Increased Mitochondria Mass In Aoa3 Atl2abr Cells Biology Essay

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The results in 2.3.2.1, revealed an increase in fluorescence intensity in the patient cells. To determine whether this was due to an increase in the mitochondrial mass, a specific dye, Nonyl Acridine Orange (NAO) was employed. The results in Figure 2.4 A) show a shift in NAO fluorescence intensity in AOA3 (ATL2ABR) cells compared to controls. Remarkably, a similar intensity shift was also observed with the cells from the father (ATLH3ABR) in the experiment illustrated in Figure 2.3 A). Quantification of the shift over repeat experiments demonstrated a consistent and fluorescence shift for AOA3 (ATL2ABR) cells compared to the other cells types (Figure 2.3B). In contrast, a greater variation in the fluorescence intensity shift was observed in the father's, P<0.05 (ATLH3ABR) cells over repeated experiments, as shown in Figure 2.4 B) Altogether, the data point to an increased mitochondrial mass in the AOA3 patient cells.

NAO FINAL

Figure 2.4 Increased mitochondrial mass in AOA3 (ATL2ABR). A. FACS profile of AOA3 (ATL2ABR), controls (C2ABR and C3ABR), mother (ATLH4ABR) and father (ATLH3ABR) after NAO and propidium iodide (PI) staining. Cells were washed, stained with NAO for 30 minutes, washed again, resuspended in 300 µl PBS and PI was added. NAO fluorescence was measured using flowcytometry (FACS Calibur, BD Biosciences, USA). B. NAO fluorescence intensity in all cell lines was obtained from 3 independent experiments (n=3). *,**, ***, p < 0.05, $ = 0.1.

2.3.3 Mitochondrial functions in AOA3 (ATL2ABR) cells

2.3.3.1 Defective mitochondrial membrane depolarization after DNA damage in AOA3

(ATL2ABR) cells

Gueven and collegues have previously shown that cells from the patient are resistant to apoptosis (Gueven et al., 2007). In addition, there was no change in the mitochondrial outer membrane potential (MMP) in AOA3 (ATL2ABR) cells after DNA damage treatment, suggesting that this could be the cause of the apoptosis resistance observed in these cells. Mitochondrial membrane depolarization is one of the early events in cellular apoptosis (Waterhouse et al., 2001). Depolarization of the mitochondrial membrane results in the release of cytochrome C from the mitochondria into the cytosol and apoptosis inducing factor (AIF) from the mitochondria into the nucleus (Waterhouse et al., 2001). These, subsequently led to the formation of apoptosome and finally apoptosis induction through caspases activation (Waterhouse et al., 2001). Mitochondrial membrane depolarization was measured using JC-1 dye, a membrane permeable dye (Molecular Probes, Invitrogen). JC-1 floresces red (aggregate form) when excited at 488 nm and upon membrane depolarization, the emmited light shifts from 530nm to 590 nm where the JC-1 flouresces green (monomeric form). Figure 2.5 shows the MMP in AOA3 (PSF) compared to control (NFF) at basal levels. In total, 50 cells were counted and the fluorescence insensity was observed. JC-1 dye stained the mitochondria red (aggregate form) in almost half of the control cells, suggesting intact MMP in these cells. In contrast, almost all AOA3 (PSF) cells have lost their MMP where the cells were stained green or orange, indicateing a defective mitochondrial function in AOA3 cells. To further confirm the defect in mitochondrial membrane depolarization, a quantitative analysis was performed using JC-1 dye and flow cytometry analysis.

