Bacterial nucleic acid synthesis in plants following bacterial contact

Summary After plants have been in contact with a suspension of bacteria one finds in plant cells self replicating bacterial DNA and replicating molecules formed of bacterial DNA combined with plant DNA. Moreover newly synthesized bacterial RNA appears in the host cell. These phenomena seem to be due to a transfer of bacterial DNA into plant cells.     

Peptide‐dominated membranes preceding the genetic takeover by RNA: latest thinking on a classic controversy

It is commonly presumed that abiotic membranes were colonized by proteins later on. Yet, hydrophobic peptides could have formed primordial protein‐dominated membranes on their own. In a metabolism‐first context, “autocatalytically closed” sets of statistical peptides could organize a self‐maintaining protometabolism, assisted by an unfolding set of ribotide‐related cofactors. Pairwise complementary ribotide cofactors may have formed docking guides for stochastic peptide formation, before replicating RNA emerged from this subset. Tidally recurring wet‐drying cycles and an early onset of photosynthetic activities are considered most likely to meet the thermodynamic requirements. Conceivably, the earliest peptide‐dominated vesicles were engaged in light harvesting, together with isoprenoid‐tethered pigments, rather than providing an external boundary. Early on, the bulk of prebiotic organic matter can have formed a contiguous layer covering the mineral sediment, held in place by colloidal coherence of a hydrogel matrix. This unconventional scenario assumes a late onset of cellular individualization – perhaps from within, resembling endosporogenesis.     

Monoclonal antibody detection of a major self peptide. MHC class II complex.

MHC class I and class II molecules transport foreign and self peptides to the cell surface and present them to T lymphocytes. Detection of these peptide:MHC complexes has thus far been limited to analysis of the response of a T cell. Previously, we showed that a mAb, Y-Ae, reacts with 10 to 15% of class II molecules on peripheral B lymphocytes and on cells in the thymus medulla but not thymus cortex in mice that express both I-Ab and I-Eb molecules. Elsewhere, we show that Y-Ae detects a self E alpha peptide bound to I-Ab molecules. Data presented here suggest that the antibody binds over the peptide binding groove of class II molecules, and, like a TCR, appears to recognize both the self peptide and polymorphic class II residues. In addition to B lymphocytes, the Y-Ae determinant is expressed at comparable levels on other APC, including macrophages and dendritic cells. Finally, the antibody does not react with invariant chain-associated class II complexes, thus providing direct evidence that invariant chain:class II complexes and peptide:class II complexes are mutually exclusive. These data provide further evidence that immunologic self is of limited complexity, and have important implications for T cell selection, self tolerance, and autoreactivity.     

Peptide signalling in plant development and self/non-self perception

Plant genomes contain very large families of genes encoding receptor-like kinases (RLKs). In recent years, several of these RLKs have been shown by biochemical and mutational analysis to represent receptors for peptides, and the emerging picture shows that peptide signalling in development and self/non-self perception is based on a similar repertoire of receptors and signalling cascades. The need to recognize multiple peptide signals in self/non-self recognition may have led to the surprising radiation and diversification of RLKs in the plant kingdom.     

Shifts in growth strategies reflect tradeoffs in cellular economics

The growth rate‐dependent regulation of cell size, ribosomal content, and metabolic efficiency follows a common pattern in unicellular organisms: with increasing growth rates, cell size and ribosomal content increase and a shift to energetically inefficient metabolism takes place. The latter two phenomena are also observed in fast growing tumour cells and cell lines. These patterns suggest a fundamental principle of design. In biology such designs can often be understood as the result of the optimization of fitness. Here we show that in basic models of self‐replicating systems these patterns are the consequence of maximizing the growth rate. Whereas most models of cellular growth consider a part of physiology, for instance only metabolism, the approach presented here integrates several subsystems to a complete self‐replicating system. Such models can yield fundamentally different optimal strategies. In particular, it is shown how the shift in metabolic efficiency originates from a tradeoff between investments in enzyme synthesis and metabolic yields for alternative catabolic pathways. The models elucidate how the optimization of growth by natural selection shapes growth strategies.     

