RyR2 Serine-2030 PKA Site Governs Ca2+ Release Termination and Ca2+ Alternans.

BACKGROUND: PKA (protein kinase A)-mediated phosphorylation of cardiac RyR2 (ryanodine receptor 2) has been extensively studied for decades, but the physiological significance of PKA phosphorylation of RyR2 remains poorly understood. Recent determination of high-resolution 3-dimensional structure of RyR2 in complex with CaM (calmodulin) reveals that the major PKA phosphorylation site in RyR2, serine-2030 (S2030), is located within a structural pathway of CaM-dependent inactivation of RyR2. This novel structural insight points to a possible role of PKA phosphorylation of RyR2 in CaM-dependent inactivation of RyR2, which underlies the termination of Ca2+ release and induction of cardiac Ca2+ alternans. METHODS: We performed single-cell endoplasmic reticulum Ca2+ imaging to assess the impact of S2030 mutations on Ca2+ release termination in human embryonic kidney 293 cells. Here we determined the role of the PKA site RyR2-S2030 in a physiological setting, we generated a novel mouse model harboring the S2030L mutation and carried out confocal Ca2+ imaging. RESULTS: We found that mutations, S2030D, S2030G, S2030L, S2030V, and S2030W reduced the endoplasmic reticulum luminal Ca2+ level at which Ca2+ release terminates (the termination threshold), whereas S2030P and S2030R increased the termination threshold. S2030A and S2030T had no significant impact on release termination. Furthermore, CaM-wild-type increased, whereas Ca2+ binding deficient CaM mutant (CaM-M [a loss-of-function CaM mutation with all 4 EF-hand motifs mutated]), PKA, and Ca2+/CaMKII (CaM-dependent protein kinase II) reduced the termination threshold. The S2030L mutation abolished the actions of CaM-wild-type, CaM-M, and PKA, but not CaMKII, in Ca2+ release termination. Moreover, we showed that isoproterenol and CaM-M suppressed pacing-induced Ca2+ alternans and accelerated Ca2+ transient recovery in intact working hearts, whereas CaM-wild-type exerted an opposite effect. The impact of isoproterenol was partially and fully reversed by the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide and the CaMKII inhibitor N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide individually and together, respectively. S2030L abolished the impact of CaM-wild-type, CaM-M, and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide-sensitive component, but not the N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide-sensitive component, of isoproterenol.

Increased Ca2+ Transient Underlies RyR2-Related Left Ventricular Noncompaction.

BACKGROUND: A loss-of-function cardiac ryanodine receptor (RyR2) mutation, I4855M+/-, has recently been linked to a new cardiac disorder termed RyR2 Ca2+ release deficiency syndrome (CRDS) as well as left ventricular noncompaction (LVNC). The mechanism by which RyR2 loss-of-function causes CRDS has been extensively studied, but the mechanism underlying RyR2 loss-of-function-associated LVNC is unknown. Here, we determined the impact of a CRDS-LVNC-associated RyR2-I4855M+/- loss-of-function mutation on cardiac structure and function. METHODS: We generated a mouse model expressing the CRDS-LVNC-associated RyR2-I4855M+/- mutation. Histological analysis, echocardiography, ECG recording, and intact heart Ca2+ imaging were performed to characterize the structural and functional consequences of the RyR2-I4855M+/- mutation. RESULTS: As in humans, RyR2-I4855M+/- mice displayed LVNC characterized by cardiac hypertrabeculation and noncompaction. RyR2-I4855M+/- mice were highly susceptible to electrical stimulation-induced ventricular arrhythmias but protected from stress-induced ventricular arrhythmias. Unexpectedly, the RyR2-I4855M+/- mutation increased the peak Ca2+ transient but did not alter the L-type Ca2+ current, suggesting an increase in Ca2+-induced Ca2+ release gain. The RyR2-I4855M+/- mutation abolished sarcoplasmic reticulum store overload-induced Ca2+ release or Ca2+ leak, elevated sarcoplasmic reticulum Ca2+ load, prolonged Ca2+ transient decay, and elevated end-diastolic Ca2+ level upon rapid pacing. Immunoblotting revealed increased level of phosphorylated CaMKII (Ca2+-calmodulin dependent protein kinases II) but unchanged levels of CaMKII, calcineurin, and other Ca2+ handling proteins in the RyR2-I4855M+/- mutant compared with wild type. CONCLUSIONS: The RyR2-I4855M+/- mutant mice represent the first RyR2-associated LVNC animal model that recapitulates the CRDS-LVNC overlapping phenotype in humans. The RyR2-I4855M+/- mutation increases the peak Ca2+ transient by increasing the Ca2+-induced Ca2+ release gain and the end-diastolic Ca2+ level by prolonging Ca2+ transient decay. Our data suggest that the increased peak-systolic and end-diastolic Ca2+ levels may underlie RyR2-associated LVNC.

