Presented at the Graduate School of Biological Sciences and UNIST Graduate School in In addition, I confirmed that CypD has the ability to bind with mitochondrial Prxs and that the binding strength is different according to the existence of ROS, in vitro condition.
1. Introduction
Prxs, the family of the hydrogen peroxide scavenging proteins are abundant in the various locations of the cell (Wood, Schroder, Harris, & Poole, 2003). Through these studies, I clarify the binding capacity between CypD and mitochondrial Prxs and propose the model for their mechanism.
2. Material & Method
2-1. Preparation of recombinant human mitochondrial CypD/PrxIII/V
Beads attached to recombinant His tag proteins were washed with washing buffer (50mM NaH2PO4, 300mM NaCl, 50mM Imidazole). Elution buffer (50mM NaH2PO4, 300mM NaCl and 250mM Imidazole) was added to the column and allowed to elute the proteins by gravity flow. Binding of recombinant CypD to PrxIII and PrxV was confirmed through immunoblotting results (Figure 7A,B). The CypD storage buffer was changed to MES buffer pH6.5 and loaded onto the SP HP cation exchange column ( Figure 8A ).
The buffer of CypD was changed to MES pH 6.5, NaCl 50 mM for cation exchange chromatography. After incubation, CypD was inserted into the syringe and PrxV was loaded into the ITC cell. Interestingly, the binding of CypD with PrxV was confirmed (Figure 9A), but the samples mixed with hydrogen peroxide show the unstable bonds (Figure 9B,C).
High-resolution kinetic analysis of CypD binding to the PrxV surface using the Biacore 3000 biosensor. If CypD function is affected by PrxV through direct binding, PrxV maintenance may be important to regulate mitochondrial membrane potential. Other proteins, peripheral benzodiazepine receptor (PBR), hexokinase (HK), and creatine kinase (CK) are now known as members of the MPTPC ( Song et al., 2011 ). The causes of the creation of MPTPC are calcium density, cell death signals and ROS, etc.
2-2. RNA interfearence
2-3. Protein purification
Purification of GST-tagged proteins: GST-tagged CypD and PrxIII/V proteins (pGEX-4T-1) were overexpressed from Escherichia coli BL21/DE3 cells grown in Luria Broth (LB) medium in the presence of ampicillin at 36 °C. The cell has reached OD. The recombinant protein was induced by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM for 24 h at 18 °C. Harvested cells were resuspended in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM MgCl2) with a final concentration of 1 mM DTT or 5 mM β-mercaptoethanol.
The sample supernatant was mixed with glutathione-sepharose beads (GE Healthcare) for 4 h at 4°C. Beads bound to recombinant GST-tagged proteins were washed with washing buffer (50mM Tris-HCl, pH 7.4, 500mM NaCl, 5mM MgCl2). When the cell reached OD, the recombinant protein was induced by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM for 24 h at 18°C.
The harvested cell was resuspended in lysis buffer (50 mM NaH PO , 300 mM NaCl, 20 mM imidazole) with a final concentration of 1 mM DTT or 5 mM β-mercaptoethanol.
2-4. Pull down assay
CypD overexpression in the bacterial system was good and stably expressed in solution (Figure 2A). From MALD-TOF MS results, ANT and Hsp90 already known as the partner of CypD and novel one, PrxV were established (Figure 3B). In mitochondria, not only PrxV is the candidate CypD partner protein, but PrxIII also exists.
Since PrxIII also exists in mitochondria, I compared the peptide sequence of PrxIII and PrxV to assess the possibility of binding to CypD. Through alignment data, the core peptide part of PrxIII and PrxV shows a small degree of sequence similarity (A), but the secondary structures there are very similar (B). Since wild-type PrxIII and PrxV have the mitochondrial targeting sequence, the forward primer of PrxIII and PrxV was designed near the targeting sequence ( Figure 5B ).
The PrxIII and PrxV genes were derived from the MDA-MB-231 breast cancer cell line. Due to the deletion of the mitochondrial target sequence, the size of mitochondrial PrxIII and PrxV is smaller than wild-type PrxIII and PrxV. CypD and PrxV storage buffer purified by GST affinity chromatography was modified using a HiTrap desalting column and further purified by ion exchange chromatography.
The purified CypD (30 µg/µl) was injected into the syringe and PrxV (3 µg/µl) was loaded into the ITC cell. Through ITC, I confirm that the binding capacity of PrxV with CypD was increased on the status of oxidation environment.
2-5. Thrombin remobval with pAminobenzamidine-Agarose
2-6. Mitochondria isolation from brain
2-7. Cell culture
2-8. Cell staining using Mitotracker and TMRM
On the other hand, the positive protein bands of binding fraction samples eluted using 10 M urea were on the mixed CypD sample and not on the control sample. Since PrxIII and PrxV have the same functional capacity and similar other structures, PrxIII can also bind to CypD. PrxIII and PrxV genes were extracted from MDA-MB-231, a human breast cancer cell line, and inserted into pGEX-4T1 GST expression vectors (Figure 5A).
