Generation of a caged lentiviral vector through an unnatural amino acid for photo-switchable transduction.

Application of viral vectors in gene delivery is attracting widespread attention but is hampered by the absence of control over transduction, which may lead to non-selective transduction with adverse side effects. To overcome some of these limitations, we proposed an unnatural amino acid aided caging-uncaging strategy for controlling the transduction capability of a viral vector. In this proof-of-principle study, we first expanded the genetic code of the lentiviral vector to incorporate an azido-containing unnatural amino acid (Nϵ-2-azidoethyloxycarbonyl-l-lysine, NAEK) site specifically within a lentiviral envelope protein. Screening of the resultant vectors indicated that NAEK incorporation at Y77 and Y116 was capable of inactivating viral transduction upon click conjugation with a photo-cleavable chemical molecule (T1). Exposure of the chimeric viral vector (Y77-T1) to UVA light subsequently removed the photo-caging group and restored the transduction capability of lentiviral vector both in vitro and in vivo. Our results indicate that the use of the photo-uncage activation procedure can reverse deactivated lentiviral vectors and thus enable regulation of viral transduction in a switchable manner. The methods presented here may be a general approach for generating various switchable vectors that respond to different stimulations and adapt to different viral vectors.

Genetic encoding of 2-aryl-5-carboxytetrazole-based protein photo-cross-linkers.

Three γ-heteroatom-substituted N-methylpyrroletetrazole-lysines (mPyTXKs) were synthesized and subsequently incorporated into proteins site-specifically via genetic code expansion. The γ-seleno-substituted derivative, mPyTSeK, showed excellent incorporation efficiency in Escherichia coli and allowed site-selective photo-cross-linking of the GST dimer. Furthermore, the mPyTSeK-cross-linked GST dimer can be cleaved under mild oxidative conditions. The incorporation of mPyTXKs into proteins in mammalian cells was also demonstrated. Lastly, the recombinantly expressed mPyTSeK-encoded Grb2 was shown to covalently capture its interaction partner, EGFR, in mammalian cell lysate, which was subsequently released after treatment with H2O2.

Multiplexed proteome analysis with neutron-encoded stable isotope labeling in cells and mice.

We describe a protocol for multiplexed proteomic analysis using neutron-encoded (NeuCode) stable isotope labeling of amino acids in cells (SILAC) or mice (SILAM). This method currently enables simultaneous comparison of up to nine treatment and control proteomes. Another important advantage over traditional SILAC/SILAM is that shorter labeling times are required. Exploiting the small mass differences that correspond to subtle differences in the neutron-binding energies of different isotopes, the amino acids used in NeuCode SILAC/SILAM differ in mass by just a few milliDaltons. Isotopologs of lysine are introduced into cells or mammals, via the culture medium or diet, respectively, to metabolically label the proteome. Labeling time is ∼2 weeks for cultured cells and 3-4 weeks for mammals. The proteins are then extracted, relevant samples are combined, and these are enzymatically digested with lysyl endopeptidase (Lys-C). The resultant peptides are chromatographically separated and then mass analyzed. During mass spectrometry (MS) data acquisition, high-resolution MS1 spectra (≥240,000 resolving power at m/z = 400) reveal the embedded isotopic signatures, enabling relative quantification, while tandem mass spectra, collected at lower resolutions, provide peptide identities. Both types of spectra are processed using NeuCode-enabled MaxQuant software. In total, the approximate completion time for the protocol is 3-5 weeks.

Conditional Chaperone-Client Interactions Revealed by Genetically Encoded Photo-cross-linkers.

