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1.
Neurobiol Aging ; 32(2): 257-71, 2011 Feb.
Article in English | MEDLINE | ID: mdl-19278755

ABSTRACT

Apolipoprotein-E (apoE) plays important roles in neurobiology and the apoE4 isoform increases risk for Alzheimer's disease (AD). ApoE peptides are biologically active and may be produced in the brain. It is unclear if apoE proteolysis is dependent on isoform or AD status and this was addressed here. Hippocampus, frontal cortex, occipital lobe and cerebellum samples were homogenized into fractions that were soluble in Tris-buffered saline (TBS), Triton X-100 or guanidine hydrochloride and analysed for apoE fragmentation by Western blotting. Approximately 20% of apoE3 was detected as fragments and this was predominantly as a 25 kDa peptide in TBS-soluble fractions. The concentration of TBS-soluble apoE fragments was two- to three-fold higher in apoE3 compared to apoE4 subjects. This difference was observed in all areas of the brain examined and was not related to AD status. Cathepsin-D treatment generated apoE fragments that were very similar to those detected in brain, however, no apoE isoform-specific differences in susceptibility to cathepsin-D proteolysis were detected. This indicates that proteolytic processing of apoE to form soluble fragments in the human brain is dependent on apoE isoform but not AD status.


Subject(s)
Alzheimer Disease/pathology , Apolipoproteins E/metabolism , Brain/metabolism , Aged , Aged, 80 and over , Alzheimer Disease/genetics , Amyloid beta-Peptides/metabolism , Apolipoprotein E3/drug effects , Apolipoprotein E3/genetics , Apolipoprotein E3/metabolism , Apolipoprotein E4/drug effects , Apolipoprotein E4/genetics , Apolipoprotein E4/metabolism , Apolipoproteins E/classification , Apolipoproteins E/drug effects , Apolipoproteins E/genetics , Cathepsin D/pharmacology , Cell Line, Tumor , Enzyme-Linked Immunosorbent Assay/methods , Female , Hemostatics/pharmacology , Humans , Male , Middle Aged , Neuroblastoma/pathology , Peptide Fragments/metabolism , Protein Isoforms/metabolism , Statistics, Nonparametric , Thrombin/pharmacology , Transfection/methods
2.
Biochemistry ; 46(28): 8392-400, 2007 Jul 17.
Article in English | MEDLINE | ID: mdl-17580963

ABSTRACT

Oxidative damage to proteins such as apolipoprotein B-100 increases the atherogenicity of low-density lipoproteins (LDL). However, little is known about the potential oxidative damage to apolipoprotein E (apoE), an exchangeable antiatherogenic apolipoprotein. ApoE plays an integral role in lipoprotein metabolism by regulating the plasma cholesterol and triglyceride levels. Hepatic uptake of lipoproteins is facilitated by apoE's ability to bind with cell surface heparan sulfate proteoglycans and to lipoprotein receptors via basic residues in its 22 kDa N-terminal domain (NT). We investigated the effect of acrolein, an aldehydic product of endogenous lipid peroxidation and a tobacco smoke component, on the conformation and function of recombinant human apoE3-NT. Acrolein caused oxidative modification of apoE3-NT as detected by Western blot with acrolein-lysine-specific antibodies, and tertiary conformational alterations. Acrolein modification impairs the ability of apoE3-NT to interact with heparin and the LDL receptor. Furthermore, acrolein-modified apoE3-NT displayed a 5-fold decrease in its ability to interact with lipid surfaces. Our data indicate that acrolein disrupts the functional integrity of apoE3, which likely interferes with its role in regulating plasma cholesterol homeostasis. These observations have implications regarding the role of apoE in the pathogenesis of smoking- and oxidative stress-mediated cardiovascular and cerebrovascular diseases.


Subject(s)
Acrolein/pharmacology , Apolipoprotein E3/chemistry , Apolipoprotein E3/drug effects , Aging , Apolipoprotein E3/metabolism , Heparin/metabolism , Humans , Oxidative Stress , Phospholipids/metabolism , Protein Structure, Tertiary , Receptors, LDL/metabolism , Smoking
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