Peptides Of Different Lengths Research Articles

The HLA class I immunopeptidomes of AAV capsid proteins.

Cellular immune responses against AAV vector capsid represent an obstacle for successful gene therapy. Previous studies have used overlapping peptides spanning the entire capsid sequence to identify T cell epitopes recognized by AAV-specific CD8+ T cells. However, the repertoire of peptides naturally displayed by HLA class I molecules for CD8 T cell recognition is unknown. Using mRNA transfected monocyte-derived dendritic cells (MDDCs) and MHC-associated peptide proteomics (MAPPs), we identified the HLA class I immunopeptidomes of AAV2, AAV6 and AAV9 capsids. MDDCs were isolated from a panel of healthy donors that have diverse alleles across the US population. mRNA-transfected MDDCs were lysed, the peptide:HLA complexes immunoprecipitated, and peptides eluted and analyzed by mass spectrometry. We identified 65 AAV capsid-derived peptides loaded on HLA class I molecules of mRNA transfected monocyte derived dendritic cells. The HLA class I peptides are distributed along the entire capsid and more than 60% are contained within HLA class II clusters. Most of the peptides are organized as single species, however we identified twelve clusters containing at least 2 peptides of different lengths. Only 9% of the identified peptides have been previously identified as T cell epitopes, demonstrating that the immunogenicity potential for the vast majority of the AAV HLA class I immunopeptidome remains uncharacterized. In contrast, 12 immunogenic epitopes identified before were not found to be naturally processed in our study. Remarkably, 11 naturally presented AAV peptides were highly conserved among the three serotypes analyzed suggesting the possibility of cross-reactive AAV-specific CD8 T cells. This work is the first comprehensive study identifying the naturally displayed HLA class I peptides derived from the capsid of AAVs. The results from this study can be used to generate strategies to assess immunogenicity risk and cross-reactivity among serotypes during gene therapies.

Deep serological profiling of the Trypanosoma cruzi TSSA antigen reveals different epitopes and modes of recognition by Chagas disease patients.

Trypanosoma cruzi, the agent of Chagas disease, displays a highly structured population, with multiple strains that can be grouped into 6-7 evolutionary lineages showing variable eco-epidemiological traits and likely also distinct disease-associated features. Previous works have shown that antibody responses to 'isoforms' of the polymorphic parasite antigen TSSA enable robust and sensitive identification of the infecting strain with near lineage-level resolution. To optimize the serotyping performance of this molecule, we herein used a combination of immunosignaturing approaches based on peptide microarrays and serum samples from Chagas disease patients to establish a deep linear B-cell epitope profiling of TSSA. Our assays revealed variations in the seroprevalence of TSSA isoforms among Chagas disease populations from different settings, hence strongly supporting the differential distribution of parasite lineages in domestic cycles across the Americas. Alanine scanning mutagenesis and the use of peptides of different lengths allowed us to identify key residues involved in antibody pairing and the presence of three discrete B-cell linear epitopes in TSSAII, the isoform with highest seroprevalence in human infections. Comprehensive screening of parasite genomic repositories led to the discovery of 9 novel T. cruzi TSSA variants and one TSSA sequence from the phylogenetically related bat parasite T. cruzi marinkellei. Further residue permutation analyses enabled the identification of diagnostically relevant or non-relevant substitutions among TSSA natural polymorphisms. Interestingly, T. cruzi marinkellei TSSA displayed specific serorecognition by one chronic Chagas disease patient from Colombia, which warrant further investigations on the diagnostic impact of such atypical TSSA. Overall, our findings shed new light into TSSA evolution, epitope landscape and modes of recognition by Chagas disease patients; and have practical implications for the design and/or evaluation of T. cruzi serotyping strategies.

