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Attachment to host tissues is a critical step in the pathogenesis of most bacterial infections. Enterotoxigenic Escherichia coli (ETEC) remains one of the principal causes of infectious diarrhea in humans. The recent identification of additional ETEC surface molecules suggests that new targets may be exploited in vaccine development. The EtpA protein identified in ETEC H10407 is a large glycosylated adhesin secreted via the two-partner secretion system. EtpA requires its putative partner EtpB for translocation across the outer membrane (OM). We investigated the biochemical and electrophysiological properties of purified EtpB. We showed that EtpB is 65-kDa heat-modifiable protein localized to the OM. Electrophysiological experiments indicated that EtpB is able to form pores in planar lipid bilayer membranes with an asymmetric current, suggesting its functional asymmetry. The pore of EtpB frequently assumes an opened conformation and fluctuates between three well-defined conductance states. In silico analysis of the EtpB amino acid sequence and molecular modeling suggest that EtpB is similar to the well-known TpsB protein FhaC from Bordetella pertussis and has a C-terminal transmembrane beta-barrel domain that is occluded by an N-terminal alpha-helix, an extracellular loop, and two periplasmic polypeptide-transport-associated (POTRA) domains. Together, these data confirm that EtpB is a pore-forming protein mainly folded into a beta-barrel conformation and indicate that EtpB presents typical features of the OM TpsB proteins. [ABSTRACT FROM AUTHOR]

[Display omitted] • The HslVU protease of Trypanosomatids is a potential drug target. • We developed LmHslU C-terminal-derived peptides binding to LmHslV and potentially inhibiting HslVU assembly. • A SAR study of the C-terminal LmHslU dodecapeptide identified a potent hexapeptide. • Conjugated to a penetrating peptide, it was found to be toxic to parasites in culture. • The fluorescently-labelled conjugate entered the promastigote form of L. infantum. It is urgent to develop less toxic and more efficient treatments for leishmaniases and trypanosomiases. We explore the possibility to target the parasite mitochondrial HslVU protease, which is essential for growth and has no analogue in the human host. For this, we develop compounds potentially inhibiting the complex assembly by mimicking the C-terminal (C-ter) segment of the ATPase HslU. We previously showed that a dodecapeptide derived from Leishmania major HslU C-ter segment (LmC12-U2, Cpd 1) was able to bind to and activate the digestion of a fluorogenic substrate by LmHslV. Here, we present the study of its structure-activity relationships. By replacing each essential residue with related non-proteinogenic residues, we obtained more potent analogues. In particular, a cyclohexylglycine residue at position 11 (cpd 24) allowed a more than three-fold gain in potency while reducing the size of compound 24 from twelve to six residues (cpd 50) without significant loss of potency, opening the way toward short HslU C-ter peptidomimetics as potential inhibitors of HslV proteolytic function. Finally, conjugates constituted of LmC6-U2 analogues and a mitochondrial penetrating peptide were found to penetrate into the promastigote form of L. infantum and to inhibit the parasite growth without showing toxicity toward human THP-1 cells at the same concentration (i.e. 30 μM). [ABSTRACT FROM AUTHOR]

Summary: We have recently described 95 predicted α-helical coiled-coil peptides derived from putative Plasmodium falciparum erythrocytic stage proteins. Seventy peptides recognized with the highest level of prevalence by sera from three endemic areas were selected for further studies. In this study, we sequentially examined antibody responses to these synthetic peptides in two cohorts of children at risk of clinical malaria in Kilifi district in coastal Kenya, in order to characterize the level of peptide recognition by age, and the role of anti-peptide antibodies in protection from clinical malaria. Antibody levels from 268 children in the first cohort (Chonyi) were assayed against 70 peptides. Thirty-nine peptides were selected for further study in a second cohort (Junju). The rationale for the second cohort was to confirm those peptides identified as protective in the first cohort. The Junju cohort comprised of children aged 1–6 years old (inclusive). Children were actively followed up to identify episodes of febrile malaria in both cohorts. Of the 70 peptides examined, 32 showed significantly (p <0.05) increased antibody recognition in older children and 40 showed significantly increased antibody recognition in parasitaemic children. Ten peptides were associated with a significantly reduced odds ratio (OR) for an episode of clinical malaria in the first cohort of children and two of these peptides (LR146 and AS202.11) were associated with a significantly reduced OR in both cohorts. LR146 is derived from hypothetical protein PFB0145c in PlasmoDB. Previous work has identified this protein as a target of antibodies effective in antibody dependent cellular inhibition (ADCI). The current study substantiates further the potential of protein PFB0145c and also identifies protein PF11_0424 as another likely target of protective antibodies against P. falciparum malaria. [Copyright &y& Elsevier]

The retromer complex is required for the sorting of acid hydrolases to lysosomes, transcytosis of the polymeric immunoglobulin receptor, Wnt gradient formation, iron transporter recycling and processing of the amyloid precursor protein. Human retromer consists of two smaller complexes: the cargo recognition VPS26–VPS29–VPS35 heterotrimer and a membrane-targeting heterodimer or homodimer of SNX1 and/or SNX2 (ref. 13). Here we report the crystal structure of a VPS29–VPS35 subcomplex showing how the metallophosphoesterase-fold subunit VPS29 (refs 14, 15) acts as a scaffold for the carboxy-terminal half of VPS35. VPS35 forms a horseshoe-shaped, right-handed, α-helical solenoid, the concave face of which completely covers the metal-binding site of VPS29, whereas the convex face exposes a series of hydrophobic interhelical grooves. Electron microscopy shows that the intact VPS26–VPS29–VPS35 complex is a stick-shaped, flexible structure, approximately 21 nm long. A hybrid structural model derived from crystal structures, electron microscopy, interaction studies and bioinformatics shows that the α-solenoid fold extends the full length of VPS35, and that VPS26 is bound at the opposite end from VPS29. This extended structure presents multiple binding sites for the SNX complex and receptor cargo, and appears capable of flexing to conform to curved vesicular membranes. [ABSTRACT FROM AUTHOR]

Polyglutamylation of tubulin has been implicated in severalfunctions of microtubules, but the identification of the responsibleenzyme(s) has been challenging. We found that the neuronal tubulinpolyglutamylase is a protein complex containing a tubulin tyrosineligase-like (TTLL) protein, TTLL1. TTLL1 is a member of a large familyof proteins with a TTL homology domain, whose members could catalyzeligations of diverse amino acids to tubulins or other substrates. In themodel protist Tetrahymena thermophita, two conserved types ofpolyglutamylases were characterized that differ in substrate preferenceand subcellular localization. [ABSTRACT FROM AUTHOR]

