Insulin Crystals Grown in Short-Peptide Supramolecular Hydrogels Show Enhanced Thermal Stability and Slower Release Profile.

Protein therapeutics have a major role in medicine in that they are used to treat diverse pathologies. Their three-dimensional structures not only offer higher specificity and lower toxicity than small organic compounds but also make them less stable, limiting their in vivo half-life. Protein analogues obtained by recombinant DNA technology or by chemical modification and/or the use of drug delivery vehicles has been adopted to improve or modulate the in vivo pharmacological activity of proteins. Nevertheless, strategies to improve the shelf-life of protein pharmaceuticals have been less explored, which has challenged the preservation of their activity. Herein, we present a methodology that simultaneously increases the stability of proteins and modulates the release profile, and implement it with human insulin as a proof of concept. Two novel thermally stable insulin composite crystal formulations intended for the therapeutic treatment of diabetes are reported. These composite crystals have been obtained by crystallizing insulin in agarose and fluorenylmethoxycarbonyl-dialanine (Fmoc-AA) hydrogels. This process affords composite crystals, in which hydrogel fibers are occluded. The insulin in both crystalline formulations remains unaltered at 50 °C for 7 days. Differential scanning calorimetry, high-performance liquid chromatography, mass spectrometry, and in vivo studies have shown that insulin does not degrade after the heat treatment. The nature of the hydrogel modifies the physicochemical properties of the crystals. Crystals grown in Fmoc-AA hydrogel are more stable and have a slower dissolution rate than crystals grown in agarose. This methodology paves the way for the development of more stable protein pharmaceuticals overcoming some of the existing limitations.

A novel cysteine carbamoyl-switch is responsible for the inhibition of formamidase, a nitrilase superfamily member.

Formamidases (EC 3.5.1.49) and amidases (EC 3.5.1.4) are paralogous cysteine-dependent enzymes which catalyze the conversion of amide substrates to ammonia and the corresponding carboxylic acid. Both enzymes have been suggested as an alternative pathway for ammonia production during urea shortage. Urea was proved key in the transcriptional regulation of formamidases/amidases, connecting urea level to amide metabolism. In addition, different amidases have also been shown to be inhibited by urea, pointing to urea-regulation at the enzymatic level. Although amidases have been widely studied due to its biotechnological application in the hydrolysis of aliphatic amides, up to date, only two formamidases have been extensively characterized, belonging to Helicobacter pylori (HpyAmiF) and Bacillus cereus (BceAmiF). In this work, we report the first structure of an acyl-intermediate of BceAmiF. We also report the inhibition of BceAmiF by urea, together with mass spectrometry studies confirming the S-carbamoylation of BceAmiF after urea treatment. X-ray studies of urea-soaked BceAmiF crystals showed short- and long-range rearrangements affecting oligomerization interfaces. Since cysteine-based switches are known to occur in the regulation of different metabolic and signaling pathways, our results suggest a novel S-carbamoylation-switch for the regulation of BceAmiF. This finding could relate to previous observations of unexplained modifications in the catalytic cysteine of different nitrilase superfamily members and therefore extending this regulation mechanism to the whole nitrilase superfamily.

High-Affinity Chemotaxis to Histamine Mediated by the TlpQ Chemoreceptor of the Human Pathogen Pseudomonas aeruginosa.

