Explore the vast range of titles available.

Gel electrophoresis is routinely used to separate and analyse macromolecules in biological systems. Although many of these electrophoretic techniques necessitate the denaturing of the analytes prior to their analysis, blue native polyacrylamide gel electrophoresis (BN-PAGE) permits the investigation of proteins/enzymes and their supramolecular structures such as the metabolon in native form. This attribute renders this analytical tool conducive to deciphering the metabolic perturbations invoked by metal toxicity. In this review, we elaborate on how BN-PAGE has led to the discovery of the dysfunctional metabolic pathways associated with disorders such as Alzheimer’s disease, Parkinson’s disease, and obesity that have been observed as a consequence of exposure to various metal toxicants.

The mitochondrial oxidative phosphorylation (OXPHOS) system plays a pivotal role in the cell’s energy conversion. The enzymes involved in OXPHOS are arranged in five protein-lipid complexes. The first four complexes (I–IV) form the mitochondrial respiratory chain, while Complex V is an F 1 F o -ATP synthase. Mutations in genes involved in the biosynthesis of the OXPHOS complexes are an important cause of metabolic diseases. Blue-native polyacrylamide gel electrophoresis (BN-PAGE), originally developed by Hermann Schägger in the 1990s, has become instrumental in gaining insights into structure/function relationships of the OXPHOS system, including: (1) the assembly pathways of the complexes, (2) the composition of higher-order respiratory chain supercomplexes and (3) pathologic mechanisms in patients with a monogenetic OXPHOS disorder. We have used BN-PAGE for >20 years and validate here our recently published step-by-step laboratory protocol. This protocol describes the manual casting of native mini-gels and sample preparation for the resolution of individual OXPHOS complexes or respiratory chain supercomplexes. In addition to BN-PAGE, we explain the closely related clear-native (CN)-PAGE and two-dimensional BN/denaturing-PAGE techniques. Downstream applications include western blot analysis and in-gel enzyme activity staining for Complexes I, II, IV and V. Limitations of the technique are the comparative insensitivity of in-gel Complex IV activity staining and the lack of in-gel Complex III activity staining. Compared to other published BN-PAGE protocols, our protocol contains a shortened sample extraction procedure, advises when to use BN-PAGE and when to use CN-PAGE, and suggests a simple enhancement step for in-gel Complex V activity staining that markedly improves sensitivity. Our protocol is adaptable and yields robust, semi-quantitative and reproducible results.

Polyacrylamide gel electrophoresis (PAGE) is a ubiquitous technique used in biochemical research laboratories to characterize protein samples. Despite its popularity, PAGE is relatively slow and provides limited separation resolution, especially for native proteins. This report describes the development of a microfluidic thermal gel transient isotachophoresis (TG-tITP) method to rapidly separate native proteins with high resolution. Thermal gels were employed as a separations matrix because of their unique ability to change viscosity in response to temperature. Proteins were added into thermal gel and loaded into a microfluidic device. Electrolyte optimization was conducted to achieve robust tITP to isotachophoretically preconcentrate proteins and then electrophoretically separate them. Electropherograms were collected through both time and distance to enable both small and large proteins to be measured within a single analysis. The effects of temperature were evaluated and found to exhibit a pronounced effect on the separation. Temperature gradients were then employed to alter thermal gel viscosity over time to maximize separation resolution between proteins. The results herein demonstrate how gradient TG-tITP achieves rapid, high-performance separations of native proteins. This analysis provided a wide mass range (6–464 kDa) with two-fold higher resolution than native PAGE while requiring 15,000-fold less protein loading and providing five-fold faster analysis times. Graphical abstract

Ammonia oxidation, as the first step of nitrification, constitutes a critical process in the global nitrogen cycle. However, fundamental knowledge of its key enzyme, the copper-dependent ammonia monooxygenase, is lacking, in particular for the environmentally abundant ammonia-oxidizing archaea (AOA). Here the structure of the enzyme is investigated by blue-native gel electrophoresis and proteomics from native membrane complexes of two AOA. Besides the known AmoABC subunits and the earlier predicted AmoX, two new protein subunits, AmoY and AmoZ, were identified. They are unique to AOA, highly conserved and co-regulated, and their genes are linked to other AMO subunit genes in streamlined AOA genomes. Modeling and in-gel cross-link approaches support an overall protomer structure similar to the distantly related bacterial particulate methane monooxygenase but also reveals clear differences in extracellular domains of the enzyme. These data open avenues for further structure-function studies of this ecologically important nitrification complex.

Store-operated Ca(2+) entry mediated by STIM1 and ORAI1 constitutes one of the major Ca(2+) entry routes in mammalian cells. The molecular choreography of STIM1-ORAI1 coupling is initiated by endoplasmic reticulum (ER) Ca(2+) store depletion with subsequent oligomerization of the STIM1 ER-luminal domain, followed by its redistribution towards the plasma membrane to gate ORAI1 channels. The mechanistic underpinnings of this inside-out Ca(2+) signalling were largely undefined. By taking advantage of a unique gain-of-function mutation within the STIM1 transmembrane domain (STIM1-TM), here we show that local rearrangement, rather than alteration in the oligomeric state of STIM1-TM, prompts conformational changes in the cytosolic juxtamembrane coiled-coil region. Importantly, we further identify critical residues within the cytoplasmic domain of STIM1 (STIM1-CT) that entail autoinhibition. On the basis of these findings, we propose a model in which STIM1-TM reorganization switches STIM1-CT into an extended conformation, thereby projecting the ORAI-activating domain to gate ORAI1 channels.