Polo-like kinase 1 (PLK1) O-GlcNAcylation is essential for dividing mammalian cells and inhibits uterine carcinoma.

The O-linked ß-N-acetylglucosamine (O-GlcNAc) transferase (OGT) mediates intracellular O-GlcNAcylation modification. O-GlcNAcylation occurs on Ser/Thr residues and is important for numerous physiological processes. OGT is essential for dividing mammalian cells and is involved in many human diseases; however, many of its fundamental substrates during cell division remain unknown. Here, we focus on the effect of OGT on polo-like kinase 1 (PLK1), a mitotic master kinase that governs DNA replication, mitotic entry, chromosome segregation, and mitotic exit. We show that PLK1 interacts with OGT and is O-GlcNAcylated. By utilizing stepped collisional energy/higher-energy collisional dissociation mass spectrometry, we found a peptide fragment of PLK1 that is modified by O-GlcNAc. Further mutation analysis of PLK1 shows that the T291A mutant decreases O-GlcNAcylation. Interestingly, T291N is a uterine carcinoma mutant in The Cancer Genome Atlas. Our biochemical assays demonstrate that T291A and T291N both increase PLK1 stability. Using stable H2B-GFP cells, we found that PLK1-T291A and PLK1-T291N mutants display chromosome segregation defects and result in misaligned and lagging chromosomes. In mouse xenograft models, we demonstrate that the O-GlcNAc-deficient PLK1-T291A and PLK1-T291N mutants enhance uterine carcinoma in animals. Hence, we propose that OGT partially exerts its mitotic function through O-GlcNAcylation of PLK1, which might be one mechanism by which elevated levels of O-GlcNAc promote tumorigenesis.

A gain-of-function mutation in the ITPR1 gating domain causes male infertility in mice.

Inositol 1,4,5-trisphosphate receptor 1 (ITPR1) is an intracellular Ca2+ release channel critical for numerous cellular processes. Despite its ubiquitous physiological significance, ITPR1 mutations have thus far been linked to primarily movement disorders. Surprisingly, most disease-associated ITPR1 mutations generate a loss of function. This leaves our understanding of ITPR1-associated pathology oddly one-sided, as little is known about the pathological consequences of ITPR1 gain of function (GOF). To this end, we generated an ITPR1 gating domain mutation (D2594K) that substantially enhanced the inositol trisphosphate (IP3 )-sensitivity of ITPR1, and a mouse model expressing this ITPR1-D2594K+/- GOF mutation. We found that heterozygous ITPR1-D2594K+/- mutant mice exhibited male infertility, azoospermia, and acrosome loss. Furthermore, we functionally characterized a human ITPR1 variant V494I identified in the UK Biobank database as potentially associated with disorders of the testis. We found that the ITPR1-V494I variant significantly enhanced IP3 -induced Ca2+ release in HEK293 cells. Thus, ITPR1 hyperactivity may increase the risk of testicular dysfunction.

Intrinsically Disordered Protein Condensate-Modified Surface for Mitigation of Biofouling and Foreign Body Response.

