Rickard Glas

Short-lived green fluorescent proteins for quantifying ubiquitin/proteasome-dependent proteolysis in living cells.

The ubiquitin/proteasome-dependent proteolytic pathway is an attractive target for therapeutics because of its critical involvement in cell cycle progression and antigen presentation. However, dissection of the pathway and development of modulators are hampered by the complexity of the system and the lack of easily detectable authentic substrates. We have developed a convenient reporter system by producing N-end rule and ubiquitin fusion degradation (UFD)-targeted green fluorescent proteins that allow quantification of ubiquitin/proteasome-dependent proteolysis in living cells. Accumulation of these reporters serves as an early predictor of G2/M arrest and apoptosis in cells treated with proteasome inhibitors. Comparison of reporter accumulation and cleavage of fluorogenic substrates demonstrates that the rate-limiting chymotrypsin-like activity of the proteasome can be substantially curtailed without significant effect on ubiquitin-dependent proteolysis. These reporters provide a new powerful tool for elucidation of the ubiquitin/proteasome pathway and for high throughput screening of compounds that selectively modify proteolysis in vivo.

A proteolytic system that compensates for loss of proteasome function

Proteolysis is essential for the execution of many cellular functions. These include removal of incorrectly folded or damaged proteins, the activation of transcription factors, the ordered degradation of proteins involved in cell cycle control, and the generation of peptides destined for presentation by class I molecules of the major histocompatibility complex. A multisubunit protease complex, the proteasome, accomplishes these tasks. Here we show that in mammalian cells inactivation of the proteasome by covalent inhibitors allows the outgrowth of inhibitor-resistant cells. The growth of such adapted cells is apparently maintained by the induction of other proteolytic systems that compensate for the loss of proteasomal activity.

Tumors Acquire Inhibitor of Apoptosis Protein (IAP)-mediated Apoptosis Resistance through Altered Specificity of Cytosolic Proteolysis

Many tumors overexpress members of the inhibitor of apoptosis protein (IAP) family. IAPs contribute to tumor cell apoptosis resistance by the inhibition of caspases, and are degraded by the proteasome to allow further progression of apoptosis. Here we show that tumor cells can alter the specificity of cytosolic proteolysis in order to acquire apoptosis resistance, which promotes formation of rapidly growing tumors. Survival of tumor cells with low proteasomal activity can occur in the presence of high expression of Tri-peptidyl-peptidase II (TPP II), a large subtilisin-like peptidase that complements proteasomal activity. We find that this state leaves tumor cells unable of effectively degrading IAPs, and that cells in this state form rapidly growing tumors in vivo. We also find, in studies of apoptosis resistant cells derived from large in vivo tumors, that these have acquired an altered peptidase activity, with up-regulation of TPP II activity and decreased proteasomal activity. Importantly, we find that growth of subcutaneous tumors is limited by maintenance of the apoptosis resistant phenotype. The apoptosis resistant phenotype was reversed by increased expression of Smac/DIABLO, an antagonist of IAP molecules. Our data suggest a reversible mechanism in regulation of apoptosis resistance that drives tumor progression in vivo. These data are relevant in relation to the multitude of therapy-resistant clinical tumors that have increased levels of IAP molecules.

