Mitochondrial Molecular Adaptations and Life History Strategies Coevolve in Plants

Messenger RNA secondary structure prevents mutations at functionally important sites. Mutations at exposed sites would cause micro-adaptations, niche-specialization, and therefore, can be thought to promote K-strategists. Exposing, rather than protecting, conserved sites, is also potentially adaptive because they probably promote macro-adaptive changes. This presumably fits r-strategists: their population dynamics tolerate decreased survival. We found that helix-forming tendencies are greater at evolutionary conserved sites of plant mitochondrial mRNAs than at evolutionary variable sites in a majority (73%) of species–gene combinations. K-strategists preferentially protect conserved sites in short genes, r-strategists protect them most in larger genes. This adaptive scenario resembles our earlier findings in chloroplast genes. Protection levels at various codon positions also display disparity with respect to life history strategies of the plants. Conserved site protection increases overall mRNA folding stabilities for some genes, while decreases it for some others. This contrast exists between homologous genes of r- and K- strategists. Such compensating interactions between variability, mRNA size, codon position, and secondary structure factors within r- and K-strategists are most likely, molecular adaptations of plants belonging to the two extreme life history strategies. Our results suggest coevolution between molecular and ecological adaptive strategies

Bioinformatic flowchart and database to investigate the origins and diversity of Clan AA peptidases

<p>Abstract</p> <p>Background</p> <p>Clan AA of aspartic peptidases relates the family of pepsin monomers evolutionarily with all dimeric peptidases encoded by eukaryotic LTR retroelements. Recent findings describing various pools of single-domain nonviral host peptidases, in prokaryotes and eukaryotes, indicate that the diversity of clan AA is larger than previously thought. The ensuing approach to investigate this enzyme group is by studying its phylogeny. However, clan AA is a difficult case to study due to the low similarity and different rates of evolution. This work is an ongoing attempt to investigate the different clan AA families to understand the cause of their diversity.</p> <p>Results</p> <p>In this paper, we describe in-progress database and bioinformatic flowchart designed to characterize the clan AA protein domain based on all possible protein families through ancestral reconstructions, sequence logos, and hidden markov models (HMMs). The flowchart includes the characterization of a major consensus sequence based on 6 amino acid patterns with correspondence with Andreeva's model, the structural template describing the clan AA peptidase fold. The set of tools is work in progress we have organized in a database within the GyDB project, referred to as Clan AA Reference Database <url>http://gydb.uv.es/gydb/phylogeny.php?tree=caard</url>.</p> <p>Conclusion</p> <p>The pre-existing classification combined with the evolutionary history of LTR retroelements permits a consistent taxonomical collection of sequence logos and HMMs. This set is useful for gene annotation but also a reference to evaluate the diversity of, and the relationships among, the different families. Comparisons among HMMs suggest a common ancestor for all dimeric clan AA peptidases that is halfway between single-domain nonviral peptidases and those coded by <it>Ty3/Gypsy </it>LTR retroelements. Sequence logos reveal how all clan AA families follow similar protein domain architecture related to the peptidase fold. In particular, each family nucleates a particular consensus motif in the sequence position related to the flap. The different motifs constitute a network where an alanine-asparagine-like variable motif predominates, instead of the canonical flap of the HIV-1 peptidase and closer relatives.</p> <p>Reviewers</p> <p>This article was reviewed by Daniel H. Haft, Vladimir Kapitonov (nominated by Jerry Jurka), and Ben M. Dunn (nominated by Claus Wilke).</p

: peel it

International audienceThree-dimensional structures of proteins are the support of their biological functions. Their folds are maintained by inter-residue interactions which are one of the main focuses to understand the mechanisms of protein folding and stability. Furthermore, protein structures can be composed of single or multiple functional domains that can fold and function independently. Hence, dividing a protein into domains is useful for obtaining an accurate structure and function determination. In previous studies, we enlightened protein contact properties according to different definitions and developed a novel methodology named Protein Peeling. Within protein structures, Protein Peeling characterizes small successive compact units along the sequence called protein units (PUs). The cutting done by Protein Peeling maximizes the number of contacts within the PUs and minimizes the number of contacts between them. This method is so a relevant tool in the context of the protein folding research and particularly regarding the hierarchical model proposed by George Rose. Here, we accurately analyze the PUs at different levels of cutting, using a non-redundant protein databank. Distribution of PU sizes, number of PUs or their accessibility are screened to determine their common and different features. Moreover, we highlight the preferential amino acid interactions inside and between PUs. Our results show that PUs are clearly an intermediate level between secondary structures and protein structural domains

