Elimination In Vivo of Developing T Cells by Natural Killer Cells

Natural killer cells gauge the absence of self class I MHC on susceptible target cells by means of inhibitory receptors such as members of the Ly49 family. To initiate killing by natural killer cells, a lack of inhibitory signals must be accompanied by the presence of activating ligands on the target cell. Although natural killer cell–mediated rejection of class I MHC–deficient bone marrow (BM) grafts is a matter of record, little is known about the targeting in vivo of specific cellular subsets by natural killer cells. We show here that development of class I MHC–negative thymocytes is delayed as a result of natural killer cell toxicity after grafting of a class I MHC–positive host with class I MHC–negative BM. Double positive thymocytes that persist in the presence of natural killer cells display an unusual T cell receptor–deficient phenotype, yet nevertheless give rise to single positive thymocytes and yield mature class I MHC–deficient lymphocytes that accumulate in the class I MHC–positive host. The resulting class I MHC–deficient CD8 T cells are functional and upon activation remain susceptible to natural killer cell toxicity in vivo. Reconstitution of class I MHC–deficient BM precursors with H2-Kb by retroviral transduction fully restores normal thymic development

ElaD, a Deubiquitinating Protease Expressed by E. Coli

Background: Ubiquitin and ubiquitin-like proteins (Ubl) are designed to modify polypeptides in eukaryotes. Covalent binding of ubiquitin or Ubls to substrate proteins can be reversed by specific hydrolases. One particular set of cysteine proteases, the CE clan, which targets ubiquitin and Ubls, has homologs in eukaryotes, prokaryotes, and viruses. Findings: We have cloned and analyzed the E. coli protein elaD, which is distantly related to eukaryotic CE clan members of the ULP/SENP protease family that are specific for SUMO and Nedd8. Previously misannotated as a putative sulfatase/phosphatase, elaD is an efficient and specific deubiquitinating enzyme in vitro. Interestingly, elaD is present in all intestinal pathogenic E. coli strains, but conspicuously absent from extraintestinal pathogenic strains (ExPECs). Further homologs of this protease can be found in Acanthamoeba Polyphaga Mimivirus, and in Alpha-, Beta-and Gammaproteobacteria. Conclusion: The expression of ULP/SENP-related hydrolases in bacteria therefore extends to plant pathogens and medically relevant strains of Escherichia coli, Legionella pneumophila, Rickettsiae, Chlamydiae, and Salmonellae, in which the elaD ortholog sseL has recently been identified as a virulence factor with deubiquitinating activity. As a counterpoint, our phylogenetic and functional examination reveals that ancient eukaryotic ULP/SENP proteases also have the potential of ubiquitin-specific hydrolysis, suggesting an early common origin of this peptidase clan

Chemoenzymatic Site-Specific Labeling of Influenza Glycoproteins as a Tool to Observe Virus Budding in Real Time

The influenza virus uses the hemagglutinin (HA) and neuraminidase (NA) glycoproteins to interact with and infect host cells. While biochemical and microscopic methods allow examination of the early steps in flu infection, the genesis of progeny virions has been more difficult to follow, mainly because of difficulties inherent in fluorescent labeling of flu proteins in a manner compatible with live cell imaging. We here apply sortagging as a chemoenzymatic approach to label genetically modified but infectious flu and track the flu glycoproteins during the course of infection. This method cleanly distinguishes influenza glycoproteins from host glycoproteins and so can be used to assess the behavior of HA or NA biochemically and to observe the flu glycoproteins directly by live cell imaging

Peptide Antagonism and T Cell Receptor Interactions with Peptide-MHC Complexes

AbstractWe describe antagonist peptides that specifically inhibit cytolytic activity of T cell clones and lines that express the antigen-specific receptor of CD8+ T lymphocyte clone 2C, which recognizes peptides in association with syngeneic (Kb) and allogeneic (Ld) MHC proteins. Addition of an antagonist peptide that can bind to Kb on 2C cells decreased the tyrosine phosphorylation of CD3 ζ chains elicited by prior exposure of the cells to an agonist peptide-Kb complex. Contrary to previous agonist-antagonist comparisons, the 2C T cell receptor had higher affinity for an antagonist peptide-Kb complex than for a weak agonist peptide-Kb complex. This difference is considered in light of evidence that antigen-specific receptor affinity values can be substantially higher when determined with the receptor on live cells than with the receptor in cell-free systems

XBP-1 specifically promotes IgM synthesis and secretion, but is dispensable for degradation of glycoproteins in primary B cells

Differentiation of B cells into plasma cells requires X-box binding protein–1 (XBP-1). In the absence of XBP-1, B cells develop normally, but very little immunoglobulin is secreted. XBP-1 controls the expression of a large set of genes whose products participate in expansion of the endoplasmic reticulum (ER) and in protein trafficking. We define a new role for XBP-1 in exerting selective translational control over high and sustained levels of immunoglobulin M (IgM) synthesis. XBP-1−/− and XBP-1+/+ primary B cells synthesize IgM at comparable levels at the onset of stimulation with lipopolysaccharide or CpG. However, later there is a profound depression in synthesis of IgM in XBP-1−/− B cells, notwithstanding similar levels of μmRNA. In marked contrast, lack of XBP-1 does not affect synthesis and trafficking of other glycoproteins, or of immunoglobulin light chains. Contrary to expectation, degradation of proteins from the ER, using TCRα or US11-mediated degradation of class I major histocompatibility complex molecules as substrates, is normal in XBP-1−/− B cells. Furthermore, degradation of membrane μ was unaffected by enforced expression of XBP-1. We conclude that in primary B cells, the XBP-1 pathway promotes synthesis and secretion of IgM, but does not seem to be involved in the degradation of ER proteins, including that of μ chains themselves

Screen for ISG15-crossreactive Deubiquitinases

Background: The family of ubiquitin-like molecules (UbLs) comprises several members, each of which has sequence, structural, or functional similarity to ubiquitin. ISG15 is a homolog of ubiquitin in vertebrates and is strongly upregulated following induction by type I interferon. ISG15 can be covalently attached to proteins, analogous to ubiquitination and with actual support of ubiquitin conjugating factors. Specific proteases are able to reverse modification with ubiquitin or UbLs by hydrolyzing the covalent bond between their C-termini and substrate proteins. The tail regions of ubiquitin and ISG15 are identical and we therefore hypothesized that promiscuous deubiquitinating proteases (DUBs) might exist, capable of recognizing both ubiquitin and ISG15. Results: We have cloned and expressed 22 human DUBs, representing the major clades of the USP protease family. Utilizing suicide inhibitors based on ubiquitin and ISG15, we have identified USP2, USP5 (IsoT1), USP13 (IsoT3), and USP14 as ISG15-reactive proteases, in addition to the bona fide ISG15-specific protease USP18 (UBP43). USP14 is a proteasome-associated DUB, and its ISG15 isopeptidase activity increases when complexed with the proteasome. Conclusions: By evolutionary standards, ISG15 is a newcomer among the UbLs and it apparently not only utilizes the conjugating but also the deconjugating machinery of its more established relative ubiquitin. Functional overlap between these two posttranslational modifiers might therefore be more extensive than previously appreciated and explain the rather innocuous phenotype of ISG15 null mice