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Small-molecule hydrophobic tagging-induced degradation of HaloTag fusion proteins.

Neklesa TK, Tae HS, Schneekloth AR, Stulberg MJ, Corson TW, Sundberg TB, Raina K, Holley SA, Crews CM - Nat. Chem. Biol. (2011)

Bottom Line: The ability to regulate any protein of interest in living systems with small molecules remains a challenge.We designed and synthesized bifunctional small molecules to bind a bacterial dehalogenase (the HaloTag protein) and present a hydrophobic group on its surface.We demonstrated the in vivo utility of hydrophobic tagging by degrading proteins expressed in zebrafish embryos and by inhibiting Hras1(G12V)-driven tumor progression in mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA.

ABSTRACT
The ability to regulate any protein of interest in living systems with small molecules remains a challenge. We hypothesized that appending a hydrophobic moiety to the surface of a protein would mimic the partially denatured state of the protein, thus engaging the cellular quality control machinery to induce its proteasomal degradation. We designed and synthesized bifunctional small molecules to bind a bacterial dehalogenase (the HaloTag protein) and present a hydrophobic group on its surface. Hydrophobic tagging of the HaloTag protein with an adamantyl moiety induced the degradation of cytosolic, isoprenylated and transmembrane HaloTag fusion proteins in cell culture. We demonstrated the in vivo utility of hydrophobic tagging by degrading proteins expressed in zebrafish embryos and by inhibiting Hras1(G12V)-driven tumor progression in mice. Therefore, hydrophobic tagging of HaloTag fusion proteins affords small-molecule control over any protein of interest, making it an ideal system for validating potential drug targets in disease models.

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Functional validation of HaloTag degradation by HyT13(a) NIH-3T3 cells were retrovirally infected with a construct expressing either HA-HaloTag-HRas(G12V) or HA-HaloTag(D106A)-HRas(G12V). The cells were then treated with vehicle or 1 μM HyT13 for 24 hours. The lysates were prepared for immunoblotting and the blots were probed with anti-HA and anti-β-actin antibodies. Full gels are available in Supplementary Results. (b) One hundred thousand NIH-3T3 cells infected with HA-HaloTag-HRas(G12V) or HA-HaloTag(D106A)-HRas(G12V) were plated in 10% FBS containing medium onto 10-cm plates. The next day, the medium was replaced with 1% FBS containing medium, along with vehicle or 1 μM HyT13. The media was refreshed every 2 days, and the plates were pictured on day 6. Bar, 5 mm. (c) Quantification of foci as described in (b). The number of foci/cm2 was counted from three separate plates, with error bars representing SEM. (d) One hundred thousand HA-HaloTag-HRasG12V-expressing NIH-3T3 cells were injected into the flank of nude mice on day 0. The mice were administered IP injections of vehicle or HyT13 daily from day 0. Tumor size was measured daily, and the tumor volume was calculated. Each treatment group employed 7 mice. Error bars represent SEM.
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Figure 3: Functional validation of HaloTag degradation by HyT13(a) NIH-3T3 cells were retrovirally infected with a construct expressing either HA-HaloTag-HRas(G12V) or HA-HaloTag(D106A)-HRas(G12V). The cells were then treated with vehicle or 1 μM HyT13 for 24 hours. The lysates were prepared for immunoblotting and the blots were probed with anti-HA and anti-β-actin antibodies. Full gels are available in Supplementary Results. (b) One hundred thousand NIH-3T3 cells infected with HA-HaloTag-HRas(G12V) or HA-HaloTag(D106A)-HRas(G12V) were plated in 10% FBS containing medium onto 10-cm plates. The next day, the medium was replaced with 1% FBS containing medium, along with vehicle or 1 μM HyT13. The media was refreshed every 2 days, and the plates were pictured on day 6. Bar, 5 mm. (c) Quantification of foci as described in (b). The number of foci/cm2 was counted from three separate plates, with error bars representing SEM. (d) One hundred thousand HA-HaloTag-HRasG12V-expressing NIH-3T3 cells were injected into the flank of nude mice on day 0. The mice were administered IP injections of vehicle or HyT13 daily from day 0. Tumor size was measured daily, and the tumor volume was calculated. Each treatment group employed 7 mice. Error bars represent SEM.

