Specific Amino Acid Sequences Research Articles

Computational Characterisation and Identification of Peptides for in silico Detection of Potentially Celiac-Toxic Proteins

This work reports on in silico analysis of celiac-toxic peptide occurrence in proteins. The toxic properties of celiac disease are linked to the presence of specific amino acid sequences and the properties of their environment. The analysed celiac-toxic peptides were found to be predominated by unordered structures of random coil and β-turns. Proline and glutamine-rich amino acid sequences from hydrophilic β-turns were exposed on the surface of the precursor proteins. The sequence motifs represented by gluten peptide epitopes or tetrapeptides with surroundings seem to represent an immunodominant structure. The application of MS BLAST software enabled identification of a few fragments with high degrees of identity to the toxic peptides in one protein sequence. Rich sources of celiac-disease-potentiating peptides were wheat gliadins, barley hordeins and rye secalins as well as low-molecular weight fractions of glutenin. In addition, amino acid sequences with a high degree of identity to the toxic peptides examined were detected in maize zein, oat avenin, protein of rice, yeast and chicken muscles, as well as β-casein and galanin.

Peptide Mapping of a Functionally Versatile Fimbrial Adhesin from Porphyromonas gingivalis

Porphyromonas gingivalis is strongly implicated in adult periodontitis. This oral pathogen expresses adhesive filamentous appendages, known as fimbriae, which constitute one of its major virulence factors. Fimbriae are composed of polymerized fimbrillin (FimA) subunits and play an indispensable role in the ability of P. gingivalis to colonize and invade periodontal tissue and to induce alveolar bone loss. The virulence potential of fimbriae is attributable to their capacity to interact with various dental or epithelial substrates, extracellular matrix proteins, other bacteria, and host immune cells. It has been puzzling whether the multifunctional adhesive ability of fimbriae results from multiple adhesion epitopes specific for each receptor, or whether fimbriae contain versatile structural motifs that are recognizable by multiple receptors. This review summarizes peptide mapping studies that have defined functional epitopes of P. gingivalis fimbriae. Available evidence suggests that the binding of fimbriae to various receptors generally involves specific amino acid sequences of the FimA subunit, although the same FimA peptide may occasionally recognize different receptors. Moreover, in cases where distinct FimA peptides interact with the same receptor, the peptides involved share common sequences. It therefore appears that the promiscuous binding reactivity of P. gingivalis fimbriae is attributable to a multitude of adhesion epitopes which however share minimal binding elements, although the overall hydrophobicity and polymeric nature of fimbriae may significantly enhance the avidity of binding interactions. Peptide mapping of fimbriae is significant also for translational purposes, such as for development of subunit vaccines that contain defined immunogenic and functionally important epitopes and for identification of peptides that can competitively inhibit virulence activities of P. gingivalis fimbriae.

Protein Particulates: Another Generic Form of Protein Aggregation?

Protein aggregation is a problem with a multitude of consequences, ranging from affecting protein expression to its implication in many diseases. Of recent interest is the specific form of aggregation leading to the formation of amyloid fibrils, structures associated with diseases such as Alzheimer’s disease. The ability to form amyloid fibrils is now regarded as a property generic to all polypeptide chains. Here we show that around the isoelectric point a different generic form of aggregation can also occur by studying seven widely different, nonrelated proteins that are also all known to form amyloid fibrils. Under these conditions gels consisting of relatively monodisperse spherical particulates are formed. Although these gels have been described before for β-lactoglobulin, our results suggest that the formation of particulates in the regime where charge on the molecules is minimal is a common property of all proteins. Because the proteins used here also form amyloid fibrils, we further propose that protein misfolding into clearly defined aggregates is a generic process whose outcome depends solely on the general properties of the state the protein is in when aggregation occurs, rather than the specific amino acid sequence. Thus under conditions of high net charge, amyloid fibrils form, whereas under conditions of low net charge, particulates form. This observation furthermore suggests that the rules of soft matter physics apply to these systems.