PEPTIDE DEPENDENCY AND SELECTIVITY OF THE NK CELL INHIBITORY RECEPTOR LY-49C

MHC class I molecules can prevent NK cell‐mediated cytotoxicity by interacting with inhibitory receptors on the effector cells. Different conclusions have been reached regarding possible peptide selectivity of these receptors. To address whether peptide selectivity is an exclusive feature of human or immunoglobulin‐superfamily receptors, we have studied a system based on the murine NK receptor Ly‐49C in the lectin‐superfamily. Loading of TAP‐deficient RMA‐S cells with the H‐2Kb‐restricted, ovalbumin‐derived peptide OVA257 – 264 (pOVA) induced their ability to bind Ly‐49C‐transfected reporter cells, and also protected them from killing by Ly‐49C+ NK cells. Other peptides that bound and stabilized H‐2Kb equally well differed in their NK protective capacity. Comparison of the MHC class I peptide complexes (crystal structures and molecular models) revealed a conformational motif encompassing the C‐terminal parts of the α1 helix (73 – 77) and the bound peptide that was common for the protective complexes. Substitution analysis of pOVA suggested that position 7 in the peptide may be critical for optimal protection as well as for the conformational motif at position 73 – 77. In conclusion, protection mediated by the murine C‐type lectin receptor Ly‐49C is peptide dependent and selective.

β2 -Microglobulin-deficient NK cells show increased sensitivity to MHC class I-mediated inhibition, but self tolerance does not depend upon target cell expression of H-2Kb and Db heavy chains

Mice lacking β2 ‐microglobulin (β2 m− mice) express greatly reduced levels of MHC class I molecules, and cells from β2 m− mice are therefore highly sensitive NK cells. However, NK cells from β2 m− mice fail to kill β2 m− normal cells, showing that they are self tolerant. In a first attempt to understand better the basis of this tolerance, we have analyzed more extensively the target cell specificity of β2 m− NK cells. In a comparison between several MHC class I‐deficient and positive target cell pairs for sensitivity to β2 m− NK cells, we made the following observations: First, β2 m− NK cells displayed a close to normal ability to kill a panel of MHC class I‐deficient tumor cells, despite their nonresponsiveness to β2 m− concanavalin A (Con A)‐activated T cell blasts. Secondly, β2 m− NK cells were highly sensitive to MHC class I‐mediated inhibition, in fact more so than β2 m+ NK cells. Third β2 m− NK cells were not only tolerant to β2 m− Con A blasts but also to Con A blasts from H‐2Kb − /Db − double deficient mice in vitro. We conclude that NK cell tolerance against MHC class I‐deficient targets is restricted to nontransformed cells and independent of target cell expression of MHC class I free heavy chains. The enhanced ability of β2 m− NK cells to distinguish between MHC class I‐negative and ‐positive target cells may be explained by increased expression of Ly49 receptors, as described previously. However, the mechanisms for enhanced inhibition by MHC class I molecules appear to be unrelated to self tolerance in β2 m− mice, which may instead operate through mechanisms involving triggering pathways.

MHC class I antigen processing regulated by cytosolic proteolysis-short cuts that alter peptide generation.

Cytotoxic T lymphocyte (CTL)-mediated immune responses rely on the efficiency of MHC class I ligand generation and presentation by antigen presenting cells (APCs). Whereas the abnormal expression of MHC molecules and transporters associated with antigen processing (TAPs) are commonly discussed as factors that modulate antigen presentation, much less is known about possible regulatory mechanisms at the level of proteolysis responsible for the generation of antigenic peptides. The ubiquitin-proteasome system is recognized as the major component responsible for this process in the cytosol and its activity can be regulated by cytokines, such as IFN-gamma. However, new evidence suggests the involvement of other proteases that can contribute to cytosolic proteolysis and therefore, to the quality and quantity of antigen production. Here, we review recent findings on an increasing number of proteolytic enzymes linked to antigen presentation, and we discuss how regulation of cytosolic protease activities might have implications for immune escape mechanisms that could be used by tumor cells and pathogens.

