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1.
Narinobu Juge Akiko Muroyama Miki Hiasa Hiroshi Omote Yoshinori Moriyama 《The Journal of biological chemistry》2009,284(50):35073-35078
The vesicular inhibitory amino acid transporter (VIAAT) is a synaptic vesicle protein responsible for the vesicular storage of γ-aminobutyrate (GABA) and glycine which plays an essential role in GABAergic and glycinergic neurotransmission. The transport mechanism of VIAAT remains largely unknown. Here, we show that proteoliposomes containing purified VIAAT actively took up GABA upon formation of membrane potential (Δψ) (positive inside) but not ΔpH. VIAAT-mediated GABA uptake had an absolute requirement for Cl− and actually accompanied Cl− movement. Kinetic analysis indicated that one GABA molecule and two Cl− equivalents were transported during one transport cycle. VIAAT in which Glu213 was specifically mutated to alanine completely lost the ability to take up both GABA and Cl−. Essentially the same results were obtained with glycine, another substrate of VIAAT. These results demonstrated that VIAAT is a vesicular Cl− transporter that co-transports Cl− with GABA or glycine in a Δψ dependent manner. It is concluded that Cl− plays an essential role in vesicular storage of GABA and glycine. 相似文献
2.
The nature of aromatic amino acid residues in Japanese-radish peroxidase a and the apoprotein was investigated by means of spectrophotometry and fluorospectrophotometry. The tyrosine residues in the holoenzyme were masked in the alkali-titration, giving an abnormally high value of 12.6, while they were exposed in the apoenzyme, exhibiting a value of 10.8. The difference spectra in the ultraviolet region between the holo-and apo-enzyme showed characteristic bands of tryptophan and phenylalanine as well as tyrosine. The perturbation of the aromatic amino acid residues by 50% ethyleneglycol was observed in the apoenzyme but not in the holoenzyme. The fluorophotometric experiments also revealed that the aromatic amino acid residues were in different environments in the holo- and apoenzyme. The difference between the conformation of peroxidase and that of the apoprotein was discussed. 相似文献
3.
4.
α-Aminoisobutyric acid is the only tertiary amino acid which is reported to occur in the proteins. Nevertheless, this amino acid has not been yet isolated from the proteins. Recently we succeeded in isolating this amino acid as white prismy crystalline substance from both acid and pepsin hydrolysate of horse hind leg muscle proteins, and this crystal was identified to be α-amino-isobutyric acid by elementary analysis, properties of this derivates, etc. 相似文献
5.
Kazuyuki Kitatani Kely Sheldon Vinodh Rajagopalan Viviana Anelli Russell W. Jenkins Ying Sun Gregory A. Grabowski Lina M. Obeid Yusuf A. Hannun 《The Journal of biological chemistry》2009,284(19):12972-12978
Activation of protein kinase C (PKC) promotes the salvage pathway of
ceramide formation, and acid sphingomyelinase has been implicated, in part, in
providing substrate for this pathway (Zeidan, Y. H., and Hannun, Y. A. (2007)
J. Biol. Chem. 282, 11549–11561). In the present study, we
examined whether acid β-glucosidase 1 (GBA1), which hydrolyzes
glucosylceramide to form lysosomal ceramide, was involved in PKC-regulated
formation of ceramide from recycled sphingosine. Glucosylceramide levels
declined after treatment of MCF-7 cells with a potent PKC activator, phorbol
12-myristate 13-acetate (PMA). Silencing GBA1 by small interfering RNAs
significantly attenuated acid glucocerebrosidase activity and decreased
PMA-induced formation of ceramide by 50%. Silencing GBA1 blocked PMA-induced
degradation of glucosylceramide and generation of sphingosine, the source for
ceramide biosynthesis. Reciprocally, forced expression of GBA1 increased
ceramide levels. These observations indicate that GBA1 activation can generate
the source (sphingosine) for PMA-induced formation of ceramide through the
salvage pathway. Next, the role of PKCδ, a direct effector of PMA, in
the formation of ceramide was determined. By attenuating expression of
PKCδ, cells failed to trigger PMA-induced alterations in levels of
ceramide, sphingomyelin, and glucosylceramide. Thus, PKCδ activation is
suggested to stimulate the degradation of both sphingomyelin and
glucosylceramide leading to the salvage pathway of ceramide formation.
