Targeting antitumoral proteins to breast cancer by local administration of functional inclusion bodies

Altres ajuts de l'Instituto de Salud Carlos III: PI15/00272, PI1702242FIS i #PI16/01224Two structurally and functionally unrelated proteins, namely Omomyc and p31, are engineered as CD44-targeted inclusion bodies produced in recombinant bacteria. In this unusual particulate form, both types of protein materials selectively penetrate and kill CD44 tumor cells in culture, and upon local administration, promote destruction of tumoral tissue in orthotropic mouse models of human breast cancer. These findings support the concept of bacterial inclusion bodies as versatile protein materials suitable for application in chronic diseases that, like cancer, can benefit from a local slow release of therapeutic protein

hnRNPDL phase separation is regulated by alternative splicing and disease-causing mutations accelerate its aggregation

Altres ajuts: .V. acknowledges funding from ICREA (ICREA-Academia 2016). IRB Barcelona is the recipient of a Severo Ochoa Award of Excellence from MINECO (government of Spain). C.B. acknowledges funding from "Ministerio de Educación y Formación ProfesionalPrion-like proteins form multivalent assemblies and phase separate into membraneless organelles. Heterogeneous ribonucleoprotein D-like (hnRNPDL) is a RNA-processing prion-like protein with three alternative splicing (AS) isoforms, which lack none, one, or both of its two disordered domains. It has been suggested that AS might regulate the assembly properties of RNA-processing proteins by controlling the incorporation of multivalent disordered regions in the isoforms. This, in turn, would modulate their activity in the downstream splicing program. Here, we demonstrate that AS controls the phase separation of hnRNPDL, as well as the size and dynamics of its nuclear complexes, its nucleus-cytoplasm shuttling, and amyloidogenicity. Mutation of the highly conserved D378 in the disordered C-terminal prion-like domain of hnRNPDL causes limb-girdle muscular dystrophy 1G. We show that D378H/N disease mutations impact hnRNPDL assembly properties, accelerating aggregation and dramatically reducing the protein solubility in the muscle of Drosophila, suggesting a genetic loss-of-function mechanism for this muscular disorde

Bottom-up instructive quality control in the biofabrication of smart protein materials

The impact of cell factory quality control on material properties is a neglected but critical issue in the fabrication of protein biomaterials, which are unique in merging structure and function. The molecular chaperoning of protein conformational status is revealed here as a potent molecular instructor of the macroscopic properties of self-assembling, cell-targeted protein nanoparticles, including biodistribution upon in vivo administration

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

Targeting antitumoral proteins to breast cancer by local administration of functional inclusion bodies

Biofabrication; Cancer therapy; Functional amyloidsBiofabricación; Terapia contra el cáncer; Amiloides funcionalesBiofabricació; Teràpia contra el càncer; Amiloides funcionalsTwo structurally and functionally unrelated proteins, namely Omomyc and p31, are engineered as CD44-targeted inclusion bodies produced in recombinant bacteria. In this unusual particulate form, both types of protein materials selectively penetrate and kill CD44+ tumor cells in culture, and upon local administration, promote destruction of tumoral tissue in orthotropic mouse models of human breast cancer. These findings support the concept of bacterial inclusion bodies as versatile protein materials suitable for application in chronic diseases that, like cancer, can benefit from a local slow release of therapeutic proteins.This study has been supported by La Fundacio Marato TV3 and NanoCanTri (CIBER-BBN) to E.V. and I.A., and partially by ISCIII (PI15/00272 and PI1702242 co-founded by Fondo Europeo de Desarrollo Regional (FEDER), to E.V. and S.S., respectively), and Agencia Estatal de Investigacion (AEI) and FEDER (BIO2016-76063-R, AEI/FEDER, UE), AGAUR (2017SGR-229) and CIBER-BBN (VENOM4CANCER) granted to A.V. Protein production and DLS have been partially performed by the ICTS "NANBIOSIS," more specifically by the Protein Production Platform of CIBER-BBN/IBB () and the Biomaterial Processing and Nanostructuring Unit (), respectively. Biodistribution and immunohistochemistry assays were performed at the ICTS "NANBIOSIS," specifically by U20/FVPR (). L.S.-G. was supported by predoctoral fellowship from AGAUR (2018FI_B2_00051). L.S. was supported by the European Research Council (CoG #617473) and the Instituto de Salud Carlos III (FIS #PI16/01224). J.S.-F. was supported by an AECC post-doctoral fellowship. A.V. received an ICREA ACADEMIA awar

Release of targeted protein nanoparticles from functional bacterial amyloids : A death star-like approach

Altres ajuts: we are indebted to CIBER de Bioingeniería, Biomateriales y Nanomedicina (projects NANOREMOTE and VENOM4CANCER) to EV and AV respectively, Marató de TV3 foundation (TV32013-132031) and CIBER (NanoMets3) to RM. Protein production has been partially performed by the ICTS "NANBIOSIS", more specifically by the Protein Production Platform of CIBER in Bioengineering, Biomaterials & Nanomedicine (CIBER-BBN)/IBB, at the UAB SepBioES scientific-technical service (http://www.nanbiosis.es/unit/u1-protein-production-platform-ppp/), whereas the in vivo biodistribution assays were performed in the NANBIOSIS Nanotoxicology platform (http://www.nanbiosis.es/unit/u18-nanotoxicology-unit/). We are also indebted to Fran Cortes from the Cell Culture and Cytometry Units of the Servei de Cultius Cel·lulars, Producció d'Anticossos i Citometria (SCAC), and to the Servei de Microscòpia at the UAB. AV received an ICREA ACADEMIA award. U.U received a Sara Borrell postdoctoral fellowship from ISCIII, MVC was supported by Miguel Servet contract from ISCIII, and JSF received and AECC postdoctoral fellowship.Sustained release of drug delivery systems (DDS) has the capacity to increase cancer treatment efficiency in terms of drug dosage reduction and subsequent decrease of deleterious side effects. In this regard, many biomaterials are being investigated but none offers morphometric and functional plasticity and versatility comparable to protein-based nanoparticles (pNPs). Here we describe a new DDS by which pNPs are fabricated as bacterial inclusion bodies (IB), that can be easily isolated, subcutaneously injected and used as reservoirs for the sustained release of targeted pNPs. Our approach combines the high performance of pNP, regarding specific cell targeting and biodistribution with the IB supramolecular organization, stability and cost effectiveness. This renders a platform able to provide a sustained source of CXCR4-targeted pNPs that selectively accumulate in tumor cells in a CXCR4 colorectal cancer xenograft model. In addition, the proposed system could be potentially adapted to any other protein construct offering a plethora of possible new therapeutic applications in nanomedicine

