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961.
Eva Forgacs Takeshi Sakamoto Suzanne Cartwright Betty Belknap Mih��ly Kov��cs Judit T��th Martin R. Webb James R. Sellers Howard D. White 《The Journal of biological chemistry》2009,284(4):2138-2149
We have determined the kinetic mechanism and motile properties of the
switch 1 mutant S217A of myosin Va. Phosphate dissociation from myosin
V-ADP-Pi (inorganic phosphate) and actomyosin V-ADP-Pi
and the rate of the hydrolysis step (myosin V-ATP → myosin
V-ADP-Pi) were all ∼10-fold slower in the S217A mutant than in
wild type (WT) myosin V, resulting in a slower steady-state rate of basal and
filamentous actin (actin)-activated ATP hydrolysis. Substrate binding and ADP
dissociation kinetics were all similar to or slightly faster in S217A than in
WT myosin V and mechanochemical gating of the rates of dissociation of ADP
between trail and lead heads is maintained. The reduction in the rate
constants of the hydrolysis and phosphate dissociation steps reduces the duty
ratio from ∼0.85 in WT myosin V to ∼0.25 in S217A and produces a motor
in which the average run length on actin at physiological concentrations of
ATP is reduced 10-fold. Thus we demonstrate that, by mutational perturbation
of the switch 1 structure, myosin V can be converted into a low duty ratio
motor that is processive only at low substrate concentrations.During the past 2 decades a considerable number of different myosins have
been discovered (1). Myosin V
is the best characterized among the so-called unconventional myosins
(i.e. those not belonging to class II), and it serves as an important
model molecule for studying actomyosin interactions and single molecule
processive motility (2). Myosin
V is a highly processive motor whose role is to transport cargo along actin
filaments or bundles inside the cell
(3–5).
The kinetic mechanism of myosin V is significantly different from that of
conventional myosins such as muscle myosin II, as it remains bound to actin
(filamentous actin) through a number of ATPase cycles
(6–8).
Myosin V has a high duty ratio: a single-headed myosin V-S1 (myosin V,
subfragment 1) is in the strongly bound AM-ADP state 80–90% of the time
during ATP hydrolysis. An additional mechanism for promoting highly processive
runs is the preferential release of ADP from the trail head because of
mechanochemical gating, which causes a drastic reduction of the rate constant
of ADP release from the lead head
(9–11).
Although there are significant differences between the ATPase mechanisms of
the different myosins, the structure of the nucleotide binding pocket
(composed of the switch 1 and 2 regions and the P-loop) is highly conserved.
The position of the Ser217 (Ser236 in
Dictyostelium myosin II) residue of the switch 1 loop (the first
serine in the NDNSSRFG sequence) is shown in
Fig. 1. It had been shown
previously by mutagenesis in Dictyostelium
(12) and in smooth muscle
myosin II (13) that the
substitution of serine 236 to alanine retains at least partial enzymatic and
motile function in these mutant myosins. Therefore, the OH group is not an
essential part of the catalytic mechanism, but the rate of steady-state ATP
hydrolysis is reduced several fold. However, neither of these studies includes
a detailed kinetic analysis to determine which steps of the catalytic
mechanism were altered by the mutation. Here we have exploited the higher
affinity of myosin V-ADP-Pi for actin to determine the effect of
the mutation on the rate constants of the product dissociation steps following
the power stroke, which could not be determined using either
Dictyostelium or smooth muscle myosin. We also conducted single
molecule motility studies using total internal reflectance fluorescence
(TIRF)5 microscopy to
determine how the changes in the kinetic mechanism affect the motile
properties of the molecule.Open in a separate windowFIGURE 1.Schematic representation of the critical residues in the ATP binding
site of myosin based on the MgADP·VO4 crystal of the
Dictyostelium motor domain (Smith and Rayment
(30)). The
serine in position 217 was mutated to alanine for these kinetic studies. The
small spheres are at the position of the oxygen of the water
molecules. 相似文献
962.
963.
