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1.
Yusuke Takahashi Gennadiy Moiseyev Zsolt Ablonczy Ying Chen Rosalie K. Crouch Jian-xing Ma 《The Journal of biological chemistry》2009,284(5):3211-3218
RPE65 is a membrane-associated protein abundantly expressed in the retinal
pigment epithelium, which converts all-trans-retinyl ester to
11-cis-retinol, a key step in the retinoid visual cycle. Although
three cysteine residues (Cys-231, Cys-329, and Cys-330) were identified to be
palmitylated in RPE65, recent studies showed that a triple mutant, with all
three Cys replaced by an alanine residue, was still palmitylated and remained
membrane-associated, suggesting that there are other yet to be identified
palmitylated Cys residues in RPE65. Here we mapped the entire RPE65 using mass
spectrometry analysis and demonstrated that a trypsin-digested RPE65 fragment
(residues 98-118), which contains two Cys residues (Cys-106 and Cys-112), was
singly palmitylated in both native bovine and recombinant human RPE65. To
determine whether Cys-106 or Cys-112 is the palmitylation site, these Cys were
separately replaced by alanine. Mass spectrometry analysis of purified
wild-type RPE65 and C106A and C112A mutants showed that mutation of Cys-106
did not affect the palmitylation status of the fragment 98-118, whereas
mutation of Cys-112 abolished palmitylation in this fragment. Subcellular
fractionation and immunocytochemistry analyses both showed that mutation of
Cys-112 dissociated RPE65 from the membrane, whereas the C106A mutant remained
associated with the membrane. In vitro isomerohydrolase activity
assay showed that C106A has an intact enzymatic activity similar to that of
wtRPE65, whereas C112A lost its enzymatic activity. These results indicate
that the newly identified Cys-112 palmitylation site is essential for the
membrane association and activity of RPE65.Both rod and cone visual pigments in vertebrates require
11-cis-retinal as the chromophore. Isomerization of
11-cis-retinal to all-trans-retinal by a photon triggers the
phototransduction cascade and initiates vision
(1,
2). Recycling of
11-cis-retinal through the retinoid visual cycle is an essential
process for the regeneration of visual pigments and for normal vision
(3,
4). The key step in the visual
cycle is to isomerize all-trans-retinyl ester to
11-cis-retinol in retinal pigment epithelium
(RPE)2
(5,
6). This isomerization process
is known to be catalyzed by an isomerohydrolase in the RPE. Several recent
lines of evidence suggest that RPE65 is the isomerohydrolase in the visual
cycle
(7-9).RPE65 is a microsomal protein, abundantly expressed in the RPE
(10-12).
RPE65 knock-out (Rpe65-/-)
mice showed a lack of 11-cis-retinoids, overaccumulation of
all-trans-retinyl ester, impaired visual function, and early
degeneration of cone photoreceptors
(7-9).
RPE65 is an iron(II)-dependent enzyme, in which an iron is coordinated by four
conserved histidine (His) residues (His-180, -241, -313, and -527) based on
molecular modeling using a crystal structure of apocarotenoid monooxygenase as
a template (8,
13-15).
RPE65 lacks any predicted transmembrane helix and is associated with the
microsomal membrane (11).
Previous studies have shown that membrane association of RPE65 is essential
for its isomerohydrolase activity
(7). However, the structural
basis for its membrane association has not been revealed. An earlier study
showed that three Cys residues (Cys-231, 329 and 330) in RPE65 were
palmitylated, which were suggested to be responsible for its membrane
association (16). However,
triple mutations of all the three Cys residues did not completely dissociate
RPE65 from the membrane (17,
18). Moreover, the triple Cys
mutant remains palmitylated
(17). These results suggested
that either the site of palmitylation responsible for the membrane association
of RPE65 had not yet been identified or other mechanisms, such as hydrophobic
interactions, anchor the protein to cellular membranes
(17,
19).In this study, we used the combination of mass spectrometric analysis and
site-directed mutagenesis to identify the palmitylated site in RPE65.
Moreover, we determined the role of this site in the membrane association and
enzymatic activity of RPE65. 相似文献
2.
Michelle Y. S. Shih Maureen A. Kane Ping Zhou C. L. Eric Yen Ryan S. Streeper Joseph L. Napoli Robert V. Farese Jr. 《The Journal of biological chemistry》2009,284(7):4292-4299
Retinoic acid (RA) is a potent signaling molecule that is essential for
many biological processes, and its levels are tightly regulated by mechanisms
that are only partially understood. The synthesis of RA from its precursor
retinol (vitamin A) is an important regulatory mechanism. Therefore, the
esterification of retinol with fatty acyl moieties to generate retinyl esters,
the main storage form of retinol, may also regulate RA levels. Here we show
that the neutral lipid synthesis enzyme acyl-CoA:diacylglycerol
acyltransferase 1 (DGAT1) functions as the major acyl-CoA:retinol
acyltransferase (ARAT) in murine skin. When dietary retinol is abundant, DGAT1
deficiency results in elevated levels of RA in skin and cyclical hair loss;
both are prevented by dietary retinol deprivation. Further, DGAT1-deficient
skin exhibits enhanced sensitivity to topically administered retinol. Deletion
of the enzyme specifically in the epidermis causes alopecia, indicating that
the regulation of RA homeostasis by DGAT1 is autonomous in the epidermis.
These findings show that DGAT1 functions as an ARAT in the skin, where it acts
to maintain retinoid homeostasis and prevent retinoid toxicity. Our findings
may have implications for human skin or hair disorders treated with agents
that modulate RA signaling.Regulation of cellular proliferation and differentiation of epithelial
tissues is crucial in embryonic development and in adult homeostasis. Retinoic
acid (RA)2 is a major
regulator of these processes
(1) through its ability to
serve as a ligand for RA nuclear receptors (RARs)
(2). Since RA is such a potent
signaling molecule, its levels must be tightly controlled. Indeed, excess RA
is highly teratogenic during embryonic development and may be toxic to adult
tissues (3). Further, RA is
used therapeutically for skin disorders, such as acne and psoriasis, and
certain cancers (4), but its
uses are often limited by local and systemic toxicity. Thus, understanding how
RA levels are regulated has important biological and clinical relevance.The synthesis of RA from its precursor retinol, or vitamin A, is a major
node in the regulation of RA levels
(5). To generate RA, retinol is
oxidized in two sequential reactions, catalyzed by retinol and retinal
dehydrogenases (5), whose
activities regulate RA homeostasis. We hypothesized that the availability of
retinol for these reactions may also be regulated by the balance between
retinol and retinyl esters. Indeed, the majority of retinol in the body is
stored as retinyl esters, which are concentrated in cytosolic lipid droplets
of cells and serve as a local source of retinol. Retinyl esters are also
stored in major organs, such as liver and white adipose tissue (WAT), from
which retinol can be mobilized to supply other tissues during increased
demand. Thus, retinol esterification may participate in regulating the retinol
pool available for RA synthesis.Retinol esterification is carried out by two distinct enzymatic activities.
