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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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Disruption of the SAGA and ADA complexes confirmed binding of the IL-1α precursor to the HAT/Core module and suggested the mutually exclusive role of Spt7 and Ahc1 in SAGA complex assembly.Co-immunoprecipitation was performed using yeast lysates with an anti-Flag antibody that recognizes the Flag tag at the N-terminus of the IL-1α precursor. Western blotting was performed with an anti-CBP antibody which identifies the TAP tag at the C-terminus of the respective HAT complex subunits. (A) Gcn5 does not bind to pre-IL-1α, and because it is not required for SAGA or ADA complex integrity, its deletion has no effect on Ahc1 or Spt8 co-immunoprecipitation with the IL-1α precursor (pre). Deletion of the AHC2 gene doesn’t impair co-IP of pre-IL-1α with Gcn5, Spt8 and Spt7. (B) The disruption of the ADA HAT complex did not affect the co-immunoprecipitation of Gcn5 and Spt8 with IL-1α. However, the interaction between Spt7 and the IL-1α precursor was significantly weakened. In experiments with TAP/Spt7,ahc1Δ strain, we received either no or very low signal (the latter is depicted) of TAP-tagged Spt7, with a success rate 3∶1, respectively. (C) The disruption of the SAGA complex abolished the interaction between Spt8 and the IL-1α precursor but had no effect on Ahc1 binding to the IL-1α precursor. Control cells (ctrl) carry the empty plasmid pYX212. For each line of the IP experiment, 3.5 mL of cell lysate prepared from 20.108 yeast cells in average was used, except TAP/Spt8,gcn5Δ, TAP/Spt8,ahc1Δ, TAP/Spt8,spt7Δ and TAP/Gcn5,ahc1Δ strains, where 1.3 mL of cell lysates from 9.108 yeast cells each were applied. Inputs contain 16.7 µL of the corresponding lysates taken before the lysates were used for immunoprecipitation.
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pone-0041801-g006: Disruption of the SAGA and ADA complexes confirmed binding of the IL-1α precursor to the HAT/Core module and suggested the mutually exclusive role of Spt7 and Ahc1 in SAGA complex assembly.Co-immunoprecipitation was performed using yeast lysates with an anti-Flag antibody that recognizes the Flag tag at the N-terminus of the IL-1α precursor. Western blotting was performed with an anti-CBP antibody which identifies the TAP tag at the C-terminus of the respective HAT complex subunits. (A) Gcn5 does not bind to pre-IL-1α, and because it is not required for SAGA or ADA complex integrity, its deletion has no effect on Ahc1 or Spt8 co-immunoprecipitation with the IL-1α precursor (pre). Deletion of the AHC2 gene doesn’t impair co-IP of pre-IL-1α with Gcn5, Spt8 and Spt7. (B) The disruption of the ADA HAT complex did not affect the co-immunoprecipitation of Gcn5 and Spt8 with IL-1α. However, the interaction between Spt7 and the IL-1α precursor was significantly weakened. In experiments with TAP/Spt7,ahc1Δ strain, we received either no or very low signal (the latter is depicted) of TAP-tagged Spt7, with a success rate 3∶1, respectively. (C) The disruption of the SAGA complex abolished the interaction between Spt8 and the IL-1α precursor but had no effect on Ahc1 binding to the IL-1α precursor. Control cells (ctrl) carry the empty plasmid pYX212. For each line of the IP experiment, 3.5 mL of cell lysate prepared from 20.108 yeast cells in average was used, except TAP/Spt8,gcn5Δ, TAP/Spt8,ahc1Δ, TAP/Spt8,spt7Δ and TAP/Gcn5,ahc1Δ strains, where 1.3 mL of cell lysates from 9.108 yeast cells each were applied. Inputs contain 16.7 µL of the corresponding lysates taken before the lysates were used for immunoprecipitation.

Mentions: The GCN5 knock-out did not abolish the interaction between pre-IL-1α and either histone acetyltransferase complex because the interaction complexes were visible in the co-immunoprecipitation experiments in the gcn5Δ yeast strains producing either the Ahc1-TAP or Spt8-TAP proteins. This result strongly suggests that the IL-1α precursor does not interact with the catalytic subunit of the HAT/Core module, and the IL-1α-binding site should thus comprise Ada2, Ada3 and Sgf29 only (Figure 6A). As expected, the disruption of the SPT7 gene, which encodes a subunit that is important for the maintenance of the integrity of the SAGA complex, did not affect the interaction between the IL-1α precursor and the ADA complex, as represented by the Ahc1 protein, but the disruption completely abolished the co-immunoprecipitation of pre-IL-1α with the Spt8 protein (Figure 6C).


