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Kelly J. Inglis David Chereau Elizabeth F. Brigham San-San Chiou Susanne Sch?bel Normand L. Frigon Mei Yu Russell J. Caccavello Seth Nelson Ruth Motter Sarah Wright David Chian Pamela Santiago Ferdie Soriano Carla Ramos Kyle Powell Jason M. Goldstein Michael Babcock Ted Yednock Frederique Bard Guriqbal S. Basi Hing Sham Tamie J. Chilcote Lisa McConlogue Irene Griswold-Prenner John P. Anderson 《The Journal of biological chemistry》2009,284(5):2598-2602
Several neurological diseases, including Parkinson disease and dementia
with Lewy bodies, are characterized by the accumulation of α-synuclein
phosphorylated at Ser-129 (p-Ser-129). The kinase or kinases responsible for
this phosphorylation have been the subject of intense investigation. Here we
submit evidence that polo-like kinase 2 (PLK2, also known as serum-inducible
kinase or SNK) is a principle contributor to α-synuclein phosphorylation
at Ser-129 in neurons. PLK2 directly phosphorylates α-synuclein at
Ser-129 in an in vitro biochemical assay. Inhibitors of PLK kinases
inhibited α-synuclein phosphorylation both in primary cortical cell
cultures and in mouse brain in vivo. Finally, specific knockdown of
PLK2 expression by transduction with short hairpin RNA constructs or by
knock-out of the plk2 gene reduced p-Ser-129 levels. These results
indicate that PLK2 plays a critical role in α-synuclein phosphorylation
in central nervous system.The importance of α-synuclein to the pathogenesis of Parkinson
disease (PD)4 and the
related disorder, dementia with Lewy bodies (DLB), is suggested by its
association with Lewy bodies and Lewy neurites, the inclusions that
characterize these diseases
(1–3),
and demonstrated by the existence of mutations that cause syndromes mimicking
sporadic PD and DLB
(4–6).
Furthermore, three separate mutations cause early onset forms of PD and DLB.
It is particularly telling that duplications or triplications of the gene
(7–9),
which increase levels of α-synuclein with no alteration in sequence,
also cause PD or DLB.α-Synuclein has been reported to be phosphorylated on serine
residues, at Ser-87 and Ser-129
(10), although to date only
the Ser-129 phosphorylation has been identified in the central nervous system
(11,
12). Phosphorylation at
tyrosine residues has been observed by some investigators
(13,
14) but not by others
(10–12).
Phosphorylation at Ser-129 (p-Ser-129) is of particular interest because the
majority of synuclein in Lewy bodies contains this modification
(15). In addition, p-Ser-129
was found to be the most extensive and consistent modification in a survey of
synuclein in Lewy bodies (11).
Results have been mixed from studies investigating the function of
phosphorylation using S129A and S129D mutations to respectively block and
mimic the modification. Although the phosphorylation mimic was associated with
pathology in studies in Drosophila
(16) and in transgenic mouse
models (17,
18), studies using
adeno-associated virus vectors to overexpress α-synuclein in rat
substantia nigra found an exacerbation of pathology with the S129A mutation,
whereas the S129D mutation was benign, if not protective
(19). Interpretation of these
studies is complicated by a recent study showing that the S129D and S129A
mutations themselves have effects on the aggregation properties of
α-synuclein independent of their effects on phosphorylation, with the
S129A mutation stimulating fibril formation
(20). Clearly, determination
of the role of p-Ser-129 phosphorylation would be helped by identification of
the responsible kinase. In addition, identification will provide a
pathologically relevant way to increase phosphorylation in a cell or animal
model.Several kinases have been proposed to phosphorylate α-synuclein,
including casein kinases 1 and 2
(10,
12,
21) and members of the
G-protein-coupled receptor kinase family
(22). In this report, we offer
evidence that a member of the polo-like kinase (PLK) family, PLK2 (or
serum-inducible kinase, SNK), functions as an α-synuclein kinase. The
ability of PLK2 to directly phosphorylate α-synuclein at Ser-129 is
established by overexpression in cell culture and by in vitro
reaction with the purified kinase. We show that PLK2 phosphorylates
α-synuclein in cells, including primary neuronal cultures, using a
series of kinase inhibitors as well as inhibition of expression with RNA
interference. In addition, inhibitor and knock-out studies in mouse brain
support a role for PLK2 as an α-synuclein kinase in vivo. 相似文献
3.
