DPPA as a Potential Cell Membrane Component Responsible for Binding Amyloidogenic Protein Human Cystatin C.

A phospholipid bilayer is a typical structure that serves crucial functions in various cells and organelles. However, it is not unusual for it to take part in pathological processes. The cell membrane may be a binding target for amyloid-forming proteins, becoming a factor modulating the oligomerization process leading to amyloid deposition-a hallmark of amyloidogenic diseases-e.g., Alzheimer's disease. The information on the mechanisms governing the oligomerization influenced by the protein-membrane interactions is scarce. Therefore, our study aims to describe the interactions between DPPA, a cell membrane mimetic, and amyloidogenic protein human cystatin C. Circular dichroism spectroscopy and differential scanning calorimetry were used to monitor (i) the secondary structure of the human cystatin C and (ii) the phase transition temperature of the DPPA, during the protein-membrane interactions. NMR techniques were used to determine the protein fragments responsible for the interactions, and molecular dynamics simulations were applied to provide a molecular structure representing the interaction. The obtained data indicate that the protein interacts with DPPA, submerging itself into the bilayer via the AS region. Additionally, the interaction increases the content of α-helix within the protein's secondary structure and stabilizes the whole molecule against denaturation.

Methods for monitoring protein-membrane binding. Comparison based on the interactions between amyloidogenic protein human cystatin C and phospholipid liposomes.

A cell membrane is an essential cellular component providing protection against the outer environment. It is also a host for proteins and carbohydrates responsible for, e.g. transporter, receptor, or enzymatic functions. In parallel, the membrane may also be implicated in pathological processes leading, e.g. to the oligomerization of amyloid-forming proteins, a hallmark of i.a. Alzheimer's disease. The increasing need for detailed information on mechanisms driving the amyloid formation and the potential role of cell membranes in the process proves the research on protein-membrane interactions biologically relevant. Considering the potential and limitations of the relatively well established and newly developed methods, this study focused on selecting methods that allow a broad and comprehensive description of interactions between amyloidogenic protein human cystatin C and lipid bilayers. In the first step, dot-blot and ELISA tests were selected as techniques allowing fast screening for protein-ligand interactions. Next, surface plasmon resonance, spectral shift, biolayer interferometry, and switchSENSE® technology were used to determine kinetic parameters and binding constants for interactions between human cystatin C and the selected lipid bilayers. Based on the obtained results we have proposed the most promising candidates for monitoring of interactions and determining affinity between amyloidogenic proteins and membrane mimetics.

Monomer-Dimer Equilibrium of Human Cystatin C During Internalization Into Cancer Cells.

Human cystatin C (hCC) is a physiologically important protein that serves as intra- and extracellular cysteine proteinase inhibitor in homeostasis. However, in pathological states it dimerizes and further oligomerizes accumulating into a toxic amyloid. HCC forms an active monomer in the extracellular space and becomes an inactive dimer when internalized in cellular organelles. However, hCC cell penetration and its oligomeric state during this process are not well understood. To determine if and how the oligomeric state influences hCC transmembrane migration, we investigated the internalization of the hCC wild type protein as well as three different mutants, which exclusively exist in the monomeric or multimeric state into HeLa cells via confocal fluorescence microscopy. Our results showed that the preferred pathway was endocytosis and that the oligomeric state did not significantly influence the internalization because both monomeric and dimeric hCC migrated into HeLa cells. Considering the differences of the active monomeric and the passive dimeric states of hCC, our findings contribute to a better understanding of the intra and extra cellular functions of hCC and their interaction with cysteine proteases.

Monitoring the interactions between POPG phospholipid bilayer and amyloid-forming protein human cystatin C. Does the bilayer influence the oligomeric state and structure of the protein?

A biological membrane is a structure characteristic for various cells and organelles present in almost all living organisms. Even though, it is one of the most common structures in organisms, where it serves crucial functions, a phospholipid bilayer may also take part in pathological processes leading to severe diseases. Research indicates that biological membranes have a profound impact on the pathological processes of oligomerization of amyloid-forming proteins. These processes are a hallmark of amyloid diseases, a group of pathological states involving, e.g., Parkinson's or Alzheimer's disease. Even though amyloidogenic diseases reap the harvest in modern societies, especially in elderly patients, the mechanisms governing the amyloid deposition are not clearly described. Therefore, the presented study focuses on the description of interactions between a model biological membrane (POPG) and one of amyloid forming proteins - human cystatin C. For the purpose of the study molecular dynamics simulations were applied to confirm interactions between the protein and POPG membrane. Next the NMR techniques were used to verify how the data obtained in solution compared to MD simulations and determine fragments of the protein responsible for interactions with POPG. Finally, circular dichroism was used to monitor the changes in secondary structure of the protein and size exclusion chromatography was used to monitor its oligomerization process. Obtained data indicates that the protein interacts with POPG submerging itself into the bilayer with the AS region. However, the presence of POPG bilayer does not significantly affect the structure or oligomerization process of human cystatin C.

