共查询到20条相似文献,搜索用时 15 毫秒
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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
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Tomoya Isaji Yuya Sato Tomohiko Fukuda Jianguo Gu 《The Journal of biological chemistry》2009,284(18):12207-12216
N-Glycosylation of integrin α5β1 plays a crucial role
in cell spreading, cell migration, ligand binding, and dimer formation, but
the detailed mechanisms by which N-glycosylation mediates these
functions remain unclear. In a previous study, we showed that three potential
N-glycosylation sites (α5S3–5) on the β-propeller of
the α5 subunit are essential to the functional expression of the
subunit. In particular, site 5 (α5S5) is the most important for its
expression on the cell surface. In this study, the function of the
N-glycans on the integrin β1 subunit was investigated using
sequential site-directed mutagenesis to remove the combined putative
N-glycosylation sites. Removal of the N-glycosylation sites
on the I-like domain of the β1 subunit (i.e. the Δ4-6
mutant) decreased both the level of expression and heterodimeric formation,
resulting in inhibition of cell spreading. Interestingly, cell spreading was
observed only when the β1 subunit possessed these three
N-glycosylation sites (i.e. the S4-6 mutant). Furthermore,
the S4-6 mutant could form heterodimers with either α5S3-5 or α5S5
mutant of the α5 subunit. Taken together, the results of the present
study reveal for the first time that N-glycosylation of the I-like
domain of the β1 subunit is essential to both the heterodimer formation
and biological function of the subunit. Moreover, because the
α5S3-5/β1S4-6 mutant represents the minimal
N-glycosylation required for functional expression of the β1
subunit, it might also be useful for the study of molecular structures.Integrin is a heterodimeric glycoprotein that consists of both an α
and a β subunit (1). The
interaction between integrin and the extracellular matrix is essential to both
physiologic and pathologic events, such as cell migration, development, cell
viability, immune homeostasis, and tumorigenesis
(2,
3). Among the integrin
superfamily, β1 integrin can combine with 12 distinct α subunits
(α1–11, αv) to form heterodimers, thereby acquiring a wide
variety of ligand specificity
(1,
4). Integrins are thought to be
regulated by inside-out signaling mechanisms that provoke conformational
changes, which modulate the affinity of integrin for the ligand
(5). However, an increasing
body of evidence suggests that cell-surface carbohydrates mediate a variety of
interactions between integrin and its extracellular environment, thereby
affecting integrin activity and possibly tumor metastasis as well
(6–8).Guo et al. (9)
reported that an increase in β1–6-GlcNAc sugar chains on the
integrin β1 subunit stimulated cell migration. In addition, elevated
sialylation of the β1 subunit, because of Ras-induced STGal-I transferase
activity, also induced cell migration
(10,
11). Conversely, cell
migration and spreading were reduced by the addition of a bisecting GlcNAc,
which is a product of N-acetylglucosaminyltransferase III
(GnT-III),2 to the
α5β1 and α3β1 integrins
(12,
13). Alterations of
N-glycans on integrins might also regulate their cis interactions
with membrane-associated proteins, including the epidermal growth factor
receptor, the galectin family, and the tetraspanin family of proteins
(14–19).In addition to the positive and negative regulatory effects of
N-glycan, several research groups have reported that
N-glycans must be present on integrin α5β1 for the
αβ heterodimer formation and proper integrin-matrix interactions.
