20,013 results match your criteria Methods in enzymology[Journal]


Preface.

Methods Enzymol 2019 ;619:xv-xvii

Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT, United States.

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http://dx.doi.org/10.1016/S0076-6879(19)30113-2DOI Listing
January 2019
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Methods for genetic analysis of mammalian ER-associated degradation.

Methods Enzymol 2019 20;619:97-120. Epub 2019 Feb 20.

Department of Biology, Stanford University, Stanford, CA, United States. Electronic address:

Identification and degradation of misfolded proteins by the ubiquitin-proteasome system (UPS) is crucial for maintaining proteostasis, but only a handful of UPS components have been linked to the recognition of specific substrates. Studies in Saccharomyces cerevisiae using systematic perturbation of nonessential genes have uncovered UPS components that recognize and ubiquitylate model substrates of the UPS; however, similar analyses in metazoans have been limited. In this chapter, we describe methods for using CRISPR/Cas9 technology combined with genome-wide high complexity single guide (sgRNA) libraries and a transcriptional shutoff strategy for phenotypic selection based on kinetic measurements of protein turnover to identify the genes required to degrade model clients of the mammalian ER-associated degradation system. Read More

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http://dx.doi.org/10.1016/bs.mie.2019.01.006DOI Listing
February 2019
1 Read

Assays for dissecting the in vitro enzymatic activity of yeast Ubc7.

Methods Enzymol 2019 7;619:71-95. Epub 2019 Feb 7.

Department of Biological Chemistry, The Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel. Electronic address:

Ubiquitin (Ub)-mediated protein degradation is a key cellular defense mechanism that detects and eliminates defective proteins. A major intracellular site of protein quality control degradation is the endoplasmic reticulum (ER), hence the term ER-associated degradation, or endoplasmic reticulum-associated degradation (ERAD). Yeast ERAD is composed of three Ub-protein conjugation complexes, named according to their E3 Ub-protein ligase components, Hrd1, Doa10, and the Asi complex, which resides at the nuclear envelope (NE). Read More

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http://dx.doi.org/10.1016/bs.mie.2018.12.035DOI Listing
February 2019

Methodologies to monitor protein turnover at the inner nuclear membrane.

Methods Enzymol 2019 8;619:47-69. Epub 2019 Feb 8.

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, United States; Department of Cell Biology, Yale School of Medicine, New Haven, CT, United States. Electronic address:

Lamin B receptor (LBR) is an inner nuclear membrane protein that associates with the nuclear lamina and harbors sterol reductase activity essential for cholesterol biosynthesis. Several LBR mutations implicated in human congenital disorders give rise to C-terminal truncations which render LBR metabolically unstable, resulting in their rapid turnover in the nucleus. These LBR variants serve as model substrates for investigating the poorly understood protein quality control pathways in the mammalian nuclear envelope (NE). Read More

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http://dx.doi.org/10.1016/bs.mie.2018.12.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6457266PMC
February 2019
2 Reads

Monitoring stress-induced autophagic engulfment and degradation of the 26S proteasome in mammalian cells.

Methods Enzymol 2019 7;619:337-366. Epub 2019 Mar 7.

Technion Integrated Cancer Center (TICC), The Rappaport Faculty of Medicine and Research Institute, Technion-Israel Institute of Technology, Haifa, Israel; Department of Biomedical Sciences, Protein Metabolism Medical Research Center, College of Medicine, Seoul National University, Seoul, South Korea.

Almost 70 years after the discovery of the lysosome, and about four decades following the unraveling of ubiquitin as a specific "mark of death," the field of protein turnover-the numerous processes it regulates, the pathologies resulting from its dysregulation, and the drugs that have been developed to target them-is still growing exponentially. Accordingly, the need for new technologies and methods is ever growing. One interesting question in the field is the mechanism(s) by which the "predators become prey". Read More

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http://dx.doi.org/10.1016/bs.mie.2018.12.022DOI Listing

Western blot analysis of the autophagosomal membrane protein LGG-1/LC3 in Caenorhabditis elegans.

Methods Enzymol 2019 4;619:319-336. Epub 2019 Feb 4.

