Spatially resolved distribution of pancreatic hormones proteoforms by MALDI-imaging mass spectrometry.

Zinc plays a crucial role both in the immune system and endocrine processes. Zinc restriction in the diet has been shown to lead to degeneration of the endocrine pancreas, resulting in hormonal imbalance within the ß-cells. Proteostasismay vary depending on the stage of a pathophysiological process, which underscores the need for tools aimed at directly analyzing biological status. Among proteomics methods, MALDI-ToF-MS can serve as a rapid peptidomics tool for analyzing extracts or by histological imaging. Here we report the optimization of MALDI imaging mass spectrometry analysis of histological thin sections from mouse pancreas. This optimization enables the identification of the major islet peptide hormones as well as the major accumulated precursors and/or proteolytic products of peptide hormones. Cross-validation of the identified peptide hormones was performed by LC-ESI-MS from pancreatic islet extracts. Mice subjected to a zinc-restricted diet exhibited a relatively lower amount of peptide intermediates compared to the control group. These findings provide evidence for a complex modulation of proteostasis by micronutrients imbalance, a phenomenon directly accessed by MALDI-MSI.

Zinc deficiency disrupts pain signaling promoting nociceptive but not inflammatory pain in mice.

Zinc (Zn) is an essential micronutrient involved in the physiology of nervous system and pain modulation. There is little evidence for the role of nutritional Zn alternations to the onset and progression of neuropathic (NP) and inflammatory pain. The study investigated the effects of a zinc restricted diet on the development of pain. Weaned mice were submitted to a regular (38 mg/kg of Zn) or Zn deficient (11 mg/kg of Zn) diets for four weeks, pain responses evaluated (mechanical, cold and heat allodynia; formalin- and carrageenan-induced inflammatory hypernociception), plasma and tissues collected for biochemical and metabolomic analysis. Zn deficient diet inhibited animal growth (37%) and changed mice sensitivity pattern, inducing an intense allodynia evoked by mechanical, cold and heat stimulus for four weeks. The inflammatory pain behavior of formalin test was drastically reduced or absent when challenged by an inflammatory stimulus. Zn restriction also reduce plasma TNF, increase neuronal activation, oxidative stress, indicating a disruption of the immune response. Liver metabolomic analyses suggest a downregulation of lipid metabolism of arachidonic acid. Zn restriction since weaned disrupts pain signaling considerably and reduce inflammatory pain. Zn could be considered a predisposing factor for the onset of chronic pain such as painful neuropathies.

The reproducible normality of the crystallographic B-factor.

Reproducibility determines the utility of a measurement. In structural biology the reproducibility permeate areas such as mechanics, data measurement, data analysis and refinement. In order to access the reproducibility of the combined contribution of these sources in uncertainties of protein crystallography we evaluated four groups of parameters from data collection to final structural model. We used lysozyme as a model, with 20 datasets collected at 1.6 Å resolution using two dissimilar x-ray diffraction setups and refined through a single automatic pipeline without arbitrary interpretation. Besides statistical differences in some structural parameters, the reproducibility of the final refined models allowed the determination of positional uncertainty, in good agreement with the Luzzati coordinate error. While the raw B-factor was found non-reproducible, an empirical scaling/normalization resulted in reproducible B-factors. The validity of this empirical scaling was corroborated by the reproducibility of normalized B-factors of independently solved datasets from proteins (insulin and myoglobin) from varying space groups available from structural database. The reproducibility of normalized B-factor may reposition this displacement parameter in the analysis of chemical (ligands, pH) and physical (pressure, temperature, space groups) variables.

Polymeric microparticle systems for modified release of glucagon-like-peptide-1 receptor agonists.

Type 2 diabetes is a fast-growing worldwide epidemic. Despite the multiple therapies available to treat type 2 diabetes, the disease is not correctly managed in over half of patients, mainly due to non-compliance with prescribed treatment regimes. The development of analogues to the glucagon-like peptide 1 (GLP-1) has resulted in the extension of its half-life and associated benefits. Further benefits in the use of peptide-based GLP-1 receptor agonists have been achieved by the use of controlled-release systems based on polymeric microparticles. In this review, we focus on commercially available formulations and others that remain in development, discussing the preparation methods and the relationship between in vitro and in vivo kinetic release behaviours.

Sustained release and pharmacologic evaluation of human glucagon-like peptide-1 and liraglutide from polymeric microparticles.

