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The development of efficient light-harvesting systems is important to understand the key aspects of solar-energy conversion processes and to utilize them in various photonic applications. Here, atomically well-defined gold nanoclusters are reported as a new platform to fabricate artificial light-harvesting systems. An efficient amide coupling method is developed to synthesize water-soluble Au 22 clusters fully protected with pyrene chromophores by taking advantage of their facile phase-transfer reaction. The synthesized Au 22 clusters with densely packed 18 pyrene chromophores (Au 22 -PyB 18 ) exhibit triple-emission in blue, green, and red wavelength regions arising respectively from pyrene monomer, pyrene excimer, and Au 22 emission, producing bright white light emission together. The photoluminescence of Au 22 is enhanced by more than tenfold, demonstrating that pyrenes at the periphery efficiently channel the absorbed energy to the luminescent Au 22 at the center. A combination of femtosecond transient absorption and anisotropy measurements of Au 22 -PyB 18 explicitly reveals three main decay components of 220 fs, 3.5 ps, and 160 ps that can be assigned to energy migration between pyrenes and energy transfer processes from pyrene monomer and excimer to the central Au 22 , respectively., (© 2021 Wiley-VCH GmbH.)

Accurate measurements of intracellular pH are of crucial importance in understanding the cellular activities and in the development of intracellular drug delivery systems. Here we report a highly sensitive pH probe based on a fluorescein-conjugated Au 22 nanocluster. Steady-state photoluminescence (PL) measurements have shown that, when conjugated to Au 22 , fluorescein exhibits more than 160-fold pH-contrasting PL in the pH range of 4.3-7.8. Transient absorption measurements show that there are two competing ultrafast processes in the fluorescein-conjugated Au 22 nanocluster: the intracore-state relaxation and the energy transfer from the nonthermalized states of Au 22 to fluorescein. The latter becomes predominant at a higher pH, leading to dramatic PL enhancement of fluorescein. In addition to the intrinsically low toxicity, fluorescein-conjugated Au 22 nanoclusters exhibit high pH sensitivity, wide dynamic range, and excellent photostability, providing a powerful tool for the study of intracellular processes.

It is a well-known fact that ZnO has been one of the most studied wide bandgap II-VI materials by the scientific community specifically due to its potential for being used as exciton-related optical devices. Hence, realizing ways to increase the efficiency of these devices is important. We discuss a plasma treatment technique to enhance the near-band-edge (NBE) excitonic emission from ZnO based nanoribbons. We observed an enhancement of the NBE peak and simultaneous quenching of the visible emission peak resulting from the removal of surface traps on these ZnO nanoribbons. More importantly, we report here the associated ultrafast carrier dynamics resulting from this surface treatment. Femtosecond transient absorption spectroscopy was performed using pump-probe differential transmission measurements shedding new light on these improved dynamics with faster relaxation times. The knowledge obtained is important for improving the application of ZnO based optoelectronic devices. We also observed how these improved carrier dynamics have a direct effect on the threshold and efficiency of random lasing from the material.

We report on a nanoinfrared (IR) imaging study of ultraconfined plasmonic hotspots inside graphene nanobubbles formed in graphene/hexagonal boron nitride (hBN) heterostructures. The volume of these plasmonic hotspots is more than one-million-times smaller than what could be achieved by free-space IR photons, and their real-space distributions are controlled by the sizes and shapes of the nanobubbles. Theoretical analysis indicates that the observed plasmonic hotspots are formed due to a significant increase of the local plasmon wavelength in the nanobubble regions. Such an increase is attributed to the high sensitivity of graphene plasmons to its dielectric environment. Our work presents a novel scheme for plasmonic hotspot formation and sheds light on future applications of graphene nanobubbles for plasmon-enhanced IR spectroscopy.

All-optical signal processing enables modulation and transmission speeds not achievable using electronics alone. However, its practical applications are limited by the inherently weak nonlinear effects that govern photon-photon interactions in conventional materials, particularly at high switching rates. Here, we show that the recently discovered nonlocal optical behaviour of plasmonic nanorod metamaterials enables an enhanced, ultrafast, nonlinear optical response. We observe a large (80%) change of transmission through a subwavelength thick slab of metamaterial subjected to a low control light fluence of 7 mJ cm(-2), with switching frequencies in the terahertz range. We show that both the response time and the nonlinearity can be engineered by appropriate design of the metamaterial nanostructure. The use of nonlocality to enhance the nonlinear optical response of metamaterials, demonstrated here in plasmonic nanorod composites, could lead to ultrafast, low-power all-optical information processing in subwavelength-scale devices.

We show that interfering surface plasmon polaritons can be excited with a focused laser beam at normal incidence to a plane metal film. No protrusions or holes are needed in this excitation scheme. Depending on the axial position of the focus, the intensity distribution on the metal surface is either dominated by interferences between counterpropagating plasmons or by a two-lobe pattern characteristic of localized surface plasmon excitation. Our experiments can be accurately explained by use of the angular spectrum representation and provide a simple means for locally exciting standing surface plasmon polaritons.

Light emission resulting from two-photon excited gold nanoparticles has been proposed to originate from the radiative decay of surface plasmon resonances. In this vein, we investigated luminescence from individual gold nanorods and found that their emission characteristics closely resemble surface plasmon behavior. In particular, we observed spectral similarities between the scattering spectra of individual nanorods and their photoluminescence emission. We also measured a blueshift of the photoluminescence peak wavelength with decreasing aspect ratio of the nanorods as well as an optically tunable shape-dependent spectrum of the photoluminescence. The emission yield of single nanorods strongly depends on the orientation of the incident polarization consistent with the properties of surface plasmons.

The local perturbation of a diffraction-limited spot by a nanometer sized gold tip in a popular apertureless scanning near-field optical microscopy (ASNOM) configuration is reproduced through topography changes in a photoresponsive polymer. Our method relies on the observation of the photochemical migration of azobenzene molecules grafted to a polymer placed beneath the tip. A local molecular displacement has been shown to be activated by a gold tip as a consequence of the lateral surface charge density present at the edges of the tip's end, resulting from a strong near-field depolarization predicted by theory.

