Profiling the Mitochondrial Proteome of

Leber's Hereditary Optic Neuropathy (LHON)

in Thailand: Down-Regulation of

Bioenergetics and Mitochondrial Protein

Quality Control Pathways in Fibroblasts with

the 11778G>A Mutation

Research of the Month

September 2014

Preclinical Research 1/12

IF = 3.534

2/12

Department: Medical Proteomics Unit, Office

for Research and Development

Field of interests: Application of Proteomics to

Nephrology and Medicine

Contribution: Corresponding Author

Prof. Visith Thongboonkerd

Profiling the Mitochondrial Proteome of Leber's Hereditary Optic Neuropathy (LHON) in Thailand: Down-

Regulation of Bioenergetics and Mitochondrial Protein Quality Control Pathways in Fibroblasts with the

11778G>A Mutation

3/12

Department: Biochemistry

Field of interests: Metabolic and

mitochondrial

Contribution: Corresponding Author

Prof.Dr. Patcharee Lertrit

Department: Biochemistry

Field of interests: Mitochondrial Biology

and Mitochondrial Genetics

Contribution: First Author

Dr. Aung Win Tun

Department: Medical Proteomics Unit, Office for

Research and Development Field of interests:

Proteomics, Kidney stone disease

Contribution: Co-author

Dr. Sakdithep Chaiyarit

Department: Biochemistry

Field of interests:

Contribution: Co-author

Supannee Kaewsutthi

Department: Biochemistry

Field of interests:

Contribution: Co-author

Wanphen Katanyoo

Department: Ophthalmology

Field of interests:

Contribution: Co-author

Prof.Dr. Wanicha Chuenkongkaew Department: Biochemistry

Field of interests: Osteoarthritis, Beta-

thalassemia syndrome Contribution: Co-author

Lect.Dr. Chayanon Peerapittayamongkol

Profiling the Mitochondrial Proteome of Leber's Hereditary Optic Neuropathy (LHON) in Thailand: Down-

Regulation of Bioenergetics and Mitochondrial Protein Quality Control Pathways in Fibroblasts with the

11778G>A Mutation

Table 1. Characteristics of LHON cases and unaffected relatives recruited in the study

4/12

Profiling the Mitochondrial Proteome of Leber's Hereditary Optic Neuropathy (LHON) in Thailand: Down-

Regulation of Bioenergetics and Mitochondrial Protein Quality Control Pathways in Fibroblasts with the

11778G>A Mutation

• Fibroblast surface protein (FSP) was used as a marker

in immunofluorescence of the cultured fibroblasts

obtained directly obtained from the skin biopsy

• The green represents fibroblasts and the nucleus was

stained with Hoechst-dye 33342 which shows blue

Figure 1. Assessment of the purity of fibroblasts

from a cultured skin biopsy

(doi:10.1371/journal.pone.0106779.g001)

• 20 µg of mitochondrial lysate and whole cell lysate from

fibroblasts were separated by 12% SDS-PAGE gel and checked

with specific antibodies against various sub-cellular organelles

• W = whole cell lysate; M = mitochondrial enriched fraction

• The same membrane for each cell type was stripped and probed

with subsequent antibodies

Figure 2. Western blot analyses for assessment of

mitochondrial enrichment and purity

(doi:10.1371/journal.pone.0106779.g002)

5/12

Profiling the Mitochondrial Proteome of Leber's Hereditary Optic Neuropathy (LHON) in Thailand: Down-

Regulation of Bioenergetics and Mitochondrial Protein Quality Control Pathways in Fibroblasts with the

11778G>A Mutation

Equal amounts of proteins (100 µg) from each fibroblast sample were

resolved by 2-DE. The numbers indicate the spot IDs of proteins whose

expression levels differed significantly between the fibroblasts of LHON

cases and controls.

