Do we need many genes for phylogenetic inference?

Fifty-six nuclear protein coding genes from Taxonomically Broad EST Database and other databases were selected for phylogenomic-based examination of alternative phylogenetic hypotheses concerning intergroup relationship between multicellular animals (Metazoa) and other representatives of Opisthokonta. The results of this work support sister group relationship between Metazoa and Choanoflagellata. Both of these groups form the taxon Holozoa along with the monophyletic Ichthyosporea or Mesomycetozoea (a group that includes Amoebidium parasiticum, Sphaeroforma arctica, and Capsaspora owczarzaki). These phylogenetic hypotheses receive high statistical support both when utilizing whole alignment and when only 5000 randomly selected alignment positions are used. The presented results suggest subdivision of Fungi into Eumycota and lower fungi, Chytridiomycota. The latter form a monophyletic group that comprises Chytridiales+Spizellomycetales+Blastocladiales (Batrachochytrium, Spizellomyces, Allomyces, Blastocladiella), contrary to the earlier reports based on the analysis of 18S rRNA and a limited set of protein coding genes. The phylogenetic distribution of genes coding for a ubiquitin-fused ribosomal protein S30 implies at least three independent cases of gene fusion: in the ancestors of Holozoa, in heterotrophic Heterokonta (Oomycetes and Blastocystis) and in the ancestors of Cryptophyta and Glaucophyta. Ubiquitin-like sequences fused with ribosomal protein S30 outside of Holozoa are not FUBI orthologs. Two independent events of FUBI replacement by the ubiquitin sequence were detected in the lineage of C. owczarzaki and in the monophyletic group of nematode worms Tylenchomorpha+Cephalobidae. Bursaphelenchus xylophilus (Aphelenchoidoidea) retains a state typical of the rest of the Metazoa. The data emphasize the fact that the reliability of phylogenetic reconstructions depends on the number of analyzed genes to a lesser extent than on our ability to recognize reconstruction artifacts.     

Internal Cleavages of the Autoinhibitory Prodomain Are Required for Membrane Type 1 Matrix Metalloproteinase Activation,although Furin Cleavage Alone Generates Inactive Proteinase

The functional activity of invasion-promoting membrane type 1 matrix metalloproteinase (MT1-MMP) is elevated in cancer. This elevated activity promotes cancer cell migration, invasion, and metastasis. MT1-MMP is synthesized as a zymogen, the latency of which is maintained by its prodomain. Excision by furin was considered sufficient for the prodomain release and MT1-MMP activation. We determined, however, that the full-length intact prodomain released by furin alone is a potent autoinhibitor of MT1-MMP. Additional MMP cleavages within the prodomain sequence are required to release the MT1-MMP enzyme activity. Using mutagenesis of the prodomain sequence and mass spectrometry analysis of the prodomain fragments, we demonstrated that the intradomain cleavage of the PGD↓L⁵⁰ site initiates the MT1-MMP activation, whereas the ¹⁰⁸RRKR¹¹¹↓Y¹¹² cleavage by furin completes the removal and the degradation of the autoinhibitory prodomain and the liberation of the functional activity of the emerging enzyme of MT1-MMP.     

Crystal structure of glucoamylase from Aspergillus awamori var. X100 to 2.2-A resolution.

The crystal structure of a catalytically active fragment of glucoamylase-I from Aspergillus awamori var. X100 has been determined to a resolution of 2.2 A. Twelve of its 13 alpha-helices are arranged into an "alpha/alpha-barrel." An inner core of six mutually parallel alpha-helices are connected to each other through a peripheral set of six alpha-helices. The peripheral helices are parallel to each other, but approximately antiparallel to the inner core of alpha-helices. The putative active site lies in the packing void of the inner set of helices. The last 30 residues of the enzyme comprise a separate domain containing 10 sites of O-glycosylation. Each instance of O-glycosylation involves a serine or threonine side chain linked to the alpha-anomer of a single mannosyl residue. The O-glycosylated domain is in an extended conformation, wrapping around the "waist" of the alpha/alpha-barrel. Two additional sites of N-glycosylation contribute well ordered glycosyl chains that lie in proximity to the belt of O-glycosylation. The model developed for glucoamylase is a rare and valuable structural example of a glycoprotein and an exo-acting amylolytic enzyme.     