• Globular domains flanking amyloid-forming regions can prevent the formation of cross-β amyloids. • Coarse-grained and rigid body simulation show that these proteins form cross-β oligomers. • The oligomerisation may provide insight into phase separation of proteins with amyloidogenic regions. • This study opens up opportunities for design of nano-oligomers with functional domains. Insoluble amyloid fibrils formed by self-assembly of amyloidogenic regions of proteins have a cross-β-structure. In this work, by using targeted molecular dynamics and rigid body simulation, we demonstrate that if a protein consists of an amyloidogenic region and a globular domain(s) and if the linker between them is short enough, such molecules cannot assemble into amyloid fibrils, instead, they form oligomers with a defined and limited number of β-strands in the cross-β core. We show that this blockage of the amyloid growth is due to the steric repulsion of the globular structures linked to amyloidogenic regions. Furthermore, we establish a relationship between the linker length and the number of monomers in such nanoparticles. We hypothesise that such oligomerisation can be a yet unrecognised way to form natural protein complexes involved in biological processes. Our results can also be used in protein engineering for designing soluble nanoparticles carrying different functional domains. [ABSTRACT FROM AUTHOR]

We investigated the effect of the point substitutions in the N-terminal domain of the yeast prion protein Sup35 (Sup35NMp) on the structure of its amyloid fibrils. As the objects of the study, proteins with mutations that have different influence on the PSI + prion propagation, but do not prevent the aggregation of Sup35NMp in vitro were chosen. The use of the wide range of physico-chemical methods allowed us to show significant differences in the structure of these aggregates, their physical size, clumping tendency. Also we demonstrated that the fluorescent probe thioflavin T (ThT) can be successfully used for investigation of subtle changes in the structural organization of fibrils formed from various Sup35NMp. The obtained results and our theoretical predictions allowed us to conclude that some of selected amino acid substitutions delimit the region of the protein that forms the core of amyloid fibrils, and change the fibrils structure. The relationship of structural features of in vitro Sup35NMp amyloid aggregates with the stability of the PSI + prion in vivo allowed us to suggest that oligopeptide repeats (R) of the amyloidogenic N-terminal domain of Sup35NMp from R0 to R2 play a key role in protein aggregation. Their arrangement rather than just presence is critical for propagation of the strong PSI + prion variants. The results confirm the suitability of the proposed combination of theoretical and empirical approaches for identifying changes in the amyloid fibrils structure, which, in turn, can significantly affect both the functional stability of amyloid fibrils and their pathogenicity. Unlabelled Image • Amino acid substitutions in Sup35p oligopeptide repeats change structure of amyloid aggregates. • The fluorescent probe ThT can be used for the investigation of subtle changes in the structural organization of amyloid. • The arrangement of the first two oligopeptide repeats of Sup35p is crucial for the propagation of strong PSI +] variants. [ABSTRACT FROM AUTHOR]

RepeatsDB (URL: https://repeatsdb.org) stands as a key resource for the classification and annotation of Structured Tandem Repeat Proteins (STRPs), incorporating data from both the Protein Data Bank (PDB) and AlphaFoldDB. This latest release features substantial advancements, including annotations for over 34 000 unique protein sequences from >2000 organisms, representing a fifteenfold increase in coverage. Leveraging state-of-the-art structural alignment tools, RepeatsDB now offers faster and more precise detection of STRPs across both experimental and predicted structures. Key improvements also include a redesigned user interface and enhanced web server, providing an intuitive browsing experience with improved data searchability and accessibility. A new statistics page allows users to explore database metrics based on repeat classifications, while API enhancements support scalability to manage the growing volume of data. These advancements not only refine the understanding of STRPs but also streamline annotation processes, further strengthening RepeatsDB's role in advancing our understanding of STRP functions., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Proteins with two similar motifs in tandem are one of the most common cases of tandem repeat proteins. The question arises: why is the first emerged repeat frequently fixed in the process of evolution, despite the ample opportunities to continue its multiplication at the DNA level? To answer this question, we systematically analyzed the structure and function of these proteins. Our analysis showed that, in the vast majority of cases, the structural repetitive units have a two-fold (C2) internal symmetry. These closed structures provide an internal structural limitation for the subsequent growth of the repeat number. Frequently, the units "swap" their secondary structure elements with each other. Moreover, the duplicated domains, in contrast to other tandem repeat proteins, form binding sites for small molecules around the axis of C2 symmetry. Thus, the closure of the C2 structures and the emergence of new functional sites around the axis of C2 symmetry provide plausible explanations for why a repeat, once appeared, becomes fixed in the evolutionary process. We have placed these structures within the general structural classification of tandem repeat proteins, classifying them as either Class IV or V depending on the size of the repetitive unit., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The development of fast, cheap and reliable methods to determine seroconversion against infectious agents is of great practical importance. In the context of the COVID-19 pandemic, an important issue is to study the rate of formation of the immune layer in the population of different regions, as well as the study of the formation of post-vaccination immunity in individuals after vaccination. Currently, the main method for this kind of research is enzyme immunoassay (ELISA, enzyme-linked immunosorbent assay). This technique is sufficiently sensitive and specific, but it requires significant time and material costs. We investigated the applicability of attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopy associated with machine learning in blood plasma to detect seroconversion against SARS-CoV-2. The study included samples of 60 patients. Clear spectral differences in plasma samples from recovered COVID-19 patients and conditionally healthy donors were identified using multivariate and statistical analysis. The results showed that ATR-FTIR spectroscopy, combined with principal components analysis (PCA) and linear discriminant analysis (LDA) or artificial neural network (ANN), made it possible to efficiently identify specimens from recovered COVID-19 patients. We built classification models based on PCA associated with LDA and ANN. Our analysis led to 87% accuracy for PCA-LDA model and 91% accuracy for ANN, respectively. Based on this proof-of-concept study, we believe this method could offer a simple, label-free, cost-effective tool for detecting seroconversion against SARS-CoV-2. This approach could be used as an alternative to ELISA., (© 2023 Wiley-VCH GmbH.)