One is mediated by lecithin:retinol acyltransferase (LRAT), which catalyzes
the covalent joining of a fatty acyl moiety from lecithin
(phosphatidylcholine) to retinol that is bound to cellular retinol-binding
protein (CRBP) (6,
7). LRAT activity is crucial
for maintaining tissue retinol stores. LRAT-null (Lrat-/-)
mice have severe reductions in hepatic and lung retinyl ester levels
(8–10),
which are accompanied by testicular hypoplasia/atrophy
(9) and blindness
(8). Retinyl ester levels are
normal in WAT and several other tissues, indicating alternative mechanisms for
retinol esterification (9,
10). This esterification is
probably mediated in part by acyl CoA:retinol acyltransferase (ARAT) enzymes,
which use fatty acyl-CoA and unbound retinol as substrates
(11). Although many tissues
exhibit ARAT activity (12),
attempts to purify and clone an ARAT gene were unsuccessful, and thus
molecular tools to study ARAT activity have been lacking. However, the enzyme
encoded by Dgat1, an acyl CoA:diacylglycerol acyltransferase (DGAT),
was recently reported to catalyze the ARAT reaction in vitro
(13,
14). Moreover, several tissues
of Dgat1-/- mice had reduced ARAT activity, and retinol
esterification was reduced in cultured murine embryonic fibroblasts lacking
DGAT1 (14). Most recently, a
study of Dgat1-/- mice demonstrated a role for the enzyme
in retinol absorption in the small intestine
(15). Thus, accumulating
evidence indicates that the retinol esterification activity of DGAT1 is of
biological, and possibly clinical, importance.In the current study, we investigated whether retinol esterification by
DGAT1 is important in murine skin. Dgat1-/- mice exhibit a
pleiotropic phenotype, which includes resistance to diet-induced obesity and
altered energy metabolism but also includes prominent phenotypic findings in
the skin
(16–19).
Retinoids play key roles in skin and hair biology
(20), and excess retinoids
induce epidermal hyperplasia, inhibit sebocyte proliferation and
differentiation, and alter hair growth
(21). Notably, the skin
manifestations of Dgat1-/- mice, which include alopecia
and sebaceous gland atrophy
(18), resemble those of
retinoid toxicity (22,
23). Thus, we hypothesized
that DGAT1 functions as an ARAT in murine skin and that the absence of DGAT1
alters retinoid homeostasis. In this study, we tested this hypothesis by
examining retinoid metabolism in the skin of DGAT1-deficient mice. 相似文献
3.
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
4.
5.
Ruben K. Dagda Salvatore J. Cherra III Scott M. Kulich Anurag Tandon David Park Charleen T. Chu 《The Journal of biological chemistry》2009,284(20):13843-13855
Mitochondrial dysregulation is strongly implicated in Parkinson disease.
Mutations in PTEN-induced kinase 1 (PINK1) are associated with familial
parkinsonism and neuropsychiatric disorders. Although overexpressed PINK1 is
neuroprotective, less is known about neuronal responses to loss of PINK1
function. We found that stable knockdown of PINK1 induced mitochondrial
fragmentation and autophagy in SH-SY5Y cells, which was reversed by the
reintroduction of an RNA interference (RNAi)-resistant plasmid for PINK1.
Moreover, stable or transient overexpression of wild-type PINK1 increased
mitochondrial interconnectivity and suppressed toxin-induced
autophagy/mitophagy. Mitochondrial oxidant production played an essential role
in triggering mitochondrial fragmentation and autophagy in PINK1 shRNA lines.
Autophagy/mitophagy served a protective role in limiting cell death, and
overexpressing Parkin further enhanced this protective mitophagic response.
The dominant negative Drp1 mutant inhibited both fission and mitophagy in
PINK1-deficient cells. Interestingly, RNAi knockdown of autophagy proteins
Atg7 and LC3/Atg8 also decreased mitochondrial fragmentation without affecting
oxidative stress, suggesting active involvement of autophagy in morphologic
remodeling of mitochondria for clearance. To summarize, loss of PINK1 function
elicits oxidative stress and mitochondrial turnover coordinated by the
autophagic and fission/fusion machineries. Furthermore, PINK1 and Parkin may
cooperate through different mechanisms to maintain mitochondrial
homeostasis.Parkinson disease is an age-related neurodegenerative disease that affects
∼1% of the population worldwide. The causes of sporadic cases are unknown,
although mitochondrial or oxidative toxins such as
1-methyl-4-phenylpyridinium, 6-hydroxydopamine
(6-OHDA),3 and
rotenone reproduce features of the disease in animal and cell culture models
(1). Abnormalities in
mitochondrial respiration and increased oxidative stress are observed in cells
and tissues from parkinsonian patients
(2,
3), which also exhibit
increased mitochondrial autophagy
(4). Furthermore, mutations in
parkinsonian genes affect oxidative stress response pathways and mitochondrial
homeostasis (5). Thus,
disruption of mitochondrial homeostasis represents a major factor implicated
in the pathogenesis of sporadic and inherited parkinsonian disorders (PD).The PARK6 locus involved in autosomal recessive and early-onset PD
encodes for PTEN-induced kinase 1 (PINK1)
(6,
7). PINK1 is a cytosolic and
mitochondrially localized 581-amino acid serine/threonine kinase that
possesses an N-terminal mitochondrial targeting sequence
(6,
8). The primary sequence also
includes a putative transmembrane domain important for orientation of the
PINK1 domain (8), a conserved
kinase domain homologous to calcium calmodulin kinases, and a C-terminal
domain that regulates autophosphorylation activity
(9,
10). Overexpression of
wild-type PINK1, but not its PD-associated mutants, protects against several
toxic insults in neuronal cells
(6,
11,
12). Mitochondrial targeting
is necessary for some (13) but
not all of the neuroprotective effects of PINK1
(14), implicating involvement
of cytoplasmic targets that modulate mitochondrial pathobiology
(8). PINK1 catalytic activity
is necessary for its neuroprotective role, because a kinase-deficient K219M
substitution in the ATP binding pocket of PINK1 abrogates its ability to
protect neurons (14). Although
PINK1 mutations do not seem to impair mitochondrial targeting, PD-associated
mutations differentially destabilize the protein, resulting in loss of
neuroprotective activities
(13,
15).Recent studies indicate that PINK1 and Parkin interact genetically
(3,
16-18)
to prevent oxidative stress
(19,
20) and regulate mitochondrial
morphology (21). Primary cells
derived from PINK1 mutant patients exhibit mitochondrial fragmentation with
disorganized cristae, recapitulated by RNA interference studies in HeLa cells
(3).Mitochondria are degraded by macroautophagy, a process involving
sequestration of cytoplasmic cargo into membranous autophagic vacuoles (AVs)
for delivery to lysosomes (22,
23). Interestingly,
mitochondrial fission accompanies autophagic neurodegeneration elicited by the
PD neurotoxin 6-OHDA (24,
25). Moreover, mitochondrial
fragmentation and increased autophagy are observed in neurodegenerative
diseases including Alzheimer and Parkinson diseases
(4,
26-28).
