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We present a potential mechanism for emergence of catalytic activity that is essential for survival, from a non-catalytic protein fold. The type B dihydrofolate reductase (DfrB) family of enzymes were first identified in pathogenic bacteria because their dihydrofolate reductase activity is sufficient to provide trimethoprim (TMP) resistance. DfrB enzymes are described as poorly evolved as a result of their unusual structural and kinetic features. No characterized protein shares sequence homology with DfrB enzymes; how they evolved to emerge in the modern resistome is unknown. In this work, we identify DfrB homologues from a database of putative and uncharacterized proteins. These proteins include an SH3-like fold homologous to the DfrB enzymes, embedded in a variety of additional structural domains. By means of functional, structural and biophysical characterization, we demonstrate that these distant homologues and their extracted SH3-like fold can display dihydrofolate reductase activity and confer TMP resistance. We provide evidence of tetrameric assembly and catalytic mechanism analogous to that of DfrB enzymes. These results contribute, to our knowledge, the first insights into a potential evolutionary path taken by this SH3-like fold to emerge in the modern resistome following introduction of TMP. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'. [ABSTRACT FROM AUTHOR]

Protein–protein interactions (PPIs) between modular binding domains and their target peptide motifs are thought to largely depend on the intrinsic binding specificities of the domains. The large family of SRC Homology 3 (SH3) domains contribute to cellular processes via their ability to support such PPIs. While the intrinsic binding specificities of SH3 domains have been studied in vitro, whether each domain is necessary and sufficient to define PPI specificity in vivo is largely unknown. Here, by combining deletion, mutation, swapping and shuffling of SH3 domains and measurements of their impact on protein interactions in yeast, we find that most SH3s do not dictate PPI specificity independently from their host protein in vivo. We show that the identity of the host protein and the position of the SH3 domains within their host are critical for PPI specificity, for cellular functions and for key biophysical processes such as phase separation. Our work demonstrates the importance of the interplay between a modular PPI domain such as SH3 and its host protein in establishing specificity to wire PPI networks. These findings will aid understanding how protein networks are rewired during evolution and in the context of mutation-driven diseases such as cancer. The SRC Homology 3 (SH3) domains mediate protein–protein interactions (PPIs). Here, the authors assess the SH3-mediated PPIs in yeast, and show that the identity of the protein itself and the position of the SH3 both affect the interaction specificity and thus the PPI-dependent cellular functions. [ABSTRACT FROM AUTHOR]

The pretreatment of biomass remains a critical requirement for bio-renewable fuel production from lignocellulose. Although current processes primarily involve chemical and physical approaches, the biological breakdown of lignin using enzymes and microorganisms is quickly becoming an interesting eco-friendly alternative to classical processes. As a result, bioprospection of wild fungi from naturally occurring lignin-rich sources remains a suitable method to uncover and isolate new species exhibiting ligninolytic activity. In this study, wild species of white rot fungi were collected from Colombian forests based on their natural wood decay ability and high capacity to secrete oxidoreductases with high affinity for phenolic polymers such as lignin. Based on high activity obtained from solid-state fermentation using a lignocellulose source from oil palm as matrix, we describe the isolation and whole-genome sequencing of Dictyopanus pusillus, a wild basidiomycete fungus exhibiting ABTS oxidation as an indication of laccase activity. Functional characterization of a crude enzymatic extract identified laccase activity as the main enzymatic contributor to fungal extracts, an observation supported by the identification of 13 putative genes encoding for homologous laccases in the genome. To the best of our knowledge, this represents the first report of an enzymatic extract exhibiting laccase activity in the Dictyopanus genera, offering means to exploit this species and its enzymes for the delignification process of lignocellulosic by-products from oil palm. [ABSTRACT FROM AUTHOR]

The 3‐(3‐hydroxyalkanoyloxy)alkanoate (HAA) synthase RhlA is an essential enzyme involved in the biosynthesis of HAAs in Pseudomonas and Burkholderia species. RhlA modulates the aliphatic chain length in rhamnolipids, conferring distinct physicochemical properties to these biosurfactants exhibiting promising industrial and pharmaceutical value. A detailed molecular understanding of substrate specificity and catalytic performance in RhlA could offer protein engineering tools to develop designer variants involved in the synthesis of novel rhamnolipid mixtures for tailored eco‐friendly products. However, current directed evolution progress remains limited due to the absence of high‐throughput screening methodologies and lack of an experimentally resolved RhlA structure. In the present work, we used comparative modeling and chimeric‐based approaches to perform a comprehensive semi‐rational mutagenesis of RhlA from Pseudomonas aeruginosa. Our extensive RhlA mutational variants and chimeric hybrids between the Pseudomonas and Burkholderia homologs illustrate selective modulation of rhamnolipid alkyl chain length in both Pseudomonas aeruginosa and Burkholderia glumae. Our results also demonstrate the implication of a putative cap‐domain motif that covers the catalytic site of the enzyme and provides substrate specificity to RhlA. This semi‐rational mutant‐based survey reveals promising 'hot‐spots' for the modulation of RL congener patterns and potential control of enzyme activity, in addition to uncovering residue positions that modulate substrate selectivity between the Pseudomonas and Burkholderia functional homologs. Database: Model data are available in the PMDB database under the accession number PM0081867. [ABSTRACT FROM AUTHOR]

The Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP), a polycationic, amphiphilic and helical neuropeptide, is well known for its neuroprotective actions and cell penetrating properties. In the present study, we evaluated the potent antibacterial property of PACAP38 and related analogs against various bacterial strains. Interestingly, PACAP38 and related analogs can inhibit the growth of various bacteria including Escherichia coli (JM109), Bacillus subtilis (PY79), and the pathogenic Burkholderia cenocepacia (J2315). Investigation of the mechanism of action suggested that a PACAP metabolite, identified as PACAP(9–38), might indeed be responsible for the observed PACAP38 antibacterial action. Surprisingly, PACAP(9–38), which does not induce haemolysis, exhibits an increased specificity toward Burkholderia cenocepacia J2315 compared to other tested bacteria. Finally, the predisposition of PACAP(9–38) to adopt a π-helix conformation rather than an α-helical conformation like PACAP38 could explain this gain in specificity. Overall, this study has revealed a new function for PACAP38 and related derivatives that can be added to its pleiotropic biological activities. This innovative study could therefore pave the way toward the development of new therapeutic agents against multiresistant bacteria, and more specifically the Burkholderia cenocepacia complex. [ABSTRACT FROM AUTHOR]

Cationic antimicrobial peptides (CAMPs) occur naturally in numerous organisms and are considered as a class of antibiotics with promising potential against multi-resistant bacteria. Herein, we report a strategy that can lead to the discovery of novel small CAMPs with greatly enhanced antimicrobial activity and retained antibiofilm potential. We geared our efforts towards i) the N-terminal cysteine functionalization of a previously reported small synthetic cationic peptide (peptide 1037, KRFRIRVRV-NH2), ii) its dimerization through a disulfide bond, and iii) a preliminary antimicrobial activity assessment of the newly prepared dimer against Pseudomonas aeruginosa and Burkholderia cenocepacia, pathogens responsible for the formation of biofilms in lungs of individuals with cystic fibrosis. This dimer is of high interest as it does not only show greatly enhanced bacterial growth inhibition properties compared to its pep1037 precursor (up to 60 times), but importantly, also displays antibiofilm potential at sub-MICs. Our results suggest that the reported dimer holds promise for its use in future adjunctive therapy, in combination with clinically-relevant antibiotics. [ABSTRACT FROM AUTHOR]

This phase I study (NCT01539889) evaluated the safety, efficacy, and dosing of PulmoBind for molecular imaging of pulmonary circulation. PulmoBind is a ligand of the adrenomedullin receptor abundantly distributed in lung capillaries. Labeled with 99mTc, it allows single-photon emission computed tomographic (SPECT) imaging of lung perfusion. In preclinical studies, PulmoBind scans enabled detection of lung perfusion defects and quantification of microcirculatory occlusion caused by pulmonary hypertension. Healthy humans (N = 20) were included into escalating groups of 5 mCi (n = 5), 10 mCi (n = 5), or 15 mCi (n = 10) 99mTc-PulmoBind. SPECT imaging was serially performed, and 99mTc-PulmoBind dosimetric analysis was accomplished. The radiochemical purity of 99mTc-PulmoBind was greater than 95%. There were no safety concerns at the three dosages studied. Imaging revealed predominant and prolonged lung uptake with a mean peak extraction of 58% ± 7%. PulmoBind was well tolerated, with no clinically significant adverse event related to the study drug. The highest dose of 15 mCi provided a favorable dosimetric profile and excellent imaging. The postural lung perfusion gradient was detectable. 99mTc-PulmoBind is safe and provides good quality lung perfusion imaging. The safety/efficacy of this agent can be tested in disorders of pulmonary circulation such as pulmonary arterial hypertension. [ABSTRACT FROM AUTHOR]

