Insights on the emerging biotechnology of histidine-rich peptides

Altres ajuts: Acord transformatiu CRUE-CSICIn the late 70's, the discovery of the restriction enzymes made possible the biological production of functional proteins by recombinant DNA technologies, a fact that largely empowered both biotechnological and pharmaceutical industries. Short peptides or small protein domains, with specific molecular affinities, were developed as purification tags in downstream processes to separate the target protein from the culture media or cell debris, upon breaking the producing cells. Among these tags, and by exploiting the interactivity of the imidazole ring of histidine residues, the hexahistidine peptide (H6) became a gold standard. Although initially used almost exclusively in protein production, H6 and related His-rich peptides are progressively proving a broad applicability in novel utilities including enzymatic processes, advanced drug delivery systems and diagnosis, through a so far unsuspected adaptation of their binding capabilities. In this context, the coordination of histidine residues and metals confers intriguing functionalities to His-rich sequences useable in the forward-thinking design of protein-based nano- and micro-materials and devices, through strategies that are comprehensively presented here

High-precision targeting and destruction of cancer-associated PDGFR-β+ stromal fibroblasts through self-assembling, protein-only nanoparticles

Altres ajuts: acords transformatius de la UABThe need for more effective and precision medicines for cancer has pushed the exploration of new materials appropriate for drug delivery and imaging, and alternative receptors for targeting. Among the most promising strategies, finding suitable cell surface receptors and targeting agents for cancer-associated platelet derived growth factor receptor β (PDGFR-β)+ stromal fibroblasts is highly appealing. As a neglected target, this cell type mechanically and biologically supports the growth, progression, and infiltration of solid tumors in non-small cell lung, breast, pancreatic, and colorectal cancers. We have developed a family of PDGFR-β-targeted nanoparticles based on biofabricated, self-assembling proteins, upon hierarchical and iterative selective processes starting from four initial candidates. The modular protein PDGFD-GFP-H6 is well produced in recombinant bacteria, resulting in structurally robust oligomeric particles that selectively penetrates into PDGFR-β+ stromal fibroblasts in a dose-dependent manner, by means of the PDGFR-β ligand PDGFD. Upon in vivo administration, these GFP-carrying protein nanoparticles precisely accumulate in tumor tissues and enlighten them for IVIS observation. When GFP is replaced by a microbial toxin, selective tumor tissue destruction is observed associated with a significant reduction in tumor volume growth. The presented data validate the PDGFR-β/PDGFD pair as a promising toolbox for targeted drug delivery in the tumor microenvironment and oligomeric protein nanoparticles as a powerful instrument to mediate highly selective biosafe targeting in cancer through non-cancer cells. Statement of significance: We have developed a transversal platform for nanoparticle-based drug delivery into cancer-associated fibroblasts. This is based on the engineered modular protein PDGFD-GFP-H6 that spontaneously self-assemble and selectively penetrates into PDGFR-β+ stromal fibroblasts in a dose-dependent manner, by means of the PDGFR-β ligand PDGFD. In vivo, these protein nanoparticles accumulate in tumor and when incorporating a microbial toxin, they destroy tumor tissues with a significant reduction in tumor volume, in absence of side toxicities. The data presented here validate the PDGFR-β/PDGFD pair as a fully versatile toolbox for targeted drug delivery in the tumor microenvironment intended as a synergistic treatment

The spectrum of building block conformers sustains the biophysical properties of clinically-oriented self-assembling protein nanoparticles

Altres ajuts: Acord transformatiu CRUE-CSICHistidine-rich peptides confer self-assembling properties to recombinant proteins through the supramolecular coordination with divalent cations. This fact allows the cost-effective, large-scale generation of microscopic and macroscopic protein materials with intriguing biomedical properties. Among such materials, resulting from the simple bioproduction of protein building blocks, homomeric nanoparticles are of special value as multivalent interactors and drug carriers. Interestingly, we have here identified that the assembly of a given His-tagged protein might render distinguishable categories of self-assembling protein nanoparticles. This fact has been scrutinized through the nanobody-containing fusion proteins EM1-GFP-H6 and A3C8-GFP-H6, whose biosynthesis results in two distinguishable populations of building blocks. In one of them, the assembling and disassembling is controllable by cations. However, a second population immediately self-assembles upon purification through a non-regulatable pathway, rendering larger nanoparticles with specific biological properties. The structural analyses of both model proteins and nanoparticles revealed important conformational variability in the building blocks. This fact renders different structural and functional categories of the final soft materials resulting from the participation of energetically unstable intermediates in the oligomerization process. These data illustrate the complexity of the His-mediated protein assembling in recombinant proteins but they also offer clues for a better design and refinement of protein-based nanomedicines, which, resulting from biological fabrication, show an architectonic flexibility unusual among biomaterials

