Tailored Chemical Reactivity Probes for Systemic Imaging of Aldehydes in Fibroproliferative Diseases.

During fibroproliferation, protein-associated extracellular aldehydes are formed by the oxidation of lysine residues on extracellular matrix proteins to form the aldehyde allysine. Here we report three Mn(II)-based, small-molecule magnetic resonance probes that contain α-effect nucleophiles to target allysine in vivo and report on tissue fibrogenesis. We used a rational design approach to develop turn-on probes with a 4-fold increase in relaxivity upon targeting. The effects of aldehyde condensation rate and hydrolysis kinetics on the performance of the probes to detect tissue fibrogenesis non-invasively in mouse models were evaluated by a systemic aldehyde tracking approach. We showed that, for highly reversible ligations, off-rate was a stronger predictor of in vivo efficiency, enabling histologically validated, three-dimensional characterization of pulmonary fibrogenesis throughout the entire lung. The exclusive renal elimination of these probes allowed for rapid imaging of liver fibrosis. Reducing the hydrolysis rate by forming an oxime bond with allysine enabled delayed phase imaging of kidney fibrogenesis. The imaging efficacy of these probes, coupled with their rapid and complete elimination from the body, makes them strong candidates for clinical translation.

Structure and mechanism of piperideine-6-carboxylate dehydrogenase from Streptomyces clavuligerus.

The core of ß-lactam antibiotics originates from amino acids of primary metabolism in certain microorganisms. ß-Lactam-producing bacteria, including Streptomyces clavuligerus, synthesize the precursor of the amino acid α-aminoadipic acid by the catabolism of lysine in two steps. The second reaction, the oxidation of piperideine-6-carboxylate (or its open-chain form α-aminoadipate semialdehyde) to α-aminoadipic acid, is catalysed by the NAD+-dependent enzyme piperideine-6-carboxylate dehydrogenase (P6CDH). This structural study, focused on ligand binding and catalysis, presents structures of P6CDH from S. clavuligerus in its apo form and in complexes with the cofactor NAD+, the product α-aminoadipic acid and a substrate analogue, picolinic acid. P6CDH adopts the common aldehyde dehydrogenase fold, consisting of NAD-binding, catalytic and oligomerization domains. The product binds in the oxyanion hole, close to the catalytic residue Cys299. Clear density is observed for the entire cofactor, including the nicotinamide riboside, in the binary complex. NAD+ binds in an extended conformation with its nicotinamide ring overlapping with the binding site of the carboxylate group of the product, implying that the conformation of the cofactor may change during catalysis. The binding site of the substrate analogue overlaps with that of the product, suggesting that the cyclic form of the substrate, piperideine-6-carboxylate, may be accepted as a substrate by the enzyme. The catalytic mechanism and the roles of individual residues are discussed in light of these results.

Collagen cross-linking mediated by lysyl hydroxylase 2: an enzymatic battlefield to combat fibrosis.

The hallmark of fibrosis is an excessive accumulation of collagen, ultimately leading to organ failure. It has become evident that the deposited collagen also exhibits qualitative modifications. A marked modification is the increased cross-linking, leading to a stabilization of the collagen network and limiting fibrosis reversibility. Not only the level of cross-linking is increased, but also the composition of cross-linking is altered: an increase is seen in hydroxyallysine-derived cross-links at the expense of allysine cross-links. This results in irreversible fibrosis, as collagen cross-linked by hydroxyallysine is more difficult to degrade. Hydroxyallysine is derived from a hydroxylysine in the telopeptides of collagen. The expression of lysyl hydroxylase (LH) 2 (LH2), the enzyme responsible for the formation of telopeptidyl hydroxylysine, is universally up-regulated in fibrosis. It is expected that inhibition of this enzyme will lead to reversible fibrosis without interfering with the normal repair process. In this review, we discuss the molecular basis of collagen modifications and cross-linking, with an emphasis on LH2-mediated hydroxyallysine cross-links, and their implications for the pathogenesis and treatment of fibrosis.

Malondialdehyde interferes with the formation and detection of primary carbonyls in oxidized proteins.

