Protein Ligation Research Articles

Enantioselective Synthesis of Chiral Sulfonimidoyl Fluorides Facilitates Stereospecific SuFEx Click Chemistry.

Sulfur-centered electrophilic 'warheads' have emerged as key components for chemical proteomic probes through sulfur-exchange chemistry (SuFEx) with protein nucleophiles. Among these functional groups, sulfonimidoyl fluorides (SIFs) stand out for their modifiable sites, tunable electrophilicities, and chiral sulfur-center, presenting exciting possibilities for new covalent chemical probes. However, the synthetic access to chiral SIFs has been a challenge, limiting their exploration and applications. In this study, we describe a convenient route to obtain chiral SIFs from readily available sulfenamides via a series of one-pot tandem reactions with high enantiomeric excess (ees). The resulting chiral SIFs were further converted into a diverse array of chiral S(VI) derivatives under mild conditions or in buffer solutions. Most significantly, the specificity of the chiral SIFs in protein ligation experiments underscored the critical role of sulfur-center chirality in the design and screening of more-selective covalent probes and therapeutics.

Enantioselective Synthesis of Chiral Sulfonimidoyl Fluorides Facilitates Stereospecific SuFEx Click Chemistry

Sulfur‐centered electrophilic 'warheads' have emerged as key components for chemical proteomic probes through sulfur‐exchange chemistry (SuFEx) with protein nucleophiles. Among these functional groups, sulfonimidoyl fluorides (SIFs) stand out for their modifiable sites, tunable electrophilicities, and chiral sulfur‐center, presenting exciting possibilities for new covalent chemical probes. However, the synthetic access to chiral SIFs has been a challenge, limiting their exploration and applications. In this study, we describe a convenient route to obtain chiral SIFs from readily available sulfenamides via a series of one‐pot tandem reactions with high enantiomeric excess (ees). The resulting chiral SIFs were further converted into a diverse array of chiral S(VI) derivatives under mild conditions or in buffer solutions. Most significantly, the specificity of the chiral SIFs in protein ligation experiments underscored the critical role of sulfur‐center chirality in the design and screening of more‐selective covalent probes and therapeutics.

Development and applications of enzymatic peptide and protein ligation.

Chemical synthesis of complex peptides and proteins continues to play increasingly important roles in industry and academia, where strategies for covalent ligation of two or more peptide fragments to produce longer peptides and proteins in convergent manners have become critical. In recent decades, efficient and site-selective ligation strategies mediated by exploiting the biocatalytic capacity of nature's diverse toolkit (i.e., enzymes) have been widely recognized as a powerful extension of existing chemical strategies. In this review, we present a chronological overview of the development of proteases, transpeptidases, transglutaminases, and ubiquitin ligases. We survey the different properties between the ligation reactions of various enzymes, including the selectivity and efficiency of the reaction, the ligation "scar" left in the product, the type of amide bond formed (natural or isopeptide), the synthetic availability of the reactants, and whether the enzymes are orthogonal to another. This review also describes how the inherent specificity of these enzymes can be exploited for peptide and protein ligation.

Developing Porous Protein Cage Nanoparticles as Cargo-Loadable and Ligand-Displayable Modular Delivery Nanoplatforms.

Protein cage nanoparticles, self-assembled from protein subunits, provide distinct exterior and interior spaces and can carry diagnostic and/or therapeutic cargo agents through chemical conjugation, in vitro disassembly/reassembly process, or assembly-mediated encapsulation. Here, we developed porous SpyCatcher-mi3 (SC-mi3) as modular delivery nanoplatforms, capable of loading cargos through pores and displaying targeting ligands using SpyCatchers (SC) as anchors for SpyTagged (ST) ligands. Fluorescent dyes (F5M and A647) and a pH-sensitive prodrug (Aldox) were conjugated to the interior surface cysteines of SC-mi3, forming F5M@SC-mi3, A647@SC-mi3, and Aldox@SC-mi3. Subsequently, EGFR-binding affibody molecules (EGFRAfb) were displayed on the exterior surface of F5M@SC-mi3 and Aldox@SC-mi3 using the SC/ST protein ligation system, forming F5M@mi3/EGFRAfb and Aldox@mi3/EGFRAfb, respectively. F5M@mi3/EGFRAfb selectively bound to EGFR-overexpressing MDA-MB-468 cells, visualizing the target cancer cells, while Aldox@mi3/EGFRAfb selectively delivered doxorubicin, leading to target-specific cancer cell death. To encapsulate large proteins within SC-mi3, biotins were initially conjugated to the interior surface (BPM@SC-mi3) and mSA2-fused protein cargo molecules (mSA2-HaloTag and mSA2-yCD) were successfully introduced through the pores and securely encapsulated, forming TMR-H@SC-mi3 and yCD@SC-mi3, respectively. Subsequent display of EGFRAfb on their surface allowed the visualization of target cancer cells using fluorescent HaloTag ligand labeling and facilitated the killing of target cancer cells by converting the prodrug 5-FC to the cytotoxic drug 5-FU. Modular functionalization of the two distinct spaces in porous SC-mi3 may offer opportunities for developing target-specific functional cargo-delivery nanoplatforms in biomedical fields.

