Amphipathic alpha-helix AH2 is a major determinant for the oligomerization of hepatitis C virus nonstructural protein 4B.

Nonstructural protein 4B (NS4B) is a key organizer of hepatitis C virus (HCV) replication complex formation. It induces a specific membrane rearrangement, designated membranous web, that serves as a scaffold for the HCV replication complex. However, the mechanisms underlying membranous web formation are poorly understood. Based on fluorescence resonance energy transfer (FRET) and confirmatory coimmunoprecipitation analyses, we provide evidence for an oligomerization of NS4B in the membrane environment of intact cells. Several conserved determinants were found to be involved in NS4B oligomerization, through homotypic and heterotypic interactions. N-terminal amphipathic ?-helix AH2, comprising amino acids 42 to 66, was identified as a major determinant for NS4B oligomerization. Mutations that affected the oligomerization of NS4B disrupted membranous web formation and HCV RNA replication, implying that oligomerization of NS4B is required for the creation of a functional replication complex. These findings enhance our understanding of the functional architecture of the HCV replication complex and may provide new angles for therapeutic intervention. At the same time, they expand the list of positive-strand RNA virus replicase components acting as oligomers.

Trichophyton rubrum secreted and membrane-associated carboxypeptidases

Dermatophytes are the most common agents of superficial mycoses, and exclusively infect stratum corneum, nails or hair. Therefore, secreted proteolytic activity is considered a virulence trait of these fungi. In a medium containing protein as a sole nitrogen and carbon source Trichophyton rubrum secretes a metallocarboxypeptidase (TruMcpA) of the M14 family according to the MEROPS proteolytic enzyme database. TruMcpA is homologous to human pancreatic carboxypeptidase A, and is synthesized as a precursor in a preproprotein form. The propeptide is removed to generate the mature active enzyme alternatively by either one of two subtilisins which are concomitantly secreted by the fungus. In addition, T. rubrum was shown to possess two genes (TruSCPA and TruSCPB) encoding serine carboxypeptidases of the S10 family which are homologues of the previously characterized Aspergillus and Penicillium secreted acid carboxypeptidases. However, in contrast to the Aspergillus and Penicillium homologues, TruScpA and TruScpB enzymes are not secreted into the environment, but are membrane-associated with a glycosylphosphatidylinositol (GPI) anchor. During infection, T. rubrum secreted and GPI-anchored carboxypeptidases may contribute to fungal virulence by cooperating with previously characterized endoproteases and aminopeptidases in the degradation of compact keratinized tissues into assimilable amino acids and short peptides.

Heat shock response in photosynthetic organisms: membrane and lipid connections.

The ability of photosynthetic organisms to adapt to increases in environmental temperatures is becoming more important with climate change. Heat stress is known to induce heat-shock proteins (HSPs) many of which act as chaperones. Traditionally, it has been thought that protein denaturation acts as a trigger for HSP induction. However, increasing evidence has shown that many stress events cause HSP induction without commensurate protein denaturation. This has led to the membrane sensor hypothesis where the membrane's physical and structural properties play an initiating role in the heat shock response. In this review, we discuss heat-induced modulation of the membrane's physical state and changes to these properties which can be brought about by interaction with HSPs. Heat stress also leads to changes in lipid-based signaling cascades and alterations in calcium transport and availability. Such observations emphasize the importance of membranes and their lipids in the heat shock response and provide a new perspective for guiding further studies into the mechanisms that mediate cellular and organismal responses to heat stress.

Small proline-rich protein 1A is a gp130 pathway- and stress-inducible cardioprotective protein.

The interleukin-6 cytokines, acting via gp130 receptor pathways, play a pivotal role in the reduction of cardiac injury induced by mechanical stress or ischemia and in promoting subsequent adaptive remodeling of the heart. We have now identified the small proline-rich repeat proteins (SPRR) 1A and 2A as downstream targets of gp130 signaling that are strongly induced in cardiomyocytes responding to biomechanical/ischemic stress. Upregulation of SPRR1A and 2A was markedly reduced in the gp130 cardiomyocyte-restricted knockout mice. In cardiomyocytes, MEK1/2 inhibitors prevented SPRR1A upregulation by gp130 cytokines. Furthermore, binding of NF-IL6 (C/EBPbeta) and c-Jun to the SPRR1A promoter was observed after CT-1 stimulation. Histological analysis revealed that SPRR1A induction after mechanical stress of pressure overload was restricted to myocytes surrounding piecemeal necrotic lesions. A similar expression pattern was found in postinfarcted rat hearts. Both in vitro and in vivo ectopic overexpression of SPRR1A protected cardiomyocytes against ischemic injury. Thus, this study identifies SPRR1A as a novel stress-inducible downstream mediator of gp130 cytokines in cardiomyocytes and documents its cardioprotective effect against ischemic stress.

