Fluorescence Assay For Activity Research Articles

Continuous Fluorescent Sirtuin Activity Assay Based on Fatty Acylated Lysines.

Lysine deacetylases, like histone deacetylases (HDACs) and sirtuins (SIRTs), are involved in many regulatory processes such as control of metabolic pathways, DNA repair, and stress responses. Besides robust deacetylase activity, sirtuin isoforms SIRT2 and SIRT3 also show demyristoylase activity. Interestingly, most of the inhibitors described so far for SIRT2 are not active if myristoylated substrates are used. Activity assays with myristoylated substrates are either complex because of coupling to enzymatic reactions or time-consuming because of discontinuous assay formats. Here we describe sirtuin substrates enabling direct recording of fluorescence changes in a continuous format. Fluorescence of the fatty acylated substrate is different when compared to the deacylated peptide product. Additionally, the dynamic range of the assay could be improved by the addition of bovine serum albumin, which binds the fatty acylated substrate and quenches its fluorescence. The main advantage of the developed activity assay is the native myristoyl residue at the lysine side chain avoiding artifacts resulting from the modified fatty acyl residues used so far for direct fluorescence-based assays. Due to the extraordinary kinetic constants of the new substrates (KM values in the low nM range, specificity constants between 175,000 and 697,000 M-1s-1) it was possible to reliably determine the IC50 and Ki values for different inhibitors in the presence of only 50 pM of SIRT2 using different microtiter plate formats.

A fast, sensitive and fluorescent LPMO activity assay.

Lytic polysaccharide monooxygenases (LPMOs) are industrially relevant enzymes that utilize a copper co-factor and an oxygen species to break down recalcitrant polysaccharides. These enzymes are secreted by microorganisms and are used in lignocellulosic refineries. As such, they are interesting from both the ecological/biological and industrial perspectives. Here we describe the development of a new fluorescence-based kinetic LPMO activity assay. The assay is based on the enzymatic production of fluorescein from its reduced counterpart. The assay can detect as little as 1 nM LPMO with optimized assay conditions. Furthermore, the reduced fluorescein substrate can also be used to identify peroxidase activity as seen by the formation of fluorescein by horseradish peroxidase. The assay was shown to work well at relatively low H2O2 and dehydroascorbate concentrations. The applicability of the assay was demonstrated.

Fluorescence-Based NAPE-PLD Activity Assay.

N-Acylphosphatidylethanolamine phospholipase D (NAPE-PLD) is regarded as the principal enzyme that generates N-acylethanolamines (NAEs), a family of signaling lipids that includes the endocannabinoid anandamide. To investigate the biological function and biosynthesis of NAEs, we sought to develop potent NAPE-PLD inhibitors. To this aim, we utilized a high-throughput screening-compatible NAPE-PLD activity assay, which uses the fluorescence-quenched substrate PED6. This assay conveniently uses membrane fractions of NAPE-PLD overexpressing HEK293T cell lysates, thus avoiding the need for protein purification. Here, we give a detailed description of the NAPE-PLD PED6 fluorescence activity assay, which has increased throughput compared to previous radioactivity- or mass-spectrometry-based assays.

Activation and Inhibition of Human Matrix Metalloproteinase-9 (MMP9) by HOCl, Myeloperoxidase and Chloramines.