Phospholipid-functionalized gold electrode for cellular membrane interface studies - interactions between DMPC bilayer and human cystatin C.

This work describes the electrochemical studies on the interactions between V57G mutant of human cystatin C (hCC V57G) and membrane bilayer immobilized on the surface of a gold electrode. The electrode was modified with 6-mercaptohexan-1-ol (MCH) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). DMPC was used as a membrane mimetic for monitoring electrochemical changes resulting from the interactions between the functionalized electrode surface and human cystatin C. The interactions between the modified electrode and hCC V57G were investigated by cyclic voltammetry and electrochemical impedance spectroscopy in a phosphate buffered saline (PBS) containing Fe(CN)63-/4- as a redox probe. The electrochemical measurements confirm that fabricated electrode is sensitive to hCC V57G at the concentration of 1 × 10-14 M. The incubation studies carried out at higher concentrations resulted in insignificant changes observed in cyclic voltammetry and electrochemical impedance spectroscopy measurements. The calculated values of surface coverage θR confirm that the electrode is equally covered at higher concentrations of hCC V57G. Measurements of wettability and surface free energy made it possible to determine the influence of individual structural elements of the modified gold electrode on its properties, and thus allowed to understand the nature of the interactions. Contact angle values confirmed the results obtained during electrochemical measurements, indicating the sensitivity of the electrode towards hCC V57G at the concentration of 1 × 10-14 M. In addition, the XPS spectra confirmed the successful anchoring of hCC V57G to the DMPC-functionalized surface.

Human cystatin C induces the disaggregation process of selected amyloid beta peptides: a structural and kinetic view.

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 (Aß), the hallmark of AD, transforms structurally from monomers into ß-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 Aß 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 Aß peptides and HCC to understand the pathological aggregation of these polypeptides into neurotoxic oligomers and then amyloid plaques. To characterize the interactions between Aß 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 Aß peptide on the ability of the associated HCC to disaggregate it.

Novel Venetin-1 nanoparticle from earthworm coelomic fluid as a promising agent for the treatment of non-small cell lung cancer.

The present research shows the antitumor activity of a protein-polysaccharide complex Venetin-1 obtained from the coelomic fluid of Dendrobaena veneta earthworms against A549 cancer cells. The investigations are a continuation of experiments on the antitumor activity of coelomic fluid obtained from this species. The Venetin-1 nanoparticle was obtained after thermal treatment of the coelomic fluid, separation from coelomocytes, filtration, and lyophilization. The preparation showed a selective effect on cancer cells, whereas normal cells were unaffected. Venetin-1 was effective against the lung cancer cells at doses of 31.3 and 62.5 µg/ml, and the results were imaged using light microscopy and scanning electron microscopy (SEM). The cells died mainly via the apoptosis pathway. Necrotic cells appeared sporadically in the microscopic view. SEM imaging revealed complete destruction of the A549 cells after the incubation with Venetin-1. The atomic force microscopy (AFM) analyses showed changes in the topography, peak force error images, and Young's modulus (elasticity) of the A549 cells after the incubation with Venetin-1. The transmission electron cryomicroscopy (Cryo-TEM) analysis indicated a polymeric nature of the analyzed preparation. The samples of Venetin-1 showed a very homogeneous size profile with the microparticle size of approximately 58.23 nm. A significant decrease in Venetin-1 binding to sphingomyelin was observed. Venetin-1 lost its pore-forming activity or deactivation of the pore-forming activity occurred. This confirms the absence of hemolytic capacity of Venetin-1 towards red blood cells. The conducted analyses show the suitability of the obtained complex for biomedical research. The next step will consist in analyses of the effect of Venetin-1 on the immune system in mice.

DMPC Phospholipid Bilayer as a Potential Interface for Human Cystatin C Oligomerization: Analysis of Protein-Liposome Interactions Using NMR Spectroscopy.

