Efficient isotopic tryptophan labeling of membrane proteins by an indole controlled process conduct

A protocol for the efficient isotopic labeling of large G protein‐coupled receptors with tryptophan in Escherichia coli as expression host was developed that sufficiently suppressed the naturally occurring L‐tryptophan indole lyase, which cleaves tryptophan into indole, pyruvate, and ammonia resulting in scrambling of the isotopic label in the protein. Indole produced by the tryptophanase is naturally used as messenger for cell–cell communication. Detailed analysis of different process conducts led to the optimal expression strategy, which mimicked cell–cell communication by the addition of indole during expression. Discrete concentrations of indole and ¹⁵N₂‐L‐tryptophan at dedicated time points in the fermentation drastically increased the isotopic labeling efficiency. Isotope scrambling was only observed in glutamine, asparagine, and arginine side chains but not in the backbone. This strategy allows producing specifically tryptophan labeled membrane proteins at high concentrations avoiding the disadvantages of the often low yields of auxotrophic E. coli strains. In the fermentation process carried out according to this protocol, we produced ～15 mg of tryptophan labeled neuropeptide Y receptor type 2 per liter medium. Biotechnol. Bioeng. 2013; 110: 1681–1690. © 2013 Wiley Periodicals, Inc.     

Affinity chromatography of the D1 dopamine receptor from rat corpus striatum

The D1 dopamine receptor from rat corpus striatum has been purified 200-250-fold by using a newly developed biospecific affinity chromatography matrix based on a derivative of the D1 selective antagonist SCH 23390. This compound, (RS)-5-(4-aminophenyl)-8-chloro-2,3,4,5-tetrahydro-3-methyl-1H-3-benz azepin-7-o l (SCH 39111), possesses high affinity for the D1 receptor and, when immobilized on Sepharose 6B through an extended spacer arm, was able to adsorb digitonin-solubilized D1 receptors. The interaction between the solubilized receptor and the affinity matrix was biospecific. Adsorption of receptor activity could be blocked in a stereoselective fashion [SCH 23390 greater than SCH 23388; (+)-butaclamol greater than (-)-butaclamol]. The elution of [3H]SCH 23390 activity from the gel demonstrated similar stereoselectivity for antagonist ligands. Agonists eluted receptor activity with a rank order of potency consistent with that of a D1 receptor [apomorphine greater than dopamine greater than (-)-epinephrine much greater than LY 171555 greater than serotonin]. SCH 39111-Sepharose absorbed 75-85% of the soluble receptor activity, and after the gel was washed extensively, 35-55% of the absorbed receptor activity could be eluted with 100 microM (+)-butaclamol with specific activities ranging from 250 to 450 pmol/mg of protein. The affinity-purified receptor retains the ligand binding characteristics of a D1 dopamine receptor. This affinity chromatography procedure should prove valuable in the isolation and molecular characterization of the D1 dopamine receptor.     

Microtubule-dependent retrograde transport of proteins into the ER in the presence of brefeldin A suggests an ER recycling pathway

Characteristics of brefeldin A (BFA)-induced redistribution of Golgi proteins into the endoplasmic reticulum (ER) and its relationship to an ER retrieval pathway were investigated. Retrograde movement of Golgi proteins into the ER occurred via long, tubulovesicular processes extending out of the Golgi along microtubules. Microtubule-disrupting agents (i.e., nocodazole), energy poisons, and reduced temperatures inhibited this pathway. In BFA-treated cells Golgi proteins appeared to cycle between the ER and an intermediate compartment marked by a 53 kd protein. Addition of nocodazole disrupted this dynamic cycle by preferentially inhibiting retrograde movement, causing Golgi proteins to accumulate in the intermediate compartment. In the absence of BFA, such an ER cycling pathway appeared to be followed normally by the 53 kd protein but not by Golgi proteins, as revealed by temperature shift experiments. We propose that BFA induces the interaction of the Golgi with an intermediate "recycling" compartment that utilizes a microtubule-dependent pathway into the ER.     

Baculovirus expression: tackling the complexity challenge

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Lithium induces autophagy by inhibiting inositol monophosphatase

Macroautophagy is a key pathway for the clearance of aggregate-prone cytosolic proteins. Currently, the only suitable pharmacologic strategy for up-regulating autophagy in mammalian cells is to use rapamycin, which inhibits the mammalian target of rapamycin (mTOR), a negative regulator of autophagy. Here we describe a novel mTOR-independent pathway that regulates autophagy. We show that lithium induces autophagy, and thereby, enhances the clearance of autophagy substrates, like mutant huntingtin and alpha-synucleins. This effect is not mediated by glycogen synthase kinase 3beta inhibition. The autophagy-enhancing properties of lithium were mediated by inhibition of inositol monophosphatase and led to free inositol depletion. This, in turn, decreased myo-inositol-1,4,5-triphosphate (IP3) levels. Our data suggest that the autophagy effect is mediated at the level of (or downstream of) lowered IP3, because it was abrogated by pharmacologic treatments that increased IP3. This novel pharmacologic strategy for autophagy induction is independent of mTOR, and may help treatment of neurodegenerative diseases, like Huntington's disease, where the toxic protein is an autophagy substrate.     

