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Vertebrates produce at least seven distinct β-tubulin isotypes that
coassemble into all cellular microtubules. The functional differences among
these tubulin isoforms are largely unknown, but recent studies indicate that
tubulin composition can affect microtubule properties and cellular
microtubule-dependent behavior. One of the isotypes whose incorporation causes
the largest change in microtubule assembly is β5-tubulin. Overexpression
of this isotype can almost completely destroy the microtubule network, yet it
appears to be required in smaller amounts for normal mitotic progression.
Moderate levels of overexpression can also confer paclitaxel resistance.
Experiments using chimeric constructs and site-directed mutagenesis now
indicate that the hypervariable C-terminal region of β5 plays no role in
these phenotypes. Instead, we demonstrate that two residues found in β5
(Ser-239 and Ser-365) are each sufficient to inhibit microtubule assembly and
confer paclitaxel resistance when introduced into β1-tubulin; yet the
single mutation of residue Ser-239 in β5 eliminates its ability to confer
these phenotypes. Despite the high degree of conservation among β-tubulin
isotypes, mutations affecting residue 365 demonstrate that amino acid
substitutions can be context sensitive; i.e. an amino acid change in
one isotype will not necessarily produce the same phenotype when introduced
into a different isotype. Modeling studies indicate that residue Cys-239 of
β1-tubulin is close to a highly conserved Cys-354 residue suggesting the
possibility that disulfide formation could play a significant role in the
stability of microtubules formed with β1- but not with
β5-tubulin.Microtubules are needed to organize the Golgi apparatus and endoplasmic
reticulum, maintain cell shape, construct ciliary and flagellar axonemes, and
ensure the accurate segregation of genetic material prior to cell division.
These cytoskeletal structures assemble from α- and β-tubulin
heterodimers to form long cylindrical filaments that exist in a state of
dynamic equilibrium characterized by stochastic episodes of slow growth and
rapid shrinkage (1). Impairment
of normal dynamic behavior has serious consequences for cell proliferation and
thus makes microtubules an attractive target for drug development
(2).Vertebrates express multiple β-tubulin genes that produce highly
homologous proteins differing most notably in their C-terminal 15–20
amino acids (3,
4). These variable C-terminal
sequences are conserved across vertebrate species and have been used to
classify β-tubulin genes into distinct isotypes
(5). In mammals, for example,
there are seven known isotypes designated by the numbers I, II, III, IVa, IVb,
V, and VI. The functional significance of the C-terminal sequences is
uncertain, but some studies suggest that they may be involved in binding or
modulating the action of microtubule-interacting proteins
(6–14).
Additional amino acid differences are scattered throughout the primary
sequence, but the functional role of these differences, if any, has not been
elucidated. Although some β-tubulin isotypes are expressed in a
tissue-specific manner (3),
evidence indicates that microtubules incorporate all available isotypes,
including transfected isotypes that are not normally produced in those cells
(5,
15–17).
Genetic experiments designed to test potential functional differences among
the various β-tubulin isotypes have only demonstrated isotype-specific
effects on the assembly of specialized microtubule-containing structures such
as flagellar axonemes in Drosophila or 15-protofilament microtubules
in Caenorhabditis elegans
(18,
19). Thus, the consequences,
if any, of producing multiple β-tubulin isoforms in vertebrate organisms
remain elusive.Our recent work showed that conditional overexpression of isotypes β1,
β2, and β4b has no effect on microtubule assembly or drug
sensitivity in transfected Chinese hamster ovary
(CHO)2 cells
(20). Similarly, expression of
neuronal-specific β4a produced very minor effects on microtubule assembly
but was able to increase sensitivity to paclitaxel, most likely through
increased binding of the drug
(21). On the other hand, high
expression of neuronal-specific β3 reduced microtubule assembly,
conferred low level resistance to paclitaxel, and inhibited cell growth
(22). The most dramatic
effects, however, were seen in cells transfected with β5, a minor but
widely expressed isotype (23).
Even modest overexpression of this isotype reduced microtubule assembly and
conferred paclitaxel resistance, whereas high levels of expression (∼50%
of total tubulin) caused fragmentation and a near complete loss of the
microtubule cytoskeleton (24).
Despite the toxicity associated with β5 overexpression, this isotype was
recently shown to be required for normal mitotic progression and cell
proliferation (25).Because of its importance for cell division, and the extreme phenotype
associated with its overexpression, we sought to identify the structural
differences between β5-tubulin and its more “normal” homolog,
β1. Although there are 40 amino acid differences between the 2 isotypes,
we report that most of the unique properties of β5 can be attributed to
the presence of serine in place of cysteine at residue 239. This residue faces
the colchicine binding pocket and is very close to a highly conserved Cys-354
residue. We propose that Ser-239 found in β5-tubulin may prevent
formation of a disulfide bond that normally stabilizes microtubules. 相似文献
4.
Mait�� Montero-Hadjadje Salah Elias Laurence Chevalier Magalie Benard Yannick Tanguy Val��rie Turquier Ludovic Galas Laurent Yon Maria M. Malagon Azeddine Driouich St��phane Gasman Youssef Anouar 《The Journal of biological chemistry》2009,284(18):12420-12431
Chromogranin A (CgA) has been proposed to play a major role in the
formation of dense-core secretory granules (DCGs) in neuroendocrine cells.
Here, we took advantage of unique features of the frog CgA (fCgA) to assess
the role of this granin and its potential functional determinants in hormone
sorting during DCG biogenesis. Expression of fCgA in the constitutively
secreting COS-7 cells induced the formation of mobile vesicular structures,
which contained cotransfected peptide hormones. The fCgA and the hormones
coexpressed in the newly formed vesicles could be released in a regulated
manner. The N- and C-terminal regions of fCgA, which exhibit remarkable
sequence conservation with their mammalian counterparts were found to be
essential for the formation of the mobile DCG-like structures in COS-7 cells.
Expression of fCgA in the corticotrope AtT20 cells increased
pro-opiomelanocortin levels in DCGs, whereas the expression of N- and
C-terminal deletion mutants provoked retention of the hormone in the Golgi
area. Furthermore, fCgA, but not its truncated forms, promoted
pro-opiomelanocortin sorting to the regulated secretory pathway. These data
demonstrate that CgA has the intrinsic capacity to induce the formation of
mobile secretory granules and to promote the sorting and release of peptide
hormones. The conserved terminal peptides are instrumental for these
activities of CgA.Eukaryotic cells share the capacity to rapidly secrete proteins through the
constitutive secretory pathway. The fundamental feature of neuroendocrine and
endocrine cells is the occurrence of dense-core secretory granules
(DCGs),3
which are key cytoplasmic organelles responsible for secretion of hormones,
neuropeptides, and neurotransmitters through the regulated secretory pathway
(RSP). Storage at high concentrations of these secretory products is required
for their finely tuned release in response to extracellular stimulation
(1,
2). DCG biogenesis starts with
the budding of immature secretory granules (ISGs) from the
trans-Golgi network (TGN) through interactions between lipid rafts
and protein components, in a similar manner to constitutive vesicle budding
(2,
3). The ISG budding is followed
by a multistep maturation process to form the mature secretory granules,
including removal of the constitutive secretory proteins and lysosomal enzymes
inadvertently packaged into ISGs
(4).Despite increasing knowledge of the various steps of DCG formation, the
nature of the sorting signals for entry of proteins into the DCGs and the
molecular machinery required to generate secretory granules are not fully
elucidated (5,
6). Several recent studies
highlighted the role of members of the granin family, which may represent the
driving force for granulogenesis in the TGN
(2), although this notion has
been a matter of debate (7).
Granins are soluble acidic proteins widely distributed in endocrine and
neuroendocrine cells, which are characterized by the ability to aggregate at
acidic pH and a high Ca2+ environment
(8,
9). These conditions are found
in the lumen of the TGN allowing granins to aggregate in this compartment and
to be segregated from constitutively secreted proteins
(10,
11). The granin aggregates are
believed to associate directly or indirectly with lipid rafts at the TGN to
induce budding and formation of the ISGs. A prominent role of chromogranin A
(CgA) in the regulation of DCG formation in endocrine and neuroendocrine cells
has been proposed. Thus, depletion of CgA in PC12 cells led to a dramatic
decrease in the number of DCGs
(12), and exogenously
expressed CgA in these depleted PC12 cells, as in DCG-deficient endocrine A35C
and 6T3 cells, restored DCG biogenesis
(12,
13). Besides, expression of
granins in non-endocrine, constitutively secreting cells such as CV-1, NIH3T3,
or COS-7 cells provoked the formation of DCG-like structures that release
their content in response to Ca2+ influx
(12,
14,
15). Further investigations
performed in CgA null mice and transgenic mice expressing antisense RNA
against CgA also revealed a reduction in the number of DCGs in chromaffin
cells that was associated with an impairment of catecholamine storage, thus
demonstrating the crucial role of CgA in normal DCG biogenesis
(16,
17). In CgA knockout mice, the
introduction of the gene expressing human CgA restored the regulated secretory
phenotype (16). A different
CgA null mice strain exhibited no discernable effect on DCG formation, but
elevated catecholamine secretion
(18), proving that CgA
deficiency is associated with hormone storage impairment in neuroendocrine
cells in vivo, a finding that was confirmed in vitro
(19). The CgA-/-
mice strain generated by Hendy et al.
(18) exhibited a compensatory
overexpression of other granins, pointing to a possible overlap in granin
function in secretory granule biogenesis.We reported previously that the frog CgA (fCgA) gene is coordinately
regulated with the pro-opiomelanocortin (POMC) gene in the pituitary pars
intermedia during the neuroendocrine reflex of skin color change, which allows
amphibia to adapt to their environment through the release of POMC-derived
melanotropic peptides (20,
21). Sequence comparison of
fCgA with its mammalian orthologs revealed a high conservation of the N- and
C-terminal domains, and far less conservation of the central part of the
protein (Fig. 1A),
suggesting that these domains may play a role in DCG formation and hormone
release in various species (9,
20,
21). To assess the role of
fCgA and its conserved N- and C-terminal regions in hormone sorting, storage,
and secretion, we engineered different constructs that produce the native
unmodified (no tag added) protein and truncated forms lacking the conserved N-
and C-terminal domains, and we developed an antibody that specifically
recognizes the central region of fCgA. Using the constitutively secreting
COS-7 cells, which are devoid of DCGs, we could demonstrate for the first time
that CgA is essential for targeting peptide hormones to newly formed mobile
DCG-like structures. In the CgA-expressing AtT20 cells, which exhibit an only
moderate capacity to sort secretory proteins to the regulated pathway
(22), the granin plays a
pivotal role in the sorting and release of POMC. The conserved terminal
peptides of CgA are instrumental for these activities.Open in a separate windowFIGURE 1.Specificity of the antibody directed against frog CgA. A,
scheme depicting the structure of fCgA and showing the high conservation of
the terminal regions and the percentages of amino acid identity between frog
and human CgA sequences. The highly conserved peptide WE14 and dibasic
cleavage sites are also indicated. B, Western blot showing that the
antibody developed against fCgA recognized the protein and several processing
intermediates in frog but not rat pituitary extracts, whereas an antibody,
directed against the WE14 conserved peptide, detected CgA and its processing
products in both rat and frog pituitary extracts. C,
immunofluorescence analysis of frog pituitary and adrenal glands, and rat
adrenal gland using the antibodies against fCgA and WE14. cx, cortex;
DL, distal lobe; IL, intermediate lobe; and m,
medulla. Scale bars equal 10 μm. 相似文献
5.
