Protein Trans-splicing Research Articles

In Silico Structural Prediction for the Generation of Novel Performant Midi-Dystrophins Based on Intein-Mediated Dual AAV Approach.

Duchenne Muscular Dystrophy (DMD) is a pediatric disorder characterized by progressive muscle degeneration and premature death, and has no current cure. The current, most promising therapeutic avenue is based on gene replacement mediated by adeno-associated viruses (AAVs) using a shortened, but still functional, version of dystrophin, known as micro-dystrophin (µDys), to fit AAV capacity. The limited improvements observed in clinical trials suggest a sub-optimal performance of µDys in the human context that could be due to the lack of key domains in the protein. Therefore, expressing larger dystrophin proteins may be necessary for a more complete correction of the disease phenotype. In this study, we developed three novel midi-dystrophin constructs using a dual-AAV approach, leveraging split-intein-based protein trans-splicing. The midi-dystrophins include additional domains compared to µDys, such as the central cytoskeleton-binding domain, nNOS and Par1b interacting domains, and a complete C-terminal region. Given the limited capacity of each AAV vector, we strategically partially reduced hinge regions while ensuring that the structural stability of the protein remains intact. We predicted the interactions between the two halves of the split midi-Dys proteins thanks to the deep learning algorithm AphaFold3. We observed strong associations between the N- and C-termini in midi-Dys 1 and 2, while a weaker interaction in midi-Dys 3 was revealed. Our subsequent experiments confirmed the efficient protein trans-splicing both in vitro and in vivo in DBA2/mdx mice of the midi-Dys 1 and 2 and not in midi-Dys 3 as expected from the structural prediction. Additionally, we demonstrated that midi-Dys 1 and 2 exhibit significant therapeutic efficacy in DBA2/mdx mice, highlighting their potential as therapeutic agents for DMD. Overall, these findings highlight the potential of deep learning-based structural modeling for the generation of intein-based dystrophin versions and pose the basis for further investigation of these new midi-dystrophins versions for clinical studies.

Proximity-triggered protein trans-splicing.

Proximity-triggered protein trans-splicing.

Split intein-mediated protein trans-splicing to express large dystrophins.

Gene replacement using adeno-associated virus (AAV) vectors is a promising therapeutic approach for many diseases1,2. However, this therapeutic modality is challenged by the packaging capacity of AAVs (approximately 4.7 kilobases)3, limiting its application for disorders involving large coding sequences, such as Duchenne muscular dystrophy, with a 14 kilobase messenger RNA. Here we developed a new method for expressing large dystrophins by utilizing the protein trans-splicing mechanism mediated by split inteins. We identified several split intein pairs that efficiently join two or three fragments to generate a large midi-dystrophin or the full-length protein. We show that delivery of two or three AAVs into dystrophic mice results in robust expression of large dystrophins and significant physiological improvements compared with micro-dystrophins. Moreover, using the potent myotropic AAVMYO4, we demonstrate that low total doses (2 × 1013 viral genomes per kg) are sufficient to express large dystrophins in striated muscles body-wide with significant physiological corrections in dystrophic mice. Our data show a clear functional superiority of large dystrophins over micro-dystrophins that are being tested in clinical trials. This method could benefit many patients with Duchenne or Becker muscular dystrophy, regardless of genotype, and could be adapted to numerous other disorders caused by mutations in large genes thatexceed the AAV capacity.

Distinct phases of cellular signaling revealed by time-resolved protein synthesis.

The post-translational regulation of protein function is involved in most cellular processes. As such, synthetic biology tools that operate at this level provide opportunities for manipulating cellular states. Here we deploy proximity-triggered protein trans-splicing technology to enable the time-resolved synthesis of target proteins from premade parts. The modularity of the strategy allows for the addition or removal of various control elements as a function of the splicing reaction, in the process permitting the cellular location and/or activity state of starting materials and products to be differentiated. The approach is applied to a diverse set of proteins, including the kinase oncofusions breakpoint cluster region-Abelson (BCR-ABL) and DNAJ-PKAc where dynamic cellular phosphorylation events are dissected, revealing distinct phases of signaling and identifying molecular players connecting the oncofusion to cancer transformation as new therapeutic targets of cancer cells. We envision that the tools and control strategies developed herein will allow the activity of both naturally occurring and designer proteins to be harnessed for basic and applied research.

