Ubiquitin-Modulated Phase Separation of Shuttle Proteins: Does Condensate Formation Promote Protein Degradation?

Liquid-liquid phase separation (LLPS) has recently emerged as a possible mechanism that enables ubiquitin-binding shuttle proteins to facilitate the degradation of ubiquitinated substrates via distinct protein quality control (PQC) pathways. Shuttle protein LLPS is modulated by multivalent interactions among their various domains as well as heterotypic interactions with polyubiquitin chains. Here, the properties of three different shuttle proteins (hHR23B, p62, and UBQLN2) are closely examined, unifying principles for the molecular determinants of their LLPS are identified, and how LLPS is connected to their functions is discussed. Evidence supporting LLPS of other shuttle proteins is also found. In this review, it is proposed that shuttle protein LLPS leads to spatiotemporal regulation of PQC activities by mediating the recruitment of PQC machinery (including proteasomes or autophagic components) to biomolecular condensates, assembly/disassembly of condensates, selective enrichment of client proteins, and extraction of ubiquitinated proteins from condensates in cells.     

A User’s Guide for Phase Separation Assays with Purified Proteins

The formation of membrane-less organelles and compartments by protein phase separation is an important way in which cells organize their cytoplasm and nucleoplasm. In vitro phase separation assays with purified proteins have become the standard way to investigate proteins that form membrane-less compartments. By now, various proteins have been purified and tested for their ability to phase separate and form liquid condensates in vitro. However, phase-separating proteins are often aggregation-prone and difficult to purify and handle. As a consequence, the results from phase separation assays often differ between labs and are not easily reproduced. Thus, there is an urgent need for high-quality proteins, standardized procedures, and generally agreed-upon practices for protein purification and conducting phase separation assays. This paper provides protocols for protein purification and guides the user through the practicalities of in vitro protein phase separation assays, including best-practice approaches and pitfalls to avoid. We believe that this compendium of protocols and practices will provide a useful resource for scientists studying the phase behavior of proteins.     

Understanding the Tendency of Amorphous Solid Dispersions to Undergo Amorphous–Amorphous Phase Separation in the Presence of Absorbed Moisture

Formulation of an amorphous solid dispersion (ASD) is one of the methods commonly considered to increase the bioavailability of a poorly water-soluble small-molecule active pharmaceutical ingredient (API). However, many factors have to be considered in designing an API–polymer system, including any potential changes to the physical stability of the API. In this study, the tendency of ASD systems containing a poorly water-soluble API and a polymer to undergo amorphous–amorphous phase separation was evaluated following exposure to moisture at increasing relative humidity. Infrared spectroscopy was used as the primary method to investigate the phase behavior of the systems. In general, it was observed that stronger drug–polymer interactions, low-ASD hygroscopicity, and a less hydrophobic API led to the formation of systems resistant to moisture-induced amorphous–amorphous phase separation. Orthogonal partial least squares analysis provided further insight into the systems, confirming the importance of the aforementioned properties. In order to design a more physically stable ASD that is resistant to moisture-induced amorphous–amorphous phase separation, it is important to consider the interplay between these properties.     

Purification and Characterization of a Cold-Adapted α-Amylase Produced by Nocardiopsis sp. 7326 Isolated from Prydz Bay, Antarctic

An actinomycete strain 7326 producing cold-adapted α-amylase was isolated from the deep sea sediment of Prydz Bay, Antarctic. It was identified as Nocardiopsis based on morphology, 16S rRNA gene sequence analysis, and physiological and biochemical characteristics. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymogram activity staining of purified amylase showed a single band equal to a molecular mass of about 55 kDa. The optimal activity temperature of Nocardiopsis sp. 7326 amylase was 35°C, and the activity decreased dramatically at temperatures above 45°C. The enzyme was stable between pH 5 and 10, and exhibited a maximal activity at pH 8.0. Ca²⁺, Mn²⁺, Mg²⁺, Cu²⁺, and Co²⁺ stimulated the activity of the enzyme significantly, and Rb²⁺, Hg²⁺, and EDTA inhibited the activity. The hydrolysates of soluble starch by the enzyme were mainly glucose, maltose, and maltotriose. This is the first report on the isolation and characterization of cold-adapted amylase from Nocardiopsis sp.     