Autocatalytic sets of proteins

This article investigates the possibility that the emergence of reflexively autocatalytic sets of peptides and polypeptides may be an essentially inevitable collective property of any sufficiently complex set of polypeptides. The central idea is based on the connectivity properties of random directed graphs. In the set of amino acid monomer and polymer species up to some maximum length, M, the number of possible polypeptides is large, but, for specifiable "legitimate" end condensation, cleavage and transpeptidation exchange reactions, the number of potential reactions by which the possible polypeptides can interconvert is very much larger. A directed graph in which arrows from smaller fragments to larger condensation products depict potential synthesis reactions, while arrows from the larger peptide to the smaller fragments depict the reverse cleavage reactions, comprises the reaction graph for such a system. Polypeptide protoenzymes are able to catalyze such reactions. The distribution of catalytic capacities in peptide space is a fundamental problem in its own right, and in its bearing on the existence of autocatalytic sets of proteins. Using an initial idealized hypothesis that an arbitrary polypeptide has a fixed a priori probability of catalyzing any arbitrary legitimate reaction to assign to each polypeptide those reactions, if any, which it catalyzes, the probability that the set of polypeptides up to length M contains a reflexively autocatalytic subset can be calculated and is a percolation problem on such reaction graphs. Because, as M increases, the ratio of reactions among the possible polypeptides to polypeptides rises rapidly, the existence of such autocatalytic subsets is assured for any fixed probability of catalysis. The main conclusions of this analysis appear independent of the idealizations of the initial model, introduce a novel kind of parallel selection for peptides catalyzing connected sequences of reactions, depend upon a new kind of minimal critical complexity whose properties are definable, and suggest that the emergence of self replicating systems may be a self organizing collective property of critically complex protein systems in prebiotic evolution. Similar principles may apply to the emergence of a primitive connected metabolism. Recombinant DNA procedures, cloning random DNA coding sequences into expression vectors, afford a direct avenue to test the distribution of catalytic capacities in peptide space, may provide a new means to select or screen for peptides with useful properties, and may ultimately lead toward the actual construction of autocatalytic peptide sets.     

Recognition of self and altered self by T cells in autoimmunity and allergy

T cell recognition of foreign peptide antigen and tolerance to self peptides is key to the proper function of the immune system. Usually, in the thymus T cells that recognize self MHC+ self peptides are deleted and those with the potential to recognize self MHC+ foreign peptides are selected to mature. However there are exceptions to these rules. Autoimmunity and allergy are two of the most common immune diseases that can be related to recognition of self. Many genes work together to lead to autoimmunity. Of those, particular MHC alleles are the most strongly associated, reflecting the key importance of MHC presentation of self peptides in autoimmunity. T cells specific for combinations of self MHC and self peptides may escape thymus deletion, and thus be able to drive autoimmunity, for several reasons: the relevant self peptide may be presented at low abundance in the thymus but at high level in particular peripheral tissues; the relevant self peptide may bind to MHC in an unusual register, not present in the thymus but apparent elsewhere; finally the relevant self peptide may be post translationally modified in a tissue specific fashion. In some types of allergy, the peptide+ MHC combination may also be fully derived from self. However the combination in question may be modified by the presence of other ligands, such as small drug molecules or metal ions. Thus these types of allergies may act like the post translationally modified peptides involved some types of autoimmunity.     

Isotope chirality in long‐armed multifunctional organosilicon (“Cephalopod”) molecules

Long‐armed multifunctional organosilicon molecules display self‐replicating and self‐perfecting behavior in asymmetric autocatalysis (Soai reaction). Two representatives of this class were studied by statistical methods aiming at determination of probabilities of natural abundance chiral isotopomers. The results, reported here, show an astonishing richness of possibilities of the formation of chiral isotopically substituted derivatives. This feature could serve as a model for the evolution of biological chirality in prebiotic and early biotic stereochemistry.     

Peptide contour length determines equilibrium secondary structure in protein‐analogous micelles

This work advances bottom‐up design of bioinspired materials built from peptide‐amphiphiles, which are a class of bioconjugates in which a biofunctional peptide is covalently attached to a hydrophobic moiety that drives self‐assembly in aqueous solution. Specifically, this work highlights the importance of peptide contour length in determining the equilibrium secondary structure of the peptide as well as the self‐assembled (i.e., micelle) geometry. Peptides used here repeat a seven‐amino acid sequence between one and four times to vary peptide contour length while maintaining similar peptide‐peptide interactions. Without a hydrophobic tail, these peptides all exhibit a combination of random coil and α‐helical structure. Upon self‐assembly in the crowded environment of a micellar corona, however, short peptides are prone to β‐sheet structure and cylindrical micelle geometry while longer peptides remain helical in spheroidal micelles. The transition to β‐sheets in short peptides is rapid, whereby amphiphiles first self‐assemble with α‐helical peptide structure, then transition to their equilibrium β‐sheet structure at a rate that depends on both temperature and ionic strength. These results identify peptide contour length as an important control over equilibrium peptide secondary structure and micelle geometry. Furthermore, the time‐dependent nature of the helix‐to‐sheet transition opens the door for shape‐changing bioinspired materials with tunable conversion rates. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 573–581, 2013.     

