Biomedical subjects

Julie A Maupin-Furlow

Publications and source records attributed to Julie A Maupin-Furlow.

15 recordsLinked to original sources

PubMed2006

Posttranslational modification of the 20S proteasomal proteins of the archaeon Haloferax volcanii.

20S proteasomes are large, multicatalytic proteases that play an important role in intracellular protein degradation. The barrel-like architecture of 20S proteasomes, formed by the stacking of four heptameric protein rings, is highly conserved from archaea to eukaryotes. The outer two rings are composed of alpha-type subunits, and the inner two rings are composed of beta-type subunits. The halophilic archaeon Haloferax volcanii synthesizes two different alpha-type proteins, alpha1 and alpha2, and one beta-type protein that assemble into at least two 20S proteasome subtypes. In this study, we demonstrate that all three of these 20S proteasomal proteins (alpha1, alpha2, and beta) are modified either post- or cotranslationally. Using electrospray ionization quadrupole time-of-flight mass spectrometry, a phosphorylation site of the beta subunit was identified at Ser129 of the deduced protein sequence. In addition, alpha1 and alpha2 contained N-terminal acetyl groups. These findings represent the first evidence of acetylation and phosphorylation of archaeal proteasomes and are one of the limited examples of post- and/or cotranslational modification of proteins in this unusual group of organisms.

Acetylation↗

PubMed2006

Trizol-based method for sample preparation and isoelectric focusing of halophilic proteins.

A persisting complication in the development of well-resolved two-dimensional PAGE maps of halophilic proteins is their natural incompatibility with isoelectric focusing (IEF). The complete desalting of samples, which is necessary for IEF, tends to aggregate halophilic proteins, often requires relatively large amounts of starting material due to significant loss of sample, and is relatively time-consuming. Here, we describe a method of preparing protein samples from the haloarchaeon Haloferax volcanii that not only desalts the samples thoroughly but also drastically reduces the amount of protein loss associated with previous sample preparation methods and prevents protein aggregation during the removal of salt. This method of sample preparation, which incorporates Trizol (phenol/guanidine isothiocyanate), can easily be extended to analyze halophilic proteins from other organisms.

Archaeal Proteins↗

PubMed2006

Proteasomes from structure to function: perspectives from Archaea.

Insight into the world of proteolysis has expanded considerably over the past decade. Energy-dependent proteases, such as the proteasome, are no longer viewed as nonspecific degradative enzymes associated solely with protein catabolism but are intimately involved in controlling biological processes that span life to death. The proteasome maintains this exquisite control by catalyzing the precisely timed and rapid turnover of key regulatory proteins. Proteasomes also interplay with chaperones to ensure protein quality and to readjust the composition of the proteome following stress. Archaea encode proteasomes that are highly related to those of eukaryotes in basic structure and function. Investigations of archaeal proteasomes coupled with those of eukaryotes has greatly facilitated our understanding of the molecular mechanisms that govern regulated protein degradation by this elaborate nanocompartmentalized machine.

Archaea↗

PubMed2006

Haloarchaeal proteases and proteolytic systems.

Proteases play key roles in many biological processes and have numerous applications in biotechnology and industry. Recent advances in the genetics, genomics and biochemistry of the halophilic Archaea provide a tremendous opportunity for understanding proteases and their function in the context of an archaeal cell. This review summarizes our current knowledge of haloarchaeal proteases and provides a reference for future research.

Archaeal Proteins↗

PubMed2005

Archaeal proteasomes and other regulatory proteases.

Numerous proteases have been shown to catalyze the precisely-timed and rapid turnover of key cellular proteins. Often these regulatory proteases are either energy-dependent or intramembrane-cleaving. In archaea, two different types of energy-dependent proteases have been characterized: 20S proteasomes associated with proteasome-activating nucleotidases and membrane-associated Lon proteases. Interestingly, homologs of all three mechanistic classes of intramembrane-cleaving proteases are widely distributed in archaea. Similar to their eucaryal and bacterial counterparts, members of these uncharacterized proteases might promote the controlled release of membrane-anchored regulatory proteins or liberate small peptide reporters and/or effectors that function in cell signaling.

Amino Acid Sequence↗

PubMed2005

Improvement of two-dimensional gel electrophoresis proteome maps of the haloarchaeon Haloferax volcanii.

Proteins of haloarchaea are remarkably unstable in low-ionic-strength solvents and tend to aggregate under standard two-dimensional (2-D) gel electrophoresis conditions, causing strong horizontal streaking. We have developed a new approach to generate 2-D maps of halophilic proteins which included washing cells with 1.5 M Tris-HCl buffer. In addition, proteins were precipitated with acetone, solubilized with urea and thiourea in the presence of the sulfobetaine detergent 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate (CHAPS), reduced with tributylphosphine (TBP), and separated with microrange strips of immobilized pH gradients (pH 3.9-5.1). This combination enabled the construction of highly reproducible 2-D maps of Haloferax volcanii proteins.

Acetone↗

PubMed2005

Construction and expression of an ethanol production operon in Gram-positive bacteria.

Pyruvate decarboxylase (PDC), an enzyme central to homoethanol fermentation, catalyses the non-oxidative decarboxylation of pyruvate to acetaldehyde with release of carbon dioxide. PDC enzymes from diverse organisms have different kinetic properties, thermal stability and codon usage that are likely to offer unique advantages for the development of desirable Gram-positive biocatalysts for use in the ethanol industry. To examine this further, pdc genes from bacteria to yeast were expressed in the Gram-positive host Bacillus megaterium. The PDC activity and protein levels were determined for each strain. In addition, the levels of pdc-specific mRNA transcripts and stability of recombinant proteins were assessed. From this analysis, the pdc gene of Gram-positive Sarcina ventriculi was found to be the most advantageous for engineering high-level synthesis of PDC in a Gram-positive host. This gene was thus selected for transcriptional coupling to the alcohol dehydrogenase gene (adh) of Geobacillus stearothermophilus. The resulting Gram-positive ethanol production operon was expressed at high levels in B. megaterium. Extracts from this recombinant were shown to catalyse the production of ethanol from pyruvate.