Tripeptidyl-peptidase II Controls DNA Damage Responses and In vivo γ-Irradiation Resistance of Tumors

Cellular responses to gamma-irradiation exposure are controlled by phosphatidylinositol 3-kinase-related kinases (PIKK) in the nucleus, and in addition, cytosolic PIKKs may have a role in such responses. Here, we show that the expression of tripeptidyl-peptidase II (TPPII), a high molecular weight cytosolic peptidase, required PIKK signaling and that TPPII was rapidly translocated into the nucleus of gamma-irradiated cells. These events were dependent on mammalian target of rapamycin, a cytosolic/mitochondrial PIKK that is activated by gamma-irradiation. Lymphoma cells with inhibited expression of TPPII failed to efficiently stabilize p53 and had reduced ability to arrest proliferation in response to gamma-irradiation. We observed that TPPII contains a BRCA COOH-terminal-like motif, contained within sequences of several proteins involved in DNA damage signaling pathways, and this motif was important for nuclear translocation of TPPII and stabilization of p53. Novel tripeptide-based inhibitors of TPPII caused complete in vivo tumor regression in mice in response to relatively low doses of gamma-irradiation (3-4 Gy/wk). This was observed with established mouse and human tumors of diverse tissue backgrounds, with no tumor regrowth after cancellation of treatment. These TPPII inhibitors had minor effects on tumor growth as single agent and had low cellular toxicity. Our data indicated that TPPII connects signaling by cytosolic/mitochondrial and nuclear PIKK-dependent pathways and that TPPII can be targeted for inhibition of tumor therapy resistance.

Inhibition of the proteasome reduces transfer-induced diabetes in nonobese diabetic mice.

Inhibition of the 26S proteasome reduces the severity of several immune‐mediated diseases. Here, we report that the proteasome also regulates transfer‐induced diabetes in nonobese mice. Treatment of recipient mice with the proteasome inhibitor Nα‐benzyloxycarbonyl‐l‐leucyl‐l‐leucyl‐l‐leucinal (MG132) resulted in a 76% reduction in transfer‐induced diabetes. The closely related inhibitor carbobenzoxy‐l‐leucyl‐l‐leucinal that inhibits calpains but not the proteasome had no protective effect, suggesting that MG132 acted via inhibition of the proteasome. MG132 decreased proliferation of transferred T cells in the pancreatic lymph nodes in vivo and prevented their expansion in a dose‐dependent manner in vitro, consistent with a direct effect by MG132 on the T cells. MG132 did not prevent migration of transferred T cells into the islets but reduced the number of mice with severe infiltration. We suggest that MG132 prevents transfer‐induced diabetes by directly targeting the autoreactive T cells and lowering their diabetogenic potential.

Peptide vinyl sulfones : inhibitors and active site probes for the study of proteasome function in vivo

Protein degradation plays an important role in te control and regulation of many biological functions, ranging from cell cycle progression to presentation of viral antigens for scrutiny by cells of the immune system. At the heart of many of these catabolic events is the multi-catalytic proteinase complex known as the proteasome. This large barrel-shaped protein complex executes a remarkable set of functions ranging from the complete destruction of abnormal and misfolded proteins to the exquisitely specific proteolytic activation of crucial signaling molecules. Inhibitors of this proteolytic complex have thus been extremely useful for perturbing its function and deciphering its role in these widely diverse biologica; processes. The first part of this work describes the synthesis of a class of peptides in which the C-terminal carboxylic acid is replaced with a vinyl sulfone moiety. Although initially described as inhibitors of cysteine proteases, peptide vinyl sulfones containing the core sequence LeuLeuLeu are capable of covalent, irreversible inhibition of proteasomal proteolysis by modification of the catalytic, threonine hydroxyl nucleophile. Peptide vinyl sulfones composed of different amino acid sequences were also synthesized and evaluated for activity against the proteasome. These structure-function studies provide further insight into the proteolytic mechanism and substrate specificity of the proteasome. The second portion of this thesis describes the in vivo application of the peptide vinyl sulfones and in their use in uncovering the role of the proteasome in the Human cytomegalovirus (HCMV)-mediated destruction of MHC class I heavy chains The genome of the Human cytomegalovirus contains two genes, US2 and US11 which are responsible for the rapid degradation of MHC class I molecules. Treatment of HCMV infected cells with peptide vinyl sulfone