m →GSTs and biotransformation: changed heading to remove redundancy |
→Role in cell signaling: made this its own section, separate from Structure; also made Implications in cancer development a subsection |
||
| Line 94: | Line 94: | ||
|} |
|} |
||
==Role in cell signaling== |
|||
[[File:MAPK-pathway-mammalian.png|thumb|A simplified overview of MAPK pathways in mammals, organised into three main signaling modules (ERK1/2, JNK/p38 and ERK5).]] |
[[File:MAPK-pathway-mammalian.png|thumb|A simplified overview of MAPK pathways in mammals, organised into three main signaling modules (ERK1/2, JNK/p38 and ERK5).]] |
||
| Line 103: | Line 103: | ||
Like GSTP, GSTμ 1 ([[Glutathione_S-transferase_Mu_1|GSTM1]]) is involved in regulating apoptotic pathways through direct protein-protein interactions, although it acts on [[ASK1]], which is upstream of JNK. The mechanism and result are similar to that of GSTP and JNK, in that GSTM1 sequesters ASK1 through complex formation and prevents its induction of the pro-apoptotic [[P38_mitogen-activated_protein_kinases|p38]] and JNK portions of the MAPK signaling cascade. Like GSTP, GSTM1 interacts with its partner in the absence of oxidative stress, although ASK1 is also involved in [[Heat_shock|heat shock]] response, which is likewise prevented during ASK1 sequestration. The fact that high levels of GST are associated with resistance to apoptosis induced by a range of substances, including chemotherapeutic agents, supports its putative role in MAPK signaling prevention.<ref name="pmid14576844">{{cite journal |author=Townsend DM, Tew KD |title=The role of glutathione-S-transferase in anti-cancer drug resistance |journal=Oncogene |volume=22 |issue=47 |pages=7369–75 |year=2003 |month=October |pmid=14576844 |doi=10.1038/sj.onc.1206940 |url=http://dx.doi.org/10.1038/sj.onc.1206940}}</ref> |
Like GSTP, GSTμ 1 ([[Glutathione_S-transferase_Mu_1|GSTM1]]) is involved in regulating apoptotic pathways through direct protein-protein interactions, although it acts on [[ASK1]], which is upstream of JNK. The mechanism and result are similar to that of GSTP and JNK, in that GSTM1 sequesters ASK1 through complex formation and prevents its induction of the pro-apoptotic [[P38_mitogen-activated_protein_kinases|p38]] and JNK portions of the MAPK signaling cascade. Like GSTP, GSTM1 interacts with its partner in the absence of oxidative stress, although ASK1 is also involved in [[Heat_shock|heat shock]] response, which is likewise prevented during ASK1 sequestration. The fact that high levels of GST are associated with resistance to apoptosis induced by a range of substances, including chemotherapeutic agents, supports its putative role in MAPK signaling prevention.<ref name="pmid14576844">{{cite journal |author=Townsend DM, Tew KD |title=The role of glutathione-S-transferase in anti-cancer drug resistance |journal=Oncogene |volume=22 |issue=47 |pages=7369–75 |year=2003 |month=October |pmid=14576844 |doi=10.1038/sj.onc.1206940 |url=http://dx.doi.org/10.1038/sj.onc.1206940}}</ref> |
||
===Implications in cancer development=== |
|||
