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1.
Joseph T. Roland Lynne A. Lapierre James R. Goldenring 《The Journal of biological chemistry》2009,284(2):1213-1223
Rab proteins influence vesicle trafficking pathways through the assembly of
regulatory protein complexes. Previous investigations have documented that
Rab11a and Rab8a can interact with the tail region of myosin Vb and regulate
distinct trafficking pathways. We have now determined that a related Rab
protein, Rab10, can interact with myosin Va, myosin Vb, and myosin Vc. Rab10
localized to a system of tubules and vesicles that have partially overlapping
localization with Rab8a. Both Rab8a and Rab10 were mislocalized by the
expression of dominant-negative myosin V tails. Interaction with Rab10 was
dependent on the presence of the alternatively spliced exon D in myosin Va and
myosin Vb and the homologous region in myosin Vc. Yeast two-hybrid assays and
fluorescence resonance energy transfer studies confirmed that Rab10 binding to
myosin V tails in vivo required the alternatively spliced exon D. In
contrast to our previous work, we found that Rab11a can interact with both
myosin Va and myosin Vb tails independent of their splice isoform. These
results indicate that Rab GTPases regulate diverse endocytic trafficking
pathways through recruitment of multiple myosin V isoforms.Eukaryotic cells are comprised of networks of highly organized membranous
structures that require the efficient and timely movement of diverse
intracellular proteins for proper function. Molecular motors provide the
physical force needed to move these materials along microtubules and actin
microfilaments. Unconventional myosin motors, such as those belonging to
classes V, VI, and VII, have roles in the trafficking and recycling of
membrane-bound structures in eukaryotic cells
(1) and are recruited to
discrete vesicle populations. Myosin VI is involved in clathrin-mediated
endocytosis (2), whereas myosin
VIIa participates in the proper development of stereocilia of inner ear hair
cells and the transport of pigment granules in retinal pigmented epithelial
cells (3,
4). Similarly, the three
members of vertebrate class V myosins, myosin Va, myosin Vb, and myosin Vc,
are required for the proper transport of a wide array of membrane cargoes,
such as the melanosomes of pigment cells, synaptic vesicles in neurons, apical
recycling endosomes in polarized epithelial cells, and bulk recycling vesicles
in non-polarized cells (5).Members of the Rab family of small GTPases regulate many cellular systems,
including membrane trafficking
(6,
7). Certain Rab proteins
associate with and regulate the function of class V myosins. Rab27a, in a
complex with the adaptor protein melanophilin/Slac2-a, is required to localize
myosin Va to the surface of melanin-filled pigment granules in vertebrates
(8-10),
whereas Rab27a and Slac2-c/MyRIP associate with both Myosin Va and myosin VIIa
(3,
11). Rab11a, in a complex with
its adaptor protein Rab11-FIP2, associates with myosin Vb on recycling
endosomes
(12-14)
where the tripartite complex regulates the recycling of a variety of cargoes
(15-19).
In addition, Rab8a associates with both myosin Vb
(20) and myosin Vc
(21) as part of the
non-clathrin-mediated tubular recycling system
(20). Recently, Rab11a has
also been shown to associate with myosin Va in the transport of AMPA receptors
in dendritic spines (22),
contributing to the model of myosin V regulation by multiple Rab proteins.Previous investigations have documented alternative splicing of myosin Va
in a tissue-specific manner
(23-28).
Alternate splicing occurs in a region lying between the coiled-coil region of
the neck of the motor and the globular tail region. Three exons in particular
are subject to alternative splicing: exons B, D, and F
(23-25).
Exon F is critical for association with melanophilin/Slac2 and Rab27a
(8,
9,
29,
30). Additionally, exon B is
required for the interaction of myosin Va with dynein light chain 2 (DLC2)
(27,
28). Currently no function for
the alternatively spliced exon D has been reported. Similar to myosin Va,
myosin Vb contains exons A, B, C, D, and E, whereas no exon F has yet been
identified in myosin Vb (Fig.
1A). In addition, exon B in myosin Vb does not resemble
the dynein light chain 2 (DLC2) binding region in myosin Va
(27,
28), and therefore, it likely
does not interact with DLC2. On the other hand, exon D is highly conserved
among Myosin Va, myosin Vb, and myosin Vc, suggesting a common function in
these molecular motors.Open in a separate windowFIGURE 1.Tissue distribution of human myosin Va and myosin Vb splice
isoforms. A, schematic of the alternative exon organization in
the tails of myosin Va and myosin Vb. It is known that exons B, D, and F are
subject to alternative splicing in myosin Va, whereas there is only evidence
that exon D is alternatively spliced in myosin Vb, which does not contain exon
F. B, alignment of exon D sequences from mouse and human myosin V''s.
myosin Va and myosin Vb both contain exon D (amino acids 1320-1346 of myosin
Va and 1315-1340 of myosin Vb), whereas myosin Vc contains an exon D-like
region (amino acids 1124-1147 of human myosin Vc) that is not known to be
alternatively spliced. Alignment of the exon D regions from all three motors
reveals a high degree of homology, especially in the center of the exon.
Asterisks indicate amino acid identities. C, PCR-based
analysis of human tissue panels reveals the alternative splicing pattern of
exon D in myosin Va and myosin Vb. Primers flanking the region encoding exon D
for both motors were used to amplify cDNA from human MTC™ panels
(Clontech). cDNA amplified from HeLa cell RNA as well as myosin Va and myosin
Vb tail constructs were used as positive controls. Variants expressing exon D
(upper bands) and lacking exon D (lower bands) were visible.
Per., peripheral; Pos., positive.Here we report that Rab10, a protein related to Rab8a and thought to have
similar function
(31-35),
localizes to a system of tubules and vesicles overlapping in distribution with
Rab8a in HeLa cells. Utilizing dominant-negative myosin V tail constructs, we
show that Rab8a and Rab10 can interact with Myosin Va, myosin Vb, and myosin
Vc in vivo. In addition, we have determined that the alternatively
spliced exon D in both myosin Va and myosin Vb is required for interaction
with Rab10. In contrast to our previous findings, we demonstrate that Rab11a
is able to interact with both myosin Va and myosin Vb tails in an exon
independent-manner. These results reveal that multiple Rab proteins
potentially regulate all three class V myosin motors. 相似文献
2.
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
3.
Michael A. Gitcho Jeffrey Strider Deborah Carter Lisa Taylor-Reinwald Mark S. Forman Alison M. Goate Nigel J. Cairns 《The Journal of biological chemistry》2009,284(18):12384-12398
Frontotemporal lobar degeneration (FTLD) with inclusion body myopathy and
Paget disease of bone is a rare, autosomal dominant disorder caused by
mutations in the VCP (valosin-containing protein) gene. The disease
is characterized neuropathologically by frontal and temporal lobar atrophy,
neuron loss and gliosis, and ubiquitin-positive inclusions (FTLD-U), which are
distinct from those seen in other sporadic and familial FTLD-U entities. The
major component of the ubiquitinated inclusions of FTLD with VCP
mutation is TDP-43 (TAR DNA-binding protein of 43 kDa). TDP-43 proteinopathy
links sporadic amyotrophic lateral sclerosis, sporadic FTLD-U, and most
familial forms of FTLD-U. Understanding the relationship between individual
gene defects and pathologic TDP-43 will facilitate the characterization of the
mechanisms leading to neurodegeneration. Using cell culture models, we have
investigated the role of mutant VCP in intracellular trafficking,
proteasomal function, and cell death and demonstrate that mutations in the
VCP gene 1) alter localization of TDP-43 between the nucleus and
cytosol, 2) decrease proteasome activity, 3) induce endoplasmic reticulum
stress, 4) increase markers of apoptosis, and 5) impair cell viability. These
results suggest that VCP mutation-induced neurodegeneration is
mediated by several mechanisms.Frontotemporal lobar degeneration
(FTLD)2
accounts for 10% of all late onset dementias and is the third most frequent
neurodegenerative disease after Alzheimer disease and dementia with Lewy
bodies (1). FTLD with
ubiquitin-immunoreactive inclusions is genetically, clinically, and
neuropathologically heterogeneous
(2,
3). FTLD-U comprises several
distinct entities, including sporadic forms and familial cases caused by
mutations in the genes encoding VCP (valosin-containing protein), GRN
(progranulin), CHMP2B (charged multivesicular body protein 2B), TDP-43 (TAR
DNA-binding protein of 43 kDa) and an unknown gene linked to chromosome 9
(2,
3). Frontotemporal dementia
with inclusion body myopathy and Paget disease of bone is a rare, autosomal
dominant disorder caused by mutations in the VCP gene located on
chromosome 9p13-p12
(4-10)
(Fig. 1). This multisystem
disease is characterized by progressive muscle weakness and atrophy, increased
osteoclastic bone resorption, and early onset frontotemporal dementia, also
called FTLD (9,
11). Mutations in VCP
are also associated with dilatative cardiomyopathy with ubiquitin-positive
inclusions (12).
