共查询到20条相似文献,搜索用时 15 毫秒
1.
Stuart Smith Andrzej Witkowski Ayesha Moghul Yuko Yoshinaga Michael Nefedov Pieter de Jong Dejiang Feng Loren Fong Yiping Tu Yan Hu Stephen G. Young Thomas Pham Carling Cheung Shana M. Katzman Martin D. Brand Casey L. Quinlan Marcel Fens Frans Kuypers Stephanie Misquitta Stephen M. Griffey Son Tran Afshin Gharib Jens Knudsen Hans Kristian Hannibal-Bach Grace Wang Sandra Larkin Jennifer Thweatt Saloni Pasta 《PloS one》2012,7(10)
A mouse model with compromised mitochondrial fatty acid synthesis has been engineered in order to assess the role of this pathway in mitochondrial function and overall health. Reduction in the expression of mitochondrial malonyl CoA-acyl carrier protein transacylase, a key enzyme in the pathway encoded by the nuclear Mcat gene, was achieved to varying extents in all examined tissues employing tamoxifen-inducible Cre-lox technology. Although affected mice consumed more food than control animals, they failed to gain weight, were less physically active, suffered from loss of white adipose tissue, reduced muscle strength, kyphosis, alopecia, hypothermia and shortened lifespan. The Mcat-deficient phenotype is attributed primarily to reduced synthesis, in several tissues, of the octanoyl precursors required for the posttranslational lipoylation of pyruvate and α-ketoglutarate dehydrogenase complexes, resulting in diminished capacity of the citric acid cycle and disruption of energy metabolism. The presence of an alternative lipoylation pathway that utilizes exogenous free lipoate appears restricted to liver and alone is insufficient for preservation of normal energy metabolism. Thus, de novo synthesis of precursors for the protein lipoylation pathway plays a vital role in maintenance of mitochondrial function and overall vigor. 相似文献
2.
3.
Dmitry Kryndushkin Natalia Pripuzova Barrington G. Burnett Frank Shewmaker 《The Journal of biological chemistry》2013,288(38):27100-27111
The formation of amyloid aggregates is implicated both as a primary cause of cellular degeneration in multiple human diseases and as a functional mechanism for providing extraordinary strength to large protein assemblies. The recent identification and characterization of several amyloid proteins from diverse organisms argues that the amyloid phenomenon is widespread in nature. Yet identifying new amyloid-forming proteins usually requires a priori knowledge of specific candidates. Amyloid fibers can resist heat, pressure, proteolysis, and denaturation by reagents such as urea or sodium dodecyl sulfate. Here we show that these properties can be exploited to identify naturally occurring amyloid-forming proteins directly from cell lysates. This proteomic-based approach utilizes a novel purification of amyloid aggregates followed by identification by mass spectrometry without the requirement for special genetic tools. We have validated this technique by blind identification of three amyloid-based yeast prions from laboratory and wild strains and disease-related polyglutamine proteins expressed in both yeast and mammalian cells. Furthermore, we found that polyglutamine aggregates specifically recruit some stress granule components, revealing a possible mechanism of toxicity. Therefore, core amyloid-forming proteins as well as strongly associated proteins can be identified directly from cells of diverse origin. 相似文献
4.