Figure 2.5 Mitochondrial membrane depolarization defect in AOA3 (PSF) using JC-1 dye and immunofluorescence. Cells were treated JC-1 dye and images were captured using a fluorescent microscope (Axioskop 2Mot plus and Plan Approchromat 63x/1.4 Oil, Zeiss, North Ryde, Australia). AOA3 (PSF) cells display loss of mitochondrial membrane potential compared to control. Scale bar: 20µm

2.3.3.2 Low mitochondrial membrane potential in AOA3 (ATL2ABR) cells

To investigate this in more detail, a quantitative analysis of MMP using JC-1 dye and flow cytometry analysis was performed. Normal mitochondrial membrane potential is shown by a decreased percentage (%) of the monomer form of the JC-1 dye. It is clear that AOA3 (ATL2ABR) cells have increased levels of JC-1 dye monomer form at basal levels (Figure 2.6A). This data suggests that AOA3 (ATL2ABR) have low mitochondrial membrane potential compared to controls (C2ABR and C3ABR) and the parent cells [mother (ATLH4ABR) and father (ATLH3ABR)]. When cells are exposed to the oxidative phosphorylation uncoupler, CCCP, there is a dramatic collapse in the membrane potential in all cells (Figure 2.6A). The quantitative approach described here revealed that MMP is already collapsed in untreated AOA3 (ATL2ABR) cells, in agreement with the qualitative approach as shown in figure 2.5. This result is consistent with an abnormality at the level of mitochondria in ATL2ABR (AOA3) cells.

MMP

Figure 2.6 Low mitochondrial membrane potential in AOA3 (ATL2ABR) cells. Cells were incubated with 2.5µg/ml JC-1 dye for 30 minutes, washed, resuspended in 300µl of PBS and analyzed using low cytometry (FACS Calibur, Beckman Coulter, CA, USA). 200µM of CCCP was used as a positive control to collapse the mitochondrial membrane potential. A. FACS profiles of JC-1 dye in AOA3 (ATL2ABR), controls (C2ABR and C3ABR), mother (ATLH4ABR) and father (ATL3ABR). Aggregates and monomer form of the JC-1 dye were shown in the upper and lower right quadrant respectively. For positive control, cells were incubated simultaneously with 2.5µg/ml JC-1 dye and 200 µM CCCP which lead to decreased MMP in all cell lines. B. Decreased MMP in AOA3 (ATL2ABR) compared to controls (C2ABR and C3ABR), mother (ATLH4ABR) and father (ATL3ABR). C. Schematic of the inhibition of the proton pumping activity by CCCP. CCCP disconnects the ETC from the production of ATP and thus leading to the collapse of the mitochondrial membrane potential.

2.3.3.3 Defective DNA damage-induced apoptosis in AOA3 (ATL2ABR) cells

MMP is an important event in the DNA damage response to ensure apoptosis occurs through the mitochondrial-dependant apoptosis pathway (intrinsic pathway) (Waterhouse et al., 2001). In this intrinsic pathway, pro-apoptotic signals such as the Bcl-2 family proteins (Bax and Bak) are activated, resulting in the release of apoptosis-inducing factor (AIF) from the mitochondria to the nucleus and translocation of cytochrome C (cyto-C) from the mitochondria to the cytoplasm (Waterhouse et al., 2001). Subsequently, the caspase-activating complex or apoptosome which is composed of cyt-C, Apaf-1, dATP and procaspase-9 is formed (Zou et al., 1999). Formation of the apoptosome results in the activation of the effector caspases which are required for most of the biochemical and morphological changes during apoptosis. Caspases or cysteine-aspartic proteases are a family of cysteine proteases which are involved in apoptosis, necrosis and inflammation (Danial & Korsmeyer, 2004). Caspases are regulated at the post-translational levels to ensure rapid activation (Danial & Korsmeyer, 2004). Caspases can activate the effector caspases that include granzyme B, death receptors and apoptosome which activates caspase-9 for apoptosis (Danial & Korsmeyer, 2004). Some final targets of caspases include nuclear lamins, inhibitor of caspase activated DNase or DNA fragmentation factor 45 (ICAD/DFF45), poly-ADP-ribose polymerase (PARP-1) and P21-activated kinase 2 (PAK2) (Danial & Korsmeyer, 2004). PARP-1 initiates a nuclear signal to the mitochondria and triggers the release of AIF, subsequently shuttles to the nucleus leading to the induction of chromatin condensation and DNA fragmentation, and ultimately apoptosis (Hong et al., 2004). During apoptosis, PARP-1 is cleaved from a 118 kDA protein to a smaller fragment at approximately 98 kDA. To investigate this further, a western blot analysis was performed with PARP-1 antibody to determine the DNA-damage-induced apoptosis in AOA3 (ATL2ABR) cells. Since AOA3 cells show increased sensitivity to ionizing radiation (IR) this agent was chosen (Gueven et al., 2007). A decreased intensity of the PARP-1 cleavage band at 98 kDa confirmed that AOA3 (ATL2ABR) cells were defective in IR-induced apoptosis compared to the control, father and mother cells (Figure 2.7). This is in agreement with previous findings (Gueven et al., 2007) and with the low mitochondrial membrane potential observed in AOA3 (ATL2ABR) compared to controls (C2ABR and C3ABR), father (ATLH3ABR) and mother (ATLH4ABR) cells.

