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

Human 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.

Cloning and Overexpressing Membrane Proteins Using Pichia pastoris (Komagataella phaffii).

Understanding the structure and function of key proteins located within biological membranes is essential for fundamental knowledge and therapeutic applications. Robust cell systems allowing their actual overexpression are required, among which stands the methylotrophic yeast Pichia pastoris. This system proves highly efficient in producing many eukaryotic membrane proteins of various functions and structures at levels and quality compatible with their subsequent isolation and molecular investigation. This article describes a set of basic guidelines and directions to clone and select recombinant P. pastoris clones overexpressing eukaryotic membrane proteins. Illustrative results obtained for a panel of mammalian membrane proteins are presented, and hints are given on a series of experimental parameters that may substantially improve the amount and/or the functionality of the expressed proteins. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Designing and cloning a P. pastoris expression vector Basic Protocol 2: Integrative transformation of P. pastoris and selection of recombinant clones Basic Protocol 3: Culturing transformed P. pastoris for membrane protein expression Basic Protocol 4: Yeast cell lysis and membrane preparation Basic Protocol 5: Immunodetection of expressed membrane proteins: western blot Alternate Protocol 1: Immunodetection of expressed membrane proteins: dot blot Alternate Protocol 2: Immunodetection of expressed membrane proteins: yeastern blot Basic Protocol 6: Activity assay: ligand-binding analysis of an expressed GPCR.

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

Membrane 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.Magic Angle Spinning (MAS) NMR is a reliable and efficient method for the determination of protein structures and dynamics and for the identification of ligand binding sites and equilibria. MAS-NMR is particularly well suited for MPs since they can be directly analysed in a native-like lipid bilayer environment but used to require aggravating large amounts of isotope enriched material. The frequent toxicity of human MP overexpression in bacterial cultures poses an additional hurdle, resulting in the need for alternative (and often more costly) expression systems. The recent development of very fast (up to 150 kHz) MAS probes has revolutionized the field of biomolecular solid-state NMR enabling higher spectral resolution with significant reduction of the required sample, rendering eukaryotic expression systems cost-effective.Here is presented a set of accessible procedures validated for the production and preparation of eukaryotic MPs for Fast-MAS 1H-detected NMR analysis. The methodology is illustrated with the human copper uptake protein hCTR1 recombinantly produced and 13C-15N uniformly labeled with the versatile and affordable Pichia pastoris system. Subsequent purification procedures allow the recovery of mg amounts that are then reconstituted into liposome formulations compatible with solid-state NMR handling and analysis.

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

We 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 (PIP2). 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 PIP2-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 PIP2. 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.

Identification of an N-acylated-DArg-Leu-NH2 Dipeptide as a Highly Selective Neuropeptide FF1 Receptor Antagonist That Potently Prevents Opioid-Induced Hyperalgesia.

RFamide-related peptide-3 (RFRP-3) and neuropeptide FF (NPFF) target two different receptor subtypes called neuropeptide FF1 (NPFF1R) and neuropeptide FF2 (NPFF2R) that modulate several functions. However, the study of their respective role is severely limited by the absence of selective blockers. We describe here the design of a highly selective NPFF1R antagonist called RF3286, which potently blocks RFRP-3-induced hyperalgesia in mice and luteinizing hormone release in hamsters. We then showed that the pharmacological blockade of NPFF1R in mice prevents the development of fentanyl-induced hyperalgesia while preserving its analgesic effect. Altogether, our data indicate that RF3286 represents a useful pharmacological tool to study the involvement of the NPFF1R/RFRP-3 system in different functions and different species. Thanks to this compound, we showed that this system is critically involved in the development of opioid-induced hyperalgesia, suggesting that NPFF1R antagonists might represent promising therapeutic tools to improve the use of opioids in the treatment of chronic pain.

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

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

VHH characterization. Comparison of recombinant with chemically synthesized anti-HER2 VHH.

In the continuous exploration of the VHH chemistry, biochemistry and therapeutic future use, we investigated two different production strategies of this small antibody-like protein, using an anti-HER2 VHH as a model. The total chemical synthesis of the 125 amino-acid peptide was performed with reasonable yield, even if optimization will be necessary to upgrade this kind of production. In parallel, we expressed the same sequence in two different hosts: Escherichia coli and Pichia pastoris. Both productions were successful and led to a fair amount of VHHs. The integrity and conformation of the VHH were characterized by complementary mass spectrometry approaches, while surface plasmon resonance experiments were used to assess the VHH recognition capacity and affinity toward its "antigen." Using this combination of orthogonal techniques, it was possible to show that the three VHHs-whether synthetic or recombinant ones-were properly and similarly folded and recognized the "antigen" HER2 with similar affinities, in the nanomolar range. This opens a route toward further exploration of modified VHH with unnatural amino acids and subsequently, VHH-drug conjugates.

