PIERGIORGIO PETTAZZONI
Relatore: Prof. Carlo Ferretti Co-Relatore Dott.ssa Stefania Pizzimenti
Data di laurea: 17 luglio 2007
Titolo: 4-Hydroxynonenal (HNE)-protein adducts identification in HL-60 human lukemic cells
4-Hydoxynonenal (HNE), one of the most important aldheydic products generated from the lipidic peroxidation of ω-6 polyunsaturated fatty acid, has been suggested to play a critical role in the pathogenesis of various diseases, such as atherosclerosis, diabetes, Alzheimer and Parkinson diseases. It has been demonstrated that HNE plays an important role in the control of tumour cell proliferation and differentiation. One micromolar HNE, a dose similar to those detected in normal cells, inhibits proliferation and induces differentiation of HL-60 human leukemic cells. This HNE concentration also induces erythroid differentiation by increasing gamma globin expression in MEL and K562 cells. Moreover, HNE causes cell cycle arrest and apoptosis in numerous cell lines. The mechanism by which HNE causes these biological effects remains unclear but several studies indicate that the ability of HNE to generate covalent adducts with cellular proteins may be directly involved.
HNE modifies proteins either by forming simple Michael adducts on lysine, histidine, and cysteine residues or through Schiff base formation with lysine residues. Several studies have already identified some HNE protein targets, such as metabolism enzymes, signal transduction proteins, structural proteins, transporters and others.
The cytostatic effect of HNE on the HL-60 cell line has been well characterized in our laboratory.
After 1 micromolar HNE treatment, the expression of several genes controlling cellular proliferation and differentiation is affected, such as c-myc, c-myb, cyclins and telomerase. The identification of HNE-adducts in this well-characterized cell line represents a new insight in the comprehension of molecular mechanisms elicited by HNE. However, the aim of this study was to identify HNE-protein adducts after exposure of HL-60 cells to non citotoxic concentrations of aldehyde. HL-60 represents a good model for HNE studies, since the lipid peroxidation process in this cell line is very low, thus it is possible to identify the HNE adducts without the interference caused by HNE endogenously produced.
For this study we used biochemical and proteomic techniques like western blotting, 2D electrophoresis, MALDI/TOF mass spectrometry, immunoprecipitation, enzymatic assays ad confocal microscopy imaging.
In the first part of our study we performed a series of western blots to choose the anti-HNE antibody that shows the highest specificity for the evaluation of HNE-protein adducts.
Secondly, we have performed 2D electrophoresis to separate proteins, obtained by HNE-treated cells. HNE-protein adducts were revealed after blotting on a PVDF membrane and by immunochemical detection with the specific anti-HNE antibody, identified in the first part of our study. Spots that reacted with the anti-HNE antibody were purified and analysed by MALDI/TOF
MS for their identification.
Our results showed the formation of HNE protein adducts with the glicolitic enzime α-enolase.
Immunoprecipitation analysis confirmed these results. This is the first evidence of the formation of this HNE-protein adduct in tumor cells. HNE/α-enolase adduct formation doesn’t affect α-enolase protein expression nor protein localisation. Nevertheless, enzymatic activity was reduced, but only after exposure of the purified protein to high doses of HNE.
We also demonstrated the formation of HNE adducts with the c-myc binding protein-1 (MBP-1), an alternative product of translation of α-enolase mRNA. MBP-1 is a negative regulator of c-myc

expression via interaction with the P2 promoter of the oncogene c-myc. The HNE/MBP-1 adducts could be involved in c-myc down-regulation, a key event in HNE-caused cell proliferation arrest.
The use of confocal microscopy confirmed the co-localisation of enolase/HNE adducts in the cytoplasm. Moreover, a diffuse nuclear fluorescence indicated the presence of several HNEadducts, that have to be characterized.
HNE/enolase and HNE/MBP-1 adducts are the first molecular targets indentified in the HL-60 cell line, and this leads to new insights in the molecular mechanism of HNE-proliferation arrest.