Ethanol↗

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

Proteasomes: perspectives from the Archaea.

The development of whole systems approaches to microbiology (e.g. genomics and proteomics) has facilitated a global view of archaeal physiology. Surprisingly, as archaea respond to environmental signals, the majority of protein concentration changes that occur are not reflected at the mRNA level. This incongruity highlights the importance of post-transcription control mechanisms in these organisms. One of the central players in proteolysis is the proteasome, a multicatalytic energy-dependent protease. Proteasomes serve both proteolytic and non-proteolytic roles in protein quality control and in the regulation of cell function. The proteolytic active sites of these enzymes are housed within a central chamber of an elaborate nanocompartment termed the 20S proteasome or core particle. Axial gates, positioned at each end of this particle, restrict the type of substrate that can access the proteolytic active sites. Assortments of regulatory AAA complexes are predicted to recognize/bind and unfold substrate proteins, open the axial gates, and translocate substrate into the 20S core particle.

Archaea↗

PubMed2004

Analysis of proteasome-dependent proteolysis in Haloferax volcanii cells, using short-lived green fluorescent proteins.

Proteasomes are energy-dependent proteases that are central to the quality control and regulated turnover of proteins in eukaryotic cells. Dissection of this proteolytic pathway in archaea, however, has been hampered by the lack of substrates that are easily detected in whole cells. In the present study, we developed a convenient reporter system by functional expression of a green fluorescent protein variant with C-terminal fusions in the haloarchaeon Haloferax volcanii. The levels of this reporter protein correlated with whole-cell fluorescence that was readily detected in culture. Accumulation of the reporter protein was dependent on the sequence of the C-terminal amino acid fusion, as well as the presence of an irreversible, proteasome-specific inhibitor (clasto-lactacystin beta-lactone). This inhibitor was highly specific for H. volcanii 20S proteasomes, with a Ki of approximately 40 nM. In contrast, phenylmethanesulfonyl fluoride did not influence the levels of fluorescent reporter protein or inhibit 20S proteasomes. Together, these findings provide a powerful tool for the elucidation of protein substrate recognition motifs and the identification of new genes which may be involved in the proteasome pathway of archaea.

Amino Acid Sequence↗

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↗

PubMed2002

Cloning and characterization of the Zymobacter palmae pyruvate decarboxylase gene (pdc) and comparison to bacterial homologues.

Pyruvate decarboxylase (PDC) is the key enzyme in all homo-ethanol fermentations. Although widely distributed among plants, yeasts, and fungi, PDC is absent in animals and rare in bacteria (established for only three organisms). Genes encoding the three known bacterial pdc genes have been previously described and expressed as active recombinant proteins. The pdc gene from Zymomonas mobilis has been used to engineer ethanol-producing biocatalysts for use in industry. In this paper, we describe a new bacterial pdc gene from Zymobacter palmae. The pattern of codon usage for this gene appears quite similar to that for Escherichia coli genes. In E. coli recombinants, the Z. palmae PDC represented approximately 1/3 of the soluble protein. Biochemical and kinetic properties of the Z. palmae enzyme were compared to purified PDCs from three other bacteria. Of the four bacterial PDCs, the Z. palmae enzyme exhibited the highest specific activity (130 U mg of protein(-1)) and the lowest Km for pyruvate (0.24 mM). Differences in biochemical properties, thermal stability, and codon usage may offer unique advantages for the development of new biocatalysts for fuel ethanol production.

Amino Acid Sequence↗

PubMed2001

Production of the Gram-positive Sarcina ventriculi pyruvate decarboxylase in Escherichia coli.

Sarcina ventriculi grows in a remarkable range of mesophilic environments from pH 2 to pH 10. During growth in acidic environments, where acetate is toxic, expression of pyruvate decarboxylase (PDC) serves to direct the flow of pyruvate into ethanol during fermentation. PDC is rare in bacteria and absent in animals, although it is widely distributed in the plant kingdom. The pdc gene from S. ventriculi is the first to be cloned and characterized from a Gram-positive bacterium. In Escherichia coli, the recombinant pdc gene from S. ventriculi was poorly expressed due to differences in codon usage that are typical of low-G+C organisms. Expression was improved by the addition of supplemental codon genes and this facilitated the 136-fold purification of the recombinant enzyme as a homo-tetramer of 58 kDa subunits. Unlike Zymomonas mobilis PDC, which exhibits Michaelis-Menten kinetics, S. ventriculi PDC is activated by pyruvate and exhibits sigmoidal kinetics similar to fungal and higher plant PDCs. Amino acid residues involved in the allosteric site for pyruvate in fungal PDCs were conserved in S. ventriculi PDC, consistent with a conservation of mechanism. Cluster analysis of deduced amino acid sequences confirmed that S. ventriculi PDC is quite distant from Z. mobilis PDC and plant PDCs. S. ventriculi PDC appears to have diverged very early from a common ancestor which included most fungal PDCs and eubacterial indole-3-pyruvate decarboxylases. These results suggest that the S. ventriculi pdc gene is quite ancient in origin, in contrast to the Z. mobilis pdc, which may have originated by horizontal transfer from higher plants.

Amino Acid Sequence↗