分子生物学

Sterol regulatory element binding proteins (SREBPs) are transcription factors that reside in the endoplasmic reticulum (ER) membrane as inactive precursors. To be active, SREBPs are translocated to the Golgi where the transcriptionally active N-terminus is cleaved and released to the nucleus to regulate gene expression. Nuclear SREBP levels can be determined by immunoblot analysis; however, this method can only determine the steady-state levels of nuclear SREBPs and does not capture the actual status of activation. The vesicle budding assay provides an alternative way to quantify the activation of SREBPs by monitoring the initiation of SREBP translocation from the ER to the Golgi through vesicles. Microsomal membranes isolated from the liver are incubated in a reaction buffer containing the necessary components to facilitate vesicle formation. Microsomal membranes and vesicles are isolated and SREBPs are quantified in each by immunoblot analysis. The amount of SREBPs found in the budded vesicles provides an assessment of the SREBP activation in the liver.

Two aconitase isoforms are present in mammalian cells: the mitochondrial aconitase (ACO2) that catalyzes the reversible isomerization of citrate to isocitrate in the citric acid cycle, and the bifunctional cytosolic enzyme (ACO1), which also plays a role as an RNA-binding protein in the regulation of intracellular iron metabolism. Aconitase activities in the different subcellular compartments can be selectively inactivated by different genetic defects, iron depletion, and oxidative or nitrative stress. Aconitase contains a [4Fe-4S]²⁺ cluster that is essential for substrate coordination and catalysis. Many Fe-S clusters are sensitive to oxidative stress, nitrative stress, and reduced iron availability, which forms the basis of redox- and iron-mediated regulation of intermediary metabolism via aconitase and other Fe-S cluster-containing metabolic enzymes, such as succinate dehydrogenase. As such, ACO1 and ACO2 activities can serve as compartment-specific surrogate markers of oxygen levels, reactive oxygen species (ROS), reactive nitrogen species (RNS), iron bioavailability, and the status of intermediary and iron metabolism. Here, we provide a protocol describing a non-denaturing polyacrylamide gel electrophoresis (PAGE)-based procedure that has been successfully used to monitor ACO1 and ACO2 aconitase activities simultaneously in human and mouse cells and tissues.

Eukaryotic cells have different types of proteasomes that differ in size. The smallest proteolytically active particle is the 20S proteasome, which degrades damaged and oxidized proteins; the most common larger particle is the 26S proteasome, which degrades ubiquitylated proteins. The 26S proteasome is formed by a 20S particle capped with one or two regulatory particles, named 19S. While proteasome particles function in the cytoplasm, endoplasmic reticulum, and nucleus, our understanding of their abundance and activity in different cellular compartments is still limited. We provide a three-step protocol that first involves detergent-based fractionation of the cytoplasmic and nuclear compartments, maintaining the integrity and activity of proteasome complexes. Second, the protocol employs native gel separation of large multiprotein complexes in the fractions and a fluorescence-based in-gel quantitation of the activity and different proteasome particles. Finally, the protocol involves protein in-gel denaturation and transfer to a PVDF membrane. Western blotting then detects and quantifies the different proteasome particles. Therefore, the protocol allows for sensitive measurements of activity and abundance of individual proteasome particles from different cellular compartments. It has been optimized for motor neurons induced from mouse embryonic stem cells but can be applied to a variety of mammalian cell lines.

Key features

• Protocol for fractionation of active nuclear and cytoplasmic proteasome complexes.

• Native electrophoresis and fluorescence-based in-gel activity assay, which allows the visualization and quantification of active complexes within the acrylamide gel matrix.

• In-gel protein denaturation followed by transfer of complexes to PVDF membrane, which allows the analysis of complexes’ abundance using antibodies.

Graphical overview

Membrane transporters and soluble binding proteins recognize particular nutrients, metabolites, vitamins, or ligands. By modifying genetically engineered single cysteine residues near the active sites of such proteins with extrinsic maleimide fluorophores, the approaches that we report create sensitive fluorescent sensors that detect, quantify, and monitor molecules that are relevant to the biochemistry, physiology, microbiology, and clinical properties of pro- and eukaryotic organisms.