Protamine‐induced condensation of peptide nanofilaments into twisted bundles with controlled helical geometry

Chiral self‐assembly of peptides is of fundamental interest in the field of biology and material science. Protamine, an alkaline biomacromolecule which is ubiquitous in fish and mammalian, plays crucial roles in directing the helical twisting of DNA. Inspired by this, we reported a bioinspired pathway to direct the hierarchical chiral self‐assembly of a short synthetic dipeptide. The peptide could self‐assemble into negatively charged chiral micelles in water that spontaneously formed a nematic liquid crystalline phase. By incorporation with protamine, the micelles condensed with the protamine into large helical bundles with precisely controlled diameter. Furthermore, to simulate the intracellular environments, we investigated macromolecular crowding on the coassembly of peptide and protamine, which leads to the formation of much thinner helical structures. The results highlight the roles of highly charged biomacromolecules and macromolecular crowding on peptide self‐assembly, which are beneficial for the practical applications of self‐assembling peptides in biomedicine and sensing.     

CD4+CD25+ regulatory T cells specific for a thymus-expressed antigen prevent the development of anaphylaxis to self

A role for CD4(+)CD25(+) regulatory T cells (Tregs) in the control of allergic diseases has been postulated. We developed a mouse model in which anaphylaxis is induced in SJL mice by immunization and challenge with the fragment of self myelin proteolipid protein (PLP)(139-151), that is not expressed in the thymus, but not with fragment 178-191 of the same protein, that is expressed in the thymus. In this study, we show that resistance to anaphylaxis is associated with naturally occurring CD4(+)CD25(+) Tregs specific for the self peptide expressed in the thymus. These cells increase Foxp3 expression upon Ag stimulation and suppress peptide-induced proliferation of CD4(+)CD25(-) effector T cells. Depletion of Tregs with anti-CD25 in vivo significantly diminished resistance to anaphylaxis to PLP(178-191), suggesting an important role for CD4(+)CD25(+) Tregs in preventing the development of allergic responses to this thymus-expressed peptide. These data indicate that naturally occurring CD4(+)CD25(+) Tregs specific for a peptide expressed under physiological conditions in the thymus are able to suppress the development of a systemic allergic reaction to self.     

Recognition of a sequestered self peptide by influenza virus-specific CD8+ cytolytic T lymphocytes

The Ag receptors on CD8+ CTL recognize foreign antigenic peptides associated with cell surface MHC class I molecules. Peptides derived from self proteins are also normally presented by MHC class I molecules. Here we report that an H-2Kd-restricted murine CD8+ CTL clone directed to an influenza hemagglutinin epitope can recognize a peptide derived from the murine mitochondrial aconitase enzyme in association with H-2Kd molecules. Surprisingly, this self peptide is not normally displayed on the cell surface associated with the restricting MHC class I molecule. Several lines of evidence suggest that this self peptide, although requiring association with the Kd molecule for CTL recognition, is not associated with this or other MHC class I allele under physiologic conditions in intact cells. Rather, it is sequestered in the cytoplasm associated with a carrier protein and is released only upon cell disruption. These results suggest a means of restricting the entry of self peptide into the class I pathway. In addition, this finding raises the possibility that self peptides sequestered within the cell can, after release from damaged cells, interact with MHC class I molecules on bystander cells and trigger autoimmune injury by virus-specific CTLs during viral infection.     

Breakdown of tolerance to a self-peptide of acetylcholine receptor alpha-subunit induces experimental myasthenia gravis in rats

Experimental autoimmune myasthenia gravis (EAMG), a model for human myasthenia (MG), is routinely induced in susceptible rat strains by a single immunization with Torpedo acetylcholine receptor (TAChR). TAChR immunization induces anti-AChR Abs that cross-react with self AChR, activate the complement cascade, and promote degradation of the postsynaptic membrane of the neuromuscular junction. In parallel, TAChR-specific T cells are induced, and their specific immunodominant epitope has been mapped to the sequence 97-116 of the AChR alpha subunit. A proliferative T cell response against the corresponding rat sequence (R97-116) was also found in TAChR-immunized rats. To test whether the rat (self) sequence can be pathogenic, we immunized Lewis rats with R97-116 or T97-116 peptides and evaluated clinical, neurophysiological, and immunological parameters. Clinical signs of the disease were noted only in R97-116-immunized animals and were confirmed by electrophysiological signs of impaired neuromuscular transmission. All animals produced Abs against the immunizing peptide, but anti-rat AChR Abs were observed only in animals immunized with the rat peptide. These findings suggested that EAMG in rats can be induced by a single peptide of the self AChR, that this sequence is recognized by T cells and Abs, and that breakdown of tolerance to a self epitope might be an initiating event in the pathogenesis of rat EAMG and MG.     