Hideki Watanabe Hiroyuki Matsumaru Ayako Ooishi YanWen Feng Takayuki Odahara Kyoko Suto Shinya Honda 《The Journal of biological chemistry》2009,284(18):12373-12383
Protein-protein interaction in response to environmental conditions enables
sophisticated biological and biotechnological processes. Aiming toward the
rational design of a pH-sensitive protein-protein interaction, we engineered
pH-sensitive mutants of streptococcal protein G B1, a binder to the IgG
constant region. We systematically introduced histidine residues into the
binding interface to cause electrostatic repulsion on the basis of a rigid
body model. Exquisite pH sensitivity of this interaction was confirmed by
surface plasmon resonance and affinity chromatography employing a clinically
used human IgG. The pH-sensitive mechanism of the interaction was analyzed and
evaluated from kinetic, thermodynamic, and structural viewpoints.
Histidine-mediated electrostatic repulsion resulted in significant loss of
exothermic heat of the binding that decreased the affinity only at acidic
conditions, thereby improving the pH sensitivity. The reduced binding energy
was partly recovered by “enthalpy-entropy compensation.” Crystal
structures of the designed mutants confirmed the validity of the rigid body
model on which the effective electrostatic repulsion was based. Moreover, our
data suggested that the entropy gain involved exclusion of water molecules
solvated in a space formed by the introduced histidine and adjacent tryptophan
residue. Our findings concerning the mechanism of histidine-introduced
interactions will provide a guideline for the rational design of pH-sensitive
protein-protein recognition.Molecular interactions govern a number of biological processes, including
metabolism, signal transduction, and immunoreaction. A better understanding of
the molecular basis for these interactions is crucial for a complete
elucidation of biological phenomena and redesign of interactions for drug
discovery and industrial biotechnology applications. Interactions between
biomolecules are generally characterized by their affinity, specificity, and
environmental responsiveness, such as sensitivity to pH. Such pH-dependent
ligand binding enables biological processes to function in an “on and
off” manner in response to environmental conditions, resulting in
sophisticated systems of regulation (e.g. pheromone production
(1,
2), immune systems
(3-5),
and mechanisms of virus survival
(6)).From an industrial perspective, pH sensitivity is advantageous to various
fields, such as drug delivery systems for medications
(7), biosensing techniques
(8,
9), and affinity chromatography
(10,
11). Although structure-based
protein design is a promising technique for improving molecular function
(12-15),
it is yet difficult to specifically modulate pH sensitivity of a
protein-protein interaction without an associated loss of inherent function
and/or structural stability. Some naturally occurring proteins undergo
substantial conformational change by pH shift, thereby achieving pH-dependent
binding for small molecules (2,
4,
16,
17). However, artificial
design of an equivalent mechanism involving conformational change is highly
problematic. Indeed, proteins have multiple degrees of freedom and consist of
a large number of atoms. Therefore, given that the resulting protein must
maintain both its innate binding ability and structural stability, the system
appears too complicated for rational design. By contrast to the method based
on conformational change, a rigid body-based model (i.e. introduction
of electrostatic repulsion or attraction into a binding interface between
rigid protein domains) could be a more promising approach for pH switching.
Naturally occurring proteins with pH sensitivity generally conserve histidine
residues
(18-21),
which function as a pH switch at slightly acidic conditions (pH ∼6.5) near
the pKa of the histidine side chain. In the presence of a
histidine residue at a binding interface, dissociation under acidic conditions
would be driven by electrostatic repulsion between rigid domains without
conformational change (Fig. 1).
This mechanism is rather simple and applicable to protein engineering
(22,
23). However, to our
knowledge, it still remains unclear how systematic design should be carried
out and, in particular, how histidine-mediated electrostatic repulsion
influences protein-protein interactions. Indeed, very little experimental data
are available for the molecular basis of histidine-introduced protein
binders.Open in a separate windowFIGURE 1.A schematic model for introduction of histidine-mediated electrostatic
repulsion into the binding interface between protein G (GB) and
Fc. Protein G residues positioned closely to basic side chains (depicted
as B) on Fc were systematically identified by distance calculations
and mutated into histidine to cause electrostatic repulsion under acidic
conditions. The inset shows an example of candidate positions for the
mutation.To better understand the design methodology for a pH-sensitive
protein-protein interaction, we generated a number of pH-sensitive
streptococcal protein G B1
(24) mutants by rationally
introducing histidine residues onto the binding surface. Protein G, a
bacterial Fc (fragment of crystallizable region) receptor to the constant
region of IgG, has been used as an affinity chromatography binder for antibody
immobilization and purification. Protein G has an acidic pH optimum for
binding relative to another bacterial Fc receptor, Staphylococcus
aureus protein A. The harsh elution conditions are likely to induce
acidic conformational changes in antibodies
(25,
26) during the purification
procedure, causing aggregation that is problematic for pharmaceutical
applications. The usefulness of the histidine-mediated electrostatic repulsion
for antibody purification was examined by constructing affinity chromatography
columns. Using the designed mutants, we analyzed the molecular basis of the
histidine-mediated interaction from a kinetic, thermodynamic, and structural
perspective. The observed data revealed functional and structural consequences
for the introduction of histidine residues. Analysis of our results provides a
guideline for the design of pH-dependent protein-protein interactions. 相似文献
6.
Lu Meng Farhad Forouhar David Thieker Zhongwei Gao Annapoorani Ramiah Heather Moniz Yong Xiang Jayaraman Seetharaman Sahand Milaninia Min Su Robert Bridger Lucas Veillon Parastoo Azadi Gregory Kornhaber Lance Wells Gaetano T. Montelione Robert J. Woods Liang Tong Kelley W. Moremen 《The Journal of biological chemistry》2013,288(48):34680-34698
Glycan structures on glycoproteins and glycolipids play critical roles in biological recognition, targeting, and modulation of functions in animal systems. Many classes of glycan structures are capped with terminal sialic acid residues, which contribute to biological functions by either forming or masking glycan recognition sites on the cell surface or secreted glycoconjugates. Sialylated glycans are synthesized in mammals by a single conserved family of sialyltransferases that have diverse linkage and acceptor specificities. We examined the enzymatic basis for glycan sialylation in animal systems by determining the crystal structures of rat ST6GAL1, an enzyme that creates terminal α2,6-sialic acid linkages on complex-type N-glycans, at 2.4 Å resolution. Crystals were obtained from enzyme preparations generated in mammalian cells. The resulting structure revealed an overall protein fold broadly resembling the previously determined structure of pig ST3GAL1, including a CMP-sialic acid-binding site assembled from conserved sialylmotif sequence elements. Significant differences in structure and disulfide bonding patterns were found outside the sialylmotif sequences, including differences in residues predicted to interact with the glycan acceptor. Computational substrate docking and molecular dynamics simulations were performed to predict and evaluate the CMP-sialic acid donor and glycan acceptor interactions, and the results were compared with kinetic analysis of active site mutants. Comparisons of the structure with pig ST3GAL1 and a bacterial sialyltransferase revealed a similar positioning of donor, acceptor, and catalytic residues that provide a common structural framework for catalysis by the mammalian and bacterial sialyltransferases. 相似文献
7.
Orwah Saleh Bertolt Gust Bj?rn Boll Hans-Peter Fiedler Lutz Heide 《The Journal of biological chemistry》2009,284(21):14439-14447
The bacterium Streptomyces anulatus 9663, isolated from the
intestine of different arthropods, produces prenylated derivatives of
phenazine 1-carboxylic acid. From this organism, we have identified the
prenyltransferase gene ppzP. ppzP resides in a gene cluster
containing orthologs of all genes known to be involved in phenazine
1-carboxylic acid biosynthesis in Pseudomonas strains as well as
genes for the six enzymes required to generate dimethylallyl diphosphate via
the mevalonate pathway. This is the first complete gene cluster of a phenazine
natural compound from streptomycetes. Heterologous expression of this cluster
in Streptomyces coelicolor M512 resulted in the formation of
prenylated derivatives of phenazine 1-carboxylic acid. After inactivation of
ppzP, only nonprenylated phenazine 1-carboxylic acid was formed.
Cloning, overexpression, and purification of PpzP resulted in a 37-kDa soluble
protein, which was identified as a 5,10-dihydrophenazine 1-carboxylate
dimethylallyltransferase, forming a C–C bond between C-1 of the
isoprenoid substrate and C-9 of the aromatic substrate. In contrast to many
other prenyltransferases, the reaction of PpzP is independent of the presence
of magnesium or other divalent cations. The Km value for
dimethylallyl diphosphate was determined as 116 μm. For
dihydro-PCA, half-maximal velocity was observed at 35 μm.
Kcat was calculated as 0.435 s-1. PpzP shows
obvious sequence similarity to a recently discovered family of
prenyltransferases with aromatic substrates, the ABBA prenyltransferases. The
present finding extends the substrate range of this family, previously limited
to phenolic compounds, to include also phenazine derivatives.The transfer of isoprenyl moieties to aromatic acceptor molecules gives
rise to an astounding diversity of secondary metabolites in bacteria, fungi,
and plants, including many compounds that are important in pharmacotherapy.
However, surprisingly little biochemical and genetic data are available on the
enzymes catalyzing the C-prenylation of aromatic substrates. Recently, a new
family of aromatic prenyltransferases was discovered in streptomycetes
(1), Gram-positive soil
bacteria that are prolific producers of antibiotics and other biologically
active compounds (2). The
members of this enzyme family show a new type of protein fold with a unique
α-β-β-α architecture
(3) and were therefore termed
ABBA prenyltransferases (1).
Only 13 members of this family can be identified by sequence similarity
searches in the data base at present, and only four of them have been
investigated biochemically
(3–6).
Up to now, only phenolic compounds have been identified as aromatic substrates
of ABBA prenyltransferases. We now report the discovery of a new member of the
ABBA prenyltransferase family, catalyzing the transfer of a dimethylallyl
moiety to C-9 of 5,10-dihydrophenazine 1-carboxylate
(dihydro-PCA).2
Streptomyces strains produce many of prenylated phenazines as natural
products. For the first time, the present paper reports the identification of
a prenyltransferase involved in their biosynthesis.Streptomyces anulatus 9663, isolated from the intestine of
different arthropods, produces several prenylated phenazines, among them
endophenazine A and B (Fig.
1A) (7).
We wanted to investigate which type of prenyltransferase might catalyze the
prenylation reaction in endophenazine biosynthesis. In streptomycetes and
other microorganisms, genes involved in the biosynthesis of a secondary
metabolite are nearly always clustered in a contiguous DNA region. Therefore,
the prenyltransferase of endophenazine biosynthesis was expected to be
localized in the vicinity of the genes for the biosynthesis of the phenazine
core (i.e. of PCA).Open in a separate windowFIGURE 1.A, prenylated phenazines from S. anulatus 9663.