Separation of adenosine diphosphate-adenosine triphosphate–exchange activity from the cerebral microsomal sodium-plus-potassium ion-stimulated adenosine triphosphatase

1. A microsomal fraction from ox cerebral cortex catalysed [(14)C]ADP-ATP exchange at a speed similar to that at which it liberated P(i) from ATP in the presence of Na(+), K(+) and Mg(2+). 2. Repeated washing the fraction with MgATP solutions solubilized most of the exchange activity and left the adenosine triphosphatase insoluble and little changed in activity. The exchange activity was accompanied by negligible adenosine-triphosphatase activity and was enriched by precipitation at chosen pH and by DEAE-Sephadex. At no stage was its activity affected by Na(+), K(+) or ouabain. 3. The washed microsomal fraction was exposed to a variety of reagents; a sodium iodide-cysteine treatment increased both adenosine-triphosphatase and exchange activities, as also did a synthetic zeolite. Preparations were obtained with exchange activities less than 3% of their Na(+)-plus-K(+)-stimulated adenosine-triphosphatase activity. Some contribution to the residual exchange activity was made by an adenylate kinase. 4. Thus over 95% of the microsomal ADP-ATP-exchange activity does not take part in the Na(+)-plus-K(+)-stimulated adenosine-triphosphatase reaction. Participation of some of the residual 3% of the ADP-ATP-exchange activity has not been excluded, but there appears no firm evidence for its participation in the adenosine triphosphatase; the bearing of this conclusion on mechanisms proposed for the Na(+)-plus-K(+)-stimulated adenosine triphosphatase is indicated.     

Purification of Xylitol from Fermented Hemicellulosic Hydrolyzate Using Liquid–Liquid Extraction and Precipitation Techniques

Xylitol was produced by Candida guilliermondii by fermentation of sugarcane bagasse hemicellulosic hydrolysate. Undesirable impurities were extracted from the broth using either ethyl acetate, chloroform or dichloromethane. The best results on clarification of the broth without xylitol loss were obtained with ethyl acetate. When ethanol, acetone or tetrahydrofuran were used for precipitation of impurities, only tetrahydrofuran clarified the fermented broth, but a high xylitol loss (~30%) was observed.     

Rieske Iron–Sulfur Proteins from Extremophilic Organisms

Proteins located on the outside of the membranes of organisms thriving under extreme conditions like high or low pH, or high salinity face special challenges maintaining their structural integrity. This review is focused on the Rieske iron-sulfur proteins from these organisms. Rieske proteins are essential subunits of the cytochrome bc-complexes, which are often of crucial importance for the energy metabolism of the cells. On the basis of the available data we propose strategies by which these proteins are able to stabilize their noncovalent bound cofactor and adapt to the function under extreme conditions.     

Seasonal Rhythms of “Acute Phase Proteins” in Humans

“Acute phase proteins” comprise a group of proteins whose concentrations increase or decrease by at least 25% after a damaging stimulus (burn, trauma, tissue damage, etc.) or during inflammation. We investigated the seasonal variation in the concentrations of several acute phase proteins—α1‐antichymotrypsin (ACT), α1‐acid glycoprotein (AGP), transferrin (Tf), α2‐macroglobulin (α2‐M), ceruloplasmin (Cp), antitrypsin (AT), and haptoglobin (Hp). Blood samples were collected from 15 healthy volunteers, who were subjected to the seasonal changes in illumination, were drawn at 08:00 h every 3 months (August, November, January/February, March/April, June/July). With the exception of Hp, all acute phase proteins showed an annual rhythm (ANOVA; p<0.01). Lowest concentrations occurred in the winter months (November through February), with the exception of Tf, which was oppositely phased.     

Structural thermal adaptation of β-tubulins from the Antarctic psychrophilic protozoan Euplotes focardii

Tubulin dimers of psychrophilic eukaryotes can polymerize into microtubules at 4°C, a temperature at which microtubules from mesophiles disassemble. This unique capability requires changes in the primary structure and/or in post-translational modifications of the tubulin subunits. To contribute to the understanding of mechanisms responsible for microtubule cold stability, here we present a computational structural analysis based on molecular dynamics (MD) and experimental data of three β-tubulin isotypes, named EFBT2, EFBT3, and EFBT4, from the Antarctic protozoon Euplotes focardii that optimal temperature for growth and reproduction is 4°C. In comparison to the β-tubulin from E. crassus, a mesophilic Euplotes species, EFBT2, EFBT3, and EFBT4 possess unique amino acid substitutions that confer different flexible properties of the polypeptide, as well as an increased hydrophobicity of the regions involved in microtubule interdimeric contacts that may overcome the microtubule destabilizing effect of cold temperatures. The structural analysis based on MD indicated that all isotypes display different flexibility properties in the regions involved in the formation of longitudinal and lateral contacts during microtubule polymerization. We also investigated the role of E. focardii β-tubulin isotypes during the process of cilia formation. The unique characteristics of the primary and tertiary structures of psychrophilic β-tubulin isotypes seem responsible for the formation of microtubules with distinct dynamic and functional properties.     

