Carolina Allende-Ballestero

Bio: Carolina Allende-Ballestero is an academic researcher from Spanish National Research Council. The author has contributed to research in topics: Capsid & RNA silencing. The author has an hindex of 2, co-authored 4 publications receiving 56 citations.

Structural Characterization of Native and Modified Encapsulins as Nanoplatforms for in Vitro Catalysis and Cellular Uptake

Abstract: Recent years have witnessed the emergence of bacterial semiorganelle encapsulins as promising platforms for bio-nanotechnology. To advance the development of encapsulins as nanoplatforms, a functional and structural basis of these assemblies is required. Encapsulin from Brevibacterium linens is known to be a protein-based vessel for an enzyme cargo in its cavity, which could be replaced with a foreign cargo, resulting in a modified encapsulin. Here, we characterize the native structure of B. linens encapsulins with both native and foreign cargo using cryo-electron microscopy (cryo-EM). Furthermore, by harnessing the confined enzyme (i.e., a peroxidase), we demonstrate the functionality of the encapsulin for an in vitro surface-immobilized catalysis in a cascade pathway with an additional enzyme, glucose oxidase. We also demonstrate the in vivo functionality of the encapsulin for cellular uptake using mammalian macrophages. Unraveling both the structure and functionality of the encapsulins allows transforming biological nanocompartments into functional systems.

The RNA-Binding Protein of a Double-Stranded RNA Virus Acts like a Scaffold Protein

Abstract: Infectious bursal disease virus (IBDV), a nonenveloped, double-stranded RNA (dsRNA) virus with a T=13 icosahedral capsid, has a virion assembly strategy that initiates with a precursor particle based on an internal scaffold shell similar to that of tailed double-stranded DNA (dsDNA) viruses In IBDV-infected cells, the assembly pathway results mainly in mature virions that package four dsRNA segments, although minor viral populations ranging from zero to three dsRNA segments also form We used cryo-electron microscopy (cryo-EM), cryo-electron tomography, and atomic force microscopy to characterize these IBDV populations The VP3 protein was found to act as a scaffold protein by building an irregular, ∼40-A-thick internal shell without icosahedral symmetry, which facilitates formation of a precursor particle, the procapsid Analysis of IBDV procapsid mechanical properties indicated a VP3 layer beneath the icosahedral shell, which increased the effective capsid thickness Whereas scaffolding proteins are discharged in tailed dsDNA viruses, VP3 is a multifunctional protein In mature virions, VP3 is bound to the dsRNA genome, which is organized as ribonucleoprotein complexes IBDV is an amalgam of dsRNA viral ancestors and traits from dsDNA and single-stranded RNA (ssRNA) virusesIMPORTANCE Structural analyses highlight the constraint of virus evolution to a limited number of capsid protein folds and assembly strategies that result in a functional virion We report the cryo-EM and cryo-electron tomography structures and the results of atomic force microscopy studies of the infectious bursal disease virus (IBDV), a double-stranded RNA virus with an icosahedral capsid We found evidence of a new inner shell that might act as an internal scaffold during IBDV assembly The use of an internal scaffold is reminiscent of tailed dsDNA viruses, which constitute the most successful self-replicating system on Earth The IBDV scaffold protein is multifunctional and, after capsid maturation, is genome bound to form ribonucleoprotein complexes IBDV encompasses numerous functional and structural characteristics of RNA and DNA viruses; we suggest that IBDV is a modern descendant of ancestral viruses and comprises different features of current viral lineages

Nanotechnological Applications Based on Bacterial Encapsulins

Abstract: Encapsulins are proteinaceous nanocontainers, constructed by a single species of shell protein that self-assemble into 20-40 nm icosahedral particles. Encapsulins are structurally similar to the capsids of viruses of the HK97-like lineage, to which they are evolutionarily related. Nearly all these nanocontainers encase a single oligomeric protein that defines the physiological role of the complex, although a few encapsulate several activities within a single particle. Encapsulins are abundant in bacteria and archaea, in which they participate in regulation of oxidative stress, detoxification, and homeostasis of key chemical elements. These nanocontainers are physically robust, contain numerous pores that permit metabolite flux through the shell, and are very tolerant of genetic manipulation. There are natural mechanisms for efficient functionalization of the outer and inner shell surfaces, and for the in vivo and in vitro internalization of heterologous proteins. These characteristics render encapsulin an excellent platform for the development of biotechnological applications. Here we provide an overview of current knowledge of encapsulin systems, summarize the remarkable toolbox developed by researchers in this field, and discuss recent advances in the biomedical and bioengineering applications of encapsulins.

