Progress 10/01/10 to 09/30/11

Outputs
OUTPUTS: Archaea are the least characterized among the three domains of life, yet represent a group of microorganisms with tremendous potential toward furthering CRIS missions to understand global nutrient cycles, discover new pathways of carbon sequestration and foster a bio-based economy. Archaea contribute to a large fraction of the Earth's biomass and have central roles in global inorganic and organic cycles including nitrogen fixation, anaerobic oxidation of methane, methanogenesis and novel pathways of CO2 fixation. Furthermore, unlike many bacteria, archaea are GRAS (Generally Recognized as Safe) and commonly encode enzymes that can withstand harsh conditions such as extreme temperatures and pH and high concentrations of solvent and salt. Thus, archaea and their enzymes are ideal platforms for petroleum-independent biocatalytic applications and sustainable chemical processes. Archaea also provide new insight into the breadth of metabolic and enzymatic potential present on this planet with as much as 50% of their genes encoding novel proteins with no obvious counterparts in bacteria or eukaryotes. The long-term goal of our research program is to elucidate global mechanisms of post-translational regulation in archaea. Investigating these types of systems is not only important in furthering our knowledge of how the three domains of life are related in cellular function, but is also important in advancing our ability to modify pathways for biotechnology applications in extremophilic microbes and facilitate further transition to a biobased economy. Understanding how to engineer tight control of post-translational mechanisms will enable the design of metabolic pathways and enzymes that undergo predictable (and even transient) alterations in activity, covalent protein structure, multisubunit complex formation, protein stability and/or cellular location. Cultivating a deeper knowledge of post-translational control mechanisms will also allow new biotechnology applications to be developed that benefit from the timed synthesis of bioproducts which may be otherwise detrimental to cell function. PARTICIPANTS: Hugo V. Miranda (graduate student) Katherine S. Rawls (graduate student) Saad Boutaiba (visiting Ph.D. scholar) Nikita Nembhard (graduate student) Jonathan Martin (graduate student) Mary Holman (graduate student) Nathaniel Hepowit (graduate student) Laia Pedro Roig (yisiting Ph.D. scholar) Dr. Laurence Prunetti (post-doctoral scholar) Dr. Micaela Toniutti (post-doctoral scholar) Dr. Sivakumar Uthandi(post-doctoral scholar) David Krause (undergraduate research student) Cortlin A. Phillips (undergraduate research student) Jonathan Pritz (undergraduate research student) Desire E Javier (undergraduate research student) Rachel Stecker (undergraduate research student) Oliver Oliveraz (undergraduate research student) Dina A. Elbanna (undergraduate research student) Alyssa Berganini (undergraduate research student) Mark Guterman (undergraduate research student) Jessica Ulloa (undergraduate research student) TARGET AUDIENCES: Target audiences for training science-based knowledge included racial and ethnic minorities from the United States as well as training scholars from disadvantaged countries including Algeria, India, China and Spain. Science-based knowledge was delivered to people through formal classroom instruction, laboratory instruction, workshops and research seminars. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Through our work on this CRIS project, we have investigated the role of proteasomes (energy-dependent proteases) in archaea and recently discovered a new type of post-translational control mechanism (termed sampylation) that appears to be biologically linked to proteasomes. In sampylation, small archaeal ubiquitin-like modifier proteins (termed SAMPs) are covalently attached through isopeptide bonds to protein targets by a process that has deep evolutionary roots with the ubiquitin-proteasome system of eukaryotes. The ubiquitin-proteasome system of eukaryotes is a central node for controlling cell division, DNA repair, inflammation, formation of protein aggregates and other important processes. Based on studies performed by our lab group, archaeal homologs of the eukaryotic ubiquitin-activating enzyme E1 and deubiquitylating enzymes of the JAMM/MPN+ superfamily appear to mediate sampylation and desampylation. Interestingly, components of the archaeal sampylation system also appear to be required for the production of sulfur-containing biomolecules such as 2-thiolated tRNA and the pyranopterin-based molybdenum cofactor (MoCo). Thus, our current working model is that ubiquitin- and E1-type proteins are at the crossroads of mediating protein conjugation and sulfur incorporation in archaea.

Publications

• Maupin-Furlow, J.A., M.A. Humbard, and P.A. Kirkland. 2012. Extreme challenges and advances in archaeal proteomics. Curr. Opin. Microbiol. in press.
• Maupin-Furlow, J.A. 2012. Archaeal proteasomes and sampylation. In D. Dougan and T. van Vlijmen (Eds.) General and Regulatory Proteolysis in Microorganisms. The Netherlands. Springer-SBM. in press.
• Maupin-Furlow, J.A. 2012. Proteasomes and protein conjugation across domains of life. Nature Rev. Microbiol. in press.
• Maupin-Furlow, J.A. and H.V. Miranda. 2012. Regulatory particle triple-A proteins (Rpt). In S. Choi (Ed.) Encyclopedia of Signaling Molecules. Springer. in press.
• Miranda, H.V., N. Nembhard, D. Su, N. Hepowit, D.J. Krause, J.R. Pritz, C. Phillips, D. Soll and J.A. Maupin-Furlow. 2011. E1- and ubiquitin-like proteins provide a direct link between protein conjugation and sulfur transfer in archaea. Proc. Natl. Acad. Sci. U.S.A. 108:4417-22.
• Rawls, K.S., J.H. Martin, and J.A. Maupin-Furlow. 2011. Activity and transcriptional regulation of bacterial protein-like glycerol-3-phosphate dehydrogenase of the haloarchaea in Haloferax volcanii. J. Bacteriol. 193:4469-76.
• S. Boutaiba, H. Hacene, K.A. Bidle and J.A. Maupin-Furlow. 2011. Microbial diversity of the hypersaline Sidi Ameur and Himalatt Salt Lakes of the Algerian Sahara. J. Arid Environ. 75:909-916.

Progress 10/01/09 to 09/30/10

Outputs
OUTPUTS: Of the three domains of life, archaea are the least characterized yet represent tremendous potential to advance human health and the development bio-based economy. Archaea have a central role in global inorganic and organic cycles including the production of methane and contribute to a large fraction of the Earth's biomass. In addition, they encode enzymes that are stable in the extreme conditions often encountered in industrial processes (e.g., boiling water, high solvent) and are designated as 'Generally Recognized as Safe' or GRAS for biocatalyst development. Archaea also provide new insight into metabolic biochemistry and the diversity of cell function with as much as 50% of their genes encoding novel proteins with no obvious counterparts in bacteria or eukaryotes. Furthermore, these organisms share deep evolutionary roots with eukaryotes and thus provide fundamental insight into cell function including the regulation of cell division by the ubiquitin-proteasome system. The long-term goal of our research is to understand how proteins are targeted by ubiquitin to proteasomes for degradation and what pathways are regulated by this proteolytic system in archaea. Investigating central processes in archaeal cells, such as the ubiquitin-proteasome system, is important in furthering our understanding of this unusual group of organisms and in advancing our ability to modify pathways for applications in biotechnology including targeting proteins for post-translational modification and modulating protein stability and/or cellular location. PARTICIPANTS: Co-authors on papers for this report include the following: 1) graduate students in lab P.A. Kirkland, K. S. Rawls, M. A. Humbard, C.J. Reuter, H.V. Miranda and B. Saad (visiting Ph.D. scholar); 2) undergraduate students in lab S.K. Yacovone, D.J. Krause, J.R. Pritz, J.A. Puentes and D.J. Cano; 3) post-doctoral scholars in the lab K. Zuobi-Hasona, G. Zhou and S. Uthandi; 4) international and national scientists J.-M. Lim, S. Chen, L. Wells, A. L. Hartman, C. Norais, J.H. Badger, S. Delmas, S. Haldenby, R. Madupu, J. Robinson, H. Khouri, Q. Ren, T.M. Lowe, M. Pohlschroder, C. Daniels, F. Pfeiffer, T. Allers and J.A. Eisen. TARGET AUDIENCES: Undergraduate, graduate and post-doctoral students in research efforts toward a biobased economy and advances in human health. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The following benchmarks were achieved: 1. Discovered a new type of post-translational modification in prokaryotic cells, the covalent attachment of beta-grasp fold proteins of the ubiquitin-like (Ubl) superfamily to target proteins using the archaeon Haloferax volcanii as a model organism. We anticipate that this type of modification is universal among archaea and may even be functional in bacteria, based on the distribution of Ubl gene homologs among prokaryotic genomes. 2. Characterized central metabolic processes likely to be modulated by archaeal proteasome function and/or Ubl post-translational modification mechanisms. We propose that glycerol metabolism (an important pathway in the generation of renewable fuels and chemicals) is regulated by proteasomes in H. volcanii based on our previous MS-based proteomic analysis. Through this work, we have discovered that glycerol is preferred over glucose as a carbon/energy source and that a DeoR/GlpR-type regulatory protein, although not responsible for this preference, does repress the transcription of genes encoding sugar metabolic enzymes during growth on glycerol. 3. Mapped and characterized the function of a number of post-translational modification sites on archaeal proteasomes including phosphorylation, Nalpha-acetylation and methylation of CP subunits (alpha1, alpha2, beta) and PAN proteins. 4. Generated a series of knockout strains in proteasomal CP and Rpt-like genes to further understand archaeal proteasome function. Through this work we found that genes encoding either alpha1 or PAN-A were required for cells to overcome stresses including N-limitation, hypo-osmotic stress, heat shock and exposure to L-canavanine (an amino acid analogue that promotes protein unfolding). We also found CP (and not Rpt-like) subtypes are required for cell division. 5. Performed MS-based shotgun (phospho)proteomics that enhanced accuracy of genome sequence annotation, mapped sites of post-translational modification and developed advanced methods to monitor the baseline proteome of H. volcanii. Over one-fourth (29%) of the proteins with detectable N-termini were Nalpha-acetylated, thus refuting previous assumptions that Nalpha-acetylation is rare in prokaryotes. 6. Identified and characterized a number of archaeal proteins that undergo post-translational modifications including: i) the sliding clamp proliferating cell nuclear antigen or PCNA, ii) a highly-stable extracellular laccase that is a glycoprotein, iii) green fluorescent protein reporters with engineered tags for enhanced protein turnover, and iv) a haloalkaliphilic extracellular protease of Natrialba magadii (Nep). 7. Furthered our understanding of the salt stress response in haloarchaea.

