Shotgun Proteomics of the Haloarchaeon <i>Haloferax volcanii</i>

Kirkland, P. Aaron; Humbard, Matthew A.; Daniels, Charles J.; Maupin-Furlow, Julie A.

doi:10.1021/pr800517a

Cited by 49 publications

(60 citation statements)

References 35 publications

(85 reference statements)

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“…Unlike bacteria, formyl-methionine has not been identified as an initiating residue in archaea (36). These final Nterminal states of a protein are thought to be governed by the nature of the first three amino acids: the initiating methionine and the penultimate and antepenultimate amino acids (10,23,26,29,35,49). Taking this into consideration, a series of amino acid substitutions were introduced into the ␣1 protein through site-directed mutagenesis of the psmA gene.…”

Section: Smentioning

confidence: 99%

“…It should also be noted that N ␣ acetylation of an initiator methionine preceding a glutamine residue (Met-Gln-), as in ␣1, is not common among proteins of eukaryotes (35) or haloarchaea (1,10,23). This motif is somewhat related to substrates of the eukaryotic protein NatB, which acetylates initiator methionine residues preceding glutamate, aspartate, asparagine, or methionine residues (Met-Glu-, Met-Asp-, MetAsn-, or Met-Met-, respectively).…”

Section: Smentioning

confidence: 99%

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The N-Terminal Penultimate Residue of 20S Proteasome α1 Influences its N ^α Acetylation and Protein Levels as Well as Growth Rate and Stress Responses of Haloferax volcanii

2009

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Proteasomes are energy-dependent proteolytic machines. We elaborate here on the previously observed N ␣ acetylation of the initiator methionine of the ␣1 protein of 20S core particles (CPs) of Haloferax volcanii proteasomes. Quantitative mass spectrometry revealed this was the dominant N-terminal form of ␣1 in H. volcanii cells. To further examine this, ␣1 proteins with substitutions in the N-terminal penultimate residue as well as deletion of the CP "gate" formed by the ␣1 N terminus were examined for their N ␣ acetylation. Both the "gate" deletion and Q2A substitution completely altered the N ␣ -acetylation pattern of ␣1, with the deletion rendering ␣1 unavailable for N ␣ acetylation and the Q2A modification apparently enhancing cleavage of ␣1 by methionine aminopeptidase (MAP), resulting in acetylation of the N-terminal alanine. Cells expressing these two ␣1 variants were less tolerant of hypoosmotic stress than the wild type and produced CPs with enhanced peptidase activity. Although ␣1 proteins with Q2D, Q2P, and Q2T substitutions were N ␣ acetylated in CPs similar to the wild type, cells expressing these variants accumulated unusually high levels of ␣1 as rings in N ␣ -acetylated, unmodified, and/or MAP-cleaved forms. More detailed examination of this group revealed that while CP peptidase activity was not impaired, cells expressing these ␣1 variants displayed higher growth rates and were more tolerant of hypoosmotic and high-temperature stress than the wild type. Overall, these results suggest that N ␣ acetylation of ␣1 is important in CP assembly and activity, high levels of ␣1 rings enhance cell proliferation and stress tolerance, and unregulated opening of the CP "gate" impairs the ability of cells to overcome salt stress.Proteolysis is important in regulation and protein quality control. Energy-dependent proteases are crucial to early stages of these proteolytic events and include proteasomes, multicatalytic proteases present in all eukaryotes and archaea and in some bacteria. The catalytic component of proteasomes, the 20S core particle (CP), consists of four heptameric rings of ␣-and ␤-type subunits stacked as a barrel in an ␣7␤7␤7␣7 configuration and is essential for growth of archaeal and eukaryotic cells (39, 54). The active sites responsible for peptide bond hydrolysis are formed by N-terminal Thr residues of ␤-type subunits and are sequestered within the central chamber of the barrel-like structure. Energy-dependent triple-A ATPases, including regulatory particle triple-A ATPases (Rpt) in eukaryotes and proteasome-activating nucleotidases (PAN) in archaea, mediate the unfolding and translocation of substrate proteins through the ␣-rings for degradation within the CP (39, 40).One major difference between eukaryotic and prokaryotic proteasomal CPs is in the crystal structure of the channel opening formed by the ␣-rings. Due to partial disorder of the ␣-subunit N termini, the site of substrate entry appears open at the ends of the cylinders of archaeal and bacterial CPs (e.g., CPs of Thermoplasma ...

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Section: Smentioning

confidence: 99%

Section: Smentioning

confidence: 99%

The N-Terminal Penultimate Residue of 20S Proteasome α1 Influences its N ^α Acetylation and Protein Levels as Well as Growth Rate and Stress Responses of Haloferax volcanii

2009

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“…These structures and their accompanying biochemical characterization have provided significant insight into the mechanisms of substrate specificity and catalysis used by NAT enzymes. Interestingly, although NatA and NatE both catalyze α-amino group acetylation, they use unique catalytic strategies and substantially different substrate amino-terminal residue binding pockets to achieve sequence-based substrate specificity.Although only six amino-terminally acetylated proteins have been identified in Escherichia coli, large-scale proteomics studies of three different archaeal species revealed that ∼14-29% of all proteins isolated are acetylated at their amino terminus (17)(18)(19)(20). Analysis of the thermophilic archaea Sulfolobus solfataricus genome resulted in the identification of a protein with 37% sequence identity to human NAA10 that is believed to be the only NAT in this species (21).…”

mentioning

confidence: 99%

“…Although only six amino-terminally acetylated proteins have been identified in Escherichia coli, large-scale proteomics studies of three different archaeal species revealed that ∼14-29% of all proteins isolated are acetylated at their amino terminus (17)(18)(19)(20). Analysis of the thermophilic archaea Sulfolobus solfataricus genome resulted in the identification of a protein with 37% sequence identity to human NAA10 that is believed to be the only NAT in this species (21).…”

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confidence: 99%

Implications for the evolution of eukaryotic amino-terminal acetyltransferase (NAT) enzymes from the structure of an archaeal ortholog

Liszczak

Marmorstein

2013

Proc. Natl. Acad. Sci. U.S.A.

