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1.
Intracellular β-Carbonic Anhydrase of the Unicellular Green
Alga Coccomyxa
: Cloning of the cDNA and Characterization of the Functional
Enzyme Overexpressed in Escherichia coli
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Thomas Hiltonen Harry Bj?rkbacka Cecilia Forsman Adrian K. Clarke G?ran Samuelsson 《Plant physiology》1998,117(4):1341-1349
Carbonic anhydrase (CA) (EC 4.2.1.1) enzymes catalyze the reversible hydration of CO2, a reaction that is important in many physiological processes. We have cloned and sequenced a full-length cDNA encoding an intracellular β-CA from the unicellular green alga Coccomyxa. Nucleotide sequence data show that the isolated cDNA contains an open reading frame encoding a polypeptide of 227 amino acids. The predicted polypeptide is similar to β-type CAs from Escherichia coli and higher plants, with an identity of 26% to 30%. The Coccomyxa cDNA was overexpressed in E. coli, and the enzyme was purified and biochemically characterized. The mature protein is a homotetramer with an estimated molecular mass of 100 kD. The CO2-hydration activity of the Coccomyxa enzyme is comparable with that of the pea homolog. However, the activity of Coccomyxa CA is largely insensitive to oxidative conditions, in contrast to similar enzymes from most higher plants. Fractionation studies further showed that Coccomyxa CA is extrachloroplastic. 相似文献
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Nidhi Ahuja Dmitry Korkin Rachna Chaba Brent O. Cezairliyan Robert T. Sauer Kyeong Kyu Kim Carol A. Gross 《The Journal of biological chemistry》2009,284(8):5403-5413
The Escherichia coli envelope stress response is controlled by the
alternative sigma factor, σE, and is induced when unfolded
outer membrane proteins accumulate in the periplasm. The response is initiated
by sequential cleavage of the membrane-spanning antisigma factor, RseA. RseB
is an important negative regulator of envelope stress response that exerts its
negative effects onσE activity through its binding to RseA.
In this study, we analyze the interaction between RseA and RseB. We found that
tight binding of RseB to RseA required intact RseB. Using programs that
performed global and local sequence alignment of RseB and RseA, we found
regions of high similarity and performed alanine substitution mutagenesis to
test the hypothesis that these regions were functionally important. This
protocol is based on the hypothesis that functionally dependent regions of two
proteins co-evolve and therefore are likely to be sequentially conserved. This
procedure allowed us to identify both an N-terminal and C-terminal region in
RseB important for binding to RseA. We extensively analyzed the C-terminal
region, which aligns with a region of RseA coincident with the major RseB
binding determinant in RseA. Both allele-specific suppression analysis and
cysteine-mediated disulfide bond formation indicated that this C-terminal
region of similarity of RseA and RseB identifies a contact site between the
two proteins. We suggest a similar protocol can be successfully applied to
pairs of non-homologous but functionally linked proteins to find specific
regions of the protein sequences that are important for establishing
functional linkage.The Escherichia coli σE-mediated envelope stress
response is the major pathway to ensure homeostasis in the envelope
compartment of the cell
(1-3).
σE regulon members encode periplasmic chaperones and
proteases, the machinery for inserting β-barrel proteins into the outer
membrane and components controlling the synthesis and assembly of LPS
(4-6).
This pathway is highly conserved among γ-proteobacteria
(6).The σE response is initiated when periplasmic protein
folding and assembly is compromised
(7-9).
During steady state growth, σE is inhibited by its antisigma
factor, RseA, a membrane-spanning protein whose cytoplasmic domain binds to
σE with picomolar affinity
(10-13).
Accumulation of unassembled porin monomers serves as a signal to activate the
DegS protease to cleave RseA in its periplasmic domain
(14,
15). This initiates a
proteolytic cascade in which RseP cleaves periplasmically truncated RseA near
or within the cytoplasmic membrane to release the
RseAcytoplasmic-σE complex, and cytoplasmic
ATP-dependent proteases complete the degradation of RseA thereby releasing
active σE
(16-19).RseB, a second negative regulator of the envelope stress response
(11,
20,
21), binds to the periplasmic
domain of RseA with nanomolar affinity. RseB is an important regulator of the
response (2,
22,
23). It prevents RseP from
degrading intact RseA, thereby ensuring that proteolysis is initiated only
when the DegS protease is activated by a stress signal
(21). Additionally, RseB
prevents activated DegS from cleaving RseA, suggesting that interaction of
RseB with RseA must be altered before the signal transduction cascade is
activated (23).The goal of the present studies was to explore how RseB binds to RseA. The
interaction partner of RseB is the unstructured periplasmic domain of RseA
(RseA-peri). Within RseA-peri, amino acids ∼169-186 constitute a major
binding determinant to RseB
(23,
24). This peptide alone binds
RseB with 6 μm affinity, and deleting this region abrogates
binding to RseB (23).