Publications

• 1. Rawls, K.S., S.K. Yacovone, and J.A. Maupin-Furlow. 2010. GlpR represses fructose and glucose metabolic enzymes at the level of transcription in the haloarchaeon Haloferax volcanii. J. Bacteriol. 192: 6251-6260.
• 2. Humbard, M.A., C.J. Reuter, K. Zuobi-Hasona, G. Zhou, and J.A. Maupin-Furlow. 2010. Phosphorylation and methylation of proteasomal proteins of the haloarcheon Haloferax volcanii. Archaea 2010:481725.
• 3. Hartman, A.L., C. Norais, J.H. Badger, S. Delmas, S. Haldenby, R. Madupu, J. Robinson, H. Khouri, Q. Ren, T.M. Lowe, J. Maupin-Furlow, M. Pohlschroder, C. Daniels, F. Pfeiffer, T. Allers, and J.A. Eisen. 2010. The complete genome sequence of Haloferax volcanii DS2 a model archaeon. PLoS ONE 5: e9605.
• 4. Humbard, M.A., H.V. Miranda, J.-M. Lim, D.J. Krause, J.R. Pritz, G. Zhou2, S. Chen, L. Wells and J.A. Maupin-Furlow. 2010. Ubiquitin-like small archaeal modifier proteins (SAMPs) in Haloferax volcanii. Nature 463: 54-60.
• 5. Uthandi, S., B. Saad, M.A. Humbard and J.A. Maupin-Furlow. 2010. LccA, an archaeal laccase secreted as a highly-stable glycoprotein into the extracellular medium of Haloferax volcanii. Appl. Environ. Microbiol. 76:733-743.
• 6. Reuter, C.J., S. Uthandi, J.A. Puentes and J.A. Maupin-Furlow. 2010. Hydrophobic carboxy-terminal residues dramatically reduce protein levels in the haloarchaeon Haloferax volcanii. Microbiology 156:248-55.
• 7. Humbard, M.A., G. Zhou and J.A. Maupin-Furlow. 2009. N-terminal penultimate residue of 20S proteasome α1 influences its Nα-acetylation and protein quantity as well as the thermotolerance and hypoosmotic stress response of Haloferax volcanii. J. Bacteriol. 191:3794-803.
• 8. Sherwood, K.E., D.J. Cano and J.A. Maupin-Furlow. 2009. Glycerol-mediated repression of glucose metabolism and glycerol kinase as the sole route of glycerol catabolism in the haloarchaeon Haloferax volcanii. J. Bacteriol. 191:4307-15.
• 9. Kirkland, P.A. and J.A. Maupin-Furlow. 2009. Stabilization of an archaeal DNA sliding clamp protein, PCNA, by proteasome-activating nucleotidase gene knockout in Haloferax volcanii. FEMS Microbiol. Lett. 294:32-6.

Progress 10/01/07 to 09/30/08

Outputs
OUTPUTS: Archaea and their enzymes often exhibit extreme properties (e.g., growth and function in saturating concentrations of salt, boiling temperatures, alkaline and acidic pH) that are desirable for the development and optimization of biocatalysts that operate at the harsh conditions required for many industrial processes (e.g., biomass saccrification, bioethanol distillation). These unusual characteristics are also of interest in furthering our understanding of basic protein structure and protein quality control. The close evolutionary relationship of archaea to eukaryotes also makes these microbes ideal for characterizing central processes important to human and animal health as well as in the optimization of plant growth. The overarching goal of my research program is to develop extremophilic archaea for use as versatile biocatalysts and for use as models to enhance our fundamental knowledge of central processes important to cell survival and proliferation (e.g. proteasomes). To achieve this goal, we are using a combination of proteomic, genetic, molecular, and biochemical methods to examine the central metabolic and proteolytic systems of Haloferax volcanii, an archaeon which thrives in hypersaline conditions prohibitive to most life. Current systems under investigation include: the proteasome (a multicatalytic protease that is a current target of chemotherapy in humans) and carbon metabolism with emphasis on glycerol metabolism (an important process in the conversion of biodiesel waste to useful products) and laccase-mediated oxidation of lignin for the generation of value-added products from biomass to ethanol. PARTICIPANTS: National and International Collaborators include: Dr. Kelly Bidle (Rider University, Lawrenceville, NJ) Drs. Lonnie O'Neil Ingram, K. T. Shanmugam, J. F. Preston, W. Nicholson, and G. Lorca (University of Florida, Dept. Microbiol. and Cell Science, Gainesville, FL) Dr. Rosana E. DeCastro (Universidad Nacional de Mar del Plata, Argentina) Research Advisees and Collaborators include: International Scholars: Dr. U. Sivakumar (Visiting Professor, 2007 - 2009) Post-doctoral Fellows: Dr. Guangyin Zhou (2005 - present) Dr. Phillip Aaron Kirkland (2007 - 2008) Graduate Scholars: Matthew A. Humbard (Ph.D., Fall 2004 - present) Katherine E. Sherwood (Ph.D., Fall 2006 - present) Undergraduate Scholars: David Cano (Fall 2007 - Spring 2008) Javier A. Velez (Fall 2007 - Spring 2008) Camile Ramos (Spring 2008) Brian Mead (Summer 2008, NSF REU) David M. Kowalczyk (2007 - Summer 2008) Keisin Wang (Fall 2007 - present; HHMI Scholar and Award Recipient) Lanie Jarvis (Summer 2008 - present) Mario Corro (Summer 2008 - present) Nicholas McGarvey (Fall 2008 - present) Cortlin A. Phillips (Fall 2008 - present; HHMI Scholar) Nikita Nembhard (Fall 2008 - present) High School Students: Sunil Rohatgi (Summer 2008, UF Student Science Training Program) TARGET AUDIENCES: My research and teaching efforts include acts and processes that delivered science-based knowledge to people through formal and informal educational programs. Examples have included: formal classroom instruction, laboratory instruction, and internships; the development of curriculum; and outreach to the K - 12 public school system. My research also resulted in the approval of patent No. 7326551. "Cloning and sequencing of pyruvate decarboxylase (pdc) genes from bacteria and uses thereof" (UF no. 10636, issued 02/05/2008). Target audiences include women and racial and ethnic minorities particularly African American and Hispanic Research Scholars. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Our research has greatly impacted the field of extremophiles as well as our understanding of proteasome structure and function and carbon metabolism in the halophilic archaea. In addition, we have recently discovered that H. volcanii thrives on and converts raw biodiesel waste and synthesizes a multicopper oxidase (laccases) that oxidizes lignin and other phenolics. We have also developed genetic engineering techniques to produce a number of thermal stable and salt-tolerant enzymes from this archaeon at high levels in recombinant Escherichia coli as well as H. volcanii. Our research success has lead to a number of international collaborations focused on enhancing H. volcanii as a model to benefit agriculture and medicine (e.g., development of drugs for cancer therapy, minimizing the aging process in plants and animals).

