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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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The SAGA and ADA complex subunits co-purify as a part of the IL-1α precursor-binding complex.Co-immunoprecipitation experiments with an anti-Flag antibody using yeast strains from a TAP tag library transformed with IL-1α expression vectors revealed that both of the HAT complexes bound pre-IL-1α (pre) but not mature IL-1α (Mat). Control cells (ctrl) carry the empty plasmid pYX212. Western blotting was performed using an anti-CBP antibody that recognizes the TAP tag at the C-terminus of the respective HAT complex subunits. For each line of the IP experiment, 1.4 mL of the cell lysate prepared from 5.108 yeast cells in average was used. Inputs contain 16.7 µL of the corresponding lysates taken before the lysates were used for immunoprecipitation.
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pone-0041801-g005: The SAGA and ADA complex subunits co-purify as a part of the IL-1α precursor-binding complex.Co-immunoprecipitation experiments with an anti-Flag antibody using yeast strains from a TAP tag library transformed with IL-1α expression vectors revealed that both of the HAT complexes bound pre-IL-1α (pre) but not mature IL-1α (Mat). Control cells (ctrl) carry the empty plasmid pYX212. Western blotting was performed using an anti-CBP antibody that recognizes the TAP tag at the C-terminus of the respective HAT complex subunits. For each line of the IP experiment, 1.4 mL of the cell lysate prepared from 5.108 yeast cells in average was used. Inputs contain 16.7 µL of the corresponding lysates taken before the lysates were used for immunoprecipitation.

Mentions: Our previous results showed that the nuclear localization and interaction of the IL-1α precursor with histone acetyltransferase complexes are similar in yeast and human cells (above and [40]). However, our structure similarity searches and serial drop tests with snf1Δ strains suggested that the IL-1α precursor may interact not only with the SAGA complex but also with the ADA complex because the Snf1 kinase was genetically shown to interact with Ahc1, the structural subunit of the ADA complex [47]. This interaction would be surprising because the expression of the Gal4BD/IL-1αNTP fusion in yeast induces a classical Ada- phenotype, and the ahc1 mutation, in contrast to gcn5Δ, ada2Δ, ada3Δ and spt7Δ, did not rescue the toxicity of Gal4BD/IL-1αNTP overproduction [40]. To examine this interaction, we took advantage of the power of yeast model and employed a yeast TAP fusion library [41]. Plasmids 212-pre-Flag and 212-Mat-Flag were separately introduced into S. cerevisiae BY4741 strains expressing selected TAP-tagged subunits of either the SAGA or ADA complex [41] and subsequently a set of co-immunoprecipitation experiments using an anti-Flag antibody against the Flag-tagged IL-1α precursor and the Flag-tagged mature IL-1α as a control was performed (Figure 5).


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)

The SAGA and ADA complex subunits co-purify as a part of the IL-1α precursor-binding complex.Co-immunoprecipitation experiments with an anti-Flag antibody using yeast strains from a TAP tag library transformed with IL-1α expression vectors revealed that both of the HAT complexes bound pre-IL-1α (pre) but not mature IL-1α (Mat). Control cells (ctrl) carry the empty plasmid pYX212. Western blotting was performed using an anti-CBP antibody that recognizes the TAP tag at the C-terminus of the respective HAT complex subunits. For each line of the IP experiment, 1.4 mL of the cell lysate prepared from 5.108 yeast cells in average was used. Inputs contain 16.7 µL of the corresponding lysates taken before the lysates were used for immunoprecipitation.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3412866&req=5

pone-0041801-g005: The SAGA and ADA complex subunits co-purify as a part of the IL-1α precursor-binding complex.Co-immunoprecipitation experiments with an anti-Flag antibody using yeast strains from a TAP tag library transformed with IL-1α expression vectors revealed that both of the HAT complexes bound pre-IL-1α (pre) but not mature IL-1α (Mat). Control cells (ctrl) carry the empty plasmid pYX212. Western blotting was performed using an anti-CBP antibody that recognizes the TAP tag at the C-terminus of the respective HAT complex subunits. For each line of the IP experiment, 1.4 mL of the cell lysate prepared from 5.108 yeast cells in average was used. Inputs contain 16.7 µL of the corresponding lysates taken before the lysates were used for immunoprecipitation.
Mentions: Our previous results showed that the nuclear localization and interaction of the IL-1α precursor with histone acetyltransferase complexes are similar in yeast and human cells (above and [40]). However, our structure similarity searches and serial drop tests with snf1Δ strains suggested that the IL-1α precursor may interact not only with the SAGA complex but also with the ADA complex because the Snf1 kinase was genetically shown to interact with Ahc1, the structural subunit of the ADA complex [47]. This interaction would be surprising because the expression of the Gal4BD/IL-1αNTP fusion in yeast induces a classical Ada- phenotype, and the ahc1 mutation, in contrast to gcn5Δ, ada2Δ, ada3Δ and spt7Δ, did not rescue the toxicity of Gal4BD/IL-1αNTP overproduction [40]. To examine this interaction, we took advantage of the power of yeast model and employed a yeast TAP fusion library [41]. Plasmids 212-pre-Flag and 212-Mat-Flag were separately introduced into S. cerevisiae BY4741 strains expressing selected TAP-tagged subunits of either the SAGA or ADA complex [41] and subsequently a set of co-immunoprecipitation experiments using an anti-Flag antibody against the Flag-tagged IL-1α precursor and the Flag-tagged mature IL-1α as a control was performed (Figure 5).

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