Mentions: We next explored the functional utility of HaloTag-based degradation of an oncogene by HyT13 both in cell culture and in mice. The small GTPase H-Ras is one of the most commonly mutated genes in cancer, with up to 90% of cancers harboring activating mutations in this gene35. Activating mutations, such as the H-RasG12V allele, lead to decreased dependence on extracellular mitogenic signals. Ectopic expression of H-RasG12V in mouse fibroblast cell line NIH-3T3 can lead to a transformed phenotype, as demonstrated by assays in cell culture and in mice. When H-RasG12V expressing cells are grown in culture under low serum conditions they lose cell-to-cell contact inhibition and form distinct foci instead of growing as a cellular monolayer. Furthermore, these transformed cells are capable of tumor formation when injected into immuno-compromised nude mice36,37. We investigated whether (1) HaloTag-H-RasG12V driven focus formation can be suppressed in NIH-3T3 cells and (2) HaloTag-H-RasG12V driven tumor burden in mice can be reduced by administration of HyT13. First, NIH-3T3 cells were stably infected with a HA-HaloTag-H-RasG12V retroviral construct. The encoded fusion protein was readily degraded with HyT13 (Fig. 3a). To test the HaloTag receptor specificity for HyT13, we generated a point mutation in the HaloTag protein (HaloTagD106A) that is unable to form a covalent bond with the reactive chloroalkane in HyT1326. Unlike HA-HaloTag-H-RasG12V, HA-HaloTag(D106A)-H-RasG12V fusion protein was unaffected by HyT13 (Fig. 3a). Next, we plated both cell lines sparsely (105 cells/10-cm plate) in 10% FBS containing media. The next day, the media was replaced with 1% FBS containing media and the cultures were treated with either vehicle or HyT13. By day 6, both vehicle-treated cell lines and HyT13-treated HA-HaloTag(D106A)-H-RasG12V expressing cells had formed many foci, whereas HA-HaloTag-H-RasG12V expressing cells treated with HyT13 had grown a normal monolayer of cells, much like the parental NIH-3T3 cells (Fig. 3b-c). In the absence of HyT13, HA-HaloTag-H-RasG12V expressing cells exhibited slightly higher number of colonies than HA-HaloTag(D106A)-H-RasG12V cells. However, we attribute this observation to slight differences in retroviral infection efficiencies, since we have observed instances where the HaloTag(D106A)-H-RasG12V cells exhibit more colonies than the HA-HaloTag-H-RasG12V cells as well (data not shown). These results demonstrate that hydrophobic tagging can be used to reduce protein activity in the context of in vitro cell culture.


Small-molecule hydrophobic tagging-induced degradation of HaloTag fusion proteins.

Neklesa TK, Tae HS, Schneekloth AR, Stulberg MJ, Corson TW, Sundberg TB, Raina K, Holley SA, Crews CM - Nat. Chem. Biol. (2011)

Functional validation of HaloTag degradation by HyT13(a) NIH-3T3 cells were retrovirally infected with a construct expressing either HA-HaloTag-HRas(G12V) or HA-HaloTag(D106A)-HRas(G12V). The cells were then treated with vehicle or 1 μM HyT13 for 24 hours. The lysates were prepared for immunoblotting and the blots were probed with anti-HA and anti-β-actin antibodies. Full gels are available in Supplementary Results. (b) One hundred thousand NIH-3T3 cells infected with HA-HaloTag-HRas(G12V) or HA-HaloTag(D106A)-HRas(G12V) were plated in 10% FBS containing medium onto 10-cm plates. The next day, the medium was replaced with 1% FBS containing medium, along with vehicle or 1 μM HyT13. The media was refreshed every 2 days, and the plates were pictured on day 6. Bar, 5 mm. (c) Quantification of foci as described in (b). The number of foci/cm2 was counted from three separate plates, with error bars representing SEM. (d) One hundred thousand HA-HaloTag-HRasG12V-expressing NIH-3T3 cells were injected into the flank of nude mice on day 0. The mice were administered IP injections of vehicle or HyT13 daily from day 0. Tumor size was measured daily, and the tumor volume was calculated. Each treatment group employed 7 mice. Error bars represent SEM.
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Related In: Results  -  Collection