Key points The renin-angiotensin system plays a key role in cardiovascular physiology and its overactivation has been implicated in the pathogenesis of several major cardiovascular diseases., There is growing evidence that angiotensin II (Ang-II) may function as an intracellular peptide to activate intracellular/nuclear receptors and their downstream signalling effectors independently of cell surface receptors., Current methods used to study intracrine Ang-II signalling are limited to indirect approaches because of a lack of selective intracellularly-acting probes., Here, we present novel photoreleasable Ang-II analogues used to probe intracellular actions with spatial and temporal precision., The photorelease of intracellular Ang-II causes nuclear and cytosolic calcium mobilization and initiates the de novo synthesis of RNA in cardiac cells, demonstrating the application of the method., Abstract Several lines of evidence suggest that intracellular angiotensin II (Ang-II) contributes to the regulation of cardiac contractility, renal salt reabsorption, vascular tone and metabolism; however, work on intracrine Ang-II signalling has been limited to indirect approaches because of a lack of selective intracellularly-acting probes. Here, we aimed to synthesize and characterize cell-permeant Ang-II analogues that are inactive without uncaging, but release active Ang-II upon exposure to a flash of UV-light, and act as novel tools for use in the study of intracrine Ang-II physiology. We prepared three novel caged Ang-II analogues, [Tyr(DMNB)4]Ang-II, Ang-II-ODMNB and [Tyr(DMNB)4]Ang-II-ODMNB, based upon the incorporation of the photolabile moiety 4,5-dimethoxy-2-nitrobenzyl (DMNB). Compared to Ang-II, the caged Ang-II analogues showed 2-3 orders of magnitude reduced affinity toward both angiotensin type-1 (AT1R) and type-2 (AT2R) receptors in competition binding assays, and greatly-reduced potency in contraction assays of rat thoracic aorta. After receiving UV-irradiation, all three caged Ang-II analogues released Ang-II and potently induced the contraction of rat thoracic aorta. [Tyr(DMNB)4]Ang-II showed the most rapid photolysis upon UV-irradiation and was the focus of subsequent characterization. Whereas Ang-II and photolysed [Tyr(DMNB)4]Ang-II increased ERK1/2 phosphorylation (via AT1R) and cGMP production (AT2R), caged [Tyr(DMNB)4]Ang-II did not. Cellular uptake of [Tyr(DMNB)4]Ang-II was 4-fold greater than that of Ang-II and significantly greater than uptake driven by the positive-control HIV TAT(48-60) peptide. Intracellular photolysis of [Tyr(DMNB)4]Ang-II induced an increase in nucleoplasmic Ca2+ ([Ca2+]n), and initiated 18S rRNA and nuclear factor kappa B mRNA synthesis in adult cardiac cells. We conclude that caged Ang-II analogues represent powerful new tools for use in the selective study of intracrine signalling via Ang-II. [ABSTRACT FROM AUTHOR]

The urotensinergic system plays central roles in the physiological regulation of major mammalian organ systems, including the cardiovascular system. As a matter of fact, this system has been linked to numerous pathophysiological states including atherosclerosis, heart failure, hypertension, diabetes as well as psychological, and neurological disorders. The delineation of the (patho)physiological roles of the urotensinergic system has been hampered by the absence of potent and selective antagonists for the urotensin II-receptor (UT). Thus, a more precise definition of the molecular functioning of the urotensinergic system, in normal conditions as well as in a pathological state is still critically needed. The recent discovery of nuclear UT within cardiomyocytes has highlighted the cellular complexity of this system and suggested that UT-associated biological responses are not only initiated at the cell surface but may result from the integration of extracellular and intracellular signaling pathways. Thus, such nuclear-localized receptors, regulating distinct signaling pathways, may represent new therapeutic targets. With the recent observation that urotensin II (UII) and urotensin II-related peptide (URP) exert different biological effects and the postulate that they could also have distinct pathophysiological roles in hypertension, it appears crucial to reassess the recognition process involving UII and URP with UT, and to push forward the development of new analogs of the UT system aimed at discriminating UII- and URP-mediated biological activities. The recent development of such compounds, i.e. urocontrin A and rUII(1-7), is certainly useful to decipher the specific roles of UII and URP in vitro and in vivo. Altogether, these studies, which provide important information regarding the pharmacology of the urotensinergic system and the conformational requirements for binding and activation, will ultimately lead to the development of potent and selective drugs. [ABSTRACT FROM AUTHOR]

Studies the development of agonists of endothelin-1 (ET-1) exhibiting selectivity towards ET[sub A] receptors. Description of the first compounds showing a significant bioactivity in an ET[sub A] pharmacological preparation; Implications for pharmacological research.