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1.
Cell death can be divided into the anti-inflammatory process of apoptosis and the
pro-inflammatory process of necrosis. Necrosis, as apoptosis, is a regulated form of cell
death, and Poly-(ADP-Ribose) Polymerase-1 (PARP-1) and Receptor-Interacting Protein (RIP)
1/3 are major mediators. We previously showed that absence or inhibition of PARP-1
protects mice from nephritis, however only the male mice. We therefore hypothesized that
there is an inherent difference in the cell death program between the sexes. We show here
that in an immune-mediated nephritis model, female mice show increased apoptosis compared
to male mice. Treatment of the male mice with estrogens induced apoptosis to levels
similar to that in female mice and inhibited necrosis. Although PARP-1 was activated in
both male and female mice, PARP-1 inhibition reduced necrosis only in the male mice. We
also show that deletion of RIP-3 did not have a sex bias. We demonstrate here that male
and female mice are prone to different types of cell death. Our data also suggest that
estrogens and PARP-1 are two of the mediators of the sex-bias in cell death. We therefore
propose that targeting cell death based on sex will lead to tailored and better treatments
for each gender. 相似文献
2.
3.
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5.
6.
Sorghum (Sorghum bicolor L. Moench) has two isozymes of the cyanogenic β-glucosidase dhurrinase: dhurrinase-1 (Dhr1) and dhurrinase-2 (Dhr2). A nearly full-length cDNA encoding dhurrinase was isolated from 4-d-old etiolated seedlings and sequenced. The cDNA has a 1695-nucleotide-long open reading frame, which codes for a 565-amino acid-long precursor and a 514-amino acid-long mature protein, respectively. Deduced amino acid sequence of the sorghum Dhr showed 70% identity with two maize (Zea mays) β-glucosidase isozymes. Southern-blot data suggested that β-glu-cosidase is encoded by a small multigene family in sorghum. Northern-blot data indicated that the mRNA corresponding to the cloned Dhr cDNA is present at high levels in the node and upper half of the mesocotyl in etiolated seedlings but at low levels in the root—only in the zone of elongation and the tip region. Light-grown seedling parts had lower levels of Dhr mRNA than those of etiolated seedlings. Immunoblot analysis performed using maize-anti-β-glucosidase sera detected two distinct dhurrinases (57 and 62 kD) in sorghum. The distribution of Dhr activity in different plant parts supports the mRNA and immunoreactive protein data, suggesting that the cloned cDNA corresponds to the Dhr1 (57 kD) isozyme and that the dhr1 gene shows organ-specific expression. 相似文献
7.
Xiaojing Wang Snezana Levic Michael Anne Gratton Karen Jo Doyle Ebenezer N. Yamoah Anthony E. Pegg 《The Journal of biological chemistry》2009,284(2):930-937
Male gyro (Gy) mice, which have an X chromosomal deletion inactivating the
SpmS and Phex genes, were found to be profoundly hearing
impaired. This defect was due to alteration in polyamine content due to the
absence of spermine synthase, the product of the SpmS gene. It was
reversed by breeding the Gy strain with CAG/SpmS mice, a transgenic line that
ubiquitously expresses spermine synthase under the control of a composite
cytomegalovirus-IE enhancer/chicken β-actin promoter. There was an almost
complete loss of the endocochlear potential in the Gy mice, which parallels
the hearing deficiency, and this was also reversed by the production of
spermine from the spermine synthase transgene. Gy mice showed a striking toxic
response to treatment with the ornithine decarboxylase inhibitor
α-difluoromethylornithine (DFMO). Within 2–3 days of exposure to
DFMO in the drinking water, the Gy mice suffered a catastrophic loss of motor
function resulting in death within 5 days. This effect was due to an inability
to maintain normal balance and was also prevented by the transgenic expression
of spermine synthase. DFMO treatment of control mice or Gy-CAG/SpmS had no
effect on balance. The loss of balance in Gy mice treated with DFMO was due to
inhibition of polyamine synthesis because it was prevented by administration
of putrescine. Our results are consistent with a critical role for polyamines
in regulation of Kir channels that maintain the endocochlear potential and
emphasize the importance of normal spermidine:spermine ratio in the hearing
and balance functions of the inner ear.Polyamines are essential for viability in mammals. Knockouts of the genes
for ornithine decarboxylase and S-adenosylmethionine decarboxylase,
which are enzymes needed for the synthesis of putrescine, spermidine, and
spermine, are lethal at early stages of embryonic development
(1,
2). There is convincing
evidence that the formation of hypusine in eIF5A, which requires spermidine as
a precursor, is essential for eukaryotes
(3). However, the function(s)
of spermine is not so well established. Yeast mutants with inactivated
spermine synthase grow at a normal rate
(4). Mammalian cells in culture
also grow normally in the presence of inhibitors of spermine synthase
(5) or after inactivation of
the spermine synthase gene (SpmS)
(6–8).
Inactivation of both of the genes that were originally described as encoding
spermine synthases in plants leads to profound developmental defects
(9–11),
but recently it was discovered that one of these genes actually encodes a
thermospermine synthase, and it appears that the lack of thermospermine may be
responsible for these defects
(12).In contrast, spermine is clearly required for normal development in
mammals. The rare human Snyder-Robinson syndrome is caused by mutations in
SpmS located in the X chromosome that drastically reduces the amount
of spermine synthase (13,
14). This leads to mental
retardation, hypotonia, cerebellar circuitry dysfunction, facial asymmetry,
thin habitus, osteoporosis, and kyphoscoliosis. Male mice, which have an X
chromosomal deletion that includes SpmS and have no detectable
spermine synthase activity, do survive but are only viable on the B6C3H
background
(15–17).
