Étude de la structure et de la fonction de la petite protéine de choc thermique DmHsp27

Les petites protéines de choc thermique (sHsps : small heat shock proteins) sont présentes en nombre variable dans tous les organismes. Le génome de Drosophila melanogaster encode 12 sHsps, avec une expression au cours de développement, une localisation intracellulaire et une spécificité de substrats différents. DmHsp27 est l’une des rares sHsps à avoir une localisation nucléaire avant et après un choc thermique. Cette localisation nucléaire est inhabituelle, d’autant plus qu’aucune fonction spécifique de cette protéine n’a encore été identifiée. Les mécanismes responsables de la localisation nucléaire de DmHsp27 ainsi que sa probable fonction dans le noyau restent peu connus. L’étude des orthologues de DmHsp27 chez les insectes a permis de déterminer que la localisation nucléaire n’est pas spécifique à DmHsp27 et d’autres sHsps chez les insectes présentent le même signal de localisation nucléaire que DmHsp27. Le réseau d’interaction de DmHsp27 nous laisse croire que cette protéine ne joue pas seulement le rôle de chaperon moléculaire, mais qu’elle est probablement impliquée dans différents processus nucléaires. Au niveau structurel, contrairement aux sHsps chez les métazoaires, DmHsp27 forme deux populations d'oligomères non en équilibre. Des mutations indépendantes de trois arginines hautement conservées au niveau du domaine alpha-cristallin (R122, R131 et R135) à la glycine affectent l’oligomérisation par formation d’une seule population de grand poids moléculaire. In vitro, l'activité de chaperon de DmHsp27WT est comparable à celle de ses deux populations isolées et à celle des mutants R122G, R131G et R135G utilisant la luciférase comme substrat. Cependant, dans un essai de protection contre l’aggrégation de l’insuline, l'activité de chaperon de DmHsp27 est inférieure à celle des mutants R122G et R131G. Une stratégie de délétion et mutation a été utilsée pour étudier le rôle de la région N-terminale sur l’oligomérisation et la fonction chaperon de DmHsp27. Tel que déterminé par chromatographie d'exclusion et gel natif, des mutations au niveau de F29, G30 et G32 présentent des structures oligomériques différentes de celle du type sauvage. Aucun de ces mutants sauf l'élimination complète de la région N-terminale n’a montré une perte d'activité chaperon, suggérant un rôle important dans la reconnaissance et liaison de substrat. Étonnamment, les deux glycines (G30 et G32) conservées chez les orthologues de DmHsp27 agissent comme régulateurs négatifs de l'activité chaperon; leurs mutations améliorent grandement la prévention d’agrégation d’insuline. La différence du rendement de la protéine sauvage et des mutants G30R et G32R à prévenir l’agrégation de l’insuline à 20 °C et à 42 °C a permis d’établir un lien entre la structure et la fonction chaperon de DmHsp27 et de définir le mode d’action de DmHsp27 suite au stress thermique. En résumé, cette étude a permis de caractériser DmHsp27 et ses mutants au niveau du domaine alpha cristallin et de la région N-terminale et a donné un aperçu d’un nouveau mécanisme de protection contre l’agrégation des sHsps. Le rôle de chaperon moléculaire joué par DmHsp27 et son induction durant le développement embryonnaire laisse cependant présumer que cette protéine remplit d’autres fonctions cellulaires importantes.Small heat shock proteins are present in varying numbers in all organisms. In Drosophila melanogaster there are 12 sHsps, which have distinctive developmental expression patterns, intracellular localizations and substrate specificities. DmHsp27 is one of the very few sHsps that have a nuclear localization before and after heat shock. This nuclear localization is unusual, especially since no specific function has yet been identified. The mechanisms responsible for the nuclear localization of DmHsp27 and its function in the nucleus remain poorly understood. First, the study of DmHsp27 orthologs helped to determine that nuclear localization is not specific to DmHsp27 and other sHsps in insects have the same nuclear localization signal as DmHsp27. The DmHsp27 interaction network leads to believe that this protein does not only play the role of chaperone, but it is also involved in various nuclear processes. Second, unlike metazoan sHsps, DmHsp27 forms two populations of oligomers not in equilibrium. Mutations of highly conserved arginine residues in the ACD domain in mammalian sHsps has been reported to be associated with protein conformational defects and intracellular aggregation. Independent mutation of three highly conserved arginines (R122, R131 and R135) to glycine in DmHsp27 results in only one population of higher molecular weight form. In vitro, the chaperone-like activity of wild type DmHsp27 was comparable with that of its two isolated populations and to the single population of the R122G, R131G and R135G using luciferase as substrate. However, using insulin, the chaperone-like activity of wild type DmHsp27 was lower than that of R122G and R131G mutants. Finaly, we established the importance of the N-terminal region for oligomerization and we investigated the heat activation under in vitro experimental conditions using size exclusion chromatography and gradient native gels electrophoresis. By deletion strategy, we have examined the role of the N-terminal region and delineated a motif (FGFG) important for the oligomeric structure and chaperone-like activity of this sHsp. Deletion of the full N-terminal domain, resulted in total loss of chaperon-like activity; intriguingly deletion of the (FGFG) at position 29 to 32 or single mutation of G30R and G32R enhanced oligomerization and chaperoning capacity under non heat shock conditions using the insulin assay suggesting the importance of this site for chaperone activity. Unlike mammalian sHsps heat activation of DmHsp27 leads to enhanced dissociation/association of oligomers to form large structures about 1000 kDa. We suggest a new mechanism of heat activation for DmHsp27. In summary, this study characterized DmHsp27 and mutant in the alpha crystallin domain and the N-terminal region and provided an overview of a new protection mechanism. The role played by DmHsp27 as molecular chaperone and its induction during embryonic development, suggest that this protein may perform other important cellular function