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Figure 3: Functional validation of HaloTag degradation by HyT13(a) NIH-3T3 cells were retrovirally infected with a construct expressing either HA-HaloTag-HRas(G12V) or HA-HaloTag(D106A)-HRas(G12V). The cells were then treated with vehicle or 1 μM HyT13 for 24 hours. The lysates were prepared for immunoblotting and the blots were probed with anti-HA and anti-β-actin antibodies. Full gels are available in Supplementary Results. (b) One hundred thousand NIH-3T3 cells infected with HA-HaloTag-HRas(G12V) or HA-HaloTag(D106A)-HRas(G12V) were plated in 10% FBS containing medium onto 10-cm plates. The next day, the medium was replaced with 1% FBS containing medium, along with vehicle or 1 μM HyT13. The media was refreshed every 2 days, and the plates were pictured on day 6. Bar, 5 mm. (c) Quantification of foci as described in (b). The number of foci/cm2 was counted from three separate plates, with error bars representing SEM. (d) One hundred thousand HA-HaloTag-HRasG12V-expressing NIH-3T3 cells were injected into the flank of nude mice on day 0. The mice were administered IP injections of vehicle or HyT13 daily from day 0. Tumor size was measured daily, and the tumor volume was calculated. Each treatment group employed 7 mice. Error bars represent SEM.
Mentions: We next explored the functional utility of HaloTag-based degradation of an oncogene by HyT13 both in cell culture and in mice. The small GTPase H-Ras is one of the most commonly mutated genes in cancer, with up to 90% of cancers harboring activating mutations in this gene35. Activating mutations, such as the H-RasG12V allele, lead to decreased dependence on extracellular mitogenic signals. Ectopic expression of H-RasG12V in mouse fibroblast cell line NIH-3T3 can lead to a transformed phenotype, as demonstrated by assays in cell culture and in mice. When H-RasG12V expressing cells are grown in culture under low serum conditions they lose cell-to-cell contact inhibition and form distinct foci instead of growing as a cellular monolayer. Furthermore, these transformed cells are capable of tumor formation when injected into immuno-compromised nude mice36,37. We investigated whether (1) HaloTag-H-RasG12V driven focus formation can be suppressed in NIH-3T3 cells and (2) HaloTag-H-RasG12V driven tumor burden in mice can be reduced by administration of HyT13. First, NIH-3T3 cells were stably infected with a HA-HaloTag-H-RasG12V retroviral construct. The encoded fusion protein was readily degraded with HyT13 (Fig. 3a). To test the HaloTag receptor specificity for HyT13, we generated a point mutation in the HaloTag protein (HaloTagD106A) that is unable to form a covalent bond with the reactive chloroalkane in HyT1326. Unlike HA-HaloTag-H-RasG12V, HA-HaloTag(D106A)-H-RasG12V fusion protein was unaffected by HyT13 (Fig. 3a). Next, we plated both cell lines sparsely (105 cells/10-cm plate) in 10% FBS containing media. The next day, the media was replaced with 1% FBS containing media and the cultures were treated with either vehicle or HyT13. By day 6, both vehicle-treated cell lines and HyT13-treated HA-HaloTag(D106A)-H-RasG12V expressing cells had formed many foci, whereas HA-HaloTag-H-RasG12V expressing cells treated with HyT13 had grown a normal monolayer of cells, much like the parental NIH-3T3 cells (Fig. 3b-c). In the absence of HyT13, HA-HaloTag-H-RasG12V expressing cells exhibited slightly higher number of colonies than HA-HaloTag(D106A)-H-RasG12V cells. However, we attribute this observation to slight differences in retroviral infection efficiencies, since we have observed instances where the HaloTag(D106A)-H-RasG12V cells exhibit more colonies than the HA-HaloTag-H-RasG12V cells as well (data not shown). These results demonstrate that hydrophobic tagging can be used to reduce protein activity in the context of in vitro cell culture.

Bottom Line: The ability to regulate any protein of interest in living systems with small molecules remains a challenge.We designed and synthesized bifunctional small molecules to bind a bacterial dehalogenase (the HaloTag protein) and present a hydrophobic group on its surface.We demonstrated the in vivo utility of hydrophobic tagging by degrading proteins expressed in zebrafish embryos and by inhibiting Hras1(G12V)-driven tumor progression in mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA.

ABSTRACT
The ability to regulate any protein of interest in living systems with small molecules remains a challenge. We hypothesized that appending a hydrophobic moiety to the surface of a protein would mimic the partially denatured state of the protein, thus engaging the cellular quality control machinery to induce its proteasomal degradation. We designed and synthesized bifunctional small molecules to bind a bacterial dehalogenase (the HaloTag protein) and present a hydrophobic group on its surface. Hydrophobic tagging of the HaloTag protein with an adamantyl moiety induced the degradation of cytosolic, isoprenylated and transmembrane HaloTag fusion proteins in cell culture. We demonstrated the in vivo utility of hydrophobic tagging by degrading proteins expressed in zebrafish embryos and by inhibiting Hras1(G12V)-driven tumor progression in mice. Therefore, hydrophobic tagging of HaloTag fusion proteins affords small-molecule control over any protein of interest, making it an ideal system for validating potential drug targets in disease models.

Show MeSH
Related in: MedlinePlus