Mitigation of biofouling and the host's foreign body response (FBR) is a critical challenge with biomedical implants. The surface coating with various anti-fouling materials provides a solution to overcome it, but limited options in clinic and their potential immunogenicity drive the development of more alternative coating materials. Herein, inspired by liquid-liquid phase separation of intrinsically disordered proteins (IDPs) to form separated condensates in physiological conditions, we develop a new type of low-fouling biomaterial based on flexible IDP of FUS protein containing rich hydrophilic residues. A chemical structure-defined FUS IDP sequence tagged with a tetra-cysteine motif (IDPFUS) was engineered and applied for covalent immobilization on various surfaces to form a uniform layer of protein tangles, which boosted strong hydration on surfaces, as revealed by molecular dynamics simulation. The IDPFUS-coated surfaces displayed excellent performance in resisting adsorption of various proteins and adhesion of different cells, platelets, and bacteria. Moreover, the IDPFUS-coated implants largely mitigated the host's FBR compared with bare implants and particularly outperformed PEG-coated implants in reducing collagen encapsulation. Thus, this novel low-fouling and anti-FBR strategy provides a potential surface coating material for biomedical implants, which will also shed light on exploring similar applications of other IDP proteins.

Genetically Encoded Phosphine Ligand for Metalloprotein Design.

Phosphine ligands are the most important class of ligands for cross-coupling reactions due to their unique electronic and steric properties. However, metalloproteins generally rely on nitrogen, sulfur, or oxygen ligands. Here, we report the genetic incorporation of P3BF, which contains a biocompatible borane-protected phosphine, into proteins. This step is followed by a straightforward one-pot strategy to perform deboronation and palladium coordination in aqueous and aerobic conditions. The genetically encoded phosphine ligand P3BF should significantly expand our ability to design functional metalloproteins.

Accumulation of Plasma-Derived Lipids in the Lipid Core and Necrotic Core of Human Atheroma: Imaging Mass Spectrometry and Histopathological Analyses.

Objective: To clarify the pathogenesis of human atheroma, the origin of deposited lipids, the developmental mechanism of liponecrotic tissue, and the significance of the oxidation of phospholipids were investigated using mass spectrometry-aided imaging and immunohistochemistry.Atherosclerotic lesions in human coronary arteries were divided into 3 groups: pathologic intimal thickening with lipid pool, atheroma with lipid core, and atheroma with necrotic core. The lipid pool and lipid core were characterized by the deposition of extracellular lipids. The necrotic core comprised extracellular lipids and liponecrotic tissue. The proportion of cholesteryl linoleate in cholesteryl linoleate+cholesteryl oleate fraction in the extracellular lipid and liponecrotic regions differed significantly from that of the macrophage foam cell-dominant region, and the plasma-derived components (apolipoprotein B and fibrinogen) were localized in the regions. The liponecrotic region was devoid of elastic and collagen fibers and accompanied by macrophage infiltration in the surrounding tissue. Non-oxidized phospholipid (Non-OxPL), OxPL, and Mox macrophages were detected in the three lesions. In the atheroma with lipid core and atheroma with necrotic core, non-OxPL tended to localize in the superficial layer, whereas OxPL was distributed evenly. Mox macrophages were colocalized with OxPL epitopes.In human atherosclerosis, plasma-derived lipids accumulate to form the lipid pool of pathologic intimal thickening, lipid core of atheroma with lipid core, and necrotic core of atheroma with necrotic core. The liponecrotic tissue in the necrotic core appears to be developed by the loss of elastic and collagen fibers. Non-OxPL in the accumulated lipids is oxidized to form OxPL, which may contribute to the lesion development through Mox macrophages.

HSP25 Vaccination Attenuates Atherogenesis via Upregulation of LDLR Expression, Lowering of PCSK9 Levels and Curbing of Inflammation.

[Figure: see text].

Heat shock protein 27 immune complex altered signaling and transport (ICAST): Novel mechanisms of attenuating inflammation.

Blood levels of heat shock protein (HSP27) and natural IgG auto-antibodies to HSP27 (AAbs) are higher in healthy controls compared to cardiovascular disease patients. Vaccination of mice with recombinant HSP25 (rHSP25, murine ortholog of human rHSP27) increased AAb levels, attenuated atherogenesis and reduced plaque inflammation and cholesterol content. We sought to determine if the HSP27 immune complex (IC) altered MΦ inflammation signaling (Toll Like Receptor 4; TLR4), and scavenger receptors involved in cholesterol uptake (SR-AI, CD-36). Combining a validated polyclonal IgG anti-HSP27 antibody (PAb) with rHSP27 enhanced binding to THP-1 MΦ cell membranes and activation of NF-κB signaling via TLR4, competing away LPS and effecting an anti-inflammatory cytokine profile. Similarly, adding the PAb with rHSP27 enhanced binding to SR-AI and CD-36, as well as lowered oxLDL binding in HEK293 cells separately transfected with SR-AI and CD-36, or THP-1 MΦ. Finally, the PAb enhanced the uptake and internalization of rHSP27 in THP-1 MΦ. Thus, the HSP27 IC potentiates HSP27 cell membrane signaling with receptors involved in modulating inflammation and cholesterol uptake, as well as HSP27 internalization. Going forward, we are focusing on the development of HSP27 Immune Complex Altered Signaling and Transport (ICAST) as a means of modulating inflammation.

