RNA and the RNA-binding protein FUS act in concert to prevent TDP-43 spatial segregation.

FUS and TDP-43 are two self-adhesive aggregation-prone mRNA-binding proteins whose pathological mutations have been linked to neurodegeneration. While TDP-43 and FUS form reversible mRNA-rich compartments in the nucleus, pathological mutations promote their respective cytoplasmic aggregation in neurons with no apparent link between the two proteins except their intertwined function in mRNA processing. By combining analyses in cellular context and at high resolution in vitro, we unraveled that TDP-43 is specifically recruited in FUS assemblies to form TDP-43-rich subcompartments but without reciprocity. The presence of mRNA provides an additional scaffold to promote the mixing between TDP-43 and FUS. Accordingly, we also found that the pathological truncated form of TDP-43, TDP-25, which has an impaired RNA-binding ability, no longer mixes with FUS. Together, these results suggest that the binding of FUS along nascent mRNAs enables TDP-43, which is highly aggregation-prone, to mix with FUS phase to form mRNA-rich subcompartments. A functional link between FUS and TDP-43 may explain their common implication in amyotrophic lateral sclerosis.

Electroneutral Polymer Nanodiscs Enable Interference-Free Probing of Membrane Proteins in a Lipid-Bilayer Environment.

Membrane proteins can be examined in near-native lipid-bilayer environments with the advent of polymer-encapsulated nanodiscs. These nanodiscs self-assemble directly from cellular membranes, allowing in vitro probing of membrane proteins with techniques that have previously been restricted to soluble or detergent-solubilized proteins. Often, however, the high charge densities of existing polymers obstruct bioanalytical and preparative techniques. Thus, the authors aim to fabricate electroneutral-yet water-soluble-polymer nanodiscs. By attaching a sulfobetaine group to the commercial polymers DIBMA and SMA(2:1), these polyanionic polymers are converted to the electroneutral maleimide derivatives, Sulfo-DIBMA and Sulfo-SMA(2:1). Sulfo-DIBMA and Sulfo-SMA(2:1) readily extract proteins and phospholipids from artificial and cellular membranes to form nanodiscs. Crucially, the electroneutral nanodiscs avert unspecific interactions, thereby enabling new insights into protein-lipid interactions through lab-on-a-chip detection and in vitro translation of membrane proteins. Finally, the authors create a library comprising thousands of human membrane proteins and use proteome profiling by mass spectrometry to show that protein complexes are preserved in electroneutral nanodiscs.

Cloning, expression, solubilization, and purification of a functionally active recombinant cAMP-dependent protein kinase catalytic subunit-like protein PKAC1 from Trypanosoma equiperdum.

The gene encoding the cAMP-dependent protein kinase (PKA) catalytic subunit-like protein PKAC1 from the Venezuelan TeAp-N/D1 strain of Trypanosoma equiperdum was cloned, and the recombinant TeqPKAC1 protein was overexpressed in bacteria. A major polypeptide with an apparent molecular mass of ∼38 kDa was detected by SDS-polyacrylamide gel electrophoresis, and immunoblotting using antibodies against the human PKA catalytic subunit α. Unfortunately, most of the expressed TeqPKAC1 was highly insoluble. Polypeptides of 36-38 kDa and 45-50 kDa were predominantly seen by immunoblotting in the bacterial particulate and cytosolic fractions, respectively. Since the incorporation of either 4% Triton X-100 or 3% sarkosyl or a mixture of 10 mM MgCl2 and 1 mM ATP (MgATP) improved the solubilization of TeqPKAC1, we used a combination of Triton X-100, sarkosyl and MgATP to solubilize the recombinant protein. TeqPKAC1 was purified by first reconstituting a hybrid holoenzyme between the recombinant protein and a mammalian poly-His-tagged PKA regulatory subunit that was immobilized on a Ni2+-chelating affinity resin, and then by eluting TeqPKAC1 using cAMP. TeqPKAC1 was functional given that it was capable of phosphorylating PKA catalytic subunit substrates, such as kemptide (LRRASLG), histone type II-AS, and the peptide SP20 (TTYADFIASGRTGRRNSIHD), and was inhibited by the peptide IP20 (TTYADFIASGRTGRRNAIHD), which contains the inhibitory motif of the PKA-specific heat-stable inhibitor PKI-α. Optimal enzymatic activity was obtained at 37 °C and pH 8.0-9.0; and the order of effectiveness of nucleotide triphosphates and divalent cations was ATP ¼ GTP â‰… ITP and Mg2+ â‰… Mn2+ â‰… Fe2+ ¼ Ca2+ â‰… Zn2, respectively.

