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Methods in Enzymology

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https://www.readbyqxmd.com/read/28065275/preface
#1
EDITORIAL
Michael H Gelb
No abstract text is available yet for this article.
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28065274/probing-the-activity-of-eukaryotic-rhomboid-proteases-in-vitro
#2
B Cordier, M K Lemberg
Proteolysis within the membrane is a recent concept in biology. Rhomboid intramembrane serine proteases are conserved in evolution and serve as key switches in diverse cellular pathways ranging from signaling to protein degradation. Since deregulation of intramembrane proteolysis can lead to severe diseases including neurodegenerative disorders, dissecting their enzymatic function and specificity becomes crucial. As membrane proteins, their solubilization, and purification are technically challenging. As a start point for a comprehensive in vitro characterization of eukaryotic rhomboid proteases, we depict in this chapter a robust workflow to find the best conditions to obtain pure and active enzymes from a bacterial expression system...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28065273/screening-and-characterization-strategies-for-nanobodies-targeting-membrane-proteins
#3
S Veugelen, M Dewilde, B De Strooper, L Chávez-Gutiérrez
The study of membrane protein function and structure requires their successful detection, expression, solubilization, and/or reconstitution, which poses a challenging task and relies on the availability of suitable tools. Several research groups have successfully applied Nanobodies in the purification, as well as the functional and structural characterization of membrane proteins. Nanobodies are small, single-chain antibody fragments originating from camelids presenting on average a longer CDR3 which enables them to bind in cavities and clefts (such as active and allosteric sites)...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28065272/activity-assays-for-rhomboid-proteases
#4
E Arutyunova, K Strisovsky, M J Lemieux
Rhomboids are ubiquitous intramembrane serine proteases that are involved in various signaling pathways. This fascinating class of proteases harbors an active site buried within the lipid milieu. High-resolution structures of the Escherichia coli rhomboid GlpG with various inhibitors revealed the catalytic mechanism for rhomboid-mediated proteolysis; however, a quantitative characterization was lacking. Assessing an enzyme's catalytic parameters is important for understanding the details of its proteolytic reaction and regulatory mechanisms...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28065271/signal-peptidase-enzymology-and-substrate-specificity-profiling
#5
R E Dalbey, D Pei, Ö D Ekici
Signal peptidases are membrane proteases that play crucial roles in the protein transport pathway of bacteria. They cleave off the signal peptide from precursor proteins that are membrane inserted by the SecYEG or Tat translocons. Signal peptide cleavage releases the translocated protein from the inner membrane allowing the protein to be exported to the periplasm, outer membrane, or secreted into the medium. Signal peptidases are very important proteins to study. They are unique serine proteases with a Ser-Lys dyad, catalyze cleavage at the membrane surface, and are promising potential antibacterial drug targets...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28065270/functional-study-of-the-vitamin-k-cycle-enzymes-in-live-cells
#6
J-K Tie, D W Stafford
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...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28065269/methods-for-structural-and-functional-analyses-of-intramembrane-prenyltransferases-in-the-ubia-superfamily
#7
Y Yang, N Ke, S Liu, W Li
The UbiA superfamily is a group of intramembrane prenyltransferases that generate lipophilic compounds essential in biological membranes. These compounds, which include various quinones, hemes, chlorophylls, and vitamin E, participate in electron transport and function as antioxidants, as well as acting as structural lipids of microbial cell walls and membranes. Prenyltransferases producing these compounds are involved in important physiological processes and human diseases. These UbiA superfamily members differ significantly in their enzymatic activities and substrate selectivities...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28065268/enzymatic-assays-for-studying-intramembrane-proteolysis
#8
D M Bolduc, D J Selkoe, M S Wolfe