Recent Progress of Nanozymes in the Detection of Pathogenic Microorganisms.

Infectious diseases are among the world's principal health problems. It is crucial to develop rapid, accurate and cost-effective methods for the detection of pathogenic microorganisms. Recently, considerable progress has been achieved in the field of inorganic enzyme mimics (nanozymes). Compared with natural enzymes, nanozymes have higher stability and lower cost. More interestingly, their properties can be designed for various demands. Herein, we introduce the latest research progress on the detection of pathogenic microorganisms by using various nanozymes. We also discuss the current challenges of nanozymes in biosensing and provide some strategies to overcome these barriers.

Fully Synthetic Invariant NKT Cell-Dependent Self-Adjuvanting Antitumor Vaccines Eliciting Potent Immune Response in Mice.

Constructing an effective therapeutic cancer vaccine is very attractive and promising for cancer immunotherapy. However, the poor immunogenicity of tumor antigens and suppression of the immune system in the tumor microenvironment are two major obstacles for developing effective cancer vaccines. Invariant NKT cells (iNKT cells), which are essential bridges between the innate and adaptive immune systems, can be rapidly activated by their agonists and, consequently, evoke whole immune systems. Herein, we conjugated a potent agonist of the iNKT cell, α-galactosylceramide (α-GalCer), with the tumor-associated MUC1 glycopeptide antigens as novel self-adjuvanting cancer vaccines through click chemistry. Immunological studies revealed that the mouse immune system was potently evoked and that high levels of tumor-specific IgG antibodies were elicited by vaccine conjugates without an external adjuvant. The produced antibodies could specifically recognize and bind to antigen-expressing cancer cells and, subsequently, induce cytotoxicity through complement-dependent cytotoxicity. Thus, the insertion of α-GalCer significantly improved the immunogenicity of the MUC1 glycopeptide and induced strong antigen-specific antitumor responses, indicating that α-GalCer is an effective built-in adjuvant for constructing potent chemical synthetic antitumor vaccines.

Unlike estrogens that increase PCSK9 levels post-menopause HSP27 vaccination lowers cholesterol levels and atherogenesis due to divergent effects on PCSK9 and LDLR.

AIMS: The estrogen-inducible protein Heat Shock Protein 27 (HSP27) as well as anti-HSP27 antibodies are elevated in healthy subjects compared to cardiovascular disease patients. Vaccination of ApoE-/- mice with recombinant HSP25 (rHSP25, the murine ortholog), boosts anti- HSP25 levels and attenuates atherogenesis. As estrogens promote HSP27 synthesis, cellular release and blood levels, we hypothesize that menopause will result in loss of HSP27 atheroprotection. Hence, the rationale for this study is to compare the efficacy of rHSP25 vaccination vs. estradiol (E2) therapy for the prevention of post-menopausal atherogenesis. METHODS AND RESULTS: ApoE-/- mice subjected to ovariectomy (OVX) showed a 65 % increase atherosclerotic burden compared to sham mice after 5 weeks of a high fat diet. Relative to vaccination with rC1, a truncated HSP27 control peptide, atherogenesis was reduced by 5-weekly rHSP25 vaccinations (-43 %), a subcutaneous E2 slow release pellet (-52 %) or a combination thereof (-82 %). Plasma cholesterol levels declined in parallel with the reductions in atherogenesis, but relative to rC1/OVX mice plasma PCSK9 levels were 52 % higher in E2/OVX and 41 % lower in rHSP25/OVX mice (p < 0.0001 for both). Hepatic LDLR mRNA levels did not change with E2 treatment but increased markedly with rHSP25 vaccination. Conversely, hepatic PCSK9 mRNA increased 148 % with E2 treatment vs. rC1/OVX but did not change with rHSP25 vaccination. In human HepG2 hepatocytes E2 increased PCSK9 promoter activity 303 %, while the combination of [rHSP27 + PAb] decreased PCSK9 promoter activity by 64 %. CONCLUSION: The reduction in post-OVX atherogenesis and cholesterol levels with rHSP25 vaccination is associated with increased LDLR but not PCSK9 expression. Surprisingly, E2 therapy attenuates atherogenesis and cholesterol levels post-OVX without altering LDLR but increases PCSK9 expression and promoter activity. This is the first documentation of increased PCSK9 expression with E2 therapy and raises questions about balancing physiological estrogenic / PCSK9 homeostasis and targeting PCSK9 in women - are there effects beyond cholesterol?

