TY - BOOK AB - Histone proteins are subjected to numerous modifications that are being catalysed by enzymes known as ‘writers’, and this phenomenon constitutes one of the main epigenetic mechanisms through which the cell regulates DNA-based processes, ultimately impacting cellular phenotypes. One such writer is N-terminal acetyltransferase 40 (NAA40) which mediates the N-terminal acetylation (Nt-Ac) of histones H4 and H2A. This epigenetic modifier belongs in the N-terminal acetyltransferase (NAT) family of enzymes, which altogether target approximately 80 % of all eukaryotic proteins. NAA40 is thought to mediate this histone modification co-translationally when associated with the ribosomes, but recent work has also suggested that NAA40 acts within the nucleus. Hence in this study, we performed affinity purification coupled to mass spectrometry (AP-MS), aiming to identify nuclear protein interactions of NAA40 in an attempt to further characterise its function within the nuclear compartment. The AP-MS described here can be applied to other NAT enzymes to enhance the understanding on their molecular functions and that of their corresponding protein N-terminal acetylation. AU - Klavaris, A.* AU - König, A.-C. AU - Demetriades, C.* AU - Hauck, S.M. AU - Kirmizis, A.* C1 - 75232 C2 - 57865 SP - 259-282 TI - Affinity purification-mass spectrometry to identify nuclear protein interactions of N-terminal acetyltransferase NAA40. JO - Methods Enzymol. VL - 719 PY - 2025 SN - 0076-6879 ER - TY - JOUR AB - Understanding the structure and dynamics of biological macromolecules is essential to decipher the molecular mechanisms that underlie cellular functions. The description of structure and conformational dynamics often requires the integration of complementary techniques. In this review, we highlight the utility of combining nuclear magnetic resonance (NMR) spectroscopy with small angle scattering (SAS) to characterize these challenging biomolecular systems. NMR can assess the structure and conformational dynamics of multidomain proteins, RNAs and biomolecular complexes. It can efficiently provide information on interaction surfaces, long-distance restraints and relative domain orientations at residue-level resolution. Such information can be readily combined with high-resolution structural data available on subcomponents of biomolecular assemblies. Moreover, NMR is a powerful tool to characterize the dynamics of biomolecules on a wide range of timescales, from nanoseconds to seconds. On the other hand, SAS approaches provide global information on the size and shape of biomolecules and on the ensemble of all conformations present in solution. Therefore, NMR and SAS provide complementary data that are uniquely suited to investigate dynamic biomolecular assemblies. Here, we briefly review the type of data that can be obtained by both techniques and describe different approaches that can be used to combine them to characterize biomolecular assemblies. We then provide guidelines on which experiments are best suited depending on the type of system studied, ranging from fully rigid complexes, dynamic structures that interconvert between defined conformations and systems with very high structural heterogeneity. AU - Delhommel, F. AU - Martinez Lumbreras, S. AU - Sattler, M. C1 - 66935 C2 - 53356 CY - 525 B Street, Suite 1900, San Diego, Ca 92101-4495 Usa SP - 263-297 TI - Combining NMR, SAXS and SANS to characterize the structure and dynamics of protein complexes. JO - Methods Enzymol. VL - 678 PB - Elsevier Academic Press Inc PY - 2023 SN - 0076-6879 ER - TY - JOUR AB - The 17β-hydroxysteroid dehydrogenase type 1 (HSD17B1) has a key role in estrogen biosynthesis as it catalyzes the reduction of estrone to the most potent estrogen, estradiol. Estradiol has a high affinity for estrogen receptors and thus stimulates their transactivation, which leads to cell proliferation and numerous other effects. HSD17B2 catalyzes the oxidation of estradiol to the less potent estrone, thereby decreasing estrogen receptor activation, which results in reduction of estrogen-associated effects. HSD17B1 and HSD17B2 overexpressing E.coli homogenates or recombinant enzymes can be used for screening and development of drugs against various pathologies such as cancer, endometriosis or osteoporosis. Here we describe the preparation of HSD17B1 and HSD17B2 bacterial homogenates and purified recombinant HSD17B1 protein as enzyme sources as well as enzymatic assays based on radiometric and mass-spectrometric detection for enzyme characterization. AU - Sinreih, M.