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The HTLV-1-encoded protein HBZ directly inhibits the acetyl transferase activity of p300/CBP.

Wurm T, Wright DG, Polakowski N, Mesnard JM, Lemasson I - Nucleic Acids Res. (2012)

Bottom Line: This effect correlated with a reduction of H3K18 acetylation, a specific target of p300/CBP, in cells expressing HBZ.The inhibitory effect of HBZ was not limited to histones, as HBZ also inhibited acetylation of the NF-κB subunit, p65, and the tumor suppressor, p53.Recent studies reported that mutations in the HAT domain of p300/CBP that cause a defect in acetylation are found in certain types of leukemia.

View Article: PubMed Central - PubMed

Affiliation: East Carolina University, Brody School of Medicine, Greenville, NC 27834, USA.

ABSTRACT
The homologous cellular coactivators p300 and CBP contain intrinsic lysine acetyl transferase (termed HAT) activity. This activity is responsible for acetylation of several sites on the histones as well as modification of transcription factors. In a previous study, we found that HBZ, encoded by the Human T-cell Leukemia Virus type 1 (HTLV-1), binds to multiple domains of p300/CBP, including the HAT domain. In this study, we found that HBZ inhibits the HAT activity of p300/CBP through the bZIP domain of the viral protein. This effect correlated with a reduction of H3K18 acetylation, a specific target of p300/CBP, in cells expressing HBZ. Interestingly, lower levels of H3K18 acetylation were detected in HTLV-1 infected cells compared to non-infected cells. The inhibitory effect of HBZ was not limited to histones, as HBZ also inhibited acetylation of the NF-κB subunit, p65, and the tumor suppressor, p53. Recent studies reported that mutations in the HAT domain of p300/CBP that cause a defect in acetylation are found in certain types of leukemia. These observations suggest that inhibition of the HAT activity by HBZ is important for the development of adult T-cell leukemia associated with HTLV-1 infection.

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HBZ inhibits p65 and p53 acetylation by p300/CBP. (A) HBZ inhibits p65 acetylation by p300 in vitro. HAT assays were performed using recombinant p65 (80 nM), p300 (4 nM) and GST-HBZ (0.3 µM) or HBZ-bZIP (0.3 µM) as indicated. (B) HBZ inhibits p53 acetylation by p300 in vitro. HAT assays were performed using recombinant p53 (50 nM), p300 (4 nM) and GST-HBZ (0.5 µM) or HBZ-bZIP (0.5 µM) as indicated. In (A) and (B), the lower panel shows the membrane from the top panel reprobed for total p65 and p53, respectively. Reactions were analyzed by western blot using the antibodies indicated. (C) HBZ inhibits p65 acetylation in vivo. Nuclear extracts (40 µg) were prepared from HeLa cell lines expressing HBZ or containing the empty pcDNA vector. Cells used in these experiments were stimulated with 50 µM etoposide (ETO) for 5 h. (D) HBZ inhibits p53 acetylation in vivo. Experiments were performed as described in (C). In (C) and (D) the middle panel shows the membrane from the upper panel reprobed for total p65 and p53, respectively. Extracts were analyzed by western blot using the antibodies indicated.
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gks244-F8: HBZ inhibits p65 and p53 acetylation by p300/CBP. (A) HBZ inhibits p65 acetylation by p300 in vitro. HAT assays were performed using recombinant p65 (80 nM), p300 (4 nM) and GST-HBZ (0.3 µM) or HBZ-bZIP (0.3 µM) as indicated. (B) HBZ inhibits p53 acetylation by p300 in vitro. HAT assays were performed using recombinant p53 (50 nM), p300 (4 nM) and GST-HBZ (0.5 µM) or HBZ-bZIP (0.5 µM) as indicated. In (A) and (B), the lower panel shows the membrane from the top panel reprobed for total p65 and p53, respectively. Reactions were analyzed by western blot using the antibodies indicated. (C) HBZ inhibits p65 acetylation in vivo. Nuclear extracts (40 µg) were prepared from HeLa cell lines expressing HBZ or containing the empty pcDNA vector. Cells used in these experiments were stimulated with 50 µM etoposide (ETO) for 5 h. (D) HBZ inhibits p53 acetylation in vivo. Experiments were performed as described in (C). In (C) and (D) the middle panel shows the membrane from the upper panel reprobed for total p65 and p53, respectively. Extracts were analyzed by western blot using the antibodies indicated.

Mentions: To gauge the efficiency with which HBZ inhibits histone acetylation, we compared its inhibitory effect to that of curcumin, which is known to specifically target p300/CBP HAT activity (76). We performed a parallel set of in vitro HAT assays, in which reactions were supplemented with increasing concentrations of full-length HBZ, HBZ-bZIP, or curcumin. Results from these experiments show a reduction in histone acetylation at lower concentrations of full-length HBZ or HBZ-bZIP than curcumin (compare Figure 7A and B to C). Quantification of the data revealed an IC50 of 0.1 µM for full-length HBZ and the bZIP domain and an IC50 of 25 µM for curcumin (Figure 7D). Importantly, the curcumin value is similar to that reported previously (76). These results demonstrate that HBZ is a more potent inhibitor of p300/CBP HAT activity than curcumin. Figure 7A shows acetylation of HBZ by p300. This modification was consistently detected with the full-length viral protein in in vitro HAT assays (Figure 8 and data not shown); however, its significance remains undefined. Importantly, acetylation of HBZ is not inconsistent with its effect on HAT activity, as E1A is similarly acetylated by p300, while functioning to inhibit the HAT activity of the coactivator (64,74,75).Figure 7.


