Cdc42-dependent formation of the ZO-1/MRCKβ complex at the leading edge controls cell migration

Zonula occludens (ZO)-1 is a multi-domain scaffold protein known to have critical roles in the establishment of cell-cell adhesions and the maintenance of stable tissue structures through the targeting, anchoring, and clustering of transmembrane adhesion molecules and cytoskeletal proteins. Here, we report that ZO-1 directly binds to MRCKβ, a Cdc42 effector kinase that modulates cell protrusion and migration, at the leading edge of migrating cells. Structural studies reveal that the binding of a β hairpin from GRINL1A converts ZO-1 ZU5 into a complete ZU5-fold. A similar interaction mode is likely to occur between ZO-1 ZU5 and MRCKβ. The interaction between ZO-1 and MRCKβ requires the kinase to be primed by Cdc42 due to the closed conformation of the kinase. Formation of the ZO-1/MRCKβ complex enriches the kinase at the lamellae of migrating cells. Disruption of the ZO-1/MRCKβ complex inhibits MRCKβ-mediated cell migration. These results demonstrate that ZO-1, a classical scaffold protein with accepted roles in maintaining cell-cell adhesions in stable tissues, also has an active role in cell migration during processes such as tissue development and remodelling.     

The Osaka FAD mutation E22Δ leads to the formation of a previously unknown type of amyloid β fibrils and modulates Aβ neurotoxicity

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by cerebral deposition of amyloid fibrils formed by the amyloid β (Aβ) peptide. Aβ has a length of 39-43 amino acid residues; the predominant Aβ isoforms are Aβ1-40 and Aβ1-42. While the majority of AD cases occur spontaneously, a subset of early-onset familial AD cases is caused by mutations in the genes encoding the Aβ precursor protein or presenilin 1/presenilin 2. Recently, a deletion of glutamic acid at position 22 within the Aβ sequence (E22Δ) was identified in Japanese patients with familial dementia, but the aggregation properties of the deletion variant of Aβ are not well understood. We investigated the aggregation characteristics and neurotoxicity of recombinantly expressed Aβ isoforms 1-40 and 1-42 with and without the E22Δ mutation. We show that the E22Δ mutation strongly accelerates the fibril formation of Aβ1-42 E22Δ compared to Aβ1-42 wild type (wt). In addition, we demonstrate that fibrils of Aβ1-40 E22Δ form a unique quaternary structure characterized by a strong tendency to form fibrillar bundles and a strongly increased thioflavin T binding capacity. Aβ1-40 E22Δ was neurotoxic in rat primary neuron cultures as compared to nontoxic Aβ1-40 wt. Aβ1-42 E22Δ was less toxic than Aβ1-42 wt, but it significantly decreased neurite outgrowth per cell in neuronal primary cultures. Because Aβ1-40 is the major Aβ form in vivo, the gain of toxic function caused by the E22 deletion may explain the development of familial AD in mutation carriers.     

Constitutive stabilization of ß-catenin in the dental mesenchyme leads to excessive dentin and cementum formation

Wnt/ß-catenin signaling plays an important role in morphogenesis and cellular differentiation during development. Essential roles of Wnt/ß-catenin signaling in tooth morphogenesis have been well known, but the involvement of Wnt/ß-catenin signaling in the dental hard tissue formation remains undefined. To understand roles of Wnt/ß-catenin signaling in dentin and cementum formation, we generated and analyzed the conditional ß-catenin stabilized mice in the dental mesenchyme. The OC-Cre;Catnb^lox(ex3)/+ mice exhibited malformed teeth characterized by aberrantly formed dentin and excessively deposited cementum. Large amount of dentin was rapidly formed with widened predentin and numerous globular calcifications in the crown. Whereas roots of molars were short and covered with the excessively formed cellular cementum. With age, the coronal pulp chamber and periodontal space were narrowed by the excessively formed dentin and cementum, respectively. To compare the changes of gene expression in the mutant mice, Col1a1 expression was increased but that of Dspp was decreased in the odontoblasts. However, both of Col1a1 and Bsp expression was increased in the cementoblasts. The gene expression changes were consistent with the localization of matrix proteins. Biglycan and PC-1 was increased but Phex was decreased in the odontoblasts and dentin matrix, respectively. TNAP was increased but Dmp1 and FGF23 was decreased in the cementoblasts and cementum matrix, respectively. Our results indicate that persistent stabilization of ß-catenin in the dental mesenchyme leads to premature differentiation of odontoblasts and differentiation of cementoblasts, and induces excessive dentin and cementum formation in vivo. These results suggest that temporospatial regulation of Wnt/ß-catenin signaling plays critical roles in the differentiation of odontoblasts and cementoblasts, and that inhibition of Wnt/ß-catenin signaling may be important for the formation of dentin and cementum during tooth development. Local modulation of Wnt/ß-catenin signaling has therapeutic potential to improve the regeneration of dentin and periodontium.     

