Native Post-translational Modifications Research Articles

Structural insights into actin isoforms.

Actin isoforms organize into distinct networks that are essential for the normal function of eukaryotic cells. Despite a high level of sequence and structure conservation, subtle differences in their design principles determine the interaction with myosin motors and actin-binding proteins. Therefore, identifying how the structure of actin isoforms relates to function is important for our understanding of normal cytoskeletal physiology. Here, we report the high-resolution structures of filamentous skeletal muscle α-actin (3.37 Å), cardiac muscle α-actin (3.07 Å), ß-actin (2.99 Å), and γ-actin (3.38 Å) in the Mg2+·ADP state with their native post-translational modifications. The structures revealed isoform-specific conformations of the N-terminus that shift closer to the filament surface upon myosin binding, thereby establishing isoform-specific interfaces. Collectively, the structures of single-isotype, post-translationally modified bare skeletal muscle α-actin, cardiac muscle α-actin, ß-actin, and γ-actin reveal general principles, similarities, and differences between isoforms. They complement the repertoire of known actin structures and allow for a comprehensive understanding of in vitro and in vivo functions of actin isoforms.

A solution to the long-standing problem of actin expression and purification

Actin is the most abundant protein in the cytoplasm of eukaryotic cells and interacts with hundreds of proteins to perform essential functions, including cell motility and cytokinesis. Numerous diseases are caused by mutations in actin, but studying the biochemistry of actin mutants is difficult without a reliable method to obtain recombinant actin. Moreover, biochemical studies have typically used tissue-purified α-actin, whereas humans express six isoforms that are nearly identical but perform specialized functions and are difficult to obtain in isolation from natural sources. Here, we describe a solution to the problem of actin expression and purification. We obtain high yields of actin isoforms in human Expi293F cells. Experiments along the multistep purification protocol demonstrate the removal of endogenous actin and the functional integrity of recombinant actin isoforms. Proteomics analysis of endogenous vs. recombinant actin isoforms confirms the presence of native posttranslational modifications, including N-terminal acetylation achieved after affinity-tag removal using the actin-specific enzyme Naa80. The method described facilitates studies of actin under fully native conditions to determine differences among isoforms and the effects of disease-causing mutations that occur in all six isoforms.

An Optimized Purification Design for Extracting Active ADAMTS13 from Conditioned Media

ADAMTS13 is a hemostatic enzyme that breaks down pro-thrombotic ultra-large multimers of von Willebrand factor (VWF). The deficiency of ADAMTS13 increases VWF-mediated thrombogenic potential and may lead to thrombotic thrombocytopenic purpura (TTP). Recently, clinical studies have shown the development of acquired TTP after COVID-19 infection and a correlation between low ADAMTS13 plasma levels and increased mortality. As a result, investigating ADAMTS13 as a potential recombinant therapeutic is of broad interest in the field of hematology. ADAMTS13 is considered challenging to purify in its biologically active state. Current purification methods utilize immobilized metal ions, which can interfere with ADAMTS13 metalloprotease activity. For this reason, we optimized an alternative strategy to isolate milligram quantities of highly active recombinant ADAMTS13 (rADAMTS13) from conditioned media after exogenous expression in human cell line, HEK293. HEK293 cells stably expressing C-terminal V5-His-tagged ADAMTS13 were grown in two parallel systems, culture bottles and flasks, for identifying an optimal cultivation strategy. Subsequently, we employed anion exchange followed by anti-V5-tag affinity chromatography to purify rADAMTS13, and extracted rADAMTS13 of high specific activity while preserving its native post-translational modifications. In addition, this process has been optimized and scaled up to produce active rADAMTS13 at levels sufficient for laboratory-scale structural, enzymatic, and biochemical studies.

Chemical Synthesis of Phosphorylated Insulin-like Growth Factor Binding Protein 2.

Chemical protein synthesis is a powerful avenue for accessing homogeneously modified proteins. While a significant number of small modified proteins bearing native post-translational modifications and non-natural modifications have been generated to date, access to larger targets has proved challenging. Herein, we describe the use of two ligation manifolds, namely, diselenide-selenoester ligation and native chemical ligation, to assemble a 31.5 kDa phosphorylated insulin-like growth factor binding protein (IGFBP-2) that comprises 290 amino acid residues, a phosphoserine post-translational modification, and nine disulfide bonds.

Diselenide-selenoester ligation for chemical protein synthesis.