Jianzhong Liu Shunqing Wang Ping Zhang Nasser Said-Al-Naief Suzanne M. Michalek Xu Feng 《The Journal of biological chemistry》2009,284(18):12512-12523
Lipopolysaccharide (LPS), a common bacteria-derived product, has long been
recognized as a key factor implicated in periodontal bone loss. However, the
precise cellular and molecular mechanisms by which LPS induces bone loss still
remains controversial. Here, we show that LPS inhibited osteoclastogenesis
from freshly isolated osteoclast precursors but stimulated osteoclast
formation from those pretreated with RANKL in vitro in tissue culture
dishes, bone slices, and a co-culture system containing osteoblasts,
indicating that RANKL-mediated lineage commitment is a prerequisite for
LPS-induced osteoclastogenesis. Moreover, the RANKL-mediated lineage
commitment is long term, irreversible, and TLR4-dependent. LPS exerts the dual
function primarily by modulating the expression of NFATc1, a master regulator
of osteoclastogenesis, in that it abolished RANKL-induced NFATc1 expression in
freshly isolated osteoclast precursors but stimulated its expression in
RANKL-pretreated cells. In addition, LPS prolonged osteoclast survival by
activating the Akt, NF-κB, and ERK pathways. Our current work has not
only unambiguously defined the role of LPS in osteoclastogenesis but also has
elucidated the molecular mechanism underlying its complex functions in
osteoclast formation and survival, thus laying a foundation for future
delineation of the precise mechanism of periodontal bone loss.LPS,2 a
common bacteria-derived product, has long been recognized as a key factor
implicated in the development of chronic periodontitis. LPS plays an important
role in periodontitis by initiating a local host response in gingival tissues
that involves recruitment of inflammatory cells, production of prostanoids and
cytokines, elaboration of lytic enzymes and activation of osteoclast formation
and function to induce bone loss
(1-3).Osteoclasts, the body''s sole bone-resorbing cells, are multinucleated giant
cells that differentiate from cells of hematopoietic lineage upon stimulation
by two critical factors: the macrophage/monocyte colony-forming factor (M-CSF)
and the receptor activator of NF-κB ligand (RANKL)
(4-6).
RANKL exerts its effects on osteoclast formation and function by binding to
its receptor, RANK (receptor activator of NF-κB) expressed on osteoclast
precursors and mature osteoclasts
(7-9).
RANKL also has a decoy receptor, osteoprotegerin, which inhibits RANKL action
by competing with RANK for binding RANKL
(10,
11).RANK is a member of the tumor necrosis factor receptor (TNFR) family
(12). Members of the TNFR
family lack intrinsic enzymatic activity, and hence they transduce
intracellular signals by recruiting various adaptor proteins including TNF
receptor-associated factors (TRAFs) through specific motifs in the cytoplasmic
domain (13,
14). It has been established
that RANK contains three functional TRAF-binding sites
(369PFQEP373, 559PVQEET564, and
604PVQEQG609) that, redundantly, play a role in
osteoclast formation and function
(15,
16). Collectively, through
these functional TRAF-binding motifs, RANK activates six major signaling
pathways, NF-κB, JNK, ERK, p38, NFATc1, and Akt, which play important
roles in osteoclast formation, function, and/or survival
(15,
17-19).
In particular, NFATc1 has been established as a master regulator of osteoclast
differentiation
(20-22).The involvement of osteoclasts in the pathogenesis of periodontal bone loss
is supported by observations that osteoclasts are physically present and
functionally involved in bone resorption in periodontal tissues
(23-27).
RANKL and RANK knockout mice develop osteopetrosis and show failure in tooth
eruption due to a lack of osteoclasts
(24,
25,
28). Moreover,
op/op mice, in which a mutation in the coding region of the
M-CSF gene generates a stop codon that leads to premature termination of
translation of M-CSF mRNA, also show osteopetrosis and failure in tooth
eruption due to a defect in osteoclast development
(26,
27).Whereas the role of osteoclasts in periodontal disease associated alveolar
bone destruction has been well established, the precise role of LPS in
osteoclastogenesis still remains controversial. The vast majority of the
previous studies demonstrated that LPS stimulates osteoclastogenesis. This is
consistent with the role that LPS, a well recognized pathogenic factor in
periodontitis, presumably plays in periodontal bone loss
(29-33).
However, two previous studies demonstrated, surprisingly, that LPS plays
bifunctional roles in osteoclastogenesis in that although it inhibits
osteoclast formation from normal osteoclast precursors, it reverses to promote
osteoclastogenesis from osteoclast precursors pretreated with RANKL
(34,
35). Given that this finding
is inconsistent with the presumed role of LPS as a pathogenic factor in
periodontal bone loss and lacks careful and further validation, the prevalent
view is still that LPS stimulates osteoclastogenesis
(1-3).