Histamine is a key biological signaling molecule. It acts as a neurotransmitter in the central and peripheral nervous systems and coordinates local inflammatory responses by modulating the activity of different immune cells. During inflammatory processes, including bacterial infections, neutrophils stimulate the production and release of histamine. Here, we report that the opportunistic human pathogen Pseudomonas aeruginosa exhibits chemotaxis toward histamine. This chemotactic response is mediated by the concerted action of the TlpQ, PctA, and PctC chemoreceptors, which display differing sensitivities to histamine. Low concentrations of histamine were sufficient to activate TlpQ, which binds histamine with an affinity of 639 nM. To explore this binding, we resolved the high-resolution structure of the TlpQ ligand binding domain in complex with histamine. It has an unusually large dCACHE domain and binds histamine through a highly negatively charged pocket at its membrane distal module. Chemotaxis to histamine may play a role in the virulence of P. aeruginosa by recruiting cells at the infection site and consequently modulating the expression of quorum-sensing-dependent virulence genes. TlpQ is the first bacterial histamine receptor to be described and greatly differs from human histamine receptors, indicating that eukaryotes and bacteria have pursued different strategies for histamine recognition.IMPORTANCE Genome analyses indicate that many bacteria possess an elevated number of chemoreceptors, suggesting that these species are able to perform chemotaxis to a wide variety of compounds. The scientific community is now only beginning to explore this diversity and to elucidate the corresponding physiological relevance. The discovery of histamine chemotaxis in the human pathogen Pseudomonas aeruginosa provides insight into tactic movements that occur within the host. Since histamine is released in response to bacterial pathogens, histamine chemotaxis may permit bacterial migration and accumulation at infection sites, potentially modulating, in turn, quorum-sensing-mediated processes and the expression of virulence genes. As a consequence, the modulation of histamine chemotaxis by signal analogues may result in alterations of the bacterial virulence. As the first report of bacterial histamine chemotaxis, this study lays the foundation for the exploration of the physiological relevance of histamine chemotaxis and its role in pathogenicity.

Seeding from silica-reinforced lysozyme crystals for neutron crystallography.

The fragility of protein crystals plays an important role in the final quality of the diffraction data and therefore that of the derived three-dimensional structural model. The growth of protein crystals in gels of various natures has been shown to overcome this problem, facilitating the manipulation of the crystals; this is probably owing, amongst other factors, to the incorporation of the gel fibres within the body of the crystal. In this study, lysozyme crystals were grown in silica gel at a wide range of concentrations of up to 22%(v/v) to quantitatively determine the amount of gel incorporated into the crystal structure by means of thermogravimetric analysis. The interaction between the silica fibres and the lysozyme molecules within the crystals was also investigated using Raman spectroscopy and the direct influence on the crystalline protein stability was analysed using differential scanning calorimetry. Finally, the benefits of the use of gel-grown crystals to overgrow protein crystals intended for neutron diffraction are highlighted.

Continuous Sensing Photonic Lab-on-a-Chip Platform Based on Cross-Linked Enzyme Crystals.

Microfluidics or lab-on-a-chip technology offer clear advantages over conventional systems such as a dramatic reduction of reagent consumption or a shorter analysis time, which are translated into cost-effective systems. In this work, we present a photonic enzymatic lab-on-a-chip reactor based on cross-linked enzyme crystals (CLECs), able to work in continuous flow, as a highly sensitive, robust, reusable, and stable platform for continuous sensing with superior performance as compared to the state of the art. The microreactor is designed to facilitate the in situ crystallization and crystal cross-linking generating enzymatically active material that can be stored for months/years. Thus, and by means of monolithically integrated micro-optics elements, continuous enzymatic reactions can be spectrophotometrically monitored. Lipase, an enzyme with industrial significance for catalyzed transesterification, hydrolysis, and esterification reactions, is used to demonstrate the potential of the microplatforms as both a continuous biosensor and a microreactor for the synthesis of high value compounds.

Identification and characterization of a bacterial hyaluronidase and its production in recombinant form.

Hyaluronidases (Hyals) are broadly used in medical applications to facilitate the dispersion and/or absorption of fluids or medications. This study reports the isolation, cloning, and industrial-scale recombinant production, purification and full characterization, including X-ray structure determination at 1.45 Å, of an extracellular Hyal from the nonpathogenic bacterium Streptomyces koganeiensis. The recombinant S. koganeiensis Hyal (rHyal_Sk) has a novel bacterial catalytic domain with high enzymatic activity, compared with commercially available Hyals, and is more thermostable and presents higher proteolytic resistance, with activity over a broad pH range. Moreover, rHyal_Sk exhibits remarkable substrate specificity for hyaluronic acid (HA) and poses no risk of animal cross-infection.