Carbonylation is one of the most remarkable expressions of the oxidative damage to proteins and the DNPH method the most common procedure to assess protein oxidation in biological samples. The present study was elicited by two hypotheses: i) is malondialdehyde, as a reactive dicarbonyl, able to induce the formation of allysine through a Maillard-type reaction? and ii) to which extent does the attachment of MDA to proteins interfere in the assessment of protein carbonyls using the DNPH method? Human serum albumin (HSA), human hemoglobin (HEM) and ß-lactoglobulin (LAC) (5 mg/mL) were incubated with MDA (0.25 mM) for 24 h at 37 °C (HSA and HEM) or 80 °C (LAC). Results showed that MDA was unable to induce oxidative deamination of lysine residues and instead, formed stable and fluorescent adducts with proteins. Such adducts were tagged by the DNPH method, accounting for most of the protein hydrazones quantified. This interfering effect was observed in a wide range of MDA concentrations (0.05-1 mM). Being aware of its limitations, protein scientists should accurately interpret results from the DNPH method, and apply, when required, other methodologies such as chromatographic methods to detect specific primary oxidation products such as allysine.

68Ga-NODAGA-Indole: An Allysine-Reactive Positron Emission Tomography Probe for Molecular Imaging of Pulmonary Fibrogenesis.

Oxidized collagen, wherein lysine residues are converted to the aldehyde allysine, is a universal feature of fibrogenesis, i.e. actively progressive fibrosis. Here we report the small molecule, allysine-binding positron emission tomography probe, 68Ga-NODAGA-indole, that can noninvasively detect and quantify pulmonary fibrogenesis. We demonstrate that the uptake of 68Ga-NODAGA-indole in actively fibrotic lungs is 7-fold higher than in control groups and that uptake is linearly correlated ( R2 = 0.98) with the concentration of lung allysine.

Formation of allysine in ß-lactoglobulin and myofibrillar proteins by glyoxal and methylglyoxal: Impact on water-holding capacity and in vitro digestibility.

The ability of α-dicarbonyls, glyoxal (GO) and methyl-glyoxal (MGO) (2 M), to induce the formation of allysine in ß-lactoglubulin (LAC), and myofibrillar proteins (MP) (2 mg/mL) during incubation at 80 °C for 48 h, was studied. Both GO and MGO induced the formation of allysine in all tested proteins with GO being more reactive (23.8 and 8.6 nmoles/mg protein in LAC and MP respectively after 6 h) than MGO (2.6 and 3.1 nmoles/mg protein at the same sampling point). LAC seemed to be more susceptible to the glycation reactions than MP. The concentration of allysine decreased at 24 h along with a concomitant increase of advanced-glycation end-products suggesting that allysine may be involved in the formation of fluorescent adducts. The water-holding capacity and trypsin-chymotrypsin digestibility of the proteins decreased during the incubation assay. The mechanisms by which α-dicarbonyls-mediated carbonylation likely influenced the impairment of such protein properties are thoroughly discussed.

Elastin is heterogeneously cross-linked.

Elastin is an essential vertebrate protein responsible for the elasticity of force-bearing tissues such as those of the lungs, blood vessels, and skin. One of the key features required for the exceptional properties of this durable biopolymer is the extensive covalent cross-linking between domains of its monomer molecule tropoelastin. To date, elastin's exact molecular assembly and mechanical properties are poorly understood. Here, using bovine elastin, we investigated the different types of cross-links in mature elastin to gain insight into its structure. We purified and proteolytically cleaved elastin from a single tissue sample into soluble cross-linked and noncross-linked peptides that we studied by high-resolution MS. This analysis enabled the elucidation of cross-links and other elastin modifications. We found that the lysine residues within the tropoelastin sequence were simultaneously unmodified and involved in various types of cross-links with different other domains. The Lys-Pro domains were almost exclusively linked via lysinonorleucine, whereas Lys-Ala domains were found to be cross-linked via lysinonorleucine, allysine aldol, and desmosine. Unexpectedly, we identified a high number of intramolecular cross-links between lysine residues in close proximity. In summary, we show on the molecular level that elastin formation involves random cross-linking of tropoelastin monomers resulting in an unordered network, an unexpected finding compared with previous assumptions of an overall beaded structure.

Molecular Magnetic Resonance Imaging of Lung Fibrogenesis with an Oxyamine-Based Probe.