Chemoselective peptide ligation methods have provided synthetic access to numerous proteins, including those bearing native post-translational modifications and unnatural labels. This protocol outlines the chemical synthesis of proteins using a recently discovered reaction (diselenide-selenoester ligation (DSL)) in a rapid, additive-free manner. After ligation, the products can be chemoselectively deselenized to produce native peptide and protein products. We describe methods for the synthesis of suitably functionalized peptide diselenide and peptide selenoester fragments via Fmoc-solid-phase peptide synthesis (SPPS) protocols, fusion of these fragments by DSL, and the chemoselective deselenization of the ligation products to generate native synthetic proteins. We demonstrate the method's utility through the total chemical synthesis of the post-translationally modified collagenous domain of the hormone adiponectin via DSL-deselenization at selenocystine (the oxidized form of selenocysteine) and the rapid preparation of two tick-derived thrombin-inhibiting proteins by DSL-deselenization at β-selenoaspartate and γ-selenoglutamate. This method should find widespread use for the rapid synthesis of proteins, including cases in which other peptide ligation methods cannot be used (or cannot be used efficiently), e.g., at sterically hindered or deactivated acyl donors. The method's speed and efficiency may render it useful in the generation of synthetic protein libraries. Each protein discussed can be synthesized within 15 working days from resin loading and can be readily produced by practitioners with master's-level experience in organic chemistry. Each synthesis using these protocols was performed independently by two labs (one academic and one industrial), which attained comparable yields of the protein products.

Development and Evaluation of an Optimal Human Single-Chain Variable Fragment-Derived BCMA-Targeted CAR T Cell Vector.

B cell maturation antigen (BCMA) has recently been identified as an important multiple myeloma (MM)-specific target for chimeric antigen receptor (CAR) Tcell therapy. In CAR Tcell therapy targeting CD19 for lymphoma, host immune anti-murine CAR responses limited the efficacy of repeat dosing and possibly long-term persistence. This clinically relevant concern can be addressed by generating a CAR incorporating a human single-chain variable fragment (scFv). We screened a human B cell-derived scFv phage display library and identified a panel of BCMA-specific clones from which human CARs were engineered. Despite a narrow range of affinity for BCMA, dramatic differences in CAR Tcell expansion were observed between unique scFvs in a repeat antigen stimulation assay. These results were confirmed by screening in a MM xenograft model, where only the top preforming CARs from the repeat antigen stimulation assay eradicated disease and prolonged survival. The results of this screening identified a highly effective CAR Tcell therapy with properties, including rapid invivo expansion (>10,000-fold, day 6), eradication of large tumor burden, and durable protection to tumor re-challenge. We generated a bicistronic construct including a second-generation CAR and a truncated-epithelial growth factor receptor marker. CAR Tcell vectors stemming from this work are under clinical investigation.

Expression and Purification of Protein Complexes Suitable for Structural Studies Using Mammalian HEK 293F Cells.

Prokaryotic expression systems have been widely used to express proteins for structural studies. Such expression systems have the advantage of being economical, straightforward and fast. However, for many eukaryotic proteins and particularly protein complexes, bacterial expression systems do not produce significant yields of soluble protein. This may result from failure to efficiently transcribe/translate the required protein or may result from the formation of insoluble aggregates known as inclusion bodies. Mammalian expression systems can often produce natively folded proteins, sometimes with native post-translational modifications. However, such expression systems are underutilized due to the perception that they are costly, technically challenging and result in limited protein yields. In fact, HEK 293F cells are straightforward to grow, transfect with high efficiency and often produce significant yields of recombinant proteins. In this unit, we describe a method to express and purify milligram quantities of a human protein complex from HEK 293F cells grown in suspension transiently transfected with the appropriate plasmids. © 2017 by John Wiley & Sons, Inc.