Although inclusion of mitochondria in autophagosomes was once believed to be a
random process, as observed during starvation, studies involving hypoxia,
mitochondrial damage, apoptotic stimuli, or limiting amounts of aerobic
substrates in facultative anaerobes support the concept of selective
mitochondrial autophagy (mitophagy)
(29,
30). In particular,
mitochondrially localized kinases may play an important role in models
involving oxidative mitochondrial injury
(25,
31,
32).Autophagy is involved in the clearance of protein aggregates
(33-35)
and normal regulation of axonal-synaptic morphology
(36). Chronic disruption of
lysosomal function results in accumulation of subtly impaired mitochondria
with decreased calcium buffering capacity
(37), implicating an important
role for autophagy in mitochondrial homeostasis
(37,
38). Recently, Parkin, which
complements the effects of PINK1 deficiency on mitochondrial morphology
(3), was found to promote
autophagy of depolarized mitochondria
(39). Conversely, Beclin
1-independent autophagy/mitophagy contributes to cell death elicited by the PD
toxins 1-methyl-4-phenylpyridinium and 6-OHDA
(25,
28,
31,
32), causing neurite
retraction in cells expressing a PD-linked mutation in leucine-rich repeat
kinase 2 (40). Whereas
properly regulated autophagy plays a homeostatic and neuroprotective role,
excessive or incomplete autophagy creates a condition of “autophagic
stress” that can contribute to neurodegeneration
(28).As mitochondrial fragmentation
(3) and increased mitochondrial
autophagy (4) have been
described in human cells or tissues of PD patients, we investigated whether or
not the engineered loss of PINK1 function could recapitulate these
observations in human neuronal cells (SH-SY5Y). Stable knockdown of endogenous
PINK1 gave rise to mitochondrial fragmentation and increased autophagy and
mitophagy, whereas stable or transient overexpression of PINK1 had the
opposite effect. Autophagy/mitophagy was dependent upon increased
mitochondrial oxidant production and activation of fission. The data indicate
that PINK1 is important for the maintenance of mitochondrial networks,
suggesting that coordinated regulation of mitochondrial dynamics and autophagy
limits cell death associated with loss of PINK1 function. 相似文献
6.
Kelvin B. Luther Hermann Schindelin Robert S. Haltiwanger 《The Journal of biological chemistry》2009,284(5):3294-3305
The Notch receptor is critical for proper development where it orchestrates
numerous cell fate decisions. The Fringe family of
β1,3-N-acetylglucosaminyltransferases are regulators of this
pathway. Fringe enzymes add N-acetylglucosamine to O-linked
fucose on the epidermal growth factor repeats of Notch. Here we have analyzed
the reaction catalyzed by Lunatic Fringe (Lfng) in detail. A mutagenesis
strategy for Lfng was guided by a multiple sequence alignment of Fringe
proteins and solutions from docking an epidermal growth factor-like
O-fucose acceptor substrate onto a homology model of Lfng. We
targeted three main areas as follows: residues that could help resolve where
the fucose binds, residues in two conserved loops not observed in the
published structure of Manic Fringe, and residues predicted to be involved in
UDP-N-acetylglucosamine (UDP-GlcNAc) donor specificity. We utilized a
kinetic analysis of mutant enzyme activity toward the small molecule acceptor
substrate 4-nitrophenyl-α-l-fucopyranoside to judge their
effect on Lfng activity. Our results support the positioning of
O-fucose in a specific orientation to the catalytic residue. We also
found evidence that one loop closes off the active site coincident with, or
subsequent to, substrate binding. We propose a mechanism whereby the ordering
of this short loop may alter the conformation of the catalytic aspartate.
Finally, we identify several residues near the UDP-GlcNAc-binding site, which
are specifically permissive toward UDP-GlcNAc utilization.Defects in Notch signaling have been implicated in numerous human diseases,
including multiple sclerosis
(1), several forms of cancer
(2-4),
cerebral autosomal dominant arteriopathy with sub-cortical infarcts and
leukoencephalopathy (5), and
spondylocostal dysostosis
(SCD)3
(6-8).
The transmembrane Notch signaling receptor is activated by members of the DSL
(Delta, Serrate, Lag2) family of ligands
(9,
10). In the endoplasmic
reticulum, O-linked fucose glycans are added to the epidermal growth
factor-like (EGF) repeats of the Notch extracellular domain by protein
O-fucosyltransferase 1
(11-13).
These O-fucose monosaccharides can be elongated in the Golgi
apparatus by three highly conserved
β1,3-N-acetylglucosaminyltransferases of the Fringe family
(Lunatic (Lfng), Manic (Mfng), and Radical Fringe (Rfng) in mammals)
(14-16).
The formation of this GlcNAc-β1,3-Fuc-α1,
O-serine/threonine disaccharide is necessary and sufficient for
subsequent elongation to a tetrasaccharide
(15,
19), although elongation past
the disaccharide in Drosophila is not yet clear
(20,
21). Elongation of
O-fucose by Fringe is known to potentiate Notch signaling from Delta
ligands and inhibit signaling from Serrate ligands
(22). Delta ligands are termed
Delta-like (Delta-like1, -2, and -4) in mammals, and the homologs of Serrate
are known as Jagged (Jagged1 and -2) in mammals. The effects of Fringe on
Drosophila Notch can be recapitulated in Notch ligand in
vitro binding assays using purified components, suggesting that the
elongation of O-fucose by Fringe alters the binding of Notch to its
ligands (21). Although Fringe
also appears to alter Notch-ligand interactions in mammals, the effects of
elongation of the glycan past the O-fucose monosaccharide is more
complicated and appears to be cell type-, receptor-, and ligand-dependent (for
a recent review see Ref.
23).The Fringe enzymes catalyze the transfer of GlcNAc from the donor substrate
UDP-α-GlcNAc to the acceptor fucose, forming the GlcNAc-β1,3-Fuc
disaccharide
(14-16).
They belong to the GT-A-fold of inverting glycosyltransferases, which includes
N-acetylglucosaminyltransferase I and β1,4-galactosyltransferase
I (17,
18). The mechanism is presumed
to proceed through the abstraction of a proton from the acceptor substrate by
a catalytic base (Asp or Glu) in the active site. This creates a nucleophile
that attacks the anomeric carbon of the nucleotide-sugar donor, inverting its
configuration from α (on the nucleotide sugar) to β (in the
product) (24,
25). The enzyme then releases
the acceptor substrate modified with a disaccharide and UDP. The Mfng
structure (26) leaves little
doubt as to the identity of the catalytic residue, which in all likelihood is
aspartate 289 in mouse Lfng (we will use numbering for mouse Lunatic Fringe
throughout, unless otherwise stated). The structure of Mfng with UDP-GlcNAc
soaked into the crystals (26)
showed density only for the UDP portion of the nucleotide-sugar donor and no
density for two loops flanking either side of the active site. The presence of
flexible loops that become ordered upon substrate binding is a common
observation with glycosyltransferases in the GT-A fold family
(18,
25). Density for the entire
donor was observed in the structure of rabbit
N-acetylglucosaminyltransferase I
(27). In this case, ordering
of a previously disordered loop upon UDP-GlcNAc binding may have contributed
to increased stability of the donor. In the case of bovine
β1,4-galactosyltransferase I, a section of flexible random coil from the
apo-structure was observed to change its conformation to α-helical upon
donor substrate binding (28).
