Steady-state Turnover Research Articles

Second-Order Biomimicry: In Situ Oxidative Self-Processing Converts Copper(I)/Diamine Precursor into a Highly Active Aerobic Oxidation Catalyst.

A homogeneous Cu-based catalyst system consisting of [Cu(MeCN)4]PF6, N,N′-di-tert-butylethylenediamine (DBED), and p-(N,N-dimethylamino)pyridine (DMAP) mediates efficient aerobic oxidation of alcohols. Mechanistic study of this reaction shows that the catalyst undergoes an in situ oxidative self-processing step, resulting in conversion of DBED into a nitroxyl that serves as an efficient cocatalyst for aerobic alcohol oxidation. Insights into this behavior are gained from kinetic studies, which reveal an induction period at the beginning of the reaction that correlates with the oxidative self-processing step, EPR spectroscopic analysis of the catalytic reaction mixture, which shows the buildup of the organic nitroxyl species during steady state turnover, and independent synthesis of oxygenated DBED derivatives, which are shown to serve as effective cocatalysts and eliminate the induction period in the reaction. The overall mechanism bears considerable resemblance to enzymatic reactivity. Most notable is the “oxygenase”-type self-processing step that mirrors generation of catalytic cofactors in enzymes via post-translational modification of amino acid side chains. This higher-order function within a synthetic catalyst system presents new opportunities for the discovery and development of biomimetic catalysts.

Macrophages and skeletal health.

Bone is in a constant state of remodeling, a process which was once attributed solely to osteoblasts and osteoclasts. Decades of research has identified many other populations of cells in the bone that participate and mediate skeletal homeostasis. Recently, osteal macrophages emerged as vital participants in skeletal remodeling and osseous repair. The exact mechanistic roles of these tissue-resident macrophages are currently under investigation. Macrophages are highly plastic in response to their micro-environment and are typically classified as being pro- or anti-inflammatory (pro-resolving) in nature. Given that inflammatory states result in decreased bone mass, proinflammatory macrophages may be negative regulators of bone turnover. Pro-resolving macrophages have been shown to release anabolic factors and may present a target for therapeutic intervention in inflammation-induced bone loss and fracture healing. The process of apoptotic cell clearance, termed efferocytosis, is mediated by pro-resolving macrophages and may contribute to steady-state bone turnover as well as fracture healing and anabolic effects of osteoporosis therapies. Parathyroid hormone is an anabolic agent in bone that is more effective in the presence of mature phagocytic macrophages, further supporting the hypothesis that efferocytic macrophages are positive contributors to bone turnover. Therapies which alter macrophage plasticity in tissues other than bone should be explored for their potential to treat bone loss either alone or in conjunction with current bone therapeutics. A better understanding of the exact mechanisms by which macrophages mediate bone homeostasis will lead to an expansion of pharmacologic targets for the treatment of osteoporosis and inflammation-induced bone loss.

Myosins Active Site Closure via Switch-1 Allosterically Regulates the Motor-Actin Interaction during the ATPase Cycle for Productive Force Generation

Myosins are essential for many cellular processes including cell motility and muscle contraction. These motors have active site motifs (P-loop, switch-1, switch-2) that bind ATP and a divalent metal cofactor to coordinate ATP hydrolysis. ATP hydrolysis is activated through allosteric communication between the ATP and polymer binding sites to couple the free energy of ATP hydrolysis to force generation. Our lab seeks to understand how changes in enzymatic properties leads to different mechanical properties in molecular motors. We hypothesize that myosins depend on active site closure via switch-1 to regulate the motor-actin interaction during the ATPase cycle for productive force generation. We developed a strategy to control enzymatic activity and motility of myosin by substituting a switch-1 serine with cysteine, diminishing its interaction with the Mg+2 metal thus inhibiting ATPase activity. Substituting Mg+2 with Mn+2, which strongly interacts with cysteine, restores metal interaction and ATPase activity. Thus swapping of the divalent metals provides a direct and experimentally reversible link between switch-1 and the actin binding cleft. We have shown that the S237C mutant of non-muscle myosin II has rapid basal steady-state ATP turnover, but upon binding actin, its MgATPase is inhibited. Mn+2 relieves this inhibition and restores ATP turnover near WT values. The acto•S237C complex has slow, rate limiting MgATP binding which is rescued to near WT activity in the presence of Mn+2. Mant-ADP release experiments show the need for switch-1 interaction with the metal for tight ADP binding. Finally, to test whether this allosteric communication is necessary for force generation, we will test S237C motility in the presence of Mg2+ and Mn2+. These results support a strong reciprocal coupling of nucleotide and F-actin binding in myosin.