Single-molecule sorting of DNA helicases

DNA helicases participate in virtually all aspects of cellular DNA metabolism by using ATP-fueled directional translocation along the DNA molecule to unwind DNA duplexes, dismantle nucleoprotein complexes, and remove non-canonical DNA structures. Post-translational modifications and helicase interacting partners are often viewed as determining factors in controlling the switch between bona fide helicase activity and other functions of the enzyme that do not involve duplex separation. The bottleneck in developing a mechanistic understanding of human helicases and their control by post-translational modifications is obtaining sufficient quantities of the modified helicase for traditional structure-functional analyses and biochemical reconstitutions. This limitation can be overcome by single-molecule analysis, where several hundred surface-tethered molecules are sufficient to obtain a complete kinetic and thermodynamic description of the helicase-mediated substrate binding and rearrangement. Synthetic oligonucleotides site-specifically labeled with Cy3 and Cy5 fluorophores can be used to create a variety of DNA substrates that can be used to characterize DNA binding, as well as helicase translocation and duplex unwinding activities. This chapter describes “single-molecule sorting”, a robust experimental approach to simultaneously quantify, and distinguish the activities of helicases carrying their native post-translational modifications. Using this technique, a DNA helicase of interest can be produced and biotinylated in human cells to enable surface-tethering for the single-molecule studies by total internal reflection fluorescence microscopy. The pool of helicases extracted from the cells is expected to contain a mixture of post-translationally modified and unmodified enzymes, and the contributions from either population can be monitored separately, but in the same experiment providing a direct route to evaluating the effect of a given modification.

Re-print of "Histone extraction protocol from the two model diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana"

Post-translational modifications of histones affect many biological processes by influencing higher order chromatin structure that affects gene and genome regulation. It is therefore important to develop methods for extracting histones while maintaining their native post-translational modifications. While histone extraction protocols have been developed in multicellular and single celled organisms such as yeast and Arabidopsis, they are inefficient in diatoms that have a silica cell wall that is likely to hinder histone extraction. We report in this work a rapid and reliable method for extraction of large amounts of high quality histones from the two model diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. The protocol is an important enabling step permitting downstream applications such as western blotting and mass spectrometry.

Single molecule sorting of the DNA repair machines (477.1)

The functional diversity and dynamic nature of the eukaryotic proteome are highly enhanced by posttranslational modifications (PTMs). DNA repair enzymes are often controlled by reversible PTMs that modulate their activities and interactions with their targets to broaden the range of repairable DNA lesions. A fraction of a DNA repair protein carrying an activating or modifying PTM may greatly influence cellular fate. To expedite the quantification, analysis and comparison of distinct posttranslationally modified pools of a protein we developed a technique to simultaneously, but separately evaluate the activities of modified and unmodified proteins extracted from the human cells. I will describe the experimental strategy behind “single‐molecule sorting”, as well as its application to the analysis of several DNA repair helicases, which produce transient single‐stranded intermediates of DNA replication, recombination and repair. Individual protein molecules isolated from human cells with their native PTMs are analyzed using total internal reflection fluorescence microscopy (TIRFM). Separation of the activity trajectories originated from modified and unmodified enzymes allows us to quantify the fraction of the protein modified in the cell and to assess the effect of each modification or a combination of modifications on the target activity.

Identification of Ligand–Target Pairs from Combined Libraries of Small Molecules and Unpurified Protein Targets in Cell Lysates

We describe the development and validation of interaction determination using unpurified proteins (IDUP), a method that selectively amplifies DNA sequences identifying ligand+target pairs from a mixture of DNA-linked small molecules and unpurified protein targets in cell lysates. By operating in cell lysates, IDUP preserves native post-translational modifications and interactions with endogenous binding partners, thereby enabling the study of difficult-to-purify targets and increasing the potential biological relevance of detected interactions compared with methods that require purified proteins. In IDUP, target proteins are associated with DNA oligonucleotide tags either non-covalently using a DNA-linked antibody or covalently using a SNAP-tag. Ligand–target binding promotes hybridization of a self-priming hairpin that is extended by a DNA polymerase to create a DNA strand that contains sequences identifying both the target and its ligand. These sequences encoding ligand+target pairs are selectively amplified by PCR and revealed by high-throughput DNA sequencing. IDUP can respond to the effect of affinity-modulating adaptor proteins in cell lysates that would be absent in ligand screening or selection methods using a purified protein target. This capability was exemplified by the 100-fold amplification of DNA sequences encoding FRB+rapamycin or FKBP+rapamycin in samples overexpressing both FRB and FKBP (FRB·rapamycin+FKBP, Kd ≈ 100 fM; FKBP·rapamycin+FRB, Kd = 12 nM). In contrast, these sequences were amplified 10-fold less efficiently in samples overexpressing either FRB or FKBP alone (rapamycin+FKBP, Kd ≈ 0.2 nM; rapamcyin+FRB, Kd = 26 μM). Finally, IDUP was used to process a model library of DNA-linked small molecules and a model library of cell lysates expressing SNAP-target fusions combined in a single sample. In this library×library experiment, IDUP resulted in enrichment of sequences corresponding to five known ligand+target pairs ranging in binding affinity from Kd = 0.2 nM to 3.2 μM out of 67,858 possible combinations, with no false positive signals enriched to the same extent as that of any of the bona fide ligand+target pairs.

