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胡艺川 《基因组学与应用生物学》2019,(3):1417-1422
乙醛脱氢酶2 (aldehyde dehydrogenase 2, ALDH2)是线粒体特异性酶,已被证明参与氧化应激诱导的细胞凋亡,而在心肌细胞中的作用知之甚少。本研究旨在通过用特异性ALDH2抑制剂大豆苷抑制ALDH2活性来研究ALDH2在抗霉素A诱导的心肌细胞凋亡中的作用。应用抗霉素A和大豆苷诱导小鼠心肌细胞,然后测定ALDH2酶活性、细胞内活性氧(reactive oxy gen species, ROS)含量和细胞凋亡,应用RT-PCR和蛋白质印迹法(Western blotting)检测ALDH2 m RNA和蛋白表达。结果表明,抗霉素A (40μg/mL)可诱导新生心肌细胞凋亡,而大豆苷(50μmol/L)能有效地抑制ALDH2活性而对细胞凋亡没有影响,并且可显著增强抗霉素A诱导的心肌细胞凋亡(53.72%~71.33%, p<0.05)。与单独用抗霉素A处理的细胞相比,抗霉素A和大豆苷共处理的心肌细胞中活化的丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)信号传导途径(p38-MAPK)的磷酸化也显著增加。本研究初步表明,改变线粒体ALDH2活性可能是减少氧化损伤诱导的心肌细胞凋亡的潜在选择。 相似文献
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下调c—erbB—2对细胞DNA修复和凋亡的影响 总被引:2,自引:0,他引:2
将有义和反义c-erbB-2逆转录病毒体分别经脂质体包裹后转染入人胚肺二倍体成纤维细胞(2BS)。Southern印迹杂交表明,外源c-erbB-2 cDNA在转染细胞中已成功整合入基因组中。Northern印迹杂交显示,有义转染细胞的erbB-2表达上升57%,反义转染细胞erbB-2表达下降48%。与对照和空载体转染细胞相比,反义转染细胞的DNA损伤修理能力显著下降,凋亡可诱导性降低。这和我们观察到的反义转染细胞提早出现衰老表型相一致。 相似文献
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Antonia L. Dow Tiffany V. Lin Elena H. Chartoff David Potter Donna L. McPhie Ashlee V. Van’t Veer Allison T. Knoll Kristen N. Lee Rachael L. Neve Tarun B. Patel Dost Ongur Bruce M. Cohen William A. Carlezon Jr 《PloS one》2015,10(3)
Both the development and relief of stress-related psychiatric conditions such as major depression (MD) and post-traumatic stress disorder (PTSD) have been linked to neuroplastic changes in the brain. One such change involves the birth of new neurons (neurogenesis), which occurs throughout adulthood within discrete areas of the mammalian brain, including the dorsal hippocampus (HIP). Stress can trigger MD and PTSD in humans, and there is considerable evidence that it can decrease HIP neurogenesis in laboratory animals. In contrast, antidepressant treatments increase HIP neurogenesis, and their efficacy is eliminated by ablation of this process. These findings have led to the working hypothesis that HIP neurogenesis serves as a biomarker of neuroplasticity and stress resistance. Here we report that local alterations in the expression of Sprouty2 (SPRY2), an intracellular inhibitor of growth factor function, produces profound effects on both HIP neurogenesis and behaviors that reflect sensitivity to stressors. Viral vector-mediated disruption of endogenous Sprouty2 function (via a dominant negative construct) within the dorsal HIP of adult rats stimulates neurogenesis and produces signs of stress resilience including enhanced extinction of conditioned fear. Conversely, viral vector-mediated elevation of SPRY2 expression intensifies the behavioral consequences of stress. Studies of these manipulations in HIP primary cultures indicate that SPRY2 negatively regulates fibroblast growth factor-2 (FGF2), which has been previously shown to produce antidepressant- and anxiolytic-like effects via actions in the HIP. Our findings strengthen the relationship between HIP plasticity and stress responsiveness, and identify a specific intracellular pathway that could be targeted to study and treat stress-related disorders. 相似文献
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Potentiated caspase-3 in Ras-transformed 10T1/2 cells 总被引:1,自引:0,他引:1
Song P Wei J Plummer H Wang HC 《Biochemical and biophysical research communications》2004,322(2):557-564
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Tingdong Tang Bin Zheng Sheng-hong Chen Anne N. Murphy Krystyna Kudlicka Huilin Zhou Marilyn G. Farquhar 《The Journal of biological chemistry》2009,284(8):5414-5424
Mitochondria are dynamic organelles that play key roles in metabolism,
energy production, and apoptosis. Coordination of these processes is essential
to maintain normal cellular functions. Here we characterized hNOA1, the human
homologue of AtNOA1 (Arabidopsis thaliana nitric oxide-associated
protein 1), a large mitochondrial GTPase. By immunofluorescence,
immunoelectron microscopy, and mitochondrial subfractionation, endogenous
hNOA1 is localized within mitochondria where it is peripherally associated
with the inner mitochondrial membrane facing the mitochondrial matrix.