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.

Towards targeted Cas9 (CRISPR-Cas) delivery: Preparation of IgG antibody-Cas9 conjugates using a split intein.

The CRISPR-Cas9 system has revolutionized the field of genetic engineering, but targeted cellular delivery remains a central problem. The delivery of the preformed ribonuclease-protein (RNP) complex has the advantages of fewer side effects and avoidance of potential permanent effects. We reasoned that an internalizing IgG antibody as a targeting device could address the delivery of Cas9-RNP. We opted for protein trans-splicing mediated by a split intein to facilitate posttranslational conjugation of the two large protein entities. We recently described the cysteine-less CL split intein that efficiently performs under oxidizing conditions and does not interfere with disulfide bonds or thiol bioconjugation chemistries. Using the CL split intein, we report for the first time the ligation of monoclonal IgG antibody precursors, expressed in mammalian cells, and a Cas9 precursor, obtained from bacterial expression. A purified IgG-Cas9 conjugate was loaded with sgRNA to form the active RNP complex and introduced a double-strand break in its target DNA in vitro. Furthermore, a synthetic peptide variant of the short N-terminal split intein precursor proved useful for chemical modification of Cas9. The split intein ligation procedure reported here for IgG-Cas9 provides the first step towards a novel CRISPR-Cas9 targeting approach involving the preformed RNP complex.

Development of a β-lactamase activity assay for detecting ligand–protein interactions using an engineered split intein and β-lactamase

Abstract Several synthetic compounds bind to proteins of interest and inhibit protein–protein interactions. To develop a detection method for the interactions between the synthetic compounds and the target proteins, we used an engineered split intein derived from Nostoc punctiforme PCC73102 (Npu) DnaE and TEM-1 β-lactamase as reporter proteins. We constructed synthetic ligands bearing a 6-residue C-terminal peptide from Npu DnaE and Cys-Trp as the C-extein, and target proteins bearing the N-terminal region of the engineered Npu DnaE and residues 24–284 of β-lactamase. Specific ligand–protein interactions such as phosphopeptide–Src homology domain 2 (SH2) of c-Src and imatinib–quinone reductase 2 (NQO2) increased the protein trans-splicing (PTS) reaction rates and yields. The PTS product showed the enhanced β-lactamase activity compared with the starting materials. The PTS-based β-lactamase activity assay was used for the quantitative analysis of the ligand–protein interactions. The signal sequence and 9-residue N-terminal sequence of Escherichia coli (E. coli) lipoprotein (Lpp) and residues 46–159 of outer membrane protein A (OmpA) (LppOmpA) were conjugated with the target proteins bearing split intein and β-lactamase to display them on live E. coli cell surfaces. PTS on live E. coli surfaces provided enhanced resistance to carbenicillin.

Protein semisynthesis underscores the role of a conserved lysine in activation and desensitization of acid-sensing ion channels

Acid-sensing ion channels (ASICs) are trimeric ion channels that open a cation-conducting pore in response to proton binding. Excessive ASIC activation during prolonged acidosis in conditions such as inflammation and ischemia is linked to pain and stroke. A conserved lysine in the extracellular domain (Lys211 in mASIC1a) is suggested to play a key role in ASIC function. However, the precise contributions are difficult to dissect with conventional mutagenesis, as replacement of Lys211 with naturally occurring amino acids invariably changes multiple physico-chemical parameters. Here, we study the contribution of Lys211 to mASIC1a function using tandem protein trans-splicing (tPTS) to incorporate non-canonical lysine analogs. We conduct optimization efforts to improve splicing and functionally interrogate semisynthetic mASIC1a. In combination with molecular modeling, we show that Lys211 charge and side-chain length are crucial to activation and desensitization, thus emphasizing that tPTS can enable atomic-scale interrogations of membrane proteins in live cells.