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)

Disruption of the SAGA and ADA complexes confirmed binding of the IL-1α precursor to the HAT/Core module and suggested the mutually exclusive role of Spt7 and Ahc1 in SAGA complex assembly.Co-immunoprecipitation was performed using yeast lysates with an anti-Flag antibody that recognizes the Flag tag at the N-terminus of the IL-1α precursor. Western blotting was performed with an anti-CBP antibody which identifies the TAP tag at the C-terminus of the respective HAT complex subunits. (A) Gcn5 does not bind to pre-IL-1α, and because it is not required for SAGA or ADA complex integrity, its deletion has no effect on Ahc1 or Spt8 co-immunoprecipitation with the IL-1α precursor (pre). Deletion of the AHC2 gene doesn’t impair co-IP of pre-IL-1α with Gcn5, Spt8 and Spt7. (B) The disruption of the ADA HAT complex did not affect the co-immunoprecipitation of Gcn5 and Spt8 with IL-1α. However, the interaction between Spt7 and the IL-1α precursor was significantly weakened. In experiments with TAP/Spt7,ahc1Δ strain, we received either no or very low signal (the latter is depicted) of TAP-tagged Spt7, with a success rate 3∶1, respectively. (C) The disruption of the SAGA complex abolished the interaction between Spt8 and the IL-1α precursor but had no effect on Ahc1 binding to the IL-1α precursor. Control cells (ctrl) carry the empty plasmid pYX212. For each line of the IP experiment, 3.5 mL of cell lysate prepared from 20.108 yeast cells in average was used, except TAP/Spt8,gcn5Δ, TAP/Spt8,ahc1Δ, TAP/Spt8,spt7Δ and TAP/Gcn5,ahc1Δ strains, where 1.3 mL of cell lysates from 9.108 yeast cells each were applied. Inputs contain 16.7 µL of the corresponding lysates taken before the lysates were used for immunoprecipitation.
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Related In: Results  -  Collection

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

pone-0041801-g006: Disruption of the SAGA and ADA complexes confirmed binding of the IL-1α precursor to the HAT/Core module and suggested the mutually exclusive role of Spt7 and Ahc1 in SAGA complex assembly.Co-immunoprecipitation was performed using yeast lysates with an anti-Flag antibody that recognizes the Flag tag at the N-terminus of the IL-1α precursor. Western blotting was performed with an anti-CBP antibody which identifies the TAP tag at the C-terminus of the respective HAT complex subunits. (A) Gcn5 does not bind to pre-IL-1α, and because it is not required for SAGA or ADA complex integrity, its deletion has no effect on Ahc1 or Spt8 co-immunoprecipitation with the IL-1α precursor (pre). Deletion of the AHC2 gene doesn’t impair co-IP of pre-IL-1α with Gcn5, Spt8 and Spt7. (B) The disruption of the ADA HAT complex did not affect the co-immunoprecipitation of Gcn5 and Spt8 with IL-1α. However, the interaction between Spt7 and the IL-1α precursor was significantly weakened. In experiments with TAP/Spt7,ahc1Δ strain, we received either no or very low signal (the latter is depicted) of TAP-tagged Spt7, with a success rate 3∶1, respectively. (C) The disruption of the SAGA complex abolished the interaction between Spt8 and the IL-1α precursor but had no effect on Ahc1 binding to the IL-1α precursor. Control cells (ctrl) carry the empty plasmid pYX212. For each line of the IP experiment, 3.5 mL of cell lysate prepared from 20.108 yeast cells in average was used, except TAP/Spt8,gcn5Δ, TAP/Spt8,ahc1Δ, TAP/Spt8,spt7Δ and TAP/Gcn5,ahc1Δ strains, where 1.3 mL of cell lysates from 9.108 yeast cells each were applied. Inputs contain 16.7 µL of the corresponding lysates taken before the lysates were used for immunoprecipitation.
Mentions: The GCN5 knock-out did not abolish the interaction between pre-IL-1α and either histone acetyltransferase complex because the interaction complexes were visible in the co-immunoprecipitation experiments in the gcn5Δ yeast strains producing either the Ahc1-TAP or Spt8-TAP proteins. This result strongly suggests that the IL-1α precursor does not interact with the catalytic subunit of the HAT/Core module, and the IL-1α-binding site should thus comprise Ada2, Ada3 and Sgf29 only (Figure 6A). As expected, the disruption of the SPT7 gene, which encodes a subunit that is important for the maintenance of the integrity of the SAGA complex, did not affect the interaction between the IL-1α precursor and the ADA complex, as represented by the Ahc1 protein, but the disruption completely abolished the co-immunoprecipitation of pre-IL-1α with the Spt8 protein (Figure 6C).

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