Institute for Genetics and CECAD Research Center, University of Cologne, Cologne, Germany. Electronic address:

Autophagy is being studied intensively in Caenorhabditis elegans in the context of protein homeostasis and aging. However, in contrast to the yeast and mammalian autophagosomal membrane proteins Atg8 and LC3, lipidation of the C. elegans ortholog LGG-1 with phosphatidylethanolamine has rarely been investigated by western blotting. Read More

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https://linkinghub.elsevier.com/retrieve/pii/S00766879183053
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http://dx.doi.org/10.1016/bs.mie.2018.12.034DOI Listing
February 2019
7 Reads

Detection of ubiquitinated targets in mammalian and Drosophila models.

Methods Enzymol 2019 11;619:293-318. Epub 2019 Feb 11.

IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Italy; Dipartimento di oncologia ed emato-oncologia, Universita' degli Studi di Milano, Milan, Italy. Electronic address:

In this chapter, we describe techniques to detect ubiquitination events occurring in vivo. We focus on methodologies capable of preserving and detecting target protein ubiquitination in physiological conditions, without overexpressing a tagged version of ubiquitin. We provide detailed protocols for mammalian and Drosophila melanogaster systems using linkage-specific antibodies against ubiquitin. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.12.030DOI Listing
February 2019
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Methods for measuring misfolded protein clearance in the budding yeast Saccharomyces cerevisiae.

Methods Enzymol 2019 8;619:27-45. Epub 2019 Feb 8.

Department of Biology, Stanford University, Stanford, CA, United States; Department of Genetics, Stanford University, Stanford, CA, United States. Electronic address:

Protein misfolding in the cell is linked to an array of diseases, including cancers, cardiovascular disease, type II diabetes, and numerous neurodegenerative disorders. Therefore, investigating cellular pathways by which misfolded proteins are trafficked and cleared ("protein quality control") is of both mechanistic and therapeutic importance. The clearance of most misfolded proteins involves the covalent attachment of one or more ubiquitin molecules; however, the precise fate of the ubiquitinated protein varies greatly, depending on the linkages present in the ubiquitin chain. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.12.039DOI Listing
February 2019

Methods for studying the regulation of membrane traffic by ubiquitin and the ESCRT pathway.

Methods Enzymol 2019 4;619:269-291. Epub 2019 Feb 4.

Weill Institute for Cell and Molecular Biology, Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, United States.

Covalent modification of proteins with ubiquitin dynamically regulates their function and fate. The ubiquitination of most plasma membrane proteins initiates endocytosis and ESCRT-mediated sorting to the lysosomal lumen for degradation. Powerful genetic approaches in the budding yeast Saccharomyces cerevisiae have been particularly instrumental in the discovery and elucidation of these molecular mechanisms, which are conserved in all eukaryotes. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.12.041DOI Listing
February 2019
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In vitro analysis of proteasome-associated USP14 activity for substrate degradation and deubiquitylation.

Methods Enzymol 2019 1;619:249-268. Epub 2019 Feb 1.

Department of New Biology, DGIST, Daegu, Republic of Korea. Electronic address:

The ubiquitin-proteasome pathway plays an essential role in maintaining protein homeostasis and regulates almost every aspect of cellular processes in eukaryotes. Emerging evidence indicates that the proteasome does not work as a simple unidirectional molecular machinery for substrate proteolysis. In fact, proteasome activity should be tightly regulated, and the proteasome itself can be dynamically engaged in the degradation cycle. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.12.028DOI Listing
February 2019

Single-molecule methods for measuring ubiquitination and protein stability.

Authors:
Jason Hon Ying Lu

Methods Enzymol 2019 4;619:225-247. Epub 2019 Feb 4.

Department of Systems Biology, Harvard Medical School, Boston, MA, United States. Electronic address:

The ubiquitin-proteasome system (UPS) contributes to changes in cell state and homeostatic maintenance in humans by modulating the stability of about a third of human proteins. For example, cell-cycle regulation requires a central ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C), which starts a ubiquitination cascade leading to the degradation of multiple targets. This targeted degradation is mediated by the 26S proteasome, a 2. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.12.031DOI Listing
February 2019
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Structural mass spectrometry approaches to study the 20S proteasome.

Methods Enzymol 2019 1;619:179-223. Epub 2019 Feb 1.

Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot, Israel. Electronic address:

The 20S proteasome is a large multisubunit proteolytic machine that is central to intracellular protein degradation. It is found in all three kingdoms of life and is ubiquitous in archaea and eukaryotes. Since its discovery, much effort employing a diverse array of structural biology methods has been applied to help understand its structure/function relationships. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.12.029DOI Listing
February 2019
1 Read

Assays for ubiquitin-like protein ligation and proteasome function in archaea.