The RhoGEF TRIO is known to play a major role in neuronal development by controlling actin cytoskeleton remodeling, primarily through the activation of the RAC1 GTPase. Numerous de novo mutations in the TRIO gene have been identified in individuals with neurodevelopmental disorders (NDDs). We have previously established the first phenotype/genotype correlation in TRIO-associated diseases, with striking correlation between the clinical features of the individuals and the opposite modulation of RAC1 activity by TRIO variants targeting different domains. The mutations hyperactivating RAC1 are of particular interest, as they are recurrently found in patients and are associated with a severe form of NDD and macrocephaly, indicating their importance in the etiology of the disease. Yet, it remains unknown how these pathogenic TRIO variants disrupt TRIO activity at a molecular level and how they affect neurodevelopmental processes such as axon outgrowth or guidance. Here we report an additional cohort of individuals carrying a pathogenic TRIO variant that reinforces our initial phenotype/genotype correlation. More importantly, by performing conformation predictions coupled to biochemical validation, we propose a model whereby TRIO is inhibited by an intramolecular fold and NDD-associated variants relieve this inhibition, leading to RAC1 hyperactivation. Moreover, we show that in cultured primary neurons and in the zebrafish developmental model, these gain-of-function variants differentially affect axon outgrowth and branching in vitro and in vivo, as compared to loss-of-function TRIO variants. In summary, by combining clinical, molecular, cellular and in vivo data, we provide compelling new evidence for the pathogenicity of novel genetic variants targeting the TRIO gene in NDDs. We report a novel mechanism whereby the fine-tuned regulation of TRIO activity is critical for proper neuronal development and is disrupted by pathogenic mutations., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

The Nipah and Hendra viruses (NiV and HeV) are biosafety level 4 human pathogens classified within the Henipavirus genus of the Paramyxoviridae family. In both NiV and HeV, the gene encoding the Phosphoprotein (P protein), an essential polymerase cofactor, also encodes the V and W proteins. These three proteins, which share an intrinsically disordered N-terminal domain (NTD) and have unique C-terminal domains (CTD), are all known to counteract the host innate immune response, with V and W acting by either counteracting or inhibiting Interferon (IFN) signaling. Recently, the ability of a short region within the shared NTD (i.e., PNT3) to form amyloid-like structures was reported. Here, we evaluated the relevance of each of three contiguous tyrosine residues located in a previously identified amyloidogenic motif (EYYY) within HeV PNT3 to the fibrillation process. Our results indicate that removal of a single tyrosine in this motif significantly decreases the ability to form fibrils independently of position, mainly affecting the elongation phase. In addition, we show that the C-terminal half of PNT3 has an inhibitory effect on fibril formation that may act as a molecular shield and could thus be a key domain in the regulation of PNT3 fibrillation. Finally, the kinetics of fibril formation for the two PNT3 variants with the highest and the lowest fibrillation propensity were studied by Taylor Dispersion Analysis (TDA). The results herein presented shed light onto the molecular mechanisms involved in fibril formation.

Most proteins fold into 3D structures that determine how they function and orchestrate the biological processes of the cell. Recent developments in computational methods for protein structure predictions have reached the accuracy of experimentally determined models. Although this has been independently verified, the implementation of these methods across structural-biology applications remains to be tested. Here, we evaluate the use of AlphaFold2 (AF2) predictions in the study of characteristic structural elements; the impact of missense variants; function and ligand binding site predictions; modeling of interactions; and modeling of experimental structural data. For 11 proteomes, an average of 25% additional residues can be confidently modeled when compared with homology modeling, identifying structural features rarely seen in the Protein Data Bank. AF2-based predictions of protein disorder and complexes surpass dedicated tools, and AF2 models can be used across diverse applications equally well compared with experimentally determined structures, when the confidence metrics are critically considered. In summary, we find that these advances are likely to have a transformative impact in structural biology and broader life-science research., (© 2022. The Author(s).)

The currently devastating pandemic of severe acute respiratory syndrome known as coronavirus disease 2019 or COVID-19 is caused by the coronavirus SARS-CoV-2. Both the virus and the disease have been extensively studied worldwide. A trimeric spike (S) protein expressed on the virus outer bilayer leaflet has been identified as a ligand that allows the virus to penetrate human host cells and cause infection. Its receptor-binding domain (RBD) interacts with the angiotensin-converting enzyme 2 (ACE2), the host-cell viral receptor, and is, therefore, the subject of intense research for the development of virus control means, particularly vaccines. In this work, we search for smaller fragments of the S protein able to elicit virus-neutralizing antibodies, suitable for production by peptide synthesis technology. Based on the analysis of available data, we selected a 72 aa long receptor binding motif (RBM 436-507 ) of RBD. We used ELISA to study the antibody response to each of the three antigens (S protein, its RBD domain and the RBM 436-507 synthetic peptide) in humans exposed to the infection and in immunized mice. The seroreactivity analysis showed that anti-RBM antibodies are produced in COVID-19 patients and immunized mice and may exert neutralizing function, although with a frequency lower than anti-S and -RBD. These results provide a basis for further studies towards the development of vaccines or treatments focused on specific regions of the S virus protein, which can benefit from the absence of folding problems, conformational constraints and other advantages of the peptide synthesis production., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Pratesi, Errante, Pacini, Peña-Moreno, Quiceno, Carotenuto, Balam, Konaté, Diakité, Arévalo-Herrera, Kajava, Rovero, Corradin, Migliorini, Papini and Herrera.)

