Multiproteic Complex Research Articles

Lhx4 surpasses its paralog Lhx3 in promoting the differentiation of spinal V2a interneurons

Paralog factors are considered to ensure the robustness of biological processes by providing redundant activity in cells where they are co-expressed. However, the specific contribution of each factor is frequently underestimated. In the developing spinal cord, multiple families of transcription factors successively contribute to differentiate an initially homogenous population of neural progenitors into a myriad of neuronal subsets with distinct molecular, morphological, and functional characteristics. The LIM-homeodomain transcription factors Lhx3, Lhx4, Isl1 and Isl2 promote the segregation and differentiation of spinal motor neurons and V2 interneurons. Based on their high sequence identity and their similar distribution, the Lhx3 and Lhx4 paralogs are considered to contribute similarly to these processes. However, the specific contribution of Lhx4 has never been studied. Here, we provide evidence that Lhx3 and Lhx4 are present in the same cell populations during spinal cord development. Similarly to Lhx3, Lhx4 can form multiproteic complexes with Isl1 or Isl2 and the nuclear LIM interactor NLI. Lhx4 can stimulate a V2-specific enhancer more efficiently than Lhx3 and surpasses Lhx3 in promoting the differentiation of V2a interneurons in chicken embryo electroporation experiments. Finally, Lhx4 inactivation in mice results in alterations of differentiation of the V2a subpopulation, but not of motor neuron production, suggesting that Lhx4 plays unique roles in V2a differentiation that are not compensated by the presence of Lhx3. Thus, Lhx4 could be the major LIM-HD factor involved in V2a interneuron differentiation during spinal cord development and should be considered for in vitro differentiation of spinal neuronal populations.

NLRP-3 inflammasome: study target in the development of epilepsy

The inflammasome is assembled in response to a wide range of patterns associated with pathogens or damage molecules inpresence of brain injury, they are multiproteic complexes that comprise a sensor, an adapter protein and a zymogenic enzyme thatactivates caspase-1 for secretion of mature proteins of IL-1β and IL-18 that trigger pyroptosis (neuroinflammation-induced celldeath) which is involved in the onset and development of several pathophysiological events. Until now, four inflammasomes havebeen described that recruit the Apoptosis-associated speck-like (ASC) adapter protein to activate caspase-1: NLRP-1, NLRP-3,NLRC4 and AIM2. The activation of the NLRP-3 inflammasomes are mediated by variations in K⁺ concentrations, increasedextracellular ATP and oxidative stress mainly. Because of this, it has been implicated in the pathogenesis of several neurologicaldisorders such as epilepsy; which is a neurological disorder characterized by the presence of spontaneous and a recurrent seizure(SRS) that affects around 70 million people in the world. SRS are generated by the establishment of the epileptome (defined area ofepileptogenesis) triggered by genetic or acquired factors that cause structural and functional modifications, such asneuroinflammation that induces oxidative stress, neuronal loss, reactive gliosis and pathological synaptic reorganization.Neuroinflammation can be preserved during epileptogenesis due activation of the NLRP-3 inflammasome. On the other hand, awide variety of inhibitors have been used to alter the secretion of IL-1β and IL-18, indirect inhibitors of NLRP-3, ASC and caspase-1; however, these only disturb the signaling pathways without directly regulating NLRP-3 and blocked the pathologicalneuroinflammatory response. For this reason, it is important to study the activation and modulation of inflammasomes like NLRP-3in epileptogenic processes to expand the view on neuroinflammation and its possible mechanisms involved in epilepsy.

Inflammasome components and obesity onset: novel combination for comorbidities reversion after bariatric surgery

Rationale: Obesity is associated with metabolic/inflammatory comorbidities, which can be reversed/improved after bariatric-surgery(BS), and influenced by obesity onset. Dysregulations in the inflammasome, a multiproteic complex that promotes cytokine maturation and induces cellular pyroptosis, have been associated to the development/stage of obesity. Whether the obesity onset affects the expression of inflammasome components and comorbidities reversal has not been yet explored.

Serum 25-OH vitamin D after bariatric surgery is correlated with changes in the molecular profile of inflammasome components

Rationale: Vitamin D deficiency (VDD) is increased in obesity and associated with obesity-related comorbidities and immune response. Dysregulations in the inflammasome, a multiproteic complex that promotes cytokine maturation and induces cellular pyroptosis, have been associated to the development of obesity. Whether serum 25OH-vitamin D influences the reversal of metabolic comorbidities and the expression of inflammasome components has not been yet explored.