Construction of IgG–Fab2 bispecific antibody via intein-mediated protein trans-splicing reaction

A bispecific antibody (bsAb) is a class of engineered antibody molecules that simultaneously binds to two different antigens by having two kinds of antigen-binding domains. One of the major obstacles for the bsAb production is the incorrect chain-pairing problem, wherein each heavy and light chain should form pairings with the correct counterpart’s chains, but the structural similarity of the incorrect partners also forms the incorrect pairings. This study aimed to demonstrate a bsAb construction method using intein-mediated protein trans-splicing to create IgG–Fab2–type bsAbs, which is a modified antibody with a structure in which two additional Fabs are linked to the N-terminus of the heavy chain of an IgG molecule. The chain-paring problem between a heavy chain and a light chain is circumvented by separate expression and purification of the IgG part and the Fab part. We found that the deletion of a possible glycosylation residue improved the reaction yield and side-reaction cleavage in the protein ligation step. The resulting bsAb, IgG–Fab2 (Her2/CD3), demonstrated target binding activity and cytotoxicity mediated by activated T cells. These results indicate that the use of the protein ligation to produce the IgG–Fab2 type bsAb will expand the bsAb production method.

Extending AAV Packaging Cargo through Dual Co-Transduction: Efficient Protein Trans-Splicing at Low Vector Doses.

Adeno-associated viral (AAV) vectors represent one of the leading platforms for gene delivery. Nevertheless, their small packaging capacity restricts their use for diseases requiring large-gene delivery. To overcome this, dual-AAV vector systems that rely on protein trans-splicing were developed, with the split-intein Npu DnaE among the most-used. However, the reconstitution efficiency of Npu DnaE is still insufficient, requiring higher vector doses. In this work, two split-inteins, Cfa and Gp41-1, with reportedly superior trans-splicing were evaluated in comparison with Npu DnaE by transient transfections and dual-AAV in vitro co-transductions. Both Cfa and Gp41-1 split-inteins enabled reconstitution rates that were over two-fold higher than Npu DnaE and 100% of protein reconstitution. The impact of different vector preparation qualities in split-intein performances was also evaluated in co-transduction assays. Higher-quality preparations increased split-inteins' performances by three-fold when compared to low-quality preparations (60-75% vs. 20-30% full particles, respectively). Low-quality vector preparations were observed to limit split-gene reconstitutions by inhibiting co-transduction. We show that combining superior split-inteins with higher-quality vector preparations allowed vector doses to be decreased while maintaining high trans-splicing rates. These results show the potential of more-efficient protein-trans-splicing strategies in dual-AAV vector co-transduction, allowing the extension of its use to the delivery of larger therapeutic genes.

Expandig Lentiviral Vectors cargo capacity with protein trans-splicing

In the last decades Lentiviral vectors (LVs) have been widely used as vehicles to stably transfer and express genes in mammalian cells in the context of monogenic diseases in both dividing and non-dividing or slowlyproliferating cells, as human CD34+ stem cells (CD34+ HSCs). However LVs cargo capacity, limited to around 8 kilobases (kb), prevents their application for the treatment of conditions requiring the delivery of larger DNA sequences and the stable expression of the transgene in actively proliferating cells. Recent studies have shown that protein trans-splicing is a powerful means to ably reconstitute large full-length proteins upon coinfection of a host cell with different vectors, each expressing a split-intein flanked portion of the full lenght- protein. The aim of our study is to translate the same technology to the platform of LVs. As proof-of-principle developed lentiviral expression plasmids (pLenti), each encoding either the N- or the C-terminal half of the EGFP reporter protein fused to the N- and C-terminal halves of the DnaE split-inteins from Nostoc punctiforme, under the control of EF1A promoter. We co-transfected HEK293 cells and we demonstrated the reconstitution of a functional full-lenght protein of the proper size. We are currently generating LVs from these pLenti that we will use to infect both lineage-negative cells from mouse bone marrow and CD34+ HSCs in order to assess if the platform of LVs is as efficient as AAVs. This system will be helpful to develop a gene therapy approach for specific inborn errors of immunity, including LRBA deficiency and Ataxia Telangiectasia, caused by mutations in genes larger than 8 kb.