Function of GRIM-19, a Mitochondrial Respiratory Chain Complex I Protein, in Innate Immunity 总被引:1,自引:0,他引:1
Chen Y Lu H Liu Q Huang G Lim CP Zhang L Hao A Cao X 《The Journal of biological chemistry》2012,287(32):27227-27235
Mitochondria respiratory chain (RC), consisting of five multisubunit complexes, is crucial for cellular energy production, reactive oxygen species generation, and regulation of apoptosis. Recently, a few mitochondrial proteins have been reported to be essential for innate immunity, but the function of mitochondrial RC in innate immunity is largely unknown. By knock-out of GRIM-19, a newly identified subunit protein of mitochondrial complex I, in mice, we found that heterogeneous mice (GRIM-19(+/-)) are prone to spontaneous urinary tract infection, mostly by Staphylococcus saprophyticus. Macrophages derived from these mice have compromised mitochondrial complex I activity and increased reactive oxygen species level. Bacterial infection induces a rapid up-regulation of GRIM-19 and complex I activity in the wild-type macrophages, but both are reduced in the macrophages from GRIM-19(+/-) mice. These cells also have decreased intracellular killing ability against S. saprophyticus. The defects for this probably occur in the fusion of bacteria to lysosome, but not in the bacterial engulfment and macrophage migration. In addition, production of proinflammatory cytokines, such as interleukin (IL)-1, IL-12, IL-6, and interferon (IFN)-γ, induced by both bacterial infection and lipopolysaccharide (LPS) and monodansylcadaverine treatment, is also decreased in the GRIM19(+/-) macrophages. Inhibition of mitochondrial RC activity by inhibitors shows a similar reduction on the cytokine production. Due to low cytokine production, the inflammatory response caused by in vivo bacterial challenge in the bladders of GRIM-19(+/-) mice is compromised. This study provides genetic evidence for a critical role of mitochondrial RC in innate immunity. 相似文献
5.
Anne Chomyn 《Journal of bioenergetics and biomembranes》2001,33(3):251-257
Sixteen years ago, we demonstrated, by immunological and biochemical approaches, that seven subunits of complex I are encoded in mitochondrial DNA (mtDNA) and synthesized on mitochondrial ribosomes in mammalian cells. More recently, we carried out a biochemical, molecular, and cellular analysis of a mutation in the gene for one of these subunits, ND4, that causes Leber's hereditary optic neuropathy (LHON). We demonstrated that, in cells carrying this mutation, the mtDNA-encoded subunits of complex I are assembled into a complex, but the rate of complex I-dependent respiration is decreased. Subsequently, we isolated several mutants affected in one or another of the mtDNA-encoded subunits of complex I by exposing established cell lines to high concentrations of rotenone. Our analyses of these mtDNA mutations affecting subunits of complex I have shown that at least two of these subunits, ND4 and ND6, are essential for the assembly of the enzyme. ND5 appears to be located at the periphery of the enzyme and, while it is not essential for assembly of the other mtDNA-encoded subunits into a complex, it is essential for complex I activity. In fact, the synthesis of the ND5 polypeptide is rate limiting for the activity of the enzyme. 相似文献
6.
Tara R. Richman Judith A. Ermer Stefan M. K. Davies Kara L. Perks Helena M. Viola Anne-Marie J. Shearwood Livia C. Hool Oliver Rackham Aleksandra Filipovska 《PLoS genetics》2015,11(3)
The evolutionary divergence of mitochondrial ribosomes from their bacterial and cytoplasmic ancestors has resulted in reduced RNA content and the acquisition of mitochondria-specific proteins. The mitochondrial ribosomal protein of the small subunit 34 (MRPS34) is a mitochondria-specific ribosomal protein found only in chordates, whose function we investigated in mice carrying a homozygous mutation in the nuclear gene encoding this protein. The Mrps34 mutation causes a significant decrease of this protein, which we show is required for the stability of the 12S rRNA, the small ribosomal subunit and actively translating ribosomes. The synthesis of all 13 mitochondrially-encoded polypeptides is compromised in the mutant mice, resulting in reduced levels of mitochondrial proteins and complexes, which leads to decreased oxygen consumption and respiratory complex activity. The Mrps34 mutation causes tissue-specific molecular changes that result in heterogeneous pathology involving alterations in fractional shortening of the heart and pronounced liver dysfunction that is exacerbated with age. The defects in mitochondrial protein synthesis in the mutant mice are caused by destabilization of the small ribosomal subunit that affects the stability of the mitochondrial ribosome with age. 相似文献
7.