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Amino-terminal acetylation is a ubiquitous modification in eukaryotes that is involved in a growing number of biological processes. There are six known eukaryotic amino-terminal acetyltransferases (NATs), which are differentiated from one another on the basis of substrate specificity. To date, two eukaryotic NATs, NatA and NatE, have been structurally characterized, of which NatA will acetylate the α-amino group of a number of nonmethionine amino-terminal residue substrates such as serine; NatE requires a substrate amino-terminal methionine residue for activity. Interestingly, these two NATs use different catalytic strategies to accomplish substrate-specific acetylation. In archaea, where this modification is less prevalent, only one NAT enzyme has been identified. Surprisingly, this enzyme is able to acetylate NatA and NatE substrates and is believed to represent an ancestral NAT variant from which the eukaryotic NAT machinery evolved. To gain insight into the evolution of NAT enzymes, we determined the X-ray crystal structure of an archaeal NAT from Sulfolobus solfataricus (ssNAT). Through the use of mutagenesis and kinetic analysis, we show that the active site of ssNAT represents a hybrid of the NatA and NatE active sites, and we highlight features of this protein that allow it to facilitate catalysis of distinct substrates through different catalytic strategies, which is a unique characteristic of this enzyme. Taken together, the structural and biochemical data presented here have implications for the evolution of eukaryotic NAT enzymes and the substrate specificities therein.structural biology | evolutionary biology | enzymology | X-ray crystallography T he cotranslational process of amino-terminal acetylation occurs on a majority of eukaryotic proteins and mediates many biological processes, including cellular apoptosis, enzymatic regulation, protein localization, and protein degradation (1-4). In humans, three major amino-terminal acetyltransferase (NAT) complexes, called NatA, NatB, and NatC, are responsible for modifying ∼85% of all proteins that undergo amino-terminal acetylation (5). These complexes are conserved in yeast, exist as obligate heterodimers, and are differentiated from one another on the basis of substrate specificity, which is dictated by the amino-terminal sequence of the substrate protein (5, 6). Each complex consists of a single unique catalytic subunit and an additional unique auxiliary subunit that has been shown to potentiate activity and alter substrate specificity of the enzymatic component, as well as anchor the complex to the ribosome during translation (6-11). Three additional human NAT enzymes, NatD-NatF, have also been identified, but they have a much more limited set of physiological substrates, appear to be independently active, and are not well characterized across eukaryotes (12)(13)(14).The only two eukaryotic NATs that have been structurally characterized are Schizosaccharomyces pombe NatA, consisting of the Naa10p catalytic subunit and Naa15p regulatory subunit, and ...

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“…Haloferax volcanii is well suited for such an approach because sRNAs predicted by comparative genomics can subsequently be characterized using various experimental approaches. Notably, deletion mutants can be constructed very easily, 32,33 it can be grown in microtiter plates enabling phenotyping of mutants, 31 transcriptome analysis using DNA microarrays has been established, 34,35 proteome analysis is routinely performed, 36,37 and many molecular genetic tools are available. [38][39][40] Here we report the application of two bioinformatic approaches to predict sRNA genes in the genome in H. volcanii by comparative genomics of intergenic regions.…”

Section: Introductionmentioning

confidence: 99%

Bioinformatic prediction and experimental verification of sRNAs in the haloarchaeonHaloferax volcanii

Babski¹,

Tjaden²,

Voss³

et al. 2011

RNA Biology

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Shotgun Proteomics of the Haloarchaeon Haloferax volcanii

Cited by 49 publications

References 35 publications

The N-Terminal Penultimate Residue of 20S Proteasome α1 Influences its N ^α Acetylation and Protein Levels as Well as Growth Rate and Stress Responses of Haloferax volcanii

The N-Terminal Penultimate Residue of 20S Proteasome α1 Influences its N ^α Acetylation and Protein Levels as Well as Growth Rate and Stress Responses of Haloferax volcanii

Implications for the evolution of eukaryotic amino-terminal acetyltransferase (NAT) enzymes from the structure of an archaeal ortholog

Bioinformatic prediction and experimental verification of sRNAs in the haloarchaeonHaloferax volcanii

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Shotgun Proteomics of the Haloarchaeon Haloferax volcanii

Cited by 49 publications

References 35 publications

The N-Terminal Penultimate Residue of 20S Proteasome α1 Influences its N α Acetylation and Protein Levels as Well as Growth Rate and Stress Responses of Haloferax volcanii

The N-Terminal Penultimate Residue of 20S Proteasome α1 Influences its N α Acetylation and Protein Levels as Well as Growth Rate and Stress Responses of Haloferax volcanii

Implications for the evolution of eukaryotic amino-terminal acetyltransferase (NAT) enzymes from the structure of an archaeal ortholog

Bioinformatic prediction and experimental verification of sRNAs in the haloarchaeonHaloferax volcanii

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The N-Terminal Penultimate Residue of 20S Proteasome α1 Influences its N ^α Acetylation and Protein Levels as Well as Growth Rate and Stress Responses of Haloferax volcanii

The N-Terminal Penultimate Residue of 20S Proteasome α1 Influences its N ^α Acetylation and Protein Levels as Well as Growth Rate and Stress Responses of Haloferax volcanii