Biomedical subjects

Paul J Lehner

Publications and source records attributed to Paul J Lehner.

12 recordsLinked to original sources

PubMed2006

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 Kaposi's 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.

Adaptor Proteins, Vesicular Transport↗

PubMed2005

Degradation of endocytosed epidermal growth factor and virally ubiquitinated major histocompatibility complex class I is independent of mammalian ESCRTII.

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.

Animals↗

PubMed2005

Loss of function of a lupus-associated FcgammaRIIb polymorphism through exclusion from lipid rafts.

Dysfunction of receptors for IgG (FcgammaRs) has been thought to be involved in the pathogenesis of systemic lupus erythematosus (SLE). We show that a recently described SLE-associated polymorphism of FcgammaRIIb (FcgammaRIIbT(232)), encoding a single transmembrane amino acid substitution, is functionally impaired. FcgammaRIIbT(232) is unable to inhibit activatory receptors because it is excluded from sphingolipid rafts, resulting in the unopposed proinflammatory signaling thought to promote SLE.

Amino Acid Substitution↗

PubMed2005

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

The mK3, K3, and K5 gene products from the gamma2 group of gamma-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.

Animals↗

PubMed2004

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 gamma-herpesvirus Kaposi's 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 Lys(63)-linked ubiquitination.

Amino Acid Sequence↗

PubMed2004

Recent developments in MHC-class-I-mediated antigen presentation.

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.

Animals↗

PubMed2004

HSP70 peptide binding mutants separate antigen delivery from dendritic cell stimulation.

Microbial heat shock proteins (HSPs) have been implicated in the induction of both the innate and adaptive arms of the immune response. We now show that human dendritic cells (DC) pulsed with peptide-loaded mycobacterial HSP70 complexes generate potent antigen-specific cytotoxic lymphocyte (CTL) responses, which are dependent on an HSP70-stimulated calcium signaling cascade. From the calculated peptide binding affinity of mycobacterial HSP70 (K(D) = 14 microM) we show that 120 pM HSP70 bound peptide is sufficient to generate a peptide-specific CTL response that is up to four orders of magnitude more efficient than peptide alone. The minimal 136 amino acid, mycobacterial HSP70 peptide binding domain can generate CTL responses, and a single amino acid mutant HSP70 designed to prevent peptide binding but retain stimulatory capacity has allowed us to separate antigen delivery from DC immunostimulation.

Animals↗

PubMed2004

Viral degradation of the MHC class I peptide loading complex.

The murine gamma-herpesvirus-68 MK3 protein inhibits CD8(+) T cell recognition by ubiquitinating the cytoplasmic tails of classical MHC class I heavy chains. Here we show that MK3 also provides the first example of a protein that degrades tapasin and TAP. The degradation was MK3 RING finger dependent and primarily affected TAP. MK3 associated with TAP1 in the absence of tapasin or TAP2, suggesting that TAP1 was a primary binding partner in the peptide loading complex. TAP2 also played a major role in MK3 stability and function. By degrading TAP, therefore, MK3 limited its own expression. However, TAP degradation also broadened the MK3 inhibitory repertoire and achieved a remarkable resistance to MHC class I upregulation by interferon-gamma, suggesting that it represents a specific adaptation to immune evasion in lymphoid tissue.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2003

Tapasin is a facilitator, not an editor, of class I MHC peptide binding.

Tapasin has been proposed to function as a peptide editor to displace lower affinity peptides and/or to favor the binding of high affinity peptides. Consistent with this, cell surface HLA-B8 molecules in tapasin-deficient cells were less stable and the peptide repertoire was substantially altered. However, the binding affinities of peptides expressed in the absence of tapasin were unexpectedly higher, not lower. The peptide repertoire from cells expressing soluble tapasin was similar in both appearance and affinity to that presented in the presence of full-length tapasin, but the HLA-B8 molecules showed altered cell surface stability characteristics. Similarly, the binding affinities of HLA-A*0201-associated peptides from tapasin(+) and tapasin(-) cells were equivalent, although steady state HLA-A*0201 cell surface expression was decreased and the molecules demonstrated reduced cell surface stability on tapasin(-) cells. These data are inconsistent with a role for tapasin as a peptide editor. Instead, we propose that tapasin acts as a peptide facilitator. In this role, it stabilizes the peptide-free conformation of class I MHC molecules in the endoplasmic reticulum and thus increases the number and variety of peptides bound to class I MHC. Full-length tapasin then confers additional stability on class I MHC molecules that are already associated with peptides.

Antigen Presentation↗

PubMed2003

The ABC-transporter signature motif is required for peptide translocation but not peptide binding by TAP.

The transporter associated with antigen processing (TAP) translocates peptides from their site of generation in the cytosol to the lumen of the endoplasmic reticulum for binding to MHC class I molecules. TAP is a member of the ATP-binding cassette (ABC) transporter family whose members utilize energy from ATP hydrolysis to translocate substrates across membranes. The highly conserved nucleotide-binding domains of ABC transporters couple ATP hydrolysis to substrate translocation by the membrane domains. The conserved 'signature motif' can be identified in the nucleotide-binding domains of all ABC transporters, and may play a role in ATP hydrolysis. Here we show that introduction of mutations into the signature motifs of either TAP1 or TAP2 inhibits the translocation of peptide without affecting binding of either peptide or ATP by TAP. We therefore conclude that the signature motifs in both TAP1 and TAP2 are required after peptide binding to facilitate peptide translocation by TAP.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2003

The calculus of immunity: quantitating antigen processing.

Peptide ligands destined for binding MHC class I molecules are generated by the proteasome in the cytoplasm and transported to the endoplasmic reticulum for loading onto newly synthesized class I molecules. A new study now provides a quantitative analysis of how total protein turnover in the cell relates to the efficiency of generating peptide epitopes presented by class I molecules.

Animals↗

PubMed2002

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

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.

DNA-Binding Proteins↗