B, biosynthetic gene cluster of endophenazine A.In Pseudomonas, an operon of seven genes named phzABCDEFG
is responsible for the biosynthesis of PCA
(8). The enzyme PhzC catalyzes
the condensation of phosphoenolpyruvate and erythrose-4-phosphate
(i.e. the first step of the shikimate pathway), and further enzymes
of this pathway lead to the intermediate chorismate. PhzD and PhzE catalyze
the conversion of chorismate to 2-amino-2-deoxyisochorismate and the
subsequent conversion to 2,3-dihydro-3-hydroxyanthranilic acid, respectively.
These reactions are well established biochemically. Fewer data are available
about the following steps (i.e. dimerization of
2,3-dihydro-3-hydroxyanthranilic acid, several oxidation reactions, and a
decarboxylation, ultimately leading to PCA via several instable
intermediates). From Pseudomonas, experimental data on the role of
PhzF and PhzA/B have been published
(8,
9), whereas the role of PhzG is
yet unclear. Surprisingly, the only gene cluster for phenazine biosynthesis
described so far from streptomycetes
(10) was found not to contain
a phzF orthologue, raising the question of whether there may be
differences in the biosynthesis of phenazines between Pseudomonas and
Streptomyces.Screening of a genomic library of the endophenazine producer strain S.
anulatus now allowed the identification of the first complete gene
cluster of a prenylated phenazine, including the structural gene of
dihydro-PCA dimethylallyltransferase. 相似文献
8.
Martin J. Sergeant Jian-Jun Li Christine Fox Nicola Brookbank Dean Rea Timothy D. H. Bugg Andrew J. Thompson 《The Journal of biological chemistry》2009,284(8):5257-5264
Members of the carotenoid cleavage dioxygenase family catalyze the
oxidative cleavage of carotenoids at various chain positions, leading to the
formation of a wide range of apocarotenoid signaling molecules. To explore the
functions of this diverse enzyme family, we have used a chemical genetic
approach to design selective inhibitors for different classes of carotenoid
cleavage dioxygenase. A set of 18 arylalkyl-hydroxamic acids was synthesized
in which the distance between an iron-chelating hydroxamic acid and an
aromatic ring was varied; these compounds were screened as inhibitors of four
different enzyme classes, either in vitro or in vivo. Potent
inhibitors were found that selectively inhibited enzymes that cleave
carotenoids at the 9,10 position; 50% inhibition was achieved at submicromolar
concentrations. Application of certain inhibitors at 100 μm to
Arabidopsis node explants or whole plants led to increased shoot
branching, consistent with inhibition of 9,10-cleavage.Carotenoids are synthesized in plants and micro-organisms as
photoprotective molecules and are key components in animal diets, an example
being β-carotene (pro-vitamin A). The oxidative cleavage of carotenoids
occurs in plants, animals, and micro-organisms and leads to the release of a
range of apocarotenoids that function as signaling molecules with a diverse
range of functions (1). The
first gene identified as encoding a carotenoid cleavage dioxygenase
(CCD)2 was the maize
Vp14 gene that is required for the formation of abscisic acid (ABA),
an important hormone that mediates responses to drought stress and aspects of
plant development such as seed and bud dormancy
(2). The VP14 enzyme cleaves at
the 11,12 position (Fig. 1) of
the epoxycarotenoids 9′-cis-neoxanthin and/or
9-cis-violaxanthin and is now classified as a
9-cis-epoxycarotenoid dioxygenase (NCED)
(3), a subclass of the larger
CCD family.Open in a separate windowFIGURE 1.Reactions catalyzed by the carotenoid cleavage dioxygenases.
a, 11,12-oxidative cleavage of 9′-cis-neoxanthin by
NCED; b, oxidative cleavage reactions on β-carotene and
zeaxanthin.Since the discovery of Vp14, many other CCDs have been shown to be
involved in the production of a variety of apocarotenoids
(Fig. 1). In insects, the
visual pigment retinal is formed by oxidative cleavage of β-carotene by
β-carotene-15,15′-dioxygenase
(4). Retinal is produced by an
orthologous enzyme in vertebrates, where it is also converted to retinoic
acid, a regulator of differentiation during embryogenesis
(5). A distinct mammalian CCD
is believed to cleave carotenoids asymmetrically at the 9,10 position
(6) and, although its function
is unclear, recent evidence suggests a role in the metabolism of dietary
lycopene (7). The plant
volatiles β-ionone and geranylacetone are produced from an enzyme that
cleaves at the 9,10 position
(8) and the pigment
α-crocin found in the spice saffron results from an 7,8-cleavage enzyme
(9).Other CCDs have been identified where biological function is unknown, for
example, in cyanobacteria where a variety of cleavage specificities have been
described
(10-12).
In other cases, there are apocarotenoids with known functions, but the
identity or involvement of CCDs have not yet been described: grasshopper
ketone is a defensive secretion of the flightless grasshopper Romalea
microptera (13),
mycorradicin is produced by plant roots during symbiosis with arbuscular
mycorrhyza (14), and
strigolactones (15) are plant
metabolites that act as germination signals to parasitic weeds such as
Striga and Orobanche
(16).Recently it was discovered that strigolactones also function as a branching
hormone in plants (17,
18). The existence of such a
branching hormone has been known for some time, but its identity proved
elusive. However, it was known that the hormone was derived from the action of
at least two CCDs, max3 and max4 (more axillary growth)
(19), because deletion of
either of these genes in Arabidopsis thaliana, leads to a bushy
phenotype (20,
21). In Escherichia
coli assays, AtCCD7 (max3) cleaves β-carotene at the 9,10 position
and the apocarotenoid product (10-apo-β-carotene) is reported to be
further cleaved at 13,14 by AtCCD8 (max4) to produce 13-apo-β-carotene
(22). Also recent evidence
suggests that AtCCD8 is highly specific, cleaving only 10-apo-β-carotene
(23). How the production of
13-apo-β-carotene leads to the synthesis of the complex strigolactone is
unknown. The possibility remains that the enzymes may have different
specificities and cleavage activities in planta. In addition, a
cytochrome P450 enzyme (24) is
believed to be involved in strigolactone synthesis and acts in the pathway
downstream of the CCD genes. Strigolactone is thought to effect branching by
regulating auxin transport
(25). Because of the
involvement of CCDs in strigolactone synthesis, the possibility arises that
plant architecture and interaction with parasitic weeds and mycorrhyza could
be controlled by the manipulation of CCD activity.Although considerable success has been obtained using genetic approaches to
probe function and substrate specificity of CCDs in their native biological
contexts, particularly in plant species with simple genetic systems or that
are amenable to transgenesis, there are many systems where genetic approaches
are difficult or impossible. Also, when recombinant CCDs are studied either
in vitro or in heterologous in vivo assays, such as in
E. coli strains engineered to accumulate carotenoids
(26), they are often active
against a broad range of substrates
(5,
21,
27), and in many cases the
true in vivo substrate of a particular CCD remains unknown. Therefore
additional experimental tools are needed to investigate both apocarotenoid and
CCD functions in their native cellular environments.In the reverse chemical genetics approach, small molecules are identified
that are active against known target proteins; they are then applied to a
biological system to investigate protein function in vivo
(28,
29). This approach is
complementary to conventional genetics since the small molecules can be
applied easily to a broad range of species, their application can be
controlled in dose, time, and space to provide detailed studies of biological
functions, and individual proteins or whole protein classes may be targeted by
varying the specificity of the small molecules. Notably, functions of the
plant hormones gibberellin, brassinosteroid, and abscisic acid have been
successfully probed using this approach by adapting triazoles to inhibit
specific cytochrome P450 monooxygenases involved in the metabolism of these
hormones (30).In the case of the CCD family, the tertiary amines abamine
(31) and the more active
abamineSG (32) were reported
as specific inhibitors of NCED, and abamine was used to show new functions of
abscisic acid in legume nodulation
(33). However, no selective
inhibitors for other types of CCD are known. Here we have designed a novel
class of CCD inhibitor based on hydroxamic acids, where variable chain length
was used to direct inhibition of CCD enzymes that cleave carotenoids at
specific positions. We demonstrate the use of such novel inhibitors to control
shoot branching in a model plant. 相似文献
9.
Obidimma C. Ezezika Noah S. Younger Jia Lu Donald A. Kaiser Zachary A. Corbin Bradley J. Nolen David R. Kovar Thomas D. Pollard 《The Journal of biological chemistry》2009,284(4):2088-2097
Expression of human profilin-I does not complement the
temperature-sensitive cdc3-124 mutation of the single profilin gene
in fission yeast Schizosaccharomyces pombe, resulting in death from
cytokinesis defects. Human profilin-I and S. pombe profilin have
similar affinities for actin monomers, the FH1 domain of fission yeast formin
Cdc12p and poly-l-proline (Lu, J., and Pollard, T. D. (2001)
Mol. Biol. Cell 12, 1161–1175), but human profilin-I does not
stimulate actin filament elongation by formin Cdc12p like S. pombe
profilin. Two crystal structures of S. pombe profilin and homology
models of S. pombe profilin bound to actin show how the two profilins
bind to identical surfaces on animal and yeast actins even though 75% of the
residues on the profilin side of the interaction differ in the two profilins.
Overexpression of human profilin-I in fission yeast expressing native profilin
also causes cytokinesis defects incompatible with viability. Human profilin-I
with the R88E mutation has no detectable affinity for actin and does not have
this dominant overexpression phenotype. The Y6D mutation reduces the affinity
of human profilin-I for poly-l-proline by 1000-fold, but
overexpression of Y6D profilin in fission yeast is lethal. The most likely
hypotheses to explain the incompatibility of human profilin-I with Cdc12p are
differences in interactions with the proline-rich sequences in the FH1 domain
of Cdc12p and wider “wings” that interact with actin.The small protein profilin not only helps to maintain a cytoplasmic pool of
actin monomers ready to elongate actin filament barbed ends
(2), but it also binds to type
II poly-l-proline helices
(3,
4). The actin
(5) and
poly-l-proline
(6–8)
binding sites are on opposite sides of the profilin molecule, so profilin can
link actin to proline-rich targets. Viability of fission yeast depends
independently on profilin binding to both actin and poly-l-proline,
although cells survive >10-fold reductions in affinity for either ligand
(1).Fission yeast Schizosaccharomyces pombe depend on formin Cdc12p
(9,
10) and profilin
(11) to assemble actin
filaments for the cytokinetic contractile ring. Formins are multidomain
proteins that nucleate and assemble unbranched actin filaments
(12). Formin FH2 domains form
homodimers that can associate processively with the barbed ends of growing
actin filaments (13,
14). FH2 dimers slow the
elongation of barbed ends
(15). Most formin proteins
have an FH1 domain linked to the FH2 domain. Binding profilin-actin to
multiple polyproline sites in an FH1 domain concentrates actin near the barbed
end of an actin filament associated with a formin FH2 homodimer. Actin
transfers very rapidly from the FH1 domains onto the filament end
(16) allowing profilin to
stimulate elongation of the filament
(15,
17).We tested the ability of human (Homo sapiens,
Hs)7 profilin-I to
complement the temperature-sensitive cdc3-124 mutation
(11) in the single fission
yeast profilin gene with the aim of using yeast to characterize human profilin
mutations. The failure of expression of Hs profilin-I to complement the
cdc3-124 mutation prompted us to compare human and fission yeast
profilins more carefully. We report here a surprising incompatibility of Hs
profilin-I with fission yeast formin Cdc12p, a crystal structure of fission
yeast profilin, which allowed a detailed comparison with Hs profilin, and
mutations that revealed how overexpression of Hs profilin-I compromises the
viability of wild-type fission yeast. 相似文献
10.
