Ratiometric gibberellin biosensors for the analysis of signaling dynamics and metabolism in plant protoplasts.

Gibberellins (GAs) are major regulators of developmental and growth processes in plants. Using the degradation-based signaling mechanism of GAs, we have built transcriptional regulator (DELLA)-based, genetically encoded ratiometric biosensors as proxies for hormone quantification at high temporal resolution and sensitivity that allow dynamic, rapid and simple analysis in a plant cell system, i.e. Arabidopsis protoplasts. These ratiometric biosensors incorporate a DELLA protein as a degradation target fused to a firefly luciferase connected via a 2A peptide to a renilla luciferase as a co-expressed normalization element. We have implemented these biosensors for all five Arabidopsis DELLA proteins, GA-INSENSITIVE, GAI; REPRESSOR-of-ga1-3, RGA; RGA-like1, RGL1; RGL2 and RGL3, by applying a modular design. The sensors are highly sensitive (in the low pm range), specific and dynamic. As a proof of concept, we have tested the applicability in three domains: the study of substrate specificity and activity of putative GA-oxidases, the characterization of GA transporters, and the use as a discrimination platform coupled to a GA agonists' chemical screening. This work demonstrates the development of a genetically encoded quantitative biosensor complementary to existing tools that allow the visualization of GA in planta.

Study design for development of novel safety biomarkers of drug-induced liver injury by the translational safety biomarker pipeline (TransBioLine) consortium: a study protocol for a nested case-control study.

A lack of biomarkers that detect drug-induced liver injury (DILI) accurately continues to hinder early- and late-stage drug development and remains a challenge in clinical practice. The Innovative Medicines Initiative's TransBioLine consortium comprising academic and industry partners is developing a prospective repository of deeply phenotyped cases and controls with biological samples during liver injury progression to facilitate biomarker discovery, evaluation, validation and qualification.In a nested case-control design, patients who meet one of these criteria, alanine transaminase (ALT) ≥ 5 × the upper limit of normal (ULN), alkaline phosphatase ≥ 2 × ULN or ALT ≥ 3 ULN with total bilirubin > 2 × ULN, are enrolled. After completed clinical investigations, Roussel Uclaf Causality Assessment and expert panel review are used to adjudicate episodes as DILI or alternative liver diseases (acute non-DILI controls). Two blood samples are taken: at recruitment and follow-up. Sample size is as follows: 300 cases of DILI and 130 acute non-DILI controls. Additional cross-sectional cohorts (1 visit) are as follows: Healthy volunteers (n = 120), controls with chronic alcohol-related or non-alcoholic fatty liver disease (n = 100 each) and patients with psoriasis or rheumatoid arthritis (n = 100, 50 treated with methotrexate) are enrolled. Candidate biomarkers prioritised for evaluation include osteopontin, glutamate dehydrogenase, cytokeratin-18 (full length and caspase cleaved), macrophage-colony-stimulating factor 1 receptor and high mobility group protein B1 as well as bile acids, sphingolipids and microRNAs. The TransBioLine project is enabling biomarker discovery and validation that could improve detection, diagnostic accuracy and prognostication of DILI in premarketing clinical trials and for clinical healthcare application.

Plasma Sphingoid Base Profiles of Patients Diagnosed with Intrinsic or Idiosyncratic Drug-induced Liver Injury.

Sphingolipids are exceptionally diverse, comprising hundreds of unique species. The bulk of circulating sphingolipids are synthesized in the liver, thereby plasma sphingolipid profiles represent reliable surrogates of hepatic sphingolipid metabolism and content. As changes in plasma sphingolipid content have been associated to exposure to drugs inducing hepatotoxicity both in vitro and in rodents, in the present study the translatability of the preclinical data was assessed by analyzing the plasma of patients with suspected drug-induced liver injury (DILI) and control subjects. DILI patients, whether intrinsic or idiosyncratic cases, had no alterations in total sphingoid base levels and profile composition compared to controls, whereby cardiovascular disease (CVD) was a confounding factor. Upon exclusion of CVD individuals, elevation of 1-deoxysphingosine (1-deoxySO) in the DILI group emerged. Notably, 1-deoxySO values did not correlate with ALT values. While 1-deoxySO was elevated in all DILI cases, only intrinsic DILI cases concomitantly displayed reduction of select shorter chain sphingoid bases. Significant perturbation of the sphingolipid metabolism observed in this small exploratory clinical study is discussed and put into context, in the consideration that sphingolipids might contribute to the onset and progression of DILI, and that circulating sphingoid bases may function as mechanistic markers to study DILI pathophysiology.

