Binding of a Diphosphorylated-ITAM peptide to spleen tyrosine kinase (Syk) induces distal conformational changes: A hydrogen exchange mass spectrometry study

Structural flexibility plays a crucial role in protein function. To assess whether specific structural changes are associated with the binding of an immunoreceptor tyrosine-based activation motif (ITAM) to the tandem Src homology-2 domains (tSH2) of the spleen tyrosine kinase [EC 2.7.7.112] (Syk), we used an approach based on protein hydrogen/deuterium exchange in the presence and absence of the diphosphorylated ITAM peptide. The protein deuterium uptake by the intact Syk protein was monitored in time by electrospray mass spectrometry, which revealed a dramatic relative decrease in deuterium uptake when the protein was bound to the ITAM peptide, suggesting an overall change in protein dynamics. Subsequently, the deuterium incorporation of individual segments of the protein was investigated using proteolysis and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) peptide mass-analysis, which revealed that several regions of Syk tSH2 are significantly more protected from exchange in the presence of the ITAM peptide. Four protected regions encompass the phosphotyrosine and hydrophobic binding sites on the SH2 domains, whereas two other protected regions are located in the inter-SH2 linker motif and do not make any direct contacts with the peptide. Interestingly, our data suggest that binding of the ITAM peptide to Syk tSH2 induces distal structural effects on the protein that stabilize the inter-SH2 linker region, possibly by raising the degree of helical structure upon binding.     

A synthetic mimic of human Fc receptors: defined chemical modification of cell surfaces enables efficient endocytic uptake of human immunoglobulin-G

Binding of ligands to macromolecular receptors on the surface of mammalian cells often results in ligand uptake through receptor-mediated endocytosis. Certain human leukocytes and epithelial cells express Fc receptors (FcRs) that bind and internalize antibodies through this mechanism. To mimic this process, we synthesized an artificial FcR comprising the membrane anchor N-alkyl-3beta-amino-5alpha-cholestane linked to a disulfide-constrained cyclic peptide, termed FcIII, known to exhibit high affinity and specificity for the Fc region of human IgG. Treatment of human Jurkat lymphocytes that lack natural FcRs with the synthetic FcR (1 microM, 1 h) installed an average of approximately 6.2 x 10(5) synthetic receptor molecules per cell surface. These treated cells gained the capacity to internalize human IgG at levels greater than human THP-1 cells that express the natural receptors FcgammaRI and FcgammaRII. By linking binding motifs for circulating ligands to membrane anchors that cycle between the cell surface and intracellular endosomes, minimalistic cell surface receptors can be used to destroy targeted ligands by endocytosis. These small mimics of macromolecular receptors may be useful for controlling the extracellular abundance of ligands involved in disease.     

Intracellular Delivery of Peptidyl Ligands by Reversible Cyclization: Discovery of a PDZ Domain Inhibitor that Rescues CFTR Activity

A general strategy was developed for the intracellular delivery of linear peptidyl ligands through fusion to a cell‐penetrating peptide and cyclization of the fusion peptides via a disulfide bond. The resulting cyclic peptides are cell permeable and have improved proteolytic stability. Once inside the cell, the disulfide bond is reduced to produce linear biologically active peptides. This strategy was applied to generate a cell‐permeable peptide substrate for real‐time detection of intracellular caspase activities during apoptosis and an inhibitor for the CFTR‐associated ligand (CAL) PDZ domain as a potential treatment for cystic fibrosis.     

Bioactive constituents from Chinese natural medicines. XX. Inhibitors of antigen-induced degranulation in RBL-2H3 cells from the seeds of Psoralea corylifolia

The ethyl acetate-soluble fraction of the methanolic extract from the seeds of Psoralea corylifolia was found to inhibit the release of beta-hexosaminidase, as a marker of antigen-IgE-induced degranulation in RBL-2H3 cells. Sixteen constituents were isolated from the ethyl acetate-soluble fraction and several constituents, Delta(1),3-hydroxybakuchiol (IC(50) = 49 microM), Delta(3),2-hydroxybakuchiol (69 microM, bavachin (58 microM), and psoralidin (ca. 100 microM), showed inhibitory activities against the antigen-induced degranulation.     