Despite the global interest and the unprecedented number of scientific studies triggered by the COVID-19 pandemic, few data are available from developing and low-income countries. In these regions, communities live under the threat of various transmissible diseases aside from COVID-19, including malaria. This study aims to determine the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seroreactivity of antibodies from COVID-19 and pre-COVID-19 samples of individuals in Mali (West Africa). Blood samples from COVID-19 patients (n = 266) at Bamako Dermatology Hospital (HDB) and pre-COVID-19 donors (n = 283) from a previous malaria survey conducted in Dangassa village were tested by ELISA to assess IgG antibodies specific to the full-length spike (S) protein, the receptor-binding domain (RBD), and the receptor-binding motif (RBM 436-507 ). Study participants were categorized by age, gender, treatment duration for COVID-19, and comorbidities. In addition, the cross-seroreactivity of samples from pre-COVID-19, malaria-positive patients against the three antigens was assessed. Recognition of the SARS-CoV-2 proteins by sera from COVID-19 patients was 80.5% for S, 71.1% for RBD, and 31.9% for RBM ( p < 0.001). While antibody responses to S and RBD tended to be age-dependent, responses to RBM were not. Responses were not gender-dependent for any of the antigens. Higher antibody levels to S, RBD, and RBM at hospital entry were associated with shorter treatment durations, particularly for RBD ( p < 0.01). In contrast, higher body weights negatively influenced the anti-S antibody response, and asthma and diabetes weakened the anti-RBM antibody responses. Although lower, a significant cross-reactive antibody response to S (21.9%), RBD (6.7%), and RBM (8.8%) was detected in the pre-COVID-19 and malaria samples. Cross-reactive antibody responses to RBM were mostly associated ( p < 0.01) with the absence of current Plasmodium falciparum infection, warranting further study., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Traoré, Guindo, Konaté, Traoré, Diakité, Kanté, Dembélé, Cissé, Incandela, Kodio, Coulibaly, Faye, Kajava, Pratesi, Migliorini, Papini, Pacini, Rovero, Errante, Diakité, Arevalo-Herrera, Herrera, Corradin and Balam.)

In the last two decades it has become increasingly evident that a large number of proteins adopt either a fully or a partially disordered conformation. Intrinsically disordered proteins are ubiquitous proteins that fulfill essential biological functions while lacking a stable 3D structure. Their conformational heterogeneity is encoded by the amino acid sequence, thereby allowing intrinsically disordered proteins or regions to be recognized based on their sequence properties. The identification of disordered regions facilitates the functional annotation of proteins and is instrumental for delineating boundaries of protein domains amenable to crystallization. This chapter focuses on the methods currently employed for predicting protein disorder and identifying intrinsically disordered binding sites., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Prokaryotes growing at high temperatures have a high proportion of charged residues in their proteins to stabilize their 3D structure. By mining 175 disparate bacterial and archaeal proteomes we found that, against the general trend for charged residues, the frequency of aspartic acid residues decreases strongly as natural growth temperature increases. In search of the explanation, we hypothesized that the reason for such unusual correlation is the deleterious consequences of spontaneous chemical transformations of aspartate at high temperatures. Our subsequent statistical analysis supported this hypothesis. This finding reveals that organisms have likely adapted to high temperatures by minimizing the harmful consequences of spontaneous chemical transformations., (© 2021 Wiley Periodicals LLC.)

The p53 isoform, Δ133p53β, is critical in promoting cancer. Here we report that Δ133p53β activity is regulated through an aggregation-dependent mechanism. Δ133p53β aggregates were observed in cancer cells and tumour biopsies. The Δ133p53β aggregation depends on association with interacting partners including p63 family members or the CCT chaperone complex. Depletion of the CCT complex promotes accumulation of Δ133p53β aggregates and loss of Δ133p53β dependent cancer cell invasion. In contrast, association with p63 family members recruits Δ133p53β from aggregates increasing its intracellular mobility. Our study reveals novel mechanisms of cancer progression for p53 isoforms which are regulated through sequestration in aggregates and recruitment upon association with specific partners like p63 isoforms or CCT chaperone complex, that critically influence cancer cell features like EMT, migration and invasion., (© 2021. The Author(s).)

The RepeatsDB database (URL: https://repeatsdb.org/) provides annotations and classification for protein tandem repeat structures from the Protein Data Bank (PDB). Protein tandem repeats are ubiquitous in all branches of the tree of life. The accumulation of solved repeat structures provides new possibilities for classification and detection, but also increasing the need for annotation. Here we present RepeatsDB 3.0, which addresses these challenges and presents an extended classification scheme. The major conceptual change compared to the previous version is the hierarchical classification combining top levels based solely on structural similarity (Class > Topology > Fold) with two new levels (Clan > Family) requiring sequence similarity and describing repeat motifs in collaboration with Pfam. Data growth has been addressed with improved mechanisms for browsing the classification hierarchy. A new UniProt-centric view unifies the increasingly frequent annotation of structures from identical or similar sequences. This update of RepeatsDB aligns with our commitment to develop a resource that extracts, organizes and distributes specialized information on tandem repeat protein structures., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) was proposed by Vitaly V. Kushnirov in the Michael D. Ter-Avanesyan's laboratory as a method to compare sizes of amyloid aggregates. Currently, this method is widely used for amyloid investigation, but mostly as a qualitative approach. In this work, we assessed the possibilities and limitations of the quantitative analysis of amyloid aggregate size distribution using SDD-AGE results. For this purpose, we used aggregates of two well-characterized yeast amyloid-forming proteins, Sup35 and Rnq1, and developed a protocol to standardize image analysis and process the result. A detailed investigation of factors that may affect the results of SDD-AGE revealed that both the cell lysis method and electrophoresis conditions can substantially affect the estimation of aggregate size. Despite this, quantitative analysis of SDD-AGE results is possible when one needs to estimate and compare the size of aggregates on the same gel, or even in different experiments, if the experimental conditions are tightly controlled and additional standards are used.

Natural selection has endorsed proteins with amazing structures and functionalities that cannot be matched by synthetic means, explaining the exponential interest in developing protein-based materials. Protein self-assembly allows fabricating complex supramolecular structures from relatively simple building blocks, a bottom-up strategy naturally employed by amyloid fibrils. However, the design of amyloid-inspired materials with biological activity is inherently difficult. Here, we exploit a modular procedure to generate functional amyloid nanostructures with tight control of their mesoscopic properties. The soft amyloid core of a yeast prion was fused to dihydrofolate reductase through flexible linkers of different sizes. This enabled us to produce, for the first time, biocompatible protein-only amyloid-like oligomeric nanoparticles with defined dimensions in which the embedded enzyme remained highly active, as assessed by biophysical and enzymatic assays. The modular design allowed one to obtain multifunctional nanoparticles by incorporating the antibody-binding Z-domain to the protein fusion. We show how these assemblies can be exploited for antibody-directed targeting of specific cell types and the localized delivery of methotrexate, resulting in the intracellular uptake of the drug by cancer cells and their death. Overall, the novel protein particles we describe in this work might find applications in areas as diverse as biocatalysis, bioimaging, or targeted therapies.