Sodium channels and pain

Voltage-gated sodium (Na+) channels play a key role in membrane excitation in neurons. These channels have a critical role in the development and maintenance of several pain syndromes, including inflammatory pain, neuropathic pain, and central pain associated with spinal cord injury.1–4 The increasing understanding of the molecular organization, function, and regulation of voltage-gated Na+ channels,5,6 their plasticity of expression following injury,4,7 and the clinical consequences of mutations8–12 provide insight into the important role of these channels in the pathophysiology of pain and rationale for development of channel subtype-specific drug therapy. Influx of Na+ through voltage-gated Na+ channels causes membrane depolarization, which is responsible for generation and conduction of the action potential in the axon and activation of presynaptic Ca2+ channels for exocytosis. Voltage-gated Na+ channels also modulate the resting membrane potential and subthreshold oscillations of the membrane potential, which determine the excitability of the neuron and its axon. The typical voltage-gated Na+ channel opens (activates) on membrane depolarization and then closes (inactivates) either rapidly on repolarization or more slowly on sustained depolarization. Fast inactivation is the primary mechanism of repolarization at the nodes of Ranvier, whereas slow inactivation regulates excitability and modulates burst discharges of neurons and axons. Toxins, local anesthetics, or mutations affecting single amino acid residue of the channel molecule affect the inactivation of voltage-gated Na+ channels.6 The voltage-gated Na+ channels are composed of an α-subunit consisting of four homologous domains (DI-DIV) and an auxiliary β-subunit5,6 (figure). Specific amino acid sequences of the α-subunit form the voltage sensor, the wall of the ion channel pore, the inactivation gate, the sites of binding for local anesthetics and toxins, and modulatory phosphorylation sites for several kinases The β-subunits modulate channel gating, inactivation, and cellular localization, …

The CFTR-derived peptides as a model of sequence-specific protein aggregation

Protein aggregation is a hallmark of a growing group of pathologies known as conformational diseases. Although many native or mutated proteins are able to form aggregates, the exact amino acid sequences involved in the process of aggregation are known only in a few cases. Hence, there is a need for different model systems to expand our knowledge in this area. The so-called ag region was previously found to cause the aggregation of the C-terminal fragment of the cystic fibrosis transmembrane conductance regulator (CFTR). To investigate whether this specific amino acid sequence is able to induce protein aggregation irrespective of the amino acid context, we altered its position within the CFTR-derived C-terminal peptide and analyzed the localization of such modified peptides in transfected mammalian cells. Insertion of the ag region into a different amino acid background affected not only the overall level of intracellular protein aggregation, but also the morphology and subcellular localization of aggregates, suggesting that sequences other than the ag region can substantially influence the peptide’s behavior. Also, the introduction of a short dipeptide (His-Arg) motif, a crucial component of the ag region, into different locations within the C-terminus of CFTR lead to changes in the aggregation pattern that were less striking, although still statistically significant. Thus, our results indicate that even subtle alterations within the aggregating peptide can affect many different aspects of the aggregation process.

Sequence-Independent Targeting of Transmembrane Proteins Synthesized within Vaccinia Virus Factories to Nascent Viral Membranes

The primary membrane of vaccinia virus, as well as those of other poxviruses, forms within a discrete cytoplasmic factory region. We recently determined the existence of an operative pathway from the endoplasmic reticulum within the virus factory to nascent viral membranes and demonstrated that a viral protein could be diverted from this pathway to Golgi membranes by the addition of COPII-binding sites (M. Husain, A. S. Weisberg, and B. Moss, Proc. Natl. Acad. Sci. USA, 103:19506-19511, 2006). Here we describe an investigation of the structural features that are required for transit of proteins to the viral membrane. Deletion of either the N-terminal domain or the C-terminal cytoplasmic tail from the conserved A9 protein did not prevent its incorporation into viral membranes, whereas deletion of the transmembrane domain resulted in its distribution throughout the cytoplasm. Nevertheless, replacement of the A9 transmembrane domain with the corresponding region of a nonpoxvirus transmembrane protein or of a vaccinia virus extracellular envelope protein allowed viral membrane targeting, indicating no requirement for a specific amino acid sequence. Remarkably, the epitope-tagged A9 transmembrane domain alone, as well as a heterologous transmembrane domain lacking a poxvirus sequence, was sufficient for viral membrane association. The data are consistent with a sequence-independent pathway in which transmembrane proteins that are synthesized within the virus factory and lack COPII or other binding sites that enable conventional endoplasmic reticulum exiting are incorporated into nascent viral membranes.