Intracellular Delivery of Native Proteins Facilitated by Cell‐Penetrating Poly(disulfide)s

Intracellular delivery of therapeutic proteins is highly challenging and in most cases requires chemical or genetic modifications. Herein, two complementary approaches for endocytosis‐independent delivery of proteins to live mammalian cells are reported. By using either a “glycan” tag naturally derived from glycosylated proteins or a “traceless” tag that could reversibly label native lysines on non‐glycosylated proteins, followed by bioorthogonal conjugation with cell‐penetrating poly(disulfide)s (CPDs), we achieved intracellular delivery of proteins (including antibodies and enzymes) which, upon spontaneous degradation of CPDs, led to successful release of their “native” functional forms with immediate bioavailability.     

An Intrinsically Disordered Peptide Facilitates Non‐Endosomal Cell Entry

Many cell‐penetrating peptides (CPPs) fold at cell surfaces, adopting α‐ or β‐structure that enable their intracellular transport. However, the same structural folds that facilitate cellular entry can also elicit potent membrane‐lytic activity, limiting their use in delivery applications. Further, a distinct CPP can enter cells through many mechanisms, often leading to endosomal entrapment. Herein, we describe an intrinsically disordered peptide (CLIP6) that exclusively employs non‐endosomal mechanisms to cross cellular membranes, while being remarkably biocompatible and serum‐stable. We show that a single anionic glutamate residue is responsible for maintaining the disordered bioactive state of the peptide, defines its mechanism of cellular entry, and is central to its biocompatibility. CLIP6 can deliver membrane‐impermeable cargo directly to the cytoplasm of cells, suggesting its broad utility for delivery of drug candidates limited by poor cell permeability and endosomal degradation.     

Effects of metal elements on beta-hexosaminidase release from rat basophilic leukemia cells (RBL-2H3).

Immediate allergy is caused by a chemical mediator released from basophile and mast cells via cell degranulation due to reaction between an immunoglobulin E (IgE) antibody, bound with the IgE receptor on the cell membrane, and an antigen. The present authors have established a new method for assaying the enzyme activity of beta-hexosaminidase as an index of chemical mediator release. Using cultured cells instead of conventional methods based on histamine release from mast cells, the present method permits highly accurate mass screening since it uses a well-established cell line of rat basophilic leukemia cells (RBL-2H3). The effects of metal elements on immediate allergic reaction were evaluated using a newly developed assay system. A total of 38 metal elements were investigated for effects on immediate allergic reactions in vitro. These elements were classified by five types on the basis of action on beta-hexosanimidase release: 1) those which showed very strong inhibitory action, such as ZnCl2 and ZrCl4, 2) those which showed relatively strong inhibitory action, such as CdCl2 and CuCl2, 3) those which showed relatively weak inhibitory action, such as CoCl2 and Pb(NO3)2, 4) those which showed neither inhibitory nor promoting action, such as MnCl2 and SrCl2, and 5) AgNO3, which alone showed promoting action.     

Reduction‐Triggered Transformation of Disulfide‐Containing Micelles at Chemically Tunable Rates

A dilemma exists between the circulation stability and cargo release/mass diffusion at desired sites when designing delivery nanocarriers and in vivo nanoreactors. Reported herein are disulfide‐crosslinked (DCL) micelles exhibiting reduction‐triggered switching of crosslinking modules and synchronized hydrophobic‐to‐hydrophilic transition. Tumor cell targeted DCL micelles undergo cytoplasmic milieu triggered disulfide cleavage and self‐immolative decaging reactions at chemically adjustable rates, generating primary amine moieties. Extensive amidation reactions with neighboring ester moieties then occur because of the high local concentration and suppression of the apparent amine pK_a value within the hydrophobic cores, thus leading to the transformation of crosslinking modules and formation of tracelessly crosslinked (TCL) micelles, with hydrophilic cores, inside live cells. We further integrate this design principle with theranostic nanocarriers for selective intracellular drug transport guided by enhanced magnetic resonance (MR) imaging performance.     