In a model of tauopathy, tau directly inhibits proteasome activity, and cognitive impairment can be prevented by activation of cAMP-PKA signaling. The ubiquitin proteasome system (UPS) degrades misfolded proteins including those implicated in neurodegenerative diseases. We investigated the effects of tau accumulation on proteasome function in a mouse model of tauopathy and in a cross to a UPS reporter mouse (line Ub-G76V-GFP). Accumulation of insoluble tau was associated with a decrease in the peptidase activity of brain 26S proteasomes, higher levels of ubiquitinated proteins and undegraded Ub-G76V-GFP. 26S proteasomes from mice with tauopathy were physically associated with tau and were less active in hydrolyzing ubiquitinated proteins, small peptides and ATP. 26S proteasomes from normal mice incubated with recombinant oligomers or fibrils also showed lower hydrolyzing capacity in the same assays, implicating tau as a proteotoxin. Administration of an agent that activates cAMP–protein kinase A (PKA) signaling led to attenuation of proteasome dysfunction, probably through proteasome subunit phosphorylation. In vivo , this led to lower levels of aggregated tau and improvements in cognitive performance.

Nucleophosmin (NPM1) is a multifunctional phospho-protein with critical roles in ribosome biogenesis, tumor suppression, and nucleolar stress response. Here we show that the N-terminal oligomerization domain of NPM1 (Npm-N) exhibits structural polymorphism by populating conformational states ranging from a highly ordered, folded pentamer to a highly disordered monomer. The monomer–pentamer equilibrium is modulated by posttranslational modification and protein binding. Phosphorylation drives the equilibrium in favor of monomeric forms, and this effect can be reversed by Npm-N binding to its interaction partners. We have identified a short, arginine-rich linear motif in NPM1 binding partners that mediates Npm-N oligomerization. We propose that the diverse functional repertoire associated with NPM1 is controlled through a regulated unfolding mechanism signaled through posttranslational modifications and intermolecular interactions.

Tripartite multidrug efflux systems of Gram-negative bacteria are composed of an inner membrane transporter, an outer membrane channel and a periplasmic adaptor protein. They are assumed to form ducts inside the periplasm facilitating drug exit across the outer membrane. Here we present the reconstitution of native Pseudomonas aeruginosa MexAB–OprM and Escherichia coli AcrAB–TolC tripartite Resistance Nodulation and cell Division (RND) efflux systems in a lipid nanodisc system. Single-particle analysis by electron microscopy reveals the inner and outer membrane protein components linked together via the periplasmic adaptor protein. This intrinsic ability of the native components to self-assemble also leads to the formation of a stable interspecies AcrA–MexB–TolC complex suggesting a common mechanism of tripartite assembly. Projection structures of all three complexes emphasize the role of the periplasmic adaptor protein as part of the exit duct with no physical interaction between the inner and outer membrane components. Tripartite efflux systems consist of inner membrane, outer membrane and periplasmic components. Here, Daury et al . reconstitute native versions of RND transporters in nanodiscs and present projection structures emphasizing the role of the periplasmic adaptor in linking the inner and outer membrane proteins.

Medium-chain specific acyl-CoA dehydrogenase (MCAD) is a mitochondrial homotetrameric flavoprotein that catalyzes the first step in fatty acid beta-oxidation. MCAD deficiency arises from variants that either impair enzymatic activity or destabilize interactions between subunits, leading to protein aggregation. Standard enzymatic assays measure the overall MCAD activity but cannot differentiate between tetramers and other protein forms—critical for understanding the impact of pathogenic variants on structure destabilization. In this study, we adapted a native gel colorimetric assay to quantify the activity of MCAD tetramers separately from other protein forms, providing novel insights into how pathogenic variants affect MCAD structure and function. The assay showed a linear correlation between protein amount and enzymatic activity for octanoyl-CoA, a physiological MCAD substrate. Applying this method to clinically relevant MCAD variants allowed us to distinguish subtle differences in protein shape, enzymatic activity, and FAD content, offering profound implications for understanding the molecular basis of MCADD. This methodology can be extended to analyze variants in other acyl-CoA dehydrogenase family members—such as glutaryl-CoA, isovaleryl-CoA or short-chain fatty acyl-CoA dehydrogenases—that are implicated in disorders of fatty acid and amino acid metabolism.

The study examines the impact of climate change-induced drought on red kidney bean ( Phaseolus vulgaris L.) cv. VL Rajma 63 and evaluates the effectiveness of 0.25% of seed chitosan application priming and foliage spray under restricted irrigation. Specifically, it assesses how chitosan treatment enhances the antioxidant potential in response to varying degrees of drought stress. The protein content of all treated leaves ranged between 12.00 and 41.55 mg/g fresh weight in primed plants. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) of leaf proteins expounded new polypeptides in the range of 69.8 to 9.9 kDa. The unprimed plants under severe drought stress showed 3.86 folds of higher hydrogen peroxide compared to control on the 70th day after sowing (DAS). The seed primed plant showed the highest superoxide dismutase (SOD) activity of 2.57 ± 0.01 U/mg protein on the 70th DAS with 20% irrigation in VL Rajma 63 (SPV20). The highest polyphenol oxidase (PPO) activity was observed with the treatment SPV20 (2.22 ± 0.01 ∆O.D./min/mg protein) on the 70th DAS. Hydrogen peroxide showed a strong positive correlation between antioxidant enzymes. Native PAGE (12%) assays revealed four, six, and four isoforms of SOD, guaiacol peroxidase (GPOX), and PPO. The isoenzymes Mn-SOD (Rf 0.14), Fe-SOD (Rf 0.24), CuZn-SOD I (Rf 0.36), CuZn-SOD II (Rf 0.46), GPOX 1–4, and PPO 1–4 were upregulated with varying degrees of drought stress. This study significantly extends the role of seed biopriming as a better option than foliage spray for augmenting drought tolerance.