Dynamic Personality of Proteins and Effect of the Molecular Environment.

Protein dynamics display distinct traits that are linked to their specific biological function. However, the interplay between intrinsic dynamics and the molecular environment on protein stability remains poorly understood. In this study, we investigate, by incoherent neutron scattering, the subnanosecond time scale dynamics of three model proteins: the mesophilic lysozyme, the thermophilic thermolysin, and the intrinsically disordered ß-casein. Moreover, we address the influence of water, glycerol, and glucose, which create progressively more viscous matrices around the protein surface. By comparing the protein thermal fluctuations, we find that the internal dynamics of thermolysin are less affected by the environment compared to lysozyme and ß-casein. We ascribe this behavior to the protein dynamic personality, i.e., to the stiffer dynamics of the thermophilic protein that contrasts the influence of the environment. Remarkably, lysozyme and thermolysin in all molecular environments reach a critical common flexibility when approaching the calorimetric melting temperature.

NMR structure of emfourin, a novel protein metalloprotease inhibitor: Insights into the mechanism of action.

Emfourin (M4in) is a protein metalloprotease inhibitor recently discovered in the bacterium Serratia proteamaculans and the prototype of a new family of protein protease inhibitors with an unknown mechanism of action. Protealysin-like proteases (PLPs) of the thermolysin family are natural targets of emfourin-like inhibitors widespread in bacteria and known in archaea. The available data indicate the involvement of PLPs in interbacterial interaction as well as bacterial interaction with other organisms and likely in pathogenesis. Arguably, emfourin-like inhibitors participate in the regulation of bacterial pathogenesis by controlling PLP activity. Here, we determined the 3D structure of M4in using solution NMR spectroscopy. The obtained structure demonstrated no significant similarity to known protein structures. This structure was used to model the M4in-enzyme complex and the complex model was verified by small-angle X-ray scattering. Based on the model analysis, we propose a molecular mechanism for the inhibitor, which was confirmed by site-directed mutagenesis. We show that two spatially close flexible loop regions are critical for the inhibitor-protease interaction. One region includes aspartic acid forming a coordination bond with catalytic Zn2+ of the enzyme and the second region carries hydrophobic amino acids interacting with protease substrate binding sites. Such an active site structure corresponds to the noncanonical inhibition mechanism. This is the first demonstration of such a mechanism for protein inhibitors of thermolysin family metalloproteases, which puts forward M4in as a new basis for the development of antibacterial agents relying on selective inhibition of prominent factors of bacterial pathogenesis belonging to this family.

Denuded Descemet's membrane supports human embryonic stem cell-derived retinal pigment epithelial cell culture.

Recent clinical studies suggest that retinal pigment epithelial (RPE) cell replacement therapy may preserve vision in retinal degenerative diseases. Scaffold-based methods are being tested in ongoing clinical trials for delivering pluripotent-derived RPE cells to the back of the eye. The aim of this study was to investigate human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells survival and behaviour on a decellularized Descemet's Membrane (DM), which may be of clinical relevance in retinal transplantation. DMs were isolated from human donor corneas and treated with thermolysin. The DM surface topology and the efficiency of the denudation method were evaluated by atomic force microscope, scanning electron microscopy and histology. hESC-RPE cells were seeded onto the endothelial-side surface of decellularized DM in order to determine the potential of the membrane to support hESC-RPE cell culture, alongside maintaining their viability. Integrity of the hESC-RPE monolayer was assessed by measuring transepithelial resistance. RPE-specific gene expression and growth factors secretion were assessed to confirm maturation and functionality of the cells over the new substrate. Thermolysin treatment did not affect the integrity of the tissue, thus ensuring a reliable method to standardize the preparation of decellularized DM. 24 hours post-seeding, hESC-RPE cell attachment and initial proliferation rate over the denuded DM were higher than hESC-RPE cells cultured on tissue culture inserts. On the new matrix, hESC-RPE cells succeeded in forming an intact monolayer with mature tight junctions. The resulting cell culture showed characteristic RPE cell morphology and proper protein localization. Gene expression analysis and VEGF secretion demonstrate DM provides supportive scaffolding and inductive properties to enhance hESC-RPE cells maturation. Decellularized DM was shown to be capable of sustaining hESC-RPE cells culture, thus confirming to be potentially a suitable candidate for retinal cell therapy.