Self-Assembly of Protein-Containing Lipid-Bilayer Nanodiscs from Small-Molecule Amphiphiles.

When membrane proteins are removed from their natural environment, the quality of the membrane-solubilizing agent used is critical for preserving their native structures and functions. Nanodiscs that retain a lipid-bilayer core around membrane proteins have attracted great attention because they offer a much more native-like environment than detergent micelles. Here, two small-molecule amphiphiles with diglucose headgroups and either a hydrocarbon or a fluorocarbon hydrophobic chain are shown to directly assemble lipids and membrane proteins to form native nanodiscs rather than mixed micelles. Self-assembly of nanodiscs of increasing complexity from both defined, artificial vesicles as well as complex, cellular membranes is demonstrated. A detailed investigation of bilayer integrity and membrane-protein activity in these nanodiscs reveals gentle effects on the encapsulated bilayer core. The fluorinated amphiphile appears particularly promising because its lipophobicity results in gentle, non-perturbing interactions with the nanoscale lipid bilayer. A sequential model of nanodisc self-assembly is proposed that proceeds through perforation of the original membrane followed by saturation and complete solubilization of the bilayer. On this basis, pseudophase diagrams are established for mixtures of lipids and nanodisc-forming diglucoside amphiphiles, and the latter are used for the extraction of a broad range of membrane proteins from cellular membranes.

mRNA Engineering for the Efficient Chaperone-Mediated Co-Translational Folding of Recombinant Proteins in Escherichia coli.

The production of soluble, functional recombinant proteins by engineered bacterial hosts is challenging. Natural molecular chaperone systems have been used to solubilize various recombinant proteins with limited success. Here, we attempted to facilitate chaperone-mediated folding by directing the molecular chaperones to their protein substrates before the co-translational folding process completed. To achieve this, we either anchored the bacterial chaperone DnaJ to the 3' untranslated region of a target mRNA by fusing with an RNA-binding domain in the chaperone-recruiting mRNA scaffold (CRAS) system, or coupled the expression of DnaJ and a target recombinant protein using the overlapping stop-start codons 5'-TAATG-3' between the two genes in a chaperone-substrate co-localized expression (CLEX) system. By engineering the untranslated and intergenic sequences of the mRNA transcript, bacterial molecular chaperones are spatially constrained to the location of protein translation, expressing selected aggregation-prone proteins in their functionally active, soluble form. Our mRNA engineering methods surpassed the in-vivo solubilization efficiency of the simple DnaJ chaperone co-overexpression method, thus providing more effective tools for producing soluble therapeutic proteins and enzymes.

Optimization of protein recovery from bovine lung by pH shift process using response surface methodology.

BACKGROUND: Response surface methodology (RSM) was used in a sequential manner to optimize solubilization and precipitation conditions in the recovery of protein from bovine lung using pH shift. RESULTS: Separate D-optimal designs were employed for protein solubilization and precipitation. Independent variables investigated for protein solubilization were time (10-120 min), temperature (4-20 °C), pH (8.0-11.0) and solvent/sample ratio (2.5-10). Variables for protein precipitation were time (0-60 min) and pH (4.25-6.00). Soluble protein yields ranged from 323 to 649 g kg-1 and the quadratic model for protein solubilization revealed a coefficient of determination R2 of 0.9958. Optimal conditions for maximum protein solubility were extraction time 140 min, temperature 19 °C, pH 10.8 and solvent/sample ratio 13.02. Protein precipitation yields varied from 407 to 667 g kg-1 , giving a coefficient of determination R2 of 0.9335. Optimal conditions for maximum protein precipitation were pH 5.03 and 60 min. Based on the RSM model, solubilization conditions were manipulated to maximize protein solubilization under reduced water and alkaline usage. These conditions were also validated. CONCLUSION: Models for solubilization and precipitation using bovine and porcine lung were validated; predicted and actual yields were in good agreement, showing cross-species applicability of the results. © 2017 Society of Chemical Industry.

