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1.
S��bastien Thomas Brigitte Ritter David Verbich Claire Sanson Lyne Bourbonni��re R. Anne McKinney Peter S. McPherson 《The Journal of biological chemistry》2009,284(18):12410-12419
Intersectin-short (intersectin-s) is a multimodule scaffolding protein
functioning in constitutive and regulated forms of endocytosis in non-neuronal
cells and in synaptic vesicle (SV) recycling at the neuromuscular junction of
Drosophila and Caenorhabditis elegans. In vertebrates,
alternative splicing generates a second isoform, intersectin-long
(intersectin-l), that contains additional modular domains providing a guanine
nucleotide exchange factor activity for Cdc42. In mammals, intersectin-s is
expressed in multiple tissues and cells, including glia, but excluded from
neurons, whereas intersectin-l is a neuron-specific isoform. Thus,
intersectin-I may regulate multiple forms of endocytosis in mammalian neurons,
including SV endocytosis. We now report, however, that intersectin-l is
localized to somatodendritic regions of cultured hippocampal neurons, with
some juxtanuclear accumulation, but is excluded from synaptophysin-labeled
axon terminals. Consistently, intersectin-l knockdown (KD) does not affect SV
recycling. Instead intersectin-l co-localizes with clathrin heavy chain and
adaptor protein 2 in the somatodendritic region of neurons, and its KD reduces
the rate of transferrin endocytosis. The protein also co-localizes with
F-actin at dendritic spines, and intersectin-l KD disrupts spine maturation
during development. Our data indicate that intersectin-l is indeed an
important regulator of constitutive endocytosis and neuronal development but
that it is not a prominent player in the regulated endocytosis of SVs.Clathrin-mediated endocytosis
(CME)4 is a
major mechanism by which cells take up nutrients, control the surface levels
of multiple proteins, including ion channels and transporters, and regulate
the coupling of signaling receptors to downstream signaling cascades
(1-5).
In neurons, CME takes on additional specialized roles; it is an important
process regulating synaptic vesicle (SV) availability through endocytosis and
recycling of SV membranes (6,
7), it shapes synaptic
plasticity
(8-10),
and it is crucial in maintaining synaptic membranes and membrane structure
(11).Numerous endocytic accessory proteins participate in CME, interacting with
each other and with core components of the endocytic machinery such as
clathrin heavy chain (CHC) and adaptor protein-2 (AP-2) through specific
modules and peptide motifs
(12). One such module is the
Eps15 homology domain that binds to proteins bearing NPF motifs
(13,
14). Another is the Src
homology 3 (SH3) domain, which binds to proline-rich domains in protein
partners (15). Intersectin is
a multimodule scaffolding protein that interacts with a wide range of
proteins, including several involved in CME
(16). Intersectin has two
N-terminal Eps15 homology domains that are responsible for binding to epsin,
SCAMP1, and numb
(17-19),
a central coil-coiled domain that interacts with Eps15 and SNAP-23 and -25
(17,
20,
21), and five SH3 domains in
its C-terminal region that interact with multiple proline-rich domain
proteins, including synaptojanin, dynamin, N-WASP, CdGAP, and mSOS
(16,
22-25).
The rich binding capability of intersectin has linked it to various functions
from CME (17,
26,
27) and signaling
(22,
28,
29) to mitogenesis
(30,
31) and regulation of the
actin cytoskeleton (23).Intersectin functions in SV recycling at the neuromuscular junction of
Drosophila and C. elegans where it acts as a scaffold,
regulating the synaptic levels of endocytic accessory proteins
(21,
32-34).
In vertebrates, the intersectin gene is subject to alternative splicing, and a
longer isoform (intersectin-l) is generated that is expressed exclusively in
neurons (26,
28,
35,
36). This isoform has all the
binding modules of its short (intersectin-s) counterpart but also has
additional domains: a DH and a PH domain that provide guanine nucleotide
exchange factor (GEF) activity specific for Cdc42
(23,
37) and a C2 domain at the C
terminus. Through its GEF activity and binding to actin regulatory proteins,
including N-WASP, intersectin-l has been implicated in actin regulation and
the development of dendritic spines
(19,
23,
24). In addition, because the
rest of the binding modules are shared between intersectin-s and -l, it is
generally thought that the two intersectin isoforms have the same endocytic
functions. In particular, given the well defined role for the invertebrate
orthologs of intersectin-s in SV endocytosis, it is thought that intersectin-l
performs this role in mammalian neurons, which lack intersectin-s. Defining
the complement of intersectin functional activities in mammalian neurons is
particularly relevant given that the protein is involved in the
pathophysiology of Down syndrome (DS). Specifically, the intersectin gene is
localized on chromosome 21q22.2 and is overexpressed in DS brains
(38). Interestingly,
alterations in endosomal pathways are a hallmark of DS neurons and neurons
from the partial trisomy 16 mouse, Ts65Dn, a model for DS
(39,
40). Thus, an endocytic
trafficking defect may contribute to the DS disease process.Here, the functional roles of intersectin-l were studied in cultured
hippocampal neurons. We find that intersectin-l is localized to the
somatodendritic regions of neurons, where it co-localizes with CHC and AP-2
and regulates the uptake of transferrin. Intersectin-l also co-localizes with
actin at dendritic spines and disrupting intersectin-l function alters
dendritic spine development. In contrast, intersectin-l is absent from
presynaptic terminals and has little or no role in SV recycling. 相似文献
2.
Jaemin Lee Xiaofan Wang Bruno Di Jeso Peter Arvan 《The Journal of biological chemistry》2009,284(19):12752-12761
The carboxyl-terminal cholinesterase-like (ChEL) domain of thyroglobulin
(Tg) has been identified as critically important in Tg export from the
endoplasmic reticulum. In a number of human kindreds suffering from congenital
hypothyroidism, and in the cog congenital goiter mouse and
rdw rat dwarf models, thyroid hormone synthesis is inhibited because
of mutations in the ChEL domain that block protein export from the endoplasmic
reticulum. We hypothesize that Tg forms homodimers through noncovalent
interactions involving two predicted α-helices in each ChEL domain that
are homologous to the dimerization helices of acetylcholinesterase. This has
been explored through selective epitope tagging of dimerization partners and
by inserting an extra, unpaired Cys residue to create an opportunity for
intermolecular disulfide pairing. We show that the ChEL domain is necessary
and sufficient for Tg dimerization; specifically, the isolated ChEL domain can
dimerize with full-length Tg or with itself. Insertion of an N-linked
glycan into the putative upstream dimerization helix inhibits homodimerization
of the isolated ChEL domain. However, interestingly, co-expression of upstream
Tg domains, either in cis or in trans, overrides the
dimerization defect of such a mutant. Thus, although the ChEL domain provides
a nidus for Tg dimerization, interactions of upstream Tg regions with the ChEL
domain actively stabilizes the Tg dimer complex for intracellular
transport.The synthesis of thyroid hormone in the thyroid gland requires secretion of
thyroglobulin (Tg)2 to
the apical luminal cavity of thyroid follicles
(1). Once secreted, Tg is
iodinated via the activity of thyroid peroxidase
(2). A coupling reaction
involving a quinol-ether linkage especially engages di-iodinated tyrosyl
residues 5 and 130 to form thyroxine within the amino-terminal portion of the
Tg polypeptide (3,
4). Preferential iodination of
Tg hormonogenic sites is dependent not on the specificity of the peroxidase
(5) but upon the native
structure of Tg (6,
7). To date, no other thyroidal
proteins have been shown to effectively substitute in this role for Tg.The first 80% of the primary structure of Tg (full-length murine Tg: 2,746
amino acids) involves three regions called I-II-III comprised of
disulfide-rich repeat domains held together by intradomain disulfide bonds
(8,
9). The final 581 amino acids
of Tg are strongly homologous to acetylcholinesterase
(10–12).