Mutational Analysis of the Carboxyl-terminal Region of the SV40 Major Capsid Protein VP1

Virus-like particles (VLPs), a promising next-generation drug delivery vehicle, can be formed in vitro using a recombinant viral capsid protein VP1 from SV40. Seventy-two VP1 pentamers interconnect to form the T = 7d lattice of SV40 capsids, through three types of C-terminal interactions, alpha-alpha'-alpha'', beta-beta' and gamma-gamma. These appear to require VP1 conformational switch, which involve in particular the region from amino acids 301-312 (herein Region I). Here we show that progressive deletions from the C-terminus of VP1, up to 34 amino acids, cause size and shape variations in the resulting VLPs, including tubular formation, whereas deletions beyond 34 amino acids simply blocked VP1 self-assembly. Mutants carrying in Region I point mutations predicted to disrupt alpha-alpha'-alpha''-type and/or beta-beta'-type interactions formed small VLPs resembling T = 1 symmetry. Chimeric VP1, in which Region I of SV40 VP1 was substituted with the homologous region from VP1 of other polyomaviruses, assembled only into small VLPs. Together, our results show the importance of the integrity of VP1 C-terminal region and the specific amino acid sequences within Region I in the assembly of normal VLPs. By understanding how to alter VLP sizes and shapes contributes to the development of drug delivery systems using VLPs.

Effects of randomizing the Sup35NM prion domain sequence on formation of amyloid fibrils in vitro

The mechanism by which proteins aggregate and form amyloid fibrils is still elusive. In order to preclude interference by cellular factors and to clarify the role of the primary sequence of Sup35p prion domain in formation of amyloid fibrils, we generated five Sup35NM variants by randomizing amino acid sequences in PrDs without altering the amino acid composition and analyzed the in vitro process of amyloid fibril formation. The results showed that each of the five Sup35NM variants polymerized into amyloid fibrils in vitro under native conditions. Furthermore, the Sup35NM variants showed differences in their aggregation time courses. These findings indicate that specific amino acid sequence features in PrD can modify the rate of conversion of Sup35p into amyloid fibrils in vitro.

Folding, activity and targeting of mutated human cathepsin D that cannot be processed into the double-chain form

The precursor of human cathepsin D (CD) is converted into the single-chain and the double-chain active polypeptides by subsequent proteolysis reactions taking place in the endosomal–lysosomal compartment and involving specific aminoacid sequences. We have mutagenized the region of aminoacids (comprising the β-hairpin loop) involved in the latter proteolytic maturation step and generated a mutant CD that cannot be converted into the mature double-chain form. This mutant CD expressed in rodent cells reaches the lysosome and is stable as single-chain polypeptide, bears high-mannose type sugars, binds to pepstatin A and is enzymatically active, indicating that it is correctly folded. The present work provides new insights on the aminoacid region involved in the terminal processing of human CD and on the function of the processing β-hairpin loop.

Analysis of the Amino Acid Sequence Specificity Determinants of the Enterococcal cCF10 Sex Pheromone in Interactions with the Pheromone-Sensing Machinery

The level of expression of conjugation genes in Enterococcus faecalis strains carrying the pheromone-responsive transferable plasmid pCF10 is determined by the ratio in the culture medium of two types of signaling peptides, a pheromone (cCF10) and an inhibitor (iCF10). Recent data have demonstrated that both peptides target the cytoplasmic receptor protein PrgX. However, the relative importance of the interaction of these peptides with the pCF10 protein PrgZ (which enhances import of cCF10) versus PrgX is not fully understood, and there is relatively little information about specific amino acid sequence determinants affecting the functional interactions of cCF10 with these proteins in vivo. To address these issues, we used a pheromone-inducible reporter gene system where various combinations of PrgX and PrgZ could be expressed in an isogenic host background to examine the biological activities of cCF10, iCF10, and variants of cCF10 isolated in a genetic screen. The results suggest that most of the amino acid sequence determinants of cCF10 pheromone activity affect interactions between the peptide and PrgX, although some sequence variants that affected peptide/PrgZ interactions were also identified. The results provide functional data to complement ongoing structural studies of PrgX and increase our understanding of the functional interactions of cCF10 and iCF10 with the pheromone-sensing machinery of pCF10.

