A growth hormone receptor SNP promotes lung cancer by impairment of SOCS2-mediated degradation

Both humans and mice lacking functional growth hormone (GH) receptors are known to be resistant to cancer. Further, autocrine GH has been reported to act as a cancer promoter. Here we present the first example of a variant of the GH receptor (GHR) associated with cancer promotion, in this case lung cancer. We show that the GHRP495T variant located in the receptor intracellular domain is able to prolong the GH signal in vitro using stably expressing mouse pro-B-cell and human lung cell lines. This is relevant because GH secretion is pulsatile, and extending the signal duration makes it resemble autocrine GH action. Signal duration for the activated GHR is primarily controlled by suppressor of cytokine signalling 2 (SOCS2), the substrate recognition component of the E3 protein ligase responsible for ubiquitinylation and degradation of the GHR. SOCS2 is induced by a GH pulse and we show that SOCS2 binding to the GHR is impaired by a threonine substitution at Pro 495. This results in decreased internalisation and degradation of the receptor evident in TIRF microscopy and by measurement of mature (surface) receptor expression. Mutational analysis showed that the residue at position 495 impairs SOCS2 binding only when a threonine is present, consistent with interference with the adjacent Thr494. The latter is key for SOCS2 binding, together with nearby Tyr487, which must be phosphorylated for SOCS2 binding. We also undertook nuclear magnetic resonance spectroscopy approach for structural comparison of the SOCS2 binding scaffold Ile455-Ser588, and concluded that this single substitution has altered the structure of the SOCS2 binding site. Importantly, we find that lung BEAS-2B cells expressing GHRP495T display increased expression of transcripts associated with tumour proliferation, epithelial–mesenchymal transition and metastases (TWIST1, SNAI2, EGFR, MYC and CCND1) at 2 h after a GH pulse. This is consistent with prolonged GH signalling acting to promote cancer progression in lung cancer

Exploiting Hydrophobicity for Efficient Production of Transmembrane Helices for Structure Determination by NMR Spectroscopy

Despite the biological and pharmaceutical significance of membrane proteins, their tertiary structures constitute less than 3% of known structures. One of the major obstacles for initiating structural studies of membrane proteins by NMR spectroscopy is the generation of high amounts of isotope-labeled protein. In this work, we have exploited the hydrophobic nature of membrane proteins to develop a simple and efficient production scheme for isotope-labeled single-pass transmembrane domains (TMDs) with or without intrinsically disordered regions. We have evaluated the applicability and limitations of the strategy using seven membrane protein variants that differ in their overall hydrophobicity and length and show a recovery for suitable variants of >70%. The developed production scheme is cost-efficient and easy to implement and has the potential to facilitate an increase in the number of structures of single-pass TMDs, which are difficult to solve by other means

A set of engineered Escherichia coli expression strains for selective isotope and reactivity labeling of amino acid side chains and flavin cofactors.

Biological reactions are facilitated by delicate molecular interactions between proteins, cofactors and substrates. To study and understand their dynamic interactions researchers have to take great care not to influence or distort the object of study. As a non-invasive alternative to a site-directed mutagenesis approach, selective isotope labeling in combination with vibrational spectroscopy may be employed to directly identify structural transitions in wild type proteins. Here we present a set of customized Escherichia coli expression strains, suitable for replacing both the flavin cofactor and/or selective amino acids with isotope enriched or chemically modified substrates. For flavin labeling we report optimized auxotrophic strains with significantly enhanced flavin uptake properties. Labeled protein biosynthesis using these strains was achieved in optimized cultivation procedures using high cell density fermentation. Finally, we demonstrate how this approach is used for a clear assignment of vibrational spectroscopic difference signals of apoprotein and cofactor of a flavin containing photoreceptor of the BLUF (Blue Light receptors Using FAD) family

Cassette for constitutive expression of flavokinases in <i>E. coli</i> (A).

<p>The enhanced flavokinase activity of the strains CpXFMN and CpXribF containing the respective expression cassette reduces the intracellular amount of free riboflavin by accumulation of FAD and FMN, respectively. Thereby the transport equilibrium is shifted towards a higher uptake (B). CpXFMN and CpXribF accordingly show an enhanced growth at riboflavin concentrations below 10 µM as compared to the riboflavin auxotroph CmpX131 (C).</p

Amino acid and cofactor specific isotope labeling using custom-made auxotrophic expression strains (A) in a high cell density fermentation setup (B).

<p>CmpX13 is rendered auxotrophic for selected amino acid and/or cofactor synthesis pathways. The resulting expression strains are cultivated under controlled conditions to achieve the highest cell density under complete consumption of unlabeled substrates. Subsequently labeled substrates are fed and protein production is induced.</p

BLUF photoactivation according to the glutamine rotamer model, which accommodates hydrogen bond switching around the flavin chromophore upon illumination (A).

<p>Using the tyrosine auxotrophic expression strain CpXΔY selective isotope labeling of the phenyl ring (black) of tyrosine side chains was obtained (B). Light-minus-dark FTIR difference spectra of unlabeled Slr1694 (grey) and ring-¹³C₆-Y labeled Slr1694 (black) in H₂O show various isotope-induced shifts, which represent the changed hydrogen bond network around the tyrosine (C).</p

Selective unlabeling of the flavin chromophore (A, light grey) upon uniform ¹³C labeling of the protein (dark grey and black) using the riboflavin auxotrophic strain CpXribF.

<p>Light-minus-dark FTIR difference spectra of unlabeled Slr1694 (grey) and apoprotein ¹³C-labeled Slr1694 (black) in H₂O are presented in B and C. The close-up of the amide frequency range shows a downshift of secondary structural changes as well as coupling of flavin and protein modes (C). </p