Oxa1 is a mitochondrial inner membrane protein with a predicted
five-transmembrane segment (TM1∼5) topology in which the N terminus and a
hydrophilic loop, L2, are exposed to the intermembrane space and the
C-terminal region and two loops, L1 and L3, are exposed to the matrix. Oxa1
mediates the insertion of mitochondrial DNA-encoded subunits of respiratory
complexes and several nuclear DNA-encoded proteins into the inner membrane
from the matrix. Compared with yeast Oxa1, little is known about the import
and function of mammalian Oxa1. Here, we investigated the topogenesis of Oxa1
in HeLa cells using systematic deletion or mutation constructs and found that
(i) the N-terminal 64-residue segment formed a presequence, and its deletion
directed the mature protein to the endoplasmic reticulum, indicating that the
presequence arrests cotranslational activation of the potential endoplasmic
reticulum-targeting signal within mature Oxa1, (ii) systematic deletion of
Oxa1 TM segments revealed that the presence of all five TMs is essential for
efficient membrane integration, (iii) the species-conserved hexapeptide
(GLPWWG) located near the N terminus of TM1 was essential for export of the
N-terminal segment and L2 into the intermembrane space from the matrix,
i.e. for correct topogenesis of Oxa1, and (iv) GLPWWG placed near the
N terminus of TM2 or TM3 in the reporter construct also supported its membrane
integration in the Nout-Cin orientation. Together, these results demonstrated
that topogenesis of Oxa1 is a cooperative event of all five TMs, and GLPWWG
followed immediately by TM1 is essential for correct Oxa1 topogenesis.Most mitochondrial proteins are nuclear DNA-coded, and their import into
mitochondrial compartments, that is, the mitochondrial outer membrane
(MOM),3 mitochondrial
inner membrane (MIM), intermembrane space (IMS), and matrix, is mediated by
five protein translocation systems: translocase of the outer membrane (TOM
complex), sorting and assembly machinery of MOM (SAM/TOB), translocases of the
inner membrane (TIM23 complex and TIM22 complex), and a fifth system in the
MIM that mediates integration of proteins from the matrix into the MIM
(1,
2). The last system, which has
been analyzed in detail in yeast, requires a membrane potential across the MIM
and matrix ATP and mediates MIM integration of the mtDNA-encoded proteins as
well as the integration of certain nuclear DNA-encoded proteins considered to
be of bacterial origin, such as cytochrome c oxidase subunit II,
F1Fo-ATPase subunit 9, and Oxa1
(3–5).
Translocation efficiency is affected by the charge difference across the
transmembrane (TM) in accordance with the positive-inside rule
(5). Furthermore, the
matrix-exposed C-terminal segment of Oxa1 is essential for binding
mitochondrial ribosomes during cotranslational integration of mtDNA-encoded
proteins (6,
7). Recent reports further
demonstrated that the MIM protein Mba1, as a ribosome receptor, cooperates
with the C-terminal ribosome binding segment of Oxa1
(8). The machinery and the
underlying mechanisms of MIM insertion from the matrix must be further
analyzed.Oxa1 protein, originally identified in yeast, is a component of the
matrix-to-MIM export system conserved from prokaryote to eukaryote and is
involved in Oxa1 biogenesis
(9–14).
YidC, a bacterial homologue of Oxa1, is involved in the biogenesis of inner
membrane proteins in a Sec-dependent or Sec-independent manner
(15,
16). In yeast, IMS export from
the matrix of the Oxa1 N-terminal segment emerging from the Tim23 channel
requires a membrane potential
(4,
17), and the export is
compromised in mitochondria isolated from a temperature-sensitive
Oxa1-expressing strain at a non-permissive temperature
(12). Herrmann and Bonnefoy
(18) reported that Oxa1
protein functions in the export of a single hydrophilic loop region that was
artificially produced by ligating the C-terminal region of cytochrome
b with cytochrome c oxidase subunit II and placed between TM
segments. Direct interaction of Oxa1 with an immature subunit in complex V was
observed during its biogenesis
(19). So far, these studies
have only been performed in yeast, and no information is available on the
mechanism of topogenesis in mammals with regard to how Oxa1 is involved in the
export of multiple regions in a protein molecule. Our in vivo study
revealed that the correct topogenesis of Oxa1 in the MIM proceeds as a result
of the cooperation of all five TMs and that the cooperation of TM1 and the
species-conserved six-residue segment (GLPWWG) in the N-terminal flanking
region is essential for export from the matrix of both the N-terminal segment
and hydrophilic L2 into the IMS. 相似文献
8.