There is a growing body of evidence supporting the role of GST, particularly GSTP, in cancer development and chemotherapeutic resistance. The link between GSTP and cancer is most obvious in the overexpression of GSTP in many cancers, but it is also supported by the fact that the transformed phenotype of tumor cells is associated with aberrantly regulated kinase signaling pathways and cellular addiction to overexpressed proteins. That most anti-cancer drugs are poor substrates for GSTP indicates that the role of elevated GSTP in many tumor cell lines is not to detoxify the compounds, but must have another purpose; this theory is also given credence by the common finding of GSTP overexpression in tumor cell lines that are not drug resistant.<ref name="pmid21558000">{{cite journal |author=Tew KD, Manevich Y, Grek C, Xiong Y, Uys J, Townsend DM |title=The role of glutathione S-transferase P in signaling pathways and S-glutathionylation in cancer |journal=Free Radic. Biol. Med. |volume=51 |issue=2 |pages=299–313 |year=2011 |month=July |pmid=21558000 |pmc=3125017 |doi=10.1016/j.freeradbiomed.2011.04.013 |url=http://linkinghub.elsevier.com/retrieve/pii/S0891-5849(11)00239-5}}</ref> |
There is a growing body of evidence supporting the role of GST, particularly GSTP, in cancer development and chemotherapeutic resistance. The link between GSTP and cancer is most obvious in the overexpression of GSTP in many cancers, but it is also supported by the fact that the transformed phenotype of tumor cells is associated with aberrantly regulated kinase signaling pathways and cellular addiction to overexpressed proteins. That most anti-cancer drugs are poor substrates for GSTP indicates that the role of elevated GSTP in many tumor cell lines is not to detoxify the compounds, but must have another purpose; this theory is also given credence by the common finding of GSTP overexpression in tumor cell lines that are not drug resistant.<ref name="pmid21558000">{{cite journal |author=Tew KD, Manevich Y, Grek C, Xiong Y, Uys J, Townsend DM |title=The role of glutathione S-transferase P in signaling pathways and S-glutathionylation in cancer |journal=Free Radic. Biol. Med. |volume=51 |issue=2 |pages=299–313 |year=2011 |month=July |pmid=21558000 |pmc=3125017 |doi=10.1016/j.freeradbiomed.2011.04.013 |url=http://linkinghub.elsevier.com/retrieve/pii/S0891-5849(11)00239-5}}</ref> |
||
Revision as of 21:40, 17 April 2013
| Glutathione S-transferase | |||||||||
|---|---|---|---|---|---|---|---|---|---|
 Crystallographic structure of glutathione S-transferase from Anopheles cracens.[1] | |||||||||
| Identifiers | |||||||||
| EC no. | 2.5.1.18 | ||||||||
| CAS no. | 50812-37-8 | ||||||||
| Databases | |||||||||
| IntEnz | IntEnz view | ||||||||
| BRENDA | BRENDA entry | ||||||||
| ExPASy | NiceZyme view | ||||||||
| KEGG | KEGG entry | ||||||||
| MetaCyc | metabolic pathway | ||||||||
| PRIAM | profile | ||||||||
| PDB structures | RCSB PDB PDBe PDBsum | ||||||||
| Gene Ontology | AmiGO / QuickGO | ||||||||
| |||||||||
The glutathione S-transferase (GST, previously known as ligandins) family of enzymes are composed of many cytosolic, mitochondrial, and microsomal (now designated as MAPEG) proteins. GSTs are present in eukaryotes and in prokaryotes, where they catalyze a variety of reactions and accept endogenous and xenobiotic substrates.[1][2][3] Members of the GST superfamily are extremely diverse in amino acid sequence, and a large fraction of the sequences deposited in public databases are of unknown function.[4] The Enzyme Function Initiative (EFI) is using GSTs as a model superfamily to identify new GST functions.
