Self assembly of human septin 2 into amyloid filaments

Septins are a conserved group of GTP-binding proteins that form hetero-oligomeric complexes which assemble into filaments. These are essential for septin function, including their role in cytokinesis, cell division, exocytosis and membrane trafficking. Septin 2 (SEPT2) is a member of the septin family and has been associated with neurofibrillary tangles and other pathological features of senile plaques in Alzheimer's disease. An in silico analysis of the amino acid sequence of SEPT2 identified regions with a significant tendency to aggregate and/or form amyloid. These were all observed within the GTP-binding domain. This was consistent with the experimental identification of a structure rich in β-sheet during temperature induced unfolding transitions observed for both the full length protein and the GTP-binding domain alone. This intermediate state is characterized by irreversible aggregation and has the ability to bind Thioflavin-T, suggesting its amyloid nature. Under electron microscopy, fibers extending for several micrometers in length could be visualized. The results shown in this study support the hypothesis that single septins, when present in excess or with unbalanced stoichiometries, may be unstable and assemble into amyloid-like structures.     

NMR solution structure of the N-terminal domain of hERG and its interaction with the S4-S5 linker

The human Ether-à-go-go Related Gene (hERG) potassium channel mediates the rapid delayed rectifier current (IKr) in the cardiac action potential. Mutations in the 135 amino acid residue N-terminal domain (NTD) cause channel dysfunction or mis-translocation. To study the structure of NTD, it was overexpressed and purified from Escherichia coli cells using affinity purification and gel filtration chromatography. The purified protein behaved as a monomer under purification conditions. Far- and near-UV, circular dichroism (CD) and solution nuclear magnetic resonance (NMR) studies showed that the purified protein was well-folded. The solution structure of NTD was obtained and the N-terminal residues 13-23 forming an amphipathic helix which may be important for the protein-protein or protein-membrane interactions. NMR titration experiment also demonstrated that residues from 88 to 94 in NTD are important for the molecular interaction with the peptide derived from the S4-S5 linker.     

The determinants that govern microtubule assembly from the atomic structure of GTP-tubulin

Tubulin alternates between a soluble curved structure and a microtubule straight conformation. GTP binding to αβ-tubulin is required for microtubule assembly, but whether this triggers conversion into a straighter structure is still debated. This is due, at least in part, to the lack of structural data for GTP-tubulin before assembly. Here, we report atomic-resolution crystal structures of soluble tubulin in the GDP and GTP nucleotide states in a complex with a stathmin-like domain. The structures differ locally in the neighborhood of the nucleotide. A loop movement in GTP-bound tubulin favors its recruitment to the ends of growing microtubules and facilitates its curved-to-straight transition, but this conversion has not proceeded yet. The data therefore argue for the conformational change toward the straight structure occurring as microtubule-specific contacts are established. They also suggest a model for the way the tubulin structure is modified in relation to microtubule assembly.     

CTP:phosphocholine cytidylyltransferase: Function,regulation, and structure of an amphitropic enzyme required for membrane biogenesis

CTP:phosphocholine cytidylyltransferase (CCT) catalyzes a rate-limiting and regulated step in the CDP-choline pathway for the synthesis of phosphatidylcholine (PC) and PC-derived lipids. Control of CCT activity is multi-layered, and includes direct regulation by reversible membrane binding involving a built-in lipid compositional sensor. Thus CCT contributes to phospholipid compositional homeostasis. CCT also modifies the curvature of its target membrane. Knowledge of CCT structure and regulation of its catalytic function are relatively advanced compared to many lipid metabolic enzymes, and are reviewed in detail. Recently the genetic origins of two human developmental and lipogenesis disorders have been traced to mutations in the gene for CCTα.     