A new method for optically exciting and visualizing surface plasmons in thin metal films is described. The technique relies on the use of a high-numerical-aperture objective lens to locally launch a broad wavelength spectrum of surface waves and to detect the leaky radiative modes associated with them. We used this approach to obtain a direct visualization of the plasmon intensity distributions, e.g., rainbow jets, and to quantify their propagation lengths throughout the visible spectrum.

We report an apertureless near-field optical study on nanostructured objects formed by J-aggregates adsorbed on silver (Ag) nanoparticles. Near-field images reveal that the enhanced near-field from the dressed particle's (DP) resonantly excited plasmon oscillation is efficiently absorbed by the J-aggregates. The sensitivity of the near-field images recorded at the harmonics of the probe vibration frequency suggests that the DP is releasing part of the absorbed energy radiatively upon interaction with the probe. The role of the probe in providing this new radiative relaxation channel is further confirmed as fluorescence from the J-aggregates on the particle is detected on the particle location only. We based the interpretation of our results on the near-field optical response from a bare Ag particle excited at the plasmon resonance as well as on far-field emission and transient absorption experiments.

Emerging areas of nanotechnology hold the promise of overcoming the limitations of existing technologies for intracellular manipulation. These new developments provide approaches for the creation of chemical-biological hybrid nanocomposites that can be introduced into cells and subsequently used to initiate intracellular processes or biochemical reactions. Such nanocomposites would advance medical biotechnology, just as they are improving microarray technology and imaging in biology and medicine, and introducing new possibilities in chemistry and material sciences. Here we describe the behaviour of 45-A nanoparticles of titanium dioxide semiconductor combined with oligonucleotide DNA into nanocomposites in vivo and in vitro. These nanocomposites not only retain the intrinsic photocatalytic capacity of TiO2 and the bioactivity of the oligonucleotide DNA (covalently attached to the TiO2 nanoparticle), but also possess the chemically and biologically unique new property of a light-inducible nucleic acid endonuclease, which could become a new tool for gene therapy.

The synthesis of a new, noncovalent anthracene-dimethylaniline dyad (ensemble I) held together via guanosine-cytidine Watson-Crick base-pairing interactions is reported. Upon excitation at 420 nm, photoinduced electron-transfer from the dimethylaniline donor to the singlet excited state of the anthracene acceptor occurs, as inferred from a combination of time-resolved fluorescence quenching and transient absorption measurements. In toluene at room temperature, the rate constants for photoinduced intraensemble electron-transfer and subsequent back-electron-transfer (charge recombination) are k(CS) = (3.5 +/- 0.03) x 10(10) s(-1) and k(CR) = (1.42 +/- 0.03) x 10(9) s(-1), respectively.

32-Indole ether derivatives of tacrolimus and ascomycin retain the potent immunosuppressive activity of their parent compounds but display reduced toxicity. In addition, their complexes with the 12-kDa FK506-binding protein (FKBP) form more stable complexes with the protein phosphatase calcineurin, the molecular target of these drugs. We have solved the three-dimensional structures of the FKBP complexes with two 32-indolyl derivatives of ascomycin. The structures of the protein and the macrolide are remarkably similar to those seen in the complexes with tacrolimus and ascomycin. The indole groups project away from the body of the complex, and multiple conformations are observed for the linkage to these groups as well as for a nearby peptide suggesting apparent flexibility in these parts of the structure. Comparison of these structures with that of the ternary complex of calcineurin, FKBP, and tacrolimus suggests that the indole groups interact with a binding site comprising elements of both the calcineurin alpha- and beta-chains and that this interaction is responsible for the increased stability of these complexes.

The synthesis of C32-O-arylethyl ether derivatives of ascomycin that possess equivalent immunosuppressant activity but reduced toxicity, compared to FK-506, is described.

A tether length study of C32-O-phenalkyl ether derivatives of ascomycin was conducted wherein it was determined that a 2-carbon tether provides optimum in vitro immunosuppressive activity. Oxygen-bearing substituents along the 2-carbon tether can further increase the potency of this design.

A series of C32-O-aralkyl ether derivatives of the FK-506 related macrolide ascomycin have been prepared based on an earlier reported C32-O-cinnamyl ether design. In the present study, the nature of the aryl tethering group was varied in an attempt to improve oral activity. An imidazol-2-yl-methyl tether was found to be superior among those investigated and has resulted in an ascomycin analog, L-733,725, with in vivo immunosuppressive activity comparable to FK-506 but with an improved therapeutic index.

Background: Tacrolimus (FK506) is an immunosuppressive drug 50-100 times more potent than cyclosporine (CsA), the current mainstay of organ transplant rejection therapy. Despite being chemically unrelated, CsA and tacrolimus exert their immunosuppressive effects through the inhibition of calcineurin (CaN), a critical signaling molecule during T-lymphocyte activation. Although numerous clinical studies have proven the therapeutic efficacy of drugs within this class, tacrolimus and CsA also have a strikingly similar profile of unwanted side effects., Method: Our objective has been to identify a less toxic immunosuppressant through the modification of ascomycin (FK520). Quantitative in vitro immunosuppression and toxicity assays have demonstrated (see the accompanying article, p. 18) that we achieved our goal with L-732,531 (indolyl-ascomycin; indolyl-ASC), a 32-O-(1-hydroxyethylindol-5-yl) ascomycin derivative with an improved therapeutic index relative to tacrolimus., Results: We report that the attributes of indolyl-ASC may result from its distinctive biochemical properties. In contrast to tacrolimus, indolyl-ASC binds poorly to FK506 binding protein 12 (FKBP12), the major cytosolic receptor for tacrolimus and related compounds. However, the stability of the interaction between the FKBP12-indolyl-ASC complex and CaN is much greater than that of the FKBP12-tacrolimus complex. These distinguishing properties of indolyl-ASC result in the potent inhibition of CaN within T lymphocytes but may lower the accumulation of the drug at sites of toxicity., Conclusions: Indolyl-ASC may define those properties needed to increase the therapeutic efficacy of a macrolactam immunoregulant for treating both human autoimmune disease and organ transplant rejection.

