Expression of Eukaryotic Membrane Proteins in Pichia pastoris.

A key point when it comes to heterologous expression of eukaryotic membrane proteins (EMPs) is the choice of the best-suited expression platform. The yeast Pichia pastoris has proven to be a very versatile system showing promising results in a growing number of cases. Indeed, its particular methylotrophic characteristics combined to the very simple handling of a eukaryotic microorganism that possesses the majority of mammalian-like machineries make it a very competitive expression system for various complex proteins, in amounts compatible with functional and structural studies. This chapter describes a set of robust methodologies routinely used for the successful expression of a variety of EMPs, going from yeast transformation with the recombinant plasmid to the analysis of the quality and quantity of the proteins produced.journal article2016importe

Carbon and arsenic metabolism in Thiomonas strains: differences revealed diverse adaptation processes

<p>Abstract</p> <p>Background</p> <p><it>Thiomonas </it>strains are ubiquitous in arsenic-contaminated environments. Differences between <it>Thiomonas </it>strains in the way they have adapted and respond to arsenic have never been studied in detail. For this purpose, five <it>Thiomonas </it>strains, that are interesting in terms of arsenic metabolism were selected: <it>T. arsenivorans</it>, <it>Thiomonas </it>spp. WJ68 and 3As are able to oxidise As(III), while <it>Thiomonas </it>sp. Ynys1 and <it>T. perometabolis </it>are not. Moreover, <it>T. arsenivorans </it>and 3As present interesting physiological traits, in particular that these strains are able to use As(III) as an electron donor.</p> <p>Results</p> <p>The metabolism of carbon and arsenic was compared in the five <it>Thiomonas </it>strains belonging to two distinct phylogenetic groups. Greater physiological differences were found between these strains than might have been suggested by 16S rRNA/<it>rpoA </it>gene phylogeny, especially regarding arsenic metabolism. Physiologically, <it>T. perometabolis </it>and Ynys1 were unable to oxidise As(III) and were less arsenic-resistant than the other strains. Genetically, they appeared to lack the <it>aox </it>arsenic-oxidising genes and carried only a single <it>ars </it>arsenic resistance operon. <it>Thiomonas arsenivorans </it>belonged to a distinct phylogenetic group and increased its autotrophic metabolism when arsenic concentration increased. Differential proteomic analysis revealed that in <it>T. arsenivorans</it>, the <it>rbc</it>/<it>cbb </it>genes involved in the assimilation of inorganic carbon were induced in the presence of arsenic, whereas these genes were repressed in <it>Thiomonas </it>sp. 3As.</p> <p>Conclusion</p> <p>Taken together, these results show that these closely related bacteria differ substantially in their response to arsenic, amongst other factors, and suggest different relationships between carbon assimilation and arsenic metabolism.</p

Shifting the limits in wheat research and breeding using a fully annotated reference genome

Introduction: Wheat (Triticum aestivum L.) is the most widely cultivated crop on Earth, contributing about a fifth of the total calories consumed by humans. Consequently, wheat yields and production affect the global economy, and failed harvests can lead to social unrest. Breeders continuously strive to develop improved varieties by fine-tuning genetically complex yield and end-use quality parameters while maintaining stable yields and adapting the crop to regionally specific biotic and abiotic stresses. Rationale: Breeding efforts are limited by insufficient knowledge and understanding of wheat biology and the molecular basis of central agronomic traits. To meet the demands of human population growth, there is an urgent need for wheat research and breeding to accelerate genetic gain as well as to increase and protect wheat yield and quality traits. In other plant and animal species, access to a fully annotated and ordered genome sequence, including regulatory sequences and genome-diversity information, has promoted the development of systematic and more time-efficient approaches for the selection and understanding of important traits. Wheat has lagged behind, primarily owing to the challenges of assembling a genome that is more than five times as large as the human genome, polyploid, and complex, containing more than 85% repetitive DNA. To provide a foundation for improvement through molecular breeding, in 2005, the International Wheat Genome Sequencing Consortium set out to deliver a high-quality annotated reference genome sequence of bread wheat. Results: An annotated reference sequence representing the hexaploid bread wheat genome in the form of 21 chromosome-like sequence assemblies has now been delivered, giving access to 107,891 high-confidence genes, including their genomic context of regulatory sequences. This assembly enabled the discovery of tissue- and developmental stage–related gene coexpression networks using a transcriptome atlas representing all stages of wheat development. The dynamics of change in complex gene families involved in environmental adaptation and end-use quality were revealed at subgenome resolution and contextualized to known agronomic single-gene or quantitative trait loci. Aspects of the future value of the annotated assembly for molecular breeding and research were exemplarily illustrated by resolving the genetic basis of a quantitative trait locus conferring resistance to abiotic stress and insect damage as well as by serving as the basis for genome editing of the flowering-time trait. Conclusion: This annotated reference sequence of wheat is a resource that can now drive disruptive innovation in wheat improvement, as this community resource establishes the foundation for accelerating wheat research and application through improved understanding of wheat biology and genomics-assisted breeding. Importantly, the bioinformatics capacity developed for model-organism genomes will facilitate a better understanding of the wheat genome as a result of the high-quality chromosome-based genome assembly. By necessity, breeders work with the genome at the whole chromosome level, as each new cross involves the modification of genome-wide gene networks that control the expression of complex traits such as yield. With the annotated and ordered reference genome sequence in place, researchers and breeders can now easily access sequence-level information to precisely define the necessary changes in the genomes for breeding programs. This will be realized through the implementation of new DNA marker platforms and targeted breeding technologies, including genome editing