Collectively, GBA1 is identified as a novel source of regulated formation of
ceramide, and PKCδ is an upstream regulator of this pathway.Sphingolipids are abundant components of cellular membranes, many of which
are emerging as bioactive lipid mediators thought to play crucial roles in
cellular responses (1,
2). Ceramide, a central
sphingolipid, serves as the main precursor for various sphingolipids,
including glycosphingolipids, gangliosides, and sphingomyelin. Regulation of
formation of ceramide has been demonstrated through the action of three major
pathways: the de novo pathway
(3,
4), the sphingomyelinase
pathway (5), and the salvage
pathway
(6–8).
The latter plays an important role in constitutive sphingolipid turnover by
salvaging long-chain sphingoid bases (sphingosine and dihydrosphingosine) that
serve as sphingolipid backbones for ceramide and dihydroceramide as well as
all complex sphingolipids (Fig.
1A).Open in a separate windowFIGURE 1.The scheme of the sphingosine salvage pathway of ceramide formation and
inhibition of PMA induction of ceramide by fumonisin B1. A, the
scheme of the sphingosine salvage pathway of ceramide formation. B,
previously published data as to effects of fumonisin B1 on ceramide mass
profiles (23) are re-plotted
as a PMA induction of ceramide. In brief, MCF-7 cells were pretreated with or
without 100 μm fumonisin B1 for 2 h followed by treatment with
100 nm PMA for 1 h. Lipids were extracted, and then the levels of
ceramide species were determined by high-performance liquid
chromatography-tandem mass spectrometry. Results are expressed as sum of
increased mass of ceramide species. Dotted or open columns
represents C16-ceramide or sum of other ceramide species
(C14-ceramide, C18-ceramide, C18:1-ceramide,
C20-ceramide, C24-ceramide, and
C24:1-ceramide), respectively. The data represent mean ±
S.E. of three to five values.Metabolically, ceramide is also formed from degradation of
glycosphingolipids (Fig.
1A) usually in acidic compartments, the lysosomes and/or
late endosomes (9). The
stepwise hydrolysis of complex glycosphingolipids eventually results in the
formation of glucosylceramide, which in turn is converted to ceramide by the
action of acid β-glucosidase 1
(GBA1)2
(9,
10). Severe defects in GBA1
activity cause Gaucher disease, which is associated with aberrant accumulation
of the lipid substrates
(10–14).
On the other hand, sphingomyelin is cleaved by acid sphingomyelinase to also
form ceramide (15,
16). Either process results in
the generation of lysosomal ceramide that can then be deacylated by acid
ceramidase (17), releasing
sphingosine that may escape the lysosome
(18). The released sphingosine
may become a substrate for either sphingosine kinases or ceramide synthases,
forming sphingosine 1-phosphate or ceramide, respectively
(3,
19–21).In a related line of investigation, our studies
(20,
22,
23) have begun to implicate
protein kinase Cs (PKC) as upstream regulators of the sphingoid base salvage
pathway resulting in ceramide synthesis. Activation of PKCs by the phorbol
ester (PMA) was shown to stimulate the salvage pathway resulting in increases
in ceramide. All the induced ceramide was inhibited by pretreatment with a
ceramide synthase inhibitor, fumonisin B1, but not by myriocin, thus negating
acute activation of the de novo pathway and establishing a role for
ceramide synthesis (20,
23). Moreover, labeling
studies also implicated the salvage pathway because PMA induced turnover of
steady state-labeled sphingolipids but did not affect de novo labeled
ceramide in pulse-chase experiments.Moreover, PKCδ, among PKC isoforms, was identified as an upstream
molecule for the activation of acid sphingomyelinase in the salvage pathway
(22). Interestingly, the
PKCδ isoform induced the phosphorylation of acid sphingomyelinase at
serine 508, leading to its activation and consequent formation of ceramide.
