Curcumin induces Tat protein degradation

Earlier reports, including ours have described the unfolded nature of Tat protein and using Foldindex program we found Tat to be completely unfolded along its entire length18,24. The intrinsically unfolded proteins are degraded through ubiquitin independent 20S proteasomal pathway. Curcumin is reported to activate the pathway as it is a competitive inhibitor of NADH NQO1 interaction11. To investigate the effect of curcumin on Tat protein, Myc-Tat transfected HEK-293T cells were treated with curcumin and Tat protein level was measured by western blotting. The level of Tat protein showed a decrease with curcumin treatment in a dose dependent manner (Fig. 1A). The p53 protein is known to be degraded by curcumin11, hence as a positive control, p53 protein level was measured which also showed similar decrease (Fig. 1A). For transfection control EGFP was transfected which was unaffected by curcumin treatment suggesting the specificity of Tat degradation. To investigate the effect of curcumin on Tat in a time dependent manner, HEK-293T cells were transfected with 1 μg Myc-Tat expressing plasmid followed by treatment with 80 μM curcumin from 0–8 hrs. Tat protein level was decreased with time in response to curcumin treatment (Fig. 1B). Unlike Tat, HIV-1 Gag is a properly folded protein as found by using Foldindex program. The effect of curcumin on Gag was investigated with curcumin treatment of HEK-293T cells that were transfected with Gag-Opt plasmid29. Immunoblotting of Gag showed that it remains largely stable with curcumin treatment as there is no significant decrease in the level of Gag protein (Fig. 1C). To study the effect of curcumin on the rate of Tat protein degradation, cycloheximide (CHX) chase assay was carried out of Myc-Tat transfected and curcumin treated HEK-293T cells. CHX treatment led to Tat protein degradation in a time-dependent manner, however the treatment of curcumin and CHX together results in rapid degradation of Tat supporting the fact that curcumin induces Tat degradation (Fig. 1D,E). To investigate the degradation pathway involved, Myc-Tat transfected HEK-293T cells were treated with curcumin alone, or curcumin along with proteasomal inhibitor MG132 or lysosomal inhibitor ammonium chloride. Tat protein levels were decreased after treatment with curcumin alone or with curcumin and ammonium chloride, however it was unaffected when the cells were treated with curcumin and MG132 suggesting the involvement of proteasomal pathway in the process (Fig. 1F). To further confirm the finding CHX chase assay was carried out of Myc-Tat transfected HEK29T cells treated with curcumin or curcumin + MG132. The degradation of Tat protein was inhibited completely when MG132 treatment was carried out confirming the involvement of proteasomal degradation of Tat in response to curcumin treatment (Fig. 1G,H). The ubiquitination of proteins can be inhibited by the treatment of ubiquitin activating enzyme E1 inhibitor Pyr-4130 or with the expression of dominant negative ubiquitin (HA-Ubiquitin KO) having all the lysine residues replaced with arginine26. To study whether the degradation of Tat by curcumin is dependent on ubiquitination of Tat, Myc-Tat transfected HEK-293T cells were treated with Pyr-41 and curcumin. Pyr-41 treatment was unable to stop the curcumin mediated Tat protein degradation suggesting for ubiquitin independent degradation (Fig. 1I). Curcumin treatment of HEK-293T cells that were earlier transfected with Myc-Tat + HA-Ubiquitin KO also resulted in the complete degradation of Tat protein (Fig. 1I). In order to study the effect of curcumin on Tat ubiquitination, HEK-293T cells were transfected with Myc-Tat and 6X-His Ubiquitin followed by curcumin treatment for 8 hrs. MG132 treatment was also carried out, which increased the extent of Tat ubiquitination. The ubiquitination of Tat got decreased in a dose dependent manner with curcumin treatment (Fig. 1J). Thus the results show that curcumin specifically promotes the degradation of Tat, which is a structurally unfolded protein, the properly folded Gag protein is unaffected by curcumin treatment. The degradation of Tat was found to be through proteasomal pathway independent of ubiquitination.

