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The serine protease inhibitors TLCK and TPCK react with the RB-binding core of HPV-18 E7 protein and abolish its
H Stöppler1, M C Stöppler, A Adduci
1Department of Pathology, Georgetown University Medical Center, Washington, DC 20007, USA.
Abstract:
The human papillomaviruses associated with cervical cancer (e.g., HPV-16 and HPV-18) express an E7 oncoprotein which mediates the immortalization of primary genital keratinocytes and the transformation of rodent cells. The 105-amino-acid HPV-18 E7 protein contains two zinc fingers as well as a conserved amino-terminal motif (Rb-binding core) which binds and alters the interactions of the retinoblastoma susceptibility gene product (Rb). We report here that two serine protease inhibitors, tosyl-L-lysine chloromethyl ketone (TLCK) and tosyl-L-phenylalanine chloromethyl ketone (TPCK), reacted with and generated an altered form of the HPV-18 E7 protein. Chemical modification of the E7 protein was initially observed during its extraction and immunoprecipitation from mammalian cells but could also be detected using E7 protein expressed in vitro by reticulocyte lysates. More importantly, TLCK and TPCK were able to modify E7 protein in live keratinocytes following their addition to the culture medium. Site-specific mutagenesis demonstrated that the E7 Rb-binding core (Leu-X-Cys-X-Glu) contained a cysteine residue which was essential for this modification and that the TLCK/TPCK-dependent alteration of the E7 protein abolished its ability to bind Rb. These studies indicate that the E7 protein can be inactivated by a specific class of protease inhibitors and that such reagents may be useful for pharmacologically regulating E7 function in vivo. In addition, these results demonstrate that care must be taken when applying these commonly used protease inhibitors in experiments evaluating E7/cellular protein interactions.
Insights
Serine protease inhibitors TLCK and TPCK modify the human papillomavirus (HPV) E7 oncoprotein, altering its structure and function. This modification inhibits E7
Area of Science:
- Oncology
- Virology
- Molecular Biology
Background:
- Cervical cancer is linked to human papillomaviruses (HPV), which express the E7 oncoprotein.
- The E7 oncoprotein plays a role in cell immortalization and transformation.
- E7 binds to and alters the function of the retinoblastoma tumor suppressor protein (Rb).
Purpose of the Study:
- To investigate the effect of serine protease inhibitors on the HPV-18 E7 oncoprotein.
- To determine if these inhibitors can alter E7 protein function and Rb binding.
- To explore the potential of protease inhibitors for regulating E7 activity in vivo.
Main Methods:
- Treatment of E7 protein with tosyl-L-lysine chloromethyl ketone (TLCK) and tosyl-L-phenylalanine chloromethyl ketone (TPCK).
- Analysis of E7 protein modification using immunoprecipitation and in vitro expression systems.
- Site-specific mutagenesis to identify the cysteine residue involved in modification.
- Assessment of E7 protein's ability to bind Rb after inhibitor treatment.
Main Results:
- TLCK and TPCK were found to react with and modify the HPV-18 E7 protein.
- Modification of E7 was observed in cell extracts, in vitro expressed protein, and live keratinocytes.
- A cysteine residue within the E7 Rb-binding core was essential for this modification.
- Inhibitor-induced E7 modification abolished its binding to Rb.
Conclusions:
- Specific serine protease inhibitors (TLCK, TPCK) can inactivate the HPV E7 oncoprotein.
- These inhibitors alter E7 by modifying a critical cysteine residue, preventing Rb binding.
- Protease inhibitors may offer a strategy for pharmacologically targeting E7 function in HPV-associated cancers.
- Caution is advised when using these inhibitors in experiments studying E7/protein interactions.