Controlling self-assembling and tumor cell-targeting of protein-only nanoparticles through modular protein engineering

Altres ajuts: EU COST Action CA 17140. Villaverde A received an ICREA ACADEMIA award. Unzueta was supported by PERIS program from the Health Department of la Generalitat de Catalunya.Modular protein engineering is suited to recruit complex and multiple functionalities in single-chain polypeptides. Although still unexplored in a systematic way, it is anticipated that the positioning of functional domains would impact and refine these activities, including the ability to organize as supramolecular entities and to generate multifunctional protein materials. To explore this concept, we have repositioned functional segments in the modular protein T22-GFP-H6 and characterized the resulting alternative fusions. In T22-GFP-H6, the combination of T22 and H6 promotes self-assembling as regular nanoparticles and selective binding and internalization of this material in CXCR4-overexpressing tumor cells, making them appealing as vehicles for selective drug delivery. The results show that the pleiotropic activities are dramatically affected in module-swapped constructs, proving the need of a carboxy terminal positioning of H6 for protein self-assembling, and the accommodation of T22 at the amino terminus as a requisite for CXCR4 cell binding and internalization. Furthermore, the failure of self-assembling as regular oligomers reduces cellular penetrability of the fusions while keeping the specificity of the T22-CXCR4 interaction. All these data instruct how multifunctional nanoscale protein carriers can be designed for smart, protein-driven drug delivery, not only for the treatment of CXCR4 human neoplasias, but also for the development of anti-HIV drugs and other pathologies in which CXCR4 is a relevant homing marker

Assembly of histidine-rich protein materials controlled through divalent cations

Nanostructured protein materials show exciting biomedical applications, since both structure and function can be genetically programmed. In particular, self-assembling histidine-rich proteins benefit from functional plasticity that allows the generation of protein-only nanoparticles for cell targeted drug delivery. However, the rational development of constructs with improved functions is limited by a poor control of the oligomerization process. By exploring cross-interactions between histidine-tagged building blocks, we have identified a critical architectonic role of divalent cations. The obtained data instruct about how histidine-rich protein materials can be assembled, disassembled and reassembled within the nanoscale through the stoichiometric manipulation of divalent ions, in a biochemical approach to biomaterials design. Statement of Significance: Divalent metal and non-metal cations such as Ni, Cu Ca and Zn have been identified as unexpected molecular tools to control the assembling, disassembling and reassembling of histidine-rich protein materials at the nanoscale. Their stoichiometric manipulation allows generating defined protein-protein cross-molecular contacts between building blocks, for a powerful nano-biochemical manipulation of the material's architecture

Fluorescent dye labeling changes the biodistribution of cell-targeted nanoparticles

Fluorescent dye labeling is a common strategy to analyze the fate of administered nanoparticles in living organisms. However, to which extent the labeling processes can alter the original nanoparticle biodistribution has been so far neglected. In this work, two widely used fluorescent dye molecules, namely, ATTO488 (ATTO) and Sulfo-Cy5 (S-Cy5), have been covalently attached to a well-characterized CXCR4-targeted self-assembling protein nanoparticle (known as T22-GFP-H6). The biodistribution of labeled T22-GFP-H6-ATTO and T22-GFP-H6-S-Cy5 nanoparticles has been then compared to that of the non-labeled nanoparticle in different CXCR4+ tumor mouse models. We observed that while parental T22-GFP-H6 nanoparticles accumulated mostly and specifically in CXCR4+ tumor cells, labeled T22-GFP-H6-ATTO and T22-GFP-H6-S-Cy5 nanoparticles showed a dramatic change in the biodistribution pattern, accumulating in non-target organs such as liver or kidney while reducing tumor targeting capacity. Therefore, the use of such labeling molecules should be avoided in target and non-target tissue uptake studies during the design and development of targeted nanoscale drug delivery systems, since their effect over the fate of the nanomaterial can lead to considerable miss-interpretations of the actual nanoparticle biodistribution