Methods Enzymol 2019 15;619:161-178. Epub 2019 Feb 15.

Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL, United States; University of Florida, Genetics Institute, Gainesville, FL, United States. Electronic address:

Ubiquitin-like protein (Ubl) ligation is common to diverse archaea and targets many cellular pathways, including those associated with sulfur mobilization, and also tags proteins as substrates for degradation by the proteasome. Here we highlight protocols to assay proteasome function and Ubl ligation in archaea. A chase assay is described to monitor the impact of proteasome function on the stability of Ubl-modified proteins in the cell. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.12.036DOI Listing
February 2019

Engineered disulfide crosslinking to measure conformational changes in the 26S proteasome.

Methods Enzymol 2019 3;619:145-159. Epub 2019 Jan 3.

Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, FL, United States. Electronic address:

The 26S proteasome is a multisubunit ATP-dependent peptidase complex mediating most regulated protein degradation in eukaryotes. The proteasome undergoes several coordinated conformational changes during catalysis that activate it for substrate processing and functionally couple distinct enzymatic activities during substrate degradation. Understanding the impact of substrate interactions and individual ATP binding events on these conformational changes is currently a major bottleneck in the study of proteasome function. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.11.006DOI Listing
January 2019
2 Reads

A dual system to manipulate protein levels for DNA replication- and cell cycle-related studies.

Methods Enzymol 2019 8;619:121-143. Epub 2019 Feb 8.

Institute of Molecular Biology (IMB), Mainz, Germany. Electronic address:

Investigation of cell cycle-regulated processes often necessitates the rapid manipulation of individual protein levels in synchronized populations over the course of a cell cycle. In the budding yeast, the two major orthogonal approaches by which this is accomplished are conditional gene expression via inducible or repressible promoters and regulated metabolic destabilization of a protein of interest via ubiquitin-mediated degradation signals. Here, we describe an application of these principles to the investigation of DNA damage signaling during replication. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.12.038DOI Listing
February 2019

Assays for protein retrotranslocation in ERAD.

Methods Enzymol 2019 15;619:1-26. Epub 2019 Feb 15.

Division of Biological Sciences, The Section of Cell and Developmental Biology, University of California San Diego, La Jolla, CA, United States.

Elimination of misfolded proteins by endoplasmic reticulum (ER)-associated protein degradation (ERAD) ensures that proteins proceeding through the secretory pathway are correctly folded and processed, which is critical to minimize ER stress. All ERAD pathways include a protein translocation process termed retrotranslocation, in which ubiquitinated misfolded substrates are extracted from the ER and degraded by the cytosolic 26S proteasome. Despite being integral to ERAD, the retrotranslocation process has been largely obscure. Read More

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http://dx.doi.org/10.1016/bs.mie.2019.01.002DOI Listing
February 2019
3 Reads

Preface.

Methods Enzymol 2019 ;618:xvii-xix

Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT, United States.

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http://dx.doi.org/10.1016/S0076-6879(19)30079-5DOI Listing
January 2019

Enzymatic preparation of monoubiquitinated FANCD2 and FANCI proteins.

Methods Enzymol 2019 11;618:73-104. Epub 2019 Feb 11.

Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom; MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences University of Dundee, Dundee, United Kingdom. Electronic address:

In higher eukaryotes, DNA damage repair response pathways are orchestrated by several molecular signals including ubiquitination. In particular the repair of DNA interstrand crosslinks, toxic to transcription and replication processes, involve the activation of the Fanconi anemia repair pathway. At the heart of this pathway lies the monoubiquitination of FANCD2 and FANCI proteins, which triggers the recruitment of DNA repair factors. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.12.021DOI Listing
February 2019
2 Reads

A genetic approach to study polyubiquitination in Saccharomyces cerevisiae.

Methods Enzymol 2019 21;618:49-72. Epub 2019 Feb 21.

Department of Biochemistry, Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA, United States. Electronic address:

Ubiquitin has seven lysines, all of which are used to generate polyubiquitin chains in the yeast Saccharomyces cerevisiae. While the biology associated with chains formed through lysines 48 and 63 is well studied, other chain types are more poorly characterized. We outline a methodology for using synthetic genetic analysis to examine ubiquitin mutants. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.12.020DOI Listing
February 2019

Assays of SUMO protease/isopeptidase activity and function in mammalian cells and tissues.

