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201.
Benjamin T. Goult Neil Bate Nicholas J. Anthis Kate L. Wegener Alexandre R. Gingras Bipin Patel Igor L. Barsukov Iain D. Campbell Gordon C. K. Roberts David R. Critchley 《The Journal of biological chemistry》2009,284(22):15097-15106
Talin is a large flexible rod-shaped protein that activates the integrin
family of cell adhesion molecules and couples them to cytoskeletal actin. It
exists in both globular and extended conformations, and an intramolecular
interaction between the N-terminal F3 FERM subdomain and the C-terminal part
of the talin rod contributes to an autoinhibited form of the molecule. Here,
we report the solution structure of the primary F3 binding domain within the
C-terminal region of the talin rod and use intermolecular nuclear Overhauser
effects to determine the structure of the complex. The rod domain (residues
1655–1822) is an amphipathic five-helix bundle; Tyr-377 of F3 docks into
a hydrophobic pocket at one end of the bundle, whereas a basic loop in F3
(residues 316–326) interacts with a cluster of acidic residues in the
middle of helix 4. Mutation of Glu-1770 abolishes binding. The rod domain
competes with β3-integrin tails for binding to F3, and the structure of
the complex suggests that the rod is also likely to sterically inhibit binding
of the FERM domain to the membrane.The cytoskeletal protein talin has emerged as a key player, both in
regulating the affinity of the integrin family of cell adhesion molecules for
ligand (1) and in coupling
integrins to the actin cytoskeleton
(2). Thus, depletion of talin
results in defects in integrin activation
(3), integrin signaling through
focal adhesion kinase, the maintenance of cell spreading, and the assembly of
focal adhesions in cultured cells
(4). In the whole organism,
studies on the single talin gene in worms
(5) and flies
(6) show that talin is
essential for a variety of integrin-mediated events that are crucial for
normal embryonic development. In vertebrates, there are two talin
genes, and mice carrying a talin1 null allele fail to complete
gastrulation (7).
Tissue-specific inactivation of talin1 results in an inability to activate
integrins in platelets (8,
9), defects in the
membrane-cytoskeletal interface in megakaryocytes
(10), and disruption of the
myotendinous junction in skeletal muscle
(11). In contrast, mice
homozygous for a talin2 gene trap allele have no phenotype, although
the allele may be hypomorphic
(12).Recent structural studies have provided substantial insights into the
molecular basis of talin action. Talin is composed of an N-terminal globular
head (∼50 kDa) linked to an extended flexible rod (∼220 kDa). The
talin head contains a
FERM2 domain (made up
of F1, F2, and F3 subdomains) preceded by a domain referred to here as F0
(2). Studies by Wegener et
al. (30) have shown how
the F3 FERM subdomain, which has a phosphotyrosine binding domain fold,
interacts with both the canonical NPXY motif and the
membrane-proximal helical region of the cytoplasmic tails of integrin
β-subunits (13). The
latter interaction apparently activates the integrin by disrupting the salt
bridge between the integrin α- and β-subunit tails that normally
keeps integrins locked in a low affinity state. The observation that the F0
region is also important in integrin activation
(14) may be explained by our
recent finding that F0 binds, albeit with low affinity,
Rap1-GTP,3 a known
activator of integrins (15,
16). The talin rod is made up
of a series of amphipathic α-helical bundles
(17–20)
and contains a second integrin binding site (IBS2)
(21), numerous binding sites
for the cytoskeletal protein vinculin
(22), at least two actin
binding sites (23), and a
C-terminal helix that is required for assembly of talin dimers
(20,
24).Both biochemical (25) and
cellular studies (16) suggest
that the integrin binding sites in full-length talin are masked, and both
phosphatidylinositol 4,5-bisphosphate (PIP2) and Rap1 have been implicated in
exposing these sites. It is well established that some members of the FERM
domain family of proteins are regulated by a head-tail interaction
(26); gel filtration,
sedimentation velocity, and electron microscopy studies all show that talin is
globular in low salt buffers, although it is more elongated (∼60 nm in
length) in high salt (27). By
contrast, the talin rod liberated from full-length talin by calpain-II
cleavage is elongated in both buffers, indicating that the head is required
for talin to adopt a more compact state. Direct evidence for an interaction
between the talin head and rod has recently emerged from NMR studies by Goksoy
et al. (28), who
demonstrated binding of 15N-labeled talin F3 to a talin rod
fragment spanning residues 1654–2344, an interaction that was confirmed
by surface plasmon resonance (Kd = 0.57 μm)
(28). Chemical shift data also
showed that this segment of the talin rod partially masked the binding site in
F3 for the membraneproximal helix of the β3-integrin tail
(28), directly implicating the
talin head-rod interaction in regulating the integrin binding activity of
talin. Goksoy et al.