Importantly, if LPS indeed has a dual function in osteoclastogenesis, the
molecular mechanism by which LPS exerts a dual effect on osteoclastogenesis
need to be further elucidated.In the present work, using various in vitro assays, we have
demonstrated independently that LPS inhibits osteoclastogenesis from normal
osteoclast precursors but promotes the development of osteoclasts from
RANKL-pretreated cells in tissue culture dishes and bone slices in single-cell
and co-culture settings, confirming the two previous observations that LPS
play a bifunctional role in osteoclastogenesis
(34,
35). Moreover, we have further
shown that the RANKL-mediated lineage commitment is long term and irreversible
in LPS-mediated osteoclastogenesis. More importantly, we have revealed that
LPS inhibits osteoclastogenesis by suppressing NFATc1 expression and JNK
activation while it prolongs osteoclast survival by activating the Akt,
NF-κB, and ERK pathways. These studies have not only unambiguously and
precisely defined the role of LPS in osteoclastogenesis but, more importantly,
may also lead to a paradigm shift in future investigation of the molecular
mechanism of periodontal bone loss. 相似文献
964.
Uta Ulrike von Borstel Ian James Heatly Duncan Anna Kate Shoveller Katrina Merkies Linda Jane Keeling Suzanne Theresa Millman 《Applied animal behaviour science》2009,116(2-4):228-236
Rollkur, the usually coercively obtained hyperflexion of the horse's neck, is employed as a training method by some dressage riders; however, its use is controversial as it may cause discomfort and adversely affect the horse's welfare. The objectives of this study were to determine: (1) if horses showed differences in stress, discomfort and fear responses as measured by heart rate and behaviour when ridden in Rollkur (R) obtained by pressure on the reins compared to regular poll flexion (i.e. with the nose-line being at or just in front of the vertical; N), and (2) if they showed a preference between the two riding styles when given the choice. Fifteen riding horses were ridden 30 times through a Y-maze randomly alternating between sides. Riding through one arm of the Y-maze was always followed by a short round ridden in R, whereas riding through the other arm was followed by a short round ridden in N. Immediately after the conditioning phase, horses were again repeatedly ridden into the maze; however, riders left it to the horse to decide which arm of the maze to enter. During R, horses moved slower and showed more often behavioural signs of discomfort, such as tail-swishing, head-tossing or attempted bucks (P < 0.05), and 14 of the 15 horses chose significantly (P < 0.05) more often the maze-arm associated with N rather than R. Subsequently, eight of the horses were also subjected to two fear tests following a short ride in N as well as a ride in R. During R, horses tended to react stronger (P = 0.092) to the fear stimuli and to take longer (P = 0.087) to approach them. These findings indicate that a coercively obtained Rollkur position may be uncomfortable for horses and that it makes them more fearful and therefore potentially more dangerous to ride. Further studies need to assess horses’ reaction to gradual training of Rollkur, as opposed to a coercively obtained hyperflexion, in order to decide whether the practice should be banned. 相似文献
965.
David M. Conrad Suzanne J. Furlong Carolyn D. Doucette Robert T.M. Boudreau David W. Hoskin 《Cellular signalling》2009,21(8):1298-1307
Thy-1 (CD90) crosslinking by monoclonal antibodies (mAb) in the context of costimulation causes the activation of mouse T-lymphocytes; however, the associated signal transduction processes have not been studied in detail. In this study we investigated the role of mitogen-activated protein kinases (MAPKs) in Thy-1-mediated T-lymphocyte activation using mAb-coated polystyrene microspheres to crosslink Thy-1 and costimulatory CD28 on murine T-lymphocytes. Concurrent Thy-1 and CD28 crosslinking induced DNA synthesis by T-lymphocytes, as well as interleukin (IL)-2 and IL-2 receptor (IL-2R) α chain (CD25) expression. Increased phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38 MAPK, and c-Jun N-terminal protein kinase (JNK) was also observed. Pharmacologic inhibition of ERK1/2 or JNK activation inhibited Thy-1-induced DNA synthesis and IL-2 production by T-lymphocytes. p38 MAPK inhibition also decreased DNA synthesis in Thy-1-stimulated T-lymphocytes; however, IL-2 production was increased in these cells. Inhibition of JNK, but not ERK1/2 or p38 MAPK, caused a marked reduction in Thy-1-induced CD25 expression. In addition, inhibition of p38 MAPK or JNK, but not ERK1/2, impaired the growth of IL-2-dependent CTLL-2 T-lymphocytes but did not substantially affect CD25 expression. Finally, exogenous IL-2 reversed the inhibitory effect of ERK1/2 or JNK inhibition on Thy-1-stimulated DNA synthesis by T-lymphocytes but did not substantially reverse JNK inhibition of CD25 expression. Collectively, these results suggest that during Thy-1-induced T-lymphocyte activation, ERK1/2 and JNK promoted IL-2 production whereas p38 MAPK negatively regulated IL-2 expression. JNK signalling was also required for CD25 expression. IL-2R signalling involved both p38 MAPK and JNK in CTLL-2 cells, whereas p38 MAPK was most important for IL-2R signalling in primary T-lymphocytes. MAPKs are therefore essential signalling intermediates for the Thy-1-driven proliferation of mouse T-lymphocytes. 相似文献
966.