Overexpression and knockdown of hNOA1 led to changes in mitochondrial shape
implying effects on mitochondrial dynamics. To identify the interaction
partners of hNOA1 and to further understand its cellular functions, we
performed immunoprecipitation-mass spectrometry analysis of endogenous hNOA1
from enriched mitochondrial fractions and found that hNOA1 interacts with both
Complex I of the electron transport chain and DAP3
(death-associated protein 3), a positive
regulator of apoptosis. Knockdown of hNOA1 reduces mitochondrial O2
consumption ∼20% in a Complex I-dependent manner, supporting a functional
link between hNOA1 and Complex I. Moreover, knockdown of hNOA1 renders cells
more resistant to apoptotic stimuli such as γ-interferon and
staurosporine, supporting a role for hNOA1 in regulating apoptosis. Thus,
based on its interactions with both Complex I and DAP3, hNOA1 may play a role
in mitochondrial respiration and apoptosis.Emerging evidence indicates that mitochondrial metabolism, apoptosis, and
dynamics (fission and fusion) are closely intertwined. Apoptosis and changes
in metabolism are associated with morphological changes in mitochondria
(1,
2). Conversely, when
mitochondrial morphology is altered either by mutations or altered expression
of mitochondrial fission or fusion proteins such as the dynamin like large G
proteins Drp1 and Opa1, the cell''s susceptibility to apoptotic agents
(3) or ability to generate ATP
(4,
5) is altered.Apoptosis is controlled by a diverse range of cell signals, which may
originate either extracellularly (extrinsic inducers) or intracellularly
(intrinsic inducers), and mitochondria play central roles in both pathways
(6). The apoptotic pathways
involve a growing list of mitochondria-associated proteins, such as Bad,
cytochrome c, Smac, AIF, Bcl-2, and others, most of which are located
either on the outer mitochondrial membrane
(OMM)3 or in the
intermembrane space (IMS) (7).
Recently, proteins of the mitochondrial matrix such as DAP3, have also been
shown to be involved in apoptosis
(8). DAP3 has been reported to
be involved in both γ-interferon-
(9) and tumor necrosis
factor-α-induced (10)
apoptosis as well as staurosporine-induced mitochondrial fragmentation
(11), but the detailed
mechanisms involved remain to be elucidated.Besides their role in apoptosis, much more is known about the functions of
mitochondria in respiration and generation of ATP. The electron transport
chain in the inner mitochondrial membrane (IMM) contains four major enzyme
complexes (Complexes I, II, III, and IV) that are involved in transferring
electrons from NADH (Complex I-linked) or FADH2 (Complex II-linked) to
O2 and in pumping protons out of the matrix to create an
electrochemical proton gradient, which is harnessed by ATP synthase to make
ATP (12).Despite the accumulating evidence showing intercommunication between
mitochondrial metabolism, apoptosis, and dynamics, how these processes are
coordinated remains to be elucidated. In this study we characterize hNOA1, the
human homologue of Arabidopsis thaliana nitric oxide-associated
protein, 1 (AtNOA1) (13).
