Paulina Juszczyk

Binding Epitopes and Interaction Structure of the Neuroprotective Protease Inhibitor Cystatin C with beta-Amyloid Revealed by Proteolytic Excision Mass Spectrometry and Molecular Docking Simulation

Human cystatin C (HCC) is a protease inhibitor with a propensity to form beta-amyloid (Abeta)-like fibrils and to coassociate with amyloidogenic proteins. Recently, a specific interaction between HCC and Abeta has been found. Here, we report the identification of the Abeta and HCC binding epitopes in the Abeta-HCC complex, using a combination of selective proteolytic excision and high resolution mass spectrometry. Proteolytic excision of Abeta(1-40) on sepharose-immobilized HCC and MALDI-MS identified the epitope Abeta(17-28). On immobilized Abeta(1-40), affinity MS of HCC fragments identified a specific C-terminal epitope, HCC(101-117). Binding specificities of both epitopes were ascertained by ELISA and surface plasmon resonance and by direct electrospray MS of the HCC-Abeta epitope peptide complexes. A structure model of the HCC-Abeta complex by molecular docking simulation showed full agreement with the identified Abeta and HCC epitopes. Inhibition studies in vitro revealed Abeta-fibril inhibiting activity of the HCC(101-117)-epitope. The Abeta-HCC interacting epitopes provide lead structures of neuroprotective inhibitors for AD and HCC amyloidosis therapy.

FTIR spectroscopic studies on aggregation process of the β-amyloid 11–28 fragment and its variants

Aggregation of Aβ peptides is a seminal event in Alzheimers disease. Detailed understanding of the Aβ assembly process would facilitate the targeting and design of fibrillogenesis inhibitors. Here, conformational studies using FTIR spectroscopy are presented. As a model peptide, the 11–28 fragment of Aβ was used. This model peptide is known to contain the core region responsible for Aβ aggregation. The structural behavior of the peptide during aggregation provoked by the addition of water to Aβ(11–28) solution in hexafluoroisopropanol was compared with the properties of its variants corresponding to natural, clinically relevant mutants at positions 21–23 (A21G, E22K, E22G, E22Q and D23N). The results showed that the aggregation of the peptides proceeds via a helical intermediate, and it is possible that the formation of α‐helical structures is preceded by creation of 310‐helix/310‐turn structures. Copyright

Interactions of Cu2+ ions with chicken prion tandem repeats.

The potentiometric and spectroscopic (EPR, UV-Vis, CD) data have shown that the chicken prion hexa-repeat (Ac-His-Asn-Pro-Gly-Tyr-Pro-NH(2)) is a very specific ligand for Cu(2+) ions. The His imidazole is an anchoring binding site, then the adjacent amide nitrogen coordinates as a second donor. The presence of Pro at position 3 induces binding of phenolate oxygen as a third donor atom. The tridentate coordination dominates around physiological pH. Similar to human octapeptide fragments, chicken tandem repeats exhibit a cooperative effect in binding Cu(2+) ions, although chicken peptides are much less effective in metal ion coordination.

The whole hexapeptide repeats domain from avian PrP displays untypical hallmarks in aspect of the Cu2+ complexes formation

Prions, the infectious agents responsible for the transmissible spongiform encephalopathies (TSEs) have defied full characterization for decades. Although the interactions of Cu2+ ions with PrP both in vivo and in vitro are well documented, there are still a lot of ambiguities concerning the biological and chemical nature of these effects. In this work, we have investigated the interactions of Cu2+ ions with whole repeat region of the copper‐binding domain (hexapeptide repeats) of chicken PrP. Our results provide explanations for the structural and chemical basis of the specific interactions of Cu2+ ions with the hexapeptide repeat region. Furthermore, we show that SOD‐like activity depends on Cu2+ complexes.

Simple and efficient synthesis of chiral amino alcohols with an amino acid-based skeleton

Sodium bis(2-methoxyethoxy)aluminum hydride, NaAlH2(OCH2CH2OCH3)2, commercially known as Vitride® or Red-Al®, enables rapid synthesis of pure optically active N-protected amino alcohols and peptide alcohols in very high yields. The method is very simple and attractive, as it does not require an additional step of N-protected amino acid derivatization and proceeds without the loss of enantiomeric homogeneity.

Fine tuning the structure of the Cu2+ complex with the prion protein chicken repeat by proline isomerization

The interaction between the single hexarepeat unit of chicken prion protein [ChPrP(54-59)] and Cu(II) was investigated by NMR, finding different coordination modes for the trans/trans and cis/trans isomers.

Synthesis of orthogonally protected vicinal diamines with amino acid-based skeleton

A convenient route to amino acid-based orthogonally protected 1,2-diamines starting from materials readily available for a peptide chemist is presented. The key step of the procedure is the Mitsunobu reaction ofN-protected aminoalcohol, obtained by the reduction of commercially available Z- or Boc-protected amino acid, with imidodicarbonate or sulfonylcarbamate related to standard amino-protecting groups used in peptide chemistry yielding triprotected vicinal diamines.

The impact of human cystatin C hinge loop L1 on its dimerization and oligomerization propensity

Gonadotropin Releasing Hormone (pGlu-His-Trp-Ser-Tyr-Gly-Leu- Arg-Pro-Gly-NH2, GnRH) plays a signifi cant role in the controlling of gonadotropins and steroids hormones. A large number of linear GnRH analogues has been synthesized and tested for several medical uses. Leuprolide acetate (pGlu-His-Trp-Ser-Tyr-(D)Leu-Leu-Arg-Pro-NHEt, LPA) is a potent GnRH agonist and is used to treat a wide range of sex hormone related disorders, including prostatic cancer, endometriosis and precocious puberty. Despite its widespread use, only limited information based on spectroscopic evidence regarding the solution conformation of Leuprolide are known. Moreover, non crystallographic data is available for the receptor of GnRH (G protein-coupled receptor). The aim of this study was to characterize the conformation of Leuprolide and its modifi ed linear analogue (pGlu-His-Trp-Ser-Tyr(OMe)-(D)Leu-Leu- Arg-Aze-NHEt) in DMSO solution (which simulates better the receptor environment) using Nuclear Magnetic Resonance (NMR) and Molecular Modeling techniques. By using both NMR and Molecular Modeling we have characterized the secondary structural preferences of these GnRH analogues.