ABSTRACT
Extracellular deposits of amyloid ß (Aß) aggregates in the brain is the hallmark of Alzheimer's disease. We present the configurations (location and conformation) and the interfacial folding and membrane insertion mechanisms of Aß fragments, wild-type Aß(25-35), Aß(35-25), and a sequence-shuffled peptide [Aß(25-35)-shuffled] from Aß(25-35) within membranes by replica-exchange molecular dynamics simulations. Although these peptides have the same amino acid composition, simulations show they have distinct locations and conformations within membranes. Moreover, our in vitro experiments show that these peptides have distinct neurotoxicities. We rationalize the distinct neurotoxicities of these peptides in terms of their simulated locations and conformations in membranes. This work provides another view that complements the general hydrophobicity-toxicity views, to better explain the neurotoxicity of Aß peptides.
Subject(s)
Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/toxicity , Cell Membrane/metabolism , Peptide Fragments/chemistry , Peptide Fragments/toxicity , Protein Aggregation, Pathological/metabolism , Amyloid beta-Peptides/metabolism , Animals , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Hydrogen Bonding , Molecular Dynamics Simulation , PC12 Cells , Peptide Fragments/metabolism , Protein Aggregation, Pathological/pathology , Protein Conformation , Rats , Structure-Activity Relationship , Water/chemistry , Water/metabolismABSTRACT
<p><b>OBJECTIVE</b>To prepare cytochrome (CYP)2E1-silenced hepatocytes by lentivirus-mediated RNA interference technology and to investigate the hepatotoxicity of trichloroethylene (TCE) in CYP2E1-silenced hepatocytes.</p><p><b>METHODS</b>Short hairpin RNA fragments were designed and synthesized and were then ligated into the lentiviral vector; single colonies were screened; the plasmid was extracted after PCR and sequence identification and then transferred into L02 hepatocytes; the CYP2E1-silenced hepatocytes were selected; real-time quantitative PCR and Western blot were used to evaluate the interference effects. The obtained CYP2E1-silenced hepatocytes, as well as normal L02 hepatocytes, were treated with TCE (0, 0.25, 0.50, 1.00, 2.00, and 4.00 mmol/L). The cell viability and half maximal inhibitory concentration (IC50) of TCE were measured; the apoptotic rate of cells was measured by flow cytometry; the mRNA expression levels of apoptosis genes and oncogenes were measured by real-time quantitative PCR.</p><p><b>RESULTS</b>The IC50s of TCE for L02 hepatocytes and CYP2E1-silenced hepatocytes were 15.1 mmol/L and 23.6 mmol/L, respectively. The apoptotic rate increased as the dose of TCE rose in the two types of cells; the CYP2E1-silenced hepatocytes hada significantly lower apoptotic rate than L02 hepatocytes when they were exposed to 2.0 and 4.0 mmol/L TCE (P < 0.05 or P < 0.01). The mRNA expression level of bcl-2 (anti-apoptosis gene) in CYP2E1-silenced hepatocytes was 15% ∼ 60% higher than that in L02 hepatocytes (P < 0.01), while the mRNA expression levels of caspase-3 and caspase-9 (apoptosis genes) in CYP2E1-silenced hepatocytes were 30% ∼ 60% lower than those in L02 hepatocytes (P < 0.01). The mRNA expression level of p53 (cancer suppressor gene) in CYP2E1-silenced hepatocytes was 81 - 278% higher than that in L02 hepatocytes (P < 0.01), while the mRNA expression levels of c-fos and k-ras (oncogenes) in CYP2E1-silenced hepatocytes were 20-68% lower than those in L02 hepatocytes (P < 0.01).</p><p><b>CONCLUSION</b>CYP2E1-silenced cells can be successfully prepared by lentivirus-mediated RNA interference technology. Silencing CYP2E1 gene can reduce the hepatotoxicity of TCE and inhibit the expression of some apoptosis genes and oncogenes, suggesting that CYP2E1 gene plays an important role in TCE metabolism and is related to the hepatotoxicity of TCE.</p>
Subject(s)