TectoRNP: self‐assembling RNAs with peptide recognition motifs as templates for chemical peptide ligation

TectoRNA, an artificial RNA with self‐assembling ability, has been employed as a structural platform for RNA nanotechnology and RNA synthetic biology. In this study, tectoRNA was applied as a specific template for chemical peptide ligation. On the basis of a self‐assembling tectoRNA, we designed and constructed a template RNA that facilitates peptide ligation depending on controlled dimer formation. Two RNA‐binding peptides were recognized by two peptide‐binding RNA motifs embedded in the template RNA, and chemical ligation was promoted because of the entropic effect of Mg²⁺‐dependent dimerization. In a series of biochemical analyses, we determined the relationship between the structures of the tectoRNA‐based templates and the extent of acceleration in peptide ligation. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Differential presentation of an altered peptide within fetal central and peripheral organs supports an avidity model for thymic T cell development and implies a peripheral readjustment for activation

Altered self peptides may drive T cell development by providing avidity of interactions low enough to potentiate positive selection but not powerful enough to trigger programmed cell death. Since the peptide repertoire in both central and peripheral organs is nearly the same, interactions of these peptides with T cells in the thymus would have to be different from those taking place in the periphery; otherwise, T cell development and maturation would result in either autoimmunity or T cell deficiency. Herein, a self and an altered self peptide were delivered to fetuses, and their presentation as well as the consequence of such presentation on T cell development were assessed. The results indicate that the self peptide was presented in both central and peripheral fetal organs and that such presentation abolished T cell responses to both peptides during adult life. However, the altered peptide, although presented in vivo as well as in vitro by splenic cells, was unable to stimulate a specific T cell clone when the presenting cells were of thymic origin and allowed offspring to be responsive to both peptides. These findings indicate that central and peripheral organs accommodate selection and peripheral survival of T cells by promoting differential altered peptide presentation.     

T cells that recognize peptide sequences of self MHC class II molecules exist in syngeneic mice.

T cell reactivity toward self MHC class II molecules has been recognized in syngeneic MLR in a number of studies, where the T cells are believed to recognize the combination of self/nonself peptide and self MHC molecule. We investigated the stimulation of T cell proliferation by synthetic peptides of sequences corresponding to the first polymorphic amino terminal domain of alpha- and beta-chains of self I-A molecules. Both unprimed and primed T cells responded to a number of peptides of alpha 1 and beta 1 domains of self I-Ad molecules. The response was dependent on the presentation of I-Ad peptides by syngeneic APC and was blocked by anti-class II MHC mAb. Upon further investigation it was observed that I-Ad peptides could inhibit the stimulation of Ag-specific MHC class II-restricted T cell hybridoma due to self presentation of peptides rather than to direct binding of free peptides to the TCR, further supporting their affinity/interaction with intact self MHC class II molecules. The peptide I-A beta d 62-78 showed high affinity toward intact self MHC II molecule as determined by the inhibition of Ag-specific T cell stimulation and yet was nonstimulatory for syngeneic T cells, therefore representing an MHC determinant that may have induced self tolerance. Thus we have shown that strong T cell proliferative responses can be generated in normal mice against the peptides derived from self MHC class II molecules and these cells are part of the normal T cell repertoire. Therefore complete tolerance toward potentially powerful immunodominant but cryptic determinants of self Ag may not be necessary to prevent autoimmune diseases.     

T cell tolerance based on avidity thresholds rather than complete deletion allows maintenance of maximal repertoire diversity