Methods Enzymol 2019 21;618:389-410. Epub 2019 Feb 21.

Institute of Biochemistry II, Goethe University, Frankfurt, Germany. Electronic address:

Covalent conjugation of the ubiquitin-related SUMO modifier to lysine residues of cellular proteins (SUMOylation) is a prevalent posttranslational modification. SUMOs are synthesized as precursor proteins that require carboxy-terminal processing prior to conjugation. Subsequently, a multistep enzymatic pathway is used for conjugation to target proteins. Read More

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http://dx.doi.org/10.1016/bs.mie.2019.01.005DOI Listing
February 2019

Profiling DUBs and Ubl-specific proteases with activity-based probes.

Methods Enzymol 2019 14;618:357-387. Epub 2019 Feb 14.

Department of Cell and Chemical Biology, Chemical Immunology, Oncode Institute, Leiden University Medical Centre, Leiden, The Netherlands. Electronic address:

Protein (poly-)ubiquitination is a posttranslational modification that plays a key role in almost all cellular processes. It involves the installment of either single ubiquitin (Ub) moieties or one of eight different polyUb linkage types, each giving a distinct cellular outcome. Deubiquitinating enzymes (DUBs) reverse Ub signaling by disassembly of one or multiple poly-Ub chain types and their malfunction is often associated with human disease. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.12.037DOI Listing
February 2019

Purification and functional characterization of the DUB domain of SdeA.

Methods Enzymol 2019 1;618:343-355. Epub 2019 Feb 1.

Department of Chemistry, Purdue University, West Lafayette, IN, United States. Electronic address:

Intracellular pathogens like Legionella pneumophila hijack the host ubiquitination network in order to create a facultative niche for their survival by means of effector molecules secreted into the host cell. Some of these effectors function as ubiquitin ligases or deubiquitinases, among other types of enzymes. Deubiquitinating enzymes (DUBs) remove ubiquitin or ubiquitin-like modifiers from conjugated substrates to regulate various cellular processes. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.12.024DOI Listing
February 2019

Evaluating enzyme activities and structures of DUBs.

Methods Enzymol 2019 22;618:321-341. Epub 2019 Feb 22.

Ubiquitin Signalling Division, The Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia. Electronic address:

Ubiquitin signaling requires tight control of all aspects of protein ubiquitination, including the timing, locale, extent, and type of modification. Dysregulation of any of these signaling features can lead to severe human disease. One key mode of regulation is through the controlled removal of the ubiquitin signal by dedicated families of proteases, termed deubiquitinases. Read More

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http://dx.doi.org/10.1016/bs.mie.2019.01.001DOI Listing
February 2019

Dual-color pulse-chase ubiquitination assays to simultaneously monitor substrate priming and extension.

Methods Enzymol 2019 11;618:29-48. Epub 2019 Feb 11.

Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN, United States; Department of Molecular Machines and Signaling, Max Planck Institute of Biochemistry, Martinsried, Germany. Electronic address:

Many fundamental discoveries in ubiquitin-proteasome research have relied on reconstitution of activities from purified or recombinantly expressed components. These include landmark discoveries of E1-E2-E3 mechanisms, in which ubiquitin (UB) is initially activated and then covalently shuttled between enzyme active sites and ultimately ligated to substrate or substrate-linked UBs during polyubiquitination. However, recent studies have unearthed enormous variations on the E1-E2-E3 theme; for example, one E3 may employ two distinct E2s, or two different E3s may act in a single assembly or in series, to prime substrates directly with UB and subsequently decorate them with myriad types of polyubiquitin chains. Read More

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https://linkinghub.elsevier.com/retrieve/pii/S00766879193000
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http://dx.doi.org/10.1016/bs.mie.2019.01.004DOI Listing
February 2019
2 Reads

Quantitative analysis of USP activity in vitro.

Methods Enzymol 2019 23;618:281-319. Epub 2019 Feb 23.

Division of Biochemistry and Oncode Institute, Netherlands Cancer Institute, Amsterdam, The Netherlands. Electronic address:

Ubiquitin-specific proteases (USPs) are an important class of deubiquitinating enzymes (DUBs) that carry out critical roles in cellular physiology and are regulated at multiple levels. Quantitative characterization of USP activity is crucial for mechanistic understanding of USP function and regulation. This requires kinetic analysis using in vitro activity assays on minimal and natural substrates with purified proteins. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.12.023DOI Listing
February 2019

Methods to analyze STUbL activity.