New insights into the membrane-binding and activation mechanism of pyruvate oxidase from Escherichia coli

Pyruvate oxidase from Escherichia coli (EcPOX) is a thiamin diphosphate- (ThDP) and FAD-dependent peripheral membrane protein that carries out the irreversible oxidative decarboxylation of pyruvate to acetate and carbon dioxide. Concomitant two-electron reduction of the flavin cofactor was suggested to induce a structural rearrangement of the C-terminus triggering recruitment of the protein from the cytosol to the cell membrane, where the electrons are eventually transferred to final electron acceptor ubiquinone 8. Binding to the membrane, or alternatively, mild proteolytic digestion leads to a multifold enhancement of both the catalytic activity and substrate affinity. Recent X-ray crystallographic studies on EcPOX in the resting state and on a C-terminal truncation variant mimicking the membrane-bound activated form have fueled our understanding of the membrane-binding mechanism and concomitant catalytic activation. In the resting state, the auto-inhibitory C-terminal membrane anchor adopts a half-barrel/helix fold that occludes the active site. Upon activation, the C-terminus is expelled and becomes structurally flexible thereby freeing the active site. Circular dichroism spectroscopic analysis revealed the isolated C-terminus to be disordered, however, formation of a helical structure was observed in the presence of micelles. Limited proteolysis experiments indicate that activation of EcPOX involves at least two sequential structural transitions: the first occurring after binding of pyruvate to ThDP and the second after two-electron reduction of the flavin.     

The structure and orientation of class-A amphipathic peptides on a phospholipid bilayer surface

The amphipathic α-helix is a recognised structural motif that is shared by membrane-associating proteins and peptides of diverse function. The aim of this paper is to determine the orientation of an α-helical amphipathic peptide on the bilayer surface. We use five amphipathic 18-residue peptide analogues of a class A amphipathic peptide that is known to associate with a bilayer surface. Tyrosine and tryptophan are used as spectroscopic probes to sense local environments in the peptide in solution and when bound to the surface of unilamellar phosphatidylcholine vesicles. In a series of peptides, tryptophan is moved progressively along the sequence from the nonpolar face (positions 3, 7, 4) to the polar face of the peptide (positions 2, 12). The local environment of the tryptophan residue at each position is determined using fluorescence spectroscopy employing quantum yield, and the wavelength of the emission maximum as indicators of micropolarity. The exposure of the tryptophan residues at each site is assessed by acrylamide quenching. On association with vesicles, the tryptophan residues at positions 3, 7 and 14 are in nonpolar water-shielded environments, and the tryptophan at position 12 is in an exposed polar environment. The tryptophan at position 2, which is located near the bilayer-water interface, exhibits intermediate behaviour. Analysis of the second-derivative absorption spectrum confirmed that the tyrosine residue at position 7 is in a nonpolar water-shielded environment in the peptide-lipid complex. We conclude that these class A amphipathic peptides lie parallel to the lipid surface and penetrate no deeper than the ester linkages of the phospholipids. Received: 8 April 1998 / Revised version: 6 July 1998 / Accepted: 7 August 1998     

Mitochondria mediate septin cage assembly to promote autophagy of Shigella

Septins, cytoskeletal proteins with well‐characterised roles in cytokinesis, form cage‐like structures around cytosolic Shigella flexneri and promote their targeting to autophagosomes. However, the processes underlying septin cage assembly, and whether they influence S. flexneri proliferation, remain to be established. Using single‐cell analysis, we show that the septin cages inhibit S. flexneri proliferation. To study mechanisms of septin cage assembly, we used proteomics and found mitochondrial proteins associate with septins in S. flexneri‐infected cells. Strikingly, mitochondria associated with S. flexneri promote septin assembly into cages that entrap bacteria for autophagy. We demonstrate that the cytosolic GTPase dynamin‐related protein 1 (Drp1) interacts with septins to enhance mitochondrial fission. To avoid autophagy, actin‐polymerising Shigella fragment mitochondria to escape from septin caging. Our results demonstrate a role for mitochondria in anti‐Shigella autophagy and uncover a fundamental link between septin assembly and mitochondria.     