This mouse strain having an X-linked dominant mutation was isolated from a
female offspring of an irradiated mouse and was termed gyro
(Gy)2 based on a
circling behavior pattern in affected males
(18). Subsequent studies have
shown that the Gy mice have a deletion of part of the X chromosome that
inactivates both Phex, a gene that regulates phosphate metabolism,
and SpmS (16,
19). The lack of SpmS
causes a total absence of spermine
(6,
7,
15,
16). Such Gy mice suffer from
hypophosphatemia, have a greatly reduced size, sterility, and neurological
abnormalities, and have a short life span
(6,
16,
18). All of these changes
except the hypophosphatemia are reversed when spermine synthase activity is
restored (20).The original characterization of Gy mice also reported preliminary
indications that these mice had hearing defects lacking the Preyer reflex
(21,
22). This is of particular
interest in the context of polyamine metabolism because a drug,
α-difluoromethylornithine (DFMO, Eflornithine), that targets ornithine
decarboxylase has been shown to cause occasional hearing loss in some patients
(23–26).
Although DFMO was ineffective for cancer treatment, it is an extremely
promising agent for cancer chemoprevention
(27,
28). When combined with
sulindac, DFMO treatment produced a substantial reduction in the recurrence of
colorectal adenomas in a large clinical trial
(27). DFMO is a major drug for
the treatment of African sleeping sickness caused by Trypanosoma
brucei (29,
30). It is also used as a
topically applied cream for treatment of unwanted facial hair in women
(31,
32). DFMO is generally well
tolerated even at high doses, but reversible hearing loss has been reported in
multiple clinical trials (25,
33), and a rarer irreversible
defect has also been reported
(34). These side effects are
not observed at lower doses of DFMO
(26,
27).Ototoxicity has been demonstrated to occur in experimental animals treated
with DFMO including rats (35),
guinea pigs (36), gerbils
(37), and mice
(38). Using
immunohistochemistry, a high level of ornithine decarboxylase was observed in
the inner ear of the rat, with the highest in the organ of Corti and lateral
wall followed by the cochlear nerve
(39). Measurements of
polyamines in the relevant structures are very difficult due to the small
amount of tissue available, but as expected, DFMO treatment reduced polyamine
levels and ornithine decarboxylase activity in the inner ear of the guinea pig
(36). A plausible explanation
for the importance of polyamines in auditory physiology is based on their well
documented role as regulators of potassium channels
(38). The inward rectification
of Kir channels is caused by blockage of the outward current by polyamines
(40–42).
Studies of the cloned mouse cochlear lateral wall-specific Kir4.1 channel
showed that inward rectification was reduced and that there was a marked
reduction in endocochlear potential (EP). It was proposed that DFMO treatment
increases the outward Kir4.1 current, resulting in a drop in EP
(38).In the experiments reported here, we have studied in more detail the role
of polyamines in auditory physiology using Gy mice and crosses of these mice
with transgenic CAG/SpmS mice
(43). These mice express
spermine synthase under the control of a composite cytomegalovirus-IE
enhancer/chicken β-actin promoter, which was designed to provide
ubiquitous expression
(44–46).
Assays of the spermine synthase activity in CAG/SpmS line 8 confirmed that
there was a high level of expression of the transgene in many different organs
and that this level was maintained for at least 1 year
(43). Our studies confirm that
Gy mice are totally deaf and that this condition is reversed by the expression
of the SpmS gene. These changes are due to a virtually complete loss
of the EP in the Gy mice. We have also examined the effect of DFMO on the Gy
mice. Unexpectedly, it was found that these mice show a rapid and profound
toxicity to this drug, leading to death within a few days. Within 5 days of
exposure to DFMO in the drinking water, the DFMO-treated mice suffered a
catastrophic loss of balance due to inner ear effects. This toxicity was also
prevented by the transgenic expression of spermine synthase in the Gy
background. 相似文献
8.
9.
Yamini S. Bynagari Bela Nagy Jr. Florin Tuluc Kamala Bhavaraju Soochong Kim K. Vinod Vijayan Satya P. Kunapuli 《The Journal of biological chemistry》2009,284(20):13413-13421
The novel class of protein kinase C (nPKC) isoform η is expressed in
platelets, but not much is known about its activation and function. In this
study, we investigated the mechanism of activation and functional implications
of nPKCη using pharmacological and gene knock-out approaches. nPKCη
was phosphorylated (at Thr-512) in a time- and concentration-dependent manner
by 2MeSADP. Pretreatment of platelets with MRS-2179, a P2Y1
receptor antagonist, or YM-254890, a Gq blocker, abolished
2MeSADP-induced phosphorylation of nPKCη. Similarly, ADP failed to
activate nPKCη in platelets isolated from P2Y1 and
Gq knock-out mice. However, pretreatment of platelets with
P2Y12 receptor antagonist, AR-C69331MX did not interfere with
ADP-induced nPKCη phosphorylation. In addition, when platelets were
activated with 2MeSADP under stirring conditions, although nPKCη was
phosphorylated within 30 s by ADP receptors, it was also dephosphorylated by
activated integrin αIIbβ3 mediated outside-in
signaling. Moreover, in the presence of SC-57101, a
αIIbβ3 receptor antagonist, nPKCη
dephosphorylation was inhibited. Furthermore, in murine platelets lacking
PP1cγ, a catalytic subunit of serine/threonine phosphatase,
αIIbβ3 failed to dephosphorylate nPKCη.