The Farnesoid X Receptor as a Master Regulator of Hepatotoxicity.

The nuclear receptor farnesoid X receptor (FXR, NR1H4) is a bile acid (BA) sensor that links the enterohepatic circuit that regulates BA metabolism and elimination to systemic lipid homeostasis. Furthermore, FXR represents a real guardian of the hepatic function, preserving, in a multifactorial fashion, the integrity and function of hepatocytes from chronic and acute insults. This review summarizes how FXR modulates the expression of pathway-specific as well as polyspecific transporters and enzymes, thereby acting at the interface of BA, lipid and drug metabolism, and influencing the onset and progression of hepatotoxicity of varying etiopathogeneses. Furthermore, this review article provides an overview of the advances and the clinical development of FXR agonists in the treatment of liver diseases.

The role of cholesterol recognition (CARC/CRAC) mirror codes in the allosterism of the human organic cation transporter 2 (OCT2, SLC22A2).

The human organic cation transporter 2 (OCT2) is a multispecific transporter with cholesterol-dependent allosteric features. The present work elucidates the role of evolutionarily conserved cholesterol recognition/interaction amino acid consensus sequences (CRAC and CARC) in the allosteric binding to 1-methyl-4-phenylpyridinium (MPP+) in human embryonic kidney 293 cells stably or transiently expressing OCT2. Molecular blind simulations docked two mirroring cholesterol molecules in the 5th putative transmembrane domain, where a CARC and a CRAC sequence lie. The impact of the conserved amino acids that may constitute the CARC/CRAC mirror code was studied by alanine-scanning mutagenesis. At a saturating extracellular concentration of substrate, at which the impact of cholesterol depletion is maximal, five mutants transported MPP+ at a significantly lower rate than the wild-type OCT2 (WT), resembling the behavior of the WT upon cholesterol depletion. MPP+ influx rate as a function of the extracellular concentration of substrate was measured for the mutants R234A, R235A, L252A and R263A. R234A kinetic behavior was similar to that of the WT, whereas R235A, L252A and R263A activity shifted from allosteric to one-binding site kinetics, very much like the WT upon cholesterol depletion. The impact of cholesterol on protein thermal stability was assessed for WT, R234A and R263A. While the thermal stability of WT and R234A was improved by the supplementation with cholesterol, R263A was not sensitive to the presence of cholesterol. To conclude, the disruption of the CARC/CRAC mirror code in the 5th putative transmembrane domain is sufficient to abolish the allosteric interaction between OCT2 and MPP+.

A Protoplast-Based Bioassay to Quantify Strigolactone Activity in Arabidopsis Using StrigoQuant.

Understanding the biological background of strigolactone (SL) structural diversity and the SL signaling pathway at molecular level requires quantitative and sensitive tools that precisely determine SL dynamics. Such biosensors may be also very helpful in screening for SL analogs and mimics with defined biological functions.Recently, the genetically encoded, ratiometric sensor StrigoQuant was developed and allowed the quantification of the activity of a wide concentration range of SLs. StrigoQuant can be used for studies on the biosynthesis, function and signal transduction of this hormone class.Here, we provide a comprehensive protocol for establishing the use of StrigoQuant in Arabidopsis protoplasts. We first describe the generation and transformation of the protoplasts with StrigoQuant and detail the application of the synthetic SL analogue GR24. We then show the recording of the luminescence signal and how the obtained data are processed and used to assess/determine SL perception.

Organic Cation Transporters in Human Physiology, Pharmacology, and Toxicology.