Aβ43 levels determine the onset of pathological amyloid deposition

About 2% of Alzheimer's disease (AD) cases have early onset (FAD) and are caused by mutations in either Presenilins (PSEN1/2) or amyloid-β precursor protein (APP). PSEN1/2 catalyze production of Aβ peptides of different length from APP. Aβ peptides are the major components of amyloid plaques, a pathological lesion that characterizes AD. Analysis of mechanisms by which PSEN1/2 and APP mutations affect Aβ peptide compositions lead to the implication of the absolute or relative increase in Aβ42 in amyloid-β plaques formation. Here, to elucidate the formation of pathogenic Aβ cocktails leading to amyloid pathology, we utilized FAD rat knock-in models carrying the Swedish APP (Apps allele) and the PSEN1 L435F (Psen1LF allele) mutations. To accommodate the differences in the pathogenicity of rodent and human Aβ, these rat models are genetically engineered to express human Aβ species as both the Swedish mutant allele and the WT rat allele (called Apph) have been humanized in the Aβ-coding region. Analysis of the eight possible FAD mutant permutations indicates that the CNS levels of Aβ43, rather than absolute or relative increases in Aβ42, determine the onset of pathological amyloid deposition in FAD knock-in rats. Notably, Aβ43 was found in amyloid plaques in late onset AD and mild cognitive impairment cases, suggesting that the mechanisms initiating amyloid pathology in FAD knock-in rat reflect disease mechanisms driving amyloid pathology in late onset AD. This study helps clarifying the molecular determinants initiating amyloid pathology and supports therapeutic interventions targeting Aβ43 in AD.

Investigation of the Fuzzy Complex between RSV Nucleoprotein and Phosphoprotein to Optimize an Inhibition Assay by Fluorescence Polarization

The interaction between Respiratory Syncytial Virus phosphoprotein P and nucleoprotein N is essential for the formation of the holo RSV polymerase that carries out replication. In vitro screening of antivirals targeting the N-P protein interaction requires a molecular interaction model, ideally consisting of a complex between N protein and a short peptide corresponding to the C-terminal tail of the P protein. However, the flexibility of C-terminal P peptides as well as their phosphorylation status play a role in binding and may bias the outcome of an inhibition assay. We therefore investigated binding affinities and dynamics of this interaction by testing two N protein constructs and P peptides of different lengths and composition, using nuclear magnetic resonance and fluorescence polarization (FP). We show that, although the last C-terminal Phe241 residue is the main determinant for anchoring P to N, only longer peptides afford sub-micromolar affinity, despite increasing mobility towards the N-terminus. We investigated competitive binding by peptides and small compounds, including molecules used as fluorescent labels in FP. Based on these results, we draw optimized parameters for a robust RSV N-P inhibition assay and validated this assay with the M76 molecule, which displays antiviral properties, for further screening of chemical libraries.

From an Hsp90 - binding protein to a peptide drug.

The Lpl proteins represent a class of lipoproteins that was first described in the opportunistic bacterial pathogen Staphylococcus aureus, where they contribute to pathogenicity by enhancing F-actin levels of host epithelial cells and thereby increasing S. aureus internalization. The model Lpl protein, Lpl1 was shown to interact with the human heat shock proteins Hsp90α and Hsp90ß, suggesting that this interaction may trigger all observed activities. Here we synthesized Lpl1-derived peptides of different lengths and identified two overlapping peptides, namely, L13 and L15, which interacted with Hsp90α. Unlike Lpl1, the two peptides not only decreased F-actin levels and S. aureus internalization in epithelial cells but they also decreased phagocytosis by human CD14+ monocytes. The well-known Hsp90 inhibitor, geldanamycin, showed a similar effect. The peptides not only interacted directly with Hsp90α, but also with the mother protein Lpl1. While L15 and L13 significantly decreased lethality of S. aureus bacteremia in an insect model, geldanamycin did not. In a mouse bacteremia model L15 was found to significantly decreased weight loss and lethality. Although the molecular bases of the L15 effect is still elusive, in vitro data indicate that simultaneous treatment of host immune cells with L15 or L13 and S. aureus significantly increase IL-6 production. L15 and L13 represent not antibiotics but they cause a significant reduction in virulence of multidrug-resistant S. aureus strains in in vivo models. In this capacity, they can be an important drug alone or additive with other agents.

SARS-Arena: Sequence and Structure-Guided Selection of Conserved Peptides from SARS-related Coronaviruses for Novel Vaccine Development.

