Purification Of Protein Complexes Research Articles

A nanobody-based strategy for rapid and scalable purification of human protein complexes.

The isolation of proteins in high yield and purity is a major bottleneck for the analysis of their three-dimensional structure, function and interactome. Here, we present a streamlined workflow for the rapid production of proteins or protein complexes using lentiviral transduction of human suspension cells, combined with highly specific nanobody-mediated purification and proteolytic elution. Application of the method requires prior generation of a plasmid coding for a protein of interest (POI) fused to an N- or C-terminal GFP or ALFA peptide tag using a lentiviral plasmid toolkit we have designed. The plasmid is then used to generate human suspension cell lines stably expressing the tagged fusion protein by lentiviral transduction. By leveraging the picomolar affinity of the GFP and ALFA nanobodies for their respective tags, the POI can be specifically captured from the resulting cell lysate even when expressed at low levels and under a variety of conditions, including detergents and mild denaturants. Finally, rapid and specific elution of the POI (in its tagged or untagged form) under native conditions is achieved within minutes at 4 °C, using the engineered SUMO protease SENPEuB. We demonstrate the wide applicability of the method by purifying multiple challenging soluble and membrane protein complexes to high purity from human cells. Our strategy is also directly compatible with many widely used GFP-expression plasmids, cell lines and transgenic model organisms. Finally, our method is faster than alternative approaches, requiring only 8 d from plasmid to purified protein, and results in substantially improved yields and purity.

Synthesis of biologically active proteins as L6KD-SUMO fusions forming inclusion bodies in Escherichia coli.

A new platform has been developed to facilitate the production of biologically active proteins and peptides in Escherichia coli. The platform includes an N-terminal self-associating L6 KD peptide fused to the SUMO protein (small ubiquitin-like protein modifier) from the yeast Saccharomyces cerevisiae, which is known for its chaperone activity. The target proteins are fused at the C termini of the L6 KD-SUMO fusions, and the resulting three-component fusion proteins are synthesized and self-assembled in E. coli into so-called active inclusion bodies (AIBs). In vivo, the L6 KD-SUMO platform facilitates the correct folding of the target proteins and directs them into AIBs, greatly simplifying their purification. In vitro, the platform facilitates the effective separation of AIBs by centrifugation and subsequent target protein release using SUMO-specific protease. The properties of the AIBs were determined using five proteins with different sizes, folding efficiencies, quaternary structure, and disulfide modifications. Electron microscopy shows that AIBs are synthesized in the form of complex fibrillar structures resembling "loofah sponges" with unusually thick filaments. The obtained results indicate that the new platform has promising features and could be developed to facilitate the synthesis and purification of target proteins and protein complexes without the use of renaturation.

Improved Cross-Linking Coverage for Protein Complexes Containing Low Levels of Lysine by Using an Enrichable Photo-Cross-Linker.

Chemical cross-linking coupled with mass spectrometry (XL-MS) is an important technique for the structural analysis of protein complexes where the coverage of amino acids and the identification of cross-linked sites are crucial. Photo-cross-linking has multisite reactivity and is valuable for the structural analysis of chemical cross-linking. However, a high degree of heterogeneity results from this multisite reactivity, which results in samples with higher complexity and lower abundance. Additionally, the applicability of photo-cross-linking is limited to purified protein complexes. In this work, we demonstrate a photo-cross-linker, alkynyl-succinimidyl-diazirine (ASD) with the reactive groups of N-hydroxysuccinimide ester and diazirine, as well as the click-enrichable alkyne group. Photo-cross-linkers can provide higher site reactivity for proteins that contain only a small number of lysine residues, thereby complementing the more commonly used lysine-targeting cross-linkers. By systematically analyzing proteins with differing lysine contents and differing flexibilities, we demonstrated clear enhancement in structure elucidation for proteins containing less lysine and with high flexibility. In addition, enrichment approaches of alkynyl-azide click chemistry conjugated with biotin-streptavidin purification (coinciding with parallel orthogonal digestion) improved the identification coverage of cross-links. We show that this photo-cross-linking approach can be used for membrane proteome-wide complex analysis. This method led to the identification of a total of 14066 lysine-X cross-linked site pairs from a total of 2784 proteins. Thus, this cross-linker is a valuable addition to a photo-cross-linking toolkit and improves the identification coverage of XL-MS in functional structure analysis.