The GLP1-receptor agonists exert regulatory key roles in diabetes, obesity and related complications. Here we aimed to develop polymeric microparticles loaded with homologous human GLP1 (7-37) or the analogue liraglutide. Peptide-loaded microparticles were prepared by a double emulsion and solvent evaporation process with a set of eight polymers based on lactide (PLA) or lactide-glycolide (PLGA), and evaluated for particle-size distribution, morphology, in vitro release and pharmacologic activity in mice. The resulting microparticles showed size distribution of about 30-50 µm. The in vitro kinetic release assays showed a sustained release of the peptides extending up to 30-40 days. In vivo evaluation in Swiss male mice revealed a similar extension of glycemic and body weight gain modulation for up to 25 days after a single subcutaneous administration of either hGLP1-microparticles or liraglutide-microparticles. Microparticles-loaded hGLP1 shows equivalent in vivo pharmacologic activity to the microparticles-loaded liraglutide.

Physico-chemical stability of co-formulation of PEGylated human amylin with insulin.

Since the discovery of amylin no combined formulation with insulin has been made available. Amylin or its triple proline analog pramlintide are not compatible in solution with insulin. The drug candidate hAmy-PEG5k is a novel monoPEGylated amylin derivative with improved physicochemical properties and retained similar pharmacological activity compared to free amylin and pramlintide. We have investigated the short- and long-term physicochemical compatibility of hAmy-PEG5k co-formulated with slow-acting human insulin analogs glargine or detemir. While human amylin promptly aggregates over a large range of pH, and both free and in the presence of regular, glargine or detemir insulin, the hAmy-PEG5k analog is stable at these conditions as shown by Thioflavin T (ThT) binding assay. When hAmy-PEG5k (100 or 500 µg/mL) was added to the commercial formulations of either insulin glargine or detemir (95 IU/mL), the combinations remained stable after 6 months stored at 4 °C, as probed by ThT, dynamic light scattering (DLS) measurements and high performance liquid chromatography (HPLC) analyses, confirming the absence of amyloid fibers, minor aggregation products or loss of material. These results suggest hAmy-PEG5k and the insulin analogs glargine and detemir are physicochemically compatible and are candidate ready-to-use fixed-dose combinations.

Oligomeric transition and dynamics of RNA binding by the HuR RRM1 domain in solution.

Human antigen R (HuR) functions as a major post-transcriptional regulator of gene expression through its RNA-binding activity. HuR is composed by three RNA recognition motifs, namely RRM1, RRM2, and RRM3. The two N-terminal RRM domains are disposed in tandem and contribute mostly to HuR interaction with adenine and uracil-rich elements (ARE) in mRNA. Here, we used a combination of NMR and electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) to characterize the structure, dynamics, RNA recognition, and dimerization of HuR RRM1. Our solution structure reveals a canonical RRM fold containing a 19-residue, intrinsically disordered N-terminal extension, which is not involved in RNA binding. NMR titration results confirm the primary RNA-binding site to the two central ß-strands, ß1 and ß3, for a cyclooxygenase 2 (Cox2) ARE I-derived, 7-nucleotide RNA ligand. We show by 15N relaxation that, in addition to the N- and C-termini, the ß2-ß3 loop undergoes fast backbone dynamics (ps-ns) both in the free and RNA-bound state, indicating that no structural ordering happens upon RNA interaction. ESI-IMS-MS reveals that HuR RRM1 dimerizes, however dimer population represents a minority. Dimerization occurs via the α-helical surface, which is oppositely orientated to the RNA-binding ß-sheet. By using a DNA analog of the Cox2 ARE I, we show that DNA binding stabilizes HuR RRM1 monomer and shifts the monomer-dimer equilibrium toward the monomeric species. Altogether, our results deepen the current understanding of the mechanism of RNA recognition employed by HuR.

The toxic nature of murine amylin and the immune responsivity of pancreatic islet to conformational antibody in mice.

The human amylin is a pancreatic peptide hormone found in hyperhormonemic state along with insulin in subclinical diabetes. Amylin has been associated with the pathology of type 2 diabetes, particularly due to its ability to assembly into toxic oligomers and amyloid specimens. On the other hand, some variants such as murine amylin has been described as non-amyloidogenic, either in vitro or in vivo. Recent data have demonstrated the amyloid propensity of murine amylin and the therapeutic analogue pramlintide, suggesting a universality for amylin amyloidosis. Here, we report the amyloidogenesis of murine amylin, which showed lower responsivity to the fluorescent probe thioflavin T compared to human amylin, but presented highly organized fibrilar amyloid material. The aggregation of murine amylin also resulted in the formation of cytotoxic specimens, as evaluated in vitro in INS-1 cells. The aggregation product from murine amylin was responsive to a specific antibody raised against amyloid oligomers, the A11 oligomer antibody. Pancreatic islets of wild-type Swiss male mice have also shown responsivity for the anti-oligomer, indicating the natural abundance of such specimen in rodents. These data provide for the first time evidences for the toxic nature of oligomeric assemblies of murine amylin and its existence in wild-type, non-transgenic mice.