Additional regions of RseA-peri also contribute to RseB binding, as intact
RseA-peri binds with 20 nm affinity to RseB
(23). Much less is known about
the regions of RseB required for interaction with RseA. RseB is homodimeric
two-domain protein, whose large N-terminal domain shares structural homology
with LolA, a protein that transports lipoproteins to outer membrane
(24,
25). The smaller C-terminal
domain is connected to the N-terminal domain by a linker, and the two domains
share a large interface, which may facilitate interdomain signaling.
Glutaraldehyde cross-linking studies indicate that the C-terminal domain
interacts with RseA, but the regions of interaction were not identified
(25).In the present report, we study the interaction of RseB and RseA. We
establish that both domains of RseB interact with RseA-peri. Using a global
sequence alignment, we discovered several regions in RseA and RseB that had
high sequence similarity, despite the low overall sequence similarity between
these two proteins, a finding that was independently confirmed by a local
sequence similarity algorithm. This suggested that these regions were
functionally dependent, and we performed a set of mutagenesis experiments
designed to test this idea. Our studies of the binding properties of these
mutants revealed that regions in both the N terminus and C terminus of RseB
modulate interaction with RseA. Moreover, genetic suppression analysis and
cysteine-mediated disulfide bond formation suggest that the region of RseA/B
with highest similarity (RseA residues 165-191 (major binding determinant in
RseA) and RseB residues 233-258) are interacting partners. 相似文献
5.
The genus Planinasus Cresson is revised and includes 18 extant and one fossil species. We clarify the status of the three previously described species and describe 15 new species as follows (type locality in parenthesis): Planinasus aenigmaticus (Colombia. Bogota: Bogota (04°35.8''N, 74°08.8''W)), Planinasus neotropicus (Panama. Canal Zone: Barro Colorado Island (09°09.1''N, 79°50.8''W)), Planinasus kotrbae (Ecuador. Orellana: Rio Tiputini Biodiversity Station (0°38.2''S, 76°08.9''W)), Planinasus miradorus (Brazil. Maranhão: Parque Estadual Mirador, Base da Geraldina (06°22.2''S, 44°21.8''W)), Planinasus tobagoensis (Trinidad and Tobago. Tobago. St. John: Parlatuvier (11°17.9''N, 60°39''W)), Planinasus xanthops (Ecuador. Orellana: Rio Tiputini Biodiversity Station (0°38.2''S, 76°8.9''W)), Planinasus argentifacies (Peru. Madre de Dios: Río Manu, Pakitza (11°56.6''S, 71°16.9''W; 250 m)), Planinasus insulanus (Dominican Republic. La Vega: near Jarabacoa, Salto Guasara (19°04.4''N, 70°42.1''W, 680 m)), Planinasus nigritarsus (Guyana. Conservation of Ecological Interactions and Biotic Associations (CEIBA; ca. 40 km S Georgetown; 06°29.9''N, 58°13.1''W)), Planinasus atriclypeus (Brazil. Rio de Janeiro: Rio de Janeiro, Floresta da Tijuca (22°57.6''S, 43°16.4''W)), Planinasus atrifrons (Bolivia. Santa Cruz: Ichilo, Buena Vista (4-6 km SSE; Hotel Flora y Fauna; 17°29.95''S, 63°33.15''W; 4-500 m)), P. flavicoxalis (West Indies. Dominica. St. David: 1.6 km N of junction of roads to Rosalie and Castle Bruce (15°23.8''N, 61°18.6''W)), Planinasus mcalpineorum (Mexico. Chiapas: Cacahoatan (7 km N; 15°04.1''N, 92°07.4''W)), Planinasus nigrifacies (Brazil. São Paulo: Mogi das Cruzes, Serra do Itapeti (23°31.5''S, 46°11.2''W)), Planinasus obscuripennis (Peru. Madre de Dios: Río Manu, Erika (near Salvación; 12°50.7''S, 71°23.3''W; 550 m)). In addition to external characters, we also describe and illustrate structures of the male terminalia and for Planinasus kotrbae
sp. n., the internal female reproductive organs. Detailed locality data and distribution maps for all species are provided. For perspective and to facilitate genus-group and species-group recognition, the family Periscelididae and subfamily Stenomicrinae are diagnosed and for the latter, a key to included genera is provided. 相似文献
6.