Onto Grid Purification and 3D Reconstruction of Protein Complexes Using Matrix-Landing Native Mass Spectrometry.

Addressing mixtures and heterogeneity in structural biology requires approaches that can differentiate and separate structures based on mass and conformation. Mass spectrometry (MS) provides tools for measuring and isolating gas-phase ions. The development of native MS including electrospray ionization allowed for manipulation and analysis of intact noncovalent biomolecules as ions in the gas phase, leading to detailed measurements of structural heterogeneity. Conversely, transmission electron microscopy (TEM) generates detailed images of biomolecular complexes that show an overall structure. Our matrix-landing approach uses native MS to probe and select biomolecular ions of interest for subsequent TEM imaging, thus unifying information on mass, stoichiometry, heterogeneity, etc., available via native MS with TEM images. Here, we prepare TEM grids of protein complexes purified via quadrupolar isolation and matrix-landing and generate 3D reconstructions of the isolated complexes. Our results show that these complexes maintain their structure through gas-phase isolation.

Utilization of Nonstop mRNA to Assess Ribosome-Associated Nascent Polypeptide Chains in Early Topogenesis of Peroxisomal Proteins.

The translation of mRNAs lacking a stop codon results in a nascent polypeptide chain still attached to the translating ribosome. When containing an exposed N-terminal targeting signal, these so-called nonstop (ns) proteins have been shown to localize to their respective organellar translocation channel, resulting in stabilized translocation intermediates. Utilizing a plasmid encoding a FLAG-tagged nonstop protein with an N-terminal targeting signal early-stage ribosome-associated protein complexes can be purified by affinity chromatography. This will be exemplified by purification of protein complexes of the peroxisomal protein import machinery using different nonstop variants of the PTS2 cargo protein Fox3p from both soluble and membrane fractions.

Npl3 functions in mRNP assembly by recruitment of mRNP components to the transcription site and their transfer onto the mRNA.

RNA-binding proteins (RBPs) control every RNA metabolic process by multiple protein-RNA and protein-protein interactions. Their roles have largely been analyzed by crude mutations, which abrogate multiple functions at once and likely impact the structural integrity of the large ribonucleoprotein particles (RNPs) these proteins function in. Using UV-induced RNA-protein crosslinking of entire cells, protein complex purification and mass spectrometric analysis, we identified >100 in vivo RNA crosslinks in 16 nuclear mRNP components in Saccharomyces cerevisiae. For functional analysis, we chose Npl3, which displayed crosslinks in its two RNA recognition motifs (RRMs) and in the connecting flexible linker region. Both RRM domains and the linker uniquely contribute to RNA recognition as revealed by NMR and structural analyses. Interestingly, mutations in these regions cause different phenotypes, indicating distinct functions of the different RNA-binding domains. Notably, an npl3-Linker mutation strongly impairs recruitment of several mRNP components to chromatin and incorporation of other mRNP components into nuclear mRNPs, establishing a so far unknown function of Npl3 in nuclear mRNP assembly. Taken together, our integrative analysis uncovers a specific function of the RNA-binding activity of the nuclear mRNP component Npl3. This approach can be readily applied to RBPs in any RNA metabolic process.

Efficient production of protein complexes in mammalian cells using a poxvirus vector.

The production of full length, biologically active proteins in mammalian cells is critical for a wide variety of purposes ranging from structural studies to preparation of subunit vaccines. Prior research has shown that Modified vaccinia virus Ankara encoding the bacteriophage T7 RNA polymerase (MVA-T7) is particularly suitable for high level expression of proteins upon infection of mammalian cells. The expression system is safe for users and 10-50 mg of full length, biologically active proteins may be obtained in their native state, from a few litres of infected cell cultures. Here we report further improvements which allow an increase in the ease and speed of recombinant virus isolation, the scale-up of protein production and the simultaneous synthesis of several polypeptides belonging to a protein complex using a single virus vector. Isolation of MVA-T7 viruses encoding foreign proteins was simplified by combining positive selection for virus recombinants and negative selection against parental virus, a process which eliminated the need for tedious plaque purification. Scale-up of protein production was achieved by infecting a BHK 21 suspension cell line and inducing protein expression with previously infected cells instead of virus, thus saving time and effort in handling virus stocks. Protein complexes were produced from infected cells by concatenating the Tobacco Etch Virus (TEV) N1A protease sequence with each of the genes of the complex into a single ORF, each gene being separated from the other by twin TEV protease cleavage sites. We report the application of these methods to the production of a complex formed on the one hand between the HIV-1 integrase and its cell partner LEDGF and on the other between the HIV-1 VIF protein and its cell partners APOBEC3G, CBFβ, Elo B and Elo C. The strategies developed in this study should be valuable for the overexpression and subsequent purification of numerous protein complexes.