PARP1

Figure 2.7 Defect in DNA damage-induced apoptosis in AOA3 (ATL2ABR) cells. PARP-1 cleavage was used as a readout of apoptosis. Briefly, cells were irradiated with 10Gy, and harvested at 8 hours post- irradiation. Total protein extracts were prepared and immunoblotting was performed using 50µg of total proteins and electrophoresed on 8% SDS-PAGE. β-actin was used as a loading control. AOA3 (ATL2ABR) cells are defective in DNA damage-induced apoptosis as indicated by an absence or reduced extent of PARP-1 cleavage product at 98kDa.

2.3.3.4 Decreased mitochondrial respiration in AOA3 (ATL2ABR) cells

The accumulation of evidence for mitochondrial dysfunction would predict a defect in mitochondrial respiration in the AOA3 patient cells. To address this, mitochondrial respiration was measured by the extent of reduction of the fluorescent dye, resazurin (Ambrose et al., 2007). As shown in Figure 2.8A the extent of mitochondrial respiration is significantly higher in the control (C2ABR) compared to that in AOA3 (ATL2ABR) cells. Cells from the patient's mother (ATLH4ABR) are similar to that of controls while there appears to be a decreased level of respiration in the father (ATLH3ABR) cells. As a positive control of inhibition of mitochondrial repiration, the cells were incubated with the respiratory chain complex inhibitor, amiodarone (Figure 2.8B). Amiodarone is an amphiphilic drug with a highly lipophilic diiodobenzoylbenzofuran moiety and a partly protonated dietylaminoethoxy side chain which has been used as antiarrythmatic agent (Fromenty et al., 2009). It serves as a mitochondrial toxin which has an uncoupling effect on oxidative phosphorylation (OXPHOS) through Ca2+ influx which leads to mitochondrial fragmentation (Fromenty et al., 2009). Amiodarone inhibits ETC in isolated mice liver mitochondria (Spaniol et al., 2001). As expected, a decrease in mitochondrial respiration to approximately the same extent in all cells was observed (Figure 2.8A). Decreased mitochondrial respiration is in agreement with defective mitochondrial functions in AOA3 cells as observed previously (Gueven et al., 2007).

Resazurin final

Figure 2.8 A.Decreased mitochondrial respiration in AOA3 (ATL2ABR) by resazurin assay. In vivo mitochondrial respiration in the AOA3 (ATL2ABR) cells compared to control (C2ABR) and mother (ATLH4ABR). Interestingly, the father (ATLH3ABR) cells showed an intermediate decrease in mitochondrial respiration. B. Amiodarone an antiarythmic agents, is an amphiphilic drug with a highly lipophilic diiodobenzoylbenzofuran moiety and a partly protonated dietylaminoethoxy side chain. It was used as a positive control. Amiodarone inhibit the ETC via Ca2+ influx through the mitochondrial membrane which subsequently led to mitochondrial fragmentation (Fromenty et al., 2009). Treatment with 100µM amiodarone was used as a positive control and resulted in decreased cell metabolic activity in all cell lines.

