Alpha repeat proteins (αRep) as expression and crystallization helpers

We have previously described a highly diverse library of artificial repeat proteins based on thermostable HEAT-like repeats, named αRep. αReps binding specifically to proteins difficult to crystallize have been selected and in several examples, they made possible the crystallization of these proteins. To further simplify the production and crystallization experiments we have explored the production of chimeric proteins corresponding to covalent association between the targets and their specific binders strengthened by a linker. Although chimeric proteins with expression partners are classically used to enhance expression, these fusions cannot usually be used for crystallization. With specific expression partners like a cognate αRep this is no longer true, and chimeric proteins can be expressed purified and crystallized. αRep selection by phage display suppose that at least a small amount of the target protein should be produced to be used as a bait for selection and this might, in some cases, be difficult. We have therefore transferred the αRep library in a new construction adapted to selection by protein complementation assay (PCA). This new procedure allows to select specific binders by direct interaction with the target in the cytoplasm of the bacteria and consequently does not require preliminary purification of target protein. αRep binders selected by PCA or by phage display can be used to enhance expression, stability, solubility and crystallogenesis of proteins that are otherwise difficult to express, purify and/or crystallize.     

COMMD1-mediated ubiquitination regulates CFTR trafficking

The CFTR (cystic fibrosis transmembrane conductance regulator) protein is a large polytopic protein whose biogenesis is inefficient. To better understand the regulation of CFTR processing and trafficking, we conducted a genetic screen that identified COMMD1 as a new CFTR partner. COMMD1 is a protein associated with multiple cellular pathways, including the regulation of hepatic copper excretion, sodium uptake through interaction with ENaC (epithelial sodium channel) and NF-kappaB signaling. In this study, we show that COMMD1 interacts with CFTR in cells expressing both proteins endogenously. This interaction promotes CFTR cell surface expression as assessed by biotinylation experiments in heterologously expressing cells through regulation of CFTR ubiquitination. In summary, our data demonstrate that CFTR is protected from ubiquitination by COMMD1, which sustains CFTR expression at the plasma membrane. Thus, increasing COMMD1 expression may provide an approach to simultaneously inhibit ENaC absorption and enhance CFTR trafficking, two major issues in cystic fibrosis.     

Nonsense-mediated mRNA decay impacts MSI-driven carcinogenesis and anti-tumor immunity in colorectal cancers

Nonsense-mediated mRNA Decay (NMD) degrades mutant mRNAs containing premature termination codon (PTC-mRNAs). Here we evaluate the consequence of NMD activity in colorectal cancers (CRCs) showing microsatellite instability (MSI) whose progression is associated with the accumulation of PTC-mRNAs encoding immunogenic proteins due to frameshift mutations in coding repeat sequences. Inhibition of UPF1, one of the major NMD factors, was achieved by siRNA in the HCT116 MSI CRC cell line and the resulting changes in gene expression were studied using expression microarrays. The impact of NMD activity was also investigated in primary MSI CRCs by quantifying the expression of several mRNAs relative to their mutational status and to endogenous UPF1 and UPF2 expression. Host immunity developed against MSI cancer cells was appreciated by quantifying the number of CD3epsilon-positive tumor-infiltrating lymphocytes (TILs). UPF1 silencing led to the up-regulation of 1251 genes in HCT116, among which a proportion of them (i.e. 38%) significantly higher than expected by chance contained a coding microsatellite (P<2x10(-16)). In MSI primary CRCs, UPF1 was significantly over-expressed compared to normal adjacent mucosa (P<0.002). Our data provided evidence for differential decay of PTC-mRNAs compared to wild-type that was positively correlated to UPF1 endogenous expression level (P = 0.02). A negative effect of UPF1 and UPF2 expression on the host's anti-tumor response was observed (P<0.01). Overall, our results show that NMD deeply influences MSI-driven tumorigenesis at the molecular level and indicate a functional negative impact of this system on anti-tumor immunity whose intensity has been recurrently shown to be an independent factor of favorable outcome in CRCs.     

