Abstract:
:Classifications and characterizations of specific proteins, such as enzymes, not only allow us to understand biosynthetic and metabolic pathways but they also help to drive our understanding of protein structure and function. How those characterizations are evaluated, however, may change our interpretations and lead us into broader and novel directions in research. Here, we will make the argument that using lipidomics as a tool for characterizing enzymatic function over more traditional toolkit options allows for these types of revelations. Using lipidomics techniques on specific brain regions with a series of enzyme knockout and disease models, we have generated a novel set of analyses from which to view protein function. Through these data, we have demonstrated that NAPE-PLD, MAG lipase, and FAAH all have broader roles throughout the brain than previously thought. Much like the data on how the extinction of specific species within an ecosystem has unpredicted outcomes, so too does the elimination of these enzymes affect the brain lipidome. From a purely biochemical standpoint, it is a fascinating story of how one change in a system can have exponential effects; however, from a drug-target standpoint, it may prove to be a cautionary tale.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
Bradshaw HB,Leishman Edoi
10.1016/bs.mie.2017.06.024subject
Has Abstractpub_date
2017-01-01 00:00:00pages
123-141eissn
0076-6879issn
1557-7988pii
S0076-6879(17)30181-7journal_volume
593pub_type
杂志文章abstract::This chapter provides the methodologies employed to study the polymorphism of human apoE. These and other related studies have advanced our understanding of the structure and function of this protein as follows: The complex array of human apoE observed by two-dimensional gel electrophoresis results from genetic variat...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/0076-6879(86)28109-4
更新日期:1986-01-01 00:00:00
abstract::Mass spectrometry (MS) has gradually replaced classical methods as a major tool in protein sequencing and characterization. However, the sample preparation repertoire has not changed very much; it has just been adjusted to the needs of the new analytical method. In this chapter frequently used in-solution enzymatic di...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)05003-2
更新日期:2005-01-01 00:00:00
abstract::Ultraviolet photodissociation time-of-flight (TOF) mass spectrometry (MS) is described as a method for determination of peptide ion primary structure. Monoisotopic selection and bond-specific activation, combined with the rapidity of TOF MS analysis, render this technique invaluable to the rapidly expanding field of p...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(05)02006-9
更新日期:2005-01-01 00:00:00
abstract::The Omega class of cytosolic glutathione transferases was initially recognized by bioinformatic analysis of human sequence databases, and orthologous sequences were subsequently discovered in mouse, rat, pig, Caenorhabditis elegans, Schistosoma mansoni, and Drosophila melanogaster. In humans and mice, two GSTO genes h...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(05)01005-0
更新日期:2005-01-01 00:00:00
abstract::Scanning transmission electron microscopy (STEM) of unstained, freeze-dried biological macromolecules in the dark-field mode provides an image based on the number of electrons elastically scattered by the constituent atoms of the macromolecule. The image of each isolated particle provides information about the project...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(08)36027-3
更新日期:2008-01-01 00:00:00
abstract::Aliphatic carbon-halogen (C-X) bonds are prevalent in modern pharmaceuticals and bioactive natural products. Three distinct chemical strategies are known in Nature to generate these structural motifs. The first is via the nucleophilic substitution at a prefunctionalized electrophilic carbon center with a halide anion ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2018.02.015
更新日期:2018-01-01 00:00:00
abstract::Reviewing the circumstances that have led to the first monoclonal antibody against the disease-associated form of PrP, we consider the availability of PrP knockout mice and recombinant PrP, as well as a reliable conformational screening protocol as being important prerequisites for a successful immunization approach. ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/s0076-6879(99)09010-2
更新日期:1999-01-01 00:00:00
abstract::We describe a method for molecular confinement and single-fluorophore sensitive measurement in aqueous nanodroplets in oil. The sequestration of individual molecules in droplets has become a useful tool in genomics and molecular evolution. Similarly, the use of single fluorophores, or pairs of fluorophores, to study b...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(10)72015-2
更新日期:2010-01-01 00:00:00
abstract::Microscopy is a simple, direct technique for examining the morphology of cells and their organelles. Embedding cells in agarose and then in paraffin as 'cell blocks' allows for them to be processed in the same manner that tissue specimens are processed for histology. This method is advantageous because numerous sectio...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-420067-8.00018-0
更新日期:2013-01-01 00:00:00
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
更新日期:2006-01-01 00:00:00
abstract::Integrin adhesion receptors mediate cell-cell and cell-substratum adhesion and provide a continuous link for the bidirectional transmission of mechanical force and biochemical signals across the plasma membrane. Integrin-dependent cellular activities such as adhesion, migration, proliferation, and survival rely upon t...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(07)26004-5
更新日期:2007-01-01 00:00:00
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
更新日期:1988-01-01 00:00:00
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
更新日期:2004-01-01 00:00:00
abstract::Hormone, hormone-like substances, and neuroendocrine peptides are natural constituents of milk, and they may have an important physiological and pharmacological role in neonate development. The concentration of these peptides in milk greatly exceeds those in serum and implies an active concentration mechanism in the m...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/0076-6879(89)68038-x
更新日期:1989-01-01 00:00:00