Methods Enzymol 2019 3;618:257-280. Epub 2019 Jan 3.

Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, United Kingdom. Electronic address:

Posttranslational modification with small ubiquitin-like modifier (SUMO) plays an important role in many biological processes. SUMO-targeted ubiquitin E3 ligases (STUbLs) are part of the really interesting new gene (RING)-type family of ubiquitin E3 ligases. STUbLs recognize their SUMO-modified substrates via SUMO-interaction motifs and ubiquitinate them via the RING domain. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.11.005DOI Listing
January 2019

Analysis of modification and proteolytic targeting by the ubiquitin-like modifier FAT10.

Methods Enzymol 2019 8;618:229-256. Epub 2019 Feb 8.

Biotechnology Institute Thurgau at the University of Konstanz, Kreuzlingen, Switzerland; Division of Immunology, Department of Biology, University of Konstanz, Konstanz, Germany. Electronic address:

The ubiquitin-like modifier FAT10 (also called ubiquitin D (UBD)) interacts noncovalently with a substantial number of proteins and also gets covalently conjugated to many substrate proteins, leading to their degradation by the 26S proteasome. FAT10 comprises two loosely folded ubiquitin-like domains that are connected by a flexible linker, and this unusual structure makes it highly prone to aggregation. Here, we report methods to purify high amounts of soluble recombinant FAT10 for various uses, such as in vitro FAT10ylation assays. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.12.040DOI Listing
February 2019

Approaches for investigating the extracellular signaling function of ISG15.

Methods Enzymol 2019 1;618:211-227. Epub 2019 Feb 1.

Department of Molecular Biosciences, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, United States. Electronic address:

ISG15 is a ubiquitin-like protein (Ubl) that is expressed in response to Type 1 Interferon (IFN-α/β) signaling. Remarkably, ISG15 has three distinct biochemical activities involved in innate immune responses to viral and/or microbial infections. The canonical function of ISG15 is as a posttranslational modifier, and protein ISGylation has been demonstrated to be antiviral. Read More

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https://linkinghub.elsevier.com/retrieve/pii/S00766879183052
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http://dx.doi.org/10.1016/bs.mie.2018.12.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6472260PMC
February 2019
2 Reads

Methods to study SUMO dynamics in yeast.

Methods Enzymol 2019 11;618:187-210. Epub 2019 Feb 11.

Institute for Genetics, Center of Molecular Biosciences, University of Cologne, Cologne, Germany. Electronic address:

Covalent modification of proteins with the small ubiquitin-related modifier (SUMO) is found in all eukaryotes and is involved in many important processes. SUMO attachment may change interaction properties, subcellular localization, or stability of a modified protein. Usually, only a small fraction of a protein is modified at a given time because sumoylation is a highly dynamic process. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.12.026DOI Listing
February 2019

Biochemical characterization of SUMO-conjugating enzymes by in vitro sumoylation assays.

Methods Enzymol 2019 11;618:167-185. Epub 2019 Feb 11.

Department of Epigenetics, Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany. Electronic address:

The small ubiquitin-related modifier (SUMO) is a protein of ~10kDa that is covalently conjugated to its substrate proteins in an enzymatic process called sumoylation. This posttranslational modification is an essential regulatory mechanism that plays crucial roles in many cellular pathways. It allows rapid adaptation to environmental changes by switching protein functions due to alternate complex assemblies, changes in intracellular localization, enzymatic activity, or stability. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.12.025DOI Listing
February 2019

Methods to study phosphoribosylated ubiquitin ligation and removal.

Methods Enzymol 2019 8;618:149-166. Epub 2019 Feb 8.

Purdue Institute for Inflammation, Immunology and Infectious Disease and Department of Biological Sciences, Purdue University, West Lafayette, IN, United States. Electronic address:

Ubiquitination is a prevalent protein modification catalyzed by E1, E2, and E3 enzymes that activate, conjugate, and ligate, respectively, the ubiquitin protein to substrate protein. In order to establish a mutualistic or parasitic relationship with their eukaryotic hosts, many microorganisms hijack different aspects of the ubiquitination machinery using bacterial proteins that function as E3 ligases or as enzymes that modify E2s or ubiquitin. Recently, the SidE family of effector proteins (SidEs) from the intracellular bacterial pathogen Legionella pneumophila was found to catalyze ubiquitination by a mechanism unrelated to the classical three-enzyme cascade. Read More

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http://dx.doi.org/10.1016/bs.mie.2019.01.007DOI Listing
February 2019

Detection of ubiquitination activity and identification of ubiquitinated substrates using TR-TUBE.