Reduction of quinones and nitroaromatic compounds by Escherichia coli nitroreductase A (NfsA): Characterization of kinetics and substrate specificity

NfsA, a major FMN-associated nitroreductase of E. coli, reduces nitroaromatic compounds via consecutive two-electron transfers. NfsA has potential applications in the biodegradation of nitroaromatic environment pollutants, e.g. explosives, and is also of interest for the anticancer strategy gene-directed enzyme prodrug therapy. However, the catalytic mechanism of NfsA is poorly characterized. Here we examined the NADPH-dependent reduction of quinones (n = 16) and nitroaromatic compounds (n = 12) by NfsA. We confirmed a general “ping-pong” reaction scheme, and preliminary rapid reaction studies of the enzyme reduction by NADPH showed that this step is much faster than the steady-state turnover number, i.e., the enzyme turnover is limited by the oxidative half-reaction. The reactivity of nitroaromatic compounds (log kcat/Km) followed a linear dependence on their single-electron reduction potential (E17), indicating a limited role for compound structure or active site flexibility in their reactivity. The reactivity of quinones was lower than that of nitroaromatics having similar E17 values, except for the significantly enhanced reactivity of 2-OH-1,4-naphthoquinones, consistent with observations previously made for the group B nitroreductase of Enterobacter cloacae. We present evidence that the reduction of quinones by NfsA is most consistent with a single-step (H−) hydride transfer mechanism.

Distinct protein and mRNA kinetics of skeletal muscle proton transporters following exercise can influence interpretation of adaptations to training.

What is the central question of this study? Following a training intervention, how is the interpretation of adaptations in skeletal muscle H+ transporters influenced by biopsy timing in the context of individual protein and mRNA kinetics after the final exercise bout? What is the main finding and its importance? We show that distinct postexercise protein and mRNA kinetics for monocarboxylate transporter1/4 and sodium-hydrogen exchanger1 indicate that timing of a single end-point biopsy after a training intervention can influence the inferences made. Furthermore, we found the intrasubject, intersample variability of the muscle buffer capacity titration assay to be greater than the typical training effect. In order to gain a better understanding of training-induced adaptations in skeletal muscle pH regulation, in this study we measured protein and mRNA kinetics of proton (H+ ) transporters for 72h following a bout of high-intensity interval exercise (HIIE), conducted after 4 weeks of similar training. We also assayed muscle buffer capacity (βm) by a titration technique (βmin vitro ) over the same period. Sixteen active men cycled for seven bouts of 2min at ∼80% of peak aerobic power, interspersed with 1min rest. Compared with the first 9h postexercise, monocarboxylate transporter (MCT)1 protein content was ∼1.3-fold greater 24-72h post-HIIE, whereas there was no such change in MCT4 protein content. Conversely, MCT1 and MCT4 mRNA expression progressively decreased 9-72h post-HIIE. Sodium-hydrogen exchanger1 (NHE1) protein content was lower 9h post-HIIE (∼0.8-fold) compared with every other postexercise time point, but NHE1 mRNA expression was 2.2 to 2.9-fold greater 24-72h post-HIIE, compared with the first 24h post-HIIE. Furthermore, we determined the intrasubject, intersample variability (11.5%) of βmin vitro for resting samples taken on consecutive days to be greater than the typical training effect (mean 6%; 95% confidence limits ±4%). In conclusion, the delay in steady-state protein turnover should inform biopsy timing in studies investigating the response to training of the H+ transport proteins, whereas the temporal resolution provided by single time points has been shown to be of limited epistemological value for their corresponding mRNA expression. Finally, our data cast doubt on the ecological validity of the βmin vitro assay for measuring true changes in βm.