* AU - Gjorgoska, M.* AU - Möller, G. AU - Adamski, J. AU - Rizner, T.L.* C1 - 68553 C2 - 54894 CY - 525 B Street, Suite 1900, San Diego, Ca 92101-4495 Usa SP - 201-234 TI - 17β-Hydroxysteroid dehydrogenases types 1 and 2: Enzymatic assays based on radiometric and mass-spectrometric detection. JO - Methods Enzymol. VL - 689 PB - Elsevier Academic Press Inc PY - 2023 SN - 0076-6879 ER - TY - JOUR AB - Genetically engineered bacterial outer membrane vesicles (OMVs) offer promising applications for gene therapy, immunotherapy, and vaccine delivery. Importantly, OMVs are biocompatible, biodegradable, and easy to engineer and produce on a large scale. In this chapter, we discuss the development and application of bioengineered OMVs for optoacoustics-guided phototherapy applications (theranostics). We provide detailed protocols for OMVs preparation, characterization, and in vitro and in vivo validation. The engineered OMVs carry the biopolymer melanin, which generates a strong optoacoustic (OA) signal and intense heat upon absorption of near-infrared (NIR) light, enabling optoacoustics-guided cancer diagnosis and photothermal therapy in vivo. AU - Gujrati, V. AU - Ntziachristos, V. C1 - 62621 C2 - 50880 CY - 525 B Street, Suite 1900, San Diego, Ca 92101-4495 Usa SP - 349-364 TI - Bioengineered bacterial vesicles for optoacoustics-guided phototherapy. JO - Methods Enzymol. VL - 657 PB - Elsevier Academic Press Inc PY - 2021 SN - 0076-6879 ER - TY - JOUR AB - Photochromic proteins and photoswitching optoacoustics (OA) are a promising combination, that allows OA imaging of even small numbers of cells in whole live animals and thus can facilitate a more wide-spread use of OA in life-science and preclinical research. The concept relies on exploiting the modulation achieved by the photoswitching to discriminate the agents' signal from the non-modulating background. Here we share our analysis approaches that can be readily used on data generated with commercial OA tomography imaging instrumentation allowing—depending on the used photoswitching agent and sample—routine visualizations of as little as several hundreds of transgene labeled cells per imaging volume in the live animal. AU - Stankevych, M. AU - Mishra, K. AU - Ntziachristos, V. AU - Stiel, A.-C. C1 - 62708 C2 - 50978 CY - 525 B Street, Suite 1900, San Diego, Ca 92101-4495 Usa SP - 365-383 TI - A practical guide to photoswitching optoacoustics tomography. JO - Methods Enzymol. VL - 657 PB - Elsevier Academic Press Inc PY - 2021 SN - 0076-6879 ER - TY - JOUR AB - Biological activity in the cell is predominantly mediated by large multiprotein and protein-nucleic acid complexes that act together to ensure functional fidelity. Nuclear magnetic resonance (NMR) spectroscopy is the only method that can provide information for high-resolution three-dimensional structures and the conformational dynamics of these complexes in solution. Mapping of binding interfaces and molecular interactions along with the characterization of conformational dynamics is possible for very large protein complexes. In contrast, de novo structure determination by NMR becomes very time consuming and difficult for protein complexes larger than 30. kDa as data are noisy and sparse.Fortunately, high-resolution structures are often available for individual domains or subunits of a protein complex and thus sparse data can be used to define their arrangement and dynamics within the assembled complex. In these cases, NMR can therefore be