Matrix metalloproteinase-9 (MMP9, gelatinase B) plays a key role in the degradation of extracellular-matrix (ECM) proteins in both normal physiology and multiple pathologies, including those linked with inflammation. MMP9 is excreted as an inactive proform (proMMP9) by multiple cells, and particularly neutrophils. The proenzyme undergoes subsequent processing to active forms, either enzymatically (e.g., via plasmin and stromelysin-1/MMP3), or via the oxidation of a cysteine residue in the prodomain (the “cysteine-switch”). Activated leukocytes, including neutrophils, generate O2− and H2O2 and release myeloperoxidase (MPO), which catalyzes hypochlorous acid (HOCl) formation. Here, we examine the reactivity of HOCl and a range of low-molecular-mass and protein chloramines with the pro- and activated forms of MMP9. HOCl and an enzymatic MPO/H2O2/Cl− system were able to generate active MMP9, as determined by fluorescence-activity assays and gel zymography. The inactivation of active MMP9 also occurred at high HOCl concentrations. Low (nM—low μM) concentrations of chloramines formed by the reaction of HOCl with amino acids (taurine, lysine, histidine), serum albumin, ECM proteins (laminin and fibronectin) and basement membrane extracts (but not HEPES chloramines) also activate proMMP9. This activation is diminished by the competitive HOCl-reactive species, methionine. These data indicate that HOCl-mediated oxidation and MMP-mediated ECM degradation are synergistic and interdependent. As previous studies have shown that modified ECM proteins can also stimulate the cellular expression of MMP proteins, these processes may contribute to a vicious cycle of increasing ECM degradation during disease development.

High-Throughput Screening Reveals New Glutaminase Inhibitor Molecules.

The glutaminase (GLS) enzyme hydrolyzes glutamine into glutamate, an important anaplerotic source for the tricarboxylic acid cycle in rapidly growing cancer cells under the Warburg effect. Glutamine-derived α-ketoglutarate is also an important cofactor of chromatin-modifying enzymes, and through epigenetic changes, it keeps cancer cells in an undifferentiated state. Moreover, glutamate is an important neurotransmitter, and deregulated glutaminase activity in the nervous system underlies several neurological disorders. Given the proven importance of glutaminase for critical diseases, we describe the development of a new coupled enzyme-based fluorescent glutaminase activity assay formatted for 384-well plates for high-throughput screening (HTS) of glutaminase inhibitors. We applied the new methodology to screen a ∼30,000-compound library to search for GLS inhibitors. The HTS assay identified 11 glutaminase inhibitors as hits that were characterized by in silico, biochemical, and glutaminase-based cellular assays. A structure-activity relationship study on the most promising hit (C9) allowed the discovery of a derivative, C9.22, with enhanced in vitro and cellular glutaminase-inhibiting activity. In summary, we discovered a new glutaminase inhibitor with an innovative structural scaffold and described the molecular determinants of its activity.

Compound heterozygosis in AADC deficiency: A complex phenotype dissected through comparison among heterodimeric and homodimeric AADC proteins

Compound heterozygosis is the most diffuse and hardly to tackle condition in aromatic amino acid decarboxylase (AADC) deficiency, a genetic disease leading to severe neurological impairment. Here, by using an appropriate vector, we succeeded in obtaining high yields of AADC protein and characterizing two new heterodimers, T69M/S147R and C281W/M362T, detected in two AADC deficiency patients. We performed an extensive biochemical characterization of the heterodimeric recombinant proteins and of the related homodimers, by a combination of dichroic and fluorescence spectroscopy and activity assays together with bioinformatic analyses. We found that T69M/S147R exhibits negative complementation in terms of activity but it is more stable than the average of the homodimeric counterparts. The heterodimer C281W/M362T retains a nearly good catalytic efficiency, whereas M362T homodimer is less affected and C281W homodimer is recovered as insoluble. These results, which are consistent with the related phenotypes, and the data emerging from previous studies, suggest that the severity of AADC deficiency is not directly explained by positive or negative complementation phenomena, but rather depends on: i) the integrity of one or both active sites; ii) the structural and functional properties of the entire pool of AADC proteins expressed. Overall, this integrated and cross-sectional approach enables proper characterization and depicts the functional result of subunit interactions in the dimeric structure and will help to elucidate the physio-pathological mechanisms in AADC deficiency.

