Pharmaco-Proteomic Study of Hydroxyurea-Induced Modifications

in the Sickle Red Blood Cell Membrane Proteome

Swati S. Ghatpande, Pankaj K. Choudhary, Charles T. Quinn, Steven R. Goodman

Advanced Cell and Molecular BiologyIsabel V. Neri

Sickle Cell Anemia (SS)

Sickle Cell Anemia (SS)

In the deoxygenated state…In the deoxygenated state…

Sickle Cell Anemia (SS)

The formation of sickle RBCs...The formation of sickle RBCs...

Vasoocclusion

Chronic Hemolysis

Ischemic tissue damage

HYDROXYUREA

Effective oral drug for the management of SS

Capacity to increase HbF levels

Inhibit polymerization of HbS Reduce sickling

HYDROXYUREA

Other benefits of HU

HYDROXYUREA

MULTICENTER STUDY OF HUMULTICENTER STUDY OF HU

Patients often improved clinically before a significant rise in HbF levels

Patients often improved clinically before a significant rise in HbF levels

HU – induced alterations of additional cellular mechanisms that are yet to be identified

HU – induced alterations of additional cellular mechanisms that are yet to be identified

HYDROXYUREA

2D - DIGE2D - DIGE Mass spectrometryMass spectrometry

To determine the elemental composition of an unknown

substance.

To determine the elemental composition of an unknown

substance.

It utilizes the charged properties of molecules to

separate them

It utilizes the charged properties of molecules to

separate them

Ability to separate complex mixtures of proteins

Ability to separate complex mixtures of proteins

MATERIALS AND METHODS

Homozygous SS patients 10ml blood samples

by venipuncture

Southwestern Comprehensive Sickle Cell Center

No patient had received any HU or blood transfusion within the preceding 4 months

SUBJECTSSUBJECTS

MATERIALS AND METHODS

1. INCUBATION OF SS WHOLE BLOOD WITH HU1. INCUBATION OF SS WHOLE BLOOD WITH HU

Equal volumes of the same SS blood sample +/- 50 μM

HU

Equal volumes of the same SS blood sample +/- 50 μM

HUincubatedincubated

37 C, 15 hours with constant shaking

37 C, 15 hours with constant shaking

2. PREPARATION OF ERYTHROCYTE MEMBRANES2. PREPARATION OF ERYTHROCYTE MEMBRANES

RBCs were sedimented at 1,000 g for 10 minutes at 4C

RBCs were sedimented at 1,000 g for 10 minutes at 4C

MATERIALS AND METHODS

2. PREPARATION OF ERYTHROCYTE MEMBRANES2. PREPARATION OF ERYTHROCYTE MEMBRANES

RBCs were resuspended in 10 volumes of PBS and

sedimented at 2,000g for 10mins.

RBCs were resuspended in 10 volumes of PBS and

sedimented at 2,000g for 10mins.

The washed RBCs were lysed in 6 volumes of lysis buffer and were

sedimented at 31,000g for 30mins.

The washed RBCs were lysed in 6 volumes of lysis buffer and were

sedimented at 31,000g for 30mins.

The pellet becomes white or light pink

MATERIALS AND METHODS

3. MINIMAL LABELING OF SS MEMBRANE PROTEINS3. MINIMAL LABELING OF SS MEMBRANE PROTEINS

4. SEPARATION OF PROTEINS IN FIRST DIMENSION 4. SEPARATION OF PROTEINS IN FIRST DIMENSION

SS membrane proteins = 100μg each of (-)HU and (+) HU samples solubilized in lysis buffer were minimally labeled with Cy3 and Cy5 fluorophores

SS membrane proteins = 100μg each of (-)HU and (+) HU samples solubilized in lysis buffer were minimally labeled with Cy3 and Cy5 fluorophores