Targeting low-density lipoprotein receptors with protein-only nanoparticles

We are grateful to the Protein Production Platform (CIBER-BBN-UAB) for helpful technical assistance and for protein production and purification services (http://​www.​ciber-bbn.​es/​en/​programas/​89-plataforma-de-produccion-de-proteinas-ppp). We are indebted to FIS PI12/01861, Marató 416/C/2013-2030 and NanoMets to RM, MINECO BIO2013-41019-P to AV, AGAUR (2014SGR-132), and CIBER de Bioingeniería, Biomateriales y Nanomedicina (project NANOPROTHER) for funding our research on protein-based therapeutics. We thank the CIBER-BBN Nanotoxicology Unit for fluorescent in vivo follow-up using the IVIS equipment. We are also indebted to the Cell Culture and Citometry Units of the Servei de Cultius Cel·lulars, Producció d'Anticossos i Citometria (SCAC), and to the Servei de Microscòpia, both at the UAB, and to the Soft Materials Service (ICMAB-CSIC/CIBER-BBN). CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. Z. X. and M. P. acknowledge financial support from China Scholarship Council and Universitat Autònoma de Barcelona through pre-doctoral fellowships, respectively. AV received an ICREA ACADEMIA award.Low-density lipoprotein receptors (LDLR) are appealing cell surface targets in drug delivery, as they are expressed in the blood-brain barrier (BBB) endothelium and are able to mediate transcytosis of functionalized drugs for molecular therapies of the central nervous system (CNS). On the other hand, brain-targeted drug delivery is currently limited, among others, by the poor availability of biocompatible vehicles, as most of the nanoparticles under development as drug carriers pose severe toxicity issues. In this context, protein nanoparticles offer functional versatility, easy and cost-effective bioproduction, and full biocompatibility. In this study, we have designed and characterized several chimerical proteins containing different LDLR ligands, regarding their ability to bind and internalize target cells and to self-organize as viral mimetic nanoparticles of about 18 nm in diameter. While the self-assembling of LDLR-binding proteins as nanoparticles positively influences cell penetration in vitro, the nanoparticulate architecture might be not favoring BBB crossing in vivo. These findings are discussed in the context of the use of nanostructured materials as vehicles for the systemic treatment of CNS diseases

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

Rational engineering of single-chain polypeptides into protein-only, BBB-targeted nanoparticles

A single chain polypeptide containing the low density lipoprotein receptor (LDLR) ligand Seq-1 with blood-brain barrier (BBB) crossing activity has been successfully modified by conventional genetic engineering to self-assemble into stable protein-only nanoparticles of 30 nm. The nanoparticulate presentation dramatically enhances in vitro, LDLR-dependent cell penetrability compared to the parental monomeric version, but the assembled protein does not show any enhanced brain targeting upon systemic administration. While the presentation of protein drugs in form of nanoparticles is in general advantageous regarding correct biodistribution, this principle might not apply to brain targeting that is hampered by particular bio-physical barriers. Irrespective of this fact, which is highly relevant to the nanomedicine of central nervous system, engineering the cationic character of defined protein stretches is revealed here as a promising and generic approach to promote the controlled oligomerization of biologically active protein species as still functional, regular nanoparticles

Structural and functional features of self-assembling protein nanoparticles produced in endotoxin-free Escherichia coli

Background: production of recombinant drugs in process-friendly endotoxin-free bacterial factories targets to a lessened complexity of the purification process combined with minimized biological hazards during product application. The development of nanostructured recombinant materials in innovative nanomedical activities expands such a need beyond plain functional polypeptides to complex protein assemblies. While Escherichia coli has been recently modified for the production of endotoxin-free proteins, no data has been so far recorded regarding how the system performs in the fabrication of smart nanostructured materials. - Results: we have here explored the nanoarchitecture and in vitro and in vivo functionalities of CXCR4-targeted, self-assembling protein nanoparticles intended for intracellular delivery of drugs and imaging agents in colorectal cancer. Interestingly, endotoxin-free materials exhibit a distinguishable architecture and altered size and target cell penetrability than counterparts produced in conventional E. coli strains. These variant nanoparticles show an eventual proper biodistribution and highly specific and exclusive accumulation in tumor upon administration in colorectal cancer mice models, indicating a convenient display and function of the tumor homing peptides and high particle stability under physiological conditions. - Discussion: the observations made here support the emerging endotoxin-free E. coli system as a robust protein material producer but are also indicative of a particular conformational status and organization of either building blocks or oligomers. This appears to be promoted by multifactorial stress-inducing conditions upon engineering of the E. coli cell envelope, which impacts on the protein quality control of the cell factory