Investigation into the Redox Regulation of the Arabidopsis β‐amylase BAM1

Starch in plants is a large polymer of glucose which stores excess glucose for times of limited photosynthesis such as at night. Starch and derivatives of starch are used in many industrial processes such as laundry and papermaking while also playing a nutritional role for humans and other animals. In plants, regulation of starch storage and degradation is a multifaceted system which allows the plant to adjust carbohydrate storage in response to a variety of environmental and stress conditions. However, we have an incomplete picture of how this process is regulated. β-amylases or BAMs hydrolyze starch to produce maltose, a key carbohydrate in plant metabolism. Plants contain many BAM proteins and each has an apparently unique function and regulatory mechanism, although this is an area of active research with many outstanding questions. Sparla and coworkers proposed that BAM1 from the model plant Arabidopsis thaliana, is regulated by the redox state of two amino acids called cysteine which form a crosslink and decrease activity. The molecular basis for this regulation is not clear and is the focus of this project. Small angle X-ray scattering (SAXS) data were collected on reduced BAM1 and the data analyzed in the program RAW to determine solution dimensions of BAM1 in the reduced state. Preliminary analysis of the SAXS data supports the plausibility of the proposed crosslink. The next step within this project is to determine the effect of forming this disulfide bond on the amylase activity. This would be done by activating the disulfide bond within the BAM1 to test the enzymes activity levels through different activity assays. The permanent crosslinks would allow the redox abilities of BAM1 to be tested. Using a fluorescent activity assay for amylases oxidized and reduced BAM1 activity will be compared to correlate our SAXS structures with biochemical function.

Fluorescent enzyme-coupled activity assay for phenylalanine ammonia-lyases

Phenylalanine ammonia-lyases (PALs) catalyse the non-oxidative deamination of l-phenylalanine to trans-cinnamic acid, while in the presence of high ammonia concentration the reverse reaction occurs. PALs have been intensively studied, however, their industrial applications for amino acids synthesis remained limited, mainly due to their decreased operational stability or limited substrate specificity. The application of extensive directed evolution procedures to improve their stability, activity or selectivity, is hindered by the lack of reliable activity assays allowing facile screening of PAL-activity within large-sized mutant libraries. Herein, we describe the development of an enzyme-coupled fluorescent assay applicable for PAL-activity screens at whole cell level, involving decarboxylation of trans-cinnamic acid (the product of the PAL reaction) by ferulic acid decarboxylase (FDC1) and a photochemical reaction of the produced styrene with a diaryltetrazole, that generates a detectable, fluorescent pyrazoline product. The general applicability of the fluorescent assay for PALs of different origin, as well as its versatility for the detection of tyrosine ammonia-lyase (TAL) activity have been also demonstrated. Accordingly, the developed procedure provides a facile tool for the efficient activity screens of large mutant libraries of PALs in presence of non-natural substrates of interest, being essential for the substrate-specificity modifications/tailoring of PALs through directed evolution-based protein engineering.

Design and in vitro analysis of SIRT2 inhibitor targeting Parkinson's disease.

Inhibition of Sirtuin2 (SIRT2) protein rescues the α-synuclein toxicity in vitro and in vivo models of Parkinson's disease (PD). Thioacetyl group can structurally mimic the acetyl group and restrain the deacetylating p53 reaction by SIRT2. This work evaluated the biological activity of designed pentapeptides inhibitor containing N-thioacetyl-lysine against SIRT2. Pentapeptide by introducing thioacetyl-lysine as an inhibitor of SIRT2 was screened by molecular docking and synthesized by solid phase method. The inhibition of pure recombinant SIRT2 as well as SIRT2 in serum of PD patients by peptide was done by fluorescent activity assay. The inhibition of SIRT2 was assessed in PC12 cell line by measuring acetylated α-tubulin level. The peptide YKK(ε-thioAc)AM and HRK(ε-thioAc)AM were found to be SIRT2 inhibitors by molecular docking. However, YKK(ε-thioAc)AM was more specific towards SIRT2 than SIRT1 (Sirtuin1). It inhibited recombinant SIRT2 by IC50 value of 0.15µM and KD values 9.92 × 10-8/M. It also inhibited serum SIRT2 of PD. It increased the acetylation of α-tubulin in PC12 neuroblastoma cells which is essential for maintaining the microtubular cell functions of brain. It can be concluded that novel peptide YKK(ε-thioAc)AM may be a platform for therapeutic agent for Parkinson's disease targeting SIRT2.