The activation of acid sphingomyelinase appeared to contribute to ∼50% of
the salvage pathway-induced increase in ceramide
(28) (also, see
Fig. 4C). This raised
the possibility that distinct routes of ceramide metabolism may account for
the remainder of ceramide generation. In this study, we investigated
glucocerebrosidase GBA1 as a candidate for one of the other routes accounting
for PKC-regulated salvage pathway of ceramide formation.Open in a separate windowFIGURE 4.Effects of knockdown of lysosomal enzymes on the generation of ceramide
after PMA treatment. A, MCF-7 cells were transfected with 5
nm siRNAs of each of four individual sequences (SCR, GBA1-a,
GBA1-b, and GBA1-c) for 48 h and then stimulated with 100 nm PMA
for 1 h. Lipids were extracted, and then the levels of the
C16-ceramide species were determined by high-performance liquid
chromatography-tandem mass spectrometry. The data represent mean ± S.E.
of three to nine values. B, MCF-7 cells were transfected with 5
nm siRNAs of SCR or GBA1-a (GBA1) for 48 h and then stimulated with
100 nm PMA for 1 h. Lipids were extracted, and then the levels of
individual ceramide species were determined by high-performance liquid
chromatography-tandem mass spectrometry. The data represent mean ± S.E.
of three to five values. C14-Cer,
C14-ceramide; C16-Cer,
C16-ceramide; C18-Cer;
C18-ceramide; C18:1-Cer,
C18:1-ceramide; C20-Cer,
C20-ceramide; C20-Cer,
C24-ceramide; C24:1-Cer,
C24:1-ceramide. C, MCF-7 cells were transfected with 5
nm siRNAs of SCR, acid sphingomyelinase (ASM), or GBA1-a
(GBA1) for 48 h following stimulation with (PMA) or without
(Control) 100 nm PMA for 1 h. Lipids were extracted, and
then the levels of ceramide species were determined by high-performance liquid
chromatography-tandem mass spectrometry. Levels of C16-ceramide are
shown. The data represent mean ± S.E. of four to five values.
Significant changes from SCR-transfected cells treated with PMA are shown in
A–C (*, p < 0.02; **,
p < 0.05; ***, p < 0.01). 相似文献
6.
Omar A. Ramírez René L. Vidal Judith A. Tello Karina J. Vargas Stefan Kindler Steffen H?rtel Andrés Couve 《The Journal of biological chemistry》2009,284(19):13077-13085
Understanding the mechanisms that control synaptic efficacy through the
availability of neurotransmitter receptors depends on uncovering their
specific intracellular trafficking routes. γ-Aminobutyric acid type B
(GABAB) receptors (GABABRs) are obligatory heteromers
present at dendritic excitatory and inhibitory postsynaptic sites. It is
unknown whether synthesis and assembly of GABABRs occur in the
somatic endoplasmic reticulum (ER) followed by vesicular transport to
dendrites or whether somatic synthesis is followed by independent transport of
the subunits for assembly and ER export throughout the somatodendritic
compartment. To discriminate between these possibilities we studied the
association of GABABR subunits in dendrites of hippocampal neurons
combining live fluorescence microscopy, biochemistry, quantitative
colocalization, and bimolecular fluorescent complementation. We demonstrate
that GABABR subunits are segregated and differentially mobile in
dendritic intracellular compartments and that a high proportion of
non-associated intracellular subunits exist in the brain. Assembled heteromers
are preferentially located at the plasma membrane, but blockade of ER exit
results in their intracellular accumulation in the cell body and dendrites. We
propose that GABABR subunits assemble in the ER and are exported
from the ER throughout the neuron prior to insertion at the plasma membrane.
Our results are consistent with a bulk flow of segregated subunits through the
ER and rule out a post-Golgi vesicular transport of preassembled
GABABRs.The efficacy of synaptic transmission depends on the intracellular
trafficking of neurotransmitter receptors
(1,
2). The trafficking of
glutamatergic and
GABAA6
receptors has been extensively studied, and their implications for synaptic
plasticity have been well documented
(3,
4). For example, differential
trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
(AMPA) receptors modifies synaptic strength and influences
experience-dependent plasticity in vivo
(5). The molecular mechanisms
that govern the trafficking of metabotropic GABABRs and their
consequences for synaptic inhibition remain less clear. In particular, limited
information is available regarding the relationship between the trafficking of
GABABRs and the topological complexity of the secretory pathway in
neurons.GABABRs mediate the slow component of synaptic inhibition by
acting on pre- and postsynaptic targets
(6–8).
