Covalent attachment of selected cellular proteins to small protein modifiers of the 2-grasp fold ubiquitin/MoaD/ThiS superfamily plays an integral role in regulating a wide variety of metabolic and developmental processes related to human health. This includes the degradation of proteins by proteasomes, autophagy and non-proteolyitc events such as protein trafficking, DNA repair and cell signaling. While all eukaryotes encode ubiquitin- and ubiquitin-like-conjugating systems, the origin of these pathways is unclear and speculated to have assembled from various prokaryotic pathways that function in related, yet distinct, chemistry such as sulfur activation. This application is based on our recent discovery of two small archaeal protein modifiers (SAMP1 and SAMP2) that cluster to the 2-grasp fold superfamily, are highly conserved among archaea and are differentially conjugated to a large number of proteins in the halophilic archaeon Haloferax volcanii. The levels of SAMPylation were found to be modulated by environmental signals and proteasomal knockout mutations, thus providing early insight into how this modification may influence cell physiology. The chemistry of SAMPylation appears analogous in chemistry to ubiquitination based on the requirement of the C-terminal di-glycine motif of SAMP in covalent modification and the isolation of E1-, E2- and rhodanese-like SAMP conjugates.
The aims of this project are to: 1) identify and classify the large group of Hfx. volcanii proteins that are conjugated to the SAMP1 and SAMP2 proteins (SAMPylated) and define the site and type of isopeptide bond formed in these modifications, 2) investigate whether the Hfx. volcanii proteins related to the E1-activating and E2-conjugating enzymes of ubiquitination that were isolated in complex with the SAMP proteins are required for SAMPylation, and 3) link the environmental signals and proteasomal gene knockouts that influence SAMP-conjugate formation to cell function. This application will test the following hypotheses: 1) SAMP1 and SAMP2 differentially conjugate a wide variety of proteins resulting in the formation of isopeptide bonds between the C-terminal carboxyl group of the SAMP protein and the 5-amino groups of the lysine residues of substrate proteins, 2) the E1 and E2 homologs of Hfx. volcanii function in the activation of SAMPs for transfer to the protein substrate and, 3) SAMPylation is likely to signal proteasome-mediated proteolysis in addition to non-proteolytic events. The proposed study of archaeal SAMPylation will provide new insight into 2- grasp Ub/MoaD/ThiS superfamily-mediated protein conjugation including an understanding of how this type of system functions in prokaryotic cells.

Public Health Relevance

This research is related to ubiquitinylation and proteasome-mediated protein degradation, both of which are of great interest in human health. These pathways are emerging as key regulators of immunity, DNA repair and cell division, and their dysregulation is linked to a growing number of diseases including cancer, neurodegenerative disorders and mental retardation. Advancing our understanding of these processes will greatly assist in targeting proteasomes and Ub/Ubl-conjugation pathways for the development of new anti-cancer and anti-inflammatory drugs.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM057498-13
Application #
8438503
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Gerratana, Barbara
Project Start
2000-04-01
Project End
2015-03-31
Budget Start
2013-04-01
Budget End
2015-03-31
Support Year
13
Fiscal Year
2013
Total Cost
$230,932
Indirect Cost
$73,299
Name
University of Florida
Department
Microbiology/Immun/Virology
Type
Schools of Earth Sciences/Natur
DUNS #
969663814
City
Gainesville
State
FL
Country
United States
Zip Code
32611
Rosnow, Joshua J; Hwang, Sungmin; Killinger, Bryan J et al. (2018) Cobalamin activity-based probe enables microbial cell growth and finds new cobalamin-protein interactions across domains. Appl Environ Microbiol :
Deng, Yue; Jiang, Beichen; Rankin, Carolyn L et al. (2018) Methionine sulfoxide reductase A (MsrA) mediates the ubiquitination of 14-3-3 protein isotypes in brain. Free Radic Biol Med 129:600-607
Fu, Xian; Adams, Zachary; Maupin-Furlow, Julie A (2018) In vitro Analysis of Ubiquitin-like Protein Modification in Archaea. Bio Protoc 8:
McMillan, Lana J; Hwang, Sungmin; Farah, Rawan E et al. (2018) Multiplex quantitative SILAC for analysis of archaeal proteomes: a case study of oxidative stress responses. Environ Microbiol 20:385-401
Hwang, Sungmin; Cordova, Bryan; Abdo, Merna et al. (2017) ThiN as a Versatile Domain of Transcriptional Repressors and Catalytic Enzymes of Thiamine Biosynthesis. J Bacteriol 199:
Fu, Xian; Adams, Zachary; Liu, Rui et al. (2017) Methionine Sulfoxide Reductase A (MsrA) and Its Function in Ubiquitin-Like Protein Modification in Archaea. MBio 8:
Fu, Xian; Maupin-Furlow, Julie A (2017) Chase Assay of Protein Stability in Haloferax volcanii. Bio Protoc 7:
Cao, Shiyun; Engilberge, Sylvain; Girard, Eric et al. (2017) Structural Insight into Ubiquitin-Like Protein Recognition and Oligomeric States of JAMM/MPN+ Proteases. Structure 25:823-833.e6
McMillan, Lana J; Hepowit, Nathaniel L; Maupin-Furlow, Julie A (2016) Archaeal Inorganic Pyrophosphatase Displays Robust Activity under High-Salt Conditions and in Organic Solvents. Appl Environ Microbiol 82:538-48
Fu, Xian; Liu, Rui; Sanchez, Iona et al. (2016) Ubiquitin-Like Proteasome System Represents a Eukaryotic-Like Pathway for Targeted Proteolysis in Archaea. MBio 7:

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