Alkaline Phosphatase Assay Based on the Chromogenic Interaction of Diethanolamine with 4-Aminophenol.

Diethanolamine (DEA) has been extensively utilized as an alkaline buffer in current assays of alkaline phosphatase (ALP) activity in the past decades. While playing the role of a buffer, the chemical reactivity of DEA has been widely ignored in such assays. Herein, we report an interesting chromogenic interaction between DEA and 4-aminophenol (AP) in the presence of H2O for the first time, which inspires us to develop a novel DEA-participated ALP activity assay by using 4-aminophenyl phosphate (APP) as a substrate. This APP/DEA-based colorimetric approach has been proved to be comparable and even superior to the conventional p-nitrophenyl phosphate (pNPP)-based one, especially in the low ALP activity region, due to its higher sensitivity. The clear response mechanism and excellent sensing performance ensure that it can be further applied to determining ALP activity in real biological samples, screening potential ALP inhibitors in vitro, establishing ALP-enabled ELISA, and even fluorophore-assisted fluorescent ALP activity assay. It is demonstrated that this strategy not only possesses a good feasibility, but also exhibits a promising outlook for a series of ALP-related and -extended detections.

Inhibition of Monoamine Oxidases (MAOs) by Green Tea Extracts

Monoamine oxidase (MAO) performs deamination of amines and is found bound to the outer mitochondrial membrane at high‐concentration in neuronal cells. There are two isoforms of MAO: MAO_A which oxidizes serotonin, noradrenaline and adrenaline, and MAO_B which oxidizes dopamine, b‐phenylethylamine (PEA), and benzylamine. Alterations in MAO activity can occur in some central and peripheral nervous system diseases. More specifically, heightened MAO_B activity in the brain occurs in Alzheimer's disease, Huntington's disease, Parkinson's disease and normal aging. Abnormal MAO_A activity has found to be associated with depression, anxiety and psychiatric disorders. Drugs have been developed and continue to be developed for both MAO_A and MAO_B as targets. MAO_A is inhibited by clorgyline and MAO_B is potently inhibited by both deprenyl and pargyline. Using these inhibitors as controls, a fluorescent activity assay was performed with commercially available catechins (green tea extracts), serotonin, and benzylamine substrates for MAO_A and MAO_B respectively, to investigate and confirm recent studies suggesting that green tea catechins (polyphenols) may be preventative for certain degenerative diseases and emotional illnesses utilizing MAOs as a target. Using a fluorescent assay, the Km value of serotonin to MAO_A was 1.75 μM and the Km value of benzylamine to MAO_B was 0.75 μM. The IC50 of clorgyline to MAO_A was 3.25 nM and that of deprenyl to MAO_B was 7.25 nM, in close agreement with the literature. The commercial catechins tested were found to have IC50s in the low‐to‐mid μM range (~50–750 μM). Efforts to purify catechins are underway to repeat these studies. Molecular docking of specific catechins into the MAO_A and MAO_B active sites resulted in binding constants in the low μM range (in agreement with experimentally determined Km values for natural substrates). Crystallization studies of MAO/catechin complexes are in progress.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Development of a novel fluorescent activity assay for lecithin:cholesterol acyltransferase.

