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Biochemistry
Generation of Human Pyruvate Carboxylase Knockout Cell Lines Using Retrovirus Expressing Short Hairpin RNA and CRISPR-Cas9 as Models to Study Its Metabolic Role in Cancer Research.
Dec 11, 2018   Methods In Molecular Biology (Clifton, N.J.)
Rattanapornsompong K, Ngamkham J, Chavalit T, Jitrapakdee S
Generation of Human Pyruvate Carboxylase Knockout Cell Lines Using Retrovirus Expressing Short Hairpin RNA and CRISPR-Cas9 as Models to Study Its Metabolic Role in Cancer Research.
Dec 11, 2018
Methods In Molecular Biology (Clifton, N.J.)
We report two protocols to generate human pyruvate carboxylase knockdown and knockout cell lines using short hairpin RNA (shRNA) and CRISPR-Cas9 technologies. The first protocol involved cloning of a shRNA cassette targeted to human pyruvate carboxylase (PC) under the control of a U6 promoter in a retrovirus-based vector. The stable knockdown cells were achieved following infection of retroviruses expressing shRNA in target cells followed by selecting these in medium containing puromycin. The second protocol describes a CRISPR Cas9-knockout cell constructed by cloning of single guide RNA (gRNA) targeted to the human pyruvate carboxylase gene placed adjacent to Cas 9 in the pSpCas9(BB)-2A-GFP vector. The knockout cells can be selected by sorting the cells expressing GFP. We also describe protocols for detecting the level of PC mRNA and protein in the knockdown or knockout cells using qPCR and Western blot analyses, respectively. The above protocols allow investigators to create PC deficient cell lines as a tool to study role of this enzyme in cancer research.
Conversion of Fibroblasts to Hepatocytes In Vitro.
Dec 11, 2018   Methods In Molecular Biology (Clifton, N.J.)
Huang P, Sun L, Zhang L, Hui L
Conversion of Fibroblasts to Hepatocytes In Vitro.
Dec 11, 2018
Methods In Molecular Biology (Clifton, N.J.)
Primary hepatocytes are widely used in regenerative medicine, drug metabolism analysis, and in vitro drug screens. To overcome the shortage of liver donors, several strategies, such as differentiation of pluripotent stem cells and transdifferentiation from somatic cells, were developed to generate hepatocytes from alternative sources. Here, we describe in detail lenti-virus-based procedure for direct conversion of human fibroblasts to hepatocytes (hiHep cells) in vitro. A detailed protocol for preparation of human fibroblasts from scar tissues is also provided. Based on this protocol, FOXA3, HNF1A, and HNF4A are introduced into SV40-large-T-antigen-expressing human scar fibroblasts by lenti-virus. It usually takes about 5-7 days to get epithelial hiHep colonies. SV40-large-T-antigen-expressing hiHep (hiHepLT) cells are proliferative and can be expanded to a large number for potential uses.
Spontaneous Symmetry Breaking in the Formation of Supramolecular Polymers: Implications for the Origin of Biological Homochirality.
Dec 11, 2018   Angewandte Chemie (International Ed. In English)
Hud NV, Schuster G, Karunakaran S, Cafferty B, Weigert-Muñoz A
Spontaneous Symmetry Breaking in the Formation of Supramolecular Polymers: Implications for the Origin of Biological Homochirality.
Dec 11, 2018
Angewandte Chemie (International Ed. In English)
Aqueous solutions of the achiral, monomeric, nucleobase mimics triaminopyrimidine (TAP) and a cyanuric acid derivative (CyCo6) spontaneously assemble into macroscopic homochiral domains of supramolecular polymers. These assemblies exhibit a high degree of chiral amplification. Addition of a small quantity of one handedness of a chiral derivative of CyCo6 generates exclusively homochiral structures. This system exhibits the highest reported degree of chiral amplification for dynamic helical polymers or supramolecular helices. Significantly, homochiral polymers comprised of hexameric rosettes with structural features that resemble nucleic acids are formed from mixtures of cyanuric acid (Cy) and a ribonucleotides (l-, d-pTARC) that arise spontaneously from the reaction of TAP with the sugars. These findings support the hypothesis that nucleic acid homochirality was a result of symmetry breaking at the supramolecular polymer level.
Linear mixed models for association analysis of quantitative traits with next-generation sequencing data.