Conditional protein splicing triggered by SUMO protease.

Conditional protein splicing is a powerful biotechnological tool that can be used to post-translationally control the activity of target proteins. Here we demonstrated a novel conditional protein splicing approach in which the small ubiquitin-like modifier (SUMO) protease induced the splicing of an atypical split intein. The engineered Ter DnaE-3 S11 split intein which has a small C-intein segment with only 6 amino acids was used in this study. A SUMO tag was fused to the N-terminus of the C-intein to inhibit the protein trans-splicing invitro. The splicing products could be detected in 15min with the addition of SUMO protease by western blotting and the splicing efficiency was ∼4-fold higher than the control without SUMO protease for overnight reaction. This engineered Ter DnaE-3 S11 split intein-mediated protein trans-splicing had been further shown to be triggered by SUMO protease in different exteins invitro. Our study provides new insights into the regulation of protein splicing and is a promising tool for the control of protein structure and function invitro.

An artificially split class 3 intein.

Inteins excise themselves from precursor polypeptides through protein splicing, joining N- and C-exteins with a peptide bond. Split inteins are expressed as separate polypeptides that undergo protein trans splicing (PTS). Here, we demonstrate PTS can be achieved using an artificially split class 3 intein. Because class 3 inteins use an internal initiating nucleophile near the C-extein junction, rather than the first residue of the intein, both catalytic nucleophiles are present on a single polypeptide. This results in a compact arrangement of catalytic nucleophiles for PTS compared to the standard arrangement for split class 1 inteins.

Structural and biochemical analysis of a novel atypically split intein reveals a conserved histidine specific to cysteine-less inteins.

Protein trans-splicing mediated by a split intein reconstitutes a protein backbone from two parts. This virtually traceless autoprocessive reaction provides the basis for numerous protein engineering applications. Protein splicing typically proceeds through two thioester or oxyester intermediates involving the side chains of cysteine or serine/threonine residues. A cysteine-less split intein has recently attracted particular interest as it can splice under oxidizing conditions and is orthogonal to disulfide or thiol bioconjugation chemistries. Here, we report the split PolB16 OarG intein, a second such cysteine-independent intein. As a unique trait, it is atypically split with a short intein-N precursor fragment of only 15 amino acids, the shortest characterized to date, which was chemically synthesized to enable protein semi-synthesis. By rational engineering we obtained a high-yielding, improved split intein mutant. Structural and mutational analysis revealed the dispensability of the usually crucial conserved motif N3 (block B) histidine as an obvious peculiar property. Unexpectedly, we identified a previously unnoticed histidine in hydrogen-bond forming distance to the catalytic serine 1 as critical for splicing. This histidine has been overlooked so far in multiple sequence alignments and is highly conserved only in cysteine-independent inteins as a part of a newly discovered motif NX. The motif NX histidine is thus likely of general importance to the specialized environment in the active site required in this intein subgroup. Together, our study advances the toolbox as well as the structural and mechanistic understanding of cysteine-less inteins.

Hearing of Otof-deficient mice restored by trans-splicing of N- and C-terminal otoferlin.

Mutations to the OTOF gene are among the most common reasons for auditory neuropathy. Although cochlear implants are often effective in restoring sound transduction, there are currently no biological treatments for individuals with variants of OTOF. Previous studies have reported the rescue of hearing in DFNB9 mice using OTOF gene replacement although the efficacy needs improvement. Here, we developed a novel dual-AAV-mediated gene therapy system based on the principles of protein trans-splicing, and we show that this system can reverse bilateral deafness in Otof -/- mice after a single unilateral injection. The system effectively expressed exogenous mouse or human otoferlin after injection on postnatal day 0-2. Human otoferlin restored hearing to near wild-type levels for at least 6months and restored the release of synaptic vesicles in inner hair cells. Our study not only provides a preferential clinical strategy for the treatment of OTOF-related auditory neuropathies, but also describes a route of development for other large-gene therapies and protein engineering techniques.

A Conserved Histidine Residue Drives Extein Dependence in an Enhanced Atypically Split Intein.