Individual cells and epithelia control the chemical exchange with the surrounding environment by the fine-tuned expression, localization, and function of an array of transmembrane proteins that dictate the selective permeability of the lipid bilayer to small molecules, as actual gatekeepers to the interface with the extracellular space. Among the variety of channels, transporters, and pumps that localize to cell membrane, organic cation transporters (OCTs) are considered to be extremely relevant in the transport across the plasma membrane of the majority of the endogenous substances and drugs that are positively charged near or at physiological pH. In humans, the following six organic cation transporters have been characterized in regards to their respective substrates, all belonging to the solute carrier 22 (SLC22) family: the organic cation transporters 1, 2, and 3 (OCT1-3); the organic cation/carnitine transporter novel 1 and 2 (OCTN1 and N2); and the organic cation transporter 6 (OCT6). OCTs are highly expressed on the plasma membrane of polarized epithelia, thus, playing a key role in intestinal absorption and renal reabsorption of nutrients (e.g., choline and carnitine), in the elimination of waste products (e.g., trimethylamine and trimethylamine N-oxide), and in the kinetic profile and therapeutic index of several drugs (e.g., metformin and platinum derivatives). As part of the Special Issue Physiology, Biochemistry, and Pharmacology of Transporters for Organic Cations, this article critically presents the physio-pathological, pharmacological, and toxicological roles of OCTs in the tissues in which they are primarily expressed.

Untargeted Metabolomics Reveals Anaerobic Glycolysis as a Novel Target of the Hepatotoxic Antidepressant Nefazodone.

Mitochondrial damage is considered a hallmark of drug-induced liver injury (DILI). However, despite the common molecular etiology, the evolution of the injury is usually unpredictable, with some cases that are mild and reversible upon discontinuation of the treatment and others characterized by irreversible acute liver failure. This suggests that additional mechanisms of damage play a role in determining the progression of the initial insult. To uncover novel pathways potentially involved in DILI, we investigated in vitro the metabolic perturbations associated with nefazodone, an antidepressant associated with acute liver failure. Several pathways associated with ATP production, including gluconeogenesis, anaerobic glycolysis, and oxidative phosphorylation, were altered in human hepatocellular carcinoma-derived (Huh7) cells after 2-hour exposure to a 50 µM extracellular concentration of nefazodone. In the presence or absence of glucose, ATP production of Huh7 cells was glycolysis- and oxidative phosphorylation-dependent, respectively. In glucose-containing medium, nefazodone-induced ATP depletion from Huh7 cells was biphasic. Huh7 cells in glucose-free medium were more sensitive to nefazodone than those in glucose-containing medium, losing the biphasic inhibition. Nefazodone-induced ATP depletion in primary cultured mouse hepatocytes, mainly dependent on oxidative phosphorylation, was monophasic. At lower extracellular concentrations, nefazodone inhibited the oxygen consumption of Huh7 cells, whereas at higher extracellular concentrations, it also inhibited the extracellular acidification. ATP content was rescued by increasing the extracellular concentration of glucose. In conclusion, nefazodone has a dual inhibitory effect on mitochondrial-dependent and mitochondrial-independent ATP production. SIGNIFICANCE STATEMENT: Mitochondrial damage is a hallmark of drug-induced liver injury, yet other collateral alterations might contribute to the severity and evolution of the injury. Our in vitro study supports previous results arguing that a deficit in hepatic glucose metabolism, concomitant to the mitochondrial injury, might be cardinal in the prognosis of the initial insult to the liver. From a drug development standpoint, coupling anaerobic glycolysis and mitochondrial function assessment might increase the drug-induced liver injury preclinical screening performance.

Flexibility of intrinsically disordered degrons in AUX/IAA proteins reinforces auxin co-receptor assemblies.

Cullin RING-type E3 ubiquitin ligases SCFTIR1/AFB1-5 and their AUX/IAA targets perceive the phytohormone auxin. The F-box protein TIR1 binds a surface-exposed degron in AUX/IAAs promoting their ubiquitylation and rapid auxin-regulated proteasomal degradation. Here, by adopting biochemical, structural proteomics and in vivo approaches we unveil how flexibility in AUX/IAAs and regions in TIR1 affect their conformational ensemble allowing surface accessibility of degrons. We resolve TIR1·auxin·IAA7 and TIR1·auxin·IAA12 complex topology, and show that flexible intrinsically disordered regions (IDRs) in the degron's vicinity, cooperatively position AUX/IAAs on TIR1. We identify essential residues at the TIR1 N- and C-termini, which provide non-native interaction interfaces with IDRs and the folded PB1 domain of AUX/IAAs. We thereby establish a role for IDRs in modulating auxin receptor assemblies. By securing AUX/IAAs on two opposite surfaces of TIR1, IDR diversity supports locally tailored positioning for targeted ubiquitylation, and might provide conformational flexibility for a multiplicity of functional states.