Both loops in Lfng are highly conserved, and we have mutated a number of
residues in each to test the hypothesis that they interact with the
substrates. The mutagenesis strategy was also guided by docking of an
EGF-O-fucose acceptor substrate into the active site of the Lfng
model as well as comparison of the Lfng model with a homology model of the
β1,3-glucosyltransferase (β3GlcT) that modifies O-fucose on
thrombospondin type 1 repeats
(29,
30). The β3GlcT is
predicted to be a GT-A fold enzyme related to the Fringe family
(17,
18,
29). 相似文献
7.
Graham H. Diering John Church Masayuki Numata 《The Journal of biological chemistry》2009,284(20):13892-13903
NHE5 is a brain-enriched Na+/H+ exchanger that
dynamically shuttles between the plasma membrane and recycling endosomes,
serving as a mechanism that acutely controls the local pH environment. In the
current study we show that secretory carrier membrane proteins (SCAMPs), a
group of tetraspanning integral membrane proteins that reside in multiple
secretory and endocytic organelles, bind to NHE5 and co-localize predominantly
in the recycling endosomes. In vitro protein-protein interaction
assays revealed that NHE5 directly binds to the N- and C-terminal cytosolic
extensions of SCAMP2. Heterologous expression of SCAMP2 but not SCAMP5
increased cell-surface abundance as well as transporter activity of NHE5
across the plasma membrane. Expression of a deletion mutant lacking the
SCAMP2-specific N-terminal cytosolic domain, and a mini-gene encoding the
N-terminal extension, reduced the transporter activity. Although both Arf6 and
Rab11 positively regulate NHE5 cell-surface targeting and NHE5 activity across
the plasma membrane, SCAMP2-mediated surface targeting of NHE5 was reversed by
dominant-negative Arf6 but not by dominant-negative Rab11. Together, these
results suggest that SCAMP2 regulates NHE5 transit through recycling endosomes
and promotes its surface targeting in an Arf6-dependent manner.Neurons and glial cells in the central and peripheral nervous systems are
especially sensitive to perturbations of pH
(1). Many voltage- and
ligand-gated ion channels that control membrane excitability are sensitive to
changes in cellular pH
(1-3).
Neurotransmitter release and uptake are also influenced by cellular and
organellar pH (4,
5). Moreover, the intra- and
extracellular pH of both neurons and glia are modulated in a highly transient
and localized manner by neuronal activity
(6,
7). Thus, neurons and glia
require sophisticated mechanisms to finely tune ion and pH homeostasis to
maintain their normal functions.Na+/H+ exchangers
(NHEs)3 were
originally identified as a class of plasma membrane-bound ion transporters
that exchange extracellular Na+ for intracellular H+,
and thereby regulate cellular pH and volume. Since the discovery of NHE1 as
the first mammalian NHE (8),
eight additional isoforms (NHE2-9) that share 25-70% amino acid identity have
been isolated in mammals (9,
10). NHE1-5 commonly exhibit
transporter activity across the plasma membrane, whereas NHE6-9 are mostly
found in organelle membranes and are believed to regulate organellar pH in
most cell types at steady state
(11). More recently, NHE10 was
identified in human and mouse osteoclasts
(12,
13). However, the cDNA
encoding NHE10 shares only a low degree of sequence similarity with other
known members of the NHE gene family, raising the possibility that
this sodium-proton exchanger may belong to a separate gene family distantly
related to NHE1-9 (see Ref.
9).NHE gene family members contain 12 putative transmembrane domains
at the N terminus followed by a C-terminal cytosolic extension that plays a
role in regulation of the transporter activity by protein-protein interactions
and phosphorylation. NHEs have been shown to regulate the pH environment of
synaptic nerve terminals and to regulate the release of neurotransmitters from
multiple neuronal populations
(14-16).
The importance of NHEs in brain function is further exemplified by the
findings that spontaneous or directed mutations of the ubiquitously expressed
NHE1 gene lead to the progression of epileptic seizures, ataxia, and
increased mortality in mice
(17,
18). The progression of the
disease phenotype is associated with loss of specific neuron populations and
increased neuronal excitability. However, NHE1-null mice appear to
develop normally until 2 weeks after birth when symptoms begin to appear.
Therefore, other mechanisms may compensate for the loss of NHE1
during early development and play a protective role in the surviving neurons
after the onset of the disease phenotype.NHE5 was identified as a unique member of the NHE gene
family whose mRNA is expressed almost exclusively in the brain
(19,
20), although more recent
studies have suggested that NHE5 might be functional in other cell
types such as sperm (21,
22) and osteosarcoma cells
(23). Curiously, mutations
found in several forms of congenital neurological disorders such as
spinocerebellar ataxia type 4
(24-26)
and autosomal dominant cerebellar ataxia
(27-29)
have been mapped to chromosome 16q22.1, a region containing NHE5.
However, much remains unknown as to the molecular regulation of NHE5 and its
role in brain function.Very few if any proteins work in isolation. Therefore identification and
characterization of binding proteins often reveal novel functions and
regulation mechanisms of the protein of interest. To begin to elucidate the
biological role of NHE5, we have started to explore NHE5-binding proteins.
Previously, β-arrestins, multifunctional scaffold proteins that play a
key role in desensitization of G-protein-coupled receptors, were shown to
directly bind to NHE5 and promote its endocytosis
(30). This study demonstrated
that NHE5 trafficking between endosomes and the plasma membrane is regulated
by protein-protein interactions with scaffold proteins. More recently, we
demonstrated that receptor for activated
C-kinase 1 (RACK1), a scaffold protein that links
signaling molecules such as activated protein kinase C, integrins, and Src
kinase (31), directly
interacts with and activates NHE5 via integrin-dependent and independent
pathways (32). These results
further indicate that NHE5 is partly associated with focal adhesions and that
its targeting to the specialized microdomain of the plasma membrane may be
regulated by various signaling pathways.Secretory carrier membrane proteins (SCAMPs) are a family of evolutionarily
conserved tetra-spanning integral membrane proteins. SCAMPs are found in
multiple organelles such as the Golgi apparatus, trans-Golgi network,
recycling endosomes, synaptic vesicles, and the plasma membrane
(33,
34) and have been shown to
play a role in exocytosis
(35-38)
and endocytosis (39).