Kazuyuki Kitatani Kely Sheldon Vinodh Rajagopalan Viviana Anelli Russell W. Jenkins Ying Sun Gregory A. Grabowski Lina M. Obeid Yusuf A. Hannun 《The Journal of biological chemistry》2009,284(19):12972-12978
Activation of protein kinase C (PKC) promotes the salvage pathway of
ceramide formation, and acid sphingomyelinase has been implicated, in part, in
providing substrate for this pathway (Zeidan, Y. H., and Hannun, Y. A. (2007)
J. Biol. Chem. 282, 11549–11561). In the present study, we
examined whether acid β-glucosidase 1 (GBA1), which hydrolyzes
glucosylceramide to form lysosomal ceramide, was involved in PKC-regulated
formation of ceramide from recycled sphingosine. Glucosylceramide levels
declined after treatment of MCF-7 cells with a potent PKC activator, phorbol
12-myristate 13-acetate (PMA). Silencing GBA1 by small interfering RNAs
significantly attenuated acid glucocerebrosidase activity and decreased
PMA-induced formation of ceramide by 50%. Silencing GBA1 blocked PMA-induced
degradation of glucosylceramide and generation of sphingosine, the source for
ceramide biosynthesis. Reciprocally, forced expression of GBA1 increased
ceramide levels. These observations indicate that GBA1 activation can generate
the source (sphingosine) for PMA-induced formation of ceramide through the
salvage pathway. Next, the role of PKCδ, a direct effector of PMA, in
the formation of ceramide was determined. By attenuating expression of
PKCδ, cells failed to trigger PMA-induced alterations in levels of
ceramide, sphingomyelin, and glucosylceramide. Thus, PKCδ activation is
suggested to stimulate the degradation of both sphingomyelin and
glucosylceramide leading to the salvage pathway of ceramide formation.
Collectively, GBA1 is identified as a novel source of regulated formation of
ceramide, and PKCδ is an upstream regulator of this pathway.Sphingolipids are abundant components of cellular membranes, many of which
are emerging as bioactive lipid mediators thought to play crucial roles in
cellular responses (1,
2). Ceramide, a central
sphingolipid, serves as the main precursor for various sphingolipids,
including glycosphingolipids, gangliosides, and sphingomyelin. Regulation of
formation of ceramide has been demonstrated through the action of three major
pathways: the de novo pathway
(3,
4), the sphingomyelinase
pathway (5), and the salvage
pathway
(6–8).
The latter plays an important role in constitutive sphingolipid turnover by
salvaging long-chain sphingoid bases (sphingosine and dihydrosphingosine) that
serve as sphingolipid backbones for ceramide and dihydroceramide as well as
all complex sphingolipids (Fig.
1A).Open in a separate windowFIGURE 1.The scheme of the sphingosine salvage pathway of ceramide formation and
inhibition of PMA induction of ceramide by fumonisin B1. A, the
scheme of the sphingosine salvage pathway of ceramide formation. B,
previously published data as to effects of fumonisin B1 on ceramide mass
profiles (23) are re-plotted
as a PMA induction of ceramide. In brief, MCF-7 cells were pretreated with or
without 100 μm fumonisin B1 for 2 h followed by treatment with
100 nm PMA for 1 h. Lipids were extracted, and then the levels of
ceramide species were determined by high-performance liquid
chromatography-tandem mass spectrometry. Results are expressed as sum of
increased mass of ceramide species. Dotted or open columns
represents C16-ceramide or sum of other ceramide species
(C14-ceramide, C18-ceramide, C18:1-ceramide,
C20-ceramide, C24-ceramide, and
C24:1-ceramide), respectively. The data represent mean ±
S.E. of three to five values.Metabolically, ceramide is also formed from degradation of
glycosphingolipids (Fig.
1A) usually in acidic compartments, the lysosomes and/or
late endosomes (9). The
stepwise hydrolysis of complex glycosphingolipids eventually results in the
formation of glucosylceramide, which in turn is converted to ceramide by the
action of acid β-glucosidase 1
(GBA1)2
(9,
10). Severe defects in GBA1
activity cause Gaucher disease, which is associated with aberrant accumulation
of the lipid substrates
(10–14).
On the other hand, sphingomyelin is cleaved by acid sphingomyelinase to also
form ceramide (15,
16). Either process results in
the generation of lysosomal ceramide that can then be deacylated by acid
ceramidase (17), releasing
sphingosine that may escape the lysosome
(18). The released sphingosine
may become a substrate for either sphingosine kinases or ceramide synthases,
forming sphingosine 1-phosphate or ceramide, respectively
(3,
19–21).In a related line of investigation, our studies
(20,
22,
23) have begun to implicate
protein kinase Cs (PKC) as upstream regulators of the sphingoid base salvage
pathway resulting in ceramide synthesis. Activation of PKCs by the phorbol
ester (PMA) was shown to stimulate the salvage pathway resulting in increases
in ceramide. All the induced ceramide was inhibited by pretreatment with a
ceramide synthase inhibitor, fumonisin B1, but not by myriocin, thus negating
acute activation of the de novo pathway and establishing a role for
ceramide synthesis (20,
23). Moreover, labeling
studies also implicated the salvage pathway because PMA induced turnover of
steady state-labeled sphingolipids but did not affect de novo labeled
ceramide in pulse-chase experiments.Moreover, PKCδ, among PKC isoforms, was identified as an upstream
molecule for the activation of acid sphingomyelinase in the salvage pathway
(22). Interestingly, the
PKCδ isoform induced the phosphorylation of acid sphingomyelinase at
serine 508, leading to its activation and consequent formation of ceramide.