Detergent-free isolation, characterization, and functional reconstitution of a tetrameric K+ channel: the power of native nanodiscs.

A major obstacle in the study of membrane proteins is their solubilization in a stable and active conformation when using detergents. Here, we explored a detergent-free approach to isolating the tetrameric potassium channel KcsA directly from the membrane of Escherichia coli, using a styrene-maleic acid copolymer. This polymer self-inserts into membranes and is capable of extracting membrane patches in the form of nanosize discoidal proteolipid particles or "native nanodiscs." Using circular dichroism and tryptophan fluorescence spectroscopy, we show that the conformation of KcsA in native nanodiscs is very similar to that in detergent micelles, but that the thermal stability of the protein is higher in the nanodiscs. Furthermore, as a promising new application, we show that quantitative analysis of the co-isolated lipids in purified KcsA-containing nanodiscs allows determination of preferential lipid-protein interactions. Thin-layer chromatography experiments revealed an enrichment of the anionic lipids cardiolipin and phosphatidylglycerol, indicating their close proximity to the channel in biological membranes and supporting their functional relevance. Finally, we demonstrate that KcsA can be reconstituted into planar lipid bilayers directly from native nanodiscs, which enables functional characterization of the channel by electrophysiology without first depriving the protein of its native environment. Together, these findings highlight the potential of the use of native nanodiscs as a tool in the study of ion channels, and of membrane proteins in general.

Preventing N- and O-formylation of proteins when incubated in concentrated formic acid.

Concentrated formic acid is among the most effective solvents for protein solubilization. Unfortunately, this acid also presents a risk of inducing chemical modifications thereby limiting its use in proteomics. Previous reports have supported the esterification of serine and threonine residues (O-formylation) for peptides incubated in formic acid. However as shown here, exposure of histone H4 to 80% formic (1 h, 20(o) C) induces N-formylation of two independent lysine residues. Furthermore, incubating a mixture of Escherichia coli proteins in formic acid demonstrates a clear preference toward lysine modification over reactions at serine/threonine. N-formylation accounts for 84% of the 225 uniquely identified formylation sites. To prevent formylation, we provide a detailed investigation of reaction conditions (temperature, time, acid concentration) that define the parameters permitting the use of concentrated formic acid in a proteomics workflow for MS characterization. Proteins can be maintained in 80% formic acid for extended periods (24 h) without inducing modification, so long as the temperature is maintained at or below -20(o) C.

Isomeric Detergent Comparison for Membrane Protein Stability: Importance of Inter-Alkyl-Chain Distance and Alkyl Chain Length.

Membrane proteins encapsulated by detergent micelles are widely used for structural study. Because of their amphipathic property, detergents have the ability to maintain protein solubility and stability in an aqueous medium. However, conventional detergents have serious limitations in their scope and utility, particularly for eukaryotic membrane proteins and membrane protein complexes. Thus, a number of new agents have been devised; some have made significant contributions to membrane protein structural studies. However, few detergent design principles are available. In this study, we prepared meta and ortho isomers of the previously reported para-substituted xylene-linked maltoside amphiphiles (XMAs), along with alkyl chain-length variation. The isomeric XMAs were assessed with three membrane proteins, and the meta isomer with a C12 alkyl chain was most effective at maintaining solubility/stability of the membrane proteins. We propose that interplay between the hydrophile-lipophile balance (HLB) and alkyl chain length is of central importance for high detergent efficacy. In addition, differences in inter-alkyl-chain distance between the isomers influence the ability of the detergents to stabilise membrane proteins.

GPCR-styrene maleic acid lipid particles (GPCR-SMALPs): their nature and potential.