4.
5.
Lisa Placanica Leonid Tarassishin Guangli Yang Erica Peethumnongsin Seong-Hun Kim Hui Zheng Sangram S. Sisodia Yue-Ming Li 《The Journal of biological chemistry》2009,284(5):2967-2977
γ-Secretase is known to play a pivotal role in the pathogenesis of
Alzheimer disease through production of amyloidogenic Aβ42 peptides.
Early onset familial Alzheimer disease mutations in presenilin (PS), the
catalytic core of γ-secretase, invariably increase the
Aβ42:Aβ40 ratio. However, the mechanism by which these mutations
affect γ-secretase complex formation and cleavage specificity is poorly
understood. We show that our in vitro assay system recapitulates the
effect of PS1 mutations on the Aβ42:Aβ40 ratio observed in cell and
animal models. We have developed a series of small molecule affinity probes
that allow us to characterize active γ-secretase complexes. Furthermore
we reveal that the equilibrium of PS1- and PS2-containing active complexes is
dynamic and altered by overexpression of Pen2 or PS1 mutants and that
formation of PS2 complexes is positively correlated with increased
Aβ42:Aβ40 ratios. These data suggest that perturbations to
γ-secretase complex equilibrium can have a profound effect on enzyme
activity and that increased PS2 complexes along with mutated PS1 complexes
contribute to an increased Aβ42:Aβ40 ratio.β-Amyloid
(Aβ)5 peptides
are believed to play a causative role in Alzheimer disease (AD). Aβ
peptides are generated from the processing of the amyloid precursor protein
(APP) by two proteases, β-secretase and γ-secretase. Although
γ-secretase generates heterogenous Aβ peptides ranging from 37 to
46 amino acids in length, significant work has focused mainly on the Aβ40
and Aβ42 peptides that are the major constituents of amyloid plaques.
γ-Secretase is a multisubunit membrane aspartyl protease comprised of at
least four known subunits: presenilin (PS), nicastrin (Nct), anterior
pharynx-defective (Aph), and presenilin enhancer 2 (Pen2). Presenilin is
thought to contain the catalytic core of the complex
(1–4),
whereas Aph and Nct play critical roles in the assembly, trafficking, and
stability of γ-secretase as well as substrate recognition
(5,
6). Lastly Pen2 facilitates the
endoproteolysis of PS into its N-terminal (NTF) and C-terminal (CTF) fragments
thereby yielding a catalytically competent enzyme
(5,
7–10).
All four proteins (PS, Nct, Aph1, and Pen2) are obligatory for
γ-secretase activity in cell and animal models
(11,
12). There are two homologs of
PS, PS1 and PS2, and three isoforms of Aph1, Aph1aS, Aph1aL, and Aph1b. At
least six active γ-secretase complexes have been reported (two
presenilins × three Aph1s)
(13,
14). The sum of apparent
molecular masses of the four proteins (PS1-NTF/CTF ≈ 53 kDa, Nct ≈ 120
kDa, Aph1 ≈ 30 kDa, and Pen2 ≈ 10kDa) is ∼200 kDa. However, active
γ-secretase complexes of varying sizes, ranging from 250 to 2000 kDa,
have been reported
(15–19).
Recently a study suggested that the γ-secretase complex contains only
one of each subunit (20).
Collectively these studies suggest that a four-protein complex around
200–250 kDa may be the minimal functional γ-secretase unit with
additional cofactors and/or varying stoichiometry of subunits existing in the
high molecular weight γ-secretase complexes. CD147 and TMP21 have been
found to be associated with the γ-secretase complex
(21,
22); however, their role in
the regulation of γ-secretase has been controversial
(23,
24).Mutations of PS1 or PS2 are associated with familial early onset AD (FAD),
although it is debatable whether these familial PS mutations act as
“gain or loss of function” alterations in regard to
γ-secretase activity
(25–27).