Currently, five isoforms of SCAMP have been identified in mammals. The
extended N terminus of SCAMP1-3 contain multiple Asn-Pro-Phe (NPF) repeats,
which may allow these isoforms to participate in clathrin coat assembly and
vesicle budding by binding to Eps15 homology (EH)-domain proteins
(40,
41). Further, SCAMP2 was shown
recently to bind to the small GTPase Arf6
(38), which is believed to
participate in traffic between the recycling endosomes and the cell surface
(42,
43). More recent studies have
suggested that SCAMPs bind to organellar membrane type NHE7
(44) and the serotonin
transporter SERT (45) and
facilitate targeting of these integral membrane proteins to specific
intracellular compartments. We show in the current study that SCAMP2 binds to
NHE5, facilitates the cell-surface targeting of NHE5, and elevates
Na+/H+ exchange activity at the plasma membrane, whereas
expression of a SCAMP2 deletion mutant lacking the N-terminal domain
containing the NPF repeats suppresses the effect. Further we show that this
activity of SCAMP2 requires an active form of a small GTPase Arf6, but not
Rab11. We propose a model in which SCAMPs bind to NHE5 in the endosomal
compartment and control its cell-surface abundance via an Arf6-dependent
pathway. 相似文献
8.
As obligate intracellular parasites, viruses exploit diverse cellular
signaling machineries, including the mitogen-activated protein-kinase pathway,
during their infections. We have demonstrated previously that the open reading
frame 45 (ORF45) of Kaposi sarcoma-associated herpesvirus interacts with p90
ribosomal S6 kinases (RSKs) and strongly stimulates their kinase activities
(Kuang, E., Tang, Q., Maul, G. G., and Zhu, F.
(2008) J. Virol. 82
,1838
-1850). Here, we define the
mechanism by which ORF45 activates RSKs. We demonstrated that binding of ORF45
to RSK increases the association of extracellular signal-regulated kinase
(ERK) with RSK, such that ORF45, RSK, and ERK formed high molecular mass
protein complexes. We further demonstrated that the complexes shielded active
pERK and pRSK from dephosphorylation. As a result, the complex-associated RSK
and ERK were activated and sustained at high levels. Finally, we provide
evidence that this mechanism contributes to the sustained activation of ERK
and RSK in Kaposi sarcoma-associated herpesvirus lytic replication.The extracellular signal-regulated kinase
(ERK)2
mitogen-activated protein kinase (MAPK) signaling pathway has been implicated
in diverse cellular physiological processes including proliferation, survival,
growth, differentiation, and motility
(1-4)
and is also exploited by a variety of viruses such as Kaposi
sarcoma-associated herpesvirus (KSHV), human cytomegalovirus, human
immunodeficiency virus, respiratory syncytial virus, hepatitis B virus,
coxsackie, vaccinia, coronavirus, and influenza virus
(5-17).
The MAPK kinases relay the extracellular signaling through sequential
phosphorylation to an array of cytoplasmic and nuclear substrates to elicit
specific responses (1,
2,
18). Phosphorylation of MAPK
is reversible. The kinetics of deactivation or duration of signaling dictates
diverse biological outcomes
(19,
20). For example, sustained
but not transient activation of ERK signaling induces the differentiation of
PC12 cells into sympathetic-like neurons and transformation of NIH3T3 cells
(20-22).
During viral infection, a unique biphasic ERK activation has been observed for
some viruses (an early transient activation triggered by viral binding or
entry and a late sustained activation correlated with viral gene expression),
but the responsible viral factors and underlying mechanism for the sustained
ERK activation remain largely unknown
(5,
8,
13,
23).The p90 ribosomal S6 kinases (RSKs) are a family of serine/threonine
kinases that lie at the terminus of the ERK pathway
(1,
24-26).
In mammals, four isoforms are known, RSK1 to RSK4. Each one has two
catalytically functional kinase domains, the N-terminal kinase domain (NTKD)
and C-terminal kinase domain (CTKD) as well as a linker region between the
two. The NTKD is responsible for phosphorylation of exogenous substrates, and
the CTKD and linker region regulate RSK activation
(1,
24,
25). In quiescent cells ERK
binds to the docking site in the C terminus of RSK
(27-29).
Upon mitogen stimulation, ERK is activated by its upstream MAPK/ERK kinase
(MEK). The active ERK phosphorylates Thr-359/Ser-363 of RSK in the linker
region (amino acid numbers refer to human RSK1) and Thr-573 in the CTKD
activation loop. The activated CTKD then phosphorylates Ser-380 in the linker
region, creating a docking site for 3-phosphoinositide-dependent protein
kinase-1. The 3-phosphoinositide-dependent protein kinase-1 phosphorylates
Ser-221 of RSK in the activation loop and activates the NTKD. The activated
NTKD autophosphorylates the serine residue near the ERK docking site, causing
a transient dissociation of active ERK from RSK
(25,
26,
28). The stimulation of
quiescent cells by a mitogen such as epidermal growth factor or a phorbol
ester such as 12-O-tetradecanoylphorbol-13-acetate (TPA) usually
results in a transient RSK activation that lasts less than 30 min. RSKs have
been implicated in regulating cell survival, growth, and proliferation.
Mutation or aberrant expression of RSK has been implicated in several human
diseases including Coffin-Lowry syndrome and prostate and breast cancers
(1,
24,
25,
30-32).KSHV is a human DNA tumor virus etiologically linked to Kaposi sarcoma,
primary effusion lymphoma, and a subset of multicentric Castleman disease
(33,
34). Infection and
reactivation of KSHV activate multiple MAPK pathways
(6,
12,
35). Noticeably, the ERK/RSK
activation is sustained late during KSHV primary infection and reactivation
from latency (5,
6,
12,
23), but the mechanism of the
sustained ERK/RSK activation is unclear. Recently, we demonstrated that ORF45,
an immediate early and also virion tegument protein of KSHV, interacts with
RSK1 and RSK2 and strongly stimulates their kinase activities
(23). We also demonstrated
that the activation of RSK plays an essential role in KSHV lytic replication
(23). In the present study we
determined the mechanism of ORF45-induced sustained ERK/RSK activation. We
found that ORF45 increases the association of RSK with ERK and protects them
from dephosphorylation, causing sustained activation of both ERK and RSK. 相似文献
9.
10.
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
11.
George Minasov Sivaraman Padavattan Ludmilla Shuvalova Joseph S. Brunzelle Darcie J. Miller Arnaud Basl�� Claudia Massa Frank R. Collart Tilman Schirmer Wayne F. Anderson 《The Journal of biological chemistry》2009,284(19):13174-13184
Cyclic di-GMP (c-di-GMP) is a ubiquitous bacterial second messenger that is
involved in the regulation of cell surface-associated traits and the
persistence of infections. Omnipresent GGDEF and EAL domains, which occur in
various combinations with regulatory domains, catalyze c-di-GMP synthesis and
degradation, respectively. The crystal structure of full-length YkuI from
Bacillus subtilis, composed of an EAL domain and a C-terminal
PAS-like domain, has been determined in its native form and in complex with
c-di-GMP and Ca2+. The EAL domain exhibits a triose-phosphate
isomerase-barrel fold with one antiparallel β-strand. The complex with
c-di-GMP-Ca2+ defines the active site of the putative
phosphodiesterase located at the C-terminal end of the β-barrel. The EAL
motif is part of the active site with Glu-33 of the motif being involved in
cation coordination. The structure of the complex allows the proposal of a
phosphodiesterase mechanism, in which the divalent cation and the general base
Glu-209 activate a catalytic water molecule for nucleophilic in-line attack on
the phosphorus. The C-terminal domain closely resembles the PAS-fold. Its
pocket-like structure could accommodate a yet unknown ligand. YkuI forms a
tight dimer via EAL-EAL and trans EAL-PAS-like domain association.