Overall energy conversion efficiency of a photosynthetic vesicle.

The chromatophore of purple bacteria is an intracellular spherical vesicle that exists in numerous copies in the cell and that efficiently converts sunlight into ATP synthesis, operating typically under low light conditions. Building on an atomic-level structural model of a low-light-adapted chromatophore vesicle from Rhodobacter sphaeroides, we investigate the cooperation between more than a hundred protein complexes in the vesicle. The steady-state ATP production rate as a function of incident light intensity is determined after identifying quinol turnover at the cytochrome bc1 complex (cytb⁢c1) as rate limiting and assuming that the quinone/quinol pool of about 900 molecules acts in a quasi-stationary state. For an illumination condition equivalent to 1% of full sunlight, the vesicle exhibits an ATP production rate of 82 ATP molecules/s. The energy conversion efficiency of ATP synthesis at illuminations corresponding to 1%-5% of full sunlight is calculated to be 0.12-0.04, respectively. The vesicle stoichiometry, evolutionarily adapted to the low light intensities in the habitat of purple bacteria, is suboptimal for steady-state ATP turnover for the benefit of protection against over-illumination.

ChemInform Abstract: Unlocking the Potential of Supported Liquid Phase Catalysts with Supercritical Fluids: Low Temperature Continuous Flow Catalysis with Integrated Product Separation

AbstractReview: 198 refs.

NET Confusion.

Neutrophils are arguably the most important white blood cell for defense against bacterial and fungal infections. These leukocytes are produced in high numbers on a daily basis in humans and are recruited rapidly to injured/infected tissues. Phagocytosis and subsequent intraphagosomal killing and digestion of microbes have historically been the accepted means by which neutrophils carry out their role in innate host defense. Indeed, neutrophils contain and produce numerous cytotoxic molecules, including antimicrobial peptides, proteases, and reactive oxygen species, that are highly effective at killing the vast majority of ingested microbes. On the other hand, it is these characteristics – high numbers and toxicity – that endow neutrophils with the potential to injure and destroy host tissues. This potential is borne out by many inflammatory processes and diseases. Therefore, it is not surprising that host mechanisms exist to control virtually all steps in the neutrophil activation process and to prevent unintended neutrophil activation and/or lysis during the resolution of inflammatory responses or during steady-state turnover. The notion that neutrophil extracellular traps (NETs) form by cytolysis as a standard host defense mechanism seems inconsistent with these aforementioned neutrophil “containment” processes. It is with this caveat in mind that we provide perspective on the role of NETs in human host defense and disease.

Stem Cells and Lung Regeneration.

Background:Tissues such as the lung, liver, and pancreas that have a low steady-state cell turnover yet can respond robustly after injury to replace damaged cells. The airway epithelium is exposed to inhaled particles and pathogens that may lead to the development of a many infectious and inflammatory respiratory diseases. Lung transplantation is an accepted modality of treatment for end-stage lung diseases. Since the early 1990 s, more than 26,000 lung transplants have been performed at centers worldwide. However, the availability of donor tissues and organs is limited, which presents a serious limitation for widespread transplantation surgery. The appearance of bioengineered lung and tracheal tissue transplants is considered a promising alternative to the classical transplantation of donor organ/tissue. Stem cells therapy arises as a new therapeutic approach, with a wide application potential.

Human naïve regulatory T-cells feature high steady-state turnover and are maintained by IL-7.