Role of actin binding protein phosphorylation in platelet cytoskeleton assembly

Actin binding protein from human blood platelets is shown to exist in the resting platelet as a phosphorylated protein and contains two residues of phosphate per 260,000 kd. Removal of one-half of these residues with E. coli alkaline phosphatase results in the loss of its ability to crosslink F-actin into a low speed sedimentable complex (its cytoskeleton) and to bind to an F-actin affinity column. Thus, phosphorylation-dephosphorylation of ABP may be an important regulatory mechanism by which the platelet regulates its shape via its cytoskeletal structure.     

Borg/septin interactions and the assembly of mammalian septin heterodimers,trimers, and filaments

Septins constitute a family of guanine nucleotide-binding proteins that were first discovered in the yeast Saccharomyces cerevisiae but are also present in many other eukaryotes. In yeast they congregate at the bud neck and are required for cell division. Their function in metazoan cells is uncertain, but they have been implicated in exocytosis and cytokinesis. Septins have been purified from cells as hetero-oligomeric filaments, but their mechanism of assembly is unknown. Further studies have been limited by the difficulty in expressing functional septin proteins in bacteria. We now show that stable, soluble septin heterodimers can be produced by co-expression from bicistronic vectors in bacteria and that the co-expression of three septins results in their assembly into filaments. Pre-assembled dimers and trimers bind guanine nucleotide and show a slow GTPase activity. The assembly of a heterodimer from monomers in vitro is accompanied by GTP hydrolysis. Borg3, a downstream effector of the Cdc42 GTPase, binds specifically to a septin heterodimer composed of Sept6 and Sept7 and to the Sept2/6/7 trimer, but not to septin monomers or to other heterodimers. Septins associate through their C-terminal coiled-coil domains, and Borg3 appears to recognize the interface between these domains in Sept6 and Sept7.     

Deciphering the rules governing assembly order of mammalian septin complexes

Septins are conserved GTP-binding proteins that assemble into lateral diffusion barriers and molecular scaffolds. Vertebrate genomes contain 9-17 septin genes that encode both ubiquitous and tissue-specific septins. Expressed septins may assemble in various combinations through both heterotypic and homotypic G-domain interactions. However, little is known regarding assembly states of mammalian septins and mechanisms directing ordered assembly of individual septins into heteromeric units, which is the focus of this study. Our analysis of the septin system in cells lacking or overexpressing selected septins reveals interdependencies coinciding with previously described homology subgroups. Hydrodynamic and single-particle data show that individual septins exist solely in the context of stable six- to eight-subunit core heteromers, all of which contain SEPT2 and SEPT6 subgroup members and SEPT7, while heteromers comprising more than six subunits also contain SEPT9. The combined data suggest a generic model for how the temporal order of septin assembly is homology subgroup-directed, which in turn determines the subunit arrangement of native heteromers. Because mammalian cells normally express multiple members and/or isoforms of some septin subgroups, our data also suggest that only a minor fraction of native heteromers are arranged as perfect palindromes.     

3D reconstruction of mammalian septin filaments

Mammalian septins are a family of guanosine triphosphate-binding proteins thought to play a role in a number of key cellular processes, such as cytokinesis, protein scaffolding and vesicle trafficking. Although their precise functions remain to be determined, electron microscopy has shown septin filament formation in vitro and a role as a cytoskeletal polymer has been proposed. Here, we present a 3D reconstruction of septin filaments determined using electron microscopy of negatively stained specimens and single-particle image processing. Septin was isolated from rat brain as an approximately 240-kDa complex, from which immunoblotting and N-terminal sequencing identified the major components as septins 3, 5 and 7. Electron microscopy and single-particle analysis indicated that the majority of the septin filaments were ∼ 27 nm long. A comparison of 3D volumes obtained using two independent starting models (a row of spheres or a helix) and projection matching techniques revealed no major differences at the final resolution of 27 Å, and this structure was highly reproducible when the entire procedure was repeated several times. The reconstruction revealed three apparent subunits, each separated by a cleft; these subunits were similar, but not identical, possibly indicating multiple isoforms within each filament. In some views a smaller cleft appeared to separate the subunits into two smaller regions, perhaps reflecting the presence of septin dimers. This is the first 3D reconstruction of the native septin assembly, and appears compatible with the hypothesis that the septin complex is a hexamer consisting of dimers or heterotrimers. Further investigations are necessary to confirm how the structure of the filaments determined in the present study correlates with the roles of septins in vivo.     

