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A reducing milieu renders cofilin insensitive to phosphatidylinositol 4,5-bisphosphate (PIP2) inhibition.

Schulte B, John I, Simon B, Brockmann C, Oelmeier SA, Jahraus B, Kirchgessner H, Riplinger S, Carlomagno T, Wabnitz GH, Samstag Y - J. Biol. Chem. (2013)

Bottom Line: However, the reducing effects on proteins leading to restored T cell activation remained unknown.Accordingly, reduction of cofilin had no effect on F-actin binding and depolymerization and did not influence the cofilin phosphorylation state.Altogether, we introduce a novel mechanism of redox regulation: reduction of the actin-remodeling protein cofilin renders it insensitive to PIP2 inhibition, resulting in enhanced actin dynamics.

View Article: PubMed Central - PubMed

Affiliation: From the Institute for Immunology, Ruprecht Karls University, D-69120 Heidelberg, Germany.

ABSTRACT
Oxidative stress can lead to T cell hyporesponsiveness. A reducing micromilieu (e.g. provided by dendritic cells) can rescue T cells from such oxidant-induced dysfunction. However, the reducing effects on proteins leading to restored T cell activation remained unknown. One key molecule of T cell activation is the actin-remodeling protein cofilin, which is dephosphorylated on serine 3 upon T cell costimulation and has an essential role in formation of mature immune synapses between T cells and antigen-presenting cells. Cofilin is spatiotemporally regulated; at the plasma membrane, it can be inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2). Here, we show by NMR spectroscopy that a reducing milieu led to structural changes in the cofilin molecule predominantly located on the protein surface. They overlapped with the PIP2- but not actin-binding sites. Accordingly, reduction of cofilin had no effect on F-actin binding and depolymerization and did not influence the cofilin phosphorylation state. However, it did prevent inhibition of cofilin activity through PIP2. Therefore, a reducing milieu may generate an additional pool of active cofilin at the plasma membrane. Consistently, in-flow microscopy revealed increased actin dynamics in the immune synapse of untransformed human T cells under reducing conditions. Altogether, we introduce a novel mechanism of redox regulation: reduction of the actin-remodeling protein cofilin renders it insensitive to PIP2 inhibition, resulting in enhanced actin dynamics.

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Analysis of the cofilin redox state and its influence on cofilin structure.A, mass spectrometry analysis of recombinant WT cofilin purified under nonreducing conditions. The His-tagged untreated molecule (left spectrum) shows a main mass peak at 21,831 atomic mass units (amu). Treatment with IAA (right spectrum) resulted in an increase in size by 114 atomic mass units (to 21,945 atomic mass units), corresponding to the addition of two IAA molecules. The accuracy of the spectrometer was ±1 atomic mass unit. B, Western blot of Mal-PEG cysteine modification assay performed with PBT lysate with and without reducing treatment. The membrane was probed for total cofilin; note that although the same amount of lysate was loaded on each lane, antibody detection decreased in Mal-PEGylated samples, probably due to epitope masking by Mal-PEG molecules. M, molecular mass markers shown in kilodaltons. C, the 15N-1H heteronuclear single quantum coherence spectrum of untreated cofilin (black) is overlaid with that of DTT-treated cofilin (red). Amino acids that exhibit distinct CSPs (>0.1 ppm) are labeled with their amino acid position. D, all CSPs occurring upon cofilin reduction. Again, all CSPs > 0.1 ppm are labeled; additionally, the four cysteines are marked in red. CSPs involving the complete disappearance of an amino acid peak in one of the spectra are marked by a negative bar. E, a three-dimensional rendering of the molecular surface of cofilin (PDB ID 1Q8G), with CSPs from the WT with/without DTT comparison colored red. Four views from around the molecule are shown by a perspective shift of 90° from one image to the next.
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Figure 1: Analysis of the cofilin redox state and its influence on cofilin structure.A, mass spectrometry analysis of recombinant WT cofilin purified under nonreducing conditions. The His-tagged untreated molecule (left spectrum) shows a main mass peak at 21,831 atomic mass units (amu). Treatment with IAA (right spectrum) resulted in an increase in size by 114 atomic mass units (to 21,945 atomic mass units), corresponding to the addition of two IAA molecules. The accuracy of the spectrometer was ±1 atomic mass unit. B, Western blot of Mal-PEG cysteine modification assay performed with PBT lysate with and without reducing treatment. The membrane was probed for total cofilin; note that although the same amount of lysate was loaded on each lane, antibody detection decreased in Mal-PEGylated samples, probably due to epitope masking by Mal-PEG molecules. M, molecular mass markers shown in kilodaltons. C, the 15N-1H heteronuclear single quantum coherence spectrum of untreated cofilin (black) is overlaid with that of DTT-treated cofilin (red). Amino acids that exhibit distinct CSPs (>0.1 ppm) are labeled with their amino acid position. D, all CSPs occurring upon cofilin reduction. Again, all CSPs > 0.1 ppm are labeled; additionally, the four cysteines are marked in red. CSPs involving the complete disappearance of an amino acid peak in one of the spectra are marked by a negative bar. E, a three-dimensional rendering of the molecular surface of cofilin (PDB ID 1Q8G), with CSPs from the WT with/without DTT comparison colored red. Four views from around the molecule are shown by a perspective shift of 90° from one image to the next.