Naïve FoxP3-expressing regulatory T-cells (Tregs) are essential to control immune responses via continuous replenishment of the activated-Treg pool with thymus-committed suppressor cells. The mechanisms underlying naïve-Treg maintenance throughout life in face of the age-associated thymic involution remain unclear. We found that in adults thymectomized early in infancy the naïve-Treg pool is remarkably well preserved, in contrast to conventional naïve CD4 T-cells. Naïve-Tregs featured high levels of cycling and pro-survival markers, even in healthy individuals, and contrasted with other circulating naïve/memory CD4 T-cell subsets in terms of their strong γc-cytokine-dependent signaling, particularly in response to IL-7. Accordingly, ex-vivo stimulation of naïve-Tregs with IL-7 induced robust cytokine-dependent signaling, Bcl-2 expression, and phosphatidylinositol 3-kinase (PI3K)-dependent proliferation, whilst preserving naïve phenotype and suppressive capacity. Altogether, our data strongly implicate IL-7 in the thymus-independent long-term survival of functional naïve-Tregs, and highlight the potential of targeting the IL-7 pathway to modulate Tregs in different clinical settings.

Evidence of Negative Cooperativity and Half-Site Reactivity within an F420-Dependent Enzyme: Kinetic Analysis of F420H2:NADP(+) Oxidoreductase.

Here, we report the very first example of half-site reactivity and negative cooperativity involving an important F420 cofactor-dependent enzyme. F420H2:NADP(+) oxidoreductase (Fno) is an F420 cofactor-dependent enzyme that catalyzes the reversible reduction of NADP(+) through the transfer of a hydride from the reduced F420 cofactor. These catalytic processes are of major significance in numerous biochemical processes. While the steady-state kinetic analysis showed classic Michaelis-Menten kinetics with varying concentrations of the F420 redox moiety, FO, such plots revealed non-Michaelis-Menten kinetic behavior when NADPH was varied. The double reciprocal plot of the varying concentrations of NADPH displays a downward concave shape, suggesting that negative cooperativity occurs between the two identical monomers. The transient state kinetic data show a burst prior to entering steady-state turnover. The burst suggests that product release is rate-limiting, and the amplitude of the burst phase corresponds to production of product in only one of the active sites of the functional dimer. These results suggest either half-site reactivity or an alternate sites model wherein the reduction of the cofactor, FO occurs at one active site at a time followed by reduction at the second active site. Thus, the data imply that Fno may be a functional regulatory enzyme.

Mechanism of Copper/Azodicarboxylate-Catalyzed Aerobic Alcohol Oxidation: Evidence for Uncooperative Catalysis.

Cooperative catalysis between Cu(II) and redox-active organic cocatalysts is a key feature of important chemical and enzymatic aerobic oxidation reactions, such as alcohol oxidation mediated by Cu/TEMPO and galactose oxidase. Nearly 20 years ago, Markó and co-workers reported that azodicarboxylates, such as di-tert-butyl azodicarboxylate (DBAD), are effective redox-active cocatalysts in Cu-catalyzed aerobic alcohol oxidation reactions [Markó, I. E., et al. Science 1996, 274, 2044], but the nature of the cooperativity between Cu and azodicarboxylates is not well understood. Here, we report a mechanistic study of Cu/DBAD-catalyzed aerobic alcohol oxidation. In situ infrared spectroscopic studies reveal a burst of product formation prior to steady-state catalysis, and gas-uptake measurements show that no O2 is consumed during the burst. Kinetic studies reveal that the anaerobic burst and steady-state turnover have different rate laws. The steady-state rate does not depend on [O2] or [DBAD]. These results, together with other EPR and in situ IR spectroscopic and kinetic isotope effect studies, reveal that the steady-state mechanism consists of two interdependent catalytic cycles that operate in sequence: a fast Cu(II)/DBAD pathway, in which DBAD serves as the oxidant, and a slow Cu(II)-only pathway, in which Cu(II) is the oxidant. This study provides significant insight into the redox cooperativity, or lack thereof, between Cu and redox-active organic cocatalysts in aerobic oxidation reactions.

Unlocking the potential of supported liquid phase catalysts with supercritical fluids: low temperature continuous flow catalysis with integrated product separation.