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.

Pichia sorbitophila, an Interspecies Yeast Hybrid, Reveals Early Steps of Genome Resolution After Polyploidization.

Polyploidization is an important process in the evolution of eukaryotic genomes, but ensuing molecular mechanisms remain to be clarified. Autopolyploidization or whole-genome duplication events frequently are resolved in resulting lineages by the loss of single genes from most duplicated pairs, causing transient gene dosage imbalance and accelerating speciation through meiotic infertility. Allopolyploidization or formation of interspecies hybrids raises the problem of genetic incompatibility (Bateson-Dobzhansky-Muller effect) and may be resolved by the accumulation of mutational changes in resulting lineages. In this article, we show that an osmotolerant yeast species, Pichia sorbitophila, recently isolated in a concentrated sorbitol solution in industry, illustrates this last situation. Its genome is a mosaic of homologous and homeologous chromosomes, or parts thereof, that corresponds to a recently formed hybrid in the process of evolution. The respective parental contributions to this genome were characterized using existing variations in GC content. The genomic changes that occurred during the short period since hybrid formation were identified (e.g., loss of heterozygosity, unilateral loss of rDNA, reciprocal exchange) and distinguished from those undergone by the two parental genomes after separation from their common ancestor (i.e., NUMT (NUclear sequences of MiTochondrial origin) insertions, gene acquisitions, gene location movements, reciprocal translocation). We found that the physiological characteristics of this new yeast species are determined by specific but unequal contributions of its two parents, one of which could be identified as very closely related to an extant Pichia farinosa strain.

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

The 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.

Lactobacillus plantarum response to inorganic carbon concentrations: PyrR2-dependent and -independent transcription regulation of genes involved in arginine and nucleotide metabolism.

Lactobacillus plantarum susbp. plantarum is a capnophilic Gram-positive heterotroph with optimal growth in 4 % CO(2)-enriched air. At low inorganic carbon (C(i)) concentrations, the pyr genes encoding the enzymes of the pyrimidine biosynthetic pathway were overexpressed, in agreement with a previous study showing that these genes are regulated at the transcription level in response to C(i) via a PyrR(2)-mediated mechanism. A previous study of high-CO(2)-requiring (HCR) mutants revealed an unknown genetic link between arginine regulation and C(i)-dependent nutritional needs. To better understand L. plantarum's adaptation to C(i) availability, additional C(i)-responsive genes were sought in the arginine biosynthetic pathway (arg and car genes) using slot-blot hybridization and a proteomic differential 2D gel electrophoresis (DIGE) global approach. Besides the nine pyr-encoded proteins, 16 new Icr (inorganic-carbon-regulated) proteins accumulated differentially in response to C(i) availability, suggesting that the C(i) response involves several metabolic pathways and adaptation processes. Among these Icr proteins only argininosuccinate lyase, encoded by argH, was involved in arginine biosynthesis. Three proteins involved in the purine biosynthetic pathway and nucleotide conversion, adenylate kinase (Adk), GMP synthase (GuaA), and IMP dehydrogenase (GuaB), accumulated differentially in response to changes in C(i) levels. Expression of the Icr protein-encoding genes argH and guaB was regulated at the transcription level or by RNA stability in response to C(i) availability, as previously demonstrated for the pyr genes. However, PyrR(2) was not essential for the C(i)-regulated transcription of argH and guaB, demonstrating that PyrR(2) modulates only a subset of C(i)-regulated genes. These results suggest that the C(i) response may involve at least two regulatory mechanisms in L. plantarum.

Expression of the pyr operon of Lactobacillus plantarum is regulated by inorganic carbon availability through a second regulator, PyrR2, homologous to the pyrimidine-dependent regulator PyrR1.

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-CO2-requiring (HCR) prototrophy. IC enrichment significantly decreased the amounts of the enzymes in the pyrimidine biosynthetic pathway encoded by the pyrR1BCAa1Ab1DFE 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. PyrR1 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 PyrR2 was investigated by allelic gene replacement. Unlike the pyrR1 mutant, the DeltapyrR2 strain acquired a demand for both pyrimidines and arginine unless bicarbonate or CO2 was present at high concentrations, which is known as an HCR phenotype. Analysis of the IC- and pyrimidine-mediated regulation in pyrR1 and pyrR2 mutants suggested that only PyrR2 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 PyrR1 and PyrR2 in the pyr regulon expression.