Regardless the overall outcome of these mutations is an increased ratio of
Aβ42:Aβ40. Clearly these mutations differentially affect
γ-secretase activity for the production of Aβ40 and Aβ42.
Despite intensive studies of Aβ peptides and γ-secretase, the
molecular mechanism controlling the specificity of γ-secretase activity
for Aβ40 and Aβ42 production has not been resolved. It has been
found that PS1 mutations affect the formation of γ-secretase complexes
(28). However, the precise
mechanism by which individual subunits alter the dynamics of γ-secretase
complex formation and activity is largely unresolved. A better mechanistic
understanding of γ-secretase activity associated with FAD mutations has
been hindered by the lack of suitable assays and probes that are necessary to
recapitulate the effect of these mutations seen in cell models and to
characterize the active γ-secretase complex.In our present studies, we have determined the overall effect of Pen2 and
PS1 expression on the dynamics of PS1- and PS2-containing complexes and their
association with γ-secretase activity. Using newly developed
biotinylated small molecular probes and activity assays, we revealed that
expression of Pen2 or PS1 FAD mutants markedly shifts the equilibrium of
PS1-containing active complexes to that of PS2-containing complexes and
results in an overall increase in the Aβ42:Aβ40 ratio in both stable
cell lines and animal models. Our studies indicate that perturbations to the
equilibrium of active γ-secretase complexes by an individual subunit can
greatly affect the activity of the enzyme. Moreover they serve as further
evidence that there are multiple and distinct γ-secretase complexes that
can exist within the same cells and that their equilibrium is dynamic.
Additionally the affinity probes developed here will facilitate further study
of the expression and composition of endogenous active γ-secretase from
a variety of model systems. 相似文献
6.
Toru Yoshihara Kazushi Sugihara Yasuhiko Kizuka Shogo Oka Masahide Asano 《The Journal of biological chemistry》2009,284(18):12550-12561
The glycosylation of glycoproteins and glycolipids is important for central
nervous system development and function. Although the roles of several
carbohydrate epitopes in the central nervous system, including polysialic
acid, the human natural killer-1 (HNK-1) carbohydrate, α2,3-sialic acid,
and oligomannosides, have been investigated, those of the glycan backbone
structures, such as Galβ1-4GlcNAc and Galβ1-3GlcNAc, are not fully
examined. Here we report the generation of mice deficient in
β4-galactosyltransferase-II (β4GalT-II). This galactosyltransferase
transfers Gal from UDP-Gal to a nonreducing terminal GlcNAc to synthesize the
Gal β1-4GlcNAc structure, and it is strongly expressed in the central
nervous system. In behavioral tests, the β4GalT-II-/- mice
showed normal spontaneous activity in a novel environment, but impaired
spatial learning/memory and motor coordination/learning. Immunohistochemistry
showed that the amount of HNK-1 carbohydrate was markedly decreased in the
brain of β4GalT-II-/- mice, whereas the expression of
polysialic acid was not affected. Furthermore, mice deficient in
glucuronyltransferase (GlcAT-P), which is responsible for the biosynthesis of
the HNK-1 carbohydrate, also showed impaired spatial learning/memory as
described in our previous report, although their motor coordination/learning
was normal as shown in this study. Histological examination showed abnormal
alignment and reduced number of Purkinje cells in the cerebellum of
β4GalT-II-/- mice. These results suggest that the
Galβ1-4GlcNAc structure in the HNK-1 carbohydrate is mainly synthesized
by β4GalT-II and that the glycans synthesized by β4GalT-II have