Solution-phase catalysis using molecular transition metal complexes is an extremely powerful tool for chemical synthesis and a key technology for sustainable manufacturing. However, as the reaction complexity and thermal sensitivity of the catalytic system increase, engineering challenges associated with product separation and catalyst recovery can override the value of the product. This persistent downstream issue often renders industrial exploitation of homogeneous catalysis uneconomical despite impressive batch performance of the catalyst. In this regard, continuous-flow systems that allow steady-state homogeneous turnover in a stationary liquid phase while at the same time effecting integrated product separation at mild process temperatures represent a particularly attractive scenario. While continuous-flow processing is a standard procedure for large volume manufacturing, capitalizing on its potential in the realm of the molecular complexity of organic synthesis is still an emerging area that requires innovative solutions. Here we highlight some recent developments which have succeeded in realizing such systems by the combination of near- and supercritical fluids with homogeneous catalysts in supported liquid phases. The cases discussed exemplify how all three levels of continuous-flow homogeneous catalysis (catalyst system, separation strategy, process scheme) must be matched to locate viable process conditions.

The α4 isoform of the Na⁺,K⁺-ATPase is tuned for changing extracellular environments.

In their journey from the male to the female reproductive tract, spermatozoa are confronted with a constantly changing environment. To cope with the associated challenges, spermatozoa express a distinct set of transporters, channels and pumps. One of the membrane proteins unique to spermatozoa is the α4 isoform of the Na(+) ,K(+) -ATPase. In addition to α4, spermatozoa express the ubiquous α1 variant. To get a detailed understanding of how α1 and α4 differ, and why spermatozoa need an additional Na(+) ,K(+) -ATPase, we have conducted an electrophysiological comparison of the rodent isoforms (rat α4 versus mouse α1-3) using the Xenopus oocyte expression system. We demonstrate isoform-specific differences in the voltage sensitivity of steady-state turnover, with α2 being the more sensitive, and α1 and α2 having faster Na(+) release kinetics than α3 and α4. Our data further show that the α1 and α2 turnover rates are fast compared with those of α3 and α4. Finally, α4 is less influenced by changes in extracellular Na(+) and temperature than α1. Based on these findings, we discuss the possibility that evolution has selected robust activity rather than rapid turnover for α4.

Reverse Electron Transfer Completes the Catalytic Cycle in a 2,3,5-Trifluorotyrosine-Substituted Ribonucleotide Reductase.

Escherichia coli class Ia ribonucleotide reductase is composed of two subunits (α and β), which form an α2β2 complex that catalyzes the conversion of nucleoside 5′-diphosphates to deoxynucleotides (dNDPs). β2 contains the essential tyrosyl radical (Y122•) that generates a thiyl radical (C439•) in α2 where dNDPs are made. This oxidation occurs over 35 Å through a pathway of amino acid radical intermediates (Y122 → [W48] → Y356 in β2 to Y731 → Y730 → C439 in α2). However, chemistry is preceded by a slow protein conformational change(s) that prevents observation of these intermediates. 2,3,5-Trifluorotyrosine site-specifically inserted at position 122 of β2 (F3Y•-β2) perturbs its conformation and the driving force for radical propagation, while maintaining catalytic activity (1.7 s–1). Rapid freeze–quench electron paramagnetic resonance spectroscopy and rapid chemical-quench analysis of the F3Y•-β2, α2, CDP, and ATP (effector) reaction show generation of 0.5 equiv of Y356• and 0.5 equiv of dCDP, both at 30 s–1. In the absence of an external reducing system, Y356• reduction occurs concomitant with F3Y reoxidation (0.4 s–1) and subsequent to oxidation of all α2s. In the presence of a reducing system, a burst of dCDP (0.4 equiv at 22 s–1) is observed prior to steady-state turnover (1.7 s–1). The [Y356•] does not change, consistent with rate-limiting F3Y reoxidation. The data support a mechanism where Y122• is reduced and reoxidized on each turnover and demonstrate for the first time the ability of a pathway radical in an active α2β2 complex to complete the catalytic cycle.