Polymorphic distribution of proteins in solution by mass spectrometry: The analysis of insulin analogues.

The characterization of conformational and oligomeric distribution of proteins is of paramount importance for the understanding of the correlation between structure and function. Among the bioanalytical approaches currently available, the electrospray ionization-mass spectrometry (ESI-MS) coupled to ion mobility spectrometry (IMS) is the best suited for high resolution identification with high sensitivity, allowing the in situ separation of oligomeric and conformational species. We tested the performance of the ESI-MS technique along with the IMS separation approach on a broad variety of insulin and insulin analogues with distinct oligomeric distribution pattern. The measurement of commercial insulin allowed the identification of species ranging from monomers to hexamers and their complexes with zinc ions. Dissimilar distribution profile for regular insulin as a function of formulation component and among the insulin analogues were observed by ESI-IMS-MS but not by ESI-MS along, crystallographic assays or size-exclusion chromatography. These data suggest the additional suitability of ESI-IMS-MS in conformational and oligomeric profiling of biomacromolecules and biopharmaceuticals. The easiness of the technique provides further motivation for its application in the characterization of both raw and formulated protein biopharmaceuticals in routine and comparability exercises.

Structural insights into the stabilization of the human immunodeficiency virus type 1 capsid protein by the cyclophilin-binding domain and implications on the virus cycle.

During infection, human immunodeficiency virus type 1 (HIV-1) interacts with the cellular host factor cyclophilin A (CypA) through residues 85-93 of the N-terminal domain of HIV-1's capsid protein (CA). The role of the CA:CypA interaction is still unclear. Previous studies showed that a CypA-binding loop mutant, Δ87-97, has increased ability to assemble in vitro. We used this mutant to infer whether the CypA-binding region has an overall effect on CA stability, as measured by pressure and chemical perturbation. We built a SAXS-based envelope model for the dimer of both WT and Δ87-97. A new conformational arrangement of the dimers is described, showing the structural plasticity that CA can adopt. In protein folding studies, the deletion of the loop drastically reduces CA stability, as assayed by high hydrostatic pressure and urea. We hypothesize that the deletion promotes a rearrangement of helix 4, which may enhance the heterotypic interaction between the N- and C-terminal domains of CA dimers. In addition, we propose that the cyclophilin-binding loop may modulate capsid assembly during infection, either in the cytoplasm or near the nucleus by binding to the nuclear protein Nup385.

Refolding, purification, and preliminary structural characterization of the DNA-binding domain of the quorum sensing receptor RhlR from Pseudomonas aeruginosa.

RhlR is a 241-residue quorum sensing receptor that controls the expression of a myriad of virulence genes in Pseudomonas aeruginosa. Here, the DNA sequence encoding the carboxi-terminal DNA-binding domain of RhlR was cloned into the pET-RP1B plasmid and expressed as an N-terminal fusion protein to the expression/purification Thio6His6 tag. The fusion construct expressed insolubly in Escherichia coli BL21 (DE3) cells. The recombinant protein was extracted from the bacterial inclusion bodies and refolded in the presence of the charged amino acids l-arginine and l-glutamate. The refolded protein was purified by a combination of Ni(+2)-affinity and size exclusion chromatography, allowing the production of 2 mg of highly purified protein (>95% purity) per 5 mg of wet cells derived from 1 L culture. (1)H 1D NMR analysis revealed that the recombinant protein is folded. Moreover, a fluorescence anisotropy DNA-binding assay showed that the refolded protein is functional, as it recognizes the rhlAB promoter. This is the first time that a domain of the quorum sensing regulator RhlR was produced in sufficient amounts for structural studies, enabling the investigation of the molecular basis for RhlR specific interaction with DNA promoters.

Molecular confinement of human amylin in lipidic nanoparticles.

