Alterations in activity and specificity of intracellular proteolysis in disease pathogenesis

Sammanfattning: The ubiquitin-proteasome pathway (UPP) plays a fundamental role in many basic cellular processes. Virtually all events in cellular physiology include control by the ubiquitin-proteasome pathway. Ubiquitinated substrates are degraded by the 26S proteasome, a large multicatalytic protease complex. Through the degradation of ubiquitinated proteins all eukaryotic cells vary the protein concentration of regulatory proteins within minutes, which is the key to the very sophisticated cellular regulations of e.g. the cell cycle and apoptosis. Low proteasomal activity can be compensated by increased activity of TPPII (Tri-peptidyl-peptidase II), another large cytosolic peptidase. Over-expression of TPPII can be sufficient to maintain cellular viability although the proteasome is inhibited. The aim of the work described in the Licentiate study is to understand the regulation of cellular physiology performed by cytosolic proteolysis in Cell- and Immuno-biology, with respect to proteasome and TPPII. In the first paper, we have examined if increased activity of TPPII in EL-4 Lymphoma cells growing with reduced proteasomal activity has any consequences for pathways controlled by the ubiquitin-proteasome proteolysis. In this study, we find that EL-4 lymphoma cells that can grow in the presence of low proteasomal activity acquire apoptosis resistance due to a failure in degradation of inhibitor of apoptosis proteins (IAPs). The rate of in vivo tumor growth of such cells was strongly increased. Rapid tumor growth, as well as a delayed degradation of IAPs, could be induced by transfection of TPPII. In addition, we observe a slower degradation of IAPs in cells derived from large in vivo tumors, as well as a reduced activity of the proteasome in combination with up-regulated TPPII activity. Our data suggest a novel mechanism for apotosis resistance in tumors. In paper II, we studied whether increased expression of TPPII was an essential response to stress, and redistribution of proteasomes. Here we found that suppression of the ubiquitin-proteasome pathway during stress, mediated at least in part by expression of TPPII, was crucial for cellular survival. Our data give further support for nuclear translocation of proteasomes in tumor cell resistance to stress, and suggest that TPPII has an essential role in this process. TPPII-mediated control of proteasome distribution may be a crucial control of the rate of ubiquitin-dependent proteolysis, which is relevant to many pathogenic processes. Furthermore, expression of TPPII was important for induction of growth arrest in response to cellular stress. Here, we established a link between redistribution of proteasome complexes and control of the rate of ubiquitin-dependent proteolysis. Several aberrant gene products involved in pathogenesis of neurodegenerative diseases interfere with function of the ubiquitin-proteasome pathway. Proteins containing extended repeats of Glutamine (Q) cause inhibition of the ubiquitin-pathway and apoptotic cell death. To study the mechanism of these effects (Paper III), we have investigated how stably expressed poly-Q proteins affects the ubiquitin-proteasome pathway in lymphoma cells in vitro by using a poly-Glutamine-extended N-end rule GFP reporter. We find a significantly reduced ability to degrade ubiquitin-conjugates in poly-Q expressing cells and an alteration of the distribution of proteasome from the cytosol into perinuclear location in the presence of stable expression of poly-Q proteins. These cells failed to resist low concentrations of proteasomal inhibitor, whereas they had delayed apoptosis during exposure to several other apoptotic stimuli. This study shows that poly-Glutamine proteins may affect distribution of proteasomes to alter the efficiency of the ubiquitine-proteasome pathway and give further support for a link between cellular protection responses and pathological mechanisms during expression of poly-Q proteins. These data suggest a novel mechanism that controls the ubiquitin-proteasome pathway during stress. These are relevant in relation to new therapies for multi-therapy-resistant clinical tumors and other human diseases.