Production and analysis of synthetic Cascade variants

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR assoziiert) ist ein adaptives Immunsystem in Archaeen und Bakterien, das fremdes genetisches Material mit Hilfe von Ribonukleoprotein-Komplexen erkennt und zerstört. Diese Komplexe bestehen aus einer CRISPR RNA (crRNA) und Cas Proteinen. CRISPR-Cas Systeme sind in zwei Hauptklassen und mehrere Typen unterteilt, abhängig von den beteiligten Cas Proteinen. In Typ I Systemen sucht ein Komplex namens Cascade (CRISPR associated complex for antiviral defence) nach eingedrungener viraler DNA während einer Folgeinfektion und bindet die zu der eingebauten crRNA komplementäre Sequenz. Anschließend wird die Nuklease/Helikase Cas3 rekrutiert, welche die virale DNA degradiert (Interferenz). Das Typ I System wird in mehrere Subtypen unterteilt, die Unterschiede im Aufbau von Cascade vorweisen. Im Fokus dieser Arbeit steht eine minimale Cascade-Variante aus Shewanella putrefaciens CN-32. Im Vergleich zur gut untersuchten Typ I-E Cascade aus Escherichia coli fehlen in diesem Komplex zwei Untereinheiten, die gewöhnlicher Weise für die Zielerkennung benötigt werden. Dennoch ist der Komplex aktiv. Rekombinante I-Fv Cascade wurde bereits aus E. coli aufgereinigt und es war möglich, den Komplex zu modifizieren, indem das Rückgrat entweder verlängert oder verkürzt wurde. Dadurch wurden synthetische Varianten mit veränderter Protein-Stöchiometrie erzeugt. In der vorliegenden Arbeit wurde I-Fv Cascade weiter mit in vitro Methoden untersucht. So wurde die Bindung von Ziel-DNA beobachtet und die 3D Struktur zeigt, dass strukturelle Veränderungen im Komplex die fehlenden Untereinheiten ersetzen, möglicherweise um viralen Anti-CRISPR Proteinen zu entgehen. Die Nuklease/Helikase dieses Systems, Cas2/3fv, ist eine Fusion des Cas3 Proteins mit dem Interferenz-unabhängigen Protein Cas2. Ein unabhängiges Cas3fv ohne Cas2 Untereinheit wurde aufgereinigt und in vitro Assays zeigten, dass dieses Protein sowohl freie ssDNA als auch Cascadegebundene Substrate degradiert. Das komplette Cas2/3fv Protein bildet einen Komplex mit dem Protein Cas1 und zeigt eine reduzierte Aktivität gegenüber freier ssDNA, möglicherweise als Regulationsmechanismus zur Vermeidung von unspezifischer Aktivität. Weiterhin wurde ein Prozess namens „RNA wrapping“ etabliert. Synthetische Cascade-Komplexe wurden erzeugt, in denen die grundlegende RNA-Bindung des charakteristischen Cas7fv RückgratProteins auf eine ausgewählte RNA gelenkt wird. Diese spezifische Komplexbildung kann in vivo durch eine Repeat-Sequenz der crRNA stromaufwärts der Zielsequenz und durch Bindung des Cas5fv Proteins initiiert werden. Die erzeugten Komplexe beinhalten die ersten 100 nt der markierten RNA, die anschließend isoliert werden kann. Innerhalb der Komplexe ist die RNA stabilisiert und geschützt vor Degradation durch RNasen. Komplexbildung kann außerdem genutzt werden, um ReportergenTranskripte stillzulegen. Zusätzlich wurden erste Hinweise geliefert, dass das Rückgrat der synthetischen Komplexe durch Fusion mit weiteren Reporterproteinen modifiziert werden kann.CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated) is an adaptive immune system of Archaea and Bacteria. It is able to target and destroy foreign genetic material with ribonucleoprotein complexes consisting of CRISPR RNAs (crRNAs) and certain Cas proteins. CRISPR-Cas systems are classified in two major classes and multiple types, according to the involved Cas proteins. In type I systems, a ribonucleoprotein complex called Cascade (CRISPR associated complex for antiviral defence) scans for invading viral DNA during a recurring infection and binds the sequence complementary to the incorporated crRNA. After target recognition, the nuclease/helicase Cas3 is recruited and subsequently destroys the viral DNA in a step termed interfere nce. Multiple subtypes of type I exist that show differences in the Cascade composition. This work focuses on a minimal Cascade variant found in Shewanella putrefaciens CN-32. In comparison to the well-studied type I-E Cascade from Escherichia coli, this complex is missing two proteins usually required for target recognition, yet it is still able to provide immunity. Recombinant I-Fv Cascade was previously purified from E. coli and it was possible to modulate the complex by extending or shortening the backbone, resulting in synthetic variants with altered protein stoichiometry. In the present study, I-Fv Cascade was further analyzed by in vitro methods. Target binding was observed and the 3D structure revealed structural variations that replace the missing subunits, potentially to evade viral anti-CRISPR proteins. The nuclease/helicase of this system, Cas2/3fv, is a fusion of the Cas3 protein with the interference-unrelated protein Cas2. A standalone Cas3fv was purified without the Cas2 domain and in vitro cleavage assays showed that Cas3fv degrades both free ssDNA as well as Cascade-bound substrates. The complete Cas2/3fv protein forms a complex with the protein Cas1 and was shown to reduce cleave of free ssDNA, potentially as a regulatory mechanism against unspecific cleavage. Furthermore, we established a process termed “RNA wrapping”. Synthetic Cascade assemblies can be created by directing the general RNA-binding ability of the characteristic Cas7fv backbone protein on an RNA of choice such as reporter gene transcripts. Specific complex formation can be initiated in vivo by including a repeat sequence from the crRNA upstream a given target sequence and binding of the Cas5fv protein. The created complexes contain the initial 100 nt of the tagged RNA which can be isolated afterwards. While incorporated in complexes, RNA is stabilized and protected from degradation by RNases. Complex formation can be used to silence reporter gene transcripts. Furthermore, we provided initial indications that the backbone of synthetic complexes can be modified by addition of reporter proteins