Proteolysis within the membrane is catalyzed by a diverse family of proteases immersed within the hydrophobic environment of cellular membranes. These ubiquitous intramembrane-cleaving proteases (I-CLiPs) hydrolyze the transmembrane domains of a large variety of membrane-embedded proteins to facilitate signaling events essential to normal biological functions found in all forms of life. The importance of this unique class of enzyme is highlighted by its central involvement in a variety of human pathologies, including Alzheimer's disease (AD), Parkinson's disease, cancer, and the virulence of a number of viral, bacterial, and fungal pathogens...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28065267/mechanism-and-inhibition-of-rhomboid-proteases
#9
K Strisovsky
Intramembrane serine proteases of the rhomboid family are widespread, and their gradually uncovered functions in different organisms already suggest medical relevance for infectious diseases and cancer. However, selective inhibitors that could serve as research tools for rhomboids, for validation of their disease relevance, or as templates for drug development are lacking. Here I summarize the current knowledge about rhomboid protease mechanism and specificity, overview the currently used inhibitors, and conclude by proposing avenues for future development of rhomboid protease inhibitors...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28065266/production-of-recombinant-rhomboid-proteases
#10
E Arutyunova, R Panigrahi, K Strisovsky, M J Lemieux
Rhomboid proteases are intramembrane enzymes that hydrolyze peptide bonds of transmembrane proteins in the lipid bilayer. They play a variety of roles in key biological events and are linked to several disease states. Over the last decade a great deal of structural and functional knowledge has been generated on this fascinating class of proteases. Both structural and kinetic analyses require milligram amounts of protein, which may be challenging for membrane proteins such as rhomboids. Here, we present a detailed protocol for optimization of expression and purification of three rhomboid proteases from Escherichia coli (ecGlpG), Haemophilus influenzae (hiGlpG), and Providencia stuartii (AarA)...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28065265/an-inducible-reconstitution-system-for-the-real-time-kinetic-analysis-of-protease-activity-and-inhibition-inside-the-membrane
#11
R P Baker, S Urban
Intramembrane proteases are an ancient and diverse group of multispanning membrane proteins that cleave transmembrane substrates inside the membrane to effect a wide range of biological processes. As proteases, a clear understanding of their function requires kinetic dissection of their catalytic mechanism, but this is difficult to achieve for membrane proteins. Kinetic measurements in detergent systems are complicated by micelle fusion/exchange, which introduces an additional kinetic step and imposes system-specific behaviors (e...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28065264/a-new-method-to-determine-the-transmembrane-conformation-of-substrates-in-intramembrane-proteolysis-by-deep-uv-resonance-raman-spectroscopy
#12
J W Cooley, A Abdine, M Brown, J Chavez, B Lada, R D JiJi, I Ubarretxena-Belandia
We present a new method based on deep-UV resonance Raman spectroscopy to determine the backbone conformation of intramembrane protease substrates. The classical amide vibrational modes reporting on the conformation of just the transmembrane region of the substrate can be resolved from solvent exchangeable regions outside the detergent micelle by partial deuteration of the solvent. In the presence of isotopically triple-labeled intramembrane protease, these amide modes can be accurately measured to monitor the transmembrane conformation of the substrate during intramembrane proteolysis...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28065263/probing-the-structure-and-function-relationships-of-presenilin-by-substituted-cysteine-accessibility-method
#13
T Tomita
Presenilin is a catalytic subunit of γ-secretase, which hydrolyzes several transmembrane proteins within the lipid bilayer, together with binding cofactors such as nicastrin, Aph-1, and Pen-2. However, the structural basis as well as molecular mechanism of this unusual proteolytic process remains unknown. We have analyzed the structure and function relationships of presenilin using the substituted-cysteine accessibility method (SCAM), which enables identification of the hydrophilic environment by the accessibility of sulfhydryl reagents to cysteine residues introduced at a desired position...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28065262/analyzing-amyloid-%C3%AE-peptide-modulation-profiles-and-binding-sites-of-%C3%AE-secretase-modulators
#14
J Trambauer, A Fukumori, B Kretner, H Steiner
