Biomedical subjects

Steven J Kaczowka

Publications and source records attributed to Steven J Kaczowka.

4 recordsLinked to original sources

PubMed2005

Recombinant production of Zymomonas mobilis pyruvate decarboxylase in the haloarchaeon Haloferax volcanii.

The unusual physiological properties of archaea (e.g., growth in extreme salt concentration, temperature and pH) make them ideal platforms for metabolic engineering. Towards the ultimate goal of modifying an archaeon to produce bioethanol or other useful products, the pyruvate decarboxylase gene of Zymomonas mobilis (Zm pdc) was expressed in Haloferax volcanii. This gene has been used successfully to channel pyruvate to ethanol in various Gram-negative bacteria, including Escherichia coli. Although the ionic strength of the H. volcanii cytosol differs over 15-fold from that of E. coli, gel filtration and circular dichroism revealed no difference in secondary structure between the ZmPDC protein isolated from either of these hosts. Like the E. coli purified enzyme, ZmPDC from H. volcanii catalyzed the nonoxidative decarboxylation of pyruvate. A decrease in the amount of soluble ZmPDC protein was detected as H. volcanii transitioned from log phase to late stationary phase that was inversely proportional to the amount of pdc-specific mRNA. Based on these results, proteins from non-halophilic organisms can be actively synthesized in haloarchaea; however, post-transcriptional mechanisms present in stationary phase appear to limit the amount of recombinant protein expressed.

Biotechnology↗

PubMed2004

Differential regulation of the PanA and PanB proteasome-activating nucleotidase and 20S proteasomal proteins of the haloarchaeon Haloferax volcanii.

The halophilic archaeon Haloferax volcanii produces three different proteins (alpha1, alpha2, and beta) that assemble into at least two 20S proteasome isoforms. This work reports the cloning and sequencing of two H. volcanii proteasome-activating nucleotidase (PAN) genes (panA and panB). The deduced PAN proteins were 60% identical with Walker A and B motifs and a second region of homology typical of AAA ATPases. The most significant region of divergence was the N terminus predicted to adopt a coiled-coil conformation involved in substrate recognition. Of the five proteasomal proteins, the alpha1, beta, and PanA proteins were the most abundant. Differential regulation of all five genes was observed, with a four- to eightfold increase in mRNA levels as cells entered stationary phase. In parallel with this mRNA increase, the protein levels of PanB and alpha2 increased severalfold during the transition from exponential growth to stationary phase, suggesting that these protein levels are regulated at least in part by mechanisms that control transcript levels. In contrast, the beta and PanA protein levels remained relatively constant, while the alpha1 protein levels exhibited only a modest increase. This lack of correlation between the mRNA and protein levels for alpha1, beta, and PanA suggests posttranscriptional mechanisms are involved in regulating the levels of these major proteasomal proteins. Together these results support a model in which the cell regulates the ratio of the different 20S proteasome and PAN proteins to modulate the structure and ultimately the function of this central energy-dependent proteolytic system.

Adenosine Triphosphatases↗

PubMed2003

Archaeal proteasomes: potential in metabolic engineering.

Archaea are a valuable source of enzymes for industrial and scientific applications because of their ability to survive extreme conditions including high salt and temperature. Thanks to advances in molecular biology and genetics, archaea are also attractive hosts for metabolic engineering. Understanding how energy-dependent proteases and chaperones function to maintain protein quality control is key to high-level synthesis of recombinant products. In archaea, proteasomes are central players in energy-dependent proteolysis and form elaborate nanocompartments that degrade proteins into oligopeptides by processive hydrolysis. The catalytic core responsible for this proteolytic activity is the 20S proteasome, a barrel-shaped particle with a central channel and axial gates on each end that limit substrate access to a central proteolytic chamber. AAA proteins (ATPases associated with various cellular activities) are likely to play several roles in mediating energy-dependent proteolysis by the proteasome. These include ATP binding/hydrolysis, substrate binding/unfolding, opening of the axial gates, and translocation of substrate into the proteolytic chamber.

Adenosine Triphosphatases↗

PubMed2003

Subunit topology of two 20S proteasomes from Haloferax volcanii.

Haloferax volcanii, a halophilic archaeon, synthesizes three different proteins (alpha1, alpha2, and beta) which are classified in the 20S proteasome superfamily. The alpha1 and beta proteins alone form active 20S proteasomes; the role of alpha2, however, is not clear. To address this, alpha2 was synthesized with an epitope tag and purified by affinity chromatography from recombinant H. volcanii. The alpha2 protein copurified with alpha1 and beta in a complex with an overall structure and peptide-hydrolyzing activity comparable to those of the previously described alpha1-beta proteasome. Supplementing buffers with 10 mM CaCl(2) stabilized the halophilic proteasomes in the absence of salt and enabled them to be separated by native gel electrophoresis. This facilitated the discovery that wild-type H. volcanii synthesizes more than one type of 20S proteasome. Two 20S proteasomes, the alpha1-beta and alpha1-alpha2-beta proteasomes, were identified during stationary phase. Cross-linking of these enzymes, coupled with available structural information, suggested that the alpha1-beta proteasome was a symmetrical cylinder with alpha1 rings on each end. In contrast, the alpha1-alpha2-beta proteasome appeared to be asymmetrical with homo-oligomeric alpha1 and alpha2 rings positioned on separate ends. Inter-alpha-subunit contacts were only detected when the ratio of alpha1 to alpha2 was perturbed in the cell using recombinant technology. These results support a model that the ratio of alpha proteins may modulate the composition and subunit topology of 20S proteasomes in the cell.

Blotting, Western↗