Demonstration That Loss of TET2 Impairs DNA Mismatch Repair Resulting in Clonally Restricted Genomic Instability

Epigenetic dysregulation is a common feature of myeloid neoplasia (MN). TET2, along with TET1 and TET3 are the only known enzymes, which progressively oxidize 5-methyl cytosine and thus facilitate active demethylation of CpG islands, a process critical for efficient gene expression and maintenance of genomic integrity. Loss of function TET2 mutations (TET2MT) are one of the most common somatic lesions in MN. To date, the precise mechanisms of TET2MT in leukemogenesis remain unclear. However, two potential mechanisms have been postulated; i) TET2MT may lead to expansion of stem cells by differentiation block and/or increased self-renewal ii) clonally restricted hyper mutations resulting in acquisition of secondary driver mutations. This functional duality may explain proleukemogenic consequences of TET2MT. Both mechanisms are compatible with their relatively moderate driver phenotype. Increased CàT transitions via deamination of 5mC in TET2MT has been postulated; however this report focuses on a novel mechanism of TET2MT- mediated impairment of mismatch repair (MMR). WES was used for analysis of the mutational status of 435 MN cases to test whether TET2MT (n= 82/435; 19%) predispose to acquisition of additional mutations. Following exclusion of dysfunction in known DNA repair genes, we compared the median SNVs in TET2MT vs. TET2WT. TET2MT cases had nearly a 2-fold increased median SNVs (p<0.0001) vs. TET2WT including a 2.1-fold increase in truncations (p=0.03) with a linear correlation (r=0.7) of SNVs vs. clonality, consistent with a gene-dose effect. ShRNA knockdown of TET2 in HeLa and CRISPR knockout in HEK293 cells resulted in a 24- and 30-fold increase in spontaneous mutations in the HPRT assay. The heightened spontaneous mutability in TET2KO/KD cells were reversed by ectopic expression of TET2. In Tet2-/- mice, ~1.5-fold increased mutagenicity at TET2-dependent active demethylation sites were detected suggesting its direct or indirect involvement in one or more DNA repair pathways. Consistent with our hypothesis, endogenous co-immunoprecipitation of TET2 followed by tandem-affinity purification and characterization of protein complexes with LCMS/MS in 4 different systems including human leukemic cell lines (K562 and CMK), murine embryonic stem cell and erythroid leukemia cells MEL identified all the major components of DNA MMR complex as a part of TET2 interactome. In every cell line, MSH6, a main component of MMR complex was an abundant TET2 interacting protein. This result was further confirmed by reverse co-immunoprecipitation using anti-MSH6 antibodies in different cell lines. Using truncating deletion constructs of TET2, MSH6 binding site was mapped on TET2 N-terminal amino acid segment 460-942. Functional MMR fluorescence reporter assay, wherein a point mutant of GFP, if repaired, is converted to BFP, demonstrated that loss of TET2 indeed compromised the rate of MMR in CRISPR generated deletion of TET2 in K562 and HEK293 cells. Ectopic expression of N-terminal (aa 460-942) MSH6 binding domain rescued this defect. The genomic instability due to MMR dysfunction in TET2MT cells may allow therapeutic targeting of DNA repair proteins in a subset of TET2MT MN. Therefore, we tested the therapeutic vulnerability of TET2MT cells against small molecule DNA repair inhibitors (e.g., clinically approved PARP antagonists) in engineered isogenic and natural TET2MT cell lines. The TET2KO/KD cells were 2-10X more sensitive to olaparib and rukaparib compared to parental TET2 proficient cells, both under steady state and under conditions of oxidative stress as observed in gH2AX assays and various apoptotic readouts. In summary, for the first time here we report that TET2 directly binds to MSH6 and recruits MMR complex to the site of mismatch around CpG islands and facilitate the repair. Loss of TET2impairs this process, explaining the accumulation of secondary hits in mutant clones, which by this virtue, progressively increases their leukemogenic fitness. Given that MSH6 is known to interact 5hmC, TET2 may guide MMR to TET2-dependent DNA loci directly linking TET2MTto genomic instability a biologic feature that can be therapeutically exploited. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