Thus, we conclude that ADP activates nPKCη via P2Y1 receptor
and is subsequently dephosphorylated by PP1γ phosphatase activated by
αIIbβ3 integrin. In addition, pretreatment of
platelets with η-RACK antagonistic peptides, a specific inhibitor of
nPKCη, inhibited ADP-induced thromboxane generation. However, these
peptides had no affect on ADP-induced aggregation when thromboxane generation
was blocked. In summary, nPKCη positively regulates agonist-induced
thromboxane generation with no effects on platelet aggregation.Platelets are the key cellular components in maintaining hemostasis
(1). Vascular injury exposes
subendothelial collagen that activates platelets to change shape, secrete
contents of granules, generate thromboxane, and finally aggregate via
activated αIIbβ3 integrin, to prevent further
bleeding (2,
3). ADP is a physiological
agonist of platelets secreted from dense granules and is involved in feedback
activation of platelets and hemostatic plug stabilization
(4). It activates two distinct
G-protein-coupled receptors (GPCRs) on platelets, P2Y1 and
P2Y12, which couple to Gq and Gi,
respectively
(5–8).
Gq activates phospholipase Cβ (PLCβ), which leads to
diacyl glycerol (DAG)2
generation and calcium mobilization
(9,
10). On the other hand,
Gi is involved in inhibition of cAMP levels and PI 3-kinase
activation (4,
6). Synergistic activation of
Gq and Gi proteins leads to the activation of the
fibrinogen receptor integrin αIIbβ3.
Fibrinogen bound to activated integrin αIIbβ3
further initiates feed back signaling (outside-in signaling) in platelets that
contributes to the formation of a stable platelet plug
(11).Protein kinase Cs (PKCs) are serine/threonine kinases known to regulate
various platelet functional responses such as dense granule secretion and
integrin αIIbβ3 activation
(12,
13). Based on their structure
and cofactor requirements, PKCs are divided in to three classes: classical
(cofactors: DAG, Ca2+), novel (cofactors: DAG) and atypical
(cofactors: PIP3) PKC isoforms
(14). All the members of the
novel class of PKC isoforms (nPKC), viz. nPKC isoforms δ, θ,
η, and ε, are expressed in platelets
(15), and they require DAG for
activation. Among all the nPKCs, PKCδ
(15,
16) and PKCθ
(17–19)
are fairly studied in platelets. Whereas nPKCδ is reported to regulate
protease-activated receptor (PAR)-mediated dense granule secretion
(15,
20), nPKCθ is activated
by outside-in signaling and contributes to platelet spreading on fibrinogen
(18). On the other hand, the
mechanism of activation and functional role of nPKCη is not addressed as
yet.PKCs are cytoplasmic enzymes. The enzyme activity of PKCs is modulated via
three mechanisms (14,
21): 1) cofactor binding: upon
cell stimulus, cytoplasmic PKCs mobilize to membrane, bind cofactors such as
DAG, Ca2+, or PIP3, release autoinhibition, and attain an active
conformation exposing catalytic domain of the enzyme. 2) phosphorylations:
3-phosphoinositide-dependent kinase 1 (PDK1) on the membrane phosphorylates
conserved threonine residues on activation loop of catalytic domain; this is
followed by autophosphorylations of serine/threonine residues on turn motif
and hydrophobic region. These series of phosphorylations maintain an active
conformation of the enzyme. 3) RACK binding: PKCs in active conformation bind
receptors for activated C kinases (RACKs) and are lead to various subcellular
locations to access the substrates
(22,
23). Although various leading
laboratories have elucidated the activation of PKCs, the mechanism of
down-regulation of PKCs is not completely understood.The premise of dynamic cell signaling, which involves protein
phosphorylations by kinases and dephosphorylations by phosphatases has gained
immense attention over recent years. PP1, PP2A, PP2B, PHLPP are a few of the
serine/threonine phosphatases reported to date. Among them PP1 and PP2
phosphatases are known to regulate various platelet functional responses
(24,
25). Furthermore, PP1c, is the
catalytic unit of PP1 known to constitutively associate with
αIIb and is activated upon integrin engagement with
fibrinogen and subsequent outside-in signaling
(26). Among various PP1
isoforms, recently PP1γ is shown to positively regulate platelet
functional responses (27).
Thus, in this study we investigated if the above-mentioned phosphatases are
involved in down-regulation of nPKCη. Furthermore, reports from other cell
systems suggest that nPKCη regulates ERK/JNK pathways
(28). In platelets ERK is
known to regulate agonist induced thromboxane generation
(29,
30). Thus, we also
investigated if nPKCη regulates ERK phosphorylation and thereby
agonist-induced platelet functional responses.In this study, we evaluated the activation of nPKCη downstream of ADP
receptors and its inactivation by an integrin-associated phosphatase
PP1γ. We also studied if nPKCη regulates functional responses in
platelets and found that this isoform regulates ADP-induced thromboxane
generation, but not fibrinogen receptor activation in platelets. 相似文献
10.
