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

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

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

Conformational and functional variants of CD44-targeted protein nanoparticles bio-produced in bacteria

Biofabrication is attracting interest as a means to produce nanostructured functional materials because of its operational versatility and full scalability. Materials based on proteins are especially appealing, as the structure and functionality of proteins can be adapted by genetic engineering. Furthermore, strategies and tools for protein production have been developed and refined steadily for more than 30 years. However, protein conformation and therefore activity might be sensitive to production conditions. Here, we have explored whether the downstream strategy influences the structure and biological activities, in vitro and in vivo, of a self-assembling, CD44-targeted protein-only nanoparticle produced in Escherichia coli. This has been performed through the comparative analysis of particles built from soluble protein species or protein versions obtained by in vitro protein extraction from inclusion bodies, through mild, non-denaturing procedures. These methods have been developed recently as a convenient alternative to the use of toxic chaotropic agents for protein resolubilization from protein aggregates. The results indicate that the resulting material shows substantial differences in its physicochemical properties and its biological performance at the systems level, and that its building blocks are sensitive to the particular protein source

Intrinsic functional and architectonic heterogeneity of tumor-targeted protein nanoparticles

Altres ajuts: CIBER de Bioingeniería, Biomateriales y Nanomedicina (project NANOPROTHER) (to AV), Marató de TV3 foundation (TV32013-132031) (TV32013-133930). 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/) and DLS measurements have been done at the Biomaterial Processing and Nanostructuring Unit of NANBIOSIS. We are also indebted to Fran Cortés from the Cell Culture and Cytometry Units of the Servei de CultiusCel·lulars, Producciód'AnticossosiCitometria (SCAC), and to the Servei de Microscòpia, both at the UAB. Strain KPM335 was kindly provided by Research Corporation Technologies, Tucson, AZ. AV received an ICREA ACADEMIA award.Self-assembling proteins are gaining attention as building blocks for application-tailored nanoscale materials. This is mostly due to the biocompatibility, biodegradability, and functional versatility of peptide chains. Such a potential for adaptability is particularly high in the case of recombinant proteins, which are produced in living cells and are suitable for genetic engineering. However, how the cell factory itself and the particular protein folding machinery influence the architecture and function of the final material is still poorly explored. In this study we have used diverse analytical approaches, including small-angle X-ray scattering (SAXS) and field emission scanning electron microscopy (FESEM) to determine the fine architecture and geometry of recombinant, tumor-targeted protein nanoparticles of interest as drug carriers, constructed on a GFP-based modular scheme. A set of related oligomers were produced in alternative Escherichia coli strains with variant protein folding networks. This resulted in highly regular populations of morphometric types, ranging from 2.4 to 28 nm and from spherical- to rod-shaped materials. These differential geometric species, whose relative proportions were determined by the features of the producing strain, were found associated with particular fluorescence emission, cell penetrability and receptor specificity profiles. Then, nanoparticles with optimal properties could be analytically identified and further isolated from producing cells for use. The cell's protein folding machinery greatly modulates the final geometry reached by the constructs, which in turn defines the key parameters and biological performance of the material

Intracellular targeting of CD44+ cells with self-assembling, protein only nanoparticles

CD44 is a multifunctional cell surface protein involved in proliferation and differentiation, angiogenesis and signaling. The expression of CD44 is up-regulated in several types of human tumors and particularly in cancer stem cells, representing an appealing target for drug delivery in the treatment of cancer. We have explored here several protein ligands of CD44 for the construction of self-assembling modular proteins designed to bind and internalize target cells. Among five tested ligands, two of them (A5G27 and FNI/II/V) drive the formation of protein-only, ring-shaped nanoparticles of about 14 nm that efficiently bind and penetrate CD44(+) cells by an endosomal route. The potential of these newly designed nanoparticles is evaluated regarding the need of biocompatible nanostructured materials for drug delivery in CD44-linked conditions