Ancestral sequence reconstruction dissects structural and functional differences among eosinophil ribonucleases.

Evolutionarily conserved structural folds can give rise to diverse biological functions, yet predicting atomic-scale interactions that contribute to the emergence of novel activities within such folds remains challenging. Pancreatic-type ribonucleases illustrate this complexity, sharing a core structure that has evolved to accommodate varied functions. In this study, we used ancestral sequence reconstruction to probe evolutionary and molecular determinants that distinguish biological activities within eosinophil members of the RNase 2/3 subfamily. Our investigation unveils functional, structural, and dynamical behaviors that differentiate the evolved ancestral ribonuclease (AncRNase) from its contemporary eosinophil RNase orthologs. Leveraging the potential of ancestral reconstruction for protein engineering, we used AncRNase predictions to design a minimal 4-residue variant that transforms human RNase 2 into a chimeric enzyme endowed with the antimicrobial and cytotoxic activities of RNase 3 members. This work provides unique insights into mutational and evolutionary pathways governing structure, function, and conformational states within the eosinophil RNase subfamily, offering potential for targeted modulation of RNase-associated functions.

Perturbing dimer interactions and allosteric communication modulates the immunosuppressive activity of human galectin-7.

The design of allosteric modulators to control protein function is a key objective in drug discovery programs. Altering functionally essential allosteric residue networks provides unique protein family subtype specificity, minimizes unwanted off-target effects, and helps avert resistance acquisition typically plaguing drugs that target orthosteric sites. In this work, we used protein engineering and dimer interface mutations to positively and negatively modulate the immunosuppressive activity of the proapoptotic human galectin-7 (GAL-7). Using the PoPMuSiC and BeAtMuSiC algorithms, mutational sites and residue identity were computationally probed and predicted to either alter or stabilize the GAL-7 dimer interface. By designing a covalent disulfide bridge between protomers to control homodimer strength and stability, we demonstrate the importance of dimer interface perturbations on the allosteric network bridging the two opposite glycan-binding sites on GAL-7, resulting in control of induced apoptosis in Jurkat T cells. Molecular investigation of G16X GAL-7 variants using X-ray crystallography, biophysical, and computational characterization illuminates residues involved in dimer stability and allosteric communication, along with discrete long-range dynamic behaviors involving loops 1, 3, and 5. We show that perturbing the protein-protein interface between GAL-7 protomers can modulate its biological function, even when the overall structure and ligand-binding affinity remains unaltered. This study highlights new avenues for the design of galectin-specific modulators influencing both glycan-dependent and glycan-independent interactions.

Lipidated peptides derived from intracellular loops 2 and 3 of the urotensin II receptor act as biased allosteric ligands.

Over the last decade, the urotensinergic system, composed of one G protein-coupled receptor and two endogenous ligands, has garnered significant attention as a promising new target for the treatment of various cardiovascular diseases. Indeed, this system is associated with various biomarkers of cardiovascular dysfunctions and is involved in changes in cardiac contractility, fibrosis, and hypertrophy contributing, like the angiotensinergic system, to the pathogenesis and progression of heart failure. Significant investment has been made toward the development of clinically relevant UT ligands for therapeutic intervention, but with little or no success to date. This system therefore remains to be therapeutically exploited. Pepducins and other lipidated peptides have been used as both mechanistic probes and potential therapeutics; therefore, pepducins derived from the human urotensin II receptor might represent unique tools to generate signaling bias and study hUT signaling networks. Two hUT-derived pepducins, derived from the second and the third intracellular loop of the receptor (hUT-Pep2 and [Trp1, Leu2]hUT-Pep3, respectively), were synthesized and pharmacologically characterized. Our results demonstrated that hUT-Pep2 and [Trp1, Leu2]hUT-Pep3 acted as biased ago-allosteric modulators, triggered ERK1/2 phosphorylation and, to a lesser extent, IP1 production, and stimulated cell proliferation yet were devoid of contractile activity. Interestingly, both hUT-derived pepducins were able to modulate human urotensin II (hUII)- and urotensin II-related peptide (URP)-mediated contraction albeit to different extents. These new derivatives represent unique tools to reveal the intricacies of hUT signaling and also a novel avenue for the design of allosteric ligands selectively targeting hUT signaling potentially.