abstract::Heterodimeric complexes between individual members of the R7 subfamily of regulators of G-protein signaling proteins and the Gbeta5 isoform of the heterotrimeric G-protein beta subunit family are strongly expressed in the cell nucleus in neurons and brain, as well as in the cytoplasm and plasma membrane. Native and re...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(04)90014-6
更新日期:2004-01-01 00:00:00
abstract::The technique of site-directed spin labeling (SDSL) provides unique information on biomolecules by monitoring the behavior of a stable radical tag (i.e., spin label) using electron paramagnetic resonance (EPR) spectroscopy. In this chapter, we describe an approach in which SDSL is integrated with computational modelin...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2015.07.007
更新日期:2015-01-01 00:00:00
abstract::Reliable modeling of RNA tertiary structures is key to both understanding these structures' roles in complex biological machines and to eventually facilitating their design for molecular computing and robotics. In recent years, a concerted effort to improve computational prediction of RNA structure through the RNA-Puz...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2014.10.051
更新日期:2015-01-01 00:00:00
abstract::Hexameric helicases couple the energy of ATP hydrolysis to processive movement along nucleic acids and are critical components of cells and many viruses. Molecular motion derives from ATP hydrolysis at up to six distinct catalytic centers, which is coupled to the coordinated action of translocation loops in the center...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-396546-2.00008-5
更新日期:2012-01-01 00:00:00
abstract::Nonribosomal peptides are natural products typically of bacterial and fungal origin. These highly complex molecules display a broad spectrum of biological activities, and have been exploited for the development of immunosuppressant, antibiotic, anticancer, and other therapeutic agents. The nonribosomal peptides are as...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(09)04814-9
更新日期:2009-01-01 00:00:00
abstract::This protocol describes a method for determining the concentration of a nucleic acid sample. ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-420037-1.00018-X
更新日期:2013-01-01 00:00:00
abstract::Modeling enzymatic reactions is a demanding task due to the complexity of the system, the many degrees of freedom involved and the complex, chemical, and conformational transitions associated with the reaction. Consequently, enzymatic reactions are not determined by precisely one reaction pathway. Hence, it is benefic...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/bs.mie.2016.05.025
更新日期:2016-01-01 00:00:00
abstract::Enzymology is approaching an era where many problems can benefit from computational studies. While ample challenges remain in quantitatively predicting behavior for many enzyme systems, the insights that often come from computations are an important asset for the enzymology community. Here we provide a primer for enzy...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2016.05.016
更新日期:2016-01-01 00:00:00
abstract::The specificity of CRISPR-Cas systems for nucleic acid targets is determined by a combination of binding and cleavage. Understanding the mechanisms by which Cas proteins specifically select their targets is critical for the development of CRISPR-Cas systems for biotechnology applications. Moreover, the specificity of ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2018.10.019
更新日期:2019-01-01 00:00:00
abstract::Epitope tagging has provided a useful experimental strategy with widespread applicability. The ample variety of epitope tag systems that have been put to use to date provide a collection of attributes relevant to virtually any experimental system. As a consequence, epitope tagging will continue to be a valuable tool f...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/s0076-6879(00)27263-7
更新日期:2000-01-01 00:00:00
abstract::We have described a method using polyvalent peptide display on filamentous phage that can be used to identify ligands that bind to G protein beta gamma subunits. Also described is how to construct phage that have known beta gamma binding sequences fused to the coat protein to allow a competition analysis to be perform...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/s0076-6879(02)44740-4
更新日期:2002-01-01 00:00:00
abstract::This chapter presents methodologies for RNA extraction from soils coupled with competitive reverse transcription-polymerase chain reaction and capillary electrophoresis techniques. Combined, these approaches provide new capabilities to quantify gene expression in different environments and can aid our understanding of...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)97019-5
更新日期:2005-01-01 00:00:00
abstract::Rab7, a member of the Rab family of small G proteins, has been shown to regulate late endocytic traffic and lysosome biogenesis, but the exact roles and the mode of actions of Rab7 are still undetermined. Accumulating evidence suggests that each Rab protein has multiple target proteins and works together with them to ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)03059-4
更新日期:2005-01-01 00:00:00
abstract::A- and B-type lamins support the nuclear envelope, contribute to heterochromatin organization, and regulate a myriad of nuclear processes. The mechanisms by which lamins function in different cell types and the mechanisms by which lamin mutations cause over a dozen human diseases (laminopathies) remain unclear. The id...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2015.08.008
更新日期:2016-01-01 00:00:00
abstract::Plots of free energy versus temperature are commonly called stability curves or Gibbs-Helmholtz curves, and they have proven to be extremely useful in protein folding and ligand-binding studies. Curvature in a Gibbs-Helmholtz or stability plot is indicative of a heat capacity change, and some of their primary uses in ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-381268-1.00009-4
更新日期:2011-01-01 00:00:00
abstract::Molecular mechanisms of dark-to-light state transitions in flavoprotein photoreceptors have been the subject of intense investigation. Blue-light sensing flavoproteins fall into three general classes that share aspects of their activation processes: LOV domains, BLUF proteins, and cryptochromes. In all cases, light-in...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2019.03.023
更新日期:2019-01-01 00:00:00