Adrien W. Schmid Diego Chiappe V��r��ne Pignat Valerie Grimminger Ivan Hang Marc Moniatte Hilal A. Lashuel 《The Journal of biological chemistry》2009,284(19):13128-13142
Tissue transglutaminase (tTG) has been implicated in the pathogenesis of
Parkinson disease (PD). However, exactly how tTG modulates the structural and
functional properties of α-synuclein (α-syn) and contributes to
the pathogenesis of PD remains unknown. Using site-directed mutagenesis
combined with detailed biophysical and mass spectrometry analyses, we sought
to identify the exact residues involved in tTG-catalyzed cross-linking of
wild-type α-syn and α-syn mutants associated with PD. To better
understand the structural consequences of each cross-linking reaction, we
determined the effect of tTG-catalyzed cross-linking on the oligomerization,
fibrillization, and membrane binding of α-syn in vitro. Our
findings show that tTG-catalyzed cross-linking of monomeric α-syn
involves multiple cross-links (specifically 2-3). We subjected tTG-catalyzed
cross-linked monomeric α-syn composed of either wild-type or Gln →
Asn mutants to sequential proteolysis by multiple enzymes and peptide mapping
by mass spectrometry. Using this approach, we identified the glutamine and
lysine residues involved in tTG-catalyzed intramolecular cross-linking of
α-syn. These studies demonstrate for the first time that
Gln79 and Gln109 serve as the primary tTG reactive
sites. Mutating both residues to asparagine abolishes tTG-catalyzed
cross-linking of α-syn and tTG-induced inhibition of α-syn
fibrillization in vitro. To further elucidate the sequence and
structural basis underlying these effects, we identified the lysine residues
that form isopeptide bonds with Gln79 and Gln109. This
study provides mechanistic insight into the sequence and structural basis of
the inhibitory effects of tTG on α-syn fibrillogenesis in vivo,
and it sheds light on the potential role of tTG cross-linking on modulating
the physiological and pathogenic properties of α-syn.Parkinson disease
(PD)2 is a progressive
movement disorder that is caused by the loss of dopaminergic neurons in the
substantia nigra, the part of the brain responsible for controlling movement.
Clinically, PD is manifested in symptoms that include tremors, rigidity, and
difficulty in initiating movement (bradykinesia). Pathologically, PD is
characterized by the presence of intraneuronal, cytoplasmic inclusions known
as Lewy bodies (LB), which are composed primarily of the protein
“α-synuclein” (α-syn)
(1) and are seen in the
post-mortem brains of PD patients with the sporadic or familial forms of the
disease (2). α-Syn is a
presynaptic protein of 140 residues with a “natively” unfolded
structure (3). Three missense
point mutations in α-syn (A30P, E46K, and A53T) are associated with the
early-onset, dominant, inherited form of PD
(4,
5). Moreover, duplication or
triplication of the α-syn gene has been linked to the familial
form of PD, suggesting that an increase in α-syn expression is
sufficient to cause PD. Together, these findings suggest that α-syn
plays a central role in the pathogenesis of PD.The molecular and cellular determinants that govern α-syn
oligomerization and fibrillogenesis in vivo remain poorly understood.
In vitro aggregation studies have shown that the mutations associated
with PD (A30P, E46K, and A53T) accelerate α-syn oligomerization, but
only E46K and A53T α-syn show higher propensity to fibrillize than
wild-type (WT) α-syn
(6-8).
This suggests that oligomerization, rather than fibrillization, is linked to
early-onset familial PD (9).
Our understanding of the molecular composition and biochemical state of
α-syn in LBs has provided important clues about protein-protein
interactions and post-translational modifications that may play a role in
modulating oligomerization, fibrillogenesis, and LB formation of the protein.
In addition to ubiquitination
(10), phosphorylation
(11,
12), nitration
(13,
14), and C-terminal truncation
(15,
16), analysis of post-mortem
brain tissues from PD and Lewy bodies in dementia patients has confirmed the
colocalization of tissue transglutaminase (tTG)-catalyzed cross-linked
α-syn monomers and higher molecular aggregates in LBs within
dopaminergic neurons (17,
18). Tissue transglutaminase
catalyzes a calcium-dependent transamidating reaction involving glutamine and
lysine residues, which results in the formation of a covalent cross-link via
ε-(γ-glutamyl) lysine bonds
(Fig. 2F). To date,
seven different isoforms of tTGs have been reported, of which only tTG2 seems
to be expressed in the human brain
(19), whereas tTG1 and tTG3
are more abundantly found in stratified squamous epithelia
(20). Subsequent
immuno-histochemical, colocalization, and immunoprecipitation studies have
shown that the levels of tTG and cross-linked α-syn species are
increased in the substantia nigra of PD brains
(17). These findings, combined
with the known role of tTG in cross-linking and stabilizing bimolecular
assemblies, led to the hypothesis that tTG plays an important role in the
initiation and propagation of α-syn fibril formation and that it
contributes to fibril stability in LBs. This hypothesis was initially
supported by in vitro studies demonstrating that tTG catalyzes the
polymerization of the α-syn-derived non-amyloid component (NAC) peptide
via intermolecular covalent cross-linking of residues Gln79 and
Lys80 (21) and by
other studies suggesting that tTG promotes the fibrillization of amyloidogenic
proteins implicated in the pathogenesis of other neurodegenerative diseases
such as Alzheimer disease, supranuclear palsy, Huntington disease, and other
polyglutamine diseases
(22-24).
However, recent in vitro studies with full-length α-syn have
shown that tTG catalyzes intramolecular cross-linking of monomeric α-syn
and inhibits, rather than promotes, its fibrillization in vitro
(25,
26). The structural basis of
this inhibitory effect and the exact residues involved in tTG-mediated
cross-linking of α-syn, as well as structural and functional
consequences of these modifications, remain poorly understood.Open in a separate windowFIGURE 2.tTG-catalyzed cross-linking of α-syn involves one to three
intramolecular cross-links. A-C, MALDI-TOF/TOF analysis of native
(—) and cross-linked (- - -) α-syn, showing that most
tTG-catalyzed cross-linking products of WT or disease-associated mutant forms
of α-syn are intramolecularly linked (predominant peak with two
cross-links), and up to three intramolecular cross-links can occur (left
shoulder). The abbreviations M and m/cl are
used to designate native and cross-linked α-synuclein, respectively.
D and E, kinetic analysis of α-syn (A30P)
cross-linking monitored by MALDI-TOF and SDS-PAGE. F, schematic
depiction of the tTG-catalyzed chemical reaction (isodipeptide formation)
between glutamine and lysine residues.In this study, we have identified the primary glutamine and lysine residues
involved in tTG-catalyzed, intramolecularly cross-linked monomeric α-syn
and investigated how cross-linking these residues affects the oligomerization,
fibrillization, and membrane binding of α-syn in vitro. Using
single-site mutagenesis and mass spectrometry applied to exhaustive
proteolytic digests of native and cross-linked monomeric α-syn, we
identified Gln109 and Gln79 as the major tTG substrates.
We demonstrate that the altered electrophoretic mobility of the
intramolecularly cross-linked α-syn in SDS-PAGE occurs as a result of
tTG-catalyzed cross-linking of Gln109 to lysine residues in the N
terminus of α-syn, which leads to the formation of more compact
monomers. Consistent with previous studies, we show that intramolecularly
cross-linked α-syn forms off-pathway oligomers that are distinct from
those formed by the wild-type protein and that do not convert to fibrils
within the time scale of our experiments (3-5 days). We also show that
membrane-bound α-syn is a substrate of tTG and that intramolecular
cross-linking does not interfere with the ability of monomeric α-syn to
adopt an α-helical conformation upon binding to synthetic membranes.
These studies provide novel mechanistic insight into the sequence and
structural basis of events that allow tTG to inhibit α-syn
fibrillogenesis, and they shed light on the potential role of tTG-catalyzed
cross-linking in modulating the physiological and pathogenic properties of
α-syn. 相似文献
11.
Linoleate (10R)-dioxygenase (10R-DOX) of Aspergillus
fumigatus was cloned and expressed in insect cells. Recombinant
10R-DOX oxidized 18:2n-6 to
(10R)-hydroperoxy-8(E),12(Z)-octadecadienoic acid
(10R-HPODE; ∼90%), (8R)-hydroperoxylinoleic acid
(8R-HPODE; ∼10%), and small amounts of
12S(13R)-epoxy-(10R)-hydroxy-(8E)-octadecenoic
acid. We investigated the oxygenation of 18:2n-6 at C-10 and C-8 by
site-directed mutagenesis of 10R-DOX and 7,8-linoleate diol synthase
(7,8-LDS), which forms ∼98% 8R-HPODE and ∼2%
10R-HPODE. The 10R-DOX and 7,8-LDS sequences differ in
homologous positions of the presumed dioxygenation sites (Leu-384/Val-330 and
Val-388/Leu-334, respectively) and at the distal site of the heme
(Leu-306/Val-256). Leu-384/Val-330 influenced oxygenation, as L384V and L384A
of 10R-DOX elevated the biosynthesis of 8-HPODE to 22 and 54%,
respectively, as measured by liquid chromatography-tandem mass spectrometry
analysis. The stereospecificity was also decreased, as L384A formed the
R and S isomers of 10-HPODE and 8-HPODE in a 3:2 ratio.
Residues in this position also influenced oxygenation by 7,8-LDS, as its V330L
mutant augmented the formation of 10R-HPODE 3-fold. Replacement of
Val-388 in 10R-DOX with leucine and phenylalanine increased the
formation of 8R-HPODE to 16 and 36%, respectively, whereas L334V of
7,8-LDS was inactive. Mutation of Leu-306 with valine or alanine had little
influence on the epoxyalcohol synthase activity. Our results suggest that
Leu-384 and Val-388 of 10R-DOX control oxygenation of
18:2n-6 at C-10 and C-8, respectively. The two homologous positions
of prostaglandin H synthase-1, Val-349 and Ser-353, are also critical for the
position and stereospecificity of the cyclooxygenase reaction.Linoleate diol synthases
(LDS)2 and linoleate
10R-DOX are fungal fatty acid dioxygenases of the myeloperoxidase
gene family
(1-3).
LDS have dual enzyme activities and transform 18:2n-6 sequentially to
8R-HPODE in an 8R-dioxygenase reaction and to 5,8-, 7,8-, or
8,11-DiHODE in hydroperoxide isomerase reactions. These oxylipins affect
sporulation, development, and pathogenicity of Aspergilli
(4-6).
Fatty acid dioxygenases of the myeloperoxidase gene family also occur in
vertebrates, plants, and algae
(7-9).
The most thoroughly investigated vertebrate enzymes are ovine PGHS-1 and mouse
PGHS-2 with known crystal structures
(10-12).