The possible regulatory significance of the EAL-EAL interface and a mechanism
for signal transduction between sensory and catalytic domains of
c-di-GMP-specific phosphodiesterases are discussed.The dinucleotide cyclic di-GMP (c-di-GMP) was discovered about 20 years ago
when it was found to regulate the activity of cellulase synthase in
Acetobacter xylinum
(1). However, its prominent
role as a global second messenger has been realized only upon the recent
recognition of the omnipresence of genes coding for domains that catalyze
c-di-GMP biosynthesis and degradation in eubacteria
(2). GGDEF domains catalyze the
condensation of two GTP molecules to the cyclic 2-fold symmetric dinucleotide
(diguanylate cyclase activity
(3-6)),
whereas EAL domains are involved in its degradation to yield the linear
dinucleotide pGpG (phosphodiesterase
(PDE)4 A activity)
(3,
7-9).
Recently, also HD-GYP domains have been implicated in c-di-GMP-specific PDE
activity (10). All the domains
have been named according to their sequence signature motifs. They are
typically found in combinations with various other, mostly sensory or
regulatory, domains. It is thought that the balance between antagonistic
diguanylate cyclase and PDE-A activities determines the cellular level of
c-di-GMP and, thus, affects a variety of physiological processes in
bacteria.It has been shown that, in general, c-di-GMP regulates cell
surface-associated traits and community behavior such as biofilm formation
(for reviews see Refs.
11-12),
and its relevance to the virulence of pathogenic bacteria has been
demonstrated (11,
13,
14). In particular, the
dinucleotide has been proposed to orchestrate the switch between acute and
persistent phase of infection.The best characterized diguanylate cyclase is PleD from Caulobacter
crescentus with a Rec-Rec-GGDEF domain architecture (Rec indicates
response regulator receiver domain). The structure of its GGDEF domain
revealed a single GTP-binding site and suggested that dimerization is the
prerequisite for enzymatic activity
(4). This has been corroborated
recently by crystallography showing directly that
modification of the first Rec
domain, mimicking phosphorylation by the cognate kinase, induces formation of
a tightly packed dimer (15).
Additionally, an upper limit of c-di-GMP levels in the cell seems to be
ensured by potent allosteric product inhibition of the PleD cyclase
(4,
15,
16). Recently, the crystal
structure of another diguanylate cyclase, WspR from Pseudomonas
aeruginosa with a Rec-GGDEF domain architecture, has been determined
(17), which showed a
tetrameric quaternary structure and active and feedback inhibition sites that
are very similar to those in PleD.For EAL domains, it has been demonstrated that genetic knock-out results in
phenotypes that are in line with the paradigm that an elevated cellular
c-di-GMP concentration corresponds to a sessile and a low concentration to a
motile bacterial life style
(13,
18,
19). Only recently,
EAL-mediated PDE-A activity has been measured in vitro
(7-9,
20-22).The Bacillus subtilis YkuI protein was targeted for structure
determination by the Midwest Center for Structural Genomics as a member of the
large sequence family that contains EAL (Pfam number PF00563) domains. Here we
report the crystal structure of YkuI showing the fold of the N-terminal EAL
domain and the C-terminal PAS-like domain. Co-crystallization with c-di-GMP
revealed the substrate binding mode and allows the proposal of a catalytic
mechanism. The PAS-like domain most probably has regulatory function, which is
discussed. Recently, another EAL structure has been deposited in the Protein
Data Bank by the Midwest Center for Structural Genomics, the EAL domain of a
GGDEF-EAL protein from Thiobacillus denitrificans (tdEAL; PDB code
2r6o). Comparison of the two structures suggests a possible regulatory
mechanism. 相似文献
12.
13.
14.
15.
16.
17.
Gunthard Stübs Volker Fingerle Bettina Wilske Ulf B. G?bel Ulrich Z?hringer Ralf R. Schumann Nicolas W. J. Schr?der 《The Journal of biological chemistry》2009,284(20):13326-13334
Borrelia burgdorferi sensu lato is the causative agent of Lyme
disease (LD), an infectious disease occurring in North America, Europe, and
Asia in different clinical stages. B. burgdorferi sensu lato
encompasses at least 12 species, with B. burgdorferi sensu stricto,
B. garinii, and B. afzelii being of highest clinical
importance. Immunologic testing for LD as well as recent vaccination
strategies exclusively refer to proteinaceous antigens. However, B.
burgdorferi sensu stricto exhibits glycolipid antigens, including
6-O-acylated cholesteryl β-d-galactopyranoside
(ACGal), and first the data indicated that this compound may act as an
immunogen. Here we investigated whether B. garinii and B.
afzelii also possess this antigen, and whether antibodies directed
against these compounds are abundant among patients suffering from different
stages of LD. Gas-liquid chromatography/mass spectroscopy and NMR spectroscopy
showed that both B. garinii and B. afzelii exhibit ACGal in
high quantities. In contrast, B. hermsii causing relapsing fever
features 6-O-acylated cholesteryl β-d-glucopyranoside
(ACGlc). Sera derived from patients diagnosed for LD contained antibodies
against ACGal, with 80% of patients suffering from late stage disease
exhibiting this feature. Antibodies reacted with ACGal from all three B.
burgdorferi species tested, but not with ACGlc from B. hermsii.
These data show that ACGal is present in all clinically important B.
burgdorferi species, and that specific antibodies against this compound
are frequently found during LD. ACGal may thus be an interesting tool for
improving diagnostics as well as for novel vaccination strategies.Lyme disease (LD)2
is caused by B. burgdorferi sensu lato (s.l.) and is transmitted by
ticks of the genus Ixodes
(1,
2). It is the most common
tick-borne disease in the U.S. with an incidence of 6 per 100,000, with
endemic areas such as Connecticut reaching 111 cases per 100,000
(3). LD is also frequent in
Asia and Europe, particularly in Germany, Austria, Slovenia, and Sweden
(2,
4). B. burgdorferi
s.l. comprises at least 12 species with B. burgdorferi sensu stricto
(Bbu), B. garinii (Bga), and B. afzelii (Baf) being of
highest clinical importance
(2). In the U.S., LD is
exclusively caused by Bbu, whereas in Europe all human pathogenic species are
found, with Bga and Baf being predominant
(2,
5,
6). LD is an infectious disease
occurring in different clinical stages: early localized infection is indicated
by erythema migrans (EM) in ∼70–90% of the patients
(7–9),
and early disseminated infection often causes neurological manifestations,
such as facial palsy, meningitis, meningoradiculitits, or meningoencephalitis
(early neuroborreliosis (NB))
(2,8,9).
