Membrane association of the PTEN tumor suppressor: molecular details of the protein-membrane complex from SPR binding studies and neutron reflection

The structure and function of the PTEN phosphatase is investigated by studying its membrane affinity and localization on in-plane fluid, thermally disordered synthetic membrane models. The membrane association of the protein depends strongly on membrane composition, where phosphatidylserine (PS) and phosphatidylinositol diphosphate (PI(4,5)P₂) act pronouncedly synergistic in pulling the enzyme to the membrane surface. The equilibrium dissociation constants for the binding of wild type (wt) PTEN to PS and PI(4,5)P₂ were determined to be K_d∼12 µM and 0.4 µM, respectively, and K_d∼50 nM if both lipids are present. Membrane affinities depend critically on membrane fluidity, which suggests multiple binding sites on the protein for PI(4,5)P₂. The PTEN mutations C124S and H93R show binding affinities that deviate strongly from those measured for the wt protein. Both mutants bind PS more strongly than wt PTEN. While C124S PTEN has at least the same affinity to PI(4,5)P₂ and an increased apparent affinity to PI(3,4,5)P₃, due to its lack of catalytic activity, H93R PTEN shows a decreased affinity to PI(4,5)P₂ and no synergy in its binding with PS and PI(4,5)P₂. Neutron reflection measurements show that the PTEN phosphatase “scoots" along the membrane surface (penetration <5 Å) but binds the membrane tightly with its two major domains, the C2 and phosphatase domains, as suggested by the crystal structure. The regulatory C-terminal tail is most likely displaced from the membrane and organized on the far side of the protein, ∼60 Å away from the bilayer surface, in a rather compact structure. The combination of binding studies and neutron reflection allows us to distinguish between PTEN mutant proteins and ultimately may identify the structural features required for membrane binding and activation of PTEN.     

Identification and quantification of concentration-dependent biomarkers in MCF-7/BOS cells exposed to 17β-estradiol by 2-D DIGE and label-free proteomics

This paper reports the identification of biomarkers resulting from the exposure of MCF-7/BOS cells to 17β-estradiol (E(2)). The biomarkers were identified using 2 independent and complementary techniques, 2-D DIGE/MALDI-TOF peptide mass fingerprint, and 2-D UPLC-ESI MS/MS. They were identified from the cytosolic fractions of cells treated for 24h with mitogenic concentrations of 1, 30 and 500 pM of 17β-estradiol. Five biomarkers were up-regulated proteins, namely HSP 74, EF2, FKBP4, EF1 and GDIB and one was a down-regulated protein, namely K2C8. Three of these proteins, EF2, FKBP4 and K2C8 are implicated in a network centered on the estrogen receptors ESR1 and ESR2 as well as on AKT1. After the discovery phase, three biomarkers were selected to test the presence of estrogens using selected reaction monitoring (SRM). They were monitored using SRM after incubation of MCF-7/BOS in the presence of E(2) for confirmation or selected xenoestrogens. Daidzein, coumestrol and enterolactone induced an up-regulation of EF2 and FKPB4 proteins, while tamoxifen and resveratrol induced a down-regulation. The exposure of all phytoestrogens induced the down-regulation of K2C8. These markers form a preliminary molecular signature that can be used when testing the estrogenic activity of xenobiotics, either pure or in mixtures.     

APC/C-mediated degradation of dsRNA-binding protein 4 (DRB4) involved in RNA silencing

Background

Selective protein degradation via the ubiquitin-26S proteasome is a major mechanism underlying DNA replication and cell division in all Eukaryotes. In particular, the APC/C (Anaphase Promoting Complex or Cyclosome) is a master ubiquitin protein ligase (E3) that targets regulatory proteins for degradation allowing sister chromatid separation and exit from mitosis. Interestingly, recent work also indicates that the APC/C remains active in differentiated animal and plant cells. However, its role in post-mitotic cells remains elusive and only a few substrates have been characterized.

Methodology/Principal Findings

In order to identify novel APC/C substrates, we performed a yeast two-hybrid screen using as the bait Arabidopsis APC10/DOC1, one core subunit of the APC/C, which is required for substrate recruitment. This screen identified DRB4, a double-stranded RNA binding protein involved in the biogenesis of different classes of small RNA (sRNA). This protein interaction was further confirmed in vitro and in plant cells. Moreover, APC10 interacts with DRB4 through the second dsRNA binding motif (dsRBD2) of DRB4, which is also required for its homodimerization and binding to its Dicer partner DCL4. We further showed that DRB4 protein accumulates when the proteasome is inactivated and, most importantly, we found that DRB4 stability depends on APC/C activity. Hence, depletion of Arabidopsis APC/C activity by RNAi leads to a strong accumulation of endogenous DRB4, far beyond its normal level of accumulation. However, we could not detect any defects in sRNA production in lines where DRB4 was overexpressed.

Conclusions/Significance

Our work identified a first plant substrate of the APC/C, which is not a regulator of the cell cycle. Though we cannot exclude that APC/C-dependent degradation of DRB4 has some regulatory roles under specific growth conditions, our work rather points to a housekeeping function of APC/C in maintaining precise cellular-protein concentrations and homeostasis of DRB4.     