Dec 11, 2018   Genetic Epidemiology
Chiu CY, Yuan F, Zhang BS, Yuan A, Li X,   . . . . . .   , Cook RJ, McMahon FJ, Amos CI, Xiong M, Fan R
Linear mixed models for association analysis of quantitative traits with next-generation sequencing data.
Dec 11, 2018
Genetic Epidemiology
We develop linear mixed models (LMMs) and functional linear mixed models (FLMMs) for gene-based tests of association between a quantitative trait and genetic variants on pedigrees. The effects of a major gene are modeled as a fixed effect, the contributions of polygenes are modeled as a random effect, and the correlations of pedigree members are modeled via inbreeding/kinship coefficients. F -statistics and χ 2 likelihood ratio test (LRT) statistics based on the LMMs and FLMMs are constructed to test for association. We show empirically that the F -distributed statistics provide a good control of the type I error rate. The F -test statistics of the LMMs have similar or higher power than the FLMMs, kernel-based famSKAT (family-based sequence kernel association test), and burden test famBT (family-based burden test). The F -statistics of the FLMMs perform well when analyzing a combination of rare and common variants. For small samples, the LRT statistics of the FLMMs control the type I error rate well at the nominal levels α = 0.01 and 0.05 . For moderate/large samples, the LRT statistics of the FLMMs control the type I error rates well. The LRT statistics of the LMMs can lead to inflated type I error rates. The proposed models are useful in whole genome and whole exome association studies of complex traits.
Melatonin inhibits the migration of human gastric carcinoma cells at least in part by remodeling tight junction.
Dec 11, 2018   Journal Of Cellular Biochemistry
Wei X, Chen S, Xu Z, Jia N, Qi Y, Zhou Q, Wang J, Qu L, Zhang S, Wang Y
Melatonin inhibits the migration of human gastric carcinoma cells at least in part by remodeling tight junction.
Dec 11, 2018
Journal Of Cellular Biochemistry
The recurrence and metastasis is one of the major reasons for malignant tumor treatment failure. Melatonin, a naturally occuring hormone, could reduce the recurrence and metastasis of various tumors. However, the underlying molecular mechanisms of melatonin on tumor metastasis inhibition have not been fully elucidated. In the present study, we explored the impact of melatonin on the migratory capability of human gastric carcinoma cells using wound healing assay, and further investigated if the inhibition on migration ability of melatonin was embodied by relocating tight junction proteins zo-1 and occludin onto the cells surface to remodel tight junction structure. Immunofluorescence assay and Western blot analysis were performed to detect the expression and cell location of the tight junction proteins. The migration distance was decreased as the cells were treated with melatonin. And melatonin increased the membrane location of tight junction proteins, zo-1 and occludin, showed by immunofluorescence staining and Western blot analysis. The results we got show that melatonin makes tight junction proteins anchored more on the cells membrane to remodel cells tight junction, which increase cells adhesion and decrease motility, resulting in the inhibition of gastric cancer cells migration and metastasis ability.
CLIP-170 spatially modulates receptor tyrosine kinase recycling to coordinate cell migration.
Dec 11, 2018   Traffic (Copenhagen, Denmark)
Zaoui K, Duhamel S, Parachoniak CA, Park M
CLIP-170 spatially modulates receptor tyrosine kinase recycling to coordinate cell migration.
Dec 11, 2018
Traffic (Copenhagen, Denmark)
Endocytic sorting of activated receptor tyrosine kinases (RTKs), alternating between recycling and degradative processes, controls signal duration, location, and surface complement of RTKs. The Microtubule (MT) plus-end tracking proteins (+TIPs) play essential roles in various cellular activities including translocation of intracellular cargo. However, mechanisms through which RTKs recycle back to the plasma membrane following internalisation in response to ligand remain poorly understood. We report that net outward-directed movement of endocytic vesicles containing the hepatocyte growth factor (HGF) Met RTK, requires recruitment of the +TIP, CLIP-170, as well as the association of CLIP-170 to MT plus-ends. In response to HGF, entry of Met into Rab4-positive endosomes results in Golgi-localized γ-ear-containing Arf-binding protein 3 (GGA3) and CLIP-170 recruitment to an activated Met RTK complex. We conclude that CLIP-170 co-ordinates the recycling and the transport of Met-positive endocytic vesicles to plus-ends of MTs towards the cell cortex, including the plasma membrane and the lamellipodia, thereby promoting cell migration. This article is protected by copyright. All rights reserved.