The cell envelope is an integral and essential component of Gram-negative bacteria. As the front line during host-pathogen interactions, it is directly challenged by host immune responses as well as other harsh extracellular stimuli. The high permeability of the outer-membrane and the lack of ATP energy system render it difficult to maintain important biological activities within the periplasmic space under stress conditions. The HdeA/B chaperone machinery is the only known acid resistant system found in bacterial periplasm, enabling enteric pathogens to survive through the highly acidic human stomach and establish infections in the intestine. These two homologous chaperones belong to a fast growing family of conditionally disordered chaperones that conditionally lose their well-defined three-dimensional structures to exert biological activities. Upon losing ordered structures, these proteins commit promiscuous binding of diverse clients in response to environmental stimulation. For example, HdeA and HdeB are well-folded inactive dimers at neutral pH but become partially unfolded to protect a wide array of acid-denatured proteins upon acid stress. Whether these conditionally disordered chaperones possess client specificities remains unclear. This is in part due to the lack of efficient tools to investigate such versatile and heterogeneous protein-protein interactions under living conditions. Genetically encoded protein photo-cross-linkers have offered a powerful strategy to capture protein-protein interactions, showing great potential in profiling protein interaction networks, mapping binding interfaces, and probing dynamic changes in both physiological and pathological settings. Despite great success, photo-cross-linkers that can simultaneously capture the promiscuous binding partners and directly identify the interaction interfaces remain technically challenging. Furthermore, methods for side-by-side profiling and comparing the condition-dependent client pools from two homologous chaperones are lacking. Herein, we introduce our recent efforts in developing a panel of versatile genetically encoded photo-cross-linkers to study the disorder-mediated chaperone-client interactions in living cells. In particular, we have developed a series of proteomic-based strategies relying on these new photo-cross-linkers to systematically compare the client profiles of HdeA and HdeB, as well as to map their interaction interfaces. These studies revealed the mode-of-action, particularly the client specificity, of these two conditionally disordered chaperones. In the end, some recent elegant work from other groups that applied the genetically encoded photo-cross-linking strategy to illuminate important protein-protein interactions within bacterial cell envelope is also discussed.

Lysines 3241 and 3260 of DNA-PKcs are important for genomic stability and radioresistance.

DNA-dependent protein kinase (DNA-PK) is a serine/threonine kinase that plays an essential role in the repair of DNA double-strand breaks (DSBs) in the non-homologous end-joining (NHEJ) pathway. The DNA-PK holoenzyme consists of a catalytic subunit (DNA-PKcs) and DNA-binding subunit (Ku70/80, Ku). Ku is a molecular sensor for double-stranded DNA and once bound to DSB ends it recruits DNA-PKcs to the DSB site. Subsequently, DNA-PKcs is activated and heavily phosphorylated, with these phosphorylations modulating DNA-PKcs. Although phosphorylation of DNA-PKcs is well studied, other post-translational modifications of DNA-PKcs are not. In this study, we aimed to determine if acetylation of DNA-PKcs regulates DNA-PKcs-dependent DSB repair. We report that DNA-PKcs is acetylated in vivo and identified two putative acetylation sites, lysine residues 3241 and 3260. Mutating these sites to block potential acetylation results in increased radiosensitive, a slight decrease in DSB repair capacity as assessed by γH2AX resolution, and increased chromosomal aberrations, especially quadriradial chromosomes. Together, our results provide evidence that acetylation potentially regulates DNA-PKcs.

Physicochemical properties of peanut protein isolate-glucomannan conjugates prepared by ultrasonic treatment.

Peanut protein isolate (PPI) was glycated with glucomannan through classical heating or ultrasound treatment in this work. The physicochemical properties of PPI-glucomannan conjugates prepared by ultrasound treatment were compared to those prepared by classical heating. Compared with classical heating, ultrasound treatment could accelerate the graft reaction between PPI and glucomannan and improve the concentration of available free amino groups of PPI. Solubility and emulsifying properties of the conjugates obtained by ultrasound treatment were both improved as compared to those obtained by classical heating and native PPI. Decreases of lysine and arginine contents during the graft reaction indicated that these two amino acid residues attended the covalent linkage between PPI and glucomannan. Structural feature analyses suggested that conjugates obtained by ultrasound treatment had less α-helix, more ß-structures and random coil, higher surface hydrophobicity and less compact tertiary structure as compared to those obtained by classical heating and native PPI.

Corneal and retinal effects of ultraviolet-B exposure in a soft contact lens mouse model.