Knocking on Heaven's Door: Are Novel Invaders Necessarily Facing Na?ve Native Species on Islands?

The impact of alien predator species on insular native biota has often been attributed to island prey naïveté (i.e. lack of, or inefficient, anti-predator behavior). Only rarely, however, has the concept of island prey naïveté been tested, and then only a posteriori (i.e. hundreds or thousands of years after alien species introduction). The presence of native or anciently introduced predators or competitors may be crucial for the recognition and development of adaptive behavior toward unknown predators or competitors of the same archetype (i.e. a set of species that occupy a similar ecological niche and show similar morphological and behavioral traits when interacting with other species). Here, we tested whether two squamates endemic to New Caledonia, a skink, Caledoniscincus austrocaledonicus, and a gecko, Bavayia septuiclavis, recognized and responded to the odor of two major invaders introduced into the Pacific islands, but not yet into New Caledonia. We chose one predator, the small Indian mongoose Herpestes javanicus and one competitor, the cane toad Rhinella marina, which belong respectively to the same archetype as the following two species already introduced into New Caledonia in the nineteenth century: the feral cat Felis catus and the golden bell frog Litoria aurea. Our experiment reveals that geckos are naïve with respect to the odors of both an unknown predator and an unknown competitor, as well as to the odors of a predator and a competitor they have lived with for centuries. In contrast, skinks seem to have lost some naïveté regarding the odor of a predator they have lived with for centuries and seem “predisposed” to avoid the odor of an unknown potential competitor. These results indicate that insular species living in contact with invasive alien species for centuries may be, although not systematically, predisposed toward developing adaptive behavior with respect to species belonging to the same archetype and introduced into their native range.     

Neural Mechanisms Underlying Breathing Complexity

Breathing is maintained and controlled by a network of automatic neurons in the brainstem that generate respiratory rhythm and receive regulatory inputs. Breathing complexity therefore arises from respiratory central pattern generators modulated by peripheral and supra-spinal inputs. Very little is known on the brainstem neural substrates underlying breathing complexity in humans. We used both experimental and theoretical approaches to decipher these mechanisms in healthy humans and patients with chronic obstructive pulmonary disease (COPD). COPD is the most frequent chronic lung disease in the general population mainly due to tobacco smoke. In patients, airflow obstruction associated with hyperinflation and respiratory muscles weakness are key factors contributing to load-capacity imbalance and hence increased respiratory drive. Unexpectedly, we found that the patients breathed with a higher level of complexity during inspiration and expiration than controls. Using functional magnetic resonance imaging (fMRI), we scanned the brain of the participants to analyze the activity of two small regions involved in respiratory rhythmogenesis, the rostral ventro-lateral (VL) medulla (pre-Bötzinger complex) and the caudal VL pons (parafacial group). fMRI revealed in controls higher activity of the VL medulla suggesting active inspiration, while in patients higher activity of the VL pons suggesting active expiration. COPD patients reactivate the parafacial to sustain ventilation. These findings may be involved in the onset of respiratory failure when the neural network becomes overwhelmed by respiratory overload We show that central neural activity correlates with airflow complexity in healthy subjects and COPD patients, at rest and during inspiratory loading. We finally used a theoretical approach of respiratory rhythmogenesis that reproduces the kernel activity of neurons involved in the automatic breathing. The model reveals how a chaotic activity in neurons can contribute to chaos in airflow and reproduces key experimental fMRI findings.     