We previously described the isolation of an FK506-binding protein, FKBP51, that is predominantly expressed in murine T cells and is capable of mediating drug-dependent calcineurin inhibition in vitro. In addition, the gene for FKBP51 is induced by glucocorticoids. Screening of a human thymus cDNA library resulted in the identification of the human homologue of FKBP51. Expression of the 3.7 kb mRNA corresponding to FKBP51 is induced by glucocorticoids in the human T cell line, C7TK.4. The 51.2 kDa protein encoded by this gene shares 87% identity to murine FKBP51 and demonstrates a similar IC50 value for the FK506-mediated inhibition of calcineurin phosphatase in vitro. The distribution and abundance of FKBP51 and FKBP12 in seventeen human tissues were compared by Western analysis. Unlike its murine counterpart, the human FKBP51 is abundantly expressed in numerous tissues and in many cases, is in molar excess over FKBP12.

The immunosuppressant, rapamycin, inhibits cell growth by interfering with the function of a novel kinase, termed mammalian target of rapamycin (mTOR). The putative catalytic domain of mTOR is similar to those of mammalian and yeast phosphatidylinositol (PI) 3-kinases. This study demonstrates that mTOR is a component of a cytokine-triggered protein kinase cascade leading to the phosphorylation of the eukaryotic initiation factor-4E (eIF-4E) binding protein, PHAS-1, in activated T lymphocytes. This event promotes G1 phase progression by stimulating eIF-4E-dependent translation initiation. A mutant YAC-1 T lymphoma cell line, which was selected for resistance to the growth-inhibitory action of rapamycin, was correspondingly resistant to the suppressive effect of this drug on PHAS-1 phosphorylation. In contrast, the PI 3-kinase inhibitor, wortmannin, reduced the phosphorylation of PHAS-1 in both rapamycin-sensitive and -resistant T cells. At similar drug concentrations (0.1-1 microM), wortmannin irreversibly inhibited the serine-specific autokinase activity of mTOR. The autokinase activity of mTOR was also sensitive to the structurally distinct PI 3-kinase inhibitor, LY294002, at concentrations (1-30 microM) nearly identical to those required for inhibition of the lipid kinase activity of the mammalian p85-p110 heterodimer. These studies indicate that the signaling functions of mTOR, and potentially those of other high molecular weight PI 3-kinase homologs, are directly affected by cellular treatment with wortmannin or LY294002.

The calcium release channels (CRC)/ryanodine receptors of skeletal (Sk) and cardiac (C) muscle sarcoplasmic reticulum (SR) are hetero-oligomeric complexes with the structural formulas (ryanodine recepter (RyR)1 protomer)4(FKBP12)4 and (RyR2 protomer)4(FKBP12.6)4, respectively, where FKBP12 and FKBP12.6 are isoforms of the 12-kDa receptor for the immunosuppressant drug FK506. The sequence similarity between the RyR protomers and FKBP12 isoforms is 63 and 85%, respectively. Using 35S-labeled FKBP12 and 35S-labeled FKBP12.6 as probes to study the interaction with CRC, we find that: 1) analogous to its action in skeletal muscle sarcoplasmic reticulum (SkMSR), FK506 (or analog FK590) dissociates FKBP12.6 from CSR; 2) both FKBP isoforms bind to FKBP-stripped SkMSR and exchange with endogenously bound FKBP12 of SkMSR; and 3) by contrast, only FKBP12. 6 exchanges with endogenously bound FKBP12.6 or rebinds to FKBP-stripped CSR. This selective binding appears to explain why the cardiac CRC is isolated as a complex with FKBP12.6, whereas the skeletal muscle CRC is isolated as a complex with FKBP12, although only FKBP12 is detectable in the myoplasm of both muscle types. Also, in contrast to the activation of the channel by removal of FKBP from skeletal muscle, no activation is detected in CRC activity in FKBP-stripped CSR. This differential action of FKBP may reflect a fundamental difference in the modulation of excitation-contraction coupling in heart versus skeletal muscle.

A 12-kDa immunophilin (FKBP12) is an integral component of the skeletal muscle ryanodine receptor (RyR). The RyR is a hetero-oligomeric complex with structural formula (FKBP)4(Ryr1)4, where Ryr1 is the 565-kDa product of the Ryr1 gene. To aid in the detection of the immunophilin's location in the receptor, we exchanged the FKBP12 present in RyR-enriched vesicles derived from sarcoplasmic reticulum with an engineered construct of FKBP12 fused to glutathione S-transferase and then isolated the complexes. Cryoelectron microscopy and image averaging of the complexes (in an orientation displaying the RyR's fourfold symmetry) revealed four symmetrically distributed, diffuse density regions that were located just outside the boundary defining the cytoplasmic assembly of the RyR. These regions are attributed to the glutathione transferase portion of the fusion protein because they are absent from receptors lacking the fusion protein. To more precisely define the location of FKBP12, we similarly analyzed complexes of RyR containing FKBP12 itself. Apparently some FKBP is lost during the purification or storage of the RyR because, to detect the receptor-bound immunophilin, it was necessary to add FKBP12 to the purified receptor before electron microscopy. Averaged images of these complexes showed a region of density that had not been observed previously in images of isolated receptors, and its position, along the edges of the transmembrane assembly, agreed with the position of the FKBP12 deduced from the experiments with the fusion protein. The proposed locations for FKBP12 are about 10 nm from the transmembrane baseplate assembly that contains the ion channel of the RyR.