Vertebrates produce at least seven distinct β-tubulin isotypes that
coassemble into all cellular microtubules. The functional differences among
these tubulin isoforms are largely unknown, but recent studies indicate that
tubulin composition can affect microtubule properties and cellular
microtubule-dependent behavior. One of the isotypes whose incorporation causes
the largest change in microtubule assembly is β5-tubulin. Overexpression
of this isotype can almost completely destroy the microtubule network, yet it
appears to be required in smaller amounts for normal mitotic progression.
Moderate levels of overexpression can also confer paclitaxel resistance.
Experiments using chimeric constructs and site-directed mutagenesis now
indicate that the hypervariable C-terminal region of β5 plays no role in
these phenotypes. Instead, we demonstrate that two residues found in β5
(Ser-239 and Ser-365) are each sufficient to inhibit microtubule assembly and
confer paclitaxel resistance when introduced into β1-tubulin; yet the
single mutation of residue Ser-239 in β5 eliminates its ability to confer
these phenotypes. Despite the high degree of conservation among β-tubulin
isotypes, mutations affecting residue 365 demonstrate that amino acid
substitutions can be context sensitive; i.e. an amino acid change in
one isotype will not necessarily produce the same phenotype when introduced
into a different isotype. Modeling studies indicate that residue Cys-239 of
β1-tubulin is close to a highly conserved Cys-354 residue suggesting the
possibility that disulfide formation could play a significant role in the
stability of microtubules formed with β1- but not with
β5-tubulin.Microtubules are needed to organize the Golgi apparatus and endoplasmic
reticulum, maintain cell shape, construct ciliary and flagellar axonemes, and
ensure the accurate segregation of genetic material prior to cell division.
These cytoskeletal structures assemble from α- and β-tubulin
heterodimers to form long cylindrical filaments that exist in a state of
dynamic equilibrium characterized by stochastic episodes of slow growth and
rapid shrinkage (1). Impairment
of normal dynamic behavior has serious consequences for cell proliferation and
thus makes microtubules an attractive target for drug development
(2).Vertebrates express multiple β-tubulin genes that produce highly
homologous proteins differing most notably in their C-terminal 15–20
amino acids (3,
4). These variable C-terminal
sequences are conserved across vertebrate species and have been used to
classify β-tubulin genes into distinct isotypes
(5). In mammals, for example,
there are seven known isotypes designated by the numbers I, II, III, IVa, IVb,
V, and VI. The functional significance of the C-terminal sequences is
uncertain, but some studies suggest that they may be involved in binding or
modulating the action of microtubule-interacting proteins
(6–14).
Additional amino acid differences are scattered throughout the primary
sequence, but the functional role of these differences, if any, has not been
elucidated. Although some β-tubulin isotypes are expressed in a
tissue-specific manner (3),
evidence indicates that microtubules incorporate all available isotypes,
including transfected isotypes that are not normally produced in those cells
(5,
15–17).
Genetic experiments designed to test potential functional differences among
the various β-tubulin isotypes have only demonstrated isotype-specific
effects on the assembly of specialized microtubule-containing structures such
as flagellar axonemes in Drosophila or 15-protofilament microtubules
in Caenorhabditis elegans
(18,
19). Thus, the consequences,
if any, of producing multiple β-tubulin isoforms in vertebrate organisms
remain elusive.Our recent work showed that conditional overexpression of isotypes β1,
β2, and β4b has no effect on microtubule assembly or drug
sensitivity in transfected Chinese hamster ovary
(CHO)2 cells
(20). Similarly, expression of
neuronal-specific β4a produced very minor effects on microtubule assembly
but was able to increase sensitivity to paclitaxel, most likely through
increased binding of the drug
(21). On the other hand, high
expression of neuronal-specific β3 reduced microtubule assembly,
conferred low level resistance to paclitaxel, and inhibited cell growth
(22). The most dramatic
effects, however, were seen in cells transfected with β5, a minor but
widely expressed isotype (23).
Even modest overexpression of this isotype reduced microtubule assembly and
conferred paclitaxel resistance, whereas high levels of expression (∼50%
of total tubulin) caused fragmentation and a near complete loss of the
microtubule cytoskeleton (24).
Despite the toxicity associated with β5 overexpression, this isotype was
recently shown to be required for normal mitotic progression and cell
proliferation (25).Because of its importance for cell division, and the extreme phenotype
associated with its overexpression, we sought to identify the structural
differences between β5-tubulin and its more “normal” homolog,
β1. Although there are 40 amino acid differences between the 2 isotypes,
we report that most of the unique properties of β5 can be attributed to
the presence of serine in place of cysteine at residue 239. This residue faces
the colchicine binding pocket and is very close to a highly conserved Cys-354
residue. We propose that Ser-239 found in β5-tubulin may prevent
formation of a disulfide bond that normally stabilizes microtubules. 相似文献
11.
12.
Lisa Placanica Leonid Tarassishin Guangli Yang Erica Peethumnongsin Seong-Hun Kim Hui Zheng Sangram S. Sisodia Yue-Ming Li 《The Journal of biological chemistry》2009,284(5):2967-2977
γ-Secretase is known to play a pivotal role in the pathogenesis of
Alzheimer disease through production of amyloidogenic Aβ42 peptides.