Figure 3. Representative proteome map of differentially expressed

proteins from the mitochondrial fractions of fibroblasts from (A)

LHON cases (n = 7) and (C) controls (n = 5).

(doi:10.1371/journal.pone.0106779.g003)

Equal amounts of proteins (100 µg) from each sample were resolved by 2-DE.

The numbers indicate the spot IDs of proteins whose expression levels differed

significantly between the fibroblasts of unaffected LHON individuals and

controls. Some spots in the same horizontal row showed the same proteins.

For example, spot IDs 300 and 301 were identified as LONP1 and IDs 448,

451, 468 as catalase.

Figure 4. Representative proteome map of differentially

expressed proteins from mitochondria fraction between

fibroblasts from (U) the unaffected LHON individuals (n = 3) and

(C) the controls (n = 5).

(doi:10.1371/journal.pone.0106779.g004)

6/12

Profiling the Mitochondrial Proteome of Leber's Hereditary Optic Neuropathy (LHON) in Thailand: Down-

Regulation of Bioenergetics and Mitochondrial Protein Quality Control Pathways in Fibroblasts with the

11778G>A Mutation

Equal amounts of proteins (100 µg) from each sample were resolved

by 2-DE. The numbers indicate the spot IDs of proteins whose

expression levels differed significantly between the LHON cases and

their unaffected relatives.

(doi:10.1371/journal.pone.0106779.g005)

Figure 5. Representative proteome map of differentially

expressed proteins from the mitochondrial fractions of

fibroblasts from (A) LHON cases (n = 7) and (U) unaffected

LHON individuals (n = 3).

Table 2. Summary of significantly altered proteins between the LHON cases and the control group

7/12

Profiling the Mitochondrial Proteome of Leber's Hereditary Optic Neuropathy (LHON) in Thailand: Down-

Regulation of Bioenergetics and Mitochondrial Protein Quality Control Pathways in Fibroblasts with the

11778G>A Mutation

Table 3. Summary of significantly altered proteins between the unaffected LHON individuals and the control group

8/12

Profiling the Mitochondrial Proteome of Leber's Hereditary Optic Neuropathy (LHON) in Thailand: Down-

Regulation of Bioenergetics and Mitochondrial Protein Quality Control Pathways in Fibroblasts with the

11778G>A Mutation

Table 3. Summary of significantly altered proteins between the unaffected LHON individuals and the control group (cont.)

9/12

Profiling the Mitochondrial Proteome of Leber's Hereditary Optic Neuropathy (LHON) in Thailand: Down-

Regulation of Bioenergetics and Mitochondrial Protein Quality Control Pathways in Fibroblasts with the

11778G>A Mutation

Table 4. Summary of significantly altered proteins between the LHON cases and the unaffected relatives

10/12

11/12

Profiling the Mitochondrial Proteome of Leber's Hereditary Optic Neuropathy (LHON) in Thailand: Down-

Regulation of Bioenergetics and Mitochondrial Protein Quality Control Pathways in Fibroblasts with the

11778G>A Mutation

Table 5. Functional categories and sub-cellular localizations of proteins identified in the study based on the

MitoMiner Database and Nextprot

12/12

Profiling the Mitochondrial Proteome of Leber's Hereditary Optic Neuropathy (LHON) in Thailand: Down-

Regulation of Bioenergetics and Mitochondrial Protein Quality Control Pathways in Fibroblasts with the

11778G>A Mutation

(A) The differentially expression of heat-shock protein 60 and

catalase between LHON cases and controls

(B) the differential expression of NDUFS1 and catalase

between unaffected mutation carriers and controls.

• VDAC = a loading control

• The two lanes represent samples from two different

individuals for each group of samples.

Figure 6. Validation of proteomic data by Western

Blot analysis

(doi:10.1371/journal.pone.0106779.g006)

Figure 7. (A) A plot of log-ratio spot intensities, comparing affected

individuals (black bars) and unaffected relatives (grey bars) versus controls