共查询到20条相似文献,搜索用时 46 毫秒
1.
Francesca Cherubino Andreea Miszner Maria Daniela Renna Rachele Sangaletti Stefano Giovannardi Elena Bossi 《Cellular and molecular life sciences : CMLS》2009,66(23):3797-3808
The effects of three tricyclic antidepressants (TCAs) and two serotonin selective reuptake inhibitors (SSRIs) have been studied
with an electrophysiological approach on Xenopus laevis oocytes expressing the rat GABA (γ-Aminobutyric-acid) transporter rGAT1. All tested TCAs and SSRIs inhibit the GABA-associated
current in a dose-dependent way with low but comparable efficacy. The pre-steady-state and uncoupled currents appear substantially
unaffected. The efficacy of desipramine, but not of the other drugs, is strongly increased in the lysine-glutamate or -aspartate
mutants K448E and K448D. Comparison of I
max and K
0.5GABA in the absence and presence of desipramine showed that both parameters are reduced by the drug in the wild-type and in the
K448E mutant. This suggests an uncompetitive inhibition, in which the drug can bind only after the substrate, an explanation
in agreement with the lack of effects on the pre-steady-state and leak currents, and with the known structural data. 相似文献
2.
Giovannardi S Fesce R Bossi E Binda F Peres A 《Cellular and molecular life sciences : CMLS》2003,60(3):550-556
The effects of reducing external Cl- on the electrophysiological properties of the Na+/Cl(-)-dependent GABA transporter rGAT1 expressed in Xenopus oocytes were investigated. In agreement with a recently proposed kinetic scheme, the effects of Cl- are complex but preserve the mutual relationship that links the transport-associated current, I(tr), measured in saturating GABA concentration, and the transient current, I(pre), recorded in the absence of GABA following a voltage step from the holding potential Vh to V. In particular, I(tr) (V) - I(tr) (Vh) = r integral I(pre) (V) dt, where r is the relaxation rate of I(pre) at the same membrane potential and Cl- concentration. The model also predicts a relationship between charge relaxation rate and apparent affinity for GABA, which is also verified in the presence of lowered Na+ or Cl- concentrations. In these conditions, the binding rate of GABA to the transporter is increased. All these effects are consistent with the hypothesis that interaction of the organic substrate with rGAT1 induces a conversion from a capacitive to a conductive mode of operation without strongly altering either the amount or the rate of charge movement. 相似文献
3.
Mari SA Soragna A Castagna M Santacroce M Perego C Bossi E Peres A Sacchi VF 《Cellular and molecular life sciences : CMLS》2006,63(1):100-111
We investigated the role of the Q291 glutamine residue in the functioning of the rat γ-aminobutyric acid (GABA) transporter
GAT-1. Q291 mutants cannot transport GABA or give rise to transient, leak and transport-coupled currents even though they
are targeted to the plasma membrane. Coexpression experiments of wild-type and Q291 mutants suggest that GAT-1 is a functional
monomer though it requires oligomeric assembly for membrane insertion. We determined the accessibility of Q291 by investigating
the impact of impermeant sulfhydryl reagents on cysteine residues engineered in close proximity to Q291. The effect of these
reagents indicates that Q291 faces the external aqueous milieu. The introduction of a steric hindrance close to Q291 by means
of [2-(trimethylammonium)ethyl] methanethiosulfonate bromide modification of C74A/T290C altered the affinity of the mutant
for cations. Taken together, these results suggest that this irreplaceable residue is involved in the interaction with sodium
or in maintaining the cation accessibility to the transporter.
Received 24 October 2005; accepted 11 November 2005 相似文献
4.
Renna MD Oyadeyi AS Bossi E Kottra G Peres A 《Cellular and molecular life sciences : CMLS》2011,68(17):2961-2975
The functional and structural basis of reverse operation of PepT1 has been studied in Xenopus oocytes expressing the wild-type
and mutated forms of this protein. Using brief pulses from a negative holding potential, wild-type and Arg282 mutants exhibit
outward currents in the presence of Gly-Gln. The reversal potential of these currents is affected by both pH and substrate
concentration, confirming coupled transport in the wild type and in the mutants as well. Long-lasting voltage and current-clamp
experiments show that the outward currents are only temporary, and reflect accumulation and/or depletion effects near the
membrane. The ability to operate in reverse mode was confirmed in all isoforms by intracellular injection of substrate. The
role of Arg282 and Asp341 in the reverse transport was also investigated using charged substrates. Positive Lys-Gly (but not
Gly-Lys) showed enhanced transport currents in the Arg282 mutants. In contrast, negative Gly-Asp and Asp-Gly elicited modest
currents in all isoforms. 相似文献
5.