The activation of acid sphingomyelinase appeared to contribute to ∼50% of
the salvage pathway-induced increase in ceramide
(28) (also, see
Fig. 4C). This raised
the possibility that distinct routes of ceramide metabolism may account for
the remainder of ceramide generation. In this study, we investigated
glucocerebrosidase GBA1 as a candidate for one of the other routes accounting
for PKC-regulated salvage pathway of ceramide formation.Open in a separate windowFIGURE 4.Effects of knockdown of lysosomal enzymes on the generation of ceramide
after PMA treatment. A, MCF-7 cells were transfected with 5
nm siRNAs of each of four individual sequences (SCR, GBA1-a,
GBA1-b, and GBA1-c) for 48 h and then stimulated with 100 nm PMA
for 1 h. Lipids were extracted, and then the levels of the
C16-ceramide species were determined by high-performance liquid
chromatography-tandem mass spectrometry. The data represent mean ± S.E.
of three to nine values. B, MCF-7 cells were transfected with 5
nm siRNAs of SCR or GBA1-a (GBA1) for 48 h and then stimulated with
100 nm PMA for 1 h. Lipids were extracted, and then the levels of
individual ceramide species were determined by high-performance liquid
chromatography-tandem mass spectrometry. The data represent mean ± S.E.
of three to five values. C14-Cer,
C14-ceramide; C16-Cer,
C16-ceramide; C18-Cer;
C18-ceramide; C18:1-Cer,
C18:1-ceramide; C20-Cer,
C20-ceramide; C20-Cer,
C24-ceramide; C24:1-Cer,
C24:1-ceramide. C, MCF-7 cells were transfected with 5
nm siRNAs of SCR, acid sphingomyelinase (ASM), or GBA1-a
(GBA1) for 48 h following stimulation with (PMA) or without
(Control) 100 nm PMA for 1 h. Lipids were extracted, and
then the levels of ceramide species were determined by high-performance liquid
chromatography-tandem mass spectrometry. Levels of C16-ceramide are
shown. The data represent mean ± S.E. of four to five values.
Significant changes from SCR-transfected cells treated with PMA are shown in
A–C (*, p < 0.02; **,
p < 0.05; ***, p < 0.01). 相似文献
7.
8.
Duvalius (sg. Neoduvalius) gejzadunayi
sp. n. from Pećina u Dubokom potoku cave ( Donje Biševo village near Rožaje, Montenegro), the first known representative of this subgenus from the territory of Montenegro is described, illustrated and compared with the related species of the subgenus Neoduvalius Müller, 1913. This new species is characterised by depigmented, medium sized body, totally reduced eyes, deep and complete frontal furrows, 3–4 pairs of discal setae in third elytral stria, as well as by the shape of aedeagus. Data on the distribution and the ecology of this remarkable species, as well as a check-list of the subgenus Neoduvalius are also provided. Recently described genera Serboduvalius Ćurčić, S. B. Pavićević & Ćurčić, B.P.M., 2001, Rascioduvalius Ćurčić, S. B. Brajković, Mitić & Ćurčić, B.P.M., 2003, Javorella Ćurčić, S. B. Brajković, Ćurčić, B.P.M. & Mitić, 2003 and Curcicia Ćurčić, S. B. & Brajković, 2003 are regarded as junior synonyms of the genus Duvalius Delarouzée. 相似文献
9.
10.
cDNA
corresponding to the GA4 gene of
Arabidopsis thaliana L. (Heynh.) was
expressed in Escherichia coli, from which cell lysates
converted [14C]gibberellin (GA)9 and
[14C]GA20 to radiolabeled GA4 and
GA1, respectively, thereby confirming that
GA4 encodes a GA 3β-hydroxylase. GA9 was
the preferred substrate, with a Michaelis value of 1 μm
compared with 15 μm for GA20. Hydroxylation
of these GAs was regiospecific, with no indication of
2β-hydroxylation or 2,3-desaturation. The capacity of the recombinant
enzyme to hydroxylate a range of other GA substrates was investigated.
In general, the preferred substrates contained a polar bridge between
C-4 and C-10, and 13-deoxy GAs were preferred to their 13-hydroxylated
analogs. Therefore, no activity was detected using
GA12-aldehyde, GA12, GA19,
GA25, GA53, or GA44 as the open
lactone (20-hydroxy-GA53), whereas GA15,
GA24, and GA44 were hydroxylated to
GA37, GA36, and GA38, respectively.
