New linear polyamine derivatives in spider venoms.

Linear free polyamines were characterized in the venom of the spiders Agelenopsis aperta, Hololena curta, and Paracoelotes birulai by RP-HPLC coupled to mass spectrometry. The several linear polyamines found were tetramine, pentamine, and hexamine derivatives. Some of these natural products were identified as N-hydroxylated, guanidylated, or acetylated compounds. In addition, the biosynthetical pathway leading to the formation of acylpolyamines in spider venoms is discussed.     

Regulation of Dishevelled DEP domain swapping by conserved phosphorylation sites

Wnt signals bind to Frizzled receptors to trigger canonical and noncanonical signaling responses that control cell fates during animal development and tissue homeostasis. All Wnt signals are relayed by the hub protein Dishevelled. During canonical (β-catenin–dependent) signaling, Dishevelled assembles signalosomes via dynamic head-to-tail polymerization of its Dishevelled and Axin (DIX) domain, which are cross-linked by its Dishevelled, Egl-10, and Pleckstrin (DEP) domain through a conformational switch from monomer to domain-swapped dimer. The domain-swapped conformation of DEP masks the site through which Dishevelled binds to Frizzled, implying that DEP domain swapping results in the detachment of Dishevelled from Frizzled. This would be incompatible with noncanonical Wnt signaling, which relies on long-term association between Dishevelled and Frizzled. It is therefore likely that DEP domain swapping is differentially regulated during canonical and noncanonical Wnt signaling. Here, we use NMR spectroscopy and cell-based assays to uncover intermolecular contacts in the DEP dimer that are essential for its stability and for Dishevelled function in relaying canonical Wnt signals. These contacts are mediated by an intrinsically structured sequence spanning a conserved phosphorylation site upstream of the DEP domain that serves to clamp down the swapped N-terminal α-helix onto the structural core of a reciprocal DEP molecule in the domain-swapped configuration. Mutations of this phosphorylation site and its cognate surface on the reciprocal DEP core attenuate DEP-dependent dimerization of Dishevelled and its canonical signaling activity in cells without impeding its binding to Frizzled. We propose that phosphorylation of this crucial residue could be employed to switch off canonical Wnt signaling.