E. coli chromosomal-driven expression of NADK2 from A. thaliana: A preferable alternative to plasmid-driven expression for challenging proteins

The expression and purification of large recombinant proteins or protein complexes is problematic for some biotechnology laboratories. Indeed, it is often difficult to obtain enough active proteins to perform biological characterization or reach commercialization, when large proteins or protein complexes are expressed in E. coli via the popular T7-based plasmid-driven expression system.There is also an industrial demand to decrease our dependence on plasmid-driven expression, because of its drawbacks, such as: i) the common use of antibiotics to maintain the plasmid, ii) the issue of plasmid copy number, and iii) the risk of overloading the expression system. Despite all these issues, alternative solutions, such as gene integration in the bacterial chromosome, are rarely employed and their advantages are still a matter of debate.Plant plastidial NAD kinases (NADK; ATP:NAD 2′-phosphotransferase, EC 2.7.1.23) are a classic example of proteins with high molecular weight, that are difficult to express and purify with traditional T7-based technology. We therefore compared plasmid-driven and chromosomal-driven expression of the Arabidopsis thaliana NADK2 protein, using a proprietary counter-selection tool, COLIBELT®, that allows scar-free and marker-free chromosomal modifications.Here we show that chromosomal-driven expression allowed recovery of more active NADK2 protein than classic T7 expression systems, as well as better production, thus confirming that expression from one single chromosomal copy is preferable to plasmid-driven expression and might be appealing for both basic and applied research.

Mirror cutting-assisted orthogonal digestion enabling large-scale and accurate protein complex characterization

蛋白质主要以复合物的形式参与各项生命活动。化学交联质谱(CXMS)技术作为近年来新兴的蛋白质复合物解析技术,不仅可实现蛋白质复合物规模化解析,而且普遍适用于任意相对分子质量和纯度的蛋白质复合物样品,因此已成为X-射线晶体衍射技术、冷冻电镜技术等蛋白质复合物解析经典技术的重要补充。目前,CXMS主要采用胰蛋白酶将交联后的蛋白质复合物进行酶切,并进行质谱鉴定。然而鉴定到的交联肽段谱图中b/y特征碎片离子的鉴定数目不足,且因肽段序列匹配连续性较差导致谱图可信度相对较低,影响了蛋白质复合物交联位点的鉴定精准度。该文基于镜像酶正交切割的互补特性,采用胰蛋白酶镜像酶与胰蛋白酶联合酶切的方法,对交联后的蛋白质复合物分别进行酶切,再利用液相色谱-质谱联用技术对酶解产生的交联肽段进行鉴定,进而实现蛋白质复合物的组成、相互作用和结构位点距离约束的解析。对牛血清白蛋白和大肠杆菌全蛋白的交联肽段的分析结果表明,该方法通过增加交联肽段谱图中特征碎裂离子的数目和连续性,鉴定到的大肠杆菌全蛋白的交联位点,较单一胰蛋白酶酶切,提高了16%。因此,镜像酶正交酶切策略能有效提高交联肽段的鉴定准确度和覆盖度,有望为实现规模化的蛋白质复合物精准解析提供新思路。

Affinity Purification and Coimmunoprecipitation of Transenvelope Protein Complexes in Gram-Negative Bacteria.

Multiprotein complexes are important machineries that organize a large number of different proteins into functional units. Studying protein-protein interactions in the complexes, rather than individual proteins, is a fundamental step to gaining functional insights into a biological process. Here, we present the sequential affinity purification and coimmunoprecipitation system that was applied to enable the efficient purification of all the proteins that compose the Lpt system complex in Escherichia coli and their identification by western blotting and mass spectrometry (MS).

X-ray Crystal Structure Analysis of VHH-Protein Antigen Complexes.

VHHs are antigen-binding domains cloned from heavy-chain antibodies found in camelids. These proteins have generated considerable interest in a variety of applications as research reagents, crystallization chaperones, and therapeutics. The evolutionary adaptations of VHHs have resulted in biophysical properties and antigen-binding modalities which are distinct from those of conventional antibodies. A detailed molecular analysis of VHH interactions with their cognate protein antigens is valuable for understanding structure-function relationships and for protein engineering. The majority of VHHs bind to folded proteins and thus recognize discontinuous three-dimensional epitopes. While multiple approaches exist for dissecting the interaction between a protein antigen and a VHH, X-ray crystallography remains the highest resolution method available. Here, we provide an updated procedure for determining and analyzing the X-ray structure of a VHH in complex with a protein antigen. We describe the recombinant expression and purification of VHHs and protein antigens, purification and analysis of protein complexes, crystallization, and optimization, X-ray structure determination by molecular replacement, and analysis of the complex.