Early onset familial Alzheimer disease mutations in presenilin (PS), the
catalytic core of γ-secretase, invariably increase the
Aβ42:Aβ40 ratio. However, the mechanism by which these mutations
affect γ-secretase complex formation and cleavage specificity is poorly
understood. We show that our in vitro assay system recapitulates the
effect of PS1 mutations on the Aβ42:Aβ40 ratio observed in cell and
animal models. We have developed a series of small molecule affinity probes
that allow us to characterize active γ-secretase complexes. Furthermore
we reveal that the equilibrium of PS1- and PS2-containing active complexes is
dynamic and altered by overexpression of Pen2 or PS1 mutants and that
formation of PS2 complexes is positively correlated with increased
Aβ42:Aβ40 ratios. These data suggest that perturbations to
γ-secretase complex equilibrium can have a profound effect on enzyme
activity and that increased PS2 complexes along with mutated PS1 complexes
contribute to an increased Aβ42:Aβ40 ratio.β-Amyloid
(Aβ)5 peptides
are believed to play a causative role in Alzheimer disease (AD). Aβ
peptides are generated from the processing of the amyloid precursor protein
(APP) by two proteases, β-secretase and γ-secretase. Although
γ-secretase generates heterogenous Aβ peptides ranging from 37 to
46 amino acids in length, significant work has focused mainly on the Aβ40
and Aβ42 peptides that are the major constituents of amyloid plaques.
γ-Secretase is a multisubunit membrane aspartyl protease comprised of at
least four known subunits: presenilin (PS), nicastrin (Nct), anterior
pharynx-defective (Aph), and presenilin enhancer 2 (Pen2). Presenilin is
thought to contain the catalytic core of the complex
(1–4),
whereas Aph and Nct play critical roles in the assembly, trafficking, and
stability of γ-secretase as well as substrate recognition
(5,
6). Lastly Pen2 facilitates the
endoproteolysis of PS into its N-terminal (NTF) and C-terminal (CTF) fragments
thereby yielding a catalytically competent enzyme
(5,
7–10).
All four proteins (PS, Nct, Aph1, and Pen2) are obligatory for
γ-secretase activity in cell and animal models
(11,
12). There are two homologs of
PS, PS1 and PS2, and three isoforms of Aph1, Aph1aS, Aph1aL, and Aph1b. At
least six active γ-secretase complexes have been reported (two
presenilins × three Aph1s)
(13,
14). The sum of apparent
molecular masses of the four proteins (PS1-NTF/CTF ≈ 53 kDa, Nct ≈ 120
kDa, Aph1 ≈ 30 kDa, and Pen2 ≈ 10kDa) is ∼200 kDa. However, active
γ-secretase complexes of varying sizes, ranging from 250 to 2000 kDa,
have been reported
(15–19).
Recently a study suggested that the γ-secretase complex contains only
one of each subunit (20).
Collectively these studies suggest that a four-protein complex around
200–250 kDa may be the minimal functional γ-secretase unit with
additional cofactors and/or varying stoichiometry of subunits existing in the
high molecular weight γ-secretase complexes. CD147 and TMP21 have been
found to be associated with the γ-secretase complex
(21,
22); however, their role in
the regulation of γ-secretase has been controversial
(23,
24).Mutations of PS1 or PS2 are associated with familial early onset AD (FAD),
although it is debatable whether these familial PS mutations act as
“gain or loss of function” alterations in regard to
γ-secretase activity
(25–27).
Regardless the overall outcome of these mutations is an increased ratio of
Aβ42:Aβ40. Clearly these mutations differentially affect
γ-secretase activity for the production of Aβ40 and Aβ42.
Despite intensive studies of Aβ peptides and γ-secretase, the
molecular mechanism controlling the specificity of γ-secretase activity
for Aβ40 and Aβ42 production has not been resolved. It has been
found that PS1 mutations affect the formation of γ-secretase complexes
(28). However, the precise
mechanism by which individual subunits alter the dynamics of γ-secretase
complex formation and activity is largely unresolved. A better mechanistic
understanding of γ-secretase activity associated with FAD mutations has
been hindered by the lack of suitable assays and probes that are necessary to
recapitulate the effect of these mutations seen in cell models and to
characterize the active γ-secretase complex.In our present studies, we have determined the overall effect of Pen2 and
PS1 expression on the dynamics of PS1- and PS2-containing complexes and their
association with γ-secretase activity. Using newly developed
biotinylated small molecular probes and activity assays, we revealed that
expression of Pen2 or PS1 FAD mutants markedly shifts the equilibrium of
PS1-containing active complexes to that of PS2-containing complexes and
results in an overall increase in the Aβ42:Aβ40 ratio in both stable
cell lines and animal models. Our studies indicate that perturbations to the
equilibrium of active γ-secretase complexes by an individual subunit can
greatly affect the activity of the enzyme. Moreover they serve as further
evidence that there are multiple and distinct γ-secretase complexes that
can exist within the same cells and that their equilibrium is dynamic.
Additionally the affinity probes developed here will facilitate further study
of the expression and composition of endogenous active γ-secretase from
a variety of model systems. 相似文献
13.
14.
