Human cystatin C (hCC), a small secretory protein, has gained attention beyond its classical role as a cysteine protease inhibitor owing to its potential involvement in neurodegenerative disorders. ...This study investigates the interaction between copper(II) ions Cu(II) and hCC, specifically targeting histidine residues known to participate in metal binding. Through various analytical techniques, including mutagenesis, circular dichroism, fluorescence assays, gel filtration chromatography, and electron microscopy, we evaluated the impact of Cu(II) ions on the structure and oligomerization of hCC. The results show that Cu(II) does not influence the secondary and tertiary structure of the studied hCC variants but affects their stability. To explore the Cu(II)‐binding site, nuclear magnetic resonance (NMR) and X‐ray studies were conducted. NMR experiments revealed notable changes in signal intensities and linewidths within the region 86His‐Asp‐Gln‐Pro‐His90, suggesting its involvement in Cu(II) coordination. Both histidine residues from this fragment were found to serve as a primary anchor of Cu(II) in solution, depending on the structural context and the presence of other Cu(II)‐binding agents. The presence of Cu(II) led to significant destabilization and altered thermal stability of the wild‐type and H90A variant, confirming differentiation between His residues in Cu(II) binding. In conclusion, this study provides valuable insights into the interaction between Cu(II) and hCC, elucidating the impact of copper ions on protein stability and identifying potential Cu(II)‐binding residues. Understanding these interactions enhances our knowledge of the role of copper in neurodegenerative disorders and may facilitate the development of therapeutic strategies targeting copper‐mediated processes in protein aggregation and associated pathologies.
This study investigates interactions between copper(II) ions, Cu(II) and human cystatin C (hCC), specifically targeting histidine residues, in the context of copper‐related impairment of proteins' structure and stability. The observed flexible and context‐depending binding of Cu(II) suggests a role for hCC as a platform for interactions with other biomolecules, potentially influencing neurodegenerative pathways. This deepens the understanding of Cu(II) contribution to protein destabilization, contributing to the research on copper's role in neurodegenerative disorders.
Neurodegenerative diseases, such as Alzheimer's disease (AD) and various types of amyloidosis, are incurable; therefore, understanding the mechanisms of amyloid decomposition is crucial to develop an ...effective drug against them for future therapies. It has been reported that one out of three people over the age of 85 are suffering from dementia as a comorbidity to AD. Amyloid beta (Aβ), the hallmark of AD, transforms structurally from monomers into β-stranded aggregates (fibrils) via multiple oligomeric states. Astrocytes in the central nervous system secrete the human cystatin C protein (HCC) in response to various proteases and cytokines. The codeposition of Aβ and HCC in the brains of patients with AD led to the hypothesis that cystatin C is implicated in the disease process. In this study, we investigate the intermolecular interactions between different atomic structures of fibrils formed by Aβ peptides and HCC to understand the pathological aggregation of these polypeptides into neurotoxic oligomers and then amyloid plaques. To characterize the interactions between Aβ and HCC, we used a complementary approach based on the combination of small-angle neutron scattering analysis, atomic force microscopy and computational modelling, allowing the exploration of the structures of multicomponent protein complexes. We report here an optimized protocol to study that interaction. The results show a dependency of the sequence length of the Aβ peptide on the ability of the associated HCC to disaggregate it.
Misfolding of the cellular prion protein (PrPC) is associated with the development of fatal neurodegenerative diseases called transmissible spongiform encephalopathies (TSEs). Metal ions appear to ...play a crucial role in PrPC misfolding. PrPC is a combined Cu(II) and Zn(II) metal-binding protein, where the main metal-binding site is located in the octarepeat (OR) region. Thus, the biological function of PrPC may involve the transport of divalent metal ions across membranes or buffering concentrations of divalent metal ions in the synaptic cleft. Recent studies have shown that an excess of Cu(II) ions can result in PrPC instability, oligomerization, and/or neuroinflammation. Here, we have used biophysical methods to characterize Cu(II) and Zn(II) binding to the isolated OR region of PrPC. Circular dichroism (CD) spectroscopy data suggest that the OR domain binds up to four Cu(II) ions or two Zn(II) ions. Binding of the first metal ion results in a structural transition from the polyproline II helix to the β-turn structure, while the binding of additional metal ions induces the formation of β-sheet structures. Fluorescence spectroscopy data indicate that the OR region can bind both Cu(II) and Zn(II) ions at neutral pH, but under acidic conditions, it binds only Cu(II) ions. Molecular dynamics simulations suggest that binding of either metal ion to the OR region results in the formation of β-hairpin structures. As the formation of β-sheet structures can be a first step toward amyloid formation, we propose that high concentrations of either Cu(II) or Zn(II) ions may have a pro-amyloid effect in TSE diseases.
In this paper, we present studies on the influence of the disulfide bridge on the copper (II) ions’ binding abilities by the cyclic His4-peptide. The studied ligand HKHPHRHC-S-S-C consists of nine ...amino acids. The cyclic structure was obtained through a disulfide bridge between two cysteinyl groups. Moreover, this peptide is characterized by the presence of four His residues in the sequence, which makes it an interesting ligand for transition metal ions. The potentiometric and spectroscopic (UV-Vis spectroscopy and circular dichroism spectroscopy (CD)) studies were carried out in various molar ligand to metal ratios: 2:1, 1:1, and 1:2, in the pH range of 2.5–11 at 25 °C. The results showed that the cyclic His4-peptide promotes dinuclear complexes in each of these systems and forms the final dinuclear species with the {NIm, 3N-amide}{NIm, 3N-amide} coordination mode. The obtained data shows that cyclization by the formation of the disulfide bond has an impact on the peptide chain flexibility and appearance of additional potential donors for metal ions and influences the copper (II) ions’ coordination.