Publications

• 1. Zhou, G., D. Kowalczyk, M. A. Humbard, S. Sohatgi and J. A. Maupin-Furlow. 2008. Proteasomal components required for cell growth and stress responses in the haloarchaeon Haloferax volcanii. J. Bacteriol. (online - in press).
• 2. Kirkland, P. A., M. A. Humbard, C. J. Daniels and J. A. Maupin-Furlow. 2008. Shotgun proteomics of the haloarchaeon Haloferax volcanii. J. Proteome Res. (online - in press).
• 3. De Castro, R. E., D. M. Ruiz, M. I. Gimenez, M. X. Silveyra, R. A. Paggi, and J.A. Maupin-Furlow. 2008. Gene cloning and heterologous synthesis of a haloalkaliphilic extracellular protease of Natrialba magadii (Nep). Extremophiles 12:677-687.
• 4. Bidle, K. A., P. A. Kirkland, J. Nannen, and J. Maupin-Furlow. 2008. Proteomic analysis of Haloferax volcanii reveals up-regulation of the transcriptional activator PspA in response to salt stress. Microbiology 154:1436-1443.
• 5. Kirkland, P. A., M. A. Gil, I. M. Karadzic, and J. A. Maupin-Furlow. 2008. Genetic and proteomic analyses of a proteasome-activating nucleotidase A mutant of the haloarchaeon Haloferax volcanii. J. Bacteriol. 190:193-205.

Progress 10/01/06 to 09/30/07

Outputs
OUTPUTS: The extremophilic properties of archaea and their enzymes (optimal growth and function in saturating concentrations of salt, boiling temperatures, alkaline and acidic pH) make these microbes ideal for the development and optimization of biocatalysts that operate under the harsh conditions encountered in many industrial processes (e.g. biomass saccrification). These unusual characteristics are also of interest in understanding protein structure and quality control. Furthermore, the close evolutionary relationship of archaea to eukaryotes makes these microbes ideal models for characterizing central processes important to human and animal health as well as optimization of plant growth. The overarching goal of my research program is to develop these extremophilic archaea for use as versatile biocatalysts and for use as models to enhance our fundamental knowledge of central process important to cell survival and proliferation (e.g. cancer). To achieve this goal, we are using a combination of proteomic, genetic, molecular, and biochemical methods to examine the central metabolic and proteolytic systems of Haloferax volcanii, an archaeon which thrives in hypersaline conditions prohibitive to most life. The central systems under investigation include the proteasome (a multicatalytic protease which is a current target of chemotherapy in humans) as well as carbon metabolism with emphasis on a dihydroxyacetone kinase-linked phosphoenolpyruvate protein transferase system which appears to be controlled by the proteasome. PARTICIPANTS: National and International Collaborators: Dr. Stanley M. Stevens Jr. (University of North Texas Health Science Center, Fort Worth, TX) Dr. Jennifer Busby (Scripps Florida, Jupiter, FL) Dr. Kelly Bidle (Rider University, Lawrenceville, NJ) Drs. Jonathan Eisen, Amber Hartman, and Jonathan Badger (University of California, Davis and TIGR) Drs. Jerry Eichler and Boaz Shaanan (Ben Gurion University, Israel) Drs. Lonnie O'Neil Ingram, K. T. Shanmugam, J. F. Preston, W. Nicholson, and G. Lorca (University of Florida, Dept. Microbiol. and Cell Science, Gainesville, FL) Dr. Rosana E. DeCastro (Universidad Nacional de Mar del Plata, Argentina) Research Advisees and Collaborators: International Scholars: Dr. U. Sivakumar (Visiting Professor, Tamil Nadu Agricultural University, Tamil Nadu, India, Government of India Associateship for Young Scientists, Specialized Training in Niche Areas of Biotechnology, Dec. 2007 - 2008) and Saad Boutaiba (Visiting Ph.D. Scholar, University of Djelfa, Algeria; Algerian Cooperative Science Commission, Nov. 8, 2006 - June 13, 2007) Post-doctoral Fellows: Dr. Guangyin Zhou, 2005 - present. Graduate Scholars: Dr. Phillip Aaron Kirkland (Ph.D., Fall 2003 - Fall 2007). Matthew A. Humbard (Ph.D., Fall 2004 - present). Katherine E. Sherwood (Ph.D., Fall 2006 - present). Undergraduate Scholars: Chris J. Tzikas (2006 - 2007, Osprey Biotechnics Inc. Employee) Kristin Toscano (2006 - 2007, U. Penn graduate school) Linda Du (2006 - 2007, medical school) Candace Bichsel (2007, UF Interdisciplinary Ph.D. Program in Biomedical Sciences) Steve Garrett (2006 - 2007, dental school) David M. Kowalczyk (Spring 2007 - present) Jessica Coleman (Summer 2007, NSF REU Program) Javier A. Velez (Fall 2007 - present) David Cano (Fall 2007 - present) Keisin Wang (Fall 2007 - present; HHMI Scholar) Adriana Penuela (Fall 2007 - present; HHMI Scholar) High School Students: Katiana Garagozlo (UF High School Student Science Training Program, Summer 2007) Sam Zakria (UF High School Student Science Training Program, Summer 2007) TARGET AUDIENCES: Target audiences include racial and ethnic minorities particularly Hispanic research scholars from Latin America and the Caribbean Islands. Research and teaching efforts have included acts and processes that delivered science-based knowledge to people through formal and informal educational programs. This has included formal classroom instruction, laboratory instruction, and internships; the development of curriculum; and outreach to the K - 12 public school system.

Impacts
Our research has greatly impacted the field of extremophiles as well as our understanding of proteasome structure and function and carbon metabolism in the halophilic archaea. In addition, we have recently discovered that H. volcanii thrives on and converts raw biodiesel waste to useful products such as butanediol. We have also developed genetic engineering techniques to produce a number of thermal stable and salt-tolerant enzymes from this archaeon at high levels in recombinant Escherichia coli as well as H. volcanii. Our research success has lead to a number of international collaborations focused on enhancing H. volcanii as a model to benefit agriculture and medicine (e.g. development of drugs for cancer therapy, minimizing the aging process in plants and animals).

Publications

• Kirkland, PA , MA Gil, IM Karadzic & JA Maupin-Furlow. 2007. Genetic and proteomic analyses of a proteasome-activating nucleotidase A mutant of the haloarchaeon Haloferax volcanii. J Bacteriol (in press/online).
• Gil, MA, KE Sherwood & JA Maupin-Furlow. 2007. Transcriptional linkage of Haloferax volcanii proteasomal genes with non-proteasomal gene neighbors including RNase P, MOSC domain and SAM-methyltransferase homologs. Microbiology 153: 3009-22.
• Kirkland, PA, CJ Reuter & JA Maupin-Furlow. 2007. Effect of proteasome inhibitor clasto-lactacystin-β-lactone on the proteome of the haloarchaeon Haloferax volcanii. Microbiology 153: 2271-80.
• Humbard, MA, G Zhou & JA Maupin-Furlow. 2007. N-terminal acetylation, methylation, and phosphorylation of Haloferax volcanii 20S proteasomes. Abs Fla Genetics Symp, Gainesville, Fla.
• Sherwood, KE & JA Maupin-Furlow. 2007. Genetic and biochemical characterization of a dihydroxyacetone kinase-linked phosphoenolpyruvate: protein transferase system in Haloferax volcanii. Abs Fla Genetics Symp, Gainesville, Fla.
• Sherwood, KE & JA Maupin-Furlow. 2007. Dihydroxyacetone kinase-linked phosphoenolpyruvate: protein transferase system of Haloferax volcanii. Abs Ann Mtg Amer Soc Microbiol Fla Branch, St. Petersburg, Fla.
• Kirkland, PA & JA Maupin-Furlow. 2007. Proteins that accumulate in proteasome-deficient haloarchaeon Haloferax volcanii cells identified by comparative 2D-PAGE, phosphoenrichment and hybrid mass spectrometry. Abs Gordon Res Conf, Archaea: Ecol, Metabol & Mol Biol, Procter Academy, NH.
• Humbard, MA & JA Maupin-Furlow. 2007. N-terminal acetylation, methylation, and phosphorylation of Haloferax volcanii 20S proteasomes. Abs Gordon Res Conf, Archaea: Ecol, Metabol & Mol Biol, Procter Academy, NH
• Bidle, KA, J Nannen, M Geigel, PA Kirkland & J Maupin-Furlow. 2007. Proteomic analysis of Haloferax volcanii reveals the up-regulation of the transcriptional activator PspA in response to salt stress. Abs Ann Mtg Amer Soc Microbiol, Toronto, Canada.
• Humbard, MA & JA Maupin-Furlow. 2007. N-alpha-acetylation and 20S proteasomes from the haloarcheon Haloferax volcanii. Abs Ann UF IFAS CALS Grad Res Symp, Gainesville, Fla.