The pandemic caused by the SARS-CoV-2 virus, the agent responsible for the COVID-19 disease, has affected millions of people worldwide. There is constant search for new therapies to either prevent or mitigate the disease. Fortunately, we have observed the successful development of multiple vaccines. Most of them are focused on one viral envelope protein, the spike protein. However, such focused approaches may contribute for the rise of new variants, fueled by the constant selection pressure on envelope proteins, and the widespread dispersion of coronaviruses in nature. Therefore, it is important to examine other proteins, preferentially those that are less susceptible to selection pressure, such as the nucleocapsid (N) protein. Even though the N protein is less accessible to humoral response, peptides from its conserved regions can be presented by class I Human Leukocyte Antigen (HLA) molecules, eliciting an immune response mediated by T-cells. Given the increased number of protein sequences deposited in biological databases daily and the N protein conservation among viral strains, computational methods can be leveraged to discover potential new targets for SARS-CoV-2 and SARS-CoV-related viruses. Here we developed SARS-Arena, a user-friendly computational pipeline that can be used by practitioners of different levels of expertise for novel vaccine development. SARS-Arena combines sequence-based methods and structure-based analyses to (i) perform multiple sequence alignment (MSA) of SARS-CoV-related N protein sequences, (ii) recover candidate peptides of different lengths from conserved protein regions, and (iii) model the 3D structure of the conserved peptides in the context of different HLAs. We present two main Jupyter Notebook workflows that can help in the identification of new T-cell targets against SARS-CoV viruses. In fact, in a cross-reactive case study, our workflows identified a conserved N protein peptide (SPRWYFYYL) recognized by CD8+ T-cells in the context of HLA-B7+. SARS-Arena is available at https://github.com/KavrakiLab/SARS-Arena.

Role of Helical Structure in MBP Immunodominant Peptides for Efficient IgM Antibody Recognition in Multiple Sclerosis.

The involvement of Myelin Basic Protein (MBP) in Multiple Sclerosis (MS) has been widely discussed in the literature. This intrinsically disordered protein has an interesting α-helix motif, which can be considered as a conformational epitope. In this work we investigate the importance of the helical structure in antibody recognition by MBP peptides of different lengths. Firstly, we synthesized the peptide MBP (81–106) (1) and observed that its elongation at both N- and C-termini, to obtain the peptide MBP (76–116) (2) improves IgM antibody recognition in SP-ELISA, but destabilizes the helical structure. Conversely, in competitive ELISA, MBP (81–106) (1) is recognized more efficiently by IgM antibodies than MBP (76–116) (2), possibly thanks to its more stable helical structure observed in CD and NMR conformational experiments. These results are discussed in terms of different performances of peptide antigens in the two ELISA formats tested.

An internal docking site stabilizes substrate binding to γ-secretase: Analysis by molecular dynamics simulations

Amyloid precursor protein (APP) is cleaved and processed sequentially by γ-secretase yielding amyloid β (Aβ) peptides of different lengths. Longer Aβ peptides are associated with the formation of neurotoxic plaques related to Alzheimer’s disease. Based on the APP substrate-bound structure of γ-secretase, we investigated the enzyme-substrate interaction using molecular dynamics simulations and generated model structures that represent the sequentially cleaved intermediates during the processing reaction. The simulations indicated an internal docking site providing strong enzyme-substrate packing interaction. In the enzyme-substrate complex, it is located close to the region where the helical conformation of the substrate is interrupted and continues toward the active site in an extended conformation. The internal docking site consists of two non-polar pockets that are preferentially filled by large hydrophobic or aromatic substrate side chains to stabilize binding. Placement of smaller residues such as glycine can trigger a shift in the cleavage pattern during the simulations or results in destabilization of substrate binding. The reduced packing by smaller residues also influences the hydration of the active site and the formation of a catalytically active state. The simulations on processed substrate intermediates and a substrate G33I mutation offer an explanation of the experimentally observed relative increase of short Aβ fragment production for this mutation. In addition, studies on a substrate K28A mutation indicate that the internal docking site opposes the tendency of substrate dissociation due to a hydrophobic mismatch at the membrane boundary caused by K28 during processing and substrate movement toward the enzyme active site. The proposed internal docking site could also be useful for the specific design of new γ-secretase modulators.