Background Lecithin:cholesterol acyltransferase (LCAT) is a plasma enzyme that esterifies cholesterol. Recombinant human LCAT (rhLCAT) is now being developed as an enzyme replacement therapy for familial LCAT deficiency and as a possible treatment for acute coronary syndrome. The current 'gold standard' assay for LCAT activity involves the use of radioisotopes, thus making it difficult for routine clinical use. Methods We have developed a novel and more convenient LCAT activity assay using fluorescence-labelled cholesterol (BODIPY-cholesterol), which is incorporated into proteoliposomes as a substrate instead of radiolabelled cholesterol. Results The apparent Km and Vmax were 31.5 µmol/L and 55.8 nmol/h/nmoL, rhLCAT, respectively, for the 3H-cholesterol method and 103.1 µmol/L and 13.4 nmol/h/nmol rhLCAT, respectively, for the BODIPY-cholesterol method. Although the two assays differed in their absolute units of LCAT activity, there was a good correlation between the two test assays ( r = 0.849, P < 1.6 × 10-7, y = 0.1378x + 1.106). The BODIPY-cholesterol assay had an intra-assay CV of 13.7%, which was superior to the intra-assay CV of 20.8% for the radioisotopic assay. The proteoliposome substrate made with BODIPY-cholesterol was stable to storage for at least 10 months. The reference range ( n = 20) for the fluorescent LCAT activity assay was 4.6-24.1 U/mL/h in healthy subjects. Conclusions In summary, a novel fluorescent LCAT activity assay that utilizes BODIPY-cholesterol as a substrate is described that yields comparable results to the radioisotopic method.

Abstract 2013: Increased MMPs activity in MDM2 overexpressing cancer cell lines

Abstract The matrix metalloprotenases (MMPs) are a family of zinc-dependent endopeptidase and have proteolysis activity which found to impact several physiological and pathophysiological conditions including cancer. In cancer, over 20 types of MMPs promote invasion, angiogenesis, metastasis, and proliferation. For instance, MMP-9 is involved in many processes such as wound healing, tissue remodeling and regulation of inflammation. So far, several studies have also shown that MMP-9 is associated with poor clinical outcome. MMP-9 can facilitate angiogenesis and metastasis by releasing cytokines, pro-angiogenic and pro-metastatic factors. Conversely, the tissue inhibitor of metalloproteinases (TIMP-1) is a well-known negative regulator of MMPs which can also modulate other biological functions independent of MMPs regulation. TIMP-1 dysregulation has been shown to impact ECM integrity and potentiate metastatic ability also. Similarly, MDM2 (Murine Double Minute) has been recognized as an important multi-domain protooncogene which is overexpressed in many types of cancer and is associated with poor prognosis in different tumor types including sarcomas and carcinomas. Previously, our group reported that MDM2 overexpression is regulating MMP-9/TIMP-1 axis in LNCaP and LNCaP-MST (MDM2 transfected) prostate cancer cells. In order to further analyze the status of MMP in MDM2 overexpressing cancers the fluorescence activity assay and the zymography assay were used for measuring the activity of MMPs including MMP-9. Immunoblotting analysis were also used to correlate the expression levels of MDM2, MMP-9, and TIMP-1 in MDM2 overexpressing cell lines with and without Nutlin-3 (20 uM) treatment. Our results indicated that MMP activity is elevated by 97.3 % in LNCaP-MST cells compared to LNCaP. This may be possibly due to the near knockdown levels of TIMP-1 that was observed in LNCaP-MST cells. However, the Nutlin 3 treatment was able to decrease the MMP-9 levels and activity only marginally, without elevating TIMP-1 levels. This suggests that, in addition to MDM2, the PTEN loss in LNCaP-MST cells may also play a role in regulating the levels of TIMP-1 and consequently the activity of MMP-9. Furthermore, in order to verify our original speculation, we analyzed the levels of TIMP-1 in SJSA1 and GI-101A cell lines, which are known to overexpress MDM2 and impart aggressive metastatic abilities. Interestingly, the levels of TIMP-1 were found to be elevated in both SJSA1 and GI-101A cell lines compared to LNCaP-MST cells. This finding indicates that TIMP-1 may paly dual role depending on cancer types and the gene expression status in the tumor microenvironment. Further studies are required to fully delineate the interplay between MDM2 and pro-metastatic mechanisms, including the expression of TIMP-1 and MMP-9. (The financial support from the Royal Dames of Cancer Research Inc., Ft. Lauderdale is gratefully acknowledged). Citation Format: Ali Alaseem, Thiagarajan Venkatesan, Thanigaivelan Kanagasabai, Khalid Alhazzani, Saad Alobid, Priya Dondapati, Appu Rathinavelu. Increased MMPs activity in MDM2 overexpressing cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2013. doi:10.1158/1538-7445.AM2017-2013