2.3.3.5 Normal mitochondrial supercomplex formation in AOA3 (ATL2ABR) cells

To determine whether the defect in respiration was due to abnormalities in the different mitochondrial electron transport complexes, blue native-polyacrylamide gel electrophoresis (BN-PAGE) was performed. BN-PAGE is the method of choice to study the individual respiratory complexes (Schagger & von Jagow, 1991). It can be used to visualize changes in the proteins of the mitochondria, for examples in the inner mitochondrial membrane or mitochondrial supercomplex (Bailey et al., 2008, Andringa et al., 2009). In this technique, the mitochondrial complexes are separated under non-denaturing condition for the first dimension, which is performed using an isoelectric focusing gel (IEF) to separate the proteins according to their isoelectric point (pI). This is followed by the second dimension via SDS-PAGE, where the proteins are resolved individually according to their molecular mass (Bailey et al., 2008). BN-PAGE did not reveal any abnormalities in various complexes in AOA3 (ATL2ABR) cells (Figure 2.9). It is notable that there is some variability in some of these complexes in the different controls. The data demonstrate that there are no gross changes to the mitochondrial electron transfer complexes structure and formation in AOA3, but do not reveal whether the activity of these complexes are compromised or not in the patient cells.

Figure 2.9 Normal mitochondrial supercomplex formation in AOA3 (ATL2ABR) cells. BN-PAGE analysis was used to compare the structure and formation of the OXPHOS respiratory complexes in AOA3 (ATL2ABR) cells as compared to control (C2ABR) cells. Variability in the mitochondrial supercomplex formation was seen in different type of control where C3ABR cells showed less in CI/CIII2/CIV, CI/CIII2, CII and CI/CIV. This data was provided by Dr. Matthew McKenzie from the Department of Biochemistry, LaTrobe University, Melbourne, Australia.BN PAGE

2.3.3.6 Normal mitochondrial complex activities in AOA3 (ATL2ABR) cells

The activities of the mitochondrial complexes were measured by our collaborator, Dr. David Thorburn at the Mitochondrial Research Laboratory, Murdoch Children's Research Institute (MCRI), Melbourne using their established protocols (Kirby et al., 2007). The mitochondrial complex activities were expressed as relative to a mitochondrial matrix enzyme, citrate synthase (CS). The results showed that there were slight increased in complex I as well as complex II in AOA3 cells. The activities of complex III and II +III were also decreased slightly in AOA3 cells compared to controls. However, these changes are not statistically significant. This is in agreement with the normal mitochondrial supercomplex formation in AOA3 compared to controls. Table 2.6 and Figure 2.10 showed normal mitochondrial complex activities in AOA3 cells comparable to control cell lines.

Table 2.6 Mitochondrial complex activities in AOA3 (ATL2ABR) cells

Lymphoblasts

I/CS

II/CS

III/CS

II+III/CS

IV/CS

Control 1

452

659

230

586

16.5

Control 2

445

831

183

537

11.4

AOA3

537

847

118

462

12.5

complex activities.tif

Figure 2.10 Normal mitochondrial complex activities in AOA3 (ATL2ABR). AOA3 cells displayed normal mitochondrial complexes activities compared to controls which are in agreement with the normal formation of mitochondrial supercomplex.