They are implicated in epilepsy, anxiety, stress, sleep disorders,
nociception, depression, and cognition
(9). They also represent
attractive targets for the treatment of withdrawal symptoms from drugs of
addiction such as cocaine
(10). They are obligatory
heteromers composed of GABABR1 and GABABR2 subunits.
GABABR1 contains an RXR-type sequence in the intracellular
C-terminal domain that functions as an ER retention motif
(11,
12). The ER retention sequence
is masked upon assembly with GABABR2 resulting in the appearance of
functional receptors at the plasma membrane. Only GABABR1 binds
GABA with high affinity, whereas G protein signaling is exclusively mediated
by the second and third intracellular loops of GABABR2
(13–15).
GABABRs are located in dendrites and axons, but their distribution
does not coincide with the active zone or the postsynaptic density. Rather,
they are adjacent to both compartments constituting perisynaptic receptors
(16,
17).If GABABR subunits are synthesized in the soma, at least two
possibilities exist for their anterograde transport, assembly, and insertion
in dendrites. First, the subunits may be synthesized in the cell body,
assembled in the somatic ER, and targeted preassembled in post-Golgi vesicles
to their site of insertion in dendrites. Alternatively, they may be
synthesized in the soma and transported through the ER membrane as
non-heteromeric subunits. In the latter scenario, newly assembled receptors
may exit the ER throughout the somatodendritic compartment prior to insertion
at the plasma membrane and diffuse laterally for retention at functional
sites. No evidence exists to discriminate between these possibilities. We
reasoned that a prevalence of associated subunits in post-Golgi vesicles in
dendrites would favor the first alternative, whereas the existence of
non-associated subunits in intracellular compartments would support a
somatodendritic assembly mechanism. Here we explore the presence of associated
GABABR subunits using fluorescence recovery after photobleaching
(FRAP), biochemistry, and quantitative colocalization. In addition, we report
for the first time the use of BiFC
(18) to study
GABABR assembly in neurons. Our results demonstrate that
GABABR subunits are differentially mobile in dendrites and that a
high proportion of non-associated subunits prevail in an intracellular
fraction of the adult brain. They also show that GABABR subunits
are heteromeric at the plasma membrane but segregated in intracellular
compartments of dendrites of hippocampal neurons. Importantly, treatment with
brefeldin A (BFA) or interference of the coatomer protein complex II impair ER
export and result in the accumulation of assembled subunits in intracellular
compartments throughout the somatodendritic arbor. We conclude that
GABABR subunits are synthesized in the soma and remain segregated
in intracellular compartments prior to somatodendritic assembly. Our
observations rule out a post-Golgi vesicular transport of preassembled
GABABRs and suggest an alternative mechanism of receptor
targeting. 相似文献
7.
8.
Raiford DW Heizer EM Miller RV Akashi H Raymer ML Krane DE 《Journal of molecular evolution》2008,67(6):621-630
Prokaryotic organisms preferentially utilize less energetically costly amino acids in highly expressed genes. Studies have
shown that the proteome of Saccharomyces cerevisiae also exhibits this behavior, but only in broad terms. This study examines the question of metabolic efficiency as a proteome-shaping
force at a finer scale, examining whether trends consistent with cost minimization as an evolutionary force are present independent
of protein function and amino acid physicochemical property, and consistently with respect to amino acid biosynthetic costs.
Inverse correlations between the average amino acid biosynthetic cost of the protein product and the levels of gene expression
in S. cerevisiae are consistent with natural selection to minimize costs. There are, however, patterns of amino acid usage that raise questions
about the strength (and possibly the universality) of this selective force in shaping S. cerevisiae’s proteome. 相似文献
9.