PGHS transforms 20:4n-6 to PGG2 in a cyclooxygenase and
PGG2 to PGH2 in a peroxidase reaction. Aspirin and other
nonsteroidal anti-inflammatory drugs inhibit the cyclooxygenase reaction. This
is of paramount medical importance
(13,
14), and PGHS-1 and -2 are
commonly known as COX-1 and -2
(15). α-DOX occur in
plants and algae, and biosynthesis of α-DOX in plants is elicited by
pathogens (7). α-DOX
oxidizes fatty acids to unstable (2R)-hydroperoxides, which readily
break down nonenzymatically to fatty acid aldehydes and CO2
(7).LDS, 10R-DOX, PGHS, and α-DOX oxygenate fatty acids to
different products, but their oxygenation mechanisms have mechanistic
similarities. Sequence alignment shows that many critical amino acid residues
for the cyclooxygenase reaction are conserved in LDS, 10R-DOX, and
α-DOX. These include the proximal histidine heme ligand, the distal
histidine, and the catalytic important tyrosine (Tyr-385) of PGHS-1. The
latter is oxidized to a tyrosyl radical, which initiates the cyclooxygenase
reaction by abstraction of the pro-S hydrogen at C-13 of
20:4n-6 (16). In
analogy, LDS and 10R-DOX catalyze stereospecific abstraction of the
pro-S hydrogen at C-8 of 18:2n-6
(3), whereas α-DOX
abstracts the pro-R hydrogen at C-2 of fatty acids
(17). Site-directed
mutagenesis of the conserved tyrosine homologues of Tyr-385 and proximal heme
ligands abolishes the dioxygenase activities of 7,8-LDS and α-DOX
(17,
18). The orientation of the
substrate at the dioxygenation site differs. The carboxyl groups of fatty
acids are positioned in a hydrophobic grove close to the tyrosine residue of
α-DOX (19). In contrast,
the ω ends of eicosanoic fatty acids are buried deep inside the
cyclooxygenase channel so that C-13 lies in the vicinity of Tyr-385
(20). Several observations
suggest that 18:2n-6 may also be positioned with its ω end
embedded in the interior of 7,8-LDS of Gaeumannomyces graminis
(18).7,8-LDS of G. graminis and Magnaporthe grisea and 5,8-LDS
of Aspergillus nidulans have been sequenced
(5,
8,
21). Gene targeting revealed
the catalytic properties of 5,8-LDS, 8,11-LDS, and 10R-DOX in
Aspergillus fumigatus and A. nidulans
(3). Homologous genes can be
found in other Aspergilli spp. Alignment of the two 7,8-LDS amino
acid sequences with 5,8-LDS, 8,11-LDS, and 10R-DOX sequences of five
Aspergilli revealed several conserved regions with single amino acid
differences between the enzymes with 8R-DOX and 10R-DOX
activities, as illustrated by the selected sequences in
Fig. 1. Leu-306, Leu-384, and
Val-388 of 10R-DOX are replaced in 5,8- and 7,8-LDS by valine,
valine, and leucine residues, respectively. Whether these amino acids are
important for the oxygenation mechanism is unknown, and this is one topic of
the present investigation. The predicted secondary structure of
10R-DOX suggests that Leu-384 of 10R-DOX can be present in
an α-helix with Val-388 close to its border. This α-helix is
homologous to helix 6 of PGHS-1, which contains Val-349 and Ser-353 at the
homologous positions of Leu-384 and Val-388
(Fig. 1).Open in a separate windowFIGURE 1.Alignments of partial amino acid sequences of five heme containing fatty
acid dioxgenases and a comparison of the predicted secondary structure of
10R-DOX with ovine PGHS-1. A, top, amino acids residues
at the presumed peroxidase and hydroperoxide isomerase sites. The last two
residues, His and Asn, are conserved in all myeloperoxidases
(1). Middle and
bottom, amino acid residues of the presumed dioxygenation sites are
shown. Conserved residues in all sequences are in boldface, and
mutated residues of 10R-DOX and/or 7,8-LDS are marked by an
asterisk. B, alignment of partial amino acid sequences of
10R-DOX with ovine PGHS-1, and a secondary structure prediction of
the 10R-DOX sequence. The secondary structure of 10R-DOX was
predicted by PSIPRED (43) and
the secondary structure of ovine PGHS-1 from its crystal structure (Protein
Data Bank code 1diy; cf. Ref
19). In short, our first
strategy for site-directed mutagenesis was to switch hydrophobic residues
between the enzymes with 10R- and 8R-DOX activities and to
assess the effects on the DOX and hydroperoxide isomerase activities
(10R-DOX/7,8-LDS: Leu-306/Val-256, Leu-384/Val-330, Val-388/Leu-334,
and Ala-426/Ile-375) and to switch one hydrophobic/charged residue
(Ala-435/Glu-384). Only catalytically active pairs would provide clear
information on their importance for the position of dioxygenation
(e.g. L384V of 10R-DOX and V330L of 7,8-LDS, both of which
were active). Unfortunately, replacements of 7,8-LDS often led to inactivation
or very low activity (e.g. V330A, V330M, I375A, E384A). Our second
strategy was to study replacements in two homologous positions of ovine PGHS-1
(Val-349 and Ser-353) with smaller and larger hydrophobic residues,
i.e. at Leu-384 and Val-388 of 10R-DOX. Abbreviations used
are as follows: oCOX-1, ovine cyclooxygenase-1; Af, A.
fumigatus; Gg, G. graminis. The GenBank™ protein sequences
were derived from , P05979, EAL89712, AAD49559, and EAL84400. The
amino acid sequences were aligned with the ClustalW algorithm (DNAStar).The overall three-dimensional structures of myeloperoxidases are conserved.
It is therefore conceivable that important residues for substrate binding in
the cyclooxygenase channel of PGHS could be conserved in LDS and
10R-DOX. The three-dimensional structure of ovine PGHS-1 shows that
Val-349 and Ser-353 are close to C-3 and C-4 of 20:4n-6, and residues
in these positions can alter both position and stereospecificity of
oxygenation
( ACL1417722-24).
Replacement of Val-349 of PGHS-1 with alanine increased the biosynthesis of
11R-HETE, whereas V349L decreased the generation of
11R-H(P)ETE and increased formation of
15(R/S)-H(P)ETE
(23,
25). V349I formed
PGG2 with 15R configuration
(22,
24). Replacement of Ser-353
with threonine reduced cyclooxygenase and peroxidase activities by over 50%
and increased the biosynthesis of 11R-HPETE and 15S-HPETE
4-5 times (23).There is little information on the hydroperoxide isomerase and peroxidase
sites of LDS (18,
26), but the latter could be
structurally related to the peroxidase site of PGHS. PGG2 and
presumably 8R-HPODE bind to the distal side of the heme group, which
can be delineated by hydrophobic amino acid residues
(27). Val-291 is one of these
residues, which form a dome over the distal heme side of COX-1. The V291A
mutant retained cyclooxygenase and peroxidase activities
(27). 5,8- and 7,8-LDS also
have valine residues in the homologous position, whereas 8,11-LDS and
10R-DOX have leucine residues
(Fig. 1). Whether these
hydrophobic residues are important for the peroxidase activities is
unknown.In this study we decided to compare the two catalytic sites of
10R-DOX of A. fumigatus and 7,8-LDS (EC 1.13.11.44) of
G. graminis (18). Our
first aim was to find a robust expression system for 10R-DOX of
A. fumigatus. The second objective was to determine whether
C16 and C20 fatty acid substrates enter the oxygenation
site of 10R-DOX “head” or “tail” first.
Unexpectedly, we found that 10R-DOX oxygenated 20:4n-6 by
hydrogen abstraction at both C-13 and C-10 with formation of two nonconjugated
and four cis-trans-conjugated HPETEs. Our third objective was to
investigate the structural differences between 10R-DOX and 7,8-LDS of
G. graminis, which could explain that oxygenation of 18:2n-6
mainly occurred at C-10 and at C-8, respectively. The strategy for
site-directed mutagenesis of 10R-DOX and 7,8-LDS is outlined in the
legend to Fig. 1; an alignment
of the amino acid sequences of 10R-DOX and 7,8-LDS is found in
supplemental material. 相似文献
12.
Yong Chen Chongguang Chen Evangelia Kotsikorou Diane L. Lynch Patricia H. Reggio Lee-Yuan Liu-Chen 《The Journal of biological chemistry》2009,284(3):1673-1685
We demonstrated previously that the protein GEC1 (glandular epithelial cell
1) bound to the human κ opioid receptor (hKOPR) and promoted cell
surface expression of the receptor by facilitating its trafficking along the
secretory pathway. Here we showed that three hKOPR residues
(Phe345, Pro346, and Met350) and seven GEC1
residues (Tyr49, Val51, Leu55,
Thr56, Val57, Phe60, and Ile64)
are indispensable for the interaction. Modeling studies revealed that the
interaction was mediated via direct contacts between the kinked hydrophobic
fragment in hKOPR C-tail and the curved hydrophobic surface in GEC1 around the
S2 β-strand. Intramolecular Leu44-Tyr109
interaction in GEC1 was important, likely by maintaining its structural
integrity. Microtubule binding mediated by the GEC1 N-terminal domain was
essential for the GEC1 effect. Expression of GEC1 also increased cell surface
levels of the GluR1 subunit and the prostaglandin EP3.f receptor, which have
FPXXM and FPXM sequences, respectively. With its widespread
distribution in the nervous system and its predominantly hydrophobic
interactions, GEC1 may have chaperone-like effects for many cell surface
proteins along the biosynthesis pathway.κ opioid receptor
(KOPR)2 is one of the
three major types of opioid receptors mediating effects of opioid drugs and
endogenous opioid peptides. Stimulation of KOPR generates many effects in
vivo, for example antinociception (especially for visceral chemical pain,
antipruritis, and water diuresis
(1). The KOPR agonist
nalfurafine (TRK-820) is used clinically in Sweden for the treatment of uremic
pruritus in kidney dialysis patients
(2). Because KOPR agonists
produce profound sedative effects, it has been proposed that KOPR agonists may
be useful in treating mania, antagonists as anti-depressants, and partial
agonists for the management of mania depression
(3). KOPR antagonists may also
be useful for curbing cocaine craving and as anti-anxiety drugs
(4,
5).KOPR, a member of the rhodopsin subfamily of the seven-transmembrane
receptor superfamily, is coupled preferentially to pertussis toxin-sensitive G
proteins, namely Gi/o proteins
(6). KOPR has been found to
interact with several non-G protein-binding partners, such as
Na+,H+-exchanger regulatory
factor-1/ezrin-radixin-moesin-binding phosphoprotein-50 and the δ opioid
receptor. These interactions have influence on signal transduction and
trafficking of the receptor
(7–9).
By yeast two-hybrid (Y2H) assay using the hKOPR C-tail to screen a human brain
cDNA library, we identified GEC1, also named GABAA
receptor-associated protein like 1 (GABARAPL1), to be a binding partner of
hKOPR (10).GEC1 cDNA was first cloned as an early estrogen-regulated mRNA from guinea
pig endometrial glandular epithelial cells by Pellerin et al.
