Preferential action of a brain detyrosinolating carboxypeptidase on polymerized tubulin

A carboxypeptidase purified from brain catalyzes the release of COOH-terminal tyrosine without further digesting tubulin. It is distinct from previously described carboxypeptidases, and appears to have specificity for tubulin as it is not inhibited by peptides and proteins with COOH-terminal tyrosine, and because, unlike carboxypeptidase A (which by removing tyrosine from aldolase causes its inactivation), this enzyme does not decrease aldolase activity. The enzyme detyrosinolates both self-assembly-competent (cycle-purified) and -incompetent (phosphocellulose-purified) tubulin. However, under assembly conditions the rate was 2-3-fold higher for competent tubulin. Preincubation of assembly-competent tubulin with podophyllotoxin or colchicine resulted in a parallel concentration-dependent inhibition of tubulin polymerization and detyrosinolation. Similarly, when incompetent tubulin was induced to polymerize by preincubation with purified microtubule-associated protein 2 (an assembly-promoting protein) or taxol, the initial rate of its detyrosinolation increased 3-5-fold, and this increase was blocked if podophyllotoxin was also added along with microtubule-associated protein 2 or taxol during the preincubation. Oligomers induced by adding vinblastine to incompetent tubulin were also detyrosinolated more rapidly, and the stimulation was abolished by maytansine, which has been shown to disperse the vinblastine-induced oligomers. When polymerized and subunit fractions were separated after a steady state mixture had been partially digested with the carboxypeptidase, the former was found to have lost 2-3 times more COOH-terminal tyrosine. Although both polymer and monomer can be detyrosinolated by the enzyme, polymeric and oligomeric forms are the preferred substrates. Carboxypeptidase appeared to release tyrosine at the same rate from populations of short and long microtubules.     

A dynein ATPase inhibitor isolated from a commercial ATP preparation.

Preparations of ATP from equine muscle contained an inhibitor of dynein Mg2+-activated ATPase. The inhibitory material was separated from the ATP by molecular sieve filtration. The several molecular species of dynein extracted from three different axonemal sources were all inhibited; myosin ATPase was not. With increasing amounts of inhibitor the inhibition did not go to completion but reached a plateau when the rate had been reduced to 1/5 the uninhibited rate. A plot of 1/[S] against 1/v at several inhibitor concentrations yielded parallel lines. There was little inhibition of dynein ATPase when Mg2+ was replaced by Ca2+. The inhibitor appeared slightly smaller in molecular size than ATP, had anionic character, and was not adsorbed to charcoal.     

An Enhanced In Vivo Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) Model for Quantification of Drug Metabolism Enzymes

