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A splice variant of ASC regulates IL-1beta release and aggregates differently from intact ASC.

Matsushita K, Takeoka M, Sagara J, Itano N, Kurose Y, Nakamura A, Taniguchi S - Mediators Inflamm. (2009)

Bottom Line: The apoptosis-associated speck-like protein containing a caspase recruit domain (ASC) is involved in apoptosis and innate immunity and is a major adaptor molecule responsible for procaspase-1 activation.Both fASC and vASC were found to activate procaspase-1 to a similar degree, but the efficiency of IL-1beta excretion was significantly higher for vASC.These results suggest that although the PGR domain is dispensable for procaspase-1 activation, it plays an important role in the regulation of the molecular structure and activity of ASC.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Oncology, Institute on Aging and Adaptation, Graduate School of Medicine, Shinshu University, Negano 390-8621, Japan.

ABSTRACT
The apoptosis-associated speck-like protein containing a caspase recruit domain (ASC) is involved in apoptosis and innate immunity and is a major adaptor molecule responsible for procaspase-1 activation. ASC mRNA is encoded by three exons: exons 1 and 3 encode a pyrin domain (PYD) and caspase recruit domain (CARD), respectively, and exon 2 encodes a proline and glycine-rich (PGR) domain. Here, we identified a variant ASC protein (vASC) lacking the PGR domain that was smaller than full length ASC (fASC) derived from fully transcribed mRNA and searched for differences in biochemical and biological nature. Both fASC and vASC were found to activate procaspase-1 to a similar degree, but the efficiency of IL-1beta excretion was significantly higher for vASC. There was also a marked structural difference observed in the fibrous aggregates formed by fASC and vASC. These results suggest that although the PGR domain is dispensable for procaspase-1 activation, it plays an important role in the regulation of the molecular structure and activity of ASC.

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Analysis of protein X by mass spectrometry. (a) HL-60 cells were subjected to SDS-PAGE and western blot analysis using antihuman ASC antibody. In addition to full length ASC (fASC), an additional polypeptide of 20 kDa (Protein X) was detected. (b) Proteins that were affinity-purified using anti-ASC antibody were stained with Coomassie Blue. HC: Ig heavy chain, LC: Ig light chain. (c) Mass spectrum of digested protein X is shown. (d) Structures of ASC and Protein X. Homologous regions are underlined. aa: amino acid. (e) Amino acid sequence of fASC from a database (GenBank Accession no. NP 037390) and Protein X is displayed. Homologous sequences are underlined. The amino acid sequence written in light characters was not detected in Protein X. (f) Sequence of ASC cDNA extracted from HL-60 cells as Protein X is shown.
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fig1: Analysis of protein X by mass spectrometry. (a) HL-60 cells were subjected to SDS-PAGE and western blot analysis using antihuman ASC antibody. In addition to full length ASC (fASC), an additional polypeptide of 20 kDa (Protein X) was detected. (b) Proteins that were affinity-purified using anti-ASC antibody were stained with Coomassie Blue. HC: Ig heavy chain, LC: Ig light chain. (c) Mass spectrum of digested protein X is shown. (d) Structures of ASC and Protein X. Homologous regions are underlined. aa: amino acid. (e) Amino acid sequence of fASC from a database (GenBank Accession no. NP 037390) and Protein X is displayed. Homologous sequences are underlined. The amino acid sequence written in light characters was not detected in Protein X. (f) Sequence of ASC cDNA extracted from HL-60 cells as Protein X is shown.

Mentions: When HL-60 cells were subjected to SDS-PAGE, anti-ASC antibody detected one band of 23 kDa, which corresponds to fASC, and another of 20 kDa, called Protein X (Figure 1(a), right lane). A mouse IgG control is shown in Figure 1(a), left lane. Affinity-purified polypeptides from HL-60 cell lysates incubated with anti-ASC antibody are displayed in Figure 1(b). The unknown protein X was then treated by trypsin ingel digestion and analyzed with MALDI-TOF MS (Figure 1(c)), and peptide mass data were subjected to a database search using MS-Fit, a peptide mass fingerprinting program from the University of California, San Francisco. Since the N and C terminal sequences of protein X were confirmed to be the same as fASC but lacked the amino acid sequence corresponding to the PGR domain (Figures 1(d) and 1(e)), we were able to identify protein X as vASC lacking a PGR domain. The sequence of vASC cDNA from mRNA extracted from HL-60 cells is displayed in Figure 1(f) and confirms deletion of exon 2 from fASC. This vASC corresponds to a protein reported in the GeneBank as association number NP 660183. Thus, protein X was not a degraded ASC but rather a variant form of ASC derived from alternatively spliced mRNA missing exon 2 of the ASC gene.