Linear and nonlinear rheology of liberase-treated breast cancer tumors.

Extracellular matrix (ECM) rigidity has been shown to increase the invasive properties of breast cancer cells, promoting transformation and metastasis through mechanotransduction. Reducing ECM stiffness via enzymatic digestion could be a promising approach to slowing breast cancer development by de-differentiation of breast cancer cells to less aggressive phenotypes and enhancing the effectiveness of existing chemotherapeutics via improved drug penetrance throughout the tumor. In this study, we examine the effects of injectable liberase (a blend of collagenase and thermolysin enzymes) treatments on the linear and nonlinear rheology of allograft 4T1 mouse mammary tumors. We perform two sets of in vivo mouse studies, in which either one or multiple treatment injections occur before the tumors are harvested for rheological analysis. The treatment groups in each study consist of a buffer control, free liberase enzyme in buffer, a thermoresponsive copolymer called LiquoGel (LQG) in buffer, and a combined, localized injection of LQG and liberase. All tumor samples exhibit gel-like linear rheological behavior with the elastic modulus significantly larger than the viscous modulus and both independent of frequency. Tumors that receive a single injection of localized liberase have significantly lower tumor volumes and lower tissue moduli at both the center and edge compared to buffer- and free liberase-injected control tumors, while tissue viscoelasticity remains relatively unaffected. Tumors injected multiple times with LQG and liberase also have lower tissue volumes but possess higher tissue moduli and lower viscoelasticities compared to the other treatment groups. We propose that a mechanotransductive mechanism could cause the formation of smaller but stiffer tumors after repeated, localized liberase injections. Large amplitude oscillatory shear (LAOS) experiments are also performed on tissues from the multiple injection study and the results are analyzed using MITlaos. LAOS analysis reveals that all 4T1 tumors from the multiple injection study exhibit nonlinear rheological behavior at high strains and strain rates. Examination of the Lissajous-Bowditch curves, Chebyshev coefficient ratios, elastic moduli, and dynamic viscosities demonstrate that the onset and type of nonlinear behavior is independent of treatment type and elastic modulus, suggesting that multiple liberase injections do not affect the nonlinear viscoelasticity of 4T1 tumors.

Comparison of human amniotic membrane decellularisation approaches for hESC-derived RPE cells culture.

OBJECTIVE: Recent clinical studies have shown that the transplantation of functional retinal pigment epithelium (RPE) cells can prevent the onset of RPE degeneration in age-related macular degeneration. This study aimed to investigate the potential of human amniotic membrane (hAM) as a viable scaffold for the growth and proliferation of pluripotent-derived RPE cells. METHODS AND ANALYSIS: Three enzymatic hAM de-epithelialisation methods (thermolysin, trypsin-EDTA and dispase II) were assessed by histological analysis and optical coherence tomography (OCT). We generated RPE cells from a human embryonic stem cell (hESC) line subjected to spontaneous differentiation in feeder-free conditions. The hESC-derived RPE cells were seeded over denuded hAM at a density of 2.0×105 cells/cm2 and maintained in culture for up to 4 weeks. Immnofluorescence was carried out to evaluate the development of a confluent monolayer of RPE cells on the top of the hAM. Conditioned medium was collected to measure pigment epithelium-derived factor (PEDF) concentration by ELISA. RESULTS: Laminin α5 and collagen IV staining confirmed the efficiency of the de-epithelialisation process. In particular, thermolysin showed good retention of tissue integrity on OCT images and greater preservation of the hAM basement membrane. The hESC-derived RPE cells formed patches of pigmented cells interspersed along the denuded hAM, but failed to form a regular sheet of RPE cells. These cells expressed typical RPE markers, such as PMEL17 and RPE65, but they secreted low levels of PEDF. CONCLUSION: The biological variability of the hAM could influence the adhesion and the expansion of hESC-derived RPE cells. Further studies are required to verify whether a non-confluent monolayer might represent a limit to transplantation.

21†Denuded descemet's membrane as potential tool to support human embryonic stem cell derived retinal pigment epithelial cells culture.