Methods Enzymol 2019 1;618:135-147. Epub 2019 Feb 1.

Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan. Electronic address:

Ubiquitination is a transient posttranslational modification; polyubiquitin chains are removed from proteins by deubiquitinating enzymes (DUBs) and many ubiquitinated proteins are degraded by the proteasome. Exogenously expressed trypsin-resistant tandem ubiquitin-binding entity (TR-TUBE) protects polyubiquitin chains from DUBs and inhibits proteasomal degradation in cells. TR-TUBE effectively binds to substrates ubiquitinated by an exogenously expressed ubiquitin ligase, and enables detection of the specific activity of a given ubiquitin ligase and isolation of its substrates. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.12.032DOI Listing
February 2019

Methods to measure ubiquitin chain length and linkage.

Methods Enzymol 2019 15;618:105-133. Epub 2019 Feb 15.

Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan. Electronic address:

To understand the biological roles of different ubiquitin chains, it is important to determine the types of ubiquitin linkages, the lengths of the polymers, and the combinations of ubiquitin chains attached to substrates. In this chapter, we describe a mass spectrometry-based quantification method of ubiquitin chains, named Ub-AQUA/PRM (ubiquitin-absolute quantification/parallel reaction monitoring), for direct and highly sensitive measurement of the stoichiometry of all eight ubiquitin-ubiquitin linkage types simultaneously. We also show a method to quantify the K48/K63 branched ubiquitin chain, a recently identified ubiquitin signal with a complex topology. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.12.019DOI Listing
February 2019
3 Reads

Semisynthesis of ubiquitinated histone H2B with a native or nonhydrolyzable linkage.

Methods Enzymol 2019 26;618:1-27. Epub 2019 Feb 26.

Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD, United States. Electronic address:

Posttranslational modifications of histone proteins regulate all biological processes requiring access to DNA. Monoubiquitination of histone H2B is a mark of actively transcribed genes in all eukaryotes that also plays a role in DNA replication and repair. Solution and structural studies of the mechanism by which histone ubiquitination modulates these processes depend on the ability to generate homogeneous preparations of nucleosomes containing ubiquitin conjugated to a specific lysine residue. Read More

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http://dx.doi.org/10.1016/bs.mie.2019.01.003DOI Listing
February 2019

Preface.

Methods Enzymol 2019 ;617:xv-xvii

University of Minnesota.

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http://dx.doi.org/10.1016/S0076-6879(19)30067-9DOI Listing
January 2019
1 Read

Repurposing peroxisomes for microbial synthesis for biomolecules.

Methods Enzymol 2019 25;617:83-111. Epub 2019 Jan 25.

Division of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China. Electronic address:

Microbial synthesis represents an alternative approach for the sustainable production of chemicals, fuels, and medicines. However, construction of biosynthetic pathways always suffers from side reactions, toxicity of intermediates, or low efficiency of substrate channeling. Subcellular compartmentalization may contribute to a more efficient production of target products by reducing side reactions and toxic effects within a compact insular space. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.12.004DOI Listing
January 2019
1 Read

Cloning, expression, and purification of intact polyketide synthase modules.

Methods Enzymol 2019 4;617:63-82. Epub 2019 Feb 4.

School of Biochemistry, Biomedical Sciences Building, University Walk, University of Bristol, Bristol, United Kingdom; BrisSynBio Synthetic Biology Research Centre, Life Sciences Building, University of Bristol, Bristol, United Kingdom. Electronic address:

Polyketides are a structurally and functionally diverse family of bioactive natural products that have proven to be a rich source of pharmaceutical and agrochemical lead compounds. Many polyketides are biosynthesized by large multifunctional megaenzymes termed type I modular polyketide synthases (PKSs). These systems possess a distinctive assembly line-like architecture, comprising a series of linearly arranged, multidomain extension modules, housed in sequence within giant polypeptide chains. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.12.018DOI Listing
February 2019
2 Reads

EMMA assembly explained: A step-by-step guide to assemble synthetic mammalian vectors.

Methods Enzymol 2019 13;617:463-493. Epub 2019 Feb 13.

