Molecular MR Imaging of Renal Fibrogenesis in Mice.

BACKGROUND: In most CKDs, lysyl oxidase oxidation of collagen forms allysine side chains, which then form stable crosslinks. We hypothesized that MRI with the allysine-targeted probe Gd-oxyamine (OA) could be used to measure this process and noninvasively detect renal fibrosis. METHODS: Two mouse models were used: hereditary nephritis in Col4a3-deficient mice (Alport model) and a glomerulonephritis model, nephrotoxic nephritis (NTN). MRI measured the difference in kidney relaxation rate, ΔR1, after intravenous Gd-OA administration. Renal tissue was collected for biochemical and histological analysis. RESULTS: ΔR1 was increased in the renal cortex of NTN mice and in both the cortex and the medulla of Alport mice. Ex vivo tissue analyses showed increased collagen and Gd-OA levels in fibrotic renal tissues and a high correlation between tissue collagen and ΔR1. CONCLUSIONS: Magnetic resonance imaging using Gd-OA is potentially a valuable tool for detecting and staging renal fibrogenesis.

Engineered RecA Constructs Reveal the Minimal SOS Activation Complex.

The SOS response is a bacterial DNA damage response pathway that has been heavily implicated in bacteria's ability to evolve resistance to antibiotics. Activation of the SOS response is dependent on the interaction between two bacterial proteins, RecA and LexA. RecA acts as a DNA damage sensor by forming lengthy oligomeric filaments (RecA*) along single-stranded DNA (ssDNA) in an ATP-dependent manner. RecA* can then bind to LexA, the repressor of SOS response genes, triggering LexA degradation and leading to induction of the SOS response. Formation of the RecA*-LexA complex therefore serves as the key "SOS activation signal." Given the challenges associated with studying a complex involving multiple macromolecular interactions, the essential constituents of RecA* that allow LexA cleavage are not well defined. Here, we leverage head-to-tail linked and end-capped RecA constructs as tools to define the minimal RecA* filament that can engage LexA. In contrast to previously postulated models, we found that as few as three linked RecA units are capable of ssDNA binding, LexA binding, and LexA cleavage. We further demonstrate that RecA oligomerization alone is insufficient for LexA cleavage, with an obligate requirement for ATP and ssDNA binding to form a competent SOS activation signal with the linked constructs. Our minimal system for RecA* highlights the limitations of prior models for the SOS activation signal and offers a novel tool that can inform efforts to slow acquired antibiotic resistance by targeting the SOS response.

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.

A Janus Chelator Enables Biochemically Responsive MRI Contrast with Exceptional Dynamic Range.

We introduce a new biochemically responsive Mn-based MRI contrast agent that provides a 9-fold change in relaxivity via switching between the Mn3+ and Mn2+ oxidation states. Interchange between oxidation states is promoted by a "Janus" ligand that isomerizes between binding modes that favor Mn3+ or Mn2+. It is the only ligand that supports stable complexes of Mn3+ and Mn2+ in biological milieu. Rapid interconversion between oxidation states is mediated by peroxidase activity (oxidation) and l-cysteine (reduction). This Janus system provides a new paradigm for the design of biochemically responsive MRI contrast agents.

FRETing about the details: Case studies in the use of a genetically encoded fluorescent amino acid for distance-dependent energy transfer.

Förster resonance energy transfer (FRET) is a valuable method for monitoring protein conformation and biomolecular interactions. Intrinsically fluorescent amino acids that can be genetically encoded, such as acridonylalanine (Acd), are particularly useful for FRET studies. However, quantitative interpretation of FRET data to derive distance information requires careful use of controls and consideration of photophysical effects. Here we present two case studies illustrating how Acd can be used in FRET experiments to study small molecule induced conformational changes and multicomponent biomolecular complexes.

Generating Efficient Methanomethylophilus alvus Pyrrolysyl-tRNA Synthetases for Structurally Diverse Non-Canonical Amino Acids.