efficiently combined with complementary solution techniques, such as small-angle X-ray or neutron scattering, to provide a comprehensive description of the structure and dynamics of protein complexes in solution. Particularly useful are NMR-derived paramagnetic relaxation enhancements (PREs), which provide long-range distance restraints (ca. 20. Å) for structural analysis of large complexes and also report on conformational dynamics in solution.Here, we describe the use of PREs from sample production to structure calculation, focusing on protein-RNA complexes. On the basis of recent examples from our own research, we demonstrate the utility, present protocols, and discuss potential pitfalls when using PREs for studying the structure and dynamic features of protein-RNA complexes. AU - Hennig, J. AU - Warner, L. AU - Simon, B.H.* AU - Geerlof, A. AU - Mackereth, C.D.* AU - Sattler, M. C1 - 44082 C2 - 36820 SP - 333-362 TI - Structural analysis of protein-RNA complexes in solution using NMR paramagnetic relaxation enhancements. JO - Methods Enzymol. VL - 558 IS - 1 PY - 2015 SN - 0076-6879 ER - TY - JOUR AB - Breakthrough technologies to measure cellular oxygen consumption and proton efflux are reigniting the study of cellular energetics by increasing the scope and pace with which discoveries are made. As we learn the variation in metabolism between cell types is large, it is helpful to continually provide additional perspectives and update our roadmap for data interpretation. In that spirit, this chapter provides the following for those conducting microplate-based oxygen consumption experiments: (i) a description of the standard parameters for measuring respiration in intact cells, (ii) a framework for data analysis and normalization, and (iii) examples of measuring respiration in permeabilized cells to follow up results observed with intact cells. Additionally, rate-based measurements of extracellular pH are increasingly used as a qualitative indicator of glycolytic flux. As a resource to help interpret these measurements, this chapter also provides a detailed accounting of proton production during glucose oxidation in the context of plate-based assays. AU - Divakaruni, A.S.* AU - Paradyse, A.* AU - Ferrick, D.A.* AU - Murphy, A.N.* AU - Jastroch, M. C1 - 42830 C2 - 35422 SP - 309-354 TI - Analysis and interpretation of microplate-based oxygen consumption and pH data. JO - Methods Enzymol. VL - 547 PY - 2014 SN - 0076-6879 ER - TY - JOUR AB - The discovery of ghrelin as the endogenous ligand for the growth hormone secretagogue receptor (GHS-R) led to subsequent studies characterizing the endogenous action of this gastrointestinal hormone. Accordingly, exogenous administration of ghrelin was found to increase food intake and adiposity in a variety of species, including rodents, nonhuman primates, and humans. Later work supported these findings and confirmed that ghrelin acts through hypothalamic neurons to mediate its effects on energy metabolism. Ghrelin acts specifically through GHS-R to promote a positive energy balance as demonstrated by loss of ghrelin action after pharmacological blockade or genetic deletion of GHS-R. More recently, ghrelin was found to be a mediator of glucose metabolism and acts to inhibit insulin secretion from pancreatic β-cells. Together, the literature highlights a predominant role of ghrelin in regulating energy and glucose metabolism. AU - Heppner, K.M.* AU - Müller, T.D. AU - Tong, J.