Neuropathologic features of FTLD with VCP mutation include frontal
and temporal lobar atrophy, neuron loss and gliosis, and ubiquitin-positive
inclusions (FTLD-U). The majority of aggregates are ubiquitin- and
TDP-43-positive neuronal intranuclear inclusions (NIIs); a smaller proportion
is made up of TDP-43-immunoreactive dystrophic neurites (DNs) and neuronal
cytoplasmic inclusions (NCIs). A small number of inclusions are
VCP-immunoreactive (5,
13). Pathologic TDP-43 in
inclusions links a spectrum of diseases in which TDP-43 pathology is a primary
feature, including FTLD-U, motor neuron disease, including amyotrophic lateral
sclerosis, FTLD with motor neuron disease, and inclusion body myopathy and
Paget disease of bone, as well as an expanding spectrum of other disorders in
which TDP-43 pathology is secondary
(14,
15).Open in a separate windowFIGURE 1.Model of pathogenic mutations and domains in valosin-containing
protein. CDC48 (magenta), located within the N terminus (residues
22-108), binds the following cofactors: p47, gp78, and Npl4-Ufd1
(23-25,
28). There are two AAA-ATPase
domains (AAA; blue) at residues 240-283 and 516-569, which
are joined by two linker regions (L1 and L2;
red).TDP-43 proteinopathy in FTLD with VCP mutation has a biochemical
signature similar to that seen in other sporadic and familial cases of FTLD-U,
including sporadic amyotrophic lateral sclerosis, FTLD-motor neuron disease,
FTLD with progranulin (GRN) mutation, and FTLD linked to chromosome
9p (3,
16). TDP-43 proteinopathy in
these disorders is characterized by hyperphosphorylation of TDP-43,
ubiquitination, and cleavage to form C-terminal fragments detected only in
insoluble brain extracts from affected brain regions
(16). Identification of TDP-43
as the major component of the ubiquitin-immunoreactive inclusions of FTLD with
VCP mutation supports the hypothesis that VCP gene mutations
cause an alteration of VCP function, leading to TDP-43 proteinopathy.VCP/p97 (valosin-containing protein) is a member of the AAA (ATPase
associated with diverse cellular activities) superfamily. The N-terminal
domain of VCP has been shown to be involved in cofactor binding (CDC48 (cell
division cycle protein 48)) and two AAA-ATPase domains that form a hexameric
complex (Fig. 1)
(17). Recently, it has been
shown that the N-terminal domain of VCP binds phosphoinositides
(18,
19). AKT (activated
serine-threonine protein kinase) phosphorylates VCP and is required for
constitutive VCP function (20,
21). AKT is activated through
phospholipid binding and phosphorylation via the phosphoinositide 3-kinase
signaling pathway, which is involved in cell survival
(22). The lipid binding domain
may recruit VCP to the cell membrane where it is phosphorylated by AKT
(19).The diversity of VCP functions is modulated, in part, by a variety of
intracellular cofactors, including p47, gp78, and Npl4-Ufd1
(23). Cofactor p47 has been
shown to play a role in the maintenance and biogenesis of both the endoplasmic
reticulum (ER) and Golgi apparatus
(24). The structure of p47
contains a ubiquitin regulatory X domain that binds the N-terminus of VCP, and
together they act as a chaperone to deliver membrane fusion machinery to the
site of adjacent membranes
(25). The function of the
p47-VCP complex is dependent upon cell division cycle 2 (CDC2)
serine-threonine kinase phosphorylation of p47
(26,
27). Also, VCP has been found
to interact with the cytosolic tail of gp78, an ER membrane-spanning E3
ubiquitin ligase that exclusively binds VCP and enhances ER-associated
degradation (ERAD) (28). The
Npl4-Ufd1-VCP complex is involved in nuclear envelope assembly and targeting
of proteins through the ubiquitin-proteasome system
(29,
30). The cell survival
response of this complex has been found to be important in DNA damage repair
though activation by phosphorylation and its recruitment to double-stranded
breaks (20,
31). The Npl4-Ufd1-VCP
cytosolic complex is also recruited to the ER membrane, interacting with
Derlin 1, VCP-interacting membrane proteins (VIMP), and other complexes. At
the ER membrane, these misfolded proteins are targeted to the proteasome via
ERAD
(32-34).
VCP also targets IKKβ for ubiquitination to the ubiquitin-proteasome
system, implicating VCP in the cell survival pathway and neuroprotection
(21,
35-37).To investigate the mechanism of neurodegeneration caused by VCP
mutations, we first tested the hypothesis that VCP mutations decrease
cell viability in vitro using a neuroblastoma SHSY-5Y cell line and
then investigated cellular pathways that are known to lead to
neurodegeneration, including decrease in proteasome activity, caspase-mediated
degeneration, and a change in cellular localization of TDP-43. 相似文献
4.
5.
Brooke K. McMichael Robert B. Wysolmerski Beth S. Lee 《The Journal of biological chemistry》2009,284(18):12266-12275
The nonmuscle myosin IIA heavy chain (Myh9) is strongly associated with
adhesion structures of osteoclasts. In this study, we demonstrate that during
osteoclastogenesis, myosin IIA heavy chain levels are temporarily suppressed,
an event that stimulates the onset of cell fusion. This suppression is not
mediated by changes in mRNA or translational levels but instead is due to a
temporary increase in the rate of myosin IIA degradation. Intracellular
activity of cathepsin B is significantly enhanced at the onset of osteoclast
precursor fusion, and specific inhibition of its activity prevents myosin IIA
degradation. Further, treatment of normal cells with cathepsin B inhibitors
during the differentiation process reduces cell fusion and bone resorption
capacity, whereas overexpression of cathepsin B enhances fusion. Ongoing
suppression of the myosin IIA heavy chain via RNA interference results in
formation of large osteoclasts with significantly increased numbers of nuclei,
whereas overexpression of myosin IIA results in less osteoclast fusion.
Increased multinucleation caused by myosin IIA suppression does not require
RANKL. Further, knockdown of myosin IIA enhances cell spreading and lessens
motility. These data taken together strongly suggest that base-line expression
of nonmuscle myosin IIA inhibits osteoclast precursor fusion and that a
temporary, cathepsin B-mediated decrease in myosin IIA levels triggers
precursor fusion during osteoclastogenesis.The final stages of osteoclastogenesis involve fusion of differentiated
precursors from the monocyte/macrophage lineage
(1). Although the membrane
structural components regulating preosteoclast fusion are not well understood,
in recent years a number of candidate cell surface molecules have been
implicated, including receptors CD44
(2,
3), CD47 and its ligand
macrophage fusion receptor (also known as signal regulatory protein α)
(4–6),
the purinergic receptor P2X7
(7), and the disintegrin and
metalloproteinase ADAM8 (8). A
recently identified receptor, the dendritic cell-specific transmembrane
protein, is essential for osteoclast fusion both in vitro and in
vivo (9,
10). More recently, the d2
subunit of proton-translocating vacuolar proton-translocating ATPases, a
membrane subunit isoform expressed predominantly in osteoclasts, similarly was
demonstrated to be required for fusion in vitro and in vivo
(11). However, elucidation of
the mechanisms by which these molecules may mediate cell fusion has proved to
be difficult.The mammalian class II myosin family consists of distinct isoforms
expressed in skeletal, smooth, and cardiac muscle, as well as three nonmuscle
forms designated IIA, IIB, and IIC
(12–14).
Although all class II molecules are composed of two heavy chains, two
essential light chains, and two regulatory chains, their unique activities are
a function of their particular heavy chain isoforms. Although the nonmuscle
heavy chain isoforms share extensive structural homology, they have been shown
to demonstrate distinct patterns of expression
(15–18),
enzyme kinetics and activation
(12,
19–21),
and cellular function
(22–24).
Knock-out of either myosin IIA or IIB results in embryonic lethality, although
death derives from defects unique to each isoform
(25,
26). In vitro, myosin
IIA, a target of Rho kinase, has been shown to be involved in a wide variety
of cellular functions, including cytokinesis, cell contractility, and adhesion
and motility.The actin cytoskeleton of osteoclasts possesses features unlike those of
most mammalian cell types. First, osteoclasts do not possess stress fibers but
instead form a meshwork of fine actin filaments throughout the cell
(27–29).
Osteoclasts express unusual attachment structures typified by the podosome, a
form of adhesion structure most typically present in cells of the
monocyte/macrophage lineage, dendritic cells, and smooth muscle cells.
Podosomes are integrin-based cell-matrix contact structures that are notable
for the presence of a short (0.5–1.0 μm) F-actin core surrounded by a
ring of adaptor proteins, kinases, small GTPases, and regulators of
endocytosis (30,
31). When cultured on glass,
mature osteoclasts generate a belt of podosomes at the cell periphery.