Activity-independent screening of secreted proteins using split GFP

The large-scale industrial production of proteins requires efficient secretion, as provided, for instance, by the Sec system of Gram-positive bacteria. Protein engineering approaches to optimize secretion often involve the screening of large libraries, e.g. comprising a target protein fused to many different signal peptides. Respective high-throughput screening methods are usually based on photometric or fluorimetric assays enabling fast and simple determination of enzymatic activities. Here, we report on an alternative method for quantification of secreted proteins based on the split GFP assay. We analyzed the secretion by Bacillus subtilis of a homologous lipase and a heterologous cutinase by determination of GFP fluorescence and enzyme activity assays. Furthermore, we identified from a signal peptide library a variant of the biotechnologically relevant B. subtilis protein swollenin EXLX1 with up to 5-fold increased secretion. Our results demonstrate that the split GFP assay can be used to monitor secretion of enzymatic and non-enzymatic proteins in B. subtilis in a high-throughput manner.

Testing the influence of surfactant-based wound dressings on proteinase activity.

Proteinases are enzymes that can digest other proteins. In chronic wounds, a sub-class of these enzymes with the ability to degrade the extracellular matrix (matrix metalloproteinases, MMPs) have been found to both inhibit healing and to be able to aid in enzymatically debriding a wound. Enzymatic debridement using the enzymes present in a wound is generally called autolytic debridement. Clinicians seeking to employ autolytic debridement typically use occlusive materials such as medical honey, alginate dressings and other occlusive dressings. A relatively new class of gel dressings comprised of surfactants are now available for clinical use. A variety of surfactants are used in the study of MMP biochemistry. Surfactants can deactivate MMPs or can enhance their activity, depending on the surfactant. In order to begin to understand how the MMPs found in chronic wounds would respond to these new dressings, we tested a serial dilution series of two of the currently available surfactant-based dressings to determine their effects on four separate MMPs. The dose-response versus MMP activity of bacterial collagenase, host-derived MMP-8 and MMPs-2 and -9 was assessed using a simple mix-and-read fluorescent peptide activity assay. The enzyme's native activity in the absence of the gel was used to compare against the surfactant-treated samples. We found that the surfactant affected the proteinase activity differently for each enzyme. The activity of the bacterial collagenase was increased at low concentrations but slightly inhibited as the concentrations increased. The host MMP-8 collagenase responded similarly in that it was inhibited at higher concentrations. Interestingly, both MMP gelatinases presented with substantially increased activities, with MMP-2 increased to 200% of native activity, while MMP-9 presented with an increase of 300% activity over the same concentration range. MMPs appear to respond to a surfactant-based gel dressing differentially, with the MMP most commonly elevated in chronic wounds having the highest boost to activity. In wounds with elevated MMPs, our data suggest that the use of these surfactant-based dressings would be expected to enhance the activity of MMPs 2 and 9 gelatinases while simultaneously inhibiting MMP-8 collagenase. Hypothetically, this imbalanced effect would support a protection of the native dermal collagen and removal of denatured materials. However, the demonstration of these anticipated consequences is still being investigated.

