Profiling Of Enzymes Research Articles

Gonadal development and transcript profiling of steroidogenic enzymes in response to 17α-methyltestosterone in the rare minnow Gobiocypris rarus

It is well known that natural and anthropogenic chemicals interfere with the hormonal system of vertebrate and invertebrate organisms. How these chemicals regulate gonadal steroidogenesis remains to be determined. The main objective of this study was to evaluate the effects of 17α-methyltestosterone (MT), a synthetic model androgen, on gene expression profiles of six key steroidogenic genes in adult rare minnow. The full-length cDNA encoding 11β-hydroxysteroid dehydrogenase-2 (11β-HSD2) was firstly isolated and characterized by RT-PCR and RACE methods. The gonadal transcript changes of StAR, cyp11a1, 3β-HSD, cyp17a1, 11β-HSD2 and cyp19a1a in 6-month adult Gobiocypris rarus exposed to MT and 17α-ethinylestradiol (EE2) for 7, 14 and 21 days were detected by qRT-PCR. To make an effort to connect the transcriptional changes of steroidogenic enzymes with effects on higher levels of biological organization and on VTG, one remarkable sensitive target of steroids, body and gonad weights, histology of gonads, and hepatic vtg mRNA level were measured. MT caused varying degree of abnormalities in ovaries and testes. The hepatic vtg mRNA level was highly inhibited in females and slightly altered in males by MT. Transcripts of several steroidogenic genes including StAR, cyp17a1, and cyp11a1 showed high responsiveness to MT exposure in G. rarus. The gene expression profiles of these steroidogenic genes in MT-treated groups were much distinct with the EE2-treated group.

Activity profiling of vacuolar processing enzymes reveals a role for VPE during oomycete infection

Vacuolar processing enzymes (VPEs) are important cysteine proteases that are implicated in the maturation of seed storage proteins, and programmed cell death during plant-microbe interactions and development. Here, we introduce a specific, cell-permeable, activity-based probe for VPEs. This probe is highly specific for all four Arabidopsis VPEs, and labeling is activity-dependent, as illustrated by sensitivity for inhibitors, pH and reducing agents. We show that the probe can be used for in vivo imaging and displays multiple active isoforms of VPEs in various tissues and in both monocot and dicot plant species. Thus, VPE activity profiling is a robust, simple and powerful tool for plant research for a wide range of applications. Using VPE activity profiling, we discovered that VPE activity is increased during infection with the oomycete pathogen Hyaloperonospora arabidopsidis (Hpa). The enhanced VPE activity is host-derived and EDS1-independent. Sporulation of Hpa is reduced on vpe mutant plants, demonstrating a role for VPE during compatible interactions that is presumably independent of programmed cell death. Our data indicate that, as an obligate biotroph, Hpa takes advantage of increased VPE activity in the host, e.g. to mediate protein turnover and nutrient release.

Gene expression profiling and pathway network analysis of hepatic metabolic enzymes targeted by baicalein

Ethnopharmacological relevanceBaicalein is a flavone originally isolated from the roots of traditional Chinese medicinal herb, Scutellaria baicalensis, which has been proved as a promising chemopreventive compound for many chronic human diseases. Aim of the studyThe present study aimed to clarify the molecular mechanism targeted by baicalein. Materials and methodsGene expression profiling of HepG2 cells treated with baicalein was carried out, using the Affymetrix 42K oligonucleotide microarray in the present study. Microarray data analyzed by Ingenuity Pathway Analysis (IPA), further study performed by real time PCR, reporter gene assay, and Western blot. ResultsAmong total 42K gene probes, baicalein treatment up-regulated the signals of 440 gene probes (1.04% of total gene probes) and down-regulated signals of 254 gene probes (0.6% of total gene probes) by ≥2-fold. These genes were categorized into 35 groups and hit for biological processes, molecular functions, and signaling pathways. The network and pathway analyses of these data further revealed that an Nrf2 (nuclear factor-erythroid 2 p45-related factor 2)-mediated ARE (antioxidant response element) pathway is involved in baicalein-induced gene expression of hepatic metabolic enzymes. The representative enzymes involved in Nrf2/ARE pathway were further confirmed at mRNA level by real time PCR and at protein level by Western blot analysis. Moreover, the ARE-reporter gene assay demonstrated that baicalein stimulated Nrf2-mediated ARE transactivation. ConclusionsOur results provide a comprehensive data for understanding the hepatic metabolism, bioactive role and the molecular mechanisms of baicalein.

