Cullin3 - BTB Interface: A Novel Target for Stapled Peptides

Cullin3 (Cul3), a key factor of protein ubiquitination, is able to interact with dozens of different proteins containing a BTB (Bric-a-brac, Tramtrack and Broad Complex) domain. We here targeted the Cul3–BTB interface by using the intriguing approach of stabilizing the α-helical conformation of Cul3-based peptides through the “stapling” with a hydrocarbon cross-linker. In particular, by combining theoretical and experimental techniques, we designed and characterized stapled Cul3-based peptides embedding the helix 2 of the protein (residues 49–68). Intriguingly, CD and NMR experiments demonstrate that these stapled peptides were able to adopt the helical structure that the fragment assumes in the parent protein. We also show that some of these peptides were able to bind to the BTB of the tetrameric KCTD11, a substrate adaptor involved in HDAC1 degradation, with high affinity (~ 300–600 nM). Cul3-derived staple peptides are also able to bind the BTB of the pentameric KCTD5. Interestingly, the affinity of these peptides is of the same order of magnitude of that reported for the interaction of full-length Cul3 with some BTB containing proteins. Moreover, present data indicate that stapling endows these peptides with an increased serum stability. Altogether, these findings indicate that the designed stapled peptides can efficiently mimic protein-protein interactions and are potentially able to modulate fundamental biological processes involving Cul3.     

Molecular basis of the PED/PEA15 interaction with the C-terminal fragment of phospholipase D1 revealed by NMR spectroscopy

PED/PEA15 is a small protein involved in many protein–protein interactions that modulates the function of a number of key cellular effectors involved in major cell functions, including apoptosis, proliferation and glucose metabolism. In particular, PED/PEA15 interacts with the phospholipase D (PLD) isoforms 1 and 2 increasing protein kinase C-α isoform activity and affects both insulin-stimulated glucose transport and glucose-stimulated insulin secretion.     

Scavenger Receptors Mediate the Role of SUMO and Ftz-f1 in Drosophila Steroidogenesis

SUMOylation participates in ecdysteroid biosynthesis at the onset of metamorphosis in Drosophila melanogaster. Silencing the Drosophila SUMO homologue smt3 in the prothoracic gland leads to reduced lipid content, low ecdysone titers, and a block in the larval–pupal transition. Here we show that the SR-BI family of Scavenger Receptors mediates SUMO functions. Reduced levels of Snmp1 compromise lipid uptake in the prothoracic gland. In addition, overexpression of Snmp1 is able to recover lipid droplet levels in the smt3 knockdown prothoracic gland cells. Snmp1 expression depends on Ftz-f1 (an NR5A-type orphan nuclear receptor), the expression of which, in turn, depends on SUMO. Furthermore, we show by in vitro and in vivo experiments that Ftz-f1 is SUMOylated. RNAi–mediated knockdown of ftz-f1 phenocopies that of smt3 at the larval to pupal transition, thus Ftz-f1 is an interesting candidate to mediate some of the functions of SUMO at the onset of metamorphosis. Additionally, we demonstrate that the role of SUMOylation, Ftz-f1, and the Scavenger Receptors in lipid capture and mobilization is conserved in other steroidogenic tissues such as the follicle cells of the ovary. smt3 knockdown, as well as ftz-f1 or Scavenger knockdown, depleted the lipid content of the follicle cells, which could be rescued by Snmp1 overexpression. Therefore, our data provide new insights into the regulation of metamorphosis via lipid homeostasis, showing that Drosophila Smt3, Ftz-f1, and SR-BIs are part of a general mechanism for uptake of lipids such as cholesterol, required during development in steroidogenic tissues.     