INTRODUCTION: Recent clinical studies suggest that RPE-cell replacement therapy may preserve vision and restore retinal structure in retinal degenerative diseases. New developments enabled the differentiation of RPE cells from pluripotent stem cells. Scaffold-based methods are being tested in ongoing clinical trials for delivering these cells to the back of the eye. Borrowed materials from donor tissues can be used as cell supports in subretinal transplantation. These biological matrices resemble the extracellular matrix microenvironment of the native tissue. The Descemet's membrane (DM) is an example of high collagen-rich basement membrane (BM). The potential of this tissue in retinal repair remains to be uncovered. AIMS: To investigate human embryonic stem cell-retinal pigment epithelium (hESC-RPE) cells survival and behaviour on a decellularized DM, which may be of clinical relevance in retinal transplantation. MATERIALS: DMs were isolated from human donor corneas and treated with thermolysin. The DM surface topology and the efficiency of the denudation method were evaluated by atomic force microscope and histology. hESC-RPE cells were seeded onto the endothelial-side surface of acellular DM in order to determine the potential of the membrane to support hESC-RPE cell culture, alongside maintaining their viability. Integrity of the hESC-RPE monolayer was assessed by measuring transepithelial resistance. RPE-specific gene, protein expression and growth factors secretion were assessed to confirm maturation and functionality of the cells over the new substrate. RESULTS: Thermolysin treatment did not affect the integrity of the tissue, thus ensuring a reliable method to standardize the preparation of decellularized DM. hESC-RPE cell attachment 6 days post-seeding and proliferation rates over the acellular DM were similar to hESC-RPE cells cultured on tissue culture inserts.On the new matrix, hESC-RPE cells succeeded in forming an intact monolayer with mature tight junctions. The resulting cell graft showed the characteristic RPE morphology. The expression of typical RPE genes, proper protein localization and key growth factor secretion further confirmed the correct RPE phenotype. The viability of the cells was maintained for up to 4 weeks in culture. CONCLUSION: Acellular DM was shown to be capable of sustaining hESC-RPE cells growth, thus confirming to be potentially a valid alternative to the Bruch's membrane.Further in vivo studies will need to verify if this product can represent a feasible tool to deliver RPE cells in the back of the eye.Our study highlights the possibility of recycling unsuitable corneal tissues, which would otherwise be discarded by the eye banks for clinical application.

Salt Mediated Modulation of Autolysis of Thermolysin-Like Proteinase, Salilysin, Isolated from a Moderate Halophile, Chromohalobacter salexigens DSM3043.

Halophilic salilysin is first synthesized as a pro-form, which has been shown autolysis activity to process pro-region (55 amino acids long) three times to form intermediate 1 (I1), intermediate 2 (I2) and final mature (M) salilysin. The autolysis of I1- to M-form salilysin in vitro was significantly accelerated with increasing NaCl concentration up to 4 M. Strong salting-out salts, (NH4)2SO4, Na2SO4 and MgSO4, were more effective, suggesting that autolysis is enhanced by inter-molecular association or structure compaction or both. However, MgCl2, a salting-in salt, was also effective, suggesting that other mechanisms, such as charge shielding and ionic binding to this halophilic protein, operated. Autolytic cleavage at site 3 resulted in mixed formation of correctly and incorrectly processed mature forms in the absence of salt, indicating that salt affected the accuracy of autolytic cleavage reaction. Far UV circular dichroism (CD) measurements indicated that E167A pro-salilysin showed an identical CD spectrum to the wild-type mature salilysin, suggesting pro-form has a proper fold for proteolytic activity. Thermal scanning indicated that E167A pro-salilysin was more heat-stable by ~ 10 °C than mature form. The CD spectra, thermal stability and modeling structure of salilysin clearly suggested that pro-salilysin is folded to the same structure as native form and is functional for autolysis.

Theoretical Evaluation of Novel Thermolysin Inhibitors from Bacillus thermoproteolyticus. Possible Antibacterial Agents.