Coomassie staining results show that equal amounts of GST and target protein-binding beads are loaded. It was confirmed by confocal microscopy that the membrane potential of the PrxV knockdown cell was reduced (Figure 10A, B and C). After a 15-minute treatment with hydrogen peroxide (50 uM), the rate of decline in the membrane potential of the PrxV knockdown H9C2 cell was faster than that of the negative control cell line.
Mitochondrial ROS levels were not affected by PrxV knockdown according to FACS data. In this study, PrxV was found to associate with CypD using MALDI TOF MS spectrometry analysis. The point is that the cause of the membrane potential drop is not caused by ROS.
I hypothesize that one of the mechanisms to resist high levels of ROS disease in tumors is to overexpress PrxV.
2-9. Immonoblotting
2-10. FPLC
Using HiTrap Q Desalting Column (GE Healthcare), the buffer in which the PrxV was stored (50mM Tris-HCl, pH 7.4, 500mM NaCl, and 5mM MgCl2) was changed to Tris buffer A. The PrxV stored with buffer A was to HiTrap loaded Q anion exchange chromatography column (GE healthcare) using FPLC (AkTa FPLC, GE healthcare).
2-11. Isothermal Titration Calorimetry (ITC)
A diverse type of Prxs family has a reactive Cys and a conserved Cys except for PrxVI, which is not yet precisely known. The number of functional subunits of Prxs is monomer or dimer, but diverse binding formation can exist depending on the environmental conditions. This causes the reactive Cys on one peptide to meet with the conserved Cys on the other peptide.
Because of this, the oxidation of reactive Cys is reduced spontaneously through its peptide bond. When reactive Cys meets ROS, it changes their formation to oxidation and produces hydrogen monoxide. After the reaction between the reactive Cys and the conserved Cys, it forms disulfide bonds between these two Cys.
The disulfide bond is reduced by an electron donor such as thioredoxin or glutathione, which vary according to the type of Prxs.
3. Results
3-1. The preparation of screening
3-2. Screening of CypD partner proteins
But the parts of similar sites exist and the secondary structures of the central active Cys group are very similar (Figure 4B). When the purified CypD sample was mixed with the small amount of PrxIII-binding bead, the CypD clearly bound to PrxIII and not to the GST control bead (Figure 6B). Isothermal titration calorimetry (ITC), which confirms binding capacity by measuring the change in thermodynamics, was necessary to verify the interactions between CypD and PrxV under different conditions.
The buffer of PrxV was changed to Tris pH 8.0, NaCl 50 mM and loaded onto MonoQ anion exchange column. Maybe cause the difference in reaction buffer and status of PrxV according to the experimental method (solution method: ITC / Binding method: SPR). The ROI value of the fluorescence regions was adjusted using the ROI value of the dark regions.
The opening of the PT pore allows an influx of ions and water into the mitochondrial matrix, causing mitochondrial swelling (Henry-Mowatt, Dive, Martinou, & James, 2004). It was confirmed by FACS data and I thought that compensatory effects against knock down of PrxV arose. In tumors it is already known that the level of ROS is higher than in normal cells and some types of tumors have the highest amount of PrxIII and V.
3-3. The PrxIII has the possibility of binding capacity with CypD
3-4. In vitro pull down assay of CypD with PrxIII and PrxV
3-5. Purification of proteins using FPLC
The nature of CypD, which readily aggregates under acidic conditions and acquires weak ionic charges under alkaline conditions, makes it difficult to determine the ion exchange state. The CypD in the MES pH6.5 buffer showed minimal aggregation and fine-binding capacity with cation-ion exchange chromatography resins. PrxV prepurified through GST affinity chromatography was finally purified using MonoQ anion exchange chromatography column.
The best conditions to obtain the highly purified PrxV was the unconfined protein state using the MonoQ anion exchange chromatography column with the buffer, Tris pH 8.0, NaCl 50 mM. The recombinant proteins that were primarily purified using glutathione-sepharose beads were secondary purified by ion exchange chromatography. The buffer-altered CypD was loaded onto an SP HP column and eluted with linear gradients of NaCl.
Other peaks in the linear gradient region of the MonoQ anion exchange column are non-specific binding proteins.
3-6. Isothermal Titration Calorimetry (ITC)
Since dithiothreitol (DTT) has an effect on ITC results, the reducing agent for purification of these proteins was beta-mercaptoethanol and not DTT.
3-7. Surface plasmon resonance (SPR)
3-8. The knockdown of PrxV affect to the mitochondrial membrane potential
The PrxV-silenced H9C2 cell was stained with Mito-Sox for 30 minutes and analyzed via FACS.
4. Discussion
Because PrxV normally inhibits CypD by direct but reversible binding, MPTP is stably regulated. If a large generation of ROS is induced, PrxV is oxidized and changes their conformation. In many cases, various types of tumors overexpress mitochondrial Prx and generate high levels of ROS in mitochondria.
Although the tumors are at the high level of ROS status, the MPTP opening is not induced. Characterization of three isoforms of mammalian peroxiredoxin that reduce peroxides in the presence of thioredoxin and their role in signal transduction. Cyclosporin A binding to mitochondrial cyclophilin inhibits the permeability transition pore and protects hearts from ischemia/reperfusion injury.