GSTs can constitute up to 10% of cytosolic protein in some mammalian organs.[5] GSTs catalyse the conjugation of reduced glutathione — via a sulfhydryl group — to electrophilic centers on a wide variety of substrates in order to make the compounds more soluble.[6][7] This activity detoxifies endogenous compounds such as peroxidised lipids, as well as breakdown of xenobiotics. GSTs may also bind toxins and function as transport proteins, which gave rise to the early term for GSTs of “ligandin”.[8][9] The mammalian GST super-family consists of cytosolic dimeric isoenzymes of 45–55 kDa size that have been assigned to at least six classes: Alpha, Mu, Pi, Theta, Zeta and Omega.[10][11]
Most mammalian isoenzymes have affinity for the substrate 1-chloro-2,4-dinitrobenzene (CDNB), and spectrophotometric assays utilising this substrate are commonly used to report GST activity.[12] However, some endogenous compounds, e.g., bilirubin, can inhibit the activity of GSTs. In mammals, GST isoforms have cell specific distributions (e.g., alpha GST in hepatocytes and pi GST in the biliary tract of the human liver).[13]
Structure
| Glutathione S-transferase, C-terminal domain | |||||||||
|---|---|---|---|---|---|---|---|---|---|
 Structure of the xenobiotic substrate binding site of rat glutathione S-transferase mu 1 bound to the GSH adduct of phenanthrene-9,10-oxide.[14] | |||||||||
| Identifiers | |||||||||
| Symbol | GST_C | ||||||||
| Pfam | PF00043 | ||||||||
| InterPro | IPR004046 | ||||||||
| SCOP2 | 2gst / SCOPe / SUPFAM | ||||||||
| CDD | cd00299 | ||||||||
| |||||||||
Protein sequence and structure are important classification methods for GSTs: while classes from the cytosolic superfamily of GSTs possess more than 40% sequence homology, those from other classes may have less than 25%. Cytosolic GSTs are divided into 13 classes based upon their structure: alpha, beta, delta, epsilon, zeta, theta, mu, nu, pi, sigma, tau, phi, and omega. Mitochondrial GSTs are in class kappa. The MAPEG superfamily of microsomal GSTs consists of subgroups designated I-IV, between which amino acid sequences share less than 20% identity.[7]
The glutathione binding site, or "G-site," is located in the thioredoxin-like domain of both cytosolic and mitochondrial GSTs. The region containing the greatest amount of variability between the assorted classes is that of helix α2, where one of three different amino acid residues interacts with the glycine residue of glutathione. Two subgroups of cytosolic GSTs have been characterized based upon their interaction with glutathione: the Y-GST group, which uses a tyrosine residue to activate glutathione, and the S/C-GST, which instead use serine or cysteine residues.[7]
The porcine pi-class enzyme pGTSP1-1 was the first GST to have its structure determined, and it is representative of other members of the cytosolic GST superfamily, which contain a thioredoxin-like N-terminus domain as well as a C-terminus domain consisting of alpha helices.[7]
Mammalian cytosolic GSTs are dimeric, both subunits being from the same class of GSTs, although not necessarily identical. The monomers are in the range of 22–30 kDa. They are active over a wide variety of substrates with considerable overlap. The following table lists all GST enzymes of each class known to exist in Homo sapiens. The maximum enzyme number for each class found across all species in the UniProtKB/Swiss-Prot database is included, shown in the Max All species column.