Insights into Structural and Dynamical Changes Experienced by Human RNase 6 upon Ligand Binding.

Ribonuclease 6 (RNase 6) is one of eight catalytically active human pancreatic-type RNases that belong to a superfamily of rapidly evolving enzymes. Like some of its human homologues, RNase 6 exhibits host defense properties such as antiviral and antibacterial activities. Recently solved crystal structures of this enzyme in its nucleotide-free form show the conservation of the prototypical kidney-shaped fold preserved among vertebrate RNases, in addition to revealing the presence of a unique secondary active site. In this study, we determine the structural and conformational properties experienced by RNase 6 upon binding to substrate and product analogues. We present the first crystal structures of RNase 6 bound to a nucleotide ligand (adenosine 5'-monophosphate), in addition to RNase 6 bound to phosphate ions. While the enzyme preserves B2 subsite ligand preferences, our results show a lack of typical B2 subsite interactions normally observed in homologous ligand-bound RNases. A comparison of the dynamical properties of RNase 6 in its apo-, substrate-, and product-bound states highlight the unique dynamical properties experienced on time scales ranging from nano- to milliseconds. Overall, our results confirm the specific evolutionary adaptation of RNase 6 relative to its unique catalytic and biological activities.

Binding of a Soluble meso-Tetraarylporphyrin to Human Galectin-7 Induces Oligomerization and Modulates Its Pro-Apoptotic Activity.

The selective targeting of protein-protein interactions remains a significant determinant for the proper modulation and regulation of cell apoptosis. Prototypic galectins such as human galectin-7 (GAL-7) are characterized by their ability to form homodimers that control the molecular fate of a cell by mediating subtle yet critical glycan-dependent interactions between pro- and anti-apoptotic molecular partners. Altering the structural architecture of GAL-7 can therefore result in resistance to apoptosis in various human cancer cells, further illustrating its importance in cell survival. In this study, we used a combination of biophysical and cellular assays to illustrate that binding of a water-soluble meso-tetraarylporphyrin molecule to GAL-7 induces protein oligomerization and modulation of GAL-7-induced apoptosis in human Jurkat T cells. Our results suggest that the integrity of the GAL-7 homodimer architecture is essential for its molecular function, in addition to providing an interesting porphyrin binding modulator for controlling apoptosis in mammalian cells.

Effect of the pituitary adenylate cyclase-activating polypeptide on the autophagic activation observed in in vitro and in vivo models of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder that leads to destruction of the midbrain dopaminergic (DA) neurons. This phenomenon is related to apoptosis and its activation can be blocked by the pituitary adenylate cyclase-activating polypeptide (PACAP). Growing evidence indicates that autophagy, a self-degradation activity that cleans up the cell, is induced during the course of neurodegenerative diseases. However, the role of autophagy in the pathogenesis of neuronal disorders is yet poorly understood and the potential ability of PACAP to modulate the related autophagic activation has never been significantly investigated. Hence, we explored the putative autophagy-modulating properties of PACAP in in vitro and in vivo models of PD, using the neurotoxic agents 1-methyl-4-phenylpyridinium (MPP(+)) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), respectively, to trigger alterations of DA neurons. In both models, following the toxin exposure, PACAP reduced the autophagic activity as evaluated by the production of LC3 II, the modulation of the p62 protein levels, and the formation of autophagic vacuoles. The ability of PACAP to inhibit autophagy was also observed in an in vitro cell assay by the blocking of the p62-sequestration activity produced with the autophagy inducer rapamycin. Thus, the results demonstrated that autophagy is induced in PD experimental models and that PACAP exhibits not only anti-apoptotic but also anti-autophagic properties.

Molecular imaging of the human pulmonary vascular endothelium in pulmonary hypertension: a phase II safety and proof of principle trial.