Consistent with this hypothesis, in the presence of the glycosylation
inhibitor, tunicamycin, normal integrin-substrate binding and transport to the
cell surface are inhibited
(20). Moreover, treatment of
purified integrin with N-glycosidase F blocked both the inherent
association of the subunits and the interaction between integrin and
fibronectin (FN) (21). These
results suggest that N-glycosylation is essential to the functional
expression of α5β1. However, because integrin α5β1
contains 26 potential N-linked glycosylation sites, 14 in the α
subunit and 12 in the β subunit, identification of the sites that are
essential to its biological functions is key to understanding the molecular
mechanisms by which N-glycans alter integrin function. Recently, our
group determined that N-glycosylation of the β-propeller domain
on the α5 subunit is essential to both heterodimerization and biological
functions of the subunit. Furthermore, we determined that sites 3–5 are
the most important sites for α5 subunit-mediated cell spreading and
migration on FN (22). The
purpose of this study was to clarify the roles of N-glycosylation of
the β1 subunit. Therefore, we performed combined substitutions in the
putative N-glycosylation sites by replacement of asparagine residues
with glutamine residues. We subsequently introduced these mutated genes into
β1-deficient epithelial cells (GE11). The results of these mutation
experiments revealed that the N-glycosylation sites on the I-like
domain of the β1 subunit, sites number 4–6 (S4-6), are essential to
both heterodimer formation and biological functions, such as cell
spreading. 相似文献
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Hye Shin Lee Mujeeburahiman Cheerathodi Sankar P. Chaki Steve B. Reyes Yanhua Zheng Zhimin Lu Helena Paidassi Celine DerMardirossian Adam Lacy-Hulbert Gonzalo M. Rivera Joseph H. McCarty 《Molecular and cellular biology》2015,35(8):1401-1413
Directional cell motility is essential for normal development and physiology, although how motile cells spatiotemporally activate signaling events remains largely unknown. Here, we have characterized an adhesion and signaling unit comprised of protein tyrosine phosphatase (PTP)-PEST and the extracellular matrix (ECM) adhesion receptor β8 integrin that plays essential roles in directional cell motility. β8 integrin and PTP-PEST form protein complexes at the leading edge of migrating cells and balance patterns of Rac1 and Cdc42 signaling by controlling the subcellular localization and phosphorylation status of Rho GDP dissociation inhibitor 1 (RhoGDI1). Translocation of Src-phosphorylated RhoGDI1 to the cell''s leading edge promotes local activation of Rac1 and Cdc42, whereas dephosphorylation of RhoGDI1 by integrin-bound PTP-PEST promotes RhoGDI1 release from the membrane and sequestration of inactive Rac1/Cdc42 in the cytoplasm. Collectively, these data reveal a finely tuned regulatory mechanism for controlling signaling events at the leading edge of directionally migrating cells. 相似文献
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Shu Tang Rehana Buriro Zhijun Liu Miao Zhang Islam Ali Abdelnasir Adam J?rg Hartung Endong Bao 《PloS one》2013,8(7)
Neonatal rat primary myocardial cells were subjected to heat stress in vitro, as a model for investigating the distribution and expression of Hsp27 and αB-crystallin. After exposure to heat stress at 42°C for different durations, the activities of enzymes expressed during cell damage increased in the supernatant of the heat-stressed myocardial cells from 10 min, and the pathological lesions were characterized by karyopyknosis and acute degeneration. Thus, cell damage was induced at the onset of heat stress. Immunofluorescence analysis showed stronger positive signals for both Hsp27 and αB-crystallin from 10 min to 240 min of exposure compared to the control cells. According to the Western blotting results, during the 480 min of heat stress, no significant variation was found in Hsp27 and αB-crystallin expression; however, significant differences were found in the induction of their corresponding mRNAs. The expression of these small heat shock proteins (sHsps) was probably delayed or overtaxed due to the rapid consumption of sHsps in myocardial cells at the onset of heat stress. Our findings indicate that Hsp27 and αB-crystallin do play a role in the response of cardiac cells to heat stress, but the details of their function remain to be investigated. 相似文献
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Kenshi Yamasaki Jun Muto Kristen R. Taylor Anna L. Cogen David Audish John Bertin Ethan P. Grant Anthony J. Coyle Amirhossein Misaghi Hal M. Hoffman Richard L. Gallo 《The Journal of biological chemistry》2009,284(19):12762-12771
Inflammation under sterile conditions is a key event in autoimmunity and
following trauma. Hyaluronan, a glycosaminoglycan released from the
extracellular matrix after injury, acts as an endogenous signal of trauma and
can trigger chemokine release in injured tissue. Here, we investigated whether
NLRP3/cryopyrin, a component of the inflammasome, participates in the
inflammatory response to injury or the cytokine response to hyaluronan. Mice
with a targeted deletion in cryopyrin showed a normal increase in Cxcl2 in
response to sterile injuries but had decreased inflammation and release of
interleukin-1β (IL-1β). Similarly, the addition of hyaluronan to
macrophages derived from cryopyrin-deficient mice increased release of Cxcl2
but did not increase IL-1β release. To define the mechanism of
hyaluronan-mediated activation of cryopyrin, elements of the hyaluronan
recognition process were studied in detail. IL-1β release was inhibited
in peritoneal macrophages derived from CD44-deficient mice, in an MH-S
macrophage cell line treated with antibodies to CD44, or by inhibitors of
lysosome function. The requirement for CD44 binding and hyaluronan
internalization could be bypassed by intracellular administration of
hyaluronan oligosaccharides (10–18-mer) in lipopolysaccharide-primed
macrophages. Therefore, the action of CD44 and subsequent hyaluronan
catabolism trigger the intracellular cryopyrin → IL-1β pathway.