Potential relation of aberrant proteolysis of human protein tyrosine kinase 7 (PTK7) chuzhoi by membrane type 1 matrix metalloproteinase (MT1-MMP) to congenital defects

Membrane PTK7 pseudo-kinase plays an essential role in planar cell polarity and the non-canonical Wnt pathway in vertebrates. Recently, a new N-ethyl-N-nitrosourea-induced mutant named chuzhoi (chz) was isolated in mice. chz embryos have severe birth defects, including a defective neural tube, defective heart and lung development, and a shortened anterior-posterior body axis. The chz mutation was mapped to the Ala-Asn-Pro tripeptide insertion into the junction region between the fifth and the sixth Ig-like domains of PTK7. Unexpectedly, chz reduced membrane localization of the PTK7 protein. We hypothesized and then proved that the chz mutation caused an insertion of an additional membrane type 1 matrix metalloproteinase cleavage site in PTK7 and that the resulting aberrant proteolysis of chz affected the migratory parameters of the cells. It is likely that aberrations in the membrane type 1 matrix metalloproteinase/PTK7 axis are detrimental to cell movements that shape the body plan and that chz represents a novel model system for increasing our understanding of the role of proteolysis in developmental pathologies, including congenital defects.     

Rosiglitazone as a regulator of innate immunity in a cell model of hyperglycemia

Epidemiological studies have shown that severe inflammatory responses occur in patients with hyperglycemia. The molecular nature of these changes is currently under intense investigations. A central role of nuclear receptors PPAR has been shown in the regulation of metabolic changes associated with hyperglycemia, a selective agonist of nuclear receptor PPARγ rosiglitazone is used as a hypoglycemic drug. Rosiglitazone is known to have anti-inflammatory effects, but its properties as an anti-inflammatory drug in hyperglycemic conditions have not been studied. This was an aim of our work. We used a human cell culture model of hyperglycemia: HeLa cells incubated in the conditions of 25 mM glucose for 3 days. Control cells were incubated with 5 mM glucose. The cells were stimulated with lipopolysaccharide (LPS) that is known to trigger innate immune response through activation of Toll-like receptor 4 and influence mRNA expression levels of three of PPAR (α, β/δ, γ) isotypes as well as cyclooxygenase (COX-1 and COX-2). We have shown that under hyperglycemic conditions expression levels of PPARα and PPARβ/δ decreased almost twofold, expression level of COX-2 also decreased, while expression levels of COX-1 and PPARγ remained unchanged compared to those under normal glucose concentration. LPS administration in control cells leads to a 1.5–2.5-fold stimulation of expression of COX-2 and PPAR isotypes. In contrast, under hyperglycemia, LPS exhibited no effect on expression of COX-2 and the PPAR isotypes, which indicates potential mechanisms of hyperglycemia-related alterations in innate immunity. Rosiglitazone, an agonist of PPARγ, decreased expression level of PPARβ/δ and abolished the effect of LPS under hyperglycemia. Rosiglitazone also reduced expression level of COX-1 and COX-2, which indicates on the agonist possible role as an anti-inflammatory agent under high glucose concentrations. These data broaden applicability of rosiglitazone as an anti-inflammatory agent in hyperglycemic conditions.     

Crystallization and preliminary X-ray study of minor glucoamylase from Aspergillus awamori variant X-100/D27.

Crystals of the reduced form of glucoamylase were obtained from polyethylene glycol 6000 solution by the hanging-drop method. The protein was treated with alpha-mannosidase to partly remove the sugar component. The crystals belong to the space group P2(1)2(1)2(1) with cell dimensions a = 116.7 A, b = 104.3 A, c = 48.5 A and diffract beyond 2.5 A resolution.     