A single amino acid change in the transmembrane domain of the VirB8 protein affects dimerization,interaction with VirB10 and Brucella suis virulence

VirB8 is a critical component of the Brucella suis type IV secretion system (T4SS). We previously showed that the transmembrane (TM) domain plays an essential role in interactions of this protein with itself and the other proteins of the T4SS. We report that a point mutation in this TM domain stabilizes homodimers of VirB8 and heterodimers with VirB10. A similar variant of Agrobacterium tumefaciens VirB8 showed the same phenotype. The B. suis VirB8 variant was unable to complement a virB8 mutant and displayed a dominant negative phenotype when expressed in wild type B. suis. We suggest that interaction of VirB8 with VirB10 could play a major role in the correct function of the B. suis VirB T4SS.Structured summary of protein interactionsAtVirB8 physically interacts with AtVirB10 by two hybrid (View interaction)TraJ physically interacts with TraJ by two hybrid (View Interaction 1, 2)AtVirB8 physically interacts with AtVirB8 by two hybrid (View interaction)VirB10 physically interacts with VirB10 by two hybrid (View interaction)VirB8 physically interacts with VirB8 by two hybrid (View Interaction 1, 2)VirB10 physically interacts with VirB8 by two hybrid (View interaction)AtVirB10 physically interacts with AtVirB10 by two hybrid (View interaction)VirB8 physically interacts with VirB10 by two hybrid (View interaction)AtVirB10 physically interacts with AtVirB8 by two hybrid (View interaction)     

Histochemical features of the Muscovy duck small intestine during development

We demonstrated for the first time the distribution and morphology of argyrophil and of goblet cells in the mucosa of the small intestine of the Muscovy duck during development using the Grimelius silver staining and alcian blue/periodic acid-Schiff (AB/PAS) staining technique. The argyrophil cells distribution was variable over the length of the small intestine from embryonic day 24 (24E) to post-hatching day 13 (13d). In the villi most argyrophil cells belonged to the open-type, while in the crypts they belonged to the closed-type. In the duodenum the density of argyrophil cells was highest at hatching, while in the jejunum and in the ileum the highest density value was at hatching and 13d. AB/PAS-positive goblet cells appeared on the villi and crypts of the duodenum and jejunum at 30E, and in the ileum at hatching. The density of AB/PAS-positive cells was the highest in the three segments at hatching. The AB-positive cells, compared with the PAS-positive cells, predominated in villi and crypts of the three segments, moreover the rate of AB-positive cells to PAS-positive cells significantly decreased from 30E to 9d. An increase in argyrophil and goblet cells number during the later incubation and at hatching, could indicate the small intestine in that period is being prepared to face a new diet.     

Modelling Co-Infection of the Cystic Fibrosis Lung by Pseudomonas aeruginosa and Burkholderia cenocepacia Reveals Influences on Biofilm Formation and Host Response

The Gram-negative bacteria Pseudomonas aeruginosa and Burkholderia cenocepacia are opportunistic human pathogens that are responsible for severe nosocomial infections in immunocompromised patients and those suffering from cystic fibrosis (CF). These two bacteria have been shown to form biofilms in the airways of CF patients that make such infections more difficult to treat. Only recently have scientists begun to appreciate the complicated interplay between microorganisms during polymicrobial infection of the CF airway and the implications they may have for disease prognosis and response to therapy.To gain insight into the possible role that interaction between strains of P. aeruginosa and B. cenocepacia may play during infection, we characterised co-inoculations of in vivo and in vitro infection models. Co-inoculations were examined in an in vitro biofilm model and in a murine model of chronic infection. Assessment of biofilm formation showed that B. cenocepacia positively influenced P. aeruginosa biofilm development by increasing biomass. Interestingly, co-infection experiments in the mouse model revealed that P. aeruginosa did not change its ability to establish chronic infection in the presence of B. cenocepacia but co-infection did appear to increase host inflammatory response.Taken together, these results indicate that the co-infection of P. aeruginosa and B. cenocepacia leads to increased biofilm formation and increased host inflammatory response in the mouse model of chronic infection. These observations suggest that alteration of bacterial behavior due to interspecies interactions may be important for disease progression and persistent infection.     

