Biological applications of protein splicing.

Protein splicing is a naturally occurring process in which a protein editor, called an intein, performs a molecular disappearing act by cutting itself out of a host protein in a traceless manner. In the two decades since its discovery, protein splicing has been harnessed for the development of several protein-engineering methods. Collectively, these technologies help bridge the fields of chemistry and biology, allowing hitherto impossible manipulations of protein covalent structure. These tools and their application are the subject of this Primer.

Detection of cell-cell interactions via photocatalytic cell tagging.

The growing appreciation of immune cell-cell interactions within disease environments has led to extensive efforts to develop immunotherapies. However, characterizing complex cell-cell interfaces in high resolution remains challenging. Thus, technologies leveraging therapeutic-based modalities to profile intercellular environments offer opportunities to study cell-cell interactions with molecular-level insight. We introduce photocatalytic cell tagging (PhoTag) for interrogating cell-cell interactions using single-domain antibodies (VHHs) conjugated to photoactivatable flavin-based cofactors. Following irradiation with visible light, the flavin photocatalyst generates phenoxy radical tags for targeted labeling. Using this technology, we demonstrate selective synaptic labeling across the PD-1/PD-L1 axis in antigen-presenting cell-T cell systems. In combination with multiomics single-cell sequencing, we monitored interactions between peripheral blood mononuclear cells and Raji PD-L1 B cells, revealing differences in transient interactions with specific T cell subtypes. The utility of PhoTag in capturing cell-cell interactions will enable detailed profiling of intercellular communication across different biological systems.

Site-Specific Antibody Drug Conjugates Using Streamlined Expressed Protein Ligation.

Antibody-Drug Conjugates (ADCs) have been shown to produce clinical benefit in cancer patient thanks to their ability to target highly cytotoxic small molecules to tumor cells. However, the development of these complex molecules faces significant challenges due to the need to combine a large biologic drug with a small molecule drug to generate the desired bioconjugate. We describe here the use of a protein ligation methodology, based on the native chemical ligation reaction to generate site-specific Antibody-Drug Conjugates, which does not require the incorporation of unnatural modifications into the antibody. Fully native antibodies, with only the desired cytotoxic molecules attached, can be generated, thus minimizing the risk that additional modifications required for the site-specific conjugation pose a risk to the antibody activity. We demonstrate that our approach can be used to generate site-specifically modified ADCs, with potent in vitro and in vivo antitumor activity in a breast cancer tumor model.

Structure of the branched intermediate in protein splicing.

Inteins are autoprocessing domains that cut themselves out of host proteins in a traceless manner. This process, known as protein splicing, involves multiple chemical steps that must be coordinated to ensure fidelity in the process. The committed step in splicing involves attack of a conserved Asn side-chain amide on the adjacent backbone amide, leading to an intein-succinimide product and scission of that peptide bond. This cleavage reaction is stimulated by formation of a branched intermediate in the splicing process. The mechanism by which the Asn side-chain becomes activated as a nucleophile is not understood. Here we solve the crystal structure of an intein trapped in the branched intermediate step in protein splicing. Guided by this structure, we use protein-engineering approaches to show that intein-succinimide formation is critically dependent on a backbone-to-side-chain hydrogen-bond. We propose that this interaction serves to both position the side-chain amide for attack and to activate its nitrogen as a nucleophile. Collectively, these data provide an unprecedented view of an intein poised to carry out the rate-limiting step in protein splicing, shedding light on how a nominally nonnucleophilic group, a primary amide, can become activated in a protein active site.

Induction of innate and adaptive immunity by delivery of poly dA:dT to dendritic cells.

Targeted delivery of antigens to dendritic cells (DCs) is a promising vaccination strategy. However, to ensure immunity, the approach depends on coadministration of an adjuvant. Here we ask whether targeting of both adjuvant and antigen to DCs is sufficient to induce immunity. Using a protein ligation method, we develop a general approach for linking the immune stimulant, poly dA:dT (pdA:dT), to a monoclonal antibody (mAb) specific for DEC205 (DEC). We show that DEC-specific mAbs deliver pdA:dT to DCs for the efficient production of type I interferon in human monocyte-derived DCs and in mice. Notably, adaptive T-cell immunity is elicited when mAbs specific for DEC-pdA:dT are used as the activation stimuli and are administered together with a DC-targeted antigen. Collectively, our studies indicate that DCs can integrate innate and adaptive immunity in vivo and suggest that dual delivery of antigen and adjuvant to DCs might be an efficient approach to vaccine development.

