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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Prokaryotic Cell and Molecular Biology Study Section (PCMB)
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Gerratana, Barbara
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University of Florida
Schools of Earth Sciences/Natur
United States
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Dantuluri, Swathi; Wu, Yifei; Hepowit, Nathaniel L et al. (2016) Proteome targets of ubiquitin-like samp1ylation are associated with sulfur metabolism and oxidative stress in Haloferax volcanii. Proteomics 16:1100-10
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:
Hepowit, Nathaniel L; de Vera, Ian Mitchelle S; Cao, Shiyun et al. (2016) Mechanistic insight into protein modification and sulfur mobilization activities of noncanonical E1 and associated ubiquitin-like proteins of Archaea. FEBS J 283:3567-3586
McMillan, Lana J; Hepowit, Nathaniel L; Maupin-Furlow, Julie A (2015) Archaeal Inorganic Pyrophosphatase Displays Robust Activity under High-Salt Conditions and in Organic Solvents. Appl Environ Microbiol 82:538-48
Cao, Shiyun; Hepowit, Nathaniel; Maupin-Furlow, Julie A (2015) Ubiquitin-Like Protein SAMP1 and JAMM/MPN+ Metalloprotease HvJAMM1 Constitute a System for Reversible Regulation of Metabolic Enzyme Activity in Archaea. PLoS One 10:e0128399
Chavarria, Nikita E; Hwang, Sungmin; Cao, Shiyun et al. (2014) Archaeal Tuc1/Ncs6 homolog required for wobble uridine tRNA thiolation is associated with ubiquitin-proteasome, translation, and RNA processing system homologs. PLoS One 9:e99104
Maupin-Furlow, Julie A (2014) Prokaryotic ubiquitin-like protein modification. Annu Rev Microbiol 68:155-75
Prunetti, Laurence; Reuter, Christopher J; Hepowit, Nathaniel L et al. (2014) Structural and biochemical properties of an extreme 'salt-loving' proteasome activating nucleotidase from the archaeon Haloferax volcanii. Extremophiles 18:283-93
Miranda, Hugo V; Antelmann, Haike; Hepowit, Nathaniel et al. (2014) Archaeal ubiquitin-like SAMP3 is isopeptide-linked to proteins via a UbaA-dependent mechanism. Mol Cell Proteomics 13:220-39
Hwang, Sungmin; Cordova, Bryan; Chavarria, Nikita et al. (2014) Conserved active site cysteine residue of archaeal THI4 homolog is essential for thiamine biosynthesis in Haloferax volcanii. BMC Microbiol 14:260

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