Michael A. Gitcho Jeffrey Strider Deborah Carter Lisa Taylor-Reinwald Mark S. Forman Alison M. Goate Nigel J. Cairns 《The Journal of biological chemistry》2009,284(18):12384-12398
Frontotemporal lobar degeneration (FTLD) with inclusion body myopathy and
Paget disease of bone is a rare, autosomal dominant disorder caused by
mutations in the VCP (valosin-containing protein) gene. The disease
is characterized neuropathologically by frontal and temporal lobar atrophy,
neuron loss and gliosis, and ubiquitin-positive inclusions (FTLD-U), which are
distinct from those seen in other sporadic and familial FTLD-U entities. The
major component of the ubiquitinated inclusions of FTLD with VCP
mutation is TDP-43 (TAR DNA-binding protein of 43 kDa). TDP-43 proteinopathy
links sporadic amyotrophic lateral sclerosis, sporadic FTLD-U, and most
familial forms of FTLD-U. Understanding the relationship between individual
gene defects and pathologic TDP-43 will facilitate the characterization of the
mechanisms leading to neurodegeneration. Using cell culture models, we have
investigated the role of mutant VCP in intracellular trafficking,
proteasomal function, and cell death and demonstrate that mutations in the
VCP gene 1) alter localization of TDP-43 between the nucleus and
cytosol, 2) decrease proteasome activity, 3) induce endoplasmic reticulum
stress, 4) increase markers of apoptosis, and 5) impair cell viability. These
results suggest that VCP mutation-induced neurodegeneration is
mediated by several mechanisms.Frontotemporal lobar degeneration
(FTLD)2
accounts for 10% of all late onset dementias and is the third most frequent
neurodegenerative disease after Alzheimer disease and dementia with Lewy
bodies (1). FTLD with
ubiquitin-immunoreactive inclusions is genetically, clinically, and
neuropathologically heterogeneous
(2,
3). FTLD-U comprises several
distinct entities, including sporadic forms and familial cases caused by
mutations in the genes encoding VCP (valosin-containing protein), GRN
(progranulin), CHMP2B (charged multivesicular body protein 2B), TDP-43 (TAR
DNA-binding protein of 43 kDa) and an unknown gene linked to chromosome 9
(2,
3). Frontotemporal dementia
with inclusion body myopathy and Paget disease of bone is a rare, autosomal
dominant disorder caused by mutations in the VCP gene located on
chromosome 9p13-p12
(4-10)
(Fig. 1). This multisystem
disease is characterized by progressive muscle weakness and atrophy, increased
osteoclastic bone resorption, and early onset frontotemporal dementia, also
called FTLD (9,
11). Mutations in VCP
are also associated with dilatative cardiomyopathy with ubiquitin-positive
inclusions (12).