hNOA1 is a large G protein closely related to dynamin that is associated with
the IMM. Perturbation of hNOA1 affects mitochondrial morphology, Complex
I-linked O2 consumption, and the cell''s susceptibility to apoptotic
stimuli, possibly through interactions with proteins such as Complex I and
DAP3. 相似文献
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John M. Harrington Sawyer Howell Stephen L. Hajduk 《The Journal of biological chemistry》2009,284(20):13505-13512
Trypanosome lytic factor (TLF) is a subclass of human high density
lipoprotein (HDL) that mediates an innate immune killing of certain mammalian
trypanosomes, most notably Trypanosoma brucei brucei, the causative
agent of a wasting disease in cattle. Mechanistically, killing is initiated in
the lysosome of the target trypanosome where the acidic pH facilitates a
membrane-disrupting activity by TLF. Here we utilize a model liposome system
to characterize the membrane binding and permeabilizing activity of TLF and
its protein constituents, haptoglobin-related protein (Hpr), apolipoprotein
L-1 (apoL-1), and apolipoprotein A-1 (apoA-1). We show that TLF efficiently
binds and permeabilizes unilamellar liposomes at lysosomal pH, whereas
non-lytic human HDL exhibits inefficient permeabilizing activity. Purified,
delipidated Hpr and apoL-1 both efficiently permeabilize lipid bilayers at low
pH. Trypanosome lytic factor, apoL-1, and apoA-1 exhibit specificity for
anionic membranes, whereas Hpr permeabilizes both anionic and zwitterionic
membranes. Analysis of the relative particle sizes of susceptible liposomes
reveals distinctly different membrane-active behavior for native TLF and the
delipidated protein components. We propose that lysosomal membrane damage in
TLF-susceptible trypanosomes is initiated by the stable association of the TLF
particle with the lysosomal membrane and that this is a property unique to
this subclass of human HDL.High density lipoproteins
(HDL)2 are complex yet
ordered macromolecules consisting of characteristic proteins embedded in a
phospholipid monolayer that surrounds a hydrophobic core of esterified
cholesterol and triglycerides. A subclass of HDL is responsible for an innate
immune killing of the African blood stream parasite Trypanosoma brucei
brucei
(1–3),
and very recently, has been shown to be cytotoxic to intracellular
Leishmania promastigotes
(4). The trypanolytic HDL
particle, termed trypanosome lytic factor (TLF), is characterized by the
presence of two proteins, apolipoprotein L-1 (apoL-1) and haptoglobin-related
protein (Hpr), as well as the HDL ubiquitous apolipoprotein A-1 (apoA-1)
(1,
5–7).
Killing of the susceptible parasite involves high affinity binding to a
cell-surface receptor, endocytosis, and trafficking of the TLF particle to the
lysosome
(8–12).
The acidic lysosomal environment facilitates a membrane-disrupting activity by
the TLF particle and subsequent cell death
(9,
13). It has been shown that
purified, delipidated apoL-1 or Hpr are sufficient for trypanosome killing.
When these proteins are incorporated into the same lipoprotein particle, a
several hundredfold increase in killing activity is exhibited
(5). In addition,
Molina-Portela et al.
(14) show that maximal
protection against T. b. brucei in a transgenic mouse model requires
the expression of human Hpr, apoL-1, and apoA-1, supporting a synergistic mode
of action.Haptoglobin-related protein evolved during primate evolution and is
restricted to apes, old world monkeys, and humans
(15). Haptoglobin-related
protein is highly similar (92%) to the acute phase serum protein haptoglobin
(Hp) (16). All mammals use Hp
as a scavenger of hemoglobin (Hb) released during hemolysis associated with
infection or trauma. Haptoglobin binds cell-free Hb with high affinity and
facilitates its removal from the circulation through a receptor-mediated
process in the liver (17).
Like Hp, Hpr binds free Hb, yet this Hpr·Hb complex is not recognized
by the requisite receptors in mammals and is thus not removed from the
circulation (18). TLF uptake
by susceptible trypanosomes requires specific binding to an Hpr·Hb
complex that facilitates trafficking of the TLF particle to the lysosome
(10). It has been proposed
that once inside the lysosomal compartment, Hpr·Hb contributes directly
to membrane disruption through the generation of oxygen radicals with the
bound Hb providing the iron necessary for Fenton chemistry
(7,
10,
19).Apolipoprotein L-1 is a unique member of the apolipoprotein L protein
family in that, unlike the remaining apoL proteins, it possesses an N-terminal
signal sequence and is thus secreted from cells. As is the case for Hpr,
apoL-1 appeared during primate evolution
(20–22).