(11). Subsequently, it was
cloned from other species, including human and house mouse
(12). Interestingly, the amino
acid sequences of GEC1 are completely conserved among all these species except
orangutan, in which Arg99 substitutes for His99.
Northern blot and immunoblotting analyses revealed that it has widespread
tissue distribution
(12–14).
In particular, GEC1 was found to be abundant in the central nervous system and
expressed throughout the rat brain
(14,
15). This wide tissue
distribution and the high sequence identity across species strongly suggest
that GEC1 has important biological functions in mammalian cells.Based on sequence similarity, GEC1 is classified as a member of
microtubule-associated proteins (MAPs), which also include GABAA
receptor-associated protein (GABARAP), Golgi-associated ATPase enhancer of 16
kDa (GATE16), GABARAP-like 3 (GABARAPL3), light chain 3 (LC3) of MAP 1A/1B,
and the yeast autophagy protein 8 (Atg8)
(12,
13). Among these homologues,
GEC1 share the highest identity with GABARAPL3 (93%), followed by GABARAP
(86%), GATE16 (61%), Atg8 (55%), and LC3 (∼30%).A growing body of evidence shows that this protein family is closely
related to two distinct biological functions. Studies mainly on GABARAP,
GATE16, and GEC1 indicate that they promote intracellular protein trafficking
by enhancing vesicle fusion
(10,
16–21).
In addition, they facilitate degradation of proteins and intracellular
organelles via autophagy-related pathways, which is bolstered largely by
research on Atg8 and LC3 (22,
23).We previously reported that GEC1 interacted with the hKOPR C-tail and
enhanced cell surface levels of hKOPR stably expressed in CHO cells. GEC1
expression enhances hKOPR expression through facilitating its anterograde
trafficking along the protein biosynthesis pathway without affecting
degradation of the receptor
(10). This represented the
first biological function reported for GEC1. Mansuy et al.
(24) demonstrated that GEC1
interacted with tubulin and promoted microtubule bundling in vitro,
and that green fluorescence protein-tagged GEC1 was localized in the
perinuclear vesicles with a scattered pattern. Our electron microscopic
studies in the rat brain showed that GEC1 was associated with ER, Golgi
apparatus, endosome-like vesicles, and plasma membranes and scattered in
cytoplasm in neurons (14). In
addition, N-ethylmaleimide-sensitive factor, a protein critical for
intracellular membrane-trafficking events, binds directly to GEC1
(10).In this study, we employed Y2H techniques to determine the amino acid
residues in both GEC1 and hKOPR C-tail involved in the interaction. Further
studies were then carried out in mammalian cells to examine if elimination of
the interaction affected the effect of GEC1 on hKOPR expression. In addition,
we generated a molecular model of GEC1 based on the x-ray crystal structure of
GABARAP and found that the residues involved in hKOPR binding formed
hydrophobic patches on the exterior surface of GEC1. Moreover, we found that
the cytosolic tail of AMPA receptor subunit GluR1 has the same FPXXM
motif as that found in the hKOPR C-tail to be involved in GEC1 binding and
that GEC1 expression up-regulated GluR1. 相似文献
13.
14.
In Escherichia coli, the periplasmic protein disulfide isomerase,
DsbC, is maintained reduced by transfer of electrons from cytoplasmic
thioredoxin-1 (Trx1) via the cytoplasmic membrane protein, DsbD. The
transmembrane domain of DsbD (DsbDβ), which comprises eight transmembrane
segments (TMs), contains two redox-active cysteines (Cys-163 and Cys-285),
each of which is water-exposed to both sides of the membrane. Cys-163 in TM1
and Cys-285 in TM4 can interact with cytoplasmic Trx1 and a periplasmic
Trx-like domain of DsbD, respectively. When Cys-163 and Cys-285 are
disulfide-bonded, the C-terminal halves of TM1 and TM4 are water-exposed,
whereas the N-terminal halves of these TMs are not. To assess possible
conformational changes of DsbDβ when its two cysteines are reduced, we
have determined the accessibility of portions of TM1 and TM4. We substituted
cysteines for amino acids in these TM segments and determined alkylation
accessibility. We find that the alkylation accessibility of single Cys
replacements in TM1 and TM4 is the same in oxidized and reduced DsbDβ,
indicating a relatively static conformation of DsbDβ between the two
redox states. We also find that the accessibility of amino acids of TM2 and
TM3 when Cys-163 and Cys-285 are oxidized or reduced shows no change.
Together, these results support a relatively static structure of DsbDβ in
the switch between the oxidized and the reduced state but raise the
possibility of conformational changes when interacting with Trx proteins. In
addition, we also find water-exposed residues in the cytoplasmic proximal
portion of TM3, allowing a more detailed characterization of the cavity in
DsbDβ.The cell envelope of most bacteria is an oxidizing environment. In many
bacteria, the main oxidant system consists of DsbA and DsbB. DsbA introduces
disulfide bonds into newly synthesized and secreted polypeptides containing
cysteines and is regenerated as an oxidative enzyme by the membrane protein
DsbB. Electrons are ultimately transferred from DsbB to the respiratory chain
(1–3).
However, there are also certain cell envelope proteins that require a
reductive enzyme to act on them. This is the case for those proteins that
contain multiple cysteines and that are often misoxidized by DsbA, thus
generating non-native disulfide bonds. The protein DsbC, a protein disulfide
isomerase, can promote rearrangement of such incorrect disulfide bonds,
resulting in a correctly folded protein
(4–7).
It does this either by using the reduced cysteine in its active site to
resolve non-native disulfide bonds and promoting the formation of the native
pairs or simply by reducing the substrate protein, which may be correctly
oxidized by DsbA and given a second chance
(8). In the latter mechanism,
DsbC becomes oxidized and must be reduced. This reduction is carried out by a
cytoplasmic membrane protein, DsbD, which receives electrons for this purpose
from thioredoxin-1
(Trx1)2 in the
cytoplasm (5,
9).DsbD is composed of three domains, each containing two redox-active
cysteines (Fig. 1). DsbDβ,
the membrane-embedded domain containing eight transmembrane segments (TMs),
receives electrons from Trx1 and then transfers them to the C-terminal
periplasmic domain, DsbDγ, which contains a Trx-like fold
(10–15).
The N-terminal periplasmic domain, DsbDα, which contains an
immunoglobulin-like fold, is then reduced by DsbDγ and transfers
electrons to DsbC (13,
16,
17).Open in a separate windowFIGURE 1.Electron transfer pathway through transmembrane domain (β) of DsbD
and its membrane topology predicted from the primary sequence. The
topology of DsbDβ was predicted using HMMTOP. The essential two cysteines
are shown in bold without a circle and numbered. The
residues indicated with a star in TM1 and TM4 are water-exposed when
the Cys-163 and Cys-285 are disulfide-bonded
(19). Studies on the residues
in TM2 and TM3 are shown in Fig.
4. The essential cysteines in the other domains (α and
γ) and interacting proteins (Trx1 and DsbC) are shown in a white
S (the sulfur of thiol) in gray circles. The tailless
arrows indicate where a signal sequence of DsbD and three hemagglutinin
(HA) epitopes are fused at the N terminus of DsbDβ, and a c-Myc
epitope is fused at the C terminus of DsbDβ. The figure in the
inset describes the model of DsbDβ, in which Cys-163 and Cys-285
form a disulfide bond in the middle of the protein and halves of C-terminal
TM1 and TM4 are water-exposed (black; C-TM1 and
C-TM4), whereas those of N-terminal ones are not (N-TM1 and
N-TM4).Electron transfer through the transmembrane domain DsbDβ is quite
unusual when compared with that of other membrane electron transport proteins.
Required extrinsic factors, for example, quinone, FAD, heme, or metal centers,
which are often used as cofactors for electron transfer, have not been found
(18). As a result, it is
proposed that thiol-disulfide exchange reactions alone promote the transfer of
electrons across the cytoplasmic membrane, utilizing the two cysteines,
Cys-163 and Cys-285 of DsbDβ. Evidence for this mechanism comes from the
detection of likely reaction intermediates including the Cys-163–Cys-285
disulfide and mixed disulfide complexes, Trx1-DsbDβ(Cys-163) and
DsbDβ(Cys-285)-DsbDγ
(13,
14,
19).We have previously studied the accessibility to the aqueous environment of
amino acids in TM1 and TM4 of DsbDβ, which contain Cys-163 and Cys-285,
respectively. Our results, in conjunction with a comparison of the amino acid
sequences of TM1–3 and TM4–6 of DsbDβ, suggest antiparallel
and pseudosymmetrical properties of TM1 and TM4
(19,
20). Cys-163 in TM1 and
Cys-285 in TM4 are water-exposed to both sides of the membrane when they are
in the reduced state and suggested to be located in the middle of the membrane
(helices). When Cys-163 and Cys-285 are disulfide-bonded, the proximal portion
of the cytoplasmic side of TM1 at the C terminus of Cys-163 and that of the
periplasmic side of TM4 at the C terminus of Cys-285 are highly water-exposed,
whereas the other portion of each TM is not. Therefore, we proposed an
hourglass-like model (Fig. 1,
inset) and suggested that the water-exposed halves of TM1 and TM4 are
cavity-located non-membrane-spanning helices and involved in the interactions
of Trx1 and DsbDγ, respectively.However, in our previous studies
(19), we did not determine
whether, when DsbDβ is in the reduced state, the arrangement of TM1 and
TM4 or other TMs would be similar to that seen when Cys-163 and Cys-285 are
disulfide-bonded. Doing this comparison is important because it has been
proposed that alternative exposure of the cysteines to the aqueous environment
depending on their redox states explains the electron transfer process across
the membrane (Refs.
21–23;
see “Discussion”). In addition, to further define the structural
features of DsbDβ, we wished to determine how TM segments other than TM1
and TM4 are arranged in terms of their water accessibility. In this study, we
examined the accessibility of many residues in TM1 and TM4, as well as
studying the arrangements of TM2 and TM3, using site-directed cysteine
alkylation in both redox states.Our data show that the four TMs studied, TM1, TM2, TM3, and TM4, have
similar accessibility properties whether DsbDβ is in the oxidized or
reduced state. We also find additional water-exposed residues in the proximal
portion of the cytoplasmic side of TM3. 相似文献
15.
Ajit K. Satapathy Donald J. Crampton Benjamin B. Beauchamp Charles C. Richardson 《The Journal of biological chemistry》2009,284(21):14286-14295
The multifunctional protein encoded by gene 4 of bacteriophage T7 (gp4)
provides both helicase and primase activity at the replication fork. T7 DNA
helicase preferentially utilizes dTTP to unwind duplex DNA in vitro
but also hydrolyzes other nucleotides, some of which do not support helicase
activity. Very little is known regarding the architecture of the nucleotide
binding site in determining nucleotide specificity. Crystal structures of the
T7 helicase domain with bound dATP or dTTP identified Arg-363 and Arg-504 as
potential determinants of the specificity for dATP and dTTP. Arg-363 is in
close proximity to the sugar of the bound dATP, whereas Arg-504 makes a
hydrogen bridge with the base of bound dTTP. T7 helicase has a serine at
position 319, whereas bacterial helicases that use rATP have a threonine in
the comparable position. Therefore, in the present study we have examined the
role of these residues (Arg-363, Arg-504, and Ser-319) in determining
nucleotide specificity. Our results show that Arg-363 is responsible for dATP,
dCTP, and dGTP hydrolysis, whereas Arg-504 and Ser-319 confer dTTP
specificity. Helicase-R504A hydrolyzes dCTP far better than wild-type
helicase, and the hydrolysis of dCTP fuels unwinding of DNA. Substitution of
threonine for serine 319 reduces the rate of hydrolysis of dTTP without
affecting the rate of dATP hydrolysis. We propose that different nucleotides
bind to the nucleotide binding site of T7 helicase by an induced fit
mechanism. We also present evidence that T7 helicase uses the energy derived
from the hydrolysis of dATP in addition to dTTP for mediating DNA
unwinding.Helicases are molecular machines that translocate unidirectionally along
single-stranded nucleic acids using the energy derived from nucleotide
hydrolysis
(1–3).
