Tandem repeat-like domain of "similar to prion protein" (StPrP) of Japanese pufferfish binds Cu(II) as effectively as the mammalian protein

The main structural domains of prion proteins, in particular the N-terminal region containing characteristic amino acid repeats, are well conserved among different species, despite divergence in primary sequence. The repeat region seems to play an important role, as verified by pathogenicity only observed in organisms having repeats composed of eight residues. In this work three different peptides belonging to the tandem repeat region of StPrP-2 from the Japanese pufferfish Takifugu rubripes have been considered; the coordination modes and conformations of their complexes with Cu(II) have been investigated by using potentiometric titrations, spectroscopic data, and restrained molecular dynamics simulations. In all cases the histidine imidazole(s) provide the anchoring site for copper, with the further involvement of amide nitrogens depending on the peptide sequence and on pH. An increase in copper binding affinity has been observed going from the shortest peptide, corresponding to a single repeat and containing two histidines, to the longest one, encompassing three repeats with six histidines.     

Conserved repeat motifs and glucan binding by glucansucrases of oral streptococci and Leuconostoc mesenteroides

Glucansucrases of oral streptococci and Leuconostoc mesenteroides have a common pattern of structural organization and characteristically contain a domain with a series of tandem amino acid repeats in which certain residues are highly conserved, particularly aromatic amino acids and glycine. In some glucosyltransferases (GTFs) the repeat region has been identified as a glucan binding domain (GBD). Such GBDs are also found in several glucan binding proteins (GBP) of oral streptococci that do not have glucansucrase activity. Alignment of the amino acid sequences of 20 glucansucrases and GBP showed the widespread conservation of the 33-residue A repeat first identified in GtfI of Streptococcus downei. Site-directed mutagenesis of individual highly conserved residues in recombinant GBD of GtfI demonstrated the importance of the first tryptophan and the tyrosine-phenylalanine pair in the binding of dextran, as well as the essential contribution of a basic residue (arginine or lysine). A microplate binding assay was developed to measure the binding affinity of recombinant GBDs. GBD of GtfI was shown to be capable of binding glucans with predominantly alpha-1,3 or alpha-1,6 links, as well as alternating alpha-1,3 and alpha-1,6 links (alternan). Western blot experiments using biotinylated dextran or alternan as probes demonstrated a difference between the binding of streptococcal GTF and GBP and that of Leuconostoc glucansucrases. Experimental data and bioinformatics analysis showed that the A repeat motif is distinct from the 20-residue CW motif, which also has conserved aromatic amino acids and glycine and which occurs in the choline-binding proteins of Streptococcus pneumoniae and other organisms.     

Cysteine residue periodicity is a conserved structural feature of variable surface proteins from Paramecium tetraurelia.

The DNA sequences of the entire coding regions of the A and C type variable surface protein genes from Paramecium tetraurelia, stock 51 have been determined. The 8151 nucleotide open reading frame of the A gene contains several tandem repeats of 210 nucleotides within the central portion of the molecule as well as a periodic structure defined by cysteine residues. The 6699 nucleotide open reading frame of the C gene does not contain any identifiable tandem repeats or internal similarity but maintains a periodicity based on the cysteine residue spacing. The deduced amino acid sequences encoded by the two genes are most similar within the 600 amino-terminal and 600 carboxyl-terminal amino acid residues, the central portions show only limited sequence similarity. We conclude that internal repeats are not a conserved feature of variable surface proteins in Paramecium and discuss the possible importance of the regular pattern of cysteine residues.     

Designed ankyrin repeat proteins as scaffolds for multivalent recognition

Ankyrin repeat (AR) proteins are composed of tandem repeats of a basic structural motif of ca. 33 amino acid residues that form a β-turn followed by two antiparallel α-helices. Multiple repeats stack together in a modular fashion to form a scaffold that is ideally suited for the presentation of multiple functional groups and/or recognition elements. Here we describe a biosynthetic strategy that takes advantage of the modular nature of these proteins to generate multivalent ligands that are both chemically homogeneous and structurally well-defined. Glycosylated AR proteins cluster the tetrameric lectin concanavalin A (Con A) at a rate that is comparable to the rate of Con A aggregation mediated by globular protein conjugates and variable density linear polymers. Thus, AR proteins define a new class of multivalent ligand scaffolds that have significant potential application in the study and control of a variety of multivalent interactions.     

