Divalent Cations: A Molecular Glue for Protein Materials.

Among inorganic materials, divalent cations modulate thousands of physiological processes that support life. Their roles in protein assembly and aggregation are less known, although they are progressively being brought to light. We review the structural roles of divalent cations here, as well as the novel protein materials that are under development, in which they are used as glue-like agents. More specifically, we discuss how mechanically stable nanoparticles, fibers, matrices, and hydrogels are generated through their coordination with histidine-rich proteins. We also describe how the rational use of divalent cations combined with simple protein engineering offers unexpected and very simple biochemical approaches to biomaterial design that might address unmet clinical needs in precision medicine.

Enhanced recombinant protein capture, purity and yield from crude bacterial cell extracts by N-Lauroylsarcosine-assisted affinity chromatography.

BACKGROUND: Recombinant proteins cover a wide range of biomedical, biotechnological, and industrial needs. Although there are diverse available protocols for their purification from cell extracts or from culture media, many proteins of interest such as those containing cationic domains are difficult to purify, a fact that results in low yields of the final functional product. Unfortunately, this issue prevents the further development and industrial or clinical application of these otherwise interesting products. RESULTS: Aiming at improving the purification of such difficult proteins, a novel procedure has been developed based on supplementing crude cell extracts with non-denaturing concentrations of the anionic detergent N-Lauroylsarcosine. The incorporation of this simple step in the downstream pipeline results in a substantial improvement of the protein capture by affinity chromatography, an increase of protein purity and an enhancement of the overall process yield, being the detergent not detectable in the final product. CONCLUSION: By taking this approach, which represents a smart repurposing of N-Lauroylsarcosine applied to protein downstream, the biological activity of the protein is not affected. Being technologically simple, the N-Lauroylsarcosine-assisted protein purification might represent a critical improvement in recombinant protein production with wide applicability, thus smothering the incorporation of promising proteins into the protein market.

An In Silico Methodology That Facilitates Decision Making in the Engineering of Nanoscale Protein Materials.

Under the need for new functional and biocompatible materials for biomedical applications, protein engineering allows the design of assemblable polypeptides, which, as convenient building blocks of supramolecular complexes, can be produced in recombinant cells by simple and scalable methodologies. However, the stability of such materials is often overlooked or disregarded, becoming a potential bottleneck in the development and viability of novel products. In this context, we propose a design strategy based on in silico tools to detect instability areas in protein materials and to facilitate the decision making in the rational mutagenesis aimed to increase their stability and solubility. As a case study, we demonstrate the potential of this methodology to improve the stability of a humanized scaffold protein (a domain of the human nidogen), with the ability to oligomerize into regular nanoparticles usable to deliver payload drugs to tumor cells. Several nidogen mutants suggested by the method showed important and measurable improvements in their structural stability while retaining the functionalities and production yields of the original protein. Then, we propose the procedure developed here as a cost-effective routine tool in the design and optimization of multimeric protein materials prior to any experimental testing.

Selecting Subpopulations of High-Quality Protein Conformers among Conformational Mixtures of Recombinant Bovine MMP-9 Solubilized from Inclusion Bodies.

A detailed workflow to analyze the physicochemical characteristics of mammalian matrix metalloproteinase (MMP-9) protein species obtained from protein aggregates (inclusion bodies-IBs) was followed. MMP-9 was recombinantly produced in the prokaryotic microbial cell factories Clearcoli (an engineered form of Escherichia coli) and Lactococcus lactis, mainly forming part of IBs and partially recovered under non-denaturing conditions. After the purification by affinity chromatography of solubilized MMP-9, four protein peaks were obtained. However, so far, the different conformational protein species forming part of IBs have not been isolated and characterized. Therefore, with the aim to link the physicochemical characteristics of the isolated peaks with their biological activity, we set up a methodological approach that included dynamic light scattering (DLS), circular dichroism (CD), and spectrofluorometric analysis confirming the separation of subpopulations of conformers with specific characteristics. In protein purification procedures, the detailed analysis of the individual physicochemical properties and the biological activity of protein peaks separated by chromatographic techniques is a reliable source of information to select the best-fitted protein populations.

