A Gene Optimization Strategy that Enhances Production of Fully Functional P-Glycoprotein in Pichia pastoris

Structural and biochemical studies of mammalian membrane proteins remain hampered by inefficient production of pure protein. We explored codon optimization based on highly expressed Pichia pastoris genes to enhance co-translational folding and production of P-glycoprotein (Pgp), an ATP-dependent drug efflux pump involved in multidrug resistance of cancers.Codon-optimized "Opti-Pgp" and wild-type Pgp, identical in primary protein sequence, were rigorously analyzed for differences in function or solution structure. Yeast expression levels and yield of purified protein from P. pastoris (∼130 mg per kg cells) were about three-fold higher for Opti-Pgp than for wild-type protein. Opti-Pgp conveyed full in vivo drug resistance against multiple anticancer and fungicidal drugs. ATP hydrolysis by purified Opti-Pgp was strongly stimulated ∼15-fold by verapamil and inhibited by cyclosporine A with binding constants of 4.2±2.2 µM and 1.1±0.26 µM, indistinguishable from wild-type Pgp. Maximum turnover number was 2.1±0.28 µmol/min/mg and was enhanced by 1.2-fold over wild-type Pgp, likely due to higher purity of Opti-Pgp preparations. Analysis of purified wild-type and Opti-Pgp by CD, DSC and limited proteolysis suggested similar secondary and ternary structure. Addition of lipid increased the thermal stability from T(m) ∼40 °C to 49 °C, and the total unfolding enthalpy. The increase in folded state may account for the increase in drug-stimulated ATPase activity seen in presence of lipids.The significantly higher yields of protein in the native folded state, higher purity and improved function establish the value of our gene optimization approach, and provide a basis to improve production of other membrane proteins

Restoration of NBD1 Thermal Stability Is Necessary and Sufficient to Correct ∆F508 CFTR Folding and Assembly

CFTR (ABCC7), unique among ABC exporters as an ion channel, regulates ion and fluid transport in epithelial tissues. Loss of function due to mutations in the cftr gene causes cystic fibrosis (CF). The most common CF-causing mutation, the deletion of F508 (ΔF508) from the first nucleotide binding domain (NBD1) of CFTR, results in misfolding of the protein and clearance by cellular quality control systems. The ΔF508 mutation has two major impacts on CFTR: reduced thermal stability of NBD1 and disruption of its interface with membrane-spanning domains (MSDs). It is unknown if these two defects are independent and need to be targeted separately. To address this question we varied the extent of stabilization of NBD1 using different second site mutations and NBD1 binding small molecules with or without NBD1/MSD interface mutation. Combinations of different NBD1 changes had additive corrective effects on ΔF508 maturation that correlated with their ability to increase NBD1 thermostability. These effects were much larger than those caused by interface modification alone and accounted for most of the correction achieved by modifying both the domain and the interface. Thus, NBD1 stabilization plays a dominant role in overcoming the ΔF508 defect. Furthermore, the dual target approach resulted in a locked-open ion channel that was constitutively active in the absence of the normally obligatory dependence on phosphorylation by protein kinase A. Thus, simultaneous targeting of both the domain and the interface, as well as being non-essential for correction of biogenesis, may disrupt normal regulation of channel function

A survey of transcutaneous blood gas monitoring among European neonatal intensive care units

BACKGROUND: PCO(2 )and PO(2 )are important monitoring parameters in neonatal intensive care units (NICU). Compared to conventional blood gas measurements that cause significant blood loss in preterms, transcutaneous (tc) measurements allow continuous, non-invasive monitoring of blood gas levels. The aim of the study was to survey the usage and opinions among German speaking NICUs concerning tc blood gas monitoring. METHODS: A questionnaire was developed and sent to 56 head nurses of different NICUs in Germany, Switzerland and Austria. RESULTS: A completely answered questionnaire was obtained from 41 NICUs. In two of these units tc measurements are not performed. In most NICUs (77%), both P(tc)O(2 )and P(tc)CO(2 )are measured simultaneously. Most units change the sensors every 3 hours; however, the recommended temperature of 44°C is used in only 15% of units. In only 8% of units are arterial blood gases obtained to validate tc values. Large variations were found concerning the targeted level of oxygen saturation [median upper limit: 95% (range 80–100%); median lower limit: 86% (range 75–93%)] and PO(2 )[median upper limit: 70 mmHg (range 45–90 mmHg); median lower limit: 44 mmHg (range 30–60 mmHg)]. CONCLUSION: Our survey shows that the use of tc monitors remains widespread among German speaking NICUs, despite earlier data suggesting that their use had been abandoned in many NICUs worldwide. In addition, we suggest that the current method of monitoring oxygenation may not prevent hyperoxemia in preterm infants