FK-506 blocks T cell activation by preventing lymphokine gene transcription through formation of a complex with FKBP12 that inhibits calcineurin phosphatase activity. Immunosuppressive FK-506 analogs (agonists) have been generated whose potency correlates with calcineurin inhibition. Nonimmunosuppressive antagonist analogs have also been identified, including L-685,818, which binds to FKBP12 but does not inhibit calcineurin. We describe a novel property of FK-506 analog, characterized as a mixed agonist/antagonist immunosuppressive activity. It is displayed by L-688,617, the 32 O-methoxyethoxymethyl derivative of the agonist L-683,590 (C21-ethyl). Although it binds to FKBP12 similarly to L-683,590, L-688,617 incompletely suppressed T cell proliferation induced by optimal activation and enhanced that induced by supraoptimal activation. In the latter situation, L-688,617 suppressed IL-2 production only partially but blocked activation-driven cell death. Moreover, a 1000-fold molar excess of L-688,617 antagonized the immunosuppressive activity of L-683,590. L-688,617 inhibited calcineurin phosphatase activity in cells only partially. The unique agonist/antagonist activity of L-688,617 may therefore reflect its high affinity for FKBP12, combined with a reduced ability of the drug-FKBP12 complex to inhibit calcineurin function. However, in a cell-free system, L-688,617 completely blocked this function when a large excess of FKBP12 over calcineurin was present, suggesting that the intracellular concentration of FKBP12 may be a limiting factor that prevents full agonist activity of L-688,617 in cells.

We have created embryonic stem (ES) cells and mice lacking the predominant isoform (alpha) of the calcineurin A subunit (CNA alpha) to study the role of this serine/threonine phosphatase in the immune system. T and B cell maturation appeared to be normal in CNA alpha -/- mice. CNA alpha -/- T cells responded normally to mitogenic stimulation (i.e., PMA plus ionomycin, concanavalin A, and anti-CD3 epsilon antibody). However, CNA alpha -/- mice generated defective antigen-specific T cell responses in vivo. Mice produced from CNA alpha -/- ES cells injected into RAG-2-deficient blastocysts had a similar defective T cell response, indicating that CNA alpha is required for T cell function per se, rather than for an activity of other cell types involved in the immune response. CNA alpha -/- T cells remained sensitive to both cyclosporin A and FK506, suggesting that CNA beta or another CNA-like molecule can mediate the action of these immunosuppressive drugs. CNA alpha -/- mice provide an animal model for dissecting the physiologic functions of calcineurin as well as the effects of FK506 and CsA.

The potent immunosuppressive drugs FK506 and rapamycin interfere with signal transduction pathways required for T cell activation and growth. The distinct inhibitory effects of these drugs on the T cell activation program are mediated through the formation of pharmacologically active complexes with members of a family of intracellular receptors termed the FK506 binding proteins (FKBPs). The FKBP12.FK506 complex specifically binds to and inhibits calcineurin, a signaling protein required for transcriptional activation of the interleukin (IL)-2 gene in response to T cell antigen receptor engagement. The FKBP12. rapamycin complex interacts with a recently defined target protein termed the mammalian target of rapamycin (mTOR). Accumulating data suggest that mTOR functions in a previously unrecognized signal transduction pathway required for the progression of IL-2-stimulated T cells from G1 into the S phase of the cell cycle. Here we review the immunopharmacology of rapamycin, with particular emphasis on the characterization of mTOR.

FK506, an immunosuppressant that prolongs allograft survival, is a co-drug with its intracellular receptor, FKBP12. The FKBP12.FK506 complex inhibits calcineurin, a critical signaling molecule during T-cell activation. FKBP12 was, until recently, the sole FKBP known to mediate calcineurin inhibition at clinically relevant FK506 concentrations. The best characterized cellular function of FKBP12 is the modulation of ryanodine receptor isoform-1, a component of the calcium release channel of skeletal muscle sarcoplasmic reticulum. Recently, a novel protein, FKBP12.6, was found to inhibit calcineurin at clinically relevant FK506 concentrations. We have cloned the cDNA encoding human FKBP12.6 and characterized the protein. In transfected Jurkat cells, FKBP12.6 is equivalent to FKBP12 at mediating the inhibitory effects of FK506. Upon binding rapamycin, FKBP12.6 complexes with the 288-kDa mammalian target of rapamycin. In contrast to FKBP12, FKBP12.6 is not associated with ryanodine receptor isoform-1 but with the distinct ryanodine receptor isoform-2 in cardiac muscle sarcoplasmic reticulum. Our results suggest that FKBP12.6 has both a unique physiological role in excitation-contraction coupling in cardiac muscle and the potential to contribute to the immunosuppressive and toxic effects of FK506 and rapamycin.

The ryanodine receptor (RyR)/calcium release channel isolated from skeletal muscle terminal cisternae (TC) of sarcoplasmic reticulum (SR) is tightly associated with FK506 binding protein of 12.0 kDa (FKBP12) (Jayaraman et al., (1992) J.Biol.Chem. 267, 9474-9477). In this study, we describe a new method of affinity chromatography for purifying the RyR from skeletal muscle SR based on: 1) its tight association with FKBP12; and 2) the finding that bound FKBP on the RyR can be exchanged with soluble FKBP12 (Timerman et al., (1995) J.Biol.Chem. 270, 2451-2459). Soluble glutathione S-transferase/FKBP12 (GST/FKBP12) fusion protein was first exchanged with bound FKBP12 on the RyR of TC. The TC were then solubilized with CHAPS and the complex of RyR.GST/FKBP12 was specifically adsorbed by glutathione Sepharose 4B and then eluted with glutathione. The RyR, purified by this method, has similar characteristics by SDS-PAGE, radioligand binding and immuno-reactivity as the RyR purified by multiple sequential column chromatography.