Microglial cell activation and senescence are characteristic of the pathology FXTAS.
Dec 11, 2018   Movement Disorders : Official Journal Of The Movement Disorder Society
Martínez Cerdeño V, Hong T, Amina S, Lechpammer M, Ariza J, Tassone F, Noctor SC, Hagerman P, Hagerman R
Microglial cell activation and senescence are characteristic of the pathology FXTAS.
Dec 11, 2018
Movement Disorders : Official Journal Of The Movement Disorder Society
BACKGROUND: Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder associated with premutation alleles of the FMR1 gene. Expansions of more than 200 CGG repeats give rise to fragile X syndrome, the most common inherited form of cognitive impairment. Fragile X-associated tremor/ataxia syndrome is characterized by cerebellar tremor and ataxia, and the presence of ubiquitin-positive inclusions in neurons and astrocytes. It has been previously suggested that fragile X-associated tremor/ataxia syndrome is associated with an inflammatory state based on signs of oxidative stress-mediated damage and iron deposition. OBJECTIVE: Determine whether the pathology of fragile X-associated tremor/ataxia syndrome involves microglial activation and an inflammatory state. METHODS: Using ionized calcium binding adaptor molecule 1 and cluster differentiation 68 antibodies to label microglia, we examined the number and state of activation of microglial cells in the putamen of 13 fragile X-associated tremor/ataxia syndrome and 9 control postmortem cases. RESULTS: Nearly half of fragile X-associated tremor/ataxia syndrome cases (6 of 13) presented with dystrophic senescent microglial cells. In the remaining fragile X-associated tremor/ataxia syndrome cases (7 of 13), the number of microglial cells and their activation state were increased compared to controls. CONCLUSIONS: The presence of senescent microglial cells in half of fragile X-associated tremor/ataxia syndrome cases suggests that this indicator could be used, together with the presence of intranuclear inclusions and the presence of iron deposits, as a biomarker to aid in the postmortem diagnosis of fragile X-associated tremor/ataxia syndrome. An increased number and activation indicate that microglial cells play a role in the inflammatory state present in the fragile X-associated tremor/ataxia syndrome brain. Anti-inflammatory treatment of patients with fragile X-associated tremor/ataxia syndrome may be indicated to slow neurodegeneration. © 2018 International Parkinson and Movement Disorder Society.
Structures and activities of widely conserved small prokaryotic Aminopeptidases P clarify classification of M24B peptidases.
Dec 11, 2018   Proteins
Are VN, Kumar A, Goyal VD, Gotad SS, Ghosh B, Gadre R, Jamdar SN, Makde RD
Structures and activities of widely conserved small prokaryotic Aminopeptidases P clarify classification of M24B peptidases.
Dec 11, 2018
Proteins
The proline-specific M24B peptidases that cleave Xaa-Pro iminopeptide bond in dipeptides are called prolidases while those that preferentially cleave this bond at the N-termini of longer peptides are called aminopeptidases-P. Escherichia coli and other bacteria have small aminopeptidases-P (36-39 kDa) which are considerably diverged from the canonical aminopeptidase-P of E. coli (50 kDa). The structure-function studies of these small aminopeptidases-P are not reported. We report the crystal structures of small aminopeptidases-P from E. coli and Deinococcus radiodurans at 2.6 Å and 1.8 Å resolutions, respectively. We also report substrate specificities of these proteins and their ortholog from Mycobacterium tuberculosis. These are indeed aminopeptidases-P but structurally closer to small prolidases (38-40 kDa). The absence of substrate selectivity loop found in the prolidases enables them to hydrolyze longer peptides. We noticed absence of this loop and a conserved arginine in the canonical archaeal prolidase from Pyrococcus furiosus (Maher et al., Biochemistry. 43, 2004, 2771-2783) and suspected that this enzyme could actually be an aminopeptidase-P. Our enzymatic assays confirm this suspicion and facilitate identification of archaeal small aminopeptidases-P. Further, our mutagenesis studies illuminate the functional importance of DXRY sequence motif in bacterial small aminopeptidases-P. Conservation of this motif in metazoan three-domain aminopeptidases-P including human XPNPEP1 and XPNPEP2 suggest their common evolutionary origin. Our analyses thus reveal the sequence and structural features that distinguish small aminopeptidases-P from closely related M24B peptidases. This article is protected by copyright. All rights reserved.