However, when cultured on bone, osteoclasts form a dense ring of podosome-like
structures that is usually internal to the cell margins
(32). This region, termed the
sealing zone, surrounds a specialized membrane domain termed the ruffled
border, from which protons and proteases are secreted to induce resorption of
bone (1). We previously
demonstrated that myosins IIA and IIB localize to distinct subcellular regions
within osteoclasts, with
MyoIIA2 strongly
segregating to both podosomes and the actin ring of the sealing zone
(28). Because of this
distribution into osteoclast adhesion structures and findings in other cells
showing MyoIIA to be associated with dynamic Rho-kinase-dependent functions,
such as adhesion and locomotion, we hypothesized that MyoIIA may play a vital
role in cell motility and the bone resorption function. In this study, we
examined cellular expression of MyoIIA during osteoclastogenesis and, along
with RNA interference-mediated suppression of the protein, have confirmed its
role in cell spreading, motility, and sealing zone formation. However, this
study also unexpectedly revealed a role for MyoIIA in regulating preosteoclast
fusion during osteoclastogenesis. 相似文献
6.
7.
8.
Lilly Y. W. Bourguignon Weiliang Xia Gabriel Wong 《The Journal of biological chemistry》2009,284(5):2657-2671
9.
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
10.
Jeffrey M. Boyd Jamie L. Sondelski Diana M. Downs 《The Journal of biological chemistry》2009,284(1):110-118
The ApbC protein has been shown previously to bind and rapidly transfer
iron-sulfur ([Fe-S]) clusters to an apoprotein (Boyd, J. M., Pierik, A. J.,
Netz, D. J., Lill, R., and Downs, D. M. (2008) Biochemistry 47,
8195–8202. This study utilized both in vivo and in
vitro assays to examine the function of variant ApbC proteins. The in
vivo assays assessed the ability of ApbC proteins to function in pathways
with low and high demand for [Fe-S] cluster proteins. Variant ApbC proteins
were purified and assayed for the ability to hydrolyze ATP, bind [Fe-S]
cluster, and transfer [Fe-S] cluster. This study details the first kinetic
analysis of ATP hydrolysis for a member of the ParA subfamily of
“deviant” Walker A proteins. Moreover, this study details the
first functional analysis of mutant variants of the ever expanding family of
ApbC/Nbp35 [Fe-S] cluster biosynthetic proteins. The results herein show that
ApbC protein needs ATPase activity and the ability to bind and rapidly
transfer [Fe-S] clusters for in vivo function.Proteins containing iron-sulfur ([Fe-S]) clusters are employed in a wide
array of metabolic functions (reviewed in Ref.
1). Research addressing the
biosynthesis of the iron-molybdenum cofactor of nitrogenase in Azotobacter
vinelandii led to the discovery of an operon
(iscAnifnifUSVcysE1) involved in the
biosynthesis of [Fe-S] clusters (reviewed in Ref.
2). Subsequent experiments led
to the finding of two more systems involved in the de novo
biosynthesis of [Fe-S] clusters, the isc and the suf systems
(3,
4). Like Escherichia
coli, the genome of Salmonella enterica serovar Typhimurium
encodes for the isc and suf [Fe-S] cluster biosynthesis
machinery.Recent studies have identified a number of additional or
non-isc/-suf-encoded proteins that are involved in bacterial
[Fe-S] cluster biosynthesis and repair. Examples include the following: CyaY,
an iron-binding protein believed to be involved in iron trafficking and iron
delivery
(5–7);
YggX, an Fe2+-binding protein that protects the cell from oxidative
stress (8,
9); ErpA, an alternate A-type
[Fe-S] cluster scaffolding protein
(10); NfuA, a proposed
intermediate [Fe-S] delivery protein
(11–13);
YtfE, a protein proposed to be involved in [Fe-S] cluster repair
(14,
15); and CsdA-CsdE, an
alternative cysteine desulferase
(16).Analysis of the metabolic network anchored to thiamine biosynthesis in
S. enterica identified lesions in three non-isc or
-suf loci that compromise Fe-S metabolism as follows: apbC,
apbE, and rseC
(17–21).
This metabolic system was subsequently used to dissect a role for
cyaY and gshA in [Fe-S] cluster metabolism
(6,
22,
23). Of these, the
apbC (mrp in E. coli) locus was identified as the
predominant site of lesions that altered thiamine synthesis by disrupting
[Fe-S] cluster metabolism (17,
18).ApbC is a member of the ParA subfamily of proteins that have a wide array
of functions, including electron transfer
(24), initiation of cell
division (25), and DNA
segregation (26,
27). Importantly, ATP
hydrolysis is required for function of all well characterized members of this
subfamily, and all members contain a “deviant” Walker A motif,
which contains two lysine residues instead of one (GKXXXGK(S/T))
(28). ApbC has been shown to
hydrolyze ATP (17).Recently, five proteins with a high degree of identity to ApbC have been
shown to be involved in [Fe-S] cluster metabolism in eukaryotes. The sequence
alignments of the central portion of these proteins and bacterial ApbC are
shown in Fig. 1. HCF101 was
demonstrated to be involved in chloroplast [Fe-S] cluster metabolism
(29,
30). The CFD1, Npb35, and
huNbp35 (formally Nubp1) proteins were demonstrated to be involved in
cytoplasmic [Fe-S] cluster metabolism
(31,
32). Ind1 was demonstrated to
be involved in the maturation of [Fe-S] clusters in the mitochondrial enzyme
NADH:ubiquinone oxidoreductase
(33). There is currently no
report of any of these proteins hydrolyzing ATP.Open in a separate windowFIGURE 1.Protein sequence alignments of members of the ApbC/Nbp35 subfamily of
ParA family of proteins. Protein alignments were assembled using the
Clustal_W method in the Lasergene® software and show only the central
portion of the proteins, which have the highest sequence conservation. The
three boxed areas highlight the Walker A box, conserved Ser residue,
and CXXC motif. Proteins listed are as follows: ApbC (S.
enterica serovar Typhimurium LT2), CFD1 (S. cerevisiae), Nbp35
(S. cerevisiae), HCF101 (Arabidopsis thaliana), huNpb35
(formally Nubp1) (Homo sapiens), and Ind1 (Candida
albicans).Biochemical analysis of ApbC indicated that it could bind and transfer
[Fe-S] clusters to Saccharomyces cerevisiae apo-isopropylmalate
isomerase (34). Additional
genetic studies indicated that ApbC has a degree of functional redundancy with
IscU, a known [Fe-S] cluster scaffolding protein
(35,
36).In this study we investigate the correlation between the biochemical
properties of ApbC (i.e. ATPase activity, [Fe-S] cluster binding, and
[Fe-S] cluster transfer rates) and the in vivo function of this
protein. This is the first detailed kinetic analysis of ATP hydrolysis for a
member of the ParA subfamily of deviant Walker A proteins and the first
functional analysis of a member of the ever expanding family of ApbC/Nbp35
proteins. Data presented indicate that noncomplementing variants have distinct
biochemical properties that place them in three distinct classes. 相似文献
11.
Leonard Kaysser Liane Lutsch Stefanie Siebenberg Emmanuel Wemakor Bernd Kammerer Bertolt Gust 《The Journal of biological chemistry》2009,284(22):14987-14996
Caprazamycins are potent anti-mycobacterial liponucleoside antibiotics
isolated from Streptomyces sp. MK730-62F2 and belong to the
translocase I inhibitor family. Their complex structure is derived from
5′-(β-O-aminoribosyl)-glycyluridine and comprises a unique
N-methyldiazepanone ring. The biosynthetic gene cluster has been
identified, cloned, and sequenced, representing the first gene cluster of a
translocase I inhibitor. Sequence analysis revealed the presence of 23 open
reading frames putatively involved in export, resistance, regulation, and
biosynthesis of the caprazamycins. Heterologous expression of the gene cluster
in Streptomyces coelicolor M512 led to the production of
non-glycosylated bioactive caprazamycin derivatives. A set of gene deletions
validated the boundaries of the cluster and inactivation of cpz21
resulted in the accumulation of novel simplified liponucleoside antibiotics
that lack the 3-methylglutaryl moiety. Therefore, Cpz21 is assigned to act as
an acyltransferase in caprazamycin biosynthesis. In vivo and in
silico analysis of the caprazamycin biosynthetic gene cluster allows a
first proposal of the biosynthetic pathway and provides insights into the
biosynthesis of related uridyl-antibiotics.Caprazamycins
(CPZs)2
(Fig. 1, 1) are
liponucleoside antibiotics isolated from a fermentation broth of
Streptomyces sp. MK730-62F2
(1,
2). They show excellent
activity in vitro against Gram-positive bacteria, in particular
against the genus Mycobacterium including Mycobacterium
intracellulare, Mycobacterium avium, and Mycobacterium
tuberculosis (3). In a
pulmonary mouse model with M. tuberculosis H37Rv, administration of
caprazamycin B exhibited a therapeutic effect but no significant toxicity
(4). Structural elucidation
(2) revealed a complex and
unique composition of elements the CPZs share only with the closely related
liposidomycins (LPMs, 2)
(5). The core skeleton is the
(+)-caprazol (5)
composed of an N-alkylated
5′-(β-O-aminoribosyl)-glycyluridine, also known from
FR-900493 (6)
(6) and the muraymycins
(7)
(7), which is cyclized to form
a rare diazepanone ring. Attached to the 3′″-OH are β-hydroxy
fatty acids of different chain length resulting in CPZs A–G
(1). They differ from
the LPMs in the absence of a sulfate group at the 2″-position of the
aminoribose and the presence of a permethylated l-rhamnose
β-glycosidically linked to the 3-methylglutaryl (3-MG) moiety.Open in a separate windowFIGURE 1.Nucleoside antibiotics of the translocase I inhibitor family.The LPMs have been shown to inhibit biosynthesis of the bacterial cell wall
by targeting the formation of lipid I
(8). The CPZs are expected to
act in the same way and are assigned to the growing number of translocase I
inhibitors that include other nucleoside antibiotics, like the tunicamycins
and mureidomycins (9). During
peptidoglycan formation, translocase I catalyzes the transfer of
UDP-MurNAc-pentapeptide to the undecaprenyl phosphate carrier to
generate lipid I (10). This
reaction is considered an unexploited and promising target for new
anti-infective drugs (11).Recent investigations indicate that the 3″-OH group
(12), the amino group of the
aminoribosyl-glycyluridine, and an intact uracil moiety
(13) are essential for the
inhibition of the Escherichia coli translocase I MraY. The chemical
synthesis of the (+)-caprazol
(5) was recently
accomplished (14), however,
this compound only shows weak antibacterial activity. In contrast, the
acylated compounds 3 and 4 exhibit strong growth inhibition of
mycobacteria, suggesting a potential role of the fatty acid side chain in
penetration of the bacterial cell
(15,
16). Apparently, the
acyl-caprazols (4)
represent the most simplified antibiotically active liponucleosides and a good
starting point for further optimization of this class of potential
therapeutics.Although chemical synthesis and biological activity of CPZs and LPMs has
been studied in some detail, their biosynthesis remains speculative and only
few data exists about the formation of other translocase I inhibitors
(17,
18). Nevertheless, we assume
that the CPZ biosynthetic pathway is partially similar to that of LPMs,
FR-90043 (6), and
muraymycins (7) and
presents a model for the comprehension and manipulation of liponucleoside
formation. Considering the unique structural features of the CPZs we also
expect some unusual biotransformations to be involved in the formation of,
e.g. the (+)-caprazol.Here we report the identification and analysis of the CPZ gene cluster, the
first cluster of a translocase I inhibitor. A set of gene disruption
experiments provide insights into the biosynthetic origin of the CPZs and
moreover, heterologous expression of the gene cluster allows the generation of
novel bioactive derivatives by pathway engineering. 相似文献
12.