Obeticholic Acid Ameliorates Valproic Acid-Induced Hepatic Steatosis and Oxidative Stress.

Farnesoid X receptor (FXR), or NR1H4, protects the liver from insults of various etiologies. A role of FXR in drug-induced liver injury has also been hypothesized yet only marginally investigated. The aim of this study was to assess the effect of FXR activation on gene expression and phenotype of the liver of mice treated with valproic acid (VPA), or 2-propylpentanoic acid, a prototypical hepatotoxic drug. Obeticholic acid (OCA) was used to activate FXR both in mice and in human hepatocellular carcinoma (Huh-7) cells. Next-generation sequencing of mouse liver tissues was performed from control, VPA, and VPA + OCA-treated mice. Pathway analysis validation was performed using real-time reverse-transcription polymerase chain reaction, Western blotting, immunohistochemistry, and fluorometric assays. FXR activation induced antioxidative pathways, which was confirmed by a marked reduction in VPA-induced lipid peroxidation and endoplasmic reticulum stress. In vitro, VPA-induced oxidative stress was independent of lipid accumulation, stemmed from the cytoplasm, and was mitigated by OCA. In the liver of the mice treated with OCA, the levels of cytochrome P450 potentially involved in VPA metabolism were increased. The hepatic lipid-lowering effect observed in animals cotreated with VPA and OCA in comparison with that of animals treated with VPA was associated with regulation of the genes involved in the steatogenic nuclear receptor peroxisome proliferator-activated γ (PPARγ) pathway. In conclusion, pronounced antioxidant activity, repression of the PPARγ pathway, and higher expression of P450 enzymes involved in VPA metabolism may underlie the hepatoprotective of FXR activation during VPA treatment. SIGNIFICANCE STATEMENT: Valproic acid-induced oxidative stress occurs in absence of lipid accumulation and is not of mitochondrial origin. Valproic acid exposure induces the expression of the steatogenic nuclear receptor peroxisome proliferator-activated γ (PPARγ) and its downstream target genes. Constitutive activation of the farnesoid X receptor (FXR) reduces PPARγ hepatic expression and induces hepatic antioxidant activity. The variability in FXR expression level/activity, for instance in individuals carrying loss-of-function genetic variants of the FXR gene, could contribute to valproic acid pharmacokinetic and toxicokinetic profile.

Renal Reabsorption of Folates: Pharmacological and Toxicological Snapshots.

Folates are water-soluble B9 vitamins that serve as one-carbon donors in the de novo synthesis of thymidylate and purines, and in the conversion of homocysteine to methionine. Due to their key roles in nucleic acid synthesis and in DNA methylation, inhibiting the folate pathway is still one of the most efficient approaches for the treatment of several tumors. Methotrexate and pemetrexed are the most prescribed antifolates and are mainly used in the treatment of acute myeloid leukemia, osteosarcoma, and lung cancers. Normal levels of folates in the blood are maintained not only by proper dietary intake and intestinal absorption, but also by an efficient renal reabsorption that seems to be primarily mediated by the glycosylphosphatidylinositol- (GPI) anchored protein folate receptor α (FRα), which is highly expressed at the brush-border membrane of proximal tubule cells. Folate deficiency due to malnutrition, impaired intestinal absorption or increased urinary elimination is associated with severe hematological and neurological deficits. This review describes the role of the kidneys in folate homeostasis, the molecular basis of folate handling by the kidneys, and the use of high dose folic acid as a model of acute kidney injury. Finally, we provide an overview on the development of folate-based compounds and their possible therapeutic potential and toxicological ramifications.

Renal glycosuria as a novel early sign of colistin-induced kidney damage in mice.