Given the flexible nature of TCR specificity, deletion or permanent disabling of all T cells with the capacity to recognize self peptides would severely limit the diversity of the repertoire and the capacity to recognize foreign Ags. To address this, we have investigated the patterns of CD8+ CTL reactivity to a naturally H-2Kb-presented self peptide derived from the elongation factor 1alpha (EF1alpha). EF1alpha occurs as two differentially expressed isoforms differing at one position of the relevant peptide. Low avidity CTLs could be raised against both variants of the EF1alpha peptide. These CTLs required 100-fold more peptide-H-2Kb complexes on the target cell compared with CTLs against a viral peptide, and did not recognize the naturally expressed levels of EF1alpha peptides. Thus, low avidity T cells specific for these self peptides escape tolerance by deletion, despite expression of both EF1alpha isoforms in dendritic cells known to mediate negative selection in the thymus. The low avidity in CTL recognition of these peptides correlated with low TCR affinity. However, self peptide-specific CTLs expressed elevated levels of CD8. Furthermore, CTLs generated against altered self peptide variants displayed intermediate avidity, indicating cross-reactivity in induction of tolerance. We interpret these data, together with results previously published by others, in an avidity pit model based on avidity thresholds for maintenance of both maximal diversity and optimal self tolerance in the CD8+ T cell repertoire.     

Controlling network topology and mechanical properties of co‐assembling peptide hydrogels

Oligopeptides are well‐known to self‐assemble into a wide array of nanostructures including β‐sheet‐rich fibers that when present above a critical concentration become entangled and form self‐supporting hydrogels. The length, quantity, and interactions between fibers influence the mechanical properties of the hydrogel formed and this is typically achieved by varying the peptide concentration, pH, ionic strength, or the addition of a second species or chemical cross‐linking agent. Here, we outline an alternative, facile route to control the mechanical properties of the self‐assembling octa‐peptide, FEFEFKFK (FEKII); simply doping with controlled quantities of its double length peptide, FEFEFKFK‐GG‐FKFKFEFE (FEKII18). The structure and properties of a series of samples were studied here (0–100 M% of FEKII18) using Fourier transform infrared, small angle X‐ray scattering, transmission electron microscopy, and oscillatory rheology. All samples were found to contain elongated, flexible fibers and all mixed samples contained Y‐shaped branch points and parallel fibers which is attributed to the longer peptide self‐assembling within two fibers, thus creating a cross‐link in the network structure. Such behavior was reflected in an increase in the elasticity of the mixed samples with increasing quantity of double peptide. Interestingly the elastic modulus increased up to 30 times the pure FEKII value simply by adding 28 M% of FEKII18. These observations provide an easy, off‐the‐shelf method for an end‐user to control the cross‐linked network structure of the peptide hydrogel, and consequently strength of the hydrogel simply by physically mixing pre‐determined quantities of two similar peptide molecules. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 669–680, 2014.     

Development of short and highly potent self‐assembling elastin‐derived pentapeptide repeats containing aromatic amino acid residues

Tropoelastin is the primary component of elastin, which forms the elastic fibers that make up connective tissues. The hydrophobic domains of tropoelastin are thought to mediate the self‐assembly of elastin into fibers, and the temperature‐mediated self‐assembly (coacervation) of one such repetitive peptide sequence (VPGVG) has been utilized in various bio‐applications. To elucidate a mechanism for coacervation activity enhancement and to develop more potent coacervatable elastin‐derived peptides, we synthesized two series of peptide analogs containing an aromatic amino acid, Trp or Tyr, in addition to Phe‐containing analogs and tested their functional characteristics. Thus, position 1 of the hydrophobic pentapeptide repeat of elastin (X¹P²G³V⁴G⁵) was substituted by Trp or Tyr. Eventually, we acquired a novel, short Trp‐containing elastin‐derived peptide analog (WPGVG)₃ with potent coacervation ability. From the results obtained during this process, we determined the importance of aromaticity and hydrophobicity for the coacervation potency of elastin‐derived peptide analogs. Generally, however, the production of long‐chain synthetic polypeptides in quantities sufficient for commercial use remain cost‐prohibitive. Therefore, the identification of (WPGVG)₃, which is a 15‐mer short peptide consisting simply of five natural amino acids and shows temperature‐dependent self‐assembly activity, might serve as a foundation for the development of various kinds of biomaterials. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Improved Fmoc‐based solid‐phase synthesis of homologous peptide fragments of human and mouse prion proteins

The synthesis of difficult peptide sequences has been a challenge since the very beginning of SPPS. The self‐assembly of the growing peptide chains has been proposed as one of the causes of this synthetic problem. However, there is an increasing need to obtain peptides and proteins that are prone to aggregate. These peptides and proteins are generally associated with diseases known as amyloidoses. We present an efficient SPPS of two homologous peptide fragments of HuPrP (106–126) and MoPrP105–125 based on the use of the PEGA resin combined with proper coupling approaches. These peptide fragments were also studied by CD and TEM to determine their ability to aggregate. On the basis of these results, we support PEG‐based resins as an efficient synthetic tool to prepare peptide sequences prone to aggregate on‐resin. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.