Background: While mutations in the cardiac type 2 ryanodine receptor (RyR2) have been linked to exercise-induced or catecholaminergic polymorphic ventricular tachycardia (CPVT), its association with polymorphic ventricular tachycardia (PMVT) occurring at rest is unclear. We aimed at constructing a patient-specific human-induced pluripotent stem cell (hiPSC) model of PMVT occurring at rest linked to a single point mutation in RyR2., Methods: Blood samples were obtained from a patient with PMVT at rest due to a heterozygous RyR2-H29D mutation. Patient-specific hiPSCs were generated from the blood samples, and the hiPSC-derived cardiomyocytes (CMs) were generated via directed differentiation. Using CRIPSR/Cas9 technology, isogenic controls were generated by correcting the RyR2-H29D mutation. Using patch-clamp, fluorescent confocal microscopy and video-image-based analysis, the molecular and functional properties of RyR2-H29D hiPSCCMs and control hiPSCCMs were compared., Findings: RyR2-H29D hiPSCCMs exhibit intracellular sarcoplasmic reticulum (SR) Ca 2+ leak through RyR2 under physiological pacing. RyR2-H29D enhances the contribution of inositol 1,4,5-trisphosphate receptors to excitation-contraction coupling (ECC) that exacerbates abnormal Ca 2+ release in RyR2-H29D hiPSCCMs. RyR2-H29D hiPSCCMs exhibit shorter action potentials, delayed afterdepolarizations, arrhythmias and aberrant contractile properties compared to isogenic controls. The RyR2-H29D mutation causes post-translational remodeling that is fully reversed with isogenic controls., Interpretation: To conclude, in a model based on a RyR2 point mutation that is associated with short-coupled PMVT at rest, RyR2-H29D hiPSCCMs exhibited aberrant intracellular Ca 2+ homeostasis, shortened action potentials, arrhythmias and abnormal contractile properties., Funding: French Muscular Dystrophy Association (AFM; project 16,073, MNM2 2012 and 20,225), "Fondation de la Recherche Médicale" (FRM; SPF20130526710), "Institut National pour la Santé et la Recherche Médicale" (INSERM), National Institutes of Health (ARM; R01 HL145473) and New York State Department of Health (NYSTEM C029156)., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

The Database of Protein Disorder (DisProt, URL: https://disprot.org) provides manually curated annotations of intrinsically disordered proteins from the literature. Here we report recent developments with DisProt (version 8), including the doubling of protein entries, a new disorder ontology, improvements of the annotation format and a completely new website. The website includes a redesigned graphical interface, a better search engine, a clearer API for programmatic access and a new annotation interface that integrates text mining technologies. The new entry format provides a greater flexibility, simplifies maintenance and allows the capture of more information from the literature. The new disorder ontology has been formalized and made interoperable by adopting the OWL format, as well as its structure and term definitions have been improved. The new annotation interface has made the curation process faster and more effective. We recently showed that new DisProt annotations can be effectively used to train and validate disorder predictors. We believe the growth of DisProt will accelerate, contributing to the improvement of function and disorder predictors and therefore to illuminate the 'dark' proteome., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The widespread occurrence of repetitive stretches of DNA in genomes of organisms across the tree of life imposes fundamental challenges for sequencing, genome assembly, and automated annotation of genes and proteins. This multi-level problem can lead to errors in genome and protein databases that are often not recognized or acknowledged. As a consequence, end users working with sequences with repetitive regions are faced with 'ready-to-use' deposited data whose trustworthiness is difficult to determine, let alone to quantify. Here, we provide a review of the problems associated with tandem repeat sequences that originate from different stages during the sequencing-assembly-annotation-deposition workflow, and that may proliferate in public database repositories affecting all downstream analyses. As a case study, we provide examples of the Atlantic cod genome, whose sequencing and assembly were hindered by a particularly high prevalence of tandem repeats. We complement this case study with examples from other species, where mis-annotations and sequencing errors have propagated into protein databases. With this review, we aim to raise the awareness level within the community of database users, and alert scientists working in the underlying workflow of database creation that the data they omit or improperly assemble may well contain important biological information valuable to others., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

A number of [ PSI + ]-no-more (PNM) mutations, eliminating [ PSI + ] prion, were previously described in SUP35 . In this study, we designed and analyzed a new PNM mutation based on the parallel in-register β-structure of Sup35 prion fibrils suggested by the known experimental data. In such an arrangement, substitution of non-charged residues by charged ones may destabilize the fibril structure. We introduced Q33K/A34K amino acid substitutions into the Sup35 protein, corresponding allele was called sup35-M0 . The mutagenized residues were chosen based on ArchCandy in silico prediction of high inhibitory effect on the amyloidogenic potential of Sup35. The experiments confirmed that Sup35-M0 leads to the elimination of [ PSI + ] with high efficiency. Our data suggested that the elimination of the [ PSI + ] prion is associated with the decreased aggregation properties of the protein. The new mutation can induce the prion with very low efficiency and is able to propagate only weak [ PSI + ] prion variants. We also showed that Sup35-M0 protein co-aggregates with the wild-type Sup35 in vivo . Moreover, our data confirmed the utility of the strategy of substitution of non-charged residues by charged ones to design new mutations to inhibit a prion formation., (Copyright © 2019 Danilov, Matveenko, Ryzhkova, Belousov, Poleshchuk, Likholetova, Sokolov, Kasyanenko, Kajava, Zhouravleva and Bondarev.)

There has been an increased interest in computational methods for amyloid and (or) aggregate prediction, due to the prevalence of these aggregates in numerous diseases and their recently discovered functional importance. To evaluate these methods, several datasets have been compiled. Typically, aggregation-prone regions of proteins, which form aggregates or amyloids in vivo, are more than 15 residues long and intrinsically disordered. However, the number of such experimentally established amyloid forming and non-forming sequences are limited, not exceeding one hundred entries in existing databases. In this work, we parsed all available NMR-resolved protein structures from the PDB and assembled a new, sevenfold larger, dataset of unfolded sequences, soluble at high concentrations. We proposed to use these sequences as a negative set for evaluating methods for predicting aggregation in vivo. We also present the results of benchmarking cutting edge tools for the prediction of aggregation versus solubility propensity., (© 2017 The Protein Society.)