The search for new antibacterial agents that could decrease bacterial resistance is a subject in continuous development. Gram-negative and Gram-positive bacteria possess a group of metalloproteins belonging to the MEROPS peptidase (M4) family, which is the main virulence factor of these bacteria. In this work, we used the previous results of a computational biochemistry protocol of a series of ligands designed in silico using thermolysin as a model for the search of antihypertensive agents. Here, thermolysin from Bacillus thermoproteolyticus, a metalloprotein of the M4 family, was used to determine the most promising candidate as an antibacterial agent. Our results from docking, molecular dynamics simulation, molecular mechanics Poisson-Boltzmann (MM-PBSA) method, ligand efficiency, and ADME-Tox properties (Absorption, Distribution, Metabolism, Excretion, and Toxicity) indicate that the designed ligands were adequately oriented in the thermolysin active site. The Lig783, Lig2177, and Lig3444 compounds showed the best dynamic behavior; however, from the ADME-Tox calculated properties, Lig783 was selected as the unique antibacterial agent candidate amongst the designed ligands.

Immobilization of thermolysin enzyme on dendronized silica supports. Evaluation of its feasibility on multiple protein hydrolysis cycles.

This work evaluates different dendrimer-silica supports for the immobilization of enzymes by multipoint covalent binding. Thermolysin was immobilized on two dendrimers (PAMAM and carbosilane) with two different generations (zero (G0) and first (G1)). Results were compared with a control, a silica support functionalized with a monofunctional molecule. Dendrimers increased the number of available sites to bind the enzyme. Despite the enzyme was immobilized on all supports, G0 dendrimers immobilized a 30% more enzyme than G1. Thermolysin immobilized on G0 dendrimer supports showed the highest activity and could be employed in three consecutive hydrolysis cycles. Optimal immobilization time was 1 h while optimal protein loading was 25 mg enzyme/100 mg support. Enzyme activity was promoted when using 5 mg of immobilized enzyme at 750 rpm, 60 °C, and 2 h of hydrolysis. Under these conditions, the activity of thermolysin increased up to the 78% of the free enzyme activity. Kinetics of the hydrolysis reaction using the immobilized thermolysin was also studied and compared with the obtained using the free thermolysin. The addition of ZnCl2 and NaCl during the immobilization procedure increased thermolysin activity in the second (22% more) and in the third (14% more) hydrolysis clycles.

α2-Macroglobulin-like protein 1 can conjugate and inhibit proteases through their hydroxyl groups, because of an enhanced reactivity of its thiol ester.

Proteins in the α-macroglobulin (αM) superfamily use thiol esters to form covalent conjugation products upon their proteolytic activation. αM protease inhibitors use theirs to conjugate proteases and preferentially react with primary amines (e.g. on lysine side chains), whereas those of αM complement components C3 and C4B have an increased hydroxyl reactivity that is conveyed by a conserved histidine residue and allows conjugation to cell surface glycans. Human α2-macroglobulin-like protein 1 (A2ML1) is a monomeric protease inhibitor but has the hydroxyl reactivity-conveying histidine residue. Here, we have investigated the role of hydroxyl reactivity in a protease inhibitor by comparing recombinant WT A2ML1 and the A2ML1 H1084N mutant in which this histidine is removed. Both of A2ML1s' thiol esters were reactive toward the amine substrate glycine, but only WT A2ML1 reacted with the hydroxyl substrate glycerol, demonstrating that His-1084 increases the hydroxyl reactivity of A2ML1's thiol ester. Although both A2ML1s conjugated and inhibited thermolysin, His-1084 was required for the conjugation and inhibition of acetylated thermolysin, which lacks primary amines. Using MS, we identified an ester bond formed between a thermolysin serine residue and the A2ML1 thiol ester. These results demonstrate that a histidine-enhanced hydroxyl reactivity can contribute to protease inhibition by an αM protein. His-1084 did not improve A2ML1's protease inhibition at pH 5, indicating that A2ML1's hydroxyl reactivity is not an adaption to its acidic epidermal environment.

Methods for studying protein targeting to and within the chloroplast.

Distinct protein complements impart each of the chloroplast's three membranes and three aqueous spaces with specific functions essential for plant growth and development. Chloroplasts capture light energy, synthesize macromolecular building blocks and specialized metabolites, and communicate environmental signals to the nucleus. Establishing and maintaining these processes requires approximately 3000 proteins derived from nuclear genes, constituting approximately 95% of the chloroplast proteome. These proteins are imported into chloroplasts from the cytosol, sorted to the correct subcompartment, and assembled into functioning complexes. In vitro import assays can reconstitute these processes in isolated chloroplasts. We describe methods for monitoring in vitro protein import using Pisum sativum chloroplasts and for protease protection, fractionation, and native protein electrophoresis that are commonly combined with the import assay. These techniques facilitate investigation of the import and sorting processes, of where a protein resides, and of how that protein functions.

