Chemical approaches for investigating phosphorylation in signal transduction networks

The power and scope of chemical synthesis offer considerable opportunities to broaden the lexicon of chemical tools that can be implemented for the study of complex biological systems. To investigate individual signaling proteins and pathways, chemical tools provide a powerful complement to existing genetic, chemical genetic and immunologic methods. In particular, understanding phosphorylation-mediated signaling in real time yields important information about the regulation of cellular function and insights into the origin of disease. Recent advances in the development of photolabile caged analogs of bioactive species and fluorescence-based sensors of protein kinase activities are useful for investigating protein phosphorylation and the roles of phosphoproteins. Photolabile caged analogs allow spatial and temporal control over the release of a compound, while fluorescence-based sensors allow the real-time visualization of kinase activity. Here, we discuss recent advances that have increased the specificity and availability of these tools.     

The preparation and in vivo applications of caged peptides and proteins

Cellular behavior, such as mitosis and motility, are controlled by both when and where specific intracellular signaling pathways are activated in response to environmental cues. Analogous temporally and spatially controlled events occur throughout the lifetime of an organism (e.g. embryogenesis). Consequently, reagents that can be switched on (or off) at any time or at any place in a cell, a tissue, or a living animal, represent the means by which the biochemical basis of spatially and temporally sensitive biological behavior can be evaluated. This review summarizes recent advances in the design and synthesis of light-activated ('caged') peptides and proteins as well as the application of these caged reagents to unanswered questions in biology.     

A collection of caged compounds for probing roles of local translation in neurobiology

Spatially localized translation plays a vital role in the normal functioning of neuronal systems and is widely believed to be involved in both learning and memory formation. It is of central interest to understand both the phenomenon and molecular mechanisms of local translation using new tools and approaches. Caged compounds can, in principle, be used as tools to investigate local translation since optical activation of bioactive molecules can achieve both spatial and temporal resolution on the micron scale and on the order of seconds or less, respectively. Successful caging of bioactive molecules requires the identification of key functional groups in appropriate molecules and the introduction of a suitable caging moiety. Here we present the design, synthesis and testing of a collection of three caged compounds: anisomycin caged with a diethylaminocoumarin moiety and dimethoxynitrobenzyl caged versions of 4E-BP and rapamycin. Whereas caged anisomycin can be used to control general translation, caged 4E-BP serves as a probe of cap-dependent translation initiation and caged rapamycin serves a probe of the role of mTORC1 in translation initiation. In vitro translation assays demonstrate that these caging strategies, in combination with the aforementioned compounds, are effective for optical control making it likely that such strategies can successfully employed in the study of local translation in living systems.     

Photo-dependent protein biosynthesis using a caged aminoacyl-tRNA

Translation systems with four-base codons provide a powerful strategy for protein engineering and protein studies because they enable site-specific incorporation of non-natural amino acids into proteins. In this study, a caged aminoacyl-tRNA with a four-base anticodon was synthesized. The caged aminoacyl-tRNA contains a photocleavable nitroveratryloxycarbonyl (NVOC) group. This study showed that the caged aminoacyl-tRNA was not deacylated, did not bind to EF-Tu, and was activated by light. Photo-dependent translation of an mRNA containing the four-base codon was demonstrated using the caged aminoacyl-tRNA.     

Characterization of a new caged proton capable of inducing large pH jumps

A new caged proton, 1-(2-nitrophenyl)ethyl sulfate (caged sulfate), is characterized by infrared spectroscopy and compared with a known caged, proton 2-hydroxyphenyl 1-(2-nitrophenyl)ethyl phosphate (caged HPP). In contrast to caged HPP, caged sulfate can induce large pH jumps and protonate groups that have pK values as low as 2.2. The photolysis mechanism of caged sulfate is analogous to that of P(3)-[1-(2-nitrophenyl)ethyl] ATP (caged ATP), and the photolysis efficiency is similar. The utility of this new caged compound for biological studies was demonstrated by its ability to drive the acid-induced conformational change of metmyoglobin. This transition from the native conformation to a partially unfolded form takes place near pH 4 and was monitored by near-UV absorption spectroscopy.     