G-protein-coupled receptors (GPCRs) form the largest class of membrane proteins and are an important target for therapeutic drugs. These receptors are highly dynamic proteins sampling a range of conformational states in order to fulfil their complex signalling roles. In order to fully understand GPCR signalling mechanisms it is necessary to extract the receptor protein out of the plasma membrane. Historically this has universally required detergents which inadvertently strip away the annulus of lipid in close association with the receptor and disrupt lateral pressure exerted by the bilayer. Detergent-solubilized GPCRs are very unstable which presents a serious hurdle to characterization by biophysical methods. A range of strategies have been developed to ameliorate the detrimental effect of removing the receptor from the membrane including amphipols and reconstitution into nanodics stabilized by membrane scaffolding proteins (MSPs) but they all require exposure to detergent. Poly(styrene-co-maleic acid) (SMA) incorporates into membranes and spontaneously forms nanoscale poly(styrene-co-maleic acid) lipid particles (SMALPs), effectively acting like a 'molecular pastry cutter' to 'solubilize' GPCRs in the complete absence of detergent at any stage and with preservation of the native annular lipid throughout the process. GPCR-SMALPs have similar pharmacological properties to membrane-bound receptor, exhibit enhanced stability compared with detergent-solubilized receptors and being non-proteinaceous in nature, are fully compatible with downstream biophysical analysis of the encapsulated GPCR.

Different recombinant forms of polyphenol oxidase A, a laccase from Marinomonas mediterranea.

Polyphenol oxidase from the marine bacterium Marinomonas mediterranea (MmPPOA) is a membrane-bound, blue, multi-copper laccase of 695 residues. It possesses peculiar properties that distinguish it from known laccases, such as a broad substrate specificity (common to tyrosinases) and a high redox potential. In order to push the biotechnological application of this laccase, the full-length enzyme was overexpressed in Escherichia coli cells with and without a C-terminal His-tag. The previous form, named rMmPPOA-695-His, was purified to homogeneity by HiTrap chelating chromatography following solubilization by 1% SDS in the lysis buffer with an overall yield of ≈1 mg/L fermentation broth and a specific activity of 1.34 U/mg protein on 2,6-dimethoxyphenol as substrate. A truncated enzyme form lacking 58 residues at the N-terminus encompassing the putative membrane binding region, namely rMmPPOA-637-His, was successfully expressed in E. coli as soluble protein and was purified by using the same procedure set-up as for the full-length enzyme. Elimination of the N-terminal sequence decreased the specific activity 15-fold (which was partially restored in the presence of 1 M NaCl) and altered the secondary and tertiary structures and the pH dependence of optimal stability. The recombinant rMmPPOA-695-His showed kinetic properties on catechol higher than for known laccases, a very high thermal stability, and a strong resistance to NaCl, DMSO, and Tween-80, all properties that are required for specific, targeted industrial applications.

Hydrophobic variants of ganglio-tripod amphiphiles for membrane protein manipulation.

Membrane proteins operate in unique cellular environments. Once removed from their native context for the purification that is required for most types of structural or functional analyses, they are prone to denature if not properly stabilized by membrane mimetics. Detergent micelles have prominently been used to stabilize membrane proteins in aqueous environments as their amphipathic nature allows for shielding of the hydrophobic surfaces of these bio-macromolecules while supporting solubility and monodispersity in water. This study expands the utility of branched diglucoside-bearing tripod agents, designated ganglio-tripod amphiphiles, with introduction of key variations in their hydrophobic sections and shows how these latter elements can be fine-tuned to maximize membrane protein solubilization while preserving characteristics of these molecules that afford stabilization of rather fragile assemblies. Their efficacy rivals benchmark detergents heavily used today, such as n-dodecyl-ß-d-maltoside.

Optimization of human dendritic cell sample preparation for mass spectrometry-based proteomic studies.

Dendritic cells (DCs) are specialized leukocytes that orchestrate the adaptive immune response. Mass spectrometry (MS)-based proteomic study of these cells presents technical challenges, especially when the DCs are human in origin due to the paucity of available biological material. Here, to maximize MS coverage of the global human DC proteome, different cell disruption methods, lysis conditions, protein precipitation, and protein pellet solubilization and denaturation methods were compared. Mechanical disruption of DC cell pellets under cryogenic conditions, coupled with the use of RIPA (radioimmunoprecipitation assay) buffer, was shown to be the method of choice based on total protein extraction and on the solubilization and identification of nuclear proteins. Precipitation by acetone was found to be more efficient than that by 10% trichloroacetic acid (TCA)/acetone, allowing in excess of 28% more protein identifications. Although being an effective strategy to eliminate the detergent residue, the acetone wash step caused a loss of protein identifications. However, this potential drawback was overcome by adding 1% sodium deoxycholate into the dissolution buffer, which enhanced both solubility of the precipitated proteins and digestion efficiency. This in turn resulted in 6 to 11% more distinct peptides and 14 to 19% more total proteins identified than using 0.5M triethylammonium bicarbonate alone, with the greatest increase (34%) for hydrophobic proteins.