Expression of the pyr Operon of Lactobacillus plantarum Is Regulated by Inorganic Carbon Availability through a Second Regulator, PyrR(2), Homologous to the Pyrimidine-Dependent Regulator PyrR(1)

Inorganic carbon (IC), such as bicarbonate or carbon dioxide, stimulates the growth of Lactobacillus plantarum. At low IC levels, one-third of natural isolated L. plantarum strains are nutritionally dependent on exogenous arginine and pyrimidine, a phenotype previously defined as high-CO(2)-requiring (HCR) prototrophy. IC enrichment significantly decreased the amounts of the enzymes in the pyrimidine biosynthetic pathway encoded by the pyrR(1)BCAa(1)Ab(1)DFE operon, as demonstrated by proteomic analysis. Northern blot and reverse transcription-PCR experiments demonstrated that IC levels regulated pyr genes mainly at the level of transcription or RNA stability. Two putative PyrR regulators with 62% amino acid identity are present in the L. plantarum genome. PyrR(1) is an RNA-binding protein that regulates the pyr genes in response to pyrimidine availability by a mechanism of transcriptional attenuation. In this work, the role of PyrR(2) was investigated by allelic gene replacement. Unlike the pyrR(1) mutant, the ΔpyrR(2) strain acquired a demand for both pyrimidines and arginine unless bicarbonate or CO(2) was present at high concentrations, which is known as an HCR phenotype. Analysis of the IC- and pyrimidine-mediated regulation in pyrR(1) and pyrR(2) mutants suggested that only PyrR(2) positively regulates the expression levels of the pyr genes in response to IC levels but had no effect on pyrimidine-mediated repression. A model is proposed for the respective roles of PyrR(1) and PyrR(2) in the pyr regulon expression

Production and Preparation of Isotopically Labeled Human Membrane Proteins in Pichia pastoris for Fast-MAS-NMR Analyses

International audienceMembrane proteins (MPs) comprise about one third of the human proteome, playing critical roles in many physiological processes and associated disorders. Consistently, they represent one of the largest classes of targets for the pharmaceutical industry. Their study at the molecular level is however particularly challenging, resulting in a severe lack of structural and dynamic information that is hindering their detailed functional characterization and the identification of novel potent drug candidates

Importance of the Choice of a Recombinant System to Produce Large Amounts of Functional Membrane Protein hERG

International audienceHuman ether-a-gogo related gene (hERG) product is the membrane potassium channel Kv11.1, which is involved in the electrical activity of the heart. As such, it is a key player in the toxicity of many drug candidates. Therefore, having this protein at hand during earlier stages of drug discovery is important for preventing later toxicity. Furthermore, having a fair quantity of functional channels may help in the development of the necessary techniques for gaining insight in this channel structure. Thus, we performed a comparative study of methods for over-expressing a mutated but functional, hERG in different orthologous hosts, such as yeast, bacteria, insect and human cell lines. We also engineered the protein to test various constructs of a functional channel. We obtained a significant amount of a functional mutant channel from HEK cells that we thoroughly characterized. The present work paves the way for the expression of large amounts of this protein, with which protein crystallization or cryo-electronic microscopy will be attempted. This will be a way to gain information on the structure of the hERG active site and its modelization to obtain data on the pauses of various reference compounds from the pharmacopeia, as well as to gain information about the thermodynamics of the hERG/ligand relationship

Cryo–electron microscopy unveils unique structural features of the human Kir2.1 channel