γ-Secretase is a key player in the pathogenesis of Alzheimer's disease (AD). The intramembrane-cleaving enzyme initially cleaves a C-terminal fragment of the amyloid precursor protein (APP) at the ɛ-site within its transmembrane domain to release the APP intracellular domain. Subsequent stepwise carboxy-terminal trimming cleavages eventually release amyloid-β (Aβ) peptides of 37-43 amino acids into the extracellular space. Aβ42 as well as the much less abundant Aβ43 species are highly aggregation prone and can deposit as plaques in the brains of affected patients, which are widely believed to be causative of AD...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28065261/expression-purification-and-enzymatic-characterization-of-intramembrane-proteases
#15
R Zhou, Y Shi, G Yang
Intramembrane proteases catalyze peptide bond hydrolysis in the lipid bilayer and play a key role in numerous cellular processes. These integral membrane enzymes consist of four classes: site-2 protease (S2P), rhomboid serine protease, Rce1-type glutamyl protease, and aspartyl protease exemplified by presenilin and signal peptide peptidase (SPP). Structural elucidation of these enzymes is important for mechanistic understanding of their functions, particularly their roles in cell signaling and debilitating diseases such as Parkinson's disease and Alzheimer's disease...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28065260/biochemical-characterization-of-function-and-structure-of-rsep-an-escherichia-coli-s2p-protease
#16
Y Hizukuri, K Akiyama, Y Akiyama
Intramembrane-cleaving proteases (I-CLiPs) are a group of membrane-associated proteases with a unique feature: they are believed to cleave their substrate within the hydrophobic lipid bilayer, even though peptide bond hydrolysis requires a water molecule. Escherichia coli RseP, which belongs to the S2P zinc metalloprotease family of I-CLiPs, plays an essential role in activation of a cell envelope stress response through cleavage of anti-σ(E) protein RseA, a single-span transmembrane protein. A recent study showed that it also cleaves remnant signal peptides generated upon membrane translocation of secretory proteins...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28063501/preface
#17
EDITORIAL
Michael H Gelb
No abstract text is available yet for this article.
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28063500/cellular-assays-for-evaluating-calcium-dependent-translocation-of-cpla2%C3%AE-to-membrane
#18
B Yun, C C Leslie
The group IVA phospholipase A2, commonly called cytosolic phospholipase A2α (cPLA2α), is a widely expressed enzyme that hydrolyzes membrane phospholipid to produce arachidonic acid and lysophospholipids, which are precursors for a number of bioactive lipid mediators. Arachidonic acid is metabolized through the cyclooxygenase and lipoxygenase pathways for production of prostaglandins and leukotrienes that regulate normal physiological processes and contribute to disease pathogenesis. cPLA2α is composed of an N-terminal C2 domain and a C-terminal catalytic domain that contains the Ser-Asp catalytic dyad...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28063499/analysis-of-phosphatidic-acid-binding-and-regulation-of-pipki-in-vitro-and-in-intact-cells
#19
L W R Tay, Z Wang, G Du
Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is a lipid second messenger that regulates a wide array of essential cellular events, such as signal transduction, vesicle trafficking, actin cytoskeleton dynamics, adhesion, and motility. To control the spatiotemporal production of PI(4,5)P2, the activity of type 1 phosphotidylinositol-4-phosphate-5-kinases (PIPKIs) is tightly regulated by small GTPases and another signaling lipid, phosphatidic acid (PA). It is of interest that PI(4,5)P2 is also a critical cofactor for the activation of the PA-generating enzyme, phospholipase D (PLD)...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28063498/preparation-of-the-full-set-of-recombinant-mouse-and-human-secreted-phospholipases-a2
#20
F Ghomashchi, V Brglez, C Payré, L Jeammet, S Bezzine, M H Gelb, G Lambeau
A family of 14-20kDa, disulfide-rich, calcium-dependent secreted phospholipases A2 (sPLA2s) that release fatty acids from the sn-2 position of glycerophospholipids can be found in mammals. They have a diverse array of tissue distribution and biological functions. In this chapter we provide detailed protocols for production of nearly all of the mouse and human sPLA2s mainly by expression in bacteria and in vitro refolding or by expression in insect cells. High-resolution mass spectrometry and enzymatic assays were, respectively, used to show that all disulfides are formed and that the enzymes are active, strongly suggesting that each sPLA2 was prepared in the structurally native form...
2017: Methods in Enzymology
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