essential roles in higher brain functions, including some that are
HNK-1-dependent and some that are not.The glycosylation of glycoproteins, proteoglycans, and glycolipids is
important for their biological activities, stability, transport, and clearance
from circulation, and cell-surface glycans participate in cell-cell and
cell-extracellular matrix interactions. In the central nervous system, several
specific carbohydrate epitopes, including polysialic acid
(PSA),3 the
human natural killer-1 (HNK-1) carbohydrate, α2,3-sialic acid, and
oligomannosides play indispensable roles in neuronal generation, cell
migration, axonal outgrowth, and synaptic plasticity
(1). Functional analyses of the
glycan backbone structures, like lactosamine core (Galβ1-4GlcNAc),
neolactosamine core (Galβ1-3GlcNAc), and polylactosamine
(Galβ1-4GlcNAcβ1-3) have been carried out using gene-deficient mice
in β4-galactosyltransferase-I (β4GalT-I)
(2,
3), β4GalT-V
(4),
β3-N-acetylglucosaminyl-transferase-II (β3GnT-II)
(5), β3GnT-III
(Core1-β3GnT) (6),
β3GnT-V (7), and Core2GnT
(8). However, the roles of
these glycan backbone structures in the nervous system have not been examined
except the olfactory sensory system
(9).β4GalTs synthesize the Galβ1-4GlcNAc structure via the
β4-galactosylation of glycoproteins and glycolipids; the β4GalTs
transfer galactose (Gal) from UDP-Gal to a nonreducing terminal
N-acetylglucosamine (GlcNAc) of N- and O-glycans
with a β-1,4-linkage. The β4GalT family has seven members
(β4GalT-I to VII), of which at least five have similar
Galβ1-4GlcNAc-synthesizing activities
(10,
11). Each β4GalT has a
tissue-specific expression pattern and substrate specificity with overlapping,
suggesting each β4GalT has its own biological role as well as redundant
functions. β4GalT-I and β4GalT-II share the highest identity (52% at
the amino acid level) among the β4GalTs
(12), suggesting these two
galactosyltransferases can compensate for each other. β4GalT-I is
strongly and ubiquitously expressed in various non-neural tissues, whereas
β4GalT-II is strongly expressed in neural tissues
(13,
14). Indeed, the β4GalT
activity in the brain of β4GalT-I-deficient (β4GalT-I-/-)
mice remains as high as 65% of that of wild-type mice, and the expression
levels of PSA and the HNK-1 carbohydrate in the brain of these mice are normal
(15). These results suggest
β4GalTs other than β4GalT-I, like β4GalT-II, are important in
the nervous system.Among the β4GalT family members, only β4GalT-I-/- mice
have been examined extensively; this was done by us and another group. We
reported that glycans synthesized by β4GalT-I play various roles in
epithelial cell growth and differentiation, inflammatory responses, skin wound
healing, and IgA nephropathy development
(2,
16-18).
Another group reported that glycans synthesized by β4GalT-I are involved
in anterior pituitary hormone function and in fertilization
(3,
19). However, no other nervous
system deficits have been reported in these mice, and the role of the
β4-galactosylation of glycoproteins and glycolipids in the nervous system
has not been fully examined.In this study, we generated β4GalT-II-/- mice and examined
them for behavioral abnormalities and biochemical and histological changes in
the central nervous system. β4GalT-II-/- mice were impaired in
spatial learning/memory and motor coordination/learning. The amount of HNK-1
carbohydrate was markedly decreased in the β4GalT-II-/- brain,
but PSA expression was not affected. These results suggest that the
Galβ1-4GlcNAc structure in the HNK-1 carbohydrate is mainly synthesized
by β4GalT-II and that glycans synthesized by β4GalT-II have
essential roles in higher brain functions, including ones that are HNK-1
carbohydrate-dependent and ones that are independent of HNK-1. 相似文献
7.
8.