Fibrogenesis is the active production of extracellular matrix in response to tissue injury. In many chronic diseases persistent fibrogenesis results in the accumulation of scar tissue, which can lead to organ failure and death. However, no non-invasive technique exists to assess this key biological process. All tissue fibrogenesis results in the formation of allysine, which enables collagen cross-linking and leads to tissue stiffening and scar formation. We report herein a novel allysine-binding gadolinium chelate (GdOA), that can non-invasively detect and quantify the extent of fibrogenesis using magnetic resonance imaging (MRI). We demonstrate that GdOA signal enhancement correlates with the extent of the disease and is sensitive to a therapeutic response.

Impact of dianionic and dicationic linkers on tumor uptake and biodistribution of [64 Cu]Cu/NOTA peptide-based gastrin-releasing peptide receptors antagonists.

In this study, we investigated for the first time the influence of 2-aminoethyl-piperazine-1-carboxylic acid (APCA) and amino-hexanedioic-1-acid (AHDA) on tumor uptake and elimination kinetics of [64 Cu]-radiolabeled gastrin releasing peptide receptors (GRPR) antagonists. Three GRPR antagonists containing the RM26 sequence were synthesized and conjugated with NOTA via different linkers (LK): polyethylene glycol (PEG-neutral), APCA (dicationic) or AHDA (dianionic). The NOTA-LK-RM26 peptides were radiolabeled with 64 Cu to assess their pharmacokinetic and positron emission tomography (PET) imaging properties using PC3 tumor-bearing athymic nude mice. The inhibition constants (Ki ) of the 3 nat Cu/NOTA-LK-RM26 peptides bearing PEG, dicationic and dianionic linkers were 0.98 ± 0.48 nM, 0.95 ± 0.21 nM, and 17.97 ± 2.79 nM, respectively. The [64 Cu] NOTA-LK-RM26 conjugates were prepared with labeling yields superior to 95% and specific activities of 67 to 77 TBq/mmol. The 3 radiopeptides were stable in vivo and showed GRPR-specific uptake in pancreas with a very fast washout of this tissue observed for [64 Cu]-NOTA-AHDA-RM26 peptide. Results from imaging studies displayed specific PC3 tumor uptake for both [64 Cu]-NOTA-APCA- and AHDA-RM26, similar kidney elimination and fast liver washout. Considering their adequate imaging characteristics, [64 Cu]-NOTA-LK-RM26 bearing APCA- and AHDA-linkers are promising candidates for GRPR-targeted PET imaging prostate cancer.

α-Aminoadipic acid protects against retinal disruption through attenuating Müller cell gliosis in a rat model of acute ocular hypertension.

OBJECTIVE: Ocular hypertension is an important risk factor for glaucoma. The purpose of this study was to investigate the gliotoxic effects of α-aminoadipic acid (AAA) in a rat model of AOH and its underlying mechanisms. MATERIALS AND METHODS: In the rat model of acute ocular hypertension (AOH), intraocular pressure was increased to 110 mmHg for 60 minutes. Animals were divided into four groups: sham operation (Ctrl), AOH, AOH + phosphate-buffered saline (PBS), and AOH + AAA. Cell apoptosis in the ganglion cell layer was detected with the terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling (TUNEL) assay, and retinal ganglion cells (RGCs) immunostained with Thy-1 were counted. Müller cell activation was detected using immunostaining with glutamine synthetase and glial fibrillary acidic protein. Tumor necrosis factor-α (TNF-α) was examined using Western blot. RESULTS: In the rat model of AOH, cell apoptosis was induced in the ganglion cell layer and the number of RGCs was decreased. Müller cell gliosis in the retinas of rats was induced, and retinal protein levels of TNF-α were increased. Intravitreal treatment of AAA versus PBS control attenuated these retinal abnormalities to show protective effects in the rat model of AOH. CONCLUSION: In the retinas of the rat model of AOH, AAA treatment attenuated retinal apoptosis in the ganglion cell layer and preserved the number of RGCs, likely through the attenuation of Müller cell gliosis and suppression of TNF-α induction. Our observations suggest that AAA might be a potential therapeutic target in glaucoma.

A novel method for simultaneous quantification of alpha-aminoadipic semialdehyde/piperideine-6-carboxylate and pipecolic acid in plasma and urine.