Controlling the prion propensity of glutamine/asparagine-rich proteins

ABSTRACT

The yeast Saccharomyces cerevisiae can harbor a number of distinct prions. Most of the yeast prion proteins contain a glutamine/asparagine (Q/N) rich region that drives prion formation. Prion-like domains, defined as regions with high compositional similarity to yeast prion domains, are common in eukaryotic proteomes, and mutations in various human proteins containing prion-like domains have been linked to degenerative diseases, including amyotrophic lateral sclerosis. Here, we discuss a recent study in which we utilized two strategies to generate prion activity in non-prion Q/N-rich domains. First, we made targeted mutations in four non-prion Q/N-rich domains, replacing predicted prion-inhibiting amino acids with prion-promoting amino acids. All four mutants formed foci when expressed in yeast, and two acquired bona fide prion activity. Prion activity could be generated with as few as two mutations, suggesting that many non-prion Q/N-rich proteins may be just a small number of mutations from acquiring aggregation or prion activity. Second, we created tandem repeats of short prion-prone segments, and observed length-dependent prion activity. These studies demonstrate the considerable progress that has been made in understanding the sequence basis for aggregation of prion and prion-like domains, and suggest possible mechanisms by which new prion domains could evolve.     

Complete sequence of the lamprey fibrinogen alpha chain

The complete amino acid sequence of the lamprey fibrinogen alpha chain has been determined by a combination of peptide sequencing and cDNA and genomic cloning. The chain, which has an apparent molecular weight by dodecyl sulfate-polyacrylamide gel electrophoresis of ca. 100,000, is composed of 961 amino acid residues and has a calculated molecular weight of 96,722. It is distinguished by a large number of 18-residue repeats in a region where mammalian fibrinogens have 13-residue repeats. The data are in accord with our previous finding that the lamprey alpha chain has a distinctive amino acid composition, almost half the residues being glycine, serine, or threonine. The chain differs from mammalian alpha chains in that there are no cysteines in the carboxy-terminal half, and thus no intrachain loop, nor are there any RGD sequences in the lamprey alpha chain. Taken together with previous data on the sequences of the beta and gamma chains, the findings bear significantly on our understanding of fibrin formation. The alpha chain also provides an interesting case of structural convergence during evolution.     

Pattern of repeating aromatic residues in synexin. Similarity to the cytoplasmic domain of synaptophysin

Synexin was isolated from bovine liver by high resolution cation exchange chromatography and fragmented with cyanogen bromide or trypsin. Peptides were isolated and their amino acid sequences partially determined. Twenty percent of the synexin sequence was determined in one contiguous sequence of 61 residues and a nonoverlapping sequence of 20 residues. The sequence is characterized by a hexapeptide repeat of the form YPXXXX occurring eight times in series, with phenylalanine substituting for tyrosine in two positions. The intervening amino acids (X) are predominantly proline, glycine and alanine. This pattern of periodic aromatic residues suggests the presence of a novel secondary structure and is similar to repeats present in synaptophysin, gliadin and type II keratin.     

A comparison of genomic coding sequences for feather and scale keratins: structural and evolutionary implications.

DNA sequences have been obtained for embryonic chick feather and scale keratin genes. Strong homologies exist between the protein coding regions of the two gene types and between the deduced amino acid sequences of the keratin proteins. Scale keratins are larger than feather keratins and the size difference is mainly attributable to four 13-amino acid repeats between residues 77 and 128 which compose a peptide sequence rich in glycine and tyrosine. The strong similarities between the two peptide structures for feather and scale in the homologous regions suggests a similar conformation within the protein filaments. A likely consequence is that the additional repeat region of the scale protein is located externally to the core filament. Tissue-specific features of filament aggregation may be attributable to this one striking sequence difference between the constituent proteins. It is believed that the genes share a common ancestry and that feather-like keratin genes may have evolved from a scale keratin gene by a single deletion event.     

