Phosphorylation of a reinitiation supporting protein,RISP, determines its function in translation reinitiation

Reinitiation supporting protein, RISP, interacts with 60S (60S ribosomal subunit) and eIF3 (eukaryotic initiation factor 3) in plants. TOR (target-of-rapamycin) mediates RISP phosphorylation at residue Ser267, favoring its binding to eL24 (60S ribosomal protein L24). In a viral context, RISP, when phosphorylated, binds the CaMV transactivator/ viroplasmin, TAV, to assist in an exceptional mechanism of reinitiation after long ORF translation. Moreover, we show here that RISP interacts with eIF2 via eIF2β and TOR downstream target 40S ribosomal protein eS6. A RISP phosphorylation knockout, RISP-S267A, binds preferentially eIF2β, and both form a ternary complex with eIF3a in vitro. Accordingly, transient overexpression in plant protoplasts of RISP-S267A, but not a RISP phosphorylation mimic, RISP-S267D, favors translation initiation. In contrast, RISP-S267D preferentially binds eS6, and, when bound to the C-terminus of eS6, can capture 60S in a highly specific manner in vitro, suggesting that it mediates 60S loading during reinitiation. Indeed, eS6-deficient plants are highly resistant to CaMV due to their reduced reinitiation capacity. Strikingly, an eS6 phosphomimic, when stably expressed in eS6-deficient plants, can fully restore the reinitiation deficiency of these plants in cellular and viral contexts. These results suggest that RISP function in translation (re)initiation is regulated by phosphorylation at Ser267.     

Evidence for a single heptahelical domain being turned on upon activation of a dimeric GPCR

G-protein-coupled receptors (GPCRs) have been shown to form dimers, but the relevance of this phenomenon in G-protein activation is not known. Among the large GPCR family, metabotropic glutamate (mGlu) receptors are constitutive dimers. Here we examined whether both heptahelical domains (HDs) are turned on upon full receptor activation. To that aim, we measured G-protein coupling efficacy of dimeric mGlu receptors in which one subunit bears specific mutations. We show that a mutation in the third intracellular loop (i3 loop) known to prevent G-protein activation in a single subunit decreases coupling efficacy. However, when a single HD is blocked in its inactive state using an inverse agonist, 2-methyl-6-(phenylethynyl)pyridine (MPEP), no decrease in receptor activity is observed. Interestingly, in a receptor dimer in which the subunit that binds MPEP is mutated in its i3 loop, MPEP enhances agonist-induced activity, reflecting a 'better' activation of the adjacent HD. These data are consistent with a model in which a single HD is turned on upon activation of such homodimeric receptors and raise important issues in deciphering the functional role of GPCR dimer formation for G-protein activation.     

Differences in hydroxylation and binding of Notch and HIF-1α demonstrate substrate selectivity for factor inhibiting HIF-1 (FIH-1)

FIH-1, factor inhibiting hypoxia-inducible factor-1 (HIF-1), regulates oxygen sensing by hydroxylating an asparagine within HIF-α. It also hydroxylates asparagines in many proteins containing ankyrin repeats, including Notch1–3, p105 and IκBα. Relative binding affinity and hydroxylation rate are crucial determinants of substrate selection and modification. We determined the contributions of substrate sequence composition and length and of oxygen concentration to the FIH-1-binding and/or hydroxylation of Notch1–4 and compared them with those for HIF-1α. We also demonstrated hydroxylation of two asparagines in Notch2 and 3, corresponding to Sites 1 and 2 of Notch1, by mass spectrometry for the first time.Our data demonstrate that substrate length has a much greater influence on FIH-1-dependent hydroxylation of Notch than of HIF-1α, predominantly through binding affinity rather than maximal reaction velocity. The K_m value of FIH-1 for Notch1, <0.2 μM, is at least 250-fold lower than that of 50 μM for HIF-1α. Site 1 of Notch1–3 appeared the preferred site of FIH-1 hydroxylation in these substrates. Interestingly, binding of Notch4 to FIH-1 was observed with an affinity almost 10-fold lower than for Notch1–3, but no hydroxylation was detected. Importantly, we demonstrate that the K_m of FIH-1 for oxygen at the preferred Site 1 of Notch1–3, 10–19 μM, is an order of magnitude lower than that for Site 2 or HIF-1α. Hence, at least during in vitro hydroxylation, Notch is likely to become efficiently hydroxylated by FIH-1 even under relatively severe hypoxic conditions, where HIF-1α hydroxylation would be reduced.     