Abstract P245: Loss of Steroid Receptor Coactivator-2 in the Heart Results in a Return to the Fetal Gene Program

Steroid Receptor Coactivator-2 (SRC-2) is an integral transcriptional mediator of metabolism throughout the body with roles in both fatty acid and carbohydrate metabolism. Based on the critical link between cardiac function and cardiac metabolism, which itself has dynamic interplay between both fatty acid and carbohydrate use, we sought to determine if SRC-2 plays a role in controlling cardiac metabolism. Hearts from SRC-2-/- mice were analyzed by extensive gene expression profiling of major metabolic enzymes in fuel usage pathways as well as metabolomic analysis of major metabolites. Our results indicate that under normal conditions, loss of SRC-2 compromises the ability of the adult heart to metabolize fatty acid as its sole fuel source. We observe a gene expression profile that mimics the fetal heart and a metabolomic profile that suggests a reliance on anaerobic conversion of glucose to lactate for energy. This fetal gene switch is paralleled with changes of sarcomeric gene expression, along with several genes altered in response to stress. Interestingly, this long-term adaptive response is adequate to maintain cardiac function under normal conditions. However, SRC-2-/- mice do show impaired cardiac function as compared to SRC-2+/+ mice under hemodynamic stress from aortic banding. Surprisingly, this is accompanied by the absence of hypertrophy, which may be explained by decreased expression of serum response factor (SRF) and several of the transcription factors with which SRF is known to act. Taken together, loss of SRC-2 results in a cardiac phenotype characterized by a return to the fetal gene program that mimics the state of a stressed and/or aging heart. Further investigation of this mouse model may play an important role in understanding the mechanisms underlying the transcriptional events surrounding cardiac metabolic remodeling.

Proteomic Profiling of Mitochondrial Enzymes during Skeletal Muscle Aging

Mitochondria are of central importance for energy generation in skeletal muscles. Expression changes or functional alterations in mitochondrial enzymes play a key role during myogenesis, fibre maturation, and various neuromuscular pathologies, as well as natural fibre aging. Mass spectrometry-based proteomics suggests itself as a convenient large-scale and high-throughput approach to catalogue the mitochondrial protein complement and determine global changes during health and disease. This paper gives a brief overview of the relatively new field of mitochondrial proteomics and discusses the findings from recent proteomic surveys of mitochondrial elements in aged skeletal muscles. Changes in the abundance, biochemical activity, subcellular localization, and/or posttranslational modifications in key mitochondrial enzymes might be useful as novel biomarkers of aging. In the long term, this may advance diagnostic procedures, improve the monitoring of disease progression, help in the testing of side effects due to new drug regimes, and enhance our molecular understanding of age-related muscle degeneration.

Review article: Role of oxidative enzymes in plant defenses against insect herbivory

Crop productivity is greatly influenced by various environmental stresses, of which insect herbivory-induced biotic stress assumes much significance. As a consequence of insect herbivory, a number of plant biochemical processes involved in the tolerance mechanism are affected. Different studies have demonstrated a diverse functional role of various plant oxidative enzymes in protecting plants against biotic stress induced by insect herbivory. Comprehensive profiling of stress-associated plant oxidative enzymes is most relevant to successful molecular breeding of stress-tolerant crop plants. Thus, better understanding of the biochemical basis of plant defense mechanisms is imperative, not only from a basic science perspective, but also for biotechnology-based pest control practice. In this review, we emphasize the potential role of various oxidative enzymes in plant defense against insect herbivory.

