Catching Proteases in Action with Microarrays

AbstractProteases regulate many essential functions in biology, yet their precise roles are only beginning to be unraveled. In this issue, two related papers describe a novel method to dissect specific protease activities from complex mixtures [1, 2]

tRNA Thiolation Links Translation to Stress Responses in Saccharomyces cerevisiae

Although tRNA modifications have been well catalogued, the precise functions of many modifications and their roles in mediating gene expression are still being elucidated. While tRNA modifications were long assumed to be constitutive, it is now apparent that the modification status of tRNAs changes in response to different environmental conditions. The URM1 pathway is required for thiolation of the cytoplasmic tRNAs tGlu[superscript UUC], tGln[superscript UUG] and tLys[superscript UUU] in Saccharomyces cerevisiae. We demonstrate that URM1 pathway mutants have impaired translation, which results in increased basal activation of the Hsf1-mediated heat shock response; we also find that tRNA thiolation levels in wild type cells decrease when cells are grown at elevated temperature. We show that defects in tRNA thiolation can be conditionally advantageous, conferring resistance to endoplasmic reticulum stress. URM1 pathway proteins are unstable, and hence are more sensitive to changes in the translational capacity of cells, which is decreased in cells experiencing stresses. We propose a model in which a stress-induced decrease in translation results in decreased levels of URM1 pathway components, which results in decreased tRNA thiolation levels, which further serves to decrease translation. This mechanism ensures that tRNA thiolation and translation are tightly coupled and coregulated according to need

Road to Ruin: Targeting Proteins for Degradation in the Endoplasmic Reticulum

Some nascent proteins that fold within the endoplasmic reticulum (ER) never reach their native state. Misfolded proteins are removed from the folding machinery, dislocated from the ER into the cytosol, and degraded in a series of pathways collectively referred to as ER-associated degradation (ERAD). Distinct ERAD pathways centered on different E3 ubiquitin ligases survey the range of potential substrates. We now know many of the components of the ERAD machinery and pathways used to detect substrates and target them for degradation. Much less is known about the features used to identify terminally misfolded conformations and the broader role of these pathways in regulating protein half-lives.National Institutes of Health (U.S.

Cathepsin S regulates class II MHC processing in human CD4+ HLA-DR+ T cells

July 15, 2010Although it has long been known that human CD4+ T cells can express functional class II MHC molecules, the role of lysosomal proteases in the T cell class II MHC processing and presentation pathway is unknown. Using CD4+ T cell clones that constitutively express class II MHC, we determined that cathepsin S is necessary for invariant chain proteolysis in T cells. CD4+HLA-DR+ T cells down-regulated cathepsin S expression and activity 18 h after activation, thereby ceasing nascent class II MHC product formation. This blockade resulted in the loss of the invariant chain fragment CLIP from the cell surface, suggesting that—like professional APC—CD4+ HLA-DR+ cells modulate self-Ag presentation as a consequence of activation. Furthermore, cathepsin S expression and activity, and concordantly cell surface CLIP expression, was reduced in HLA-DR+ CD4+ T cells as compared with B cells both in vitro and ex vivo

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

Massively Parallel Microfluidic Cell-Pairing Platform for the Statistical Study of Immunological Cell-Cell Interactions

Variability in cell-cell interactions is ubiquitous and particularly relevant for the immune system, where the reliability of cell-cell interactions is critical for the prevention of disease. This variability is poorly understood mainly due to the limitations of current methods. We have therefore designed a highly parallel microfluidic cell-pairing device and optimized its pairing efficiency using fluids modeling. The optimized device can hydrodynamically pair hundreds of primary mouse immune-cells at an efficiency of ~50%. We measured T cell activation dynamics of ~130 primary mouse T cells paired with B cells. Our findings represent the first time that variation has been observed in T cell activation dynamics.National Institutes of Health (U.S.) (NIH (EB008550))Singapore-MIT Allianc

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