* AU - Tschöp, M.H. C1 - 11129 C2 - 30511 SP - 249-260 TI - Ghrelin in the control of energy, lipid, and glucose metabolism. JO - Methods Enzymol. VL - 514 PB - Elsevier Academic Press PY - 2012 SN - 0076-6879 ER - TY - JOUR AB - RNA interference (RNAi)-mediated gene knockdown has developed into a routine method to assess gene function in cultured mammalian cells in a fast and easy manner. For the use of RNAi in mice, short hairpin (sh) RNAs expressed from transgenic vectors are a fast alternative to conventional knockout approaches. We describe our strategy to elicit body-wide, cell type-specific, or inducible gene silencing in mice by control of shRNA expression through Cre recombinase or doxycycline. For reproducible expression of shRNAs, vectors are placed into the Rosa26 locus of ES cells by recombinase-mediated cassette exchange and transmitted through the germ line of chimeric mice. The site specific insertion of single copy shRNA vectors allows to expedite and reproducible production of knockdown mice and provides a simple approach to assess gene function in vivo. AU - Kleinhammer, A. AU - Wurst, W. AU - Kühn, R. C1 - 1447 C2 - 27366 SP - 387-414 TI - Gene knockdown in the mouse through RNAi. JO - Methods Enzymol. VL - 477 IS - C PB - Elsevier PY - 2010 SN - 0076-6879 ER - TY - JOUR AB - Defining the mitochondrial proteome is a prerequisite for fully understanding the organelles function as well as mechanisms underlying mitochondrial pathology. The core functions of mitochondria include oxidative phosphorylation, amino acid metabolism, fatty acid oxidation, and ion homeostasis. In addition to these well-known functions, many crucial properties in cell signaling, cell differentiation and cell death are only now being elucidated, and with them the proteins involved. With the wealth of information arriving from single protein studies and sophisticated genome-wide approaches, MitoP2 was designed and is maintained to consolidate knowledge on mitochondrial proteins in one comprehensive database, thus making all pertinent data readily accessible (http://www.mitop2.de). Although the identification of the human mitochondrial proteome is ultimately the prime objective, integration of other species includes Saccharomyces cerevisiae, mouse, Arabidopsis thaliana, and Neurospora crassa so orthology between these species can be interrogated. Data from genome-wide studies can be individually retrieved and are also processed by a support vector machine (SVM) to generate a score that indicates the likelihood of a candidate protein having a mitochondrial location. Manually validated proteins constitute the reference set of the database that contains over 590 yeast, 920 human, and 1020 mouse entries, and that is used for benchmarking the SVM score. Multiple search options allow for the interrogation of the reference set, candidates, disease related proteins, chromosome locations as well as availability of mouse models. Taken together, MitoP2 is a valuable tool for basic scientists, geneticists, and clinicians who are investigating mitochondrial physiology and dysfunction. AU - Elstner, M. AU - Andreoli, C.* AU - Klopstock, T.* AU - Meitinger, T. AU - Prokisch, H. A2 - Allison, W.S.* ; Murphy, A.N.* C1 - 1955 C2 - 26370 SP - 3-20 TI - The mitochondrial proteome database: MitoP2. JO - Methods Enzymol. VL - 457 IS - B PB - Academic Press PY - 2009 SN - 0076-6879 ER - TY - JOUR AB - Posttranslational protein modifications affect the function or the activity of proteins and exhibit important mechanisms in regulating cellular events. A broad spectrum of modifications is known, including redox-dependent alterations. During the last decade, covalent binding of nitric oxide (NO) to protein cysteines, termed S-nitrosylation, seems especially an evident process for redox-related signaling. To reveal potential target proteins for S-nitrosylation, the biotin switch method gains more and more in importance. This technique is a tool used for analyzing the nitrosylome as well as the examination of single candidates. It is based on substitution of the NO group by a biotin linker that simplifies the detection and the purification of recently S-nitrosylated proteins in a three-step procedure. AU - Sell, S. AU - Lindermayr, C. AU - Durner, J. C1 - 2943 C2 - 25348 SP - 283-293 TI - Identification of S-nitrosylated proteins in plants. JO - Methods Enzymol. VL - 440 PB - Academ. Press PY - 2008 SN - 0076-6879 ER - TY - JOUR AU - Fend, F. AU - Specht, K. AU - Kremer, M. AU - Quintanilla-Martinez, L. C1 - 21996 C2 - 20529 SP - 196-206 TI - Laser Capture Microdissection in Pathology. JO - Methods Enzymol. VL - 356 PY - 2002 SN - 0076-6879 ER - TY - JOUR AU - Wuertz, St.