Cysteine-Rich Domains of Muc3 Intestinal Mucin Promote Cell Migration, Inhibit Apoptosis, and Accelerate Wound Healing

Muc3 intestinal mucin contains an extracellular cysteine-rich domain with 2 epidermal growth factor (EGF)-like motifs. The aim of this study was to determine the functional properties of Muc3 proteins. Glutathione S-transferase-fusion proteins containing both Muc3 EGF-like domains (m3EGF1,2) or truncated versions (m3EGF1 and m3EGF2) were purified from Escherichia coli. Mouse colon (young adult mouse colon) and human A431 and LoVo cells were examined for migration and tyrosine phosphorylation in response to recombinant proteins. LoVo cells were transfected with a human MUC3A transmembrane-EGF1,2 construct and a stable clone was isolated (LhM3c14). Endogenous MUC3A in LoVo was inhibited by specific small interfering RNA transfection. Apoptosis was quantitated by nuclear morphology or terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labeling assay. Colitis was induced in mice by oral 5% dextran sodium sulfate or rectal 5% acetic acid, followed by enema treatments. m3EGF1,2 stimulated cell migration in all cell lines, but did not induce proliferation. Migration was inhibited by a tyrosine phosphorylation inhibitor, genistein, but not by the EGF receptor inhibitor, tyrphostin (AG1478). Inhibition of endogenous MUC3A in LoVo reduced baseline migration. Tyrosine phosphorylation of ErbB receptors was not observed after treatment of cells with m3EGF1,2. LoVo cells pretreated with m3EGF1,2 and transfected LhM3c14 cells showed reduced apoptosis in response to tumor necrosis factor alpha or Fas-receptor stimulation. Administration of m3EGF1,2 per rectum significantly reduced mucosal ulceration and apoptosis in experimental acute colitis. Truncated proteins m3EGF1 and m3EGF2 had no effect. The Muc3 mucin cysteine-rich domain plays an active role in epithelial restitution, and represents a potential novel therapeutic agent for intestinal wound healing.

An Angiotensin II Type 1 Receptor Mutant Lacking Epidermal Growth Factor Receptor Transactivation Does Not Induce Angiotensin II–Mediated Cardiac Hypertrophy

We have shown previously that tyrosine 319 in a conserved YIPP motif in the C terminus of angiotensin II (Ang II) type 1 receptors (AT(1)Rs) is essential for transactivation of epidermal growth factor receptor (EGFR) in vitro. We hypothesized that the signaling mechanism mediated through the specific amino acid sequence in the G protein-coupled receptor plays an important role in mediating cardiac hypertrophy in vivo. Transgenic mice with cardiac-specific overexpression of wild-type AT(1)R (Tg-WT) and an AT(1)R with a mutation in the YIPP motif (Tg-Y319F) were studied. Tg-Y319F mice developed no significant cardiac hypertrophy, in contrast to the significant development of hypertrophy in Tg-WT mice. Expression of fetal-type genes, such as atrial natriuretic factor, was also significantly lower in Tg-Y319F than in Tg-WT mice. Infusion of Ang II caused an enhancement of hypertrophy in Tg-WT mice but failed to induce hypertrophy in Tg-Y319F mice. Left ventricular myocardium in Tg-Y319F mice developed significantly less apoptosis and fibrosis than that in Tg-WT mice. EGFR phosphorylation was significantly inhibited in Tg-Y319F mice, confirming that EGFR was not activated in Tg-Y319F mouse hearts. In contrast, activation/phosphorylation of protein kinase C, STAT3, extracellular signal-regulated kinase, and Akt and translocation of Galphaq/11 to the cytosolic fraction were maintained in Tg-Y319F hearts. Furthermore, a genetic cross between Tg-WT and transgenic mice with cardiac-specific overexpression of dominant negative EGFR mimicked the phenotype of Tg-Y319F mice. In conclusion, overexpression of AT(1)-Y319F in cardiac myocytes diminished EGFR transactivation and inhibited a pathological form of cardiac hypertrophy. The YIPP motif in the AT(1)R plays an important role in mediating cardiac hypertrophy in vivo.

