Functional Study of the Vitamin K Cycle Enzymes in Live Cells.

Abstract:

:Vitamin K-dependent carboxylation, an essential posttranslational modification catalyzed by gamma-glutamyl carboxylase, is required for the biological functions of proteins that control blood coagulation, vascular calcification, bone metabolism, and other important physiological processes. Concomitant with carboxylation, reduced vitamin K (KH2) is oxidized to vitamin K epoxide (KO). KO must be recycled back to KH2 by the enzymes vitamin K epoxide reductase and vitamin K reductase in a pathway known as the vitamin K cycle. Our current knowledge about the enzymes of the vitamin K cycle is mainly based on in vitro studies of each individual enzymes under artificial conditions, which are of limited usefulness in understanding how the complex carboxylation process is carried out in the physiological environment. In this chapter, we review the current in vitro activity assays for vitamin K cycle enzymes. We describe the rationale, establishment, and application of cell-based assays for the functional study of these enzymes in the native cellular milieu. In these cell-based assays, different vitamin K-dependent proteins were designed and stably expressed in mammalian cells as reporter proteins to accommodate the readily used enzyme-linked immunosorbent assay for carboxylation efficiency evaluation. Additionally, recently emerged genome-editing techniques TALENs and CRISPR-Cas9 were used to knock out the endogenous enzymes in the reporter cell lines to eliminate the background. These cell-based assays are easy to scale up for high-throughput screening of inhibitors of vitamin K cycle enzymes and have been successfully used to clarify the genotypes and their clinical phenotypes of enzymes of the vitamin K cycle.

journal_name

Methods Enzymol

journal_title

Methods in enzymology

authors

Tie JK,Stafford DW

doi

10.1016/bs.mie.2016.10.015

subject

Has Abstract

pub_date

2017-01-01 00:00:00

pages

349-394

eissn

0076-6879

issn

1557-7988

pii

S0076-6879(16)30379-2

journal_volume

584

pub_type

杂志文章
  • Methods to monitor ROS production by fluorescence microscopy and fluorometry.

    abstract::Mitochondria are considered one of the main sources of reactive oxygen species (ROS). The overgeneration of ROS can evoke an intracellular state of oxidative stress, leading to permanent cell damage. Thus, the intracellular accumulation of ROS may not only disrupt the functions of specific tissues and organs but also ...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-416618-9.00013-3

    authors: Wojtala A,Bonora M,Malinska D,Pinton P,Duszynski J,Wieckowski MR

    更新日期:2014-01-01 00:00:00

  • Protein-fragment complementation assays (PCA) in small GTPase research and drug discovery.

    abstract::Small GTPases of the Ras and Rho families are among the most studied signaling proteins and represent promising therapeutic targets for human neoplastic disease. Despite the high level of interest in these proteins, direct analysis of most aspects of Ras protein biology in living cells has not been possible, because m...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(05)07032-1

    authors: Westwick JK,Michnick SW

    更新日期:2006-01-01 00:00:00

  • Measurement of dynamic protein binding to chromatin in vivo, using photobleaching microscopy.

    abstract::We have described procedures for collecting, processing, and analyzing kinetic data obtained by photobleaching microscopy of GFP-tagged chromatin proteins in nuclei of cultured living cells. These procedures are useful for characterizing the in vivo binding of chromatin proteins to their natural template--unperturbed,...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/s0076-6879(03)75025-3

    authors: Phair RD,Gorski SA,Misteli T

    更新日期:2004-01-01 00:00:00

  • Computational methods in tumor immunology.

    abstract::The remarkable success of cancer immunotherapies, especially the checkpoint blocking antibodies, in a subset of patients has reinvigorated the study of tumor-immune crosstalk and its role in heterogeneity of response. High-throughput sequencing and imaging technologies can help recapitulate various aspects of the tumo...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2020.01.001

    authors: Bhinder B,Elemento O

    更新日期:2020-01-01 00:00:00

  • GTPases and the control of neuronal polarity.

    abstract::Neurons are probably the most highly polarized cell type and typically develop a single axon and several dendrites. The establishment of a polarized morphology and the functional specialization of axonal and dendritic compartments are essential steps in the differentiation of neurons. Primary cultures of dissociated h...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(06)06056-3

    authors: Schwamborn JC,Li Y,Püschel AW

    更新日期:2006-01-01 00:00:00

  • Forward genetic screens to identify circadian rhythm mutants in mice.

