Structural Characterization of Covalently Stabilized Human Cystatin C Oligomers

Human cystatin C (HCC), a cysteine-protease inhibitor, exists as a folded monomer under physiological conditions but has the ability to self-assemble via domain swapping into multimeric states, including oligomers with a doughnut-like structure. The structure of the monomeric HCC has been solved by X-ray crystallography, and a covalently linked version of HCC (stab-1 HCC) is able to form stable oligomeric species containing 10−12 monomeric subunits. We have performed molecular modeling, and in conjunction with experimental parameters obtained from atomic force microscopy (AFM), transmission electron microscopy (TEM) and small-angle X-ray scattering (SAXS) measurements, we observe that the structures are essentially flat, with a height of about 2 nm, and the distance between the outer edge of the ring and the edge of the central cavity is ~5.1 nm. These dimensions correspond to the height and diameter of one stab-1 HCC subunit and we present a dodecamer model for stabilized cystatin C oligomers using molecular dynamics simulations and experimentally measured parameters. Given that oligomeric species in protein aggregation reactions are often transient and very highly heterogeneous, the structural information presented here on these isolated stab-1 HCC oligomers may be useful to further explore the physiological relevance of different structural species of cystatin C in relation to protein misfolding disease

Next-generation sequencing of a combinatorial peptide phage library screened against ubiquitin identifies peptide aptamers that can inhibit the in vitro ubiquitin transfer cascade

Defining dynamic protein–protein interactions in the ubiquitin conjugation reaction is a challenging research area. Generating peptide aptamers that target components such as ubiquitin itself, E1, E2, or E3 could provide tools to dissect novel features of the enzymatic cascade. Next-generation deep sequencing platforms were used to identify peptide sequences isolated from phage-peptide libraries screened against Ubiquitin and its ortholog NEDD8. In over three rounds of selection under differing wash criteria, over 13,000 peptides were acquired targeting ubiquitin, while over 10,000 peptides were selected against NEDD8. The overlap in peptides against these two proteins was less than 5% suggesting a high degree in specificity of Ubiquitin or NEDD8 toward linear peptide motifs. Two of these ubiquitin-binding peptides were identified that inhibit both E3 ubiquitin ligases MDM2 and CHIP. NMR analysis highlighted distinct modes of binding of the two different peptide aptamers. These data highlight the utility of using next-generation sequencing of combinatorial phage-peptide libraries to isolate peptide aptamers toward a protein target that can be used as a chemical tool in a complex multi-enzyme reaction

Structural Analysis of a Peptide Fragment of Transmembrane Transporter Protein Bilitranslocase

Using a combination of genomic and post-genomic approaches is rapidly altering the number of identified human influx carriers. A transmembrane protein bilitranslocase (TCDB 2.A.65) has long attracted attention because of its function as an organic anion carrier. It has also been identified as a potential membrane transporter for cellular uptake of several drugs and due to its implication in drug uptake, it is extremely important to advance the knowledge about its structure. However, at present, only the primary structure of bilitranslocase is known. In our work, transmembrane subunits of bilitranslocase were predicted by a previously developed chemometrics model and the stability of these polypeptide chains were studied by molecular dynamics (MD) simulation. Furthermore, sodium dodecyl sulfate (SDS) micelles were used as a model of cell membrane and herein we present a high-resolution 3D structure of an 18 amino acid residues long peptide corresponding to the third transmembrane part of bilitranslocase obtained by use of multidimensional NMR spectroscopy. It has been experimentally confirmed that one of the transmembrane segments of bilitranslocase has alpha helical structure with hydrophilic amino acid residues oriented towards one side, thus capable of forming a channel in the membrane

Why does the UL49.5 of herpes simplex 1 virus fail to inhibit the TAP-dependent antigen presentation?