2.3.4 Oxidative stress in AOA3 (ATL2ABR) cells

2.3.4.1 Oxidative protein damage (Nitrotyrosine), lipid peroxidation (4-HNE) and oxidative DNA damage (8-oxo-dG) in AOA3

Oxidative stress has been associated with mitochondrial dysfunction in neurodegenerative diseases (Trushina & McMurray, 2007). To investigate whether the mitochondrial dysfunction observed in AOA3 (ATL2ABR) cells may be due to increased levels of oxidative stress, immunofluoresence with oxidative stress markers that include oxidative protein damage (nitrotyrosine), oxidative DNA damage (8-oxo-dG) and lipid peroxidation (4-HNE) was performed. AOA3 (PSF) and the mother fibroblasts (ATLH4ABRF) showed an increase in all oxidative stress markers compared to control. Figure 2.11 shows increased oxidative protein damage in AOA3 and the mother fibroblasts compared to controls. AOA3 and the mother fibroblasts also showed high levels of lipid peroxidation compared to controls (Figure 2.12). Oxidative DNA damage was observed only in AOA3 and the mother cells (Figure 2.13A). For 8-oxo-dG, treatment of the cells with 2 mM H2O2 was used as a positive control. As expected, the addition of H2O2 resulted in increased oxidative DNA damage formation in the cells as shown by increased fluorescence intensity. The increase of oxidative DNA damage in AOA3 cells compared to controls and the parent's cells is represented in Figure 2.13B Fibroblasts from the patient's father (ATLH3ABR) exhibited a lack of oxidative stress as shown by the absence of oxidative protein damage, lipid peroxidation and oxidative DNA damage.

Figure 2.11 Increased oxidative protein damage (Nitrotyrosine) in AOA3 (PSF) cells. AOA3 (PSF) and fibroblasts from the mother (ATLH4ABRF) show increased oxidative protein damage. In contrast, the father's fibroblasts (ATLH3ABRF) show low levels of oxidative protein damage comparable to that of the control (NFF) cells. Immunofluorescence (IF) was performed using nitrotyrosine (no 9691, Cell Signaling, 1:200) and images were captured by fluorescent microscope (Axioskop 2Mot plus and Plan Approchromat 63x/1.4 Oil, Zeiss, North Ryde, Australia). The left panel indicates the nitrotyrosine only, the middle panel showed the nuclear staining with DAPI and the right panel is the merge images. Scale bar: 20µm

Figure 2.12 High levels of lipid peroxidation (4-HNE) in AOA3 (PSF) cells. Increased lipid peroxidation was observed in the AOA3 (PSF) and the fibroblast from the mother (ATLH4ABRF). On the other hand, lipid peroxidation was absent in the fathers fibroblasts (ATLH3ABRF) comparable to control (NFF). Immunoflourescence was performed using 4-HNE Micheal adducts (no 393207, Calbiochem, 1:200) and images were taken as described previously. Scale bar: 20µm

Figure 2.13A. Increased oxidative DNA damage (8-oxo-dG) in AOA3 (PSF) cells. Increased lipid peroxidation was observed in the AOA3 (PSF) and the mother's fibroblast (ATLH4ABRF). The fibroblast from the father (ATLH3ABRF) showed normal levels of oxidative DNA damage as in control (NFF). 8-oxo-dG (4355-MC100, Trevigen, 1:250) was used as an indicator for oxidative DNA damage and images were taken as described above. As expected, treatment with 2mM H2O2 led to a further increase in the oxidative DNA damage in all cell lines. Scale bar: 20µm

Figure 2.13B. Increased oxidative DNA damage in AOA3 (PSF) cells. AOA3 (PSF) and the mother's (ATLH4ABRF) fibroblasts showed an increase in 8-oxo-dG fluorescence intensity compared to the control (NFF) and the father (ATLH3ABR). Quantification of the fluorescence signal intensity was performed on the raw images using Image J version 1.34s software (NIH, USA).