涎泪腺炎病毒对AH66腹水型肝癌的促增殖作用及对荷瘤大鼠细胞免疫的影响

涎泪腺炎病毒已鉴定为实验大鼠的病原体之一（简称ＳＤＡＶ）。鼻内接种２ｄ内可引起鼻炎。唾液腺、泪腺小管和腺泡上皮坏死。临床上表现为隐性感染的ＳＤＡＶ,感染可导致宿主的一时性的兔疫功能低下。本实验将ＡＨ６６腹水型肝癌细胞接种给感染ＳＤＡｖ和未感染ＳＤＡＶ大鼠,以期观察ＳＤＡＶ感染对ＡＨ６６癌细胞存活性和荷瘤大鼠细胞免疫的影响．５０只（雌性、ＳＰＦ、近交系、６周龄）Ｄｏｎｒｙｎ大鼠被接种ＳＤＡＶ和ＡＨ６６癌细胞。采用组织病理学,血清学,血中Ｔ细胞亚群分析等实验方法。结果ＡＨ６６癌细胞在ＳＤＡＶ感染组大鼠肺内的存活率（９０％）明显高于未感染ＳＤＡＶ组（４０％）,血中Ｔ细胞亚群在ＳＤＡＶ感染组ＣＤ４平均值的比值低于对照组。ＡＨ６６癌细胞接种组ＣＤ４平均值的比值高于ＳＤＡＶ＋ＡＨ６６癌细胞接种组。同时ＳＤＡＶ感染＋ＡＨ６６癌细胞接种组的ＣＤ４／ＣＤ８的比值明显增大并倒置。以上的结果表明ＳＤＡＶ感染后,病毒干扰宿主细胞免疫功能从而对ＡＨ６６癌细胞有促增殖作用。     

Antibody-induced shedding of CD44 from adherent cells is linked to the assembly of the cytoskeleton.

CD44 is a widely expressed integral membrane glycoprotein that serves as a specific adhesion receptor for the extracellular matrix glycosaminoglycan hyaluronan. CD44 participates in a variety of physiological and pathological processes through its role in cell adhesion. Under appropriate conditions, the ectodomain of CD44 is proteolytically removed from the cell surface. In this study we show that excessive CD44 shedding can be induced in mouse fibroblasts and monocytes upon exposure of these cells to a CD44-specific Ab immobilized on plastic, whereas treatment with phorbol ester induces significantly enhanced CD44 release from the monocytes only. CD44 shedding proceeds normally in fibroblasts and monocytes deficient in TNF-alpha converting enzyme (TACE), a sheddase involved in the processing of several substrates. Conversely, activation of the CD44 protease has no effect on the release of TNF-alpha from TACE-expressing cells, although the same metalloprotease inhibitor effectively blocks both TACE and the CD44 sheddase. Concomitant with anti-CD44 Ab- or phorbol ester-induced CD44 shedding, dramatic changes are observed in cell morphology and the structure of the actin cytoskeleton. Disruption of actin assembly with cytochalasin reduces CD44 shedding, but not the release of TNF-alpha. Moreover, pharmacological activation of Rho family GTPases Rac1 and Cdc42, which regulate actin filament assembly into distinct cytoskeletal structures, has a profound effect on CD44 release. We conclude that the CD44 sheddase and TACE are distinct enzymes, and that Ab- and phorbol ester-enhanced cleavage of CD44 is controlled in a cell type-dependent fashion by Rho GTPases through the cytoskeleton.     