Progress 10/01/05 to 09/30/06

Outputs
The extremophilic properties of archaea (growth in saturating concentrations of salt, boiling temperatures, alkaline and acidic pH) make these microbes ideal for generating biocatalysts that are optimal in the harsh conditions often encountered in industrial processes. Developing these extremophiles as versatile biocatalysts, however, will require understanding how their central regulatory protease, the proteasome, functions in protein quality control and regulation of metabolic pathways. This research project is based on the following hypotheses: (a) archaeal proteasome-dependent turnover is modulated in part by the phosphorylation state of substrate proteins, (b) subtypes of AAA ATPases recognize overlapping as well as unique sets of substrates for proteasome-mediated degradation, (c) 20S core particle subtypes of different alpha subunit composition differ in their affinity for these AAA ATPase subtypes, (d) the levels and post-translational modification of the AAA ATPases and alpha proteins are regulated to influence the timing and specificity of protein turnover. To achieve the objectives of this study we are using a combination of genetic, molecular, and biochemical methods to examine proteasomes in a halophilic archaeon isolated from the Dead Sea, Haloferax volcanii. This haloarchaeon provides an excellent model system for elucidating how proteasomes recognize and degrade proteins based our: construction of isogenic mutants lacking individual proteasomal proteins; development of proteomic methods to mutant and parent strains; discovery of over 30 unique phosphoproteins that accumulate in proteasome mutants; the purification and characterization of 20S proteasome and PAN subtypes; development of an in vivo GFP reporter system to rapidly screen motifs mediating protein degradation; and detection of changes in the levels of mRNA, protein and post-transcriptional modification of proteasomes in Hfx. volcanii.

Impacts
Our research has greatly impacted the field of extremophiles as well as our understanding of proteasome structure and function. We have identified and characterized this multicatalytic protease from several archaeal organisms that survive in extreme environmental conditions (e.g. hydrothermal vents, the Dead Sea). We have also developed genetic engineering techniques to produce these thermal stable and salt-tolerant proteasome enzymes at high levels in recombinant Escherichia coli. This has lead to international collaborations with X-ray crystallographers and cell physiologists to elucidate basic structural features of this enzyme that is expected to benefit agriculture and medicine (e.g. development of drugs for cancer therapy, minimizing the aging process in plants and animals).

Publications

• Dinitz-Bab, E., H. Shmuely, J. Maupin-Furlow, J. Eichler and B. Shaanan. 2006. Haloferax volcanii PitA: An example of functional interaction between the Pfam chlorite dismutase and antibiotic biosynthesis monooxygenase families? Bioinformatics 22:671-675.
• Kirkland, P. A.2, J. Busby, S. Stevens, and J. A. Maupin-Furlow. 2006. Trizol-based method for sample preparation and isoelectric focusing of halophilic proteins. Anal. Biochem. 351: 254-259.
• Maupin-Furlow, J. A., M. A. Humbard, P. A. Kirkland, W. Li, C. J. Reuter, A. J. Wright, and G. Zhou. 2006. Proteasomes from structure to function: perspectives from archaea. Curr. Top. Dev. Biol. 75: 125-169 (front cover photo).
• Maupin-Furlow, J. A., M. A. Gil, M. A. Humbard, P. A. Kirkland, W. Li, C. J. Reuter, and A. J. Wright. 2006. Proteasomes and other nanocompartmentalized proteases in archaea. J. M. Shively (ed.) Springer-Verlag. Microbiology Monographs: Complex Intracellular Structures in Prokaryotes pp. 23-46.
• De Castro, R. E., J. A. Maupin-Furlow, M. I. Gimenez, M. K. Herrera Seitz, and J. J. Sanchez. 2006. Haloarchaeal proteases and proteolytic systems. FEMS Microbiol. Rev. 30:17-35.
• Talarico, L. A., M. A. Gil, L. P. Yomano, L. O. Ingram, and J. A. Maupin-Furlow. 2006. Construction and expression of ethanol production operon in gram-positive bacteria. Agricultural and Environmental Biotechnology Abstracts.
• Kirkland, P. A., S. M. Stevens Jr., J. Busby, and J. A. Maupin-Furlow. 2006. Determination of the baseline proteome of the halophlic archaeaon Haloferax volcanii. Abstracts of the First Annual DOE Joint Genome Institute User Meeting, Walnut Creek, CA.
• Kirkland, P. A., S. M. Stevens Jr., J. A. C. Busby, and J. A. Maupin-Furlow. May 2006. Comparative proteomics to identify native substrates of the haloarchaeal 20S proteasome. Abstracts of the Meeting of the University of Florida Shands Cancer Center. Gainesville, FL.
• Zhou, G., M. Humbard, and J. Maupin-Furlow. May 2006. Generation of isogenic protease mutants of the halophilic archaeon Haloferax volcanii. Abstracts of the Meeting of the University of Florida Shands Cancer Center. Gainesville, FL.
• Kirkland, P.A., and J. A. Maupin-Furlow. 2006. Degradomics to identify native substrates of 20S proteasomes of Haloferax volcanii. Abstracts of the Sixth Annual IFAS College of Agricultural and Life Sciences, Gainesville, FL.
• Humbard, M. A., S. M. Stevens Jr., and J. A. Maupin-Furlow. 2006. Post-translational modification of 20S proteasomes and associated proteins from Haloferax volcanii. Abstracts of the Sixth Annual IFAS College of Agricultural and Life Sciences, Gainesville, FL.
• Humbard, M. A., S. M. Stevens Jr., K. Zuobi-Hasona, and J. A. Maupin-Furlow. 2006. Post-translational modifications of 20S proteasomes from Haloferax volcanii. Ann. Mtg. Amer. Soc. Microbiol. Abs., Orlando, FL.
• Stevens, S. M., Jr., A. Chung, M. Chow, P. A. Kirkland, M. A. Gil, and J. A. Maupin-Furlow. 2006. Advancements in posttranslational modification analysis using a fluorescent affinity tag and mass spectrometry. Assoc. Biomolecular Resource Facilities, Long Beach, CA.
• Humbard, M. A., S. Stevens Jr., and J. A. Maupin-Furlow. 2006. Post-translational modification of the 20S proteasomal proteins of the archaeon Haloferax volcanii. J. Bacteriol. 188:7521-30.

Progress 10/01/04 to 09/30/05

Outputs
The extremophilic properties of archaea (growth in saturating concentrations of salt, boiling temperatures, alkaline and acidic pH) make these microbes ideal for engineering biocatalysis and biotransformations under extreme conditions. Understanding how 20S proteasome and proteasome-activating nucleotidase AAA ATPase subtypes function to maintain protein quality control is fundamental to understanding protein quality control and developing these extremophiles as versatile biocatalysts for the 21st century. This research project is based on the following hypotheses: (a) archaeal proteasome-dependent turnover is modulated in part by the phosphorylation state of substrate proteins, (b) subtypes of AAA ATPases recognize overlapping as well as unique sets of substrates for proteasome-mediated degradation, (c) 20S core particle subtypes of different alpha subunit composition differ in their affinity for these AAA ATPase subtypes, (d) the levels and post-translational modification of the AAA ATPases and alpha proteins are regulated to influence the timing and specificity of protein turnover. To achieve the objectives of this study we are using a combination of genetic, molecular, and biochemical methods to examine proteasomes in a halophilic archaeon isolated from the Dead Sea, Haloferax volcanii. This haloarchaeon provides an excellent model system for elucidating how proteasomes recognize and degrade proteins based our: construction of isogenic mutants lacking individual proteasomal proteins; development of methods to compare 2D-proteome maps of mutant and parent strains; discovery of over 30 unique phosphoproteins that accumulate in proteasome mutants; the purification and characterization of 20S proteasome and PAN subtypes; development of an in vivo GFP reporter system to rapidly screen motifs mediating protein degradation; and detection of changes in the levels of mRNA, protein and post-transcriptional modification of proteasomes in Hfx. volcanii.