Metal ion and light sequentially induced sol-gel-sol transition of a responsive peptide-hydrogel.

We developed a new responsive peptide hydrogel FmocFFpSC(oNB)-PEG, which could achieve gel formation induced by calcium ions and sequential dissolution stimulated by light. It provides a potential delivery system for the efficient encapsulation of drugs and their controlled release in a spatial and temporal way.

Colorimetric determination of ascorbic acid using a polyallylamine-stabilized IrO2/graphene oxide nanozyme as a peroxidase mimic.

The authors describe a peroxidase-mimicking nanozyme composed of IrO2 and graphene oxide (GO). It was synthesized from monodisperse IrO2 nanoparticles with an average diameter of 1.7 ± 0.3 nm that were prepared by pulsed laser ablation in ethanol. The nanoparticles were then placed on polyallylamine-modified GO nanosheets through electrostatic interaction. The peroxidase-like activity of the resulting nanocomposites was evaluated by catalytic oxidation of 3,3',5,5'-tetramethylbenzidine in the presence of H2O2. Kinetic results demonstrated that the catalytic behavior of the nanocomposites follows Michaelis-Menten kinetics. Experiments performed with terephthalic acid and cytochrome C confirmed that the peroxidase-like activity originated from the electron transfer mechanism rather than from generation of hydroxy radicals. The peroxidase-like activity is inhibited in the presence of ascorbic acid (AA). Based on this property, a colorimetric assay was developed for the determination of AA by exploiting the peroxidase-like activity of IrO2/GO nanocomposites. The linear relationship between absorbance at 652 nm and the concentration of AA was acquired. The limit of detection for AA is 324 nM. Further applications of the method for AA detection in real samples were also successfully demonstrated. Graphical abstractSchematic of the preparation of polyallylamine (PAH)-stabilized IrO2/GO nanocomposites and the colorimetric detection of AA based on the peroxidase-like activity of IrO2/GO nanocomposites.

Investigation of the Assembly Behavior of an Amphiphilic Lipopeptide at the Liquid Crystal-Aqueous Interface.

In this article, we designed an amphiphilic lipopeptide molecule, 5(6)-carboxyfluorescein-KKKKKKSKTK-Cys(C12H25)-OMe (FAM-lipopeptide-C12), and studied its assembly behavior at the 4-cyano-4'-pentylbiphenyl (5CB)-aqueous interface. The ordering transitions of liquid crystals (LCs) revealed that FAM-lipopeptide-C12 can assemble at the LC-aqueous interface (both planar and curved interfaces). The assembly can be destroyed by adding trypsin, which catalyzes the hydrolysis of lipopeptides. Fluorescence measurements further confirmed the assembly and deassembly behavior of FAM-lipopeptide-C12 at the LC-aqueous interface. Overall, our work provides a general method for the construction of a biointerface by directly assembling amphiphilic lipopeptides at the LC-aqueous interface, which can potentially be used in selectively detecting the activity of specific enzymes and other biomolecular interactions.

Stereoselective synthesis of a phosphonate pThr mimetic via palladium-catalyzed γ-C(sp3)-H activation for peptide preparation.

We report a facile synthetic strategy toward CH2-substituted phosphothreonine mimetics. Herein, inexpensive valine with a directing group was converted into homothreonine via palladium-catalyzed γ-methyl C(sp3)-H bond activation, followed by construction of a phosphorus-carbon bond via the well-developed Appel reaction and Michaelis-Becker reaction with a total yield of 30%. Furthermore, the derived mimetic was applied for solid-phase synthesis of two phosphopeptide inhibitors. This efficient synthesis provides a chance to prepare not only phosphopeptides but also phosphoproteins resistant to phosphatases.