The immunosuppressive drugs FK506 and cyclosporin A block T-lymphocyte proliferation by inhibiting calcineurin, a critical signaling molecule for activation. Multiple intracellular receptors (immunophilins) for these drugs that specifically bind either FK506 and rapamycin (FK506-binding proteins [FKBPs]) or cyclosporin A (cyclophilins) have been identified. We report the cloning and characterization of a new 51-kDa member of the FKBP family from murine T cells. The novel immunophilin, FKBP51, is distinct from the previously isolated and sequenced 52-kDa murine FKBP, demonstrating 53% identity overall. Importantly, Western blot (immunoblot) analysis showed that unlike all other FKBPs characterized to date, FKBP51 expression was largely restricted to T cells. Drug binding to recombinant FKBP51 was demonstrated by inhibition of peptidyl prolyl isomerase activity. As judged from peptidyl prolyl isomerase activity, FKBP51 had a slightly higher affinity for rapamycin than for FK520, an FK506 analog. FKBP51, when complexed with FK520, was capable of inhibiting calcineurin phosphatase activity in an in vitro assay system. Inhibition of calcineurin phosphatase activity has been implicated both in the mechanism of immunosuppression and in the observed toxic side effects of FK506 in nonlymphoid cells. Identification of a new FKBP that can mediate calcineurin inhibition and is restricted in its expression to T cells suggests that new immunosuppressive drugs may be identified that, by virtue of their specific interaction with FKBP51, would be targeted in their site of action.

Reduced-stringency PCR was used to isolate a cDNA encoding a novel human FK506-binding protein (FKBP) homolog. The encoded 38-kDa protein (FKBPr38) contains at its N-terminus a domain that is 33% identical to FKBP12. FKBPr38 is a member of a subclass of immunophilins, whose other members include FKBP52 and CyP40 (cyclophilin 40), that contain a three-unit tetratricopeptide repeat (TPR). In addition, FKBPr38 contains a consensus leucine-zipper repeat. The presence of the TPR domain and leucine zipper suggest that FKBPr38 may form homo-multimers or interact with other, as yet unidentified, proteins.

FKBP-12 (FKBP), the soluble receptor for the immunosuppresant drug FK-506, is tightly bound to the calcium release channel (CRC)/ryanodine receptor (RyR) of skeletal muscle terminal cisternae (TC) of sarcoplasmic reticulum with a stoichiometry of 4 mol of FKBP per tetrameric RyR complex. FKBP displays cis/trans-peptidyl-prolyl isomerase (PPIase) activity which is inhibited by FK-590 or rapamycin. In skeletal muscle TC, FK-590 or rapamycin binds to and dissociates FKBP from the RyR in a time- and temperature-dependent manner which increases the open probability of the channel. Therefore, the net energized Ca2+ uptake rate of TC vesicles devoid of FKBP is reduced due to the increased leak of Ca2+ from the TC specifically via the RyR, which is reversed upon rebinding of FKBP. Thus, the RyR is modulated by FKBP (Timerman, A. P., Ogunbumni, E., Freund, E. A., Wiederrecht, G., Marks, A. R., and Fleischer, S. (1993) J. Biol. Chem. 268, 22922-22999; Mayrleitner, M., Timerman, A. P., Wiederrecht, G., and Fleischer S. (1994) Cell Calcium 15, 99-108). We now find that FKBP can be displaced from the FKBP.RyR complex by exchange with FKBP in solution. The EC50 for exchange is 0.30 microM for wild type FKBP versus 0.6 to 2.4 microM for three different site-directed mutants that are practically devoid of any measurable PPIase activity. Substitution of wild-type FKBP on the RyR complex with these PPIase-deficient mutants did not alter the Ca2+ flux of TC vesicles, whereas dissociation of FKBP from TC with FK-590 increased the Ca2+ leak rate. Our studies show that, in vivo, the FKBP.RyR complex is in equilibrium with the cytosolic pool of FKBP (approximately 3 microM) and suggest that modulation of the CRC by FKBP is independent of PPIase activity.

The immunosuppressive drug, rapamycin, interferes with an undefined signaling pathway required for the progression of G1-phase T-cells into S phase. Genetic analyses in yeast indicate that binding of rapamycin to its intracellular receptor, FKBP12, generates a toxic complex that inhibits cell growth in G1 phase. These analyses implicated two related proteins, TOR1 and TOR2, as targets of the FKBP12-rapamycin complex in yeast. In this study, we have used a glutathione S-transferase (GST)-FKBP12-rapamycin affinity matrix to isolate putative mammalian targets of rapamycin (mTOR) from tissue extracts. In the presence of rapamycin, immobilized GST-FKBP12 specifically precipitates similar high molecular mass proteins from both rat brain and murine T-lymphoma cell extracts. Binding experiments performed with rapamycin-sensitive and -resistant mutant clones derived from the YAC-1 T-lymphoma cell line demonstrate that the GST-FKBP12-rapamycin complex recovers significantly lower amounts of the candidate mTOR from rapamycin-resistant cell lines. The latter results suggest that mTOR is a relevant target of rapamycin in these cells. Finally, we report the isolation of a full-length mTOR cDNA that encodes a direct ligand for the FKBP12-rapamycin complex. The deduced amino acid sequence of mTOR displays 42 and 45% identity to those of yeast TOR1 and TOR2, respectively. These results strongly suggest that the FKBP12-rapamycin complex interacts with homologous ligands in yeast and mammalian cells and that the loss of mTOR function is directly related to the inhibitory effect of rapamycin on G1- to S-phase progression in T-lymphocytes and other sensitive cell types.

The immunosuppressant drug, rapamycin (RAP), is a potent inhibitor of IL-2-dependent T-cell proliferation. The antiproliferative effect of RAP is mediated through the formation of an active complex with its cytosolic receptor protein, FKBP12. The molecular target of the FKBP12.RAP complex is a putative lipid kinase termed the mammalian Target Of Rapamycin (mTOR). This review will discuss recent findings suggesting that mTOR is a novel regulator of G1- to S-phase progression in eukaryotic cells.