Impacts
Our research has greatly impacted the field of extremophiles as well as our understanding of proteasome structure and function. We have identified and characterized this multicatalytic protease from several archaeal organisms that survive in extreme environmental conditions (e.g. hydrothermal vents, the Dead Sea). We have also developed genetic engineering techniques to produce these thermal stable and salt-tolerant proteasome enzymes at high levels in recombinant Escherichia coli. This has lead to international collaborations with X-ray crystallographers and cell physiologists to elucidate basic structural features of this enzyme that is expected to benefit agriculture and medicine (e.g. development of drugs for cancer therapy, minimizing the aging process in plants and animals).

Publications

• Reuter, C. J. and J. A. Maupin-Furlow. 2005. Unraveling factors responsible for proteolytic substrate recognition by proteasome-associated chaperones in the haloarchaeon Haloferax volcanii. Abstracts of the Annual Meeting of the Southeastern Branch American Society for Microbiology, St. Petersburg, FL.
• Humbard, M. A., Zuobi-Hasona, K., and J. A. Maupin-Furlow. 2005. Growth-dependent subunit isoforms of Haloferax volcanii 20S proteasomes. Abstracts of the Annual Meeting of the Southeastern Branch American Society for Microbiology, St. Petersburg, FL.
• Cuddy, K. K. and J. A. Maupin-Furlow. 2005. Green fluorescent protein reporter to probe Haloferax volcanii promoters. Abstracts of the UF Student Science Training Program, Gainesville, FL.
• Maupin-Furlow, J. A., M. A. Gil, M. Humbard, P. A. Kirkland, C. A. Reuter, and A. J. Wright. 2005. Biocatalysis in the extreme. Session: Frontiers in Biocatalysis and Biotransformations. Abstracts of the Annual Meeting of the American Chemical Society, San Diego, CA.
• Kirkland, P. A. and J. A. Maupin-Furlow. 2005. A proteomic approach to identifying native 20S proteasomal substrates of the haloarchaeon Haloferax volcanii. Abstracts of the Annual Meeting of the Southeastern Branch American Society for Microbiology, St. Petersburg, FL.
• Wright, A. J. and J. A. Maupin-Furlow. 2005. Molecular characterization of Haloferax volcanii 20S proteasomes reveals an intramolecular mechanism of -protein processing. Abstracts of the Annual Meeting of the American Society for Microbiology, Atlanta, GA.
• Gil, M. A. and J. A. Maupin-Furlow. 2005. Co-transcription of proteasome and tRNA modification genes of the haloarchaeon Haloferax volcanii. Abstracts of the Annual Meeting of the American Society for Microbiology, Atlanta, GA.
• Reuter, C. J. and J. A. Maupin-Furlow. 2005. Molecular characterization of the proteasome-activating nucleotidase subtypes of the haloarchaeon Haloferax volcanii. Abstracts of the Annual Meeting of the American Society for Microbiology, Atlanta, GA.
• Kirkland, P. A. and J. A. Maupin-Furlow. 2005. Identification of native proteasomal substrates of Haloferax volcanii using a degadomic approach. Abstracts of the Annual Meeting of the American Society for Microbiology, Atlanta, GA.
• Maupin-Furlow, J. A., M. A. Gil, P. A. Kirkland, M. A. Humbard, C. J. Reuter, and A. J. Wright. 2006. Proteasomes and other nanocompartmentalized proteases in archaea. J. M. Shively (ed.) Springer-Verlag. Microbiology Monographs: Complex Intracellular Structures in Prokaryotes (in press).
• Rosana E. De Castro, Julie A. Maupin-Furlow, Maria Ines Gimenez, Maria Karina Herrera Seitz, and Jorge J. Sanchez. 2006. Haloarchaeal proteases and proteolytic systems. FEMS Microbiol. Rev. (in press).
• Maupin-Furlow, J. A., M. A. Gil, P. A. Kirkland, M. A. Humbard, W. Li, C. J. Reuter, and A. J. Wright. 2005. Archaeal proteasomes and other regulatory proteases. Curr. Opin. Microbiol. 8: (in press).
• Karadzic, I. M. and J. A. Maupin-Furlow. 2005. Improvement of two-dimensional gel electrophoresis proteome maps of the haloarchaeon Haloferax volcanii. Proteomics 5:354-359.
• Kaczowka, S. J., C. J. Reuter, L. A. Talarico and J. A. Maupin-Furlow. 2005. Recombinant production of Zymomonas mobilis pyruvate decarboxylase in the haloarchaeon Haloferax volcanii. Archaea 1:327-34.
• Talarico, L. A., M. A. Gil, L. O. Ingram, and J. A. Maupin-Furlow. 2005. Construction and expression of ethanol production operon in gram-positive bacteria. Microbiology (in press).
• Stevens, S. M., Jr., Chung, A., Chow, M., Kirkland, P.A., Gil, M.A., and J. A. Maupin-Furlow. 2006. Advancements in posttranslational modification analysis using a fluorescent affinity tag and mass spectrometry. Abstracts of the Association of Biomolecular Resource Facilities Meeting, Long Beach, CA.
• Gil, M.A. and J. A. Maupin-Furlow. 2005. Co-transcription of proteasome and tRNA modification genes of the haloarchaeon Haloferax volcanii. Abstracts of the Florida Bioinformatics Workshop. Gainesville, FL.
• Wright, A. J. and J. A. Maupin-Furlow. 2005. Assembly and processing of 20S proteasomes in Haloferax volcanii. Abstracts of the 5th Annual IFAS Graduate Research Symposium. Gainesville, Florida.
• Kirkland, P. A. and J. A. Maupin-Furlow. 2005. Proteomics and the halophilic archaea. Abstracts of the 5th Annual IFAS Graduate Research Symposium. Gainesville, Florida.
• Gil, M. A. and J. A. Maupin-Furlow. 2005. Connect the dots: organization of proteasomal operons of the haloarchaeon Haloferax volcanii. Abstracts of the 5th Annual IFAS Graduate Research Symposium. Gainesville, Florida.
• Wright, A. J. and J. A. Maupin-Furlow. 2005. Molecular characterization of Haloferax volcanii 20S proteasomes reveals an intramolecular mechanism of beta-protein processing. Abstracts of the Florida Bioinformatics Workshop. Gainesville, FL.

Progress 10/01/03 to 09/30/04

Outputs
The unusual physiological properties of archaea (growth in extreme salt, temperature and pH) make these microbes ideal for engineering biocatalysis and biotransformations under extreme conditions. Understanding how 20S proteasome and proteasome-activating nucleotidase AAA ATPase subtypes function to maintain protein quality control is fundamental to our understanding of protein quality control and to our development of these extremophiles as versatile biocatalysts. This research project is based on the following hypotheses: (a) archaeal proteasome-dependent turnover is modulated in part by the phosphorylation state of substrate proteins, (b) subtypes of AAA ATPases recognize overlapping as well as unique sets of substrates for proteasome-mediated degradation, (c) 20S core particle subtypes of different alpha subunit composition differ in their affinity for these AAA ATPase subtypes, (d) the levels and post-translational modification of the AAA ATPases and alpha proteins are regulated to influence the timing and specificity of protein turnover. To achieve the objectives of this study we are using a combination of genetic, molecular, and biochemical methods to examine proteasomes in a halophilic archaeon isolated from the Dead Sea, Haloferax volcanii. This haloarchaeon provides an excellent model system for elucidating how proteasomes recognize and degrade proteins based our: construction of isogenic mutants lacking individual proteasomal proteins; development of methods to compare 2D-proteome maps of mutant and parent strains; discovery of over 30 unique phosphoproteins that accumulate in proteasome mutants; the purification and characterization of 20S proteasome and PAN subtypes; development of an in vivo GFP reporter system to rapidly screen motifs mediating protein degradation; and detection of changes in the levels of mRNA, protein and post-transcriptional modification of proteasomes in Hfx. volcanii.

Impacts
Our research has greatly impacted the field of extremophiles as well as our understanding of proteasome structure and function. We have identified and characterized this multicatalytic protease from several archaeal organisms that survive in extreme environmental conditions (e.g. hydrothermal vents, the Dead Sea). We have also developed genetic engineering techniques to produce these thermal stable and salt-tolerant proteasome enzymes at high levels in recombinant Escherichia coli. This has lead to international collaborations with X-ray crystallographers and cell physiologists to elucidate basic structural features of this enzyme that is expected to benefit agriculture and medicine (e.g. development of drugs for cancer therapy, minimizing the aging process in plants and animals).