Hong Ha Nguyen Claire Bouthier de la Tour Magali Toueille Françoise Vannier Suzanne Sommer Pascale Servant 《Molecular microbiology》2009,73(2):240-252
The nucleoid of radioresistant bacteria, including D . radiodurans , adopts a highly condensed structure that remains unaltered after exposure to high doses of irradiation. This structure may contribute to radioresistance by preventing the dispersion of DNA fragments generated by irradiation. In this report, we focused our study on the role of HU protein, a nucleoid-associated protein referred to as a histone-like protein, in the nucleoid compaction of D. radiodurans. We demonstrate, using a new system allowing conditional gene expression, that HU is essential for viability in D. radiodurans . Using a tagged HU protein and immunofluorescence microscopy, we show that HU protein localizes all over the nucleoid and that when HU is expressed from a thermosensitive plasmid, its progressive depletion at the non-permissive temperature generates decondensation of DNA before fractionation of the nucleoid into several entities and subsequent cell lysis. We also tested the effect of the absence of Dps, a protein also involved in nucleoid structure. In contrast to the drastic effect of HU depletion, no change in nucleoid morphology and cell viability was observed in dps mutants compared with the wild-type, reinforcing the major role of HU in nucleoid organization and DNA compaction in D. radiodurans . 相似文献
967.
Seung-Hwan Kim Antonia H. Holway Suzanne Wolff Andrew Dillin W. Matthew Michael 《The Journal of cell biology》2009,184(4):613-627
Yb regulates the proliferation of both germline and somatic stem cells in the Drosophila melanogaster ovary by activating piwi and hh expression in niche cells. In this study, we show that Yb protein is localized as discrete cytoplasmic spots exclusively in the somatic cells of the ovary and testis. These spots, which are different from all known cytoplasmic structures in D. melanogaster, are evenly electron-dense spheres 1.5 µm in diameter (herein termed the Yb body). The Yb body is frequently associated with mitochondria and a less electron-dense sphere of similar size that appears to be RNA rich. There are one to two Yb bodies/cell, often located close to germline cells. The N-terminal region of Yb is required for hh expression in niche cells, whereas the C-terminal region is required for localization to Yb bodies. The entire Yb protein is necessary for piwi expression in niche cells. A double mutant of Yb and a novel locus show male germline loss, revealing a function for Yb in male germline stem cell maintenance. 相似文献
968.
Activation of heterotrimeric G proteins is generally believed to induce dissociation of Gα and Gβγ subunits, which are then free to bind to and change the catalytic activity of a variety of intracellular enzymes. We have previously found that in cells, Gαq subunits remain complexed with its major effector, phospholipase Cβ1, through the activation cycle. To determine whether this behavior may be operative in other systems, we carried out Förster resonance energy transfer studies and found that eYFP-Gαi and eCFP-Gβγ remain associated after stimulation in HEK293 cells. We also found that the level of Forster resonance energy transfer between Alexa546-phospholipase Cβ2 and eGFP-Gβγ is significant and unchanged upon activation in HEK293 cells, thus showing that these proteins can localize into stable signaling complexes. To understand the basis for this stabilization, we carried out in vitro studies using a series of single-Cys mutants labeled with fluorescence tags and monitored their interaction with Gβγ subunits and changes in their fluorescence properties and accessibility upon activation and Gβγ binding. Our studies suggest a significant change in the orientation between G protein subunits upon activation that allows the G proteins to remain complexed while activating effectors. 相似文献
969.
Plasma membrane glutathione transporters and their roles in cell physiology and pathophysiology 总被引:2,自引:0,他引:2
Reduced glutathione (GSH) is critical for many cellular processes, and both its intracellular and extracellular concentrations are tightly regulated. Intracellular GSH levels are regulated by two main mechanisms: by adjusting the rates of synthesis and of export from cells. Some of the proteins responsible for GSH export from mammalian cells have recently been identified, and there is increasing evidence that these GSH exporters are multispecific and multifunctional, regulating a number of key biological processes. In particular, some of the multidrug resistance-associated proteins (Mrp/Abcc) appear to mediate GSH export and homeostasis. The Mrp proteins mediate not only GSH efflux, but they also export oxidized glutathione derivatives (e.g., glutathione disulfide (GSSG), S-nitrosoglutathione (GS-NO), and glutathione-metal complexes), as well as other glutathione S-conjugates. The ability to export both GSH and oxidized derivatives of GSH, endows these transporters with the capacity to directly regulate the cellular thiol-redox status, and therefore the ability to influence many key signaling and biochemical pathways. Among the many processes that are influenced by the GSH transporters are apoptosis, cell proliferation, and cell differentiation. This report summarizes the evidence that Mrps contribute to the regulation of cellular GSH levels and the thiol-redox state, and thus to the many biochemical processes that are influenced by this tripeptide. 相似文献
970.