High-efficiency derivation of human embryonic stem cell lines following pre-implantation genetic diagnosis

Pre-implantation genetic diagnosis allows the characterisation of embryos that carry a gene responsible for a severe monogenic disease and to transfer to the mother’s uterus only the unaffected one(s). The genetically affected embryos can be used to establish human embryonic stem cell (hESC) lines. We are currently establishing a cell bank of ESC lines carrying specific disease-causing mutant genes. These cell lines are available to the scientific community. For this purpose, we have designed a technique that requires only minimal manipulation of the embryos. At the blastocyst stage, we just removed the zona pellucida before seeding the embryo as a whole on a layer of feeder cells. This approach gave a good success rate (>20%), whatever the quality of the embryos, and allowed us to derive 11 new hESC lines, representing seven different pathologies. Full phenotypic validation of the cell lines according to ISCI guidelines confirmed their pluripotent nature, as they were positive for hESC markers and able to differentiate in vitro in all three germ layers derivatives. Nine out of 11 stem cell lines had normal karyotypes. Our results indicate that inner cell mass isolation is not mandatory for hESC derivation and that minimal manipulation of embryos can lead to high success rate.     

In Vitro Production of Biotrophic-Like Cultures of Crinipellis perniciosa, the Causal Agent of Witches’ Broom Disease of Theobroma cacao

Witches’ broom disease (WBD) of cacao, caused by the hemibiotrophic fungus, Crinipellis perniciosa, exhibits a succession of symptoms that are caused by the biotrophic phase of the fungus. However, the study of this biotrophic phase is limited by its exclusive growth inside the plant or in the presence of callus. Here we report for the first time a method for the growth and maintenance of the biotrophic-like phase of C. perniciosa on a defined medium with metabolites found in the diseased tissues. Our results suggest that glycerol is a key carbon source for this interaction. This is a crucial achievement toward understanding the biology of this fungus during the infectious phase of WBD.     

Monoclonal antibodies biosimilarity assessment using transient isotachophoresis capillary zone electrophoresis-tandem mass spectrometry

Out of all categories, monoclonal antibody (mAb) therapeutics attract the most interest due to their strong therapeutic potency and specificity. Six of the 10 top-selling drugs are antibody-based therapeutics that will lose patent protection soon. The European Medicines Agency has pioneered the regulatory framework for approval of biosimilar products and approved the first biosimilar antibodies by the end of 2013. As highly complex glycoproteins with a wide range of micro-variants, mAbs require extensive characterization through multiple analytical methods for structure assessment rendering manufacturing control and biosimilarity studies particularly product and time-consuming. Here, capillary zone electrophoresis coupled to mass spectrometry by a sheathless interface (CESI-MS) was used to characterize marketed reference mAbs and their respective biosimilar candidate simultaneously over different facets of their primary structure. CESI-MS/MS data were compared between approved mAbs and their biosimilar candidates to prove/disconfirm biosimilarity regarding recent regulation directives. Using only a single sample injection of 200 fmol, CESI-MS/MS data enabled 100% amino acids (AA) sequence characterization, which allows a difference of even one AA between 2 samples to be distinguished precisely. Simultaneously glycoforms were characterized regarding their structures and position through fragmentation spectra and glycoforms semiquantitative analysis was established, showing the capacity of the developed methodology to detect up to 16 different glycans. Other posttranslational modifications hotspots were characterized while their relative occurrence levels were estimated and compared to biosimilars. These results proved the value of using CESI-MS because the separation selectivity and ionization efficiency provided by the system allowed substantial improvement in the characterization workflow robustness and accuracy. Biosimilarity assessment could be performed routinely with a single injection of each candidate enabling improvements in the biosimilar development pipeline.