Mentions: Cofilin contains four cysteines (at positions 39, 80, 139, and 147) and has been reported to be highly sensitive to oxidation and to form an intramolecular disulfide bridge under oxidative conditions. However, these observations were made on the basis of recombinant cofilin purified under reducing conditions. Therefore, here, we ask whether cofilin in its native state, purified under nonreducing conditions, already exists in an oxidized state. Thus, we analyzed the tagging of free cysteines with the alkylating agent IAA by mass spectrometry. IAA-treated recombinant cofilin showed an increase in mass of 114.4 (±1) atomic mass units (Fig. 1A), corresponding to the addition of two IAA molecules to cysteine side chains. This implies that two cysteines in cofilin are occupied and therefore could take part in a disulfide bridge.


A reducing milieu renders cofilin insensitive to phosphatidylinositol 4,5-bisphosphate (PIP2) inhibition.

Schulte B, John I, Simon B, Brockmann C, Oelmeier SA, Jahraus B, Kirchgessner H, Riplinger S, Carlomagno T, Wabnitz GH, Samstag Y - J. Biol. Chem. (2013)

Analysis of the cofilin redox state and its influence on cofilin structure.A, mass spectrometry analysis of recombinant WT cofilin purified under nonreducing conditions. The His-tagged untreated molecule (left spectrum) shows a main mass peak at 21,831 atomic mass units (amu). Treatment with IAA (right spectrum) resulted in an increase in size by 114 atomic mass units (to 21,945 atomic mass units), corresponding to the addition of two IAA molecules. The accuracy of the spectrometer was ±1 atomic mass unit. B, Western blot of Mal-PEG cysteine modification assay performed with PBT lysate with and without reducing treatment. The membrane was probed for total cofilin; note that although the same amount of lysate was loaded on each lane, antibody detection decreased in Mal-PEGylated samples, probably due to epitope masking by Mal-PEG molecules. M, molecular mass markers shown in kilodaltons. C, the 15N-1H heteronuclear single quantum coherence spectrum of untreated cofilin (black) is overlaid with that of DTT-treated cofilin (red). Amino acids that exhibit distinct CSPs (>0.1 ppm) are labeled with their amino acid position. D, all CSPs occurring upon cofilin reduction. Again, all CSPs > 0.1 ppm are labeled; additionally, the four cysteines are marked in red. CSPs involving the complete disappearance of an amino acid peak in one of the spectra are marked by a negative bar. E, a three-dimensional rendering of the molecular surface of cofilin (PDB ID 1Q8G), with CSPs from the WT with/without DTT comparison colored red. Four views from around the molecule are shown by a perspective shift of 90° from one image to the next.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 1: Analysis of the cofilin redox state and its influence on cofilin structure.A, mass spectrometry analysis of recombinant WT cofilin purified under nonreducing conditions. The His-tagged untreated molecule (left spectrum) shows a main mass peak at 21,831 atomic mass units (amu). Treatment with IAA (right spectrum) resulted in an increase in size by 114 atomic mass units (to 21,945 atomic mass units), corresponding to the addition of two IAA molecules. The accuracy of the spectrometer was ±1 atomic mass unit. B, Western blot of Mal-PEG cysteine modification assay performed with PBT lysate with and without reducing treatment. The membrane was probed for total cofilin; note that although the same amount of lysate was loaded on each lane, antibody detection decreased in Mal-PEGylated samples, probably due to epitope masking by Mal-PEG molecules. M, molecular mass markers shown in kilodaltons. C, the 15N-1H