Expression profiling of antioxidant enzymes in the gill of disk abalone after viral hemorrhagic septicemia virus (VHSV) infection

Expression profiling of antioxidant enzymes in the gill of disk abalone after viral hemorrhagic septicemia virus (VHSV) infection

TRP-2 expression protects HEK cells from dopamine- and hydroquinone-induced toxicity

We previously reported that melanogenic enzyme TRP-2 (or DCT for DOPAchrome tautomerase) expression in WM35 melanoma cells resulted in increased intracellular GSH levels, reduction in DNA damage induced by free radicals, and decreased cell sensitivity to oxidative stress. These effects seemed to depend on a particular cellular context, because none of them were found to occur in HEK epithelial cells. We postulated that the TRP-2 beneficial effect observed in WM35 cells in the oxidative stress situation may relate to quinone metabolization and, more precisely, to the ability of TRP-2 to clear off related toxic metabolites, resulting in a global redox status modification. Here, a comparative protein expression profiling of catecholamine biosynthesis enzymes and detoxification enzymes was conducted in WM35 melanoma cells and in HEK epithelial cells, in comparison with normal human melanocytes. Results showed that WM35 cells, but not HEK cells, expressed enzymes involved in catecholamine biosynthesis, suggesting that their quinone-related toxic metabolites were present in WM35 cells but not in HEK cells. To address the issue of a possible TRP-2 beneficial effect toward quinone toxicity, cell survival experiments were then conducted in HEK cells using dopamine and hydroquinone at toxic concentrations. We showed that TRP-2 expression significantly reduced HEK cell sensitivity to both compounds. This beneficial property of TRP-2 was likely to depend on the integrity of its DOPAchrome tautomerase catalytic site, because both TRP-2 R194Q and TRP-2 H189G, which have lost their DOPAchrome tautomerase activity, failed to modify the HEK cell response to dopamine and hydroquinone. These results suggest that TRP-2 acts on quinone metabolites other than DOPAchrome, e.g., in the catecholamine pathway, and limits their deleterious effects.

Fluorescent Profiling of Modular Biosynthetic Enzymes by Complementary Metabolic and Activity Based Probes

The study of the enzymes responsible for natural product biosynthesis has proven a valuable source of new enzymatic activities and been applied to a number of biotechnology applications. Protein profiling could prove highly complementary to genetics based approaches by allowing us to understand the activity, transcriptional control, and post-translational modification of these enzymes in their native and dynamic proteomic environments. Here we present a method for the fluorescent profiling of PKS, NRPS, and FAS multidomain modular synthases in their whole proteomes using complementary metabolic and activity based probes. After first examining the reactivity of these activity based probes with a variety of purified recombinant PKS, NRPS, and FAS enzymes in vitro, we apply this duel labeling strategy to the analysis of modular synthases in a human breast cancer cell line and two strains of the natural product producer Bacillus subtilis. Collectively, these studies demonstrate that complementary protein profiling approaches can prove highly useful in the identification and assignment of inhibitor specificity and domain structure of these modular biosynthetic enzymes.

Profiling of proteolytic enzymes in the gut of the tick Ixodes ricinus reveals an evolutionarily conserved network of aspartic and cysteine peptidases

BackgroundTicks are vectors for a variety of viral, bacterial and parasitic diseases in human and domestic animals. To survive and reproduce ticks feed on host blood, yet our understanding of the intestinal proteolytic machinery used to derive absorbable nutrients from the blood meal is poor. Intestinal digestive processes are limiting factors for pathogen transmission since the tick gut presents the primary site of infection. Moreover, digestive enzymes may find practical application as anti-tick vaccine targets.ResultsUsing the hard tick, Ixodes ricinus, we performed a functional activity scan of the peptidase complement in gut tissue extracts that demonstrated the presence of five types of peptidases of the cysteine and aspartic classes. We followed up with genetic screens of gut-derived cDNA to identify and clone genes encoding the cysteine peptidases cathepsins B, L and C, an asparaginyl endopeptidase (legumain), and the aspartic peptidase, cathepsin D. By RT-PCR, expression of asparaginyl endopeptidase and cathepsins B and D was restricted to gut tissue and to those developmental stages feeding on blood.ConclusionOverall, our results demonstrate the presence of a network of cysteine and aspartic peptidases that conceivably operates to digest host blood proteins in a concerted manner. Significantly, the peptidase components of this digestive network are orthologous to those described in other parasites, including nematodes and flatworms. Accordingly, the present data and those available for other tick species support the notion of an evolutionary conservation of a cysteine/aspartic peptidase system for digestion that includes ticks, but differs from that of insects relying on serine peptidases.