E3 Ubiquitin Ligase NEDD4 Promotes Influenza Virus Infection by Decreasing Levels of the Antiviral Protein IFITM3.

Interferon (IFN)-induced transmembrane protein 3 (IFITM3) is a cell-intrinsic factor that limits influenza virus infections. We previously showed that IFITM3 degradation is increased by its ubiquitination, though the ubiquitin ligase responsible for this modification remained elusive. Here, we demonstrate that the E3 ubiquitin ligase NEDD4 ubiquitinates IFITM3 in cells and in vitro. This IFITM3 ubiquitination is dependent upon the presence of a PPxY motif within IFITM3 and the WW domain-containing region of NEDD4. In NEDD4 knockout mouse embryonic fibroblasts, we observed defective IFITM3 ubiquitination and accumulation of high levels of basal IFITM3 as compared to wild type cells. Heightened IFITM3 levels significantly protected NEDD4 knockout cells from infection by influenza A and B viruses. Similarly, knockdown of NEDD4 in human lung cells resulted in an increase in steady state IFITM3 and a decrease in influenza virus infection, demonstrating a conservation of this NEDD4-dependent IFITM3 regulatory mechanism in mouse and human cells. Consistent with the known association of NEDD4 with lysosomes, we demonstrate for the first time that steady state turnover of IFITM3 occurs through the lysosomal degradation pathway. Overall, this work identifies the enzyme NEDD4 as a new therapeutic target for the prevention of influenza virus infections, and introduces a new paradigm for up-regulating cellular levels of IFITM3 independently of IFN or infection.

Engineered and Native Coenzyme B12-dependent Isovaleryl-CoA/Pivalyl-CoA Mutase

Adenosylcobalamin-dependent isomerases catalyze carbon skeleton rearrangements using radical chemistry. We have recently demonstrated that an isobutyryl-CoA mutase variant, IcmF, a member of this enzyme family that catalyzes the interconversion of isobutyryl-CoA and n-butyryl-CoA also catalyzes the interconversion between isovaleryl-CoA and pivalyl-CoA, albeit with low efficiency and high susceptibility to inactivation. Given the biotechnological potential of the isovaleryl-CoA/pivalyl-CoA mutase (PCM) reaction, we initially attempted to engineer IcmF to be a more proficient PCM by targeting two active site residues predicted based on sequence alignments and crystal structures, to be key to substrate selectivity. Of the eight mutants tested, the F598A mutation was the most robust, resulting in an ∼17-fold increase in the catalytic efficiency of the PCM activity and a concomitant ∼240-fold decrease in the isobutyryl-CoA mutase activity compared with wild-type IcmF. Hence, mutation of a single residue in IcmF tuned substrate specificity yielding an ∼4000-fold increase in the specificity for an unnatural substrate. However, the F598A mutant was even more susceptible to inactivation than wild-type IcmF. To circumvent this limitation, we used bioinformatics analysis to identify an authentic PCM in genomic databases. Cloning and expression of the putative AdoCbl-dependent PCM with an α2β2 heterotetrameric organization similar to that of isobutyryl-CoA mutase and a recently characterized archaeal methylmalonyl-CoA mutase, allowed demonstration of its robust PCM activity. To simplify kinetic analysis and handling, a variant PCM-F was generated in which the αβ subunits were fused into a single polypeptide via a short 11-amino acid linker. The fusion protein, PCM-F, retained high PCM activity and like PCM, was resistant to inactivation. Neither PCM nor PCM-F displayed detectable isobutyryl-CoA mutase activity, demonstrating that PCM represents a novel 5'-deoxyadenosylcobalamin-dependent acyl-CoA mutase. The newly discovered PCM and the derivative PCM-F, have potential applications in bioremediation of pivalic acid found in sludge, in stereospecific synthesis of C5 carboxylic acids and alcohols, and in the production of potential commodity and specialty chemicals.