Rosanna Pescini Gobert Monique van den Eijnden Cedric Szyndralewiez Catherine Jorand-Lebrun Dominique Swinnen Linfeng Chen Corine Gillieron Fiona Pixley Pierre Juillard Patrick Gerber Caroline Johnson-L��ger Serge Halazy Montserrat Camps Agnes Bombrun Margaret Shipp Pierre-Alain Vitte Vittoria Ardissone Chiara Ferrandi Dominique Perrin Christian Rommel Rob Hooft van Huijsduijnen 《The Journal of biological chemistry》2009,284(17):11385-11395
15.
Gonzalo Izaguirre Alireza R. Rezaie Steven T. Olson 《The Journal of biological chemistry》2009,284(3):1550-1558
We have previously shown that residues Tyr-253 and Glu-255 in the serpin
antithrombin function as exosites to promote the inhibition of factor Xa and
factor IXa when the serpin is conformationally activated by heparin. Here we
show that functional exosites can be engineered at homologous positions in a
P1 Arg variant of the serpin α1-proteinase inhibitor
(α1PI) that does not require heparin for activation. The
combined effect of the two exosites increased the association rate constant
for the reactions of α1PI with factors Xa and IXa
11–14-fold, comparable with their rate-enhancing effects on the
reactions of heparin-activated antithrombin with these proteases. The effects
of the engineered exosites were specific, α1PI inhibitor
reactions with trypsin and thrombin being unaffected. Mutation of Arg-150 in
factor Xa, which interacts with the exosite residues in heparin-activated
antithrombin, abrogated the ability of the engineered exosites in
α1PI to promote factor Xa inhibition. Binding studies showed
that the exosites enhance the Michaelis complex interaction of
α1PI with S195A factor Xa as they do with the
heparin-activated antithrombin interaction. Replacement of the P4-P2 AIP
reactive loop residues in the α1PI exosite variant with a
preferred IEG substrate sequence for factor Xa modestly enhanced the
reactivity of the exosite mutant inhibitor with factor Xa by ∼2-fold but
greatly increased the selectivity of α1PI for inhibiting
factor Xa over thrombin by ∼1000-fold. Together, these results show that a
specific and selective inhibitor of factor Xa can be engineered by
incorporating factor Xa exosite and reactive site recognition determinants in
a serpin.The ubiquitous proteins of the serpin superfamily share a common structure
and mostly function as inhibitors of intracellular and extracellular serine
and cysteine-type proteases in a vast array of physiologic processes
(1,
2). Serpins inhibit their
target proteases by a suicide substrate inhibition mechanism in which an
exposed reactive loop of the serpin is initially recognized as a substrate by
the protease. Subsequent cleavage of the reactive loop by the protease up to
the acyl-intermediate stage of proteolysis triggers a massive conformational
change in the serpin that kinetically traps the acyl-intermediate
(3,
4). Although it is well
established that serpins recognize their cognate proteases through a specific
reactive loop “bait” sequence, it has more recently become clear
that serpin exosites outside the reactive loop provide crucial determinants of
protease specificity
(5–7).
In the case of the blood clotting regulator antithrombin and its target
proteases, physiological rates of protease inhibition are only possible with
the aid of exosites generated upon activation of the serpin by heparin binding
(5). Mutagenesis studies have
shown that the antithrombin exosites responsible for promoting the interaction
of heparin-activated antithrombin with factor Xa and factor IXa map to two key
residues, Tyr-253 and Glu-255, in strand 3 of β-sheet C
(8,
9). Parallel mutagenesis
studies of factor Xa and factor IXa have shown that the protease residues that
interact with the antithrombin exosites reside in the autolysis loop, arginine
150 in this loop being most important
(10,
11). The crystal structures of
the Michaelis complexes of heparin-activated antithrombin with catalytically
inactive S195A variants of thrombin and factor Xa have confirmed that these
complexes are stabilized by exosites in antithrombin and in heparin
(12–14).
In particular, the Michaelis complex with S195A factor Xa revealed that
Tyr-253 of antithrombin and Arg-150 of factor Xa comprise a critical
protein-protein interaction of the antithrombin exosite, in agreement with
mutagenesis studies. Binding studies of antithrombin interactions with S195A
proteases have shown that the exosites in heparin-activated antithrombin
increase the binding affinity for proteases minimally by ∼1000-fold in the
Michaelis complex (15,
16).In this study, we have grafted the two exosites in strand 3 of β-sheet
C of antithrombin onto their homologous positions in a P1 Arg variant of
α1-proteinase inhibitor
(α1PI)2
and shown that the exosites are functional in promoting α1PI
inhibition of factor Xa and factor IXa. The exosites specifically promote
factor Xa and factor IXa inhibition and do not affect the inhibition of
trypsin or thrombin. Moreover, mutation of the complementary exosite residue
in factor Xa, Arg-150, largely abrogates the rate-enhancing effect of the
engineered exosites in α1PI on factor Xa inhibition. Binding
studies show that the exosites function by promoting the binding of
α1PI and factor Xa in the Michaelis complex. Replacing the
P4-P2 residues of the P1 Arg α1PI with an IEG factor Xa
recognition sequence modestly enhances the reactivity of the exosite mutant of
α1PI with factor Xa and greatly increases the selectivity of
the mutant α1PI for inhibiting factor Xa over thrombin. These
findings demonstrate that a potent and selective inhibitor of factor Xa can be
engineered by grafting exosite and reactive site determinants for the protease
on a serpin scaffold. 相似文献
16.