Saija Kiljunen Neeta Datta Svetlana V. Dentovskaya Andrey P. Anisimov Yuriy A. Knirel Jos�� A. Bengoechea Otto Holst Mikael Skurnik 《Journal of bacteriology》2011,193(18):4963-4972
φA1122 is a T7-related bacteriophage infecting most isolates of Yersinia pestis, the etiologic agent of plague, and used by the CDC in the identification of Y. pestis. φA1122 infects Y. pestis grown both at 20°C and at 37°C. Wild-type Yersinia pseudotuberculosis strains are also infected but only when grown at 37°C. Since Y. pestis expresses rough lipopolysaccharide (LPS) missing the O-polysaccharide (O-PS) and expression of Y. pseudotuberculosis O-PS is largely suppressed at temperatures above 30°C, it has been assumed that the phage receptor is rough LPS. We present here several lines of evidence to support this. First, a rough derivative of Y. pseudotuberculosis was also φA1122 sensitive when grown at 22°C. Second, periodate treatment of bacteria, but not proteinase K treatment, inhibited the phage binding. Third, spontaneous φA1122 receptor mutants of Y. pestis and rough Y. pseudotuberculosis could not be isolated, indicating that the receptor was essential for bacterial growth under the applied experimental conditions. Fourth, heterologous expression of the Yersinia enterocolitica O:3 LPS outer core hexasaccharide in both Y. pestis and rough Y. pseudotuberculosis effectively blocked the phage adsorption. Fifth, a gradual truncation of the core oligosaccharide into the Hep/Glc (l-glycero-d-manno-heptose/d-glucopyranose)-Kdo/Ko (3-deoxy-d-manno-oct-2-ulopyranosonic acid/d-glycero-d-talo-oct-2-ulopyranosonic acid) region in a series of LPS mutants was accompanied by a decrease in phage adsorption, and finally, a waaA mutant expressing only lipid A, i.e., also missing the Kdo/Ko region, was fully φA1122 resistant. Our data thus conclusively demonstrated that the φA1122 receptor is the Hep/Glc-Kdo/Ko region of the LPS core, a common structure in Y. pestis and Y. pseudotuberculosis. 相似文献
9.
Long-lived trees from tropical Australasia are a potential source of information about internal variability of the El Niño-Southern Oscillation (ENSO), because they occur in a region where precipitation variability is closely associated with ENSO activity. We measured tree-ring width and oxygen isotopic composition (O) of -cellulose from Agathis robusta (Queensland Kauri) samples collected in the Atherton Tablelands, Queensland, Australia. Standard ring-width chronologies yielded low internal consistency due to the frequent presence of false ring-like anatomical features. However, in a detailed examination of the most recent 15 years of growth (1995–2010), we found significant correlation between O and local precipitation, the latter associated with ENSO activity. The results are consistent with process-based forward modeling of the oxygen isotopic composition of -cellulose. The O record also enabled us to confirm the presence of a false growth ring in one of the three samples in the composite record, and to determine that it occurred as a consequence of anomalously low rainfall in the middle of the 2004/5 rainy season. The combination of incremental growth and isotopic measures may be a powerful approach to development of long-term (150+ year) ENSO reconstructions from the terrestrial tropics of Australasia. 相似文献
10.
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
11.
12.
13.
14.
15.
Mammalian defensins are cationic antimicrobial peptides that play a central
role in host innate immunity and as regulators of acquired immunity. In
animals, three structural defensin subfamilies, designated as α, β,
and θ, have been characterized, each possessing a distinctive
tridisulfide motif. Mature α- and β-defensins are produced by
simple proteolytic processing of their prepropeptide precursors. In contrast,
the macrocyclic θ-defensins are formed by the head-to-tail splicing of
nonapeptides excised from a pair of prepropeptide precursors. Thus,
elucidation of the θ-defensin biosynthetic pathway provides an
opportunity to identify novel factors involved in this unique process. We
incorporated the θ-defensin precursor, proRTD1a, into a bait construct
for a yeast two-hybrid screen that identified rhesus macaque stromal
cell-derived factor 2-like protein 1 (SDF2L1), as an interactor. SDF2L1 is a
component of the endoplasmic reticulum (ER) chaperone complex, which we found
to also interact with α- and β-defensins. However, analysis of the
SDF2L1 domain requirements for binding of representative α-, β-,
and θ-defensins revealed that α- and β-defensins bind SDF2L1
similarly, but differently from the interactions that mediate binding of
SDF2L1 to pro-θ-defensins. Thus, SDF2L1 is a factor involved in
processing and/or sorting of all three defensin subfamilies.Mammalian defensins are tridisulfide-containing antimicrobial peptides that
contribute to innate immunity in all species studied to date. Defensins are
comprised of three structural subfamilies: the α-, β-, and
θ-defensins (1). α-
and β-Defensins are peptides of about 29–45-amino acid residues
with similar three-dimensional structures. Despite their similar tertiary
conformations, the disulfide motifs of α- and β-defensins differ.
