Phase separation modulates the functional amyloid assembly of human CPEB3.

How functional amyloids are regulated to restrict their activity is poorly understood. The cytoplasmic polyadenylation element-binding protein 3 (CPEB3) is an RNA-binding protein that adopts an amyloid state key for memory persistence. Its monomer represses the translation of synaptic target mRNAs while phase separated, whereas its aggregated state acts as a translational activator. Here, we have explored the sequence-driven molecular determinants behind the functional aggregation of human CPEB3 (hCPEB3). We found that the intrinsically disordered region (IDR) of hCPEB3 encodes both an amyloidogenic and a phase separation domain, separated by a poly-A-rich region. The hCPEB3 amyloid core is composed by a hydrophobic region instead of the Q-rich stretch found in the Drosophila orthologue. The hCPEB3 phase separation domain relies on hydrophobic interactions with ionic strength dependence, and its droplet ageing process leads to a liquid-to-solid transition with the formation of a non-fibril-based hydrogel surrounded by starburst droplets. Furthermore, we demonstrate the differential behavior of the protein depending on its environment. Under physiological-like conditions, hCPEB3 can establish additional electrostatic interactions with ions, increasing the stability of its liquid droplets and driving a condensation-based amyloid pathway.

Structural traits and catalytic versatility of the lipases from the Candida rugosa-like family: A review.

Lipases and sterol esterases are enzymes with broad biotechnological applications, which catalyze the hydrolysis or synthesis of long-chain acylglycerols and sterol esters, respectively. In this paper, we review the current knowledge on the so-called Candida rugosa-like family of enzymes, whose members display in most cases affinity against the two substrates mentioned above. The family includes proteins with the α/ß-hydrolase folding, sharing conserved motifs in their sequences, and common structural features. We will go through their production and purification, relate their described structures and catalytic activity, and discuss the influence of the hydrophobic character of these lipases on their aggregation state and activity. On the basis of the few crystal structures available, the role of each of the functional areas in catalysis will be analyzed. Considering the particular characteristics of this group, we propose their classification as "Versatile Lipases" (EC 3.1.1.x).

Properties, structure, and applications of microbial sterol esterases.

According to their substrate preferences, carboxylic ester hydrolases are organized in smaller clusters. Among them, sterol esterases (EC 3.1.1.13), also known as cholesterol esterases, act on fatty acid esters of cholesterol and other sterols in aqueous media, and are also able to catalyze synthesis by esterification or transesterification in the presence of organic solvents. Mammalian cholesterol esterases are intracellular enzymes that have been extensively studied since they are essential in lipid metabolism and cholesterol absorption, and the natural role of some microbial sterol esterases is supposed to be similar. However, besides these intracellular enzymes, a number of microbes produce extracellular sterol esterases, which show broad stability, selectivity, or wide substrate specificity, making them interesting for the industry. In spite of this, there is little information about microbial sterol esterases, and only a small amount of them have been characterized. Some of the most commercially exploited cholesterol esterases are produced by Pseudomonas species and by Candida rugosa, although in the last case they are usually described and named as "high substrate versatility lipases." From a structural point of view, most of them belong to the α/ß-hydrolase superfamily and have a conserved "catalytic triad" formed by His, an acidic amino acid and a Ser residue that is located in a highly conserved GXSXG sequence. In this review, the information available on microbial sterol esterases has been gathered, taking into account their origin, production and purification, heterologous expression, structure, stability, or substrate specificity, which are the main properties that make them attractive for different applications. Moreover, a comprehensive phylogenetic analysis on available sequences of cholesterol esterases has been done, including putative sequences deduced from public genomes.

Expression and properties of three novel fungal lipases/sterol esterases predicted in silico: comparison with other enzymes of the Candida rugosa-like family.