Genetic code expansion (GCE) technologies commonly use the pyrrolysyl-tRNA synthetase (PylRS)/tRNAPyl pairs from Methanosarcina mazei (Mm) and Methanosarcina barkeri (Mb) for site-specific incorporation of non-canonical amino acids (ncAAs) into proteins. Recently a homologous PylRS/tRNAPyl pair from Candidatus Methanomethylophilus alvus Mx1201 (Ma) was developed that, lacking the N-terminal tRNA-recognition domain of most PylRSs, overcomes insolubility, instability, and proteolysis issues seen with Mb/Mm PylRSs. An open question is how to alter Ma PylRS specificity to encode specific ncAAs with high efficiency. Prior work focused on "transplanting" ncAA substrate specificity by reconstructing the same active site mutations found in functional Mm/Mb PylRSs in Ma PylRS. Here, we found that this strategy produced low-efficiency Ma PylRSs for encoding three structurally diverse ncAAs: acridonyl-alanine (Acd), 3-nitro-tyrosine, and m-methyl-tetrazinyl-phenylalanine (Tet3.0-Me). On the other hand, efficient Ma PylRS variants were generated by a conventional life/death selection process from a large library of active site mutants: for Acd encoding, one variant was highly functional in HEK293T cells at just 10 µM Acd; for nitroY encoding, two variants also encoded 3-chloro, 3-bromo-, and 3-iodo-tyrosine at high efficiency; and for Tet-3.0-Me, all variants were more functional at lower ncAA concentrations. All Ma PylRS variants identified through selection had at least two different active site residues when compared with their Mb PylRS counterparts. We conclude that Ma and Mm/Mb PylRSs are sufficiently different that "active site transplantation" yields suboptimal Ma GCE systems. This work establishes a paradigm for expanding the utility of the promising Ma PylRS/tRNAPyl GCE platform.

Synthesis and characterization of fluorescent amino acid dimethylaminoacridonylalanine.

Fluorescent amino acids are powerful biophysical tools as they can be used in structural or imaging studies of a given protein without significantly perturbing its native fold or function. Here, we have synthesized and characterized 7-(dimethylamino)acridon-2-ylalanine (Dad), a red-shifted derivative of the genetically-incorporable amino acid, acridon-2-ylalanine. Alkylation increases the quantum yield and fluorescence lifetime of Dad relative to a previously published amino acid, 7-aminoacridon-2-ylalanine (Aad). These properties of Dad make it a potentially valuable protein label, and we have performed initial testing of its ability to be genetically incorporated using an evolved aminoacyl tRNA synthetase.

An improved fluorescent noncanonical amino acid for measuring conformational distributions using time-resolved transition metal ion FRET.

With the recent explosion in high-resolution protein structures, one of the next frontiers in biology is elucidating the mechanisms by which conformational rearrangements in proteins are regulated to meet the needs of cells under changing conditions. Rigorously measuring protein energetics and dynamics requires the development of new methods that can resolve structural heterogeneity and conformational distributions. We have previously developed steady-state transition metal ion fluorescence resonance energy transfer (tmFRET) approaches using a fluorescent noncanonical amino acid donor (Anap) and transition metal ion acceptor to probe conformational rearrangements in soluble and membrane proteins. Here, we show that the fluorescent noncanonical amino acid Acd has superior photophysical properties that extend its utility as a donor for tmFRET. Using maltose-binding protein (MBP) expressed in mammalian cells as a model system, we show that Acd is comparable to Anap in steady-state tmFRET experiments and that its long, single-exponential lifetime is better suited for probing conformational distributions using time-resolved FRET. These experiments reveal differences in heterogeneity in the apo and holo conformational states of MBP and produce accurate quantification of the distributions among apo and holo conformational states at subsaturating maltose concentrations. Our new approach using Acd for time-resolved tmFRET sets the stage for measuring the energetics of conformational rearrangements in soluble and membrane proteins in near-native conditions.

Genetic encoding of a highly photostable, long lifetime fluorescent amino acid for imaging in mammalian cells.