Publications

• Reuter, C. J. and J. A. Maupin-Furlow. 2004. Analysis of proteasome-dependent proteolysis in Haloferax volcanii cells using short-lived green fluorescent proteins. Appl. Envir. Microbiol. 70:(in press).
• Kaczowka, S. J., C. J. Reuter, L. A. Talarico and J. A. Maupin-Furlow. 2004. Recombinant production of Zymomonas mobilis pyruvate decarboxylase in the haloarchaeon Haloferax volcanii. Archaea 1:(in press).
• Reuter, C. J., S. J. Kaczowka and J. A. Maupin-Furlow. 2004. Differential regulation of PanA and PanB proteasome-activating nucleotidase and 20S proteasome proteins of the haloarchaeon Haloferax volcanii. J. Bacteriol. 186:7763-7772.
• Karadzic, I. M. and J. A. Maupin-Furlow. 2004. Improvement of two-dimensional gel electrophoresis proteome maps of the haloarchaeon Haloferax volcanii. Proteomics 5:(in press).
• Maupin-Furlow, J. A., M. A. Gil, I. M. Karadzic, P. A. Kirkland, and C. J. Reuter. 2004. Proteasomes: perspectives from the archaea [update 2004]. Front Biosci. 9:1743-1758.
• Karadzic, I. M., M. A. Gil and J. A. Maupin-Furlow. 2004. Physiological characterization of a proteasome-deficient mutant of Haloferax volcanii by a differential proteomic approach. Abstracts of the 5th International Conference on Extremophiles, Cambridge, MD.
• Reuter, C. J. and J. A. Maupin-Furlow. 2004. Analysis of substrate recognition by Pan/20S Proteasome isoforms in the haloarchaeon Haloferax volcanii. Abstracts of the 5th International Conference on Extremophiles, Cambridge, MD.
• Kirkland, A. P. and J. A. Maupin-Furlow. 2004. Development of green fluorescent protein-based expression vectors to identify regulated promoters of Haloferax volcanii. Abstracts of the 5th International Conference on Extremophiles, Cambridge, MD.
• Wright, A. J. and J. A. Maupin-Furlow. 2004. Mutational analysis of the Thr active site of the beta-protein in 20S proteasomes reveals beta-protein processing via an intramolecular mechanism. Abstracts of the 5th International Conference on Extremophiles, Cambridge, MD.

Progress 10/01/02 to 10/01/03

Outputs
Archaea are a metabolically diverse group of microorganisms that often thrive in extreme environmental conditions such as hydrothermal vents and the Dead Sea. Thus, this group has tremendous potential for use in biotechnology applications that require high temperature, low pH and/or high salt. Unfortunately, little is know regarding how these cells regulate protein turnover. This has complicated our ability to engineer recombinant archaea for expression of foreign proteins. Based on bioinformatics, we hypothesize that the proteasome, an energy-dependent protease, is the central player in the quality control and regulated turnover of proteins in this unusual domain of life. The main objective of this study is to determine how the proteasome functions in an archaeal cell. The results are expected to broaden the use of archaea and other related extremophiles in metabolic engineering for biotechnology applications. To achieve the objectives of this study we are using a combination of genetic, molecular, and biochemical methods to examine proteasomes in an archaeon isolated from the Dead Sea, Haloferax volcanii. Proteasome mRNA transcripts and protein levels are being analyzed under a variety of culture conditions. This is providing information on the regulation, promoters, and operons of the proteasomes (e.g. the proteasome appears to be co-transcribed with a RNA processing machine termed the exosome, thus, providing a link between protein and RNA metabolism). We are also modifying the levels of active proteasomes through chromosomal mutagenesis and homologous expression from plasmids. This is enabling us to link the in vivo level of the various proteasome proteins with cell phenotypes such as cell growth, stress response, 2D PAGE profiles, etc. Genetic methods are also being used to identify foreign and native substrates of the proteasome. These tasks are focused on understanding the biology of proteolysis as a mechanism of post-transcriptional regulation in this unusual group of organisms. Based on our work, it is now know that many of the halophilic archaea encode multiple isoforms of the proteasome catalytic core and ATPase regulatory particle. These studies are providing mechanistic information on why a cell would require multiple forms of the proteasome. Interestingly, Haloferax volcanii synthesizes at least two different 20S core particles and three proteasome-activating nucleotidase (Pan) complexes. We hypothesize that the diversification of Pan and other related AAA family members enhances the number of different motifs recognized as substrates for degradation by the 20S proteasome. If so, the haloarchaea encode a tremendous number of AAA proteins that may be used in different combinations with 20S proteasome isoforms for the regulated turnover of proteins. This would be similar to the new paradigm of gene regulation recently proposed for the haloarchaea in which a diversity of transcription factors interact in different combinations to recognize a large set of promoters.

Impacts
This research project has greatly impacted the field of proteasome structure and function by identifying and characterizing this multicatalytic protease from several archaeal organisms that survive in extreme environmental conditions (e.g. hydrothermal vents, the Dead Sea). We have also developed genetic engineering techniques to produce these thermal stable and salt-tolerant proteasome enzymes at high levels in recombinant Escherichia coli. This has lead to international collaborations with X-ray crystallographers and cell physiologists to elucidate basic structural features of this enzyme that is expected to benefit agriculture and medicine (e.g. development of drugs for cancer therapy, minimizing the aging process in plants and animals).

Publications

• Kaczowka, S. J. and J. A. Maupin-Furlow. Proteasomes: perspectives from the archaea [update 2004]. Front Biosci. in press.
• Maupin-Furlow, J. A., S. J. Kaczowka, C. J. Reuter, K. Zuobi-Hasona, and M. A. Gil. 2003. Archaeal proteasomes: potential in metabolic engineering. Metab. Eng. 5:151-163.
• Kaczowka, S. J. and J. A. Maupin-Furlow. 2003. Subunit topology of the multiple 20S proteasomes from Haloferax volcanii. J. Bacteriol. 185:165-174.
• Kaczowka, S. J., L. A. Talarico, and J. A. Maupin-Furlow. 2003. Recombinant production of Zymomonas mobilis pyruvate decarboxylase in the archaeon Haloferax volcanii. Ann. Mtg. Amer. Soc. Microbiol. Abs., Washington, DC.
• Reuter, C. J. and J. A. Maupin-Furlow. 2003. Proteasome-activating nucleotidases of Haloferax volcanii: Subunit complexity and activity. Ann. Mtg. Amer. Soc. Microbiol. Abs., Washington, DC.
• Zuobi-Hasona, K., S. J. Kaczowka and J. A. Maupin-Furlow. 2003. Growth-dependent post-translational modification of the a1 subunit of Haloferax volcanii 20S proteasomes. Ann. Mtg. Amer. Soc. Microbiol. Abs., Washington, DC.

Progress 10/01/01 to 10/01/02

Outputs
Archaea are a metabolically diverse group of bacteria that often grow in extreme environments such as hydrothermal vents and the Dead Sea. Halophilic archaea such as Haloferax volcanii have adapted to high salt ecosystems by modifying their protein structure. The majority of proteins from this organism have a highly charged surface that interacts with salt to stabilize the protein. Energy-dependent proteases, such as the proteasome, are central to the quality control of proteins and are needed for many regulatory responses. The major objective of this study is to determine how proteasomes function in the archaeal cell. To achieve this objective we are using genetic, molecular, and biochemical methods to examine proteasomes in H. volcanii. Proteasome mRNA transcripts and protein levels have been analyzed under a variety of conditions. This has provided information on the regulation, promoters and operons of the proteasomes. The levels of active proteasome are being modified through chromosomal mutagenesis as well as homologous expression from plasmids. This is enabling us to link the in vivo level of the various proteasome proteins with cell phenotypes such as cell growth, stress response, proteome as determined 2D PAGE, etc. Genetic methods are also being used to identify foreign and native substrates of the proteasome. These tasks are focused on understanding the biology of proteolysis as a mechanism of post-transcriptional regulation in this unusual group of organisms.

Impacts
Proteasomes are central players in maintaining quality control of proteins in many cells. Understanding the relationship of the structure and function of these large energy-dependent proteases is having a profound impact on medicine, biotechnology and other areas of national interest. Controling the hydrolysis of proteins in cells has direct application to metabolic engineering novel biocatalysts in host organisms (green chemistry). The archaea serve as ideal model systems for understanding the role of the proteasome in protein quality control.

