Toshihide Takeuchi

High-resolution multi-dimensional NMR spectroscopy of proteins in human cells

In-cell NMR is an isotope-aided multi-dimensional NMR technique that enables observations of conformations and functions of proteins in living cells at the atomic level. This method has been successfully applied to proteins overexpressed in bacteria, providing information on protein–ligand interactions and conformations. However, the application of in-cell NMR to eukaryotic cells has been limited to Xenopus laevis oocytes. Wider application of the technique is hampered by inefficient delivery of isotope-labelled proteins into eukaryote somatic cells. Here we describe a method to obtain high-resolution two-dimensional (2D) heteronuclear NMR spectra of proteins inside living human cells. Proteins were delivered to the cytosol by the pyrenebutyrate-mediated action of cell-penetrating peptides linked covalently to the proteins. The proteins were subsequently released from cell-penetrating peptides by endogenous enzymatic activity or by autonomous reductive cleavage. The heteronuclear 2D spectra of three different proteins inside human cells demonstrate the broad application of this technique to studying interactions and protein processing. The in-cell NMR spectra of FKBP12 (also known as FKBP1A) show the formation of specific complexes between the protein and extracellularly administered immunosuppressants, demonstrating the utility of this technique in drug screening programs. Moreover, in-cell NMR spectroscopy demonstrates that ubiquitin has much higher hydrogen exchange rates in the intracellular environment, possibly due to multiple interactions with endogenous proteins.

Cellular Internalization and Distribution of Arginine-Rich Peptides as a Function of Extracellular Peptide Concentration, Serum, and Plasma Membrane Associated Proteoglycans

The exact mechanisms by which arginine-rich cell-penetrating peptides enter cells are still the subject of debate. Here, we have analyzed in detail the effects of serum and extracellular concentration on the internalization of oligoarginines (R n; n = 4, 8, 12, 16). The presence of serum in the incubation medium had a major influence on the uptake of R12 and R16 peptides but did not affect the uptake of R4 and R8 significantly. Incubation of cells at 37 degrees C with R12 and R16 peptides in serum-containing medium showed that the majority of labeling was confined to punctate endocytic structures. Performing the same experiments in serum-free media led to a dramatic increase in cytosolic labeling, and similarly diffuse R12 and R16 labeling was observed in cells treated with peptides at 4 degrees C. This suggests, in both cases, that the peptides were entering via a nonendocytic mechanism. Further studies on R12 peptide suggest that the initiation of nonendocytic uptake and cytosolic labeling is also dependent on serum concentration and extracellular peptide concentration. At relatively low concentrations, the peptide labels endocytic structures, but upon raising the peptide concentration, the fraction labeling the cytosol increases dramatically and this accompanies a nonlinear increase in total cellular fluorescence. Membrane-associated proteoglycans also contribute to increasing the peptide concentration at the cell surface by enhancing their recruitment via electrostatic interactions. These results demonstrate that uptake mechanisms of these compounds are highly dependent on both the presence of serum and the effective extracellular peptide concentration.

Enhanced intracellular delivery using arginine-rich peptides by the addition of penetration accelerating sequences (Pas)

Cell penetrating peptides (CPPs), including arginine-rich peptides, are attractive tools for the intracellular delivery of various bioactive molecules with a low membrane permeability. We showed that the accelerated intracellular delivery of arginine-rich peptides was achieved by the addition of a short peptide segment (penetration accelerating sequence, Pas) to arginine-rich CPPs. The cytosolic release of the Pas-attached arginine-rich CPPs was observed within 5 min after the treatment of the cells with the peptides even in the presence of serum. Effectiveness of the Pas segment in the intracellular delivery of bioactive peptides using arginine-rich CPPs was exemplified through the enhanced growth inhibition activity of the malignant glioma cells by a retro-inverso peptide derived from the p53 C-terminal 22-amino-acid segment (positions 361-382).

Temperature-, concentration- and cholesterol-dependent translocation of L- and D-octa-arginine across the plasma and nuclear membrane of CD34+ leukaemia cells