Nucleotide pocket thermodynamics measured by EPR reveal how energy partitioning relates myosin speed to efficiency

We have used spin-labeled ADP to investigate the dynamics of the nucleotide-binding pocket in a series of myosins, which have a range of velocities. Electron paramagnetic resonance spectroscopy reveals that the pocket is in equilibrium between open and closed conformations. In the absence of actin, the closed conformation is favored. When myosin binds actin, the open conformation becomes more favored, facilitating nucleotide release. We found that faster myosins favor a more closed pocket in the actomyosin•ADP state, with smaller values of ΔH⁰ and ΔS⁰, even though these myosins release ADP at a faster rate. A model involving a partitioning of free energy between work-generating steps prior to rate-limiting ADP release explains both the unexpected correlation between velocity and opening of the pocket and the observation that fast myosins are less efficient than slow myosins.     

Osmium tetroxide, used in the treatment of arthritic joints, is a fast mimic of superoxide dismutase

Aqueous solutions of osmium tetroxide (OsO4) have been injected into arthritic knees for the past 45 years to chemically destroy diseased tissue, in a procedure termed "chemical synovectomy." Arthritis is an inflammatory disease. The primary inflammatory chemical species are the superoxide anion radical (O2.-) and nitric oxide (.NO), which combine to form the peroxynitrite anion (ONOO-). Here we show that OsO4 does not react with ONOO- but very efficiently catalyzes the dismutation of O2.- to O2 and H2O2. Using the pulse-radiolysis technique, the catalytic rate constant has been determined to be (1.43+/-0.04) x 10(9) M-1 s-1, independent of the pH in the 5.1-8.7 range. This value is about half that for the natural Cu,Zn-superoxide dismutase (Cu,Zn-SOD). Per unit mass, OsO4 is about 60 times more active than Cu,Zn-SOD. The catalytically active couple is OsVIII/OsVII, OsVIII oxidizing O2.- to O2 with a bimolecular rate constant of k=(2.6+/-0.1)x10(9) M-1 s-1 and OsVII reducing it to H2O2 with a bimolecular rate constant of (1.0+/-0.1)x10(9) M-1 s-1. Although lower valent osmium species are intrinsically poor catalysts, they are activated through oxidation by O2.- to the catalytic OsVIII/OsVII redox couple. The OsVIII/OsVII catalyst is stable to biochemicals other than proteins and peptides comprising histidine, cysteine, and dithiols.     

A hepatocellular carcinoma cell line producing mature hepatitis B viral particles

Current in vitro models for hepatitis B virus (HBV) are based on human hepatoblastoma cell lines transfected with HBV genome. The objective of this work was to develop an in vitro, hepatocellular carcinoma (HCC)-based system supporting HBV full replication and producing mature viral particles. The FLC4 human HCC cell line was stably transfected with a plasmid carrying a head-to-tail dimer of the adwHBV genome. One of the clones, FLC4A10II, exhibited prolonged expression of HBV, as was demonstrated by secreted levels of HBsAg, HBeAg, and HBV DNA in the culture medium of the growing cells. Furthermore, the cells produced HBV particles that were detected by a cesium chloride density gradient performed on the culture medium. Analysis by Southern blot revealed that HBV DNA has integrated into the FLC4A10II cell genome. The presence of HBV in the FLC4A10II cells did not cause alterations in cell morphology and the cells continued to resemble mature hepatocytes. They do exhibit a high mitotic activity. The new HBV stably transfected cell line, FLC4A10II, can serve as an important tool for further exploration of HBV host-pathogen interaction, viral life cycle, and for assessing new antiviral agents.     

CD46 is a cellular receptor for human herpesvirus 6

Human herpesvirus 6 (HHV-6) is the etiologic agent of exanthema subitum, causes opportunistic infections in immunocompromised patients, and has been implicated in multiple sclerosis and in the progression of AIDS. Here, we show that the two major HHV-6 subgroups (A and B) use human CD46 as a cellular receptor. Downregulation of surface CD46 was documented during the course of HHV-6 infection. Both acute infection and cell fusion mediated by HHV-6 were specifically inhibited by a monoclonal antibody to CD46; fusion was also blocked by soluble CD46. Nonhuman cells that were resistant to HHV-6 fusion and entry became susceptible upon expression of recombinant human CD46. The use of a ubiquitous immunoregulatory receptor opens novel perspectives for understanding the tropism and pathogenicity of HHV-6.