Beneficial effect of daidzin in dry eye rat model through the suppression of inflammation and oxidative stress in the cornea.

Present investigation evaluates the effect of daidzin in dry eye rat model through the suppression of inflammation and oxidative stress in the cornea. Briefly, electron spine resonance was used for the estimation of radical scavenging activity of daidzin and COX Fluorescent Activity Assay Kit was used for the estimation of PGS activity. Dry eye rat model was developed by removing the lacrimal gland and effect of daidzin was evaluated in dry eye rat model by estimating the fluorescein score, tear volume and expressions of heme oxigenase (HO-1), TNF α, Interlukin 6 (IL-6), matrix metallopeptidase 9 (MMP-9) and PGS-2. Result of the present study suggested that daidzin possess tyrosyl radical scavenging activity and thereby decreases the oxidative stress. Activity of PGS significantly increases in dry eye which was inhibited by daidzin treatment due to competitive inhibition of PGS. It also recovers the tear volume in dry eye rat model in which lacrimal gland was removed. Thus corneal erosion was improved by daidzin in dry eye rat model. Thus present study concludes that treatment with daidzin protects the cornea in dry eye rat model by suppression inflammation and oxidative stress.

Design, synthesis of allosteric peptide activator for human SIRT1 and its biological evaluation in cellular model of Alzheimer's disease

Sirtuin 1 (SIRT1) is one of the member of the mammalian proteins of the Sirtuin family of NAD+ dependent deacetylases, has recently been shown to attenuate amyloidogenic processing of amyloid protein precursor (APP) in in-vitro cell culture studies and transgenic mouse models of Alzheimer's disease (AD). SIRT1 has been shown to have a protective role against (AD). It has been reported earlier that increasing SIRT1 activity can prevent AD in mice model. Tripeptide as an activator of SIRT1 were screened on the basis of structural information by molecular docking and synthesized by solid phase method. The enhancement of biochemical activity of pure recombinant SIRT1 as well as SIRT1 in serum of AD patients in presence of tripeptide was done by Fluorescent Activity Assay. The activity of SIRT1 by peptide was assessed in IMR-32 cell line by measuring acetylated p53 level. Further the protective effect of SIRT1 activator in cellular model of AD was analyzed by MTT assay. We find CWR tripeptide as a SIRT1 activator by molecular docking, enhanced the activity of SIRT1 protein by lowering the Michaelis constant, Km by allosteric mechanism. The activity of serum SIRT1 of AD was also increases by CWR. It also decreased the acetylation of p53 in IMR32 neuroblastoma cells and protected the cell death caused by Aβ amyloid fragments in cell line model of AD. Thus, it can be concluded that CWR may serve as platform to elucidate further small molecule activator as a therapeutic agent for AD targeting SIRT1.

The proteasome regulates collagen-induced platelet aggregation via nuclear-factor-kappa-B (NFĸB) activation

IntroductionPlatelets possess critical hemostatic functions in the system of thrombosis and hemostasis, which can be affected by a multitude of external factors. Previous research has shown that platelets have the capacity to synthesize proteins de novo and more recently a multicatalytic protein complex, the proteasome, has been discovered in platelets. Due to its vital function for cellular integrity, the proteasome has become a therapeutic target for anti-proliferative drug therapies in cancer. Clinically thrombocytopenia is a frequent side-effect, but the aggregatory function of platelets also appears to be affected. Little is known however about underlying regulatory mechanisms and functional aspects of proteasome inhibition on platelets. Our study aims to investigate the role of the proteasome in regulating collagen-induced platelet aggregation and its interaction with NFkB in this context. Material and methodsUsing fluorescence activity assays, platelet aggregometry and immunoblotting, we investigate regulatory interactions of the proteasome and Nuclear-factor-kappa-B (NFkB) in collagen-induced platelet aggregation. ResultsWe show that collagen induces proteasome activation in platelets and collagen-induced platelet aggregation can be reduced with proteasome inhibition by the specific inhibitor epoxomicin. This effect does not depend on Rho-kinase/ROCK activation or thromboxane release, but rather depends on NFkB activation. Inhibition of the proteasome prevented cleavage of NFκB-inhibitor protein IκBα and decreased NFκB activity after collagen stimulation. Inhibition of the NFκB-pathway in return reduced collagen-induced platelet proteasome activity and cleavage of proteasome substrates. ConclusionsThis work offers novel explanations how the proteasome influences collagen-dependent platelet aggregation by involving non-genomic functions of NFkB.