The effect of exogenous factors on the polyenzymatic activity of RuBisCO and ATP synthase of chloroplasts from pea leaves

Aim. To isolate and purify protein complexes – ATP synthase and RuBisCO – from pea leaf chloroplasts and study the effect of a microbiological fertilizer “Extracon” and sulfonamide inhibitors acetazolamide and ethoxyzolamide on the enzymatic activity of these proteins.Materials and methods. Chloroplasts were isolated from the leaves of two-week-old pea sprouts, protein complexes of purified thylakoid membranes were solubilized with digitonin (10 mg of digitonin per 1 mg of protein), the protein concentration was determined according to Lowry. Native electrophoresis with displacement of the charge of the soluble protein fraction from the chloroplast stroma, as well as membrane proteins, was carried out in the modified system of Anderson et al., Kolisnichenko et al. A modified Lemmley system was applied to the protein electrophoresis in the polyacrylamide gel in the presence of sodium dodecyl sulfate. The methods of Alain and Hintsik, as well as Gomorrah were used to determine the ATPase activity in the polyacrylamide gel. Visualization of the carbonic anhydrase activity in the polyacrylamide gel was performed by the method of Edwards and Petton. Results and discussion. Using physicochemical methods of potentiometry, spectrophotometry the ATPase, carbonic anhydrase and esterase activities of the enzymes were studied. The results obtained indicate that specific carbonic anhydrase inhibitors (acetazolamide and ethoxyzolamide) also block the esterase and ATPase activity of the enzyme complexes. “Extracon” (a multifunctional microbiological preparation) almost 1.5 times increases the activity of the enzymes, showing a complex activating effect of the fertilizer on both light and dark reactions of photosynthesis.Conclusions. The method of identification and isolation of RuBisCO and ATP synthase on the basis of two-dimensional electrophoresis and electrophoretic elution has been proposed. It allows determining the presence of certain enzyme activity of complexes at first in SDS plates (express analysis) and further to study the effect of various factors of endogenous and exogenous origin on the enzymatic properties of electrophoretically pure enzymes. The use of two-dimensional electrophoresis as a tool for assessing the impact of various factors of endogenous and exogenous origin on the plant cell and the plant as a whole through constant monitoring of the work and activity of enzyme systems of the plant cell is promising.

Purification of MAP-kinase protein complexes and identification of candidate components by XL-TAP-MS.

The purification of low-abundance protein complexes and detection of in vivo protein-protein interactions in complex biological samples remains a challenging task. Here, we devised crosslinking and tandem affinity purification coupled to mass spectrometry (XL-TAP-MS), a quantitative proteomics approach for analyzing tandem affinity-purified, crosslinked protein complexes from plant tissues. We exemplarily applied XL-TAP-MS to study the MKK2-Mitogen-activated protein kinase (MPK4) signaling module in Arabidopsis thaliana. A tandem affinity tag consisting of an in vivo-biotinylated protein domain flanked by two hexahistidine sequences was adopted to allow for the affinity-based isolation of formaldehyde-crosslinked protein complexes under fully denaturing conditions. Combined with 15N stable isotopic labeling and tandem MS we captured and identified a total of 107 MKK2-MPK4 module-interacting proteins. Consistent with the role of the MPK signaling module in plant immunity, many of the module-interacting proteins are involved in the biotic and abiotic stress response of Arabidopsis. Validation of binary protein-protein interactions by in planta split-luciferase assays and in vitro kinase assays disclosed several direct phosphorylation targets of MPK4. Together, the XL-TAP-MS approach purifies low abundance protein complexes from biological samples and discovers previously unknown protein-protein interactions.