PURPOSE: The adrenomedullin receptor is densely expressed in the pulmonary vascular endothelium. PulmoBind, an adrenomedullin receptor ligand, was developed for molecular diagnosis of pulmonary vascular disease. We evaluated the safety of PulmoBind SPECT imaging and its capacity to detect pulmonary vascular disease associated with pulmonary hypertension (PH) in a human phase II study. METHODS: Thirty patients with pulmonary arterial hypertension (PAH, n = 23) or chronic thromboembolic PH (CTEPH, n = 7) in WHO functional class II (n = 26) or III (n = 4) were compared to 15 healthy controls. Lung SPECT was performed after injection of 15 mCi 99mTc-PulmoBind in supine position. Qualitative and semi-quantitative analyses of lung uptake were performed. Reproducibility of repeated testing was evaluated in controls after 1 month. RESULTS: PulmoBind injection was well tolerated without any serious adverse event. Imaging was markedly abnormal in PH with ∼50% of subjects showing moderate to severe heterogeneity of moderate to severe extent. The abnormalities were unevenly distributed between the right and left lungs as well as within each lung. Segmental defects compatible with pulmonary embolism were present in 7/7 subjects with CTEPH and in 2/23 subjects with PAH. There were no segmental defects in controls. The PulmoBind activity distribution index, a parameter indicative of heterogeneity, was elevated in PH (65% ± 28%) vs. controls (41% ± 13%, p = 0.0003). In the only subject with vasodilator-responsive idiopathic PAH, PulmoBind lung SPECT was completely normal. Repeated testing 1 month later in healthy controls was well tolerated and showed no significant variability of PulmoBind distribution. CONCLUSIONS: In this phase II study, molecular SPECT imaging of the pulmonary vascular endothelium using 99mTc-PulmoBind was safe. PulmoBind showed potential to detect both pulmonary embolism and abnormalities indicative of pulmonary vascular disease in PAH. Phase III studies with this novel tracer and direct comparisons to lung perfusion agents such as labeled macro-aggregates of albumin are needed. CLINICAL TRIAL: ClinicalTrials.gov, NCT02216279.

Photoreleasable ligands to study intracrine angiotensin II signalling.

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 Ca(2+) ([Ca(2+)]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.

Molecular Imaging of the Human Pulmonary Vascular Endothelium Using an Adrenomedullin Receptor Ligand.

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.

Molecular imaging of the human pulmonary vascular endothelium using an adrenomedullin receptor ligand.

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.

Receptor-independent cellular uptake of pituitary adenylate cyclase-activating polypeptide.

Pituitary adenylate cyclase-activating polypeptide (PACAP), a hypophysiotropic neurohormone, participates in the regulation of pleiotropic functions. The recent discovery of intracellular PACAP receptors in the brain and the testis as well as the physico-chemical characteristics of PACAP, i.e. extended α-helix containing basic residues, prompted us to evaluate the propensity of PACAP to cross the plasma membrane in a receptor-independent manner. Using confocal microscopy and flow cytometry, we demonstrated the ability of FITC-conjugated PACAP to efficiently penetrate into the internal cell compartment by direct translocation and endocytosis through clathrin-coated pits and macropinocytosis. Our study also revealed that, once inside the cells, PACAP38 is not entirely degraded by intracellular enzymes and that a significant amount of intact PACAP38 is also able to exit cells. Moreover, using binding assay on rat nuclear fractions from various tissues, PACAP nuclear receptors were identified. We also found that PACAP stimulates calcium release in rat testis nuclei. Interestingly, PACAP27 and PACAP38 but not VIP were able to upregulate de novo DNA synthesis in testis nuclei and that this effect was abolished by PACAP(6-38). These results support the presence of PAC1 receptors at the nuclear membrane and raise questions about their role in the biological activity of the peptide. These findings contribute to the characterization of PACAP as an intracrine factor and suggest that these intracellular PAC1 binding sites, probably associated with specific biological activities, should be taken into account during the development of PACAP-based drugs.