Astragaloside IV inhibits cell proliferation in vulvar squamous cell carcinoma through the TGF-β/Smad signaling pathway.
Dec 11, 2018   Dermatologic Therapy
Zhao Y, Wang L, Wang Y, Dong S, Yang S, Guan Y, Wu X
Astragaloside IV inhibits cell proliferation in vulvar squamous cell carcinoma through the TGF-β/Smad signaling pathway.
Dec 11, 2018
Dermatologic Therapy
OBJECTIVE: To explore the inhibition of the proliferation of vulvar squamous cell carcinoma (VSCC) by astragaloside IV. METHODS: MTT examined the cell proliferation of VSCC. Flow cytometry analyzed cell cycle and apoptosis. Western blot assay detected the expression of some relevant proteins. RESULTS: AS-IV reduced the proliferation of SW962 cells in a concentration- and time-dependent manner, induced cell-cycle arresting in G0/G1 phase, as demonstrated by the up-regulation of P53 and P21 expression, and the down-regulation of cyclin D1 expression. AS-IV enhanced the expression of Bax and cleaved-caspase 3, and suppressed Bcl-2 and Bcl-xl expression, which resulted in apoptosis increased. Furthermore, the expression of Beclin-1 and LC3-B was upregulated and that of P62 was downregulated, which suggested that AS-IV could increase the incidence of autophagy in SW962 cells. After inhibiting autophagy by 3-methyladenine, cell apoptosis decreased upon AS-IV treatment. Similarly, TGF-β1 stimulated SW962 cells, cell proliferation enhanced, and the expression of TGF-βRII and Smad4 was decreased. Furthermore, the expression of proteins that promote apoptosis and autophagy decreased. After AS-IV treatment, the expression levels of the above proteins exhibited the opposite effect. CONCLUSION: AS-IV inhibits cell proliferation and induces apoptosis and autophagy through the TGF-β/Smad signaling pathway in VSCC. This article is protected by copyright. All rights reserved.
Perturbed expression pattern of the immediate early gene Arc in the dentate gyrus of GluA1 C-terminal palmitoylation-deficient mice.
Dec 11, 2018   Neuropsychopharmacology Reports
Itoh M, Okuno H, Yamada D, Yamashita M, Abe M, Natsume R, Kaizuka T, Sakimura K, Hoshino M, Mishina M, Wada K, Sekiguchi M, Hayashi T
Perturbed expression pattern of the immediate early gene Arc in the dentate gyrus of GluA1 C-terminal palmitoylation-deficient mice.
Dec 11, 2018
Neuropsychopharmacology Reports
BACKGROUND: AMPA receptors predominantly mediate fast excitatory synaptic transmission in the mammalian brain. Post-translational protein S-palmitoylation of AMPA receptor GluA subunits at their C-termini reversibly controls the receptors trafficking to and from excitatory glutamatergic synapses. Excitatory inputs to neurons induce the expression of immediate early genes (IEGs), including Arc, with particular spatial patterns. In the hippocampal dentate gyrus, Arc is mainly expressed in the upper (dorsal) blade at the basal state. GluA1 C-terminal palmitoylation-deficient (GluA1C811S) mice showed enhanced seizure susceptibility and disturbed synaptic plasticity without impaired gross anatomy or basal synaptic transmission. These mutant mice also exhibited an increased expression of IEG products, c-Fos and Arc proteins, in the hippocampus and cerebral cortex. In this report, we further analyzed excitability and Arc expression pattern in the dentate gyrus of GluA1C811S mice. METHODS AND RESULTS: Electrophysiological analysis of granule neurons to measure the evoked excitatory postsynaptic current/evoked inhibitory postsynaptic current ratio revealed that excitatory/inhibitory (E/I) balance was normal in GluA1C811S mice. In contrast, immunohistochemical staining showed an abnormal distribution of Arc-positive cells between upper and lower (ventral) blades of the dentate gyrus in these mutant mice. These data suggest that deficiency of GluA1 palmitoylation causes perturbed neuronal inputs from the entorhinal cortex to the dentate gyrus, which potentially underlies the excessive excitability in response to seizure-inducing stimulation. CONCLUSION: Our findings conclude that an appropriate regulation of Arc expression in the dentate gyrus, ensured by AMPA receptor palmitoylation, may be critical for stabilizing hippocampal neural circuits and may suppress excess excitation.