Rate-limiting steps in the overall process of Tg secretion involve its
structural maturation within the endoplasmic reticulum (ER)
(13). Interactions between
regions I-II-III and the cholinesterase-like (ChEL) domain have recently been
suggested to be important in this process, with ChEL functioning as an
intramolecular chaperone and escort for I-II-III
(14). In addition, Tg
conformational maturation culminates in Tg homodimerization
(15,
16) with progression to a
cylindrical, and ultimately, a compact ovoid structure
(17–19).In human congenital hypothyroidism with deficient Tg, the ChEL domain is a
commonly affected site of mutation, including the recently described A2215D
(20,
21), R2223H
(22), G2300D, R2317Q
(23), G2355V, G2356R, and the
skipping of exon 45 (which normally encodes 36 amino acids), as well as the
Q2638stop mutant (24) (in
addition to polymorphisms including P2213L, W2482R, and R2511Q that may be
associated with thyroid overgrowth
(25)). As best as is currently
known, all of the congenital hypothyroidism-inducing Tg mutants are defective
for intracellular transport
(26). A homozygous G2300R
mutation (equivalent to residue 2,298 of mouse Tg) in the ChEL domain is
responsible for congenital hypothyroidism in rdw rats
(27,
28), whereas we identified the
Tg-L2263P point mutation as the cause of hypothyroidism in the cog
mouse (29). Such mutations
perturb intradomain structure
(30), and interestingly, block
homodimerization (31).
Acquisition of quaternary structure has long been thought to be required for
efficient export from the ER
(32) as exemplified by
authentic acetylcholinesterase
(33,
34) in which dimerization
enhances protein stability and export
(35).Tg comprised only of regions I-II-III (truncated to lack the ChEL domain)
is blocked within the ER (30),
whereas a secretory version of the isolated ChEL domain of Tg devoid of
I-II-III undergoes rapid and efficient intracellular transport and secretion
(14). A striking homology
positions two predicted α-helices of the ChEL domain to the identical
relative positions of the dimerization helices in acetylcholinesterase. This
raises the possibility that ChEL may serve as a homodimerization domain for
Tg, providing a critical function in maturation for Tg transport to the site
of thyroid hormone synthesis
(1).In this study, we provide unequivocal evidence for homodimerization of the
ChEL domain and “hetero”-dimerization of that domain with
full-length Tg, and we provide significant evidence that the predicted ChEL
dimerization helices provide a nidus for Tg assembly. On the other hand, our
data also suggest that upstream Tg regions known to interact with ChEL
(14) actively stabilize the Tg
dimer complex. Together, I-II-III and ChEL provide unique contributions to the
process of intracellular transport of Tg through the secretory pathway. 相似文献
3.
4.
Rosanna Pescini Gobert Monique van den Eijnden Cedric Szyndralewiez Catherine Jorand-Lebrun Dominique Swinnen Linfeng Chen Corine Gillieron Fiona Pixley Pierre Juillard Patrick Gerber Caroline Johnson-L��ger Serge Halazy Montserrat Camps Agnes Bombrun Margaret Shipp Pierre-Alain Vitte Vittoria Ardissone Chiara Ferrandi Dominique Perrin Christian Rommel Rob Hooft van Huijsduijnen 《The Journal of biological chemistry》2009,284(17):11385-11395
5.
Parmil K. Bansal Amanda Nourse Rashid Abdulle Katsumi Kitagawa 《The Journal of biological chemistry》2009,284(6):3586-3592
The kinetochore, which consists of DNA sequence elements and structural
proteins, is essential for high-fidelity chromosome transmission during cell
division. In budding yeast, Sgt1 and Hsp90 help assemble the core kinetochore
complex CBF3 by activating the CBF3 components Skp1 and Ctf13. In this study,
we show that Sgt1 forms homodimers by performing in vitro and in
vivo immunoprecipitation and analytical ultracentrifugation analyses.
Analyses of the dimerization of Sgt1 deletion proteins showed that the
Skp1-binding domain (amino acids 1–211) contains the Sgt1
homodimerization domain. Also, the Sgt1 mutant proteins that were unable to
dimerize also did not bind Skp1, suggesting that Sgt1 dimerization is
important for Sgt1-Skp1 binding. Restoring dimerization activity of a
dimerization-deficient sgt1 mutant (sgt1-L31P) by using the
CENP-B (centromere protein-B) dimerization
domain suppressed the temperature sensitivity, the benomyl sensitivity, and
the chromosome missegregation phenotype of sgt1-L31P. These results
strongly suggest that Sgt1 dimerization is required for kinetochore
assembly.Spindle microtubules are coupled to the centromeric region of the
chromosome by a structural protein complex called the kinetochore
(1,
2). The kinetochore is thought
to generate a signal that arrests cells during mitosis when it is not properly
attached to microtubules, thereby preventing aberrant chromosome transmission
to the daughter cells, which can lead to tumorigenesis
(3,
4). The kinetochore of the
budding yeast Saccharomyces cerevisiae has been characterized
thoroughly, genetically and biochemically; thus, its molecular structure is
the most well detailed to date. More than 70 different proteins comprise the
budding yeast kinetochore, and several of those are conserved in mammals
(2).The budding yeast centromere DNA is a 125-bp region that contains three
conserved regions, CDEI, CDEII, and CDEIII
(5,
6). CDEI is bound by Cbf1
(7–9).
CDEIII (25 bp) is essential for centromere function
(10) and is the site where
CBF3 binds to centromeric DNA. CBF3 contains four proteins: Ndc10, Cep3, Ctf13
(11–18),
and Skp1 (17,
18), all of which are
essential for viability. Mutations in any of the four CBF3 proteins abolish
the ability of CDEIII to bind to CBF3
(19,
20). All of the described
kinetochore proteins, except the CDEI-binding Cbf1, localize to kinetochores
dependent on the CBF3 complex
(2). Therefore, the CBF3
complex is the fundamental structure of the kinetochore, and the mechanism of
CBF3 assembly is of major interest.We previously isolated SGT1, the skp1-4
kinetochore-defective mutant dosage suppressor
(21). Sgt1 and Skp1 activate
Ctf13; thus, they are required for assembly of the CBF3 complex
(21). The molecular chaperone
Hsp90 is also required for the formation of the Skp1-Ctf13 complex
(22). Sgt1 has two highly
conserved motifs that are required for protein-protein interaction, the
tetratricopeptide repeat
(TPR)2
(21) and the CS
(CHORD protein- and Sgt1-specific) motif. We and others
(23–26)
have found that both domains are important for the interaction with Hsp90. The
Sgt1-Hsp90 interaction is required for the assembly of the core kinetochore
complex; this interaction is an initial step in kinetochore assembly
(24,
26,
27) that is conserved between
yeast and humans (28,
29).In this study, we further characterized the molecular mechanism of this
assembly process. We found that Sgt1 forms dimers in vivo, and our
results strongly suggest that Sgt1 dimerization is required for kinetochore
assembly in budding yeast. 相似文献
6.
John W. Hardin Francis E. Reyes Robert T. Batey 《The Journal of biological chemistry》2009,284(22):15317-15324
In archaea and eukarya, box C/D ribonucleoprotein (RNP) complexes are
responsible for 2′-O-methylation of tRNAs and rRNAs. The
archaeal box C/D small RNP complex requires a small RNA component (sRNA)
possessing Watson-Crick complementarity to the target RNA along with three
proteins: L7Ae, Nop5p, and fibrillarin. Transfer of a methyl group from
S-adenosylmethionine to the target RNA is performed by fibrillarin,
which by itself has no affinity for the sRNA-target duplex. Instead, it is
targeted to the site of methylation through association with Nop5p, which in
turn binds to the L7Ae-sRNA complex. To understand how Nop5p serves as a
bridge between the targeting and catalytic functions of the box C/D small RNP
complex, we have employed alanine scanning to evaluate the interaction between
the Pyrococcus horikoshii Nop5p domain and an L7Ae box C/D RNA
complex. From these data, we were able to construct an isolated RNA-binding
domain (Nop-RBD) that folds correctly as demonstrated by x-ray crystallography
and binds to the L7Ae box C/D RNA complex with near wild type affinity. These
data demonstrate that the Nop-RBD is an autonomously folding and functional
module important for protein assembly in a number of complexes centered on the
L7Ae-kinkturn RNP.Many biological RNAs require extensive modification to attain full
functionality in the cell (1).