The cardinal manifestation of late stage LD in the U.S. is Lyme arthritis
(LA), with ∼70% of the untreated EM cases developing this syndrome
(10,
11). In Europe, next to
arthritis, acrodermatitis chronica atrophicans (ACA) is a frequent late
manifestation, and has been associated with Baf
(11).Currently, diagnosis of LD is generally based on assessment of clinical
features in combination with immunologic serum testing, where both ELISA and a
confirming immunoblot are required
(12,
13). However, because in
Europe and Asia at least three species are causing LD, there is a substantial
variation of immunodominant antigens, which requires the combination of
various homologous antigens for effective serodiagnosis
(14–16).
Immunologic evaluation in these areas is therefore complicated, and no
consensus has been established yet
(12). In comparison to
diagnostic procedures, vaccination strategies directed against LD so far have
also been based on proteinaceous antigens: in the 1990s, recombinant vaccines
based on OspA were found to be effective
(17), but the production was
discontinued, one reason being the high production costs in comparison to
early treatment (2). Another
concern raised against this approach was a potential triggering of autoimmune
diseases by vaccination with Osps due to a similarity between an
immunodominant epitope in OspA and human leukocyte function-associated
antigen-1 (18).In contrast to proteins, information on membrane glycolipids in
Borrelia available today is rather scarce. In 1978, a preliminary
compositional analysis of lipid extracts of B. hermsii causing
relapsing fever (RF) indicated the presence of monoglucosyldiacylglycerol and
acylated as well as non-acylated cholesteryl glucosides
(19). Later, studies on Bbu
indicated the presence of complex glycolipids as well, but no chemical
analysis was performed (20,
21). A more recent study
identified mono-α-d-galactosyldiacylglycerol (MGalD) in Bbu,
and first data indicated that antibodies present in sera obtained from LD
patients detected this antigen
(22). We and others were
recently able to show that Bbu furthermore exhibits cholesteryl
6-O-acyl-β-d-galactopyranoside (ACGal) as well as its
non-acylated counterpart, cholesteryl β-d-galactopyranoside
(βCGal) (23,
24). Patient sera reacted with
ACGal more frequently as compared with MGalD
(23), and antibodies could be
raised in mice by intraperitoneal injection
(24), indicating that this
compound is a strong immunogen.The aim of this study was to elucidate whether ACGal is a common structure
present in the most relevant B. burgdorferi species of clinical
importance, and whether it is a specific feature of Borrelia causing
LD. Furthermore, we aimed at defining the frequency of the occurrence of
antibodies against this antigen in patients suffering from LD. To this end, we
performed a comparative structural analysis of glycolipid fractions of Bbu as
well as the two other B. burgdorferi s.l. species of clinical
importance, Baf and Bga, in comparison with B. hermsii (Bhe), the
causative agent of relapsing fever. We found ACGal to be present in all B.
burgdorferi species tested, whereas Bhe exhibited cholesteryl
6-O-acyl-β-d-glucopyranoside (ACGlc) instead.
Antibodies against ACGal could be detected in the majority of patients
diagnosed for arthritis or acrodermatitis, and these failed to cross-react
with ACGlc. These data demonstrate that ACGal is an abundant, but still highly
specific antigen in B. burgdorferi and thus a promising candidate for
vaccine development and improvement of serologic methods. 相似文献
18.
Kuen-Feng Chen Pei-Yen Yeh Chiun Hsu Chih-Hung Hsu Yen-Shen Lu Hsing-Pang Hsieh Pei-Jer Chen Ann-Lii Cheng 《The Journal of biological chemistry》2009,284(17):11121-11133
Hepatocellular carcinoma (HCC) is one of the most common and aggressive
human malignancies. Recombinant tumor necrosis factor-related
apoptosis-inducing ligand (TRAIL) is a promising anti-tumor agent. However,
many HCC cells show resistance to TRAIL-induced apoptosis. In this study, we
showed that bortezomib, a proteasome inhibitor, overcame TRAIL resistance in
HCC cells, including Huh-7, Hep3B, and Sk-Hep1. The combination of bortezomib
and TRAIL restored the sensitivity of HCC cells to TRAIL-induced apoptosis.
Comparing the molecular change in HCC cells treated with these agents, we
found that down-regulation of phospho-Akt (P-Akt) played a key role in
mediating TRAIL sensitization of bortezomib. The first evidence was that
bortezomib down-regulated P-Akt in a dose- and time-dependent manner in
TRAIL-treated HCC cells. Second, , a PI3K inhibitor, also sensitized
resistant HCC cells to TRAIL-induced apoptosis. Third, knocking down Akt1 by
small interference RNA also enhanced TRAIL-induced apoptosis in Huh-7 cells.
Finally, ectopic expression of mutant Akt (constitutive active) in HCC cells
abolished TRAIL sensitization effect of bortezomib. Moreover, okadaic acid, a
protein phosphatase 2A (PP2A) inhibitor, reversed down-regulation of P-Akt in
bortezomib-treated cells, and PP2A knockdown by small interference RNA also
reduced apoptosis induced by the combination of TRAIL and bortezomib,
indicating that PP2A may be important in mediating the effect of bortezomib on
TRAIL sensitization. Together, bortezomib overcame TRAIL resistance at
clinically achievable concentrations in hepatocellular carcinoma cells, and
this effect is mediated at least partly via inhibition of the PI3K/Akt
pathway.Hepatocellular carcinoma
(HCC) LY2940022 is currently
the fifth most common solid tumor worldwide and the fourth leading cause of
cancer-related death. To date, surgery is still the only curative treatment
but is only feasible in a small portion of patients
(1). Drug treatment is the
major therapy for patients with advanced stage disease. Unfortunately, the
response rate to traditional chemotherapy for HCC patients is unsatisfactory
(1). Novel pharmacological
therapy is urgently needed for patients with advanced HCC. In this regard, the
approval of sorafenib might open a new era of molecularly targeted therapy in
the treatment of HCC patients.Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a
type II transmembrane protein and a member of the TNF family, is a promising
anti-tumor agent under clinical investigation
(2). TRAIL functions by
engaging its receptors expressed on the surface of target cells. Five
receptors specific for TRAIL have been identified, including DR4/TRAIL-R1,
DR5/TRAIL-R2, DcR1, DcR2, and osteoprotegerin. Among TRAIL receptors, only DR4
and DR5 contain an effective death domain that is essential to formation of
death-inducing signaling complex (DISC), a critical step for TRAIL-induced
apoptosis. Notably, the trimerization of the death domains recruits an adaptor
molecule, Fas-associated protein with death domain (FADD), which subsequently
recruits and activates caspase-8. In type I cells, activation of caspase-8 is
sufficient to activate caspase-3 to induce apoptosis; however, in another type
of cells (type II), the intrinsic mitochondrial pathway is essential for
apoptosis characterized by cleavage of Bid and release of cytochrome
c from mitochondria, which subsequently activates caspase-9 and
caspase-3 (3).Although TRAIL induces apoptosis in malignant cells but sparing normal
cells, some tumor cells are resistant to TRAIL-induced apoptosis. Mechanisms
responsible for the resistance include receptors and intracellular resistance.