These findings support the hypothesis that hyaluronan works through IL-1β
and the cryopyrin system to signal sterile inflammation.Inflammation, as defined by changes in vascular permeability and leukocyte
recruitment, is an essential step for the control of microbial invasion.
Specific microbial products trigger this process through a diverse array of
innate immune pattern recognition receptors. However, an inflammatory response
independent of infection is also an important process for maintenance of
biological homeostasis. For example, normal wound healing requires a
controlled inflammatory response to enable the recruitment of monocytes and
the release of growth factors required for repair. This response can occur in
the absence of microbial stimuli. Furthermore, inflammation and the release of
proinflammatory mediators is also associated with many diseases such as
rheumatoid arthritis and Crohn disease
(1). These diseases are not
well understood in terms of their triggers but rather are described by the
subsequent release of proinflammatory mediators. Identification of the
triggers of sterile inflammation represents an important goal with immediate
diagnostic and therapeutic significance.Recent work has begun to elucidate pathways of inflammation that occur in
the absence of microbial stimuli. Stress signals such as heat-shock proteins,
intracellular components of necrotic cells not normally seen by immune cells,
and components of the extracellular matrix have all been implicated as
endogenous triggers of injury
(2–4).
Among this group is the glycosaminoglycan hyaluronan
(HA),6 an important
structural component of the extracellular matrix that is also a common
component of bacterial surfaces. HA is synthesized at the cell surface and
typically exists as a high molecular mass polymer greater than 106
Da and composed of repeating disaccharide units of
N-acetylglucosamine and glucuronic acid
(5,
6). Unlike other
glycosaminoglycans such as heparan sulfate or chondroitin sulfates that encode
specific activity by use of a diverse disaccharide sequence, HA is not
sulfated or epimerized, and only changes in HA size, concentration, and
location affect function.We have previously developed murine models of sterile injury to identify
the innate elements that recognize and mediate sterile inflammation
(7). Our results demonstrated
that (a) the initiation of a sterile intrinsic inflammatory process
is dependent on TLR4 activation, (b) sterile injury induces HA
accumulation at the injured site, and (c) sterile intrinsic
inflammation resembles signaling events that are activated by HA. Furthermore,
we have defined a novel alternative recognition complex for HA that involves
TLR4, MD-2, and CD44 (7). Taken
together with other work associating HA and innate pattern recognition
(4,
8–10),
these observations have provided new insight into mechanisms responsible for
sterile inflammation.Recently, the NLR (nucleotide-binding domain and leucine rich
repeat-containing) family has been extensively analyzed as a group of
intracellular pattern recognition receptors
(11). NLRs have a leucine-rich
repeat that recognizes pathogen-associated molecular patterns including
bacterial cell wall components and viral nucleic acids. NOD2 and NLR family,
pyrin containing 3 (NLRP3)/cryopyrin are two of the best
characterized NLRs. NOD2 recognizes the bacterial peptidoglycan-derived
molecule muramyl dipeptide and activates the NF-κB pathway to induce
inflammatory responses (12).