A Click-Chemistry Linked 2'3'-cGAMP Analog.
Dec 11, 2018   Chemistry (Weinheim An Der Bergstrasse, Germany)
Dialer CR, Stazzoni S, Drexler DJ, Müller FM, Veth S, Pichler A, Okamura H, Witte G, Hopfner KP, Carell T
A Click-Chemistry Linked 2'3'-cGAMP Analog.
Dec 11, 2018
Chemistry (Weinheim An Der Bergstrasse, Germany)
2'3'-cGAMP is an uncanonical cyclic dinucleotide where one A and one G base are connected via a 3'-5' and a unique 2'-5' linkage. The molecule is produced by the cyclase cGAS in response to cytosolic DNA binding. cGAMP activates STING and hence one of the most powerful pathways of innate immunity. cGAMP analogs with uncharged linkages that feature better cellular penetrability are currently highly desired. Here, we report the synthesis of a cGAMP analog with one amide and one triazole linkage. The molecule is best prepared via a first Cu(I) catalysed click reaction which establishes the triazole, while the cyclization is achieved by macrolactamization.
Characterization of the Citrobacter rodentium Cpx regulon and its role in host infection.
Dec 11, 2018   Molecular Microbiology
Vogt SL, Scholz R, Peng Y, Guest RL, Scott NE, Woodward SE, Foster LJ, Raivio TL, Finlay BB
Characterization of the Citrobacter rodentium Cpx regulon and its role in host infection.
Dec 11, 2018
Molecular Microbiology
Envelope-localized proteins, such as adhesins and secretion systems, play critical roles in host infection by Gram-negative pathogens. As such, their folding is monitored by envelope stress response systems. Previous studies demonstrated that the Cpx envelope stress response is required for virulence of Citrobacter rodentium, a murine pathogen used to model infections by the human pathogens enteropathogenic and enterohemorrhagic Escherichia coli; however, the mechanisms by which the Cpx response promotes host infection were previously unknown. Here, we characterized the C. rodentium Cpx regulon in order to identify genes required for host infection. Using transcriptomic and proteomic approaches, we found that the Cpx response upregulates envelope-localized protein folding and degrading factors but downregulates pilus genes and type III secretion effectors. Mouse infections with C. rodentium strains lacking individual Cpx-regulated genes showed that the chaperone/protease DegP and the disulfide bond oxidoreductase DsbA were essential for infection, but Cpx regulation of these genes did not fully account for attenuation of C. rodentium ΔcpxRA. Both deletion of dsbA and treatment with the reducing agent dithiothreitol activated the C. rodentium Cpx response, suggesting that it may sense disruption of disulfide bonding. Our results highlight the importance of envelope protein folding in host infection by Gram-negative pathogens. This article is protected by copyright. All rights reserved.
Isolation of Synthetic Antibodies Against BCL-2-Associated X Protein (BAX).
Dec 11, 2018   Methods In Molecular Biology (Clifton, N.J.)
Dai Z, Lai JR
Isolation of Synthetic Antibodies Against BCL-2-Associated X Protein (BAX).
Dec 11, 2018
Methods In Molecular Biology (Clifton, N.J.)
The BCL-2 protein family plays central roles in the mitochondrial pathway of cell apoptosis. The BCL-2-Associated X protein (BAX), along with other proapoptotic proteins, induces cell death in response to a variety of stress stimuli. Upon receipt of killing signals, cytosolic BAX is activated and translocates to mitochondria where it causes mitochondrial outer membrane permeabilization (MOMP) and initials a series of cellular events that eventually lead to cell destruction. Despite recent progress toward understanding the structure, function, and activation mechanism of BAX, detailed information about how cytosolic BAX can be inhibited is still limited. Here we describe a method of selecting synthetic antibody fragments (Fabs) against BAX using phage display. Synthetic antibodies discovered from the selection have been used as structural probes to gain novel mechanistic details on BAX inhibition. This synthetic antibody selection method could be potentially applied to other BCL-2 proteins.
The Use of Primary Hepatocytes in Assessment of Drug Safety and Toxicity.
Dec 11, 2018   Methods In Molecular Biology (Clifton, N.J.)