The open lactone of GA15 (20-hydroxy-GA12) was
hydroxylated but less efficiently than GA15. In contrast to
the free acid, GA25 19,20-anhydride was 3β-hydroxylated
to give GA13. 2,3-Didehydro-GA9 and
GA5 were converted by recombinant GA4 to the corresponding
epoxides 2,3-oxido-GA9 and GA6.Dwarf mutants with reduced biosynthesis of the GA plant hormones
have been valuable tools in studies of the function of these compounds
(Ross, 1994). In Arabidopsis thaliana, mutations
at six loci (GA1-GA6) that result in reduced GA
biosynthesis have been identified (Koorneef and van der Veen, 1980;
Sponsel et al., 1997), and three of these loci have recently been
cloned. The GA1 locus was isolated by genomic subtraction
(Sun et al., 1992) and shown by heterologous expression in
Escherichia coli to encode the enzyme that cyclizes
geranylgeranyl diphosphate to copalyl diphosphate (Sun and Kamiya,
1994). This enzyme was formerly referred to as ent-kaurene
synthase A but has been renamed copalyl diphosphate synthase
(Hedden and Kamiya, 1997; MacMillan, 1997). The GA5
locus was shown to correspond to one of the GA 20-oxidase genes (Xu et
al., 1995), the products of which catalyze the conversion of
GA12 to GA9 and
GA53 to GA20 (Phillips et
al., 1995; Xu et al., 1995). GA 20-oxidases are
2-oxoglutarate-dependent dioxygenases that are encoded by small
multigene families, members of which are differentially expressed in
plant tissues (Phillips et al., 1995; Garcia-Martinez et al., 1997).The GA4 locus was isolated by T-DNA tagging and, on the
basis of the derived amino acid sequence, was also shown to encode a
dioxygenase (Chiang et al., 1995). Several lines of evidence indicate
that the GA4 gene encodes a GA 3β-hydroxylase. Shoots of a
ga4 mutant, all alleles of which are semidwarf, contained
reduced concentrations of the 3β-hydroxy GAs
GA1, GA4, and
GA8 compared with the Landsberg erecta
wild type, whereas levels of immediate precursors to these GAs were
elevated (Talon et al., 1990). Furthermore, metabolism of
[13C]GA20 to
[13C]GA1 was
substantially less in the mutant than in the wild type (Kobayashi et
al., 1994). In the present paper we confirm by functional expression of
its cDNA in E. coli that GA4 encodes a GA
3β-hydroxylase. In addition, we determine the substrate specificity
of recombinant GA4 using a number of C20- and
C19-GAs and show by kinetic analysis that the enzyme
has a higher affinity for GA9 than for
GA20, which is consistent with the
non-13-hydroxylation pathway predominating in Arabidopsis (Talon et
al., 1990). 相似文献
11.
Probing the Conformation of the Fibronectin III1�C2
Domain by Fluorescence Resonance Energy
Transfer
Nancy W. Karuri Zong Lin Hays S. Rye Jean E. Schwarzbauer 《The Journal of biological chemistry》2009,284(6):3445-3452
Fibronectin (FN) matrix is crucial for cell and tissue functions during
embryonic development, wound healing, and oncogenesis. Assembly of FN matrix
fibrils requires FN domains that mediate interactions with integrin receptors
and with other FN molecules. In addition, regulation of FN matrix assembly
depends on the first two FN type III modules, III1 and
III2, which harbor FN-binding sites. We propose that interactions
between these two modules sequester FN-binding sites in soluble FN and that
these sites become exposed by FN conformational changes during assembly. To
test the idea that III1–2 has a compact conformation, we
constructed CIIIY, a conformational sensor of III1–2 based on
fluorescent resonance energy transfer between cyan and yellow fluorescent
proteins conjugated at its N and C termini. We demonstrate energy transfer in
CIIIY and show that fluorescent resonance energy transfer was eliminated by
proteolysis and by treatment with mild denaturants that disrupted
intramolecular interactions between the two modules. We also show that
mutations of key charged residues resulted in conformational changes that
exposed binding sites for the N-terminal 70-kDa FN fragment. Collectively,
these results support a conformation-dependent mechanism for the regulation of
FN matrix assembly by III1–2.Fibronectin (FN)3
is a 500-kDa modular dimeric protein and a major component of the
extracellular matrix. It exists in the blood and other body fluids as a
soluble compact molecule and undergoes cell-mediated assembly to form an
insoluble three-dimensional fibrillar matrix (reviewed in Ref.
1). The process of FN matrix
assembly has been implicated in embryonic development, wound healing, and
cancer
(2–4).
