The hyperthermophilic archaeon Pyrococcus furiosus genome encodes three proteasome component proteins: one ␣ protein (PF1571) and two ␤ proteins (␤1-PF1404 and ␤2-PF0159), as well as an ATPase (PF0115), referred to as proteasome-activating nucleotidase. Transcriptional analysis of the P. furiosus dynamic heat shock response (shift from 90 to 105°C) showed that the ␤1 gene was up-regulated over twofold within 5 minutes, suggesting a specific role during thermal stress. Consistent with transcriptional data, two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that incorporation of the ␤1 protein relative to ␤2 into the 20S proteasome (core particle [CP]) increased with increasing temperature for both native and recombinant versions. For the recombinant enzyme, the ␤2/␤1 ratio varied linearly with temperature from 3.8, when assembled at 80°C, to 0.9 at 105°C. The recombinant ␣؉␤1؉␤2 CP assembled at 105°C was more thermostable than either the ␣؉␤1؉␤2 version assembled at 90°C or the ␣؉␤2 version assembled at either 90°C or 105°C, based on melting temperature and the biocatalytic inactivation rate at 115°C. The recombinant CP assembled at 105°C was also found to have different catalytic rates and specificity for peptide hydrolysis, compared to the 90°C assembly (measured at 95°C). Combination of the ␣ and ␤1 proteins neither yielded a large proteasome complex nor demonstrated any significant activity. These results indicate that the ␤1 subunit in the P. furiosus 20S proteasome plays a thermostabilizing role and influences biocatalytic properties, suggesting that ␤ subunit composition is a factor in archaeal proteasome function during thermal stress, when polypeptide turnover is essential to cell survival.

Abstractα 2 -macroglobulin (α 2 M) is a 718 kDa homotetrameric proteinase inhibitor which undergoes a large conformational change upon activation. This conformational change can occur either by proteolytic attack on an ~40 amino acid stretch, the bait region, which results in the rupture of the four thioester bonds in α 2 M, or by direct nucleophilic attack on these thioesters by primary amines. Amine activation circumvents both bait region cleavage and protein incorporation, which occurs by proteolytic activation. These different activation methods allow for examination of the roles bait region cleavage and thioester rupture play in α 2 M stability. Differential scanning calorimetry and urea gel electrophoresis demonstrate that both bait region cleavage and covalent incorporation of protein ligands in the thioester pocket play critical roles in the stability of α 2 M complexes. Keywordsα 2 M; α 2 -macroglobulin; α 2 -macroglobulin and proteinase incorporation; α 2 -macroglobulin and nonproteolytic incorporation of lysozyme; α 2 -macroglobulin stabilization