| GST Class | Homo sapiens GST Class Members (22) | Max All Species |
|---|---|---|
| Alpha | GSTA1, GSTA2, GSTA3, GSTA4, GSTA5 | GSTA6 |
| Delta | GSTD12 | |
| Kappa | GSTK1 | GSTK2 |
| Mu | GSTM1, GSTM1L (RNAi), GSTM2, GSTM3, GSTM4, GSTM5 | GSTM7 |
| Omega | GSTO1, GSTO2 | GSTO3 |
| Pi | GSTP1 | GSTP2 |
| Theta | GSTT1, GSTT2, GSTT4 | GSTT7 |
| Zeta | GSTZ1 (aka GSTZ1 MAAI-Maleylacetoacetate isomerase) | GSTZ3 |
| Microsomal | MGST1, MGST2, MGST3 | MGST3 |
Role in cell signaling
Although best known for their ability to conjugate GSH and thereby detoxify cellular environments, GSTs are also capable of binding nonsubstrate ligands, with important cell signaling implications. Several GST isozymes from various classes have been shown to inhibit the function of a kinase involved in the MAPK pathway that regulates cell proliferation and death, preventing the kinase from carrying out its role in facilitating the signaling cascade.[15]
The cytosolic π-class GST composed of subunit 1 homodimers (GSTP1-1), a well-characterized isozyme of the mammalian GST family, is expressed primarily in heart, lung, and brain tissues; in fact, it is the most common GST expressed outside the liver.[15] Based on its overexpression in a majority of human tumor cell lines and prevalence in chemotherapeutic-resistant tumors, GSTP1-1 is thought to play a role in the development of cancer and its potential resistance to drug treatment. Further evidence for this comes from the knowledge that GSTP can selectively inhibit C-jun phosphorylation by JNK, preventing apoptosis.[15] During times of low cellular stress, a complex forms through direct protein-protein interactions between GSTP and the C-terminus of JNK, effectively preventing the action of JNK and thus its induction of the JNK pathway. Cellular oxidative stress causes the dissociation of the complex, oligomerization of GSTP, and induction of the JNK pathway, resulting in apoptosis.[16] The connection between GSTP inhibition of the pro-apoptotic JNK pathway and the isozyme's overexpression in drug-resistant tumor cells may itself account for the tumor cells' ability to escape apoptosis mediated by drugs that are not substrates of GSTP.[15]
Like GSTP, GSTμ 1 (GSTM1) is involved in regulating apoptotic pathways through direct protein-protein interactions, although it acts on ASK1, which is upstream of JNK. The mechanism and result are similar to that of GSTP and JNK, in that GSTM1 sequesters ASK1 through complex formation and prevents its induction of the pro-apoptotic p38 and JNK portions of the MAPK signaling cascade. Like GSTP, GSTM1 interacts with its partner in the absence of oxidative stress, although ASK1 is also involved in heat shock response, which is likewise prevented during ASK1 sequestration. The fact that high levels of GST are associated with resistance to apoptosis induced by a range of substances, including chemotherapeutic agents, supports its putative role in MAPK signaling prevention.[16]
Implications in cancer development
There is a growing body of evidence supporting the role of GST, particularly GSTP, in cancer development and chemotherapeutic resistance. The link between GSTP and cancer is most obvious in the overexpression of GSTP in many cancers, but it is also supported by the fact that the transformed phenotype of tumor cells is associated with aberrantly regulated kinase signaling pathways and cellular addiction to overexpressed proteins. That most anti-cancer drugs are poor substrates for GSTP indicates that the role of elevated GSTP in many tumor cell lines is not to detoxify the compounds, but must have another purpose; this theory is also given credence by the common finding of GSTP overexpression in tumor cell lines that are not drug resistant.[17]
Biotransformation
Glutathione S-transferases are considered, among several others, to contribute to the phase II metabolism of xenobiotics. Drugs, poisons, and other compounds not traditionally listed in either groups are usually modified by the phase I and/or phase II mechanisms, and finally excreted from the body. GSTs contribute to this type of metabolism by conjugating these compounds (often electrophilic and somewhat lipophilic in nature) with reduced glutathione to facilitate dissolution in the aqueous cellular and extracellular media, and, from there, out of the body.
GST-tags and the GST pull-down assay
Genetic engineers have used glutathione S-transferase to create the GST gene fusion system. This system is used to purify and detect proteins of interest. In a GST gene fusion system, the GST sequence is incorporated into an expression vector alongside the gene sequence encoding the protein of interest. Induction of protein expression from the vector's promoter results in expression of a fusion protein: the protein of interest fused to the GST protein. This GST-fusion protein can then be purified from cells through its high affinity for glutathione.
Fusion proteins offer an important biological assay for direct protein-to-protein interactions. For instance, to demonstrate that protein X binds to protein Y a GST-X fusion protein would be generated. Assay beads, coated with the tripeptide glutathione, strongly bind the GST fusion protein (GST-X). Therefore if X binds Y, then GST-X will also bind Y, and Y will be present on assay beads.