    abstract::This article describes the methods and techniques used to produce mutagenized mice to conduct high-throughput forward genetic screens for circadian rhythm mutants in the mouse. In particular, we outline methods to safely prepare and administer the chemical mutagen N-nitroso-N-ethylurea (ENU) to mice. We also discuss t...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(05)93007-3

    authors: Siepka SM,Takahashi JS

    更新日期:2005-01-01 00:00:00

  • Prediction of alpha helices and T cell-presented sequences in proteins with algorithms based on strip-of-helix hydrophobicity index.

    abstract::Recurrent aliphatic hydrophobic amino acids which occur in the sequence of a protein or a peptide at positions which form an axial, hydrophobic strip when the sequence is coiled as an alpha helix might stabilize coiling against hydrophobic surfaces. That effect can lead to helix formation against hydrophobic cores of ...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/0076-6879(91)02013-y

    authors: Reyes VE,Lew RA,Lu S,Humphreys RE

    更新日期:1991-01-01 00:00:00

  • Reconstituting Plant Secondary Metabolism in Saccharomyces cerevisiae for Production of High-Value Benzylisoquinoline Alkaloids.

    abstract::Benzylisoquinoline alkaloids (BIAs) constitute a diverse class of plant secondary metabolites that includes the opiate analgesics morphine and codeine. Collectively, BIAs exhibit a myriad of pharmacological activities, including antimicrobial, antitussive, antispasmodic, and anticancer properties. Despite 2500 known B...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2016.02.011

    authors: Pyne ME,Narcross L,Fossati E,Bourgeois L,Burton E,Gold ND,Martin VJ

    更新日期:2016-01-01 00:00:00

  • Modeling small molecule-compound binding to G-protein-coupled receptors.

    abstract::G-protein-coupled receptors (GPCRs) form a superfamily of membrane proteins that play a crucial role in mediating physiological processes as well as pathogenesis of many critical diseases. They are one of the most successful drug targets, accounting for more than 30% of prescription drugs on the market today. Three-di...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(09)05213-6

    authors: Vaidehi N,Pease JE,Horuk R

    更新日期:2009-01-01 00:00:00

  • Quantitative polymerase chain reaction and solid-phase capture nucleic acid detection.

    abstract::The combination of PCR amplification and chemiluminescent detection of PCR products provides a highly sensitive system for the quantitation of DNA and RNA. The broad dynamic range of the chemiluminescent detection assay simplifies the selection of cycling and concentration parameters critical to harnessing the quantit...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/s0076-6879(00)05507-5

    authors: Martin CS,Voyta JC,Bronstein I

    更新日期:2000-01-01 00:00:00

  • Current standards, variations, and pitfalls for the determination of constitutive TSHR activity in vitro.

    abstract::Constitutively activating mutations of the TSHR are the major cause for nonautoimmune hyperthyroidism, which is based on ligand independent, permanent receptor activation. Several reports have highlighted the difficulties to determine whether a TSHR mutation is constitutively active or not especially for borderline ca...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-381296-4.00023-3

    authors: Mueller S,Jaeschke H,Paschke R

    更新日期:2010-01-01 00:00:00

  • Whole-genome genotyping.

    abstract::We have developed an array-based whole-genome genotyping (WGG) assay (Infinium) using our BeadChip platform that effectively enables unlimited multiplexing and unconstrained single nucleotide polymorphism (SNP) selection. A single tube whole-genome amplification reaction is used to amplify the genome, and loci of inte...

    journal_title:Methods in enzymology

    pub_type: 杂志文章,评审

    doi:10.1016/S0076-6879(06)10017-8

    authors: Gunderson KL,Steemers FJ,Ren H,Ng P,Zhou L,Tsan C,Chang W,Bullis D,Musmacker J,King C,Lebruska LL,Barker D,Oliphant A,Kuhn KM,Shen R

    更新日期:2006-01-01 00:00:00

  • Computational methods for high-throughput comparative analyses of natural microbial communities.

    abstract::One of the most widely employed methods in metagenomics is the amplification and sequencing of the highly conserved ribosomal RNA (rRNA) genes from organisms in complex microbial communities. rRNA surveys, typically using the 16S rRNA gene for prokaryotic identification, provide information about the total diversity a...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-407863-5.00018-6

    authors: Preheim SP,Perrotta AR,Friedman J,Smilie C,Brito I,Smith MB,Alm E

    更新日期:2013-01-01 00:00:00

  • Study of Chemotaxis and Cell-Cell Interactions in Cancer with Microfluidic Devices.