Role of the cytoskeleton in choanoflagellate lorica assembly

ABSTRACT. Cell division in Acanthoeca spectabilis produces a "naked" motile daughter cell (juvenile) that settles onto a surface and deposits siliceous costal strips that are stored extracellularly in bundles. When complete, the bundles of strips are assembled in a single continuous movement to form a basket-like lorica. Assembly can be divided into four overlapping stages. Stage 1 entails the left-handed rotation of strips at the anterior end while the posterior end remains stationary. Stage 2 includes the posterior protrusion of the cell to form a stalk. Stage 3 involves the anterior extension of the spines, and Stage 4 the dilation of the lorica chamber and deposition of the organic investment. Scanning electron microscopic images reveal a one-to-one association between the moving bundles of strips and the anterior ring of lorica-assembling tentacles. Treatment with microtubule inhibitors produces "dwarf" cells that lack stalks, have their spines extended, and possess collars but lack flagella. Treatment with microfilament (actin) inhibitors prevents extension of the anterior spines. These experiments demonstrate that posterior cell extension is primarily mediated by microtubules whereas extension of the spines is controlled by the actin cytoskeleton. The processes of cytoskeletal rotation and extracellular costal strip movement are compared, respectively, with rotation of nuclei in animal embryos and movement of mammalian cells over surfaces.     

The role of Cdc42p GTPase-activating proteins in assembly of the septin ring in yeast

The septins are a conserved family of GTP-binding, filament-forming proteins. In the yeast Saccharomyces cerevisiae, the septins form a ring at the mother-bud neck that appears to function primarily by serving as a scaffold for the recruitment of other proteins to the neck, where they participate in cytokinesis and a variety of other processes. Formation of the septin ring depends on the Rho-type GTPase Cdc42p but appears to be independent of the actin cytoskeleton. In this study, we investigated further the mechanisms of septin-ring formation. Fluorescence-recovery-after-photobleaching (FRAP) experiments indicated that the initial septin structure at the presumptive bud site is labile (exchanges subunits freely) but that it is converted into a stable ring as the bud emerges. Mutants carrying the cdc42V36G allele or lacking two or all three of the known Cdc42p GTPase-activating proteins (GAPs: Bem3p, Rga1p, and Rga2p) could recruit the septins to the cell cortex but were blocked or delayed in forming a normal septin ring and had accompanying morphogenetic defects. These phenotypes were dramatically enhanced in mutants that were also defective in Cla4p or Gin4p, two protein kinases previously shown to be important for normal septin-ring formation. The Cdc42p GAPs colocalized with the septins both early and late in the cell cycle, and overexpression of the GAPs could suppress the septin-organization and morphogenetic defects of temperature-sensitive septin mutants. Taken together, the data suggest that formation of the mature septin ring is a process that consists of at least two distinguishable steps, recruitment of the septin proteins to the presumptive bud site and their assembly into the stable septin ring. Both steps appear to depend on Cdc42p, whereas the Cdc42p GAPs and the other proteins known to promote normal septin-ring formation appear to function in a partially redundant manner in the assembly step. In addition, because the eventual formation of a normal septin ring in a cdc42V36G or GAP mutant was invariably accompanied by a switch from an abnormally elongated to a more normal bud morphology distal to the ring, it appears that the septin ring plays a direct role in determining the pattern of bud growth.     

Fibrillin: from microfibril assembly to biomechanical function

Fibrillins form the structural framework of a unique and essential class of extracellular microfibrils that endow dynamic connective tissues with long-range elasticity. Their biological importance is emphasized by the linkage of fibrillin mutations to Marfan syndrome and related connective tissue disorders, which are associated with severe cardiovascular, ocular and skeletal defects. These microfibrils have a complex ultrastructure and it has proved a major challenge both to define their structural organization and to relate it to their biological function. However, new approaches have at last begun to reveal important insights into their molecular assembly, structural organization and biomechanical properties. This paper describes the current understanding of the molecular assembly of fibrillin molecules, the alignment of fibrillin molecules within microfibrils and the unique elastomeric properties of microfibrils.