The protein-polysaccharide fraction (AAF) isolated from the coelomic fluid of the earthworm Dendrobaena veneta destroys C. albicans cells by changing their morphology, disrupting cell division, and ...leading to cell death. Morphological changes in C. albicans cells induced by treatment with AAF were documented using DIC, SEM, and AFM. Congo Red staining showed that the fungal wall structure was changed after incubation with AAF. The effect on C. albicans cell walls was shown by AFM analysis of the surface roughness of fungal cell walls and changes in the wall thickness were visualized using Cryo-SEM. The FTIR analysis of C. albicans cells incubated with AAF indicated attachment of protein or peptide compounds to the fungal walls. The intact LC-ESI-MS analysis allowed accurate determination of the masses of molecules present in AAF. As shown by the chromatographic study, the fraction does not cross biological membranes. The Cryo-TEM analysis of AAF demonstrated the ability of smaller subunits to combine into larger agglomerates. AAF is thermally stable, which was confirmed by Raman spectroscopy. AAF can be considered as a potential antifungal antibiotic with activity against clinical C. albicans strains.
Amyloid fibrils have been known for many years. Unfortunately, their fame stems from negative aspects related to amyloid diseases. Nevertheless, due to their properties, they can be used as ...interesting nanomaterials. Apart from their remarkable stability, amyloid fibrils may be regarded as a kind of a storage medium and as a source of active peptides. In many cases, their structure may guarantee a controlled and slow release of peptides in their active form; therefore, they can be used as a potential nanomaterial in drug delivery systems. In addition, amyloid fibrils display controllable stiffness, flexibility, and satisfactory mechanical strength. In addition, they can be modified and functionalized very easily. Understanding the structure and genesis of amyloid assemblies derived from a broad range of amyloidogenic proteins could help to better understand and use this unique material. One of the factors responsible for amyloid aggregation is the steric zipper. Here, we report the discovery of steric zipper-forming peptides in the sequence of the amyloidogenic protein, human cystatin C (HCC). The ability of short peptides derived from this fragment of HCC to form fibrillar structures with defined self-association characteristics and the factors influencing this aggregation are also presented in this paper.
It is recognized that interactions between most materials are governed by their surface properties and manifest themselves at the interface formed between them. To gain more insight into this thin ...layer, several methods have been deployed. Among them, spectroscopic methods have been thoroughly evaluated. Due to their exceptional sensitivity, data acquisition speed, and broad material tolerance they have been proven to be invaluable tools for surface analysis, used by scientists in many fields, for example, implant studies. Today, in modern medicine the use of implants is considered standard practice. The past two decades of constant development has established the importance of implants in dentistry, orthopedics, as well as extended their applications to other areas such as aesthetic medicine. Fundamental to the success of implants is the knowledge of the biological processes involved in interactions between an implant and its host tissue, which are directly connected to the type of implant material and its surface properties. This review aims to demonstrate the broad applications of spectroscopic methods in implant material studies, particularly discussing hard implants, surface composition studies, and surface-cell interactions.
Human cystatin C (HCC), a small protein, plays a crucial role in inhibition of cysteine proteases. The most common structural form of human cystatin C in crystals is a dimer, which has been evidenced ...both for the native protein and its mutants. In these structures, HCC dimers were formed through the mechanism of domain swapping. The structure of the monomeric form of human cystatin C was determined for V57N mutant and the mutant with the engineered disulfide bond (L47C)–(G69C) (known as stab1-HCC). On the basis of stab1-HCC, a number of covalently stabilized oligomers, including also dimers have been obtained. The aim of this study was to analyze the structure of the covalently stabilized dimer HCC in solution by the small angle X-ray scattering (SAXS) technique and synchrotron radiation. Experimental data confirmed that in solution this protein forms a dimer, which is characterized by the radius of gyration RG=3.1nm and maximum intramolecular distance Dmax=10.3nm. Using the ab initio method and program DAMMIN, we propose a low resolution structure of stabilized covalently cystatin C in solution. Stab-HCC dimer adopts in solution an elongated conformation, which is well reconstructed by the ab initio model.
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.
Nine peptides derived from the sequence of human cystatin C fragment fulfilling the requirements for effective steric zipper formation were synthesized, and their fibrillization propensities were assessed. The obtained results confirmed the presence of 2 short amino acid stretches, located next to each other, forming one super‐steric zipper motif. This hCC fragment might be responsible for the enhanced amyloidogenic properties of dimeric or partially unfolded hCC.
Nucleases are an important group of hydrolases that degrade nucleic acids, with broad spectrum of applications in science and industry. In this paper, we report the identification and ...characterization of the nuclease from extremely psychrophilic bacterium
Psychromonas ingrahamii
that grows exponentially at 5 °C, but may also grow at even lower temperatures (down to − 12 °C). The putative endonuclease I gene, identified in the genome of
P. ingrahamii
, was cloned and expressed in
Pichia pastoris
. The recombinant protein was purified and its nucleolytic features were studied. The new enzyme, named by us as
Pin
Nuc, displays the features characteristic for the nonselective endonucleases, and has the ability to degrade different forms of nucleic acids. It is very active at room temperature in low ion-strength buffer and in the presence of low concentrations of magnesium ions. The enzyme, which possesses six cysteine residues, the most likely all engaged in disulphide bridges, is active only in oxidized form, and can be efficiently inactivated by the addition of low amounts of a reducing agent. According to our knowledge, it is the first nuclease, belonging to endonuclease I family, isolated from such extremely psychrophilic organism.
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