Nanostructure empowers active tumor targeting in ligand-based molecular delivery

Altres ajuts: to EU COST Action CA 17140 and ICREA AcademiaCell-selective targeting is expected to enhance effectiveness and minimize side effects of cytotoxic agents. Functionalization of drugs or drug nanoconjugates with specific cell ligands allows receptor-mediated selective cell delivery. However, it is unclear whether the incorporation of an efficient ligand into a drug vehicle is sufficient to ensure proper biodistribution upon systemic administration, and also at which extent biophysical properties of the vehicle may contribute to the accumulation in target tissues during active targeting. To approach this issue, structural robustness of self-assembling, protein-only nanoparticles targeted to the tumoral marker CXCR4 is compromised by reducing the number of histidine residues (from six to five) in a histidine-based architectonic tag. Thus, the structure of the resulting nanoparticles, but not of building blocks, is weakened. Upon intravenous injection in animal models of human CXCR4 colorectal cancer, the administered material loses the ability to accumulate in tumor tissue, where it is only transiently found. It instead deposits in kidney and liver. Therefore, precise cell-targeted delivery requires not only the incorporation of a proper ligand that promotes receptor-mediated internalization, but also, unexpectedly, its maintenance of a stable multimeric nanostructure that ensures high ligand exposure and long residence time in tumor tissue

Biofabrication of Self-Assembling Covalent Protein Nanoparticles through Histidine-Templated Cysteine Coupling

Altres ajuts: CERCA Programme/Generalitat de CatalunyaNanoscale protein materials show increasing applications in biotechnology and biomedicine, addressing catalysis, drug delivery, or tissue engineering. Although protein oligomerization is reachable through several engineering approaches, including the use of divalent cations for histidine-rich stretches, the effectiveness of cation-His binding is influenced by protein conformation, media composition, and chelating agents. Thus, looking for powerful, green, cross-linker-free, and transversal oligomerization platforms, we have built a histidine-templated cysteine-coupling concept. On this basis, we have engineered a Cys-containing, H6-derived His-Cys hybrid tag that enables the spontaneous and efficient self-assembling of tagged proteins into monodisperse nanoparticles through a highly ordered covalent binding process. Although the generated nanostructures are supported by disulfide bridge formation and exclusively reversed by reducing agents but not by chelating agents, the presence of cysteine residues does not disrupt the metal-binding abilities of histidine residues within the tag. This fact allows one to combine the one-step IMAC-based protein purification and, also, the Zn2+-induced formation of higher-order microparticulate materials as nanoparticle-releasing protein-only depots. The dual mode of cross-molecular interactivity shown by the hybrid tag and the structural robustness and stability of the resulting nanoparticles offer wide applicability of the green biofabrication concept proposed here for the further development of clinically usable protein materials

Biofabrication of functional protein nanoparticles through simple His-tag engineering

Altres ajuts: Acord transformatiu CRUE-CSICWe have developed a simple, robust, and fully transversal approach for thea-la-cartefabrication of functional multimeric nanoparticles with potential biomedical applications, validated here by a set of diverse and unrelated polypeptides. The proposed concept is based on the controlled coordination between Znions and His residues in His-tagged proteins. This approach results in a spontaneous and reproducible protein assembly as nanoscale oligomers that keep the original functionalities of the protein building blocks. The assembly of these materials is not linked to particular polypeptide features, and it is based on an environmentally friendly and sustainable approach. The resulting nanoparticles, with dimensions ranging between 10 and 15 nm, are regular in size, are architecturally stable, are fully functional, and serve as intermediates in a more complex assembly process, resulting in the formation of microscale protein materials. Since most of the recombinant proteins produced by biochemical and biotechnological industries and intended for biomedical research are His-tagged, the green biofabrication procedure proposed here can be straightforwardly applied to a huge spectrum of protein species for their conversion into their respective nanostructured formats

Engineering secretory amyloids for remote and highly selective destruction of metastatic foci

Altres ajuts: to EU COST Action CA 17140Functional amyloids produced in bacteria as nanoscale inclusion bodies are intriguing but poorly explored protein materials with wide therapeutic potential. Since they release functional polypeptides under physiological conditions, these materials can be potentially tailored as mimetic of secretory granules for slow systemic delivery of smart protein drugs. To explore this possibility, bacterial inclusion bodies formed by a self-assembled, tumor-targeted Pseudomonas exotoxin (PE24) are administered subcutaneously in mouse models of human metastatic colorectal cancer, for sustained secretion of tumor-targeted therapeutic nanoparticles. These proteins are functionalized with a peptidic ligand of CXCR4, a chemokine receptor overexpressed in metastatic cancer stem cells that confers high selective cytotoxicity in vitro and in vivo. In the mouse models of human colorectal cancer, time-deferred anticancer activity is detected after the subcutaneous deposition of 500 µg of PE24-based amyloids, which promotes a dramatic arrest of tumor growth in the absence of side toxicity. In addition, long-term prevention of lymphatic, hematogenous, and peritoneal metastases is achieved. These results reveal the biomedical potential and versatility of bacterial inclusion bodies as novel tunable secretory materials usable in delivery, and they also instruct how therapeutic proteins, even with high functional and structural complexity, can be packaged in this convenient format