The HTLV-1-encoded protein HBZ directly inhibits the acetyl transferase activity of p300/CBP.

Wurm T, Wright DG, Polakowski N, Mesnard JM, Lemasson I - Nucleic Acids Res. (2012)

HBZ inhibits p65 and p53 acetylation by p300/CBP. (A) HBZ inhibits p65 acetylation by p300 in vitro. HAT assays were performed using recombinant p65 (80 nM), p300 (4 nM) and GST-HBZ (0.3 µM) or HBZ-bZIP (0.3 µM) as indicated. (B) HBZ inhibits p53 acetylation by p300 in vitro. HAT assays were performed using recombinant p53 (50 nM), p300 (4 nM) and GST-HBZ (0.5 µM) or HBZ-bZIP (0.5 µM) as indicated. In (A) and (B), the lower panel shows the membrane from the top panel reprobed for total p65 and p53, respectively. Reactions were analyzed by western blot using the antibodies indicated. (C) HBZ inhibits p65 acetylation in vivo. Nuclear extracts (40 µg) were prepared from HeLa cell lines expressing HBZ or containing the empty pcDNA vector. Cells used in these experiments were stimulated with 50 µM etoposide (ETO) for 5 h. (D) HBZ inhibits p53 acetylation in vivo. Experiments were performed as described in (C). In (C) and (D) the middle panel shows the membrane from the upper panel reprobed for total p65 and p53, respectively. Extracts were analyzed by western blot using the antibodies indicated.
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gks244-F8: HBZ inhibits p65 and p53 acetylation by p300/CBP. (A) HBZ inhibits p65 acetylation by p300 in vitro. HAT assays were performed using recombinant p65 (80 nM), p300 (4 nM) and GST-HBZ (0.3 µM) or HBZ-bZIP (0.3 µM) as indicated. (B) HBZ inhibits p53 acetylation by p300 in vitro. HAT assays were performed using recombinant p53 (50 nM), p300 (4 nM) and GST-HBZ (0.5 µM) or HBZ-bZIP (0.5 µM) as indicated. In (A) and (B), the lower panel shows the membrane from the top panel reprobed for total p65 and p53, respectively. Reactions were analyzed by western blot using the antibodies indicated. (C) HBZ inhibits p65 acetylation in vivo. Nuclear extracts (40 µg) were prepared from HeLa cell lines expressing HBZ or containing the empty pcDNA vector. Cells used in these experiments were stimulated with 50 µM etoposide (ETO) for 5 h. (D) HBZ inhibits p53 acetylation in vivo. Experiments were performed as described in (C). In (C) and (D) the middle panel shows the membrane from the upper panel reprobed for total p65 and p53, respectively. Extracts were analyzed by western blot using the antibodies indicated.
Mentions: To gauge the efficiency with which HBZ inhibits histone acetylation, we compared its inhibitory effect to that of curcumin, which is known to specifically target p300/CBP HAT activity (76). We performed a parallel set of in vitro HAT assays, in which reactions were supplemented with increasing concentrations of full-length HBZ, HBZ-bZIP, or curcumin. Results from these experiments show a reduction in histone acetylation at lower concentrations of full-length HBZ or HBZ-bZIP than curcumin (compare Figure 7A and B to C). Quantification of the data revealed an IC50 of 0.1 µM for full-length HBZ and the bZIP domain and an IC50 of 25 µM for curcumin (Figure 7D). Importantly, the curcumin value is similar to that reported previously (76). These results demonstrate that HBZ is a more potent inhibitor of p300/CBP HAT activity than curcumin. Figure 7A shows acetylation of HBZ by p300. This modification was consistently detected with the full-length viral protein in in vitro HAT assays (Figure 8 and data not shown); however, its significance remains undefined. Importantly, acetylation of HBZ is not inconsistent with its effect on HAT activity, as E1A is similarly acetylated by p300, while functioning to inhibit the HAT activity of the coactivator (64,74,75).Figure 7.

Bottom Line: This effect correlated with a reduction of H3K18 acetylation, a specific target of p300/CBP, in cells expressing HBZ.The inhibitory effect of HBZ was not limited to histones, as HBZ also inhibited acetylation of the NF-κB subunit, p65, and the tumor suppressor, p53.Recent studies reported that mutations in the HAT domain of p300/CBP that cause a defect in acetylation are found in certain types of leukemia.

View Article: PubMed Central - PubMed

Affiliation: East Carolina University, Brody School of Medicine, Greenville, NC 27834, USA.

ABSTRACT
The homologous cellular coactivators p300 and CBP contain intrinsic lysine acetyl transferase (termed HAT) activity. This activity is responsible for acetylation of several sites on the histones as well as modification of transcription factors. In a previous study, we found that HBZ, encoded by the Human T-cell Leukemia Virus type 1 (HTLV-1), binds to multiple domains of p300/CBP, including the HAT domain. In this study, we found that HBZ inhibits the HAT activity of p300/CBP through the bZIP domain of the viral protein. This effect correlated with a reduction of H3K18 acetylation, a specific target of p300/CBP, in cells expressing HBZ. Interestingly, lower levels of H3K18 acetylation were detected in HTLV-1 infected cells compared to non-infected cells. The inhibitory effect of HBZ was not limited to histones, as HBZ also inhibited acetylation of the NF-κB subunit, p65, and the tumor suppressor, p53. Recent studies reported that mutations in the HAT domain of p300/CBP that cause a defect in acetylation are found in certain types of leukemia. These observations suggest that inhibition of the HAT activity by HBZ is important for the development of adult T-cell leukemia associated with HTLV-1 infection.

Show MeSH
Related in: MedlinePlus