Cleavage of the outer membrane protein OmpX by protealysin regulates Serratia proteamaculans invasion.

Protealysin is a thermolysin-like protease of Serratia proteamaculans capable of specifically cleaving actin, which correlates with the invasive activity of these bacteria. Here, we show that inactivation of the protealysin gene does not inhibit invasion but, in contrast, leads to a twofold increase in the S. proteamaculans invasive activity. By mass spectrometry, we identified the outer membrane protein OmpX as a substrate of protealysin. Recombinant E. coli carrying the OmpX gene truncated by 40 N-terminal residues or both the OmpX and protealysin genes, in contrast to the full-length OmpX, do not increase adhesion of these bacteria, indicating that the 40 N-terminal residues of OmpX are indispensable for S. proteamaculans invasion. Our results show that both protealysin and its substrates can stimulate Serratia invasion.

The extremophile Anoxybacillus sp. PC2 isolated from Brazilian semiarid region (Caatinga) produces a thermostable keratinase.

The aim of this study was to select and identify thermophilic bacteria from Caatinga biome (Brazil) able to produce thermoactive keratinases and characterize the keratinase produced by the selected isolate. After enrichment in keratin culture media, an Anoxybacillus caldiproteolyticus PC2 was isolated. This thermotolerant isolate presents a remarkable feature producing a thermostable keratinase at 60°C. The partially purified keratinase, identified as a thermolysin-like peptidase, was active at a pH range of 5.0-10.0 with maximal activity at a temperature range of 50-80°C. The optimal activity was observed at pH 7.0 and 50-60°C. These characteristics are potentially useful for biotechnological purposes such as processing and bioconversion of keratin.

Effect of enzyme immobilization and in vitro digestion on the immune-reactivity and sequence of IgE epitopes in egg white proteins.

The effects of hydrolysis by free and immobilized forms of Neutrase (FN, IN, respectively) and Thermolysin (FT, IT, respectively) and in vitro digestion on the degree of hydrolysis (DH) of egg white proteins, molecular weight distribution of peptides, immune-reactivity and IgE epitopes of egg white proteins were investigated. With FT and IT in the intestinal digests, the proteolysis followed by in vitro digestion produced peptides smaller than 10 kDa. Hydrolysis with the immobilized enzymes had a greater effect than the free enzymes on increasing surface hydrophobicity. The lowest IgE-binding capacity was observed for the intestinal digest of IT-derived hydrolysates (3.3 ± 1.9%). Compared to in vitro digestion, proteolysis showed a significant effect on the immune-reactivity reduction of egg white proteins. Liquid chromatography-tandem mass spectrometry data showed that the most resistant epitopes to enzymatic hydrolysis and in vitro digestion were in ovomucoid, where epitope fragments 1-10, 1-14, 1-20, 4-20, 11-20, 61-74, 71-75 and 101-105 remained intact. Overall, the IgE-binding capacities of egg white proteins were not completely removed after the enzymatic hydrolysis and in vitro digestion due to the presence of intact proteins such as lysozyme and also due to the several immunoreactive peptides derived from egg white proteins.

Optimization of Porcine Ovarian Follicle Isolation Methods for Better Developmental Potential.