Mutations of the NOD2 gene were identified in individuals with
chronic inflammatory disorders such as Crohn disease
(13,
14) and Blau syndrome
(15). Mouse knockin mutants of
NOD2, which have the same mutation in NOD2 as human patients
with Crohn disease, showed elevated proinflammatory cytokines following
muramyl dipeptide challenge or dextran sodium sulfate-induced bowel
inflammation (16).
NLRP3, also known as cyropyrin, CIAS1, NALP3, PYPAF1, forms
an “inflammasome” with ASC (apoptosis-associated speck-like
protein containing a CARD) and caspase-1 to convert pro-IL-1β to active
IL-1β (17). Mutations in
NLRP3 were identified in individuals with familial cold
autoinflammatory syndrome (FCAS), Muckle-Wells syndrome, and neonatal onset
multisystem inflammatory disease
(18–20).
These individuals have recurrent or chronic inflammatory symptoms, including
fever, arthritis, and a urticaria-like eruption characterized by neutrophilic
infiltration. In FCAS, symptoms can be elicited by cold provocation by a
mechanism that appears to be mediated through the skin
(15,
21).Because disorders associated with mutations in NLRP3 are examples
of inflammation under sterile conditions and HA has been shown to be a trigger
of sterile inflammation, we sought to further understand the mechanism of the
response to HA by examining the role of cryopyrin during injury and after
exposure to HA. Our results show that cryopyrin and IL-1β are integral to
sterile inflammation and the response to HA. These observations provide new
insight into the function of HA as a “danger signal” of
injury. 相似文献
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Scott A. Barros Chutima Srimaroeng Jennifer L. Perry Ramsey Walden Neetu Dembla-Rajpal Douglas H. Sweet John B. Pritchard 《The Journal of biological chemistry》2009,284(5):2672-2679
Organic anion transporters (OATs) play a pivotal role in the clearance of
small organic anions by the kidney, yet little is known about how their
activity is regulated. A yeast two-hybrid assay was used to identify putative
OAT3-associated proteins in the kidney. Atypical protein kinase Cζ
(PKCζ) was shown to bind to OAT3. Binding was confirmed in
immunoprecipitation assays. The OAT3/PKCζ interaction was investigated in
rodent renal cortical slices from fasted animals. Insulin, an upstream
activator of PKCζ, increased both OAT3-mediated uptake of estrone sulfate
(ES) and PKCζ activity. Both effects were abolished by a
PKCζ-specific pseudosubstrate inhibitor. Increased ES transport was not
observed in renal slices from OAT3-null mice. Transport of the shared
OAT1/OAT3 substrate, ρ-aminohippurate, behaved similarly, except that
stimulation was reduced, not abolished, in the OAT3-null mice. This suggested
that OAT1 activity was also modified by PKCζ, subsequently confirmed
using an OAT1-specific substrate, adefovir. Inhibition of PKCζ also
blocked the increase in ES uptake seen in response to epidermal growth factor
and to activation of protein kinase A. Thus, PKCζ acted downstream of the
epidermal growth factor to protein kinase A signaling pathway. Activation of
transport was accompanied by an increase in Vmax and was
blocked by microtubule disruption, indicating that activation may result from
trafficking of OAT3 into the plasma membrane. These data demonstrate that
PKCζ activation up-regulates OAT1 and OAT3 function, and that
protein-protein interactions play a central role controlling these two
important renal drug transporters.Organic anion transporters
(OATs)7 are members of
the solute carrier 22A family and play a pivotal role in the renal clearance
of small (<500 Dalton) anionic drugs, xenobiotics, and their metabolites.