Constructing Head-to-Tail Cyclic Peptide DNA-Encoded Libraries Using Two-Directional Synthesis Strategy.

Macrocyclic peptides are an important class of therapeutic agents for the biological targets that are difficult to modulate by small-molecule compounds. Meanwhile, DNA-encoded library technology (DELT) provides a powerful platform for hits discovery. The unity of both fields has proven highly productive in finding cyclic peptide hits against diverse pharmaceutical proteins. Many researchers have extended the chemical toolbox for constructing head-to-tail macrocyclic DNA-encoded libraries with various ring sizes. However, the linear peptides of different lengths necessitate tuning the distance between closing sites and DNA-linked sites to perform the macrocyclization process, presumably due to the constrained conformation of linear precursors. To tackle this issue and streamline the synthetic workflow, we report a two-directional synthesis strategy. This method starts from a trifunctional reagent and prepares DNA-linked macrocyclic peptides of ring size between 15 (5-mer) and 24 (8-mer) via amide bond formation reaction, a common method to create macrocyclic peptides.

A pathway to peptides in space through the condensation of atomic carbon

Organic molecules are widely present in the dense interstellar medium, and many have been synthesized in the laboratory on Earth under the conditions typical for an interstellar environment. Until now, however, only relatively small molecules of biological interest have been demonstrated to form experimentally under typical space conditions. Here we prove experimentally that the condensation of carbon atoms on the surface of cold solid particles (cosmic dust) leads to the formation of isomeric polyglycine monomers (aminoketene molecules). Following encounters between aminoketene molecules, they polymerize to produce peptides of different lengths. The chemistry involves three of the most abundant species (CO, C and NH3) present in star-forming molecular clouds, and proceeds via a novel pathway that skips the stage of amino acid formation in protein synthesis. The process is efficient, even at low temperatures, without irradiation or the presence of water. The delivery of biopolymers formed by this chemistry to rocky planets in the habitable zone might be an important element in the origins of life.

Biophysical Characterization of Pro-apoptotic BimBH3 Peptides Reveals an Unexpected Capacity for Self-Association

Bcl-2 proteins orchestrate the mitochondrial pathway of apoptosis, pivotal for cell death. Yet, the structural details of the conformational changes of pro- and antiapoptotic proteins and their interactions remain unclear. Pulse dipolar spectroscopy (double electron-electron resonance [DEER], also known as PELDOR) in combination with spin-labeled apoptotic Bcl-2 proteins unveils conformational changes and interactions of each protein player via detection of intra- and inter-protein distances. Here, we present the synthesis and characterization of pro-apoptotic BimBH3 peptides of different lengths carrying cysteines for labeling with nitroxide or gadolinium spin probes. We show by DEER that the length of the peptides modulates their homo-interactions in the absence of other Bcl-2 proteins and solve by X-ray crystallography the structure of a BimBH3 tetramer, revealing the molecular details of the inter-peptide interactions. Finally, we prove that using orthogonal labels and three-channel DEER we can disentangle the Bim-Bim, Bcl-xL-Bcl-xL, and Bim-Bcl-xL interactions in a simplified interactome.

Aminopeptidase N inhibitory peptides derived from hen eggs: Virtual screening, inhibitory activity, and action mechanisms

Aminopeptidase N (APN) is an important zinc-dependent metalloproteinase expressed on cell surfaces. This study was done to find APN inhibitory peptides using virtual screening and molecular docking. Hen egg proteins were cleaved using pepsin and trypsin in silico to peptides of different lengths. Dipeptides and tripeptides are more easily absorbed in the gastrointestinal tract (GIT). The bioactivity, solubility, absorption, distribution, metabolism, excretion, and toxicity properties of the dipeptides and tripeptides were then predicted. APN was used as a molecular docking target for potential peptides. The tripeptides CNR, CDR and GEF were docked with the APN residues, His388, His392, Glu411, Glu355 and Tyr477. An in vitro APN inhibition assay showed that the IC50 values of CNR, CDR and GEF against APN were 8.94, 6.42, and 61.7 mM, respectively.