Paired activation of two components within muscarinic M3 receptor dimers is required for recruitment of beta-arrestin-1 to the plasma membrane.

beta-Arrestins regulate the functioning of G protein-coupled receptors in a variety of cellular processes including receptor-mediated endocytosis and activation of signaling molecules such as ERK. A key event in these processes is the G protein-coupled receptor-mediated recruitment of beta-arrestins to the plasma membrane. However, despite extensive knowledge in this field, it is still disputable whether activation of signaling pathways via beta-arrestin recruitment entails paired activation of receptor dimers. To address this question, we investigated the ability of different muscarinic receptor dimers to recruit beta-arrestin-1 using both co-immunoprecipitation and fluorescence microscopy in COS-7 cells. Experimentally, we first made use of a mutated muscarinic M(3) receptor, which is deleted in most of the third intracellular loop (M(3)-short). Although still capable of activating phospholipase C, this receptor loses almost completely the ability to recruit beta-arrestin-1 following carbachol stimulation in COS-7 cells. Subsequently, M(3)-short was co-expressed with the M(3) receptor. Under these conditions, the M(3)/M(3)-short heterodimer could not recruit beta-arrestin-1 to the plasma membrane, even though the control M(3)/M(3) homodimer could. We next tested the ability of chimeric adrenergic muscarinic alpha(2)/M(3) and M(3)/alpha(2) heterodimeric receptors to co-immunoprecipitate with beta-arrestin-1 following stimulation with adrenergic and muscarinic agonists. beta-Arrestin-1 co-immunoprecipitation could be induced only when carbachol or clonidine were given together and not when the two agonists were supplied separately. Finally, we tested the reciprocal influence that each receptor may exert on the M(2)/M(3) heterodimer to recruit beta-arrestin-1. Remarkably, we observed that M(2)/M(3) heterodimers recruit significantly greater amounts of beta-arrestin-1 than their respective M(3)/M(3) or M(2)/M(2) homodimers. Altogether, these findings provide strong evidence in favor of the view that binding of beta-arrestin-1 to muscarinic M(3) receptors requires paired stimulation of two receptor components within the same receptor dimer.

A novel TMPRSS3 missense mutation in a DFNB8/10 family prevents proteolytic activation of the protein.

Pathogenic mutations in TMPRSS3, which encodes a transmembrane serine protease, cause non-syndromic deafness DFNB8/10. Missense mutations map in the low density-lipoprotein receptor A (LDLRA), scavenger-receptor cysteine-rich (SRCR), and protease domains of the protein, indicating that all domains are important for its function. TMPRSS3 undergoes proteolytic cleavage and activates the ENaC sodium channel in a Xenopus oocyte model system. To assess the importance of this gene in non-syndromic childhood or congenital deafness in Turkey, we screened for mutations affected members of 25 unrelated Turkish families. The three families with the highest LOD score for linkage to chromosome 21q22.3 were shown to harbor P404L, R216L, or Q398X mutations, suggesting that mutations in TMPRSS3 are a considerable contributor to non-syndromic deafness in the Turkish population. The mutant TMPRSS3 harboring the novel R216L missense mutation within the predicted cleavage site of the protein fails to undergo proteolytic cleavage and is unable to activate ENaC, thus providing evidence that pre-cleavage of TMPRSS3 is mandatory for normal function.

