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Protein prenylation: enzymes, therapeutics, and biotechnology applications.

Palsuledesai CC, Distefano MD - ACS Chem. Biol. (2014)

Bottom Line: It is essential for the proper cellular activity of numerous proteins, including Ras family GTPases and heterotrimeric G-proteins.Inhibition of prenylation has been extensively investigated to suppress the activity of oncogenic Ras proteins to achieve antitumor activity.Finally, we discuss recent progress in utilizing protein prenylation for site-specific protein labeling for various biotechnology applications.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Minnesota , Minneapolis, Minnesota 55455, United States.

ABSTRACT
Protein prenylation is a ubiquitous covalent post-translational modification found in all eukaryotic cells, comprising attachment of either a farnesyl or a geranylgeranyl isoprenoid. It is essential for the proper cellular activity of numerous proteins, including Ras family GTPases and heterotrimeric G-proteins. Inhibition of prenylation has been extensively investigated to suppress the activity of oncogenic Ras proteins to achieve antitumor activity. Here, we review the biochemistry of the prenyltransferase enzymes and numerous isoprenoid analogs synthesized to investigate various aspects of prenylation and prenyltransferases. We also give an account of the current status of prenyltransferase inhibitors as potential therapeutics against several diseases including cancers, progeria, aging, parasitic diseases, and bacterial and viral infections. Finally, we discuss recent progress in utilizing protein prenylation for site-specific protein labeling for various biotechnology applications.

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Key features of catalysis by protein farnesyltransferase.(A) Schematic representation of transition state showing thiol activationby Zn2+, diphosphate stabilization by Mg2+,and partial bonding to leaving group and incoming nucleophile (adaptedfrom ref (23)). (B)Structural model for transition state based on kinetic isotope effectmeasurements and DFT calculations. The model reaction used for computation(shown in these images) employed ethanethiol and dimethylallyl diphosphate.(C) Electrostatic potential map of transition state based on the samemodel shown in panel B (images B and C images adapted from ref (24)). Color scheme for B:carbon (green), hydrogen (white), oxygen (red), phosphorus (magenta),and sulfur (yellow). Color scheme for C: red represents more negativepotential, blue represents less negative potential, and green is intermediate.
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fig5: Key features of catalysis by protein farnesyltransferase.(A) Schematic representation of transition state showing thiol activationby Zn2+, diphosphate stabilization by Mg2+,and partial bonding to leaving group and incoming nucleophile (adaptedfrom ref (23)). (B)Structural model for transition state based on kinetic isotope effectmeasurements and DFT calculations. The model reaction used for computation(shown in these images) employed ethanethiol and dimethylallyl diphosphate.(C) Electrostatic potential map of transition state based on the samemodel shown in panel B (images B and C images adapted from ref (24)). Color scheme for B:carbon (green), hydrogen (white), oxygen (red), phosphorus (magenta),and sulfur (yellow). Color scheme for C: red represents more negativepotential, blue represents less negative potential, and green is intermediate.

Mentions: Protein prenylation is catalyzed by threedistinct prenyltransferase enzymes that all exist as heterodimersand have very similar topologies (Figure 3A,B).While FTase and GGTase-I share a common α-subunit, the α-subunitof rat GGTase-II has only 22% sequence similarity to the rat FTaseα-subunit.11 In rat-derived enzymes,the β-subunit of FTase is only 25% and 32% identical to thatof GGTase-I and GGTase-II, respectively.12 The reaction catalyzed by GGTase-II requires an additional escortprotein for activity. Most mechanistic analyses have focused on farnesylationwith a more limited number of studies probing geranylgeranylation.Early kinetic analysis demonstrated that farnesylation proceeds viaan ordered mechanism in which FPP binds first.13,14 After binary complex formation occurs, the CaaX-box-containing substratebinds, and C–S bond formation occurs. At that point, a newFPP molecule binds, while the farnesylated protein remains bound,followed by product dissociation either prior to or concerted withbinding of a new CaaX-box substrate protein. All of these intermediateshave been observed crystallographically, providing a clear view ofthe events occurring during catalysis; interestingly, minimal differencesin the protein conformation are observed in these different structures.15 Single turnover kinetic experiments and calculationssuggest that a conformational change in the enzyme occurs prior toC–S bond formation although no evidence for this has been notedin any of the crystal structures solved to date.16,17 Stereochemical analysis of the enzymatic process using deuteratedisotopomers of FPP (3, Figure 4) revealed that the reaction proceeds with clean inversion of configurationof stereochemistry at C-1 of the isoprenoid18,19 suggesting that attack of the sulfur nucleophile is concerted withdeparture of the diphosphate leaving group. Work with isoprenoid analogsincorporating electron withdrawing fluorine substituents (4) provides evidence that the transition state involves some carbocationiccharacter20 although analogs including 5 designed to trap such intermediates failed to do so.21 Kinetic isotope effect measurements with both 2H- and 13C-isotopomers suggest a transition statethat involves participation of the incoming sulfur nucleophile withsignificant development of positive charge at C-1 of the isoprenoid(Figure 5A–C);22−24 QM/MM computationalexperiments are in accord with this since no evidence for a discretecarbocationic species was observed.25 Forefficient catalysis, kinetic experiments indicate that the enzymeactivates the sulfur nucleophile as a Zn-thiolate.26,27 Such a species is consistent with what has been observed crystallographicallyas well as via EXAFS spectroscopy.28


Protein prenylation: enzymes, therapeutics, and biotechnology applications.