13.
Bryson W. Katona Shrikant Anant Douglas F. Covey William F. Stenson 《The Journal of biological chemistry》2009,284(5):3354-3364
Bile acids are steroid detergents that are toxic to mammalian cells at high
concentrations; increased exposure to these steroids is pertinent in the
pathogenesis of cholestatic disease and colon cancer. Understanding the
mechanisms of bile acid toxicity and apoptosis, which could include
nonspecific detergent effects and/or specific receptor activation, has
potential therapeutic significance. In this report we investigate the ability
of synthetic enantiomers of lithocholic acid (ent-LCA),
chenodeoxycholic acid (ent-CDCA), and deoxycholic acid
(ent-DCA) to induce toxicity and apoptosis in HT-29 and HCT-116
cells. Natural bile acids were found to induce more apoptotic nuclear
morphology, cause increased cellular detachment, and lead to greater capase-3
and -9 cleavage compared with enantiomeric bile acids in both cell lines. In
contrast, natural and enantiomeric bile acids showed similar effects on
cellular proliferation. These data show that bile acid-induced apoptosis in
HT-29 and HCT-116 cells is enantiospecific, hence correlated with the absolute
configuration of the bile steroid rather than its detergent properties. The
mechanism of LCA- and ent-LCA-induced apoptosis was also investigated
in HT-29 and HCT-116 cells. These bile acids differentially activate initiator
caspases-2 and -8 and induce cleavage of full-length Bid. LCA and
ent-LCA mediated apoptosis was inhibited by both pan-caspase and
selective caspase-8 inhibitors, whereas a selective caspase-2 inhibitor
provided no protection. LCA also induced increased CD95 localization to the
plasma membrane and generated increased reactive oxygen species compared with
ent-LCA. This suggests that LCA/ent-LCA induce apoptosis
enantioselectively through CD95 activation, likely because of increased
reactive oxygen species generation, with resulting procaspase-8 cleavage.Bile acids are physiologic steroids that are necessary for the proper
absorption of fats and fat-soluble vitamins. Their ability to aid in these
processes is largely due to their amphipathic nature and thus their ability to
act as detergents. Despite the beneficial effects, high concentrations of bile
acids are toxic to cells
(1-11).
High fat western diets induce extensive recirculation of the bile acid pool,
resulting in increased exposure of the colonic epithelial cells to these toxic
steroids (12,
13). A high fat diet is also a
risk factor for colon carcinogenesis; increased bile acid exposure is
responsible for some of this risk. Bile acids can contribute to both colon
cancer formation and progression, and their effects on colonic proliferation
and apoptosis aid this process by disrupting the balance between cell growth
and cell death, as well as helping to select for bile acid-resistant cells
(14,
15).In colonocyte-derived cell lines bile acid-induced apoptosis is thought to
proceed through mitochondrial destabilization with resulting mitochondrial
permeability transition formation and cytochrome c release as well as
generation of oxidative stress
(1,
9-11).
Bile acid-induced apoptosis has also been extensively explored in hepatocyte
derived cell lines with mechanisms including mitochondria dysfunction
(16-23),
endoplasmic reticulum stress
(24), ligand-independent
activation of death receptor pathways
(18,
25-28),
and modulation of cellular apoptotic and anti-apoptotic Bcl-2 family proteins
(29).Although ample evidence exists for multiple mechanisms of bile acid-induced
apoptosis, the precise interactions responsible for initiating these apoptotic
pathways are still unclear. Bile acids have been shown to interact directly
with specific receptors (30,
31). These steroids can also
initiate cellular signaling through nonspecific membrane perturbations
(32), and evidence exists
showing that other simple detergents (i.e. Triton X-100) are capable
of inducing caspase cleavage nonspecifically with resultant apoptosis
(33). Therefore, hydrophobic
bile acids may interact nonspecifically with cell membranes to alter their
physical properties, bind to receptors specific for these steroids, or utilize
a combination of both specific and nonspecific interactions to induce
apoptosis.Bile acid enantiomers could be useful tools for elucidating mechanisms of
bile acid toxicity and apoptosis. These enantiomers, known as
ent-bile acids, are synthetic nonsuperimposable mirror images of
natural bile acids with identical physical properties except for optical
rotation. Because bile acids are only made in one absolute configuration
naturally, ent-bile acids must be constructed using a total synthetic
approach. Recently we reported the first synthesis of three enantiomeric bile
acids: ent-lithocholic acid
(ent-LCA),2
ent-chenodeoxycholic acid (ent-CDCA), and
ent-deoxycholic acid (ent-DCA)
(Fig. 1)
(34,
35). Enantiomeric bile acids
have unique farnesoid X receptor, vitamin D receptor, pregnane X receptor, and
TGR5 receptor activation profiles compared with the corresponding natural bile
acids (34). This illustrates
that natural and enantiomeric bile acids interact differently within chiral
environments because of their distinct three-dimensional configurations
(Fig. 1). Despite these
differences in chiral interactions, ent-bile acids have physical
properties identical to those of their natural counterparts including
solubility and critical micelle concentrations
(34,
35). With different receptor
interaction profiles and identical physical properties compared with natural
bile acids, ent-bile acids are ideal compounds to differentiate
between the receptor-mediated and the non-receptor-mediated functions of
natural bile acids.Open in a separate windowFIGURE 1.Natural and enantiomeric bile acids. Structures and
three-dimensional projection views of natural LCA, CDCA, DCA, and their
enantiomers (ent-LCA, ent-CDCA, and ent-DCA). The
three-dimensional ent-steroid structure is rotated 180° around
the long axis for easier comparison with the natural steroid.In this study we explore the enantioselectivity of LCA-, CDCA-, and
DCA-mediated toxicity and apoptosis in two human colon adenocarcinoma cell
lines, HT-29 and HCT-116. Because the mechanism of natural LCA induced
apoptosis has never been characterized, we then examined in more detail LCA-
and ent-LCA-mediated apoptosis in colon cancer cells. These studies
will not only explore the LCA apoptotic mechanism but will also determine
whether ent-LCA signals through similar cellular pathways. 相似文献
14.