Mechanism of antiproliferative action of a new d-secoestrone-triazole derivative in cervical cancer cells and its effect on cancer cell motility

Cervical cancer is the fourth most frequently diagnosed tumor and the fourth leading cause of cancer death in females worldwide. Cervical cancer is predominantly related with human papilloma virus (HPV) infection, with the most oncogenic types being HPV-18 and -16. Our previous studies demonstrated that some d-secoestrone derivatives exert pronounced antiproliferative activity. The aim of the current investigation was to characterize the mechanism of action of d-secoestrone-triazole (D-SET) on three cervical cancer cell lines with different pathological backgrounds.The growth-inhibitory effects of D-SET were determined by a standard MTT assay. We have found that D-SET exerts a pronounced growth-inhibitory effect on HPV 18-positive HeLa and HPV-negative C-33 A cells, but it has no substantial inhibitory activity on HPV 16-positive SiHa or on intact fibroblast MRC-5 cell lines. After 24h incubation, cells showed the morphological and biochemical signs of apoptosis determined by fluorescent double staining, flow cytometry and caspase-3 activity assay. Besides the elevation of the ratio of cells in the subG1 phase, flow cytometric analysis revealed a cell cycle arrest at G2/M in both HeLa and C-33 A cell lines. To distinguish the G2/M cell population immunocytochemical flow cytometric analysis was performed on HeLa cells. The results show that D-SET significantly increases the ratio of phosphorylated histone H3, indicating cell accumulation in the M phase. Additionally, D-SET significantly increased the maximum rate of microtube formation measured by an in vitro tubulin polymerization assay. Besides its direct antiproliferative activity, the antimigratory property of D-SET has been investigated. Our results demonstrate that D-SET significantly inhibits the migration and invasion of HeLa cells after 24h incubation.These results suggests that D-SET is a potent antiproliferative agent against HPV 16+ and HPV-negative cervical cancer cell lines, with an efficacious motility-inhibiting activity against HPV 16+ cells. Accordingly D-SET can be regarded as a potential drug candidate with a promising new mechanism of action among the antiproliferative steroids, potentially allowing for the design of novel anticancer agents.

Single-column purification of the tag-free, recombinant form of the neuronal calcium sensor protein, hippocalcin expressed in Escherichia coli

Hippocalcin is a 193 aa protein that is a member of the neuronal calcium sensor protein family, whose functions are regulated by calcium. Mice that lack the function of this protein are compromised in the long term potentiation aspect of memory generation. Recently, mutations in the gene have been linked with dystonia in human. The protein has no intrinsic enzyme activity but is known to bind to variety of target proteins. Very little information is available on how the protein executes its critical role in signaling pathways, except that it is regulated by binding of calcium. Further delineation of its function requires large amounts of pure protein. In this report, we present a single-step purification procedure that yields high quantities of the bacterially expressed, recombinant protein. The procedure may be adapted to purify the protein from inclusion bodies or cytosol in its myristoylated or non-myristoylated forms. MALDI-MS (in source decay) analyses demonstrates that the myristoylation occurs at the glycine residue. The protein is also biologically active as measured through tryptophan fluorescence, mobility shift and guanylate cyclase activity assays. Thus, further analyses of hippocalcin, both structural and functional, need no longer be limited by protein availability.