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N-terminal domain of nuclear IL-1α shows structural similarity to the C-terminal domain of Snf1 and binds to the HAT/core module of the SAGA complex.

Zamostna B, Novak J, Vopalensky V, Masek T, Burysek L, Pospisek M - PLoS ONE (2012)

Bottom Line: Interestingly, a significant proportion of IL-1α is translocated to the cell nucleus, in which it interacts with histone acetyltransferase complexes.We also predicted the 3-D structure of the IL-1α N-terminal domain, and by employing structure similarity searches, we found a similar structure in the C-terminal regulatory region of the catalytic subunit of the AMP-activated/Snf1 protein kinases, which interact with HAT complexes both in mammals and yeast, respectively.Finally, the careful evaluation of our data together with other published data in the field allows us to hypothesize a new function for the ADA complex in SAGA complex assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Prague, Czech Republic.

ABSTRACT
Interleukin-1α (IL-1α) is a proinflammatory cytokine and a key player in host immune responses in higher eukaryotes. IL-1α has pleiotropic effects on a wide range of cell types, and it has been extensively studied for its ability to contribute to various autoimmune and inflammation-linked disorders, including rheumatoid arthritis, Alzheimer's disease, systemic sclerosis and cardiovascular disorders. Interestingly, a significant proportion of IL-1α is translocated to the cell nucleus, in which it interacts with histone acetyltransferase complexes. Despite the importance of IL-1α, little is known regarding its binding targets and functions in the nucleus. We took advantage of the histone acetyltransferase (HAT) complexes being evolutionarily conserved from yeast to humans and the yeast SAGA complex serving as an epitome of the eukaryotic HAT complexes. Using gene knock-out technique and co-immunoprecipitation of the IL-1α precursor with TAP-tagged subunits of the yeast HAT complexes, we mapped the IL-1α-binding site to the HAT/Core module of the SAGA complex. We also predicted the 3-D structure of the IL-1α N-terminal domain, and by employing structure similarity searches, we found a similar structure in the C-terminal regulatory region of the catalytic subunit of the AMP-activated/Snf1 protein kinases, which interact with HAT complexes both in mammals and yeast, respectively. This finding is further supported with the ability of the IL-1α precursor to partially rescue growth defects of snf1Δ yeast strains on media containing 3-Amino-1,2,4-triazole (3-AT), a competitive inhibitor of His3. Finally, the careful evaluation of our data together with other published data in the field allows us to hypothesize a new function for the ADA complex in SAGA complex assembly.

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Related in: MedlinePlus

Heterologous expression and immunoprecipitation of the IL-1α proteins in yeast.“pre” represents the IL-1α precursor produced from the plasmid 212-pre-Flag, and “Mat” represents mature interleukin-1α produced from 212-Mat-Flag. A yeast strain transformed with empty vector was used as the control (ctrl). Immunoprecipitation (IP) and western blotting was performed using an anti-Flag antibody recognizing the Flag tag at the N-terminus of the IL-1α proteins. Asterisks indicate the bands corresponding to the heavy and light chains of the anti-Flag antibody. Molecular size marker positions are shown at the right.
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pone-0041801-g002: Heterologous expression and immunoprecipitation of the IL-1α proteins in yeast.“pre” represents the IL-1α precursor produced from the plasmid 212-pre-Flag, and “Mat” represents mature interleukin-1α produced from 212-Mat-Flag. A yeast strain transformed with empty vector was used as the control (ctrl). Immunoprecipitation (IP) and western blotting was performed using an anti-Flag antibody recognizing the Flag tag at the N-terminus of the IL-1α proteins. Asterisks indicate the bands corresponding to the heavy and light chains of the anti-Flag antibody. Molecular size marker positions are shown at the right.

Mentions: In order to find suitable conditions for co-immunoprecipitation of yeast HAT complexes via interleukin-1α as their interacting partner, we prepared a set of yeast low- and high-copy number expression vectors allowing us to produce intracellularly either pre-IL-1α or the matured interleukin-1α with various N- and C-terminal epitopes. For the rest of this study we chose N-terminal tagging of the interleukin-1α peptides by Flag epitope and vectors based on yeast expression multicopy plasmid pYX212 (Ingenius), which contains strong constitutive triose phosphate isomerase promoter. The expression of the Flag-tagged IL-1α precursor and Flag-tagged mature IL-1α produced from the plasmids 212-pre-Flag and 212-Mat-Flag in yeast cells and a possibility to immunoprecipitate both proteins from a yeast cell lysate with low background were verified by western blotting (Figure 2).