    abstract::Microfluidic devices have very broad applications in biological assays from simple chemotaxis assays to much more complicated 3D bioreactors. In this chapter, we describe the design and methods for performing chemotaxis assays using simple microfluidic chemotaxis chambers. With these devices, using real-time video mic...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2015.09.023

    authors: Sai J,Rogers M,Hockemeyer K,Wikswo JP,Richmond A

    更新日期:2016-01-01 00:00:00

  • Quantifying chromatin-associated interactions: the HI-FI system.

    abstract::Chromatin plays a vital role in regulating cellular processes that occur on the DNA. Modulation of chromatin structure is conducted through interactions with binding factors that direct critical actions such as posttranslational modifications, nucleosome remodeling, and incorporation of histone variants. Specific fact...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-391940-3.00011-1

    authors: Winkler DD,Luger K,Hieb AR

    更新日期:2012-01-01 00:00:00

  • Assessment and Impacts of Phosphorylation on Protein Flexibility of the α-d-Phosphohexomutases.

    abstract::Enzymes in the α-d-phosphohexomutase (PHM) superfamily catalyze a multistep reaction, entailing two successive phosphoryl transfers. Key to this reaction is a conserved phosphoserine in the active site, which serves alternately as a phosphoryl donor and acceptor during the catalytic cycle. In addition to its role in t...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2018.04.003

    authors: Stiers KM,Beamer LJ

    更新日期:2018-01-01 00:00:00

  • A Guide to Differential Scanning Calorimetry of Membrane and Soluble Proteins in Detergents.

    abstract::Differential scanning calorimetry (DSC) detects protein thermal unfolding by directly measuring the heat absorbed. Simple DSC experiments that require relatively small amounts of pure material can provide a wealth of information related to structure, especially with respect to domain architecture, without the need for...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2015.08.014

    authors: Yang Z,Brouillette CG

    更新日期:2016-01-01 00:00:00

  • Controlling expression of genes in the unicellular alga Chlamydomonas reinhardtii with a vitamin-repressible riboswitch.

    abstract::Chloroplast genomes of land plants and algae contain generally between 100 and 150 genes. These genes are involved in plastid gene expression and photosynthesis and in various other tasks. The function of some chloroplast genes is still unknown and some of them appear to be essential for growth and survival. Repressib...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2014.10.035

    authors: Ramundo S,Rochaix JD

    更新日期:2015-01-01 00:00:00

  • Reactions of nitric oxide and oxygen with the regulator of fumarate and nitrate reduction, a global transcriptional regulator, during anaerobic growth of Escherichia coli.

    abstract::The Escherichia coli fumarate and nitrate reductase (FNR) regulator protein is an important transcriptional regulator that controls the expression of a large regulon of more than 100 genes in response to changes in oxygen availability. FNR is active when it acquires a [4Fe-4S](2+) cluster under anaerobic conditions. T...

    journal_title:Methods in enzymology

    pub_type: 杂志文章,评审

    doi:10.1016/S0076-6879(07)37011-0

    authors: Crack JC,Le Brun NE,Thomson AJ,Green J,Jervis AJ

    更新日期:2008-01-01 00:00:00

  • A Tethered Vesicle Assay for High-Throughput Quantification of Membrane Fission.

    abstract::Membrane fission, which divides membrane surfaces into separate compartments, is essential to diverse cellular processes including membrane trafficking and cell division. Quantitative assays are needed to elucidate the physical mechanisms by which proteins drive membrane fission. Toward this goal, several experimental...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2018.08.014

    authors: Snead WT,Stachowiak JC

    更新日期:2018-01-01 00:00:00

  • Studying RNA-RNA and RNA-protein interactions by isothermal titration calorimetry.

    abstract::Isothermal Titration Calorimetry (ITC) provides a sensitive and accurate means by which to study the thermodynamics of RNA folding, RNA binding to small molecules, and RNA-protein interactions. The advent of extremely sensitive instrumentation and the increasing availability of ITC in shared facilities have made it in...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(09)68019-8

    authors: Feig AL

    更新日期:2009-01-01 00:00:00

  • Liposome-mediated therapy of neuroblastoma.