The Database of Protein Disorder (DisProt, URL: www.disprot.org) has been significantly updated and upgraded since its last major renewal in 2007. The current release holds information on more than 800 entries of IDPs/IDRs, i.e. intrinsically disordered proteins or regions that exist and function without a well-defined three-dimensional structure. We have re-curated previous entries to purge DisProt from conflicting cases, and also upgraded the functional classification scheme to reflect continuous advance in the field in the past 10 years or so. We define IDPs as proteins that are disordered along their entire sequence, i.e. entirely lack structural elements, and IDRs as regions that are at least five consecutive residues without well-defined structure. We base our assessment of disorder strictly on experimental evidence, such as X-ray crystallography and nuclear magnetic resonance (primary techniques) and a broad range of other experimental approaches (secondary techniques). Confident and ambiguous annotations are highlighted separately. DisProt 7.0 presents classified knowledge regarding the experimental characterization and functional annotations of IDPs/IDRs, and is intended to provide an invaluable resource for the research community for a better understanding structural disorder and for developing better computational tools for studying disordered proteins., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Neurodegenerative diseases and other amyloidoses are linked to the formation of amyloid fibrils. It has been shown that the ability to form these fibrils is coded by the amino acid sequence. Existing methods for the prediction of amyloidogenicity generate an unsatisfactory high number of false positives when tested against sequences of the disease-related proteins., Methods: Recently, it has been shown that the three-dimensional structure of a majority of disease-related amyloid fibrils contains a β-strand-loop-β-strand motif called β-arch. Using this information, we have developed a novel bioinformatics approach for the prediction of amyloidogenicity., Results: The benchmark results show the superior performance of our method over the existing programs., Conclusions: As genome sequencing becomes more affordable, our method provides an opportunity to create individual risk profiles for the neurodegenerative, age-related, and other diseases ushering in an era of personalized medicine. It will also be used in the large-scale analysis of proteomes to find new amyloidogenic proteins., (Copyright © 2015 The Alzheimer's Association. Published by Elsevier Inc. All rights reserved.)

Numerous studies have shown that the ability to form amyloid fibrils is an inherent property of the polypeptide chain. This has lead to the development of several computational approaches to predict amyloidogenicity by amino acid sequences. Here, we discuss the principles governing these methods, and evaluate them using several datasets. They deliver excellent performance in the tests made using short peptides (~6 residues). However, there is a general tendency towards a high number of false positives when tested against longer sequences. This shortcoming needs to be addressed as these longer sequences are linked to diseases. Recent structural studies have shown that the core element of the majority of disease-related amyloid fibrils is a β-strand-loop-β-strand motif called β-arch. This insight provides an opportunity to substantially improve the prediction of amyloids produced by natural proteins, ushering in an era of personalized medicine based on genome analysis., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Tandem repeats (TRs) represent one of the most prevalent features of genomic sequences. Due to their abundance and functional significance, a plethora of detection tools has been devised over the last two decades. Despite the longstanding interest, TR detection is still not resolved. Our large-scale tests reveal that current detectors produce different, often nonoverlapping inferences, reflecting characteristics of the underlying algorithms rather than the true distribution of TRs in genomic data. Our simulations show that the power of detecting TRs depends on the degree of their divergence, and repeat characteristics such as the length of the minimal repeat unit and their number in tandem. To reconcile the diverse predictions of current algorithms, we propose and evaluate several statistical criteria for measuring the quality of predicted repeat units. In particular, we propose a model-based phylogenetic classifier, entailing a maximum-likelihood estimation of the repeat divergence. Applied in conjunction with the state of the art detectors, our statistical classification scheme for inferred repeats allows to filter out false-positive predictions. Since different algorithms appear to specialize at predicting TRs with certain properties, we advise applying multiple detectors with subsequent filtering to obtain the most complete set of genuine repeats.

The review covers the development of synthetic peptides as vaccine candidates for Plasmodium falciparum- and Plasmodium vivax-induced malaria from its beginning up to date and the concomitant progress of solid phase peptide synthesis (SPPS) that enables the production of long peptides in a routine fashion. The review also stresses the development of other complementary tools and actions in order to achieve the long sought goal of an efficacious malaria vaccine., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Amyloid fibrils are filamentous protein aggregates that accumulate in diseases such as Alzheimer's or type II diabetes. The amyloid-forming protein is disease specific. Amyloids may also be formed in vitro from many other proteins, after first denaturing them. Unlike the diverse native folds of these proteins, their amyloids are fundamentally similar in being rigid, smooth-sided, and cross-beta-structured, that is, with beta strands running perpendicular to the fibril axis. In the absence of high-resolution fibril structures, increasingly credible models are being derived by integrating data from a crossfire of experimental techniques. Most current models of disease-related amyloids invoke "beta arcades," columnar structures produced by in-register stacking of "beta arches." A beta arch is a strand-turn-strand motif in which the two beta strands interact via their side chains, not via the polypeptide backbone as in a conventional beta hairpin. Crystal structures of beta-solenoids, a class of proteins with amyloid-like properties, offer insight into the beta-arc turns found in beta arches. General conformational and thermodynamic considerations suggest that complexes of 2 or more beta arches may nucleate amyloid fibrillogenesis in vivo. The apparent prevalence of beta arches and their components have implications for identifying amyloidogenic sequences, elucidating fibril polymorphisms, predicting the locations and conformations of beta arcs within amyloid fibrils, and refining existing fibril models.

The vast majority of protein sequences are aperiodic; they do not have any strong bias in the amino acid composition, and they use a subtle mixture of all or most of the 20 amino acid residues to code a great number of various structures and functions. In this context, homorepeats, runs of a single amino acid residue, represent unusual, eye-catching motifs in proteins. Despite the sequence simplicity and relatively small size, the homorepeat runs have a strong potential for molecular interactions due to the excessively high local concentration of a certain physico-chemical property. Appearance of such runs within proteins may give them new structural and functional features. An increasing number of studies demonstrate the abundance of these motifs in proteins, their important roles in biological processes, and their link to a number of hereditary and age-related diseases. In this chapter, we summarize data on the distribution of homorepeats in proteomes and on their structural properties, evolution, and functions., (Copyright 2010 Elsevier Inc. All rights reserved.)