Binding studies of truncated variants of the Aβ peptide to the V-domain of the RAGE receptor reveal Aβ residues responsible for binding

Alzheimer's disease (AD) symptoms correlate with the concentration of soluble, although not necessarily monomeric forms of Aβ peptide in the brain parenchyma. The RAGE receptor has been implicated as the protein responsible for active transport of Aβ from blood circulation to the brain. In murine models of AD, inhibition of the Aβ:RAGE interaction decreases the levels of Aβ in the brain. Inhibition of the Aβ:RAGE interaction would be a promising alternative for the therapy of AD. Rational design of an Aβ:RAGE interaction blocker requires detailed knowledge of the structure of the complex. However, the binding domain of RAGE is natively unfolded in physiological conditions, which severely hampers the application of classic methods of protein structure analysis to the design of an antagonist. Here, alternative methods are used to characterize the structural properties of the RAGE-ligand binding domain and to monitor the binding of a series of truncated variants of Aβ. Using intrinsic RAGE tryptophan fluorescence and mass spectrometry of non-covalent protein-ligand complexes we have identified shorter versions of Aβ that bind to the RAGE V-domain. We have also shown in cell culture experiments that a selected shortened version of Aβ effectively inhibits full-length Aβ, RAGE-mediated, cell uptake. Thus, a truncated version of Aβ capable of blocking its receptor-mediated internalization was established, revealing the binding code and providing the lead compound in the process of drug design.     

Relevance of N-terminal residues for amyloid-β binding to platelet integrin αIIbβ3, integrin outside-in signaling and amyloid-β fibril formation

A pathological hallmark of Alzheimer's disease (AD) is the aggregation of amyloid-β peptides (Aβ) into fibrils, leading to deposits in cerebral parenchyma and vessels known as cerebral amyloid angiopathy (CAA). Platelets are major players of hemostasis but are also implicated in AD. Recently we provided strong evidence for a direct contribution of platelets to AD pathology. We found that monomeric Aβ40 binds through its RHDS sequence to integrin α_IIbβ₃, and promotes the formation of fibrillar Aβ aggregates by the secretion of adenosine diphosphate (ADP) and the chaperone protein clusterin (CLU) from platelets. Here we investigated the molecular mechanisms of Aβ binding to integrin α_IIbβ₃ by using Aβ11 and Aβ16 peptides. These peptides include the RHDS binding motif important for integrin binding but lack the central hydrophobic core and the C-terminal sequence of Aβ. We observed platelet adhesion to truncated N-terminal Aβ11 and Aβ16 peptides that was not mediated by integrin α_IIbβ₃. Thus, no integrin outside-in signaling and reduced CLU release was detected. Accordingly, platelet mediated Aβ fibril formation was not observed. Taken together, the RHDS motif of Aβ is not sufficient for Aβ binding to platelet integrin α_IIbβ₃ and platelet mediated Aβ fibril formation but requires other recognition or binding motifs important for platelet mediated processes in CAA. Thus, increased understanding of the molecular mechanisms of Aβ binding to platelet integrin α_IIbβ₃ is important to understand the role of platelets in amyloid pathology.     

Cortical deficiency of laminin γ1 impairs the AKT/GSK-3β signaling pathway and leads to defects in neurite outgrowth and neuronal migration

Laminins have dramatic and varied actions on neurons in vitro. However, their in vivo function in brain development is not clear. Here we show that knockout of laminin γ1 in the cerebral cortex leads to defects in neuritogenesis and neuronal migration. In the mutant mice, cortical layer structures were disrupted, and axonal pathfinding was impaired. During development, loss of laminin expression impaired phosphorylation of FAK and paxillin, indicating defects in integrin signaling pathways. Moreover, both phosphorylation and protein levels of GSK-3β were significantly decreased, but only phosphorylation of AKT was affected in the mutant cortex. Knockout of laminin γ1 expression in vitro, dramatically inhibited neurite growth. These results indicate that laminin regulates neurite growth and neuronal migration via integrin signaling through the AKT/GSK-3β pathway, and thus reveal a novel mechanism of laminin function in brain development.     