Single‐Molecule Interaction of Peptides with a Biological Nanopore for Identification of Protease Activity

AbstractThe facile and sensitive detection of peptides is essential for drug screening, pathogen detection, and protein sequencing. There are still challenges for the real‐time single‐molecule sensing and detection of peptides due to their versatile shape, structure, and charges brought by amino acids. Nanopore sensing is an emerging technology for sensing of biomolecules including DNA, RNA, and proteins. In this study, the interaction between peptides of different lengths (N6–N10) and charges with an engineered Mycobacterium smegmatis porin A nanopore are systematically studied, and two types blockage events can be identified by quantifying their dwell times and amplitude of blockades. The findings are further applied to the label‐free and real‐time quantification of protease activity of caseinolytic protease P at nanomolar concentration in 14 min. The protease activity with inhibitor can also be monitored real time by nanopore assay. In summary, this nanopore‐based sensing platform shows promising capacity for peptide detection, protease activities assay, and inhibitor screening.

Terminal charges modulate the pore forming activity of cationic amphipathic helices

KIA peptides are a series of designer-made cationic amphipathic α-helical antimicrobial peptides of different lengths, based on the repetitive sequence [KIAGKIA]. They can form toroidal pores in membranes, wherein the helices are aligned in a transmembrane orientation. Solid-state 15N NMR is used here to differentiate between the surface-bound and transmembrane states. We find that the pore-forming activity increases when the peptides carry a positive charge (Lys residue) at the N-terminus, compared to a hydrophobic Ile-Ala N-terminal motif. In contrast, a positive charge at the C-terminus gives a lower membrane activity compared to C-terminal Ile-Ala. For peptides with otherwise identical sequence, a more than ten-fold difference in vesicle leakage can be observed, depending on which terminus carries the charge. This difference is attributed to a shift in the equilibrium between peptide helices oriented on the membrane surface and those inserted into the membrane in a pore-forming state. We show that the 3D hydrophobic moment can be used to predict which peptide sequence is more prone to form pores and will thereby show a higher membranolytic activity.

Familial L723P Mutation Can Shift the Distribution between the Alternative APP Transmembrane Domain Cleavage Cascades by Local Unfolding of the Ε-Cleavage Site Suggesting a Straightforward Mechanism of Alzheimer's Disease Pathogenesis.

Alzheimer's disease is an age-related pathology associated with accumulation of amyloid-β peptides, products of enzymatic cleavage of amyloid-β precursor protein (APP) by secretases. Several familial mutations causing early onset of the disease have been identified in the APP transmembrane (TM) domain. The mutations influence production of amyloid-β, but the molecular mechanisms of this effect are unclear. The "Australian" (L723P) mutation located in the C-termini of APP TM domain is associated with autosomal-dominant, early onset Alzheimer's disease. Herein, we describe the impact of familial L723P mutation on the structural-dynamic behavior of APP TM domain studied by high-resolution NMR in membrane-mimicking micelles and augmented by molecular dynamics simulations in explicit lipid bilayer. We found L723P mutation to cause local unfolding of the C-terminal turn of the APP TM domain helix and increase its accessibility to water required for cleavage of the protein backbone by γ-secretase in the ε-site, thus switching between alternative ("pathogenic" and "non-pathogenic") cleavage cascades. These findings suggest a straightforward mechanism of the pathogenesis associated with this mutation, and are of generic import for understanding the molecular-level events associated with APP sequential proteolysis resulting in accumulation of the pathogenic forms of amyloid-β. Moreover, age-related onset of Alzheimer's disease can be explained by a similar mechanism, where the effect of mutation is emulated by the impact of local environmental factors, such as oxidative stress and/or membrane lipid composition. Knowledge of the mechanisms regulating generation of amyloidogenic peptides of different lengths is essential for development of novel treatment strategies of the Alzheimer's disease.