Relation of Photochemical Internalization to Heat,pH and Ca2+ Ions

The inefficient endosomal escape of drugs or macromolecules is a major obstacle to achieving successful delivery to therapeutic targets. An efficient approach to circumvent this barrier is photochemical internalization (PCI), which uses light and photosensitizers for endosomal escape of the delivered macromolecules. The PCI mechanism is related to photogenerated singlet oxygen, but the mechanism is still unclear. In this study, we examined the relation of PCI to heat, pH and Ca²⁺ ions using cell penetrating peptide (CPP)‐cargo‐photosensitizer (Alexa546 or Alexa633) conjugates. A cell temperature changing experiment demonstrated that heat (thermal mechanism) does not significantly contribute to the photoinduced endosomal escape. Inhibition of V‐ATPase proton pump activity and endosomal pH upregulation indicated that PCI‐mediated endosomal escape needs endosomal acidification prior to photoirradiation. Imaging of the CPP‐cargo‐photosensitizer and Ca²⁺ ions during photostimulation showed that intracellular calcium increase is not the cause of the endosomal escape of the complex. The increment is mainly due to Ca²⁺ influx. These findings show the importance of extra‐ and intracellular milieu conditions in the PCI mechanism and enrich our understanding of PCI‐related changes in cell.     

Lipoic Acid-Modified Oligoethyleneimine-Mediated miR-34a Delivery to Achieve the Anti-Tumor Efficacy

MiR-34a, an important tumor suppressor, has been demonstrated to possess great potential in tumor gene therapy. To achieve the upregulation of miR-34a expression level, an oligoethyleneimine (OEI) derivative was constructed and employed as the carrier through the modification with lipoic acid (LA), namely LA-OEI. In contrast to OEI, the derivative LA-OEI exhibited superior transfection efficiency measured by confocal laser scanning microscopy and flow cytometry, owing to rapid cargo release in the disulfide bond-based reduction sensitive pattern. The anti-proliferation and anti-migration effects were tested after the miR-34a transfection to evaluate the anti-tumor response, using human cervical carcinoma cell line HeLa as a model. The delivery of LA-OEI/miR-34a nanoparticles could achieve obvious anti-proliferative effect caused by the induction of cell apoptosis and cell cycle arrest at G1 phase. In addition, it could inhibit the migration of tumor cells via the downregulation of MMP-9 and Notch-1 level. Overall, the LA-OEI-mediated miR-34a delivery was potential to be used as an effective way in the tumor gene therapy.     

Activation of T lymphocytes by the Fc portion of immunoglobulin

T lymphocytes are stimulated to release T-cell-replacing factors in response to Fc fragments of human IgG, Lyt 1+23- T cells are directly triggered to factor production by Fc subfragments, derived from intact Fc fragments by macrophage-dependent enzymatic cleavage. These factor(s) replace T cell function in two Fc-mediated immune responses; induction of polyclonal antibody synthesis, and potentiation of anti-SRBC responses.     

Mitochondria‐Targeting,Intracellular Delivery of Native Proteins Using Biodegradable Silica Nanoparticles

Mitochondria are key organelles in mammalian cells whose dysfunction is linked to various diseases. Drugs targeting mitochondrial proteins provide a highly promising strategy for potential therapeutics. Methods for the delivery of small‐molecule drugs to the mitochondria are available, but these are not suitable for macromolecules, such as proteins. Herein, we report the delivery of native proteins and antibodies to the mitochondria using biodegradable silica nanoparticles (BS–NPs). The modification of the nanoparticle surface with triphenylphosphonium (TPP) and cell‐penetrating poly(disulfide)s (CPD) facilitated their rapid intracellular uptake with minimal endolysosomal trapping, providing sufficient time for effective mitochondrial localization followed by glutathione‐triggered biodegradation and of native, functional proteins into the mitochondria.     