Willett CS 《Journal of molecular evolution》2000,50(2):175-183
Convergence in amino acid sequences between proteins can be strong evidence for selection. Here, I look for evidence of convergence
in the amino acid sequences of pheromone binding protein (PBP) in response to convergence in pheromones. PBPs are involved
in sex pheromone reception by the antennae of male moths. In this role PBPs may selectively bind pheromone components and
experience convergent selection in response to convergence in pheromone components. However, examination of the PBPs of the
taxa that have converged upon the use of (E)- or (Z)-11-tetradecenyl acetate as their major pheromone component reveals little evidence for convergence in the PBPs identified
from these taxa. A few sites show a pattern consistent with convergence or parallelism; however, it cannot be ruled out that
these sites share the ancestral state. Two of these sites fall within the proposed binding region of PBPs. These results suggest
that PBPs either have not converged in sequence or have converged at very few sites in response to convergence on the same
pheromone component.
Received: 29 July 1999 / Accepted: 8 November 1999 相似文献
10.
We studied 10 protein-coding mitochondrial genes from 19 mammalian species to evaluate the effects of 10 amino acid properties
on the evolution of the genetic code, the amino acid composition of proteins, and the pattern of nonsynonymous substitutions.
The 10 amino acid properties studied are the chemical composition of the side chain, two polarity measures, hydropathy, isoelectric
point, volume, aromaticity, aliphaticity, hydrogenation, and hydroxythiolation. The genetic code appears to have evolved toward
minimizing polarity and hydropathy but not the other seven properties. This can be explained by our finding that the presumably
primitive amino acids differed much only in polarity and hydropathy, but little in the other properties. Only the chemical
composition (C) and isoelectric point (IE) appear to have affected the amino acid composition of the proteins studied, that
is, these proteins tend to have more amino acids with typical C and IE values, so that nonsynonymous mutations tend to result
in small differences in C and IE. All properties, except for hydroxythiolation, affect the rate of nonsynonymous substitution,
with the observed amino acid changes having only small differences in these properties, relative to the spectrum of all possible
nonsynonymous mutations.
Received: 2 January 1998 / Accepted: 25 April 1998 相似文献
11.
Gonzales AL Lee W Spencer SR Oropeza RA Chapman JV Ku JY Eskandari S 《The Journal of membrane biology》2007,220(1-3):33-51
We combined electrophysiological and freeze-fracture methods to estimate the unitary turnover rate of the γ-aminobutyric acid
(GABA) transporter GAT1. Human GAT1 was expressed in Xenopus laevis oocytes, and individual cells were used to measure and correlate the macroscopic rate of GABA transport and the total number
of transporters in the plasma membrane. The two-electrode voltage-clamp method was used to measure the transporter-mediated
macroscopic current evoked by GABA (
), macroscopic charge movements (Q
NaCl) evoked by voltage pulses and whole-cell capacitance. The same cells were then examined by freeze-fracture and electron microscopy
in order to estimate the total number of GAT1 copies in the plasma membrane. GAT1 expression in the plasma membrane led to
the appearance of a distinct population of 9-nm freeze-fracture particles which represented GAT1 dimers. There was a direct
correlation between Q
NaCl and the total number of transporters in the plasma membrane. This relationship yielded an apparent valence of 8 ± 1 elementary
charges per GAT1 particle. Assuming that the monomer is the functional unit, we obtained 4 ± 1 elementary charges per GAT1
monomer. This information and the relationship between and Q
NaCl were used to estimate a GAT1 unitary turnover rate of 15 ± 2 s−1 (21°C, −50 mV). The temperature and voltage dependence of GAT1 were used to estimate the physiological turnover rate to be 79–93 s−1 (37°C, −50 to −90 mV). 相似文献
12.
Effects of Amino Acid Substitutions at the Active Site in Escherichia Coli β-Galactosidase 总被引:2,自引:2,他引:0
Forty-nine amino acid substitutions were made at four positions in the Escherichia coli enzyme β-galactosidase; three of the four targeted amino acids are thought to be part of the active site. Many of the substitutions were made by converting the appropriate codon in lacZ to an amber codon, and using one of 12 suppressor strains to introduce the replacement amino acid. Glu-461 and Tyr-503 were replaced, independently, with 13 amino acids. All 26 of the strains containing mutant enzymes are Lac(-). Enzyme activity is reduced to less than 10% of wild type by substitutions at Glu-461 and to less than 1% of wild type by substitutions at Tyr-503. Many of the mutant enzymes have less than 0.1% wild-type activity. His-464 and Met-3 were replaced with 11 and 12 amino acids, respectively. Strains containing any one of these mutant proteins are Lac(+). The results support previous evidence that Glu-461 and Tyr-503 are essential for catalysis, and suggest that His-464 is not part of the active site. Site-directed mutagenesis was facilitated by construction of an f1 bacteriophage containing the complete lacZ gene on a single EcoRI fragment. 相似文献
13.