Visualization of Specific γ-Secretase Complexes using Bimolecular Fluorescence Complementation

γ-Secretase is involved in the regulated intramembrane proteolysis of amyloid-β protein precursor (AβPP) and of many other important physiological substrates. γ-secretase is a multiproteic complex made of four main core components, namely presenilin 1 or 2, APH-1, PEN-2, and Nicastrin. Since APH-1 exists as different variants, combinations of these proteins can theoretically yield distinct γ-secretase complexes. Whether γ-secretase complexes trafficking and targeting to either similar or distinct subcellular compartments depend upon their molecular composition remains unknown. A differential complex-specific distribution may drive a narrow specificity for a subset of substrates that would traffic within the same cellular compartments. Here, we generated bigenic expression vectors to co-express untagged nicastrin or presenilin 1 together with either PEN-2 or distinct variants of APH-1 (aL, aS and b) tagged with complementary fragments of the fluorescent protein Venus. We show that these constructs allow the formation of functional γ-secretase complexes and their visualization with bimolecular fluorescence complementation (BiFC). BiFC can be detected at the plasma membrane as well as in endosomes/lysosomes in addition to the endoplasmic reticulum (ER) of COS-7 cells transfected with the different variants of APH-1. However, the majority of cells co-transfected with APH-1b presented BiFC signal only in the ER, suggesting enhanced retention/retrieval of APH-1b-containing γ-secretase complexes. Therefore, the new tools described here should be helpful to decipher the precise subcellular trafficking of γ-secretase complexes and to delineate the distinct variant-linked pathways in various cellular systems.

Rôle de l’inflammasome dans les pathologies cardiovasculaires

NOD-like receptors (NLRs) constitute a recently identified family of intracellular pattern recognition receptors which contains more than 20 members in mammals. Some of the NLRs, the NALP subfamily, constituted from 14 members, many of them without actual identified role, form multiproteic complex known as inflammasome, that initiate inflammation by processing inactive pro-caspase-1 to its active form, allowing the cleavage and subsequent activation of pro-IL-1β and pro-IL-18. We review the identified roles of NLRs in pathologies and argue for the role of inflammasome in the development of cardiovascular diseases. The atherogenic cytokines IL-1β and IL-18 are matured in NLRPs inflammasomes. Immunocytochemistry shows that Nlrp3 inflammasome is expressed in plaques, upregulated and activated in the CD11b +Gr1 high atherosclerosis-prone monocyte subset and modulated by oxLDL in murine macrophages. These results provide an unexpected role for Nlrp3 inflammasome in atherosclerosis.

ADP-Ribosylation Factor 6 Regulates Mammalian Myoblast Fusion through Phospholipase D1 and Phosphatidylinositol 4,5-Bisphosphate Signaling Pathways

Myoblast fusion is an essential step during myoblast differentiation that remains poorly understood. M-cadherin-dependent pathways that signal through Rac1 GTPase activation via the Rho-guanine nucleotide exchange factor (GEF) Trio are important for myoblast fusion. The ADP-ribosylation factor (ARF)6 GTPase has been shown to bind to Trio and to regulate Rac1 activity. Moreover, Loner/GEP(100)/BRAG2, a GEF of ARF6, has been involved in mammalian and Drosophila myoblast fusion, but the specific role of ARF6 has been not fully analyzed. Here, we show that ARF6 activity is increased at the time of myoblast fusion and is required for its implementation in mouse C2C12 myoblasts. Specifically, at the onset of myoblast fusion, ARF6 is associated with the multiproteic complex that contains M-cadherin, Trio, and Rac1 and accumulates at sites of myoblast fusion. ARF6 silencing inhibits the association of Trio and Rac1 with M-cadherin. Moreover, we demonstrate that ARF6 regulates myoblast fusion through phospholipase D (PLD) activation and phosphatidylinositol 4,5-bis-phosphate production. Together, these data indicate that ARF6 is a critical regulator of C2C12 myoblast fusion and participates in the regulation of PLD activities that trigger both phospholipids production and actin cytoskeleton reorganization at fusion sites.