Studies revolving around mechanisms responsible for the development of amyloid-based diseases lay the foundations for the recognition of molecular targets of future to-be-developed treatments. However, the vast number of peptides and proteins known to be responsible for fibril formation, combined with their complexity and complexity of their interactions with various cellular components, renders this task extremely difficult and time-consuming. One of these proteins, human cystatin C (hCC), is a well-known and studied cysteine-protease inhibitor. While being a monomer in physiological conditions, under the necessary stimulus-usually a mutation, it tends to form fibrils, which later participate in the disease development. This process can potentially be regulated (in several ways) by many cellular components and it is being hypothesized that the cell membrane might play a key role in the oligomerization pathway. Studies involving cell membranes pose several difficulties; therefore, an alternative in the form of membrane mimetics is a very attractive solution. Here, we would like to present the first study on hCC oligomerization under the influence of phospholipid liposomes, acting as a membrane mimetic. The protein-mimetic interactions are studied utilizing circular dichroism, nuclear magnetic resonance, and size exclusion chromatography.

The Influence of the Mixed DPC:SDS Micelle on the Structure and Oligomerization Process of the Human Cystatin C.

Human cystatin C (hCC), a member of the superfamily of papain-like cysteine protease inhibitors, is the most widespread cystatin in human body fluids. Physiologically active hCC is a monomer, which dimerization and oligomerization lead to the formation of the inactive, insoluble amyloid form of the protein, strictly associated with cerebral amyloid angiopathy, a severe state causing death among young patients. It is known, that biological membranes may accelerate the oligomerization processes of amyloidogenic proteins. Therefore, in this study, we describe an influence of membrane mimetic environment-mixed dodecylphosphocholine:sodium dodecyl sulfate (DPC:SDS) micelle (molar ratio 5:1)-on the effect of the hCC oligomerization. The hCC-micelle interactions were analyzed with size exclusion chromatography, circular dichroism, and nuclear magnetic resonance spectroscopy. The experiments were performed on the wild-type (WT) cystatin C, and two hCC variants-V57P and V57G. Collected experimental data were supplemented with molecular dynamic simulations, making it possible to highlight the binding interface and select the residues involved in interactions with the micelle. Obtained data shows that the mixed DPC:SDS micelle does not accelerate the oligomerization of protein and even reverses the hCC dimerization process.

NMR and crystallographic structural studies of the extremely stable monomeric variant of human cystatin C with single amino acid substitution.

Human cystatin C (hCC), a member of the superfamily of papain-like cysteine protease inhibitors, is the most widespread cystatin in human body fluids. This small protein, in addition to its physiological function, is involved in various diseases, including cerebral amyloid angiopathy, cerebral hemorrhage, stroke, and dementia. Physiologically active hCC is a monomer. However, all structural studies based on crystallization led to the dimeric structure formed as a result of a three-dimensional exchange of the protein domains (3D domain swapping). The monomeric structure was obtained only for hCC variant V57N and for the protein stabilized by an additional disulfide bridge. With this study, we extend the number of models of monomeric hCC by an additional hCC variant with a single amino acid substitution in the flexible loop L1. The V57G variant was chosen for the X-ray and NMR structural analysis due to its exceptional conformational stability in solution. In this work, we show for the first time the structural and dynamics studies of human cystatin C variant in solution. We were also able to compare these data with the crystal structure of the hCC V57G and with other cystatins. The overall cystatin fold is retained in the solute form. Additionally, structural information concerning the N terminus was obtained during our studies and presented for the first time. DATABASE: Crystallographic structure: structural data are available in PDB databases under the accession number 6ROA. NMR structure: structural data are available in PDB and BMRB databases under the accession numbers 6RPV and 34399, respectively.

Proteins, peptides and peptidomimetics as active agents in implant surface functionalization.

The recent impact of implants on improving the human life quality has been enormous. During the past two decades we witnessed major advancements in both material and structural development of implants. They were driven mainly by the increasing patients' demand and the need to address the major issues that come along with the initially underestimated complexity of the bone-implant interface. While both, the materials and design of implants reached a certain, balanced state, recent years brought a shift in focus towards the bone-implant interface as the weakest link in the increasing implant long-term usability. As a result, several approaches were developed. They aimed at influencing and enhancing the implant osseointegration and its proper behavior when under load and stress. With this review, we would like to discuss the recent advancements in the field of implant surface modifications, emphasizing the importance of chemical methods, focusing on proteins, peptides and peptidomimetics as promising agents for titanium surface coatings.

Human cystatin C monomer, dimer, oligomer, and amyloid structures are related to health and disease.

Human cystatin C (hCC) is a small protein belonging to the cystatin family of papain-like cysteine proteinase inhibitors. We review the recent literature concerning structural aspects of hCC related to disease. We focus on the mechanisms of hCC dimerization, oligomerization, and amyloid formation. Amyloid formation is associated with a number of neurodegenerative diseases that affect the independence and quality of life of aging populations. hCC is one of the second-wave proteins that have been found to undergo amyloidosis associated with disease. For hCC, this includes cerebral amyloid angiopathy, as well as a disorder resulting in reduced male fertility.