Streamlined expressed protein ligation using split inteins.

Chemically modified proteins are invaluable tools for studying the molecular details of biological processes, and they also hold great potential as new therapeutic agents. Several methods have been developed for the site-specific modification of proteins, one of the most widely used being expressed protein ligation (EPL) in which a recombinant α-thioester is ligated to an N-terminal Cys-containing peptide. Despite the widespread use of EPL, the generation and isolation of the required recombinant protein α-thioesters remain challenging. We describe here a new method for the preparation and purification of recombinant protein α-thioesters using engineered versions of naturally split DnaE inteins. This family of autoprocessing enzymes is closely related to the inteins currently used for protein α-thioester generation, but they feature faster kinetics and are split into two inactive polypeptides that need to associate to become active. Taking advantage of the strong affinity between the two split intein fragments, we devised a streamlined procedure for the purification and generation of protein α-thioesters from cell lysates and applied this strategy for the semisynthesis of a variety of proteins including an acetylated histone and a site-specifically modified monoclonal antibody.

Ultrafast protein splicing is common among cyanobacterial split inteins: implications for protein engineering.

We describe the first systematic study of a family of inteins, the split DnaE inteins from cyanobacteria. By measuring in vivo splicing efficiencies and in vitro kinetics, we demonstrate that several inteins can catalyze protein trans-splicing in tens of seconds rather than hours, as is commonly observed for this autoprocessing protein family. Furthermore, we show that when artificially fused, these inteins can be used for rapid generation of protein α-thioesters for expressed protein ligation. This comprehensive survey of split inteins provides indispensable information for the development and improvement of intein-based tools for chemical biology.

Conjugation of a Blood Brain Barrier Peptide Shuttle to an Fc Domain for Brain Delivery of Therapeutic Biomolecules.

The frequency of brain disease has increased significantly in the past years. After diagnosis, therapeutic options are usually limited, which demands the development of innovative therapeutic strategies. The use of antibody-drug conjugates (ADCs) is promising but highly limited by the existence of the blood-brain barrier (BBB). To overcome the impermeability of this barrier, antibody fragments can be engineered and conjugated to BBB peptide shuttles (BBBpS), which are capable of brain penetration. Herein, we linked the highly efficient BBBpS, PepH3, to the IgG fragment crystallizable (Fc) domain using the streamlined expressed protein ligation (SEPL) method. With this strategy, we obtained an Fc-PepH3 scaffold that can carry different payloads. Fc-PepH3 was shown to be nontoxic, capable of crossing an in vitro cellular BBB model, and able to bind to the neonatal Fc receptor (FcRn), which is responsible for antibody long half-life (t 1/2). Overall, we demonstrated the potential of Fc-PepH3 as a versatile platform readily adaptable to diverse drugs of therapeutic value to treat different brain conditions.

Galectin-1 is a novel functional receptor for tissue plasminogen activator in pancreatic cancer.

BACKGROUND & AIMS: Tissue plasminogen activator (tPA) exerts many different functions in addition to its role in fibrinolysis. In pancreatic ductal adenocarcinoma (PDA), tPA is overexpressed and plays an important role in proliferation, invasion, and angiogenesis. tPA interaction with cell membrane receptors has been related to increased proteolytic activity and to signal transduction through nonenzymatic mechanisms. The aim was to analyze the role of galectin-1 (Gal-1), an endogenous lectin that also is overexpressed in PDA, as a new functional receptor for tPA. METHODS: Gal-1/tPA interaction was analyzed using surface plasmon resonance and pull-down assays. Pancreatic cells and tumors were used to study Gal-1 expression and localization by Western blot and immunostaining. Down-regulation of Gal-1 by small interference RNA was used to analyze the involvement of Gal-1/tPA interaction in extracellular signal-regulated kinase 1/2 activation, cell proliferation, and invasion in pancreatic and fibroblastic cells. RESULTS: Gal-1/tPA interaction is direct, specific, and of high affinity. Gal-1 moderately increases the catalytic activity of tPA. High Gal-1 levels were detected in PDA cells in culture, where it concentrates at the migration front, and in tissues, where it is expressed in epithelial cells and in the stroma. Down-regulation of Gal-1 abolished the effects of tPA on extracellular signal-regulated kinase 1/2 activation, cell proliferation, and invasion, both in pancreatic and in tumor-derived fibroblasts. CONCLUSIONS: These findings support a new molecular mechanism by which Gal-1 interaction with tPA contributes to PDA progression involving both transformed epithelial cells and tumor fibroblasts.