Separation and properties of α-mannosidase and mannanase from the basidiomycetePhellinus abietis

Proteins of a crude enzyme preparation obtained from the cultivation medium of the basidiomycetePhellinus abietis were separated by gel filtration and ion-exchange chromatography. The preparation contained a minimum of three enzymes capable of splitting α-d-mannosidic bonds: α-mannosidase, exomannanase, and endomannanase, which were separated. Some properties of the mannanase complex of the crude enzyme preparation, and of a partially purified α-mannosidase were examined. The mannanase complex exhibited two pH optima, its temperature optimum being at 46 °C The pH optimum of purified α-mannosidase was at pH 5.0, the temperature optimum was at 60 °C; the enzyme had a relatively high heat stability. The K_m of α-mannosidase forp-nitrophenyl α-d-mannopyranoside was 1.5 x 10⁻⁵ M. Pure α-mannosidase did not split mannan.     

Liquid–vapour interface varying the softness and range of the interaction potential

Molecular dynamics simulations in a canonical ensemble were carried out for simple fluids. The inter-particles interaction law is described by the Morse function plus a repulsive term. This kind of combination allows to tune the repulsive term of the interaction function by fitting the range of the attractive well and vice versa. As a relevant result, we show that for an inhomogeneous system the particle softness affects the vapour pressure, the surface tension and also the equilibrium densities of a simple fluid. Lower numerical values for these same properties were obtained by using a more repulsive interaction potential. The differences among these same interfacial properties are bigger when the range of the attractive interaction is longer. The surface tension written in terms of the corresponding critical parameters, such as scaled surface tension, was plotted for different softness degrees. And from this comparison, a unique master curve was not found.     

Subunit Composition of the Zinc Proteins α- and β-Lipovitellin from Chicken

Chicken α- and β-lipovitellin are derived from parent vitellogenin proteins and contain four subunits (125, 80, 40, and 30 kDa) and two subunits (125 and 30 kDa), respectively. Metal analyses demonstrate both are zinc proteins containing 2.1 ± 0.2 mol of zinc/275 kDa per α-lipovitellin and 1.4 ± 0.2 mol of zinc/155 kDa per β-lipovitellin, respectively. The subunits of β-lipovitellin, Lv 1 (MW 125 kDa) and Lv 2 (MW 30 kDa), are separated by gel exclusion chromatography in the presence of zwittergent 3–16. Zinc elutes with Lv 1, suggesting that this subunit binds zinc in the absence of Lv 2. The subunits of α- and β-lipovitellin were separated by SDS-PAGE, digested with trypsin, and mapped by reverse-phase HPLC. The peptide maps of the 125-kDa subunits from α- and β-lipovitellin are essentially identical. Similar results are obtained for the 30-kDa subunits of both lipovitellins. The sequences of five and four peptides of the 125-kDa subunit of α- and β-Lv, respectively, and two peptides of the 30-kDa subunit of α- and β-lipovitellin were determined and match those predicted from the gene for vitellogenin II, Vtg II. Comparison of the amino acid composition of the 125- and 30-kDa subunits of α- and β-lipovitellin support the conclusion that they originate from the same gene. The sequences of peptides from the 80- and 40-kDa subunits of α-lipovitellin have not been found in the NCBI nonredundant data bank. The 27-amino acid N-terminal sequence of the 40-kDa protein is 56% similar to the last third of the Lv 1-coding region of the Vtg II gene, suggesting it may come from an analogous region of the Vtg I gene. We propose a scheme for the precursor—product relationship of Vtg I.     

Separation and purification of α-ketoglutarate and pyruvate from the fermentation broth of Yarrowia lipolytica

Bioprocess and Biosystems Engineering - A strategy to achieve the efficient co-production of α-ketoglutarate (KGA) and pyruvate (PYR) via Yarrowia lipolytica fermentation was established in...     

Development of Dispersive Liquid–Liquid Microextraction Based on Solidification of Floating Organic Drop for the Determination of Trace Nickel

A liquid-phase microextraction technique was developed using dispersive liquid-liquid microextraction based on solidification of floating organic drop combined with flame atomic absorption spectrometry, for the extraction and determination of trace amounts of nickel in water samples. Microextraction efficiency factors, such as the type and volume of extraction and dispersive solvents, pH, extraction time, the chelating agent amount, and ionic strength, were investigated and optimized. Under optimum conditions, the calibration graph was linear in the range of 4.23-250?μg?L(-1) with a detection limit of 1.27?μg?L(-1). The relative standard deviation for ten replicate measurements of 10 and 100?μg?L(-1) of nickel were 3.21% and 2.55%, respectively. The proposed method was assessed through the analysis of certified reference water or recovery experiments.