Engineering Protein Nanoparticles Out from Components of the Human Microbiome.

Nanoscale protein materials are highly convenient as vehicles for targeted drug delivery because of their structural and functional versatility. Selective binding to specific cell surface receptors and penetration into target cells require the use of targeting peptides. Such homing stretches should be incorporated to larger proteins that do not interact with body components, to prevent undesired drug release into nontarget organs. Because of their low interactivity with human body components and their tolerated immunogenicity, proteins derived from the human microbiome are appealing and fully biocompatible building blocks for the biofabrication of nonreactive, inert protein materials within the nanoscale. Several phage and phage-like bacterial proteins with natural structural roles are produced in Escherichia coli as polyhistidine-tagged recombinant proteins, looking for their organization as discrete, nanoscale particulate materials. While all of them self-assemble in a variety of sizes, the stability of the resulting constructs at 37 °C is found to be severely compromised. However, the fine adjustment of temperature and Zn2+ concentration allows the formation of robust nanomaterials, fully stable in complex media and under physiological conditions. Then, microbiome-derived proteins show promise for the regulatable construction of scaffold protein nanomaterials, which can be tailored and strengthened by simple physicochemical approaches.

Recruiting potent membrane penetrability in tumor cell-targeted protein-only nanoparticles.

The membrane pore-forming activities of the antimicrobial peptide GWH1 have been evaluated in combination with the CXCR4-binding properties of the peptide T22, in self-assembling protein nanoparticles with high clinical potential. The resulting materials, of 25 nm in size and with regular morphologies, show a dramatically improved cell penetrability into CXCR4+ cells (more than 10-fold) and enhanced endosomal escape (the lysosomal degradation dropping from 90% to 50%), when compared with equivalent protein nanoparticles lacking GWH1. These data reveal that GWH1 retains its potent membrane activity in form of nanostructured protein complexes. On the other hand, the specificity of T22 in the CXCR4 receptor binding is subsequently minimized but, unexpectedly, not abolished by the presence of the antimicrobial peptide. The functional combination T22-GWH1 results in 30% of the nanoparticles entering cells via CXCR4 while also exploiting pore-based uptake. Such functional materials are capable to selectively deliver highly potent cytotoxic drugs upon chemical conjugation, promoting CXCR4-dependent cell death. These data support the further development of GWH1-empowered cell-targeted proteins as nanoscale drug carriers for precision medicines. This is a very promising approach to overcome lysosomal degradation of protein nanostructured materials with therapeutic value.

Selective CXCR4+ Cancer Cell Targeting and Potent Antineoplastic Effect by a Nanostructured Version of Recombinant Ricin.

Under the unmet need of efficient tumor-targeting drugs for oncology, a recombinant version of the plant toxin ricin (the modular protein T22-mRTA-H6) is engineered to self-assemble as protein-only, CXCR4-targeted nanoparticles. The soluble version of the construct self-organizes as regular 11 nm planar entities that are highly cytotoxic in cultured CXCR4+ cancer cells upon short time exposure, with a determined IC50 in the nanomolar order of magnitude. The chemical inhibition of CXCR4 binding sites in exposed cells results in a dramatic reduction of the cytotoxic potency, proving the receptor-dependent mechanism of cytotoxicity. The insoluble version of T22-mRTA-H6 is, contrarily, moderately active, indicating that free, nanostructured protein is the optimal drug form. In animal models of acute myeloid leukemia, T22-mRTA-H6 nanoparticles show an impressive and highly selective therapeutic effect, dramatically reducing the leukemia cells affectation of clinically relevant organs. Functionalized T22-mRTA-H6 nanoparticles are then promising prototypes of chemically homogeneous, highly potent antitumor nanostructured toxins for precise oncotherapies based on self-mediated intracellular drug delivery.

Conformational Conversion during Controlled Oligomerization into Nonamylogenic Protein Nanoparticles.