Photoactivatable fluorophores and techniques for biological imaging applications

Photoactivatable fluorophores (PAFs) are powerful imaging probes for tracking molecular and cellular dynamics with high spatiotemporal resolution in biological systems. Recent developments in biological microscopy have raised new demands for engineering new PAFs with improved properties, such as high two photon excitation efficiency, reversibility, cellular delivery and targeting. Here we review the history and some of the recent developments in this area, emphasizing our efforts in developing a new class of caged coumarins and related imaging methods for studying dynamic cell-cell communication through gap junction channels, and in extending the application of these caged coumarins to new areas including spatiotemporal control of microRNA activity in vivo.     

Caged single and double strand breaks

Ionizing radiation and radiomimetic drugs such as bleomycin, calichieamycin, neocarzinostatin chromophore, and other synthetic agents can produce both single and double strand breaks in DNA. The ability to study the structure-activity relationships of single and double-strand break repair, lethality, and mutagenesis in vivo is complicated by the numerous types and sites of DNA cleavage products that can be induced by such agents. The ability to "cage" such breaks in DNA might help to further such studies and additionally afford a mechanism for activating and deactivating nucleic acid based drugs and probes. The major type of single strand break induced by ionizing radiation is a 3'- and 5'-phosphate terminated single nucleotide gap. Previously, a caged strand break of this type had been developed that was designed to produce the 5'-phosphate directly upon irradiation with 366 nm light, and the 3'-phosphate by a subsequent beta-elimination reaction [Ordoukhanian, P., and Taylor, J.-S. (1995) J. Am. Chem. Soc. 117, 9570]. Unfortunately, the release of the 3'-phosphate group was quite slow at pH 7. To circumvent this problem, a second caged strand break has been developed that produces the 3'-phosphate directly upon irradiation, and the 5'-phosphate by a subsequent beta-elimination reaction. When this caged strand break was used in tandem with the previous caged strand break, 5'- and 3'-phosphate terminated gaps could be directly produced by irradiation with 366 nm light. These caged single strand breaks were also incorporated in tandem into hairpin substrates to demonstrate that they could be used to cage double strand breaks. These caged single strand breaks should be generally useful for generating site-specific DNA single and double strand breaks and gaps, using wavelengths and doses of light that are nondetrimental to biological systems. Because the position of the single strand break can be varied, it should now be possible to examine the effect of the sequence context and cleavage pattern of single and double strand breaks on the lethality and mutagenicity of this important class of DNA damage.     

Using photolabile protecting groups for the controlled release of bioactive volatiles

To develop their biological activity, bioactive volatile compounds, such as pheromones or fragrances, have to evaporate from surfaces. Because these surfaces are usually exposed to natural daylight, the preparation of non-volatile precursors using photoremovable protecting groups is an ideal tool to control the release of caged volatile molecules from various surfaces by light-induced covalent bond cleavage. Many photoreactions occur under mild environmental conditions and are highly selective. To break covalent bonds under typical application conditions, the photoreaction has to proceed at ambient daylight, to tolerate the presence of oxygen and to run in polar media (e.g. in water). The amount of volatiles generated from photochemical delivery systems depends on the light intensity to which the systems are exposed. Both photoisomerisations and photofragmentations have successfully been investigated for the slow release of caged pheromones and fragrances from their corresponding precursors.     

Molecular reaction mechanisms of proteins monitored by time-resolved FTIR-spectroscopy.

Time-resolved FTIR difference spectroscopy can provide a valuable insight into the molecular reaction mechanisms of proteins, especially membrane proteins. Isotopic labeling and site-directed mutagenesis allows an unequivocal assignment of IR absorption bands. Studies are presented which give insight into the proton pump mechanisms of proteins, especially bacteriorhodopsin. H-bonded network proton transfer via internal water molecules seems to be a general feature in proteins, also found in cytochrome c oxidase. Using caged GTP the intrinsic and GAP catalyzed GTPase activity of H-ras p21 is studied. Furthermore, protein folding reactions can be recorded with ns time-resolution.     