Acridonylalanine (Acd) is a fluorescent amino acid that is highly photostable, with a high quantum yield and long fluorescence lifetime in water. These properties make it superior to existing genetically encodable fluorescent amino acids for monitoring protein interactions and conformational changes through fluorescence polarization or lifetime experiments, including fluorescence lifetime imaging microscopy (FLIM). Here, we report the genetic incorporation of Acd using engineered pyrrolysine tRNA synthetase (RS) mutants that allow for efficient Acd incorporation in both E. coli and mammalian cells. We compare protein yields and amino acid specificity for these Acd RSs to identify an optimal construct. We also demonstrate the use of Acd in FLIM, where its long lifetime provides strong contrast compared to endogenous fluorophores and engineered fluorescent proteins, which have lifetimes less than 5 ns.

Quantitative, noninvasive MRI characterization of disease progression in a mouse model of non-alcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is an increasing cause of chronic liver disease characterized by steatosis, inflammation, and fibrosis which can lead to cirrhosis, hepatocellular carcinoma, and mortality. Quantitative, noninvasive methods for characterizing the pathophysiology of NASH at both the preclinical and clinical level are sorely needed. We report here a multiparametric magnetic resonance imaging (MRI) protocol with the fibrogenesis probe Gd-Hyd to characterize fibrotic disease activity and steatosis in a common mouse model of NASH. Mice were fed a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) to induce NASH with advanced fibrosis. Mice fed normal chow and CDAHFD underwent MRI after 2, 6, 10 and 14 weeks to measure liver T1, T2*, fat fraction, and dynamic T1-weighted Gd-Hyd enhanced imaging of the liver. Steatosis, inflammation, and fibrosis were then quantified by histology. NASH and fibrosis developed quickly in CDAHFD fed mice with strong correlation between morphometric steatosis quantification and liver fat estimated by MRI (r = 0.90). Sirius red histology and collagen quantification confirmed increasing fibrosis over time (r = 0.82). Though baseline T1 and T2* measurements did not correlate with fibrosis, Gd-Hyd signal enhancement provided a measure of the extent of active fibrotic disease progression and correlated strongly with lysyl oxidase expression. Gd-Hyd MRI accurately detects fibrogenesis in a mouse model of NASH with advanced fibrosis and can be combined with other MR measures, like fat imaging, to more accurately assess disease burden.

Protein labeling for FRET with methoxycoumarin and acridonylalanine.

Site-specific protein labeling can be used to monitor protein motions and interactions in real time using Förster resonance energy transfer (FRET). While there are many fluorophores available for protein labeling, few FRET pairs are suitable for monitoring intramolecular protein motions without being disruptive to protein folding and function. Here, we describe the synthesis and use of a minimally perturbing FRET pair comprised of methoxycoumarin maleimide (Mcm-Mal) and acridonylalanine (Acd). Acd can be incorporated into a protein through unnatural amino acid mutagenesis. Mcm-Mal is fluorogenic when reacted with cysteine and can label cysteine/Acd double mutant proteins. This labeling strategy provides an easy to install FRET pair with a working range or 15-40Å, making it ideal for monitoring most intramolecular motions. Additionally, Mcm/Acd FRET can be combined with tryptophan fluorescence for monitoring multiple protein motions via three color FRET.

The Kinetic and Molecular Basis for the Interaction of LexA and Activated RecA Revealed by a Fluorescent Amino Acid Probe.

The bacterial DNA damage response (the SOS response) is a key pathway involved in antibiotic evasion and a promising target for combating acquired antibiotic resistance. Activation of the SOS response is controlled by two proteins: the repressor LexA and the DNA damage sensor RecA. Following DNA damage, direct interaction between RecA and LexA leads to derepression of the SOS response. However, the exact molecular details of this interaction remain unknown. Here, we employ the fluorescent unnatural amino acid acridonylalanine (Acd) as a minimally perturbing probe of the E. coli RecA:LexA complex. Using LexA labeled with Acd, we report the first kinetic model for the reversible binding of LexA to activated RecA. We also characterize the effects that specific amino acid truncations or substitutions in LexA have on RecA:LexA binding strength and demonstrate that a mobile loop encoding LexA residues 75-84 comprises a key recognition interface for RecA. Beyond insights into SOS activation, our approach also further establishes Acd as a sensitive fluorescent probe for investigating the dynamics of protein-protein interactions in other complex systems.