Activation of type III interferon genes by pathogenic bacteria in infected epithelial cells and mouse placenta

Bacterial infections trigger the expression of type I and II interferon genes but little is known about their effect on type III interferon (IFN-λ) genes, whose products play important roles in epithelial innate immunity against viruses. Here, we studied the expression of IFN-λ genes in cultured human epithelial cells infected with different pathogenic bacteria and in the mouse placenta infected with Listeria monocytogenes. We first showed that in intestinal LoVo cells, induction of IFN-λ genes by L. monocytogenes required bacterial entry and increased further during the bacterial intracellular phase of infection. Other Gram-positive bacteria, Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis, also induced IFN-λ genes when internalized by LoVo cells. In contrast, Gram-negative bacteria Salmonella enterica serovar Typhimurium, Shigella flexneri and Chlamydia trachomatis did not substantially induce IFN-λ. We also found that IFN-λ genes were up-regulated in A549 lung epithelial cells infected with Mycobacterium tuberculosis and in HepG2 hepatocytes and BeWo trophoblastic cells infected with L. monocytogenes. In a humanized mouse line permissive to fetoplacental listeriosis, IFN-λ2/λ3 mRNA levels were enhanced in placentas infected with L. monocytogenes. In addition, the feto-placental tissue was responsive to IFN-λ2. Together, these results suggest that IFN-λ may be an important modulator of the immune response to Gram-positive intracellular bacteria in epithelial tissues.     

Leucine and citrulline modulate muscle function in malnourished aged rats

Protein energy malnutrition in the elderly causes preferential loss of muscle mass which is associated with poor functional states. Leucine and citrulline are able to stimulate muscle protein synthesis in aged rats but no study has been undertaken to evaluate their effect on muscle function. Sprague–Dawley male rats aged 23 months were used in the experiment. Part of them were subjected to a dietary restriction for 12 weeks and then assigned to four groups: a group was euthanized (restricted group), and the others were refed for 1 week with either a leucine-, a citrulline-supplemented diet, or a standard diet. The other rats were fed ad libitum. Muscle mass and motor activity significantly increased during the refeeding with either leucine or citrulline (respectively, +51 and +37% for muscle mass, P < 0.05). The improvement of muscle mass and of motor activity induced by leucine and citrulline was highly associated with that of maximal tetanic isometric force (r = 0.769, P < 0.0001; r = 0.389, P < 0.05, respectively) but only leucine improved maximal tetanic isometric force (+101%, P < 0.05). In conclusion, this is the first study to demonstrate the ability of two amino acids (leucine and citrulline) to modulate muscle function.     

Specific GFP-binding artificial proteins (αRep): a new tool for in?vitro to live cell applications

A family of artificial proteins, named αRep, based on a natural family of helical repeat was previously designed. αRep members are efficiently expressed, folded and extremely stable proteins. A large αRep library was constructed creating proteins with a randomized interaction surface. In the present study, we show that the αRep library is an efficient source of tailor-made specific proteins with direct applications in biochemistry and cell biology. From this library, we selected by phage display αRep binders with nanomolar dissociation constants against the GFP. The structures of two independent αRep binders in complex with the GFP target were solved by X-ray crystallography revealing two totally different binding modes. The affinity of the selected αReps for GFP proved sufficient for practically useful applications such as pull-down experiments. αReps are disulfide free proteins and are efficiently and functionally expressed in eukaryotic cells: GFP-specific αReps are clearly sequestrated by their cognate target protein addressed to various cell compartments. These results suggest that αRep proteins with tailor-made specificity can be selected and used in living cells to track, modulate or interfere with intracellular processes.     

Micropearls and other intracellular inclusions of amorphous calcium carbonate: an unsuspected biomineralization capacity shared by diverse microorganisms

An unsuspected biomineralization process, which produces intracellular inclusions of amorphous calcium carbonate (ACC), was recently discovered in unicellular eukaryotes. These mineral inclusions, called micropearls, can be highly enriched with other alkaline-earth metals (AEM) such as Sr and Ba. Similar intracellular inclusions of ACC have also been observed in prokaryotic organisms. These comparable biomineralization processes involving phylogenetically distant microorganisms are not entirely understood yet. This review gives a broad vision of the topic in order to establish a basis for discussion on the possible molecular processes behind the formation of the inclusions, their physiological role, the impact of these microorganisms on the geochemical cycles of AEM and their evolutionary relationship. Finally, some insights are provided to guide future research.