Single Cell Fate Mapping in Zebrafish

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, lymphatic, and blood vessel lineages arise in developing embryos requires fate mapping and lineage tracing of undifferentiated precursor cells. Recently, photoactivatable proteins which include: Eos^{1, 2}, PAmCherry³, Kaede^4-7, pKindling⁸, and KikGR^{9, 10} have received wide interest as cell tracing probes. The fluorescence spectrum of these photosensitive proteins can be easily converted with UV excitation, allowing a population of cells to be distinguished from adjacent ones. However, the photoefficiency of the activated protein may limit long-term cell tracking¹¹. As an alternative to photoactivatable proteins, caged fluorescein-dextran has been widely used in embryo model systems^{7, 12-14}. Traditionally, to uncage fluorescein-dextran, UV excitation from a fluorescence lamp house or a single photon UV laser has been used; however, such sources limit the spatial resolution of photoactivation. Here we report a protocol to fate map, lineage trace, and detect single labeled cells. Single cells in embryos injected with caged fluorescein-dextran are photoactivated with near-infrared laser pulses produced from a titanium sapphire femtosecond laser. This laser is customary in all two-photon confocal microscopes such as the LSM 510 META NLO microscope used in this paper. Since biological tissue is transparent to near-infrared irradiation¹⁵, the laser pulses can be focused deep within the embryo without uncaging cells above or below the selected focal plane. Therefore, non-linear two-photon absorption is induced only at the geometric focus to uncage fluorescein-dextran in a single cell. To detect the cell containing uncaged fluorescein-dextran, we describe a simple immunohistochemistry protocol¹⁶ to rapidly visualize the activated cell. The activation and detection protocol presented in this paper is versatile and can be applied to any model system. Note: The reagents used in this protocol can be found in the table appended at the end of the article.     

Correlative Photoactivated Localization and Scanning Electron Microscopy

The ability to localize proteins precisely within subcellular space is crucial to understanding the functioning of biological systems. Recently, we described a protocol that correlates a precise map of fluorescent fusion proteins localized using three-dimensional super-resolution optical microscopy with the fine ultrastructural context of three-dimensional electron micrographs. While it achieved the difficult simultaneous objectives of high photoactivated fluorophore preservation and ultrastructure preservation, it required a super-resolution optical and specialized electron microscope that is not available to many researchers. We present here a faster and more practical protocol with the advantage of a simpler two-dimensional optical (Photoactivated Localization Microscopy (PALM)) and scanning electron microscope (SEM) system that retains the often mutually exclusive attributes of fluorophore preservation and ultrastructure preservation. As before, cryosections were prepared using the Tokuyasu protocol, but the staining protocol was modified to be amenable for use in a standard SEM without the need for focused ion beam ablation. We show the versatility of this technique by labeling different cellular compartments and structures including mitochondrial nucleoids, peroxisomes, and the nuclear lamina. We also demonstrate simultaneous two-color PALM imaging with correlated electron micrographs. Lastly, this technique can be used with small-molecule dyes as demonstrated with actin labeling using phalloidin conjugated to a caged dye. By retaining the dense protein labeling expected for super-resolution microscopy combined with ultrastructural preservation, simplifying the tools required for correlative microscopy, and expanding the number of useful labels we expect this method to be accessible and valuable to a wide variety of researchers.     

Synthesis of potentially caged sphingolipids, possible precursors of cellular modulators and second messengers

An increasing number of sphingolipids, glycosphingolipids and some of their degradation products have been recognized in recent years as second messengers involved in signal transduction and as modulators of numerous cellular functions. These can be converted into inert, caged compounds, introduced into cells and tissues and subsequently photolysed to active compounds thus enabling the study of fast biological processes. The novel, potentially caged compounds synthesized here are substituted 2-nitrobenzyl urethans and 2-nitrobenzyl amines derived from sphingosine, dihydrosphingosine, N-methylsphingosine, N-methyldihydrosphingosine, psychosine and glucosylsphingosine. Upon irradiation of the afore mentioned compounds they release, or are expected to release, the free biologically active amines.     