Publications

• Kaczowka, S. J. and J. A. Maupin-Furlow. 2002. Subunit arrangement of 20S proteasomes from Haloferax volcanii. Ann. Mtg. SE Branch Amer. Soc. Microbio. Abs.
• Reuter, C. J. and J. A. Maupin-Furlow. 2002. Biochemical characterization and subunit arrangement of Pan proteins from the halophilic archaeon Haloferax volcanii. Ann. Mtg. SE Branch Amer. Soc. Microbio. Abs.
• Kaczowka, S. J., H. C. Aldrich, and J. A. Maupin-Furlow. 2001. Localization of the alpha-1 and alpha-2 subunits of 20S proteasomes from a halophilic archaeon, Haloferax volcanii. Gordon Research Archaea: Ecology, Metabolism, and Molecular Biology Abs.
• Kaczowka, S. J., H. C. Aldrich, and J. A. Maupin-Furlow. 2001. Structure and function of proteasomes from a halophilic archaeon, Haloferax volcanii. Ann. Mtg. Amer. Soc. Microbiol. Abs.
• Kaczowka, S. J. and J. A. Maupin-Furlow. 2002. Subunit topology of the multiple 20S proteasomes from Haloferax volcanii. J. Bacteriol. (in press)
• Maupin-Furlow, J. A., Kaczowka, S. J., Ou, M. S., and H. L. Wilson. 2001. Archaeal proteasomes: nanocompartments of the cell. P. Blum (ed.) Academic Press. Adv. Appl. Microbiol. 50: 279-338.
• Wilson, H. L., H. C. Aldrich, and J. A. Maupin-Furlow. 2001. Purification and characterization of the 20S proteasome and PAN from Methanococcus maripaludis. Ann. Mtg. Amer. Soc. Microbiol. Abs.
• Ou, M. S., and J. A. Maupin-Furlow. 2001. Influence of the alpha subunits on the proteolytic activity of 20S proteasomes. Ann. Mtg. Amer. Soc. Microbiol. Abs.
• Kaczowka, S. J., H. C. Aldrich, and J. A. Maupin-Furlow. 2001. Structure and function of proteasomes from a halophilic archaeon, Haloferax volcanii. Interfacing Microbiol. & Biotechnol. Abs.
• Wilson, H. L., H. C. Aldrich, and J. A. Maupin-Furlow. 2001. Purification and characterization of the 20S proteasome and PAN from Methanococcus maripaludis. Interfacing Microbiol. & Biotechnol. Abs.
• Ou, M. S., and J. A. Maupin-Furlow. 2001. Influence of the alpha subunits on the proteolytic activity of 20S proteasomes. Interfacing Microbiol. & Biotechnol. Abs.

Progress 10/01/00 to 10/01/01

Outputs
The long-term goal of this research project is to better understand energy-dependent proteolysis mediated by the proteasome. Little is known about the physiological role or mechanism of protein degradation in the archaea. Furthering our understanding in this important area is likely to broaden our ability to use the archaea and other related extremophiles in biotechnology applications. The 20S proteasome is composed of four stacked heptameric rings made up of two families of subunits designated alpha and beta. In the eucarya the complex is composed of 14 different subunits; whereas, in the archaea the majority of 20S proteasomes are composed of a single alpha and beta subunit. Likewise, archaeal proteasome-activating nucleotidase (PAN) protein is typically only composed of one subunit while six different subunits form the base of the related complex in eucarya. Due to this simpler composition, as compared to their eucaryal counterparts, the archaea have become central models in examining proteasome-mediated degradation. Our work has resulted in the purification and characterization of proteasomes from two Methanococcus species: the hyperthermophilic M. jannaschii and mesophilic M. maripaludis. Purified PAN stimulated 20S proteasomes in the hydrolysis of proteins only when nucleotide triphosphates were present. Thus, an active energy-dependent proteolytic system has been reconstituted in vitro. In order to examine the proteasome system in vivo, M. maripaludis transformants have been generated that synthesize high-levels of PAN and PAN mutants (i.e.His-tagged and active-site modifications). In addition, strains that synthesize reporter proteins (i.e. b-galactosidase and destabilized variants) have been generated to examine the proteasome system as it functions in the cell. Based on our work, it is now know that many of the halophilic archaea encode proteasomes of intermediate complexity. These studies are providing mechanistic information on why a cell would require multiple forms of the proteasome. The compositions of proteasomes isolated from Haloferax volcanii reveal protein paralogs for the alpha and PAN proteins associate with a single beta-type protein. It is the beta protein that is responsible for catalyzing the hydrolysis of peptide bonds. Current work is focused on determining if these proteins are assembled in an energy-dependent proteolytic system. In addition, we are examining when the protein paralogs are synthesized in the cell. This information is providing insight into cell physiology. For example, the levels of the alpha1 protein gradually increase as H. volcanii enters stationary phase which is a stressful time for cells. In contrast, the levels of the alpha2 and PAN1 proteins are rapidly increased as cells reach stationary phase. These results suggest that stress induces changes in the composition of proteasomes and increases their levels. This may be necessary for the stressed cells to recognize new substrate proteins and/or increase the rate of protein degradation to increase the levels of free amino acids.

Impacts
This research project has greatly impacted the field of proteasome structure and function by identifying and characterizing this multicatalytic protease from several archaeal organisms that survive in extreme environmental conditions (e.g. hydrothermal vents, the Dead Sea). We have also developed genetic engineering techniques to produce these thermal stable and salt-tolerant proteasome enzymes at high levels in recombinant Escherichia coli. This has lead to international collaborations with X-ray crystallographers and cell physiologists to elucidate basic structural features of this enzyme that is expected to benefit agriculture and medicine (e.g. development of drugs for cancer therapy, minimizing the aging process in plants and animals).

Publications

• Maupin-Furlow, J. A., Kaczowka, S. J., Ou, M. S., and H. L. Wilson. 2001. Archaeal proteasomes: nanocompartments of the cell. P. Blum (ed.) Adv. Appl. Microbiol., Academic Press. vol. 50, p. 279-338.

Progress 01/01/00 to 12/31/00

Outputs
The long term aim of our research is to better understand energy-dependent proteolysis in the cell, especially as it relates to the metabolically diverse archaea. Although putative energy-dependent proteases such as the proteasome have been isolated from archaea, little is known about their physiological role and mechanism. It is proposed that furthering our understanding in this important area will broaden our ability to use these organisms in biotechnology. Although thermodynamically peptide-bond hydrolysis does not require energy, a small group of proteases are energy-dependent and are central to the regulation of cell division, metabolism, differentiation, as well as other functions of the cell. These proteases have a self-compartmentalized structure which confines the proteolytic active-sites to an inner chamber which is barricaded from cytosolic proteins by narrow openings. Thus, additional ATPase proteins or protein-domains are required for the recognition and/or unfolding of substrate proteins which are fed into the proteolytic compartment. During the past year, our lab has determined the physical and biochemical properties of the 20S proteasome from the hyperthermophilic methanogen Methanococcus jannaschii. The 20S particle was found to be a self-compartmentalized proteolytic cylinder which was unable to degrade substrate proteins unless they were partially unfolded. We also purified a 550-kDa complex from Methanococcus jannaschii, denoted as PAN for proteasome-activating nucleotidase, which stimulates the hydrolysis of proteins by the 20S core particle in the presence of ATP or CTP. The PAN protein was found to be a member of the AAA family of ATPases, many of which fold and/or unfold proteins in a chaperone-like manner. Based on electron micrographs, the 20S proteasome and PAN form a complex in the presence of ATP. Thus, we propose that the PAN protein facilitates the energy-dependent recognition and/or unfolding of substrate proteins for entry into the central, proteolytic chamber of the 20S proteasome. This is the first example of energy-dependent proteolysis mediated by archaeal proteins and suggests that this type of mechanism occurs in the archaeal cell. To investigate the role of the proteasome and PAN proteins in vivo, we are performing genetic studies using the methanogen Methanococcus maripaludis as well as the halophile Haloferax volcanii. Both of these organisms are mesophilic archaea with genetic tools available. Based on our studies in H. volcanii, the proteasome is apparently involved in the turnover of abnormal proteins produced either in the presence of amino acid analogues or by thermal denaturation during heat shock. To further these studies, we have constructed chromosomal mutations in proteasome genes and have generated plasmids which overexpress antigen-tagged proteasome proteins in H. volcanii. We are now in the process of analyzing the physiological effects of genetic modifications in the proteasome proteins in the archaea.