IFNβ impairs extracellular matrix formation leading to inhibition of mineralization by effects in the early stage of human osteoblast differentiation

Osteoimmunology is an emerging field of research focused on the interaction of the immune system and bone. In this study we demonstrate that human osteoblasts are sensitive to the immune cytokine interferon (IFN)β. Osteoblasts respond to IFNβ as shown by the induction of several known IFN target genes such as interferon-induced (IFI) proteins (IFIT1, IFI44L), interferon-stimulated gene factor 3 (ISGF3) complex and the induction of IFNβ itself. We demonstrated that IFNβ has severe inhibitory effects on mineralization of osteoblast-derived extracellular matrix (ECM). Analysis of the timing of the IFNβ effects revealed that committed osteoblasts in early stage of differentiation are most sensitive to IFNβ inhibition of mineralization. A single IFNβ treatment was as effective as multiple treatments. During the progress of differentiation osteoblasts become desensitized for IFNβ. This pinpoints to a complex pattern of IFNβ sensitivity in osteoblasts. Focusing on early osteoblasts, we showed that IFNβ decreased gene expression of ECM-related genes, such as type I Collagen (COL1A1), fibronectin (FN1), fibullin (FBLN1), fibrillin (FBN2), and laminin (LAMA1). Additionally, ECM produced by IFNβ-treated osteoblasts contained less collagen protein. IFNβ stimulated gene expression of osteopontin (OPN), annexin2 (ANXA2), and hyaluronan synthase 1 (HAS1), which are important factors in the adhesion of hematopoietic stem cells (HSC) in the HSC niche. In conclusion, IFNβ directly modifies human osteoblast function by inhibiting ECM synthesis eventually resulting in delayed bone formation and mineralization and induces a HSC niche supporting phenotype. These effects are highly dependent on timing of treatment in the early phase of osteoblast differentiation.     

A dimeric form of N-methoxycarbonyl-2-amino-1,8-naphthyridine bound to the A–A mismatch in the CAG/CAG base triad in dsRNA

A dimeric form of N-methoxycarbonyl-2-amino-1,8-naphthyridine (MCND) connected at the C2 position with a three-atom linker was examined for the binding to mismatches in double stranded RNA. Despite the fully complementary hydrogen bonding groups to guanine, MCND did not bind to guanine–guanine mismatch but did to adenine–adenine mismatch. The base pairs flanking the mismatch had weak effect on the binding, with showing the strongest binding to the A–A mismatch in the CAG/CAG sequence. The A–A mismatch in the GAC/GAC sequence was a poor substrate for the MCND binding. A monomeric derivative of MCND and another derivative lacking a methylcarbamate group showed negligilble binding to the A–A mismatch and the sequence selectivity. These results are important clues for the better molecular design of RNA binding small molecules.     

Coxsackievirus mutants that can bypass host factor PI4KIIIβ and the need for high levels of PI4P lipids for replication

RNA viruses can rapidly mutate and acquire resistance to drugs that directly target viral enzymes, which poses serious problems in a clinical context. Therefore, there is a growing interest in the development of antiviral drugs that target host factors critical for viral replication, since they are unlikely to mutate in response to therapy. We recently demonstrated that phosphatidylinositol-4-kinase IIIβ (PI4KIIIβ) and its product phosphatidylinositol-4-phosphate (PI4P) are essential for replication of enteroviruses, a group of medically important RNA viruses including poliovirus (PV), coxsackievirus, rhinovirus, and enterovirus 71. Here, we show that enviroxime and GW5074 decreased PI4P levels at the Golgi complex by directly inhibiting PI4KIIIβ. Coxsackievirus mutants resistant to these inhibitors harbor single point mutations in the non-structural protein 3A. These 3A mutations did not confer compound-resistance by restoring the activity of PI4KIIIβ in the presence of the compounds. Instead, replication of the mutant viruses no longer depended on PI4KIIIβ, since their replication was insensitive to siRNA-mediated depletion of PI4KIIIβ. The mutant viruses also did not rely on other isoforms of PI4K. Consistently, no high level of PI4P could be detected at the replication sites induced by the mutant viruses in the presence of the compounds. Collectively, these findings indicate that through specific single point mutations in 3A, CVB3 can bypass an essential host factor and lipid for its propagation, which is a new example of RNA viruses acquiring resistance against antiviral compounds, even when they directly target host factors.     