OAT substrates include a variety of drugs such as β-lactam antibiotics,
non-steroidal anti-inflammatory drugs, diuretics, and chemotherapeutics
(1). OATs are predominantly
expressed in renal proximal tubule, with OATs 1–3 localized to the
basolateral membrane and OAT4 and URAT1 on the apical membrane. OATs 1 and 3
are dicarboxylate exchangers, and are indirectly coupled to the sodium
gradient maintained by Na,K-ATPase through sodium/dicarboxylate co-transport
to drive the uphill basolateral step in renal organic anion secretion
(2).Although the ionic gradients, electrophysiology, and underlying kinetics
that drive transport by OATs 1 and 3 are well characterized, physiologically
important interactions of these basolateral OATs with membrane or cytosolic
proteins have yet to be identified
(1). Nevertheless, there is
clear evidence that other plasma membrane transporters do interact with
protein partners, influencing a diverse array of functions including transport
itself, cytoskeletal structure, vesicle formation, and trafficking, as well as
signaling (3). Among the
transporters with activity modulated by protein-protein interactions,
particularly by the PDZ proteins, PDZK1 and NHERFs 1 and 2, are apical drug
transporters of the SLC22A family, including OCTN1, OCTN2, OAT4, and URAT1
(4–6).In the present study, we have used a yeast two-hybrid assay to identify
putative protein partners that interact directly with OAT3. The C-terminal 81
amino acids of OAT3 were used as bait to screen a human cDNA kidney library.
Among the 23 positive clones (putative binding partners) was a clone encoding
the C-terminal 141 amino acids of atypical protein kinase Cζ (PKCζ).
Functional consequences of the putative OAT3/PKCζ interaction were
investigated in rodent renal slices. The resulting data indicate that
activation of PKCζ by insulin or epidermal growth factor (EGF) increased
OAT3- and OAT1-mediated transport. Thus, PKCζ controls function of both
major secretory organic anion transporters expressed at the basolateral face
of the renal proximal tubule, positioning it to regulate the efficacy of renal
drug elimination. 相似文献
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Jihee Kim Seungkirl Ahn Keshava Rajagopal Robert J. Lefkowitz 《The Journal of biological chemistry》2009,284(18):11953-11962
Recent studies in receptor-transfected cell lines have demonstrated that
extracellular signal-regulated kinase (ERK) activation by angiotensin type 1A
receptor and other G protein-coupled receptors can be mediated by both G
protein-dependent and β-arrestin-dependent mechanisms. However, few
studies have explored these mechanisms in primary cultured cells expressing
endogenous levels of receptors. Accordingly, here we utilized the
β-arrestin biased agonist for the angiotensin type 1A receptor,
SII-angiotensin (SII), and RNA interference techniques to investigate
angiotensin II (ANG)-activated β-arrestin-mediated mitogenic signaling
pathways in rat vascular smooth muscle cells. Both ANG and SII induced DNA
synthesis via the ERK activation cascade. Even though SII cannot induce
calcium influx (G protein activation) after receptor stimulation, it does
cause ERK activation, although less robustly than ANG. Activation by both
ligands is diminished by depletion of β-arrestin2 by small interfering
RNA, although the effect is more complete with SII. ERK activation at early
time points but not later time points is strongly inhibited by those protein
kinase C inhibitors that can block protein kinase Cζ. Moreover, ANG- and
SII-mediated ERK activation require transactivation of the epidermal growth
factor receptor via metalloprotease 2/9 and Src kinase. β-Arrestin2
facilitates ANG and SII stimulation of Src-mediated phosphorylation of Tyr-845
on the EGFR, a known site for Src phosphorylation. These studies delineate a
convergent mechanism by which G protein-dependent and
β-arrestin-dependent pathways can independently mediate ERK-dependent
transactivation of the EGFR in vascular smooth muscle cells thus controlling
cellular proliferative responses.G protein-coupled receptors, also known as seven transmembrane
(7TM)2 receptors,
control virtually all known physiological processes in mammals
(1). The various functions of
these receptors are mediated and modulated by three families of proteins,
which share the property that they interact virtually universally with the
receptors in a strictly stimulus-dependent way
(1). These three families of
proteins are the heterotrimeric G proteins, the G protein-coupled receptor
kinases (GRKs), and the β-arrestins. Activation of the receptors
stimulates classical G protein-dependent signaling, often involving regulation
of levels of second messengers such as cAMP and diacyglycerol. However, as has
been known for many years, interaction of activated receptors with GRKs
leading to their phosphorylation, and subsequent interaction with
β-arrestins leads to desensitization of G protein signaling.In recent years, however, it has become increasingly clear that the
β-arrestin-GRK system is in fact bifunctional
(2). Thus, even as it
desensitizes G protein signaling by the receptors, it also serves as a signal
transduction system in its own right, activating a growing list of signaling
pathways. These positive signaling functions are often mediated by the ability
of β-arrestin to serve as an adaptor or scaffold molecule, bringing
