Synthesis and physicochemical studies of amyloidogenic hexapeptides derived from human cystatin C

Abstract

Human cystatin C (hCC) is a low molecular mass protein that belongs to the cystatin superfamily. It is an inhibitor of extracellular cysteine proteinases, present in all human body fluids. At physiological conditions, hCC is a monomer, but it has a tendency to dimerization. Naturally occurring hCC mutant, with leucine in position 68 substituted by glutamine (L68Q), is directly involved in the formation of amyloid deposits, independently of other proteins. This process is the primary cause of hereditary cerebral amyloid angiopathy, observed mainly in the Icelandic population. Oligomerization and fibrillization processes of hCC are not explained equally well, but it is proposed that domain swapping is involved in both of them. Research carried out on the fibrillization process led to new hypothesis about the existence of a steric zipper motif in amyloidogenic proteins. In the hCC sequence, there are 2 fragments which may play the role of a steric zipper: the loop L1 region and the C-terminal fragment. In this work, we focused on the first of these. Nine hexapeptides covering studied hCC fragment were synthesized, and their fibrillogenic potential was assessed using an array of biophysical methods. The obtained results showed that the studied hCC fragment has strong profibrillogenic propensities because it contains 2 fragments fulfilling the requirements for an effective steric zipper located next to each other, forming 1 super-steric zipper motif. This hCC fragment might therefore be responsible for the enhanced amyloidogenic properties of dimeric or partially unfolded hCC.

Identification of a Steric Zipper Motif in the Amyloidogenic Core of Human Cystatin C and Its Use for the Design of Self-Assembling Peptides

“…In our previous work, we identified two consecutive steric zipper motives in the sequence of human cystatin C. They are located back to back in the region 55–65 and are comprised of six amino acid residues each. According to Eisenberg’s hypothesis, the steric zipper properties depend on a particular amino acid sequence, and its change (“scrambling”) should strongly impact the self-assembling properties [ 11 , 25 , 27 ]. Therefore, we decided to obtain two shuffled peptides with the sequences VIGAQV (CysZ11) and QAGIVV (CysZ13) and check their ability to form fibrils.…”

“…Some of the peptides studied by us previously had the tendency to form microcrystal-like assemblies [ 25 ]. Therefore, we decided to check whether they can be crystallized.…”

“…Our earlier studies showed that there is indeed a steric zipper motif region in human cystatin C spanning residues from Ala 52 to Asn 65 that coincide with so-called hinge loop L1, involved as a structural switch in the domain swapping ( Figure 1 ) [ 25 ]. However, without detailed structural studies, it is difficult to determine exactly which sequence forms the steric zipper motif.…”

“…However, without detailed structural studies, it is difficult to determine exactly which sequence forms the steric zipper motif. Based on our studies—aggregation test [ 25 ] and crystallization trials (data not published) [ 26 ]—we selected the two most promising peptides, 55 QIVAGV 60 (CysZ4) and 60 VNYFLD 65 (CysZ9), for detailed studies aimed at the evaluation of their potential to serve as an effective steric zipper driving the self-association and fibrillization of human cystatin C. To this end, structural studies such as atomic force microscopy (AFM), crystallization trials, and X-ray fiber diffraction were used, and the results of this part of our research are presented in this publication.…”

The Positive Side of the Alzheimer’s Disease Amyloid Cross-Interactions: The Case of the Aβ 1-42 Peptide with Tau, TTR, CysC, and ApoA1

“…Particularly, Ala-52 to Asp-65 fragment has been proved to have high fibrillization propensity and potentially to be able to form a steric zipper. In the protein structure (3GAX), this fragment is located in the first hairpin and consists of sequences of β-strands 2 and 3 and the loop L1 which connects these strands [189]. At the moment, nothing is known about the implication of the C-terminal fragment on the amyloid behavior of CysC but much more should be studied about this fragment because of its characteristic β-harpin conformation and the fulfilling conditions for being an effective steric zipper, probably the one that can recognize the α-helix intermediate conformation of Aβ.…”