Palsuledesai CC, Distefano MD - ACS Chem. Biol. (2014)

Key features of catalysis by protein farnesyltransferase.(A) Schematic representation of transition state showing thiol activationby Zn2+, diphosphate stabilization by Mg2+,and partial bonding to leaving group and incoming nucleophile (adaptedfrom ref (23)). (B)Structural model for transition state based on kinetic isotope effectmeasurements and DFT calculations. The model reaction used for computation(shown in these images) employed ethanethiol and dimethylallyl diphosphate.(C) Electrostatic potential map of transition state based on the samemodel shown in panel B (images B and C images adapted from ref (24)). Color scheme for B:carbon (green), hydrogen (white), oxygen (red), phosphorus (magenta),and sulfur (yellow). Color scheme for C: red represents more negativepotential, blue represents less negative potential, and green is intermediate.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4301080&req=5

fig5: Key features of catalysis by protein farnesyltransferase.(A) Schematic representation of transition state showing thiol activationby Zn2+, diphosphate stabilization by Mg2+,and partial bonding to leaving group and incoming nucleophile (adaptedfrom ref (23)). (B)Structural model for transition state based on kinetic isotope effectmeasurements and DFT calculations. The model reaction used for computation(shown in these images) employed ethanethiol and dimethylallyl diphosphate.(C) Electrostatic potential map of transition state based on the samemodel shown in panel B (images B and C images adapted from ref (24)). Color scheme for B:carbon (green), hydrogen (white), oxygen (red), phosphorus (magenta),and sulfur (yellow). Color scheme for C: red represents more negativepotential, blue represents less negative potential, and green is intermediate.
Mentions: Protein prenylation is catalyzed by threedistinct prenyltransferase enzymes that all exist as heterodimersand have very similar topologies (Figure 3A,B).While FTase and GGTase-I share a common α-subunit, the α-subunitof rat GGTase-II has only 22% sequence similarity to the rat FTaseα-subunit.11 In rat-derived enzymes,the β-subunit of FTase is only 25% and 32% identical to thatof GGTase-I and GGTase-II, respectively.12 The reaction catalyzed by GGTase-II requires an additional escortprotein for activity. Most mechanistic analyses have focused on farnesylationwith a more limited number of studies probing geranylgeranylation.Early kinetic analysis demonstrated that farnesylation proceeds viaan ordered mechanism in which FPP binds first.13,14 After binary complex formation occurs, the CaaX-box-containing substratebinds, and C–S bond formation occurs. At that point, a newFPP molecule binds, while the farnesylated protein remains bound,followed by product dissociation either prior to or concerted withbinding of a new CaaX-box substrate protein. All of these intermediateshave been observed crystallographically, providing a clear view ofthe events occurring during catalysis; interestingly, minimal differencesin the protein conformation are observed in these different structures.15 Single turnover kinetic experiments and calculationssuggest that a conformational change in the enzyme occurs prior toC–S bond formation although no evidence for this has been notedin any of the crystal structures solved to date.16,17 Stereochemical analysis of the enzymatic process using deuteratedisotopomers of FPP (3, Figure 4) revealed that the reaction proceeds with clean inversion of configurationof stereochemistry at C-1 of the isoprenoid18,19 suggesting that attack of the sulfur nucleophile is concerted withdeparture of the diphosphate leaving group. Work with isoprenoid analogsincorporating electron withdrawing fluorine substituents (4) provides evidence that the transition state involves some carbocationiccharacter20 although analogs including 5 designed to trap such intermediates failed to do so.21 Kinetic isotope effect measurements with both 2H- and 13C-isotopomers suggest a transition statethat involves participation of the incoming sulfur nucleophile withsignificant development of positive charge at C-1 of the isoprenoid(Figure 5A–C);22−24 QM/MM computationalexperiments are in accord with this since no evidence for a discretecarbocationic species was observed.25 Forefficient catalysis, kinetic experiments indicate that the enzymeactivates the sulfur nucleophile as a Zn-thiolate.26,27 Such a species is consistent with what has been observed crystallographicallyas well as via EXAFS spectroscopy.28

Bottom Line: It is essential for the proper cellular activity of numerous proteins, including Ras family GTPases and heterotrimeric G-proteins.Inhibition of prenylation has been extensively investigated to suppress the activity of oncogenic Ras proteins to achieve antitumor activity.Finally, we discuss recent progress in utilizing protein prenylation for site-specific protein labeling for various biotechnology applications.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Minnesota , Minneapolis, Minnesota 55455, United States.

ABSTRACT
Protein prenylation is a ubiquitous covalent post-translational modification found in all eukaryotic cells, comprising attachment of either a farnesyl or a geranylgeranyl isoprenoid. It is essential for the proper cellular activity of numerous proteins, including Ras family GTPases and heterotrimeric G-proteins. Inhibition of prenylation has been extensively investigated to suppress the activity of oncogenic Ras proteins to achieve antitumor activity. Here, we review the biochemistry of the prenyltransferase enzymes and numerous isoprenoid analogs synthesized to investigate various aspects of prenylation and prenyltransferases. We also give an account of the current status of prenyltransferase inhibitors as potential therapeutics against several diseases including cancers, progeria, aging, parasitic diseases, and bacterial and viral infections. Finally, we discuss recent progress in utilizing protein prenylation for site-specific protein labeling for various biotechnology applications.

Show MeSH
Related in: MedlinePlus