Figure 1 Curcumin decreased HIV-1 Tat protein. (A) HEK-293T cells were transfected with 1 μg of Myc-Tat expressing plasmid and after 36 hrs treated with curcumin for 8 hrs, lysed and probed for Tat, p53 and GAPDH. pEGFP-N1 (50 ng) was also transfected as transfection control. The blot shown is a representative of three independent experiments.(B) Myc-Tat was transfected in HEK-293T cells and curcumin treatment was performed for increasing time period. The blot is a representative of three independent experiments. (C) Gag-Opt (1 μg) was transfected and curcumin treatment was performed followed by immmuno-blotting for Gag protein in HHEK-293T cells. (D) Myc-Tat transfected HEK-293T cells were treated with CHX alone or with curcumin for time periods as indicated and Tat protein level was measured by western blotting. (E) The mean value of Tat protein from three independent experiments was plotted with respect to treatment period. P value was calculated by a two-tailed t-test (*P < 0.05, **P < 0.01; NS, not significant). (F) The Myc-Tat transfected HEK-293T cells were treated with curcumin, along with proteasomal and lysosomal inhibitors MG132 and ammonium chloride for 8 hrs, subsequently Tat level was measured. (G) Myc-Tat transfected HEK-293T cells were treated with curcumin and CHX in the absence or presence of MG132 for different time periods followed by western blotting for Tat protein. (H) Densitometry of Tat bands was carried out by using image J and plotted with respect to treatment period. (I) HEK-293T cells were transfected with 1 μg of Myc-Tat (lanes 1–3) for 36 hrs followed by treatment with curcumin and Pyr-41 for 6 hrs subsequently the western blotting was done for Tat protein. HEK-293T cells were transfected with 1 μg of Myc-Tat and 2 μg of HA-Ub KO, 36 hrs of transfection curcumin treatment was done for 6 hrs and Tat protein was blotted. (J) HEK-293T cells were transfected with 1 μg of Myc-Tat and 2 μg of 6X-His Ubiquitin plasmid, after 36 hrs the cells were treated with increasing dose of curcumin or MG132. The ubiquitinated proteins were purified using Ni-NTA affinity chromatography and ubiquitinated Tat was blotted using anti-Myc antibody. The original uncropped blot images for (A–D,F,G,I,J) are shown in supplementary information as supplementary figure. Full size image

Curcumin inhibits the functional activity of Tat

To study the effect of curcumin on Tat mRNA, Myc-Tat transfected HEK-293T cells were treated with curcumin for 8 hrs followed by total RNA isolation and performing semi-quantitative RT-PCR assay. The results clearly showed that there is no change in the Tat cDNA level after curcumin treatment. As a control GAPDH mRNA was also amplified the level of which is also not changed (Fig. 2A,B). To completely rule out the role of curcumin on Tat transcription, Myc-Tat expressing plasmid was transfected in HEK-293T cells and the cell culture medium that contains Tat protein was applied to a fresh plate of HEK-293T cells followed by treatment with curcumin. Curcumin treatment reduced the Tat level suggesting the Tat protein degradation by curcumin occurs independent of its mRNA (Fig. 2C). Effect of curcumin on Tat functional activity was carried out using TZM-bl cell line harbouring an integrated HIV-1 LTR promoter upstream of luciferase gene27. The luciferase activity was measured in Myc-Tat transfected TZM-bl cells treated with curcumin for 12 hrs. Expression of Tat in TZM-bl cells resulted in an increase in luciferase activity by 9 folds which was reduced by 60% with 20 μM and 80% with 40 μM curcumin (Fig. 2D). Effect of curcumin on Tat independent HIV-1 LTR activation was also carried out by curcumin treatment of TZM-bl cells. There is a 20% decrease in LTR promoter activity at 40 μM and 30% decrease at 80 μM curcumin which is significantly less in comparison to Tat induced LTR activity (Fig. 2E). The role of curcumin on Tat gene transcription was ruled out by performing semi-quantitative RT-PCR as well as studying the effect of curcumin on Tat protein treated cells. The functional effect of curcumin on Tat was confirmed by performing LTR-luciferase assay in Tat transfected and curcumin treated cells.