    abstract::Neuroblastoma (NB) is the most common extracranial solid tumor in childhood and the most frequently diagnosed neoplasm during infancy. Despite of aggressive treatment strategies, the 5-year survival rate for metastatic disease is still less than 60% and, consequently, novel therapeutic approaches are needed. For incre...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(09)65012-6

    authors: Di Paolo D,Loi M,Pastorino F,Brignole C,Marimpietri D,Becherini P,Caffa I,Zorzoli A,Longhi R,Gagliani C,Tacchetti C,Corti A,Allen TM,Ponzoni M,Pagnan G

    更新日期:2009-01-01 00:00:00

  • Phylogenetic analysis and experimental approaches to study color vision in vertebrates.

    abstract::To elucidate the molecular mechanisms of vertebrate color vision, it is essential to establish associations between amino acid substitutions and the directions of lambda max shifts of visual pigments. In this way, we can identify critical amino acid changes that may be responsible for lambda max shifts of visual pigme...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/s0076-6879(00)15851-3

    authors: Yokoyama S

    更新日期:2000-01-01 00:00:00

  • A guide for drug inducible transcriptional activation with HIT systems.

    abstract::Precise investigation and manipulation of gene function often require modulation in a controlled and dynamic manner. In this chapter, we describe the methods to apply HIT systems for drug inducible transcriptional activation or simultaneous activation and genome editing in human cells. Together with those for editing,...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2019.02.032

    authors: Zhao C,Wei S,Wang Y

    更新日期:2019-01-01 00:00:00

  • The delitto perfetto approach to in vivo site-directed mutagenesis and chromosome rearrangements with synthetic oligonucleotides in yeast.

    abstract::In vivo genome manipulation through site-directed mutagenesis and chromosome rearrangements has been hindered by the difficulty in achieving high frequencies of targeting and the intensive labor required to create altered genomes that do not contain any heterologous sequence. Here we describe our approach, referred to...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(05)09019-1

    authors: Storici F,Resnick MA

    更新日期:2006-01-01 00:00:00

  • Cas9-based genome editing in zebrafish.

    abstract::Genome editing using the Cas9 endonuclease of Streptococcus pyogenes has demonstrated unprecedented efficacy and facility in a wide variety of biological systems. In zebrafish, specifically, studies have shown that Cas9 can be directed to user-defined genomic target sites via synthetic guide RNAs, enabling random or h...

    journal_title:Methods in enzymology

    pub_type: 杂志文章,评审

    doi:10.1016/B978-0-12-801185-0.00018-0

    authors: Gonzales AP,Yeh JR

    更新日期:2014-01-01 00:00:00

  • Real-time cell assays of phospholipase A(2)s using fluorogenic phospholipids.

    abstract::Phospholipase A(2)s (PLA(2)s) are a superfamily of enzymes involved in production of a wide variety of lipid mediators, including arachidonic acid, lysophospholipids, platelet activation factor, and eicosanoids. Fluorescence-based, real-time cellular activity assays for PLA(2)s have been developed as a tool for studyi...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(07)34002-0

    authors: Manna D,Cho W

    更新日期:2007-01-01 00:00:00

  • Metal-free chromatographic media.

    abstract::The three major considerations in reducing metal-gel binding are (1) the type of gel to be used, (2) the ionic strength of the eluent, and (3) alkaline reduction of the gel. Each of these parameters should be considered in advance of an experiment. A method is offered for determining the optimal gel type, gel treatmen...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/0076-6879(88)58044-8

    authors: Martin MT

    更新日期:1988-01-01 00:00:00

  • Measurement of ROS homeostasis in isolated mitochondria.

    abstract::In this chapter, we describe the currently most advanced methods applied for the quantitative assessment of ROS homeostasis inside the mitochondrion. These techniques are of particular interest in the field of oxidative stress. After discussing the importance of quantifying mitochondrial ROS homeostasis, three major a...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-801415-8.00012-6

    authors: Tretter L,Ambrus A

    更新日期:2014-01-01 00:00:00

  • Comparative Biochemistry and In Vitro Pathway Reconstruction as Powerful Partners in Studies of Metabolic Diversity.

    abstract::There are estimated to be >300,000 plant species, producing >200,000 metabolites. Many of these metabolites are restricted to specific plant lineages and are referred to as "specialized" metabolites. These serve varied functions in plants including defense against biotic and abiotic stresses, plant-plant and plant-mic...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2016.02.023

    authors: Fan P,Moghe GD,Last RL

    更新日期:2016-01-01 00:00:00