Direct and Ultrasensitive Bioluminescent Detection of Intact Respiratory Viruses.

Respiratory viruses such as SARS-CoV-2, influenza, and respiratory syncytial virus (RSV) represent pressing health risks. Rapid diagnostic tests for these viruses detect single antigens or nucleic acids, which do not necessarily correlate with the amount of the intact virus. Instead, specific detection of intact respiratory virus particles may be more effective at assessing the contagiousness of a patient. Here, we report GLOVID, a modular biosensor platform to detect intact virions against a background of "free" viral proteins in solution. Our approach harnesses the multivalent display of distinct proteins on the surface of a viral particle to template the reconstitution of a split luciferase, allowing specific, single-step detection of intact influenza A and RSV virions corresponding to 0.1-0.3 fM of genomic units. The protein ligation system used to assemble GLOVID sensors is compatible with a broad range of binding domains, including nanobodies, scFv fragments, and cyclic peptides, which allows straightforward adjustment of the sensor platform to target different viruses.

Promotion of RNF168-Mediated Nucleosomal H2A Ubiquitylation by Structurally Defined K63-Polyubiquitylated Linker Histone H1.

The chemical synthesis of histones with homogeneous modifications is a powerful approach for quantitatively deciphering the functional crosstalk between different post-translational modifications (PTMs). In this study, we developed an expedient site-specific (poly)ubiquitylation strategy (CAEPL, Cysteine Aminoethylation coupled with Enzymatic Protein Ligation), which integrates the Cys-aminoethylation reaction with the process of ubiquitin-activating enzyme UBA1-assisted native chemical ligation. Using this strategy, we successfully prepared monoubiquitylated and K63-linked di- and tri-ubiquitylated linker histone H1.0 proteins, which were incorporated into individual chromatosomes. Quantitative biochemical analysis of different RNF168 constructs on H1 ubiquitylated chromatosomes with different ubiquitin chain lengths demonstrated that K63-linked polyubiquitylated H1.0 could directly stimulate RNF168 ubiquitylation activity by enhancing the affinity between RNF168 and the chromatosome. Subsequent cryo-EM structural analysis of the RNF168/UbcH5c-Ub/H1.0-K63-Ub3 chromatosome complex revealed the potential recruitment orientation between RNF168 UDM1 domain and K63-linked ubiquitin chain on H1.0. Finally, we explored the impact of H1.0 ubiquitylation on RNF168 activity in the context of asymmetric H1.0-K63-Ub3 di-nucleosome substrate, revealing a comparable stimulation effect of both the inter- and intra-nucleosomal crosstalk. Overall, our study highlights the significance of access to structurally defined polyubiquitylated H1.0 by the CAEPL strategy, enabling in-depth mechanistic investigations of in-trans PTM crosstalk between linker histone H1.0 and core histone H2A ubiquitylation.

