Studies from the Laboratory of Biochemistry (NHLBI) have shown that there is an accumulation of oxidized proteins with age and in some disease states. These damaged proteins are degraded by the multicatalytic protease (MCP), a multicatalytic proteolytic complex found in eukaryotic cell. In order to gain insight into the mechanism of damaged protein degradation, we have started to examine this process with two enzymes, E. coli glutamine synthetase (GS) and L. mesenteroides glucose-6- phosphate dehydrogenase (Glu-6-PDH), that have been modified by different ways: metal catalyzed oxidations, treatment by ozone and reaction with 4-hydroxy-2-nonenal (HNE), a lipid peroxidation product. These damages which inactivate the enzymes lead, in most cases, to an enhanced susceptibility of the modified protein for proteolysis by the MCP. However, treatment with HNE results only in a small increase of proteolytic susceptibility with GS and no increase with Glu-6-PDH. In the case of the latter enzyme, we have shown that, in contrast to the HNE treated enzyme, the increase of proteolytic susceptibility of the oxidized enzyme is related to a decrease of its structural stability: thermolability and binding of the hydrophobic probe 8-anilino-1- naphthalene sulfonic acid (ANSA). Modified forms of GS exhibit also an increased ability to bind ANSA which correlates with their increased susceptibility to proteolysis by MCP. However, it remains to be determined if the signal for degradation is simply due to structural destabilization and/or is due also to more specific recognition patterns. Therefore, we have looked for the ability of the MCP to degrade various forms of GS in the presence of inhibitors specific to different proteolytic activities. No striking differences have been found so far, although some of the modified forms can be efficiently degraded even in the presence of inhibitor. We are currently investigating new ways for inhibiting specifically given proteolytic activities of the MCP and other modifications of the enzymes.