Collagen mimetic peptide-conjugated photopolymerizable PEG hydrogel

Collagen mimetic peptide (CMP) with a specific amino acid sequence, -(Pro-Hyp-Gly)(x)-, forms a triple helix conformation that resembles the native protein structure of natural collagens. CMP previously has been shown to associate with type I collagen molecules and fibers via a strand invasion process. We hypothesized that when poly(ethylene glycol) (PEG) hydrogel, a non-adhesive tissue engineering scaffold, is conjugated with CMP, it may retain cell-secreted collagens and also form physical crosslinks that can be manipulated by cells. A photopolymerizable CMP derivative was synthesized and copolymerized with poly(ethylene oxide) diacrylate to create a novel PEG hydrogel. In a model retention experiment, diffusional loss of type I collagen that was added to the hydrogel was limited. Chondrocytes were encapsulated in the hydrogel to examine its use as a tissue engineering scaffold. After 2 weeks, the biochemical analysis of the CMP-conjugated PEG gel revealed an 87% increase in glycosaminoglycan content and a 103% increase in collagen content compared to that of control PEG hydrogels. The histology and immunohistochemistry analyses also showed increased staining of extracellular matrix. These results indicate that the CMP enhances the tissue production of cells encapsulated in the PEG hydrogel by providing cell-manipulated crosslinks and collagen binding sites that simulate natural extracellular matrix.

Predicting the Subcellular Localization of Human Proteins Using Machine Learning and Exploratory Data Analysis

Identifying the subcellular localization of proteins is particularly helpful in the functional annotation of gene products. In this study, we use Machine Learning and Exploratory Data Analysis (EDA) techniques to examine and characterize amino acid sequences of human proteins localized in nine cellular compartments. A dataset of 3,749 protein sequences representing human proteins was extracted from the SWISS-PROT database. Feature vectors were created to capture specific amino acid sequence characteristics. Relative to a Support Vector Machine, a Multi-layer Perceptron, and a Naïve Bayes classifier, the C4.5 Decision Tree algorithm was the most consistent performer across all nine compartments in reliably predicting the subcellular localization of proteins based on their amino acid sequences (average Precision=0.88; average Sensitivity=0.86). Furthermore, EDA graphics characterized essential features of proteins in each compartment. As examples, proteins localized to the plasma membrane had higher proportions of hydrophobic amino acids; cytoplasmic proteins had higher proportions of neutral amino acids; and mitochondrial proteins had higher proportions of neutral amino acids and lower proportions of polar amino acids. These data showed that the C4.5 classifier and EDA tools can be effective for characterizing and predicting the subcellular localization of human proteins based on their amino acid sequences.

Common attributes of native-state structures of proteins, disordered proteins, and amyloid.

We show that a framework derived from the common character of globular proteins can be used to understand the design of protein sequences, the behavior of intrinsically unstructured proteins, and the formation of amyloid fibrils in a unified manner. Our studies provide compelling support for the idea that protein native-state structures, the structures adopted by intrinsically unstructured proteins on binding as well as those of amyloid aggregates, all reside in a physical state of matter in which the free energy landscape is sculpted not by the specific sequence of amino acids, but rather by considerations of geometry and symmetry. We elucidate the key role played by sequence design in selecting the structure of choice from the predetermined menu of putative native-state structures.

Engineering of β-propeller protein scaffolds by multiple gene duplication and fusion of an idealized WD repeat

The ability to design specific amino acid sequences that fold into desired structures is central to engineering novel proteins. Protein design is also a good method to assess our understanding of sequence–structure and structure–function relationships. While β-sheet structures are important elements of protein architecture, it has traditionally been more difficult to design β-proteins than α-helical proteins. Taking advantage of the tandem repeated sequences that form the structural building blocks in a group of β-propeller proteins; we have used a consensus design approach to engineer modular and relatively large scaffolds. An idealized WD repeat was designed from a structure-based sequence alignment with a set of structural guidelines. Using a plasmid sequential ligation strategy, artificial concatemeric genes with up to 10 copies of this idealized repeat were then constructed. Corresponding proteins with 4 through to 10 WD repeats were soluble when over-expressed in Escherichia coli. Notably, they were sufficiently stable in vivo surviving attack from endogenous proteases, and maintained a homogeneous, non-aggregated form in vitro. The results show that the β-propeller scaffold is an attractive platform for future engineering work, particularly in experiments in which directed evolution techniques might improve the stability of the molecules and/or tailor them for a specific function.