Currently there are over 100 known RNA modification types ranging from small
functional group substitutions to the addition of large multi-cyclic ring
structures (2). Transfer RNA,
one of many functional RNAs targeted for modification
(3-6),
possesses the greatest modification type diversity, many of which are
important for proper biological function
(7). Ribosomal RNA, on the
other hand, contains predominantly two types of modified nucleotides:
pseudouridine and 2′-O-methylribose
(8). The crystal structures of
the ribosome suggest that these modifications are important for proper folding
(9,
10) and structural
stabilization (11) in
vivo as evidenced by their strong tendency to localize to regions
associated with function (8,
12,
13). These roles have been
verified biochemically in a number of cases
(14), whereas newly emerging
functional modifications are continually being investigated.Box C/D ribonucleoprotein
(RNP)3 complexes serve
as RNA-guided site-specific 2′-O-methyltransferases in both
archaea and eukaryotes (15,
16) where they are referred to
as small RNP complexes and small nucleolar RNPs, respectively. Target RNA
pairs with the sRNA guide sequence and is methylated at the 2′-hydroxyl
group of the nucleotide five bases upstream of either the D or D′ box
motif of the sRNA (Fig. 1,
star) (17,
18). In archaea, the internal
C′ and D′ motifs generally conform to a box C/D consensus sequence
(19), and each sRNA contains
two guide regions ∼12 nucleotides in length
(20). The bipartite
architecture of the RNP potentially enables the complex to methylate two
distinct RNA targets (21) and
has been shown to be essential for site-specific methylation
(22).Open in a separate windowFIGURE 1.Organization of the archaeal box C/D complex. The protein components
of this RNP are L7Ae, Nop5p, and fibrillarin, which together bind a box C/D
sRNA. The regions of the Box C/D sRNA corresponding to the conserved C, D,
C′, and D′ boxes are labeled. The target RNA binds the sRNA
through Watson-Crick pairing and is methylated by fibrillarin at the fifth
nucleotide from the D/D′ boxes (star).In addition to the sRNA, the archaeal box C/D complex requires three
proteins for activity (23):
the ribosomal protein L7Ae
(24,
25), fibrillarin, and the
Nop56/Nop58 homolog Nop5p (Fig.
1). L7Ae binds to both box C/D and the C′/D′ motifs
(26), which respectively
comprise kink-turn (27) or
k-loop structures (28), to
initiate the assembly of the RNP
(29,
30). Fibrillarin performs the
methyl group transfer from the cofactor S-adenosylmethionine to the
target RNA
(31-33).
For this to occur, the active site of fibrillarin must be positioned precisely
over the specific 2′-hydroxyl group to be methylated. Although
fibrillarin methylates this functional group in the context of a Watson-Crick
base-paired helix (guide/target), it has little to no binding affinity for
double-stranded RNA or for the L7Ae-sRNA complex
(22,
26,
33,
34). Nop5p serves as an
intermediary protein bringing fibrillarin to the complex through its
association with both the L7Ae-sRNA complex and fibrillarin
(22). Along with its role as
an intermediary between fibrillarin and the L7Ae-sRNA complex, Nop5p possesses
other functions not yet fully understood. For example, Nop5p self-dimerizes
through a coiled-coil domain
(35) that in most archaea and
eukaryotic homologs includes a small insertion sequence of unknown function
(36,
37). However, dimerization and
fibrillarin binding have been shown to be mutually exclusive in
Methanocaldococcus jannaschii Nop5p, potentially because of the
presence of this insertion sequence
(36). Thus, whether Nop5p is a
monomer or a dimer in the active RNP is still under debate.In this study, we focus our attention on the Nop5p protein to investigate
its interaction with a L7Ae box C/D RNA complex because both the
fibrillarin-Nop5p and the L7Ae box C/D RNA interfaces are known from crystal
structures (29,
35,
38). Individual residues on
the surface of a monomeric form of Nop5p (referred to as mNop5p)
(22) were mutated to alanine,
and the effect on binding affinity for a L7Ae box C/D motif RNA complex was
assessed through the use of electrophoretic mobility shift assays. These data
reveal that residues important for binding cluster within the highly conserved
NOP domain (39,
40). To demonstrate that this
domain is solely responsible for the affinity of Nop5p for the preassembled
L7Ae box C/D RNA complex, we expressed and purified it in isolation from the
full Nop5p protein. The isolated Nop-RBD domain binds to the L7Ae box C/D RNA
complex with nearly wild type affinity, demonstrating that the Nop-RBD is
truly an autonomously folding and functional module. Comparison of our data
with the crystal structure of the homologous spliceosomal hPrp31-15.5K
protein-U4 snRNA complex (41)
suggests the adoption of a similar mode of binding, further supporting a
crucial role for the NOP domain in RNP complex assembly. 相似文献
7.
Aggregation of the Ure2 protein is at the origin of the [URE3]
prion trait in the yeast Saccharomyces cerevisiae. The N-terminal
region of Ure2p is necessary and sufficient to induce the [URE3]
phenotype in vivo and to polymerize into amyloid-like fibrils in
vitro. However, as the N-terminal region is poorly ordered in the native
state, making it difficult to detect structural changes in this region by
spectroscopic methods, detailed information about the fibril assembly process
is therefore lacking. Short fibril-forming peptide regions (4–7
residues) have been identified in a number of prion and other amyloid-related
proteins, but such short regions have not yet been identified in Ure2p. In
this study, we identify a unique cysteine mutant (R17C) that can greatly
accelerate the fibril assembly kinetics of Ure2p under oxidizing conditions.
We found that the segment QVNI, corresponding to residues 18–21 in
Ure2p, plays a critical role in the fast assembly properties of R17C,
suggesting that this segment represents a potential amyloid-forming region. A
series of peptides containing the QVNI segment were found to form fibrils
in vitro. Furthermore, the peptide fibrils could seed fibril
formation for wild-type Ure2p. Preceding the QVNI segment with a cysteine or a
hydrophobic residue, instead of a charged residue, caused the rate of assembly
into fibrils to increase greatly for both peptides and full-length Ure2p. Our
results indicate that the potential amyloid stretch and its preceding residue
can modulate the fibril assembly of Ure2p to control the initiation of prion
formation.The [URE3] phenotype of Saccharomyces cerevisiae arises
because of conversion of the Ure2 protein to an aggregated propagatable prion
state (1,
2). Ure2p contains two regions:
a poorly structured N-terminal region and a compactly folded C-terminal region
(3,
4). The N-terminal region is
rich in Asn and Gln residues, is highly flexible, and is without any
detectable ordered secondary structure
(4–6).
This region is necessary and sufficient for prion behavior in vivo
(2) and amyloid-forming
capacity in vitro (5,
7), so it is referred to as the
prion domain (PrD).2
The C-terminal region has a fold similar to the glutathione
S-transferase superfamily
(8,
9) and possesses
glutathione-dependent peroxidase activity
(10). Upon fibril formation,
the N-terminal region undergoes a significant conformational change from an
unfolded to a thermally resistant conformation
(11), whereas the glutathione
S-transferase-like C-terminal domain retains its enzymatic activity,
suggesting that little conformational change occurs
(10,
12). Ure2p fibrils show
various morphologies, including variations in thickness and the presence or
absence of a periodic twist
(13–16).
The overall structure of the fibrils imaged by cryoelectron microscopy
suggests that the intact fibrils contain a 4-nm amyloid filament backbone
surrounded by C-terminal globular domains
(17).It is widely accepted that disulfide bonds play a critical role in
maintaining protein stability
(18–21)
and also affect the process of protein folding by influencing the folding
pathway
(22–25).
A recent study shows that the presence of a disulfide bond in a protein can
markedly accelerate the folding process
(26). Therefore, a disulfide
bond is a useful tool to study protein folding. In the study of prion and
other amyloid-related proteins, cysteine scanning has been widely used to
study the structure of amyloid fibrils, the driving force of amyloid
formation, and the plasticity of amyloid fibrils
(13,
27–31).Short segments from amyloid-related proteins, including IAPP
(islet amyloid polypeptide),
β2-microglobulin, insulin, and the amyloid-β peptide,
show amyloid-forming capacity
(32–34).
Hence, the amyloid stretch hypothesis has been proposed, which suggests that a
short amino acid stretch bearing a highly amyloidogenic motif might supply
most of the driving force needed to trigger the self-catalytic assembly
process of a protein to form fibrils
(35,
36). In support of this
hypothesis, it was found that the insertion of an amyloidogenic stretch into a
non-amyloid-related protein can trigger the amyloidosis of the protein
(36). At the same time, the
structural information obtained from microcrystals formed by amyloidogenic
stretches and bearing cross-β-structure has contributed significantly to
our understanding of the structure of intact fibrils at the atomic level
(34,
37). However, no amyloidogenic
stretches <10 amino acids have so far been identified in the yeast prion
protein Ure2.In this study, we performed a cysteine scan within the N-terminal PrD of
Ure2p and found a unique cysteine mutant (R17C) that eliminates the lag phase
of the Ure2p fibril assembly reaction upon the addition of oxidizing agents.