14.
Samir K. Maji Rachel R. Ogorzalek Loo Mohammed Inayathullah Sean M. Spring Sabrina S. Vollers Margaret M. Condron Gal Bitan Joseph A. Loo David B. Teplow 《The Journal of biological chemistry》2009,284(35):23580-23591
Understanding the structural and assembly dynamics of the amyloid β-protein (Aβ) has direct relevance to the development of therapeutic agents for Alzheimer disease. To elucidate these dynamics, we combined scanning amino acid substitution with a method for quantitative determination of the Aβ oligomer frequency distribution, photo-induced cross-linking of unmodified proteins (PICUP), to perform “scanning PICUP.” Tyr, a reactive group in PICUP, was substituted at position 1, 10, 20, 30, or 40 (for Aβ40) or 42 (for Aβ42). The effects of these substitutions were probed using circular dichroism spectroscopy, thioflavin T binding, electron microscopy, PICUP, and mass spectrometry. All peptides displayed a random coil → α/β → β transition, but substitution-dependent alterations in assembly kinetics and conformer complexity were observed. Tyr1-substituted homologues of Aβ40 and Aβ42 assembled the slowest and yielded unusual patterns of oligomer bands in gel electrophoresis experiments, suggesting oligomer compaction had occurred. Consistent with this suggestion was the observation of relatively narrow [Tyr1]Aβ40 fibrils. Substitution of Aβ40 at the C terminus decreased the population conformational complexity and substantially extended the highest order of oligomers observed. This latter effect was observed in both Aβ40 and Aβ42 as the Tyr substitution position number increased. The ability of a single substitution (Tyr1) to alter Aβ assembly kinetics and the oligomer frequency distribution suggests that the N terminus is not a benign peptide segment, but rather that Aβ conformational dynamics and assembly are affected significantly by the competition between the N and C termini to form a stable complex with the central hydrophobic cluster.Alzheimer disease (AD)4 is the most common cause of late-life dementia (1) and is estimated to afflict more than 27 million people worldwide (2). An important etiologic hypothesis is that amyloid β-protein (Aβ) oligomers are the proximate neurotoxins in AD. Substantial in vivo and in vitro evidence supports this hypothesis (3–12). Neurotoxicity studies have shown that Aβ assemblies are potent neurotoxins (5, 13–20), and the toxicity of some oligomers can be greater than that of the corresponding fibrils (21). Soluble Aβ oligomers inhibit hippocampal long term potentiation (4, 5, 13, 15, 17, 18, 22) and disrupt cognitive function (23). Compounds that bind and disrupt the formation of oligomers have been shown to block the neurotoxicity of Aβ (24, 25). Importantly, recent studies in higher vertebrates (dogs) have shown that substantial reduction in amyloid deposits in the absence of decreases in oligomer concentration has little effect on recovery of neurological function (26).Recent studies of Aβ oligomers have sought to correlate oligomer size and biological activity. Oligomers in the supernatants of fibril preparations centrifuged at 100,000 × g caused sustained calcium influx in rat hippocampal neurons, leading to calpain activation and dynamin 1 degradation (27). Aβ-derived diffusible ligand-like Aβ42 oligomers induced inflammatory responses in cultured rat astrocytes (28). A 90-kDa Aβ42 oligomer (29) has been shown to activate ERK1/2 in rat hippocampal slices (30) and bind avidly to human cortical neurons (31), in both cases causing apoptotic cell death. A comparison of the time dependence of the toxic effects of the 90-kDa assembly with that of Aβ-derived diffusible ligands revealed a 5-fold difference, Aβ-derived diffusible ligands requiring more time for equivalent effects (31). A 56-kDa oligomer, “Aβ*56,” was reported to cause memory impairment in