Tunable Surface Charge Enables the Electrostatic Adsorption-Controlled Release of Neuroprotective Peptides from a Hydrogel-Nanoparticle Drug Delivery System.

We exploit the electrostatic interactions between the positively charged neuroprotective peptide, pituitary adenylate cyclase-activating polypeptide (PACAP), and negatively charged poly(lactic-co-glycolic acid) (PLGA) nanoparticles to control PACAP release from the surface of nanoparticles dispersed in a hyaluronan-methylcellulose (HAMC) hydrogel composite. PACAP is a promising therapeutic for the treatment of neurological disorders, yet it has been difficult to deliver in vivo. Herein, the PACAP release rate was tuned by manipulating peptide adsorption onto the surface of blank nanoparticles by modifying either nanoparticle loading in the hydrogel or nanoparticle surface charge. This peptide-nanoparticle interaction was controlled by the pH-responsive carboxylic acid end terminal groups of PLGA. We further validated this system with the controlled release of a novel stabilized PACAP analog: Ac-[Ala15, Ala20]PACAP-propylamide, which masks its recognition to peptidases in circulation. Both wild-type and stabilized PACAP released from the vehicle increased the production of neuroprotective Interleukin-6 from cultured primary astrocytes. Using computational fluid dynamics methods, PACAP release from the composite was predicted based on experimentally derived adsorption isotherms, which exhibited similar release profiles to experimental data. This versatile adsorption-based system was used to deliver PACAP locally to the brains of stroke-injured mice over a 10 day period in vivo, highlighting its effectiveness for the controlled release of PACAP to the central nervous system.

Conformational exchange divergence along the evolutionary pathway of eosinophil-associated ribonucleases.

The evolutionary role of conformational exchange in the emergence and preservation of function within structural homologs remains elusive. While protein engineering has revealed the importance of flexibility in function, productive modulation of atomic-scale dynamics has only been achieved on a finite number of distinct folds. Allosteric control of unique members within dynamically diverse structural families requires a better appreciation of exchange phenomena. Here, we examined the functional and structural role of conformational exchange within eosinophil-associated ribonucleases. Biological and catalytic activity of various EARs was performed in parallel to mapping their conformational behavior on multiple timescales using NMR and computational analyses. Despite functional conservation and conformational seclusion to a specific domain, we show that EARs can display similar or distinct motional profiles, implying divergence rather than conservation of flexibility. Comparing progressively more distant enzymes should unravel how this subfamily has evolved new functions and/or altered their behavior at the molecular level.

A conserved SH3-like fold in diverse putative proteins tetramerizes into an oxidoreductase providing an antimicrobial resistance phenotype.

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'.

Cardiotoxin-I: an unexpectedly potent insulinotropic agent.

Insulin secretion from pancreatic ß-cells is a complex process, involving the integration and interaction of multiple external and internal stimuli, in which glucose plays a major role. Understanding the physiology leading to insulin release is a crucial step toward the identification of new targets. In this study, we evaluated the presence of insulinotropic metabolites in Naja kaouthia snake venom. Only one fraction, identified as cardiotoxin-I (CTX-I) was able to induce insulin secretion from INS-1E cells without affecting cell viability and integrity, as assessed by MTT and LDH assays. Interestingly, CTX-I was also able to stimulate insulin secretion from INS-1E cells even in the absence of glucose. Although cardiotoxins have been characterized as potent hemolytic agents and vasoconstrictors, CTX-I was unable to induce direct hemolysis of human erythrocytes or to induce potent vasoconstriction. As such, this newly identified insulin-releasing toxin will surely enrich the pool of existing tools to study ß-cell physiology or even open a new therapeutic avenue.

Protein context shapes the specificity of SH3 domain-mediated interactions in vivo.

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.

Genome sequencing and functional characterization of a Dictyopanus pusillus fungal enzymatic extract offers a promising alternative for lignocellulose pretreatment of oil palm residues.

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.

Molecular imaging of monocrotaline-induced pulmonary vascular disease with radiolabeled linear adrenomedullin.