The gene 4 protein encoded by bacteriophage T7 consists of a helicase domain
and a primase domain, located in the C-terminal and N-terminal halves of the
protein, respectively (4). The
T7 helicase functions as a hexamer and has been used as a model to study
ring-shaped replicative helicases. In the presence of dTTP, T7 helicase binds
to single-stranded DNA
(ssDNA)3 as a hexamer
and translocates 5′ to 3′ along the DNA strand using the energy of
hydrolysis of dTTP
(5–7).
T7 helicase hydrolyzes a variety of ribo and deoxyribonucleotides; however,
dTTP hydrolysis is optimally coupled to DNA unwinding
(5).Most hexameric helicases use rATP to fuel translocation and unwind DNA
(3). T7 helicase does hydrolyze
rATP but with a 20-fold higher Km as compared with dTTP
(5,
8). It has been suggested that
T7 helicase actually uses rATP in vivo where the concentration of
rATP is 20-fold that of dTTP in the Escherichia coli cell
(8). However, hydrolysis of
rATP, even at optimal concentrations, is poorly coupled to translocation and
unwinding of DNA (9). Other
ribonucleotides (rCTP, rGTP, and rUTP) are either not hydrolyzed or the poor
hydrolysis observed is not coupled to DNA unwinding
(8). Furthermore, Patel et
al. (10) found that the
form of T7 helicase found in vivo, an equimolar mixture of the
full-length gp4 and a truncated form lacking the zinc binding domain of the
primase, prefers dTTP and dATP. Therefore, in the present study we have
restricted our examination of nucleotides to the deoxyribonucleotides.The nucleotide binding site of the replicative DNA helicases, such as T7
gene 4 protein, bind nucleotides at the subunit interface
(Fig. 1) located between two
RecA-like subdomains that bind ATP
(11,
12). The location of the
nucleotide binding site at the subunit interface provides multiple
interactions of residues with the bound NTP. A number of cis- and
trans-acting amino acids stabilize the bound nucleotide in the
nucleotide binding site and also provide for communication between subunits
(13–15).
Earlier reports revealed that the arginine finger (Arg-522) in T7 helicase is
positioned to interact with the γ-phosphate of the bound nucleotide in
the adjacent subunit (12,
16). However, His-465
(phosphate sensor), Glu-343 (catalytic base), and Asp-424 (Walker motif B)
interacts with the γ-phosphate of the bound nucleotide in the same
subunit (12,
17,
18). The arginine finger and
the phosphate sensor have been proposed to couple NTP hydrolysis to DNA
unwinding. Substitution of Glu-343, the catalytic base, eliminates dTTP
hydrolysis (19), and
substitution of Asp-424 with Asn leads to a severe reduction in dTTP
hydrolysis (20). The conserved
Lys-318 in Walker motif A interacts with the β-phosphate of the bound
nucleotide and plays an important role in dTTP hydrolysis
(21).Open in a separate windowFIGURE 1.Crystal structure of T7 helicase. A, crystal structure of
the hexameric helicase C-terminal domain of gp4
(17). The structure reveals a
ring-shaped molecule with a central core through which ssDNA passes. The
inset shows the interface between two subunits of the helicase with
adenosine 5′-{β,γ-imidol}-triphosphate in the nucleotide
binding site. B, the nucleotide binding site of a monomer of the gp4
with the crucial amino acid residues reported earlier and in the present study
is shown in sticks. The crystal structures of the T7 gene 4 helicase
domain (12) with bound dTTP
(C) and dATP (D). The structures shown are the nucleotide
binding site of T7 helicase as viewed in Pymol by analyzing the PDB files 1cr1
and 1cr2 (12). Arg-504 and
Tyr-535 sandwiches the base of the bound dNTP. Additionally, Arg-504 forms a
hydrogen bridge with dTTP. Arg-363 interacts specifically with the 3-OH group
of bound dATP. AMPPNP, adenosine
5′-(β,γ-imino)triphosphate.Considering the wealth of information on the above residues that are
involved in the hydrolysis of dTTP and the coupling of hydrolysis to
unwinding, it is intriguing that little information is available on nucleotide
specificity. Several crystal structures of T7 helicase in complex with a
nucleotide triphosphate are available. However, most of structures were
crystallized with a non-hydrolyzable analogue of dTTP or the nucleotide was
diffused into the crystal. The crystal structure of the T7 helicase domain
bound with dTTP or dATP was reported by Sawaya et al.
(12). These structures
assisted us in identifying two basic residues (Arg-363 and Arg-504) in close
proximity to the sugar and base of the bound nucleotide whose orientation
suggested that these residues could be involved in nucleotide selection.
Arg-504 together with Tyr-535 sandwich the base of the bound nucleotide at the
subunit interface of the hexameric helicase
(Fig. 1). Arg-504 and Tyr-535
are structurally well conserved in various helicases
(12). However, Arg-504 could
make a hydrogen bridge with the OH group of thymidine, thus suggesting a role
in dTTP specificity. On the other hand, Arg-363 is in close proximity
(∼3.4 Å) to the sugar 3′-OH of bound dATP, whereas in the
dTTP-bound structure this residue is displaced by 7.12 Å
(Fig. 1) from the equivalent
position. Consequently Arg-363 could play a role in dATP binding. The crystal
structures do not provide any information on different interaction of residues
with the phosphates of dATP and dTTP. However, alignment of the residues in
the P-loops of different hexameric helicases reveals that the serine adjacent
to the invariant lysine at position 319 (Ser-319) is conserved in
bacteriophages, whereas bacterial helicases have a conserved threonine in the
equivalent position (supplemental Fig. 1). Bacterial helicases use rATP in the
DNA unwinding reactions. whereas T7 helicase preferentially uses dTTP, and
bacteriophage T4 gene 41 uses rGTP or rATP
(22).Although considerable information is available on the role of residues in
nucleotide binding and dTTP hydrolysis, very little is known on the
determinants of nucleotide specificity. In the present study we made an
attempt to address the role of a few selected residues (Arg-363, Arg-504, and
Ser-319) in determining nucleotide specificity, especially dTTP and dATP, both
of which are hydrolyzed and mediate DNA unwinding. We show that under
physiological conditions T7 helicase uses the energy derived from the
hydrolysis of dATP in addition to dTTP for mediating DNA unwinding. 相似文献
16.
Michael J. Bailey Steven L. Coon David A. Carter Ann Humphries Jong-so Kim Qiong Shi Pascaline Gaildrat Fabrice Morin Surajit Ganguly John B. Hogenesch Joan L. Weller Martin F. Rath Morten M?ller Ruben Baler David Sugden Zoila G. Rangel Peter J. Munson David C. Klein 《The Journal of biological chemistry》2009,284(12):7606-7622
17.
Ishfaq Ahmed Sheikh Amit Kumar Singh Nagendra Singh Mau Sinha S. Baskar Singh Asha Bhushan Punit Kaur Alagiri Srinivasan Sujata Sharma Tej P. Singh 《The Journal of biological chemistry》2009,284(22):14849-14856
The crystal structure of the complex of lactoperoxidase (LPO) with its
physiological substrate thiocyanate (SCN–) has been
determined at 2.4Å resolution. It revealed that the
SCN– ion is bound to LPO in the distal heme cavity. The
observed orientation of the SCN– ion shows that the sulfur
atom is closer to the heme iron than the nitrogen atom. The nitrogen atom of
SCN– forms a hydrogen bond with a water (Wat) molecule at
position 6′. This water molecule is stabilized by two hydrogen bonds
with Gln423 Nε2 and Phe422 oxygen. In
contrast, the placement of the SCN– ion in the structure of
myeloperoxidase (MPO) occurs with an opposite orientation, in which the
nitrogen atom is closer to the heme iron than the sulfur atom. The site
corresponding to the positions of Gln423, Phe422 oxygen,
and Wat6′ in LPO is occupied primarily by the side chain of
Phe407 in MPO due to an entirely different conformation of the loop
corresponding to the segment Arg418–Phe431 of LPO.
This arrangement in MPO does not favor a similar orientation of the
SCN– ion. The orientation of the catalytic product
OSCN– as reported in the structure of
LPO·OSCN– is similar to the orientation of
SCN– in the structure of LPO·SCN–.
Similarly, in the structure of
LPO·SCN–·CN–, in which
CN– binds at Wat1, the position and orientation of
the SCN– ion are also identical to that observed in the
structure of LPO·SCN.Lactoperoxidase
(LPO4; EC 1.11.1.7) is
a Fe3+ heme enzyme that belongs to the mammalian peroxidase family
(1). The family of mammalian
peroxidases comprises lactoperoxidase
(2), eosinophil peroxidase
(3), thyroid peroxidase
(4), and myeloperoxidase (MPO)
(5). LPO, eosinophil
peroxidase, and MPO are responsible for antimicrobial function and innate
immune responses
(6–8),
whereas thyroid peroxidase plays a key role in thyroid hormone biosynthesis
(9). These peroxidases are
different from plant and fungal peroxidases because unlike plant and fungal
enzymes, the prosthetic heme group in mammalian peroxidases is covalently
linked to the protein (10).
There are also several striking structural and functional differences among
the mammalian peroxidases
(11). The heme group in MPO is
attached to the protein via three covalent linkages
(12), whereas LPO
(12,
13), eosinophil peroxidase
(12), and thyroid peroxidase
(12) contain only two ester
linkages. These covalent and various non-covalent linkages contribute
differentially to the high stability of the heme core as well as for the
peculiar values of their redox potentials
(2,
14). Furthermore, MPO consists
of two disulfide-linked protein chains, whereas LPO, eosinophil peroxidase,
and thyroid peroxidase are single chain proteins, although their chain lengths
differ greatly. In addition, their sequences contain several critical amino
acid differences that may also contribute to the variations in the
stereochemical environments of the substrate-binding sites. As a consequence
of these differences, the mammalian enzymes oxidize various inorganic ions
such as SCN–, Br–, Cl–, and
I– with differing specificities and potencies. Biochemical
studies have shown that LPO catalyzes preferentially the conversion of
SCN– to OSCN–
(15,
16), whereas MPO uses halides
(17,
18) with a preference for
chloride ion as the substrate. The preferences of eosinophil peroxidase and
thyroid peroxidase are bromide and iodide, respectively. However, the
stereochemical basis of the reported preferences for the substrates by
mammalian heme peroxidases is still unclear. So far, the structures of only
two mammalian enzymes, MPO and LPO, have been determined
(12,
13). It is of considerable
importance to identify the structural parameters that are responsible for the
subtle specificities. In the present work, we have attempted to address this
question through the new crystal structures of LPO complexes with
SCN– ions using goat, bovine, and buffalo lactoperoxidases.