OBJECTIVES: Elevated levels of pipecolic acid (PA), α-aminoadipic semialdehyde (AASA) and its cyclic form Δ1-piperideine-6-carboxylate (P6C) are characteristic of pyridoxine dependent epilepsy (PDE), a rare disorder of inborn error of metabolism. Recent studies showed the effectiveness of dietary therapy in PDE patients and emphasized the importance of the assessment of these metabolites for monitoring treatment efficacy. The objective of this study was to develop a robust and sensitive method for simultaneous quantification of AASA-P6C and PA in plasma and urine. DESIGN AND METHODS: Plasma and urine samples were derivatized with 3N HCl in n-butanol (v/v) and injected onto ACQUITY BEH-C18 column. A gradient of water/methanol containing 0.1% formic acid was used for the chromatographic separation of AASA, P6C and PA. The analytes' concentrations were calculated using their calibration curves and the sum of AASA and P6C (AASA-P6C) was calculated. To evaluate the clinical utility of this test, samples from unaffected controls and patients with confirmed PDE were analyzed. RESULTS: The performance characteristics of the assay as well as sample stability and interferences were determined. The intra- and inter- assay CVs were ≤2.9% and ≤10.9% for AASA-P6C, and ≤3.3% and ≤12.6% for PA, respectively. Reference ranges for AASA-P6C and PA in plasma and urine were established. Comparison of values obtained from unaffected controls and PDE patients showed high clinical sensitivity and specificity of the assay. CONCLUSIONS: This novel method for the simultaneous quantification of AASA-P6C and PA in plasma and urine can be used in a clinical laboratory setting for the diagnosis and monitoring of patients with PDE.

Molecular characterization of lysR-lysXE, gcdR-gcdHG and amaR-amaAB operons for lysine export and catabolism: a comprehensive lysine catabolic network in Pseudomonas aeruginosa PAO1.

Among multiple interconnected pathways for l-Lysine catabolism in pseudomonads, it has been reported that Pseudomonas aeruginosa PAO1 employs the decarboxylase and the transaminase pathways. However, up until now, knowledge of several genes involved in operation and regulation of these pathways was still missing. Transcriptome analyses coupled with promoter activity measurements and growth phenotype analyses led us to identify new members in l-Lys and d-Lys catabolism and regulation, including gcdR-gcdHG for glutarate utilization, dpkA, amaR-amaAB and PA2035 for d-Lys catabolism, lysR-lysXE for putative l-Lys efflux and lysP for putative l-Lys uptake. The gcdHG operon encodes an acyl-CoA transferase (gcdG) and glutaryl-CoA dehydrogenase (gcdH) and is under the control of the transcriptional activator GcdR. Growth on l-Lys was enhanced in the mutants of lysX and lysE, supporting the operation of l-Lys efflux. The transcriptional activator LysR is responsible for l-Lys specific induction of lysXE and the PA4181-82 operon of unknown function. The putative operator sites of GcdR and LysR were deduced from serial deletions and comparative genomic sequence analyses, and the formation of nucleoprotein complexes was demonstrated with purified His-tagged GcdR and LysR. The amaAB operon encodes two enzymes to convert pipecolate to 2-aminoadipate. Induction of the amaAB operon by l-Lys, d-Lys and pipecolate requires a functional AmaR, supporting convergence of Lys catabolic pathways to pipecolate. Growth on pipecolate was retarded in the gcdG and gcdH mutants, suggesting the importance of glutarate in pipecolate and 2-aminoadipate utilization. Furthermore, this study indicated links in the control of interconnected networks of lysine and arginine catabolism in P. aeruginosa.

Oxidative degradation of N(ε)-fructosylamine-substituted peptides in heated aqueous systems.

Glycation, or non-enzymatic glycosylation, is a common protein modification formed by reactions between reducing sugars (i.e. aldoses and ketoses) with protein amino groups. Resulting Amadori and Heyns compounds, respectively, can be oxidatively degraded yielding a structurally heterogeneous group of advanced glycation end-products. We have studied this process in aqueous conditions at 95 °C in terms of appearing products and their formation kinetics in the presence or absence of reactive oxygen species (ROS)-generating systems (iron(II) sulfate). RP-HPLC-ESI-MS revealed 20 products, 12 of which were confirmed after synthesis by identical retention times and fragmentation patterns. These products accumulated during the incubation period of 4 h (N(ε)-carboxymethyl-, N(ε)-formyl- and N(ε)-methyl lysine) or appeared intermediately (2-aminoadipic semialdehyde, N(ε)-ethanalyl lysine). Acidic and basic amino acid residues near the glycation site and elevated ROS levels in the reaction mixture had significant effects on both product formation and degradation kinetics.