A design study of hydrazine and biowaste resistojets

A generalized modeling program was adapted in BASIC on a personal computer to compare the performance of four types of biowaste resistojets and two types of hydrazine augmenters. Analyzed biowaste design types were: (1) an electrically conductive ceramic heater-exchanger of zirconia; (2) a truss heater of platinum in cross flow; (3) an immersed bicoiled tubular heater-exchanger; and (4) a nonexposed, refractory metal, radiant heater in a central cavity within a heat exchanger case. Concepts 2 and 3 are designed to have an efficient, stainless steel outer pressure case. The hydrazine design types are: (5) an immersed bicoil heater exchanger and (6) a nonexposed radiant heater now with a refractory metal case. The ceramic biowaste resistojet has the highest specific impulse growth potential at 2000 K of 192.5 (CO2) and 269 s (H2O). The bicoil produces the highest augmenter temperature of 1994 K for a 2073 K heater giving 317 s at .73 overall efficiency. Detailed temperature profiles of each of the designs are shown. The scaled layout drawings of each are presented with recommended materials and fabrication methods

DE NOVO CATIONIC ANTIMICROBIAL PEPTIDES AS THERAPEUTICS AGAINST PSEUDOMONAS AERUGINOSA

Cationic antimicrobial peptides (CAPs) are a very diverse group of amphipathic agents that demonstrate broad activity against Gram-positive and -negative bacteria. To overcome the obstacle of drug resistance among bacterial pathogens, CAPs have been extensively investigated as a potential source of new antimicrobials with novel mechanisms of action that may complement current antibiotic regimens. However, the suppression of antimicrobial activity in biological conditions (e.g., physiological salt concentrations, serum) constitutes a major challenge to the successful development of CAPs for clinical applications. We hypothesized that CAPs with optimized amphipathic structures can be designed de novo to enhance antibacterial activity and selective toxicity in environments that are generally challenging to host-derived peptides. Furthermore, the antibacterial efficacy will positively correlate with length, charge, Trp content, and helicity. Three specific aims were developed to address this hypothesis. The first aim addressed the design of amphipathic peptides to evaluate the influence of helicity, length, and Trp content on activity. Using a base unit peptide approach, we synthesized a multimeric series of 12-residue lytic base unit (LBU) composed of Arg and Val residues, positioned to form idealized amphipathic ƒÑ-helices. Another series of LBU derivatives (WLBU) was engineered by substituting Trp residues on the hydrophobic face. The correlation between length and helicity was established by circular dichroism analysis. Bacterial killing assays revealed no appreciable increase in activity for peptides longer than 24 residues. In addition, the inclusion of Trp residues in the hydrophobic face increased potency and selectivity in a novel co-culture system utilizing bacteria and primary cell lines, which led to the selection of WLBU2 as the shortest peptide (24 residues in length) with the highest potency. The second objective was to examine the selective toxicity of WLBU2 in biological or biologically-derived media. In contrast to the human CAP LL37, the peptide WLBU2 displayed potent activity against Pseudomonas aeruginosa in the presence of human serum and human blood ex-vivo, with no detectable red blood cell lysis or toxicity to human monocytes at all test concentrations. Finally, we demonstrated potent activity of WLBU2 in intraperitoneal and intravenous mouse models of Pseudomonas aeruginosa infection. WLBU2 not only protected mice prophylactically but also eradicated P. aeruginosa from the blood and other tissues at 3 to 4mg/kg. Furthermore, WLBU2 displayed only a minor stimulatory effect on inflammatory cytokines, notably IL1-£] and TNF-ƒÑ. Consistent with our in vitro studies, the in vivo data provide strong evidence for the potential application of WLBU2 in the treatment of systemic infection due to P. aeruginosa