elements of diverse signaling pathways into proximity with one another and the
receptors and thereby facilitating their activation. This new paradigm for
understanding the previously unrecognized signaling properties of the
β-arrestin-GRK system has been explored in a wide variety of transfected
cultured cell systems.However, to date, relatively little investigation of these novel signaling
pathways has been carried out in primary cell culture systems expressing
endogenous levels of 7TM receptors. In seeking such a system in which to
characterize and compare β-arrestin and G protein-mediated signaling
pathways from a typical 7TM receptor, our attention was drawn to cultured rat
vascular smooth muscle cells (VSMCs). Several features of rat VSMCs suggest
this to be a relevant system for these purposes. Rat VSMCs express a variety
of physiologically important 7TM receptors including the angiotensin II type
1A receptor (AT1R) (3). This
receptor has been the focus of extensive study in transfected cell systems
with respect to its β-arrestin-mediated signaling to a variety of
pathways, most particularly extracellular signal-regulated kinase (ERK).
Moreover, the AT1R mediates the physiologically important effects of
angiotensin II (ANG) on vascular tone as well as on proliferation and
chemotaxis (4,
5). Pathophysiologically, ANG
stimulation of this receptor has been implicated in VSMC proliferation and
chemotaxis, which are thought to play an important role in such important
disease processes as atherosclerosis and restenosis after angioplasty
(6,
7). Moreover, a ligand has been
characterized
[Sar1,Ile4,Ile8](SII)-angiotensin (SII), a
triply mutated angiotensin octapeptide that, in transfected cell systems, acts
as a specific agonist for β-arrestin-mediated signaling, although not
activating G protein-mediated signaling
(8).Accordingly, in the studies described here, we set out to investigate the
characteristics of activation of ERK in rat VSMCs that might be mediated
through G protein as well as β-arrestin signaling. The results not only
demonstrate the importance of β-arrestin-mediated signaling in
ERK-mediated proliferative responses of these cells, but also shed new light
on the molecular mechanisms and interrelationships between the β-arrestin
and classical G protein-mediated activation of these pathways. 相似文献
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Quinol oxidation at center P of the cytochrome bc1
complex involves bifurcated electron transfer to the Rieske iron-sulfur
protein and cytochrome b. It is unknown whether both electrons are
transferred from the same domain close to the Rieske protein, or if an
unstable semiquinone anion intermediate diffuses rapidly to the vicinity of
the bL heme. We have determined the pre-steady state rate
and activation energy (Ea) for quinol oxidation in
purified yeast bc1 complexes harboring either a Y185F
mutation in the Rieske protein, which decreases the redox potential of the FeS
cluster, or a E272Q cytochrome b mutation, which eliminates the
proton acceptor in cytochrome b. The rate of the bifurcated reaction
in the E272Q mutant (<10% of the wild type) was even lower than that of the
Y185F enzyme (∼20% of the wild type). However, the E272Q enzyme showed the
same Ea (61 kJ mol-1) with respect to the wild
type (62 kJ mol-1), in contrast with the Y185F mutation, which
increased Ea to 73 kJ mol-1. The rate and
Ea of the slow reaction of quinol with oxygen that are
observed after cytochrome b is reduced were unaffected by the E272Q
substitution, whereas the Y185F mutation modified only its rate. The
Y185F/E272Q double mutation resulted in a synergistic decrease in the rate of
quinol oxidation (0.7% of the wild type). These results are inconsistent with
a sequential “movable semiquinone” mechanism but are consistent
with a model in which both electrons are transferred simultaneously from the
same domain in center P.The cytochrome bc1 complex couples the oxidation of a
two-electron carrier molecule of quinol to the movement of protons across the
inner mitochondrial or bacterial membrane. The key reaction in this
energy-conserving mechanism, known as the Q-cycle
(1,
2), is the bifurcation of
electrons at the active site located closer to the positive side of the
membrane, termed center P or Qo site. One of the electrons from
quinol is transferred to a chain of one-electron carriers with relatively high
redox potentials that include the FeS cluster of the Rieske protein and the
hemes of cytochromes c1 and c. The other electron
is donated to the low potential (bL) heme of cytochrome
b, from which it crosses most of the membrane width to the high
potential bH heme, located close to another active site
(center N or Qi site), where quinone is reduced to quinol after two
center P turnovers. Proton release and uptake at each active site are achieved
by taking advantage of the chemistry of quinol and quinone, which can only
stably exist at physiological pH in the protonated and deprotonated forms,
respectively.Critical to the electron bifurcation reaction at center P is the
arrangement of protonatable groups (His181 of the Rieske protein
and Glu272 of cytochrome b) close to the electron
acceptors at opposite sides of the substrate (see
Fig. 1). However, the exact
mechanism of electron bifurcation at center P is still an unresolved issue.
