Protein Carbonylation in Patients with Myelodysplastic Syndromes

Introduction:

Myelodysplastic syndromes (MDS) are a heterogenous group of oncohematologic diseases, characterized by bone marrow hypercellularity, dysplasia of myeloid lineage cells, peripheral cytopenias, and an increased risk of evolution to acute myeloid leukemia (AML). Pathophysiology of MDS is not fully understood, however it has been described that oxidative stress plays an important role in initiation and disease progression. Oxidative stress is defined as an imbalance between prooxidative and antioxidative processes that prefers the production of reactive oxygen species (ROS) over an antioxidant defence. The association between MDS and oxidative stress was discussed in several studies, where functional mitochondrial abnormalities in MDS patients have been demonstrated as one of the possible sources of oxidative stress. Another mechanism could be associated with mitochondrial dysfunction via iron overloading, mitochondrial DNA mutation, systemic inflammation, and bone marrow stromal defects. Protein carbonylation is ROS-mediated protein oxidation and it is defined as an irreversible post-translational oxidative modification. The detection of protein carbonyl groups is a specific useful marker of oxidative stress.

The aim of this study was to determine the levels of carbonylated proteins in plasma of MDS patients resp. in plasma of individual subtype of MDS patients and evaluate changes of carbonylated plasma proteome in MDS patients compared with healthy controls.

Methods:

The content of protein carbonyls was measured after derivatization with dinitrophenylhydrazine by monitoring absorbance between 250 and 500nm. Carbonylated proteins of plasma samples were purified after reaction with biotin hydrazide. The negative controls were processed using the exact same procedure, except biotin hydrazide labeling. The dialyzed biotin hydrazide labeled samples and the negative controls were isolated using monomeric avidin resin. All the samples were prepared in doublets. One part of samples was used for the on bead tryptic digestion and the second part of samples was separated by SDS-PAGE after eluation with Laemmli buffer. The obtained carbonylated proteins were identified by mass spectrometry (LC-MS/MS).

Results:

We have examined 32 patients with different MDS subtype (10 patients RAEB-1,2; 12 RCMD; 10 RARS) and 20 healthy controls. Diagnoses of different subgroups of MDS were established according to the WHO classification criteria. The median age of patients group was 64,5 and presented 17 males (57 %); the median age of healthy controls group was 43 and presented 10 males (50 %).

We have found significantly (P <0.02) increased level of protein carbonyls in plasma of MDS patients compared to healthy controls, the increased level of protein carbonyls was determinated in all tested MDS subtypes. The difference was elevated significantly in RARS patients (45,8 ± 0,5 %, P <0.005) in comparison with healthy controls. We also observed proteome changes in isolated carbonylated proteins of RARS patients and healthy controls.

Conclusion:

Protein carbonylation also plays an important role in signalling processes. Therefore protein carbonylation in MDS disease could lead not only to the loss of protein function by oxidative modification but also can influence protein expression playing significant role in MDS disease. We suggest that protein carbonylation is not "only" a consequence of oxidative stress in MDS disease, but also plays an active role as an important cause of pathogenic changes related to angiogenesis, myelomonocytic lineage development and complement impairment in MDS.

Acknowledgment:

This work was supported by the project of the Ministry of Health of the Czech Republic for conceptual development of the research organization 00023736, by Grant from the Academy of Sciences, Czech Republic (P205/12/G118), and by ERDF OPPK CZ.2.16/3.1.00/28007.