Purification and immunodetection of the complete recombinant HER-2[neu] receptor produced in yeast.

For the first time, the full length recombinant HER-2[neu] receptor has been produced in a yeast (Arxula adeninivorans). It is one of the most studied membrane receptors in oncology and is involved in aggressive tumor formation. A yeast integration rDNA cassette containing the human gene coding for the HER-2[neu] protein was constructed and a screening procedure was performed to select the most productive transformant. Different detergents were tested for efficient solubilization of the membrane bound protein, with CHAPS giving the best results. To increase the yield of the recombinant protein from HER-2[neu] producing A. adeninivorans, optimal culture parameters were established for cultivation in bioreactor. The recombinant protein was subsequently assayed using ELISA and SPR immunoassays systems with antibodies raised against two different epitopes of the human receptor. In both cases, elution fractions containing the recombinant HER-2[neu] receptor successfully reacted with the immunoassays with limits of quantification below 100ngml(-1). These results demonstrate that the full length recombinant HER-2[neu] reported here has the potential to be a new standard for the detection of HER-2 type cancer.

Phenol-Based Protein Extraction Method for Plant Proteomic Studies.

The protein extraction method based on the phenol solution and combined with protein precipitation with ammonium acetate in methanol and purification in the same solution, and additionally in acetone and ethanol, is recommended for proteomic studies of plant tissues. The obtained protein samples do not require additional nucleic acid digestion and removal of interfering contaminations. The presented protocol was used to analyze the proteome of common buckwheat flowers and leaves.

Complete solubilization of mammalian cells in lysates.

In conventional cell lysate protocols, cell debris is typically discarded to obtain a cleaner lysate. However, this approach has limitations, as it may overlook vital cellular components. By discarding cell debris, researchers may inadvertently exclude crucial elements. Retaining all cellular components offers several advantages for studying molecular biology within various cellular compartments. Firstly, it provides a more accurate representation of the cellular environment. Secondly, it enables the study of complex cellular interactions, including those involving cellular structures and signaling pathways associated with debris. This shift in perspective highlights the importance of a holistic approach to lysate preparation. By obtaining lysates that include all cellular components, researchers can gain deeper insights into cellular processes, leading to more accurate data and a better understanding of cellular function and dysfunction. This study aimed to develop a protocol for the preparation of total cell lysates that retain all cellular components, including debris. Our method involves:•A three-step solubilization process using a combination of detergents, saccharides, and chelators, coupled with sonication, in contrast to the classical one-step approach using an all-detergent cocktail.•A comprehensive strategy ensuring the solubilization of all cellular components, providing a more complete lysate for analysis.

Strategies for the Purification of Membrane Proteins.

Membrane proteins account for approximately 30% of the coding regions of all sequenced genomes, and they play crucial roles in many fundamental cell processes. However, there are relatively few membrane proteins with known three-dimensional structures. This is likely due to technical challenges associated with membrane protein extraction, solubilization, and purification. Membrane proteins are classified based on the level of interaction with membrane lipid bilayers, with peripheral membrane proteins associating non-covalently with the membrane, and integral membrane proteins associating more strongly by means of hydrophobic interactions. Generally speaking, peripheral membrane proteins can be purified by milder techniques than integral membrane proteins, with the latter's extraction requiring phospholipid bilayer disruption using detergents or organic solvents. In this chapter, important considerations for membrane protein purification are addressed, with a focus on the initial stages of membrane protein solubilization, where problems are most frequently encountered. Protocols are outlined for the extraction of peripheral membrane proteins, solubilization of integral membrane proteins, and sample clean-up and concentration.

Evaluation of in vitro whey protein digestibility in a protein-catechins model system mimicking milk chocolate: Interaction with flavonoids does not hinder protein bioaccessibility.