Membrane protein stability can be compromised by detergent interactions with the extramembranous soluble domains: Detergents Destabilize Extramembranous Soluble Domains

Detergent interaction with extramembranous soluble domains (ESDs) is not commonly considered an important determinant of integral membrane protein (IMP) behavior during purification and crystallization, even though ESDs contribute to the stability of many IMPs. Here we demonstrate that some generally nondenaturing detergents critically destabilize a model ESD, the first nucleotide-binding domain (NBD1) from the human cystic fibrosis transmembrane conductance regulator (CFTR), a model IMP. Notably, the detergents show equivalent trends in their influence on the stability of isolated NBD1 and full-length CFTR. We used differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy to monitor changes in NBD1 stability and secondary structure, respectively, during titration with a series of detergents. Their effective harshness in these assays mirrors that widely accepted for their interaction with IMPs, i.e., anionic > zwitterionic > nonionic. It is noteworthy that including lipids or nonionic detergents is shown to mitigate detergent harshness, as will limiting contact time. We infer three thermodynamic mechanisms from the observed thermal destabilization by monomer or micelle: (i) binding to the unfolded state with no change in the native structure (all detergent classes); (ii) native state binding that alters thermodynamic properties and perhaps conformation (nonionic detergents); and (iii) detergent binding that directly leads to denaturation of the native state (anionic and zwitterionic). These results demonstrate that the accepted model for the harshness of detergents applies to their interaction with an ESD. It is concluded that destabilization of extramembranous soluble domains by specific detergents will influence the stability of some IMPs during purification

Successful Cognitive Aging in Rats: A Role for mGluR5 Glutamate Receptors, Homer 1 Proteins and Downstream Signaling Pathways

Normal aging is associated with impairments in cognition, especially learning and memory. However, major individual differences are known to exist. Using the classical Morris Water Maze (MWM) task, we discriminated a population of 24-months old Long Evans aged rats in two groups - memory-impaired (AI) and memory-unimpaired (AU) in comparison with 6-months old adult animals. AI rats presented deficits in learning, reverse memory and retention. At the molecular level, an increase in metabotropic glutamate receptors 5 (mGluR5) was observed in post-synaptic densities (PSD) in the hippocampus of AU rats after training. Scaffolding Homer 1b/c proteins binding to group 1 mGluR facilitate coupling with its signaling effectors while Homer 1a reduces it. Both Homer 1a and 1b/c levels were up-regulated in the hippocampus PSD of AU animals following MWM task. Using immunohistochemistry we further demonstrated that mGluR5 as well as Homer 1b/c stainings were enhanced in the CA1 hippocampus sub-field of AU animals. In fact mGluR5 and Homer 1 isoforms were more abundant and co-localized in the hippocampal dendrites in AU rats. However, the ratio of Homer 1a/Homer 1b/c bound to mGluR5 in the PSD was four times lower for AU animals compared to AI rats. Consequently, AU animals presented higher PKCγ, ERK, p70S6K, mTOR and CREB activation. Finally the expression of immediate early gene Arc/Arg3.1 was shown to be higher in AU rats in accordance with its role in spatial memory consolidation. On the basis of these results, a model of successful cognitive aging with a critical role for mGluR5, Homer 1 proteins and downstream signalling pathways is proposed here

“Essential—Passion for Music”: Affirming, Critiquing, and Practising Passionate Work in Creative Industries

This chapter considers “passion” as an enthusiastic orientation to work within creative worlds: work motivated by intense attachments to the products of work and their conditions of production. Drawing on Luc Boltanski’s pragmatic sociology of critique and justification, the chapter argues that the passionate lens most usefully trains our sights on normative questions: not what or how—but why such work is undertaken. Embedded in research on cultural and creative industries, the contemporary recorded music sector is presented as a “passionate” industry in transformation. Interviews with workers, who both criticize and defend their industry, act as a springboard to explore three possible interpretive approaches: affirmative, critical, and pragmatic. Theoretical flexibility is needed to keep “passion” open to future inquiry—particularly regarding inequalities in creative work