Guest PC
The Use of Primary Hepatocytes in Assessment of Drug Safety and Toxicity.
Dec 11, 2018
Methods In Molecular Biology (Clifton, N.J.)
The identification of biomarkers for toxicity is becoming increasingly important for drug discovery and development. This chapter describes the preparation and utilization of primary rat hepatocytes as a cellular model of steatosis. A protocol is presented for dosing the cells with the steatosis-inducing compound amiodarone, along with the conduction of assays for measuring lipid accumulation and mitochondrial function. A differential solubility extraction procedure is also presented, which can be used for proteomic profiling analysis.
Quantification of the Interactions Between BCL-2 Proteins by Fluorescence Correlation Spectroscopy.
Dec 11, 2018   Methods In Molecular Biology (Clifton, N.J.)
Murad F, Garcia-Saez AJ
Quantification of the Interactions Between BCL-2 Proteins by Fluorescence Correlation Spectroscopy.
Dec 11, 2018
Methods In Molecular Biology (Clifton, N.J.)
The proteins of the Bcl-2 family regulate apoptosis by forming a complex interaction network whose output determines whether mitochondrial outer membrane permeabilization is executed. Quantification of complex formation between Bcl-2 proteins in solution and in membranes is therefore key to understand how the hierarchy of interactions controls cell death induction. Fluorescence correlation spectroscopy (FCS) is a noninvasive, nondestructive method to investigate the mobility and the association of fluorescently labeled biomolecules that has provided useful insight into the binding affinity of the Bcl-2 interactome. FCS is based on the detection of fluorescence fluctuations caused by the diffusion of individual molecules through a very tiny observation volume of the detection system. Scanning FCS (SFCS) solves some of the practical challenges of acquiring FCS in membranes and expands the application scope of the method. In this chapter, we explain the principle of FCS and describe protocols how it can be used to quantify interactions between Bcl-2 proteins in solution and in model membrane systems.
Rapid Imaging of BCL-2 Family Interactions in Live Cells Using FLIM-FRET.
Dec 11, 2018   Methods In Molecular Biology (Clifton, N.J.)
Osterlund EJ, Hirmiz N, Tardif C, Andrews DW
Rapid Imaging of BCL-2 Family Interactions in Live Cells Using FLIM-FRET.
Dec 11, 2018
Methods In Molecular Biology (Clifton, N.J.)
The Bcl-2 proteins control cell death via interchanging interactions within the Bcl-2 family. Fluorescence lifetime imaging microscopy (FLIM) is used to detect Förster resonance energy transfer (FRET) between two fluorescent-fusion proteins in live cells. FLIM-FRET has been used to detect specific interactions and their disruption, for Bcl-2 family proteins. To date, this has been possible only in low throughput, due to the time required for serial data acquisition. We developed an automated optical system with eight parallel detectors for rapid and efficient data collection. Here we describe how to use this system for FLIM-FRET imaging of Bcl-2 family protein interactions in a 384-well plate format.
CW EPR and DEER Methods to Determine BCL-2 Family Protein Structure and Interactions: Application of Site-Directed Spin Labeling to BAK Apoptotic Pores.
Dec 11, 2018   Methods In Molecular Biology (Clifton, N.J.)
Mandal T, Hustedt EJ, Song L, Oh KJ
CW EPR and DEER Methods to Determine BCL-2 Family Protein Structure and Interactions: Application of Site-Directed Spin Labeling to BAK Apoptotic Pores.
Dec 11, 2018
Methods In Molecular Biology (Clifton, N.J.)
The continuous wave (CW) and pulse electron paramagnetic resonance (EPR) methods enable the measurement of distances between spin-labeled residues in biopolymers including proteins, providing structural information. Here we describe the CW EPR deconvolution/convolution method and the four-pulse double electron-electron resonance (DEER) approach for distance determination, which were applied to elucidate the organization of the BAK apoptotic pores formed in the lipid bilayers.
BCL-2 Protein Family Interaction Analysis by Nuclear Magnetic Resonance Spectroscopy.
Dec 11, 2018   Methods In Molecular Biology (Clifton, N.J.)
Garner TP, Gavathiotis E
BCL-2 Protein Family Interaction Analysis by Nuclear Magnetic Resonance Spectroscopy.
Dec 11, 2018
Methods In Molecular Biology (Clifton, N.J.)