Within the circulation of primates, apoL-1 is exclusively associated with HDL,
and the majority of the protein is in the TLF subclass
(5). The apoL family members
are all predicted to adopt amphipathic α-helical conformations,
suggesting that their physiological role involves membrane interaction
(20). Apolipoprotein L-1
shares limited homology with channel-forming colicins and, consistent with
this observation, has been shown to function as an ion channel when
incorporated into lipid bilayers
(23).The ultimate fate of TLF-targeted lysosomal membranes is not firmly
established. Several studies employing both in vivo cellular analysis
and artificial membrane systems address this point with conflicting results.
Electron microscopy studies with gold-conjugated TLF revealed accumulation of
TLF in intracellular vesicles and subsequent vesicle membrane breakdown and
appearance of gold particles in the cytoplasm
(9). Widener et al.
(10) observed efflux of
lysosomally localized large molecular mass dextrans (500 kDa) in TLF-treated
T. b. brucei. These data suggest that the lysosomal membrane
experiences large scale disruption. In contrast, Perez-Morga et al.
(23) and Vanhollebeke et
al. (24) report
uncontrollable lysosomal swelling in susceptible trypanosomes treated with
normal human serum, suggesting stability of the lamellar structure of the
lysosomal membrane after TLF attack. Swelling is attributed to apoL-1-mediated
influx of Cl– ions and concomitant osmotic flow of water into
the lysosome. However, Molina-Portela et al.
(25) observed the formation of
cation-selective pores in TLF-treated planar lipid bilayers composed of
trypanosome lipids. The diversity of activities reported for TLF and normal
human serum may reflect the packaging of multiple toxins within the same
complex that can act synergistically to provide optimal killing activity
(5,
14).Here we utilize model liposomes to monitor the membrane activity of TLF and
its protein constituents. We describe the effects of TLF, delipidated Hpr,
apoL-1, and apoA-1 on the permeability of unilamellar liposomes. Additionally,
we show that TLF, apoL-1, and apoA-1 exhibit lipid specificity and that Hpr,
apoL-1, and apoA-1 induce large scale changes in the geometry of liposomes.
These results provide a molecular basis for the recognition of lysosomal
membranes by this toxic HDL and support a multicomponent mechanism for
trypanosome killing. 相似文献
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Zemfira Karamysheva Laura A. Diaz-Martinez Sara E. Crow Bing Li Hongtao Yu 《The Journal of biological chemistry》2009,284(3):1772-1780
Shugoshin 1 (Sgo1) protects centromeric sister-chromatid cohesion in early
mitosis and, thus, prevents premature sister-chromatid separation. The protein
level of Sgo1 is regulated during the cell cycle; it peaks in mitosis and is
down-regulated in G1/S. Here we show that Sgo1 is degraded during
the exit from mitosis, and its degradation depends on the anaphase-promoting
complex/cyclosome (APC/C). Overexpression of Cdh1 reduces the protein levels
of ectopically expressed Sgo1 in human cells. Sgo1 is ubiquitinated by APC/C
bound to Cdh1 (APC/CCdh1) in vitro. We have further
identified two functional degradation motifs in Sgo1; that is, a KEN
(Lys-Glu-Asn) box and a destruction box (D box). Although removal of either
motif is not sufficient to stabilize Sgo1, Sgo1 with both KEN box and D box
deleted is stable in cells. Surprisingly, mitosis progresses normally in the
presence of non-degradable Sgo1, indicating that degradation of Sgo1 is not
required for sister-chromatid separation or mitotic exit. Finally, we show
that the spindle checkpoint kinase Bub1 contributes to the maintenance of Sgo1
steady-state protein levels in an APC/C-independent mechanism.Loss of sister-chromatid cohesion triggers chromosome segregation in
mitosis and occurs in two steps in vertebrate cells
(1-3).
In prophase, cohesin is phosphorylated by mitotic kinases including Plk1 and
removed from chromosome arms
(1,
4). Then, cleavage of
centromeric cohesin by separase takes place at the metaphase-to-anaphase
transition to allow sister-chromatid separation
(5). The shugoshin (Sgo) family
of proteins plays an important role in the protection of centromeric cohesion
(6,
7). Human cells depleted of
Sgo1 by RNAi undergo massive chromosome missegregation
(8-11).