Furthermore, we identified a 4-residue region adjacent to Arg17 as
a potential amyloid stretch in Ure2p. 相似文献
8.
Siying Wang Wen-Mei Yu Wanming Zhang Keith R. McCrae Benjamin G. Neel Cheng-Kui Qu 《The Journal of biological chemistry》2009,284(2):913-920
Mutations in SHP-2 phosphatase (PTPN11) that cause hyperactivation
of its catalytic activity have been identified in Noonan syndrome and various
childhood leukemias. Recent studies suggest that the gain-of-function (GOF)
mutations of SHP-2 play a causal role in the pathogenesis of these diseases.
However, the molecular mechanisms by which GOF mutations of SHP-2 induce these
phenotypes are not fully understood. Here, we show that GOF mutations in
SHP-2, such as E76K and D61G, drastically increase spreading and migration of
various cell types, including hematopoietic cells, endothelial cells, and
fibroblasts. More importantly, in vivo angiogenesis in SHP-2 D61G
knock-in mice is also enhanced. Mechanistic studies suggest that the increased
cell migration is attributed to the enhanced β1 integrin outside-in
signaling. In response to β1 integrin cross-linking or fibronectin
stimulation, activation of ERK and Akt kinases is greatly increased by SHP-2
GOF mutations. Also, integrin-induced activation of RhoA and Rac1 GTPases is
elevated. Interestingly, mutant cells with the SHP-2 GOF mutation (D61G) are
more sensitive than wild-type cells to the suppression of cell motility by
inhibition of these pathways. Collectively, these studies reaffirm the
positive role of SHP-2 phosphatase in cell motility and suggest a new
mechanism by which SHP-2 GOF mutations contribute to diseases.SHP-2, a multifunctional SH2 domain-containing protein-tyrosine phosphatase
implicated in diverse cell signaling processes
(1–3),
plays a critical role in cellular function. Homozygous deletion of Exon
2 (4) or Exon 3
(5) of the SHP-2 gene
(PTPN11) in mice leads to early embryonic lethality prior to and at
midgestation, respectively. SHP-2 null mutant mice die much earlier, at
peri-implantation (4). Exon
3 deletion mutation of SHP-2 blocks hematopoietic potential of embryonic
stem cells both in vitro and in vivo
(6–8),
whereas SHP-2 null mutation causes inner cell mass death and diminished
trophoblast stem cell survival
(4). Recent studies on SHP-2
conditional knock-out or tissue-specific knock-out mice have further revealed
an array of important functions of this phosphatase in various physiological
processes
(9–12).
The phenotypes demonstrated by loss of SHP-2 function are apparently
attributed to the role of SHP-2 in the cell signaling pathways induced by
growth factors/cytokines. SHP-2 generally promotes signal transmission in
growth factor/cytokine signaling in both catalytic-dependent and -independent
fashion
(1–3).
The positive role of SHP-2 in the intracellular signaling processes, in
particular, the ERK3
and PI3K/Akt kinase pathways, has been well established, although the
underlying mechanism remains elusive, in particular, the signaling function of
the catalytic activity of SHP-2 in these pathways is poorly understood.In addition to the role of SHP-2 in cell proliferation and differentiation,
the phenotypes induced by loss of SHP-2 function may be associated with its
role in cell migration. Indeed, dominant negative SHP-2 disrupts
Xenopus gastrulation, causing tail truncations
(13,
14). Targeted Exon 3
deletion mutation in SHP-2 results in decreased cell spreading, migration
(15,
16), and impaired limb
development in the chimeric mice
(7). The role of SHP-2 in cell
adhesion and migration has also been demonstrated by catalytically inactive
mutant SHP-2-overexpressing cells
(17–20).
The molecular mechanisms by which SHP-2 regulates these cellular processes,
however, have not been well defined. For example, the role of SHP-2 in the
activation of the Rho family small GTPases that is critical for cell motility
is still controversial. Both positive
(19,
21,
22) and negative roles
(18,
23) for SHP-2 in this context
have been reported. Part of the reason for this discrepancy might be due to
the difference in the cell models used. Catalytically inactive mutant SHP-2
was often used to determine the role of SHP-2 in cell signaling. In the
catalytically inactive mutant SHP-2-overexpressing cells, the catalytic
activity of endogenous SHP-2 is inhibited. However, as SHP-2 also functions
independent of its catalytic activity, overexpression of catalytically
deficient SHP-2 may also increase its scaffolding function, generating complex
effects.The critical role of SHP-2 in cellular function is further underscored by
the identification of SHP-2 mutations in human diseases. Genetic lesions in
PTPN11 that cause hyperactivation of SHP-2 catalytic activity have
been identified in the developmental disorder Noonan syndrome
(24) and various childhood
leukemias, including juvenile myelomonocytic leukemia (JMML), B cell acute
lymphoblastic leukemia, and acute myeloid leukemia
(25,
26). In addition, activating
mutations in SHP-2 have been identified in sporadic solid tumors
(27). The SHP-2 mutations
appear to play a causal role in the development of these diseases as SHP-2
mutations and other JMML-associated Ras or Neurofibromatosis 1 mutations are
mutually exclusive in the patients
(24–27).
Moreover, single SHP-2 gain-of-function (GOF) mutations are sufficient to
induce Noonan syndrome, cytokine hypersensitivity in hematopoietic progenitor
cells, and JMML-like myeloproliferative disease in mice
(28–32).
Gain-of-function cell models derived from the newly available SHP-2 GOF
mutation (D61G) knock-in mice
(28) now provide us with a
good opportunity to clarify the role of SHP-2 in cell motility. Unlike the
dominant negative approach in which overexpression of mutant forms of SHP-2
generates complex effects, the SHP-2 D61G knock-in model eliminates this
possibility as the mutant SHP-2 is expressed at the physiological level
(28). Additionally, defining
signaling functions of GOF mutant SHP-2 in cell movement can also help
elucidate the molecular mechanisms by which SHP-2 mutations contribute to the
relevant diseases. 相似文献
9.
10.
Ming-hon Yau Yu Wang Karen S. L. Lam Jialiang Zhang Donghai Wu Aimin Xu 《The Journal of biological chemistry》2009,284(18):11942-11952
Lipoprotein lipase (LPL) is a principal enzyme responsible for the
clearance of chylomicrons and very low density lipoproteins from the
bloodstream. Two members of the Angptl (angiopoietin-like protein) family,
namely Angptl3 and Angptl4, have been shown to inhibit LPL activity in
vitro and in vivo. Here, we further investigated the structural
basis underlying the LPL inhibition by Angptl3 and Angptl4. By multiple
sequence alignment analysis, we have identified a highly conserved 12-amino
acid consensus motif that is present within the coiled-coil domain (CCD) of
both Angptl3 and Angptl4, but not other members of the Angptl family.
Substitution of the three polar amino acid residues (His46,
Gln50, and Gln53) within this motif with alanine
abolishes the inhibitory effect of Angptl4 on LPL in vitro and also
abrogates the ability of Angptl4 to elevate plasma triglyceride levels in
mice. The CCD of Angptl4 interacts with LPL and converts the catalytically
active dimers of LPL to its inactive monomers, whereas the mutant protein with
the three polar amino acids being replaced by alanine loses such a property.