2.3.5 Oxidative stress in mitochondrial proteins of AOA3 (ATL2ABR) cells

2.3.5.1 Mitochondrial protein isolation in control (C3ABR) and AOA3 (ATL2ABR)

Immunofluorescence results demonstrated the presence of oxidative stress in AOA3 (ATL2ABR) as revealed by oxidative protein damage, oxidative DNA damage and lipid peroxidation. Given that mitochondria are the major producer of ROS within the cells, and that AOA3 display mitochondrial dysfunction, the possible role of oxidative stress on mitochondrial proteins of AOA3 (ATL2ABR) was investigated, specifically whether the mitochondrial proteins of AOA3 were modified. Oxidative damage could be due to increased levels of ROS which is produced in the mitochondria. ROS are highly toxic molecules which can cause damage to macromolecules such as proteins, lipids and DNA (Sies, 1991). In addition, lipid peroxidation products such as 4-HNE can bind covalently to proteins through Michael addition (Sultana & Butterfield, 2009). These affect many cellular mechanisms including inhibition of DNA and protein synthesis, inhibition of Ca2+ homeostasis, membrane damage and apoptosis. Oxidative damage to proteins could also lead to structural changes of the protein which may affect the stability and ultimately the function of the protein (Sultana & Butterfield, 2009). For example, in AD, modification of aconitase and ATP synthase which are important for energy production led to impaired energy metabolism (Perluigi et al., 2009). Aconitase which contains a Fe-S cluster which is the important enzyme for Kreb's cycle as well as ATP synthase are highly sensitive to oxidative stress (Perluigi et al., 2009). Modification of ATP synthase could inhibit the complex activity and induce leakage of the electrons hence producing more superoxide production (Perluigi et al., 2009). 4-HNE modified aconitase and ATP synhthase results in reduced enzyme activity in AD brain (Butterfield et al., 2006, Sultana et al., 2006). To approach this, mitochondrial proteins from AOA3 and control cells were first isolated (Marchenko et al., 2000). The proteins were then resolved by their isoelectric focusing points (pI 3-10) for the first dimension. A broad pH was chosen to cover the whole ranges of mitochondrial proteins. Subsequently, the proteins were separated according to their molecular mass through SDS-PAGE for the second dimension. Using this technique, approximately 200-300 mitochondrial proteins could be resolved (Bailey et al., 2008). Approximately 100 and 150 proteins spots were observed in the control (C3ABR) and AOA3 (ATLH2ABR) cells with Coomassie blue staining (Figure 2.14 and 2.15). Coomassie blue staining was chosen to avoid differences in the staining duration using silver stain technique. Given that isolation of the mitochondrial proteins involves many steps to separate the cytoplasmic, nuclear and mitochondrial proteins, it is crucial to determine the purity of the mitochondrial proteins extracted. Thus, all the proteins spots in the control (C3ABR) and AOA3 (ATLH2ABR) were excised, trypsin digested and subjected to mass spectrometry analysis. In total, 63 and 50 protein spots were excised from the control (C3ABR) and AOA3 (ATLH2ABR) respectively. The successful rate for identification of the protein in control (C3ABR) was 21/63 (33.3%) and 28/50 (56%) in AOA3 (ATLH2ABR). The low successful rate for proteins identification may be due to the low abundance and/or poor extraction of the protein in these samples. 2D gel electrophoresis of mitochondrial proteins from control (C3ABR) and AOA3 (ATL2ABR) cells were shown in Figure 2.13 and 2.14, respectively. Almost 80% of the proteins identified by mass spectrometry were of mitochondrial origin (Tables 2.7 and 2.8). Because the isolation of mitochondrial proteins invovles several separation steps, contamination with non-mitochondrial origin proteins is generally observed (Scheffler, 2001). Mitochondrial and several cytoplasmic proteins were identified in control (C3ABR) and AOA3 (ATL2ABR) as shown in Table 2.7 and 2.8, respectively. Mowse algorithm accurately models the behavior of a proteolytic enzyme, and provides a value of whether or not the proteins identified are significant. This value is known as the Mowse score (Pappin et al., 1993). A Mowse score greater than 67 is significant where the p-value is <0.05. Proteins identified in control (C3ABR) cells had Mowse scores ranging from 54 to 715 whereas proteins identified in AOA3 (ATL2ABR) had Mowse scores ranging between 41 and 439. To summarize, the patterns of the protein spots were similar between the control (C3ABR) and AOA3 (ATLH2ABR) cells. Most of the proteins were distributed between isoelectric point (pI), 5 to 8 which is consistent with mitochondrial protein isolated from the SH-SY5Y neuroblastoma cell line reported by Scheffler and collegues (Scheffler, 2001). Approximately 300-400 mitochondrial proteins were detected by silver staining from 0.5 mg proteins isolated via differential centrifugation and multiple-step percoll/metrizamide gradient (Scheffler, 2001). In contrast, here we used approximately 0.3 mg mitochondrial proteins and isolated about 150 proteins, showing that both methods are able to detect a large number of proteins. Although only several proteins were detected through mass spectrometry (MALDI-TOF-TOF), most of them were of mitochondrial origin.