Alcohol induced structural and dynamic changes in β-lactoglobulin in aqueous solution: A neutron scattering study

Structural and dynamic properties of β-lactoglobulin (β-LG) were revealed as a function of alcohol concentration in ethanol- and trifluoroethanol(TFE)-water mixtures with circular dichroism (CD), small-angle neutron scattering (SANS) and quasi-elastic neutron scattering (QENS). The CD spectra showed that an increase in TFE concentration promotes the formation of the β-sheet structure of β-LG. The SANS-intensities were fitted using form factors for two attached spheres for the native and native-like states of the protein. At higher alcohol concentrations, where aggregation takes place, a form factor modelling diffusion limited colloidal aggregation (DLCA) was employed. The QENS-data were analyzed in terms of internal motions for all alcohol concentrations. While low concentrations of TFE (10% (v/v)) lead to an increase of the mean square amplitudes of vibrations < u²> and a retention of a native-like structure — but not to an increase of the characteristic radius of proton diffusion processes a. Addition of 20% (v/v) of TFE induces aggregation, going along with a further increase of < u²>. Further increase of TFE concentration to 30% (v/v) changes the nanoscale structure of the oligomeric nucleate, but induces no further significant changes in < u²>. The present study underlines the necessity of methods sensitive to the dynamics of a system to obtain a complete picture of a molecular process.     

Rivalité acoustique et hiérarchie chez l'ephippiger (insecte,orthoptère,Tettigoniidea,)

Sans résuméDedié à M. le Professeur H. Autrum à l'occasion de son 60^eme anniversaire.     

Protection of Recombinant Mammalian Antibodies from Development-Dependent Proteolysis in Leaves of Nicotiana benthamiana

The expression of clinically useful proteins in plants has been bolstered by the development of high-yielding systems for transient protein expression using agroinfiltration. There is a need now to know more about how host plant development and metabolism influence the quantity and quality of recombinant proteins. Endogenous proteolysis is a key determinant of the stability and yield of recombinant proteins in plants. Here we characterised cysteine (C1A) and aspartate (A1) protease profiles in leaves of the widely used expression host Nicotiana benthamiana, in relation with the production of a murine IgG, C5-1, targeted to the cell secretory pathway. Agroinfiltration significantly altered the distribution of C1A and A1 proteases along the leaf age gradient, with a correlation between leaf age and the level of proteolysis in whole-cell and apoplast protein extracts. The co-expression of tomato cystatin SlCYS8, an inhibitor of C1A proteases, alongside C5-1 increased antibody yield by nearly 40% after the usual 6-days incubation period, up to ∼3 mg per plant. No positive effect of SlCYS8 was observed in oldest leaves, in line with an increased level of C1A protease activity and a very low expression rate of the inhibitor. By contrast, C5-1 yield was greater by an additional 40% following 8- to 10-days incubations in younger leaves, where high SlCYS8 expression was maintained. These findings confirm that the co-expression of recombinant protease inhibitors is a promising strategy for increasing recombinant protein yields in plants, but that further opportunity exists to improve this approach by addressing the influence of leaf age and proteases of other classes.     

Regulation of NADPH Oxidase Activity in Phagocytes: RELATIONSHIP BETWEEN FAD/NADPH BINDING AND OXIDASE COMPLEX ASSEMBLY*

The X⁺-linked chronic granulomatous disease (X⁺-CGD) variants are natural mutants characterized by defective NADPH oxidase activity but with normal Nox2 expression. According to the three-dimensional model of the cytosolic Nox2 domain, most of the X⁺-CGD mutations are located in/or close to the FAD/NADPH binding regions. A structure/function study of this domain was conducted in X⁺-CGD PLB-985 cells exactly mimicking 10 human variants: T341K, C369R, G408E, G408R, P415H, P415L, Δ507QKT509-HIWAinsert, C537R, L546P, and E568K. Diaphorase activity is defective in all these mutants. NADPH oxidase assembly is normal for P415H/P415L and T341K mutants where mutation occurs in the consensus sequences of NADPH- and FAD-binding sites, respectively. This is in accordance with their buried position in the three-dimensional model of the cytosolic Nox2 domain. FAD incorporation is abolished only in the T341K mutant explaining its absence of diaphorase activity. This demonstrates that NADPH oxidase assembly can occur without FAD incorporation. In addition, a defect of NADPH binding is a plausible explanation for the diaphorase activity inhibition in the P415H, P415L, and C537R mutants. In contrast, Cys-369, Gly-408, Leu-546, and Glu-568 are essential for NADPH oxidase complex assembly. However, according to their position in the three-dimensional model of the cytosolic domain of Nox2, only Cys-369 could be in direct contact with cytosolic factors during oxidase assembly. In addition, the defect in oxidase assembly observed in the C369R, G408E, G408R, and E568K mutants correlates with the lack of FAD incorporation. Thus, the NADPH oxidase assembly process and FAD incorporation are closely related events essential for the diaphorase activity of Nox2.     

Better to Live with Allogenerics Than to Live Alone? The Case of Single Male Cercopithecus pogonias in Troops of Colobus satanas

We report the integration of single male crowned guenons (Cercopithecus pogonias) into troops of black colobus (Colobus satanas). We observed one male Cercopithecus pogonias in three troops of Colobus satanas on 30% of observation days (n = 231). Activities of single males guenons did not differ significantly from those of the colobus with which they associated. Moreover, both species performed simultaneously the same activities more often than expected by chance. Interspecific grooming occurred on several occasions. Furthermore, single male guenons spent as much in time social activities when part of a colobus troop, as they typically do when part of a conspecific group. Unlike solitary male crowned guenons, which are silent, a male that is integrated into a troop of colobus is vocal and emits social alarm calls to which colobus monkeys respond. During the single file movements of colobus troops, single male crowned guenons were integrated in the core of the troop and used the same branches at the same height with the colobus. Thus, the life of a single male crowned guenon with black colobus was social. We suggest that the main benefits that he gained is the possibility to live in a social context. Social interactions could be the key element to explain why single males Cercopithecus pogonias join troops of monkeys so different from their natal groups.