Abstract 08: Functional Characterisation Of Uromodulin Promoter Risk Variants Associated With Blood Pressure

Using a genome wide association study (GWAS), we identified a locus on the promoter of the human uromodulin (UMOD) gene associating with primary hypertension. In this study we aimed to identify genotype-differential protein binding partners which influence UMOD expression and to examine pathways linking UMOD expression to blood pressure variability in humans.We examined a variant on this locus, by incubating 50mer oligonucleotides featuring alternate alleles (C/G) with HEK-293 nuclear protein extract. Subsequently, we performed an electrophoretic mobility shift assay (EMSA) and purified protein complexes to analyse by Tandem-mass-spectroscopy (MS2). Filtering for peptide matches &gt;20, we interpreted these data by Ingenuity Pathway Analysis (IPA). From a bank of 98 human renal biopsy samples, we assessed UMOD expression by qRT-PCR. RNA-seq was performed using the NextSeq-500 system on a subset of high and low UMOD expressors (n=3/ group). Differentially expressed (false-discovery-rate (FDR) &lt;0.05) genes were identified, with strong correlations in these data validated by qRT-PCR. Implicated pathways were interpreted using both IPA and gene set enrichment analysis (GSEA).The C allele at our risk variant displayed greater binding affinity versus the G allele; 0.77 (0.13, 1.40, 95%CI). In contrast no difference in binding affinity was observed between the T and C alleles of our control variant, 0.08 ( -1.22, 1.39, 95%CI). Mass spectroscopy of EMSA complexes identified PARP1 binding at this target. By IPA we identified a complex between PARP1 and HNF1β as driving expression of UMOD. RNA-sequencing identified positive correlation in expression of UMOD and the Na-K-Cl cotransporter (NKCC2) (FDR=2.58e-9, log2-fold-change [L2FC]= 3.3), the renal outer medullary potassium channel (ROMK) (FDR=3.69e-2, L2FC= 2.79) and the epithelial sodium channel (ENaC) (FDR=1.83e-2, L2FC= 1.25). We validated this coexpression between UMOD and NKCC2 by qRT-PCR and show a significant correlation (p=2.2e-16, R2= 0.844, n=84).Together these data suggest our risk variant contributes to differences in UMOD expression via a PARP1:HNF1β transcriptional complex and these differences associate with blood pressure through sodium homeostasis in the human kidney.

SNP rs4971059 predisposes to breast carcinogenesis and chemoresistance via TRIM46-mediated HDAC1 degradation.

Identification of the driving force behind malignant transformation holds the promise to combat the relapse and therapeutic resistance of cancer. We report here that the single nucleotide polymorphism (SNP) rs4971059, one of 65 new breast cancer risk loci identified in a recent genome-wide association study (GWAS), functions as an active enhancer of TRIM46 expression. Recreating the G-to-A polymorphic switch caused by the SNP via CRISPR/Cas9-mediated homologous recombination leads to an overt upregulation of TRIM46. We find that TRIM46 is a ubiquitin ligase that targets histone deacetylase HDAC1 for ubiquitination and degradation and that the TRIM46-HDAC1 axis regulates a panel of genes, including ones critically involved in DNA replication and repair. Consequently, TRIM46 promotes breast cancer cell proliferation and chemoresistance invitro and accelerates tumor growth invivo. Moreover, TRIM46 is frequently overexpressed in breast carcinomas, and its expression is correlated with lower HDAC1 expression, higher histological grades, and worse prognosis of the patients. Together, our study links SNP rs4971059 to replication and to breast carcinogenesis and chemoresistance and support thepursuit of TRIM46 as a potential target for breast cancer intervention.

Proteomic analysis identifies key differences in the cardiac interactomes of dystrophin and micro-dystrophin.

ΔR4-R23/ΔCT micro-dystrophin (μDys) is a miniaturized version of dystrophin currently evaluated in a Duchenne muscular dystrophy (DMD) gene therapy trial to treat skeletal and cardiac muscle disease. In pre-clinical studies, μDys efficiently rescues cardiac histopathology, but only partially normalizes cardiac function. To gain insights into factors that may impact the cardiac therapeutic efficacy of μDys, we compared by mass spectrometry the composition of purified dystrophin and μDys protein complexes in the mouse heart. We report that compared to dystrophin, μDys has altered associations with α1- and β2-syntrophins, as well as cavins, a group of caveolae-associated signaling proteins. In particular, we found that membrane localization of cavin-1 and cavin-4 in cardiomyocytes requires dystrophin and is profoundly disrupted in the heart of mdx5cv mice, a model of DMD. Following cardiac stress/damage, membrane-associated cavin-4 recruits the signaling molecule ERK to caveolae, which activates key cardio-protective responses. Evaluation of ERK signaling revealed a profound inhibition, below physiological baseline, in the mdx5cv mouse heart. Expression of μDys in mdx5cv mice prevented the development of cardiac histopathology but did not rescue membrane localization of cavins nor did it normalize ERK signaling. Our study provides the first comparative analysis of purified protein complexes assembled in vivo by full-length dystrophin and a therapeutic micro-dystrophin construct. This has revealed disruptions in cavins and ERK signaling that may contribute to DMD cardiomyopathy. This new knowledge is important for ongoing efforts to prevent and treat heart disease in DMD patients.