Granules de stress cytoplasmiques à ARN induits par le rayonnement ultraviolet (UV)

Chez les eucaryotes, différents types de granules à ARN sont des acteurs importants dans les mécanismes de la régulation post-transcriptionnelle de l'expression des gènes. L’irradiation aux UV induit la formation des petits granules cytoplasmiques (GUV) qui ne sont pas des processing bodies et qui semblent être une nouvelle sous classe de granules de stress. Ces granules n’ont pas la même cinétique de formation et de disparition ainsi que la taille, le nombre et la capacité de fusion que les granules de stress classiques. D’autre part, la formation de ces granules UV ne semble pas affecter le niveau de traduction, ni d’induire la réponse au stress. Toutefois, nous avons observé que l’apparition des granules coïncide avec l’arrêt de la prolifération cellulaire. En effet, dans les conditions expérimentales utilisées, la prolifération est décalée de 24 à 48 h selon la dose d’irradiation. L’ensemble de ces observations suggère fortement l'existence, d'une nouvelle sous classe de granules de stress induit par les UV, dont le rôle semble être la répression de la traduction des ARNm codant pour des facteurs importants de prolifération cellulaire

Thermal acclimation of photosynthetic activity and RuBisCO content in two hybrid poplar clones

The mechanistic bases of thermal acclimation of net photosynthetic rate (A n ) are still difficult to discern, and the data sets available are scarce, particularly for hybrid poplar. In the present study, we examined the contribution of a number of biochemical and biophysical traits on thermal acclimation of A n for two hybrid poplar clones. We grew cuttings of Populus maximowiczii × Populus nigra (M×N) and Populus maximowiczii × Populus balsamifera (M×B) clones under two day/night temperature of 23°C/18°C and ◦33°C /27°C and under low and high soil nitrogen level. After ten weeks, we measured leaf RuBisCO (RAR) and RuBisCO activase (RARCA) amounts and the temperature response of A n , dark respiration (R d ), stomatal conductance, (g s ), apparent maximum carboxylation rate of CO 2 (V cmax ) and apparent photosynthetic electron transport rate (J). Results showed that a 10°C increase in growth temperature resulted in a shift in thermal optimum (T opt ) of A n of 6.2±1.6 °C and 8.0±1.2 °C for clone M×B and M×N respectively, and an increased A n and g s at the growth temperature for clone M×B but not M×N. RuBisCO amount was increased by N level but was insensitive to growth temperature while RARCA amount and the ratio of its short to long isoform was stimulated by the warm condition for clone M×N and at low N for clone M×B. The activation energy of apparent V cmax and apparent J decreased under the warm condition for clone M×B and remained unchanged for clone M×N. Our study demonstrated the involvement of both RARCA, the activation energy of apparent V cmax and stomatal conductance in thermal acclimation of A n

CFTR interactome mapping using the mammalian membrane two-hybrid high-throughput screening system

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a chloride and bicarbonate channel in secretory epithelia with a critical role in maintaining fluid homeostasis. Mutations in CFTR are associated with Cystic Fibrosis (CF), the most common lethal autosomal recessive disorder in Caucasians. While remarkable treatment advances have been made recently in the form of modulator drugs directly rescuing CFTR dysfunction, there is still considerable scope for improvement of therapeutic effectiveness. Here, we report the application of a high-throughput screening variant of the Mammalian Membrane Two-Hybrid (MaMTH-HTS) to map the protein-protein interactions of wild-type (wt) and mutant CFTR (F508del), in an effort to better understand CF cellular effects and identify new drug targets for patient-specific treatments. Combined with functional validation in multiple disease models, we have uncovered candidate proteins with potential roles in CFTR function/CF pathophysiology, including Fibrinogen Like 2 (FGL2), which we demonstrate in patient-derived intestinal organoids has a significant effect on CFTR functional expression

Phosphorylation effect on oligomerization and localization of DmHsp27.

<p>A- Native gradient (4–12%) polyacrylamide gel electrophoresis of recombinant DmHsp27 wild-type, phosphomimetic and nonphosphorylatable serine mutants. Positions of protein markers with molecular weights are shown on the left. B and C- Preventing aggregation of luciferase and insulin using phosphomimetic and nonphosphorylatable mutants. Data are representative of three independent experiments with error bars corresponding to the standard error of the mean. D- Intracellular localization of DmHSP27 and its phosphomimetic and nonphosphorylatable mutants in transfected Hela cells. Forty-eight hours post-transfection, HeLa cells were fixed, permeabilized, and processed for immunofluorescence using antibodies against DmHsp27 (green). Nuclei were counterstained with DAPI. Scale bar is 10 μm.</p

Sequence analysis of the N-terminal region of different sHsp.

