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Modulation of the stability and activities of HIV-1 Tat by its ubiquitination and carboxyl-terminal region.

Zhang L, Qin J, Li Y, Wang J, He Q, Zhou J, Liu M, Li D - Cell Biosci (2014)

Bottom Line: In this study, we find that Tat undergoes lysine 48-linked ubiquitination and is targeted to proteasome-dependent degradation.Moreover, the 72-, 86- and 101-residue forms of Tat also exhibit different stability and aforementioned activities.Our findings demonstrate that the ubiquitination and carboxyl-terminal region of Tat are critical determinants of its stability and activities.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin, 300071 China.

ABSTRACT

Background: The transactivator of transcription (Tat) protein of human immunodeficiency virus type 1 (HIV-1) is known to undergo ubiquitination. However, the roles of ubiquitination in regulating Tat stability and activities are unclear. In addition, although the 72- and 86-residue forms are commonly used for in vitro studies, the 101-residue form is predominant in the clinical isolates of HIV-1. The influence of the carboxyl-terminal region of Tat on its functions remains unclear.

Results: In this study, we find that Tat undergoes lysine 48-linked ubiquitination and is targeted to proteasome-dependent degradation. Expression of various ubiquitin mutants modulates Tat activities, including the transactivation of transcription, induction of apoptosis, interaction with tubulin, and stabilization of microtubules. Moreover, the 72-, 86- and 101-residue forms of Tat also exhibit different stability and aforementioned activities.

Conclusions: Our findings demonstrate that the ubiquitination and carboxyl-terminal region of Tat are critical determinants of its stability and activities.

No MeSH data available.


Related in: MedlinePlus

Tat-induced apoptosis is modulated by its carboxyl-terminal region. (A) HeLa cells were transfected with GFP-Tat101, GFP-Tat86, GFP-Tat72 or GFP alone. 72 hours post-transfection, cells were treated with (+) or without (-) MG132 and incubated for additional 24 hours. Apoptotic cells were examined via the fluorescence microscope by analyzing cell morphology of GFP-positive cells. (B) Quantification of the results in (A). Experiments were done in duplicate, six fields per group were counted, and the results were expressed as the mean ± SD. (C) HeLa cells were transfected and treated as in (A), and the apoptotic cells were examined by Annexin V-APC staining coupled with flow cytometry. (D) Quantification of the results in (C). Bars represent the relative folds of Tat-induced apoptosis normalized to the untreated GFP vector transfection group. Mean and standard deviations were derived from two independent experiments done in duplicate. (E) HeLa cells were transfected and treated as in (A) and collected 96 hours post-transfection. Cell lysates were subjected to immunoblot analysis with antibodies specific for cleaved caspase-3, α-tubulin, or GFP, respectively. (F) HeLa cells were transfected and treated as in (A), and stained with anti-cleaved caspase-3 antibodies and the DNA dye DAPI. Apoptotic cells were indicated by hollow arrows. (G) Quantification of the results in (F). Bars represent the relative folds of Tat-induced apoptosis normalized to the untreated GFP vector transfection group. Experiments were done in duplicate, 80 GFP-positive cells per group were counted, and the results were expressed as the mean ± SD. Two-tailed Student’s t-test for all graphs. *P < 0.05, **P <0.01, ***P < 0.001; ns, not significant. Cropped blots are used in this figure, and the gels were run under the same experimental conditions.
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Fig5: Tat-induced apoptosis is modulated by its carboxyl-terminal region. (A) HeLa cells were transfected with GFP-Tat101, GFP-Tat86, GFP-Tat72 or GFP alone. 72 hours post-transfection, cells were treated with (+) or without (-) MG132 and incubated for additional 24 hours. Apoptotic cells were examined via the fluorescence microscope by analyzing cell morphology of GFP-positive cells. (B) Quantification of the results in (A). Experiments were done in duplicate, six fields per group were counted, and the results were expressed as the mean ± SD. (C) HeLa cells were transfected and treated as in (A), and the apoptotic cells were examined by Annexin V-APC staining coupled with flow cytometry. (D) Quantification of the results in (C). Bars represent the relative folds of Tat-induced apoptosis normalized to the untreated GFP vector transfection group. Mean and standard deviations were derived from two independent experiments done in duplicate. (E) HeLa cells were transfected and treated as in (A) and collected 96 hours post-transfection. Cell lysates were subjected to immunoblot analysis with antibodies specific for cleaved caspase-3, α-tubulin, or GFP, respectively. (F) HeLa cells were transfected and treated as in (A), and stained with anti-cleaved caspase-3 antibodies and the DNA dye DAPI. Apoptotic cells were indicated by hollow arrows. (G) Quantification of the results in (F). Bars represent the relative folds of Tat-induced apoptosis normalized to the untreated GFP vector transfection group. Experiments were done in duplicate, 80 GFP-positive cells per group were counted, and the results were expressed as the mean ± SD. Two-tailed Student’s t-test for all graphs. *P < 0.05, **P <0.01, ***P < 0.001; ns, not significant. Cropped blots are used in this figure, and the gels were run under the same experimental conditions.