Distinct Amino Acid Compositional Requirements for Formation and Maintenance of the [PSI+] Prion in Yeast

Multiple yeast prions have been identified that result from the structural conversion of proteins into a self-propagating amyloid form. Amyloid-based prion activity in yeast requires a series of discrete steps. First, the prion protein must form an amyloid nucleus that can recruit and structurally convert additional soluble proteins. Subsequently, maintenance of the prion during cell division requires fragmentation of these aggregates to create new heritable propagons. For the Saccharomyces cerevisiae prion protein Sup35, these different activities are encoded by different regions of the Sup35 prion domain. An N-terminal glutamine/asparagine-rich nucleation domain is required for nucleation and fiber growth, while an adjacent oligopeptide repeat domain is largely dispensable for prion nucleation and fiber growth but is required for chaperone-dependent prion maintenance. Although prion activity of glutamine/asparagine-rich proteins is predominantly determined by amino acid composition, the nucleation and oligopeptide repeat domains of Sup35 have distinct compositional requirements. Here, we quantitatively define these compositional requirements in vivo. We show that aromatic residues strongly promote both prion formation and chaperone-dependent prion maintenance. In contrast, nonaromatic hydrophobic residues strongly promote prion formation but inhibit prion propagation. These results provide insight into why some aggregation-prone proteins are unable to propagate as prions.     

Acceleration of alpha-synuclein aggregation by homologous peptides

alpha-Synuclein (alpha-Syn), amyloid beta-protein and prion protein are among the amyloidogenic proteins that are associated with the neurodegenerative diseases. These three proteins share a homologous region with a consensus sequence mainly consisting of glycine, alanine and valine residues (accordingly named as the GAV motif), which was proposed to be the critical core for the fibrillization and cytotoxicity. To understand the role of the GAV motif in protein amyloidogenesis, we studied the effects of the homologous peptides corresponding to the sequence of GAV motif region (residues 66-74) on alpha-Syn aggregation. The result shows that these peptides can promote fibrillization of wild-type alpha-Syn and induce that of the charge-incorporated mutants but not the GAV-deficient alpha-Syn mutant. The acceleration of alpha-Syn aggregation by the homologous peptides is under a sequence-specific manner. The interplay between the GAV peptide and the core regions in alpha-Syn may accelerate the aggregation process and stabilize the fibrils. This finding provides clues for developing peptide mimics that could promote transforming the toxic oligomers or protofibrils into the inert mature fibrils.     

Glycine loops in proteins: their occurrence in certain intermediate filament chains, loricrins and single-stranded RNA binding proteins.

Quasi-repetitive, glycine-rich peptide sequences are widespread in at least three distinct families of proteins: the keratins and other intermediate filament proteins, including nuclear lamins; loricrins, which are major envelope components of terminally differentiated epithelial cells; and single-stranded RNA binding proteins. We propose that such sequences comprise a new structural motif termed the 'glycine loop'. The defining characteristics of glycine loop sequences are: (1) they have the form x(y)n, where x is usually an aromatic or occasionally a long-chain aliphatic residue; y is usually glycine but may include polar residues such as serine, asparagine, arginine, cysteine, and rarely other residues; and the value of n is highly variable, ranging from 1 to 35 in examples identified to date. (2) Glycine-loop-containing domains are thought to form when at least two and to date, as many as 18, such quasi-repeats are configured in tandem, so that the entire domain in a protein may be 50-150 residues long. (3) The average value of n, the pattern of residues found in the x position and the non-glycine substitutions in the y position appear to be characteristic of a given glycine loop containing domain, whereas the actual number of repeats is less constrained. (4) Glycine loop sequences display a high degree of evolutionary sequence variability and even allelic variations among different individuals of the same vertebrate species. (5) Glycine loop sequences are expected to be highly flexible, but possess little other regular secondary structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

PSI+] maintenance is dependent on the composition, not primary sequence, of the oligopeptide repeat domain

[PSI(+)], the prion form of the yeast Sup35 protein, results from the structural conversion of Sup35 from a soluble form into an infectious amyloid form. The infectivity of prions is thought to result from chaperone-dependent fiber cleavage that breaks large prion fibers into smaller, inheritable propagons. Like the mammalian prion protein PrP, Sup35 contains an oligopeptide repeat domain. Deletion analysis indicates that the oligopeptide repeat domain is critical for [PSI(+)] propagation, while a distinct region of the prion domain is responsible for prion nucleation. The PrP oligopeptide repeat domain can substitute for the Sup35 oligopeptide repeat domain in supporting [PSI(+)] propagation, suggesting a common role for repeats in supporting prion maintenance. However, randomizing the order of the amino acids in the Sup35 prion domain does not block prion formation or propagation, suggesting that amino acid composition is the primary determinant of Sup35's prion propensity. Thus, it is unclear what role the oligopeptide repeats play in [PSI(+)] propagation: the repeats could simply act as a non-specific spacer separating the prion nucleation domain from the rest of the protein; the repeats could contain specific compositional elements that promote prion propagation; or the repeats, while not essential for prion propagation, might explain some unique features of [PSI(+)]. Here, we test these three hypotheses and show that the ability of the Sup35 and PrP repeats to support [PSI(+)] propagation stems from their amino acid composition, not their primary sequences. Furthermore, we demonstrate that compositional requirements for the repeat domain are distinct from those of the nucleation domain, indicating that prion nucleation and propagation are driven by distinct compositional features.     