<p>The alignment was made using Muscle [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177821#pone.0177821.ref079" target="_blank">79</a>]. The conserved residues are darkly highlighted in the alignment. A- Multiple sequence alignment of the NTR of DmHsp27 with l(2)efl from <i>Drosophila melanogaster</i>, human (HspB1, HspB4, HspB5 and HspB6), Mouse (CryAA and CryAB), zebrafish (CryAA_DANRE and CryAB_DANRE) and <i>Methanococcus maripaludis</i> C6 (Hsp20_METM6). B- Multiple sequence alignment of the NTR of sHsps obtained using blast similarity of NTR-DmHsp27. Sequence from <i>Drosophila melqnogaster</i>_Hsp27, <i>Drosophila simulans</i>_Hsp27, <i>Drosophila yakuba</i>_Hsp27, <i>Drosophila erecta</i>_Hsp27, <i>Drosophila ananassae</i>_Hsp27, <i>Drosophila persimilis</i>_Hsp27, <i>Drosophila willistoni</i>_Hsp27, <i>Dosophila albomicans</i>_Hsp27, <i>Drosophila busckii</i>_Hsp27, <i>Drosophila buzzatii</i>_Hsp27, <i>Drosophila sulfurigaster-albostrigata</i>_Hsp27, <i>Drosophila virilis</i>_Hsp27, <i>Drosophila mojavensis</i>_Hsp27, <i>Drosophila grimshawi</i>_Hsp27, <i>Drosophila repletoides_</i>Hsp27, <i>Bactrocera dorsalis</i>_Hsp20, Ceratitis <i>capitata</i>_Hsp27, <i>Bactrocera dorsalis</i>_Hsp27, <i>Musca domestica</i>_Hsp27, <i>Stomoxys calcitrans</i>_Hsp27, <i>Lucilia cuprina_</i>Hsp27, <i>Sarcophaga crassipalpis</i>_Hsp25.</p

Effect of N-terminal region of nuclear <i>Drosophila melanogaster</i> small heat shock protein DmHsp27 on function and quaternary structure

<div><p>The importance of the N-terminal region (NTR) in the oligomerization and chaperone-like activity of the <i>Drosophila melanogaster</i> small nuclear heat shock protein DmHsp27 was investigated by mutagenesis using size exclusion chromatography and native gel electrophoresis. Mutation of two sites of phosphorylation in the N-terminal region, S58 and S75, did not affect the oligomerization equilibrium or the intracellular localization of DmHsp27 when transfected into mammalian cells. Deletion or mutation of specific residues within the NTR region delineated a motif (FGFG) important for the oligomeric structure and chaperone-like activity of this sHsp. While deletion of the full N-terminal region, resulted in total loss of chaperone-like activity, removal of the (FGFG) at position 29 to 32 or single mutation of F29A/Y, G30R and G32R enhanced oligomerization and chaperoning capacity under non-heat shock conditions in the insulin assay suggesting the importance of this site for chaperone activity. Unlike mammalian sHsps DmHsp27 heat activation leads to enhanced association of oligomers to form large structures of approximately 1100 kDa. A new mechanism of thermal activation for DmHsp27 is presented.</p></div

Primers sequences used to construct DmHsp27 NTR mutants.

<p>Primers sequences used to construct DmHsp27 NTR mutants.</p

Effects of FGFG residues on oligomerization and chaperon-like activity.

<p>A and B- Native gradient (4–12%) polyacrylamide gel electrophoresis of recombinant DmHsp27 and NTR mutants (F29A; F29Y, F31A, F31Y, G30A, G30R, G32A and G32R) at 20°C. Positions of standard protein markers are shown on the left. C, D, E and F- Preventing aggregation of luciferase, insulin, citrate synthase and L-malate dehydrogenase using NTR mutants (F29A; F29Y, F31A, F31Y, G30A, G30R, G32A and G32R). Data are representatives of three independent experiments. ** indicates P<0.01; *** indicates P<0.001, **** indicates P<0.0001.</p

Size exclusion chromatography analysis of DmHsp27 N-terminal mutants F29A, F29Y, G30R and G32R.

<p>Size exclusion chromatography (SEC) analysis using a superose 6 10/300 GL (GE Life Sciences) column, with IGM (900 kDa), thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa) and ovalbumin (43 kDa) Blue dextran (2000 kDa) was used to determine the void volume of the column V₀. A- Profile on column of 300 μg (black line) of DmHsp27F29A compared to 300 μg (dashed line) of DmHsp27. B- Profile on column of 300 μg (black line) of DmHsp27F29Y compared to 300 μg (dashed line) of DmHsp27. C- Profile on column of 300 μg (black line) of DmHsp27G30R compared to 300 μg (dashed line) of DmHsp27. D- Profile on column of 300 μg (black line) of DmHsp27G32R compared to 300 μg (dashed line) of DmHsp27.</p