The polymixin colistin represents a last resort antibiotic for multidrug resistant infections, but its use is limited by the frequent onset of acute drug-induced kidney injury (DIKI). It is essential to closely monitor kidney function prior to and during colistin treatment in order to pinpoint early signs of injury and minimise long-term renal dysfunction. To facilitate this, a mouse model of colistin-induced nephrotoxicity was used to uncover novel early markers of colistin-induced DIKI. Increased urinary levels of kidney injury molecule 1 (Kim-1) as well as glycosuria were observed in colistin-treated mice, where alterations of established clinical markers of acute kidney injury (serum creatinine and albuminuria) and emerging markers such as cystatin C were inaccurate in flagging renal damage as confirmed by histology. A direct interaction of colistin with renal glucose reabsorption was ruled out by a cis-inhibition assay in mouse brush border membrane vesicles (BBMV). Immunohistochemical examination and protein quantification by western blotting showed a marked reduction in the protein amount of sodium-glucose transporter 2 (Sglt2), the main kidney glucose transporter, in renal tissue from colistin-treated mice in comparison to control animals. Consistently, BBMV isolated from treated mouse kidneys also showed a reduction in ex vivo glucose uptake when compared to BBMV isolated from control kidneys. These findings support pathology observations of colistin-induced proximal tubule damage at the site of the brush border membrane, where Sglt2 is expressed, and open avenues for the study of glycosuria as a sensitive, specific, and accessible marker of DIKI during colistin therapy.

Molecular Mechanisms of Colistin-Induced Nephrotoxicity.

The emergence of multidrug resistant (MDR) infections and the shortage of new therapeutic options have made colistin, a polymyxin antibiotic, the main option for the treatment of MDR Gram-negative bacterial infections in the last decade. However, the rapid onset of renal damage often prevents the achievement of optimal therapeutic doses and/or forces the physicians to interrupt the therapy, increasing the risk of drug resistance. The proper management of colistin-induced nephrotoxicity remains challenging, mostly because the investigation of the cellular and molecular pharmacology of this drug, off the market for decades, has been largely neglected. For years, the renal damage induced by colistin was considered a mere consequence of the detergent activity of this drug on the cell membrane of proximal tubule cells. Lately, it has been proposed that the intracellular accumulation is a precondition for colistin-mediated renal damage, and that mitochondria might be a primary site of damage. Antioxidant approaches (e.g., ascorbic acid) have shown promising results in protecting the kidney of rodents exposed to colistin, yet none of these strategies have yet reached the bedside. Here we provide a critical overview of the possible mechanisms that may contribute to colistin-induced renal damage and the potential protective strategies under investigation.

Auxin methylation is required for differential growth in Arabidopsis.

Asymmetric auxin distribution is instrumental for the differential growth that causes organ bending on tropic stimuli and curvatures during plant development. Local differences in auxin concentrations are achieved mainly by polarized cellular distribution of PIN auxin transporters, but whether other mechanisms involving auxin homeostasis are also relevant for the formation of auxin gradients is not clear. Here we show that auxin methylation is required for asymmetric auxin distribution across the hypocotyl, particularly during its response to gravity. We found that loss-of-function mutants in Arabidopsis IAA CARBOXYL METHYLTRANSFERASE1 (IAMT1) prematurely unfold the apical hook, and that their hypocotyls are impaired in gravitropic reorientation. This defect is linked to an auxin-dependent increase in PIN gene expression, leading to an increased polar auxin transport and lack of asymmetric distribution of PIN3 in the iamt1 mutant. Gravitropic reorientation in the iamt1 mutant could be restored with either endodermis-specific expression of IAMT1 or partial inhibition of polar auxin transport, which also results in normal PIN gene expression levels. We propose that IAA methylation is necessary in gravity-sensing cells to restrict polar auxin transport within the range of auxin levels that allow for differential responses.

Quantitatively Understanding Plant Signaling: Novel Theoretical-Experimental Approaches.