Oxidative protein damage negatively affects protein-protein interaction: The case of KRAS-cRAF

Protein-protein interactions (PPIs) play crucial roles in cellular signaling, transmitting signals from the cell surface to its interior. One of the most important signaling cascades is the RAS-RAF-MEK-ERK pathway. This pathway is initiated by various upstream signaling reactions, including receptor tyrosine kinase (RTK) activation, and it controls many biological functions like cell proliferation, differentiation, and survival. Once RAS is activated, it binds RAF and relays the signal to downstream proteins. The RAS-binding domain (RBD) in RAF protein plays a crucial role in this process, facilitating the RAS-ERK pathway signaling.In this study, we explored the effect of oxidative stress induced by UV radiation on the KRAS-RBD interaction. Using the Split Intein-Mediated Protein Ligation (SIMPL) method, we assessed the impact of different UV doses on KRAS-RBD interactions and observed a disruption of this interaction at higher doses. UV-treated samples exhibited high levels of protein carbonylation, as detected by Oxime Blot and mass spectrometry (MS) analysis, indicating oxidative damage. The MS results provided detailed insights into specific carbonylation modifications on the KRAS protein.Our study demonstrates that protein oxidation and carbonylation can disrupt protein-protein interactions, specifically the KRAS/c-RAF interaction. These findings highlight the impact of oxidative stress on signaling pathways, such as those triggered by UV irradiation. A deeper understanding of these molecular changes may aid in developing therapies targeting diseases linked to oxidative stress, including cancer.

Next Generation SICLOPPS Screening for the Identification of Inhibitors of the HIF-1α/HIF-1β Protein-Protein Interaction.

Split-intein circular ligation of proteins and peptides (SICLOPPS) is a method for generating intracellular libraries of cyclic peptides that has yielded several first-in-class inhibitors. Here, we detail a revised high-content, high-throughput SICLOPPS screening protocol that utilizes next-generation sequencing, biopanning, and computational tools to identify hits against a given protein-protein interaction. We used this platform for the identification of inhibitors of the HIF-1α/HIF-1β protein-protein interaction. The revised platform resulted in a significantly higher positive hit rate than that previously reported for SICLOPPS screens, and the identified cyclic peptides were more active in vitro and in cells than our previously reported inhibitors. The platform detailed here may be used for the identification of inhibitors of a wide range of other targets.

Ultrasonic Control of Protein Splicing by Split Inteins.

Utilizing ultrasound as an external stimulus to remotely modulate the activity of proteins is an important aspect of sonopharmacology and establishes the basis for the emerging field of sonogenetics. Here, we describe an ultrasound-responsive protein splicing system that enables spatiotemporal control of split-intein-mediated protein ligation. The system utilizes engineered split inteins that are caged and can be activated by thrombin released from a high molar mass DNA-based carrier under focused ultrasound sonication. This approach represents a general method for controlling the functions of proteins of interest by ultrasound, as demonstrated here by the controlled synthesis of the superfolder green fluorescence protein (GFP) and calcitonin. Furthermore, calcitonin receptor-mediated signal transduction in cells was triggered by this system in vitro without harming cell viability. By expanding the sonogenetic toolbox with protein splicing technologies, this study provides a possible pathway to deploy ultrasound for remotely controlling a variety of protein functions in deep tissue in the future.

Bioluminescence Resonance Energy Transfer (BRET)-Mediated Protein Release from Self-Illuminating Photoresponsive Biomaterials.

Phototriggered release of various cargos, including soluble protein factors and small molecules, has the potential to correct aberrant biological events by offering spatiotemporal control over local therapeutic levels. However, the poor penetration depth of light historically limits implementation to subdermal regions, necessitating alternative methods of light delivery to achieve the full potential of photodynamic therapeutic release. Here, we introduce a strategy exploiting bioluminescence resonance energy transfer (BRET)-an energy transfer process between light-emitting Nanoluciferase (NLuc) and a photosensitive acceptor molecule-to drive biomolecule release from hydrogel biomaterials. Through a facile, one-pot, and high-yielding synthesis (60-70%), we synthesized a heterobifunctional ruthenium cross-linker bearing an aldehyde and an azide (CHO-Ru-N3), a compound that we demonstrate undergoes predictable exchange of the azide-bearing ligand under blue-green light irradiation (>550 nm). Following site-specific conjugation to NLuc via sortase-tag enhanced protein ligation (STEPL), the modified protein was covalently attached to a poly(ethylene glycol) (PEG)-based hydrogel via strain-promoted azide-alkyne cycloaddition (SPAAC). Leveraging the high photosensitivity of Ru compounds, we demonstrate rapid and equivalent release of epidermal growth factor (EGF) via either direct illumination or via BRET-based bioluminolysis. As NLuc-originated luminescence can be controlled equivalently throughout the body, we anticipate that this unique protein release strategy will find use for locally triggered drug delivery following systemic administration of a small molecule.