Photosynthetic conversion of light energy into chemical potential begins in reaction center protein complexes, where rapid charge separation occurs with nearly unit quantum efficiency. Primary charge separation was studied in isolated photosystem II reaction centers from spinach containing 6 chlorophyll a, 2 pheophytin a (Pheo), 1 cytochrome b559, and 2 beta-carotene molecules. Time-resolved pump-probe kinetic spectroscopy was carried out with 105-fs time resolution and with the pump laser polarized parallel, perpendicular, and at the magic angle (54.7 degrees) relative to the polarized probe beam. The time evolution of the transient absorption changes due to the formation of the oxidized primary electron donor P680+ and the reduced primary electron acceptor Pheo- were measured at 820 nm and 545 nm, respectively. In addition, kinetics were obtained at 680 nm, the wavelength ascribed to the Qy transition of the primary electron donor P680 in the reaction center. At each measured probe wavelength the kinetics of the transient absorption changes can be fit to two major kinetic components. The relative amplitudes of these components are strongly dependent on the polarization of the pump beam relative to that of the probe. At the magic angle, where no photoselection occurs, the amplitude of the 3-ps component, which is indicative of the charge separation, dominates. When the primary electron acceptor Pheo is reduced prior to P680 excitation, the 3-ps component is eliminated.

The immunosuppressant FK-506 (tacrolimus) forms a complex with a ubiquitous intracellular receptor, FK-506 binding protein (FKBP12), and this complex inhibits the heterodimeric Ca2+/calmodulin-dependent phosphatase, calcineurin, an essential component of the T-cell receptor signal transduction pathway. Using a series of truncated calcineurin catalytic subunits, we show here that a region within the catalytic subunit that regulates phosphatase activity, the autoinhibitory domain, also regulates the Ca(2+)-dependent interaction of calcineurin with the FK-506.FKBP12 complex. Deletion of this domain produces constitutive activation of the phosphatase as demonstrated by transient transfection experiments in which expression of the truncated protein permitted Ca(2+)-independent induction of interleukin-2 transcription. Thus, deletion of the autoinhibitory domain is necessary and sufficient to constitutively activate calcineurin (CaN). Furthermore, CaN A467-492, an inhibitory peptide based on the autoinhibitory domain from calcineurin (ITSFEEAKGLDRINERMPPRRDAMP), inhibited dephosphorylation of the RII peptide substrate competitively with a Ki = 4 microM, consistent with binding of the autoinhibitory domain at the active site of the enzyme. To assess the role of the autoinhibitory domain in regulating the interaction of CaN with the FK-506.FKBP12 complex, we reconstituted wild type and mutant phosphatase heterodimers using in vitro transcribed and translated subunits. Association of the reconstituted calcineurin heterodimers with FKBP12 was dependent on FK-506. In the case of the wild type heterodimer, association with the FK-506.FKBP12 complex was also dependent upon Ca2+; however, mutant catalytic subunits, in which the autoinhibitory domains were deleted, associated with the drug-binding protein complex in the presence of 10 mM EGTA. These results indicate that the conserved autoinhibitory domain regulates both Ca(2+)-dependent phosphatase activity and association with the FK-506.FKBP12 complex.

The calcium release channel/ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum is tightly associated with the immunophilin FK-506 binding protein (FKBP-12). The immunosuppressant drug FK-506 effectively dissociates FKBP-12 from the calcium release channel of terminal cisternae (TC) vesicles. Furthermore, calcium flux measurements of TC indicate that FKBP-12 stabilizes the closed conformation of the calcium release channel of TC [Timerman AP, Ogunbunmi E, Freund EA, Wiederrecht G, Marks AM, Fleischer S. (1993) J. Biol. Chem., 268, 22992-22999]. In this report, the effect of FKBP on single channel recordings of the calcium release channel/ryanodine receptor of TC is measured directly. Single channel recordings of the ryanodine receptor were obtained by fusion of TC vesicles into planar bilayers. The channel devoid of FKBP, retains key diagnostic features. That is, activation by Ca2+ and ryanodine, inhibition by Mg2+ (mM) and ruthenium red (microM), and its unitary conductance remain the same. Recordings of the calcium release channel obtained from the FKBP-deficient TC vesicles, as compared with control TC, have greater open probability and longer mean open times in a free calcium concentration range of 70 nM to 1.2 microM. The sensitivity of the channel to caffeine is also enhanced by the removal of FKBP. The enhanced channel activation of FKBP-deficient TC is reversed by rebinding recombinant FKBP-12 in a cyclical fashion. We conclude that FKBP modifies the channel behavior of the calcium release channel of skeletal muscle sarcoplasmic reticulum.

We find that the purified ryanodine receptor/calcium release channel (CRC) isolated from canine heart sarcoplasmic reticulum (SR) is tightly associated with an FK-506 binding protein which is distinct from human recombinant FKBP-12. Fractions from both skeletal and heart muscle were probed by Western Blot analysis using sequence specific antibody raised against the N-terminus of human recombinant FKBP-12. Only FKBP-12 was detected in rabbit and dog skeletal muscle SR as well as in dog heart cytosol. Canine heart SR and the purified canine heart ryanodine receptor contained an immunoreactive band with a somewhat slower relative mobility than that of human recombinant FKBP-12. These observations indicate that the association between the cardiac CRC and this novel FK-506 binding protein is specific.

FK-506 and cyclosporin A (CsA) are potent immunosuppressive agents used clinically to prevent tissue rejection. Interest in the development of more effective immunosuppressive drugs has led to an intense effort toward understanding their biochemical mechanism of action with the result that these compounds have now become powerful tools used in deciphering the signal transduction events in T lymphocyte activation. Although chemically unrelated, FK-506 and CsA exert nearly identical biological effects in cells by inhibiting the same subset of early calcium-associated events involved in lymphokine expression, apoptosis, and degranulation. FK-506 binds to a family of intracellular receptors termed the FK-506 binding proteins (FKBPs). CsA binds to another family of intracellular receptors, the cyclophilins (Cyps), distinct from the FKBPs. The similarities between the mechanisms of action of CsA and FK-506 converge upon the calcium- and calmodulin-dependent serine-threonine protein phosphatase calcineurin (CaN). Both the FKBP/FK-506 complex and the Cyp/CsA complex can bind to calcineurin, thereby inhibiting its phosphatase activity. Calcineurin, a component of the signal transduction pathway resulting in IL-2 expression, catalyzes critical dephosphorylation events required for early lymphokine gene transcription.