Biomolecular nuclear magnetic resonance (NMR) is a powerful and versatile method for studying both protein-protein interactions (PPIs) and protein-small molecule binding. NMR has been used extensively in the investigation of BCL-2 family proteins revealing the structure of key family members, identifying binding partners and interaction sites, and screening small molecule modulators. In this chapter we discuss the application of NMR to identify interaction sites and structure determination of protein-protein and protein-small molecule complexes using two examples.
Assessment of Dynamic BCL-2 Protein Shuttling Between Outer Mitochondrial Membrane and Cytosol.
Dec 11, 2018   Methods In Molecular Biology (Clifton, N.J.)
Lauterwasser J, Fimm-Todt F, Edlich F
Assessment of Dynamic BCL-2 Protein Shuttling Between Outer Mitochondrial Membrane and Cytosol.
Dec 11, 2018
Methods In Molecular Biology (Clifton, N.J.)
BCL-2 proteins control stress-dependent commitment to the programmed cell death apoptosis. In nonapoptotic cells the proapoptotic BCL-2 proteins BAX and BAK but also prosurvival family members, like BCL-xL or MCL-1, translocate to the outer mitochondrial membrane (OMM) and retrotranslocate from the mitochondria back into the cytosol. The resulting equilibrium produces a broad range of localization pattern observed for BAX and BAK in human cells and shows correlation between relative BAX and BAK localizations and cellular predisposition to apoptosis. The retrotranslocation of BCL-2 proteins from the OMM can be measured using fluorescence-labeled protein in intact cells or endogenous protein from isolated heavy membrane fractions.
Photocrosslinking Approach to Investigate Protein Interactions in the BCL-2 Family.
Dec 11, 2018   Methods In Molecular Biology (Clifton, N.J.)
Lin J, Johnson AE, Zhang Z
Photocrosslinking Approach to Investigate Protein Interactions in the BCL-2 Family.
Dec 11, 2018
Methods In Molecular Biology (Clifton, N.J.)
The Bcl-2 family of proteins regulates mitochondrial outer membrane permeability thereby making life or death decisions for cells. Most of Bcl-2 proteins contain hydrophobic regions that are embedded in intracellular membranes such as mitochondria. These membrane proteins are difficult to express and purify thereby preluding biochemical and biophysical characterizations. Here, we describe a photocrosslinking approach based on in vitro synthesis of Bcl-2 proteins with photoreactive amino acid analogs incorporated at specific locations. These photoreactive proteins are reconstituted into liposomal membranes with defined phospholipids or mitochondrial membranes isolated from animals, and their interactions with other Bcl-2 proteins are detected by photocrosslinking.
Liposomal Permeabilization Assay to Study the Functional Interactions of the BCL-2 Family.
Dec 11, 2018   Methods In Molecular Biology (Clifton, N.J.)
Reyna DE, Gavathiotis E
Liposomal Permeabilization Assay to Study the Functional Interactions of the BCL-2 Family.
Dec 11, 2018
Methods In Molecular Biology (Clifton, N.J.)
Apoptosis, a form of programmed cell death that is important for development and homeostasis, is regulated by the BCL-2 family of proteins. Over twenty BCL-2 family members have been classified in three groups based on structural homology and function. The multidomain antiapoptotic proteins promote survival, whereas the multidomain and the BH3-only proapoptotic members induce cell death. Because the interaction among the BCL-2 family members occurs primarily at the mitochondrial outer membrane, biochemical assays using artificial liposomes have been developed to study the functional relationship between these proteins. The liposomal permeabilization assay is a cell-free system that relies on the ability of multidomain pro-apoptotic members to promote membrane permeabilization upon activation. By encapsulating a fluorophore and a quencher into liposomes, the degree of permeabilization can be quantified by the increase in fluorescence intensity as the fluorophore and quencher dissociate. The liposomal permeabilization assay has been used to delineate interactions among BCL-2 family members as well as to characterize peptides, small molecules, and lipids that modulate the function of BCL-2 family of proteins. Here, we describe in detail the permeabilization of liposomes induced by the interaction between BAX and BH3-only activator tBID.
Application of Mito-Priming to Generate BCL-2 Addicted Cells.
Dec 11, 2018   Methods In Molecular Biology (Clifton, N.J.)
