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Ligand binding reveals a role for heme in translationally-controlled tumor protein dimerization.

Lucas AT, Fu X, Liu J, Brannon MK, Yang J, Capelluto DG, Finkielstein CV - PLoS ONE (2014)

Bottom Line: Mutation in both His residues to Ala prevents hemin from binding and abrogates oligomerization, suggesting that the ligand site localizes at the interface of the oligomer.Unlike heme, binding of Ca2+ ligand to TCTP does not alter its monomeric state; although, Ca2+ is able to destabilize an existing TCTP dimer created by hemin addition.In agreement with TCTP's proposed buffer function, ligand binding occurs at high concentration, allowing the "buffer" condition to be dissociated from TCTP's role as a component of signal transduction mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Integrated Cellular Responses Laboratory, Virginia Bioinformatics Institute, Department of Biological Sciences, Virginia Tech, Blacksburg, Virginia, United States of America; Protein Signaling Domains Laboratory, Virginia Bioinformatics Institute, Department of Biological Sciences, Virginia Tech, Blacksburg, Virginia, United States of America.

ABSTRACT
The translationally-controlled tumor protein (TCTP) is a highly conserved, ubiquitously expressed, abundant protein that is broadly distributed among eukaryotes. Its biological function spans numerous cellular processes ranging from regulation of the cell cycle and microtubule stabilization to cell growth, transformation, and death processes. In this work, we propose a new function for TCTP as a "buffer protein" controlling cellular homeostasis. We demonstrate that binding of hemin to TCTP is mediated by a conserved His-containing motif (His76His77) followed by dimerization, an event that involves ligand-mediated conformational changes and that is necessary to trigger TCTP's cytokine-like activity. Mutation in both His residues to Ala prevents hemin from binding and abrogates oligomerization, suggesting that the ligand site localizes at the interface of the oligomer. Unlike heme, binding of Ca2+ ligand to TCTP does not alter its monomeric state; although, Ca2+ is able to destabilize an existing TCTP dimer created by hemin addition. In agreement with TCTP's proposed buffer function, ligand binding occurs at high concentration, allowing the "buffer" condition to be dissociated from TCTP's role as a component of signal transduction mechanisms.

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Cellular TCTP oligomerization is ligand-dependent.CHO cells were transfected with pCS2+myc-TCTP in serum-free medium containing 5 mM succinylacetone for 24 h prior to harvesting to prevent de novo synthesis of heme. Extracts were incubated with recombinant GST-TCTP bound beads in the absence or presence of hemin (1 µM, 100 µM, 1 mM), and/or CaCl2 (2.5 or 25 mM) and described in “Materials and Methods”. Bound complexes were resolved by SDS-PAGE and bound proteins detected by immunoblotting (upper panel). The expression of recombinant myc-TCTP in cells and GST-TCTP in the assay are shown in the middle and lower panels for each treatment, respectively. Molecular mass markers (in kDa) are indicated on the left.
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pone-0112823-g007: Cellular TCTP oligomerization is ligand-dependent.CHO cells were transfected with pCS2+myc-TCTP in serum-free medium containing 5 mM succinylacetone for 24 h prior to harvesting to prevent de novo synthesis of heme. Extracts were incubated with recombinant GST-TCTP bound beads in the absence or presence of hemin (1 µM, 100 µM, 1 mM), and/or CaCl2 (2.5 or 25 mM) and described in “Materials and Methods”. Bound complexes were resolved by SDS-PAGE and bound proteins detected by immunoblotting (upper panel). The expression of recombinant myc-TCTP in cells and GST-TCTP in the assay are shown in the middle and lower panels for each treatment, respectively. Molecular mass markers (in kDa) are indicated on the left.

Mentions: We then asked whether endogenous TCTP would be able to form oligomers as predicted by our in vitro studies. As a result, we expressed a myc-tagged form of TCTP in CHO cells maintained in serum-free medium containing succinylacetone (SA), an inhibitor of the second enzyme (δ-aminolevulinic acid dehydratase) of the heme biosynthetic pathway. Treatment of CHO cells with SA led to a progressive decline in the endogenous heme concentration and, based on our model, should result in accumulation of monomeric TCTP. Extracts from SA-treated cells were then incubated with GST-bound TCTP to detect myc-TCTP binding in the presence of various ligand concentrations. Accordingly, GST-TCTP was only able to dimerize with endogenous myc-TCTP when the concentration of hemin added to the reaction surpassed the KD value by several fold (Figure 7). In agreement with our in vitro findings, Ca2+ alone did not promote TCTP dimerization and increasing concentration of this ligand competed off myc-TCTP bound to GST-TCTP in the presence of hemin. Overall, our results support a model where TCTP helps maintain cellular homeostasis by acting as a buffer molecule that sequesters the unwanted excess of ligand in a soluble oligomeric form.