Flavonoids are largely present in plant food such as cocoa and derived products. These compounds can interact with proteins inherently contained in the food matrix and/or the proteolytic enzymes involved in gastrointestinal digestion. The flavonoid/protein interaction might hamper protein bioaccessibility and digestibility, affecting the nutritional quality. However, information on the digestion fate of proteins in food matrices containing both proteins and flavonoids is limited. The aim of this work was to evaluate the interaction between proteins and flavonoids and verify the potential effects of this interaction on protein digestibility. Taking milk chocolate as model, first a simple whey proteins/catechins mixed system was evaluated, and then the effects on digestibility were also verified in a real sample of commercial milk chocolate. The effects of the catechins/whey proteins interaction in the model system were evaluated by optical and chiro-optical spectroscopy, outlining a slight protein structure modification upon interaction with catechins. The digestibility of the protein fraction both in the model system, with and without catechins, and also in milk chocolate, was then determined by the application of INFOGEST in vitro digestion method: the bioaccessibility was evaluated in terms of protein hydrolysis and protein solubilisation, and major peptides generated by the digestion were also determined by LC/HR-MS. Despite the slight interaction with proteins, flavonoids were found to not hinder nor modify protein solubilization, protein hydrolysis and peptide profile by digestive enzymes. Also protein digestibility in milk chocolate, evaluated by SDS-PAGE, was found to be complete. The present data clearly indicate that the interaction of the proteins with the flavonoids present in the cocoa matrix does not to affect protein bioaccessibility during digestion.

A chromatographic network for the purification of detergent-solubilized six-transmembrane epithelial antigen of the prostate 1 from Komagataella pastoris mini-bioreactor lysates.

The Six-Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) is an integral membrane protein involved in cellular communications, in the stimulation of cell proliferation by increasing Reactive Oxygen Species levels, and in the transmembrane-electron transport and reduction of extracellular metal-ion complexes. The STEAP1 is particularly over-expressed in prostate cancer, in contrast with non-tumoral tissues and vital organs, contributing to tumor progression and aggressiveness. However, the current understanding of STEAP1 lacks experimental data on the respective molecular mechanisms, structural determinants, and chemical modifications. This scenario highlights the relevance of exploring the biosynthesis of STEAP1 and its purification for further bio-interaction and structural characterization studies. In this work, recombinant hexahistidine-tagged human STEAP1 (rhSTEAP1-His6) was expressed in Komagataella pastoris (K. pastoris) mini-bioreactor methanol-induced cultures and successfully solubilized with Nonidet P-40 (NP-40) and n-Decyl-ß-D-Maltopyranoside (DM) detergents. The fraction capacity of Phenyl-, Butyl-, and Octyl-Sepharose hydrophobic matrices were evaluated by manipulating the ionic strength of binding and elution steps. Alternatively, immobilized metal affinity chromatography packed with nickel or cobalt were also studied in the isolation of rhSTEAP1-His6 from lysate extracts. Overall, the Phenyl-Sepharose and Nickel-based resins provided the desired selectivity for rhSTEAP1-His6 capture from NP-40 and DM detergent-solubilized K. pastoris extracts, respectively. After a polishing step using the anion-exchanger Q-Sepharose, a highly pure, fully solubilized, and immunoreactive 35 kDa rhSTEAP1-His6 fraction was obtained. Altogether, the established reproducible strategy for the purification of rhSTEAP1-His6 paves the way to gather additional insights on structural, thermal, and environmental stability characterization significantly contributing for the elucidation of the functional role and oncogenic behavior of the STEAP1 in prostate cancer microenvironment.

Effect of combined high pressure and thermal treatment on myofibrillar proteins solubilization of beef muscle.

The effects of high pressure (to 600 MPa) at different temperatures (20 to 60 °C) for 20 min on protein solubilization and electrophoretic pattern in beef post-rigor longissimus dorsi muscle were studied. The results showed that protein solubilization increased with increasing temperature, especially from 40 °C to 60 °C. A regular trend of protein solubilization was found when isolated myofibrils were subjected to high pressure at different temperatures, an increase was observed with increasing pressure up to about 400 MPa, solubility then decreasing to 600 MPa. Electrophoretic profiles showed that myosin light chains and actin thin filaments were sensitive to pressure, and were released from myofibrils subjected to 100 MPa and higher pressures at the different temperatures.