Synthesis and antibody recognition of cyclic epitope peptides, together with their dimer and conjugated derivatives based on residues 9-22 of herpes simplex virus type 1 glycoprotein D.

The synthesis of new cyclic peptides comprising the 9-22 epitope (9)LKMADPNRFRGKDL(22) sequence derived from HSV gD-1 is reported. In addition, we describe procedures for the preparation of cyclic peptide dimers and conjugates with an oligotuftsin derivative carrier. The binding of a monoclonal antibody, Mab A16, to the synthesized compounds was determined by enzyme-linked immunosorbent assay. It was demonstrated that cyclization decreased the binding activity of the antibody to the epitope. However, dimerization and conjugation could significantly increase the binding capacity of the cyclic epitope peptides. The attachment site in dimers and conjugates, as well as the topology of the construct, had a significant influence on the antibody recognition, while replacement of Met in position 11 by Nle had no marked effect.

Characterisation of the 5 kDa growth hormone isoform.

OBJECTIVES: The 5 kDa N-terminal fragment of 43 amino acids of human growth hormone (GH) shows a specific and significant in-vivo insulin-like activity. This isoform can be easily obtained by solid phase synthesis methods. Our objective in this study is to describe this procedure in detail and to provide structural information of the protein. METHODS: Solid phase synthesis was employed for the synthesis of the 5 kDa GH isoform. Circular dichroism and limited proteolysis have been carried out to provide structural information about the folded state of the protein in solution. Surface plasmon resonance was used to compare the structural equivalence between the synthetic protein and a proteolytic homologue at an antibody binding level. For this purpose, a murine monoclonal antibody specific for the 5 kDa isoform was generated and characterised employing this and several other GH isoforms. RESULTS: Circular dichroism and proteolysis results suggested that the C-terminal segment of the 5 kDa protein folds in an alpha-helix. The comparison of the synthetic product to its proteolytic homologue at an antibody binding level suggested structural equivalency. A highly specific antibody against the 5 kDa GH isoform was generated with null cross-reactivity for 17, 20 and 22 kDa isoforms. Kinetic data on the interaction with the synthetic 5 kDa GH was obtained. CONCLUSIONS: The structure of the protein appears to be different in comparison to when it is included within the 22 kDa GH isoform. Finally, a highly specific antibody has been generated. The possible significance of the 5 kDa protein as a potential agent for obesity-related diseases is discussed.

Synthesis and biological properties of beta-turned Abeta(31-35) constrained analogues.

A series of constrained pentapeptide analogues of the fragment Abeta(31-35) has been prepared using solid phase synthesis protocols. The results of conformational studies and surface plasmon resonance (SPR) experiments seem to indicate that the affinity of these constrained analogues for immobilized Abeta(25-35) peptide could be related to their ability to adopt a Leu34N-Ile31O beta-turn-like folded conformation.

Tunable photoactivation of a post-translationally modified signaling protein and its unmodified counterpart in live cells.

An ideal technology for direct imaging of post-translationally modified proteins would be one in which the appearance of a fluorescent signal is linked to a modification dependent protein-activation event. Herein, we utilize the protein semisynthesis technique, expressed protein ligation (EPL), to prepare caged analogues of the signaling protein Smad2; the function and fluorescence of the analogues were then photocontrolled in a correlated fashion. We show that this strategy permits titration of the cellular levels of active phosphorylated Smad2 in its biologically relevant, full-length form. We also prepared a nonphosphorylated, caged full-length Smad2 analogue labeled with an orthogonal fluorophore, and simultaneously imaged the phosphorylated and nonphosphorylated forms of the protein in the same cell. This strategy should enable the dissection of the cellular consequences of post-translational modifications (PTMs) by direct comparison of the behavior of the modified and unmodified forms of the protein following uncaging.

A minimalist design approach to antimicrobial agents based on a thionin template.