Figure 2 Curcumin does not modulate the level of Tat mRNA. (A) Myc-Tat transfected HEK-293T cells were treated with curcumin for 8 hrs and total RNA was isolated using TRIZOL reagent followed by RT-PCR using Tat and GAPDH primers. The gel image is a representative of three independent experiments. (B) Using ImageJ the band intensity of Tat and GAPDH was quantified and plotted as Tat/GAPDH. P value was calculated by a two-tailed t-test (*P < 0.05, **P < 0.01; NS, not significant). (C) Myc-Tat was transfected to HEK-293T cells, 24 hrs post transfection the cell culture medium was applied on fresh HEK-293T cells followed by treatment with curcumin for 6 hrs. Western blotting was done to detect the Tat protein. (D) In a 24 well plate format TZM-bl cells were transfected with 0.2 μg of Myc Tat, after 36 hrs treated with increasing dose of curcumin for 12 hrs and luciferase activity was measured and the mean of three independent experiments was plotted. P value was calculated by a two-tailed t-test (*P < 0.05, **P < 0.01; NS, not significant). (E) Similarly un-transfected TZM-bl cells were also treated with curcumin and luciferase activity was measured and the mean of three independent experiments was plotted. P value was calculated by a two-tailed t-test (*P < 0.05, **P < 0.01; NS, not significant). Full size image

The HIV-1 virion production is inhibited with curcumin treatment

Curcumin is known to target HIV-1 protease, integrase and cellular NF-κB protein for the downregulation of HIV-1 replication19,20,21,22. However since our results showed its role in the degradation of Tat, its effect on the HIV-1 virion production was investigated. pNL4-3 was transfected in HEK-293T cells followed by curcumin treatment for 12 hrs. Cells were lysed and probed for p24 protein and the cell culture medium was saved and used for infecting TZM-bl cells. In the pNL4-3 transfected HEK-293T cells p24 levels decreased in a dose dependent manner with curcumin treatment (Fig. 3A). The p24 level of virus infected TZM-bl cells also decreased by 30% at 20 μM curcumin and reached upto 90% at 80 μM concentration (Fig. 3B). We further examined the effect of curcumin on the viral replication and virion production from chronically infected human T cell line J1.1. It is a Jurkat E6.1 derived human T lymphocyte, chronically infected with HIV-1 LAI strain. Under normal growth conditions there is limited viral replication and release from these cells, which increases tremendously upon stimulation with TNF-α28. To investigate the effect of curcumin on HIV-1 production and release, these cells were treated with TNF-α for 48 hours to induce HIV-1 expression. Subsequently the medium was replaced with fresh medium containing TNF-α and curcumin and incubated further for 12 hours. The viral replication was measured by p24 quantification through immuno-blotting. The viral supernatant was used to measure released virions from these cells using direct ELISA as described in methods. Curcumin treatment resulted in the dose dependent reduction of p24 level in the cell lysate of J1.1 cells (Fig. 3C), as well as in the supernatant (Fig. 3D). These results clearly confirm the inhibitory effect of curcumin on HIV-1 production from T cells. To rule out the possibility of Tat protein being degraded by curcumin was due to cell death of treated cells, Myc-Tat transfected HEK-293T cells were treated with curcumin for 8 hrs, subsequently the media was replaced with complete fresh DMEM and further incubated the cells for 8 hrs. As shown in Fig. 3E, treatment of 80 μM curcumin led to complete degradation of Tat, however removal of it for 8 hrs resulted in the re-appearance of Tat protein. Similarly when pNL4-3 transfected HEK-293T cells were treated with 80 μM curcumin for 8 hrs there was ~70% decrease in p24 level, whereas removal of curcumin for 12 hrs resulted in a complete rebound of virus replication as measured by p24 level, suggesting that curcumin treatment did not damage the protein synthesis machinery (Fig. 3F). To measure apoptosis in curcumin treated cells, HEK-293T cells were treated with increasing dose of curcumin for 8 hrs followed by measurement of cleavage of Poly (ADP-Ribose) Polymerase (PARP)32. There was no cleavage of PARP even with 120 uM curcumin treatment (Fig. 3G). However, when treatment was extended for 30 hrs cleavage in PARP protein was observed with 80 μM curcumin (Fig. 3H). As a positive control of apoptosis the cells were treated with hydrogen peroxide that also led to PARP cleavage33. The reduction of Gag level both in pNL4-3 transfected HEK-293T cells and chronically infected J1.1 cells clearly showed the effect of Tat degradation on HIV-1 virion production. The reversibility of the effect of curcumin on both Tat level as well Gag level in pNL4-3 transfected cells confirms that the effect of curcumin on Tat is not due to cell death which was further confirmed by measuring PARP cleavage in curcumin treated cells.