Split intein-mediated protein trans-splicing (PTS) is widely applied in chemical biology and biotechnology to carry out traceless and specific protein ligation. However, the external residues immediately flanking the intein (exteins) can reduce the splicing rate, thereby limiting certain applications of PTS. Splicing by a recently developed intein with atypical split architecture ("Cat") exhibits a stark dependence on the sequence of its N-terminal extein residues. Here, we further developed Cat using error-prone polymerase chain reaction (PCR) and a cell-based selection assay to produce Cat*, which exhibits greatly enhanced PTS activity in the presence of unfavorable N-extein residues. We then applied solution nuclear magnetic resonance spectroscopy and molecular dynamics simulations to explore how the dynamics of a conserved B-block histidine residue (His78) contribute to this extein dependence. The enhanced extein tolerance of Cat* reported here should expand the applicability of atypically split inteins, and the mechanism highlights common principles that contribute to extein dependence.

Bifunctional hetero di-disc for broad-spectrum influenza neutralization

Nanodiscs containing sialic acid, which binds the hemagglutinin of the influenza virus, rupture the viral envelope and entrap viral ribonucleoproteins in the endolysosome. While nanodiscs are potent antiviral platforms, ganglioside GD1a containing α2,3-sialic acid does not cover all virus strains. When two nanodiscs containing different receptors 6′-sialyllactose and GD1a were mixed, one nanodisc inhibited the function of the other. A nanodisc loaded with two different receptors exhibited a biased activity toward only one receptor precluding the generation of a multifunctional nanodisc. Here, we suggest hetero di-disc, in which two nanodiscs loaded with each receptor were conjugated through protein trans-splicing for a broad-spectrum antiviral. The hetero di-disc showed strong antiviral activity in vitro and in vivo. Our results suggested that hetero di-discs not only expanded the inhibitory spectrum of nanodiscs but also enabled nanodisc-based delivery of multiple ligands without interference.

KCNQ2 and KCNQ5 form heteromeric channels independent of KCNQ3

KCNQ2 and KCNQ3 channels are associated with multiple neurodevelopmental disorders and are also therapeutic targets for neurological and neuropsychiatric diseases. For more than two decades, it has been thought that most KCNQ channels in the brain are either KCNQ2/3 or KCNQ3/5 heteromers. Here, we investigated the potential heteromeric compositions of KCNQ2-containing channels. We applied split-intein protein trans-splicing to form KCNQ2/5 tandems and coexpressed these with and without KCNQ3. Unexpectedly, we found that KCNQ2/5 tandems form functional channels independent of KCNQ3 in heterologous cells. Using mass spectrometry, we went on to demonstrate that KCNQ2 associates with KCNQ5 in native channels in the brain, even in the absence of KCNQ3. Additionally, our functional heterologous expression data are consistent with the formation of KCNQ2/3/5 heteromers. Thus, the composition of KCNQ channels is more diverse than has been previously recognized, necessitating a re-examination of the genotype/phenotype relationship of KCNQ2 pathogenic variants.

Castanospermine suppresses CD44 ectodomain cleavage as revealed by transmembrane bioluminescent sensors.

Cluster of differentiation 44 (CD44) is a single-pass transmembrane glycoprotein that is a widely distributed cell-surface adhesion molecule. CD44 undergoes ectodomain cleavage by membrane-associated metalloproteinases in breast cancer cells. Cleavage plays a critical role in cancer cell migration by mediating the interaction between CD44 and the extracellular matrix. To explore inhibitors of CD44 ectodomain cleavage, we developed two bioluminescent sensors for the detection of CD44 ectodomain cleavage. The sensors were designed as two-transmembrane proteins with split-luciferase fragments, one of which was cyclized by protein trans-splicing of a DnaE intein. These two sensors emit light by the cyclization or the spontaneous complementation of the luciferase fragments. The luminescence intensities decreased upon cleavage of the ectodomain in breast cancer cells. The sensors revealed that castanospermine, an α-glucosidase inhibitor, suppressed the ectodomain cleavage of endogenous CD44 in breast cancer cells. Castanospermine also inhibited breast cancer cell invasion. Thus, the sensors are beneficial tools for evaluating the effects of different inhibitors.