heteronuclear single quantum coherence spectrum of untreated cofilin (black) is overlaid with that of DTT-treated cofilin (red). Amino acids that exhibit distinct CSPs (>0.1 ppm) are labeled with their amino acid position. D, all CSPs occurring upon cofilin reduction. Again, all CSPs > 0.1 ppm are labeled; additionally, the four cysteines are marked in red. CSPs involving the complete disappearance of an amino acid peak in one of the spectra are marked by a negative bar. E, a three-dimensional rendering of the molecular surface of cofilin (PDB ID 1Q8G), with CSPs from the WT with/without DTT comparison colored red. Four views from around the molecule are shown by a perspective shift of 90° from one image to the next.
Mentions: Cofilin contains four cysteines (at positions 39, 80, 139, and 147) and has been reported to be highly sensitive to oxidation and to form an intramolecular disulfide bridge under oxidative conditions. However, these observations were made on the basis of recombinant cofilin purified under reducing conditions. Therefore, here, we ask whether cofilin in its native state, purified under nonreducing conditions, already exists in an oxidized state. Thus, we analyzed the tagging of free cysteines with the alkylating agent IAA by mass spectrometry. IAA-treated recombinant cofilin showed an increase in mass of 114.4 (±1) atomic mass units (Fig. 1A), corresponding to the addition of two IAA molecules to cysteine side chains. This implies that two cysteines in cofilin are occupied and therefore could take part in a disulfide bridge.

Bottom Line: However, the reducing effects on proteins leading to restored T cell activation remained unknown.Accordingly, reduction of cofilin had no effect on F-actin binding and depolymerization and did not influence the cofilin phosphorylation state.Altogether, we introduce a novel mechanism of redox regulation: reduction of the actin-remodeling protein cofilin renders it insensitive to PIP2 inhibition, resulting in enhanced actin dynamics.

View Article: PubMed Central - PubMed

Affiliation: From the Institute for Immunology, Ruprecht Karls University, D-69120 Heidelberg, Germany.

ABSTRACT
Oxidative stress can lead to T cell hyporesponsiveness. A reducing micromilieu (e.g. provided by dendritic cells) can rescue T cells from such oxidant-induced dysfunction. However, the reducing effects on proteins leading to restored T cell activation remained unknown. One key molecule of T cell activation is the actin-remodeling protein cofilin, which is dephosphorylated on serine 3 upon T cell costimulation and has an essential role in formation of mature immune synapses between T cells and antigen-presenting cells. Cofilin is spatiotemporally regulated; at the plasma membrane, it can be inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2). Here, we show by NMR spectroscopy that a reducing milieu led to structural changes in the cofilin molecule predominantly located on the protein surface. They overlapped with the PIP2- but not actin-binding sites. Accordingly, reduction of cofilin had no effect on F-actin binding and depolymerization and did not influence the cofilin phosphorylation state. However, it did prevent inhibition of cofilin activity through PIP2. Therefore, a reducing milieu may generate an additional pool of active cofilin at the plasma membrane. Consistently, in-flow microscopy revealed increased actin dynamics in the immune synapse of untransformed human T cells under reducing conditions. Altogether, we introduce a novel mechanism of redox regulation: reduction of the actin-remodeling protein cofilin renders it insensitive to PIP2 inhibition, resulting in enhanced actin dynamics.

Show MeSH