Neuropathologic features of FTLD with VCP mutation include frontal
and temporal lobar atrophy, neuron loss and gliosis, and ubiquitin-positive
inclusions (FTLD-U). The majority of aggregates are ubiquitin- and
TDP-43-positive neuronal intranuclear inclusions (NIIs); a smaller proportion
is made up of TDP-43-immunoreactive dystrophic neurites (DNs) and neuronal
cytoplasmic inclusions (NCIs). A small number of inclusions are
VCP-immunoreactive (5,
13). Pathologic TDP-43 in
inclusions links a spectrum of diseases in which TDP-43 pathology is a primary
feature, including FTLD-U, motor neuron disease, including amyotrophic lateral
sclerosis, FTLD with motor neuron disease, and inclusion body myopathy and
Paget disease of bone, as well as an expanding spectrum of other disorders in
which TDP-43 pathology is secondary
(14,
15).Open in a separate windowFIGURE 1.Model of pathogenic mutations and domains in valosin-containing
protein. CDC48 (magenta), located within the N terminus (residues
22-108), binds the following cofactors: p47, gp78, and Npl4-Ufd1
(23-25,
28). There are two AAA-ATPase
domains (AAA; blue) at residues 240-283 and 516-569, which
are joined by two linker regions (L1 and L2;
red).TDP-43 proteinopathy in FTLD with VCP mutation has a biochemical
signature similar to that seen in other sporadic and familial cases of FTLD-U,
including sporadic amyotrophic lateral sclerosis, FTLD-motor neuron disease,
FTLD with progranulin (GRN) mutation, and FTLD linked to chromosome
9p (3,
16). TDP-43 proteinopathy in
these disorders is characterized by hyperphosphorylation of TDP-43,
ubiquitination, and cleavage to form C-terminal fragments detected only in
insoluble brain extracts from affected brain regions
(16). Identification of TDP-43
as the major component of the ubiquitin-immunoreactive inclusions of FTLD with
VCP mutation supports the hypothesis that VCP gene mutations
cause an alteration of VCP function, leading to TDP-43 proteinopathy.VCP/p97 (valosin-containing protein) is a member of the AAA (ATPase
associated with diverse cellular activities) superfamily. The N-terminal
domain of VCP has been shown to be involved in cofactor binding (CDC48 (cell
division cycle protein 48)) and two AAA-ATPase domains that form a hexameric
complex (Fig. 1)
(17). Recently, it has been
shown that the N-terminal domain of VCP binds phosphoinositides
(18,
19). AKT (activated
serine-threonine protein kinase) phosphorylates VCP and is required for
constitutive VCP function (20,
21). AKT is activated through
phospholipid binding and phosphorylation via the phosphoinositide 3-kinase
signaling pathway, which is involved in cell survival
(22). The lipid binding domain
may recruit VCP to the cell membrane where it is phosphorylated by AKT
(19).The diversity of VCP functions is modulated, in part, by a variety of
intracellular cofactors, including p47, gp78, and Npl4-Ufd1
(23). Cofactor p47 has been
shown to play a role in the maintenance and biogenesis of both the endoplasmic
reticulum (ER) and Golgi apparatus
(24). The structure of p47
contains a ubiquitin regulatory X domain that binds the N-terminus of VCP, and
together they act as a chaperone to deliver membrane fusion machinery to the
site of adjacent membranes
(25). The function of the
p47-VCP complex is dependent upon cell division cycle 2 (CDC2)
serine-threonine kinase phosphorylation of p47
(26,
27). Also, VCP has been found
to interact with the cytosolic tail of gp78, an ER membrane-spanning E3
ubiquitin ligase that exclusively binds VCP and enhances ER-associated
degradation (ERAD) (28). The
Npl4-Ufd1-VCP complex is involved in nuclear envelope assembly and targeting
of proteins through the ubiquitin-proteasome system
(29,
30). The cell survival
response of this complex has been found to be important in DNA damage repair
though activation by phosphorylation and its recruitment to double-stranded
breaks (20,
31). The Npl4-Ufd1-VCP
cytosolic complex is also recruited to the ER membrane, interacting with
Derlin 1, VCP-interacting membrane proteins (VIMP), and other complexes. At
the ER membrane, these misfolded proteins are targeted to the proteasome via
ERAD
(32-34).
VCP also targets IKKβ for ubiquitination to the ubiquitin-proteasome
system, implicating VCP in the cell survival pathway and neuroprotection
(21,
35-37).To investigate the mechanism of neurodegeneration caused by VCP
mutations, we first tested the hypothesis that VCP mutations decrease
cell viability in vitro using a neuroblastoma SHSY-5Y cell line and
then investigated cellular pathways that are known to lead to
neurodegeneration, including decrease in proteasome activity, caspase-mediated
degeneration, and a change in cellular localization of TDP-43. 相似文献
9.