FN is composed of type I–III modules, and sets of these modules comprise
binding domains for cells and for other extracellular matrix components (see
Fig. 1A). Three of
these binding domains are essential for matrix assembly
(1). Integrin receptor
interactions with the cell-binding domain tether disulfide-bonded FN dimers to
the cell surface, where FN-FN interactions involving the N-terminal assembly
domain form dimers into fibrils. In addition to these essential domains, other
FN-binding sites have been implicated in assembly. In particular, the
III1–2 FN-binding domain plays a regulatory role in matrix
assembly. Within this domain reside a cryptic FN-binding site in
III1 and a site available for FN binding in the native form of
III2
(5–8).
Recombinant FN lacking III1 is assembled into a matrix at wild-type
levels, but that lacking the III1–2 domain results in short
immature FN fibrils (8).
Peptides derived from the III1–2 domain or antibodies against
III1–2 block matrix assembly by cultured cells
(9–11).
Furthermore, FN binding to this region is enhanced when FN is mechanically
stretched (12). Taken
together, these results suggest that conformational changes in the
III1–2 domain may control its interactions during FN
assembly.Open in a separate windowFIGURE 1.The FN III1–2 FRET conformational sensor.
A, representation of the domain structure of FN and major interaction
sites. FN is composed of repeating modules that form binding domains for other
FN molecules, cell receptors, and other extracellular matrix components as
indicated. The first two type III modules III1 and III2
(black), have FN-binding sites and regulate FN matrix assembly. The
N-terminal 70-kDa region contains a matrix assembly domain with FN-binding
activity. The cell-binding domain (cell), the heparin-binding domain
(heparin), the dimerization site (SS), and the alternatively
spliced type IIIA (A), IIIB (B), and variable regions
(V) are indicated. 70kD, N-terminal 70-kDa FN fragment.
B, schematic of proposed model of III1–2 domain
conformation. Panel i, in solution, the FN-binding sites in
III1 and III2 (hatched areas) are sequestered
through domain orientations that are facilitated by the linker between modules
(thin line). Panel ii, binding sites are exposed through
conformational changes resulting from cell-mediated extension of FN
(arrows). The length of the linker and the height and width of the
modules are drawn to scale for a linear peptide and published data on FN type
III modules, respectively. C, ribbon diagram representation of CIIIY,
a FRET sensor of the model in B (panel i), oriented with N
and C termini 50 Å apart. CIIIY consists of the III1–2
domain with CFP at the N terminus and YFP at the C terminus.To more fully understand the roles of native and cryptic FN-binding sites
in matrix assembly, the conformational dynamics of III1–2
must be characterized. One approach to this problem is to tag
III1–2 with fluorescent probes, which, in conjunction with
fluorescent resonance energy transfer (FRET), create a molecular
conformational sensor. FRET involves the radiationless transfer of energy from
an excited donor fluorophore to an acceptor fluorophore, a process that is
very sensitive to the distance between the two fluorophores
(13–15).
Two fluorescent protein variants, cyan fluorescent protein (CFP) and yellow
fluorescent protein (YFP), are highly related to green fluorescent protein
(GFP). Because the emission spectrum of CFP is well matched to the excitation
spectrum of YFP, these two fluorophores have been widely used as a
donor-acceptor pair in FRET studies
(13–15).In this study, we describe a FRET conformational sensor designed to test
the idea that intramolecular interactions between III1 and
III2 sequester key FN-binding and assembly sites. We show that
III1–2 with CFP and YFP fused to the N and C termini,
respectively, displays a clear FRET signal, indicating that the attached
fluorescent proteins and thus the ends of III1–2 are in close
proximity. FRET data from III1–2 mutants support the presence
of a stabilizing intermodule salt bridge that regulates FN-binding
activity. 相似文献
12.
13.
The DSM-IV major depression "bereavement exclusion" (BE), which recognizes that depressive symptoms are sometimes normal in recently bereaved individuals, is proposed for elimination in DSM-5. Evidence cited for the BE's invalidity comes from two 2007 reviews purporting to show that bereavement-related depression is similar to other depression across various validators, and a 2010 review of subsequent research. We examined whether the 2007 and 2010 reviews and subsequent relevant literature support the BE's invalidity. Findings were: a) studies included in the 2007 reviews sampled bereavement-related depression groups most of whom were not BE-excluded, making them irrelevant for evaluating BE validity; b) three subsequent studies cited by the 2010 review as supporting BE elimination did examine BE-excluded cases but were in fact inconclusive; and c) two more recent articles comparing recurrence of BE-excluded and other major depressive disorder cases both support the BE's validity. We conclude that the claimed evidence for the BE's invalidity does not exist. The evidence in fact supports the BE's validity and its retention in DSM-5 to prevent false positive diagnoses. We suggest some improvements to increase validity and mitigate risk of false negatives. 相似文献
14.