Numerous studies have been devoted to the stabilization of secondary structure elements to improve receptor-ligand recognition. We report a novel application of this principle to create new antimicrobial agents using the highly folded thionin from Pyrularia puberaas a template. Non-native disulfide bonds have been used to induce two short linear segments of the protein into an amphipathic helix. The resulting 13- and 9-residue peptides are significantly more active than their linear counterparts and have an activity similar to that of native thionin.

Chemical tagging and customizing of cellular chromatin states using ultrafast trans-splicing inteins.

Post-translational modification of the histone proteins in chromatin plays a central role in the epigenetic control of DNA-templated processes in eukaryotic cells. Developing methods that enable the structure of histones to be manipulated is, therefore, essential to understand the biochemical mechanisms that underlie genomic regulation. Here we present a synthetic biology method to engineer histones that bear site-specific modifications on cellular chromatin using protein trans-splicing (PTS). We genetically fused the N-terminal fragment of ultrafast split intein to the C terminus of histone H2B, which, on reaction with a complementary synthetic C intein, generated labelled histone. Using this approach, we incorporated various non-native chemical modifications into chromatin in vivo with temporal control. Furthermore, the time and concentration dependence of PTS performed in nucleo enabled us to examine differences in the accessibility of the euchromatin and heterochromatin regions of the epigenome. Finally, we used PTS to semisynthesize a native histone modification, H2BK120 ubiquitination, in isolated nuclei and showed that this can trigger downstream epigenetic crosstalk of H3K79 methylation.

Synthetic and structural studies on Pyrularia pubera thionin: a single-residue mutation enhances activity against Gram-negative bacteria.

The thionin from Pyrularia pubera (Pp-TH), a 47-residue peptide with four internal disulfide bonds, was efficiently produced by chemical synthesis. Its antimicrobial activity in vitro against several representative pathogens (EC(50)=0.3-3.0 microM) was identical to that of natural Pp-TH. This peptide has a unique Asp(32) instead of the consensus Arg found in other thionins of the same family. In order to evaluate the effect of this mutation, the Arg(32) analogue (Pp-TH(D32R)) was also synthesized and showed a significant increase in antibiotic activity against several Gram-negative bacteria, whereas it retained the same activity against other pathogens. The overall structure of Pp-TH(D32R) was maintained, though a slight decrease in the helical content of the peptide was observed.

Tuning protein autoinhibition by domain destabilization.

Activation of many multidomain signaling proteins requires rearrangement of autoinhibitory interdomain interactions that occlude activator binding sites. In one model for activation, the major inactive conformation exists in equilibrium with activated-like conformations that can be stabilized by ligand binding or post-translational modifications. We established the molecular basis for this model for the archetypal signaling adaptor protein Crk-II by measuring the thermodynamics and kinetics of the equilibrium between autoinhibited and activated-like states. We used fluorescence and NMR spectroscopies together with segmental isotopic labeling by means of expressed protein ligation. The results demonstrate that intramolecular domain-domain interactions both stabilize the autoinhibited state and induce the activated-like conformation. A combination of favorable interdomain interactions and unfavorable intradomain structural changes fine-tunes the population of the activated-like conformation and allows facile response to activators. This mechanism suggests a general strategy for optimization of autoinhibitory interactions of multidomain proteins.

Effect of a serine-to-aspartate replacement on the recognition of chitin oligosaccharides by truncated hevein. A 3D view by using NMR.

The interaction of a synthetically prepared mutant peptide of hevein (a well known chitin-binding lectin) Hev32S19D with chitin oligosaccharides (and chitosan analogues) has allowed us to estimate their affinity constants and associated thermodynamic data. The mutant peptide is able to bind chitin oligomers, but with significant decreases in the association constants with chito-oligosaccharides. The determination of the three-dimensional structure of the peptide mutant, by using NMR, has permitted us to deduce that the topology of the backbone is very similar to that of the parent Hev32 peptide. The same is true regarding the orientations of the key aromatic residues Trp21, Trp23, and Tyr30. The decrease in the association constants can be attributed to the different topological orientation of key side chains and to the importance of protein-sugar intermolecular essential hydrogen bonds and CH-pi stacking interactions. The analysis has permitted us to infer the free energy of binding associated with these interactions as well as to estimate the corresponding binding enthalpy.