In the present study, we present a comparative analysis among the outputs of porcine follicle isolation using either mechanical technique alone or in combination with enzymes, proposing an optimized protocol useful for all further applications related to follicle in vitro growth and reproductive tissue engineering. The porcine follicles were isolated using mechanical technique alone (hand blender and scalpels) or in combination with collagenase or Liberase Dispase High (DH) at different doses applying different protocols. Finally, the number, morphology, and stage of isolated follicles were compared between the protocols. Moreover, the follicle viability (live/dead assay) and morphology (rhodamine phalloidin and 4',6-diamidino-2-phenylindole staining and scanning electron microscopy analysis) were evaluated after 10 days of culture. We found an optimum protocol for intact follicle isolation using the mechanical technique in combination with enzymes at a concentration of 0.5 mg/mL. However, the number of total isolated follicles and primordial follicles was significantly higher when collagenase was used compared to Liberase DH (p < 0.05), while Liberase DH could isolate a significantly higher percentage of preantral follicles. After 10 days of culture, the morphology and health status of follicles were statistically higher when Liberase DH was used in comparison with collagenase. Moreover, on the follicles extracted with Liberase DH, it was possible to observe theca cells covering part of the follicle surface. In conclusion, we demonstrated that the intact primary or secondary follicles could not be obtained using only mechanical methods, which led to the isolation of denuded oocytes and dramatically damaged follicles. We concluded that the collagenase-based follicle isolation could negatively affect the morphology and developmental potential of the follicles. Moreover, the incubation of ovarian cortex tissues with Liberase DH solution is an optimized protocol for porcine ovarian follicle isolation with developmental competence. Impact statement Isolation and in vitro maturation of follicles can pave the way for activities on reproductive tissue engineering (REPROTEN) and developing an artificial ovary. In this regard, the standardization and optimization of the extraction methods are pivotal for the design of experiment of follicle in vitro growth. In the present study, we provided a comparative analysis among the outputs of porcine follicle isolation using either mechanical technique alone or in combination with collagenase or Liberase DH, proposing an optimized protocol useful for all further applications related to follicles' in vitro growth and REPROTEN.

The tripartite architecture of the eukaryotic integral membrane protein zinc metalloprotease Ste24.

Ste24 enzymes, a family of eukaryotic integral membrane proteins, are zinc metalloproteases (ZMPs) originally characterized as "CAAX proteases" targeting prenylated substrates, including a-factor mating pheromone in yeast and prelamin A in humans. Recently, Ste24 was shown to also cleave nonprenylated substrates. Reduced activity of the human ortholog, HsSte24, is linked to multiple disease states (laminopathies), including progerias and lipid disorders. Ste24 possesses a unique "α-barrel" structure consisting of seven transmembrane (TM) α-helices encircling a large intramembranous cavity (~14 000 Å3 ). The catalytic zinc, coordinated via a HExxH…E/H motif characteristic of gluzincin ZMPs, is positioned at one of the cavity's bases. The interrelationship between Ste24 as a gluzincin, a long-studied class of soluble ZMPs, and as a novel cavity-containing integral membrane protein protease has been minimally explored to date. Informed by homology to well-characterized soluble, gluzincin ZMPs, we develop a model of Ste24 that provides a conceptual framework for this enzyme family, suitable for development and interpretation of structure/function studies. The model consists of an interfacial, zinc-containing "ZMP Core" module surrounded by a "ZMP Accessory" module, both capped by a TM helical "α-barrel" module of as yet unknown function. Multiple sequence alignment of 58 Ste24 orthologs revealed 38 absolutely conserved residues, apportioned unequally among the ZMP Core (18), ZMP Accessory (13), and α-barrel (7) modules. This Tripartite Architecture representation of Ste24 provides a unified image of this enzyme family.

An Internally Quenched Fluorescent Peptide Substrate for Protealysin.

Protealysin, a metalloprotease of Serratia proteamaculans, is the prototype of a subgroup of the M4 peptidase family. Protealysin-like proteases (PLPs) are widely spread in bacteria but also occur in fungi and certain archaea. The interest in PLPs is primarily due to their putative involvement in the bacterial pathogenesis in animals and plants. Studying PLPs requires an efficient quantitative assay for their activity; however, no such assay has been reported so far. Here, we used the autoprocessing site sequence of the protealysin precursor to construct an internally quenched fluorescent peptide substrate 2-aminobenzoyl-L-arginyl-L-seryl-L-valyl-L-isoleucyl-L-(ε-2,4-dinitrophenyl)lysine. Protealysin and thermolysin, the prototype of the M4 family, proved to hydrolyze only the Ser-Val bond of the substrate. The substrate exhibited a KM = 35 ± 4 µM and kcat = 21 ± 1 s-1 for protealysin as well as a KM = 33 ± 8 µM and kcat = 7 ± 1 s-1 for thermolysin at 37 °C. Comparison of the effect of different enzymes (thermolysin, trypsin, chymotrypsin, savinase, and pronase E) on the substrate has demonstrated that it is not strictly specific for protealysin; however, this enzyme has higher molar activity even compared to the closely related thermolysin. Thus, the proposed substrate can be advantageous for quantitative studies of protealysin as well as for activity assays of other M4 peptidases.

