Protegrin-1 (PG-1) is an 18 residues long, cysteine-rich beta-sheet antimicrobial peptide (AMP). PG-1 induces strong cytotoxic activities on cell membrane and acts as a potent antibiotic agent. Earlier we reported that its cytotoxicity is mediated by its channel-forming ability. In this study, we have examined the amyloidogenic fibril formation properties of PG-1 in comparison with a well-defined amyloid, the amyloid-beta (Abeta(1-42)) peptide. We have used atomic force microscopy (AFM) and thioflavin-T staining to investigate the kinetics of PG-1 fibrils growth and molecular dynamics simulations to elucidate the underlying mechanism. AFM images of PG-1 on a highly hydrophilic surface (mica) show fibrils with morphological similarities to Abeta(1-42) fibrils. Real-time AFM imaging of fibril growth suggests that PG-1 fibril growth follows a relatively fast kinetics compared to the Abeta(1-42) fibrils. The AFM results are in close agreement with results from thioflavin-T staining data. Furthermore, the results indicate that PG-1 forms fibrils in solution. Significantly, in contrast, we do not detect fibrillar structures of PG-1 on an anionic lipid bilayer 2-dioleoyl-sn-glycero-3-phospho-L-serine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine;only small PG-1 oligomers can be observed. Molecular dynamics simulations are able to identify the presence of these small oligomers on the membrane bilayer. Thus, our current results show that cytotoxic AMP PG-1 is amyloidogenic and capable of forming fibrils. Overall, comparing beta-rich AMPs and amyloids such as Abeta, in addition to cytotoxicity and amyloidogenicity, they share a common structural motif, and are channel forming. These combined properties support a functional relationship between amyloidogenic peptides and beta-sheet-rich cytolytic AMPs, suggesting that amyloids channels may have an antimicrobial function. Elucidating the structure of Abeta(1-40) fibrils is of interest in Alzheimer's disease research because it is required for designing therapeutics that target Abeta(1-40) fibril formation at an early stage of the disease. M35 is a crucial residue because of its potential oxidation and its strong interactions across beta-strands and across beta-sheets in Abeta fibrils. Experimentally, data for the three-fold symmetry structure of the Abeta(9-40) fibril suggest formation of tight hydrophobic core through M35 interactions across the fibril axis and strong I31-V39 interactions between different cross-beta units. Herein, on the basis of experimental data, we probe conformers with three-fold symmetry of the full-length Abeta(1-40). Our all-atom molecular dynamics simulations in explicit solvent of conformers based on the ssNMR data reproduced experimental observations of M35-M35 and I31-V39 distances. Our interpretation of the experimental data suggests that the observed 5-7 Angstrom M35-M35 distance in the fibril three-fold symmetry structure is likely to relate to M35 interactions along the fibril axis, rather than across the fibril axis, since our measured M35-M35 distances across the fibril axis are consistently above 15 Angstrom. Consequently, we revealed that the unique Abeta(1-40) triangular structure has a large cavity along the fibril axis and that the N-termini can assist in the stabilization of the fibril by interacting with the U-turn domains or with the C-termini domains. Our findings, together with the recent cyroEM characterization of the hollow core in Abeta(1-42) fibrils, point to the relevance of a cavity in Abeta(1-40/1-42) oligomers which should be considered when targeting oligomer toxicity. We addressed the mechanism through which transcription factors (TFs) assemble specifically along the enhancer DNA. The IFN-beta enhanceosome provides a good model system: it is small;its components crystal structures are available;and there are biochemical and cellular data. In the IFN-beta enhanceosome, there are few protein-protein interactions even though consecutive DNA response elements (REs) overlap. Our molecular dynamics (MD) simulations on different motif combinations from the enhanceosome illustrate that cooperativity is achieved via unique organization of the REs: specific binding of one TF can enhance the binding of another TF to a neighboring RE and restrict others, through overlap of REs;the order of the REs can determine which complexes will form;and the alternation of consensus and non-consensus REs can regulate binding specificity by optimizing the interactions among partners. Our observations offer an explanation of how specificity and cooperativity can be attained despite the limited interactions between neighboring TFs on the enhancer DNA. To date, when addressing selective TF binding, attention has largely focused on RE sequences. Yet, the order of the REs on the DNA and the length of the spacers between them can be a key factor in specific combinatorial assembly of the TFs on the enhancer and thus in function. Our results emphasize cooperativity via RE binding sites organization.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010440-10
Application #
8349004
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
10
Fiscal Year
2011
Total Cost
$642,558
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Lu, Shaoyong; Jang, Hyunbum; Muratcioglu, Serena et al. (2016) Ras Conformational Ensembles, Allostery, and Signaling. Chem Rev 116:6607-65
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Lu, Shaoyong; Jang, Hyunbum; Gu, Shuo et al. (2016) Drugging Ras GTPase: a comprehensive mechanistic and signaling structural view. Chem Soc Rev 45:4929-52
Chakrabarti, Mayukh; Jang, Hyunbum; Nussinov, Ruth (2016) Comparison of the Conformations of KRAS Isoforms, K-Ras4A and K-Ras4B, Points to Similarities and Significant Differences. J Phys Chem B 120:667-79
Xu, Liang; Nussinov, Ruth; Ma, Buyong (2016) Allosteric stabilization of the amyloid-? peptide hairpin by the fluctuating N-terminal. Chem Commun (Camb) 52:1733-6
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