Wnt signaling cascades are ancient cell communication pathways that regulate cell fates during embryonic development and tissue homeostasis (1, 2). Extracellular Wnt ligands bind to seven-pass transmembrane Frizzled receptors and transduce signals to downstream effectors through Dishevelled, an intracellular hub protein that binds to Frizzled and assembles dynamic signaling complexes termed “signalosomes” (1, 3). Dishevelled pivots between alternative Wnt signaling branches to specify distinct outcomes (4). These branches are broadly defined as canonical (or β-catenin–dependent), typically driving cellular proliferation or differentiation (5, 6), and noncanonical, comprising a collection of signaling branches that coordinate cellular properties such as planar cell polarity (PCP) (7) and morphogenetic processes such as convergent extension (8, 9).Dishevelled has three well-conserved domains: an N-terminal Dishevelled and Axin (DIX) domain; central Postsynaptic density protein-95, Disk large tumor suppressor, Zonula occludens-1 (PDZ) domain; and C-terminal Dishevelled, Egl-10, and Pleckstrin (DEP) domain. Dishevelled is recruited to Frizzled by the DEP domain and assembles Wnt signalosomes via self-association of both its DIX and DEP domains (Fig. 1 A and B). The DIX domain undergoes reversible head-to-tail polymerization (10) to generate dynamic filaments of Dishevelled that are stably cross-linked by dimerization through the DEP domain (11). This rapidly increases the local concentration of Dishevelled and boosts its avidity for low-affinity effectors such as Axin, enabling Dishevelled to interact with these effectors even if present at a low cellular concentration (1, 3, 12).Open in a separate windowFig. 1.Cartoon of DVL domain architecture and function in canonical Wnt signaling. (A) Dishevelled with its three well-conserved domains, DIX, PDZ, and DEP. The DEP domain binds Frizzled directly through its prominent “DEP finger,” formed from the hinge loop separating H1 and H2 in the monomeric configuration. (B) Wnt signals cause DEP to dimerize by domain swapping. During this process, H1 from one DEP molecule is exchanged with H1 from another through extension of the hinge loop. As a consequence, the “DEP finger” undergoes a conformational change, resulting in a structurally distinct β-sheet connecting the two DEP molecules, which cannot bind to Frizzed. Domain swapping therefore promotes detaching of Dishevelled from the receptor complex.The DEP domain is a small globular domain composed of three α-helices and a flexible hinge loop between the first (H1) and second helix (H2), which, in the monomeric configuration, folds back on itself to form a prominent “DEP finger” that is responsible for binding to Frizzled (Fig. 1A) (13). DEP dimerization involves a highly unusual mechanism called “domain swapping” (14). During this process, H1 of one DEP monomer is exchanged with H1 from a reciprocal one through outward motions of the hinge loops, replacing intra- with intermolecular contacts. This results in a dimer whose DEP cores, almost identical in structure to the monomer, are connected by a β-sheet formed between the two hinge loops (Fig. 2A) (11). In other words, these hinge loops, which form the “DEP finger” in the monomer, undergo a major conformational change engaging in new intermolecular interactions that are likely to stabilize the dimeric configuration. Functional assays in Dishevelled null-mutant cells based on structure-designed mutants have revealed that this mechanism is essential for Wnt/β-catenin signaling (15). There are several examples of domain swapping underlying pathological processes (e.g., in neurodegenerative disease); however, the DEP domain of Dishevelled represents a rare example of a domain undergoing physiologically relevant domain swapping (16).Open in a separate windowFig. 2.Mutations attenuating DEP dimerization and DVL2-dependent signaling. (A) Structure of the DEP dimer showing domain swapping (molecule A, dark turquoise; molecule B, light turquoise) superimposed on the DEP monomer (gray, “DEP finger”). S418 and S435 are shown (balls). (B) SuperTOP assays of HEK293T cells, expressing wt or mutant FLAG-DVL2 (as indicated; above, corresponding Western blot); ev, empty vector control; error bars, SEM of >3 independent experiments. (C) Western blots of immunoprecipitants (IPs) of polymerization-deficient DVL2 (M2M4-GFP) after coexpression with wt or DEP-mutant FLAG-DVL2 in transiently transfected HEK293T cells, probed with antibodies as indicated on the right; M2M4 was used instead of wt DVL2 to guard against confounding effects of DIX-dependent polymerization on DEP-dependent coIP (11). (D) Quantitative analysis of SNAP-FZD5-dependent recruitment of wt or mutant DEP-GFP to the plasma membrane (n = 100 cells scored in each case). (E) SuperTOP assays of HEK293T cells, monitoring the blocking of endogenous signaling in response to Wnt3a stimulation by overexpressed wt or mutant DEP-GFP (as indicated; above, corresponding Western blot); ev, GFP control; WCM, Wnt3a-conditioned medium (applied 6 h before lysis); error bars, SEM of >3 independent experiments. ****P < 0.0001; one-way ANOVA with repeated measures.A consequence of domain swapping is that the Frizzled binding “DEP finger” undergoes a conformational change that is structurally incompatible with Frizzled binding (Fig. 1B) (13). Therefore, domain swapping blocks binding between DEP and Frizzled, which could cause detachment of Dishevelled from Frizzled. In turn, this would terminate Wnt signal transduction, as the signal relay depends on continued association between Frizzled and Dishevelled. For example, PCP signaling in Drosophila requires apical recruitment of Dishevelled (Dsh) by Frizzled to be maintained for many hours, even days, in stable, membrane-localized signalosomes that are clearly visible by confocal microscopy (17, 18). In contrast, Wnt/β-catenin signaling appears to need only a transient association between Frizzled and Dsh. This is illustrated by dsh¹ flies that bear a mutation in the DEP finger (K417M), which reduces the membrane localization of Dishevelled and thus causes PCP defects without apparently affecting canonical Wnt signaling (7, 19, 20). The same mutation in Dvl2 (K446M) causes PCP and convergent extension defects when introduced into Dvl1^−/− Dvl2^−/− transgenic mice (21). Thus, either DEP domain swapping needs to be attenuated in PCP signalosomes or, if domain-swapped Dishevelled molecules were to remain within signalosomes, adaptor proteins would be required to mediate continued association between Dishevelled and Frizzled receptor complexes.One mechanism by which the DEP domain could be regulated is by Wnt-induced phosphorylation (19, 20, 22), which accompanies the “activation” of Dishevelled (23, 24). During PCP signaling in flies, phosphorylation of Dishevelled correlates with its membrane recruitment by Frizzled (19, 20). Furthermore, its phosphorylation by Discs Overgrown (Dco), the Drosophila casein kinase-1ε (CK1ε) ortholog, promotes asymmetric localization of Dishevelled along the apical plasma membrane (25) and its stable association with junctional complexes (26). Dishevelled is phosphorylated on numerous serine (Ser) and threonine (Thr) residues, but it is unclear which of these are biologically important, as the vast majority of phosphorylations detected by mass spectrometry (MS) are not required for function or are functionally redundant (22, 25, 27, 28). However, we previously reported that single point mutations of two conserved Ser residues in the DEP domain (S418 and S435) reduce Wnt/β-catenin signaling (11) (SI Appendix, Fig. S1), suggesting that the phosphorylation of either Ser could regulate DEP domain function. Importantly, S418 is clearly a substrate for phosphorylation since high-throughput analysis of phosphorylation sites in breast cancer samples detected S418 phosphorylation by MS (29) (PhosphoSite). In addition, a recent comparative analysis of human Dishevelled-3 (DVL3) phosphorylation revealed that several Ser/Thr kinases are capable of phosphorylating S407 (the equivalent of DVL2 S418) in cells (30). Whether S435 is a bona fide substrate for phosphorylation remains to be determined; however, phosphorylation of its equivalent in flies, S406, was detected by MS in cells undergoing PCP signaling (28). Based on this evidence, we decided to investigate whether these or any other phospho-sites in the DEP domain affect signaling by altering DEP domain swapping.Here, we show that these two highly conserved Ser residues (S418 and S435) are required for the stability of the DVL2 domain-swapped DEP dimer. We used NMR spectroscopy to demonstrate that S418, located immediately upstream of H1, engages in crucial noncovalent interactions with a structured loop that connects H2 and H3. As a consequence, a β-sheet forms that clamps down each H1 onto its reciprocal DEP core within a domain-swapped dimer, thereby providing stability to this DEP dimer without, however, affecting the binding between DEP monomer and Frizzled. An important corollary is that the phosphorylation of the key residue S418 within this clamp, for example, during noncanonical Wnt signaling, attenuates domain swapping, thereby allowing a stable association between DEP monomers and Frizzled. Our work has uncovered a pivotal residue within Dishevelled that negatively regulates DEP domain swapping, thereby antagonizing canonical Wnt signaling.     