Molecular evolution of rbcL in three gymnosperm families: identifying adaptive and coevolutionary patterns

<p>Abstract</p> <p>Background</p> <p>The chloroplast-localized ribulose-1, 5-biphosphate carboxylase/oxygenase (Rubisco), the primary enzyme responsible for autotrophy, is instrumental in the continual adaptation of plants to variations in the concentrations of CO₂. The large subunit (LSU) of Rubisco is encoded by the chloroplast <it>rbcL </it>gene. Although adaptive processes have been previously identified at this gene, characterizing the relationships between the mutational dynamics at the protein level may yield clues on the biological meaning of such adaptive processes. The role of such coevolutionary dynamics in the continual fine-tuning of RbcL remains obscure.</p> <p>Results</p> <p>We used the timescale and phylogenetic analyses to investigate and search for processes of adaptive evolution in <it>rbcL </it>gene in three gymnosperm families, namely Podocarpaceae, Taxaceae and Cephalotaxaceae. To understand the relationships between regions identified as having evolved under adaptive evolution, we performed coevolutionary analyses using the software CAPS. Importantly, adaptive processes were identified at amino acid sites located on the contact regions among the Rubisco subunits and on the interface between Rubisco and its activase. Adaptive amino acid replacements at these regions may have optimized the holoenzyme activity. This hypothesis was pinpointed by evidence originated from our analysis of coevolution that supported the correlated evolution between Rubisco and its activase. Interestingly, the correlated adaptive processes between both these proteins have paralleled the geological variation history of the concentration of atmospheric CO₂.</p> <p>Conclusions</p> <p>The gene <it>rbcL </it>has experienced bursts of adaptations in response to the changing concentration of CO₂in the atmosphere. These adaptations have emerged as a result of a continuous dynamic of mutations, many of which may have involved innovation of functional Rubisco features. Analysis of the protein structure and the functional implications of such mutations put forward the conclusion that this evolutionary scenario has been possible through a complex interplay between adaptive mutations, often structurally destabilizing, and compensatory mutations. Our results unearth patterns of evolution that have likely optimized the Rubisco activity and uncover mutational dynamics useful in the molecular engineering of enzymatic activities.</p> <p>Reviewers</p> <p>This article was reviewed by Prof. Christian Blouin (nominated by Dr W Ford Doolittle), Dr Endre Barta (nominated by Dr Sandor Pongor), and Dr Nicolas Galtier.</p

Structural and Conformational Analysis of B-cell Epitopes − component to guide peptide vaccine design

Peptide vaccines have many potential advantages including low cost, lack of need for cold-chain storage and safety. However, it is well known that approximately 90% of B-cell Epitopes (BCEs) are discontinuous in nature making it difficult to mimic them for creating vaccines. To perform a detailed structural analysis of these epitopes, they needs to be mapped onto antigen structures that are complexed with antibody. In order to obtain a clean dataset of antibody-antigen complex crystal structures, a pipeline was designed to process automatically and clean the antibody related structures from the PDB. To store this processed antibody structural data, a database (AbDb) was built and made available online. The degree of discontinuity in B-cell epitopes and their conformational nature was studied by mapping epitopes in the antibody-antigen dataset. The discontinuity of B-cell epitopes was analysed by defining extended ‘regions’ (R, consisting of at least 3 antibody-contacting residues each separated by ≤ 3 residues) and small fragments (F, antibody-contacting residues that do not satisfy the requirements for a region). Secondly, an algorithm was developed to classify region shape as linear, curved or folded. Molecular dynamics simulations were carried out on isolated epitope regions (wild type and mutant peptides). The mutant peptides have been designed by mutating non-contacting and hydrophobic residues in epitopes. Two types of mutations (hy- drophobic to alanine and hydrophobic to glutamine) have been studied using molec- ular dynamics simulations. Furthermore, the effect of end-capping on wild type and mutant epitope regions has been studied. Simulation studies were carried out on 5 linear and 5 folded shape regions. Out of these, 2 epitopes (one linear and one folded), along with their mutants and derivatives, were tested experimentally for conformational stability by CD spectroscopy and NMR. The binding of isolated epitopes with antibody was also validated by ELISA and SPR