N-terminal domain of nuclear IL-1α shows structural similarity to the C-terminal domain of Snf1 and binds to the HAT/core module of the SAGA complex.

Zamostna B, Novak J, Vopalensky V, Masek T, Burysek L, Pospisek M - PLoS ONE (2012)

Heterologous expression and immunoprecipitation of the IL-1α proteins in yeast.“pre” represents the IL-1α precursor produced from the plasmid 212-pre-Flag, and “Mat” represents mature interleukin-1α produced from 212-Mat-Flag. A yeast strain transformed with empty vector was used as the control (ctrl). Immunoprecipitation (IP) and western blotting was performed using an anti-Flag antibody recognizing the Flag tag at the N-terminus of the IL-1α proteins. Asterisks indicate the bands corresponding to the heavy and light chains of the anti-Flag antibody. Molecular size marker positions are shown at the right.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3412866&req=5

pone-0041801-g002: Heterologous expression and immunoprecipitation of the IL-1α proteins in yeast.“pre” represents the IL-1α precursor produced from the plasmid 212-pre-Flag, and “Mat” represents mature interleukin-1α produced from 212-Mat-Flag. A yeast strain transformed with empty vector was used as the control (ctrl). Immunoprecipitation (IP) and western blotting was performed using an anti-Flag antibody recognizing the Flag tag at the N-terminus of the IL-1α proteins. Asterisks indicate the bands corresponding to the heavy and light chains of the anti-Flag antibody. Molecular size marker positions are shown at the right.
Mentions: In order to find suitable conditions for co-immunoprecipitation of yeast HAT complexes via interleukin-1α as their interacting partner, we prepared a set of yeast low- and high-copy number expression vectors allowing us to produce intracellularly either pre-IL-1α or the matured interleukin-1α with various N- and C-terminal epitopes. For the rest of this study we chose N-terminal tagging of the interleukin-1α peptides by Flag epitope and vectors based on yeast expression multicopy plasmid pYX212 (Ingenius), which contains strong constitutive triose phosphate isomerase promoter. The expression of the Flag-tagged IL-1α precursor and Flag-tagged mature IL-1α produced from the plasmids 212-pre-Flag and 212-Mat-Flag in yeast cells and a possibility to immunoprecipitate both proteins from a yeast cell lysate with low background were verified by western blotting (Figure 2).

Bottom Line: Interestingly, a significant proportion of IL-1α is translocated to the cell nucleus, in which it interacts with histone acetyltransferase complexes.We also predicted the 3-D structure of the IL-1α N-terminal domain, and by employing structure similarity searches, we found a similar structure in the C-terminal regulatory region of the catalytic subunit of the AMP-activated/Snf1 protein kinases, which interact with HAT complexes both in mammals and yeast, respectively.Finally, the careful evaluation of our data together with other published data in the field allows us to hypothesize a new function for the ADA complex in SAGA complex assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Prague, Czech Republic.

ABSTRACT
Interleukin-1α (IL-1α) is a proinflammatory cytokine and a key player in host immune responses in higher eukaryotes. IL-1α has pleiotropic effects on a wide range of cell types, and it has been extensively studied for its ability to contribute to various autoimmune and inflammation-linked disorders, including rheumatoid arthritis, Alzheimer's disease, systemic sclerosis and cardiovascular disorders. Interestingly, a significant proportion of IL-1α is translocated to the cell nucleus, in which it interacts with histone acetyltransferase complexes. Despite the importance of IL-1α, little is known regarding its binding targets and functions in the nucleus. We took advantage of the histone acetyltransferase (HAT) complexes being evolutionarily conserved from yeast to humans and the yeast SAGA complex serving as an epitome of the eukaryotic HAT complexes. Using gene knock-out technique and co-immunoprecipitation of the IL-1α precursor with TAP-tagged subunits of the yeast HAT complexes, we mapped the IL-1α-binding site to the HAT/Core module of the SAGA complex. We also predicted the 3-D structure of the IL-1α N-terminal domain, and by employing structure similarity searches, we found a similar structure in the C-terminal regulatory region of the catalytic subunit of the AMP-activated/Snf1 protein kinases, which interact with HAT complexes both in mammals and yeast, respectively. This finding is further supported with the ability of the IL-1α precursor to partially rescue growth defects of snf1Δ yeast strains on media containing 3-Amino-1,2,4-triazole (3-AT), a competitive inhibitor of His3. Finally, the careful evaluation of our data together with other published data in the field allows us to hypothesize a new function for the ADA complex in SAGA complex assembly.

Show MeSH
Related in: MedlinePlus