Lilly Y. W. Bourguignon Weiliang Xia Gabriel Wong 《The Journal of biological chemistry》2009,284(5):2657-2671
17.
Hong Cao Jing Chen Eugene W. Krueger Mark A. McNiven 《Molecular and cellular biology》2010,30(3):781-792
The mechanisms by which epithelial cells regulate clathrin-mediated endocytosis (CME) of transferrin are poorly defined and generally viewed as a constitutive process that occurs continuously without regulatory constraints. In this study, we demonstrate for the first time that endocytosis of the transferrin receptor is a regulated process that requires activated Src kinase and, subsequently, phosphorylation of two important components of the endocytic machinery, namely, the large GTPase dynamin 2 (Dyn2) and its associated actin-binding protein, cortactin (Cort). To our knowledge these findings are among the first to implicate an Src-mediated endocytic cascade in what was previously presumed to be a nonregulated internalization process.Iron is an essential element for all mammalian organisms that plays essential roles in hemoglobin and myoglobin production (23). Altered iron transport can lead to disease states such as hemochromatosis (23), anemia (5, 23), and neuronal disorders (23). The transferrin receptor (TfR) is an important component of iron regulation in cells. There are two distinct TfRs in humans sharing 45% identity that are homodimeric and bind iron-associated transferrin (Tf) at markedly different affinities (26). While significant attention has been paid toward understanding the basic endocytic machinery that supports the efficient internalization and recycling of the TfR1 and its associated iron-bound ligand, it has been assumed that this transport process is constitutive in nature. This is in direct contrast to the highly regulated internalization pathway used by members of the receptor tyrosine kinase family (RTKs) and the family of G-coupled protein receptors (GPCRs) that utilize phosphorylation and/or ubiquination as signaling modules to regulate internalization.To test if TfR1 internalization might be regulated in a similar fashion, we focused on two essential components of the endocytic machinery: the large GTPase Dyn2 that mediates endocytic vesicle scission (35) and Cort that binds to Dyn2 via an SH3-PRD interaction and has been postulated to regulate actin dynamics to facilitate vesicle invagination and release (36, 40). Both Dyn2 and Cort have shown to be phosphorylated in vivo and in vitro by a variety of kinases (51, 58). Dyn1 interacts with (17) and is phosphorylated by Src in neuronal cells and in other excitable cells in response to activation of GPCRs and epidermal growth factor (EGF) (1, 2). While the Src phosphorylation motifs of dynamin are conserved in the epithelial expressed form of Dyn2, it is unclear if Dyn2 is phosphorylated in response to ligands that induce clathrin-based endocytosis.Cort possesses a series of C-terminal tyrosines that are heavily Src-phosphorylated and implicated in regulating actin remodeling during cell motility (20). In this study, we demonstrate that addition of Tf to cultured epithelial cells results in an internalization of the TfR1 mediated by a Src kinase-dependent phosphoactivation of the Dyn2-Cort-based endocytic machinery. In support of these findings, dominant negative forms of c-Src kinase, when expressed in a hepatocyte-derived cell line (Clone 9), attenuate Tf internalization. Remarkably, cells exposed to Tf showed a 3- to 4-fold increase in Dyn2 and Cort phosphorylation compared to that shown by untreated cells, an increase exceeding that observed in cells treated with EGF. These findings provide new insights into the regulation of what was thought to be a constitutive endocytic process. 相似文献
18.
Nicole C. Grieder Emmanuel Caussinus David S. Parker Kenneth Cadigan Markus Affolter Stefan Luschnig 《PloS one》2008,3(9)
Background
There is increasing evidence that tissue-specific modifications of basic cellular functions play an important role in development and disease. To identify the functions of COPI coatomer-mediated membrane trafficking in Drosophila development, we were aiming to create loss-of-function mutations in the γCOP gene, which encodes a subunit of the COPI coatomer complex.Principal Findings
We found that γCOP is essential for the viability of the Drosophila embryo. In the absence of zygotic γCOP activity, embryos die late in embryogenesis and display pronounced defects in morphogenesis of the embryonic epidermis and of tracheal tubes. The coordinated cell rearrangements and cell shape changes during tracheal tube morphogenesis critically depend on apical secretion of certain proteins. Investigation of tracheal morphogenesis in γCOP loss-of-function mutants revealed that several key proteins required for tracheal morphogenesis are not properly secreted into the apical lumen. As a consequence, γCOP mutants show defects in cell rearrangements during branch elongation, in tube dilation, as well as in tube fusion. We present genetic evidence that a specific subset of the tracheal defects in γCOP mutants is due to the reduced secretion of the Zona Pellucida protein Piopio. Thus, we identified a critical target protein of COPI-dependent secretion in epithelial tube morphogenesis.Conclusions/Significance
These studies highlight the role of COPI coatomer-mediated vesicle trafficking in both general and tissue-specific secretion in a multicellular organism. Although COPI coatomer is generally required for protein secretion, we show that the phenotypic effect of γCOP mutations is surprisingly specific. Importantly, we attribute a distinct aspect of the γCOP phenotype to the effect on a specific key target protein. 相似文献19.
20.
