenic factors leading to a switch from non-cycling quiescent endothelial cells to proliferating endothelium. As a result, endothelial cells of existing vessels degrade the extracellular matrix (ECM) and invade the surrounding tissue, which leads to vascularization of tumors. During the angiogenic switch, the pattern of endothelial gene expression is modified, and the level of transcripts encoding protein s involved in cell migration or cell division is affected. For example, the balance between proteases/antiproteases such as PA/PAI1 is changed, leading to increased endothelial cell motility (8). Moreover, the treatment of endothelial cells with angiogenic factors results in a fourfold increase in αVβ3 integrin expression, an adhesion molecule implicated in cell migration (9). In addition, angiogenesis was shown to modify the endothelial inflammatory response leading to abnormal expression of inflammatory adhesion molecules (10, 11). These examples do not constitute an exhaustive list, but support the hypothesis that during the angiogenic switch, the global adhesion behavior of endothelial cells is changed.

Solution Structure of the Kaposi's Sarcoma-associated Herpesvirus K3 N-terminal Domain Reveals a Novel E2-binding C4HC3-type RING Domain

RING domains are found in a large number of eukaryotic proteins. Most function as E3 ubiquitin-protein ligases, catalyzing the terminal step in the ubiquitination process. Structurally, these domains have been characterized as binding two zinc ions in a stable cross-brace motif. The tumorigenic human γ-herpesvirus Kaposis sarcoma-associated herpesvirus encodes a ubiquitin-protein ligase termed K3, which functions as an immune evasion molecule by ubiquitinating major histocompatibility complex class I. K3 possesses at its N terminus a domain related to cellular RING domains but with an altered zinc ligand arrangement. This domain was initially characterized as a plant homeodomain, a structure not previously known to function as an E3. Here, it is conclusively demonstrated that the K3 N-terminal domain is a variant member of the RING domain family and not a plant homeodomain. The domain is found to interact with the cellular ubiquitin-conjugating enzymes UbcH5A to -C and UbcH13, which dock to the equivalent surface as on classical cellular RING domains. Interaction with UbcH13 suggests a possible role for K3 in catalyzing Lys63-linked ubiquitination.

The RNA‐binding E3 ubiquitin ligase MEX‐3C links ubiquitination with MHC‐I mRNA degradation

RNA‐binding E3 ubiquitin ligases were recently identified, though their function remains unclear. While studying the regulation of the MHC class I (MHC‐I) pathway, we here characterize a novel role for ubiquitin in mRNA degradation. MHC‐I molecules provide ligands for both cytotoxic T‐lymphocytes as well as natural killer (NK) cells, and play a central role in innate and adaptive immunity. MHC‐I cell‐surface expression is closely monitored by NK cells, whose killer immunoglobulin‐like receptors encode MHC‐I‐specific activatory and inhibitory receptors, implying that MHC‐I expression needs to be tightly regulated. In a functional siRNA ubiquitome screen we identified MEX‐3C, a novel RNA‐binding ubiquitin E3 ligase, as responsible for the post‐transcriptional, allotype‐specific regulation of MHC‐I. MEX‐3C binds the 3′UTR of HLA‐A2 mRNA, inducing its RING‐dependent degradation. The RING domain of MEX‐3C is not required for HLA‐A2 cell‐surface downregulation, but regulates the degradation of HLA‐A2 mRNA. We have therefore uncovered a novel post‐transcriptional pathway for regulation of HLA‐A allotypes and provide a link between ubiquitination and mRNA degradation.

Tapasin-related protein TAPBPR is an additional component of the MHC class I presentation pathway

Tapasin is an integral component of the peptide-loading complex (PLC) important for efficient peptide loading onto MHC class I molecules. We investigated the function of the tapasin-related protein, TAPBPR. Like tapasin, TAPBPR is widely expressed, IFN-γ–inducible, and binds to MHC class I coupled with β2-microglobulin in the endoplasmic reticulum. In contrast to tapasin, TAPBPR does not bind ERp57 or calreticulin and is not an integral component of the PLC. β2-microglobulin is essential for the association between TAPBPR and MHC class I. However, the association between TAPBPR and MHC class I occurs in the absence of a functional PLC, suggesting peptide is not required. Expression of TAPBPR decreases the rate of MHC class I maturation through the secretory pathway and prolongs the association of MHC class I on the PLC. The TAPBPR:MHC class I complex trafficks through the Golgi apparatus, demonstrating a function of TAPBPR beyond the endoplasmic reticulum/cis-Golgi. The identification of TAPBPR as an additional component of the MHC class I antigen-presentation pathway demonstrates that mechanisms controlling MHC class I expression remain incompletely understood.

Haploid Genetic Screens Identify an Essential Role for PLP2 in the Downregulation of Novel Plasma Membrane Targets by Viral E3 Ubiquitin Ligases

The Kaposis sarcoma-associated herpesvirus gene products K3 and K5 are viral ubiquitin E3 ligases which downregulate MHC-I and additional cell surface immunoreceptors. To identify novel cellular genes required for K5 function we performed a forward genetic screen in near-haploid human KBM7 cells. The screen identified proteolipid protein 2 (PLP2), a MARVEL domain protein of unknown function, as essential for K5 activity. Genetic loss of PLP2 traps the viral ligase in the endoplasmic reticulum, where it is unable to ubiquitinate and degrade its substrates. Subsequent analysis of the plasma membrane proteome of K5-expressing KBM7 cells in the presence and absence of PLP2 revealed a wide range of novel K5 targets, all of which required PLP2 for their K5-mediated downregulation. This work ascribes a critical function to PLP2 for viral ligase activity and underlines the power of non-lethal haploid genetic screens in human cells to identify the genes involved in pathogen manipulation of the host immune system.

Fluorescence-based phenotypic selection allows forward genetic screens in haploid human cells.

The isolation of haploid cell lines has recently allowed the power of forward genetic screens to be applied to mammalian cells. The interest in applying this powerful genetic approach to a mammalian system is only tempered by the limited utility of these screens, if confined to lethal phenotypes. Here we expand the scope of these approaches beyond live/dead screens and show that selection for a cell surface phenotype via fluorescence-activated cell sorting can identify the key molecules in an intracellular pathway, in this case MHC class I antigen presentation. Non-lethal haploid genetic screens are widely applicable to identify genes involved in essentially any cellular pathway.