GST is commonly used to create fusion proteins. The tag has the size of 220 amino acids (roughly 26 KDa), which, compared to other tags like the myc- or the FLAG-tag, is quite big. It is fused to the N-terminus of a protein. However, many commercially-available sources of GST-tagged plasmids include a thrombin domain for cleavage of the GST tag during protein purification.
A GST-tag is often used to separate and purify proteins that contain the GST-fusion. GST-fusion proteins can be produced in Escherichia coli, as recombinant proteins. The GST part binds its substrate, glutathione. Agarose beads can be coated with glutathione, and such glutathione-Agarose beads bind GST-proteins. These beads are then washed, to remove contaminating bacterial proteins. Adding free glutathione to beads that bind purified GST-proteins will release the GST-protein in solution.
See also
- Maltose-binding protein
- Glutathione S-transferase, C-terminal domain
- Bacterial glutathione transferase
- GSTP1
- Glutathione S-transferase Mu 1
- Affinity chromatography
References
- ^ a b PDB: 1R5A; Udomsinprasert R, Pongjaroenkit S, Wongsantichon J, Oakley AJ, Prapanthadara LA, Wilce MC, Ketterman AJ (2005). "Identification, characterization and structure of a new Delta class glutathione transferase isoenzyme". Biochem. J. 388 (Pt 3): 763–71. doi:10.1042/BJ20042015. PMC 1183455. PMID 15717864.
{{cite journal}}: Unknown parameter|month=ignored (help)CS1 maint: multiple names: authors list (link) Cite error: The named reference "pmid15717864" was defined multiple times with different content (see the help page). - ^ Sheehan D, Meade G, Foley VM, Dowd CA (2001). "Structure, function and evolution of glutathione transferases: implications for classification of non-mammalian members of an ancient enzyme superfamily". Biochem. J. 360 (Pt 1): 1–16. doi:10.1042/0264-6021:3600001. PMC 1222196. PMID 11695986.
{{cite journal}}: Unknown parameter|month=ignored (help)CS1 maint: multiple names: authors list (link) - ^ Allocati N, Federici L, Masulli M, Di Ilio C (2009). "Glutathione transferases in bacteria". FEBS J. 276 (1): 58–75. doi:10.1111/j.1742-4658.2008.06743.x. PMID 19016852.
{{cite journal}}: Unknown parameter|month=ignored (help)CS1 maint: multiple names: authors list (link) - ^ Atkinson, HJ (2009 Nov 24). "Glutathione transferases are structural and functional outliers in the thioredoxin fold". Biochemistry. 48 (46): 11108–16. doi:10.1021/bi901180v. PMC 2778357. PMID 19842715.
{{cite journal}}: Check date values in:|date=(help); Unknown parameter|coauthors=ignored (|author=suggested) (help) - ^ Boyer TD (1989). "The glutathione S-transferases: an update". Hepatology. 9 (3): 486–96. doi:10.1002/hep.1840090324. PMID 2646197.
{{cite journal}}: Unknown parameter|month=ignored (help) - ^ Douglas KT (1987). "Mechanism of action of glutathione-dependent enzymes". Adv. Enzymol. Relat. Areas Mol. Biol. 59: 103–67. PMID 2880477.
- ^ a b c d Oakley A (2011). "Glutathione transferases: a structural perspective". Drug Metab. Rev. 43 (2): 138–51. doi:10.3109/03602532.2011.558093. PMID 21428697.
{{cite journal}}: Unknown parameter|month=ignored (help) Cite error: The named reference "pmid21428697" was defined multiple times with different content (see the help page). - ^ Leaver MJ, George SG (1998). "A piscine glutathione S-transferase which efficiently conjugates the end-products of lipid peroxidation". Marine Environmental Research. 46 (1–5): 71–74. doi:10.1016/S0141-1136(97)00071-8.