Ligand binding reveals a role for heme in translationally-controlled tumor protein dimerization.

Lucas AT, Fu X, Liu J, Brannon MK, Yang J, Capelluto DG, Finkielstein CV - PLoS ONE (2014)

Cellular TCTP oligomerization is ligand-dependent.CHO cells were transfected with pCS2+myc-TCTP in serum-free medium containing 5 mM succinylacetone for 24 h prior to harvesting to prevent de novo synthesis of heme. Extracts were incubated with recombinant GST-TCTP bound beads in the absence or presence of hemin (1 µM, 100 µM, 1 mM), and/or CaCl2 (2.5 or 25 mM) and described in “Materials and Methods”. Bound complexes were resolved by SDS-PAGE and bound proteins detected by immunoblotting (upper panel). The expression of recombinant myc-TCTP in cells and GST-TCTP in the assay are shown in the middle and lower panels for each treatment, respectively. Molecular mass markers (in kDa) are indicated on the left.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4232476&req=5

pone-0112823-g007: Cellular TCTP oligomerization is ligand-dependent.CHO cells were transfected with pCS2+myc-TCTP in serum-free medium containing 5 mM succinylacetone for 24 h prior to harvesting to prevent de novo synthesis of heme. Extracts were incubated with recombinant GST-TCTP bound beads in the absence or presence of hemin (1 µM, 100 µM, 1 mM), and/or CaCl2 (2.5 or 25 mM) and described in “Materials and Methods”. Bound complexes were resolved by SDS-PAGE and bound proteins detected by immunoblotting (upper panel). The expression of recombinant myc-TCTP in cells and GST-TCTP in the assay are shown in the middle and lower panels for each treatment, respectively. Molecular mass markers (in kDa) are indicated on the left.
Mentions: We then asked whether endogenous TCTP would be able to form oligomers as predicted by our in vitro studies. As a result, we expressed a myc-tagged form of TCTP in CHO cells maintained in serum-free medium containing succinylacetone (SA), an inhibitor of the second enzyme (δ-aminolevulinic acid dehydratase) of the heme biosynthetic pathway. Treatment of CHO cells with SA led to a progressive decline in the endogenous heme concentration and, based on our model, should result in accumulation of monomeric TCTP. Extracts from SA-treated cells were then incubated with GST-bound TCTP to detect myc-TCTP binding in the presence of various ligand concentrations. Accordingly, GST-TCTP was only able to dimerize with endogenous myc-TCTP when the concentration of hemin added to the reaction surpassed the KD value by several fold (Figure 7). In agreement with our in vitro findings, Ca2+ alone did not promote TCTP dimerization and increasing concentration of this ligand competed off myc-TCTP bound to GST-TCTP in the presence of hemin. Overall, our results support a model where TCTP helps maintain cellular homeostasis by acting as a buffer molecule that sequesters the unwanted excess of ligand in a soluble oligomeric form.

Bottom Line: Mutation in both His residues to Ala prevents hemin from binding and abrogates oligomerization, suggesting that the ligand site localizes at the interface of the oligomer.Unlike heme, binding of Ca2+ ligand to TCTP does not alter its monomeric state; although, Ca2+ is able to destabilize an existing TCTP dimer created by hemin addition.In agreement with TCTP's proposed buffer function, ligand binding occurs at high concentration, allowing the "buffer" condition to be dissociated from TCTP's role as a component of signal transduction mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Integrated Cellular Responses Laboratory, Virginia Bioinformatics Institute, Department of Biological Sciences, Virginia Tech, Blacksburg, Virginia, United States of America; Protein Signaling Domains Laboratory, Virginia Bioinformatics Institute, Department of Biological Sciences, Virginia Tech, Blacksburg, Virginia, United States of America.

ABSTRACT
The translationally-controlled tumor protein (TCTP) is a highly conserved, ubiquitously expressed, abundant protein that is broadly distributed among eukaryotes. Its biological function spans numerous cellular processes ranging from regulation of the cell cycle and microtubule stabilization to cell growth, transformation, and death processes. In this work, we propose a new function for TCTP as a "buffer protein" controlling cellular homeostasis. We demonstrate that binding of hemin to TCTP is mediated by a conserved His-containing motif (His76His77) followed by dimerization, an event that involves ligand-mediated conformational changes and that is necessary to trigger TCTP's cytokine-like activity. Mutation in both His residues to Ala prevents hemin from binding and abrogates oligomerization, suggesting that the ligand site localizes at the interface of the oligomer. Unlike heme, binding of Ca2+ ligand to TCTP does not alter its monomeric state; although, Ca2+ is able to destabilize an existing TCTP dimer created by hemin addition. In agreement with TCTP's proposed buffer function, ligand binding occurs at high concentration, allowing the "buffer" condition to be dissociated from TCTP's role as a component of signal transduction mechanisms.

Show MeSH
Related in: MedlinePlus