Fig_Enrich Mito protein_control_Fig 2

Figure 2.14 2D-gel electrophoresis of mitochondrial proteins isolated from control (C3ABR) cells. Mitochondrial proteins were isolated and electrophoresed on 2D gel. In the first dimension, proteins were separated according to their isoelectric point. pI 3-10 was chosen for identification of a wide spectrum of mitochondrial proteins. This was followed by the second dimension where proteins were separated according to their mass. Protein spots were excised, trypsin digested and tryptic peptides were subjected to mass spectrometry (MALDI-TOF-TOF) to determine the purity of the mitochondrial fraction.

Table 2.7 Proteins identified in control (C3ABR) cells by mass spectrometry

Spots

Proteins

kDa (~)

Mowse score

1

Calreticulin

60

61

2

Protein disulphide isomerase (PDI)

65

342

3

Tubulin b chain

55

256

4

ATP synthase subunit b

55

433

5

ACTB actin, cytoplasmic

45

99

6

Prohibitin

20

196

7

TuFm tu translation elongation factor, mitochondrial precursor

45

359

8

Protein disulphide isomerase (PDI)

55

715

9

HSPA9 Stress-70 protein, mitochondrial

72

190

10

HSPA5 heat shock cognate, 71kDa

72

270

11

Endoplamin HSP90B1

120

280

12

HYOU Hypoxia upregulated protein 1

170

117

13

IMMT isoform 1 of mitochondrial inner membrane protein

100

54

14

Cytochrome B-C1 complex subunit 2

43

150

15

Serine hydroxymethyltransferase, mitochondrial

55

82

16

ACTB Actin, cytoplasmic 1

43

61

Fig_Enrich Mito protein_AOA3l_Fig 2

Figure 2.15 2D gel separation of mitochondrial proteins isolated from AOA3 (ATL2ABR) cells. Mitochondrial proteins were isolated, electrophoresed on 2D gel electrophoresis. Samples were processed as described previously. Mass spectrometry (MALDI-TOF-TOF) was employed for identification of the proteins.

Table 2.8 Proteins identified in AOA3 (ATL2ABR) cells by mass spectrometry

Spots

Proteins

kDa(~)

Mowse score

1

HSP A8 Isoform 1 of heat shock cognate

60

192

2

HSPA9, Stress protein

60

231

3

Protein disulphide isomerase (PDI)

80

380

4

ATP5B ATP synthase subunit beta

70

439

5

ACTB actin cytoplasmic 1

45

212

6

ACTB actin cytoplasmic 1

45

100

7

IDH 3A Isoform 1 isocitrate dehydrogenase (NAD) subunit a, mitochondrial

35

160

8

Eno 1 Isoform a-enolase of a enolase

50

161

9

Glud 1-Glutamate dehydrogenase, mitochondrial

55

129

10

FSCN1 Fascin

60

98

11

P4HP Protein disulphide isomerase (PDI)