Expression of human α-defensins is tissue-specific. Four myeloid
α-defensins (HNP1–4) are expressed predominantly by neutrophils
and monocytes wherein they are packaged in granules, while two enteric
α-defensins (HD-5 and HD-6) are expressed at high levels in Paneth cells
of the small intestine. Myeloid α-defensins constitute about 5% of the
protein mass of human neutrophils. HNPs are discharged into the phagosome
during phagocytic ingestion of microbial particles. HD-5 and HD-6 are produced
and stored as propeptides in Paneth cell granules and are processed
extracellularly by intestinal trypsin
(2). β-Defensins are
produced primarily by various epithelia (e.g. skin, urogenital tract,
airway) and are secreted by the producing cells in their mature forms. In
contrast to pro-α-defensins, which contain a conserved prosegment of
∼40 amino acids, the prosegments in β-defensins vary in length and
sequence. θ-Defensins are found only in Old World monkeys and orangutans
and are the only known circular peptides in animals. These 18-residue
macrocyclic peptides are formed by ligation of two nonamer sequences excised
from two precursor polypeptides, which are truncated versions of ancestral
α-defensins. Like myeloid α-defensins, θ-defensins are
stored primarily in neutrophil and monocyte granules
(3).Numerous laboratories have demonstrated that the antimicrobial properties
of defensins derive from their ability to bind and disrupt target cell
membranes (4), and studies have
shown defensins to be active against Gram-positive and -negative bacteria
(5), viruses
(6–9),
fungi (10,
11), and parasites such as
Giardia lamblia (12).
Defensins also play a regulatory role in acquired immunity as they are known
to chemoattract T lymphocytes, monocytes, and immature dendritic cells
(13,
14), act as adjuvants,
stimulate B cell responses, and up-regulate proliferation and cytokine
production by spleen cells and T helper cells
(15,
16).Defensins are produced as pre-propeptides and undergo post-translational
processing to form the mature peptides. While much has been learned about
regulation of defensin expression, little is known about the factors involved
in their biosynthesis. Valore and Ganz
(17) investigated the
processing of defensins in cultured cells and demonstrated that maturation of
HNPs occurs through two proteolytic steps that lead to formation of mature
α-defensins, but the proteases involved have yet to be identified.
Moreover, there are virtually no published data regarding endoplasmic
reticulum (ER)2
factors that are responsible for the folding, processing, and sorting steps
necessary for defensin maturation and secretion or trafficking to the proper
subcellular compartment. It is likely that several chaperones, proteases, and
protein-disulfide isomerase (PDI) family proteins are involved. Consistent
with this possibility, Gruber et al.
(18) recently demonstrated the
role of a PDI in biosynthesis of cyclotides, small ∼30-residue macrocyclic
peptides produced by plants.The primary structures of α- and θ-defensin precursors are
closely related. We therefore undertook studies to identify proteins that
interact with representative propeptides of each defensin subfamily with the
goal of determining common and unique processes that regulate biosynthesis of
α- and θ-defensins. We used two-hybrid analysis to first identify
interactors of the θ-defensin precursor, proRTD1a. As described, we
identified SDF2L1, a component of the ER-chaperone complex as an interactor,
and showed that it also specifically interacts with α- and
β-defensins. This suggests that SDF2L1 is involved in the
maturation/trafficking of defensins at a step common to all three subfamilies
of mammalian defensins. 相似文献
16.
17.
18.
19.
Mar��a-Natalia Lisa Lars Hemmingsen Alejandro J. Vila 《The Journal of biological chemistry》2010,285(7):4570-4577
Metallo-β-lactamases (MβLs) stand as one of the main
mechanisms of bacterial resistance toward carbapenems. The rational design of an
inhibitor for MβLs has been limited by an incomplete knowledge of
their catalytic mechanism and by the structural diversity of their active sites.
Here we show that the MβL GOB from Elizabethkingia
meningoseptica is active as a monometallic enzyme by using
different divalent transition metal ions as surrogates of the native Zn(II) ion.