International audienceWe present the first structure of the human Kir2.1 channel containing both transmembrane domain (TMD) and cytoplasmic domain (CTD). Kir2.1 channels are strongly inward-rectifying potassium channels that play a key role in maintaining resting membrane potential. Their gating is modulated by phosphatidylinositol 4,5-bisphosphate (PIP 2 ). Genetically inherited defects in Kir2.1 channels are responsible for several rare human diseases, including Andersen’s syndrome. The structural analysis (cryo–electron microscopy), surface plasmon resonance, and electrophysiological experiments revealed a well-connected network of interactions between the PIP 2 -binding site and the G-loop through residues R312 and H221. In addition, molecular dynamics simulations and normal mode analysis showed the intrinsic tendency of the CTD to tether to the TMD and a movement of the secondary anionic binding site to the membrane even without PIP 2 . Our results revealed structural features unique to human Kir2.1 and provided insights into the connection between G-loop and gating and the pathological mechanisms associated with this channel

Cryo–electron microscopy unveils unique structural features of the human Kir2.1 channel

International audienceWe present the first structure of the human Kir2.1 channel containing both transmembrane domain (TMD) and cytoplasmic domain (CTD). Kir2.1 channels are strongly inward-rectifying potassium channels that play a key role in maintaining resting membrane potential. Their gating is modulated by phosphatidylinositol 4,5-bisphosphate (PIP 2 ). Genetically inherited defects in Kir2.1 channels are responsible for several rare human diseases, including Andersen’s syndrome. The structural analysis (cryo–electron microscopy), surface plasmon resonance, and electrophysiological experiments revealed a well-connected network of interactions between the PIP 2 -binding site and the G-loop through residues R312 and H221. In addition, molecular dynamics simulations and normal mode analysis showed the intrinsic tendency of the CTD to tether to the TMD and a movement of the secondary anionic binding site to the membrane even without PIP 2 . Our results revealed structural features unique to human Kir2.1 and provided insights into the connection between G-loop and gating and the pathological mechanisms associated with this channel

Biochemical, biophysical, and structural investigations of two mutants (C154Y and R312H) of the human Kir2.1 channel involved in the Andersen-Tawil syndrome

Posted February 12, 2024 on bioRxiv.International audienceInwardly rectifying potassium (Kir) channels play a pivotal role in physiology by establishing, maintaining, and regulating the resting membrane potential of the cells, particularly contributing to the cellular repolarization of many excitable cells. Dysfunction in Kir2.1 channels is implicated in several chronic and debilitating human diseases for which there are currently no effective treatments. Specifically, Kir2.1-R312H and Kir2.1-C154Y mutations are associated with Andersen-Tawil syndrome (ATS) in humans. We have performed structural studies and have investigated the impact of these two mutants in the trafficking of the channel to the cell membrane and function in Xenopus laevis oocytes. Despite both mutations being successfully trafficked to the cell membrane and capable of binding PIP 2 (phosphatidylinositol-4,5-bisphosphate), the main modulator for channel activity, they resulted in defective channels that do not display K + current, albeit through different molecular mechanisms. Co-expression studies showed that R312H and C154Y are expressed and associated with the WT subunits. While WT subunits could rescue R312H dysfunction, the presence of a unique C154Y subunit disrupts the function of the entire complex, which is a typical feature of mutations with a dominant-negative effect. Molecular dynamics simulations showed that Kir2.1-C154Y mutation induces a loss in the structural plasticity of the selectivity filter, impairing the K + flow. Furthermore, the cryo-EM structure of the Kir2.1-R312H mutant cytoplasmic region confirmed previous findings that this mutation leads to a loss of the inter-subunit interaction network between residues R312, H221, and E303, thereby impairing channel gating at the G-loop level. Therefore, this study identified the molecular mechanisms by which two ATS-causing mutations impact Kir2.1 channel function and provide valuable insights that can guide potential strategies for the development of future therapeutic interventions for ATS

Enhanced structural and functional genome elucidation of the arsenite-oxidizing strain Herminiimonas arsenicoxydans by proteomics data.

International audienceThe arsenite-oxidizing strain Herminiimonas arsenicoxydans proteome was investigated with gel electrophoresis and tandem mass spectrometry analyses. The comparison of experimental and theoretical M(r) and pI, as well as that of peptide sequences identified by MS and predicted protein sequences, allowed the correction of five protein annotations. More importantly, the functional analysis of SDS- and 2D-PAGE proteome maps obtained in the presence of arsenic, combined with partial transcriptomic results indicate that H. arsenicoxydans expressed genes and proteins required not only for arsenic detoxification or stress response but also involved in motility, exopolysaccharide synthesis, phosphate import or energetic metabolism. This study provides therefore new insights into the adaptation processes of H. arsenicoxydans in response to arsenic