Although the cell surface expression of DR4 or DR5 is absolutely required for
TRAIL-induced apoptosis, tumor cells expressing these death receptors are not
always sensitive to TRAIL due to intracellular mechanisms. For example, the
cellular FLICE-inhibitory protein (c-FLIP), a homologue to caspase-8 but
without protease activity, has been linked to TRAIL resistance in several
studies (4,
5). In addition, inactivation
of Bax, a proapoptotic Bcl-2 family protein, resulted in resistance to TRAIL
in MMR-deficient tumors (6,
7), and reintroduction of Bax
into Bax-deficient cells restored TRAIL sensitivity
(8), indicating that the Bcl-2
family plays a critical role in intracellular mechanisms for resistance of
TRAIL.Bortezomib, a proteasome inhibitor approved clinically for multiple myeloma
and mantle cell lymphoma, has been investigated intensively for many types of
cancer (9). Accumulating
studies indicate that the combination of bortezomib and TRAIL overcomes the
resistance to TRAIL in various types of cancer, including acute myeloid
leukemia (4), lymphoma
(10–13),
prostate
(14–17),
colon (15,
18,
19), bladder
(14,
16), renal cell carcinoma
(20), thyroid
(21), ovary
(22), non-small cell lung
(23,
24), sarcoma
(25), and HCC
(26,
27). Molecular targets
responsible for the sensitizing effect of bortezomib on TRAIL-induced cell
death include DR4 (14,
27), DR5
(14,
20,
22–23,
28), c-FLIP
(4,
11,
21–23,
29), NF-κB
(12,
24,
30), p21
(16,
21,
25), and p27
(25). In addition, Bcl-2
family also plays a role in the combinational effect of bortezomib and TRAIL,
including Bcl-2 (10,
21), Bax
(13,
22), Bak
(27), Bcl-xL
(21), Bik
(18), and Bim
(15).Recently, we have reported that Akt signaling is a major molecular
determinant in bortezomib-induced apoptosis in HCC cells
(31). In this study, we
demonstrated that bortezomib overcame TRAIL resistance in HCC cells through
inhibition of the PI3K/Akt pathway. 相似文献
19.
Formin-homology (FH) 2 domains from formin proteins associate processively
with the barbed ends of actin filaments through many rounds of actin subunit
addition before dissociating completely. Interaction of the actin
monomer-binding protein profilin with the FH1 domain speeds processive barbed
end elongation by FH2 domains. In this study, we examined the energetic
requirements for fast processive elongation. In contrast to previous
proposals, direct microscopic observations of single molecules of the formin
Bni1p from Saccharomyces cerevisiae labeled with quantum dots showed
that profilin is not required for formin-mediated processive elongation of
growing barbed ends. ATP-actin subunits polymerized by Bni1p and profilin
release the γ-phosphate of ATP on average >2.5 min after becoming
incorporated into filaments. Therefore, the release of γ-phosphate from
actin does not drive processive elongation. We compared experimentally
observed rates of processive elongation by a number of different FH2 domains
to kinetic computer simulations and found that actin subunit addition alone
likely provides the energy for fast processive elongation of filaments
mediated by FH1FH2-formin and profilin. We also studied the role of FH2
structure in processive elongation. We found that the flexible linker joining
the two halves of the FH2 dimer has a strong influence on dissociation of
formins from barbed ends but only a weak effect on elongation rates. Because
formins are most vulnerable to dissociation during translocation along the
growing barbed end, we propose that the flexible linker influences the
lifetime of this translocative state.Formins are multidomain proteins that assemble unbranched actin filament
structures for diverse processes in eukaryotic cells (reviewed in Ref.
1). Formins stimulate
nucleation of actin filaments and, in the presence of the actin
monomer-binding protein profilin, speed elongation of the barbed ends of
filaments
(2-6).
The ability of formins to influence elongation depends on the ability of
single formin molecules to remain bound to a growing barbed end through
multiple rounds of actin subunit addition
(7,
8). To stay associated during
subunit addition, a formin molecule must translocate processively on the
barbed end as each actin subunit is added
(1,
9-12).
This processive elongation of a barbed end by a formin is terminated when the
formin dissociates stochastically from the growing end during translocation
(4,
10).The formin-homology
(FH)2 1 and
2 domains are the best conserved domains of formin proteins
(2,
13,
14). The FH2 domain is the
signature domain of formins, and in many cases, is sufficient for both
nucleation and processive elongation of barbed ends
(2-4,
7,
15). Head-to-tail homodimers
of FH2 domains (12,
16) encircle the barbed ends
of actin filaments (9). In
vitro, association of barbed ends with FH2 domains slows elongation by
limiting addition of free actin monomers. This “gating” behavior
is usually explained by a rapid equilibrium of the FH2-associated end between
an open state competent for actin monomer association and a closed state that
blocks monomer binding (4,
9,
17).Proline-rich FH1 domains located N-terminal to FH2 domains are required for
profilin to stimulate formin-mediated elongation. Individual tracks of
polyproline in FH1 domains bind 1:1 complexes of profilin-actin and transfer
the actin directly to the FH2-associated barbed end to increase processive
elongation rates
(4-6,
8,
10,
17).Rates of elongation and dissociation from growing barbed ends differ widely
for FH1FH2 fragments from different formin homologs
(4). We understand few aspects
of FH1FH2 domains that influence gating, elongation or dissociation. In this
study, we examined the source of energy for formin-mediated processive
elongation, and the influence of FH2 structure on elongation and dissociation
from growing ends. In contrast to previous proposals
(6,
18), we found that fast
processive elongation mediated by FH1FH2-formins is not driven by energy from
the release of the γ-phosphate from ATP-actin filaments. Instead, the
data show that the binding of an actin subunit to the barbed end provides the
energy for processive elongation. We found that in similar polymerizing
conditions, different natural FH2 domains dissociate from growing barbed ends
at substantially different rates. We further observed that the length of the
flexible linker between the subunits of a FH2 dimer influences dissociation
much more than elongation. 相似文献
20.
Mario Perkovi? Stanislaw Schmidt Daniela Marino Rebecca A. Russell Benjamin Stauch Henning Hofmann Ferdinand Kopietz Bj?rn-Philipp Kloke J?rg Zielonka Heike Str?ver Johannes Hermle Dirk Lindemann Vinay K. Pathak Gisbert Schneider Martin L?chelt Klaus Cichutek Carsten Münk 《The Journal of biological chemistry》2009,284(9):5819-5826