Novel Signaling Hub of Insulin Receptor Dystrophin Glycoprotein Complex and Plakoglobin Regulates Muscle Size

Signaling through the insulin receptor governs central physiological functions related to cell growth and metabolism. Here we show by tandem native protein complex purification approach and super-resolution STED microscopy that insulin receptor activity requires association with the fundamental structural module in muscle, the dystrophin glycoprotein complex (DGC), and the desmosomal component plakoglobin (γ-catenin). These protein assemblies are also present in heart and liver. The integrity of this high-molecular-mass assembly renders skeletal muscle susceptibility to insulin, because DGC-insulin receptor dissociation by plakoglobin downregulation reduces insulin signaling and causes atrophy. Furthermore, low insulin receptor activity in muscles from diabetic or fasted mice decreases plakoglobin-DGC-insulin receptor content on the plasma membrane, but not when plakoglobin is overexpressed. By masking β-dystroglycan LIR domains, plakoglobin prevents autophagic clearance of plakoglobin-DGC-insulin receptor co-assemblies and maintains their function. Our findings establish DGC as a signaling hub, and provide a possible mechanism for the insulin resistance in Duchenne Muscular Dystrophy, and for the cardiomyopathies seen with plakoglobin mutations.

Discovery and Characterization of an ALFA-Tag-Specific Affinity Resin Optimized for Protein Purification at Low Temperatures in Physiological Buffer.

Epitope tags are widely employed as tools to detect, purify and manipulate proteins in various experimental systems. We recently introduced the ALFA-tag together with two ALFA-specific single-domain antibodies (sdAbs), NbALFA and NbALFAPE, featuring high or intermediate affinity, respectively. Together, the ALFA system can be employed for a broad range of applications in microscopy, cell biology and biochemistry requiring either extraordinarily stable binding or mild competitive elution at room temperature. In order to further enhance the versatility of the ALFA system, we, here, aimed at developing an sdAb optimized for efficient elution at low temperatures. To achieve this, we followed a stringent selection scheme tailored to the specific application. We found candidates combining a fast capture of ALFA-tagged proteins with an efficient competitive elution at 4 °C in physiological buffer. Importantly, by employing a structure-guided semisynthetic library based on well-characterized NbALFA variants, the high specificity and consistent binding of proteins harboring ALFA-tags at either terminus could be maintained. ALFA SelectorCE, a resin presenting the cold-elutable NbALFACE, is an ideal tool for the one-step purification of sensitive protein complexes or temperature-labile enzymes. We believe that the general approach followed during the selection and screening can be transferred to other challenging sdAb discovery projects.

Tandem Affinity Purification (TAP) of Low-Abundance Protein Complexes in Filamentous Fungi Demonstrated Using Magnaporthe oryzae.

Protein-protein interactions underlie cellular structure and function. In recent years, a number of methods have been developed for the identification of protein complexes and component proteins involved in the control of various biological pathways. Tandem affinity purification (TAP) coupled with mass spectrometry (MS) is a powerful method enabling the isolation of high-purity native protein complexes under mild conditions by performing two sequential purification steps using two different epitope tags. In this protocol, we describe a TAP-MS methodology for identifying protein-protein interactions present at very low levels in the fungal cell. Using the 6xHis-3xFLAG double tag, we start the affinity purification process for our protein of interest using high-capacity Ni2+ columns. This allows for greatly increased sample input compared to antibody-based first-step purification in conventional TAP protocols and provides a large amount of highly concentrated and preliminarily purified protein complexes to be used in a second purification step involving FLAG immunoprecipitation. The second step greatly facilitates the capture of low-level interacting partners under in vivo conditions. Our TAP-MS method has been proven to secure the characterization of low-abundance protein complexes under physiological conditions with high efficiency, specificity, and economy in the filamentous fungus Magnaporthe oryzae and might benefit gene function and proteomics studies in plants and other research fields.