(28) subdivided the F3 binding
site in this rod fragment into two sites with higher affinity
(Kd ∼3.6 μm; residues 1654–1848)
and lower affinity (Kd ∼78 μm; residues
1984–2344). Here, we define the rod domain boundaries and determine the
NMR structure of residues 1655–1822, a five-helix bundle. We further
show that this domain binds F3 predominantly via surface-exposed residues on
helix 4, with an affinity similar to the high affinity site reported by Goksoy
et al. (28). We also
report the structure of the complex between F3 and the rod domain and show
that the latter masks the known binding site in F3 for the β3-integrin
tail and is expected to inhibit the association of the talin FERM domain with
the membrane. 相似文献
202.
Alexandra Brand Keunsook Lee Veronica Veses Neil A. R. Gow 《Molecular microbiology》2009,71(5):1155-1164
Hyphae of the dimorphic fungus, Candida albicans , exhibit directional tip responses when grown in contact with surfaces. On hard surfaces or in liquid media, the trajectory of hyphal growth is typically linear, with tip re-orientation events limited to encounters with topographical features (thigmotropism). In contrast, when grown on semisolid surfaces, the tips of C. albicans hyphae grow in an oscillatory manner to form regular two-dimensional sinusoidal curves and three-dimensional helices. We show that, like thigmotropism, initiation of directional tip oscillation in C. albicans hyphae is severely attenuated when Ca2+ homeostasis is perturbed. Chelation of extracellular Ca2+ or deletion of the Ca2+ transporters that modulate cytosolic [Ca2+ ] (Mid1, Cch1 or Pmr1) did not affect hyphal length but curve formation was severely reduced in mid1 Δ and cch1 Δ and abolished in pmr1 Δ. Sinusoidal hypha morphology was altered in the mid1 Δ, chs3 Δ and heterozygous pmr1 Δ/ PMR1 strains. Treatments that affect cell wall integrity, changes in surface mannosylation or the provision of additional carbon sources had significant but less pronounced effects on oscillatory growth. The induction of two- and three-dimensional sinusoidal growth in wild-type C. albicans hyphae is therefore the consequence of mechanisms that involve Ca2+ influx and signalling rather than gross changes in the cell wall architecture. 相似文献
203.
Bender RP Jablonksy MJ Shadid M Romaine I Dunlap N Anklin C Graves DE Osheroff N 《Biochemistry》2008,47(15):4501-4509
Etoposide is a widely prescribed anticancer agent that stabilizes topoisomerase II-mediated DNA strand breaks. The drug contains a polycyclic ring system (rings A-D), a glycosidic moiety at C4, and a pendant ring (E-ring) at C1. A recent study that focused on yeast topoisomerase II demonstrated that the H15 geminal protons of the etoposide A-ring, the H5 and H8 protons of the B-ring, and the H2', H6', 3'-methoxyl, and 5'-methoxyl protons of the E-ring contact topoisomerase II in the binary enzyme-drug complex [ Wilstermann et al. (2007) Biochemistry 46, 8217-8225 ]. No interactions with the C4 sugar were observed. The present study used DNA cleavage assays, saturation transfer difference [ (1)H] NMR spectroscopy, and enzyme-drug binding studies to further define interactions between etoposide and human topoisomerase IIalpha. Etoposide and three derivatives that lacked the C4 sugar were analyzed. Except for the sugar, 4'-demethyl epipodophyllotoxin is identical to etoposide, epipodophyllotoxin contains a 4'-methoxyl group on the E-ring, and 6,7- O, O-demethylenepipodophyllotoxin replaces the A-ring with a diol. Results suggest that etoposide-topoisomerase IIalpha binding is driven by interactions with the A- and B-rings and potentially by stacking interactions with the E-ring. We propose that the E-ring pocket on the enzyme is confined, because the addition of bulk to this ring adversely affects drug function. The A- and E-rings do not appear to contact DNA in the enzyme-drug-DNA complex. Conversely, the sugar moiety subtly alters DNA interactions. The identification of etoposide substituents that contact topoisomerase IIalpha in the binary complex has predictive value for drug behavior in the enzyme-etoposide-DNA complex. 相似文献
204.