Sub-10 nm Resolution Patterning of Pockets for Enzyme Immobilization with Independent Density and Quasi-3D Topography Control.

The ability to precisely control the localization of enzymes on a surface is critical for several applications including biosensing, bionanoreactors, and single molecule studies. Despite recent advances, fabrication of enzyme patterns with resolution at the single enzyme level is limited by the lack of lithography methods that combine high resolution, compatibility with soft, polymeric structures, ease of fabrication, and high throughput. Here, a method to generate enzyme nanopatterns (using thermolysin as a model system) on a polymer surface is demonstrated using thermochemical scanning probe lithography (tc-SPL). Electrostatic immobilization of negatively charged sulfonated enzymes occurs selectively at positively charged amine nanopatterns produced by thermal deprotection of amines along the side-chain of a methacrylate-based copolymer film via tc-SPL. This process occurs simultaneously with local thermal quasi-3D topographical patterning of the same polymer, offering lateral sub-10 nm resolution, and vertical 1 nm resolution, as well as high throughput (5.2 × 104 µm2/h). The obtained single-enzyme resolution patterns are characterized by atomic force microscopy (AFM) and fluorescence microscopy. The enzyme density, the surface passivation, and the quasi-3D arbitrary geometry of these patterned pockets are directly controlled during the tc-SPL process in a single step without the need of markers or masks. Other unique features of this patterning approach include the combined single-enzyme resolution over mm2 areas and the possibility of fabricating enzymes nanogradients.

Impact of tissue enzymatic digestion on analysis of immune cells in mouse reproductive mucosa with a focus on γδ T cells.

Mucosal tissues are enriched in γδ T lymphocytes, which maintain epithelial homeostasis, however, the homeostatic mechanisms are still incompletely understood. To elucidate their role in the tissue integrity governance within the female genital mucosa we employed flow cytometry, which is a powerful tool used for the characterization of tissue-resident immune cells, however, often requiring cell release upon tissue enzymatic disaggregation. Here, we analyzed the impact of various proteolytic enzymes in their ability to effectively isolate viable immune cells from the reproductive system of non-pregnant mice. Murine vaginas and uteri were digested using commercially available enzyme blends (liberases) and single enzymes (dispase II and collagenase IV). Among tested enzymes, liberases released the highest number of cells from digested tissues while dispase II and collagenase IV led to a significant decrease in the number of isolated live cells. Also, liberases had only minor detrimental effects on cell viability and detection of CD45, CD3ε, γδ TCR and CD11c positive cells. We found that a single liberase blend called Liberase TL was the most suited for the analysis of γδ T cells in the reproductive tract. By examining two distinct phases of the estrous cycle - estrus and diestrus, characterized by high and low epithelial stratification, respectively, we showed that higher numbers of γδ T lymphocytes were present in the latter cycle phase in vagina and uterus. Interestingly, the diestrus-associated increase in γδ T lymphocyte number was also observed in reproductive tract draining lumbar lymph nodes but not in more distant, inguinal lymph nodes. Our data indicate that enzymes used for reproductive mucosa digestion have profound effects on the cell viability and isolation efficiency, which consequently influence the phenotypic and quantitative analysis of immune cells.

Effect of nonenzymatic deamidation on the structure stability of Camelus dromedarius α-lactalbumin.

Camelid α-lactalbumin is the only known protein that can undergo nonenzymatic deamidation on two Asn residues. This leads to the generation of a mixture of unusual isoAsp and d-Asp residues that may impact health. The effect of deamidation on camel α-lactalbumin instability was investigated. Circular dichroism showed that the altered protein acquired secondary structure resulting in an increase in α-helix content. In good agreement, the 3D structure of camel α-lactalbumin determined by X-ray crystallography, displayed a short additional α-helix probably induced by deamidation, compared to the human and bovine counterparts. This α-helix was located in the C-terminal region and included residues 101-106. Differential scanning calorimetry together with the susceptibility to thermolysin showed that the deamidation process reinforced the structural stability of the α-lactalbumin at high temperature and its resistance toward proteolysis.