The addition of rotenone (inhibitor of respiratory complex I), 3-nitropropionic acid (complex II inhibitor), harmine (inhibitor
of complexes I and II) and cyclosporin A (CsA, an inhibitor of the mitochondrial permeability transition) reduced the nuclear
damage, loss in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3,
increase in the formation of reactive oxygen species and depletion of GSH in differentiated PC12 cells treated with MG132,
a proteasome inhibitor. Meanwhile, rotenone, 3-nitropropionic acid and harmine did not affect the inhibitory effect of CsA
or trifluoperazine (an inhibitor of the mitochondrial permeability transition and calmodulin antagonist) on the cytotoxicity
of MG132. The results suggest that proteasome inhibition-induced mitochondrial dysfunction and cell injury may be attenuated
by the inhibitions of respiratory chain complex I and II. The cytoprotective effect of the mitochondrial permeability transition
prevention not appears to be modulated by respiratory complex inhibition. 相似文献
10.
11.
Abstract: The catecholaminergic neurotoxin 6-hydroxydopamine causes parkinsonian symptoms in animals and it has been proposed that reactive oxygen species and oxidative stress, enhanced by iron, may play a key role in its toxicity. The present results demonstrate that 6-hydroxydopamine reversibly inhibits complex I (NADH dehydrogenase) of brain mitochondrial respiratory chain in isolated mitochondria. 6-Hydroxydopamine itself, rather than its oxidative products, was responsible for the inhibition. Iron(III) did not enhance inhibition but decreased it by stimulating the nonenzyme oxidation of 6-hydroxydopamine. Inhibition was potentiated to some extent by calcium ion. Desferrioxamine protected complex I activity against the inhibition, but it was not due to its chelator or antioxidative properties. Desferrioxamine was also shown to activate NADH dehydrogenase in the absence of 6-hydroxydopamine. Activation of mitochondrial respiration by desferrioxamine may contribute to the enhanced neuron survival in the presence of desferrioxamine in some neurodegenerative conditions. 相似文献
12.
ACP(Acyl carrier protein,酰基载体蛋白)参与高度不饱和脂肪酸的PKS(Polyketide synthase)生物合成途径.从Schizochytrium sp.FJU-512 cDNA文库中获得了ACP基因的cDNA克隆.该序列开放读码框全长429 bp,编码142个氨基酸,等电点为5.04,具有4'-磷酸泛酰巯基乙胺(4'-PP)的结合位点.利用BamH Ⅰ/Hind Ⅲ双酶切,并连接到原核表达载体pET-30a,构建了pET-30a/acp表达载体,转化宿主菌E.coll BL21(DE3),IPTG诱导表达.SDS-PAGE分析表明该蛋白得到高效表达. 相似文献
13.
Acyl carrier protein (ACP coli) was isolated from commercially grown Escherichia coli B and was acetylated by chemical methods. Biological activity of the synthesized acetyl-ACP coli was checked in an in vitro fatty acid-synthesizing system isolated from E. coli B. Since acetyl-ACP is preferred over acetyl-coenzyme A (CoA) as a substrate in these reactions, the possibility that it may substitute for acetyl-CoA in biosynthetically and oxidatively important cellular pathways (glyoxylate and Krebs cycles, respectively) was examined. Acetyl-ACP was tested for substrate activity with the enzyme of each cycle which has been found to utilize acetyl-CoA. Crystalline citrate synthase (EC 4.1.3.7) of porcine origin (Calbiochem) was found to be inactive with acetyl-ACP coli, which acted neither as a substrate nor as an inhibitor in the presence of acetyl-CoA. Malate synthase (EC 4.1.3.2) of the acetate type was isolated from acetate-grown cells of a mutant of E. coli K-12 (VGD(3)H(5)) and was also found to be inactive with acetyl-ACP coli. The significance of these results and of the recent discovery of another phospho-pantetheine-containing protein are discussed. 相似文献
14.
目的研究鱼藤酮帕金森模型大鼠呼吸链复合酶Ⅰ、Ⅳ的变化。方法雄性Wistar大鼠每日颈背部皮下注射鱼藤酮葵花油乳化液2 mg/(kg.d)连续3~5周制备鱼藤酮帕金森模型大鼠;按行为学评分标准记分。模型动物分成高分组、低分组、模型4周组。分光光度法测定呼吸链复合酶Ⅰ、Ⅳ。结果模型低分组肌肉呼吸链复合酶Ⅰ受到明显抑制,停给鱼藤酮4周后肌肉和黑质呼吸链复合酶Ⅰ显著低于正常。而模型高分组肌肉呼吸链复合酶Ⅰ升高,模型各组肌肉呼吸链复合酶Ⅳ均见升高,但黑质未见升高。结论鱼藤酮帕金森模型大鼠肌肉和黑质呼吸链复合酶Ⅰ明显抑制。肌肉见呼吸链复合酶Ⅳ代偿性升高而黑质未见。 相似文献
15.