PURPOSE: To investigate the lipid and DNA oxidative stress as well as corneal and retinal effects after ultraviolet B (UV-B) exposure in mice, with or without silicon hydrogel soft contact lenses (SCL). METHODS: Twenty-eight C57BL6-strain male mice were divided into four groups: group I, control group with no SCL (SCL [-]) and no UV-B exposure (UV-B [-]); group II, senofilcon A SCL (senofilcon [+]) with UV-B exposure (UV-B [+]); group III, lotrafilcon A SCL (lotrafilcon [+]) with UV-B exposure (UV-B [+]); and group IV, no SCL (SCL [-]), but with UV-B exposure (UV-B [+]). All mice except group I received UV-B exposure for 5 days for a total dose of 2.73 J/cm(2). All mice underwent tear hexanoyl-lysine (HEL) and tear cytokine ELISA measurements, and fluorescein and rose bengal corneal staining before and after UV-B exposure. Corneal specimens underwent immunohistochemistry staining with CD45, HEL, 4-hydroxynonenal (4-HNE), and 8-hydroxy-2'-deoxyguanosine (8-OHdG) antibodies and evaluation with electron microscopy. RESULTS: All mice without SCL but exposed to UV-B developed corneal edema, ulcers, or epithelial damage compared with mice with senofilcon A SCL and exposure to UV-B. Tear HEL and cytokine levels significantly increased in mice without SCL after UV-B exposure. Immunohistochemistry showed a significantly higher number of cells positively stained for CD45, 8-OHdG, HEL, and 4-HNE in the corneas of mice without SCLs compared with those with senofilcon A after UV-B exposure. CONCLUSION: Silicon hydrogel SCL showed corneal and retinal protective effects, owing to UV blocking properties, against oxidative stress-related membrane lipid and cellular DNA damage.

UV B-irradiation enhances the racemization and isomerizaiton of aspartyl residues and production of Nε-carboxymethyl lysine (CML) in keratin of skin.

UV-B irradiation is one of the risk factors in age-related diseases. We have reported that biologically uncommon D-ß-Asp residues accumulate in proteins from sun-exposed elderly human skin. A previous study also reported that carboxymethyl lysine (CML; one of the advanced glycation end products (AGEs)) which is produced by the oxidation of glucose and peroxidation of lipid, also increases upon UV B irradiation. The formation of D-ß-Asp and CML were reported as the alteration of proteins in UV B irradiated skin, independently. In this study, in order to clarify the relationship between the formation of D-ß-Asp and CML, immunohistochemical analysis using anti-D-ß-Asp containing peptide antibodies and anti-CML antibodies was performed in UV B irradiated mice. Immunohistochemical analyses clearly indicated that an anti-D-ß-Asp containing peptide antibody and anti-CML antibody reacted at a common area in UV B irradiated skin. Western blot analyses of the proteins isolated from UV B irradiated skin demonstrated that proteins of 50-70 kDa were immunoreactive towards antibodies for both D-ß-Asp containing peptide and CML. These proteins were identified by proteomic analysis as members of the keratin families including keratin-1, keratin-6B, keratin-10, and keratin-14.

Photoaddition of fluphenazine to nucleophiles in peptides and proteins. Possible cause of immune side effects.

By the action of UVA light, fluphenazine reacted with nucleophiles through a mechanism involving defluorination of its trifluoromethyl group, giving rise to carboxylic acid derivatives that were easily detected by electrospray mass spectrometry. This photoreaction took place with alcohols, sulphydryls, and amines. When irradiation of fluphenazine was carried out in the presence of an amino acid at pH 7.4, the alpha-amino group was covalently bound to the drug. With amino acids possessing a further nucleophilic residue on the side chain, such as lysine, tyrosine, and cysteine--but not serine--both groups reacted, resulting in a fluphenazine-amino acid-fluphenazine diadduct. The same occurred with the physiological peptide glutathione (gamma-glutamylcysteinylglycine). By means of MALDI mass spectrometry, it was shown that fluphenazine also covalently bound to peptides and proteins such as calmodulin. This binding may result in the formation of antibodies, ultimately leading to the destruction of the granulocytes and thus suggesting that photoactivation of this drug may play a role in its clinical side effects, such as agranulocytosis.

Phototransformations of advanced glycation end products in the human eye lens due to ultraviolet A light irradiation.

Previous studies from this laboratory have shown that ultraviolet A (UVA) light can bleach the yellow advanced glycation end products (AGEs) of aged and cataractous human lenses. The AGEs OP-lysine and argpyrimidine are two UVA-absorbing posttranslational modifications that are abundant in the eye lens. The purpose of this study was to outline the changes in these two AGEs due to UVA irradiation. The changes of OP-lysine, OP-phenethylamine (a phenethylamine analogue of OP-lysine), and argpyrimidine due to irradiation with UVA light in the presence or absence of air and ascorbic acid were followed by different spectral methods. Aged human lenses were similarly irradiated in artificial aqueous humor. The amounts of OP-lysine in the irradiated lenses and in the corresponding dark controls were determined by HPLC. Both OP-lysine and argpyrimidine decreased 20% when irradiated with UVA light in the absence of ascorbic acid. Under the same conditions, OP-lysine was bleached 80% in the presence of ascorbic acid during irradiation experiments. In contrast, argpyrimidine UVA light bleaching was not affected by the presence of ascorbic acid. Interestingly the major product of OP-phenethylamine after UVA irradiation in the presence of ascorbic acid was phenethylamine, which indicates that the entire heterocycle of this AGE was cleaved and the initial amino group was restored. Some AGEs in the human eye lens can be transformed by UVA light.