Christian Rosker Gargi Meur Emily J. A. Taylor Colin W. Taylor 《The Journal of biological chemistry》2009,284(8):5186-5194
Ryanodine receptors (RyR) are Ca2+ channels that mediate
Ca2+ release from intracellular stores in response to diverse
intracellular signals. In RINm5F insulinoma cells, caffeine, and
4-chloro-m-cresol (4CmC), agonists of RyR, stimulated Ca2+
entry that was independent of store-operated Ca2+ entry, and
blocked by prior incubation with a concentration of ryanodine that inactivates
RyR. Patch-clamp recording identified small numbers of large-conductance
(γK = 169 pS) cation channels that were activated by
caffeine, 4CmC or low concentrations of ryanodine. Similar channels were
detected in rat pancreatic β-cells. In RINm5F cells, the channels were
blocked by cytosolic, but not extracellular, ruthenium red. Subcellular
fractionation showed that type 3 IP3 receptors (IP3R3)
were expressed predominantly in endoplasmic reticulum, whereas RyR2 were
present also in plasma membrane fractions. Using RNAi selectively to reduce
expression of RyR1, RyR2, or IP3R3, we showed that RyR2 mediates
both the Ca2+ entry and the plasma membrane currents evoked by
agonists of RyR. We conclude that small numbers of RyR2 are selectively
expressed in the plasma membrane of RINm5F pancreatic β-cells, where they
mediate Ca2+ entry.Ryanodine receptors
(RyR)3 and inositol
1,4,5-trisphosphate receptors (IP3R)
(1,
2) are the archetypal
intracellular Ca2+ channels. Both are widely expressed, although
RyR are more restricted in their expression than IP3R
(3,
4). In common with many cells,
pancreatic β-cells and insulin-secreting cell lines express both
IP3R (predominantly IP3R3)
(5,
6) and RyR (predominantly RyR2)
(7). Both RyR and
IP3R are expressed mostly within membranes of the endoplasmic (ER),
where they mediate release of Ca2+. Functional RyR are also
expressed in the secretory vesicles
(8,
9) or, and perhaps more likely,
in the endosomes of β-cells
(10). Despite earlier
suggestions (11),
IP3R are probably not present in the secretory vesicles of
β-cells (8,
12,
13).All three subtypes of IP3R are stimulated by IP3 with
Ca2+ (1), and the
three subtypes of RyR are each directly regulated by Ca2+. However,
RyR differ in whether their most important physiological stimulus is
depolarization of the plasma membrane (RyR1), Ca2+ (RyR2) or
additional intracellular messengers like cyclic ADP-ribose. The latter
stimulates both Ca2+ release and insulin secretion in β-cells
(8,
14). The activities of both
families of intracellular Ca2+ channels are also modulated by many
additional signals that act directly or via phosphorylation
(15,
16). Although they commonly
mediate release of Ca2+ from the ER, both IP3R and RyR
select rather poorly between Ca2+ and other cations (permeability
ratio, PCa/PK ∼7)
(1,
17). This may allow
electrogenic Ca2+ release from the ER to be rapidly compensated by
uptake of K+ (18),
and where RyR or IP3R are expressed in other membranes it may allow
them to affect membrane potential.Both Ca2+ entry and release of Ca2+ from
intracellular stores contribute to the oscillatory increases in cytosolic
Ca2+ concentration ([Ca2+]i) that
stimulate exocytosis of insulin-containing vesicles in pancreatic β-cells
(7). Glucose rapidly
equilibrates across the plasma membrane (PM) of β-cells and its oxidative
metabolism by mitochondria increases the cytosolic ATP/ADP ratio, causing
KATP channels to close
(19). This allows an
unidentified leak current to depolarize the PM
(20) and activate
voltage-gated Ca2+ channels, predominantly L-type Ca2+
channels (21). The resulting
Ca2+ entry is amplified by Ca2+-induced Ca2+
release from intracellular stores
(7), triggering exocytotic
release of insulin-containing dense-core vesicles
(22). The importance of this
sequence is clear from the widespread use of sulfonylurea drugs, which close
KATP channels, in the treatment of type 2 diabetes. Ca2+
uptake by mitochondria beneath the PM further stimulates ATP production,
amplifying the initial response to glucose and perhaps thereby contributing to
the sustained phase of insulin release
(23). However, neither the
increase in [Ca2+]i nor the insulin release
evoked by glucose or other nutrients is entirely dependent on Ca2+
entry (7,
24) or closure of
KATP channels (25).