Rapid neurotransmitter uncaging in spatially defined patterns

Light-sensitive 'caged' molecules provide a means of rapidly and noninvasively manipulating biochemical signals with submicron spatial resolution. Here we describe a new optical system for rapid uncaging in arbitrary patterns to emulate complex neural activity. This system uses TeO(2) acousto-optical deflectors to steer an ultraviolet beam rapidly and can uncage at over 20,000 locations per second. The uncaging beam is projected into the focal plane of a two-photon microscope, allowing us to combine patterned uncaging with imaging and electrophysiology. By photolyzing caged neurotransmitter in brain slices we can generate precise, complex activity patterns for dendritic integration. The method can also be used to activate many presynaptic neurons at once. Patterned uncaging opens new vistas in the study of signal integration and plasticity in neuronal circuits and other biological systems.     

Characterization of caged compounds binding to proteins by NMR spectroscopy

Photolysable caged ligands are used to investigate protein function and activity. Here, we investigate the binding properties of caged nucleotides and their photo released products to well established but evolutionary and structurally unrelated nucleotide-binding proteins, rabbit muscle creatine kinase (RMCK) and human annexin A6 (hAnxA6), using saturation transfer difference NMR spectroscopy. We detect the binding of the caged nucleotides and discuss the general implications on interpreting data collected with photolysable caged ligands using different techniques. Strategies to avoid non-specific binding of caged compound to certain proteins are also suggested.     

Caged vanilloid ligands for activation of TRPV1 receptors by 1- and 2-photon excitation

Nociceptive neurons in the peripheral nervous system detect noxious stimuli and report the information to the central nervous system. Most nociceptive neurons express the vanilloid receptor, TRPV1, a nonselective cation channel gated by vanilloid ligands such as capsaicin, the pungent essence of chili peppers. Here, we report the synthesis and biological application of two caged vanilloids: biologically inert precursors that, when photolyzed, release bioactive vanilloid ligands. The two caged vanilloids, Nb-VNA and Nv-VNA, are photoreleased with quantum efficiency of 0.13 and 0.041, respectively. Under flash photolysis conditions, photorelease of Nb-VNA and Nv-VNA is 95% complete in approximately 40 micros and approximately 125 micros, respectively. Through 1-photon excitation with ultraviolet light (360 nm), or 2-photon excitation with red light (720 nm), the caged vanilloids can be photoreleased in situ to activate TRPV1 receptors on nociceptive neurons. The consequent increase in intracellular free Ca(2+) concentration ([Ca(2+)](i)) can be visualized by laser-scanning confocal imaging of neurons loaded with the fluorescent Ca(2+) indicator, fluo-3. Stimulation results from TRPV1 receptor activation, because the response is blocked by capsazepine, a selective TRPV1 antagonist. In Ca(2+)-free extracellular medium, photoreleased vanilloid can still elevate [Ca(2+)](i), which suggests that TRPV1 receptors also reside on endomembranes in neurons and can mediate Ca(2+) release from intracellular stores. Notably, whole-cell voltage clamp measurements showed that flash photorelease of vanilloid can activate TRPV1 channels in <4 ms at 22 degrees C. In combination with 1- or 2-photon excitation, caged vanilloids are a powerful tool for probing morphologically distinct structures of nociceptive sensory neurons with high spatial and temporal precision.     

''Caged cytoskeletons'': a rapid method for the isolation of microtubule-associated proteins from synchronized plant suspension cells

In the cytoskeleton method for isolating microtubule-associated proteins MAP65, DcKRP120-1 and DcKRP120-2, carrot cells are first converted to protoplasts but this method cannot be used to isolate mitotic MAPs as mitotic synchrony is eroded during lengthy cellulase treatment. Anti-microtubule cycle blocks would also be unsuitable. We report here a method for overcoming these problems. Cellulase degradation of tobacco BY-2 cells for only several minutes allows extraction of detergent-soluble proteins, leaving synchronized "caged cytoskeletons" for depolymerization and enabling affinity purification of MAPs on neurotubules. This rapid and simple method should be of general utility: it can be bulked up, avoids anti-microtubule blocks, and is applicable to other cell suspensions. The effectiveness of the caged cytoskeleton method is demonstrated by comparing known MAPs (the 65 kDa structural MAPs and the kinesin-related protein, TKRP125) in synchronized cells taken at the mitotic peak with those in unsynchronized cells.     