Motivation: Over the last years a number of evidences have been accumulated about high incidence of tandem repeats in proteins carrying fundamental biological functions and being related to a number of human diseases. At the same time, frequently, protein repeats are strongly degenerated during evolution and, therefore, cannot be easily identified. To solve this problem, several computer programs which were based on different algorithms have been developed. Nevertheless, our tests showed that there is still room for improvement of methods for accurate and rapid detection of tandem repeats in proteins., Results: We developed a new program called T-REKS for ab initio identification of the tandem repeats. It is based on clustering of lengths between identical short strings by using a K-means algorithm. Benchmark of the existing programs and T-REKS on several sequence datasets is presented. Our program being linked to the Protein Repeat DataBase opens the way for large-scale analysis of protein tandem repeats. T-REKS can also be applied to the nucleotide sequences., Availability: The algorithm has been implemented in JAVA, the program is available upon request at http://bioinfo.montp.cnrs.fr/?r=t-reks. Protein Repeat DataBase generated by using T-REKS is accessible at http://bioinfo.montp.cnrs.fr/?r=repeatDB.

Background: The availability of the P. falciparum genome has led to novel ways to identify potential vaccine candidates. A new approach for antigen discovery based on the bioinformatic selection of heptad repeat motifs corresponding to alpha-helical coiled coil structures yielded promising results. To elucidate the question about the relationship between the coiled coil motifs and their sequence conservation, we have assessed the extent of polymorphism in putative alpha-helical coiled coil domains in culture strains, in natural populations and in the single nucleotide polymorphism data available at PlasmoDB., Methodology/principal Findings: 14 alpha-helical coiled coil domains were selected based on preclinical experimental evaluation. They were tested by PCR amplification and sequencing of different P. falciparum culture strains and field isolates. We found that only 3 out of 14 alpha-helical coiled coils showed point mutations and/or length polymorphisms. Based on promising immunological results 5 of these peptides were selected for further analysis. Direct sequencing of field samples from Papua New Guinea and Tanzania showed that 3 out of these 5 peptides were completely conserved. An in silico analysis of polymorphism was performed for all 166 putative alpha-helical coiled coil domains originally identified in the P. falciparum genome. We found that 82% (137/166) of these peptides were conserved, and for one peptide only the detected SNPs decreased substantially the probability score for alpha-helical coiled coil formation. More SNPs were found in arrays of almost perfect tandem repeats. In summary, the coiled coil structure prediction was rarely modified by SNPs. The analysis revealed a number of peptides with strictly conserved alpha-helical coiled coil motifs., Conclusion/significance: We conclude that the selection of alpha-helical coiled coil structural motifs is a valuable approach to identify potential vaccine targets showing a high degree of conservation.

To identify malaria antigens for vaccine development, we selected alpha-helical coiled coil domains of proteins predicted to be present in the parasite erythrocytic stage. The corresponding synthetic peptides are expected to mimic structurally "native" epitopes. Indeed the 95 chemically synthesized peptides were all specifically recognized by human immune sera, though at various prevalence. Peptide specific antibodies were obtained both by affinity-purification from malaria immune sera and by immunization of mice. These antibodies did not show significant cross reactions, i.e., they were specific for the original peptide, reacted with native parasite proteins in infected erythrocytes and several were active in inhibiting in vitro parasite growth. Circular dichroism studies indicated that the selected peptides assumed partial or high alpha-helical content. Thus, we demonstrate that the bioinformatics/chemical synthesis approach described here can lead to the rapid identification of molecules which target biologically active antibodies, thus identifying suitable vaccine candidates. This strategy can be, in principle, extended to vaccine discovery in a wide range of other pathogens.

Many virulence factors of gram-negative bacteria are secreted by the Type V secretion system via the autotransporter (AT) and two-partner secretion (TPS) pathways. AT proteins effect their own secretion. They comprise three domains: the amino-terminal leader sequence; the secreted passenger domain; and the translocator domain that forms the secretory channel. In the TPS pathway, the passenger and translocator domains are translated as separate proteins. In a previous publication, we proposed a beta-helical structure for the TPS passenger domain of the filamentous hemagglutinin (FHA) of Bordetella pertussis which contains two tracts, R1 and R2, of 19-residue sequence repeats and built molecular models for the R1 and R2 beta-helices. Here, we compare the structure predicted for R1 with the recently determined crystal structure of a fragment containing three R1 repeats and find close agreement, with an RMSD of 1.1A. In the interim, the number of known AT and TPS protein sequences has increased to >1000. To investigate the incidence of beta-helical structures among them, we carried out a sequence-based analysis and conclude that, despite wide diversity in the sizes and sequences of passenger domains, most of them contain beta-solenoids that we classify into thirteen types based on distinctive properties of their beta-coils (repeat length, numbers and lengths of beta-strands and turns, cross-sectional shape, presence of specific residues in certain positions) summarized in a 2D coil template. Some coil types are typical for conventional AT proteins, others, for TPS or trimeric AT proteins. Some beta-solenoids consist of stacked subdomains with coils of different types. To illustrate model-building from a coil template, we modeled a type-T4 beta-solenoid for TibA of Escherichia coli which is predicted to have two conserved polar residues, Thr and Gln, in interior positions.

Integrin receptors are the main mediators of cell adhesion to the extracellular matrix. They bind to their ligands by interacting with short amino acid sequences, such as the RGD sequence. Soluble, small RGD-based peptides have been used to block integrin-binding to ligands, thereby interfering with cell adhesion, migration and survival, while substrate-immobilized RGD sequences have been used to enhance cell binding to artificial surfaces. This approach has several important medical applications, e.g. in suppression of tumor angiogenesis or stimulation of bone formation around implants. However, the relatively weak affinity of short RGD-containing peptides often results in incomplete integrin inhibition or ineffective ligation. In this work, we designed and synthesized several new multivalent RGD-containing molecules and tested their ability to inhibit or to promote integrin-dependent cell adhesion when used in solution or immobilized on substrates, respectively. These molecules consist of an oligomeric structure formed by alpha-helical coiled coil peptides fused at their amino-terminal ends with an RGD-containing fragment. When immobilized on a substrate, these peptides specifically promoted integrin alphaVbeta3-dependent cell adhesion, but when used in solution, they blocked alphaVbeta3-dependent cell adhesion to the natural substrates fibronectin and vitronectin. One of the peptides was nearly 10-fold more efficient than fibronectin or vitronectin in promoting cell adhesion, and almost 100-fold more efficient than a linear RGD tripeptide in blocking adhesion. These results indicate that alpha-helical coiled coil peptides carrying an amino-terminal RGD motif can be used as soluble antagonists or surface-immobilized agonists to efficiently inhibit or promote integrin alphaVbeta3-mediated cell adhesion, respectively., (Copyright (c) 2005 European Peptide Society and John Wiley & Sons, Ltd.)