middle-aged transgenic mice expressing human amyloid precursor protein (32). A nonamer also had adverse effects. Impaired long term potentiation in rat brain slices has been attributed to Aβ trimers identified in media from cultured cells expressing human amyloid precursor protein (33). Dimers and trimers from this medium also have been found to cause progressive loss of synapses in organotypic rat hippocampal slices (10). In mice deficient in neprilysin, an enzyme that has been shown to degrade Aβ in vivo (34), impairment in neuronal plasticity and cognitive function correlated with significant increases in Aβ dimer levels and synapse-associated Aβ oligomers (35).The potent pathologic effects of Aβ oligomers provide a compelling reason for elucidating the mechanism(s) of their formation. This has been a difficult task because of the metastability and polydispersity of Aβ assemblies (36). To obviate these problems, we introduced the use of the method of photo-induced cross-linking of unmodified proteins (PICUP) to rapidly (<1 s) and covalently stabilize oligomer mixtures (for reviews see Refs. 37, 38). Oligomers thus stabilized no longer exist in equilibrium with monomers or each other, allowing determination of oligomer frequency distributions by simple techniques such as SDS-PAGE (37). Recently, to obtain population-average information on contributions to fibril formation of amino acid residues at specific sites in Aβ, we employed a scanning intrinsic fluorescence approach (39). Tyr was used because it is a relatively small fluorophore, exists natively in Aβ, and possesses the side chain most reactive in the PICUP chemistry (40). Using this approach, we found that the central hydrophobic cluster region (Leu17–Ala21) was particularly important in controlling fibril formation of Aβ40, whereas the C terminus was the predominant structural element controlling Aβ42 assembly (39). Here we present results of studies in which key strategic features of the two methods have been combined to enable execution of “scanning PICUP” and the consequent revelation of site-specific effects on Aβ oligomerization. 相似文献
15.
Lisa M. Petti Kristina Talbert-Slagle Megan L. Hochstrasser Daniel DiMaio 《The Journal of biological chemistry》2013,288(38):27273-27286
Receptors for PDGF play an important role in cell proliferation and migration and have been implicated in certain cancers. The 44-amino acid E5 protein of bovine papillomavirus binds to and activates the PDGFβ receptor (PDGFβR), resulting in oncogenic transformation of cultured fibroblasts. Previously, we isolated an artificial 36-amino acid transmembrane protein, pTM36-4, which transforms cells because of its ability to activate the PDGFβR despite limited sequence similarity to E5. Here, we demonstrated complex formation between the PDGFβR and three pTM36-4 mutants: T21E, T21Q, and T21N. T21Q retained wild type transforming activity and activated the PDGFβR in a ligand-independent manner as a consequence of binding to the transmembrane domain of the PDGFβR, but T21E and T21N were severely defective. In fact, T21N substantially inhibited E5-induced PDGFβR activation and transformation in both mouse and human fibroblasts. T21N did not prevent E5 from binding to the receptor, and genetic evidence suggested that T21N and E5 bind to nonidentical sites in the transmembrane domain of the receptor. T21N also inhibited transformation and PDGFβR activation induced by v-Sis, a viral homologue of PDGF-BB, as well as PDGF-induced mitogenesis and signaling by preventing phosphorylation of the PDGFβR at particular tyrosine residues. These results demonstrated that T21N acts as a novel inhibitor of the PDGFβR and validated a new strategy for designing highly specific short transmembrane protein inhibitors of growth factor receptors and possibly other transmembrane proteins. 相似文献
16.