Furthermore, a synthetic peptide consisting of the 12-amino acid consensus
motif is sufficient to inhibit LPL activity, although the potency is
much lower than the recombinant CCD of Angptl4. In summary, our data suggest
that the 12-amino acid consensus motif within the CCD of Angptl4, especially
the three polar residues within this motif, is responsible for its interaction
with and inhibition of LPL by blocking the enzyme dimerization.Lipoprotein lipase
(LPL)3 is an
endothelium-bound enzyme that catalyzes the hydrolysis of plasma triglyceride
(TG) associated with chylomicrons and very low density lipoproteins
(1,
2). This enzyme plays a major
role in maintaining lipid homeostasis by promoting the clearance of TG-rich
lipoproteins from the bloodstream. Abnormality in LPL functions has been
associated with a number of pathological conditions, including
atherosclerosis, dyslipidemia associated with diabetes, and Alzheimer disease
(1).LPL is expressed in a wide variety of cell types, particularly in
adipocytes and myocytes (2). As
a rate-limiting enzyme for clearance of TG-rich lipoproteins, the activity of
LPL is tightly modulated by multiple mechanisms in a tissue-specific manner in
response to nutritional changes
(3,
4). The enzymatic activity of
LPL in adipose tissue is enhanced after feeding to facilitate the storage of
TG, whereas it is down-regulated during fasting to increase the utilization of
TG by other tissues (5). The
active form of LPL is a noncovalent homodimer with the subunits associated in
a head-to-tail manner, and the dissociation of its dimeric form leads to the
formation of a stable inactive monomeric conformation and irreversible enzyme
inactivation (6). At the
post-translational level, the LPL activity is regulated by numerous
apolipoprotein co-factors. For instance, apoCII, a small apolipoprotein
consisting of 79 amino acid residues in human, activates LPL by directly
binding to the enzyme (7,
8). By contrast, several other
apolipoproteins such as apoCI, apo-CIII, and apoE have been shown to inhibit
the LPL activity in vitro
(3).Angiopoietin-like proteins (Angptl) are a family of secreted proteins
consisting of seven members, Angptl1 to Angptl7
(9,
10). All the members of the
Angptl family share a similar domain organization to those of angiopoietins,
with an NH2-terminal coiled-coil domain (CCD) and a COOH-terminal
fibrinogen-like domain. Among the seven family members, only Angptl3 and
Angptl4 have been shown to be involved in regulating triglyceride metabolism
(10,
11). The biological functions
of Angptl3 in lipid metabolism were first discovered by Koishi et al.
(12) in their positional
cloning of the recessive mutation gene responsible for the hypolipidemia
phenotype in a strain of obese mouse KK/snk. Subsequent studies have
demonstrated that Angptl3 increases plasma TG levels by inhibiting the LPL
enzymatic activity
(13–15).
Angptl4, also known as fasting-induced adipocyte factor, hepatic
fibrinogen/angiopoietin-related protein, or peroxisome proliferator-activated
receptor-γ angiopoietin-related, is a secreted glycoprotein abundantly
expressed in adipocyte, liver, and placenta
(16–18).
In addition to its role in regulating angiogenesis, a growing body of evidence
demonstrated that Angptl4 is an important player of lipid metabolism
(10,
11). Elevation of circulating
Angptl4 by transgenic or adenoviral overexpression, or by direct
supplementation of recombinant protein, leads to a marked elevation in the
levels of plasma TG and low density lipoprotein cholesterol in mice
(19–22).
By contrast, Angptl4 knock-out mice exhibit much lower plasma TG and
cholesterol levels compared with the wild type littermates
(19,
20). Notably, treatment of
several mouse models (such as C57BL/6J, ApoE–/–,
LDLR–/–, and db/db obese/diabetic mice) with a
neutralizing antibody against Angptl4 recapitulate the lipid phenotype found
in Angptl4 knock-out mice
(19). The role of Angptl4 as a
physiological inhibitor of LPL is also supported by the finding that its
expression levels in adipose tissue change rapidly during the fed-to-fasting
transitions and correlate inversely with LPL activity
(23). In humans, a genetic
variant of the ANGPTL4 gene (E40K) has been found to be associated
with significantly lower plasma TG levels and higher high density lipoprotein
cholesterol concentrations in several ethnic groups
(24–26).Angptl3 and Angptl4 share many common biochemical and functional properties
(10). In both humans and
rodents, Angptl3 and Angptl4 are proteolytically cleaved at the linker region
and circulate in plasma as two truncated fragments, including
NH2-terminal CCD and COOH-terminal fibrinogen-like domain
(14,
27–29).
The effects of both Angptl3 and Angptl4 on elevating plasma TG levels are
mediated exclusively by their NH2-terminal CCDs
(15,
22,
23,
27,
30). The CCDs of Angptl3 and
Angptl4 have been shown to inhibit the LPL activity in vitro as well
as in mice
(23,30,31).
Angptl4 inhibits LPL by promoting the conversion of the catalytically active
LPL dimers into catalytically inactive LPL monomers, thereby leading to the
inactivation of LPL (23,
31). However, the detailed
structural and molecular basis underlying the LPL inhibition by Angptl3 and
Angptl4 remain poorly characterized at this stage.In this study, we analyzed all known amino acid sequences of Angptl3 and
Angptl4 from various species and found a short motif,
LAXGLLXLGXGL (where X represents polar
amino acid residues), which corresponds to amino acid residues 46–57 and
44–55 of human Angptl3 and Angptl4, respectively, is highly conserved
despite the low degree of their overall homology (∼30%). Using both in
vitro and in vivo approaches, we demonstrated that this 12-amino
acid sequence motif, in particular the three polar amino acid residue within
this motif, is essential for mediating the interactions between LPL and
Angpt4, which in turn disrupts the dimerization of the enzyme. 相似文献
11.
Rebecca A. Chanoux Bu Yin Karen A. Urtishak Amma Asare Craig H. Bassing Eric J. Brown 《The Journal of biological chemistry》2009,284(9):5994-6003
Chromosomal abnormalities are frequently caused by problems encountered
during DNA replication. Although the ATR-Chk1 pathway has previously been
implicated in preventing the collapse of stalled replication forks into
double-strand breaks (DSB), the importance of the response to fork collapse in
ATR-deficient cells has not been well characterized. Herein, we demonstrate
that, upon stalled replication, ATR deficiency leads to the phosphorylation of
H2AX by ATM and DNA-PKcs and to the focal accumulation of Rad51, a marker of
homologous recombination and fork restart. Because H2AX has been shown to play
a facilitative role in homologous recombination, we hypothesized that H2AX
participates in Rad51-mediated suppression of DSBs generated in the absence of
ATR. Consistent with this model, increased Rad51 focal accumulation in
ATR-deficient cells is largely dependent on H2AX, and dual deficiencies in ATR
and H2AX lead to synergistic increases in chromatid breaks and translocations.
Importantly, the ATM and DNA-PK phosphorylation site on H2AX
(Ser139) is required for genome stabilization in the absence of
ATR; therefore, phosphorylation of H2AX by ATM and DNA-PKcs plays a pivotal
role in suppressing DSBs during DNA synthesis in instances of ATR pathway
failure. These results imply that ATR-dependent fork stabilization and
H2AX/ATM/DNA-PKcs-dependent restart pathways cooperatively suppress
double-strand breaks as a layered response network when replication
stalls.Genome maintenance prevents mutations that lead to cancer and age-related
diseases. A major challenge in preserving genome integrity occurs in the
simple act of DNA replication, in which failures at numerous levels can occur.
Besides the mis-incorporation of nucleotides, it is during this phase of the
cell cycle that the relatively stable double-stranded nature of DNA is
temporarily suspended at the replication fork, a structure that is susceptible
to collapse into
DSBs.2 Replication
fork stability is maintained by a variety of mechanisms, including activation
of the ATR-dependent checkpoint pathway.The ATR pathway is activated upon the generation and recognition of
extended stretches of single-stranded DNA at stalled replication forks
(1-4).
Genome maintenance functions for ATR and orthologs in yeast were first
indicated by increased chromatid breaks in ATR-/- cultured cells
(5) and by the
“cut” phenotype observed in Mec1 (Saccharomyces
cerevisiae) and Rad3 (Schizosaccharomyces pombe) mutants
(6-9).
Importantly, subsequent studies in S. cerevisiae demonstrated that
mutation of Mec1 or the downstream checkpoint kinase Rad53 led to increased
chromosome breaks at regions of the genome that are inherently difficult to
replicate (10), and a
decreased ability to reinitiate replication fork progression following DNA
damage or deoxyribonucleotide depletion
(11-14).In vertebrates, similar replication fork stabilizing functions have been
demonstrated for ATR and the downstream protein kinase Chk1
(15-20).