Monitoring of proteinase activation in cell apoptosis by capillary electrophoresis with bioengineered fluorescent probe

In this article, capillary electrophoresis (CE) was demonstrated as a novel means to monitor the activation of proteinase during cell apoptosis. A unique fluorescent probe that fused a specific amino acid sequence DEVD with two fluorescent proteins enhanced cyan fluorescent protein (ECFP) and red fluorescent protein (DsRed), ECFP-DEVD-DsRed, was engineered in HeLa cells as a substrate of proteinase caspase-3. Molecular imaging in vivo was conducted to evaluate the stability and reliability of this fusion protein probe expressed in HeLa cells. With treatment of a certain dose of cisplatin to HeLa cells, apoptosis was initiated, and then caspase-3 was activated that could specifically recognize the DEVD site and would subsequently cleave the constructed fluorescent probe into two pieces of protein fragments. Analyzing the cell lysates with CE in vitro at a series of time points gave a clear description of activation process of caspase-3 in cell apoptosis. Several parameters that influenced CE separation quality were optimized, such as buffer type, pH value, concentration and applied voltage. The employment of two color fluorescent proteins significantly simplified the separation and identification of the residues of the cleavage reaction.

Flk prevents premature secretion of the anti‐σ factor FlgM into the periplasm

The flk locus of Salmonella typhimurium was identified as a regulator of flagellar gene expression in strains defective in P- and l-ring formation. Flk acts as a regulator of flagellar gene expression by modulating the protein levels of the anti-sigma28 factor FlgM. Evidence is presented which suggests that Flk is a cytoplasmic-facing protein anchored to the inner membrane by a single, C-terminal transmembrane-spanning domain (TMS). The specific amino acid sequence of the TMS is not essential for Flk activity, but membrane anchoring is essential. Membrane fractionation and visualization of protein fusions of green fluorescent protein derivatives to Flk suggested that the Flk protein is present in the membrane as punctate spots in number that are much greater than the number of flagellar basal structures. The turnover of the anti-sigma28 factor FlgM was increased in flk mutant strains. Using FlgM-beta-lactamase fusions we show the increased turnover of FlgM in flk null mutations is due to FlgM secretion into the periplasm where it is degraded. Our data suggest that Flk inhibits FlgM secretion by acting as a braking system for the flagellar-associated type III secretion system. A model is presented to explain a role for Flk in flagellar assembly and gene regulatory processes.

Molecular Mechanics of Single Molecules

The mechanics of single molecules of bacteriorhodopsin interacting with purple membrane have been investigated from both sides of the membrane by Kessler and Gaub (2006) in this issue of Structure. Remarkably, barriers can be associated with specific amino acid sequences to an accuracy of ±3 amino acids.

Unconventional Signalling Mechanisms Mediated by the Angiotensin II Type 1 Receptor in Cardiovascular Cell Types

Angiotensin II (Ang II) is an important effector of the renin-angiotensin system, which plays a critical role in mediating hypertension, cardiac hypertrophy and heart failure. These pathological effects of Ang II on the heart are primarily mediated by the Ang II type 1 (AT1) receptor, a seven transmembrane G protein-coupled receptor. The signalling mechanism of the AT1 receptor is traditionally described as dependent on heterotrimeric G proteins. However, accumulating lines of evidence have suggested that some aspects of AT1 receptor-mediated signalling apparently occur independently of G protein activation. The AT1 receptor also uses a part of its amino acid sequence, the YIPP (tyrosine [Y] isoleucine [I] proline [P] proline [P]) motif, in addition to conventional G protein-dependent mechanisms, to activate tyrosine kinases, such as the epidermal growth factor receptor and Janus kinases (JAK). The AT1 receptor also directly associates with other intracellular molecules, such as AT1 receptor-associated protein. Interaction between the AT1 receptor and these molecules affects the cellular functions of each. Such mechanisms, relying on the specific amino acid sequences of the AT1 receptor, provide the AT1 receptor with a unique modality to communicate with downstream signalling molecules. This review summarises some of these unconventional signalling mechanisms initiated by the AT1 receptor, and their functional significance in cardiovascular diseases.