Salamanders are infrequently mentioned in analyses of tetrapod limb formation, as their development varies considerably from that of amniotes. However, urodeles provide an opportunity to study how limb ontogeny varies with major differences in life history. Here we assess limb development in Desmognathus aeneus, a direct-developing salamander, and compare it to patterns seen in salamanders with larval stages (e.g., Ambystoma mexicanum). Both modes of development result in a limb that is morphologically indistinct from an amniote limb. Developmental series of A. mexicanum and D. aeneus were investigated using Type II collagen immunochemistry, Alcian Blue staining, and whole-mount TUNEL staining. In A. mexicanum, as each digit bud extends from the limb palette Type II collagen and proteoglycan secretion occur almost simultaneously with mesenchyme condensation. Conversely, collagen and proteoglycan secretion in digits of D. aeneus occur only after the formation of an amniote-like paddle. Within each species, Type II collagen expression patterns resemble those of proteoglycans. In both, distal structures form before more proximal structures. This observation is contrary to the proximodistal developmental pattern of other tetrapods and may be unique to urodeles. In support of previous findings, no cell death was observed during limb development in A. mexicanum. However, apoptotic cells that may play a role in digit ontogeny occur in the limbs of D. aeneus, thereby suggesting that programmed cell death has evolved as a developmental mechanism at least twice in tetrapod limb evolution. 相似文献
205.
206.
Jahoda CA Kljuic A O'Shaughnessy R Crossley N Whitehouse CJ Robinson M Reynolds AJ Demarchez M Porter RM Shapiro L Christiano AM 《Genomics》2004,83(5):747-756
Desmosomal cadherins are essential cell adhesion molecules present throughout the epidermis and other organs, whose major function is to provide mechanical integrity and stability to epithelial cells in a wide variety of tissues. We recently identified a novel desmoglein family member, Desmoglein 4 (Dsg4), using a positional cloning approach in two families with localized autosomal recessive hypotrichosis (LAH) and in the lanceolate hair (lah) mouse. In this study, we report cloning and identification of the rat Dsg4 gene, in which we discovered a missense mutation in a naturally occurring lanceolate hair (lah) rat mutant. Phenotypic analysis of lah/lah mutant rats revealed a striking hair shaft defect with the appearance of a lance head within defective hair shafts. The mutation disrupts a critical calcium binding site bridging the second and third extracellular domains of Dsg4, likely disrupting extracellular interactions of the protein. 相似文献
207.
Mulvihill MJ Kan JL Cooke A Bhagwat S Beck P Bittner M Cesario C Keane D Lazarescu V Nigro A Nillson C Panicker B Smith V Srebernak M Sun FL O'Connor M Russo S Fischetti G Vrkljan M Winski S Castelhano AL Emerson D Gibson NW 《Bioorganic & medicinal chemistry letters》2006,16(10):2729-2733
3-[6-(2-Dimethylamino-1-imidazol-1-yl-butyl)-naphthalen-2-yloxy]-2,2-dimethyl-propionic acid and analogs were designed and synthesized as highly potent and selective CYP26 inhibitors, serving as retinoic acid metabolic blocking agents (RAMBAs), with demonstrated in vivo efficacy to increase the half-life of exogenous atRA. 相似文献
208.
Berlin A Balakshin M Gilkes N Kadla J Maximenko V Kubo S Saddler J 《Journal of biotechnology》2006,125(2):198-209
The conversion of lignocellulosic biomass to fuel ethanol typically involves a disruptive pretreatment process followed by enzyme-catalyzed hydrolysis of the cellulose and hemicellulose components to fermentable sugars. Attempts to improve process economics include protein engineering of cellulases, xylanases and related hydrolases to improve their specific activity or stability. However, it is recognized that enzyme performance is reduced during lignocellulose hydrolysis by interaction with lignin or lignin-carbohydrate complex (LCC), so the selection or engineering of enzymes with reduced lignin interaction offers an alternative means of enzyme improvement. This study examines the inhibition of seven cellulase preparations, three xylanase preparations and a beta-glucosidase preparation by two purified, particulate lignin preparations derived from softwood using an organosolv pretreatment process followed by enzymatic hydrolysis. The two lignin preparations had similar particle sizes and surface areas but differed significantly in other physical properties and in their chemical compositions determined by a 2D correlation HSQC NMR technique and quantitative 13C NMR spectroscopy. The various cellulases differed by up to 3.5-fold in their inhibition by lignin, while the xylanases showed less variability (< or = 1.7-fold). Of all the enzymes tested, beta-glucosidase was least affected by lignin. 相似文献
209.
Culp TD Cladel NM Balogh KK Budgeon LR Mejia AF Christensen ND 《Journal of virology》2006,80(22):11381-11384
Papillomaviruses (PVs) demonstrate both tissue and species tropisms. Because PVs replicate only in terminally differentiating epithelium, the recent production of infectious PV particles in 293 cells marks an important breakthrough. In this article, we demonstrate that infectious PV particles produced in 293TT cells can cause papillomatous growths in the natural host animal. Moreover, we show that species-matched PV genomes can be successfully delivered in vivo by a heterologous, species-mismatched PV capsid. Additionally, our results indicate that the addition of the simian virus 40 origin of replication to the papillomavirus genome increases the production of infectious papillomavirus particles by increasing genome amplification in the transfected 293TT cells. 相似文献