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|Title: ||Identification, Characterization, and Physiologic Analysis of Proteolytic Enzymes in Hyperthermophilic Organisms|
|Authors: ||Michel, Joshua Klaus|
|Advisors: ||Robert M. Kelly, Committee Chair|
Todd R. Klaenhammer, Committee Member
David F. Ollis, Committee Member
Jason M. Haugh, Committee Member
|Issue Date: ||6-Dec-2008|
|Discipline: ||Chemical Engineering|
|Abstract: ||Capable of growth at or above 80°C, hyperthermophilic organisms encode a myriad of proteolytic enzymes, including a number of homo- and hetero-multimeric complexes. These large hyperthermophilic proteases are often comprised of fewer distinct subunits compared to the less thermophilic bacterial and archaeal homologs; thus they provide an attractive model system for study. Whole genome transcriptional response analysis was used to survey both previously characterized and putative proteases in the hyperthermophilic archaea <i>Pyrococcus furiosus</i> and <i>Sulfolobus solfataricus</i> and hyperthermophilic bacterium <i>Thermotoga maritima</i>. The proteolytic transcriptional response of these three organisms demonstrated a complex synergistic relationship between the ATP-dependent proteases (responsible for initial degradation of proteins) and the ATP-independent proteases that liberate free amino acids from smaller peptides. Additionally, all three proteolytic systems showed up-regulation of protease genes involved in the degradation of misfolded and regulatory proteins during cellular stress response to changes in environmental pH and temperature. To a lesser extent, the ATP-dependent proteases (e.g. Clp) were also involved in the response of <i>T. maritima</i> to increased levels of extracellular acetate; this was accompanied by decreased transcription of metabolic genes and entry into stationary-phase.
Thermal stress conditions also affected expression and multi-subunit composition in the <i>P. furiosus</i> proteosome, yielding a more thermostable complex. The <i>P. furiosus</i> genome encodes three proteasome component proteins: one α (PF1571) and two β proteins (β1-PF1404; β2-PF0159), as well as an ATPase (PF0115), referred to as Proteasome-Activating Nucleosidase (PAN). Proteosome assembly and characteristics were found to be highly dependent on the environmental growth conditions. Increased growth temperature (shift from 90 to 105°C) resulted in a 2-fold up-regulation of β1 mRNA within five minutes, suggesting a specific role during thermal stress. Consistent with this data, two-dimensional SDS PAGE revealed that incorporation of the β1 protein relative to β2 into the 20S proteasome (or core particle, CP) increased with increasing temperature for both native and recombinant versions. The recombinant form of PFα+PFβ1+PFβ2 CP assembled at 105°C was found to be more thermostable and have different catalytic rates and substrate specificities, when compared with a recombinant form of PFα+PFβ1+PFβ2 assembled at 90°C or the PFα+PFβ2 version assembled at either 90°C or 105°C. These results indicate that the β1 subunit in the <i>P. furiosus</i> 20S proteasome plays a thermostabilizing role in archaeal proteasome function during thermal stress when polypeptide turnover is essential to cell survival.
In contrast to <i>P. furiosus</i>, the hyperthermophilic archaeon <i>Archaeoglobus fulgidus</i> produces a 20S proteasome comprised of two distinct subunits, α (AF0490) and β (AF0481). Combination of <i>A. fulgidus</i> &alpha and <i>P. furiosus</i> β1 and/or β2 yielded hybrid proteasome CPs that display characteristics different then the wild-type enzymes. Notably, <i>A. fulgidus</i> α was found to preferentially assemble with <i>P. furiosus</i> β1, even in the presence of AFα. The <i>A. fulgidus</i> recombinant proteasome exhibited comparable biochemical properties to the <i>P. furiosus</i> complex (α+β2 or &alpha+β1+β2), albeit with a reduced optimal temperature. However, the recombinant <i>A. fulgidus</i> 20S proteasome and hybrid CPs were not substrate-inhibited as was the case for the recombinant <i>P. furiosus</i> 20S proteasome. Taken together, these results demonstrate that proteasomes can be constructed with subunits from different hyperthermophiles, and that subunit composition influences biochemical and biophysical properties. The fact that hybrid inter-generic versions can be created in vitro also suggests that CPs in particular archaea may have arisen from common sources. Furthermore, the ability to interchange subunits and alter composition of the proteasome suggests that this system may provide a useful platform for designing proteases with unique activities or specific biophysical properties required for any biotechnological application.|
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