Quantitative Electrochemical Analysis of Cathepsin Bactivity Using Carbon Nanofiber Nanoelectrode Arrays Withoptimized Peptide Substrate Length and Temperature

It is well known that the over-expression, increased activity and altered localization of many proteases are associated with tumor progression. Many clinical diagnoses target proteases as biomarkers. However, the quantitative analyses of the activity of tumor-related protease are challenging, because of the network interaction among the large set of proteases. Hence, there is a strong demand to develop a bioanalytical technique that can rapidly detect protease activities with high sensitivity and specificity. In this presentation, I will report an efficient electrochemical quantitative analyses method for detecting the cathepsin B activity by using a peptide-functionalized nanoelectrode array. The nanoelectrode array (NEA) was fabricated with vertically aligned carbon nanofibers (VACNFs), which serves as a unique electrochemical platform to enhance the protease detection. VACNFs of ~100-150 nm in diameter and ~5 mm in length are grown on chromium coated silicon wafer in uniform vertical alignment and are fully separated from each other to form a brush-like structure. With further processes to encapsulate the VACNFs in SiO2 by chemical vapor deposition (CVD) followed by mechanical polishing and reactive ion etching (RIE), ~100 nm long VACNF tips can be exposed above the SiO2 matrix, forming a stable NEA. By covalently attaching the exposed tip with proper peptide probes containing a ferrocene (Fc) redox tag at the distal, the VACNF NEA can be used to detect proteolysis with AC voltammetry. Such NEAs showed improved temporal resolution and reduced steric hindrance. As a result, reliable real-time proteolytic kinetics are recorded, from which the protease activity is quantitatively derived based on the heterogeneous Michalis-Menten model. In this work, we systematically investigated the cathepsin B activity with peptides of different length (including tetrapeptide, hexapeptide, and octapeptide) and at different temperatures (at 19.3, 29.5, 38.6, and 44.2 °C) to reveal their effects on the kinetic proteolysis rate by cathepsin B. The hexapeptide gave the highest proteolysis rate over tetrapeptide and octapeptide, as reflected by the smallest exponential decay time constant t. Meanwhile, 38.6 °C gave the highest proteolysis rate over the other 3 temperatures. At 38.6 °C, the optimized temperature, the limit of detection of cathepsin B activity and concentration has been determined to be 2.49 X 10-4 s-1 and 0.32 nM, respectively. Furthermore, the Fc-hexapeptide functionalized VACNF NEA shows very high selectivity, with essentially no measurable cross-reaction with 6.0 nM of other two cancer-related proteases, ADAM10 and ADAM17. This study demonstrates the potential to develop this method into a multiplex electronic chip for rapidly profiling the activity of up to 9 proteases simultaneously, which would facilitate accurate diagnoses of cancers and fast careening effective protease inhibitors as drug candidates. It would be a useful tool toward developing precision medicine.

Structural Modeling of γ-Secretase Aβ n Complex Formation and Substrate Processing.

The intramembrane aspartyl protease γ-secretase (GSEC) cleaves single-span transmembrane helices including the C-terminal fragment of the amyloid precursor protein (APP). This substrate is initially cleaved at the ϵ-site followed by successive processing (trimming) events mostly in steps of three amino acids. GSEC is responsible for the formation of N-terminal APP amyloid-β (Aβ) peptides of different length (e.g., Aβ42) that can form aggregates involved in Alzheimer's disease pathogenesis. The molecular mechanism of GSEC-APP substrate recognition is key for understanding how different peptide products are formed and could help in designing APP-selective modulators. Based on the known structure of apo GSEC and the APP-C99 fragment we have generated putative structural models of the initial binding in three different possible modes using extensive molecular dynamics (MD) simulations. The binding mode with the substrate helix located in a cleft between the transmembrane helices 2 and 3 of the presenilin subunit was identified as a most likely binding mode. Based on this arrangement, the processing steps were investigated using restraint MD simulations to pull the scissile bond (for each processing step) into a transition like (cleavable) state. This allowed us to analyze in detail the motions and energetic contributions of participating residues. The structural model agrees qualitatively well with the influence of many mutations in GSEC and C99. It also explains the effects of inhibitors, cross-linking, as well as spectroscopic data on GSEC substrate binding and can serve as working model for the future planning of structural and biochemical studies.

Intrinsic Disorder in Proteins with Pathogenic Repeat Expansions.