Activity profiling of human deSUMOylating enzymes (SENPs) with synthetic substrates suggests an unexpected specificity of two newly characterized members of the family

SENPs [Sentrin/SUMO (small ubiquitin-related modifier)-specific proteases] include proteases that activate the precursors of SUMOs, or deconjugate SUMOs attached to target proteins. SENPs are usually assayed on protein substrates, and for the first time we demonstrate that synthetic substrates can be convenient tools in determining activity and specificity of these proteases. We synthesized a group of short synthetic peptide fluorogenic molecules based on the cleavage site within SUMOs. We demonstrate the activity of human SENP1, 2, 5, 6, 7 and 8 on these substrates. A parallel positional scanning approach using a fluorogenic tetrapeptide library established preferences of SENPs in the P3 and P4 positions that allowed us to design optimal peptidyl reporter substrates. We show that the specificity of SENP1, 2, 5 and 8 on the optimal peptidyl substrates matches their natural protein substrates, and that the presence of the SUMO domain enhances catalysis by 2-3 orders of magnitude. We also show that SENP6 and 7 have an unexpected specificity that distinguishes them from other members of the family, implying that, in contrast to previous predictions, their natural substrate(s) may not be SUMO conjugates.

Assessing Enzyme Activities Using Stable Isotope Labeling and Mass Spectrometry

Activity-based protein profiling has emerged as a valuable technology for labeling, enriching, and assessing protein activities from complex mixtures. This is primarily accomplished via a two-step identification and quantification process. Here we show a highly quantitative and streamlined method, termed catch-and-release activity profiling of enzymes (CAPE), which reduces this procedure to a single step. Furthermore the CAPE approach has the ability to detect small quantitative changes that may have been missed by alternative mass spectrometry-based techniques.

Citations

Citations

High-throughput profiling of posttranslational modification enzymes by phage display

Phage display has been used as a high-throughput platform for identifying proteins or peptides with desired binding or catalytic activities from a complex proteome. Recently, phage display has been applied to profile the catalytic activities of posttranslational modification (PTM) enzymes. Here, we highlight recent work elucidating the downstream targets of PTM enzymes by phage display, including the genome-wide profiling of biosynthetic enzymes subject to phosphopantetheinyl transferase (PPTase) modification.

Frameshift proteins in Alzheimer's disease and in other conformational disorders: Time for the ubiquitin-proteasome system

Neuronal homeostasis requires a constant balance between biosynthetic and catabolic processes. Eukaryotic cells primarily use two distinct mechanisms for degradation: the proteasome and autophagy of aggregates by the lysosomes. We focused on the ubiquitin-proteasome system (UPS) and discovered a frameshift protein for ubiquitin (UBB+1), that accumulates in the neuritic plaques and tangles in patients with Alzheimer's disease (AD). UBB+1, unable to tag proteins to be degraded, has been shown to be a substrate for ubiquitination and subsequent proteasomal degradation. If UBB+1 is accumulated, it inhibits the proteasome, which may result in neuronal death. We showed that UB+1 is also present in other tauopathies (e.g. Pick's disease) and in several polyglutamine diseases, but remarkably not in synucleinopathies (e.g. Parkinson's disease). Accumulation of UBB+1-being a reporter for proteasomal dysfunctioning- thus differentiates between these conformational diseases. The accumulation of UBB+1 causes a dysfunctional UPS in these multifactorial neurodegenerative diseases. Novel transgenic mouse models and large-scale expression profiling and functional analyses of enzymes of the UPS compounds - enabling us to identify the targets of the UPS in these conformational diseases - may now pave the way for intervention and treatment of AD.

