Biochemical and biophysical characterization of compartmentalizing proteases from the hyperthermophilic microorganism Pyrococcus furiosus

Show full item record

Title: Biochemical and biophysical characterization of compartmentalizing proteases from the hyperthermophilic microorganism Pyrococcus furiosus
Author: Chang, Lara Samofal
Advisors: Steven W. Peretti, Committee Member
David F. Ollis, Committee Member
Todd R. Klaenhammer, Committee Member
Robert M. Kelly, Committee Chair
Abstract: Proteases catalyze the cleavage of peptide bonds in peptides, polypeptides, and proteins using a hydrolysis reaction. From a biological standpoint, these enzymes are critical for cellular survival, particularly in removal of denatured proteins during stress events or of proteins that have completed their functions. Various proteases play distinct roles in the degradation of proteins, including proteinases that break down proteins and peptidases that break down the resulting oligopeptide products to single residues. The hyperthermophilic versions of proteases are useful for several reasons: they are easier to study because of their relative structural simplicity and, compared to their mesophilic counterparts, they are more stable in harsh conditions such as high heat. The focus of this study was on the biochemical and biophysical characteristics of two multi-subunit compartmentalizing proteases from the hyperthermophilic archaeon Pyrococcus furiosus (T[subscript opt]=100°C). The first protease was an oligopeptidase, PfpI (Pyrococcus furiosus protease I), and the second was a proteinase, called the proteasome. Both proteases are ubiquitous in all domains of life. However, they are theorized to have distinctly different roles within P. furiosus. The proteasome may be one of the primary proteinases, with access to its active sites tightly controlled by ATPase regulators that appear to be dependent on cellular environment. In contrast, the role of PfpI may be degradation of the smaller peptides that result from proteasome and other proteinase action. PfpI is a homo-multimer of 18.8-kDa subunits that assemble into hexameric rings. These rings then stack to form dodocamers and higher forms, with three active sites buried in hindered positions within each ring. Trimer, hexamer, and dodecamer forms were purified separately, with the dodecamer at least three-fold more specifically active than the smaller forms. It was also found that PfpI was only able to cleave oligopeptides up to 17 residues, preferring aromatic residues at the P₁ position. As the substrate length was increased, the cleavage by PfpI became less specific and confined to the C- and N-termini. The precise role of PfpI in P. furiosus still remains to be determined, with a particular need for studies of recombinantly expressed versions. The 20S proteasome, along with a theorized ATP-dependent regulator PAN (proteasome-activating nucleotidase), was investigated from several angles. Both enzymes, including native and recombinant forms, were tested for biochemical and biophysical characteristics as isolated structures and in combination. In particular, the PAN ATPase activity was tested primarily to observe its effects on different forms of the proteasome. Furthermore, both were subjected to targeted cDNA microarray experiments during heat shock of native P. furiosus. The P. furiosus proteasome was the first archaeal form investigated that contains two forms of the beta subunit instead of one. Subsequently, one of the primary focuses of the study was to elucidate the roles of the two (48% identical) beta subunits. Distinct differences in activity, stability, and level of ATPase-based stimulation were observed for the various proteasome forms. These differences were based on the presence or absence of one of the three subunits and the assembly temperature. The beta-2 subunit appeared to be the catalytic center for proteinase activity, while the beta-1 subunit played a stabilizing role. PAN was able to stimulate the native form of the proteasome during degradation of polypeptides but inhibited the native heat-shocked form in the same reactions. It was concluded that PAN, which is highly up-regulated during heat shock, may stimulate the native proteasome form, while the heat-shocked proteasome (containing higher levels of beta-1) may associate with a different set of regulating proteins.
Date: 2004-06-10
Degree: PhD
Discipline: Chemical Engineering
URI: http://www.lib.ncsu.edu/resolver/1840.16/4776


Files in this item

Files Size Format View
etd.pdf 4.155Mb PDF View/Open

This item appears in the following Collection(s)

Show full item record