Manchester Institute of Biotechnology (MIB), School of Chemistry, The University of Manchester, Manchester, United Kingdom. Electronic address:

Construction of expression vectors is imperative for many areas of biological research and the biotechnology industry. Modular cloning systems for expression vector construction offer a labor- and cost-effective alternative to overcome drawbacks associated with traditional cloning methods. We developed an Extensible Mammalian Modular Assembly toolkit (EMMA) as an efficient and versatile tool to facilitate the construction of functionally diverse mammalian expression vectors from a standardized library of DNA parts. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.12.017DOI Listing
February 2019
2 Reads

Metabolon formation by chemotaxis.

Authors:
Xi Zhao Ayusman Sen

Methods Enzymol 2019 4;617:45-62. Epub 2019 Feb 4.

Department of Chemistry, The Pennsylvania State University, University Park, PA, United States. Electronic address:

Enzymes that participate in reaction cascades have been shown to assemble into metabolons, in the presence of the first enzyme's substrate. However, the mechanism of metabolon formation is still an open question. We applied microfluidic and fluorescent spectroscopy techniques to study the coordinated movement of the first four enzymes of the glycolysis cycle: hexokinase, phosphoglucose isomerase, phosphofructokinase, and aldolase and showed that each enzyme independently follows its own specific substrate gradient, which is produced by the preceding enzymatic reaction. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.12.003DOI Listing
February 2019
1 Read

Insider information on successful covalent protein coupling with help from SpyBank.

Methods Enzymol 2019 25;617:443-461. Epub 2019 Jan 25.

Department of Biochemistry, University of Oxford, Oxford, United Kingdom. Electronic address:

New biological properties can stem from the freedom to link, multimerize, or multiplex protein building blocks. The peptide SpyTag on one protein irreversibly reacts with SpyCatcher on another protein, through spontaneous isopeptide bond formation. Reaction is specific in a wide range of cellular environments and all components are genetically encoded, making this chemistry accessible to molecular biologists. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.12.010DOI Listing
January 2019
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Combinatorial biosynthesis of small molecules in plants: Engineering strategies and tools.

Methods Enzymol 2019 25;617:413-442. Epub 2019 Jan 25.

Plant Biotechnology and Metabolic Engineering, Technische Universität Darmstadt, Darmstadt, Germany. Electronic address:

Biosynthetic capacity of plants, rooted in a near inexhaustible supply of photosynthetic energy and founded upon an intricate matrix of metabolic networks, makes them versatile chemists producing myriad specialized compounds. Along with tremendous success in elucidation of several plant biosynthetic routes, their reestablishment in heterologous hosts has been a hallmark of recent bioengineering endeavors. However, current efforts in the field are, in the main, aimed at grafting the pathways to fermentable recipient organisms, like bacteria or yeast. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.12.005DOI Listing
January 2019
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On-pot and cell-free biocatalysis using coimmobilized enzymes on advanced materials.

Methods Enzymol 2019 8;617:385-411. Epub 2019 Feb 8.

Heterogeneous Biocatalysis Laboratory, Instituto de Síntesis Química y Catálisis Homogénea (ISQCH-CSIC), University of Zaragoza, Zaragoza, Spain; ARAID, Aragon I+D Foundation, Zaragoza, Spain. Electronic address:

Complex synthetic schemes catalyzed by multienzyme systems immobilized on solid materials are gaining momentum in chemical biomanufacturing. These systems harness the high chemo-, regio-, and stereoselectivity of the enzymes and the recyclability of the heterogeneous catalysts. Moreover, when the enzymes become part of a solid material, they can be easily integrated into packed-bed reactor for continuous biotransformations. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.12.013DOI Listing
February 2019

Artificial scaffolds for enhanced biocatalysis.

Methods Enzymol 2019 25;617:363-383. Epub 2019 Jan 25.

Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, United States. Electronic address:

Proteins are not designed to be standalone entities and must coordinate their collective action for optimum performance. Nature has developed through evolution the ability to colocalize the functional partners of a cascade enzymatic reaction in order to ensure efficient exchange of intermediates. Inspired by these natural designs, synthetic scaffolds have been created to enhance the overall biological pathway performance. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.12.007DOI Listing
January 2019
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Protein-based scaffolds for enzyme immobilization.

Methods Enzymol 2019 10;617:323-362. Epub 2019 Feb 10.