TRAIL-Based High Throughput Screening Reveals a Link between TRAIL-Mediated Apoptosis and Glutathione Reductase,a Key Component of Oxidative Stress Response

A high throughput screen for compounds that induce TRAIL-mediated apoptosis identified ML100 as an active chemical probe, which potentiated TRAIL activity in prostate carcinoma PPC-1 and melanoma MDA-MB-435 cells. Follow-up in silico modeling and profiling in cell-based assays allowed us to identify NSC130362, pharmacophore analog of ML100 that induced 65-95% cytotoxicity in cancer cells and did not affect the viability of human primary hepatocytes. In agreement with the activation of the apoptotic pathway, both ML100 and NSC130362 synergistically with TRAIL induced caspase-3/7 activity in MDA-MB-435 cells. Subsequent affinity chromatography and inhibition studies convincingly demonstrated that glutathione reductase (GSR), a key component of the oxidative stress response, is a target of NSC130362. In accordance with the role of GSR in the TRAIL pathway, GSR gene silencing potentiated TRAIL activity in MDA-MB-435 cells but not in human hepatocytes. Inhibition of GSR activity resulted in the induction of oxidative stress, as was evidenced by an increase in intracellular reactive oxygen species (ROS) and peroxidation of mitochondrial membrane after NSC130362 treatment in MDA-MB-435 cells but not in human hepatocytes. The antioxidant reduced glutathione (GSH) fully protected MDA-MB-435 cells from cell lysis induced by NSC130362 and TRAIL, thereby further confirming the interplay between GSR and TRAIL. As a consequence of activation of oxidative stress, combined treatment of different oxidative stress inducers and NSC130362 promoted cell death in a variety of cancer cells but not in hepatocytes in cell-based assays and in in vivo, in a mouse tumor xenograft model.     

Crystal structure of C5b-6 suggests structural basis for priming assembly of the membrane attack complex

The complement membrane attack complex (MAC) forms transmembrane pores in pathogen membranes. The first step in MAC assembly is cleavage of C5 to generate metastable C5b, which forms a stable complex with C6, termed C5b-6. C5b-6 initiates pore formation via the sequential recruitment of homologous proteins: C7, C8, and 12–18 copies of C9, each of which comprises a central MAC-perforin domain flanked by auxiliary domains. We recently proposed a model of pore assembly, in which the auxiliary domains play key roles, both in stabilizing the closed conformation of the protomers and in driving the sequential opening of the MAC-perforin β-sheet of each new recruit to the growing pore. Here, we describe an atomic model of C5b-6 at 4.2 Å resolution. We show that C5b provides four interfaces for the auxiliary domains of C6. The largest interface is created by the insertion of an interdomain linker from C6 into a hydrophobic groove created by a major reorganization of the α-helical domain of C5b. In combination with the rigid body docking of N-terminal elements of both proteins, C5b becomes locked into a stable conformation. Both C6 auxiliary domains flanking the linker pack tightly against C5b. The net effect is to induce the clockwise rigid body rotation of four auxiliary domains, as well as the opening/twisting of the central β-sheet of C6, in the directions predicted by our model to activate or prime C6 for the subsequent steps in MAC assembly. The complex also suggests novel small molecule strategies for modulating pathological MAC assembly.     

Structure of complement C6 suggests a mechanism for initiation and unidirectional, sequential assembly of membrane attack complex (MAC)

The complement membrane attack complex (MAC) is formed by the sequential assembly of C5b with four homologous proteins as follows: one copy each of C6, C7, and C8 and 12-14 copies of C9. Together these form a lytic pore in bacterial membranes. C6 through C9 comprise a MAC-perforin domain flanked by 4-9 "auxiliary" domains. Here, we report the crystal structure of C6, the first and longest of the pore proteins to be recruited by C5b. Comparisons with the structures of the C8αβγ heterodimer and perforin show that the central domain of C6 adopts a "closed" (perforin-like) state that is distinct from the "open" conformations in C8. We further show that C6, C8α, and C8β contain three homologous subdomains ("upper," "lower," and "regulatory") related by rotations about two hinge points. In C6, the regulatory segment includes four auxiliary domains that stabilize the closed conformation, inhibiting release of membrane-inserting elements. In C8β, rotation of the regulatory segment is linked to an opening of the central β-sheet of its clockwise partner, C8α. Based on these observations, we propose a model for initiation and unidirectional propagation of the MAC in which the auxiliary domains play key roles: in the assembly of the C5b-8 initiation complex; in driving and regulating the opening of the β-sheet of the MAC-performin domain of each new recruit as it adds to the growing pore; and in stabilizing the final pore. Our model of the assembled pore resembles those of the cholesterol-dependent cytolysins but is distinct from that recently proposed for perforin.