This suggests that glucose metabolism may also more directly activate RyR
(7,
26) and/or IP3R
(27) to cause release of
Ca2+ from intracellular stores. A change in the ATP/ADP ratio is
one means whereby nutrient metabolism may be linked to opening of
intracellular Ca2+ channels because both RyR
(28) and IP3R
(1) are stimulated by ATP.The other major physiological regulators of insulin release are the
incretins: glucagon-like peptide-1 and glucose-dependent insulinotropic
hormone (29). These hormones,
released by cells in the small intestine, stimulate synthesis of cAMP in
β-cells and thereby potentiate glucose-evoked insulin release
(30). These pathways are also
targets of drugs used successfully to treat type 2 diabetes
(29). The responses of
β-cells to cAMP involve both cAMP-dependent protein kinase and epacs
(exchange factors activated by cAMP)
(31,
32). The effects of the latter
are, at least partly, due to release of Ca2+ from intracellular
stores via RyR
(33–35)
and perhaps also via IP3R
(36). The interplays between
Ca2+ and cAMP signaling generate oscillatory changes in the
concentrations of both messengers
(37). RyR and IP3R
are thus implicated in mediating responses to each of the major physiological
regulators of insulin secretion: glucose and incretins.Here we report that in addition to expression in intracellular stores,
which probably include both the ER and secretory vesicles and/or endosomes,
functional RyR2 are also expressed in small numbers in the PM of RINm5F
insulinoma cells and rat pancreatic β-cells. 相似文献
15.
16.
17.
Eugenijus ?imoliūnas Laura Kaliniene Lidija Truncait? Aurelija Zajan?kauskait? Juozas Staniulis Algirdas Kaupinis Marija Ger Mindaugas Valius Rolandas Me?kys 《PloS one》2013,8(4)
At 346 kbp in size, the genome of a jumbo bacteriophage vB_KleM-RaK2 (RaK2) is the largest Klebsiella infecting myovirus genome sequenced to date. In total, 272 out of 534 RaK2 ORFs lack detectable database homologues. Based on the similarity to biologically defined proteins and/or MS/MS analysis, 117 of RaK2 ORFs were given a functional annotation, including 28 RaK2 ORFs coding for structural proteins that have no reliable homologues to annotated structural proteins in other organisms. The electron micrographs revealed elaborate spike-like structures on the tail fibers of Rak2, suggesting that this phage is an atypical myovirus. While head and tail proteins of RaK2 are mostly myoviridae-related, the bioinformatics analysis indicate that tail fibers/spikes of this phage are formed from podovirus-like peptides predominantly. Overall, these results provide evidence that bacteriophage RaK2 differs profoundly from previously studied viruses of the Myoviridae family. 相似文献
18.
The tiger beetle fauna of the Balkan Peninsula is one of the richest in Europe and includes 19 species or 41% of the European tiger beetle fauna. Assembled by their biogeographical origins, the Balkan tiger beetle species fall into 14 different groups that include, Mediterranean, Middle Oriental, Central Asiatic, Euro-Siberian, South and East European, Pannonian-Sarmatian, West Palaearctic, Turano-European and Afrotropico Indo-Mediterranean species. The Mediterranean Sclerophyl and the Pontian Steppe are the Balkan biogeographical provinces with the highest species richness, while the Balkan Highlands has the lowest Cicindelidae diversity. Most species are restricted to single habitat types in lowland areas of the Balkan Peninsula and only Calomera aulica aulica and Calomera littoralis nemoralis occur in respectively 3 and 4 different types of habitat. About 60% of all Balkan Cicindelidae species are found in habitats potentially endangered by human activity. 相似文献
19.
20.
Rhamnogalacturonan α-d-Galactopyranosyluronohydrolase
: An Enzyme That Specifically Removes the Terminal Nonreducing
Galacturonosyl Residue in Rhamnogalacturonan Regions of
Pectin1
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Margien Mutter Gerrit Beldman Stuart M. Pitson Henk A. Schols Alphons G.J. Voragen 《Plant physiology》1998,117(1):153-163
A new enzyme, rhamnogalacturonan (RG) α-d-galactopyranosyluronohydrolase (RG-galacturonohydrolase), able to release a galacturonic acid residue from the nonreducing end of RG chains but not from homogalacturonan, was purified from an Aspergillus aculeatus enzyme preparation. RG-galacturonohydrolase acted with inversion of anomeric configuration, initially releasing β-d-galactopyranosyluronic acid. The enzyme cleaved smaller RG substrates with the highest catalytic efficiency. A Michaelis constant of 85 μm and a maximum reaction rate of 160 units mg−1 was found toward a linear RG fragment with a degree of polymerization of 6. RG-galacturonohydrolase had a molecular mass of 66 kD, an isoelectric point of 5.12, a pH optimum of 4.0, and a temperature optimum of 50°C. The enzyme was most stable between pH 3.0 and 6.0 (for 24 h at 40°C) and up to 60°C (for 3 h). 相似文献