Proposed models have ranged from strictly concerted mechanisms in which both
electrons from quinol are extracted simultaneously
(3,
4) to those that postulate a
highly stabilized semiquinone intermediate
(5). Between these two extremes
are mechanisms that propose the formation of an unstable semiquinone
intermediate after a first electron transfer from quinol to the Rieske protein
(6–8),
which seem to be supported by recent reports that claim to have detected low
concentrations of semiquinone at center P when reoxidation of cytochrome
b is impeded under special conditions
(9,
10). One version of the
unstable semiquinone mechanism proposes that this intermediate diffuses from
the vicinity of the Rieske protein to a location within center P located
closer to the bL heme, which would allow non-rate-limiting
rates of bL reduction to occur even at very low
semiquinone occupancy (11). In
this proposal, the movement of the unstable semiquinone would be allowed by
protonation and rotation of Glu272 in cytochrome b, which
occupies different conformations in crystallographic structures
(Fig. 1)
(11–14).Open in a separate windowFIGURE 1.Electron and proton acceptors involved in quinol oxidation at center
P. Crystallographic structures 1EZV
(12) and 1P84
(13) show stigmatellin
(A) or 5-n-heptyl-6-hydroxy-4,7-dioxobenzothiazole
(B) bound at center P forming a hydrogen bond to the
His181 residue of the Rieske protein, which is a ligand to the FeS
cluster. The Tyr185 residue in the Rieske protein influences the
Em value of the FeS cluster
(22). On the side pointing to
the bL heme, a bound water molecule is also
hydrogen-bonded to the inhibitor, either to the Glu272 carboxylate
in cytochrome b (A), or to its backbone amino group
(B), when the side chain is rotated toward a water network that
connects to the propionate of the bL heme and to
Arg79 of cytochrome b.An important prediction of the movable semiquinone model
(11) is that mutation of
Glu272 should impede diffusion of the anionic semiquinone, forcing
electron transfer to the bL heme to occur through a longer
distance from the position closer to the Rieske FeS cluster
(15), thereby shifting the
rate-limiting step from the first to the second electron transfer. Although it
has already been reported that different mutations at Glu272
partially slow down quinol oxidation at center P
(15–17),
no effort has been made so far to evaluate whether the rate-limiting step
changes upon inhibition of the deprotonation of quinol (or of a putative
semiquinone intermediate) by mutation of the cytochrome b
Glu272. In the present work, we analyze the energy of activation of
quinol oxidation at center P and show that the rate-limiting step when
Glu272 is mutated to glutamine, although slower, is still
determined by the driving force for electron transfer to the Rieske protein.
We also show that decreasing this driving force enhances the relative
inhibition caused by mutating Glu272, suggesting a tight coupling
of reactions involved in quinol oxidation and deprotonation. In contrast,
reactions with oxygen that bypass the electron bifurcation at center P, which
are likely to involve a semiquinone intermediate, are independent of
Glu272 and go through an energetic barrier different from that of
the bifurcated reaction. We discuss how these results support a mechanism in
which both electron transfer events from quinol to the Rieske protein and the
bL heme occur from the same position and at the same
time. 相似文献
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