CD13-targeting and TRAIL-displaying Protein Nanoparticles Effectively Induce Apoptotic Cell Death of Acute Myeloid Leukemia, Prolonging Survival in Mouse Models

Acute myeloid leukemia (AML) is a rapidly proliferating blood cancer, necessitating treatments that specifically target and swiftly eradicate it. In this study, we develop an AML-specific, apoptotic cell death-inducing protein nanoparticle, AaLS/TRAIL/aCD13Nb, by simultaneously displaying multiple CD13-binding nanobodies (aCD13Nb) and Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) molecules on a single AaLS protein nanoparticle using the SpyCatcher/SpyTag protein ligation system. AaLS/TRAIL/aCD13Nb selectively binds to various CD13-overexpressing AML cell lines and effectively accumulates near U937 AML tumor sites through systemic administration, demonstrating its AML targeting capabilities. The tight binding of AaLS/TRAIL/aCD13Nb to CD13-overexpressing AML cells, mediated by aCD13Nb, results in close and continuous contact between TRAIL molecules and death receptors, triggering robust apoptotic cell death. Systemic administrations of AaLS/TRAIL/aCD13Nb into U937 AML-engrafted NSG mice significantly reduce the AML burden and nearly double the mice’s survival period, especially under advanced and severe AML conditions. Collectively, our study paves the way for targeted therapies in AML, utilizing protein nanoparticles as nanoplatforms. Substantial therapeutic efficacy across various cancers can be achieved by strategically combining cancer-specific targeting ligands with apoptotic cancer cell death-inducing molecules, tailored to specific cancer types.

SiMPl-GS: Advancing Cell Line Development via Synthetic Selection Marker for Next-Generation Biopharmaceutical Production.

Rapid and efficient cell line development (CLD) process is essential to expedite therapeutic protein development. However, the performance of widely used glutamine-based selection systems is limited by low selection efficiency, stringency, and the inability to select multiple genes. Therefore, an AND-gate synthetic selection system is rationally designed using split intein-mediated protein ligation of glutamine synthetase (GS) (SiMPl-GS). Split sites of the GS are selected using a computational approach and validated with GS-knockout Chinese hamster ovary cells for their potential to enable cell survival in a glutamine-free medium. In CLD, SiMPl-GS outperforms the wild-type GS by selectively enriching high producers. Unlike wild-type GS, SiMPl-GS results in cell pools in which most cells produce high levels of therapeutic proteins. Harnessing orthogonal split intein pairs further enables the selection of four plasmids with a single selection, streamlining multispecific antibody-producing CLD. Taken together, SiMPl-GS is a simple yet effective means to expedite CLD for therapeutic protein production.

Controlled labelling of tracer antibodies for time-resolved fluorescence-based immunoassays

Tracer antibodies, which are labelled with fluorescent or other type of reporter molecules, are widely employed in diagnostic immunoassays. Time-resolved fluorescence immunoassay (TRFIA), recognized as one of the most sensitive immunoassay techniques, utilizes tracers labelled with lanthanide ion (Ln) chelates. The conventional approach for conjugating isothiocyanate (ITC) Ln-chelates to antibodies involves random chemical targeting of the primary amino group of Lys residues, requiring typically overnight exposure to an elevated pH of 9–9.3 and leading to heterogeneity. Moreover, efforts to enhance the sensitivity of the assays by introducing a higher number of Ln-chelates per tracer antibody are associated with an elevated risk of targeting critical amino acid residues in the binding site, compromising the binding properties of the antibody. Herein, we report a method to precisely label recombinant antibodies with a defined number of Ln-chelates in a well-controlled manner by employing the SpyTag/SpyCatcher protein ligation technology. We demonstrate the functionality of the method with a full-length recombinant antibody (IgG) as well as an antibody fragment by producing site-specifically labelled antibodies for TRFIA for cardiac troponin I (cTnI) detection with a significant improvement in assay sensitivity compared to that with conventionally labelled tracer antibodies. Overall, our data clearly illustrates the benefits of the site-specific labelling strategy for generating high-performing tracer antibodies for TRF immunoassays.