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, St. Paul, MN, United States. Electronic address:

Biocatalysis is emerging as an alternative approach to chemical synthesis of industrially relevant complex molecules. To obtain suitable yields of compounds in a cost-effective manner, biocatalytic reaction cascades must be efficient, robust, and self-sufficient. One approach is to immobilize biocatalysts on a solid support, stabilizing the enzymes and providing optimal microenvironments for reaction sequences. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.12.016DOI Listing
February 2019
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Tricarboxylic acid metabolon.

Methods Enzymol 2019 25;617:29-43. Epub 2019 Jan 25.

Department of Chemistry, University of Utah, Salt Lake City, UT, United States. Electronic address:

This chapter focuses on the experimental protocols used to determine the structure of the TCA metabolon. Since the TCA metabolon is quite large and cannot be crystallized, X-ray crystallography and NMR-based structural biology techniques cannot be utilized. This chapter will include a discussion of the interactomics technique of cross-linking mass spectrometry used for determining the TCA metabolon structure, including detailed procedures and the resulting metabolon structures. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.12.002DOI Listing
January 2019
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DNA binding adaptors to assemble proteins of interest on DNA scaffold.

Methods Enzymol 2019 13;617:287-322. Epub 2019 Feb 13.

Institute of Advanced Energy, Kyoto University, Uji, Kyoto, Japan. Electronic address:

DNA nanostructures serve as the ideal scaffolds to assemble materials of interest. Among these, proteins are of particularly interesting class of molecules to assemble because of their huge functional variability. Sequence-specific DNA binding proteins have been applied as adaptors to stably locate the fused proteins at defined positions of DNA scaffold in high loading yields. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.12.014DOI Listing
February 2019
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Enzyme colocalization in protein-based hydrogels.

Methods Enzymol 2019 8;617:265-285. Epub 2019 Feb 8.

Department of Chemical and Environmental Engineering, University of California, Riverside, CA, United States. Electronic address:

The development of biomaterials with embedded enzymatic activities has been driven by a range of applications including tissue engineering, biosensors, and bioenergy applications. Advances in the design and production of peptide-based biomaterials have inspired protein engineers to begin creating enzymes with self-assembling, biomaterial forming capabilities. Outfitting enzymes with cross-link forming domains allows biomaterials to be created with a range of benefits including simple low-cost production, homogenous dispersion of activity in the hydrogels, and the ability to colocalize enzymes to create multistep cascades in the hydrogels. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.12.006DOI Listing
February 2019
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Cell-surface display of designer cellulosomes by Lactobacillus plantarum.

Methods Enzymol 2019 25;617:241-263. Epub 2019 Jan 25.

Department of Biomolecular Sciences, The Weizmann Institute of Science, Rehovot, Israel. Electronic address:

Cell-surface display of designer cellulosomes complexes has attracted increased interest in recent years. These engineered microorganisms can efficiently degrade lignocellulosic biomass that represents an abundant resource for conversion into fermentable sugars, suitable for production of biofuels. The designer cellulosome is an artificial enzymatic complex that mimics the architecture of the natural cellulosome and allows the control of the positions, type, and copy number of the cellulosomal enzymes within the complex. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.12.011DOI Listing
January 2019
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TXTL-based approach to synthetic cells.

Methods Enzymol 2019 30;617:217-239. Epub 2019 Jan 30.

School of Physics and Astronomy, University of Minnesota, Minneapolis, MN, United States. Electronic address:

Cell-free transcription-translation (TXTL) has recently emerged as a versatile technology to engineer biological systems. In this chapter, we show how an all E. coli TXTL system can be used to build synthetic cell prototypes. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.12.015DOI Listing
January 2019
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Bacterial bioreactors: Outer membrane vesicles for enzyme encapsulation.

Methods Enzymol 2019 25;617:187-216. Epub 2019 Jan 25.

U.S. Naval Research Laboratory, Washington, DC, United States. Electronic address:

Bacterial membrane vesicles, whether naturally occurring or engineered for enhanced functionality, have significant potential as tools for bioremediation, enzyme catalysis, and the development of therapeutics such as vaccines and adjuvants. In many instances, the vesicles themselves and the naturally occurring proteins are sufficient to lend functionality. Alternatively, additional function can be conveyed to these biological nanoparticles through the directed packaging of peptides and proteins, specifically recombinant enzymes chosen to mediate a specific reaction or facilitate a controlled response. Read More

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https://linkinghub.elsevier.com/retrieve/pii/S00766879183050
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http://dx.doi.org/10.1016/bs.mie.2018.12.012DOI Listing
January 2019
6 Reads