CagA Associates with c‐Met, E‐Cadherin, and p120‐Catenin in a Multiproteic Complex That SuppressesHelicobacter pylori–Induced Cell‐Invasive Phenotype

Helicobacter pylori induces an invasive phenotype in gastric epithelial cells through a mechanism that requires the type IV secretion system and the phosphorylation of c-Met. The E-cadherin-catenin complex is a major component of the adherens junctions and functions as an invasion suppressor. We investigated whether E-cadherin has a role in H. pylori-induced, c-Met phosphorylation-dependent cell-invasive phenotype. AGS cells that lack E-cadherin and that are invasive to H. pylori stimulation were transduced with E-cadherin and infected with H. pylori. NCI-N87 cells, which endogenously express E-cadherin, were also used for infection experiments. E-cadherin was sufficient to suppress not only H. pylori-mediated cell-invasive phenotype but also c-Met and p120-catenin tyrosine phosphorylation. H. pylori infection led to increased interactions between E-cadherin and p120-catenin, c-Met and E-cadherin, and c-Met and p120-catenin. Using in vitro infection assays, we showed that H. pylori CagA interacts with E-cadherin, p120-catenin, and c-Met. Finally, using small interfering RNA, we showed that interactions between CagA and E-cadherin and between CagA and p120-catenin were established through c-Met. We suggest that H. pylori alters the E-cadherin-catenin complex, leading to formation of a multiproteic complex composed of CagA, c-Met, E-cadherin, and p120-catenin. This complex abrogates c-Met and p120-catenin tyrosine phosphorylation and suppresses the cell-invasive phenotype induced by H. pylori.

M-Cadherin Activates Rac1 GTPase through the Rho-GEF Trio during Myoblast Fusion

Cadherins are transmembrane glycoproteins that mediate Ca(2+)-dependent homophilic cell-cell adhesion and play crucial role during skeletal myogenesis. M-cadherin is required for myoblast fusion into myotubes, but its mechanisms of action remain unknown. The goal of this study was to cast some light on the nature of the M-cadherin-mediated signals involved in myoblast fusion into myotubes. We found that the Rac1 GTPase activity is increased at the time of myoblast fusion and it is required for this process. Moreover, we showed that M-cadherin-dependent adhesion activates Rac1 and demonstrated the formation of a multiproteic complex containing M-cadherin, the Rho-GEF Trio, and Rac1 at the onset of myoblast fusion. Interestingly, Trio knockdown efficiently blocked both the increase in Rac1-GTP levels, observed after M-cadherin-dependent contact formation, and myoblast fusion. We conclude that M-cadherin-dependent adhesion can activate Rac1 via the Rho-GEF Trio at the time of myoblast fusion.

Subcomplexes from the Xcp secretion system of Pseudomonas aeruginosa

In Gram-negative bacteria, most of the sec-dependent exoproteins are secreted via the type II secretion system (T2SS or secreton). In Pseudomonas aeruginosa, T2SS consists of 12 Xcp proteins (XcpA and XcpP to XcpZ) organized as a multiproteic complex within the envelope. In this study, by a co-purification approach using a His-tagged XcpZ as a bait, XcpY and XcpZ were found associated together to constitute the most stable functional unit so far isolated from the P. aeruginosa secreton. This subcomplex was also found to interact with XcpR and XcpS to form a XcpRSYZ complex which was isolated under native conditions. Another component, XcpP was not found to be associated to the complex but the results suggest that it can transiently interact with the XcpYZ subcomplex in vivo.

Role of XcpP in the functionality of the Pseudomonas aeruginosa secreton

In Gram-negative bacteria, most signal-peptide-dependent exoproteins are secreted via the type II secretion system (T2SS or secreton). In Pseudomonas aeruginosa, T2SS consists of twelve Xcp proteins (XcpA and XcpP to XcpZ) thought to be organized as a multiproteic complex within the envelope. Although well conserved, T2SS are known to be species-specific, namely for distant organisms, and this characteristic was thought to involve XcpP. To check which domain of XcpP could be involved in the species specificity, hybrid proteins were generated using protein domain swapping between P. aeruginosa XcpP and homolog proteins of either Erwinia chrysanthemi or Pseudomonas alcaligenes. The results obtained with hybrid proteins constructed by exchanging the C-terminal domains of P. aeruginosa and E. chrysanthemi suggested that XcpP interacts with XcpQ, probably via its C-terminal domain. More interestingly, the data obtained with a hybrid protein containing the C-terminal part of the P. alcaligenes XcpP homolog, showed that the wild-type C-terminal end plays a very important role in the function of the protein and is required both for a correct interaction with XcpQ and for modulating the opening of the secreton channel.