Of the metal derivatives in which Zn(II) is replaced, Co(II) and Cd(II) give
rise to the most active enzymes and are shown to occupy the same binding site as
the native ion. However, Zn(II) is the only metal ion capable of stabilizing an
anionic intermediate that accumulates during nitrocefin hydrolysis, in which the
C–N bond has already been cleaved. This finding demonstrates that the
catalytic role of the metal ion in GOB is to stabilize the formation of this
intermediate prior to nitrogen protonation. This role may be general to all
MβLs, whereas nucleophile activation by a Zn(II) ion is not a
conserved mechanistic feature. 相似文献
20.
Kazuyuki Kitatani Kely Sheldon Vinodh Rajagopalan Viviana Anelli Russell W. Jenkins Ying Sun Gregory A. Grabowski Lina M. Obeid Yusuf A. Hannun 《The Journal of biological chemistry》2009,284(19):12972-12978
Activation of protein kinase C (PKC) promotes the salvage pathway of
ceramide formation, and acid sphingomyelinase has been implicated, in part, in
providing substrate for this pathway (Zeidan, Y. H., and Hannun, Y. A. (2007)
J. Biol. Chem. 282, 11549–11561). In the present study, we
examined whether acid β-glucosidase 1 (GBA1), which hydrolyzes
glucosylceramide to form lysosomal ceramide, was involved in PKC-regulated
formation of ceramide from recycled sphingosine. Glucosylceramide levels
declined after treatment of MCF-7 cells with a potent PKC activator, phorbol
12-myristate 13-acetate (PMA). Silencing GBA1 by small interfering RNAs
significantly attenuated acid glucocerebrosidase activity and decreased
PMA-induced formation of ceramide by 50%. Silencing GBA1 blocked PMA-induced
degradation of glucosylceramide and generation of sphingosine, the source for
ceramide biosynthesis. Reciprocally, forced expression of GBA1 increased
ceramide levels. These observations indicate that GBA1 activation can generate
the source (sphingosine) for PMA-induced formation of ceramide through the
salvage pathway. Next, the role of PKCδ, a direct effector of PMA, in
the formation of ceramide was determined. By attenuating expression of
PKCδ, cells failed to trigger PMA-induced alterations in levels of
ceramide, sphingomyelin, and glucosylceramide. Thus, PKCδ activation is
suggested to stimulate the degradation of both sphingomyelin and
glucosylceramide leading to the salvage pathway of ceramide formation.
Collectively, GBA1 is identified as a novel source of regulated formation of
ceramide, and PKCδ is an upstream regulator of this pathway.Sphingolipids are abundant components of cellular membranes, many of which
are emerging as bioactive lipid mediators thought to play crucial roles in
cellular responses (1,
2). Ceramide, a central
sphingolipid, serves as the main precursor for various sphingolipids,
including glycosphingolipids, gangliosides, and sphingomyelin. Regulation of
formation of ceramide has been demonstrated through the action of three major
pathways: the de novo pathway
(3,
4), the sphingomyelinase
pathway (5), and the salvage
pathway
(6–8).
The latter plays an important role in constitutive sphingolipid turnover by
salvaging long-chain sphingoid bases (sphingosine and dihydrosphingosine) that
serve as sphingolipid backbones for ceramide and dihydroceramide as well as
all complex sphingolipids (Fig.
1A).Open in a separate windowFIGURE 1.The scheme of the sphingosine salvage pathway of ceramide formation and
inhibition of PMA induction of ceramide by fumonisin B1. A, the
scheme of the sphingosine salvage pathway of ceramide formation. B,
previously published data as to effects of fumonisin B1 on ceramide mass
profiles (23) are re-plotted
as a PMA induction of ceramide. In brief, MCF-7 cells were pretreated with or
without 100 μm fumonisin B1 for 2 h followed by treatment with
100 nm PMA for 1 h. Lipids were extracted, and then the levels of
ceramide species were determined by high-performance liquid
chromatography-tandem mass spectrometry. Results are expressed as sum of
increased mass of ceramide species. Dotted or open columns
represents C16-ceramide or sum of other ceramide species
(C14-ceramide, C18-ceramide, C18:1-ceramide,
C20-ceramide, C24-ceramide, and
C24:1-ceramide), respectively. The data represent mean ±
S.E. of three to five values.Metabolically, ceramide is also formed from degradation of
glycosphingolipids (Fig.