Mitochondria‐Targeting,Intracellular Delivery of Native Proteins Using Biodegradable Silica Nanoparticles

Mitochondria are key organelles in mammalian cells whose dysfunction is linked to various diseases. Drugs targeting mitochondrial proteins provide a highly promising strategy for potential therapeutics. Methods for the delivery of small‐molecule drugs to the mitochondria are available, but these are not suitable for macromolecules, such as proteins. Herein, we report the delivery of native proteins and antibodies to the mitochondria using biodegradable silica nanoparticles (BS–NPs). The modification of the nanoparticle surface with triphenylphosphonium (TPP) and cell‐penetrating poly(disulfide)s (CPD) facilitated their rapid intracellular uptake with minimal endolysosomal trapping, providing sufficient time for effective mitochondrial localization followed by glutathione‐triggered biodegradation and of native, functional proteins into the mitochondria.     

Cell‐Penetrating Poly(disulfide) Assisted Intracellular Delivery of Mesoporous Silica Nanoparticles for Inhibition of miR‐21 Function and Detection of Subsequent Therapeutic Effects

The design of drug delivery systems capable of minimal endolysosomal trapping, controlled drug release, and real‐time monitoring of drug effect is highly desirable for personalized medicine. Herein, by using mesoporous silica nanoparticles (MSNs) coated with cell‐penetrating poly(disulfide)s and a fluorogenic apoptosis‐detecting peptide (DEVD‐AAN), we have developed a platform that could be uptaken rapidly by mammalian cells via endocytosis‐independent pathways. Subsequent loading of these MSNs with small molecule inhibitors and antisense oligonucleotides resulted in intracellular release of these drugs, leading to combination inhibition of endogenous miR‐21 activities which was immediately detectable by the MSN surface‐coated peptide using two‐photon fluorescence microscopy.     

Reduction-sensitive liposomes from a multifunctional lipid conjugate and natural phospholipids: reduction and release kinetics and cellular uptake

The development of targeted and triggerable delivery systems is of high relevance for anticancer therapies. We report here on reduction-sensitive liposomes composed of a novel multifunctional lipidlike conjugate, containing a disulfide bond and a biotin moiety, and natural phospholipids. The incorporation of the disulfide conjugate into vesicles and the kinetics of their reduction were studied using dansyl-labeled conjugate 1 in using the dansyl fluorescence environmental sensitivity and the Fo?rster resonance energy transfer from dansyl to rhodamine-labeled phospholipids. Cleavage of the disulfide bridge (e.g., by tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), l-cysteine, or glutathione (GSH)) removed the hydrophilic headgroup of the conjugate and thus changed the membrane organization leading to the release of entrapped molecules. Upon nonspecific uptake of vesicles by macrophages, calcein release from reduction-sensitive liposomes consisting of the disulfide conjugate and phospholipids was more efficient than from reduction-insensitive liposomes composed only of phospholipids. The binding of streptavidin to the conjugates did not interfere with either the subsequent reduction of the disulfide bond of the conjugate or the release of entrapped molecules. Breast cancer cell line BT-474, overexpressing the HER2 receptor, showed a high uptake of the reduction-sensitive doxorubicin-loaded liposomes functionalized with the biotin-tagged anti-HER2 antibody. The release of the entrapped cargo inside the cells was observed, implying the potential of using our system for active targeting and delivery.     