Tissue integration and biodegradation of soft tissue substitutes with and without compression: an experimental study in the rat

Objectives

To analyze the influence of compression on tissue integration and degradation of soft tissue substitutes.

Material and methods

Six subcutaneous pouches in twenty-eight rats were prepared and boxes made of Al₂O₃ were implanted and used as carriers for soft tissue substitutes: a collagen matrix (MG), two volume-stable collagen matrices (FG/MGA), and a polycaprolactone scaffold(E). The volume-stable materials (FG/MGA/E) were further implanted with a twofold (2) and a fourfold (4) compression, created by the stacking of additional layers of the substitute materials. The samples were retrieved at 1, 2, and 12 weeks (10 groups, 3 time points, n?=?5 per time point and group, overall, 150 samples). The area fraction of infiltrated fibroblasts and inflammatory cells was evaluated histologically. Due to within-subject comparisons, mixed models were conducted for the primary outcome. The level of significance was set at 5%.

Results

The area fraction of fibroblasts increased in all groups over time. At 12 weeks, the densely compressed materials FG4 (1.1%), MGA4 (1.7%), and MGA2 (2.5%) obtained lower values as compared to the other groups, ranging between 4.7 (E2) and 6.5% (MG). Statistically significant differences (p?≤?0.05) were observed between groups FG4 vs MG/FG2/E/E4 as well as between MGA4 vs MG/FG2/E/E4 and E vs MGA2.

Conclusions

Higher levels of compression led to delayed tissue integration. The effect of different compression levels was more distinct when compared to the differences between the materials.