Graphical abstract:

When performing renal biopsy, it is necessary to identify the cortex, where glomeruli are exclusively distributed, to ensure the quality of the specimen for histological diagnosis. However, conventional methods using a stereomicroscope or magnifying lens often fail to clarify the quality of the specimen. We have established a fluorescent-based imaging technique for the on-site assessment of renal biopsy specimens. The fluorescent images can be easily obtained by adding an optical filter to the microscope and with a short incubation of an activatable fluorescent probe. This novel imaging technique can be applied to renal biopsy specimens for distinguishing the renal cortex.

The complement system is a central component of innate immunity, responsible for recognition and killing of bacteria by tagging invaders through opsonisation, thereby promoting phagocytosis, and by direct lysis. Complement activity is routinely measured using functional assays that utilise erythrocytes as targets. The classical pathway haemolytic assay (CH50) with antibody sensitised sheep erythrocytes as target is used worldwide in clinical and research laboratories to measure complement activity in human and rodent sera. While there are no particular limitations in the human assay, measuring complement in mouse serum is more difficult and usually requires large amounts of serum, which is challenging to collect in experiments. In particular, it is challenging to measure the activities of individual mouse complement proteins. To overcome this hurdle, we have developed protocols that employ human sera depleted of single complement proteins as the source of the other complement proteins and test mouse serum to restore the relevant component. This simple haemolytic assay is a useful tool for confirming natural or engineered complement deficiencies and complement dysregulation in mouse models.

Inteins garner significant interest from both basic and applied researchers due to their unique catalytic abilities. Herein, we describe a protocol for accurately monitoring protein splicing without purification using in-gel fluorescence immediately following Tris-Glycine SDS-PAGE. Following expression in Escherichia coli, cells are lysed by sonication, cell supernatants are separated using Tris-Glycine SDS-PAGE, and superfolder GFP (sfGFP) fluorescence is directly visualized within gels. This method is rapid, with sfGFP immediately imaged following SDS-PAGE without staining. Further, sfGFP can be specifically detected in complex samples such as E. coli cell supernatants, proteins run at expected masses, and all steps can be performed at ambient temperature. This strategy is broadly applicable beyond the study of protein splicing and can be used for sensitive and specific visualization of superfolder sfGFP-tagged proteins in-gel.

It is important to experimentally determine how membrane proteins are integrated into biomembranes to unveil the roles of the integration factors, and to understand the functions and structures of membrane proteins. We have developed a reconstitution system for membrane protein integration in E. coli using purified factors, in which the integration reaction in vivo is highly reproducible. This system enabled not only analysis of membrane-embedded factors including glycolipid MPIase, but also elucidation of the detailed mechanisms underlying membrane protein integration. Using the system, the integration of membrane proteins can be evaluated in vitro through a protease-protection assay. We report here how to prepare (proteo)liposomes and to determine the activities of membrane protein integration.

This protocol describes a simple xanthine/xanthine oxidase enzymatic equilibration method for determination of the redox potential of a flavin. As an example of the use of this method, we determine the reduction potential of the covalently bound FAD cofactor (E_m = -55 mV) in the SdhA flavoprotein subunit of succinate dehydrogenase from Escherichia coli. In principle, this method can be used routinely to determine the redox potential of flavin cofactors in any simple flavoprotein from equilibrium concentrations with an appropriate reference dye of known E_m without the use of sophisticated electrochemical equipment.

Heme oxygenase-1 (HO-1) is a stress responsive enzyme that metabolizes heme and releases free iron, carbon monoxide (CO), and biliverdin (BV), which rapidly undergoes conversion to bilirubin (BL). Estimation of bilirubin is the basis of HO-1 assay. HO-1 activity is widely employed to determine antioxidant response of cells under different physiological stress environment. Intra-macrophage infection often acts as such a stress inducer and measurement of HO-1 activity in infected cells indicates the ability of pathogens towards modulating oxidative response of host. The present protocol describes analysis of HO-1 activity in infected macrophages by spectrophotometric method, which is much less complex and therefore advantageous over other methods like high-performance liquid chromatography, radiochemical methods and detection of CO by gas chromatography. The main steps include: (1) Preparation of macrophage microsomal fraction containing HO-1 (2) Isolation of rat liver cytosolic fraction containing biliverdin reductase and (3) Assessment of heme oxygenase-1 activity by spectrophotometric detection of bilirubin. This method provides a simple and sensitive approach to measure cellular antioxidant response under infected condition.