Functions of the human papillomavirus type 16 E4 protein

1. Abstract Cervical cancer is thought to be the consequence of infection by human papillomaviruses (HPV). In the majority of cases, DNA from HPV type 16 (HPV16) is found in malignant cervical lesions. The initial steps leading to transformation of an infected cell are not clearly understood but in most cases, disruption and integration of the episomal viral DNA must take place. As a consequence, the E2 and E4 genes are usually not expressed whereas the E6 and E7 oncogenes are highly expressed. However, in a normal infection in which the viral DNA is maintained as an episome, all viral genes are expressed. The pattern according to which the viral proteins are made, and therefore the life cycle of the virus, is tightly linked to the differentiation process of the host keratinocyte. The study of the viral oncogenes E6 and E7 has revealed crucial functions in the process of malignant transformation such as degradation of the p53 tumor suppressor protein, deregulation of the Retinoblastoma protein pathway and activation of the telomerase ribonucleoprotein. All these steps are necessary for cancerous lesions to develop. However, the loss of the E2 gene product seems to be necessary for sufficient expression of E6 and E7 in order to achieve such effects. In normal infections, the E4 protein is made abundantly in the later stages of the viral life cycle. Though extensive amounts of work have been carried out to define the function of E4, it still remains unclear. In this study, several approaches have been used to try and determine the functions of E4. First, a cell-penetrating fusion protein was designed and produced in order to circumvent the chronic difficulties of expressing E4 in mammalian cells. Unfortunately, this approach was not successful due to precipitation of the purified fusion protein. Second, the observation that E4 accumulates in cells having modified their adhesion properties led to the hypothesis that E4 might be involved in the differentiation process of keratinocytes. Preliminary results suggest that E4 triggers differentiation. Last, as E4 has been reported to collapse the cytokeratin network of keratinocytes, a direct approach using atomic force microscopy has allowed us to test the potential modification of mechanical properties of cells harboring reorganized cytokeratin networks. If so, a potential role for E4 in viral particle release could be hypothesized. 2. Résumé Il a été établi que le cancer du col de l'utérus se développe essentiellement à la suite d'une infection par le virus du papillome humain (HPV). Dans la majorité des cas analysés, de l'ADN du HPV de type 16 (HPV16) est détecté. Les étapes initiales de la transformation d'une cellule infectée sont mal connues mais il semble qu'une rupture du génome viral, normalement épisomal, suivi d'une intégration dans le génome de la cellule hôte soient des étapes nécessaires dans la plupart des cas. Or il semble qu'il y ait une sélection pour les cas où l'expression des oncogènes viraux E6 et E7 soit favorisée alors que l'expression des gènes E2 et E4 est en général impossible. Par contre, dans une infection dite normale où le génome viral n'est pas rompu, il n'y pas développement de cancer et tous les gènes viraux sont exprimés. L'ordre dans lequel les protéines virales sont produites, et donc le cycle de réplication du virus, est intimement lié au processus de différentiation de la cellule hôte. L'étude des protéines oncogènes E6 et E7 a révélé des fonctions clés dans le processus de transformation des cellules infectées telles que la dégradation du suppresseur de tumeur p53, la dérégulation de la voie de signalisation Rb ainsi que l'activation de la télomérase. Toutes ces activités sont nécessaires au développement de lésions cancéreuses. Toutefois, il semble que l'expression du gène E2 doit être empêchée afin que suffisamment des protéines E6 et E7 soient produites. Lorsque le gène E2 est exprimé, et donc lorsque le génome viral n'est pas rompu, les protéines E6 et E7 n'entraînent pas de telles conséquences. Le gène E4, qui se trouve dans la séquence codante de E2, a aussi besoin d'un génome viral intact pour être exprimé. Dans une infection normale, le gène E4 est exprimé abondamment dans les dernières étapes de la réplication du virus. Bien que de nombreuses études aient été menées afin de déterminer la fonction virale à E4, aucun résultat n'apparaît évident. Dans ce travail, plusieurs approches ont été utilisées afin d'adresser cette question. Premièrement, une protéine de fusion TAT-E4 a été produite et purifiée. Cette protéine, pouvant entrer dans les cellules vivantes par diffusion au travers de la membrane plasmique, aurait permis d'éviter ainsi les problèmes chroniques rencontrés lors de l'expression de E4 dans les cellules mammifères. Malheureusement, cette stratégie n'a pas pu être utilisée à cause de la précipitation de la protéine purifiée. Ensuite, l'observation que E4 s'accumule dans les cellules ayant modifié leurs propriétés d'adhésion a suggéré que E4 pourrait être impliqué dans le procédé de différentiation des kératinocytes. Des résultats préliminaires supportent cette possibilité. Enfin, il a été montré que E4 pouvait induire une réorganisation du réseau des cytokératines. Une approche directe utilisant le microscope à force atomique nous a ainsi permis de tester une potentielle modification des propriétés mécaniques de cellules ayant modifié leur réseau de cytokératines en présence de E4. Si tel est le cas, un rôle dans la libération de particules virales peut être proposé pour E4.

Microtubule-associated protein 6 mediates neuronal connectivity through Semaphorin 3E-dependent signalling for axonal growth.

Structural microtubule associated proteins (MAPs) stabilize microtubules, a property that was thought to be essential for development, maintenance and function of neuronal circuits. However, deletion of the structural MAPs in mice does not lead to major neurodevelopment defects. Here we demonstrate a role for MAP6 in brain wiring that is independent of microtubule binding. We find that MAP6 deletion disrupts brain connectivity and is associated with a lack of post-commissural fornix fibres. MAP6 contributes to fornix development by regulating axonal elongation induced by Semaphorin 3E. We show that MAP6 acts downstream of receptor activation through a mechanism that requires a proline-rich domain distinct from its microtubule-stabilizing domains. We also show that MAP6 directly binds to SH3 domain proteins known to be involved in neurite extension and semaphorin function. We conclude that MAP6 is critical to interface guidance molecules with intracellular signalling effectors during the development of cerebral axon tracts.

The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin.

Uromodulin is the most abundant protein in the urine. It is exclusively produced by renal epithelial cells and it plays key roles in kidney function and disease. Uromodulin mainly exerts its function as an extracellular matrix whose assembly depends on a conserved, specific proteolytic cleavage leading to conformational activation of a Zona Pellucida (ZP) polymerisation domain. Through a comprehensive approach, including extensive characterisation of uromodulin processing in cellular models and in specific knock-out mice, we demonstrate that the membrane-bound serine protease hepsin is the enzyme responsible for the physiological cleavage of uromodulin. Our findings define a key aspect of uromodulin biology and identify the first in vivo substrate of hepsin. The identification of hepsin as the first protease involved in the release of a ZP domain protein is likely relevant for other members of this protein family, including several extracellular proteins, as egg coat proteins and inner ear tectorins.