Amylin is a pancreatic hormone involved in the regulation of glucose metabolism and homeostasis. Restoration of the post-prandial and basal levels of human amylin in diabetic individuals is a key in controlling glycemia, controlling glucagon, reducing the insulin dose and increasing satiety, among other physiologic functions. Human amylin has a high propensity to aggregate. We have addressed this issue by designing a liposomal human amylin formulation. Nanoparticles of multilamellar liposomes comprising human amylin were obtained with 53% encapsulation efficiency. The in vitro kinetic release assay shows a biphasic profile. The stabilization of the lipidic nanoparticle against freeze-drying was achieved by using mannitol as a cryoprotectant, as evidenced by morphological characterization. The effectiveness of the human amylin entrapped in lipidic nanoparticles was tested by the measurement of its pharmacological effect in vivo after subcutaneous administration in mice. Collectively these results demonstrate the compatibility of human amylin with the lipidic interface as an effective pharmaceutical delivery system.

Allosteric regulation of the Plasmodium falciparum cysteine protease falcipain-2 by heme.

During the erythrocytic cycle of Plasmodium falciparum malaria parasites break down host hemoglobin, resulting in the release of free heme (ferriprotoporphyrin IX). Heme is a generator of free radicals that cause oxidative stress, but it is detoxified by crystallization into hemozoin inside the food vacuole. We evaluated the interaction of heme and heme analogues with falcipain-2, a P. falciparum food vacuole cysteine protease that plays a key role in hemoglobin digestion. Heme bound to falcipain-2 with a 1:1 stoichiometry, and heme inhibited falcipain-2 activity against both human hemoglobin and chromogenic peptide substrates through a noncompetitive-like mechanism. A series of porphyrin analogues was screened for inhibition of falcipain-2, demonstrating a minor contribution of iron to heme-falcipain-2 interaction, and revealing dependence on both propionic and vinyl groups for inhibition of falcipain-2 by heme. Docking and molecular dynamics simulation unveiled a novel, inducible heme-binding moiety in falcipain-2 adjacent to the catalytic site. Kinetic data suggested that the noncompetitive-like inhibition was substrate inhibition induced by heme. Collectively these data suggest that binding of heme to falcipain-2 may limit the accumulation of free heme in the parasite food vacuole, providing a means of heme detoxification in addition to crystallization into hemozoin.

Falcipain-2 inhibition by suramin and suramin analogues.

Falcipain-2 is a cysteine protease of the malaria parasite Plasmodium falciparum that plays a key role in the hydrolysis of hemoglobin, a process that is required by intraerythrocytic parasites to obtain amino acids. In this work we show that the polysulfonated napthylurea suramin is capable of binding to falcipain-2, inhibiting its catalytic activity at nanomolar concentrations against both synthetic substrates and the natural substrate hemoglobin. Kinetic measurements suggest that the inhibition occurs through an noncompetitive allosteric mechanism, eliciting substrate inhibition. Smaller suramin analogues and those with substituted methyl groups also showed inhibition within the nanomolar range. Our results identify the suramin family as a potential starting point for the design of falcipain-2 inhibitor antimalarials that act through a novel inhibition mechanism.

Allosteric function and dysfunction of the prion protein.

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases associated with progressive oligo- and multimerization of the prion protein (PrP(C)), its conformational conversion, aggregation and precipitation. We recently proposed that PrP(C) serves as a cell surface scaffold protein for a variety of signaling modules, the effects of which translate into wide-range functional consequences. Here we review evidence for allosteric functions of PrP(C), which constitute a common property of scaffold proteins. The available data suggest that allosteric effects among PrP(C) and its partners are involved in the assembly of multi-component signaling modules at the cell surface, impose upon both physiological and pathological conformational responses of PrP(C), and that allosteric dysfunction of PrP(C) has the potential to entail progressive signal corruption. These properties may be germane both to physiological roles of PrP(C), as well as to the pathogenesis of the TSEs and other degenerative/non-communicable diseases.

Inhibition of human transthyretin aggregation by non-steroidal anti-inflammatory compounds: a structural and thermodynamic analysis.