Mentions: Given that activation of caspase-3 is involved in the process of Tat-induced apoptosis [25], we then assessed the percentage of cleaved caspase-3 (the active form of caspase-3) positive cells 96 hours post-transfection. As shown in Figure 4E, HeLa cells with overexpressed GFP-Tat101, which were positive for cleaved caspase-3, were indicated by hollow arrows. The percentage of cleaved caspase-3 positive cells in ubiquitin-K29 cotransfection group was higher than that of the other four groups in the absence or presence of MG132 respectively (Figure 4F), which concurred with the results in Figure 4B and D. Cotransfection with ubiquitin-WT, ubiquitin-K48, or ubiquitin-K63 led to an increase in Tat-induced apoptosis when compared to the ubiquitin-K0 cotransfection groups (Figure 4F), which concurred with the data in Figure 4D.With similar approaches, we assessed the effects of the carboxyl-terminal region of Tat on its ability to induce apoptosis. By fluorescence microscopy and flow cytometry, we found that Tat72 was the most potent variant of Tat to trigger apoptosis (Figure 5A-D). The induction of apoptosis by the three forms of Tat was scarcely affected by MG132 treatment (Figure 5D). Immunoblot analysis and immunofluorescence microscopy also showed that GFP-Tat72 overexpression caused the most robust activation of caspase-3 (Figure 5E-G). Thus, these results indicate that Tat-induced apoptosis is regulated by its ubiquitination and carboxyl-terminal region.Figure 5


Modulation of the stability and activities of HIV-1 Tat by its ubiquitination and carboxyl-terminal region.

Zhang L, Qin J, Li Y, Wang J, He Q, Zhou J, Liu M, Li D - Cell Biosci (2014)

Tat-induced apoptosis is modulated by its carboxyl-terminal region. (A) HeLa cells were transfected with GFP-Tat101, GFP-Tat86, GFP-Tat72 or GFP alone. 72 hours post-transfection, cells were treated with (+) or without (-) MG132 and incubated for additional 24 hours. Apoptotic cells were examined via the fluorescence microscope by analyzing cell morphology of GFP-positive cells. (B) Quantification of the results in (A). Experiments were done in duplicate, six fields per group were counted, and the results were expressed as the mean ± SD. (C) HeLa cells were transfected and treated as in (A), and the apoptotic cells were examined by Annexin V-APC staining coupled with flow cytometry. (D) Quantification of the results in (C). Bars represent the relative folds of Tat-induced apoptosis normalized to the untreated GFP vector transfection group. Mean and standard deviations were derived from two independent experiments done in duplicate. (E) HeLa cells were transfected and treated as in (A) and collected 96 hours post-transfection. Cell lysates were subjected to immunoblot analysis with antibodies specific for cleaved caspase-3, α-tubulin, or GFP, respectively. (F) HeLa cells were transfected and treated as in (A), and stained with anti-cleaved caspase-3 antibodies and the DNA dye DAPI. Apoptotic cells were indicated by hollow arrows. (G) Quantification of the results in (F). Bars represent the relative folds of Tat-induced apoptosis normalized to the untreated GFP vector transfection group. Experiments were done in duplicate, 80 GFP-positive cells per group were counted, and the results were expressed as the mean ± SD. Two-tailed Student’s t-test for all graphs. *P < 0.05, **P <0.01, ***P < 0.001; ns, not significant. Cropped blots are used in this figure, and the gels were run under the same experimental conditions.
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Related In: Results  -  Collection