70

242

12

ECHSI Enoyl-CoA hyrdratase, mitochondrial

25

112

13

ATP5A1 ATP synthase subunit a, mitochondrial

55

172

14

ACO2 - Aconitate hydratase, mitochondrial

100

66

15

IMMT Isoform 1 of mitochondrial inner membrane protein

120

177

16

HYOU1 Hypoxia upregulated protein 1

150

97

17

EZR Ezrin

100

273

18

ACTBL2 Beta actin like-protein 2

55

41

19

ACTB actin cytoplasmic 1

45

163

20

ANXA4 Annexin IV

25

51

21

PHB Prohibitin

25

122

2.3.5.2 Oxidative modification of mitochondrial proteins in AOA3 (ATL2ABR) cells

To determine whether oxidative stress may affect mitochondrial proteins through modification in AOA3 (ATL2ABR), 2D gel electrophoresis combined with western blot analysis for anti-nitrotyrosine and anti-4-HNE was performed. The mitochondrial proteins were isolated, followed by 2D gel electrophoresis and the proteins were partially transferred. Only half of the proteins were transferred for western blotting and the remaining proteins were stained using Coomassie blue. Overlay between the western blotting and Coomassie blue gel allows identification of the corresponding modified proteins. The modified proteins were excised from Coomassie blue gel, trypsin digested and subjected to mass spectrometry analysis (MALDI-TOF-TOF) for identification. Thus, identification of the modified proteins is depending on the amount of proteins remaining in the Coomassie blue gel. Therefore, it is crucial to optimize the partial transfer conditions so that enough protein remains in the gel to ensure identification. The optimum conditions for proteins transfer was at 70V for 15 minutes. The results in Figure 2.16 (upper panel) demonstrate the presence of multiple 4-HNE modified proteins in the AOA3 (ATL2ABR) sample. In total, 17 protein spots were excised and 8 proteins were identified using mass spectrometry (MALDI-TOF-TOF). However, only 6 of the proteins showed Mowse score >65. Several modified mitochondrial proteins in AOA3 are shown in Figure 2.17. Under these conditions, no 4-HNE modified proteins were detected in the control (C3ABR) sample. The majority of the modified proteins (5/7) were of mitochondrial origin with different functions ranging from energy production through OXPHOS, calcium homeostasis and RNA synthesis (Table 2.9). These includeed ATP synthase H+ transporting mitochondria F1 complex, mitochondrial acetoacetyl CoA-thiolase, elongation factor Tu, calreticulin and heat shock protein 60 (HSP60). To determine whether protein oxidation was also occurring in the mitochondria, membranes were immunoblotted with an anti-nitroY antibody. Surprisingly, no oxidative protein damage was detected in control (C3ABR) or AOA3 (ATL2ABR) cells indicating the absence of nitrosination of proteins in these cells (figures not shown).

Oxidative stress in AOA3_4HNE_ Fig 2.3.7.5.tif

Figure 2.16 4-HNE modification of mitochondrial proteins from AOA3 (ATL2ABR) cells. Oxidative stress can be indirectly measured by the presence of modification on proteins. To determine whether the heightened levels of oxidative stress present in AOA3 could affect the mitochondrial proteins, mitochondrial proteins were separated by 2D gel electrophoresis and partially transferred onto nitrocellulose membrane to detect the presence of potential oxidative modifications in the mitochondrial proteins of AOA3. Western blotting with anti-4-HNE and anti-nitro-Y was subsequently performed. Western blot analysis with 4-HNE (top panel) and coomassie blue staining (lower panel) of mitochondrial proteins from control (C3ABR) and the AOA3 patient (ATL2ABR). Mitochondrial proteins in AOA3 (ATL2ABR) show increased signals for 4-HNE compared to control (C3ABR).

Figure 2.17 4-HNE modified mitochondrial proteins in AOA3 (ATL2ABR) cells. Proteins were identified via mass spectrometry. Mass spectra data was analysed using Mascot Science software for proteins identification. Approximately, 17 protein spots were excised for mass spectrometry, however only 8 proteins were identified. 6/8 proteins showed Mowse score >65, which is considered as significant. 5/7 of the proteins were of mitochondrial origin, including HSP60, ATP synthase, mitochondrial acetoacetyl CoA-thiolase, elongation factor Tu and calreticulin.

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