Many of the enzymes involved in xenobiotic metabolism are maintained at a low basal level and are only synthesized in response to activation of upstream sensor/effector proteins. This induction can have implications in a variety of contexts, particularly during the study of the pharmacokinetics, pharmacodynamics, and drug–drug interaction profile of a candidate therapeutic compound. Previously, we combined in vivo SILAC material with a targeted high resolution single ion monitoring (tHR/SIM) LC-MS/MS approach for quantification of 197 peptide pairs, representing 51 drug metabolism enzymes (DME), in mouse liver. However, as important enzymes (for example, cytochromes P450 (Cyp) of the 1a and 2b subfamilies) are maintained at low or undetectable levels in the liver of unstimulated metabolically labeled mice, quantification of these proteins was unreliable. In the present study, we induced DME expression in labeled mice through synchronous ligand-mediated activation of multiple upstream nuclear receptors, thereby enhancing signals for proteins including Cyps 1a, 2a, 2b, 2c, and 3a. With this enhancement, 115 unique, lysine-containing, Cyp-derived peptides were detected in the liver of a single animal, as opposed to 56 in a pooled sample from three uninduced animals. A total of 386 peptide pairs were quantified by tHR/SIM, representing 68 Phase I, 30 Phase II, and eight control proteins. This method was employed to quantify changes in DME expression in the hepatic cytochrome P450 reductase null (HRN) mouse. We observed compensatory induction of several enzymes, including Cyps 2b10, 2c29, 2c37, 2c54, 2c55, 2e1, 3a11, and 3a13, carboxylesterase (Ces) 2a, and glutathione S-transferases (Gst) m2 and m3, along with down-regulation of hydroxysteroid dehydrogenases (Hsd) 11b1 and 17b6. Using DME-enhanced in vivo SILAC material with tHR/SIM, therefore, permits the robust analysis of multiple DME of importance to xenobiotic metabolism, with improved utility for the study of drug pharmacokinetics, pharmacodynamics, and of chemically treated and genetically modified mouse models.Metabolism is listed as the primary clearance mechanism for approximately three quarters of prescribed drugs (1). Of this metabolism, three quarters is carried out by enzymes of the CYP superfamily (1). The US Food and Drug Administration and European Medicines Agency recommend the assessment of metabolism by CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A as early as possible during the preclinical development of a novel therapeutic agent (2, 3). As the expression of most of these CYP isoforms (and of other DMEs (drug metabolizing enzymes)¹ and transporters) can be induced in response to chemical challenge, with potential implications for the pharmacokinetic and pharmacodynamic properties of both the agent being administered, and of other drugs (drug–drug interaction), it is therefore also recommended that the capacity of the candidate agent to induce DME expression be assessed at an early stage (2, 3).The majority of clinically relevant drug-mediated CYP induction is known to occur through activation of two closely related nuclear receptors; the pregnane x receptor (PXR) and the constitutive androstane receptor (CAR). These receptors are highly promiscuous, mediating the induction of multiple Phase I and Phase II enzymes in response to diverse exogenous and endogenous stimuli (4–8). Their activity is assessed through measurement of the levels of specific downstream targets, primarily CYP3A and CYP2B6 (2, 3). A third receptor, the aryl hydrocarbon receptor (AHR) is less promiscuous, with a target battery more distinct from those of PXR and CAR (4, 8), but is a central mediator of CYP1A induction, and its activity can be inferred from the levels of these enzymes (2, 3). An alternative approach to the direct measurement of CYP protein is assay of metabolic conversion of specific probe substrates (e.g. 1-hydroxylation of midazolam for CYP3A), often in the presence or absence of specific inhibitors (e.g. ketoconazole) or inducers (e.g. rifampicin). This approach is of utility in vivo, but is limited to the small number of CYP for which these specific agents are available and is not generally applicable to other DMEs, although efforts to identify probes specific for members of the UDP glucuronosyltransferase (UGT) subfamily have met with some success (9, 10).Aside from CYP, most of the remaining Phase I reactions are carried out by alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), aldo-keto reductase (AKR), carbonyl reductase (CBR), epoxide hydrolase (EPHX), esterase (e.g. CES), flavin-containing monooxygenase (FMO), and hydroxysteroid/retinoid dehydrogenase (HSD/RDH) superfamilies (1, 11). Subsequent Phase II (conjugation) reactions are carried out by enzymes including UGT, GST, sulfotransferase (SULT), and methyltransferase (e.g. COMT) (1, 11). In the context of drug development, greater emphasis is being placed on understanding non-CYP mediated interactions, such as with FMO, ADH, and ALDH, but particularly with UGT (2). Currently, the US Food and Drug Administration recommends assessment of the interaction with UGTs 1A1, 1A3, 1A4, 1A6, 1A9, 2B7, and 2B15 (2).Because of the unavailability of specific probe substrates, DME levels are usually assessed by Western blotting and microarray/RT-PCR. However, concerns relating to the cross-reactivity of antibodies within highly homologous protein subfamilies, and to the potential discordance between mRNA and protein levels (12), have led to a growing interest in the development of LC-MS/MS based methodology for the quantification of DMEs (13–18) and drug transporters (19–22) in various species. Among these techniques, the most common is multiple reaction monitoring (MRM)-based absolute quantification (AQUA) (23). The main drawback of this approach is the inability to control the recovery of peptides during sample processing steps prior to the addition of stable isotope peptide(s), potentially leading to quantification inaccuracies. Such bias can be severe when in-gel or filter-assisted protocols are followed (24). In order to circumvent this problem, we have previously developed a DME-targeted in vivo SILAC-based method wherein 197 peptide pairs, representing 51 DMEs, were quantified in a single sample (two LC-MS/MS analyses) (25). The workflow followed the principles of stable isotope dilution LC-MS commonly employed in the analytical laboratory for small molecule analysis, with in vivo SILAC material (¹³C₆-lysine-labeled liver lysate) used as an internal standard to control recovery and ionization efficiencies. The ratio(s) of light to heavy peptides in defined retention time and m/z window(s) were used to quantify protein expression. This method served to circumvent the uncertainties generated during sample preparation in AQUA, as both light and heavy analytes share near-identical chemical properties, environment, and processing. The number of peptides measured, and hence the confidence of protein quantification, was greater than in a typical AQUA analysis. Furthermore, stress tests such as dilution linearity enabled the removal of peptides with poor analytical performance, providing a high level of consistency and reproducibility (25). It was, however, noted that this workflow omitted key Cyps because of their low level of constitutive expression in the heavy internal standard liver. The objective of the current study was, therefore, to generate a metabolically labeled mouse model which would allow direct quantification of as many DME, both inducible and basally expressed, of relevance to xenobiotic metabolism as possible. This “DME-enhanced” in vivo SILAC model was characterized in detail and its utility was demonstrated in the proteomic characterization of the compensatory effects that occur following the deletion of P450 reductase in the hepatocyte specific P450 reductase null (HRN) mouse (26).     