Fanelli F Mauri M Capra V Raimondi F Guzzi F Ambrosio M Rovati GE Parenti M 《Cellular and molecular life sciences : CMLS》2011,68(18):3109-3120
The structure-based design of a mutant form of the thromboxane A(2) prostanoid receptor (TP) was instrumental in characterizing the structural determinants of the hetero-dimerization process of this G protein coupled receptor (GPCR). The results suggest that the hetero-dimeric complexes between the TPα and β isoforms are characterized by contacts between hydrophobic residues in helix 1 from both monomers. Functional characterization confirms that TPα-TPβ hetero-dimerization serves to regulate TPα function through agonist-induced internalization, with important implications in cardiovascular homeostasis. The integrated approach employed in this study can be adopted to gain structural and functional insights into the dimerization/oligomerization process of all GPCRs for which the structural model of the monomer can be achieved at reasonable atomic resolution. 相似文献
6.
δ-Protocadherins constitute a group of cadherins characterized by several conserved motifs in their cytoplasmic domains. We
present a phylogenetic analysis that further divides this group into δ1-protocadherins (comprising protocadherin-1, −7, −9
and −11 or -X/Y) and δ2-protocadherins (comprising protocadherin-8, −10, −17, −18 and −19). The δ-protocadherin genes, which
are located on different chromosomes in man and mouse, have a similar gene structure. They are expressed as multiple splice
forms, differing mostly in their cytoplasmic domains. Some δ-protocadherins were reported to mediate weak cell-cell adhesion
in vitro and cell sorting in vivo. In addition, individual δ-protocadherins might play important roles in signaling pathways,
as they bind to proteins such as TAF1/Set, protein phosphatase-1α and the Frizzled 7 receptor. The spatiotemporally restricted
expression of δ-protocadherins in different tissues and species and the results of their functional analysis, mainly in Xenopus, suggest that they play multiple, tightly regulated roles in vertebrate development.
Received 18 July 2005; received after revision 26 August 2005; accepted 2 September 2005 相似文献
7.
Gao S Fu W Dürrenberger M De Geyter C Zhang H 《Cellular and molecular life sciences : CMLS》2005,62(9):1015-1024
hBok is a human pro-apoptotic member of the Bcl-2 family. By fluorescence in situ hybridization and in silico analysis, hBok was found to be located on chromosome 2q37.3. Its expression was detected in various organs and several hormonally regulated cancer cells. Expression of hBok was shown to be upregulated in estrogen-dependent breast cancer by estrogen deprivation and in myocardial cells during hypoxia. Confocal laser scanning microscopy examinations and subcellular fractionation studies showed that hBok was distributed in both the cytosol and intracellular membranes of healthy cells. Upon overexpression of hBok or stimulation of apoptosis, hBok became integrated into the membrane. Furthermore, apoptosis and oligomerization were promoted by BH3-only proteins, such as Bid, Bnip3 and p53, but prevented by BFL-1. hBok was found to interact with Bnip3. Our findings suggest that functional BH3-only proteins facilite the oligomerization and insertion of hBok into the membrane to activate it.Received 7 December 2004; received after revision 23 February 2005; accepted 4 March 2005 相似文献
8.
Yoshida Y Ohkuri T Kino S Ueda T Imoto T 《Cellular and molecular life sciences : CMLS》2005,62(9):1047-1055
We investigated the activity and the internal motions of a stabilized mutant hen lysozyme (HEL) in which the residues M12 and L56 were mutated to L and F, respectively (LF mutant HEL). The result of the activity measurements against glycol chitin at various temperatures suggested that the temperature dependence of the activity of LF mutant HEL shifted to the high-temperature side compared with that of wild-type HEL. The detailed internal motions of LF mutant HEL in the absence and presence of a substrate analogue, (NAG)3, were examined by model-free analysis at 35°C. The results showed that the internal motions of LF mutant HEL in the presence of (NAG)3 were drastically restricted compared with those in wild-type HEL. Our findings thus suggested that the mutation to the stabilized lysozyme restricted internal motions required for the enzymatic reaction.Received 8 February 2005; accepted 10 March 2005Y. Yoshida and T. Ohkuri contributed equally to this work. 相似文献
9.