Because the overall structures of complexes of SCN– with LPO
from all three species were found to be identical, the structure of the
complex of buffalo LPO with SCN– and the ternary complex with
SCN– and CN– will be discussed here, and
buffalo LPO will be termed hereafter as LPO. To highlight the factors
pertaining to binding specificity of SCN–, a comparison of
the structures of LPO·SCN– and
MPO·SCN– has also been made, revealing many valuable
differences pertaining to the observed orientations of the common substrate,
SCN– ion, when bound at the substrate-binding site in the
distal heme cavity of the two structures. The structures of
LPO·SCN– and MPO·SCN– clearly
show that the bound SCN– ions are present in the distal heme
cavity of two enzymes with opposite orientations. In the structure of
LPO·SCN–, the sulfur atom is closer to the heme iron
than the nitrogen atom, whereas in that of MPO·SCN–,
the nitrogen atom is closer to the heme iron than the sulfur atom. As a result
of this, the interactions of the SCN– ion in the distal site
of two proteins differ drastically. Gln423, a conserved water (Wat)
molecule at position 6′, and a well aligned carbonyl oxygen of
Phe422 in the proximity of the substrate-binding site in LPO
against a protruding Phe407 in MPO seem to play the key roles in
inducing the observed orientations of SCN– ions in LPO and
MPO. The structure of LPO·SCN– has also been compared
with the structure of its ternary complex with SCN– and
CN– ions. 相似文献
18.
Usha Padmanabhan D. Eric Dollins Peter C. Fridy John D. York C. Peter Downes 《The Journal of biological chemistry》2009,284(16):10571-10582
19.
Vishukumar Aimanianda C��cile Clavaud Catherine Simenel Thierry Fontaine Muriel Delepierre Jean-Paul Latg�� 《The Journal of biological chemistry》2009,284(20):13401-13412
Despite its essential role in the yeast cell wall, the exact composition of
the β-(1,6)-glucan component is not well characterized. While
solubilizing the cell wall alkali-insoluble fraction from a wild type strain
of Saccharomyces cerevisiae using a recombinant
β-(1,3)-glucanase followed by chromatographic characterization of the
digest on an anion exchange column, we observed a soluble polymer that eluted
at the end of the solvent gradient run. Further characterization indicated
this soluble polymer to have a molecular mass of ∼38 kDa and could be
hydrolyzed only by β-(1,6)-glucanase. Gas chromatographymass spectrometry
and NMR (1H and 13C) analyses confirmed it to be a
β-(1,6)-glucan polymer with, on average, branching at every fifth residue
with one or two β-(1,3)-linked glucose units in the side chain. This
polymer peak was significantly reduced in the corresponding digests from
mutants of the kre genes (kre9 and kre5) that are
known to play a crucial role in the β-(1,6)-glucan biosynthesis. In the
current study, we have developed a biochemical assay wherein incubation of
UDP-[14C]glucose with permeabilized S. cerevisiae yeasts
resulted in the synthesis of a polymer chemically identical to the branched
β-(1,6)-glucan isolated from the cell wall. Using this assay, parameters
essential for β-(1,6)-glucan synthetic activity were defined.The cell wall of Saccharomyces cerevisiae and other yeasts
contains two types of β-glucans. In the former yeast, branched
β-(1,3)-glucan accounts for ∼50–55%, whereas
β-(1,6)-glucan represents 10–15% of the total yeast cell wall
polysaccharides, each chain of the latter extending up to 140–350
glucose residues in length. The amount of 3,6-branched glucose residues varies
with the yeast species: 7, 15, and 75% in S. cerevisiae, Candida
albicans, and Schizosaccharomyces pombe, respectively
(1). β-(1,6)-Glucan
stabilizes the cell wall, since it plays a central role as a linker for
specific cell wall components, including β-(1,3)-glucan, chitin, and
mannoproteins (2,
3). However, the exact
structure of the β-(1,6)-glucan and the mode of biosynthesis of this
polymer are largely unknown. In S. pombe, immunodetection studies
suggested that synthesis of this polymer backbone begins in the endoplasmic
reticulum, with extension occurring in the Golgi
(4) and final processing at the
plasma membrane. In S. cerevisiae, Montijn and co-workers
(5), by immunogold labeling,
detected β-(1,6)-glucan at the plasma membrane, suggesting that the
synthesis takes place largely at the cell surface.More than 20 genes, including the KRE gene family (14 members) and
their homologues, SKN1 and KNH1, have been reported to be
involved in β-(1,6)-glucan synthesis in S. cerevisiae, C.
albicans, and Candida glabrata
(6–10).
Among all of these genes, the ones that seem to play the major synthetic role
are KRE5 and KRE9, since their disruption caused significant
reduction (100 and 80%, respectively, relative to wild type) in the cell wall
β-(1,6)-glucan content
(11–13).To date, the biochemical reaction responsible for the synthesis of
β-(1,6)-glucan and the product synthesized remained unknown. Indeed, in
most cases, when membrane preparations are incubated with UDP-glucose, only
linear β-(1,3)-glucan polymers are produced, although some studies have
reported the production of low amounts of β-(1,6)-glucans by membrane
preparations
(14–17).
These data suggest that disruption of the fungal cell prevents or at least has
a strong negative effect on β-(1,6)-glucan synthesis. The use of
permeabilized cells, which allows substrates, such as nucleotide sugar
precursors, to be readily transported across the plasma membrane, is an
alternative method to study in situ cell wall enzyme activities
(18–22).
A number of methods have been developed to permeabilize the yeast cell wall
(23), of which osmotic shock
was successfully used to demonstrate β-(1,3)-glucan and chitin synthase
activities (20,
24). Herein, we describe the
biochemical activity responsible for β-(1,6)-glucan synthesis using
permeabilized S. cerevisiae cells and UDP-[14C]glucose as
a substrate. We also have analyzed the physicochemical parameters of this
activity and chemically characterized the end product and its structural
organization within the mature yeast cell wall. 相似文献
20.
Xiuhong Zhai Margarita L. Malakhova Helen M. Pike Linda M. Benson H. Robert Bergen III Istv��n P. Sug��r Lucy Malinina Dinshaw J. Patel Rhoderick E. Brown 《The Journal of biological chemistry》2009,284(20):13620-13628
Glycolipid transfer proteins (GLTPs) are small, soluble proteins that
selectively accelerate the intermembrane transfer of glycolipids. The GLTP
fold is conformationally unique among lipid binding/transfer proteins and
serves as the prototype and founding member of the new GLTP superfamily. In
the present study, changes in human GLTP tryptophan fluorescence, induced by
membrane vesicles containing glycolipid, are shown to reflect glycolipid
binding when vesicle concentrations are low. Characterization of the
glycolipid-induced “signature response,” i.e. ∼40%
decrease in Trp intensity and ∼12-nm blue shift in emission wavelength
maximum, involved various modes of glycolipid presentation, i.e.
microinjection/dilution of lipid-ethanol solutions or phosphatidylcholine
vesicles, prepared by sonication or extrusion and containing embedded
glycolipids. High resolution x-ray structures of apo- and holo-GLTP indicate
that major conformational alterations are not responsible for the
glycolipid-induced GLTP signature response. Instead, glycolipid binding alters
the local environment of Trp-96, which accounts for ∼70% of total emission
intensity of three Trp residues in GLTP and provides a stacking platform that
aids formation of a hydrogen bond network with the ceramide-linked sugar of
the glycolipid headgroup. The changes in Trp signal were used to
quantitatively assess human GLTP binding affinity for various lipids including
glycolipids containing different sugar headgroups and homogenous acyl chains.
The presence of the glycolipid acyl chain and at least one sugar were
essential for achieving a low-to-submicromolar dissociation constant that was
only slightly altered by increased sugar headgroup complexity.Glycolipid transfer protein
(GLTP)4 is a soluble
(∼24-kDa) protein that selectively transfers glycosphingolipids (GSLs)
between membranes. GSLs play key roles in cell recognition, adhesion,
differentiation, proliferation, and programmed death in normal and disease
states
(1–8).
Phylogenetic/evolutionary analyses show GLTP to be highly conserved among
vertebrates
(9–11).
The conformational uniqueness of the GLTP fold when compared with other lipid
binding/transfer proteins
(12–14)
has resulted in GLTP being designated the prototype and founding member of the
GLTP superfamily (15,
16). GLTP employs a novel
two-layer “sandwich motif,” dominated by α-helices and
achieved without intramolecular disulfide bridges, to accommodate glycolipid
within a single lipid binding site and to form a membrane-interaction domain
that differs from other known membrane targeting/translocation domains,
i.e. C1, C2, PH, PX, and FYVE
(9,
13,
17–21).
The glycolipid binding site of GLTP consists of a sugar headgroup recognition
center that anchors the ceramide-linked sugar to the protein surface via
multiple hydrogen bonds and a hydrophobic tunnel that accommodates the
hydrocarbon chains of ceramide. The crystal structures of glycolipid-free GLTP
and of GLTP complexed with a half-dozen glycolipids differing in sugar
headgroup and/or lipid acyl composition reveal the basis for specific
recognition and adaptive accommodation of various GSLs. A conserved, concerted
sequence of events, initiated by anchoring of the GSL headgroup to the sugar
headgroup recognition center, seems to facilitate entry and exit of the lipid
chains in the membrane-associated state
(13). Glycolipid uptake occurs
via a cleft-like gating mechanism involving conformational changes to one
α-helix and two interhelical loops
(12). The selectivity of GLTP
for glycolipids makes this protein a prime candidate for molecular
manipulation of GSL-enriched microdomains in membranes as well as a potential
vehicle for selectively delivering glycolipids to cells. However, the binding
affinity of various glycolipids for GLTP and the time frame of GSL uptake by
GLTP remain unclear. In the present study, these issues are investigated using
fluorescence approaches.GLTP is intrinsically fluorescent by virtue of having 3 Trp and 10 Tyr
residues among its 209 amino acids. All 3 Trp residues reside on or near the
surface of GLTP
(12–14,
17,
22,
23), where they could help
form a membrane-interaction site. Only one, Trp-96, is directly involved in
glycolipid binding
(12–14).
Given the likely roles in membrane interaction and GSL binding, our goal was
to define the relative contributions of the Trp fluorescence changes caused by
membrane interaction versus glycolipid binding. A signature Trp
emission response, indicative of GSL binding by WT-GLTP, has been identified
and characterized using select GLTP point mutants and different modes of
glycolipid presentation, i.e. ethanol injection of pure GSLs and
titration with membrane vesicles (LUVs and SUVs) containing GSLs as minor
components. The signature Trp emission response has been used to
comprehensively assess the glycolipid binding affinity of the novel GLTP fold
for the first time, focusing on the impact of compositional variation of the
sugar headgroup and nonpolar acyl chain moieties of the glycolipid. 相似文献