Impacts
Our laboratory has developed biotechnology methods to produce large quantities of archaeal proteasomes which are now used internationally to elucidate the mechanism of energy-dependent proteolysis and screen for drugs which may inhibitor the growth of cancer cells. In addition, proteasome antibodies from our research may be used for monitoring stress in microbial consortiums established for bioremediation, desalination, and wastewater treatment.

Publications

• Maupin-Furlow, J. A., Kaczowka, S., Ou, M. S., and H. L. Wilson. 2000. Archaeal proteasomes and the stress response. Front Biosci. 5, in press.
• Wilson, H. L., Ou, M.S., Aldrich, H. C. and J. A. Maupin-Furlow. 2000. Biochemical and physical properties of the Methanococcus jannaschii 20S proteasome and, a homolog the ATPase (Rpt) subunits of the eucaryal 26S proteasome. J. Bact. 182, in press.
• Wilson, H. L., Aldrich, H. C., and J. A. Maupin-Furlow. 1999. Halophilic 20S proteasomes of the archaeon Haloferax volcanii: purification, characterization, and gene sequence analysis. J. Bact. 181: 5814-5824.
• Maupin-Furlow, J. A., Aldrich, H. C., and J. G. Ferry. 1998. Biochemical characterization of the 20S proteasome from the methanoarchaeon Methanosarcina thermophila. J. Bacteriol. 180: 1480-1487.

Progress 10/01/99 to 09/30/00

Outputs
Our long-term goal is to better understand energy-dependent proteolysis mediated by the proteasome. In eucarya, this enzyme influences a variety of central cellular processes including stress survival, cell division, development, and the immune system. Although 20S proteasomes and proteasome-activating nucleotidase (PAN) proteins have been isolated from archaea, little is known about their physiological role or mechanism of protein degradation. It is proposed that furthering our understanding in this important area will broaden our ability to use these and other related extremophiles in biotechnology. The 20S proteasome is composed of four stacked heptameric rings made up of two families of subunits designated alpha and beta. In the eucarya the complex is composed of 14 different subunits; whereas, in the archaea the majority of 20S proteasomes are composed of a single alpha and beta subunit. Likewise, archaeal PAN which activates the proteasome is typically only composed of one subunit while six different subunits form the base of the related complex in eucarya. Due to this simpler composition, as compared to their eucaryal counterparts, archaeal 20S proteasome and PAN proteins have become model systems for the study of the structure and function relationship of proteasome-mediated degradation. We previously discovered that the archaeon Haloferax volcanii is unique in that it contains two alpha subunits and a single beta subunit. During this past year, we found the alpha1, alpha 2, and beta subunits copurify as active 20S proteasomes. The two alpha subunits associate together in heterogeneous rings. A time course of H. volcanii grown in batch culture indicates the levels of alpha1 protein are constitutive throughout growth, whereas the alpha2 protein only starts to appear at significant levels during stationary phase. Previously, we reported the purification and characterization of the recombinant Methanococcus jannaschii 20S proteasome and PAN. PAN stimulated 20S proteasome-mediated protein hydrolysis in the presence of nucleotide triphosphates. During this past year, Methanococcus maripaludis was chosen as a model organism due to its close relationship to M. jannaschii, genetic tools available, and our ability to grow this organism at high cell density. The M. maripaludis pan gene was isolated and expressed at high-levels in recombinant Escherichia coli for purification of the PAN protein. The recombinant M. maripaludis PAN formed a 600-kDa complex which was much less heat-stable than the M. jannaschii protein, aggregating at 50 degrees C. During attempts to isolate PAN directly from the M. maripaludis, two complexes of 200 kDa with NEM-inhibitable nucleotidase activity were identified. However, neither of these proteins cross-reacted with the anti-PAN antibody. A 20S proteasome was also purified from M. maripaludis, which hydrolyzed the substrate LLVY-Amc at an optimum of 80 degrees C. In order to optimize purification of PAN from M. maripaludis, the genes expressing His6-tagged PAN from both methanogens have been placed into expression shuttle vectors and will be synthesized in M. maripaludis for further characterization.

Impacts
This research project has greatly impacted the field of proteasome structure and function by identifying and characterizing this multicatalytic protease from several archaeal organisms that survive in extreme environmental conditions (e.g. hydrothermal vents, the Dead Sea). We have also developed genetic engineering techniques to produce these thermal stable and salt-tolerant proteasome enzymes at high levels in recombinant Escherichia coli. This has lead to international collaborations with X-ray crystallographers to elucidate basic structural features of this enzyme that is expected to benefit agriculture and medicine (e.g. development of drugs for cancer therapy, minimizing the aging process in plants and animals).

Publications

• Wilson, H. L., Ou, M.S., Aldrich, H. C. and J. A. Maupin-Furlow. 2000. Biochemical and physical properties of the Methanococcus jannaschii 20S proteasome and, a homolog the ATPase (Rpt) subunits of the eucaryal 26S proteasome. J. Bact. 182: 1680-1692.
• Maupin-Furlow, J. A., Kaczowka, S. J., Ou, M.S., and H. L. Wilson. 2000. Archaeal proteasomes: nanocompartments of the cell. Adv. Appl. Microbiol. (accepted).
• Maupin-Furlow, J. A., Wilson, H. L., Kaczowka, S., and M. S. Ou. 2000. Archaeal proteasomes: from structure to function. Front Biosci. 5: D837-865.

Progress 10/01/97 to 09/30/98

Outputs
Anaerobic microorganisms, which account for over 25% of the protoplasm on earth, play a major role in the global carbon cycle and production of green-house gases. Methane gas, a contributor to global warming, has more than doubled in atmospheric concentrations over the past 300 years and is increasing at a rate of almost 1% per year. Fortunately, when properly controlled, biomass conversion to methane provides inexpensive energy for Third World communities as well as innovative projects in the U.S. Thus, scientific research efforts are aimed at developing ways to improve the biological production of methane in controlled systems as well as understand the physiology of methane production and its global impact. Our laboratory in the Microbiology and Cell Science Department at the University of Florida is focused on understanding the microbial conversion of biomass to methane. The rationale is that a better understanding of the physiology of the methanogens will provide insight into how their metabolic pathways can be modified for increased economic benefit. Through these studies an ATP-dependent protease has been identified which may be involved in regulating the levels of the central enzyme of acetotrophic methanogenesis, the carbon monoxide dehydrogenase complex. The methanoarchaeal protease or proteasome is also providing an ideal model for understanding the analogous 26S proteasome of higher organisms. The 26S proteasome is now known to be involved in a diversity of functions including cell growth and the generation of peptides which are presented to the immune system. Through biotechnology, our laboratory now produces large quantities of methanogen proteasomes which are used to screen protease inhibitors developed by the pharmaceutical industry. In fact, low doses of proteasome inhibitors are showing much promise as future drugs in the inhibition of dividing cancer cells and may even prove effective in modulating the immune system. The methanogen proteasomes in particular are useful for analyzing protease inhibitors because of their multicatalytic peptide hydrolyzing activity which is analogous to the proteasome of higher organisms. Another application of our research includes the use of proteasome antibodies to monitor microbial stress responses in anaerobic consortiums employed for bioremediation, desalination, and wastewater treatment. Thus, investigation of methane production by our laboratory has provided a better understanding of metabolism while making useful contributions to industry.

Impacts
(N/A)

Publications

• Maupin-Furlow, J. A., Aldrich, H. C., and J. G. Ferry. 1998. Biochemical characterization of the 20S proteasome from the methanoarchaeon Methanosarcina thermophila. J. Bacteriol. 180: 1480-1487.
• Maupin-Furlow, J. A., Ou, M.S., Aldrich, H.A., and J. G. Ferry. 1998. Characterization of an active, cylindrical archaeal beta subunit proteasome complex. Ann. Meet. Amer. Soc. Microbiol, Abstract 11344.
• Wilson, H. L. and J. A. Maupin-Furlow. 1998. Regulation of proteasome levels in the Archaea. Ann. Meet. Amer. Soc. Microbiol, Abstract 11785.
• Wilson, H. L. and J. A. Maupin-Furlow. 1998. An archaeal homologue of the 26S proteasome ATPase regulatory subunits. Ann. Meet. Southeastern Amer. Soc. Microbiol., Abstract.
• Maupin-Furlow, J. A. and H. L. Wilson 1997. Proteasomes in the halophilic Archaea. Ann. Meet. Amer. Soc. Microbiol, Abstract I-053.