Prokaryotic gene fusion expression systems and their use in structural and functional studies of proteins

The ability to express and purify large quantity of proteins in bacteria has greatly impacted many aspects of biological research. These include their use as a source of reagent for biochemical and biophysical studies as well as a source of antigen for antibody production. Currently many different expression systems are available and new ones are being developed. These systems allow inducible expression of a desired protein as a fusion with an affinity tag for simple purification. The affinity tags can generally be removed by specific proteases which recognize cleavage sites engineered between the affinity tag and the desired protein. Presence of tags that encode epitopes of specific antibodies provide additional means for identification of recombinant proteins. This review provides an overview of some of the most commonly utilized expression systems and examples of the use of these proteins in biochemical and biophysical studies. I will also describe other available systems which may provide suitable alternative for expression of recombinant proteins.     

Rapid and efficient MALDI-TOF MS peak detection of 2-nitrobenzenesulfenyl-labeled peptides using the combination of HPLC and an automatic spotting apparatus.

In this paper, we report MALDI-TOF ms analysis of 2-nitrobenzenesulfenyl (NBS) labeled peptides with the powerful aid of an LC-automatic spotting system. using this approach we analyzed mammalian sera (rat and mouse) as biological samples to demonstrate performance. The labeling was carried out using a binary set of 2-nitrobenzenesulfenyl chloride (heavy and light), which modified tryptophan residues in sample proteins. Approximately 1600 doublet peaks were detected in the mass spectrum, some of which had more than threefold differences in their intensities. systematic separation/spotting followed by mass analysis of the NBS-labeled peptides derived from biological samples is described for the first time. This method has proved to be an effective application of NBS-labeled peptides and can be a powerful technique for quantitative analysis of proteins expressed in biological systems.     

Synthesis and biological evaluation of a novel photo-activated histone deacetylase inhibitor

Hydroxamic acid-based histone deacetylase inhibitors (HDACi) are a class of epigenetic agents with potentially broad therapeutic application to several disease states including post angioplasty mediated neointimal hyperplasia (NIH). Precise spatiotemporal control over the release of HDACi at the target blood vessel site is required for the safe and successful therapeutic use of HDACi in the setting of drug eluting balloon catheter (DEBc) angioplasty treatment of NIH. We aimed to develop and characterise a novel photoactive HDACi, as a potential coating agent for DEBc.Metacept-3 1 was caged with a photo-labile protecting group (PPG) to synthesise a novel UV365nm active HDACi, caged metacept-3 15. Conversion of caged metacept-3 15 to active/native metacept-3 1 by UV365nm was achievable in significant quantities and at UV365nm power levels in the milliwatt (mW) range.In vitro evaluation of the biological activity of pre and post UV365nm activation of caged metacept-3 15 identified significant HDACi activity in samples exposed to short duration, mW range UV365nm. Toxicity studies performed in human umbilical vein endothelial cells (HUVEC’s) identified significantly reduced toxicity of caged metacept-3 15 pre UV365nm exposure compared with native metacept-3 1 and paclitaxel (PTX).Taken together these findings identify a novel photo-activated HDACi, caged metacept-3 15, with pharmacokinetic activation characteristics and biological properties which may make it suitable for evaluation as a novel coating for targeted DEBc angioplasty interventions.     

SNARE protein analog‐mediated membrane fusion

Fusion of lipid membranes to form a single bilayer is an essential process for life and provides important biological functions including neurotransmitter release. Membrane fusion proteins facilitate approximation of interacting membranes to overcome the energy barrier. In case of synaptic transmission, proteins involved are known as soluble N‐ethylmaleimide‐sensitive‐factor attachment receptor (SNARE) proteins. The SNAREs from synaptic vesicles interact with the SNAREs from the target membrane to form a coiled‐coil bundle of four helices, thus pulling the membranes tightly together and initiating fusion. However, it remains unclear how these proteins function at molecular level. Natural systems are often too complex to obtain unambiguous results. Simple model systems mimicking natural proteins in synthetic lipid bilayers are powerful tools for obtaining insights into this essential biological process. An important advantage of such systems is their well‐defined composition, which can be systematically varied in order to fully understand events at molecular level. In this review, selected model systems are presented based upon specific interactions between recognition units embedded in separate lipid bilayers mimicking native SNARE protein‐mediated membrane fusion. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.