Harry S. Courtney Yi Li Waleed O. Twal W. Scott Argraves 《The Journal of biological chemistry》2009,284(19):12966-12971
The adhesion of bacteria to host tissues is often mediated by interactions
with extracellular matrices. Herein, we report on the interactions of the
group A streptococcus, Streptococcus pyogenes, with the extracellular
matrix protein fibulin-1. S. pyogenes bound purified fibulin-1 in a
dose-dependent manner. Genetic ablation of serum opacity factor (SOF), a
virulence determinant of S. pyogenes, reduced binding by ∼50%,
and a recombinant peptide of SOF inhibited binding of fibulin-1 to
streptococci by ∼45%. Fibulin-1 bound to purified SOF2 in a dose-dependent
manner with high affinity (Kd = 1.6 nm). The
fibulin-1-binding domain was localized to amino acid residues 457–806 of
SOF2, whereas the fibronectin-binding domain is contained within residues
807–931 of SOF2, indicating that these two domains are separate and
distinct. Fibulin-1 bound to recombinant SOF from M types 2, 4, 28, and 75 of
S. pyogenes, indicating that the fibulin-1-binding domain is likely
conserved among SOF from different serotypes. Mixed binding experiments
suggested that gelatin, fibronectin, fibulin-1, and SOF form a quaternary
molecular complex that enhanced the binding of fibulin-1. These data indicate
that S. pyogenes can interact with fibulin-1 and that SOF is a major
streptococcal receptor for fibulin-1 but not the only receptor. Such
interactions with fibulin-1 may be involved in the adhesion of S.
pyogenes to extracellular matrices of the host.Adhesion of bacteria to host surfaces is the first stage in establishing
bacterial infections in the human host, and a variety of molecular mechanisms
are utilized to initiate adhesion. A common mechanism for adhesion involves
interactions between bacterial adhesins and components of the extracellular
matrices of the host. The identification and characterization of microbial
surface components recognizing adhesive matrix molecules (MSCRAMM) has led to
important advances in vaccines and immunotherapies for preventing and treating
bacterial infections (1).The group A streptococcus, Streptococcus pyogenes, is a major
human pathogen causing diseases ranging from relative minor infections such as
pharyngitis and cellulitis to severe infections with high levels of morbidity
and mortality such as necrotizing fasciitis and toxic shock syndrome
(2). This pathogen expresses
adhesins that interact with various components of the extracellular matrix
including laminin, elastin, fibronectin, fibrinogen, and collagen
(3–7).
The interactions between fibronectin and S. pyogenes have been
intensely studied, and these investigations have revealed at least 10
different streptococcal proteins that bind fibronectin
(4).Serum opacity factor
(SOF)2 is a major
fibronectin-binding protein that is involved in adhesion to host cells
(8–11).
SOF is a virulence determinant that is expressed by approximately half of the
clinical isolates of S. pyogenes
(8). SOF opacifies serum by
binding and displacing apoA-I in high density lipoproteins
(8,
12–15).
SOF is covalently linked to the streptococcal cell wall via an LPSTG sortase
recognition site and is also released in a soluble form. SOF has two
functionally distinct domains, an N-terminal domain that opacifies serum and a
C-terminal domain that binds fibronectin. The role of SOF in adhesion involves
both its C-terminal fibronectin-binding domain and an N-terminal region (see
Fig. 1 for a schematic of
structure) (9,
11). However, the nature of
the interactions between the N-terminal region of SOF and host components is
not well characterized.Open in a separate windowFIGURE 1.A, a schematic of the structure of SOF and its functional domains
is shown. The assignment of functional domains are based on the findings of
Rakonjac et al. (33),
Kreikemeyer et al.
(34), Courtney et al.
(8,
13), and results presented in
this work. Fn, fibronectin. B, the data for the binding of
SOF peptides to fibronectin are from previous publications
(8,
13), and the data for
fibulin-1 are from the present work.Herein, we report on the interactions between a truncated form of SOF in
which its fibronectin-binding domain has been deleted and the extracellular
matrix protein fibulin-1. Fibulin-1 is a member of the fibulin family that
currently consists of seven glycoproteins. All fibulins contain epidermal
growth factor-like repeats and a unique fibulin-type module at its C terminus
that define this family (16,
17). Fibulin-1 is found within
the extracellular matrices and in human plasma at 30–50 μg/ml
(18). It interacts with many
of the components of the extracellular matrix including fibronectin, laminin,
fibrinogen, nidogen-1, endostatin, aggrecan, and versican
(16,
19). Due to its intimate
relationship with the extracellular matrix, it is not surprising that the
defects in fibulin-1 have a wide-ranging impact. Genetic evidence suggests
that fibulin-1 is involved in tissue organization, the maturation and
maintenance of blood vessels, and multiple embryonic pathways
(16,
20–22).Although it has been established that many of the other components of the
extracellular matrix can interact with bacteria, there has been no previous
report on the binding of fibulins to bacteria. Our findings indicate that
fibulin-1 does bind to streptococci and that SOF is a major streptococcal
receptor for fibulin-1. 相似文献
15.
Justin F. Shaffer Robert W. Kensler Samantha P. Harris 《The Journal of biological chemistry》2009,284(18):12318-12327
Cardiac myosin-binding protein C (cMyBP-C) is a regulatory protein
expressed in cardiac sarcomeres that is known to interact with myosin, titin,
and actin. cMyBP-C modulates actomyosin interactions in a
phosphorylation-dependent way, but it is unclear whether interactions with
myosin, titin, or actin are required for these effects. Here we show using
cosedimentation binding assays, that the 4 N-terminal domains of murine
cMyBP-C (i.e. C0-C1-m-C2) bind to F-actin with a dissociation
constant (Kd) of ∼10 μm and a molar
binding ratio (Bmax) near 1.0, indicating 1:1 (mol/mol)
binding to actin. Electron microscopy and light scattering analyses show that
these domains cross-link F-actin filaments, implying multiple sites of
interaction with actin. Phosphorylation of the MyBP-C regulatory motif, or
m-domain, reduced binding to actin (reduced Bmax) and
eliminated actin cross-linking. These results suggest that the N terminus of
cMyBP-C interacts with F-actin through multiple distinct binding sites and
that binding at one or more sites is reduced by phosphorylation. Reversible
interactions with actin could contribute to effects of cMyBP-C to increase
cross-bridge cycling.Cardiac myosin-binding protein C
(cMyBP-C)2
is a thick filament accessory protein that performs both structural and
regulatory functions within vertebrate sarcomeres. Both roles are likely to be
essential in deciphering how a growing number of mutations found in the
cMyBP-C gene, i.e. MYBPC3, lead to cardiomyopathies and heart failure
in a substantial number of the world''s population
(1,
2).Considerable progress has recently been made in determining the regulatory
functions of cMyBP-C and it is now apparent that cMyBP-C normally limits
cross-bridge cycling kinetics and is critical for cardiac function
(3-5).
Phosphorylation of cMyBP-C is essential for its regulatory effects because
elimination of phosphorylation sites (serine to alanine substitutions)
abolishes the ability of protein kinase A (PKA) to accelerate cross-bridge
cycling kinetics and blunts cardiac responses to inotropic stimuli
(6). The substitutions further
impair cardiac function, reduce contractile reserve, and cause cardiac
hypertrophy in transgenic mice
(6,
7). By contrast, substitution
of aspartic acids at these sites to mimic constitutive phosphorylation is
benign or cardioprotective
(8).Although a role for cMyBP-C in modulating cross-bridge kinetics is
supported by several transgenic and knock-out mouse models
(6,
7,
9,
10), the precise mechanisms by
which cMyBP-C exerts these effects are not completely understood. For
instance, the unique regulatory motif or “m-domain” of cMyBP-C
binds to the S2 subfragment of myosin in vitro
(11) and binding is abolished
by PKA-mediated phosphorylation of the m-domain
(12). These observations have
led to the idea that (un)binding of the m-domain from myosin S2 mediates
PKA-induced increases in cross-bridge cycling kinetics. Consistent with this
idea, Calaghan and colleagues
(13) showed that S2 added to
transiently permeabilized myocytes increased their contractility, presumably
because added S2 displaced cMyBP-C from binding endogenous S2. However, other
reports indicate that cMyBP-C can influence actomyosin interactions through
mechanisms unrelated to S2 binding, because either purified cMyBP-C
(14) or recombinant N-terminal
domains of cMyBP-C (15)
affected acto-S1 filament sliding velocities and ATPase rates in the absence
of myosin S2. These results thus raise the possibility that interactions with
ligands other than myosin S2, such as actin or myosin S1, contribute to
effects of cMyBP-C on cross-bridge interaction kinetics.The idea that cMyBP-C interacts with actin to influence cross-bridge
cycling kinetics is supported by several studies that implicate the regulatory
m-domain or sequences near it in actin binding
(16-19).
cMyBP-C is a member of the immunoglobulin (Ig) superfamily of proteins and
consists of 11 repeating domains that bear homology to either Ig or
fibronectin-like folds. Domains are numbered sequentially from the N terminus
of cMyBP-C as C0 through C10. The m-domain, a unique sequence of ∼100
amino acids, is located between domains C1 and C2 and is phosphorylated on at
least 3 serine residues by PKA
(12). Although the precise
structure of the m-domain is not known, small angle x-ray scattering data
suggest that it is compact and folded in solution and is thus similar in size
and dimensions to the surrounding Ig domains
(20). Recombinant proteins
encompassing the m-domain and/or a combination of adjacent domains including
C0, C1, C2, and a proline-alanine-rich sequence that links C0 to C1 have been
shown to bind actin (16,
18,
19).The purpose of the present study was to characterize binding interactions
of the N terminus of cMyBP-C with actin and to determine whether interactions
with actin are influenced by phosphorylation of the m-domain. Results
demonstrate that the N terminus of cMyBP-C binds to F-actin and to native thin
filaments with affinities similar to that reported for cMyBP-C binding to
myosin S2 (11). Furthermore,
actin binding was reduced by m-domain phosphorylation, suggesting that
reversible interactions of cMyBP-C with actin could contribute to modulation
of cross-bridge kinetics. 相似文献
16.