Several possible mechanisms have been put forward to explain how ATR-Chk1 and
orthologous pathways in yeast maintain replication fork stability, including
maintenance of replicative polymerases (α, δ, and ε) at forks
(17,
21), regulation of branch
migrating helicases, such as Blm
(22-25),
and regulation of homologous recombination, either positively or negatively
(26-29).Consistent with the role of the ATR-dependent checkpoint in replication
fork stability, common fragile sites, located in late-replicating regions of
the genome, are significantly more unstable (5-10-fold) in the absence of ATR
or Chk1 (19,
20). Because these sites are
favored regions of instability in oncogene-transformed cells and preneoplastic
lesions (30,
31), it is possible that the
increased tumor incidence observed in ATR haploinsufficient mice
(5,
32) may be related to subtle
increases in genomic instability. Together, these studies indicate that
maintenance of replication fork stability may contribute to tumor
suppression.It is important to note that prevention of fork collapse represents an
early response to problems occurring during DNA replication. In the event of
fork collapse into DSBs, homologous recombination (HR) has also been
demonstrated to play a key role in genome stability during S phase by
catalyzing recombination between sister chromatids as a means to re-establish
replication forks (33).
Importantly, a facilitator of homologous recombination, H2AX, has been shown
to be phosphorylated under conditions that cause replication fork collapse
(18,
34).Phosphorylation of H2AX occurs predominantly upon DSB formation
(34-38)
and has been reported to require ATM, DNA-PKcs, or ATR, depending on the
context
(37-42).
Although H2AX is not essential for HR, studies have demonstrated that H2AX
mutation leads to deficiencies in HR
(43,
44), and suppresses events
associated with homologous recombination, such as the focal accumulation of
Rad51, BRCA1, BRCA2, ubiquitinated-FANCD2, and Ubc13-mediated chromatin
ubiquitination (43,
45-51).
Therefore, through its contribution to HR, it is possible that H2AX plays an
important role in replication fork stability as part of a salvage pathway to
reinitiate replication following collapse.If ATR prevents the collapse of stalled replication forks into DSBs, and
H2AX facilitates HR-mediated restart, the combined deficiency in ATR and H2AX
would be expected to dramatically enhance the accumulation of DSBs upon
replication fork stalling. Herein, we utilize both partial and complete
elimination of ATR and H2AX to demonstrate that these genes work cooperatively
in non-redundant pathways to suppress DSBs during S phase. As discussed, these
studies imply that the various components of replication fork protection and
regeneration cooperate to maintain replication fork stability. Given the large
number of genes involved in each of these processes, it is possible that
combined deficiencies in these pathways may be relatively frequent in humans
and may synergistically influence the onset of age-related diseases and
cancer. 相似文献
12.
13.
14.
Nodar Makharashvili Tian Mi Olga Koroleva Sergey Korolev 《The Journal of biological chemistry》2009,284(3):1425-1434
RecF pathway proteins play an important role in the restart of stalled
replication and DNA repair in prokaryotes. Following DNA damage, RecF, RecR,
and RecO initiate homologous recombination (HR) by loading of the RecA
recombinase on single-stranded (ss) DNA, protected by ssDNA-binding protein.
The specific role of RecF in this process is not well understood. Previous
studies have proposed that RecF directs the RecOR complex to boundaries of
damaged DNA regions by recognizing single-stranded/double-stranded (ss/ds) DNA
junctions. RecF belongs to ABC-type ATPases, which function through an
ATP-dependent dimerization. Here, we demonstrate that the RecF of
Deinococcus radiodurans interacts with DNA as an ATP-dependent dimer,
and that the DNA binding and ATPase activity of RecF depend on both the
structure of DNA substrate, and the presence of RecR. We found that RecR
interacts as a tetramer with the RecF dimer. RecR increases the RecF affinity
to dsDNA without stimulating ATP hydrolysis but destabilizes RecF binding to
ssDNA and dimerization, likely due to increasing the ATPase rate. The
DNA-dependent binding of RecR to the RecF-DNA complex occurs through specific
protein-protein interactions without significant contributions from RecR-DNA
interactions. Finally, RecF neither alone nor in complex with RecR
preferentially binds to the ss/dsDNA junction. Our data suggest that the
specificity of the RecFOR complex toward the boundaries of DNA damaged regions
may result from a network of protein-protein and DNA-protein interactions,
rather than a simple recognition of the ss/dsDNA junction by RecF.Homologous recombination
(HR)2 is one of the
primary mechanisms by which cells repair dsDNA breaks (DSBs) and ssDNA gaps
(SSGs), and is important for restart of stalled DNA replication
(1). HR is initiated when
RecA-like recombinases bind to ssDNA forming an extended nucleoprotein
filament, referred to as a presynaptic complex
(2). The potential for genetic
rearrangements dictates that HR initiation is tightly regulated at multiple
levels (1). During replication,
the ssDNA-binding protein (SSB) protects transiently unwound DNA chains,
preventing interactions with recombinases. Following DNA damage, recombination
mediator proteins (RMPs) initiate HR by facilitating the formation of the
recombinase filaments with ssDNA, while removing SSB
(3,
4). Mutations in human proteins
involved in HR initiation are linked to cancer predisposition, chromosome
instability, UV sensitivity, and premature aging diseases
(4–8).
To date, little is known about the mechanism by which RMPs regulate the
formation of the recombinase filaments on the SSB-protected ssDNA.In Escherichia coli, there are two major recombination pathways,
RecBCD and RecF (9,
10). A helicase/nuclease
RecBCD complex processes DSBs and recruits RecA on ssDNA in a
sequence-specific manner
(11–13).
The principle players in the RecF pathway are the RecF, RecO, and RecR
proteins, which form an epistatic group that is important for SSG repair, for
restart of stalled DNA replication, and under specific conditions, can also
process DSBs
(14–20).
Homologs of RecF, -O, and -R are present in the majority of known bacteria
(21), including
Deinococcus radiodurans, extremely radiation-resistant bacteria that
lacks the RecBCD pathway, yet is capable of repairing thousands of DSBs
(22,
23). In addition, the sequence
or functional homologs of RecF pathway proteins are involved in similar
pathways in eukaryotes that include among others WRN, BLM, RAD52, and BRCA2
proteins
(4–8).The involvement of all three RecF, -O, and -R proteins in HR initiation is
well documented by genetic and cellular approaches
(18,
24–30),
yet their biochemical functions in the initiation process remain unclear,
particularly with respect to RecF. RecO and RecR proteins are sufficient to
promote formation of the RecA filament on SSB-bound ssDNA in vitro
(27). The UV-sensitive
phenotype of recF mutants can be suppressed by RecOR overexpression,
suggesting that RecF may direct the RMP complex to DNA-damaged regions where
HR initiation is required
(31). In agreement with this
hypothesis, RecF dramatically increases the efficiency of the RecA loading at
ds/ssDNA junctions with a 3′ ssDNA extension under specific conditions
(32). RecF and RecR proteins
also prevent the RecA filaments from extending into dsDNA regions adjacent to
SSGs (33). These data suggest
that RecF may directly recognize an ss/dsDNA junction structure
(34). However, DNA binding
experiments have not provided clear evidence to support such a hypothesis
(11).The targeting promoted by RecF may also occur through more complex
processes. RecF shares a high structural similarity with the head domain of
Rad50, an ABC-type ATPase that recognizes DSBs and initiates repair in archaea
and eukaryotes (35). All known
ABC-type ATPases function as oligomeric complexes in which a sequence of
inter- and intra-molecular interactions is triggered by the ATP-dependent
dimerization and the dimer-dependent ATP hydrolysis
(36–39).
RecF is also an ATP-dependent DNA-binding protein and a weak DNA-dependent
ATPase (11,
40). RecF forms an
ATP-dependent dimer and all three conserved motifs (Walker A, Walker B, and
“signature”) of RecF are important for ATP-dependent dimerization,
ATP hydrolysis, and functional resistance to DNA damage
(35). Thus, RecF may function
in recombination initiation through a complex pathway of protein-protein and
DNA-protein interactions regulated by ATP-dependent RecF dimerization.In this report, we present a detailed characterization of the RecF
dimerization, and its role in the RecF interaction with various DNA
substrates, with RecR, and in ATP hydrolysis. Our data outline the following
key findings. First, RecF interacts with DNA as a dimer. Second, neither RecF
alone nor the RecFR complex preferentially binds the ss/dsDNA junction.