Burkholderia cenocepacia strains isolated from cystic fibrosis patients are apparently more invasive and more virulent than rhizosphere strains

Given the widespread presence of Burkholderia cenocepacia in the rhizosphere it is important to determine whether rhizosphere strains are pathogenic for cystic fibrosis patients or not. Eighteen B. cenocepacia strains of rhizosphere and clinical origin were typed by multi-locus sequence typing (MLST) analysis and compared for their ability to invade pulmonary epithelial cells and their virulence in a mouse model of airway infection. Although there was great variability, clinical strains were the most invasive in vitro. Almost all the rhizosphere and two clinical strains were defined as non-invasive, six clinical strains as invasive, and two strains of both clinical and environmental origin as indeterminate. Exposure of murine airways to clinical strains caused higher acute mortality than that seen after challenge with rhizosphere strains. Furthermore, both clinical and environmental strains were able to persist in the lungs of infected mice, with no significant differences in bacterial loads and localization 14 days after challenge. DNA dot blot analyses of AHL synthase, porin and amidase genes, which play a role in B. cenocepacia virulence, showed that they were present in B. cenocepacia strains irrespective of their origin. Overall, our results suggest that rhizosphere strains do not differ from clinical strains in some pathogenic traits.     

The analysis of the different functions of starch‐phosphorylating enzymes during the development of Arabidopsis thaliana plants discloses an unexpected role for the cytosolic isoform GWD2

The genome of Arabidopsis thaliana encodes three glucan, water dikinases. Glucan, water dikinase 1 (GWD1; EC 2.7.9.4) and phosphoglucan, water dikinase (PWD; EC 2.7.9.5) are chloroplastic enzymes, while glucan, water dikinase 2 (GWD2) is cytosolic. Both GWDs and PWD catalyze the addition of phosphate groups to amylopectin chains at the surface of starch granules, changing its physicochemical properties. As a result, GWD1 and PWD have a positive effect on transitory starch degradation at night. Because of its cytosolic localization, GWD2 does not have the same effect. Single T‐DNA mutants of either GWD1 or PWD or GWD2 have been analyzed during the entire life cycle of A. thaliana. We report that the three dikinases are all important for proper seed development. Seeds from gwd2 mutants are shrunken, with the epidermal cells of the seed coat irregularly shaped. Moreover, gwd2 seeds contain a lower lipid to protein ratio and are impaired in germination. Similar seed phenotypes were observed in pwd and gwd1 mutants, except for the normal morphology of epidermal cells in gwd1 seed coats. The gwd1, pwd and gwd2 mutants were also very similar in growth and flowering time when grown under continuous light and all three behaved differently from wild‐type plants. Besides pinpointing a novel role of GWD2 and PWD in seed development, this analysis suggests that the phenotypic features of the dikinase mutants in A. thaliana cannot be explained solely in terms of defects in leaf starch degradation at night.     

New Starch Phenotypes Produced by TILLING in Barley

Barley grain starch is formed by amylose and amylopectin in a 1∶3 ratio, and is packed into granules of different dimensions. The distribution of granule dimension is bimodal, with a majority of small spherical B-granules and a smaller amount of large discoidal A-granules containing the majority of the starch. Starch granules are semi-crystalline structures with characteristic X-ray diffraction patterns. Distinct features of starch granules are controlled by different enzymes and are relevant for nutritional value or industrial applications. Here, the Targeting-Induced Local Lesions IN Genomes (TILLING) approach was applied on the barley TILLMore TILLING population to identify 29 new alleles in five genes related to starch metabolism known to be expressed in the endosperm during grain filling: BMY1 (Beta-amylase 1), GBSSI (Granule Bound Starch Synthase I), LDA1 (Limit Dextrinase 1), SSI (Starch Synthase I), SSIIa (Starch Synthase IIa). Reserve starch of nine M3 mutant lines carrying missense or nonsense mutations was analysed for granule size, crystallinity and amylose/amylopectin content. Seven mutant lines presented starches with different features in respect to the wild-type: (i) a mutant line with a missense mutation in GBSSI showed a 4-fold reduced amylose/amylopectin ratio; (ii) a missense mutations in SSI resulted in 2-fold increase in A:B granule ratio; (iii) a nonsense mutation in SSIIa was associated with shrunken seeds with a 2-fold increased amylose/amylopectin ratio and different type of crystal packing in the granule; (iv) the remaining four missense mutations suggested a role of LDA1 in granule initiation, and of SSIIa in determining the size of A-granules. We demonstrate the feasibility of the TILLING approach to identify new alleles in genes related to starch metabolism in barley. Based on their novel physicochemical properties, some of the identified new mutations may have nutritional and/or industrial applications.