Octapeptide repeat insertions increase the rate of protease-resistant prion protein formation

A central feature of transmissible spongiform encephalopathies (TSE or prion diseases) involves the conversion of a normal, protease-sensitive glycoprotein termed prion protein (PrP-sen) into a pro-tease-resistant form, termed PrP-res. The N terminus of PrP-sen has five copies of a repeating eight amino acid sequence (octapeptide repeat). The presence of one to nine extra copies of this motif is associated with a heritable form of Creutzfeld-Jakob disease (CJD) in humans. An increasing number of octapeptide repeats correlates with earlier CJD onset, suggesting that the rate at which PrP-sen misfolds into PrP-res may be influenced by these mutations. In order to determine if octapeptide repeat insertions influence the rate at which PrP-res is formed, we used a hamster PrP amyloid-forming peptide (residues 23-144) into which two to 10 extra octapeptide repeats were inserted. The spontaneous formation of protease-resistant PrP amyloid from these peptides was more rapid in response to an increased number of octapeptide repeats. Furthermore, experiments using full-length glycosylated hamster PrP-sen demonstrated that PrP-res formation also occurred more rapidly from PrP-sen molecules expressing 10 extra copies of the octapeptide repeat. The rate increase for PrP-res formation did not appear to be due to any influence of the octapeptide repeat region on PrP structure, but rather to more rapid binding between PrP molecules. Our data from both models support the hypothesis that extra octapeptide repeats in PrP increase the rate at which protease resistant PrP is formed which in turn may affect the rate of disease onset in familial forms of CJD.     

The formation of Escherichia coli curli amyloid fibrils is mediated by prion-like peptide repeats

Amyloid fibril formation is the hallmark of major human maladies including Alzheimer's disease, type II diabetes, and prion diseases. Prion-like phenomena were also observed in yeast. Although not evolutionarily related, one similarity between the animal PrP and the yeast Sup35 prion proteins is the occurrence of short peptide repeats that are assumed to play a key role in the assembly of the amyloid structures. It was recently demonstrated that typical amyloid fibril formation is associated with biofilm formation by Escherichia coli. Here, we note the functional and structural similarity between oligopeptide repeats of the major curli protein and those of animal and yeast prions. We demonstrate that synthetic peptides corresponding to the repeats form fibrillar structures. Furthermore, conjugation of beta-breaker elements to the prion-like repeat significantly inhibits amyloid formation and cell invasion of curli-expressing bacteria. This implies a functional role of the repeat in the self-assembly of the fibrils. Since mammal prion, yeast prion, and curli protein are evolutionarily distinct, the conserved peptide repeats most likely define an optimized self-association motif that was independently evolved by diverse systems.     

Identification and analysis of novel amino acid sequence repeats and domains in Pyrobaculum aerophilum using computational tools

We have identified four repeats and five domains that are novel in proteins encoded by the Pyrobaculum aerophilum str. IM2 proteome using automated in silico methods. A "repeat" corresponds to a region comprising less than 55 amino acid residues that occurs more than once in the protein sequence and sometimes present in tandem. A "domain" corresponds to a conserved region comprising greater than 55 amino acid residues and may be present as single or multiple copies in the protein sequence. These correspond to (1) 85 amino acid residues AAG domain, (2) 72 amino acid residues GFGN domain, (3) 43 amino acid residues KGG repeat, (4) 25 amino acid residues RWE repeat, (5) 25 amino acid residues RID repeat, (6) 108 amino acid residues NDFA domain, (7) 140 amino acid residues VxY domain, (8) 35 amino acid residues LLPN repeat and (9) 98 amino acid residues GxY domain. A repeat or domain is characterized by specific conserved sequence motifs. We discuss the presence of these repeats and domains in proteins from other genomes and their probable secondary structure.     