Physiological and pathological roles of Apaf1 and the apoptosome.

Different cellular pathways can lead to apoptosis. Apaf1 is the molecular core of the apoptosome, a multiproteic complex mediating the so-called mitochondrial pathway of cell death. The importance of this pathway during development has been clearly demonstrated by knocking out key genes. Also, the relevance of Apaf1 dosage during development has been recently underlined. Moreover, a growing body of evidences seems to point out a possible role of the mitochondria-dependent apoptosis in different pathologies. In particular, we discuss here some recent evidences regarding the putative role of the apoptosome in neurodegeneration and cancer.

Drosophila crooked-neck protein co-fractionates in a multiprotein complex with splicing factors

The Drosophila crooked neck ( crn) gene encodes an unusual TPR-containing protein whose function is essential for embryonic development. Homology with other TPR-proteins involved in cell cycle control, initially led to the proposal that Crn might play a critical role in regulation of embryonic cell divisions. Here, we show that Crn does not have a cell cycle function in the embryo. By using specific antibodies we also show that the Crn protein is a nuclear protein which localizes in “speckles” which could correspond to preferential localization of several other splicing factors. Fractionation of nuclear extracts on sucrose gradients revealed Crn in a 900 kDa multiproteic complex together with snRNPs, suggesting that Crn participates in the assembly of the splicing machinery in vivo.

Molecular Basis for Designing Selective Modulators of Retinoic Acid Receptor Transcriptional Activities

Retinoic acid receptors are ligand-regulated transcription factors belonging to the nuclear receptor superfamily, which comprises 49 members in the human genome. all-trans retinoic acid and 9-cis retinoic acid receptors (RARs and RXRs) are each encoded by three distinct genes and several isoforms arise from alternative splicing and the use of different promoters. While RXRs are promiscuous dimerization partners of several other nuclear receptors, RARs are active, in-vivo, when associated to RXRs. Retinoids are therefore regulators of multiple physiological processes, from embryogenesis to metabolism. Different combinations of RXR:RAR heterodimers occur as a function of their tissue-specific expression and their activity is mostly conditioned by the activation status of RAR. These heterodimers are defined as non permissive heterodimers, in opposition to permissive dimers whose transcriptional activity may be modulated through RXR and its dimerization partner. The transcriptional activity of these dimers also relies on their ability to recruit nuclear coactivators and corepressors, which function as multi proteic complexes harboring several enzymatic activities (acetylases, kinases). The structure of the ligand bound to the RAR moiety of the dimer, as well as the nature of the DNA sequence to which dimers are bound, dictate the relative affinity of dimers for coactivators and thus its overall transcriptional activity. RARs are also able to repress the activity of unrelated transcription factors such as AP1 and NF-kappa-B, and therefore have potent anti proliferative and anti inflammatory properties. This review summarizes our current view of molecular mechanisms governing these various activities and emphasizes the need for a detailed understanding of how retinoids may dictate transactivating and transrepressive properties of RARs and RXRs, which may be considered as highly valuable therapeutic targets in many diseases such as cancer, skin hyperproliferation and metabolical disorders (diabetes, atherosclerosis etc).

Expression and Human Chromosomal Localization to 17q25 of the Growth-Regulated Gene Encoding the Mitochondrial Ribosomal Protein MRPL12

Mitochondrial activity requires the expression of nuclear genes, whose products are part of multiproteic complexes leading to ATP production and delivery. We recently characterized a growth-activated mRNA encoding the human mitochondrial ribosomal MRPL12 protein, which is thought to act as a translational regulator of mitochondrial mRNAs. We show here that MRPL12 mRNA expression is enhanced in growth-stimulated cells as a result of transcriptional activation, a feature lost in transformed cell lines. MRPL12 mRNA is highly expressed in the colon, in which a reduction in mitochondrial activity was shown to be associated with tumor formation. The human MRPL12 protein is encoded by a unique gene located on chromosome 17 (q25–qter). As no predisposition to colon cancer linked to this chromosomal region was hitherto reported, the MRPL12 gene might be involved in the process of differentiation of colonic epithelial cells.