Delineating the mechanisms by which cell-penetrating peptides, such as HIV-Tat peptide, oligoarginines and penetratin, gain access to cells has recently received intense scrutiny. Heightened interest in these entities stems from their ability to enhance cellular delivery of associated macromolecules, such as genes and proteins, suggesting that they may have widespread applications as drug-delivery vectors. Proposed uptake mechanisms include energy-independent plasma membrane translocation and energy-dependent vesicular uptake and internalization through endocytic pathways. In the present study, we investigated the effects of temperature, peptide concentration and plasma membrane cholesterol levels on the uptake of a model cell-penetrating peptide, L-octa-arginine (L-R8) and its D-enantiomer (D-R8) in CD34+ leukaemia cells. We found that, at 4-12 degrees C, L-R8 uniformly labels the cytoplasm and nucleus, but in cells incubated with D-R8 there is additional labelling of the nucleolus which is still prominent at 30 degrees C incubations. At temperatures between 12 and 30 degrees C, the peptides are also localized to endocytic vesicles which consequently appear as the only labelled structures in cells incubated at 37 degrees C. Small increases in the extracellular peptide concentration in 37 degrees C incubations result in a dramatic increase in the fraction of the peptide that is localized to the cytosol and promoted the binding of D-R8 to the nucleolus. Enhanced labelling of the cytosol, nucleus and nucleolus was also achieved by extraction of plasma membrane cholesterol with methyl-beta-cyclodextrin. The data argue for two, temperature-dependent, uptake mechanism for these peptides and for the existence of a threshold concentration for endocytic uptake that when exceeded promotes direct translocation across the plasma membrane.

Arginine-rich peptides and their internalization mechanisms

As the versatility and use of CPPs (cell-penetrating peptides) as intracellular delivery vectors have been widely accepted, the cellular uptake mechanisms that enable their efficient internalization have become the subject of much interest. Arginine-rich peptides, including HIV-1 Tatp (transactivator of transcription peptide), are regarded as a representative class of CPPs. Evidence suggests that macropinocytosis plays a crucial role in the cellular uptake of these peptides. We have recently shown that treatment of cells with arginine-rich peptides induces activation of Rac protein leading to F-actin (filamentous actin) organization and macropinocytosis. We have also shown that depletion of membrane-associated proteoglycans results in the failure of this signalling pathway, suggesting that membrane-associated proteoglycans may act as a potential receptor for the induction of macropinocytic uptake of arginine-rich peptides. However, when the macropinocytic pathway is inhibited at a low temperature or by cholesterol depletion, these peptides can be internalized by alternative mechanisms, one of which appears to be direct translocation of the peptides through the plasma membrane. This review summarizes the current theories on both endocytic and non-endocytic aspects of internalization of arginine-rich peptides.

Direct Observation of Anion-Mediated Translocation of Fluorescent Oligoarginine Carriers into and across Bulk Liquid and Anionic Bilayer Membranes

The recent hypothesis that counteranion‐mediated dynamic inversion of charge and solubility might contribute to diverse functions of oligoarginines in biomembranes was tested with two fluorescently labelled oligomers, FL‐R8, one of the most active cell‐penetrating peptides, and its longer version, FL‐R16. We report evidence for counteranion‐mediated phase transfer from water into bulk chloroform and anionic lipid‐bilayer membranes as well as reverse‐phase transfer from bulk chloroform and across intact lipid‐bilayer membranes into water. The differences found between FL‐R8 and FL‐R16 with regard to location in the bilayer and reverse‐phase transfer from bulk and lipid‐bilayer membranes into water implied that the reported results may be relevant for biological function.

Cell-surface Accumulation of Flock House Virus-derived Peptide Leads to Efficient Internalization via Macropinocytosis

Arginine-rich cell-penetrating peptides (CPPs), including human immunodeficiency virus type 1 (HIV-1) Tat (48-60) and oligoarginines, have been applied as carriers for delivery of cargo molecules, because of their capacity to internalize into cells and penetrate biological membranes. Despite the fact that they have been extensively studied, the factors required for the efficient internalization of CPPs are still unclear. In this report, we evaluated the internalization efficiencies of seven CPPs derived from DNA/RNA-binding peptides, and discovered that a peptide derived from the flock house virus (FHV) coat protein was internalized most efficiently into Chinese hamster ovary (CHO-K1), HeLa, and Jurkat cells. Comparison of the factors facilitating the internalization with those of the Tat peptide revealed that the FHV peptide induces macropinocytosis much more efficiently than the Tat peptide, which leads to its high cellular uptake efficiency. Additionally, the strong adsorption of the FHV peptide on cell membranes via glycosaminoglycans (GAGs) was shown to be a key factor for induction of macropinocytosis, and these steps were successfully monitored by live imaging of the peptide internalization into cells in relation to the actin organization. The remarkable methods of FHV peptide internalization thus highlighted the critical factors for internalizations of the arginine-rich CPPs.