Heather B. Claxton David L. Akey Monica K. Silver Suzanne J. Admiraal Janet L. Smith 《The Journal of biological chemistry》2009,284(8):5021-5029
Two thioesterases are commonly found in natural product biosynthetic
clusters, a type I thioesterase that is responsible for removing the final
product from the biosynthetic complex and a type II thioesterase that is
believed to perform housekeeping functions such as removing aberrant units
from carrier domains. We present the crystal structure and the kinetic
analysis of RifR, a type II thioesterase from the hybrid nonribosomal peptide
synthetases/polyketide synthase rifamycin biosynthetic cluster of
Amycolatopsis mediterranei. Steady-state kinetics show that RifR has
a preference for the hydrolysis of acyl units from the phosphopantetheinyl arm
of the acyl carrier domain over the hydrolysis of acyl units from the
phosphopantetheinyl arm of acyl-CoAs as well as a modest preference for the
decarboxylated substrate mimics acetyl-CoA and propionyl-CoA over malonyl-CoA
and methylmalonyl-CoA. Multiple RifR conformations and structural similarities
to other thioesterases suggest that movement of a helical lid controls access
of substrates to the active site of RifR.Assembly line complexes, which include modular polyketide synthases
(PKS)3 and
nonribosomal peptide synthetases (NRPS), are multifunctional proteins composed
of modules that work in succession to synthesize secondary metabolites, many
of which are precursors of potent antibiotics, immunosuppressants, anti-tumor
agents, and other bioactive compounds. Rifamycin, the precursor to the
anti-tuberculosis drug rifampicin, is produced by the rifamycin assembly line
complex, which is an NRPS/PKS hybrid system composed of one NRPS-like and 10
PKS modules (1). Each module in
an assembly line complex extends and modifies the intermediate compound before
passing it on to the next module in the series
(Fig. 1A). The
intermediate compounds are covalently attached through a thioester linkage to
the phosphopantetheine arm (Ppant) of carrier domains, one associated with
each module, until they are released from the synthase, usually by a type I
thioesterase (TEI) (2,
3).Open in a separate windowFIGURE 1.Proposed functions of thioesterase II proteins. A, chain
elongation by a PKS module. The chain elongation intermediate is transferred
from the ACP of the upstream module to the ketosynthase (KS) domain.
The acyltransferase (AT) domain transfers an acyl group building
block from CoA to the ACP within the module. The KS domain catalyzes
condensation of the new building block with the intermediate, releasing
CO2. B, production of a decarboxylated acyl unit by the
ketosynthase domain and the subsequent hydrolysis by a TEII. C,
mispriming of a PKS by transfer of an acyl-phosphopantetheine arm by a
promiscuous phosphopantetheinyl transferase (Pptase) and the
subsequent hydrolysis by a TEII. D, hydrolysis of an amino acid
derivative by a TEII from an NRPS module comprising an adenylation domain
(A) and a peptide carrier protein (PCP) domain.TEIs are usually integrated into the final module of the assembly line
complex and remove the final product through macrocyclization or hydrolysis.
Occasionally, tandem type I thioesterases are integrated at the C terminus of
the final module of NRPS pathways
(4).Although TEIs are covalently attached to the terminal module and generally
process only the final product of an assembly line complex, type II
thioesterases (TEIIs) are discrete proteins that can remove intermediates from
any module in the complex. A variety of functions have been attributed to
TEIIs, the most prevalent of which is a “housekeeping function,”
the removal of aberrant acyl units from carrier domains. These aberrant acyl
units may be due to premature decarboxylation by a PKS ketosynthase domain
(5)
(Fig. 1B) or to
mispriming of the carrier domain by a promiscuous phosphopantetheinyl
transferase
(6–8)
(Fig. 1C). Other
proposed functions for TEIIs include the removal of intermediates from the
synthase as in the case of the mammary gland rat fatty acid synthase (FAS)
TEII in lactating rats, which removes medium chain
C8-C12 fatty acids from the ACP domain
(9) and the removal of amino
acid derivatives from a carrier domain
(10–13),
allowing these derivatives to be incorporated into the natural product by a
later module in the assembly line complex
(Fig. 1D).Disruption of the TEI function results in a complete loss of product,
whereas disruption of TEII function results in a significant decrease in
product yield (30–95%)
(4,
14–24).
Removal of the TEII from the rifamycin assembly line resulted in a 60%
decrease in product yield
(25). Neither TEIs nor TEIIs
may rescue the disrupted function of the other
(6), but a TEII from another
pathway may rescue the function of a disrupted TEII
(26).Two models have been proposed for the TEII housekeeping function
(5). In the high specificity
model, the TEII scans the complex and efficiently removes only aberrant acyl
units. In the low specificity model, the TEII removes both correct and
incorrect acyl units from the Ppant arm at an inefficient rate. Correct acyl
units are quickly incorporated into the growing intermediate compound. In
contrast, incorrect acyl units stall the assembly line, providing a longer
window of opportunity for removal by a TEII. Thus a slow, low specificity
enzyme can be effective.TEIIs from different pathways have differing specificities, but general
trends include a preference for decarboxylated acyl units over carboxylated
acyl units (5,
6,
27), substrates linked to a
carrier domain over substrates linked to CoA or the phosphopantetheine mimic
N-acetylcysteamine (7,
28), and single amino acids
over di- or tri-peptides (6,
7). TEIIs are able to hydrolyze
substrates attached to carrier domains from their native pathway as well as
other pathways (6,
20,
28).PKS/NRPS/FAS thioesterases belong to the α/β hydrolase family.
Structures are reported for seven PKS/NRPS/FAS thioesterases: crystal
structures for the TEIs from the pikromycin (PikTE) PKS
(29), 6-deoxyerythronolide B
(DEBSTE) PKS (30), surfactin
NRPS (SrfTE) (31), fengycin
NRPS (FenTE) (32), and human
fatty acid synthase (hFasTE)
(33) systems, and NMR
structures for enterobactin TEI
(34), and surfactin TEII
(35). Like PKS modules, PKS
TEIs are dimers. The dimer interface comprises two N-terminal helices that are
unique to the PKS TEIs. NRPS TEIs are monomeric, like NRPS modules. The NRPS
TEII of surfactin is also monomeric
(31). Although the FAS complex
is dimeric, the FAS TEI is a monomer
(33). All of the TEs have an
α-helical insertion after strand β5 that forms a lid over the
active site. Additionally, in the PKS TEIs, the N-terminal dimer-forming
helices contribute to the lid structure, forming a fixed channel that runs the
length of the TE and contains the active site. In contrast, the active site
pocket of monomeric NRPS TEIs and TEIIs is flexible; two conformations of the
lid and active site pocket were observed in the surfactin TEI (SrfTEI) crystal
structure (7), and chemical
shift observations suggested greater flexibility for residues of the lid
region in the surfactin TEII (SrfTEII) solution structure
(35). These movements seem to
be of functional importance, because a movement of a linker peptide in SrfTEI
determines the shape of the active site pocket and a movement of the first lid
helix appears to modulate access to the active site
(31).We report the structure and activity of recombinant RifR, the TEII of the
rifamycin biosynthetic cluster. Steady-state kinetic analysis of the
hydrolytic activity of RifR on a wide range of acyl-CoA and acyl-ACP
substrates demonstrates that acyl-ACP substrates are preferred over the
acyl-CoAs. Aberrant, decarboxylated acyl units are processed more efficiently
than are the natural rifamycin building blocks. We report the crystal
structure of RifR, the first for any hybrid PKS/NRPS TEII. The size and shape
of the substrate chamber are variable, because one of the elements forming the
chamber, an extended linker segment, is highly flexible, and different crystal
forms reveal different shapes for the substrate binding site. Access to the
active site is severely restricted, and structural comparisons with other
thioesterases suggest that a conformational change in the lid and the flexible
linker region is required for access to the substrate pocket. 相似文献
17.