Histone extraction protocol from the two model diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana

Post-translational modifications of histones affect many biological processes by influencing higher order chromatin structure that affects gene and genome regulation. It is therefore important to develop methods for extracting histones while maintaining their native post-translational modifications. While histone extraction protocols have been developed in multicellular and single celled organisms such as yeast and Arabidopsis, they are inefficient in diatoms that have a silica cell wall that is likely to hinder histone extraction. We report in this work a rapid and reliable method for extraction of large amounts of high quality histones from the two model diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. The protocol is an important enabling step permitting downstream applications such as western blotting and mass spectrometry.

Single-molecule sorting reveals how ubiquitylation affects substrate recognition and activities of FBH1 helicase

DNA repair helicases function in the cell to separate DNA duplexes or remodel nucleoprotein complexes. These functions are influenced by sensing and signaling; the cellular pool of a DNA helicase may contain subpopulations of enzymes carrying different post-translational modifications and performing distinct biochemical functions. Here, we report a novel experimental strategy, single-molecule sorting, which overcomes difficulties associated with comprehensive analysis of heterologously modified pool of proteins. This methodology was applied to visualize human DNA helicase F-box–containing DNA helicase (FBH1) acting on the DNA structures resembling a stalled or collapsed replication fork and its interactions with RAD51 nucleoprotein filament. Individual helicase molecules isolated from human cells with their native post-translational modifications were analyzed using total internal reflection fluorescence microscopy. Separation of the activity trajectories originated from ubiquitylated and non-ubiquitylated FBH1 molecules revealed that ubiquitylation affects FBH1 interaction with the RAD51 nucleoprotein filament, but not its translocase and helicase activities.

Identification and characterization of posttranslational modifications of proteins by MALDI ion trap mass spectrometry.

Matrix-assisted laser desorption/ionization (MALDI) ion trap mass spectrometry is shown to be a powerful tool for the elucidation of protein modifications. Low-energy covalent bonds that originate from certain posttranslational modifications dissociate preferentially to produce characteristic mass spectrometric signatures that prove useful for the accurate, confident identification and characterization of such modifications. Because the MALDI ion trap is an authentic tandem mass spectrometer, it proves feasible to acquire secondary information to test hypotheses as to the nature and site of the putative modifications--further increasing the reliability of the tool. The method combines the advantageous features of MALDI (i.e., the ability to measure the same sample repeatedly, to measure unfractionated complex mixtures without the need for sample cleaning, and to determine peptide mixtures with subpicomole sensitivity) with the ease and the speed of the ion trap measurement. We demonstrate how the unique properties of MALDI ion trap MS can be used to address problems involving the determination of both native posttranslational modifications of proteins (e.g., disulfide mapping, glycosylation determination, and phosphorylation determination) and non-native chemical modifications of proteins (e.g., methionine oxidation and photo-cross-linking of proteins with DNA).

Expression of a Recombinant Baculovirus for Vitronectin in Insect Cells: Purification, Characterization of Post-translational Modifications and Functional Studies of the Recombinant Protein

Vitronectin (VN) is a cell attachment glycoprotein present in plasma and the extracellular matrix that also has multiple regulatory roles in the complement, coagulation, and fibrinolytic systems. VN stabilizes plasminogen activator inhibitor-1 (PAI-1) with potential binding sites reported at both the somatomedin B and heparin binding domains. In order to perform detailed structure-function analyses of VN, we have produced and expressed a recombinant baculovirus containing the cDNA of human VN. Recombinant VN (rVN) was secreted as a single chain 70-kDa protein into the serum-free culture medium of infected Spodoptera frugiperda cells. Maximum rVN levels of 33.9 mg/liter were obtained 48 hours post-infection. Purification of rVN from serum-free medium was achieved with single-step anion-exchange chromatography. Several native post-translational modifications were demonstrated in rVN including the presence of N-linked oligosaccharides, tyrosine sulfation, and serine phosphorylation. Recombinant VN retained cell and PAI-1 binding capacity. Therefore, rVN expressed for the first time in insect cells retains many post-translational modifications and functional activities and will be useful for additional structure-function analyses.