Intercellular chaperone transmission via exosomes contributes to maintenance of protein homeostasis at the organismal level

Significance The heat shock response (HSR), a transcriptional response that up-regulates molecular chaperones upon heat shock, is known to be activated in a cell type-specific manner. Despite such imbalanced HSR upon stress, it is unclear as to how organismal protein homeostasis (proteostasis) is maintained. Here, we show that elevated expression of molecular chaperones in cells non-cell autonomously improves proteostasis in other cells. We further show that exosome-mediated secretion and intercellular transmission of chaperones are responsible for this non–cell-autonomous improvement of proteostasis. Our study reveals a molecular mechanism of non–cell-autonomous maintenance of organismal proteostasis that could functionally compensate for the imbalanced HSR among different cells, and also provides a novel physiological function of exosomes that contributes to maintenance of proteostasis. The heat shock response (HSR), a transcriptional response that up-regulates molecular chaperones upon heat shock, is necessary for cell survival in a stressful environment to maintain protein homeostasis (proteostasis). However, there is accumulating evidence that the HSR does not ubiquitously occur under stress conditions, but largely depends on the cell types. Despite such imbalanced HSR among different cells and tissues, molecular mechanisms by which multicellular organisms maintain their global proteostasis have remained poorly understood. Here, we report that proteostasis can be maintained by molecular chaperones not only in a cell-autonomous manner but also in a non–cell-autonomous manner. We found that elevated expression of molecular chaperones, such as Hsp40 and Hsp70, in a group of cells improves proteostasis in other groups of cells, both in cultured cells and in Drosophila expressing aggregation-prone polyglutamine proteins. We also found that Hsp40, as well as Hsp70 and Hsp90, is physiologically secreted from cells via exosomes, and that the J domain at the N terminus is responsible for its exosome-mediated secretion. Addition of Hsp40/Hsp70-containing exosomes to the culture medium of the polyglutamine-expressing cells results in efficient suppression of inclusion body formation, indicating that molecular chaperones non-cell autonomously improve the protein-folding environment via exosome-mediated transmission. Our study reveals that intercellular chaperone transmission mediated by exosomes is a novel molecular mechanism for non–cell-autonomous maintenance of organismal proteostasis that could functionally compensate for the imbalanced state of the HSR among different cells, and also provides a novel physiological role of exosomes that contributes to maintenance of organismal proteostasis.

Syndecan-4 Is a Receptor for Clathrin-Mediated Endocytosis of Arginine-Rich Cell-Penetrating Peptides

Arginine-rich cell-penetrating peptides (CPPs) such as Tat and oligoarginine peptides have been widely used as carriers for intracellular delivery of bioactive molecules. Despite accumulating evidence for involvement of endocytosis in the cellular uptake of arginine-rich CPPs, the primary cell-surface receptors for these peptide carriers that would initiate endocytic processes leading to intracellular delivery of bioactive cargoes have remained poorly understood. Our previous attempt to identify membrane receptors for octa-arginine (R8) peptide, one of the representative arginine-rich CPPs, using the photo-cross-linking probe bearing a photoreactive diazirine was not successful due to considerable amounts of cellular proteins nonspecifically bound to the affinity beads. To address this issue, here we developed a photo-cross-linking probe in which a cleavable linker of a diazobenzene moiety was employed to allow selective elution of cross-linked proteins by reducing agent-mediated cleavage. We demonstrated that introduction of the diazobenzene moiety into the photoaffinity probe enables efficient purification of cross-linked proteins with significant reduction of nonspecific binding proteins, leading to successful identification of 17 membrane-associated proteins that would interact with R8 peptide. RNAi-mediated knockdown experiments in combination with the pharmacological inhibitors revealed that, among the proteins identified, syndecan-4, one of the heparan sulfate proteoglycans, is an endogenous membrane-associated receptor for the cellular uptake of R8 peptide via clathrin-mediated endocytosis. This syndecan-4-dependent pathway was also involved in the intracellular delivery of bioactive proteins mediated by R8 peptide. These results reveal that syndecan-4 is a primary cell-surface target for R8 peptide that allows intracellular delivery of bioactive cargo molecules via clathrin-mediated endocytosis.

Cytosolic antibody delivery by lipid-sensitive endosomolytic peptide

One of the major obstacles in intracellular targeting using antibodies is their limited release from endosomes into the cytosol. Here we report an approach to deliver proteins, which include antibodies, into cells by using endosomolytic peptides derived from the cationic and membrane-lytic spider venom peptide M-lycotoxin. The delivery peptides were developed by introducing one or two glutamic acid residues into the hydrophobic face. One peptide with the substitution of leucine by glutamic acid (L17E) was shown to enable a marked cytosolic liberation of antibodies (immunoglobulins G (IgGs)) from endosomes. The predominant membrane-perturbation mechanism of this peptide is the preferential disruption of negatively charged membranes (endosomal membranes) over neutral membranes (plasma membranes), and the endosomolytic peptide promotes the uptake by inducing macropinocytosis. The fidelity of this approach was confirmed through the intracellular delivery of a ribosome-inactivation protein (saporin), Cre recombinase and IgG delivery, which resulted in a specific labelling of the cytosolic proteins and subsequent suppression of the glucocorticoid receptor-mediated transcription. We also demonstrate the L17E-mediated cytosolic delivery of exosome-encapsulated proteins.