100μg each of the control and drug tested samples

were mixed together

100μg each of the control and drug tested samples

were mixed together

Rehydration buffer

Rehydration buffer

Final protein mixture

Final protein mixture

MATERIALS AND METHODS

4. SEPARATION OF PROTEINS IN FIRST DIMENSION 4. SEPARATION OF PROTEINS IN FIRST DIMENSION

The sample, included in the rehydration

solution

The sample, included in the rehydration

solution

IPG (Immobilized pH Gradient) strip holder

IPG (Immobilized pH Gradient) strip holder

Immobiline Dry-Strip gel was

placed over the sample

Immobiline Dry-Strip gel was

placed over the sample

IPG cover fluidIPG cover fluid

Minimize evaporation and

urea crystallization

Minimize evaporation and

urea crystallization

REHYDRATION – 12 hours at 20 C

MATERIALS AND METHODS

5. REDUCTION OF ALKYLATION OF THE IEF SEPARATED PROTEINS5. REDUCTION OF ALKYLATION OF THE IEF SEPARATED PROTEINS

6. SEPARATION OF PROTEINS IN SECOND DIMENSION 6. SEPARATION OF PROTEINS IN SECOND DIMENSION

Immobiline strips with separated proteins were

equilibriated and reduced

Immobiline strips with separated proteins were

equilibriated and reduced

Equilibriation and Alkylation at room

temperature

Equilibriation and Alkylation at room

temperature

Proteins were further separated on a 10% SDS-

polyacrylamide gel

Proteins were further separated on a 10% SDS-

polyacrylamide gel

MATERIALS AND METHODS

7. GEL IMAGING AND ANALYSIS7. GEL IMAGING AND ANALYSIS

8. STATISTICAL ANALYSIS 8. STATISTICAL ANALYSIS

Cy3 and Cy5 fluorophores were detected using 2920

2D-Master Imager

Cy3 and Cy5 fluorophores were detected using 2920

2D-Master Imager

DeCyder DIA software

DeCyder DIA software

Detects protein spots

Quantifies the spot volume for

each imageExpresses volumes in

ratio

GOALGOALTo discover protein spots whose mean

ratios differed significantly from one

To discover protein spots whose mean

ratios differed significantly from one

MATERIALS AND METHODS

9. PROTEIN IDENTIFICATION BY TANDEM MASS SPECTROMETRY AND DATABASE SEARCH9. PROTEIN IDENTIFICATION BY TANDEM MASS SPECTROMETRY AND DATABASE SEARCH

10. IMMUNOBLOT ANALYSIS 10. IMMUNOBLOT ANALYSIS

Also known as MS/MS or MS2 involves multiple steps of mass spectrometry

selection

Also known as MS/MS or MS2 involves multiple steps of mass spectrometry

selection

2D-electrophoresis

Proteins were

electroblotted

Proteins were

electroblotted

Nitrocellulose membrane in transfer buffer overnight at 4C

and 25 V

Membranes were blocked for an hour

Membranes were incubated overnight

Immunoreactive spots were detected

RESULTS

2D DIGE + mass spectrometry2D DIGE + mass spectrometry

To identify alterations in the sickle RBC membrane proteome in response to a

clinically relevant dose of HU

To identify alterations in the sickle RBC membrane proteome in response to a

clinically relevant dose of HU

RESULTS

Each circle represents a protein spot whose position is determined by its log volume on the X axis and its intensity on the Y axis and the spots lying within the 2.5-fold threshold are shown in green.Blue – increase in protein content by more than 2.5-foldRed – decrease in protein content by more than 2.5-fold

Each circle represents a protein spot whose position is determined by its log volume on the X axis and its intensity on the Y axis and the spots lying within the 2.5-fold threshold are shown in green.Blue – increase in protein content by more than 2.5-foldRed – decrease in protein content by more than 2.5-fold

RESULTS

RESULTS

IMMUNOBLOT ANALYSIS OF CATALASEIMMUNOBLOT ANALYSIS OF CATALASE

Positions of SS RBC membrane proteins

that showed a statistically significant

change in their composition on

exposure to 50 μM HU.

Positions of SS RBC membrane proteins

that showed a statistically significant

change in their composition on

exposure to 50 μM HU.

RESULTS

DISCUSSION

HU

DISCUSSION

FACTORS INVOLVED IN IMPROVING THE PATHOLOGY OF RBC MEMBRANE AFTER HU TREATMENT

FACTORS INVOLVED IN IMPROVING THE PATHOLOGY OF RBC MEMBRANE AFTER HU TREATMENT

These indicate the likelihood of HU

targeting the membrane and acting

through cellular mechanisms that are

independent of elevated HbF and are

yet to be identified

These indicate the likelihood of HU

targeting the membrane and acting

through cellular mechanisms that are

independent of elevated HbF and are

yet to be identified

With these, the researchers used a proteomics approach to identify HU-induced changes in the SS RBC membrane

With these, the researchers used a proteomics approach to identify HU-induced changes in the SS RBC membrane

DISCUSSION

DISCUSSION

2D-DIGE coupled with LC/MS/MS

-this was used since mature RBCs are enucleated cells lacking active protein synthesis

- To quantitate post-translational protein modifications in SS RBC membranes with and without physiologic concentrations of HU

-Based on the results and literature, the researchers hypothesized that HU induces oxidative stress at a concentration of 50 μM.

-Their hypothesis is further strengthened by the fact that equal amounts of the same SS RBC membrane sample +/- HU were subjected to 2D-DIGE such that the

levels of oxidative stress before and after HU exposure were comparable.

DISCUSSION

Thus, HU stimulated increased tyrosine phosphorylation of catalase presents an attractive model for potential activation of catalase in mature sickle RBCs that do

not contain the necessary machinery for new protein synthesis

This work is the first attempt to identify and characterize in vitro HU-induced protein modifications in SS RBC membranes using 2D-DIGE protein profiling and

tandem mass spectrometry,

The End