Effect of charged amino acid side chain length on lateral cross-strand interactions between carboxylate- and guanidinium-containing residues in a ß-hairpin.

ß-Sheet is one of the major protein secondary structures. Oppositely charged residues are frequently observed across neighboring strands in antiparallel sheets, suggesting the importance of cross-strand ion pairing interactions. The charged amino acids Asp, Glu, Arg, and Lys have different numbers of hydrophobic methylenes linking the charged functionality to the backbone. To investigate the effect of side chain length of guanidinium- and carboxylate-containing residues on lateral cross-strand ion pairing interactions at non-hydrogen-bonded positions, ß-hairpin peptides containing Zbb-Agx (Zbb = Asp, Glu, Aad in increasing length; Agx = Agh, Arg, Agb, Agp in decreasing length) sequence patterns were studied by NMR methods. The fraction folded population and folding energy were derived from the chemical shift deviation data. Peptides with high fraction folded populations involved charged residue side chain lengths that supported high strand propensity. Double mutant cycle analysis was used to determine the interaction energy for the potential lateral ion pairs. Minimal interaction was observed between residues with short side chains, most likely due to the diffused positive charge on the guanidinium group, which weakened cross-strand electrostatic interactions with the carboxylate side chain. Only the Aad-Arg/Agh interactions with long side chains clearly exhibited stabilizing energetics, possibly relying on hydrophobics. A survey of a non-redundant protein structure database revealed that the statistical sheet pair propensity followed the trend Asp-Arg < Glu-Arg, implying the need for matching long side chains. This suggested the need for long side chains on both guanidinium-bearing and carboxylate-bearing residues to stabilize the ß-hairpin motif.

Structural insight into amino group-carrier protein-mediated lysine biosynthesis: crystal structure of the LysZ·LysW complex from Thermus thermophilus.

In the biosynthesis of lysine by Thermus thermophilus, the metabolite α-ketoglutarate is converted to the intermediate α-aminoadipate (AAA), which is protected by the 54-amino acid acidic protein LysW. In this study, we determined the crystal structure of LysZ from T. thermophilus (TtLysZ), an amino acid kinase that catalyzes the second step in the AAA to lysine conversion, which was in a complex with LysW at a resolution of 1.85 Å. A crystal analysis coupled with isothermal titration calorimetry of the TtLysZ mutants for TtLysW revealed tight interactions between LysZ and the globular and C-terminal extension domains of the LysW protein, which were mainly attributed to electrostatic forces. These results provided structural evidence for LysW acting as a protecting molecule for the α-amino group of AAA and also as a carrier protein to guarantee better recognition by biosynthetic enzymes for the efficient biosynthesis of lysine.

Effect of side chain length on intrahelical interactions between carboxylate- and guanidinium-containing amino acids.

The charge-containing hydrophilic functionalities of encoded charged amino acids are linked to the backbone via different numbers of hydrophobic methylenes, despite the apparent electrostatic nature of protein ion pairing interactions. To investigate the effect of side chain length of guanidinium- and carboxylate-containing residues on ion pairing interactions, α-helical peptides containing Zbb-Xaa (i, i + 3), (i, i + 4) and (i, i + 5) (Zbb = carboxylate-containing residues Aad, Glu, Asp in decreasing length; Xaa = guanidinium residues Agh, Arg, Agb, Agp in decreasing length) sequence patterns were studied by circular dichroism spectroscopy (CD). The helicity of Aad- and Glu-containing peptides was similar and mostly pH independent, whereas the helicity of Asp-containing peptides was mostly pH dependent. Furthermore, the Arg-containing peptides consistently exhibited higher helicity compared to the corresponding Agp-, Agb-, and Agh-containing peptides. Side chain conformational analysis by molecular mechanics calculations showed that the Zbb-Xaa (i, i + 3) and (i, i + 4) interactions mainly involved the χ 1 dihedral combinations (g+, g+) and (g-, g+), respectively. These low energy conformations were also observed in intrahelical Asp-Arg and Glu-Arg salt bridges of natural proteins. Accordingly, Asp and Glu provides variation in helix characteristics associated with Arg, but Aad does not provide features beyond those already delivered by Glu. Importantly, nature may have chosen the side chain length of Arg to support helical conformations through inherent high helix propensity coupled with stabilizing intrahelical ion pairing interactions with the carboxylate-containing residues.