In cells with compromised Sgo1 function, centromeric cohesin is improperly
phosphorylated and removed (4,
11), resulting in premature
sister-chromatid separation. It has been shown recently that Sgo1 collaborates
with PP2A to counteract the action of Plk1 and other mitotic kinases and to
protect centromeric cohesin from premature removal
(12-14).
In addition, Sgo1 has also been shown to promote stable
kinetochore-microtubule attachment and sense tension across sister
kinetochores (8,
15). Thus, Sgo1 is crucial for
mitotic progression and chromosome segregation.Orderly progression through mitosis is regulated by the anaphase-promoting
complex/cyclosome
(APC/C),2 a large
multiprotein ubiquitin ligase that targets key mitotic regulators for
destruction by the proteasome
(16). APC/C selects substrates
for ubiquitination by using the Cdc20 or Cdh1 activator proteins to recognize
specific sequences called APC/C degrons within target proteins
(17). Several APC/C degrons
have been characterized, including the destruction box (D box) and the
Lys-Glu-Asn box (KEN box) (18,
19). The D box, with the
consensus amino acid sequence of RXXLXXXN(X
indicates any amino acid), are found in many APC/C substrates, including
mitotic cyclins and are essential for their ubiquitin-mediated destruction.
The KEN box, which contains a consensus KEN motif, is also found in several
APC/C substrates and is preferentially but not exclusively recognized by
APC/CCdh1. When APC/C is active, it directs progression through and
exit from mitosis by catalyzing the ubiquitination and timely destruction of
mitotic regulators, including cyclin A, cyclin B, and the separase inhibitor
securin (16). The APC/C
activity needs to be tightly controlled to prevent unscheduled substrate
degradation. An important mechanism for APC/C regulation is the spindle
checkpoint, which prevents the activation of APC/C and destruction of its
substrates in response to kinetochores that have not properly attached to the
mitotic spindle (20).Recent evidence shows that Sgo1 is a substrate of APC/C, and its protein
levels oscillate during the cell cycle
(8,
9). In this article we study
the degradation of Sgo1 in human cells. We show that Sgo1 is degraded during
mitotic exit, and this degradation depends on APC/CCdh1. We further
show that both KEN and D boxes are required for Sgo1 degradation in
vivo and ubiquitination in vitro. Removal of these motifs
stabilizes Sgo1 in vivo. The prolonged presence of stable Sgo1
protein in human cells does not change the kinetics of chromosome segregation
and mitotic exit. Therefore, a timely scheduled degradation of Sgo1 takes
place but is not required for mitotic exit. Finally, we show that Bub1
regulates Sgo1 protein levels through a mechanism that does not involve
APC/C-mediated degradation. 相似文献
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Balaji Parameswaran Huai-Chin Chiang Yunzhe Lu Julia Coates Chu-Xia Deng Richard Baer Hui-Kuan Lin Rong Li Tanya T Paull Yanfen Hu 《Cell cycle (Georgetown, Tex.)》2015,14(3):437-448
The BRCA1 tumor suppressor plays an important role in homologous recombination
(HR)-mediated DNA double-strand-break (DSB) repair. BRCA1 is phosphorylated by Chk2 kinase
upon γ-irradiation, but the role of Chk2 phosphorylation is not understood. Here, we
report that abrogation of Chk2 phosphorylation on BRCA1 delays end resection and the
dispersion of BRCA1 from DSBs but does not affect the assembly of Mre11/Rad50/NBS1 (MRN)
and CtIP at DSBs. Moreover, we show that BRCA1 is ubiquitinated by SCFSkp2 and
that abrogation of Chk2 phosphorylation impairs its ubiquitination. Our study suggests
that BRCA1 is more than a scaffold protein to assemble HR repair proteins at DSBs, but
that Chk2 phosphorylation of BRCA1 also serves as a built-in clock for HR repair of DSBs.
BRCA1 is known to inhibit Mre11 nuclease activity. SCFSkp2 activity appears at
late G1 and peaks at S/G2, and is known to ubiquitinate phosphodegron motifs. The removal
of BRCA1 from DSBs by SCFSkp2-mediated degradation terminates BRCA1-mediated
inhibition of Mre11 nuclease activity, allowing for end resection and restricting the
initiation of HR to the S/G2 phases of the cell cycle. 相似文献