Humans , Apoptosis , Genetics , Cell Line , Cell Survival , Genetics , Cytochrome P-450 CYP2E1 , Genetics , Metabolism , Genetic Vectors , Hepatocytes , Metabolism , Lentivirus , Genetics , RNA Interference , Trichloroethylene , ToxicityABSTRACT
The mechanisms of interfacial folding and membrane insertion of the Alzheimer's amyloid-beta fragment Abeta(25-35) and its less toxic mutant, N27A-Abeta(25-35) and more toxic mutant, M35A-Abeta(25-35), are investigated using replica-exchange molecular dynamics in an implicit water-membrane environment. This study simulates the processes of interfacial folding and membrane insertion in a spontaneous fashion to identify their general mechanisms. Abeta(25-35) and N27A-Abeta(25-35) peptides share similar mechanisms: the peptides are first located in the membrane hydrophilic region where their C-terminal residues form helical structures. The peptides attempt to insert themselves into the membrane hydrophobic region using the C-terminal or central hydrophobic residues. A small portion of peptides can successfully enter the membrane's hydrophobic core, led by their C-terminal residues, through the formation of continuous helical structures. No detectable amount of M35A-Abeta(25-35) peptides appeared to enter the membrane's hydrophobic core. The three studied peptides share a similar helical structure for their C-terminal five residues, and these residues mainly buried within the membrane's hydrophobic region. In contrast, their N-terminal properties are markedly different. With respect to the Abeta(25-35), the N27A-Abeta(25-35) forms a more structured helix and is buried deeper within the membrane, which may result in a lower degree of aggregation and a lower neurotoxicity; in contrast, the less structured and more water-exposed M35A-Abeta(25-35) is prone to aggregation and has a higher neurotoxicity. Understanding the mechanisms of Abeta peptide interfacial folding and membrane insertion will provide new insights into the mechanisms of neurodegradation and may give structure-based clues for rational drug design preventing amyloid associated diseases.
Subject(s)
Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/toxicity , Cell Membrane/metabolism , Mutant Proteins/chemistry , Neurotoxins/toxicity , Peptides/toxicity , Protein Folding/drug effects , Amyloid beta-Peptides/metabolism , Cell Membrane/drug effects , Humans , Magnetic Resonance Spectroscopy , Models, Molecular , Mutant Proteins/metabolism , Mutant Proteins/toxicity , Neurotoxins/chemistry , Neurotoxins/metabolism , Peptides/chemistry , Peptides/metabolism , Protein Structure, Quaternary , Protein Structure, Secondary , Protons , Water/chemistryABSTRACT
@# ObjectiveTo study the characteristic of apoptosis and the expression of inducible nitric oxide synthase(iNOS),as well as relation between them after spinal cord injury (SCI) in rats.Methods84 adult SD rats were randomly divided into three groups,and made into mild,moderate and severe acute SCI models.The rats were killed at 4h,8h,1d,3d,7d,14d and 21d after surgery.The histopathology changes in spinal cord tissue were observed with HE staining microscopic examination.The expression of iNOS was determined by immunohistochemistry and the apoptosis was labeled by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling(TUNEL).ResultsThe histopathology changes in spinal cord tissue deteriorated with increasing in injury degree.A little iNOS immunoreactivity (iNOS-IR) was detected in nerve cells,gliocytes,ependymocytes and blood vessel endotheliocytes of normal and vertebra shelf operated controls,increasing at 8 h, and in flood tide in 7 d. The expression of Bax and the rates of TUNEL positive cells were similar with that of iNOS after SCI. There was positive correlation between expression of iNOS and degree of primary SCI ,apoptosis index(r=0.414,P<0.01;r=0.854,P<0.01).Conclusions Expression of iNOS and Bax increase and a large number of TUNEL positive cells present after SCI in rats. There is positive correlation between expression of iNOS and degree of primary SCI,apoptosis index.