Jingting Wang Parijat Sengupta Yuanjian Guo Urszula Golebiewska Suzanne Scarlata 《The Journal of biological chemistry》2009,284(25):16906-16913
It is well known that Gαi1(GDP) binds strongly to Gβγ subunits to form the Gαi1(GDP)-Gβγ heterotrimer, and that activation to Gαi1(GTP) results in conformational changes that reduces its affinity for Gβγ subunits. Previous studies of G protein subunit interactions have used stoichiometric amounts of the proteins. Here, we have found that Gαi1(GDP) can bind a second Gβγ subunit with an affinity only 10-fold weaker than the primary site and close to the affinity between activated Gαi1 and Gβγ subunits. Also, we find that phospholipase Cβ2, an effector of Gβγ, does not compete with the second binding site implying that effectors can be bound to the Gαi1(GDP)-(Gβγ)2 complex. Biophysical measurements and molecular docking studies suggest that this second site is distant from the primary one. A synthetic peptide having a sequence identical to the putative second binding site on Gαi1 competes with binding of the second Gβγ subunit. Injection of this peptide into cultured cells expressing eYFP-Gαi1(GDP) and eCFP-Gβγ reduces the overall association of the subunits suggesting this site is operative in cells. We propose that this second binding site serves to promote and stabilize G protein subunit interactions in the presence of competing cellular proteins.The plasma membranes of cells are organized as a series of protein-rich and lipid-rich domains (1–3). Many of the protein-rich domains, in particular those organized by caveolin proteins, are thought to be complexes of functionally related proteins that transduce extracellular signals (2). There is increasing evidence that heterotrimeric G proteins exist in pre-formed membrane complexes with their receptors and their intracellular effectors (4–8).The G protein signaling system is initiated when an extracellular agonist binds to its specific G protein-coupled receptor (for review see Refs. 9–12). The ligand-bound receptor will then catalyze the exchange of GTP for GDP on the Gα subunit in the G protein heterotrimer. In the basal state, Gα(GDP) binds strongly to Gβγ, but in the GTP-bound state this affinity is reduced, allowing Gα(GTP) and Gβγ subunits to individually bind to a host of specific intracellular enzymes and change their catalytic activity.Although the interactions between G protein subunits have been studied extensively in vitro, their behavior in cells may differ. For example, in pure or semi-pure systems, activation of Gα(GDP) sufficiently weakens its affinity for Gβγ resulting in dissociation (13). However, in cells separation of the heterotrimer is observed under some circumstances, but not others (7, 14–17). The reason for these differences in behavior is not clear. There are four families of Gα subunits that each contain several members, and, additionally, there are many subtypes of Gβγ subunits (18). It is possible that differences in dissociation behavior reflect differences in affinity between G protein subunit subtypes (19), the presence of various protein partners, and/or differences in post-synthetic modifications of the subunits (20).The mechanism that allows activated G proteins to remain bound is not apparent from the crystal structure (21, 22). If G protein subunits do not dissociate in cells, then their interaction must change in such a manner as to expose the effector interaction site(s). We have found that phospholipase Cβ1 (PLCβ1),4 an important effector of Gαq (23), is bound to Gαq prior to activation and throughout the activation cycle (6) implying that Gαq(GDP) interacts with PLCβ1 in a non-functional manner.We have evidence that signaling complexes are stabilized by a series of secondary interactions. Using purified proteins and model membranes, we have found that membranes of the Gαq-Gβγ/PLCβ1/RGS4 signaling system have secondary, weaker binding sites to members of this signaling system in addition to their high affinity site(s) to their functional partner(s). We speculate that secondary contacts allow for self-scaffolding of signaling proteins. To understand the nature of these secondary contacts, we have studied the ability of the Gαi1(GDP)-Gβγ heterotrimer to remain complexed through the activation cycle (24). Here, we present evidence that Gαi1(GDP) has two distinct Gβγ binding sites that only differ in affinity by an order of magnitude and may allow for continued association between the subunits upon activation. We also find that this site plays an important role in stabilizing G protein associations in cells and provides a mechanism of self-scaffolding. 相似文献