Ellen J. Tisdale Fouad Azizi Cristina R. Artalejo 《The Journal of biological chemistry》2009,284(9):5876-5884
Rab2 requires glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and atypical
protein kinase Cι (aPKCι) for retrograde vesicle formation from
vesicular tubular clusters that sort secretory cargo from recycling proteins
returned to the endoplasmic reticulum. However, the precise role of GAPDH and
aPKCι in the early secretory pathway is unclear. GAPDH was the first
glycolytic enzyme reported to co-purify with microtubules (MTs). Similarly,
aPKC associates directly with MTs. To learn whether Rab2 also binds directly
to MTs, a MT binding assay was performed. Purified Rab2 was found in a
MT-enriched pellet only when both GAPDH and aPKCι were present, and
Rab2-MT binding could be prevented by a recombinant fragment made to the Rab2
amino terminus (residues 2-70), which directly interacts with GAPDH and
aPKCι. Because GAPDH binds to the carboxyl terminus of α-tubulin,
we characterized the distribution of tyrosinated/detyrosinated α-tubulin
that is recruited by Rab2 in a quantitative membrane binding assay.
Rab2-treated membranes contained predominantly tyrosinated α-tubulin;
however, aPKCι was the limiting and essential factor.
Tyrosination/detyrosination influences MT motor protein binding; therefore, we
determined whether Rab2 stimulated kinesin or dynein membrane binding.
Although kinesin was not detected on membranes incubated with Rab2, dynein was
recruited in a dose-dependent manner, and binding was aPKCι-dependent.
These combined results suggest a mechanism by which Rab2 controls MT and motor
recruitment to vesicular tubular clusters.The small GTPase Rab2 is essential for membrane trafficking in the early
secretory pathway and associates with vesicular tubular
clusters
(VTCs)2 located
between the endoplasmic reticulum (ER) and the cis-Golgi compartment
(1,
2). VTCs are pleomorphic
structures that sort anterograde-directed cargo from recycling proteins and
trafficking machinery retrieved to the ER
(3-6).
Rab2 bound to a VTC microdomain stimulates recruitment of soluble factors that
results in the release of vesicles containing the recycling protein p53/p58
(7). In that regard, we have
previously reported that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and
atypical PKC ι (aPKCι) are Rab2 effectors that interact directly
with the Rab2 amino terminus and with each other
(8,
9). Their interaction requires
Src-dependent tyrosine phosphorylation of GAPDH and aPKCι
(10). Moreover, GAPDH is a
substrate for aPKCι (11).
GAPDH catalytic activity is not required for ER to Golgi transport indicating
that GAPDH provides a specific function essential for membrane trafficking
from VTCs independent of glycolytic function
(9). Indeed, phospho-GAPDH
influences MT dynamics in the early secretory pathway
(11).GAPDH was the first glycolytic enzyme reported to co-purify with
microtubules (MTs) (12) and
subsequently was shown to interact with the carboxyl terminus of
α-tubulin (13). The
binding of GAPDH to MTs promotes formation of cross-linked parallel MT arrays
or bundles (14,
15). GAPDH has also been
reported to possess membrane fusogenic activity, which is inhibited by tubulin
(16). Similarly, aPKC
associates directly with tubulin and promotes MT stability and MT remodeling
at specific intracellular sites
(17-21).
It may not be coincidental that these two Rab2 effectors influence MT dynamics
because recent studies indicate that the cytoskeleton plays a central role in
the organization and operation of the secretory pathway
(22).MTs are dynamic structures that grow or shrink by the addition or loss of
α- and β-tubulin heterodimers from the ends of protofilaments
(23). Their assembly and
stability is regulated by a variety of proteins traditionally referred to as
microtubule-associated proteins (MAPs). In addition to the multiple
α/β isoforms that are present in eukaryotes, MTs undergo an
assortment of post-translational modifications, including acetylation,
glycylation, glutamylation, phosphorylation, palmitoylation, and
detyrosination, which further contribute to their biochemical heterogeneity
(24,
25). It has been proposed that
these tubulin modifications regulate intracellular events by facilitating
interaction with MAPs and with other specific effector proteins
(24). For example, the
reversible addition of tyrosine to the carboxyl terminus of α-tubulin
regulates MT interaction with plus-end tracking proteins (+TIPs) containing
the cytoskeleton-associated protein glycine-rich (CAP-Gly) motif and with
dynein-dynactin
(27-29).
Additionally, MT motility and cargo transport rely on the cooperation of the
motor proteins kinesin and dynein
(30). Kinesin is a plus-end
directed MT motor, whereas cytoplasmic dynein is a minus-end MT-based motor,
and therefore the motors transport vesicular cargo toward the opposite end of
a MT track (31).Although MT assembly does not appear to be directly regulated by small
GTPases, Rab proteins provide a molecular link for vesicle movement along MTs
to the appropriate target (22,
32-34).
In this study, the potential interaction of Rab2 with MTs and motor proteins
was characterized. We found that Rab2 does not bind directly to preassembled
MTs but does associate when both GAPDH and aPKCι are present and bound to
MTs. Moreover, the MTs predominantly contained tyrosinated α-tubulin
(Tyr-tubulin) suggesting that a dynamic pool of MTs that differentially binds
MAPs/effector proteins/motors associates with VTCs in response to Rab2. To
that end, we determined that Rab2-promoted dynein/dynactin binding to
membranes and that the recruitment required aPKCι. 相似文献