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Fig5: Tat-induced apoptosis is modulated by its carboxyl-terminal region. (A) HeLa cells were transfected with GFP-Tat101, GFP-Tat86, GFP-Tat72 or GFP alone. 72 hours post-transfection, cells were treated with (+) or without (-) MG132 and incubated for additional 24 hours. Apoptotic cells were examined via the fluorescence microscope by analyzing cell morphology of GFP-positive cells. (B) Quantification of the results in (A). Experiments were done in duplicate, six fields per group were counted, and the results were expressed as the mean ± SD. (C) HeLa cells were transfected and treated as in (A), and the apoptotic cells were examined by Annexin V-APC staining coupled with flow cytometry. (D) Quantification of the results in (C). Bars represent the relative folds of Tat-induced apoptosis normalized to the untreated GFP vector transfection group. Mean and standard deviations were derived from two independent experiments done in duplicate. (E) HeLa cells were transfected and treated as in (A) and collected 96 hours post-transfection. Cell lysates were subjected to immunoblot analysis with antibodies specific for cleaved caspase-3, α-tubulin, or GFP, respectively. (F) HeLa cells were transfected and treated as in (A), and stained with anti-cleaved caspase-3 antibodies and the DNA dye DAPI. Apoptotic cells were indicated by hollow arrows. (G) Quantification of the results in (F). Bars represent the relative folds of Tat-induced apoptosis normalized to the untreated GFP vector transfection group. Experiments were done in duplicate, 80 GFP-positive cells per group were counted, and the results were expressed as the mean ± SD. Two-tailed Student’s t-test for all graphs. *P < 0.05, **P <0.01, ***P < 0.001; ns, not significant. Cropped blots are used in this figure, and the gels were run under the same experimental conditions.
Mentions: Given that activation of caspase-3 is involved in the process of Tat-induced apoptosis [25], we then assessed the percentage of cleaved caspase-3 (the active form of caspase-3) positive cells 96 hours post-transfection. As shown in Figure 4E, HeLa cells with overexpressed GFP-Tat101, which were positive for cleaved caspase-3, were indicated by hollow arrows. The percentage of cleaved caspase-3 positive cells in ubiquitin-K29 cotransfection group was higher than that of the other four groups in the absence or presence of MG132 respectively (Figure 4F), which concurred with the results in Figure 4B and D. Cotransfection with ubiquitin-WT, ubiquitin-K48, or ubiquitin-K63 led to an increase in Tat-induced apoptosis when compared to the ubiquitin-K0 cotransfection groups (Figure 4F), which concurred with the data in Figure 4D.With similar approaches, we assessed the effects of the carboxyl-terminal region of Tat on its ability to induce apoptosis. By fluorescence microscopy and flow cytometry, we found that Tat72 was the most potent variant of Tat to trigger apoptosis (Figure 5A-D). The induction of apoptosis by the three forms of Tat was scarcely affected by MG132 treatment (Figure 5D). Immunoblot analysis and immunofluorescence microscopy also showed that GFP-Tat72 overexpression caused the most robust activation of caspase-3 (Figure 5E-G). Thus, these results indicate that Tat-induced apoptosis is regulated by its ubiquitination and carboxyl-terminal region.Figure 5

Bottom Line: In this study, we find that Tat undergoes lysine 48-linked ubiquitination and is targeted to proteasome-dependent degradation.Moreover, the 72-, 86- and 101-residue forms of Tat also exhibit different stability and aforementioned activities.Our findings demonstrate that the ubiquitination and carboxyl-terminal region of Tat are critical determinants of its stability and activities.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin, 300071 China.

ABSTRACT

Background: The transactivator of transcription (Tat) protein of human immunodeficiency virus type 1 (HIV-1) is known to undergo ubiquitination. However, the roles of ubiquitination in regulating Tat stability and activities are unclear. In addition, although the 72- and 86-residue forms are commonly used for in vitro studies, the 101-residue form is predominant in the clinical isolates of HIV-1. The influence of the carboxyl-terminal region of Tat on its functions remains unclear.

Results: In this study, we find that Tat undergoes lysine 48-linked ubiquitination and is targeted to proteasome-dependent degradation. Expression of various ubiquitin mutants modulates Tat activities, including the transactivation of transcription, induction of apoptosis, interaction with tubulin, and stabilization of microtubules. Moreover, the 72-, 86- and 101-residue forms of Tat also exhibit different stability and aforementioned activities.

Conclusions: Our findings demonstrate that the ubiquitination and carboxyl-terminal region of Tat are critical determinants of its stability and activities.

No MeSH data available.


Related in: MedlinePlus