Eoin Barrett Patrick Fitzgerald Timothy G. Dinan John F. Cryan R. Paul Ross Eamonn M. Quigley Fergus Shanahan Barry Kiely Gerald F. Fitzgerald Paul W. O'Toole Catherine Stanton 《PloS one》2012,7(11)
The aim of this study was to compare the impact of dietary supplementation with a Bifidobacterium breve strain together with linoleic acid & α-linolenic acid, for 7 weeks, on colonic sensitivity and fatty acid metabolism in rats. Maternally separated and non-maternally separated Sprague Dawley rats (n = 15) were orally gavaged with either B. breve DPC6330 (109 microorganisms/day) alone or in combination with 0.5% (w/w) linoleic acid & 0.5% (w/w) α-linolenic acid, daily for 7 weeks and compared with trehalose and bovine serum albumin. Tissue fatty acid composition was assessed by gas-liquid chromatography and visceral hypersensitivity was assessed by colorectal distension. Significant differences in the fatty acid profiles of the non-separated controls and maternally separated controls were observed for α-linolenic acid and arachidonic acid in the liver, oleic acid and eicosenoic acid (c11) in adipose tissue, and for palmitoleic acid and docosahexaenoic acid in serum (p<0.05). Administration of B. breve DPC6330 to MS rats significantly increased palmitoleic acid, arachidonic acid and docosahexaenoic acid in the liver, eicosenoic acid (c11) in adipose tissue and palmitoleic acid in the prefrontal cortex (p<0.05), whereas feeding B. breve DPC6330 to non separated rats significantly increased eicosapentaenoic acid and docosapentaenoic acid in serum (p<0.05) compared with the NS un-supplemented controls. Administration of B. breve DPC6330 in combination with linoleic acid and α-linolenic acid to maternally separated rats significantly increased docosapentaenoic acid in the serum (p<0.01) and α-linolenic acid in adipose tissue (p<0.001), whereas feeding B. breve DPC6330 with fatty acid supplementation to non-separated rats significantly increased liver and serum docosapentaenoic acid (p<0.05), and α-linolenic acid in adipose tissue (p<0.001). B. breve DPC6330 influenced host fatty acid metabolism. Administration of B. breve DPC6330 to maternally separated rats significantly modified the palmitoleic acid, arachidonic acid and docosahexaenoic acid contents in tissues. The effect was not observed in non-separated animals. 相似文献
17.
Masaji Ogura Eiichi Katsunuma Tomoko Akabane Seiichi Ogawa 《Bioscience, biotechnology, and biochemistry》2013,77(1):235-236
An aminopeptidase was purified from an aqueous extract of mullet roe in the presence of 2-mercaptoethanol by fractionation with ammonium sulfate and column chromatography on DEAE-cellulose and Sephadex G-200. The molecular weight of the enzyme was 184,000 by gel filtration, and the enzyme appeared to consist of two homogenous subunits. The optimal pH and optimal temperature for activity were 7.4 and 45°C, respectively. Puromycin, p-chloromercuribenzoic acid, and o-phenanthroline inhibited the enzyme n on-competitively (their Ki = 1.34 μm, 0.113mm and 0.145 mm, respectively), while 2-mercaptoethylamine was competitive (Ki = 0.056 mm). The enzyme was also inhibited by l-amino acids, in particular glutamic acid. The enzyme could hydrolyze a variety of α-aminoacyl β-naphthylamides and was most active on l-alanyl-β-naphthylamide. Judging from these properties, the mullet roe aminopeptidase resembles soluble alanyl amino-peptidase [EC 3.4.11.14]. 相似文献
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Youji Sakagami Akira Isogai Akinori Suzuki Saburo Tamura Eiko Tsuchiya Sakuzo Fukui 《Bioscience, biotechnology, and biochemistry》2013,77(6):1301-1302
κ-Casein components having various carbohydrate contents were prepared by diethylaminoethyl-cellulose chromatography and the interactions of each κ-casein component both with αs1-casein and with β-casein were examined by Sepharose 4B gel chromatography, ultra-centrifugal experiments and viscosity measurements. Each κ-casein component could form complex with αs1- and β-casein in the absence and presence of CaCl2. Molecular weight of complexes of unfractionated κ-casein both with αs1-casein and with κ-casein were about 70 × 104 at 37°C in the absence of CaCl2, while those of complexes of each κ-casein component with αs1 and β-casein were about 50 × 104. Stokes radii of complexes increased with increasing calcium ion. While sedimentation coefficient at 37°C of complex with β-casein had almost the same value, those of complexes with αs1-casein decreased with increase of carbohydrate content of κ-casein components. Intrinsic viscosity of complex of κ-casein component having much carbohydrate was almost the same among tested temperatures. It is suggested that heterogeneity of κ-casein is necessary to form large complex and that the carbohydrate moiety of κ-casein contributes the stability of casein complex. 相似文献