Clinical relevance

All biomaterials demonstrated tissue integration and a minimal concomitant inflammatory reaction. Clinically, it might be more favorable to obtain a sufficient flap release or to reduce the material size to improve the tissue integration processes.

     

Membrane association of hepatitis C virus nonstructural proteins and identification of the membrane alteration that harbors the viral replication complex

Hepatitis C virus (HCV) replicates its genome in a membrane-associated complex composed of viral proteins, replicating RNA, and altered cellular membranes. Determinants for membrane association of the HCV nonstructural proteins involved in genome replication have been defined. In addition, a specific membrane alteration, designated membranous web, was recently identified as the site of viral RNA synthesis and, therefore, represents the HCV replication complex. These findings add to our current understanding of the HCV life cycle and may ultimately allow to design novel antiviral strategies against hepatitis C.     

A cytopathic and a cell culture adapted hepatitis A virus strain differ in cell killing but not in intracellular membrane rearrangements

Aside from a common gene organization shared with other picornaviruses, hepatitis A virus (HAV) is characterized by its slow-growth phenotype, the inability to shut off host macromolecular synthesis, and, in general, lack of cytopathic (cp) effects in permissive cell cultures. Nevertheless, several cp HAV strains have been isolated during the past decade. In FRhK-4 cells infected with HM175/24a, a fast-growing cp strain, increasing amounts of viral RNA, detected by fluorescence in situ hybridization, indicated viral RNA replication. An ultrastructural analysis of the infected cells revealed a tubular-vesicular network in close proximity to the rough endoplasmic reticulum. Infection of the same cell type with a cell culture adapted (cc) strain, HM175/P35, divulged membrane alterations indistinguishable from the network induced by the cp strain. The overall appearance of the tubular-vesicular network resembles membrane alterations induced by other picornaviruses. However, the shape of the vesicle-like structures is rather oblong and tubular and, thus, seems to be specific for HAV. By electron microscopic immunocytochemistry (IEM), proteins 2B and 2C were found exclusively on the membranes of the network. Proteins expressed from the open reading frame of the cc HAV variant or 2B proteins originating from HM175 cp, cc, or the wt strain expressed in the absence of other HAV proteins induced membrane alterations resembling those seen in HAV-infected cells. The induction of similar structures suggests that protein 2B is involved in the rearrangement of cellular membranes. In all cases, IEM demonstrated that the 2B protein was closely associated with altered membranes. The extent of membrane changes did not seem to increase for both the cp strain and the cc strain during the infectious cycle. Late in the infection and shortly before the culture died off, a large number of cells infected with HM175/24a showed typical signs of apoptosis, whereas the cc strain did not induce cell killing in the same type of cells. Therefore, we conclude that cell death in HM175/24a-infected cells is induced by apoptosis rather than by cytopathology.     

Glycoprotein V is not the thrombin activation receptor on human blood platelets

Thrombin activation of platelets involves two receptors: glycoprotein Ib (GPIb), which affects the kinetics of the response; and, as a strong candidate for the second, essential receptor, GPV, a hydrophobic, 82-kd glycoprotein with an isoelectric point (pI) of pH 5.85 to 6.55. Whole platelets were treated with endogenous platelets calcium-activated proteases, yielding a major fragment, GPV8, with molecular weight (mol wt) of 79 kilodaltons (kd). The fragment was purified by affinity chromatography on wheat germ agglutinin followed by ion exchange chromatography on DEAE-Sephacel using first a 0 to 0.7-mol/L and then a 0 to 0.3-mol/L NaCl gradient. A rabbit was immunized with the purified GPV8 for preparation of polyclonal antibodies. Crossed immunoelectrophoresis and two-dimensional polyacrylamide gel electrophoresis (PAGE) electrophoretic blotting with the separate phases of a Triton X-114 phase partition of human platelets showed the characteristic pattern of GPV in the hydrophobic phase. During thrombin- induced platelet aggregation GPV is hydrolysed, releasing a fragment, GPVf1, to the supernatant. The fragment GPVf1 still contains a thrombin- binding site. Anti-GPV antibodies blocked GPV proteolysis, but did not inhibit platelet activation induced by thrombin. We conclude that proteolysis of GPV by thrombin is not essential for platelet activation.