Cryo-EM structure of monomeric photosystem II at 2.78 Å resolution reveals factors important for the formation of dimer

Photosystem II (PSII) functions mainly as a dimer to catalyze the light energy conversion and water oxidation reactions. However, monomeric PSII also exists and functions in vivo in some cases. The crystal structure of monomeric PSII has been solved at 3.6 Å resolution, but it is still not clear which factors contribute to the formation of the dimer. Here, we solved the structure of PSII monomer at a resolution of 2.78 Å using cryo-electron microscopy (cryo-EM). From our cryo-EM density map, we observed apparent differences in pigments and lipids in the monomer-monomer interface between the PSII monomer and dimer. One β-carotene and two sulfoquinovosyl diacylglycerol (SQDG) molecules are found in the monomer-monomer interface of the dimer structure but not in the present monomer structure, although some SQDG and other lipid molecules are found in the analogous region of the low-resolution crystal structure of the monomer, or cryo-EM structure of an apo-PSII monomer lacking the extrinsic proteins from Synechocystis sp. PCC 6803. In the current monomer structure, a large part of the PsbO subunit was also found to be disordered. These results indicate the importance of the β-carotene, SQDG and PsbO in formation of the PSII dimer.     

Granzyme K inhibits replication of influenza virus through cleaving the nuclear transport complex importin α1/β dimer of infected host cells

The influenza A virus is a causative agent of influenza, which infects human cells and uses host factors to accomplish viral genome replication as part of its life cycle. The nucleoprotein (NP) and PB2 of the influenza virus associate with importin α1 to gain access to the host nucleus through a ternary import complex. Killer cell-mediated cytotoxicity is the primary mechanism of eliminating the influenza virus. Here, we showed that lymphokine-activated killer cells participated in the elimination of the influenza virus. Granzyme (Gzm) K inhibition elevated viral replication in vitro and aggravated viral infection in vivo. We identified that importin α1 and its transport partner protein importin β are physiological substrates of GzmK. Proteolysis of these two substrates wrecked their association to generate the importin α1/β dimer and disrupted transportation of viral NP to the nucleus, leading to inhibition of influenza virus replication.     

A Ribosomal S-6 Kinase–Mediated Signal to C/EBP-β Is Critical for the Development of Liver Fibrosis

Background

In response to liver injury, hepatic stellate cell (HSC) activation causes excessive liver fibrosis. Here we show that activation of RSK and phosphorylation of C/EBPβ on Thr217 in activated HSC is critical for the progression of liver fibrosis.

Methodology/Principal Findings

Chronic treatment with the hepatotoxin CCl₄ induced severe liver fibrosis in C/EBPβ^+/+ mice but not in mice expressing C/EBPβ-Ala217, a non-phosphorylatable RSK-inhibitory transgene. C/EBPβ-Ala217 was present within the death receptor complex II, with active caspase 8, and induced apoptosis of activated HSC. The C/EBPβ-Ala217 peptides directly stimulated caspase 8 activation in a cell-free system. C/EBPβ^+/+ mice with CCl₄-induced severe liver fibrosis, while continuing on CCl₄, were treated with a cell permeant RSK-inhibitory peptide for 4 or 8 weeks. The peptide inhibited RSK activation, stimulating apoptosis of HSC, preventing progression and inducing regression of liver fibrosis. We found a similar activation of RSK and phosphorylation of human C/EBPβ on Thr266 (human phosphoacceptor) in activated HSC in patients with severe liver fibrosis but not in normal livers, suggesting that this pathway may also be relevant in human liver fibrosis.

Conclusions/Significance

These data indicate that the RSK-C/EBPβ phosphorylation pathway is critical for the development of liver fibrosis and suggest a potential therapeutic target.     

A mutation in the start codon of γ-crystallin D leads to nuclear cataracts in the Dahl SS/Jr-Ctr strain

Cataracts are a major cause of blindness. The most common forms of cataracts are age- and UV-related and develop mostly in the elderly, while congenital cataracts appear at birth or in early childhood. The Dahl salt-sensitive (SS/Jr) rat is an extensively used model of salt-sensitive hypertension that exhibits concomitant renal disease. In the mid-1980s, cataracts appeared in a few animals in the Dahl S colony, presumably the result of a spontaneous mutation. The mutation was fixed and bred to establish the SS/Jr-Ctr substrain. The SS/Jr-Ctr substrain has been used exclusively by a single investigator to study the role of steroids and hypertension. Using a classical positional cloning approach, we localized the cataract gene with high resolution to a less than 1-Mbp region on chromosome 9 using an F₁(SS/Jr-Ctr × SHR) × SHR backcross population. The 1-Mbp region contained only 13 genes, including 4 genes from the γ-crystallins (Cryg) gene family, which are known to play a role in cataract formation. All of the γ-crystallins were sequenced and a novel point mutation in the start codon (ATG → GTG) of the Crygd gene was identified. This led to the complete absence of the CRYGD protein in the eyes of the SS/Jr-Ctr strain. In summary, the identification of the genetic cause in this novel cataract model may provide an opportunity to better understand the development of cataracts, particularly in the context of hypertension.