Steady-state kinetic mechanism of LodA, a novel cysteine tryptophylquinone-dependent oxidase.

LodA is a novel lysine-ε-oxidase which possesses a cysteine tryptophylquinone cofactor. It is the first tryptophylquinone enzyme known to function as an oxidase. A steady-state kinetic analysis shows that LodA obeys a ping-pong kinetic mechanism with values of kcat of 0.22±0.04 s(-1), Klysine of 3.2±0.5 µM and KO2 of 37.2±6.1 µM. The kcat exhibited a pH optimum at 7.5 while kcat/Klysine peaked at 7.0 and remained constant to pH 8.5. Alternative electron acceptors could not effectively substitute for O2 in the reaction. A mechanism for the reductive half reaction of LodA is proposed that is consistent with the ping-pong kinetics.

Temperature of frozen storage affects the nature and consequences of protein oxidation in beef patties.

The effect of three frozen storage temperatures (-8, -18 and -80 °C) on protein oxidation in beef patties was studied through the analysis of novel oxidation markers. Additionally, the connection between lipid and protein oxidation and the impact of the latter on particular quality traits (water holding capacity, color and texture) of subsequently processed beef patties (cooking/cold-stored) were investigated. Protein oxidation was measured as the loss of tryptophan fluorescence and formation of diverse lysine oxidation products (α-aminoadipic semialdehyde, α-aminoadipic acid and Schiff bases). Lipid oxidation was assessed by levels of thiobarbituric acid reactive substances and hexanal. A significant effect of storage temperature on protein oxidation was detected. Frozen storage increased the susceptibility of meat proteins to undergo further oxidation during processing. Timely interactions were found between lipid and protein oxidation. Plausible mechanisms by which oxidative damage to proteins may have an impact in particular quality traits are thoroughly discussed.

6-Alkynyl- and 6-aryl-substituted (R)-pipecolic acid derivatives.

(R)-α-Aminoadipic acid is a readily available enantiomerically pure starting material for the synthesis of (R)-pipecolic acid and its derivatives. Sonogashira or Suzuki cross-coupling reactions of an N-formyl pipecolate-derived vinyl bromide furnish 6-alkynyl or aryl derivatives. Reduction with sodium cyanoborohydride and subsequent N-deformylation provide 6-alkynyl substituted (R)-pipecolic acid derivatives, valuable building blocks for amino acid and peptide chemistry.

Carbonylation of myofibrillar proteins through the maillard pathway: effect of reducing sugars and reaction temperature.

Carbonylation is recognized as one of the most remarkable chemical modifications in oxidized proteins and is generally ascribed to the direct attack of free radicals to basic amino acid residues. The purpose of this work was to investigate the formation of specific carbonyls, α-aminoadipic and γ-glutamic semialdehydes (AAS and GGS, respectively), in myofibrillar proteins (MP) through a Maillard-type pathway in the presence of reducing sugars. The present study confirmed the concurrent formation of protein carbonyls and advanced glycation end-products (AGEs) during incubation (80 °C/48 h) of MP (4 mg/mL) in the presence of reducing sugars (0.5 M). Copper irons (10 µM) were found to promote the formation of protein carbonyls, and a specific inhibitor of the Maillard reaction (0.02 M pyridoxamine) blocked the carbonylation process which emphasize the occurrence of a Maillard-type pathway. The Maillard-mediated carbonylation occurred in a range of reducing sugars (0.02-0.5 M) and reaction temperatures (4-110 °C) compatible with food systems. Upcoming studies on this topic may contribute further to shed light on the complex interactions between protein oxidation and the Maillard reaction and the impact of the protein damage on food quality and human health.