McCLEC, a robust and stable enzymatic based microreactor platform.

A microfluidic chip for cross-linked enzyme crystals (McCLEC) is presented and demonstrated to be a stable, reusable and robust biocatalyst-based device with very promising biotechnological applications. The cost-effective microfluidic platform allows in situ crystallization, cross-linking and enzymatic reaction assays on a single device. A large number of enzymatic reuses of the McCLEC platform were achieved and a comparative analysis is shown illustrating the efficiency of the process and its storage stability for more than one year.

Influence of the chirality of short peptide supramolecular hydrogels in protein crystallogenesis.

For the first time the influence of the chirality of the gel fibers in protein crystallogenesis has been studied. Enantiomeric hydrogels 1 and 2 were tested with model proteins lysozyme and glucose isomerase and a formamidase extracted from B. cereus. Crystallization behaviour and crystal quality of these proteins in both hydrogels are presented and compared.

A multiple path photonic lab on a chip for parallel protein concentration measurements.

We propose a PDMS-based photonic system for the accurate measurement of protein concentration with minute amounts of the sample. As opposed to the state of the art approach, in the multiple path photonic lab on a chip (MPHIL), analyte concentration or molar absorptivity is obtained with a single injection step, by performing simultaneous parallel optical measurements varying the optical path length. Also, as opposed to the standard calibration protocol, the MPHIL approach does not require a series of measurements at different concentrations. MPHIL has three main advantages: firstly the possibility of dynamically selecting the path length, always working in the absorbance vs. concentration linear range for each target analyte. Secondly, a dramatic reduction of the total volume of the sample required to obtain statistically reliable results. Thirdly, since only one injection is required, the measurement time is minimized, reducing both contamination and signal drifts. These characteristics are clearly advantageous when compared to commercial micro-spectrophotometers. The MPHIL concept was validated by testing three commercial proteins, lysozyme (HEWL), glucose isomerase (d-xylose-ketol-isomerase, GI) and Aspergillus sp. lipase L (BLL), as well as two proteins expressed and purified for this study, B. cereus formamidase (FASE) and dihydropyrimidinase from S. meliloti CECT41 (DHP). The use of MPHIL is also proposed for any spectrophotometric measurement in the UV-VIS range, as well as for its integration as a concentration measurement platform in more advanced photonic lab on a chip systems.

Cloning, expression, purification, crystallization and preliminary X-ray characterization of allantoinase from Bacillus licheniformis ATCC 14580.

Allantoinase, a member of the amidohydrolase superfamily, exists in a wide variety of organisms, including bacteria, fungi, plants and a few animals, such as fishes and amphibians. Allantoinase catalyzes the reversible hydrolysis of allantoin into allantoate by hydrolytic cleavage of the N1-C2 amide bond of the five-membered hydantoin ring. Allantoinase from Bacillus licheniformis (AllBali) presents an inverted enantioselectivity towards allantoin (R-enantioselective), which is a distinguishable feature that is not observed for other allantoinases. In this work, B. licheniformis ATCC 14580 allantoinase (AllBali) containing a C-terminal His6 tag was overproduced in Escherichia coli and purified to homogeneity. Crystals of AllBali were obtained by the vapour-diffusion method using 0.1 M potassium thiocyanate, 20%(w/v) polyethylene glycol 3350 as a crystallization solution. X-ray diffraction data were collected to a resolution of 3.5 Šwith an Rmerge of 29.2% from a crystal belonging to space group P1211, with unit-cell parameters a=54.93, b=164.74, c=106.89 Å, ß=98.49°. There are four molecules in the asymmetric unit with a solvent content of 47% as estimated from the Matthews coefficient (VM=2.34 Å3 Da(-1)).