Expressed Protein Ligation in Flow.

The development of a flow chemistry platform for the generation of modified protein targets via expressed protein ligation (EPL) is described. The flow EPL platform enables efficient ligation reactions with high recoveries of target protein products and superior reaction rates compared to corresponding batch processes. The utility of the flow EPL technology was first demonstrated through the semisynthesis of the tick-derived chemokine-binding protein ACA-01 containing two tyrosine sulfate modifications. Full-length, sulfated ACA-01 could be efficiently assembled by ligating a recombinantly expressed C-terminal protein fragment and a synthetic sulfopeptide thioester in flow. Following folding, the semisynthetic sulfoprotein was shown to exhibit potent binding to a variety of pro-inflammatory chemokines. In a second modified protein target, we employed an in-line flow EPL-photodesulfurization strategy to generate both unmodified and phosphorylated forms of human β-synuclein by fusing a recombinant protein thioester, generated through cleavage of an intein fusion protein, and a synthetic (phospho)peptide. The semisynthetic proteins were assembled in 90 min in flow, a significant improvement over corresponding batch protein assembly, and enabled access to tens of milligrams of high purity material. Flow EPL has the potential to serve as a robust technology to streamline access to homogeneously modified proteins for a variety of applications in both academia, as well as in the pharmaceutical and biotechnology sector.

Robust and Irreversible Sortase-Mediated Ligation by Empolyment of Sarkosyl.

Sortase-mediated ligation (SML) is a widely used method for peptide and protein ligation due to ease of substrate preparation and fast enzymatic kinetics. But there are drawbacks that limit broader applications. Sorting motif in substrates may not be exposed to sortase efficiently due to folding or aggregation. In addition, the ligation is reversible under transpeptidation equilibrium that restricts ligation yield. Here we report a simple but robust method to overcome such limitations. By employment of sarkosyl, the detergent alters substrate conformation to raise sorting motif accessibility for sortase catalysis. Moreover, transpeptidation becomes irreversible presumably by formation of micelle to shield ligation products from sortase. In consequence, excellent yields were achieved from sortase variants with different substrate specificity. Notably, this method is compatible with peptides or proteins capable of forming liquid-liquid phase separation (LLPS), presenting a powerful approach for the conjugation of aggregation-prone substrates. Therefore, we believe the sarkosyl-enhanced SML could be widely applied in peptide and protein chemistry and the unique irreversible transpeptidation mechanism offers an insight to detergent-driven equilibrium.

The role of circHmbox1(3,4) in ferroptosis-mediated cognitive impairments induced by manganese

Excessive environmental exposure to manganese (Mn) has been linked to cognitive impairments, circular RNAs (circRNAs) have been recognized for their roles in epigenetic regulation in various biological processes, including neurological pathogenesis. Previous studies found that ferroptosis, an iron ion-dependent programmed cell death, may be involved in cognitive impairments. However, specific mechanisms underlying the relationship among circRNA, ferroptosis, and neurotoxicity of Mn are not well-understood. In the current study, RNA sequencing was performed to profile RNA expression in Neuro-2a (N2a) cells that were treated with 300 μM Mn. The potential molecular mechanisms of circHmbox1(3,4) in Mn-induced cognitive impairments were investigated via various experiments, such as Western blot and intracerebroventricular injection in mice. We observed a significant decrease in the expression of circHmbox1(3,4) both in vitro and in vivo following Mn treatment. The results of Y maze test and Morris water maze test demonstrated an improvement in learning and memory abilities following circHmbox1(3,4) overexpression in Mn treated mice. Mn treatment may reduce circHmbox1(3,4) biogenesis through lowered expression of E2F1/QKI. Inhibiting circHmbox1(3,4) expression led to GPX4 protein degradation through protein ligation and ubiquitination. Overall, the current study showed that Mn exposure-induced cognitive dysfunction may be mediated through ferroptosis regulated by circHmbox1(3,4).