Bacterial Valorization of Lignin: Strains, Enzymes, Conversion Pathways, Biosensors, and Perspectives.

Abstract: Lignin, an aromatic polymer found in plants, has been studied for years in many biological fields. Initially, when biofuel was produced from lignocellulosic biomass, lignin was regarded as waste generated by the biorefinery and had to be removed, because of its inhibitory effects on fermentative bacteria. Although it has since proven to be a natural resource for bio-products with considerable potential, its utilization is confined by its complex structure. Hence, the microbial degradation of lignin has attracted researchers' interest to overcome this problem. From this perspective, the studies have primarily focused on fungal systems, such as extracellular peroxidase and laccase from white- and brown-rot fungi. However, recent reports have suggested that bacteria play an increasing role in breaking down lignin. This paper, therefore, reviews the role of bacteria in lignin and lignin-related research. Several reports on bacterial species in soil that can degrade lignin and their enzymes are included. In addition, a cellulolytic anaerobic bacterium capable of solubilizing lignin and carbohydrate simultaneously has recently been identified, even though the enzyme involved has not been discovered yet. The assimilation of lignin-derived small molecules and their conversion to renewable chemicals by bacteria, such as muconic acid and polyhydroxyalkanoates, including genetic modification to enhance their capability was discussed. This review also covers the indirect use of bacteria for lignin degradation, which is concerned with whole-cell biosensors designed to detect the aromatic chemicals released from lignin transformation.

Prokaryotic nanocompartments form synthetic organelles in a eukaryote.

Abstract: Compartmentalization of proteins into organelles is a promising strategy for enhancing the productivity of engineered eukaryotic organisms. However, approaches that co-opt endogenous organelles may be limited by the potential for unwanted crosstalk and disruption of native metabolic functions. Here, we present the construction of synthetic non-endogenous organelles in the eukaryotic yeast Saccharomyces cerevisiae, based on the prokaryotic family of self-assembling proteins known as encapsulins. We establish that encapsulins self-assemble to form nanoscale compartments in yeast, and that heterologous proteins can be selectively targeted for compartmentalization. Housing destabilized proteins within encapsulin compartments afford protection against proteolytic degradation in vivo, while the interaction between split protein components is enhanced upon co-localization within the compartment interior. Furthermore, encapsulin compartments can support enzymatic catalysis, with substrate turnover observed for an encapsulated yeast enzyme. Encapsulin compartments therefore represent a modular platform, orthogonal to existing organelles, for programming synthetic compartmentalization in eukaryotes.

Bioengineering Strategies for Protein-Based Nanoparticles.

Abstract: In recent years, the practical application of protein-based nanoparticles (PNPs) has expanded rapidly into areas like drug delivery, vaccine development, and biocatalysis. PNPs possess unique features that make them attractive as potential platforms for a variety of nanobiotechnological applications. They self-assemble from multiple protein subunits into hollow monodisperse structures; they are highly stable, biocompatible, and biodegradable; and their external components and encapsulation properties can be readily manipulated by chemical or genetic strategies. Moreover, their complex and perfect symmetry have motivated researchers to mimic their properties in order to create de novo protein assemblies. This review focuses on recent advances in the bioengineering and bioconjugation of PNPs and the implementation of synthetic biology concepts to exploit and enhance PNP's intrinsic properties and to impart them with novel functionalities.

Cryo-electron microscopy for the study of virus assembly.

Abstract: Although viruses are extremely diverse in shape and size, evolution has led to a limited number of viral classes or lineages, which is probably linked to the assembly constraints of a viable capsid. Viral assembly mechanisms are restricted to two general pathways, (i) co-assembly of capsid proteins and single-stranded nucleic acids and (ii) a sequential mechanism in which scaffolding-mediated capsid precursor assembly is followed by genome packaging. Cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET), which are revolutionizing structural biology, are central to determining the high-resolution structures of many viral assemblies as well as those of assembly intermediates. This wealth of cryo-EM data has also led to the development and redesign of virus-based platforms for biomedical and biotechnological applications. In this Review, we will discuss recent viral assembly analyses by cryo-EM and cryo-ET showing how natural assembly mechanisms are used to encapsulate heterologous cargos including chemicals, enzymes, and/or nucleic acids for a variety of nanotechnological applications.