Martin J. Sergeant Jian-Jun Li Christine Fox Nicola Brookbank Dean Rea Timothy D. H. Bugg Andrew J. Thompson 《The Journal of biological chemistry》2009,284(8):5257-5264
Members of the carotenoid cleavage dioxygenase family catalyze the
oxidative cleavage of carotenoids at various chain positions, leading to the
formation of a wide range of apocarotenoid signaling molecules. To explore the
functions of this diverse enzyme family, we have used a chemical genetic
approach to design selective inhibitors for different classes of carotenoid
cleavage dioxygenase. A set of 18 arylalkyl-hydroxamic acids was synthesized
in which the distance between an iron-chelating hydroxamic acid and an
aromatic ring was varied; these compounds were screened as inhibitors of four
different enzyme classes, either in vitro or in vivo. Potent
inhibitors were found that selectively inhibited enzymes that cleave
carotenoids at the 9,10 position; 50% inhibition was achieved at submicromolar
concentrations. Application of certain inhibitors at 100 μm to
Arabidopsis node explants or whole plants led to increased shoot
branching, consistent with inhibition of 9,10-cleavage.Carotenoids are synthesized in plants and micro-organisms as
photoprotective molecules and are key components in animal diets, an example
being β-carotene (pro-vitamin A). The oxidative cleavage of carotenoids
occurs in plants, animals, and micro-organisms and leads to the release of a
range of apocarotenoids that function as signaling molecules with a diverse
range of functions (1). The
first gene identified as encoding a carotenoid cleavage dioxygenase
(CCD)2 was the maize
Vp14 gene that is required for the formation of abscisic acid (ABA),
an important hormone that mediates responses to drought stress and aspects of
plant development such as seed and bud dormancy
(2). The VP14 enzyme cleaves at
the 11,12 position (Fig. 1) of
the epoxycarotenoids 9′-cis-neoxanthin and/or
9-cis-violaxanthin and is now classified as a
9-cis-epoxycarotenoid dioxygenase (NCED)
(3), a subclass of the larger
CCD family.Open in a separate windowFIGURE 1.Reactions catalyzed by the carotenoid cleavage dioxygenases.
a, 11,12-oxidative cleavage of 9′-cis-neoxanthin by
NCED; b, oxidative cleavage reactions on β-carotene and
zeaxanthin.Since the discovery of Vp14, many other CCDs have been shown to be
involved in the production of a variety of apocarotenoids
(Fig. 1). In insects, the
visual pigment retinal is formed by oxidative cleavage of β-carotene by
β-carotene-15,15′-dioxygenase
(4). Retinal is produced by an
orthologous enzyme in vertebrates, where it is also converted to retinoic
acid, a regulator of differentiation during embryogenesis
(5). A distinct mammalian CCD
is believed to cleave carotenoids asymmetrically at the 9,10 position
(6) and, although its function
is unclear, recent evidence suggests a role in the metabolism of dietary
lycopene (7). The plant
volatiles β-ionone and geranylacetone are produced from an enzyme that
cleaves at the 9,10 position
(8) and the pigment
α-crocin found in the spice saffron results from an 7,8-cleavage enzyme
(9).Other CCDs have been identified where biological function is unknown, for
example, in cyanobacteria where a variety of cleavage specificities have been
described
(10-12).
In other cases, there are apocarotenoids with known functions, but the
identity or involvement of CCDs have not yet been described: grasshopper
ketone is a defensive secretion of the flightless grasshopper Romalea
microptera (13),
mycorradicin is produced by plant roots during symbiosis with arbuscular
mycorrhyza (14), and
strigolactones (15) are plant
metabolites that act as germination signals to parasitic weeds such as
Striga and Orobanche
(16).Recently it was discovered that strigolactones also function as a branching
hormone in plants (17,
18). The existence of such a
branching hormone has been known for some time, but its identity proved
elusive. However, it was known that the hormone was derived from the action of
at least two CCDs, max3 and max4 (more axillary growth)
(19), because deletion of
either of these genes in Arabidopsis thaliana, leads to a bushy
phenotype (20,
21). In Escherichia
coli assays, AtCCD7 (max3) cleaves β-carotene at the 9,10 position
and the apocarotenoid product (10-apo-β-carotene) is reported to be
further cleaved at 13,14 by AtCCD8 (max4) to produce 13-apo-β-carotene
(22). Also recent evidence
suggests that AtCCD8 is highly specific, cleaving only 10-apo-β-carotene
(23). How the production of
13-apo-β-carotene leads to the synthesis of the complex strigolactone is
unknown. The possibility remains that the enzymes may have different
specificities and cleavage activities in planta. In addition, a
cytochrome P450 enzyme (24) is
believed to be involved in strigolactone synthesis and acts in the pathway
downstream of the CCD genes. Strigolactone is thought to effect branching by
regulating auxin transport
(25). Because of the
involvement of CCDs in strigolactone synthesis, the possibility arises that
plant architecture and interaction with parasitic weeds and mycorrhyza could
be controlled by the manipulation of CCD activity.Although considerable success has been obtained using genetic approaches to
probe function and substrate specificity of CCDs in their native biological
contexts, particularly in plant species with simple genetic systems or that
are amenable to transgenesis, there are many systems where genetic approaches
are difficult or impossible. Also, when recombinant CCDs are studied either
in vitro or in heterologous in vivo assays, such as in
E. coli strains engineered to accumulate carotenoids
(26), they are often active
against a broad range of substrates
(5,
21,
27), and in many cases the
true in vivo substrate of a particular CCD remains unknown. Therefore
additional experimental tools are needed to investigate both apocarotenoid and
CCD functions in their native cellular environments.In the reverse chemical genetics approach, small molecules are identified
that are active against known target proteins; they are then applied to a
biological system to investigate protein function in vivo
(28,
29). This approach is
complementary to conventional genetics since the small molecules can be
applied easily to a broad range of species, their application can be
controlled in dose, time, and space to provide detailed studies of biological
functions, and individual proteins or whole protein classes may be targeted by
varying the specificity of the small molecules. Notably, functions of the
plant hormones gibberellin, brassinosteroid, and abscisic acid have been
successfully probed using this approach by adapting triazoles to inhibit
specific cytochrome P450 monooxygenases involved in the metabolism of these
hormones (30).In the case of the CCD family, the tertiary amines abamine
(31) and the more active
abamineSG (32) were reported
as specific inhibitors of NCED, and abamine was used to show new functions of
abscisic acid in legume nodulation
(33). However, no selective
inhibitors for other types of CCD are known. Here we have designed a novel
class of CCD inhibitor based on hydroxamic acids, where variable chain length
was used to direct inhibition of CCD enzymes that cleave carotenoids at
specific positions. We demonstrate the use of such novel inhibitors to control
shoot branching in a model plant. 相似文献
18.
Mait�� Montero-Hadjadje Salah Elias Laurence Chevalier Magalie Benard Yannick Tanguy Val��rie Turquier Ludovic Galas Laurent Yon Maria M. Malagon Azeddine Driouich St��phane Gasman Youssef Anouar 《The Journal of biological chemistry》2009,284(18):12420-12431
Chromogranin A (CgA) has been proposed to play a major role in the
formation of dense-core secretory granules (DCGs) in neuroendocrine cells.
Here, we took advantage of unique features of the frog CgA (fCgA) to assess
the role of this granin and its potential functional determinants in hormone
sorting during DCG biogenesis. Expression of fCgA in the constitutively
secreting COS-7 cells induced the formation of mobile vesicular structures,
which contained cotransfected peptide hormones. The fCgA and the hormones
coexpressed in the newly formed vesicles could be released in a regulated
manner. The N- and C-terminal regions of fCgA, which exhibit remarkable
sequence conservation with their mammalian counterparts were found to be
essential for the formation of the mobile DCG-like structures in COS-7 cells.
Expression of fCgA in the corticotrope AtT20 cells increased
pro-opiomelanocortin levels in DCGs, whereas the expression of N- and
C-terminal deletion mutants provoked retention of the hormone in the Golgi
area. Furthermore, fCgA, but not its truncated forms, promoted
pro-opiomelanocortin sorting to the regulated secretory pathway. These data
demonstrate that CgA has the intrinsic capacity to induce the formation of
mobile secretory granules and to promote the sorting and release of peptide
hormones. The conserved terminal peptides are instrumental for these
activities of CgA.Eukaryotic cells share the capacity to rapidly secrete proteins through the
constitutive secretory pathway. The fundamental feature of neuroendocrine and
endocrine cells is the occurrence of dense-core secretory granules
(DCGs),3
which are key cytoplasmic organelles responsible for secretion of hormones,
neuropeptides, and neurotransmitters through the regulated secretory pathway
(RSP). Storage at high concentrations of these secretory products is required
for their finely tuned release in response to extracellular stimulation
(1,
2). DCG biogenesis starts with
the budding of immature secretory granules (ISGs) from the
trans-Golgi network (TGN) through interactions between lipid rafts
and protein components, in a similar manner to constitutive vesicle budding
(2,
3). The ISG budding is followed
by a multistep maturation process to form the mature secretory granules,
including removal of the constitutive secretory proteins and lysosomal enzymes
inadvertently packaged into ISGs
(4).Despite increasing knowledge of the various steps of DCG formation, the
nature of the sorting signals for entry of proteins into the DCGs and the
molecular machinery required to generate secretory granules are not fully
elucidated (5,
6). Several recent studies
highlighted the role of members of the granin family, which may represent the
driving force for granulogenesis in the TGN
(2), although this notion has
been a matter of debate (7).