Protein materials are rapidly gaining interest in materials sciences and nanomedicine because of their intrinsic biocompatibility and full biodegradability. The controlled construction of supramolecular entities relies on the controlled oligomerization of individual polypeptides, achievable through different strategies. Because of the potential toxicity of amyloids, those based on alternative molecular organizations are particularly appealing, but the structural bases on nonamylogenic oligomerization remain poorly studied. We have applied spectrofluorimetry and spectropolarimetry to identify the conformational conversion during the oligomerization of His-tagged cationic stretches into regular nanoparticles ranging around 11 nm, useful for tumor-targeted drug delivery. We demonstrate that the novel conformation acquired by the proteins, as building blocks of these supramolecular assemblies, shows different extents of compactness and results in a beta structure enrichment that enhances their structural stability. The conformational profiling presented here offers clear clues for understanding and tailoring the process of nanoparticle formation through the use of cationic and histidine rich stretches in the context of protein materials usable in advanced nanomedical strategies.

[Body dissatisfaction, self-esteem, depressive symptoms, and nutritional status in adolescents]. / Insatisfacción corporal, autoestima, sintomatologia depresiva y estado nutricional en adolescentes.

In adolescence and especially in females, greater body dissatisfaction has been evidenced, which is defined as a negative evaluation of one's own body, being a strong predictor of eating disorders and obesity. OBJECTIVE: To relate body dissatisfaction with self-esteem, depression, and body mass index in adolescents. SUBJECTS AND METHOD: Quantitative, correlational, and cross-sectional study in a sample of 397 school adolescents (180 males and 217 females) from Concepción, Chile, aged 10 to 19 years, to whom the following instruments were applied: Body Shape Questionnaire (BSQ) to assess body dissatisfaction, Rosenberg Self-Esteem Scale, Beck's Depression Inventory-II for those older than 14 years, and Birleson Depression Self-Rating Scale for those younger than 14 years. Body mass index z-score was determined. Spearman's correlation coefficient was estimated for all variables. RESULTS: Body dissatisfaction was reported in 54.9 % of females and 18.3 % of males. Body dissatisfaction was positively correlated with age, z-BMI, and depression (p < 0.01) and negatively correlated with self-esteem (p < 0.01). When body dissatisfaction was differentiated by sex, the same significant correlations remained, except for age. CONCLUSIONS: The results confirm the relationship between body dissatisfaction with self-esteem, depression, and BMI. The importance of promoting healthy self-esteem and body image from an early age to prevent eating disorders and obesity is emphasized.

Structural Stabilization of Clinically Oriented Oligomeric Proteins During their Transit through Synthetic Secretory Amyloids.

Developing time-sustained drug delivery systems is a main goal in innovative medicines. Inspired by the architecture of secretory granules from the mammalian endocrine system it has generated non-toxic microscale amyloid materials through the coordination between divalent metals and poly-histidine stretches. Like their natural counterparts that keep the functionalities of the assembled protein, those synthetic structures release biologically active proteins during a slow self-disintegration process occurring in vitro and upon in vivo administration. Being these granules formed by a single pure protein species and therefore, chemically homogenous, they act as highly promising time-sustained drug delivery systems. Despite their enormous clinical potential, the nature of the clustering process and the quality of the released protein have been so far neglected issues. By using diverse polypeptide species and their protein-only oligomeric nanoscale versions as convenient models, a conformational rearrangement and a stabilization of the building blocks during their transit through the secretory granules, being the released material structurally distinguishable from the original source is proved here. This fact indicates a dynamic nature of secretory amyloids that act as conformational arrangers rather than as plain, inert protein-recruiting/protein-releasing granular depots.

StarD7 behaves as a fusogenic protein in model and cell membrane bilayers.