Small heat shock proteins: molecular structure and chaperone function 总被引:17,自引:0,他引:17
Small heat shock proteins (sHSPs) associate with nuclei, cytoskeleton and membranes, and as molecular chaperones they bind
partially denatured proteins, thereby preventing irreversible protein aggregation during stress. sHSP monomers consist of
a conserved α-crystallin domain of approximately 90 amino acid residues, bordered by variable amino- and carboxy-terminal
extensions. The sHSPs undergo dynamic assembly into mono- and poly-disperse oligomers where the rate of disassembly affects
chaperoning. The α-crystallin domain contains several β-strands organized into two β-sheets responsible for dimer formation,
the basic building block of most sHSPS. The amino-terminal extension modulates oligomerization, subunit dynamics and substrate
binding, whereas the flexible carboxy-terminal extension promotes solubility, chaperoning and oligomerization, the latter
by inter-subunit linkage. Crystallization studies have revealed sHSP structure and function. Additionally, site-directed mutagenesis,
biophysical investigations, functional studies and the discovery of relationships between mutated sHSPs and diseases have
illuminated the role of sHSP within cells.
Received 8 May 2005; received after revision 24 June 2005; accepted 19 July 2005 相似文献
10.
Molecular and functional heterogeneity of GABAergic synapses 总被引:1,自引:1,他引:0
Fritschy JM Panzanelli P Tyagarajan SK 《Cellular and molecular life sciences : CMLS》2012,69(15):2485-2499
Knowledge of the functional organization of the GABAergic system, the main inhibitory neurotransmitter system, in the CNS has increased remarkably in recent years. In particular, substantial progress has been made in elucidating the molecular mechanisms underlying the formation and plasticity of GABAergic synapses. Evidence available ascribes a key role to the cytoplasmic protein gephyrin to form a postsynaptic scaffold anchoring GABA(A) receptors along with other transmembrane proteins and signaling molecules in the postsynaptic density. However, the mechanisms of gephyrin scaffolding remain elusive, notably because gephyrin can auto-aggregate spontaneously and lacks PDZ protein interaction domains found in a majority of scaffolding proteins. In addition, the structural diversity of GABA(A) receptors, which are pentameric channels encoded by a large family of subunits, has been largely overlooked in these studies. Finally, the role of the dystrophin-glycoprotein complex, present in a subset of GABAergic synapses in cortical structures, remains ill-defined. In this review, we discuss recent results derived mainly from the analysis of mutant mice lacking a specific GABA(A) receptor subtype or a core protein of the GABAergic postsynaptic density (neuroligin-2, collybistin), highlighting the molecular diversity of GABAergic synapses and its relevance for brain plasticity and function. In addition, we discuss the contribution of the dystrophin-glycoprotein complex to the molecular and functional heterogeneity of GABAergic synapses. 相似文献
11.
Receptor-mediated transport and deposition of complement component C3 into developing chicken oocytes 总被引:1,自引:0,他引:1
Recheis B Rumpler H Schneider WJ Nimpf J 《Cellular and molecular life sciences : CMLS》2005,62(16):1871-1880
Immunological resistance of the chick embryo is dependent upon IgG present in the yolk of the layed egg. Here we show that complement factor 3 (C3), a key component of the humoral complement system, is a yolk component of chicken eggs. C3 is transported into oocytes by LR8-mediated endocytosis. LR8 also binds and transports other major yolk components such as vitellogenin, very-low-density lipoprotein, and α2-macroglobulin. Expression studies of LR8 during chicken development and oocyte maturation, in combination with studies on the uptake of individual yolk components, suggest the following model for oocyte maturation in the chicken: all oocytes present in the ovary contain high levels of LR8 mRNA and protein long before the onset of oocyte maturation. Selected oocytes gain access to yolk precursors, and LR8 binds, internalizes, and deposits the major yolk components in the ratio of their relative abundance in the accessible pool.Received 9 May 2005; received after revision 6 June 2005; accepted 13 June 2005 相似文献
12.
The metabotropic GABA receptor: molecular insights and their functional consequences 总被引:2,自引:0,他引:2
Recent years have seen rapid and significant advances in our understanding of the G-protein-coupled gamma-amino butyric acid, B-type (GABA(B)) receptor, which could be a therapeutic target in conditions as diverse as epilepsy and hypertension. This progress originated with the ground-breaking work of Bernhard Bettler's team at Novartis who cloned the DNA encoding a GABA(B) receptor in 1997. Currently, the receptor is thought to be an unusual, possibly unique, example of a heterodimer composed of homologous, seven-transmembrane-domain (7TMD) subunits (named GABA(B) R1 and GABA(B) R2), neither of which is fully functional when expressed alone. The large N-terminal domain of the GABA(B) R1 subunit projects extracellularly and contains a ligand binding site. The similarity of the amino acid sequence of this region to some bacterial periplasmic amino acid-binding proteins of known structure has enabled structural and functional modelling of the N-terminal domain, and the identification of residues whose substitution modulates agonist/antagonist binding affinities. The intracellular C-terminal domains of the R1 and R2 subunits appear to constitute an important means of contact between the two subunits. Alternative splice variants, a common and functionally important feature of 7TMD proteins, have been demonstrated for the R1 subunit. Notably GABA(B) R1a differs from GABA(B) R1b by the possession of an N-terminal extension containing two complement protein modules (also called SCRs, or sushi domains) of unknown function. The levels at which each of the respective variants is expressed are not equal to one another, with variations occurring over the course of development and throughout the central nervous system. It is not yet clear, however, whether one variant is predominantly presynaptically located and the other postsynaptically located. The existence of as yet unidentified splice variants, additional receptor subtypes and alternative quaternary composition has not been ruled out as a source of receptor heterogeneity. 相似文献
13.
