Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of an ASCH domain-containing protein from<i>Zymomonas mobilis</i>ZM4

Park, Suk-Youl; Park, Jeong-Hoh; Kim, Ji Soo

doi:10.1107/s1744309110053467

Cited by 4 publications

(4 citation statements)

References 18 publications

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“…These structural features indicate that EOLA1 is an intracellular protein and may interact with other unknown proteins in the cytoplasm and nucleus to regulate cell function. The bioinformatics analysis reviewed that EOLA1 gene belongs to the activating signal cointegrator-1 (ASC-1) homology (ASCH) superfamily, which is frequently observed in many organisms [ 3 , 4 ].…”

Section: Introductionmentioning

confidence: 99%

EOLA1 Inhibits Lipopolysaccharide-Induced Vascular Cell Adhesion Molecule-1 Expression by Association with MT2A in ECV304 Cells

Leng

Lei

Meng

et al. 2015

International Journal of Inflammation

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Our research group firstly discovered endothelial-overexpressed lipopolysaccharide-associated factor 1 (EOLA1, GenBank number AY074889) as a lipopolysaccharide (LPS) responsive gene in ECV304 cells. The previous studies have further demonstrated the association of EOLA1 with metallothionein 2A (MT2A), while the role of EOLA1 during LPS-induced inflammatory response in ECV304 cells is unknown. In this report, we determined the subcellular localization of EOLA1 and the regulatory capacity of EOLA1 on vascular cell adhesion molecule-1 (VCAM-1) in response to LPS in ECV304 cells. Our results show that EOLA1 is broadly diffuse in the cells, and EOLA1 expression is dramatically induced by LPS. EOLA1 knockdown results in significant enhancement of LPS-induced VCAM-1 production. Consistent with this, overexpression of EOLA1 leads to the reduction of LPS-induced VCAM-1 production. Furthermore, MT2A knockdown reduces LPS-induced VCAM-1 production. Collectively, our results demonstrate a negative regulatory role of EOLA1 on LPS-induced VCAM-1 expression involving its association with MT2A in ECV304 cells.

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Section: Introductionmentioning

confidence: 99%

EOLA1 Inhibits Lipopolysaccharide-Induced Vascular Cell Adhesion Molecule-1 Expression by Association with MT2A in ECV304 Cells

Leng

Lei

Meng

et al. 2015

International Journal of Inflammation

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“…The cloning and purification steps have been described in detail elsewhere 13 . Briefly, the cloned, full-length Zm ASCH (ZMO0922) with an N-terminal hexa-His fusion tag was transformed into E .…”

Section: Methodsmentioning

confidence: 99%

Crystal structure of an ASCH protein from Zymomonas mobilis and its ribonuclease activity specific for single-stranded RNA

Kim

Shin

et al. 2017

Sci Rep

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Activating signal cointegrator-1 homology (ASCH) domains were initially reported in human as a part of the ASC-1 transcriptional regulator, a component of a putative RNA-interacting protein complex; their presence has now been confirmed in a wide range of organisms. Here, we have determined the trigonal and monoclinic crystal structures of an ASCH domain-containing protein from Zymomonas mobilis (ZmASCH), and analyzed the structural determinants of its nucleic acid processing activity. The protein has a central β-barrel structure with several nearby α-helices. Positively charged surface patches form a cleft that runs through the pocket formed between the β-barrel and the surrounding α-helices. We further demonstrate by means of in vitro assays that ZmASCH binds nucleic acids, and degrades single-stranded RNAs in a magnesium ion-dependent manner with a cleavage preference for the phosphodiester bond between the pyrimidine and adenine nucleotides. ZmASCH also removes a nucleotide at the 5′-end. Mutagenesis studies, guided by molecular dynamics simulations, confirmed that three residues (Tyr47, Lys53, and Ser128) situated in the cleft contribute to nucleic acid-binding and RNA cleavage activities. These structural and biochemical studies imply that prokaryotic ASCH may function to control the cellular RNA amount.

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“…In a previous study, a hypothetical ASCH domain-containing protein from Zymomonas mobilis was described as a monomeric ribonuclease that binds nucleic acids and degrades single-stranded RNA molecules in vitro [ 5 , 6 ]. Recently, the Escherichia coli ASCH domain-containing protein YqfB was characterized as the smallest known amidohydrolase with only 103 amino acids [ 7 ].…”

Section: Introductionmentioning

confidence: 99%

Comparative Analysis of Mesophilic YqfB-Type Amidohydrolases

et al. 2022

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The widespread superfamily of the human activating signal cointegrator homology (ASCH) domain was identified almost 20 years ago; however, the amount of experimental data regarding the biological function of the domain is scarce. With this study, we aimed to determine the putative cellular functions of four hypothetical ASCH domain-containing amidohydrolase YqfB analogues by investigating their activity towards various N-acylated cytosine derivatives, including potential nucleoside-derived prodrugs, as well as their ability to bind/degrade nucleic acids in vitro. According to determined kinetic parameters, N4-acetylcytidine is assumed to be the primary substrate for amidohydrolases. Despite the similarity to the proteins containing the PUA domain, no nucleic acid binding activity was detected for YqfB-like proteins, suggesting that, in vivo, these enzymes are a part of the pyrimidine salvage pathway. We also demonstrate the possibility of the expression of YqfB-type amidohydrolases in both prokaryotic and eukaryotic hosts. The small protein size and remarkable halotolerance of YqfB-type amidohydrolases are of great interest for further fundamental research and biotechnological applications

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Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of an ASCH domain-containing protein fromZymomonas mobilisZM4

Cited by 4 publications

References 18 publications

EOLA1 Inhibits Lipopolysaccharide-Induced Vascular Cell Adhesion Molecule-1 Expression by Association with MT2A in ECV304 Cells

EOLA1 Inhibits Lipopolysaccharide-Induced Vascular Cell Adhesion Molecule-1 Expression by Association with MT2A in ECV304 Cells

Crystal structure of an ASCH protein from Zymomonas mobilis and its ribonuclease activity specific for single-stranded RNA

Comparative Analysis of Mesophilic YqfB-Type Amidohydrolases

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