Putting proteomics on target: activity-based profiling of ubiquitin and ubiquitin-like processing enzymes

Modification of proteins with ubiquitin (Ub) and Ub-like modifiers (Ubls) plays a fundamental role in cell biology. As a consequence, proteomics-based efforts were developed to characterize proteins that are modified by Ub or Ubls. A more focused functional proteomics strategy relies on active-site probes based on the Ub/Ubl scaffold, which specifically targets Ub/Ubl-processing enzymes. Activity-based profiling with such tools led to the identification of novel gene products with Ub/Ubl-processing activity and uncovered novel control mechanisms regulating their activity. This review discusses recent advances in chemistry-based functional proteomics applications, and how this information can provide a framework for drug development against Ub/Ubl-processing enzymes.

Expression Profiling of Homocysteine Junction Enzymes in the NCI60 Panel of Human Cancer Cell Lines

Methionine metabolism provides two key cellular reagents: S-adenosylmethionine and glutathione, derived from the common intermediate, homocysteine. A majority of cancer cells exhibit a methionine-dependent phenotype whereby they are unable to grow in medium in which methionine is replaced by its precursor, homocysteine. Additionally, CpG island hypermethylation of tumor suppressor gene promoters is observed in a background of global hypomethylation in cancerous cells. In this study, we have profiled the expression levels of the homocysteine junction enzymes, methionine synthase (MS), MS reductase (MSR), and cystathionine beta-synthase (CBS) in the NCI60 panel of cancer cell lines. The doubling time of non-small lung cell cancer lines, which exhibit the lowest levels of MS within the panel, was significantly correlated with expression of MS. The ratio of MS to MSR varied over a 5-fold range in the different cell types, which may modulate methionine synthesis. Interestingly, markedly reduced CBS expression was seen in the methionine-dependent prostate cancer cell line, PC-3, but not in the methionine-independent cell line, DU-145. However, neither provision of the transsulfuration pathway product, cysteine, nor overexpression of CBS rescued the growth impairment, indicating that reduced CBS was not responsible for the methionine-dependent phenotype in this cell line.

Immunohistochemical analysis of NAD(P)H:quinone oxidoreductase and NADPH cytochrome P450 reductase in human superficial bladder tumours: relationship between tumour enzymology and clinical outcome following intravesical mitomycin C therapy.

A central theme within the concept of enzyme-directed bioreductive drug development is the potential to predict tumour response based on the profiling of enzymes involved in the bioreductive activation process. Mitomycin C (MMC) is the prototypical bioreductive drug that is reduced to active intermediates by several reductases including NAD(P)H:quinone oxidoreductase (NQO1) and NADPH cytochrome P450 reductase (P450R). The purpose of our study was to determine whether NQO1 and P450R protein expression in a panel of low-grade, human superficial bladder tumours correlates with clinical response to MMC. A retrospective clinical study was conducted in which the response to MMC of 92 bladder cancer patients was compared to the immunohistochemical expression of NQO1 and P450R protein in archived paraffin-embedded bladder tumour specimens. A broad spectrum of NQO1 protein levels exists in bladder tumours between individual patients, ranging from intense to no immunohistochemical staining. In contrast, levels of P450R were similar with most tumours having moderate to high levels. All patients were chemotherapy naïve prior to receiving MMC and clinical response was defined as the time to first recurrence. A poor correlation exists between clinical response and NQO1, P450R or the expression patterns of various combinations of the 2 proteins. The results of our study demonstrate that the clinical response of superficial bladder cancers to MMC cannot be predicted on the basis of NQO1 and/or P450R protein expression and suggest that other factors (other reductases or post DNA damage events) have a significant bearing on tumour response.