StarD7 is a surface active protein, structurally related with the START lipid transport family. So, the present work was aimed at elucidating a potential mechanism of action for StarD7 that could be related to its interaction with a lipid-membrane interface. We applied an assay based on the fluorescence de-quenching of BD-HPC-labeled DMPC-DMPS 4:1 mol/mol SUVs (donor liposomes) induced by the dilution with non-labeled DMPC-DMPS 4:1 mol/mol LUVs (acceptor liposomes). Recombinant StarD7 accelerated the dilution of BD-HPC in a concentration-dependent manner. This result could have been explained by either a bilayer fusion or monomeric transport of the labeled lipid between donor and acceptor liposomes. Further experiments (fluorescence energy transfer between DPH-HPC/BD-HPC, liposome size distribution analysis by dynamic light scattering, and the multinuclear giant cell formation induced by recombinant StarD7) strongly indicated that bilayer fusion was the mechanism responsible for the StarD7-induced lipid dilution. The efficiency of lipid dilution was dependent on StarD7 electrostatic interactions with the lipid-water interface, as shown by the pH- and salt-induced modulation. Moreover, this process was favored by phosphatidylethanolamine which is known to stabilize non-lamellar phases considered as intermediary in the fusion process. Altogether these findings allow postulate StarD7 as a fusogenic protein.

The role of water in reactions catalysed by hydrolases under conditions of molecular crowding.

Under macromolecular crowding (MC) conditions such as cellular, extracellular, food and other environments of biotechnological interest, the thermodynamic activity of the different macromolecules present in the system is several orders of magnitude higher than in dilute solutions. In this state, the diffusion rates are affected by the volume exclusion induced by the crowders. Immiscible liquid phases, which may arise in MC by liquid-liquid phase separation, may induce a dynamic confinement of reactants, products and/or enzymes, tuning reaction rates. In cellular environments and other crowding conditions, membranes and macromolecules provide, on the whole, large surfaces that can perturb the solvent, causing its immobilisation by adsorption in the short range and also affecting the solvent viscosity in the long range. The latter phenomenon can affect the conformation of a protein and/or the degree of association of its protomers and, consequently, its activity. Changes in the water structure can also alter the enzyme-substrate interaction, and, in the case of hydrolytic enzymes, where water is one of the substrates, it also affects the reaction mechanism. Here, we review the evidence for how macromolecular crowding affects the catalysis induced by hydrolytic enzymes, focusing on the structure and dynamics of water.

Protein features instruct the secretion dynamics from metal-supported synthetic amyloids.

Hexahistidine-tagged proteins can be clustered by divalent cations into self-containing, dynamic protein depots at the microscale, which under physiological conditions leak functional protein. While such protein granules show promise in clinics as time-sustained drug delivery systems, little is known about how the nature of their components, that is, the protein and the particular cation used as cross-linker, impact on the disintegration of the material and on its secretory performance. By using four model proteins and four different cation formulations to control aggregation, we have here determined a moderate influence of the used cation and a potent impact of some protein properties on the release kinetics and on the final fraction of releasable protein. In particular, the electrostatic charge at the amino terminus and the instability and hydropathicity indexes determine the disintegration profile of the depot. These data offer clues for the fabrication of efficient and fully exploitable secretory granules that being biocompatible and chemically homogenous allow their tailored use as drug delivery platforms in biological systems.

A Novel Generation of Tailored Antimicrobial Drugs Based on Recombinant Multidomain Proteins.

Antibiotic resistance has exponentially increased during the last years. It is necessary to develop new antimicrobial drugs to prevent and treat infectious diseases caused by multidrug- or extensively-drug resistant (MDR/XDR)-bacteria. Host Defense Peptides (HDPs) have a versatile role, acting as antimicrobial peptides and regulators of several innate immunity functions. The results shown by previous studies using synthetic HDPs are only the tip of the iceberg, since the synergistic potential of HDPs and their production as recombinant proteins are fields practically unexplored. The present study aims to move a step forward through the development of a new generation of tailored antimicrobials, using a rational design of recombinant multidomain proteins based on HDPs. This strategy is based on a two-phase process, starting with the construction of the first generation molecules using single HDPs and further selecting those HDPs with higher bactericidal efficiencies to be combined in the second generation of broad-spectrum antimicrobials. As a proof of concept, we have designed three new antimicrobials, named D5L37ßD3, D5L37D5L37 and D5LAL37ßD3. After an in-depth exploration, we found D5L37D5L37 to be the most promising one, since it was equally effective against four relevant pathogens in healthcare-associated infections, such as methicillin-susceptible (MSSA) and methicillin-resistant (MRSA) Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis (MRSE) and MDR Pseudomonas aeruginosa, being MRSA, MRSE and P. aeruginosa MDR strains. The low MIC values and versatile activity against planktonic and biofilm forms reinforce the use of this platform to isolate and produce unlimited HDP combinations as new antimicrobial drugs by effective means.