Finally, RecR changes the ATPase activity and the DNA binding of RecF by
destabilizing the interaction with ssDNA, and greatly enhancing the
interaction with dsDNA. Our results suggest that the specificity of RecF for
the boundaries of SSGs is likely to result from a sequence of protein-protein
interaction events rather than a simple RecF ss/dsDNA binding, underlining a
highly regulated mechanism of the HR initiation by the RecFOR proteins. 相似文献
15.
Raymond W. Bourdeau Enrico Malito Alexandre Chenal Brian L. Bishop Mark W. Musch Mitch L. Villereal Eugene B. Chang Elise M. Mosser Richard F. Rest Wei-Jen Tang 《The Journal of biological chemistry》2009,284(21):14645-14656
Anthrolysin O (ALO) is a pore-forming, cholesterol-dependent cytolysin
(CDC) secreted by Bacillus anthracis, the etiologic agent for
anthrax. Growing evidence suggests the involvement of ALO in anthrax
pathogenesis. Here, we show that the apical application of ALO decreases the
barrier function of human polarized epithelial cells as well as increases
intracellular calcium and the internalization of the tight junction protein
occludin. Using pharmacological agents, we also found that barrier function
disruption requires increased intracellular calcium and protein degradation.
We also report a crystal structure of the soluble state of ALO. Based on our
analytical ultracentrifugation and light scattering studies, ALO exists as a
monomer. Our ALO structure provides the molecular basis as to how ALO is
locked in a monomeric state, in contrast to other CDCs that undergo
antiparallel dimerization or higher order oligomerization in solution. ALO has
four domains and is globally similar to perfringolysin O (PFO) and
intermedilysin (ILY), yet the highly conserved undecapeptide region in domain
4 (D4) adopts a completely different conformation in all three CDCs.
Consistent with the differences within D4 and at the D2-D4 interface, we found
that ALO D4 plays a key role in affecting the barrier function of C2BBE cells,
whereas PFO domain 4 cannot substitute for this role. Novel structural
elements and unique cellular functions of ALO revealed by our studies provide
new insight into the molecular basis for the diverse nature of the CDC
family.Cholesterol-dependent cytolysins
(CDCs)4 are a family
of pore-forming toxins from many organisms, including but not limited to the
genera Archanobacterium, Bacillus, Clostridium, Listeria, and
Streptococcus. Recently, work in vertebrates has revealed that CDCs
and membrane attack complex/perforin superfamily domain-containing proteins
share a similar fold, suggesting that vertebrates use a similar mechanism for
defense against infection (1,
2). A common feature of the CDC
family is the requirement of cholesterol in the membrane to form pores
(3). In addition to
cholesterol, certain members of the family also require a cellular receptor,
such as CD59 for the toxin ILY from Streptococcus intermedius
(4). The specific mechanism by
which CDCs form pores is not completely resolved; however, what is generally
known is that ring-shaped oligomerization at the cellular membrane is followed
by large conformational changes in each unit of the oligomer, resulting in the
insertion of a β-barrel into the cellular membrane
(5). Pore formation results in
a variety of downstream signaling effects, including but not limited to the
influx of Ca2+ into the cell
(6).A good deal is known about structures of the prepore conformation of CDCs.
The crystal structures of prepore PFO, from Clostridium perfringens,
and ILY have previously been elucidated
(7,
8). Each structure shows a
characteristic four-domain architecture, in which domain 4 (D4) is involved in
membrane recognition, domain 3 (D3) is involved in β-sheet insertion, and
domain 2 (D2) is the hinge region that undergoes a large conformational change
(9-11).
Nevertheless, despite the similarities, structural differences in D4
orientation and the conformation of a highly conserved segment named the
undecapeptide region confer functional differences to PFO and ILY
(8). Noting these differences,
we decided to explore the structure and function of another member of the CDC
family, anthrolysin O (ALO).ALO is secreted by Bacillus anthracis, the etiologic agent for
anthrax. ALO is chromosomally encoded by a gene whose regulation is poorly
understood, and it is highly homologous to other members of the CDC family
(12). ALO has been shown to
have hemolytic and cytolytic activity
(13,
14). Although clinical studies
have shown that B. anthracis is weakly hemolytic
(15), anthrax bacteria do
produce biologically relevant amounts of hemolytic ALO, although the levels of
expression are under complex regulation and are dependent on the culture media
and growth conditions (12,
13,
16). At lower concentrations,
ALO can disrupt cell signaling
(13,
14). Search for a cellular
receptor of ALO has lead to the conclusion that it is a TLR4 agonist
(17). However, it is not known
that ALO binds to TLR4 directly and, if so, whether ALO also binds other
cellular receptors.In addition to ALO, B. anthracis secrete ∼400 proteins, termed
the anthrax secretome (18). Of
those, two exotoxins, edema toxin (ET) and lethal toxin (LT) have been
characterized in greatest detail. ET raises intracellular cAMP to pathologic
levels, whereas LT impairs mitogenic and stress responses by inactivating
mitogen-activating protein kinase kinase
(19,
20). The complex interplay
between these two toxins on various aspects of host cellular functions have
been demonstrated
(20-25).
ALO could also work in conjunction with other anthrax virulence factors to
modulate their cellular toxicity. For example, ALO and LF together induce
macrophage apoptosis, whereas ALO and PLC play a redundant role in a murine
inhalation anthrax model (17,
26). Interplay among anthrax
secreted factors on cells relevant to anthrax infection is just beginning to
be understood. This network of interactions is vital to the molecular basis of
how anthrax bacteria interact with the hosts during anthrax infection.Anthrax infection initiates when B. anthracis spores enter the
host through one of three routes: cutaneous, inhalational, or gastrointestinal
(GI) (27,
28). All three routes of
infection can lead to systemic infection and are ultimately lethal. Different
from inhalational anthrax, spores are ingested and germinate on or within the
epithelium of the GI tract in GI anthrax
(29). This is primarily based
on pathological observations that primary lesions of the GI tract are found in
GI anthrax, whereas no primary lesions of the lung are found in inhalational
anthrax (29). Inhalational
anthrax is a disease of choice for biological weapons because of its high
infectivity and mortality
(30). The initiation of GI
anthrax requires much higher doses of spores than inhalational anthrax, and
the molecular basis for the initiation of GI anthrax remains elusive
(31).Since the primary function of GI epithelia is to control the flux of
material into the body, disruption of this barrier can lead to movement of
bacteria into the surrounding tissue
(32). The barrier is produced
by a matrix of transmembrane and membrane-associated proteins. These cell to
cell contacts, or tight junctions, are sometimes altered during bacterial
infection to specifically disrupt the barrier function of epithelial cells.
Using a functional model for the gut epithelium, human gut epithelial Caco-2
brush border expressor (C2BBE) cells, we report that ALO decreases the barrier
function of C2BBE cells through disruption of tight junctions. We also show
that ALO disruption of barrier function is dependent on epithelial cell
polarity. We also present the crystal structure of the soluble state of ALO
and compare it with the known structures of other CDCs. In addition, we show
that ALO exists primarily as a monomer, in contrast to its closely related
homologue PFO, which exists as a dimer. Finally, we used domain swapping to
examine the structural components that confer specificity of ALO to gut
epithelial cells. 相似文献
16.
17.