The ryanodine receptor/calcium release channel (CRC) of rabbit skeletal muscle terminal cisternae (TC) of sarcoplasmic reticulum (SR) has been found to be tightly associated with FK-506 binding protein (FKBP-12), the cytosolic receptor (immunophilin) for the immunosuppressant drug FK-506 (Jayaraman, T., Brillantes, A. M., Timerman, A. P., Fleischer, S., Erdjument-Bromage, H., Tempst, P., and Marks, A. (1992) J. Biol. Chem. 267, 9474-9477). In this study, a procedure is described to dissociate FKBP from TC and reconstitute human recombinant FKBP-12 back to the ryanodine receptor so that the role of the immunophilin on CRC activity can be assessed. Titration of TC vesicles with FK-506 dissociates FKBP from the ryanodine receptor. Sedimentation of FK-506-treated vesicles effectively separates the TC from the soluble FKBP-FK506 complex which remains in the supernatant. The FKBP-deficient TC vesicles have altered functional characteristics: 1) the ATP-stimulated calcium uptake rate of TC vesicles is reduced 2-fold; and 2) the threshold concentration of caffeine required to induce calcium release from TC vesicles is decreased. These changes appear to reflect modification of the calcium release channel since: 1) severalfold higher concentrations of FK-506 do not alter the calcium uptake rate of either longitudinal tubules of SR, or TC vesicles in the presence of ruthenium red; 2) human recombinant FKBP reassociates with FKBP-deficient TC but not with control TC or longitudinal tubules of SR; and 3) the reduced Ca2+ uptake rate in FKBP-deficient TC is restored to control values in the FKBP-reconstituted TC. These studies demonstrate that FKBP-12 modulates the CRC of rabbit skeletal muscle sarcoplasmic reticulum.

The immunosuppressant rapamycin (RAP) is a potent inhibitor of the entry of interleukin (IL)-2-stimulated T cells into S-phase. Earlier results indicated that RAP treatment arrested the growth of the murine IL-2-dependent T cell line CTLL-2 in late G1-phase. To explore further the interactions of RAP with the cell cycle control machinery in T cells, we examined the effects of RAP treatment on the activation of the cyclin-dependent kinases p34cdc2 and p33cdk2 in G1-phase CTLL-2 cells. Stimulation of factor-deprived cells with IL-2 led to the assembly of high molecular weight complexes containing active p34cdc2 and p33cdk2. The appearance of these complexes was explained, at least in part, by the association of both cyclin-dependent kinases with IL-2-induced cyclin A. RAP treatment profoundly inhibited both cyclin A expression and the appearance of active cyclin A-cyclin-dependent kinase complexes in IL-2-stimulated, late G1-phase CTLL-2 cells. Although p34cdc2 activation was largely dependent on association with cyclin A, a significant proportion of the active p33cdk2 pool was complexed with cyclin E. In contrast to cyclin A, the IL-2-induced accumulation of cyclin E in G1-phase cells was only partially suppressed by RAP, and cyclin E-p33cdk2 complexes were readily detected in drug-treated cells. These cyclin E-cyclin-dependent kinase complexes were nonetheless devoid of histone H1 kinase activity. The inhibitory effects of RAP on the activation of cyclin E- and cyclin A-associated cyclin-dependent kinases suggest that one or both events participate in the regulation of T cell entry into S-phase.

L-685,818 differs only slightly in structure from the immunosuppressive drug FK-506, and both compounds bind with comparable affinity to the 12-kDa FK-506-binding protein (FKBP12), the major intracellular receptor for the drug. Despite these similarities, L-685,818 is a potent antagonist of both the immunosuppressive and toxic effects of the drug. Here, we present a structural analysis of this problem. Although FK-506 and L-685,818 differ greatly in pharmacology, we have found that the three-dimensional structures of their complexes with FKBP12 are essentially identical. Approximately half of each ligand is in contact with the receptor protein, and half is exposed to solvent; the exposed region includes the two sites where the compounds differ. These results indicate that the profound differences in the pharmacology of these two compounds are not caused by any difference in their interaction with FKBP12. Rather, these effects arise because relatively minor changes in the exposed part of a bound ligand have a strong effect on how FKBP12-ligand complexes interact with calcineurin, their putative intracellular target. In addition, FK-506 complexes with FKBP12 proteins from several species all inhibit mammalian calcineurin. Analysis of the three-dimensional structure of the complex with respect to residues conserved among these proteins suggests a small number of surface residues near the bound ligands that may play a critical role in interactions between the protein-drug complex and calcineurin.

The macrolide rapamycin (RAP) is a potent inhibitor of interleukin-2 (IL-2)-induced T-cell proliferation. Current models suggest that RAP, when complexed to its intracellular receptor, FK506-binding protein, interferes with an IL-2 receptor-coupled signaling pathway required for cell-cycle progression from G1- to S-phase. Here we show that RAP treatment inhibits the growth of an IL-2-dependent cytotoxic T-cell line, CTLL-2, in late G1-phase, just prior to entry of the cells into S-phase. In contrast, RAP-treated CTLL-2 cells retained the ability to respond to IL-2 with enhanced cytolytic activity, indicating that RAP was not a general suppressant of cellular responsiveness to IL-2. Subsequent studies revealed that IL-2 stimulation triggered a delayed activation of the p34cdc2 kinase, the timing of which correlated with the G1- to S-phase transition. The IL-2-dependent increase in p34cdc2 kinase activity was blocked by RAP. The RAP sensitivity of the p34cdc2 activation mechanism implicates this signaling pathway in the control of S-phase commitment in IL-2-stimulated T-cells.

The microscopic pathway along which ions or molecules in a crystal move during a structural phase transition can often be described in terms of a collective vibrational mode of the lattice. In many cases, this mode, called a "soft" phonon mode because of its characteristically low frequency near the phase transition temperature, is difficult to characterize through conventional frequency-domain spectroscopies such as light or neutron scattering. A femtosecond time-domain analog of light-scattering spectroscopy called impulsive stimulated Raman scattering (ISRS) has been used to examine the soft modes of two perovskite ferroelectric crystals. The low-frequency lattice dynamics of KNbO(3) and BaTiO(3) are clarified in a manner that permits critical evaluation of microscopic models for their ferroelectric transitions. The results illustrate the advantages of ISRS over conventional Raman spectroscopy of low-frequency, heavily damped soft modes.