Recombinant Proteins for Assembling as Nano- and Micro-Scale Materials for Drug Delivery: A Host Comparative Overview.

By following simple protein engineering steps, recombinant proteins with promising applications in the field of drug delivery can be assembled in the form of functional materials of increasing complexity, either as nanoparticles or nanoparticle-leaking secretory microparticles. Among the suitable strategies for protein assembly, the use of histidine-rich tags in combination with coordinating divalent cations allows the construction of both categories of material out of pure polypeptide samples. Such molecular crosslinking results in chemically homogeneous protein particles with a defined composition, a fact that offers soft regulatory routes towards clinical applications for nanostructured protein-only drugs or for protein-based drug vehicles. Successes in the fabrication and final performance of these materials are expected, irrespective of the protein source. However, this fact has not yet been fully explored and confirmed. By taking the antigenic RBD domain of the SARS-CoV-2 spike glycoprotein as a model building block, we investigated the production of nanoparticles and secretory microparticles out of the versions of recombinant RBD produced by bacteria (Escherichia coli), insect cells (Sf9), and two different mammalian cell lines (namely HEK 293F and Expi293F). Although both functional nanoparticles and secretory microparticles were effectively generated in all cases, the technological and biological idiosyncrasy of each type of cell factory impacted the biophysical properties of the products. Therefore, the selection of a protein biofabrication platform is not irrelevant but instead is a significant factor in the upstream pipeline of protein assembly into supramolecular, complex, and functional materials.

Efficient Delivery of Antimicrobial Peptides in an Innovative, Slow-Release Pharmacological Formulation.

Both nanostructure and multivalency enhance the biological activities of antimicrobial peptides (AMPs), whose mechanism of action is cooperative. In addition, the efficacy of a particular AMP should benefit from a steady concentration at the local place of action and, therefore, from a slow release after a dynamic repository. In the context of emerging multi-resistant bacterial infections and the urgent need for novel and effective antimicrobial drugs, we tested these concepts through the engineering of four AMPs into supramolecular complexes as pharmacological entities. For that purpose, GWH1, T22, Pt5, and PaD, produced as GFP or human nidogen-based His-tagged fusion proteins, were engineered as self-assembling oligomeric nanoparticles ranging from 10 to 70 nm and further packaged into nanoparticle-leaking submicron granules. Since these materials slowly release functional nanoparticles during their time-sustained unpacking, they are suitable for use as drug depots in vivo. In this context, a particular AMP version (GWH1-NIDO-H6) was selected for in vivo validation in a zebrafish model of a complex bacterial infection. The GWH1-NIDO-H6-secreting protein granules are protective in zebrafish against infection by the multi-resistant bacterium Stenotrophomonas maltophilia, proving the potential of innovative formulations based on nanostructured and slowly released recombinant AMPs in the fight against bacterial infections.

The Diphtheria Toxin Translocation Domain Impairs Receptor Selectivity in Cancer Cell-Targeted Protein Nanoparticles.