Intrinsically disordered proteins and proteins with intrinsically disordered regions have been shown to be highly prevalent in disease. Furthermore, disease-causing expansions of the regions containing tandem amino acid repeats often push repetitive proteins towards formation of irreversible aggregates. In fact, in disease-relevant proteins, the increased repeat length often positively correlates with the increased aggregation efficiency and the increased disease severity and penetrance, being negatively correlated with the age of disease onset. The major categories of repeat extensions involved in disease include poly-glutamine and poly-alanine homorepeats, which are often times located in the intrinsically disordered regions, as well as repeats in non-coding regions of genes typically encoding proteins with ordered structures. Repeats in such non-coding regions of genes can be expressed at the mRNA level. Although they can affect the expression levels of encoded proteins, they are not translated as parts of an affected protein and have no effect on its structure. However, in some cases, the repetitive mRNAs can be translated in a non-canonical manner, generating highly repetitive peptides of different length and amino acid composition. The repeat extension-caused aggregation of a repetitive protein may represent a pivotal step for its transformation into a proteotoxic entity that can lead to pathology. The goals of this article are to systematically analyze molecular mechanisms of the proteinopathies caused by the poly-glutamine and poly-alanine homorepeat expansion, as well as by the polypeptides generated as a result of the microsatellite expansions in non-coding gene regions and to examine the related proteins. We also present results of the analysis of the prevalence and functional roles of intrinsic disorder in proteins associated with pathological repeat expansions.

Origins of PDZ Binding Specificity. A Computational and Experimental Study Using NHERF1 and the Parathyroid Hormone Receptor.

Na+/H+ exchanger regulatory factor-1 (NHERF1) is a scaffolding protein containing two PSD95/discs large protein/ZO1 (PDZ) domains that modifies the signaling, trafficking, and function of the parathyroid hormone receptor (PTHR), a family B G-protein-coupled receptor. PTHR and NHERF1 bind through a PDZ-ligand-recognition mechanism. We show that PTH elicits phosphorylation of Thr591 in the canonical -ETVM binding motif of PTHR. Conservative substitution of Thr591 with Cys does not affect PTH(1-34)-induced cAMP production or binding of PTHR to NHERF1. The findings suggested the presence of additional sites upstream of the PDZ-ligand motif through which the two proteins interact. Structural determinants outside the canonical NHERF1 PDZ-PTHR interface that influence binding have not been characterized. We used molecular dynamics (MD) simulation to predict residues involved in these interactions. Simulation data demonstrate that the negatively charged Glu side chains at positions -3, -5, and -6 upstream of the PDZ binding motif are involved in PDZ-PTHR recognition. Engineered mutant peptides representing the PTHR C-terminal region were used to measure the binding affinity with NHERF1 PDZ domains. Comparable micromolar affinities for peptides of different length were confirmed by fluorescence polarization, isothermal titration calorimetry, and surface plasmon resonance. Binding affinities measured for Ala variants validate MD simulations. The linear relation between the change in enthalpy and entropy following Ala substitutions at upstream positions -3, -5, and -6 of the PTHR peptide provides a clear example of the thermodynamic compensation rule. Overall, our data highlight sequences in PTHR that contribute to NHERF1 interaction and can be altered to prevent phosphorylation-mediated inhibition.

Anti-Biofilm Activity of a Self-Aggregating Peptide against Streptococcus mutans.

Streptococcus mutans is the primary agent of dental cavities, in large part due to its ability to adhere to teeth and create a molecular scaffold of glucan polysaccharides on the tooth surface. Disrupting the architecture of S. mutans biofilms could help undermine the establishment of biofilm communities that cause cavities and tooth decay. Here we present a synthetic peptide P1, derived from a tick antifreeze protein, which significantly reduces S. mutans biofilm formation. Incubating cells with this peptide decreased biofilm biomass by approximately 75% in both a crystal violet microplate assay and an in vitro tooth model using saliva-coated hydroxyapatite discs. Bacteria treated with peptide P1 formed irregular biofilms with disconnected aggregates of cells and exopolymeric matrix that readily detached from surfaces. Peptide P1 can bind directly to S. mutans cells but does not possess bactericidal activity. Anti-biofilm activity was correlated with peptide aggregation and β-sheet formation in solution, and alternative synthetic peptides of different lengths or charge distribution did not inhibit biofilms. This anti-biofilm peptide interferes with S. mutans biofilm formation and architecture, and may have future applications in preventing bacterial buildup on teeth.