Beta-rolls and beta-helices belong to a larger group of topologically similar proteins with solenoid folds: because their regular secondary structure elements are exclusively beta-strands, they are referred to as beta-solenoids. The number of beta-solenoids whose structures are known is now large enough to support a systematic analysis. Here we survey the distinguishing structural features of beta-solenoids, also documenting their notable diversity. Appraisal of these structures suggests a classification based on handedness, twist, oligomerization state, and coil shape. In addition, beta-solenoids are distinguished by the number of chains that wind around a common axis: the majority are single-stranded but there is a recently discovered subset of triple-stranded beta-solenoids. This survey has revealed some relationships of the amino acid sequences of beta-solenoids with their structures and functions-in particular, the repetitive character of the coil sequences and conformations that recur in tracts of tandem repeats. We have proposed the term beta-arc for the distinctive turns found in beta-solenoids and beta-arch for the corresponding strand-turn-strand motifs. The evolutionary mechanisms underlying these proteins are also discussed. This analysis has direct implications for sequence-based detection, structural prediction, and de novo design of other beta-solenoid proteins. The abundance of virulence factors, toxins and allergens among beta-solenoids, as well as commonalities of beta-solenoids with amyloid fibrils, imply that this class of folds may have a broader role in human diseases than was previously recognized. Thus, identification of genes with putative beta-solenoid domains promises to be a fertile direction in the search for viable targets in the development of new antibiotics and vaccines.

The beta-form of protein folding, one of the earliest protein structures to be defined, was originally observed in studies of silks. It was then seen in early studies of synthetic polypeptides and, of course, is now known to be present in a variety of guises as an essential component of globular protein structures. However, in the last decade or so it has become clear that the beta-conformation of chains is present not only in many of the amyloid structures associated with, for example, Alzheimer's Disease, but also in the prion structures associated with the spongiform encephalopathies. Furthermore, X-ray crystallography studies have revealed the high incidence of the beta-fibrous proteins among virulence factors of pathogenic bacteria and viruses. Here we describe the basic forms of the beta-fold, summarize the many different new forms of beta-structural fibrous arrangements that have been discovered, and review advances in structural studies of amyloid and prion fibrils. These and other issues are described in detail in later chapters.

In its prion form, Ure2p, a regulator of nitrogen catabolism in Saccharomyces cerevisiae, polymerizes into filaments whereby its C-terminal regulatory domain is inactivated but retains its native fold. The filament has an amyloid fibril backbone formed by the Asn-rich, N-terminal, "prion" domain. The prion domain is also capable of forming fibrils when alone or when fused to other proteins. We have developed a model for the fibril that we call a parallel superpleated beta-structure. In this model, the prion domain is divided into nine seven-residue segments, each with a four-residue strand and a three-residue turn, that zig-zag in a planar serpentine arrangement. Serpentines are stacked axially, in register, generating an array of parallel beta-sheets, with a small and potentially variable left-hand twist. The interior of the filament is mostly stabilized not by packing of apolar side chains but by H-bond networks generated by the stacking of Asn side chains: charged residues are excluded. The model is consistent with current biophysical, biochemical, and structural data (notably, mass-per-unit-length measurements by scanning transmission electron microscopy that gave one subunit rise per 0.47 nm) and is readily adaptable to other amyloids, for instance the core of Sup35p filaments and glutamine expansions in huntingtin.

Self-assembling peptides present attractive platforms for engineering materials with controlled nanostructures. Recently, an alpha-helical fibril forming peptide (alphaFFP) was designed that self-assembles into nanofibrils at acid pH. Circular dichroism spectroscopy, electron-microscopy and x-ray fibre diffraction data showed that the most likely structure of alphaFFP fibrils is a five-stranded coiled coil rope. In the present study, scanning transmission electron microscopy (STEM) was used to improve our understanding of the alphaFFP fibril structure. The measurements of fibril mass per length suggest that there are ten alpha-helices in transverse sections of the fibrils. Based on the known data, it is proposed that a predominant fibrillar structure of alphaFFP is a dimer of alpha-helical five stranded protofilaments wrapped around a common axis. It is shown that these structures have an axial dimension of 58 +/- 16 nm and a width of 4 +/- 1 nm. A small number of thin fibrils is also observed in the negative stained preparation and STEM images. The thin fibrils may correspond to the single protofilament.

The alpha-helical solenoid proteins adopt a variety of elongated curved structures. They have been examined to identify the interactions that determine their curvature. A sequence pattern characteristic for strongly curved alpha-helical solenoids has been constructed and was found to match protein sequences containing the proteasome/cyclosome repeats. Based on this, a structural model of the repeat-containing domains of the Rpn1/S2 and Rpn2/S1 proteins, which represent the largest subunits of the 26 S proteasome, has been proposed. The model has a novel architecture resembling an alpha-helical toroid. Molecular modeling shows that these toroids have a central pore that would allow passage of an unfolded protein substrate through it. This implies that the Rpn1 and Rpn2 toroids are aligned along the common axial pores of the ATPase hexamer and form an "antechamber" of the 26 S proteasome. The proposed quaternary structure agrees with the available experimental data. It is suggested that the function of this antechamber is assistance to the ATPases in the unfolding of protein substrates prior to proteolysis. An evolutionary link between the PC repeat-containing proteins and tetratricopeptide repeat proteins is proposed.

Terminally differentiating stratified squamous epithelial cells assemble a specialized protective barrier structure on their periphery termed the cornified cell envelope (CE). It is composed of numerous structural proteins that become cross-linked by several transglutaminase enzymes into an insoluble macromolecular assembly. Several proteins are involved in the initial stages of CE assembly, but only certain proteins from a choice of more than 20 different proteins are used in the final stages of CE reinforcement, apparently to meet tissue-specific requirements. In addition, a variable selection of proteins may be upregulated in response to genetic defects of one of the CE proteins or tissue injury, in an effort to maintain an effective barrier. Additionally, in the epidermis and hair fiber cuticle, a layer of lipids is covalently attached to the proteins, which provides essential water barrier properties. Here we describe our current understanding of CE structure, a possible mechanism of its assembly, and various disorders that cause a defective barrier.