- ^ Litwack G, Ketterer B, Arias IM (1971). "Ligandin: a hepatic protein which binds steroids, bilirubin, carcinogens and a number of exogenous organic anions". Nature. 234 (5330): 466–7. doi:10.1038/234466a0. PMID 4944188.
{{cite journal}}: Unknown parameter|month=ignored (help)CS1 maint: multiple names: authors list (link) - ^ Beckett GJ, Hayes JD (1993). "Glutathione S-transferases: biomedical applications". Adv Clin Chem. Advances in Clinical Chemistry. 30: 281–380. doi:10.1016/S0065-2423(08)60198-5. ISBN 978-0-12-010330-0. PMID 8237562.
- ^ Wilce MC, Parker MW (1994). "Structure and function of glutathione S-transferases". Biochim. Biophys. Acta. 1205 (1): 1–18. doi:10.1016/0167-4838(94)90086-8. PMID 8142473.
{{cite journal}}: Unknown parameter|month=ignored (help) - ^ Habig WH, Pabst MJ, Fleischner G, Gatmaitan Z, Arias IM, Jakoby WB (1974). "The Identity of Glutathione S-Transferase B with Ligandin, a Major Binding Protein of Liver". Proc. Natl. Acad. Sci. U.S.A. 71 (10): 3879–82. doi:10.1073/pnas.71.10.3879. PMC 434288. PMID 4139704.
{{cite journal}}: Unknown parameter|month=ignored (help)CS1 maint: multiple names: authors list (link) - ^ Beckett GJ, Hayes JD (1987). "Glutathione S-transferase measurements and liver disease in man". Journal of Clinical Biochemistry and Nutrition. 2: 1–24. doi:10.3164/jcbn.2.1.
- ^ PDB: 2GST; Ji X, Johnson WW, Sesay MA, Dickert L, Prasad SM, Ammon HL, Armstrong RN, Gilliland GL (1994). "Structure and function of the xenobiotic substrate binding site of a glutathione S-transferase as revealed by X-ray crystallographic analysis of product complexes with the diastereomers of 9-(S-glutathionyl)-10-hydroxy-9,10-dihydrophenanthrene". Biochemistry. 33 (5): 1043–52. doi:10.1021/bi00171a002. PMID 8110735.
{{cite journal}}: Unknown parameter|month=ignored (help)CS1 maint: multiple names: authors list (link) - ^ a b c d Laborde E (2010). "Glutathione transferases as mediators of signaling pathways involved in cell proliferation and cell death". Cell Death Differ. 17 (9): 1373–80. doi:10.1038/cdd.2010.80. PMID 20596078.
{{cite journal}}: Unknown parameter|month=ignored (help) - ^ a b Townsend DM, Tew KD (2003). "The role of glutathione-S-transferase in anti-cancer drug resistance". Oncogene. 22 (47): 7369–75. doi:10.1038/sj.onc.1206940. PMID 14576844.
{{cite journal}}: Unknown parameter|month=ignored (help) - ^ Tew KD, Manevich Y, Grek C, Xiong Y, Uys J, Townsend DM (2011). "The role of glutathione S-transferase P in signaling pathways and S-glutathionylation in cancer". Free Radic. Biol. Med. 51 (2): 299–313. doi:10.1016/j.freeradbiomed.2011.04.013. PMC 3125017. PMID 21558000.
{{cite journal}}: Unknown parameter|month=ignored (help)CS1 maint: multiple names: authors list (link)
External links
- Overview of Glutathione-S-Transferases
- UMich Orientation of Proteins in Membranes families/superfamily-199 - MAPEG (Eicosanoid and Glutathione metabolism proteins) family
- Glutathione+S-Transferase at the U.S. National Library of Medicine Medical Subject Headings (MeSH)
- EC 2.5.1.18
- Preparation of GST Fusion Proteins