The immunoregulant FK-506 potently inhibits particular calcium-associated signal transduction events that occur early during T-lymphocyte activation and during IgE receptor-mediated exocytosis in mast cells. FK-506 binds to a growing family of receptors termed FK-506-binding proteins (FKBPs), the most abundant being a 12-kDa cytosolic receptor, FKBP12. To date, there is no formal evidence proving that FKBP12 is the sole receptor mediating the immunosuppressive effects or toxic side effects of FK-506. Using gel filtration chromatography as an assay for novel FK-506-binding proteins, we identified FK-506 binding activities in extracts prepared from calf brain and from JURKAT cells. Both of these new activities comigrated with apparent molecular masses of 110 kDa. However, further characterization of both binding activities revealed that the two are not identical. The 110-kDa activity observed in brain extracts appears to be the FKBP12.FK-506.calcineurin (CaN) complex previously reported (Liu, J., Farmer, J., Lane, W., Friedman, J., Weissman, I., and Schreiber, S. (1991) Cell 66, 807-815) while the 110 kDa activity observed in JURKAT cells is a novel FK-506-binding protein. Our characterization of the FKBP12.FK-506.CaN complex reveals a dependence upon calmodulin (CaM) for formation of the complex and demonstrates that the peptidyl-prolyl cis-trans isomerase (PPIase) activity of FKBP12 is not required for binding of FKBP12.FK-506 to CaN or for inhibition of CaN phosphatase activity. The novel FK-506-binding protein in JURKAT cells has been purified to homogeneity, migrates with an apparent mass of 51 kDa on denaturing gels, and has been termed FKBP51. Like FKBP12, FKBP51 has PPIase activity, but, unlike FKBP12.FK-506, FKBP51.FK-506 does not complex with or inhibit the phosphatase activity of, CaN. These results indicate that complex formation with CaN may not be a general property of the FKBPs. Peptide sequencing reveals that FKBP51 may be similar, if not identical, to hsp56, a component of non-transformed steroid receptors.

Recently, the nearly complete peptide sequence of a 25 kDa rapamycin and FK-506 binding protein that had been isolated from calf thymus, brain, and spleen was reported (1). Based upon the amino acid sequence of this bovine protein, bFKBP25, we have isolated from a JURKAT cDNA library the cDNA encoding the human homolog, hFKBP25. Translation of the open reading frame contained within this cDNA clone yields a sequence that, in its C-terminal half, is 41% identical to the major human FK-506 binding protein, hFKBP12, and 43% identical to hFKBP13. The N-terminal half of hFKBP25 is unrelated to any known protein.

Recently, the amino acid sequence of a 12 Kd endogenous protein inhibitor of protein kinase C (PKC-I 2) has been shown to be identical to that of the 12 KDa receptor for the immunosuppressive drug, FK-506. In view of this observation we examined the effects of recombinant and native human FKBP on protein kinase C (PKC) activity. FKBP, at molar concentrations up to 1900-fold over that of PKC, failed to inhibit PKC phosphorylation of histone H1 and failed to block the auto-phosphorylation of PKC. Interestingly, FKBP is phosphorylated by PKC in these reactions. The phosphorylation of FKBP by PKC appears to be specific since the catalytic subunit of cAMP-dependent protein kinase fails to phosphorylate the binding protein. Our results fail to support a role for FKBP as an inhibitor of protein kinase C.

FK 506, a powerful immunosuppressant that blocks allograft rejection by preventing T-cell activation, binds to an 11-kDa protein called the FK 506-binding protein (FKBP). Like cyclophilin, a cytosolic protein that binds another immunosuppressant, cyclosporin A, FKBP possesses peptidylprolyl cis-trans isomerase activity. We have isolated a genomic clone encoding the yeast FKBP (FKB1). The gene encodes a protein of 114 amino acids having a calculated Mr of 12,158. Disruption of the gene shows that FKB1 is not essential for growth. A search of translated nucleic acid data bases revealed bacterial FKBP homologs in Neisseria meningiditis and Pseudomonas aeruginosa. Comparison of the conserved amino acids in FKBP homologs with the conserved amino acids in the cyclophilins has revealed a region of similarity that we speculate to be a homologous domain related to the functional similarities of the two proteins.

We have recently isolated an abundant cytosolic protein from human T-cells which specifically binds the immunosuppressive agent, FK-506. The FK-506-binding protein (FKBP) is a member of a novel class of proteins possessing peptidyl-prolyl cis-trans isomerase activity. These proteins are believed to play an important role in accelerating the rate at which proteins fold into their native conformations. In the present study, we demonstrate that FKBP is not a lymphoid-specific protein, but is widely distributed and phylogenically conserved. FKBP, purified from three sources (a human T-lymphocyte cell line JURKAT, bovine calf thymus, and Saccharomyces cerevisiae) exhibit identical molecular weights, immunological cross-reactivities, and a high degree of NH2-terminal amino acid sequence homology. In addition, FKBP from all sources possesses peptidyl-prolyl cis-trans isomerase activity which can be specifically inhibited by FK-506. We conclude that FKBP may serve an important biological function in all eukaryotic cells.

Transcription of heat shock genes is induced by exposure of cells to elevated temperatures or other stress conditions. In yeast, it is thought that induction of transcription is mediated by conversion of a DNA-bound transcriptionally inactive form of the heat shock transcription factor (HSTF) to a DNA-bound transcriptionally active form. We have identified domains in HSTF involved in transcriptional activation and in repression of transcriptional activation at non-shock temperatures. We present evidence that a temperature-regulated transcriptional activation domain exists in HSTF and that this domain is essential for survival of yeast cells at heat shock temperatures. We propose a model for temperature-regulated transcriptional activation by a derepression mechanism.