Delivery of bioactive,gel-isolated proteins into live cells

The delivery of proteins into live cells is a promising strategy for the targeted modulation of protein-protein interactions and the manipulation of specific cellular functions. Cellular delivery can be facilitated by complexing the protein of interest with carrier molecules. Recently, an amphipatic peptide was identified, Pep-1 (KETWWETWWTE WSQPKKKRKV), which crosses the plasma membrane of many cell types to carry and deliver proteins as large as antibodies. Pep-1 effectively delivers proteins in solution; but Pep-1 is not suitable for delivering sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) isolated proteins because Pep-1 complexes with cargo proteins are destroyed by SDS. Here, we report cellular delivery of SDS-PAGE-isolated proteins, without causing cellular damage, by using a nonionic detergent, Triton X-100, as carrier. To determine the specificity of our method, we separated antibodies against different intracellular targets by nonreducing SDS-PAGE. Following electrophoresis, the antibody bands were detected by zinc-imidazole reverse staining, excised, in-gel refolded with Triton X-100, and eluted in detergent-free phosphate-buffered saline. When overlaid on cultured NIH 3T3 cells, the antibodies penetrated the cells localizing to their corresponding intracellular targets. These results are proof-of-principle for the delivery of gel-isolated bioactive proteins into cultured cells and suggest new ways for experimental protein therapy and for studying protein-protein interactions using gel-isolated protein.     

Intracellular Delivery of Functional Native Antibodies under Hypoxic Conditions by Using a Biodegradable Silica Nanoquencher

Antibodies are important biopharmaceuticals, but almost all existing antibody‐based drugs are limited to targeting antigens located at the cell exterior because of the inability of antibodies to enter the cell interior. Available methods for intracellular delivery of antibodies have major shortcomings. Herein, we report an approach to encapsulate native antibodies in a biodegradable silica nanoquencher (BS‐qNP), which could undergo efficient cellular uptake and intracellular degradation to release antibodies only under hypoxic conditions. By coating the surface of BS‐qNP with cell‐penetrating poly(disulfide)s (CPD), the delivered antibodies (or other proteins) avoided endolysosomal trapping. Doping of the silica coating with a fluorescent dye and a dark hole quencher further endowed BS‐qNP with hypoxia‐responsive fluorescence turn‐on property. Our antibody delivery system thus provides the first platform capable of stable encapsulation, efficient uptake, on‐demand antibody release, and imaging of release/cell state.     

Bioactive saponins and glycosides. XXVIII. New triterpene saponins, foliatheasaponins I, II, III, IV, and V, from Tencha (the leaves of Camellia sinensis)

New triterpene saponins, foliatheasaponins I-V, were isolated from the methanolic extract of Tencha [the leaves of Camellia sinensis (L.) O. KUNTZE (Theaceae)]. The chemical structures of these new saponins were elucidated on the basis of chemical and physicochemical evidence. Among the new saponins, foliatheasaponins II and III, were found to inhibit release of beta-hexosaminidase, as a marker of antigen-induced degranulation, in RBL-2H3 cells.     

Cell-penetrating peptides as delivery vehicles for a protein-targeted terbium complex

Release after transmission: Arginine-rich, cell-penetrating peptides (CPPs) mediate cytoplasmic delivery of trimethoprim (TMP)-terbium complex conjugates and selective, intracellular labeling of E.?coli dihydrofolate reductase (eDHFR) fusion proteins. A disulfide bond linking CPP and cargo is reduced following uptake. CPP conjugation can be used to deliver otherwise cell-impermeable, ligand-fluorophore conjugates.     

Magnetically Guided Protein Transduction by Hybrid Nanogel Chaperones with Iron Oxide Nanoparticles

Protein pharmaceuticals show great therapeutic promise, but effective intracellular delivery remains challenging. To address the need for efficient protein transduction systems, we used a magnetic nanogel chaperone (MC): a hybrid of a polysaccharide nanogel, a protein carrier with molecular chaperone‐like properties, and iron oxide nanoparticles, enabling magnetically guided delivery. The MC complexed with model proteins, such as BSA and insulin, and was not cytotoxic. Cargo proteins were delivered to the target HeLa cell cytosol using a magnetic field to promote movement of the protein complex toward the cells. Delivery was confirmed by fluorescence microscopy and flow cytometry. Delivered β‐galactosidase, inactive within the MC complex, became enzymatically active within cells to convert a prodrug. Thus, cargo proteins were released from MC complexes through exchange interactions with cytosolic proteins. The MC is a promising tool for realizing the therapeutic potential of proteins.