Engineered reversible inhibition of SpyCatcher reactivity enables rapid generation of bispecific antibodies

The precise regulation of protein function is essential in biological systems and a key goal in chemical biology and protein engineering. Here, we describe a straightforward method to engineer functional control into the isopeptide bond-forming SpyTag/SpyCatcher protein ligation system. First, we perform a cysteine scan of the structured region of SpyCatcher. Except for two known reactive and catalytic residues, none of these mutations abolish reactivity. In a second screening step, we modify the cysteines with disulfide bond-forming small molecules. Here we identify 8 positions at which modifications strongly inhibit reactivity. This inhibition can be reversed by reducing agents. We call such a reversibly inhibitable SpyCatcher “SpyLock”. Using “BiLockCatcher”, a genetic fusion of wild-type SpyCatcher and SpyLock, and SpyTagged antibody fragments, we generate bispecific antibodies in a single, scalable format, facilitating the screening of a large number of antibody combinations. We demonstrate this approach by screening anti-PD-1/anti-PD-L1 bispecific antibodies using a cellular reporter assay.

One-Step Purification and N-Terminal Functionalization of Bioactive Proteins via Atypically Split Inteins.

Site-specific installation of non-natural functionality onto proteins has enabled countless applications in biotechnology, chemical biology, and biomaterials science. Though the N-terminus is an attractive derivatization location, prior methodologies targeting this site have suffered from low selectivity, a limited selection of potential chemical modifications, and/or challenges associated with divergent protein purification/modification steps. In this work, we harness the atypically split VidaL intein to simultaneously N-functionalize and purify homogeneous protein populations in a single step. Our method─referred to as VidaL-tagged expression and protein ligation (VEPL)─enables modular and scalable production of N-terminally modified proteins with native bioactivity. Demonstrating its flexibility and ease of use, we employ VEPL to combinatorially install 4 distinct (multi)functional handles (e.g., biotin, alkyne, fluorophores) to the N-terminus of 4 proteins that span three different classes: fluorescent (Enhanced Green Fluorescent Protein, mCherry), enzymatic (β-lactamase), and growth factor (epidermal growth factor). Moving forward, we anticipate that VEPL's ability to rapidly generate and isolate N-modified proteins will prove useful across the growing fields of applied chemical biology.

Efficient Segmental Isotope Labeling of Integral Membrane Proteins for High-Resolution NMR Studies.

High-resolution structural NMR analyses of membrane proteins are challenging due to their large size, resulting in broad resonances and strong signal overlap. Among the isotope labeling methods that can remedy this situation, segmental isotope labeling is a suitable strategy to simplify NMR spectra and retain high-resolution structural information. However, protein ligation within integral membrane proteins is complicated since the hydrophobic protein fragments are insoluble, and the removal of ligation side-products is elaborate. Here, we show that a stabilized split-intein system can be used for rapid and high-yield protein trans-splicing of integral membrane proteins under denaturing conditions. This setup enables segmental isotope labeling experiments within folded protein domains for NMR studies. We show that high-quality NMR spectra of markedly reduced complexity can be obtained in detergent micelles and lipid nanodiscs. Of note, the nanodisc insertion step specifically selects for the ligated and correctly folded membrane protein and simultaneously removes ligation byproducts. Using this tailored workflow, we show that high-resolution NMR structure determination is strongly facilitated with just two segmentally isotope-labeled membrane protein samples. The presented method will be broadly applicable to structural and dynamical investigations of (membrane-) proteins and their complexes by solution and solid-state NMR but also other structural methods where segmental labeling is beneficial.

Novel protein ligase based on dual split intein

Inteins are unique single-turnover enzymes that can excise themselves from the precursor protein without the aid of any external cofactors or energy. In most cases, inteins are covalently linked with the extein sequences and protein splicing happens spontaneously. In this study, a novel protein ligation system was developed based on two atypical split inteins without cross reaction, in which the large segments of one S1 and one S11 split intein fusion protein acted as a protein ligase, the small segments (only several amino acids long) was fused to the N-extein and C-extein, respectively. The splicing activity was demonstrated in E. coli and in vitro with different extein sequences, which showed ∼15% splicing efficiency in vitro. The protein trans-splicing in vitro was further optimized, and possible reaction explanations were explored. As a proof of concept, we expect this approach to expand the scope of trans-splicing-based protein engineering and provide new clues for intein based protein ligase.