Mutations that replace aromatic side chains promote aggregation of the Alzheimer's Aβ peptide

The aggregation of polypeptides into amyloid fibrils is associated with a number of human diseases. Because these fibrils--or intermediates on the aggregation pathway--play important roles in the etiology of disease, considerable effort has been expended to understand which features of the amino acid sequence promote aggregation. One feature suspected to direct aggregation is the π-stacking of aromatic residues. Such π-stacking interactions have also been proposed as the targets for various aromatic compounds that are known to inhibit aggregation. In the case of Alzheimer's disease, the aromatic side chains Phe19 and Phe20 in the wild-type amyloid beta (Aβ) peptide have been implicated. To explicitly test whether the aromaticity of these side chains plays a role in aggregation, we replaced these two phenylalanine side chains with leucines or isoleucines. These residues have similar sizes and hydrophobicities as Phe but are not capable of π-stacking. Thioflavin-T fluorescence and electron microscopy demonstrate that replacement of residues 19 and 20 by Leu or Ile did not prevent aggregation, but rather enhanced amyloid formation. Further experiments showed that aromatic inhibitors of aggregation are as effective against Ile- and Leu-substituted versions of Aβ42 as they are against wild-type Aβ. These results suggest that aromatic π-stacking interactions are not critical for Aβ aggregation or for the inhibition of Aβ aggregation.     

ST proteins, a new family of plant tandem repeat proteins with a DUF2775 domain mainly found in Fabaceae and Asteraceae

Background

Many proteins with tandem repeats in their sequence have been described and classified according to the length of the repeats: I) Repeats of short oligopeptides (from 2 to 20 amino acids), including structural cell wall proteins and arabinogalactan proteins. II) Repeats that range in length from 20 to 40 residues, including proteins with a well-established three-dimensional structure often involved in mediating protein-protein interactions. (III) Longer repeats in the order of 100 amino acids that constitute structurally and functionally independent units. Here we analyse ShooT specific (ST) proteins, a family of proteins with tandem repeats of unknown function that were first found in Leguminosae, and their possible similarities to other proteins with tandem repeats.

Results

ST protein sequences were only found in dicotyledonous plants, limited to several plant families, mainly the Fabaceae and the Asteraceae. ST mRNAs accumulate mainly in the roots and under biotic interactions. Most ST proteins have one or several Domain(s) of Unknown Function 2775 (DUF2775). All deduced ST proteins have a signal peptide, indicating that these proteins enter the secretory pathway, and the mature proteins have tandem repeat oligopeptides that share a hexapeptide (E/D)FEPRP followed by 4 partially conserved amino acids, which could determine a putative N-glycosylation signal, and a fully conserved tyrosine. In a phylogenetic tree, the sequences clade according to taxonomic group. A possible involvement in symbiosis and abiotic stress as well as in plant cell elongation is suggested, although different STs could play different roles in plant development.

Conclusions

We describe a new family of proteins called ST whose presence is limited to the plant kingdom, specifically to a few families of dicotyledonous plants. They present 20 to 40 amino acid tandem repeat sequences with different characteristics (signal peptide, DUF2775 domain, conservative repeat regions) from the described group of 20 to 40 amino acid tandem repeat proteins and also from known cell wall proteins with repeat sequences. Several putative roles in plant physiology can be inferred from the characteristics found.     

Plasmodium berghei: cloning of the circumsporozoite protein gene

A DNA fragment encoding the carboxy terminal 80% of the Plasmodium berghei circumsporozoite protein was selected from a genomic DNA expression library. Sequencing revealed that the P. berghei circumsporozoite protein was similar in overall structure to circumsporozoite proteins from other malaria species, although the central repeat region was unique in comprising two different blocks of tandem peptide repeats: 11 eight amino acid repeats with predominant sequence DPAPPNAN were followed by 16 two amino repeats, predominantly PQ. The P. berghei circumsporozoite protein exhibited limited, but about equal amino acid homology to circumsporozoite proteins from P. knowlesi, P. vivax, and P. falciparum, indicating that P. berghei is not closely related to any of these other malaria species. Cloning of the P. berghei circumsporozoite protein gene will allow direct testing of sporozoite vaccines in mice.