Protein-based materials intended as nanostructured drugs or drug carriers are progressively gaining interest in nanomedicine, since their structure, assembly and cellular interactivity can be tailored by recruiting functional domains. The main bottleneck in the development of deliverable protein materials is the lysosomal degradation that follows endosome maturation. This is especially disappointing in the case of receptor-targeted protein constructs, which, while being highly promising and in demand in precision medicines, enter cells via endosomal/lysosomal routes. In the search for suitable protein agents that might promote endosome escape, we have explored the translocation domain (TD) of the diphtheria toxin as a functional domain in CXCR4-targeted oligomeric nanoparticles designed for cancer therapies. The pharmacological interest of such protein materials could be largely enhanced by improving their proteolytic stability. The incorporation of TD into the building blocks enhances the amount of the material detected inside of exposed CXCR4+ cells up to around 25-fold, in absence of cytotoxicity. This rise cannot be accounted for by endosomal escape, since the lysosomal degradation of the new construct decreases only moderately. On the other hand, a significant loss in the specificity of the CXCR4-dependent cellular penetration indicates the unexpected role of the toxin segment as a cell-penetrating peptide in a dose-dependent and receptor-independent fashion. These data reveal that the diphtheria toxin TD displayed on receptor-targeted oligomeric nanoparticles partially abolishes the exquisite receptor specificity of the parental material and it induces nonspecific internalization in mammalian cells.

Quality Control of Proteins Solubilized from Inclusion Bodies.

Despite substantial development of production and purification protocols for heterologous recombinant proteins, some proteins are difficult to produce or, when produced, are accumulated in inclusion bodies (IBs). Nondenaturing protocols can be used to recover the entrapped protein from these protein aggregates. In this chapter, we provide a detailed procedure to analyze the physicochemical properties of one of those proteins produced in prokaryotic expression systems. Serum amyloid A3 (SAA3) was recovered from inclusion bodies (IBs) and its secondary structure associated to thermal stability and size was determined by circular dichroism (CD) and dynamic light scattering (DLS), respectively. These techniques were also applied to evaluate the SAA3 interaction with model membranes. These results show the importance of the structural analysis of proteins released from inclusion bodies under nondenaturing procedures, although similar approaches can be extended to any type of recombinant protein preparation.

His-tag ß-galactosidase supramolecular performance.

ß-Galactosidase is an important biotechnological enzyme used in the dairy industry, pharmacology and in molecular biology. In our laboratory we have overexpressed a recombinant ß-galactosidase in Escherichia coli (E. coli). This enzyme differs from its native version (ß-GalWT) in that 6 histidine residues have been added to the carboxyl terminus in the primary sequence (ß-GalHis), which allows its purification by immobilized metal affinity chromatography (IMAC). In this work we compared the functionality and structure of both proteins and evaluated their catalytic behavior on the kinetics of lactose hydrolysis. We observed a significant reduction in the enzymatic activity of ß-GalHis with respect to ß-GalWT. Although, both enzymes showed a similar catalytic profile as a function of temperature, ß-GalHis presented a higher resistance to the thermal inactivation compared to ß-GalWT. At room temperature, ß-GalHis showed a fluorescence spectrum compatible with a partially unstructured protein, however, it exhibited a lower tendency to the thermal-induced unfolding with respect to ß-GalWT. The distinctively supramolecular arranges of the proteins would explain the effect of the presence of His-tag on the enzymatic activity and thermal stability.

The Poly-Histidine Tag H6 Mediates Structural and Functional Properties of Disintegrating, Protein-Releasing Inclusion Bodies.

The coordination between histidine-rich peptides and divalent cations supports the formation of nano- and micro-scale protein biomaterials, including toxic and non-toxic functional amyloids, which can be adapted as drug delivery systems. Among them, inclusion bodies (IBs) formed in recombinant bacteria have shown promise as protein depots for time-sustained protein release. We have demonstrated here that the hexahistidine (H6) tag, fused to recombinant proteins, impacts both on the formation of bacterial IBs and on the conformation of the IB-forming protein, which shows a higher content of cross-beta intermolecular interactions in H6-tagged versions. Additionally, the addition of EDTA during the spontaneous disintegration of isolated IBs largely affects the protein leakage rate, again protein release being stimulated in His-tagged materials. This event depends on the number of His residues but irrespective of the location of the tag in the protein, as it occurs in either C-tagged or N-tagged proteins. The architectonic role of H6 in the formation of bacterial IBs, probably through coordination with divalent cations, offers an easy approach to manipulate protein leakage and to tailor the applicability of this material as a secretory amyloidal depot in different biomedical interfaces. In addition, the findings also offer a model to finely investigate, in a simple set-up, the mechanics of protein release from functional secretory amyloids.