A splice variant of ASC regulates IL-1beta release and aggregates differently from intact ASC.

Matsushita K, Takeoka M, Sagara J, Itano N, Kurose Y, Nakamura A, Taniguchi S - Mediators Inflamm. (2009)

Analysis of protein X by mass spectrometry. (a) HL-60 cells were subjected to SDS-PAGE and western blot analysis using antihuman ASC antibody. In addition to full length ASC (fASC), an additional polypeptide of 20 kDa (Protein X) was detected. (b) Proteins that were affinity-purified using anti-ASC antibody were stained with Coomassie Blue. HC: Ig heavy chain, LC: Ig light chain. (c) Mass spectrum of digested protein X is shown. (d) Structures of ASC and Protein X. Homologous regions are underlined. aa: amino acid. (e) Amino acid sequence of fASC from a database (GenBank Accession no. NP 037390) and Protein X is displayed. Homologous sequences are underlined. The amino acid sequence written in light characters was not detected in Protein X. (f) Sequence of ASC cDNA extracted from HL-60 cells as Protein X is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2744265&req=5

fig1: Analysis of protein X by mass spectrometry. (a) HL-60 cells were subjected to SDS-PAGE and western blot analysis using antihuman ASC antibody. In addition to full length ASC (fASC), an additional polypeptide of 20 kDa (Protein X) was detected. (b) Proteins that were affinity-purified using anti-ASC antibody were stained with Coomassie Blue. HC: Ig heavy chain, LC: Ig light chain. (c) Mass spectrum of digested protein X is shown. (d) Structures of ASC and Protein X. Homologous regions are underlined. aa: amino acid. (e) Amino acid sequence of fASC from a database (GenBank Accession no. NP 037390) and Protein X is displayed. Homologous sequences are underlined. The amino acid sequence written in light characters was not detected in Protein X. (f) Sequence of ASC cDNA extracted from HL-60 cells as Protein X is shown.
Mentions: When HL-60 cells were subjected to SDS-PAGE, anti-ASC antibody detected one band of 23 kDa, which corresponds to fASC, and another of 20 kDa, called Protein X (Figure 1(a), right lane). A mouse IgG control is shown in Figure 1(a), left lane. Affinity-purified polypeptides from HL-60 cell lysates incubated with anti-ASC antibody are displayed in Figure 1(b). The unknown protein X was then treated by trypsin ingel digestion and analyzed with MALDI-TOF MS (Figure 1(c)), and peptide mass data were subjected to a database search using MS-Fit, a peptide mass fingerprinting program from the University of California, San Francisco. Since the N and C terminal sequences of protein X were confirmed to be the same as fASC but lacked the amino acid sequence corresponding to the PGR domain (Figures 1(d) and 1(e)), we were able to identify protein X as vASC lacking a PGR domain. The sequence of vASC cDNA from mRNA extracted from HL-60 cells is displayed in Figure 1(f) and confirms deletion of exon 2 from fASC. This vASC corresponds to a protein reported in the GeneBank as association number NP 660183. Thus, protein X was not a degraded ASC but rather a variant form of ASC derived from alternatively spliced mRNA missing exon 2 of the ASC gene.

Bottom Line: The apoptosis-associated speck-like protein containing a caspase recruit domain (ASC) is involved in apoptosis and innate immunity and is a major adaptor molecule responsible for procaspase-1 activation.Both fASC and vASC were found to activate procaspase-1 to a similar degree, but the efficiency of IL-1beta excretion was significantly higher for vASC.These results suggest that although the PGR domain is dispensable for procaspase-1 activation, it plays an important role in the regulation of the molecular structure and activity of ASC.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Oncology, Institute on Aging and Adaptation, Graduate School of Medicine, Shinshu University, Negano 390-8621, Japan.

ABSTRACT
The apoptosis-associated speck-like protein containing a caspase recruit domain (ASC) is involved in apoptosis and innate immunity and is a major adaptor molecule responsible for procaspase-1 activation. ASC mRNA is encoded by three exons: exons 1 and 3 encode a pyrin domain (PYD) and caspase recruit domain (CARD), respectively, and exon 2 encodes a proline and glycine-rich (PGR) domain. Here, we identified a variant ASC protein (vASC) lacking the PGR domain that was smaller than full length ASC (fASC) derived from fully transcribed mRNA and searched for differences in biochemical and biological nature. Both fASC and vASC were found to activate procaspase-1 to a similar degree, but the efficiency of IL-1beta excretion was significantly higher for vASC. There was also a marked structural difference observed in the fibrous aggregates formed by fASC and vASC. These results suggest that although the PGR domain is dispensable for procaspase-1 activation, it plays an important role in the regulation of the molecular structure and activity of ASC.

Show MeSH