UNLABELLED: No test currently exists for molecular imaging of pulmonary arterial hypertension (PAH). Adrenomedullin is a vasodilator peptide predominantly cleared by pulmonary endothelial receptors. We developed a linear adrenomedullin derivative radiolabeled with (99m)Tc ((99m)Tc-AM-L) for imaging of pulmonary circulation and tested its capacity to detect anomalies of pulmonary circulation caused by PAH. METHODS: PAH was induced by monocrotaline in rats and compared with controls. After 5 wk, (99m)Tc-AM-L was injected intravenously. Plasma kinetics were measured, lung activity was determined in vivo after 30 min using a nuclear camera, and lung activity was determined ex vivo in explanted lungs. Expression of adrenomedullin receptors was measured in lung homogenates. RESULTS: The plasma levels of (99m)Tc-AM-L significantly increased in PAH by approximately 2-fold. Uptake by the lungs was homogeneous but greatly reduced in PAH by about 70%. In vivo retention was 14% +/- 1% (mean +/- SD) of the injected dose in controls and 4% +/- 1% in PAH (P < 0.0001). A similar reduction was measured ex vivo (6.0 +/- 1.6 percentage injected dose per gram [%ID/g] vs. 0.95 +/- 0.21 %ID/g, P < 0.0001). The expression of the heterodimeric component of the adrenomedullin receptor, receptor activity modifying protein 2, was also greatly reduced in PAH lungs (P < 0.001). Interestingly, right ventricular uptake of (99m)Tc-AM-L was increased by PAH (P = 0.02) and correlated with the degree of right ventricular hypertrophy (r = 0.83, P = 0.001). CONCLUSION: Pulmonary uptake of (99m)Tc-AM-L is greatly reduced in monocrotaline-induced PAH. This novel molecular imaging agent may be useful in the diagnosis and follow-up of pulmonary vascular disorders.

Semi-rational evolution of the 3-(3-hydroxyalkanoyloxy)alkanoate (HAA) synthase RhlA to improve rhamnolipid production in Pseudomonas aeruginosa and Burkholderia glumae.

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.

Design and biological assessment of membrane-tethering neuroprotective peptides derived from the pituitary adenylate cyclase-activating polypeptide type 1 receptor.

BACKGROUND: The pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1), a class B G protein-coupled receptor (GPCR), has emerged as a promising target for treating neurodegenerative conditions. Unfortunately, despite years of research, no PAC1-specific agonist has been discovered, as activity on two other GPCRs, VPAC1 and VPAC2, is retained with current analogs. Cell signaling is related to structural modifications in the intracellular loops (ICLs) of GPCRs. Thus, we hypothesized that peptides derived from the ICLs (called pepducins) of PAC1 might initiate, as allosteric ligands, signaling cascades after recognition of the parent receptor and modulation of its conformational landscape. METHODS: Three pepducins were synthesized and evaluated for their ability to 1) promote cell survival; 2) stimulate various signaling pathways associated with PAC1 activation; 3) modulate selectively PAC1, VPAC1 or VPAC2 activation; and 4) sustain mobility and prevent death of dopaminergic neurons in a zebrafish model of neurodegeneration. RESULTS: Assays demonstrated that these molecules promote SH-SY5Y cell survival, a human neuroblastoma cell line expressing PAC1, and activate signaling via Gαs and Gαq, with distinct potencies and efficacies. Also, PAC1-Pep1 and PAC1-Pep2 activated selectively PAC1-mediated Gαs stimulation. Finally, experiments, using a zebrafish neurodegeneration model, showed a neuroprotective action with all three pepducins and in particular, revealed the ability of PAC1-Pep1 and PAC1-Pep3 to preserve fish mobility and tyrosine hydroxylase expression in the brain. CONCLUSION: We have developed the first neuroprotective pepducins derived from PAC1, a class B GPCR. GENERAL SIGNIFICANCE: PAC1-derived pepducins represent attractive templates for the development of innovative neuroprotecting molecules.