* AU - Hendrickx, L.* C1 - 10000 C2 - 21870 SP - 129-143 TI - In situ quantification of gene transfer in biofilms. JO - Methods Enzymol. VL - 336 PY - 2001 SN - 0076-6879 ER - TY - JOUR AU - Langebartels, C. AU - Ernst, D. AU - Kangasjärvi, J. AU - Sandermann, H. C1 - 21810 C2 - 20012 SP - 520-535 TI - Ozone Effects on Plant Defense. JO - Methods Enzymol. VL - 319 PY - 2000 SN - 0076-6879 ER - TY - JOUR AU - Maier, K.L. AU - Lenz, A.-G. AU - Beck-Speier, I. AU - Costabel, U. C1 - 28745 C2 - 33540 SP - 455-461 TI - Analysis of methionine sulfoxide in proteins. JO - Methods Enzymol. VL - 251 PY - 1995 SN - 0076-6879 ER - TY - JOUR AB - This chapter describes the flavonoids that are plant secondary metabolites having a polyphenol structure, occurring mostly as glycosides or methoxylated derivatives, and sometimes as aglycones. It has been assumed for years that they act as antioxidants, primarily based on the fact that they extend the shelf-life of fat-containing foodstuffs. In contrast, an antioxidative function in plants themselves is still a matter of debate, even though protective effects during plant photooxidative processes. The biochemical background for the antioxidative effect of flavonoids is inhibition of lipid peroxidation, which has been observed on numerous occasions. Owing to the polyphenol structure, this inhibition can be brought about either by chelating of transition metals or by scavenging of free radicals with the formation of less reactive flavonoid aroxyl radicals. At present, radical scavenging is clearly the favored mechanism as evidenced by the lopsided ratio of reports on scavenging versus chelating properties of flavonoids. The chapter discusses the methods for oxygen radical generation. Radiolytic, photolytic, chemical, and enzymatic systems may be used as sources of oxygen radicals. AU - Bors, W. AU - Michel, C. AU - Saran, M. C1 - 20413 C2 - 13611 SP - 420-429 TI - Flavonoid Antioxidants: Rate Constants for Reactions with Oxygen Radicals. JO - Methods Enzymol. VL - 234 PY - 1994 SN - 0076-6879 ER - TY - BOOK AU - Saran, M. AU - Bors, W. A2 - Packer, L.* C1 - 20765 C2 - 16191 SP - 20-34 TI - Pulse radiolysis for investigation of nitric oxide-related reactions. JO - Methods Enzymol. VL - 233 PB - San Diego: Academic Press PY - 1994 SN - 0076-6879 ER - TY - JOUR AU - Saran, M. AU - Bors, W. A2 - Packer, L.* C1 - 20414 C2 - 13612 SP - 20-34 TI - Pulse Radiolysis Method for the Investigation of Nitric Oxide-Related Reactions. JO - Methods Enzymol. VL - 233 PB - San Diego: Academic Press PY - 1993 SN - 0076-6879 ER - TY - JOUR AU - Summer, K.H. AU - Klein, D.A. C1 - 40777 C2 - 0 SP - 57-60 TI - Determination of metallothionein in biological materials. JO - Methods Enzymol. VL - 205 PY - 1991 SN - 0076-6879 ER - TY - JOUR AU - Bors, W. AU - Heller, W. AU - Michel, C. AU - Saran, M. C1 - 18617 C2 - 11754 SP - 343-355 TI - Flavonoids as Antioxidants: Determination of Radical-Scavenging Efficiencies. JO - Methods Enzymol. VL - 186 PY - 1990 SN - 0076-6879 ER - TY - JOUR AU - Bors, W. AU - Heller, W.E. AU - Michel, C. AU - Saran, M. C1 - 42597 C2 - 0 SP - 343-355 TI - Flavonoids as antioxidants: Determination of radical-scavenging efficiencies. JO - Methods Enzymol. VL - 186 PY - 1990 SN - 0076-6879 ER - TY - JOUR AU - Lenz, A.-G. AU - Levine, R.L. AU - Gorland, D. AU - Oliver, C.N. AU - Amici, A. AU - Climent, I. C1 - 17572 C2 - 10889 SP - 464-478 TI - Determination of Carbonyl Content in Oxidatively Modified Proteins. JO - Methods Enzymol. VL - 186 PY - 1990 SN - 0076-6879 ER - TY - JOUR AU - Summer, K.H. AU - Klein, D. C1 - 18338 C2 - 11529 TI - Determination of MT in Biological Materials. JO - Methods Enzymol. PY - 1990 SN - 0076-6879 ER - TY - JOUR AU - Heller, W. AU - Bors, W. AU - Michel, C. AU - Saran, M. C1 - 17534 C2 - 10460 TI - Flavonoids as Antioxidants : Determination of their Radical Scavenging Efficiencies. JO - Methods Enzymol. PY - 1988 SN - 0076-6879 ER -