With the need to respond to and integrate a multitude of external and internal stimuli, plant signaling is highly complex, exhibiting signaling component redundancy and high interconnectedness between individual pathways. We review here novel theoretical-experimental approaches in manipulating plant signaling towards the goal of a comprehensive understanding and targeted quantitative control of plant processes. We highlight approaches taken in the field of synthetic biology used in other systems and discuss their applicability in plants. Finally, we introduce existing tools for the quantitative analysis and monitoring of plant signaling and the integration of experimentally obtained quantitative data into mathematical models. Incorporating principles of synthetic biology into plant sciences more widely will lead this field forward in both fundamental and applied research.

Variation in auxin sensing guides AUX/IAA transcriptional repressor ubiquitylation and destruction.

Auxin is a small molecule morphogen that bridges SCFTIR1/AFB-AUX/IAA co-receptor interactions leading to ubiquitylation and proteasome-dependent degradation of AUX/IAA transcriptional repressors. Here, we systematically dissect auxin sensing by SCFTIR1-IAA6 and SCFTIR1-IAA19 co-receptor complexes, and assess IAA6/IAA19 ubiquitylation in vitro and IAA6/IAA19 degradation in vivo. We show that TIR1-IAA19 and TIR1-IAA6 have distinct auxin affinities that correlate with ubiquitylation and turnover dynamics of the AUX/IAA. We establish a system to track AUX/IAA ubiquitylation in IAA6 and IAA19 in vitro and show that it occurs in flexible hotspots in degron-flanking regions adorned with specific Lys residues. We propose that this signature is exploited during auxin-mediated SCFTIR1-AUX/IAA interactions. We present evidence for an evolving AUX/IAA repertoire, typified by the IAA6/IAA19 ohnologues, that discriminates the range of auxin concentrations found in plants. We postulate that the intrinsic flexibility of AUX/IAAs might bias their ubiquitylation and destruction kinetics enabling specific auxin responses.

StrigoQuant: A genetically encoded biosensor for quantifying strigolactone activity and specificity.

Strigolactones are key regulators of plant development and interaction with symbiotic fungi; however, quantitative tools for strigolactone signaling analysis are lacking. We introduce a genetically encoded hormone biosensor used to analyze strigolactone-mediated processes, including the study of the components involved in the hormone perception/signaling complex and the structural specificity and sensitivity of natural and synthetic strigolactones in Arabidopsis, providing quantitative insights into the stereoselectivity of strigolactone perception. Given the high specificity, sensitivity, dynamic range of activity, modular construction, ease of implementation, and wide applicability, the biosensor StrigoQuant will be useful in unraveling multiple levels of strigolactone metabolic and signaling networks.

Unearthing the transition rates between photoreceptor conformers.

BACKGROUND: Obtaining accurate estimates of biological or enzymatic reaction rates is critical in understanding the design principles of a network and how biological processes can be experimentally manipulated on demand. In many cases experimental limitations mean that some enzymatic rates cannot be measured directly, requiring mathematical algorithms to estimate them. Here, we describe a methodology that calculates rates at which light-regulated proteins switch between conformational states. We focus our analysis on the phytochrome family of photoreceptors found in cyanobacteria, plants and many optogenetic tools. Phytochrome proteins change between active (P A ) and inactive (P I ) states at rates that are proportional to photoconversion cross-sections and influenced by light quality, light intensity, thermal reactions and dimerisation. This work presents a method that can accurately calculate these photoconversion cross-sections in the presence of multiple non-light regulated reactions. RESULTS: Our approach to calculating the photoconversion cross-sections comprises three steps: i) calculate the thermal reversion reaction rate(s); ii) develop search spaces from which all possible sets of photoconversion cross-sections exist, and; iii) estimate extinction coefficients that describe our absorption spectra. We confirm that the presented approach yields accurate results through the use of simulated test cases. Our test cases were further expanded to more realistic scenarios where noise, multiple thermal reactions and dimerisation are considered. Finally, we present the photoconversion cross-sections of an Arabidopsis phyB N-terminal fragment commonly used in optogenetic tools. CONCLUSIONS: The calculation of photoconversion cross-sections has implications for both photoreceptor and synthetic biologists. Our method allows, for the first time, direct comparisons of photoconversion cross-sections and response speeds of photoreceptors in different cellular environments and synthetic tools. Due to the generality of our procedure, as shown by the application to multiple test cases, the photoconversion cross-sections and quantum yields of any photoreceptor might now, in principle, be obtained.