Radiolytic modification of basic amino acid residues in peptides: probes for examining protein-protein interactions.

Protein footprinting utilizing hydroxyl radicals coupled with mass spectrometry has become a powerful technique for mapping the solvent accessible surface of proteins and examining protein-protein interactions in solution. Hydroxyl radicals generated by radiolysis or chemical methods efficiently react with many amino acid residue side chains, including the aromatic and sulfur-containing residues along with proline and leucine, generating stable oxidation products that are valuable probes for examining protein structure. In this study, we examine the radiolytic oxidation chemistry of histidine, lysine, and arginine for comparison with their metal-catalyzed oxidation products. Model peptides containing arginine, histidine, and lysine were irradiated using white light from a synchrotron X-ray source or a cesium-137 gamma-ray source. The rates of oxidation and the radiolysis products were primarily characterized by electrospray mass spectrometry including tandem mass spectrometry. Arginine is very sensitive to radiolytic oxidation, giving rise to a characteristic product with a 43 Da mass reduction as a result of the loss of guanidino group and conversion to gamma-glutamyl semialdehyde, consistent with previous metal-catalyzed oxidation studies. Histidine was oxidized to generate a mixture of products with characteristic mass changes primarily involving rupture of and addition to the imidazole ring. Lysine was converted to hydroxylysine or carbonylysine by radiolysis. The development of methods to probe these residues due to their high frequency of occurrence, their typical presence on the protein surface, and their frequent participation in protein-protein interactions considerably extends the utility of protein footprinting.

Sunlight induces N epsilon-(carboxymethyl)lysine formation from glycated polylysine-iron(III) complex.

Sunlight was found to strongly induce the formation of N epsilon-(carboxymethyl)lysine (CML) from glycated polylysine in the presence of Fe(III) ion. The initial step of this Fe(III)-catalyzed CML formation was noted to be similar to that of blueprint photography as was confirmed by the production of Turnbull's blue in sunlight-exposed glycated human serum albumin ferricyanide solution in the presence of Fe(III). Based on this, photoinduced oxidative C-C bond cleavage of the Amadori compound was assumed to be initiated by photochemical single electron transfer front ligand to Fe(III) in the Fe(III)-Amadori compound complex affording the Fe(II)-Amadori compound radical intermediate, which eventually yields either CML or active oxygen species. CML is thus a useful oxidative stress marker. The mechanism proposed here would explain the high accumulation of CML in lens protein and skin actinic elastosis.

[New approach to the study of interaction of amino acid side groups with aryl azides]. / Novyi podkhod k izucheniiu vzaimodeistviia aminokislot po ikh bokovym radikalam s arilazidami.

A new approach to the study of the interaction of amino acid side chains with photoreactive aryl azides was proposed. This approach was based on the drawing together of the reacting groups by the attachment of the reacting compounds to complementary oligonucleotides. Cystamine, histamine, and 1,6-hexamethylenediamine mimicking the cystine, histidine, and lysine residues, respectively, were attached to the 3'-terminal phosphate of the oligonucleotide GGTATCp through a phosphamide bond and used as the targets for photomodification. Derivatives of the oligonucleotide pGATACCAA with the fragment N3C6H4NH- attached directly to its 5'-end by a phosphamide bond or through the spacer -(CH2)nNH- (where n is 2, 4, and 6) were used as photoreagents. Their derivatives containing the same spacer and the N3C6F4CO-NH(CH2)3NH- or 2-N3,5-NO2-C6H3CO-NH(CH2)3NH- residues were also used. The duplexes were photomodified by irradiation with 300-350 nm wavelength light. The maximal yields of the photo-cross-linking were from 22 to 68%. The reagents containing p-azidoaniline residue were found to be the most effective toward the targets. The maximum yields of the photomodification products modeling the side chains of cysteine and lysine were found to vary from 40 to 67% and to depend on the length and the structure of the spacers used. The duplex with the target bearing the imidazole residue (the histidine model) manifested a yield decreased to 25%. This fact was in a good agreement with the data of computer modeling that indicated an unfavorable mutual displacement of the imidazole residue and the photoreactive group.