TDP-43 specific reduction induced by Di-hydrophobic tags conjugated peptides.

TAR DNA binding protein 43 (TDP-43) is a key target in amyotrophic lateral sclerosis (ALS) treatment. Here, based on hydrophobic tagging strategy, we designed and synthesized a series of single or double hydrophobic tags conjugated peptides D1-D8. Among them, it was found that D4 displayed strongest ability to induce TDP-43 degradation in cells. D4 could reduce TDP-43 induced cytotoxicity. Besides, D4 could reduce TDP-43 levels in a transgenic drosophila model.

Reduced expression of cardiac ryanodine receptor protects against stress-induced ventricular tachyarrhythmia, but increases the susceptibility to cardiac alternans.

Reduced protein expression of the cardiac ryanodine receptor type 2 (RyR2) is thought to affect the susceptibility to stress-induced ventricular tachyarrhythmia (VT) and cardiac alternans, but direct evidence for the role of RyR2 protein expression in VT and cardiac alternans is lacking. Here, we used a mouse model (crrm1) that expresses a reduced level of the RyR2 protein to determine the impact of reduced RyR2 protein expression on the susceptibility to VT, cardiac alternans, cardiac hypertrophy, and sudden death. Electrocardiographic analysis revealed that after the injection of relatively high doses of caffeine and epinephrine (agents commonly used for stress test), wild-type (WT) mice displayed long-lasting VTs, whereas the crrm1 mutant mice exhibited no VTs at all, indicating that the crrm1 mutant mice are resistant to stress-induced VTs. Intact heart Ca2+ imaging and action potential (AP) recordings showed that the crrm1 mutant mice are more susceptible to fast-pacing induced Ca2+ alternans and AP duration alternans compared with WT mice. The crrm1 mutant mice also showed an increased heart-to-body-weight ratio and incidence of sudden death at young ages. Furthermore, the crrm1 mutant hearts displayed altered Ca2+ transients with increased time-to-peak and decay time (T50), increased ventricular wall thickness and ventricular cell area compared with WT hearts. These results indicate that reduced RyR2 protein expression suppresses stress-induced VTs, but enhances the susceptibility to cardiac alternans, hypertrophy, and sudden death.

Phosphorylation at Ser8 as an Intrinsic Regulatory Switch to Regulate the Morphologies and Structures of Alzheimer's 40-residue ß-Amyloid (Aß40) Fibrils.

Polymorphism of amyloid-ß (Aß) fibrils, implying different fibril structures, may play important pathological roles in Alzheimer's disease (AD). Morphologies of Aß fibrils were found to be sensitive to fibrillation conditions. Herein, the Ser8-phosphorylated Aß (pAß), which is assumed to specially associate with symptomatic AD, is reported to modify the morphology, biophysical properties, cellular toxicity, and structures of Aß fibrils. Under the same fibrillation conditions, pAß favors the formation of fibrils (Fpß), which are different from the wild-type Aß fibrils (Fß). Both Fß and Fpß fibrils show single predominant morphologies. Compared with Fß, Fpß exhibits higher propagation efficiency and higher neuronal cell toxicity. The residue-specific structural differences between the Fß- and Fpß-seeded Aß fibrils were identified using magic angle spin NMR. Our results suggest a potential regulatory mechanism of phosphorylation on Aß fibril formation in AD and imply that the post-translationally modified Aß, especially the phosphorylated Aß, may be an important target for the diagnosis or treatment of AD at specific stages.

A site-specific branching poly-glutamate tag mediates intracellular protein delivery by cationic lipids.

Intracellular protein delivery is of significance for cellular protein analysis and therapeutic development, but remains challenging technically. Herein, we report a general and highly potent strategy for intracellular protein delivery based on commercially available cationic lipids. In this strategy, a designed double branching poly-glutamate tag is site-specifically attached onto the C-terminal of protein cargos via expressed protein ligation (EPL), which mediates the entrapment of proteins into cationic liposomes driven by electrostatic interaction. The resultant protein-lipid complexes can enter into cytosol with a high efficiency even at the low protein concentration while maintaining protein's biological activity.