1A) usually in acidic compartments, the lysosomes and/or
late endosomes (9). The
stepwise hydrolysis of complex glycosphingolipids eventually results in the
formation of glucosylceramide, which in turn is converted to ceramide by the
action of acid β-glucosidase 1
(GBA1)2
(9,
10). Severe defects in GBA1
activity cause Gaucher disease, which is associated with aberrant accumulation
of the lipid substrates
(10–14).
On the other hand, sphingomyelin is cleaved by acid sphingomyelinase to also
form ceramide (15,
16). Either process results in
the generation of lysosomal ceramide that can then be deacylated by acid
ceramidase (17), releasing
sphingosine that may escape the lysosome
(18). The released sphingosine
may become a substrate for either sphingosine kinases or ceramide synthases,
forming sphingosine 1-phosphate or ceramide, respectively
(3,
19–21).In a related line of investigation, our studies
(20,
22,
23) have begun to implicate
protein kinase Cs (PKC) as upstream regulators of the sphingoid base salvage
pathway resulting in ceramide synthesis. Activation of PKCs by the phorbol
ester (PMA) was shown to stimulate the salvage pathway resulting in increases
in ceramide. All the induced ceramide was inhibited by pretreatment with a
ceramide synthase inhibitor, fumonisin B1, but not by myriocin, thus negating
acute activation of the de novo pathway and establishing a role for
ceramide synthesis (20,
23). Moreover, labeling
studies also implicated the salvage pathway because PMA induced turnover of
steady state-labeled sphingolipids but did not affect de novo labeled
ceramide in pulse-chase experiments.Moreover, PKCδ, among PKC isoforms, was identified as an upstream
molecule for the activation of acid sphingomyelinase in the salvage pathway
(22). Interestingly, the
PKCδ isoform induced the phosphorylation of acid sphingomyelinase at
serine 508, leading to its activation and consequent formation of ceramide.
The activation of acid sphingomyelinase appeared to contribute to ∼50% of
the salvage pathway-induced increase in ceramide
(28) (also, see
Fig. 4C). This raised
the possibility that distinct routes of ceramide metabolism may account for
the remainder of ceramide generation. In this study, we investigated
glucocerebrosidase GBA1 as a candidate for one of the other routes accounting
for PKC-regulated salvage pathway of ceramide formation.Open in a separate windowFIGURE 4.Effects of knockdown of lysosomal enzymes on the generation of ceramide
after PMA treatment. A, MCF-7 cells were transfected with 5
nm siRNAs of each of four individual sequences (SCR, GBA1-a,
GBA1-b, and GBA1-c) for 48 h and then stimulated with 100 nm PMA
for 1 h. Lipids were extracted, and then the levels of the
C16-ceramide species were determined by high-performance liquid
chromatography-tandem mass spectrometry. The data represent mean ± S.E.
of three to nine values. B, MCF-7 cells were transfected with 5
nm siRNAs of SCR or GBA1-a (GBA1) for 48 h and then stimulated with
100 nm PMA for 1 h. Lipids were extracted, and then the levels of
individual ceramide species were determined by high-performance liquid
chromatography-tandem mass spectrometry. The data represent mean ± S.E.
of three to five values. C14-Cer,
C14-ceramide; C16-Cer,
C16-ceramide; C18-Cer;
C18-ceramide; C18:1-Cer,
C18:1-ceramide; C20-Cer,
C20-ceramide; C20-Cer,
C24-ceramide; C24:1-Cer,
C24:1-ceramide. C, MCF-7 cells were transfected with 5
nm siRNAs of SCR, acid sphingomyelinase (ASM), or GBA1-a
(GBA1) for 48 h following stimulation with (PMA) or without
(Control) 100 nm PMA for 1 h. Lipids were extracted, and
then the levels of ceramide species were determined by high-performance liquid
chromatography-tandem mass spectrometry. Levels of C16-ceramide are
shown. The data represent mean ± S.E. of four to five values.
Significant changes from SCR-transfected cells treated with PMA are shown in
A–C (*, p < 0.02; **,
p < 0.05; ***, p < 0.01). 相似文献