[Synthesis of oligopeptides from the polar amino acids in water media by the combined action of weak electric and magnetic fields]. / Sintez oligopeptidov iz poliarnykh aminokislot v vodnoi srede pri kombinirovannom deistvii slabykh élektricheskikh i magnitnykh polei.

The effect of weak electromagnetic fields on aqueos solutions of polar amino acids leads to the formation of stable products of the amino acid condensation reaction. These products have a definite amino acid composition and retention time in high-performance liquid chromatography.

Photochemical bleaching of fluorescent glycosylation products.

We have examined the effect of visible light and ultraviolet on the non-enzymatic glycosylation of lysine in vitro. Glucose and L-lysine were mixed and incubated under white light, UV-A, UV-B, or in the dark. During 15 days of incubation in the dark, a heterogeneous mixture of intensely brown chromophores developed, with a dominant fluorescence excitation maximum at 350 nm and emission maxima near 425 nm. The process was delayed or inhibited to a moderate extent by white light, whereas under UV-A and UV-B this effect was more pronounced. We conclude that non-enzymatic glycosylation can be photochemically modulated by both visible light and ultraviolet.

Solid state NMR study of [epsilon-13C]Lys-bacteriorhodopsin: Schiff base photoisomerization.

Previous solid state 13C-NMR studies of bacteriorhodopsin (bR) have inferred the C = N configuration of the retinal-lysine Schiff base linkage from the [14-13C]retinal chemical shift (1-3). Here we verify the interpretation of the [14-13C]-retinal data using the [epsilon-13C]lysine 216 resonance. The epsilon-Lys-216 chemical shifts in bR555 (48 ppm) and bR568 (53 ppm) are consistent with a C = N isomerization from syn in bR555 to anti in bR568. The M photointermediate was trapped at pH 10.0 and low temperatures by illumination of samples containing either 0.5 M guanidine-HCl or 0.1 M NaCl. In both preparations, the [epsilon-13C]Lys-216 resonance of M is 6 ppm downfield from that of bR568. This shift is attributed to deprotonation of the Schiff base nitrogen and is consistent with the idea that the M intermediate contains a C = N anti chromophore. M is the only intermediate trapped in the presence of 0.5 M guanidine-HCl, whereas a second species, X, is trapped in the presence of 0.1 M NaCl. The [epsilon-13C]Lys-216 resonance of X is coincident with the signal for bR568, indicating that X is either C = N anti and protonated or C = N syn and deprotonated.

Evidence for light-induced lysine conformational changes during the primary event of the bacteriorhodopsin photocycle.

Fourier transform infrared difference spectroscopy is used to examine the role of lysine in the primary event of the bacteriorhodopsin photocycle. Isotopically labeled lysine is used to tentatively assign the lysine modes in the BR and K species. The results suggest that the lysine side-chain undergoes conformational changes in concert with the known light-induced chromophore structural alterations.

[Localization of lysine residue in histone H3 forming the thymine-lysine cross-link after UV-irradiation of deoxyribonucleoprotein]. / Lokalizatsiia ostatka lizina v gistone H3, obrazuiushchego timin-lizinovuiu sshivku pri obluchenii dezoksiribonukleoproteida UF-svetom.

A thymine-modified derivative of histone H3 formed as a result of thermal treatment of UV-irradiated (lambda = 254 nm) solution of deoxyribonucleoprotein from calf thymus at low ionic strength was isolated. The peptides obtained by tryptic hydrolysis of modified histone H3 were separated by high pressure liquid chromatography. The amino acid sequence of the peptide containing a lysine residue with covalently linked thymine was determined by the Edman method. It was found that Lys localized at the N-terminus of the histone H3 molecule interacts with DNA within the composition of the deoxyribonucleoprotein.

[The role of histones H3 and H1 in the formation of thymine-lysine cross-links during UV-irradiation of deoxyribonucleoprotein]. / Uchastie gistonov H3 i H1 v obrazovanii timin-lizinovoi shivki pri UF-obluchenii deoksiribonukleoproteida.

The effect of UV light (lambda = 254 nm) on calf thymus DNP at low ionic strengths was studied. It was found that at the irradiation doses used the protein in the DNA-protein complex increases as the irradiation dose rises. Thermal treatment and acid hydrolysis resulted in a predominant release of histones H3 and H1 from the complex. Data from liquid high performance chromatography, amino acid analysis, thin-layer chromatography point to the induction by UV-light of a thymine-lysine bond, whose formation involves DNA thymines and histone lysine residues, predominantly H3 and H1 fractions.