Herpes simplex virus type 1 (HSV-1) is a well-studied herpesvirus that causes a number of human diseases. The HSV-1, like other herpesviruses, produces transmembrane glycoprotein N (gN / UL49.5 protein). Although this protein is conserved throughout the herpesvirus family, little is known about its function in HSV-1. The amino-acid sequence and length of UL49.5 proteins differ between herpesvirus species. It is, therefore, crucial to determine whether and to what extent the spatial structure of UL49.5 orthologs that are TAP inhibitors (i.e., of BoHV-1 virus) differs from that of non-TAP inhibitors (i.e., of HSV-1 virus). As a result, the primary goal of our study was to examine the 3D structure of HSV-1-encoded UL49.5 protein in an advanced model of the endoplasmic reticulum (ER) membrane. Circular dichroism (CD), 2D nuclear magnetic resonance (NMR), and multiple-microsecond all-atom molecular dynamics (MD) simulations in the ER membrane mimetic environment were used to determine the final structure of the HSV-1 UL49.5 protein. According to our findings, the N-terminus of HSV-1 UL49.5 adopts a highly flexible, unordered structure in the extracellular part due to the presence of a large number of Pro and Gly residues. In contrast to the UL49.5 protein from BoHV-1, the transmembrane region of HSV-1-encoded UL49.5 is formed by a single long transmembrane -helix, rather than two helices oriented perpendicularly, while the cytoplasmic part of the protein (C-terminus) has a short unordered structure. Our findings provide experimental structural information on HSV-1-encoded UL49.5 protein and structure-based insight into its lack of biological activity in inhibiting the TAP-independent antigen presentation pathway

Antibacterial Peptides in Dermatology–Strategies for Evaluation of Allergic Potential

During recent decades, the market for peptide-based drugs, including antimicrobial peptides, has vastly extended and evolved. These drugs can be useful in treatment of various types of disorders, e.g., cancer, autoimmune diseases, infections, and non-healing wounds. Although peptides are less immunogenic than other biologic therapeutics, they can still induce immune responses and cause allergies. It is important to evaluate the immunogenic and allergic potential of peptides before they are forwarded to the expensive stages of clinical trials. The process of the evaluation of immunogenicity and cytotoxicity is complicated, as in vitro models and bioinformatics tools cannot fully simulate situations in the clinic. Nevertheless, several potentially promising tests for the preclinical evaluation of peptide drugs have been implemented (e.g., cytotoxicity assays, the basophil activation test, and lymphocyte activation assays). In this review, we focus on strategies for evaluation of the allergic potential of peptide-based therapeutics

Spectroscopic Methods Used in Implant Material Studies

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

Governing the monomer-dimer ratio of human cystatin C by single amino acid substitution in the hinge region

Three dimensional domain swapping is one of the mechanisms involved in formation of insoluble aggregates of some amyloidogenic proteins. It has been proposed that proteins able to swap domains may share some common structural elements like conformationally constrained flexible turns/loops. We studied the role of loop L1 in the dimerization of human cystatin C using mutational analysis. Introduction of turn-favoring residues such as Asp or Asn into the loop sequence (in position 57) leads to a significant reduction of the dimer fraction in comparison with the wild type protein. On the other hand, introduction of a proline residue in position 57 leads to efficient dimer formation. Our results confirm the important role of the loop L1 in the dimerization process of human cystatin C and show that this process can be to some extent governed by single amino acid substitution

A structural model of the immune checkpoint CD160-HVEM complex derived from HDX-mass spectrometry and molecular modeling.

CD160 is a T cell coinhibitory molecule that interacts with the herpes virus entry mediator (HVEM) on antigen-presenting cells to provide an inhibitory signal to T cells. To date, the structure of CD160 and its complex with HVEM are unknown. Here, we have identified the fragments of CD160 interacting with HVEM using ELISA tests, hydrogen/deuterium studies, affinity chromatography and mass spectrometry (MS). By combining hydrogen/deuterium exchange and mass spectrometry (HDX-MS) we obtained key information about the tertiary structure of CD160, predicting the 3D structure of the CD160-HVEM complex. Our results provide insights into the molecular architecture of this complex, serving as a useful basis for designing inhibitors for future immunotherapies

Differential effects of various soy isoflavone dietary supplements (nutraceuticals) on bacterial growth and human fibroblast viability

Flavonoids, polyphenolic compounds present in many food products, affect growth of different bacterial species when tested as purified or synthetic substances. They can also influence gene expression in human cells, like fibroblasts. Here, we asked if soy isoflavone extracts, commonly used in many products sold as anti-menopausal dietary supplements, influence bacterial growth similarly to a synthetic isoflavone, genistein. Four commercially available products were tested in amounts corresponding to genistein concentrations causing inhibition of growth of Vibrio harveyi (a model bacterium sensitive to this isoflavone) and Escherichia coli (a model bacterium resistant to genistein). Differential effects of various extracts on V. harveyi and E. coli growth, from stimulation, to no changes, to inhibition, were observed. Moreover, contrary to genistein, the tested extracts caused a decrease (to different extent) in viability of human dermal fibroblasts. These results indicate that effects of various soy isoflavone extracts on bacterial growth and viability of human cells are different, despite similar declared composition of the commercially available products