Lopez J, Tait SWG
Application of Mito-Priming to Generate BCL-2 Addicted Cells.
Dec 11, 2018
Methods In Molecular Biology (Clifton, N.J.)
The majority of apoptotic stimuli trigger cell death through the mitochondrial pathway of apoptosis. Invariably, mitochondrial apoptosis requires engagement of mitochondrial outer membrane permeabilization or MOMP to initiate cell death. We have developed a new method, called mito-priming, that allows for rapid and synchronous induction of mitochondrial apoptosis in an on-target manner. Mito-priming uses coexpression of pro- and antiapoptotic Bcl-2 proteins to render cells sensitive to the addition of Bcl-2 targeting BH3-mimetic drugs. This chapter describes how to design mito-priming constructs and apply them to generate mito-primed lines. Second, we describe how to validate cell death sensitivity of mito-primed lines using different methods. Finally, we describe how to generate MOMP-resistant cell lines, using CRISPR-Cas9 mediated deletion of BAX and BAK. Facilitating the investigation of mitochondrial apoptosis, mito-priming provides a clean, robust way to induce mitochondrial apoptosis both in vitro and in vivo.
A Complete Proteomic Workflow to Study Brain-Related Disorders via Postmortem Tissue.
Dec 11, 2018   Methods In Molecular Biology (Clifton, N.J.)
Reis-de-Oliveira G, Fioramonte M, Martins-de-Souza D
A Complete Proteomic Workflow to Study Brain-Related Disorders via Postmortem Tissue.
Dec 11, 2018
Methods In Molecular Biology (Clifton, N.J.)
Here we describe a mass spectrometry-based proteomics workflow to discovery proteins differentially regulated in brains collected postmortem from mental, neurological, or substance abuse disorders (MNS) patients. One way to maximize protein detection is to carry out enrichment of cellular compartments such as the nucleus, mitochondria and cytosol. Subcellular fractionation improves proteome coverage and may shed light on the role of these organelles in the pathophysiology of MNS.
A Rat Eye Lens Model of Cataract Formation.
Dec 11, 2018   Methods In Molecular Biology (Clifton, N.J.)
Guest PC
A Rat Eye Lens Model of Cataract Formation.
Dec 11, 2018
Methods In Molecular Biology (Clifton, N.J.)
This chapter describes the use of lenses obtained from rats as a model of cataractogenesis. At the molecular level, this is visualized as reduced activity of oxidative reductive enzymes such as aldose reductase and increased proteolysis of lens structural proteins including vimentin. In this chapter, protocols for assessment of these two pathways are presented. Specifically, this analysis shows a comparison of aldose reductase activity and vimentin cleavage in male and female rat lenses. This is because female rats are more susceptible to cataract formation compared to males.
The pKa values of the catalytic residues in the retaining glycoside hydrolase T26H mutant of T4 lysozyme.
Dec 11, 2018   Protein Science : A Publication Of The Protein Society
Brockerman JA, Okon M, Withers SG, McIntosh LP
The pKa values of the catalytic residues in the retaining glycoside hydrolase T26H mutant of T4 lysozyme.
Dec 11, 2018
Protein Science : A Publication Of The Protein Society
T4 phage lysozyme (T4L) is an enzyme that cleaves bacterial cell wall peptidoglycan. Remarkably, the single substitution of the active site Thr26 to a His (T26H) converts T4L from an inverting to a retaining glycoside hydrolase with transglycosylase activity. It has been proposed that T26H-T4L follows a double displacement mechanism with His26 serving as a nucleophile to form a covalent glycosyl-enzyme intermediate (Kuroki et al. (1999) PNAS 96: 8949-8954). To gain further insights into this or alternative mechanisms, we used NMR spectroscopy to measure the acid dissociation constants (pKa values) and/or define the ionization states of the Asp, Glu, His, and Arg residues in the T4L mutant. Most notably, the pKa value of the putative nucleophile His26 is 6.8 ± 0.1, whereas that of the general acid Glu11 is 4.7 ± 0.1. If the proposed mechanism holds true, then T26H-T4L follows a reverse protonation pathway in which only a minor population of free enzyme is in its catalytically competent ionization state with His26 deprotonated and Glu11 protonated. Our studies also confirm that all arginines in T26H-T4L, including the active site Arg145, are positively charged under neutral pH conditions. This article is protected by copyright. All rights reserved.

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