Identification of new substrates for the CYP106A1-mediated 11-oxidation and investigation of the reaction mechanism

CYP106A1 from Bacillus megaterium DSM319 was recently shown to catalyze steroid and terpene hydroxylations. Besides producing hydroxylated steroid metabolites at positions 6β, 7β, 9α and 15β, the enzyme displayed previously unknown 11-oxidase activity towards 11β-hydroxysteroids. Novel examples for 11-oxidation were identified and confirmed by 1H and 13C NMR for prednisolone, dexamethasone and 11β-hydroxyandrostenedione. However, only 11β-hydroxyandrostenedione formed a single 11-keto product. The latter reaction was chosen to investigate the kinetic solvent isotope effect on the steady-state turnover of the CYP106A1-mediated 11-oxidation. Our results reveal a large inverse kinetic isotope effect (∼0.44) suggesting the involvement of the ferric peroxoanion as a reactive intermediate.

Amperometric Detection of Aqueous Silver Ions by Inhibition of Glucose Oxidase Immobilized on Nitrogen-Doped Carbon Nanotube Electrodes.

An amperometric glucose biosensor based on immobilization of glucose oxidase on nitrogen-doped carbon nanotubes (N-CNTs) was successfully developed for the determination of silver ions. Upon exposure to glucose, a steady-state enzymatic turnover rate was detected through amperometric oxidation of the H2O2 byproduct, directly related to the concentration of glucose in solution. Inhibition of the steady-state enzymatic glucose oxidase reaction by heavy metals ions such as Ag(+), produced a quantitative decrease in the steady-state rate, subsequently creating an ultrasensitive metal ion biosensor through enzymatic inhibition. The Ag(+) biosensor displayed a sensitivity of 2.00 × 10(8) ± 0.06 M(-1), a limit of detection (σ = 3) of 0.19 ± 0.04 ppb, a linear range of 20-200 nM, and sample recovery at 101 ± 2%, all acquired at a low-operating potential of 0.05 V (vs Hg/Hg2SO4). Interestingly, the biosensor does not display a loss in sensitivity with continued use due to the % inhibition based detection scheme: loss of enzyme (from continued use) does not influence the % inhibition, only the overall current associated with the activity loss. The heavy metals Cu(2+) and Co(2+) were also detected using the enzyme biosensor but found to be much less inhibitory, with sensitivities of 1.45 × 10(6) ± 0.05 M(-1) and 2.69 × 10(3) ± 0.07 M(-1), respectively. The mode of GOx inhibition was examined for both Ag(+) and Cu(2+) using Dixon and Cornish-Bowden plots, where a strong correlation was observed between the inhibition constants and the biosensor sensitivity.

The Tumor Suppressor NKX3.1 Is Targeted for Degradation by DYRK1B Kinase.

NKX3.1 is a prostate-specific homeodomain protein and tumor suppressor whose expression is reduced in the earliest phases of prostatic neoplasia. NKX3.1 expression is not only diminished by genetic loss and methylation, but the protein itself is a target for accelerated degradation caused by inflammation that is common in the aging prostate gland. NKX3.1 degradation is activated by phosphorylation at C-terminal serine residues that mediate ubiquitination and protein turnover. Because NKX3.1 is haploinsufficient, strategies to increase its protein stability could lead to new therapies. Here, a high-throughput screen was developed using an siRNA library for kinases that mediate NKX3.1 degradation. This approach identified several candidates, of which DYRK1B, a kinase that is subject to gene amplification and overexpression in other cancers, had the greatest impact on NKX3.1 half-life. Mechanistically, NKX3.1 and DYRK1B were shown to interact via the DYRK1B kinase domain. In addition, an in vitro kinase assay showed that DYRK1B phosphorylated NKX3.1 at serine 185, a residue critical for NKX3.1 steady-state turnover. Lastly, small-molecule inhibitors of DYRK1B prolonged NKX3.1 half-life. Thus, DYRK1B is a target for enzymatic inhibition in order to increase cellular NKX3.1. DYRK1B is a promising and novel kinase target for prostate cancer treatment mediated by enhancing NKX3.1 levels.