Transthyretin (TTR) is a homotetrameric protein that circulates in plasma and cerebral spinal fluid (CSF) whose aggregation into amyloid fibrils has been associated with at least two different amyloid diseases: senile systemic amyloidosis (SSA) and familial amyloid polyneuropathy (FAP). In SSA aggregates are composed of WT-TTR, while in FAP more than 100 already-described variants have been found in deposits. Until now, TTR-related diseases have been untreatable, although a new drug called Tafamidis has been approved only in Europe to specifically treat V30M patients. Thus, new strategies are still necessary to treat FAP caused by other variants of TTR. TTR has two channels in the dimer interface that bind to the hormone thyroxin and that have been used to accommodate anti-amyloidogenic compounds. These compounds stabilize the tetramers, rendering TTR less amyloidogenic. Here, we investigated the effects of three non-steroidal anti-inflammatory compounds-sulindac (SUL), indomethacin (IND) and lumiracoxib (LUM)-as tetramer stabilizers and aggregation inhibitors. WT-TTR and the very aggressive TTR variant L55P were used as models. These compounds were able to stabilize TTR against high hydrostatic pressure (HHP), increasing the ΔGf by several kcal. They were also effective in inhibiting WT-TTR and L55P acid- or HHP-induced aggregation; in particular, LUM and IND were very effective, inhibiting almost 100% of the aggregation of both proteins under certain conditions. The species formed when aggregation was performed in the presence of these compounds were much less toxic to cells in culture. The crystal structures of WT-TTR bound to the three compounds were solved at high resolution, allowing the identification of the relevant protein:drug interactions. We discuss here the ligand-binding features of LUM, IND and SUL to TTR, emphasizing the critical interactions that render the protein more stable and less amyloidogenic.

Preparation and characterization of PEGylated amylin.

Amylin is a pancreatic hormone that plays important roles in overall metabolism and in glucose homeostasis. The therapeutic restoration of postprandial and basal amylin levels is highly desirable for patients with diabetes who need to avoid glucose excursions. Protein conjugation with polyethylene glycol (PEG) has long been known to be a convenient approach for extending the biological effects of biopharmaceuticals. We have investigated the reactivity of amylin with methoxy polyethylene glycol succinimidyl carbonate and methoxy polyethylene glycol succinimidyl propionate, which have an average molecular weight of 5 kDa. The reaction, which was conducted in both aqueous and organic (dimethyl sulfoxide) solvents, occurred within a few minutes and resulted in at least four detectable products with distinct kinetic phases. These results suggest a kinetic selectivity for PEGylation by succinimidyl derivatives; these derivatives exhibit enhanced reactivity with primary amine groups, as indicated by an evaluation of the remaining amino groups using fluorescamine. The analysis of tryptic fragments from mono- and diPEGylated amylin revealed that conjugation occurred within the 1-11 amino acid region, most likely at the two amine groups of Lys(1). The reaction products were efficiently separated by C-18 reversed phase chromatography. Binding assays confirmed the ability of mono- and diPEGylated amylin to interact with the amylin co-receptor receptor activity-modifying protein 2. Subcutaneous administration in mice revealed the effectiveness of monoPEG-amylin and diPEG-amylin in reducing glycemia; both compounds exhibited prolonged action compared to unmodified amylin. These features suggest the potential use of PEGylated amylin to restore basal amylin levels.

Intriguing nucleic-acid-binding features of mammalian prion protein.

In transmissible spongiform encephalopathies, the infectious material consists chiefly of a protein, the scrapie prion protein PrP(Sc), that carries no genetic coding material; however, prions are likely to have accomplices that chaperone their activity and promote the conversion of the cellular prion protein PrP(C) into the disease-causing isoform (PrP(Sc)). Recent studies from several laboratories indicate that PrP(C) recognizes many nucleic acids (NAs) with high affinities, and we correlate these findings with a possible pathophysiological role for this interaction. Thus, of the chaperones, NA is the most likely candidate for prions. The participation of NAs in prion propagation opens new avenues for developing new diagnostic tools and therapeutics to target prion diseases, as well as for understanding the function of PrP(C), probably as a NA chaperone.

Linking B-factor and temperature-induced conformational transition.

The crystallographic B-factor, also called temperature factor or Debye-Waller factor, has long been used as a surrogate for local protein flexibility. However, the use of the absolute B-factor as a probe for protein motion requires reproducible validation against conformational changes against chemical and physical variables. Here we report the investigation of the thermal dependence of the crystallographic B-factor and its correlation with conformational changes of the protein. We obtained the crystal protein structure coordinates and B-factors at high resolution (1.5 Å) over a broad temperature range (100 K to 325 K). The exponential thermal dependence of B-factor as a function of temperature was equal for both the diffraction intensity data (Wilson B-factor) and for all modeled atoms of the system (protein and non-protein atoms), with a thermal diffusion constant of about 0.0045 K-1, similar for all atoms. The extrapolated B-factor at zero Kelvin (or zero-point fluctuation) varies among the atoms, although with no apparent correlation with temperature-dependent protein conformational changes. These data suggest that the thermal vibration of the atom does not necessarily correlate with the conformational dynamics of the protein.