Lipases from the Candida rugosa-like family are enzymes with great biotechnological interest. In a previous work, several enzymes from this family were identified by in silico mining of fungal genomes. Here, we describe the cloning, expression, and characterization of putative lipases from the genomes of Nectria haematococca, Trichoderma reesei, and Aspergillus niger and compared their catalytic properties with those of OPE, a well-characterized sterol esterase/lipase from Ophiostoma piceae. All of them hydrolyzed p-nitrophenol esters and triglycerides with different efficiency, but their activity against sterol esters was dissimilar, and the enzyme from A. niger was unable of hydrolyzing these substrates while OPE showed the best k cat values, which in general leads to an improved catalytic efficiency. Similarly, OPE was the best catalyst in the synthesis of ß-sitostanyl oleate, followed by the commercial CRL from C. rugosa, while the A. niger enzyme was unable to produce this compound. When the enzymes were evaluated for caprolactone oligomerization, the A. niger enzyme gave similar results than CRL, being OPE slightly more efficient. The expression of the putative selected proteins allowed their functional validation, suggesting that the hydrophobicity of the lid region may be an important factor, although the enzymatic efficiency is also influenced by other parameters, as the aggregation state and the size and morphology of the tunnel, where substrate recognition and catalysis takes place.

Heterologous expression of a fungal sterol esterase/lipase in different hosts: Effect on solubility, glycosylation and production.

Ophiostoma piceae secretes a versatile sterol-esterase (OPE) that shows high efficiency in both hydrolysis and synthesis of triglycerides and sterol esters. This enzyme produces aggregates in aqueous solutions, but the recombinant protein, expressed in Komagataella (synonym Pichia) pastoris, showed higher catalytic efficiency because of its higher solubility. This fact owes to a modification in the N-terminal sequence of the protein expressed in Pichia pastoris, which incorporated 4-8 additional amino acids, affecting its aggregation behavior. In this study we present a newly engineered P. pastoris strain with improved protein production. We also produced the recombinant protein in the yeast Saccharomyces cerevisiae and in the prokaryotic host Escherichia coli, corroborating that the presence of these N-terminal extra amino acids affected the protein's solubility. The OPE produced in the new P. pastoris strain presented the same physicochemical properties than the old one. An inactive form of the enzyme was produced by the bacterium, but the recombinant esterase from both yeasts was active even after its enzymatic deglycosylation, suggesting that the presence of N-linked carbohydrates in the mature protein is not essential for enzyme activity. Although the yield in S. cerevisiae was lower than that obtained in P. pastoris, this work demonstrates the importance of the choice of the heterologous host for successful production of soluble and active recombinant protein. In addition, S. cerevisiae constitutes a good engineering platform for improving the properties of this biocatalyst.

Crystal structures of Ophiostoma piceae sterol esterase: structural insights into activation mechanism and product release.

Sterol esterases are able to efficiently hydrolyze both sterol esters and triglycerides and to carry out synthesis reactions in the presence of organic solvents. Their high versatility makes them excellent candidates for biotechnological purposes. Sterol esterase from fungus Ophiostoma piceae (OPE) belongs to the family abH03.01 of the Candida rugosa lipase-like proteins. Crystal structures of OPE were solved in this study for the closed and open conformations. Enzyme activation involves a large displacement of the conserved lid, structural rearrangements of loop α16-α17, and formation of a dimer with a large opening. Three PEG molecules are placed in the active site, mimicking chains of the triglyceride substrate, demonstrating the position of the oxyanion hole and the three pockets that accommodate the sn-1, sn-2 and sn-3 fatty acids chains. One of them is an internal tunnel, connecting the active center with the outer surface of the enzyme 30 Å far from the catalytic Ser220. Based on our structural and biochemical results we propose a mechanism by which a great variety of different substrates can be hydrolyzed in OPE paving the way for the construction of new variants to improve the catalytic properties of these enzymes and their biotechnological applications.

Sugar recoveries from wheat straw following treatments with the fungus Irpex lacteus.

Irpex lacteus is a white-rot fungus capable of increasing sugar recovery from wheat straw; however, in order to incorporate biopretreatment in bioethanol production, some process specifications need to be optimized. With this objective, I. lacteus was grown on different liquid culture media for use as inoculums. Additionally, the effect of wheat straw particle size, moisture content, organic and inorganic supplementations, and mild alkali washing during solid-state fermentation (SSF) on sugar yield were investigated. Wheat thin stillage was the best medium for producing inoculums. Supplementation of wheat straw with 0.3mM Mn(II) during SSF resulted in glucose yields of 68% as compared to yields of 62% and 33% for cultures grown without supplementation or on untreated raw material, respectively after 21 days. Lignin loss, wheat straw digestibility, peroxidase activity, and fungal biomass were also correlated with sugar yields in the search for biopretreatment efficiency indicators.