Peroxidase Sensitive Amplifiable Probe for Molecular Magnetic Resonance Imaging of Pulmonary Inflammation.

An amplifiable magnetic resonance imaging (MRI) probe that combines the stability of the macrocyclic Gd-DOTAGA core with a peroxidase-reactive 5-hydroxytryptamide (5-HT) moiety is reported. The incubation of the complex under enzymatic oxidative conditions led to a 1.7-fold increase in r1 at 1.4 T that was attributed to an oligomerization of the probe upon oxidation. This probe, Gd-5-HT-DOTAGA, provided specific detection of lung inflammation by MRI in bleomycin-injured mice.

Chiral DOTA chelators as an improved platform for biomedical imaging and therapy applications.

Despite established clinical utilisation, there is an increasing need for safer, more inert gadolinium-based contrast agents, and for chelators that react rapidly with radiometals. Here we report the syntheses of a series of chiral DOTA chelators and their corresponding metal complexes and reveal properties that transcend the parent DOTA compound. We incorporated symmetrical chiral substituents around the tetraaza ring, imparting enhanced rigidity to the DOTA cavity, enabling control over the range of stereoisomers of the lanthanide complexes. The Gd chiral DOTA complexes are shown to be orders of magnitude more inert to Gd release than [GdDOTA]-. These compounds also exhibit very-fast water exchange rates in an optimal range for high field imaging. Radiolabeling studies with (Cu-64/Lu-177) also demonstrate faster labelling properties. These chiral DOTA chelators are alternative general platforms for the development of stable, high relaxivity contrast agents, and for radiometal complexes used for imaging and/or therapy.

High sensitivity HPLC method for determination of the allysine concentration in tissue by use of a naphthol derivative.

Common to all fibrotic and metastatic diseases is the uncontrollable remodeling of tissue that leads to the accumulation of fibrous connective tissue components such as collagen and elastin. Build-up of fibrous tissue occurs through the cross-linking of collagen or elastin monomers, which is initiated through the oxidation of lysine residues to form α-aminoadipic-δ-semialdehyde (allysine). To provide a measure of the extent of collagen oxidation in disease models of fibrosis or metastasis, a rapid, sensitive HPLC method was developed to quantify the amount of allysine present in tissue. Allysine was reacted with sodium 2-naphthol-7-sulfonate under conditions typically applied for acid hydrolysis of tissues (6M HCl, 110°C, 24h) to prepare AL-NP, a fluorescent bis-naphthol derivative of allysine. High performance liquid chromatography was applied for analysis of allysine content. Under optimal reaction and detection conditions, successful separation of AL-NP was achieved with excellent analytical performance attained. Good linear relationship (R2=0.994) between peak area and concentration for AL-NP was attained for 0.35-175pmol of analyte. A detection limit of 0.02pmol in the standard sample with a 20µL injection was achieved for AL-NP, with satisfactory recovery from 88 to 100% determined. The method was applied in the quantification of allysine in healthy and fibrotic mouse lung tissue, with the fibrotic tissue showing a 2.5 fold increase in the content of allysine.

Molecular imaging of oxidized collagen quantifies pulmonary and hepatic fibrogenesis.

Fibrosis results from the dysregulation of tissue repair mechanisms affecting major organ systems, leading to chronic extracellular matrix buildup, and progressive, often fatal, organ failure. Current diagnosis relies on invasive biopsies. Noninvasive methods today cannot distinguish actively progressive fibrogenesis from stable scar, and thus are insensitive for monitoring disease activity or therapeutic responses. Collagen oxidation is a universal signature of active fibrogenesis that precedes collagen crosslinking. Biochemically targeting oxidized lysine residues formed by the action of lysyl oxidase on collagen with a small-molecule gadolinium chelate enables targeted molecular magnetic resonance imaging. This noninvasive direct biochemical elucidation of the fibrotic microenvironment specifically and robustly detected and staged pulmonary and hepatic fibrosis progression, and monitored therapeutic response in animal models. Furthermore, this paradigm is translatable and generally applicable to diverse fibroproliferative disorders.