Granins are soluble acidic proteins widely distributed in endocrine and
neuroendocrine cells, which are characterized by the ability to aggregate at
acidic pH and a high Ca2+ environment
(8,
9). These conditions are found
in the lumen of the TGN allowing granins to aggregate in this compartment and
to be segregated from constitutively secreted proteins
(10,
11). The granin aggregates are
believed to associate directly or indirectly with lipid rafts at the TGN to
induce budding and formation of the ISGs. A prominent role of chromogranin A
(CgA) in the regulation of DCG formation in endocrine and neuroendocrine cells
has been proposed. Thus, depletion of CgA in PC12 cells led to a dramatic
decrease in the number of DCGs
(12), and exogenously
expressed CgA in these depleted PC12 cells, as in DCG-deficient endocrine A35C
and 6T3 cells, restored DCG biogenesis
(12,
13). Besides, expression of
granins in non-endocrine, constitutively secreting cells such as CV-1, NIH3T3,
or COS-7 cells provoked the formation of DCG-like structures that release
their content in response to Ca2+ influx
(12,
14,
15). Further investigations
performed in CgA null mice and transgenic mice expressing antisense RNA
against CgA also revealed a reduction in the number of DCGs in chromaffin
cells that was associated with an impairment of catecholamine storage, thus
demonstrating the crucial role of CgA in normal DCG biogenesis
(16,
17). In CgA knockout mice, the
introduction of the gene expressing human CgA restored the regulated secretory
phenotype (16). A different
CgA null mice strain exhibited no discernable effect on DCG formation, but
elevated catecholamine secretion
(18), proving that CgA
deficiency is associated with hormone storage impairment in neuroendocrine
cells in vivo, a finding that was confirmed in vitro
(19). The CgA-/-
mice strain generated by Hendy et al.
(18) exhibited a compensatory
overexpression of other granins, pointing to a possible overlap in granin
function in secretory granule biogenesis.We reported previously that the frog CgA (fCgA) gene is coordinately
regulated with the pro-opiomelanocortin (POMC) gene in the pituitary pars
intermedia during the neuroendocrine reflex of skin color change, which allows
amphibia to adapt to their environment through the release of POMC-derived
melanotropic peptides (20,
21). Sequence comparison of
fCgA with its mammalian orthologs revealed a high conservation of the N- and
C-terminal domains, and far less conservation of the central part of the
protein (Fig. 1A),
suggesting that these domains may play a role in DCG formation and hormone
release in various species (9,
20,
21). To assess the role of
fCgA and its conserved N- and C-terminal regions in hormone sorting, storage,
and secretion, we engineered different constructs that produce the native
unmodified (no tag added) protein and truncated forms lacking the conserved N-
and C-terminal domains, and we developed an antibody that specifically
recognizes the central region of fCgA. Using the constitutively secreting
COS-7 cells, which are devoid of DCGs, we could demonstrate for the first time
that CgA is essential for targeting peptide hormones to newly formed mobile
DCG-like structures. In the CgA-expressing AtT20 cells, which exhibit an only
moderate capacity to sort secretory proteins to the regulated pathway
(22), the granin plays a
pivotal role in the sorting and release of POMC. The conserved terminal
peptides of CgA are instrumental for these activities.Open in a separate windowFIGURE 1.Specificity of the antibody directed against frog CgA. A,
scheme depicting the structure of fCgA and showing the high conservation of
the terminal regions and the percentages of amino acid identity between frog
and human CgA sequences. The highly conserved peptide WE14 and dibasic
cleavage sites are also indicated. B, Western blot showing that the
antibody developed against fCgA recognized the protein and several processing
intermediates in frog but not rat pituitary extracts, whereas an antibody,
directed against the WE14 conserved peptide, detected CgA and its processing
products in both rat and frog pituitary extracts. C,
immunofluorescence analysis of frog pituitary and adrenal glands, and rat
adrenal gland using the antibodies against fCgA and WE14. cx, cortex;
DL, distal lobe; IL, intermediate lobe; and m,
medulla. Scale bars equal 10 μm. 相似文献
19.
C. Martin Lawrence Smita Menon Brian J. Eilers Brian Bothner Reza Khayat Trevor Douglas Mark J. Young 《The Journal of biological chemistry》2009,284(19):12599-12603
Viruses populate virtually every ecosystem on the planet, including the
extreme acidic, thermal, and saline environments where archaeal organisms can
dominate. For example, recent studies have identified crenarchaeal viruses in
the hot springs of Yellowstone National Park and other high temperature
environments worldwide. These viruses are often morphologically and
genetically unique, with genomes that show little similarity to genes of known
function, complicating efforts to understand their viral life cycles. Here, we
review progress in understanding these fascinating viruses at the molecular
level and the evolutionary insights coming from these studies.The last decade has seen resurgent interest in the study of viruses that
lie outside traditional agricultural and medical interests. One reason is the
growing appreciation of the enormous abundance and impact of viruses on the
greater biosphere. For example, the oceans are thought to contain
∼1031 viruses, a truly astronomical number
(1), making viruses the most
abundant biological entities in this ecosystem, where they catalyze turnover
of 20% of the oceanic biomass per day
(1). Remarkably, the virosphere
has now been shown to extend to almost every known environment on earth,
including the extreme acidic, thermal, and saline environments where archaeal
organisms can be dominant. Thus, because of their abundance and variety,
viruses are now thought to represent the greatest reservoir of genetic
diversity on the planet
(2).A second reason to study archaeal viruses is a growing appreciation for the
roles viruses play in evolution. Remarkably with >500 cellular genomes
sequenced to date, most show a significant amount of viral or virus-like
sequence within their genome, further evidence that viruses play a central
role in horizontal gene transfer and help drive the evolution of their hosts.
Roles for viruses in cellular evolution are also being considered. Current
hypotheses contend that viruses have catalyzed several major evolutionary
transitions, including the invention of DNA and DNA replication mechanisms
(3), the origin of the
eukaryotic nucleus (4), and
thus a role in the formation of the three domains of life. In addition, there
is also considerable interest in viral genesis and evolution in and of itself.
To evaluate these hypotheses and to analyze evolutionary relationships among
viruses, knowledge of viruses infecting the archaea is essential, yet these
viruses are vastly understudied. Finally, interest in archaeal viruses stems
also from the exceptional molecular insight viruses have traditionally
provided into host processes; archaeal viruses are certain to provide new
insights into the molecular biology of this poorly understood domain of
life.Pioneering studies by Wolfram Zillig et al.
(5) identified the first
archaeal viruses. Although initial studies suggested that viruses infecting
the euryarchaea (principally halophiles and methanogens) were similar to
head-tail bacteriophage, studies of viruses infecting the hyperthermophilic
crenarchaea revealed morphologies suggesting new viral families. Indeed, work
by several laboratories has led to the identification of seven new viral
families infecting the crenarchaea, the Globuloviridae, Guttaviridae,
Fuselloviridae, Bicaudaviridae, Ampullaviridae, Rudiviridae, and
Lipothrixviridae (Fig. 1)
(6,
7), with
STIV3
(8) and STSV1
(9) awaiting assignment. All of
these viruses contain double-stranded DNA genomes ranging in size from 13.7 to
75.3 kilobase pairs, encoding 31–74 ORFs. Although many package a
circular genome, the filamentous Lipothrixviridae and rod-shaped Rudiviridae
are notable exceptions and are the only viruses in any domain known to
encapsidate linear double-stranded DNA. Although most crenarchaeal viruses are
enveloped, the Rudiviridae are devoid of lipid, and with the
exception of the Fuselloviridae, they employ a lytic life cycle,
although only STIV and ATV (Bicaudaviridae) are known to cause cell
lysis
(11).4Open in a separate windowFIGURE 1.Morphological diversity in crenarchaeal viruses. A,
clockwise, beginning at upper left: STIV
(8), a PSV-like virus,
Sulfolobus neozealandicus droplet-shaped virus (SNDV)
(47), SSV1
(48), STSV1
(9), an ATV-like virus, an SIRV
virus, and S. icelandicus filamentous virus (SIFV)
(10). Micrographs of SIRV,
PSV-like, and ATV-like viruses from Yellowstone National Park are the courtesy
of M. J. Y. Other panels are reproduced, with permission, from Refs.
8–10,
47, and
48. B, cryoelectron
microscopy reconstruction of the STIV particle
(8) showing a cutaway view
(20) of the T = 31
icosahedral capsid with turret-like projections that extend from each of the
5-fold vertices. Portions of the protein shell (blue) and inner lipid
layer (yellow) have been removed to reveal the interior.The exceptional morphology of these viruses has been reviewed
(6,
7) and thus is only summarized
here (Fig. 1). For the
rod-shaped Rudiviridae, plugs are seen at both ends, from which three
short tail fibers emanate, whereas the Lipothrixviridae show mop- or claw-like
structures at both ends (6).
Similarly, the non-tailed icosahedral viruses, STIV and euryarchaeal SH1, have
large turrets or spikes that project from the surface
(8,
12). In each case, these
structures are thought to facilitate virus-host interactions. In contrast,
other crenarchaeal viruses utilize a fusiform or lemon-shaped virion, a
morphology unique to archaeal viruses. These fusiform viruses generally
contain tail fibers or an extended tail on one end that is also involved in
host recognition. For ATV, however, nascent particles are devoid of tails when
released from the host (13).
Remarkably, extended tails develop at both ends of the virion in an
extracellular maturation process. Finally, Acidianus bottle-shaped
virus (Ampullaviridae) shows an exceptional morphology that differs in its
basic architecture from any known virus. 相似文献
20.
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