16.
Fu-Chia Yang Ya-Huei Lin Wei-Hao Chen Jing-Yi Huang Hsin-Yun Chang Su-Hui Su Hsiao-Ting Wang Chun-Yi Chiang Pang-Hung Hsu Ming-Daw Tsai Bertrand Chin-Ming Tan Sheng-Chung Lee 《The Journal of biological chemistry》2013,288(47):33861-33872
Salt-inducible kinase 2 (SIK2) is an important regulator of cAMP response element-binding protein-mediated gene expression in various cell types and is the only AMP-activated protein kinase family member known to interact with the p97/valosin-containing protein (VCP) ATPase. Previously, we have demonstrated that SIK2 can regulate autophagy when proteasomal function is compromised. Here we report that physical and functional interactions between SIK2 and p97/VCP underlie the regulation of endoplasmic reticulum (ER)-associated protein degradation (ERAD). SIK2 co-localizes with p97/VCP in the ER membrane and stimulates its ATPase activity through direct phosphorylation. Although the expression of wild-type recombinant SIK2 accelerated the degradation and removal of ERAD substrates, the kinase-deficient variant conversely had no effect. Furthermore, down-regulation of endogenous SIK2 or mutation of the SIK2 target site on p97/VCP led to impaired degradation of ERAD substrates and disruption of ER homeostasis. Collectively, these findings highlight a mechanism by which the interplay between SIK2 and p97/VCP contributes to the regulation of ERAD in mammalian cells. 相似文献
17.
Thankiah Sudhaharan Ping Liu Yong Hwee Foo Wenyu Bu Kim Buay Lim Thorsten Wohland Sohail Ahmed 《The Journal of biological chemistry》2009,284(20):13602-13609
The RhoGTPase Cdc42 coordinates cell morphogenesis, cell cycle, and cell
polarity decisions downstream of membrane-bound receptors through distinct
effector pathways. Cdc42-effector protein interactions represent important
elements of cell signaling pathways that regulate cell biology in systems as
diverse as yeast and humans. To derive mechanistic insights into cell
signaling pathways, it is vital that we generate quantitative data from in
vivo systems. We need to be able to measure parameters such as protein
concentrations, rates of diffusion, and dissociation constants
(KD) of protein-protein interactions in vivo.
Here we show how single wavelength fluorescence cross-correlation spectroscopy
in combination with Förster resonance energy transfer analysis can be
used to determine KD of Cdc42-effector interactions in
live mammalian cells. Constructs encoding green fluorescent protein or
monomeric red fluorescent protein fusion proteins of Cdc42, an effector domain
(CRIB), and two effectors, neural Wiskott-Aldrich syndrome protein (N-WASP)
and insulin receptor substrate protein (IRSp53), were expressed as pairs in
Chinese hamster ovary cells, and concentrations of free protein as well as
complexed protein were determined. The measured KD for
Cdc42V12-N-WASP, Cdc42V12-CRIB, and Cdc42V12-IRSp53 was 27, 250, and 391
nm, respectively. The determination of KD for
Cdc42-effector interactions opens the way to describe cell signaling pathways
quantitatively in vivo in mammalian cells.Over the last 2 decades, we have been successful in describing a myriad of
cell signaling pathways that regulate the biology of cells. These pathways are
made of elements incorporating protein-protein, protein-lipid and
protein-ligand interactions. With the advent of
GFP2
(1,
2) and its variants
(3), it is now possible to
genetically encode fluorescent probes into any protein of interest. GFP fusion
proteins can be used in live cells giving spatial and temporal resolution to
cell signaling pathways (4). To
gain mechanistic insights into cellular processes, it is crucial that we
measure quantitative parameters to describe cell signaling. In this study, we
present an approach based on fluorescence cross-correlation spectroscopy
(FCCS) (5,
6) and Förster resonance
energy transfer (FRET) to determine quantitative parameters of cell signaling