Impaired basophil induction leads to an age-dependent innate defect in type 2 immunity during helminth infection in mice

Parasitic-infection studies on rhesus macaque monkeys have shown juvenile animals to be more susceptible to infection than adults, but the immunological mechanism for this is not known. In this study, we investigated the age-dependent genesis of helminth-induced type 2 immune responses using adult (6-8-wk-old) and juvenile (21-28-d-old) mice. Following infection with the parasitic nematode Nippostrongylus brasiliensis, juvenile mice had increased susceptibility to infection relative to adult mice. Juvenile mice developed a delayed type 2 immune response with decreased Th2 cytokine production, IgE Ab responses, mouse mast cell protease 1 levels, and intestinal goblet cell induction. This innate immune defect in juvenile mice was independent of TLR signaling, dendritic cells, or CD4(+) cell function. Using IL-4-eGFP mice, it was demonstrated that the numbers of IL-4-producing basophil and eosinophils were comparable in young and adult naive mice; however, following helminth infection, the early induction of these cells was impaired in juvenile mice relative to older animals. In nonhelminth models, there was an innate in vivo defect in activation of basophils, but not eosinophils, in juvenile mice compared with adult animals. The specific role for basophils in this innate defect in helminth-induced type 2 immunity was confirmed by the capacity of adoptively transferred adult-derived basophils, but not eosinophils, to restore the ability of juvenile mice to expel N. brasiliensis. The defect in juvenile mice with regard to helminth-induced innate basophil-mediated type 2 response is relevant to allergic conditions.     

Upregulation of Retinal Dehydrogenase 2 in Alternatively Activated Macrophages during Retinoid-dependent Type-2 Immunity to Helminth Infection in Mice

Although the vitamin A metabolite retinoic acid (RA) plays a critical role in immune function, RA synthesis during infection is poorly understood. Here, we show that retinal dehydrogenases (Raldh), required for the synthesis of RA, are induced during a retinoid-dependent type-2 immune response elicited by Schistosoma mansoni infection, but not during a retinoid-independent anti-viral immune response. Vitamin A deficient mice have a selective defect in T_H2 responses to S. mansoni, but retained normal LCMV specific T_H1 responses. A combination of in situ imaging, intra-vital imaging, and sort purification revealed that alternatively activated macrophages (AAMφ) express high levels of Raldh2 during S. mansoni infection. IL-4 induces Raldh2 expression in bone marrow-derived macrophages in vitro and peritoneal macrophages in vivo. Finally, in vivo derived AAMφ have an enhanced capacity to induce Foxp3 expression in CD4+ cells through an RA dependent mechanism, especially in combination with TGF-β. The regulation of Raldh enzymes during infection is pathogen specific and reflects differential requirements for RA during effector responses. Specifically, AAMφ are an inducible source of RA synthesis during helminth infections and T_H2 responses that may be important in regulating immune responses.