Yuusuke Maruyama Toshihiko Ogura Kazuhiro Mio Kenta Kato Takeshi Kaneko Shigeki Kiyonaka Yasuo Mori Chikara Sato 《The Journal of biological chemistry》2009,284(20):13676-13685
The Ca2+ release-activated Ca2+ channel is a
principal regulator of intracellular Ca2+ rise, which conducts
various biological functions, including immune responses. This channel,
involved in store-operated Ca2+ influx, is believed to be composed
of at least two major components. Orai1 has a putative channel pore and
locates in the plasma membrane, and STIM1 is a sensor for luminal
Ca2+ store depletion in the endoplasmic reticulum membrane. Here we
have purified the FLAG-fused Orai1 protein, determined its tetrameric
stoichiometry, and reconstructed its three-dimensional structure at 21-Å
resolution from 3681 automatically selected particle images, taken with an
electron microscope. This first structural depiction of a member of the Orai
family shows an elongated teardrop-shape 150Å in height and 95Å in
width. Antibody decoration and volume estimation from the amino acid sequence
indicate that the widest transmembrane domain is located between the round
extracellular domain and the tapered cytoplasmic domain. The cytoplasmic
length of 100Å is sufficient for direct association with STIM1. Orifices
close to the extracellular and intracellular membrane surfaces of Orai1 seem
to connect outside the molecule to large internal cavities.Ca2+ is an intracellular second messenger that plays important
roles in various physiological functions such as immune response, muscle
contraction, neurotransmitter release, and cell proliferation. Intracellular
Ca2+ is mainly stored in the endoplasmic reticulum
(ER).2 This ER system
is distributed through the cytoplasm from around the nucleus to the cell
periphery close to the plasma membrane. In non-excitable cells, the ER
releases Ca2+ through the inositol 1,4,5-trisphosphate
(IP3) receptor channel in response to various signals, and the
Ca2+ store is depleted. Depletion of Ca2+ then induces
Ca2+ influx from outside the cell to help in refilling the
Ca2+ stores and to continue Ca2+ rise for several
minutes in the cytoplasm (1,
2). This Ca2+ influx
was first proposed by Putney
(3) and was named
store-operated Ca2+ influx. In the immune system, store-operated
Ca2+ influx is mainly mediated by the Ca2+
release-activated Ca2+ (CRAC) current, which is a highly
Ca2+-selective inwardly rectified current with low conductance
(4,
5). Pathologically, the loss of
CRAC current in T cells causes severe combined immunodeficiency
(6) where many Ca2+
signal-dependent gene expressions, including cytokines, are interrupted
(7). Therefore, CRAC current is
necessary for T cell functions.Recently, Orai1 (also called CRACM1) and STIM1 have been physiologically
characterized as essential components of the CRAC channel
(8–12).
They are separately located in the plasma membrane and in the ER membrane;
co-expression of these proteins presents heterologous CRAC-like currents in
various types of cells (10,
13–15).
Both of them are shown to be expressed ubiquitously in various tissues
(16–18).
STIM1 senses Ca2+ depletion in the ER through its EF hand motif
(19) and transmits a signal to
Orai1 in the plasma membrane. Although Orai1 is proposed as a regulatory
component for some transient receptor potential canonical channels
(20,
21), it is believed from the
mutation analyses to be the pore-forming subunit of the CRAC channel
(8,
22–24).
In the steady state, both Orai1 and STIM1 molecules are dispersed in each
membrane. When store depletion occurs, STIM1 proteins gather into clusters to
form puncta in the ER membrane near the plasma membrane
(11,
19). These clusters then
trigger the clustering of Orai1 in the plasma membrane sites opposite the
puncta (25,
26), and CRAC channels are
activated (27).Orai1 has two homologous genes, Orai2 and Orai3
(8). They form the Orai family
and have in common the four transmembrane (TM) segments with relatively large
N and C termini. These termini are demonstrated to be in the cytoplasm,
because both N- and C-terminally introduced tags are immunologically detected
only in the membrane-permeabilized cells
(8,
9). The subunit stoichiometry
of Orai1 is as yet controversial: it is believed to be an oligomer, presumably
a dimer or tetramer even in the steady state
(16,
28–30).Despite the accumulation of biochemical and electrophysiological data,
structural information about Orai1 is limited due to difficulties in
purification and crystallization. In this study, we have purified Orai1 in its
tetrameric form and have reconstructed the three-dimensional structure from
negatively stained electron microscopic (EM) images. 相似文献
18.
19.
20.
Eun-Yeong Bergsdorf Anselm A. Zdebik Thomas J. Jentsch 《The Journal of biological chemistry》2009,284(17):11184-11193
Members of the CLC gene family either function as chloride channels or as
anion/proton exchangers. The plant AtClC-a uses the pH gradient across the
vacuolar membrane to accumulate the nutrient
in this organelle. When AtClC-a was
expressed in Xenopus oocytes, it mediated
exchange
and less efficiently mediated Cl–/H+ exchange.
Mutating the “gating glutamate” Glu-203 to alanine resulted in an
uncoupled anion conductance that was larger for Cl– than
. Replacing the “proton
glutamate” Glu-270 by alanine abolished currents. These could be
restored by the uncoupling E203A mutation. Whereas mammalian endosomal ClC-4
and ClC-5 mediate stoichiometrically coupled
2Cl–/H+ exchange, their
transport is largely uncoupled from
protons. By contrast, the AtClC-a-mediated
accumulation in plant vacuoles
requires tight
coupling. Comparison of AtClC-a and ClC-5 sequences identified a proline in
AtClC-a that is replaced by serine in all mammalian CLC isoforms. When this
proline was mutated to serine (P160S), Cl–/H+
exchange of AtClC-a proceeded as efficiently as
exchange, suggesting a role of this residue in
exchange. Indeed, when the corresponding serine of ClC-5 was replaced by
proline, this Cl–/H+ exchanger gained efficient
coupling. When inserted into the model Torpedo chloride channel
ClC-0, the equivalent mutation increased nitrate relative to chloride
conductance. Hence, proline in the CLC pore signature sequence is important
for
exchange and conductance both in
plants and mammals. Gating and proton glutamates play similar roles in
bacterial, plant, and mammalian CLC anion/proton exchangers.CLC proteins are found in all phyla from bacteria to humans and either
mediate electrogenic anion/proton exchange or function as chloride channels
(1). In mammals, the roles of
plasma membrane CLC Cl– channels include transepithelial
transport
(2–5)
and control of muscle excitability
(6), whereas vesicular CLC
exchangers may facilitate endocytosis
(7) and lysosomal function
(8–10)
by electrically shunting vesicular proton pump currents
(11). In the plant
Arabidopsis thaliana, there are seven CLC isoforms
(AtClC-a–AtClC-g)2
(12–15),
which may mostly reside in intracellular membranes. AtClC-a uses the pH
gradient across the vacuolar membrane to transport the nutrient nitrate into
that organelle (16). This
secondary active transport requires a tightly coupled
exchange. Astonishingly, however, mammalian ClC-4 and -5 and bacterial EcClC-1
(one of the two CLC isoforms in Escherichia coli) display tightly
coupled Cl–/H+ exchange, but anion flux is largely
uncoupled from H+ when
is transported
(17–21).
The lack of appropriate expression systems for plant CLC transporters
(12) has so far impeded
structure-function analysis that may shed light on the ability of AtClC-a to
perform efficient
exchange. This dearth of data contrasts with the extensive mutagenesis work
performed with CLC proteins from animals and bacteria.The crystal structure of bacterial CLC homologues
(22,
23) and the investigation of
mutants (17,
19–21,
24–29)
have yielded important insights into their structure and function. CLC
proteins form dimers with two largely independent permeation pathways
(22,
25,
30,
31). Each of the monomers
displays two anion binding sites
(22). A third binding site is
observed when a certain key glutamate residue, which is located halfway in the
permeation pathway of almost all CLC proteins, is mutated to alanine
(23). Mutating this gating
glutamate in CLC Cl– channels strongly affects or even
completely suppresses single pore gating
(23), whereas CLC exchangers
are transformed by such mutations into pure anion conductances that are not
coupled to proton transport
(17,
19,
20). Another key glutamate,
located at the cytoplasmic surface of the CLC monomer, seems to be a hallmark
of CLC anion/proton exchangers. Mutating this proton glutamate to
nontitratable amino acids uncouples anion transport from protons in the
bacterial EcClC-1 protein (27)
but seems to abolish transport altogether in mammalian ClC-4 and -5
(21). In those latter
proteins, anion transport could be restored by additionally introducing an
uncoupling mutation at the gating glutamate
(21).The functional complementation by AtClC-c and -d
(12,
32) of growth phenotypes of a
yeast strain deleted for the single yeast CLC Gef1
(33) suggested that these
plant CLC proteins function in anion transport but could not reveal details of
their biophysical properties. We report here the first functional expression
of a plant CLC in animal cells. Expression of wild-type (WT) and mutant
AtClC-a in Xenopus oocytes indicate a general role of gating and
proton glutamate residues in anion/proton coupling across different isoforms
and species. We identified a proline in the CLC signature sequence of AtClC-a
that plays a crucial role in
exchange. Mutating it to serine, the residue present in mammalian CLC proteins
at this position, rendered AtClC-a Cl–/H+ exchange
as efficient as
exchange. Conversely, changing the corresponding serine of ClC-5 to proline
converted it into an efficient
exchanger. When proline replaced the critical serine in Torpedo
ClC-0, the relative conductance of
this model Cl– channel was drastically increased, and
“fast” protopore gating was slowed. 相似文献