pathways, including the determination of the KD for
Cdc42-effector interactions in live CHO-K-1 (hereafter referred to as CHO)
mammalian cells.The RhoGTPase Cdc42 (7,
8) regulates pathways that
coordinate cell cycle, morphogenesis, and polarity. Cdc42 is a molecular
switch that cycles between an inactive (GDP-bound) and active (GTP-bound)
state. The V12 Cdc42 point mutation freezes the protein in an activated
GTP-bound form, which binds effectors strongly. In contrast, Cdc42N17 is a
dominant negative protein that is GDP-bound and interacts with effectors
weakly if at all (9). A major
Cdc42 binding site/domain in effector proteins is known as Cdc42- and
Rac-interacting binding region
(CRIB)3 and was
originally found in activated Cdc42 kinase, p21 activated kinase (PAK), and
neural Wiskott-Aldrich syndrome protein (N-WASP)
(10). The inverse
Bin-amphiphysins-Rvs domain adaptor protein IRSp53 is also an effector but
binds Cdc42 through a partial CRIB domain
(11,
12). Cdc42 interaction with
its effectors has two main consequences, which are not mutually exclusive: (i)
unfolding of effector to expose the active site and (ii) relocalization of
effector to membrane compartments. Thus Cdc42-effector interactions serve as a
good model for cell signaling as a whole.Fluorescence correlation spectroscopy and FCCS measure fluctuations in
fluorescence of a small number of molecules as they pass through a defined
confocal volume, respectively
(13,
14,
15). Since the number of
molecules in the confocal volume and the confocal volume itself can be
determined, concentrations of protein can be measured by fluorescence
correlation spectroscopy. Single wavelength fluorescence cross-correlation
spectroscopy (SW-FCCS) is an FCCS variant in which excitation of two or more
probes is achieved by single wavelength one-photon excitation. To date SW-FCCS
has been used successfully to follow receptors and receptor-ligand
interactions in vitro and in vivo
(6,
16,
17).In the present analysis, we take a two-step approach to determining the
KD of Cdc42 binding to CRIB (domain of PAK), N-WASP, and
IRSp53. First, we show that the proteins under investigation are indeed
interacting with each other directly in vivo by FRET analysis. Here
we use acceptor photobleaching (AP)-FRET as well as changes in lifetime
(through fluorescence lifetime imaging microscopy (FLIM)) as indicators of
FRET. Second, we use SW-FCCS to determine the KD of Cdc42
interacting with its effectors by measuring the concentration of free protein
versus complexed protein. Thus, the combined use of FRET and FCCS
allows quantitative analysis of cell signaling pathways in vivo. 相似文献
18.
19.
Anne-Claire Cazalé Marie-Aude Rouet-Mayer Hélène Barbier-Brygoo Yves Mathieu Christiane Laurière 《Plant physiology》1998,116(2):659-669
Oxidative burst constitutes an early response in plant defense reactions toward pathogens, but active oxygen production may also be induced by other stimuli. The oxidative response of suspension-cultured tobacco (Nicotiana tabacum cv Xanthi) cells to hypoosmotic and mechanical stresses was characterized. The oxidase involved in the hypoosmotic stress response showed similarities by its NADPH dependence and its inhibition by iodonium diphenyl with the neutrophil NADPH oxidase. Activation of the oxidative response by hypoosmotic stress needed protein phosphorylation and anion effluxes, as well as opening of Ca2+ channels. Inhibition of the oxidative response impaired Cl− efflux, K+ efflux, and extracellular alkalinization, suggesting that the oxidative burst may play a role in ionic flux regulation. Active oxygen species also induced the cross-linking of a cell wall protein, homologous to a soybean (Glycine max L.) extensin, that may act as part of cell volume and turgor regulation through modification of the physical properties of the cell wall. 相似文献