Ancient origin of reggie (flotillin), reggie-like, and other lipid-raft proteins: convergent evolution of the SPFH domain 总被引:1,自引:0,他引:1
Rivera-Milla E Stuermer CA Málaga-Trillo E 《Cellular and molecular life sciences : CMLS》2006,63(3):343-357
Reggies (flotillins) are detergent-resistant microdomains involved in the scaffolding of large heteromeric complexes that
signal across the plasma membrane. Based on the presence of an evolutionarily widespread motif, reggies/flotillins have been
included within the SPFH (stomatin-prohibitin-flotillin-HflC/K) protein superfamily. To better understand the origin and evolution
of reggie/flotillin structure and function, we searched databases for reggie/flotillin and SPFH-like proteins in organisms
at the base and beyond the animal kingdom, and used the resulting dataset to compare their structural and functional domains.
Our analysis shows that the SPFH grouping has little phylogenetic support, probably due to convergent evolution of its members.
We also find that reggie/flotillin homologues are highly conserved among metazoans but are absent in plants, fungi and bacteria,
where only proteins with ‘reggie-like’ domains can be found. However, despite their low sequence similarities, reggie/flotillin
and ‘reggie-like’ domains appear to subserve related functions, suggesting that their basic biological role was acquired independently
during evolution.
Received 21 September 2005; received after revision 14 November 2005; accepted 21 November 2005 相似文献
14.
Lamprin: A new vertebrate protein comprising the major structural protein of adult lamprey cartilage
G. M. Wright F. W. Keeley J. H. Youson 《Cellular and molecular life sciences : CMLS》1983,39(5):495-497
Summary Chemical analysis of lamprey cartilage showed that its major constituent was a newly defined structural protein termed lamprin. Amino acid analysis of lamprin revealed that it has a unique composition which is distinct from previously identified structural proteins.Acknowledgment. This work was supported by grants U0105 to G.M.W. and A5945 to J.H.Y. from the Natural Sciences and Engineering Research Council of Canada. 相似文献
15.
Ricchelli F Drago D Filippi B Tognon G Zatta P 《Cellular and molecular life sciences : CMLS》2005,62(15):1724-1733
We investigated the structural effects induced by Al3+ on different β-amyloid (Aβ) fragments at pH 7.4 and T= 25°C, with particular attention given to the sequences 1–40 and 1–42. Al3+ caused peptide enrichment in β sheet structure and formation of solvent-exposed hydrophobic clusters. These intermediates evolved to polymeric aggregates which organized in fibrillar forms in the case of the Al3+-Aβ(1–42) complex. Comparative studies showed that Zn2+ and Cu2+ were much less efficient than Al3+ in stimulating the spontaneous aggregation/fibrillogenesis of Aβs. Studies with liposomes as membrane models showed dramatic changes in the structural properties of the lipid bilayer in the presence of Al3+-Aβ complexes, suggesting a major role of Al3+ in Aβ-induced cell dysfunction. Al3+ effects were abolished by desferrioxamine mesylate (DFO) only in solution. We concluded that, in vivo, DFO may act as a protective agent by preventing or reverting Aβ aggregation in the extracellular spaces.Received 29 March 2005; received after revision 10 May 2005; accepted 25 May 2005 相似文献
16.
Eudes R Lehn P Férec C Mornon JP Callebaut I 《Cellular and molecular life sciences : CMLS》2005,62(18):2112-2123
Defective function of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) causes CF, the most frequent lethal inherited disease among the Caucasian population. The structure of this chloride ion channel includes two nucleotide-binding domains (NBDs), whose ATPase activity controls channel gating. Recently, the experimental structures of mouse and human CFTR NBD1 and our model of the human CFTR NBD1/NBD2 heterodimer have provided new insights into specific structural features of the CFTR NBD dimer. In the present work, we provide a structural classification of CF-causing mutations which may complement the existing functional classification. Our analysis also identified amino acid residues which may play a critical role in interdomain interaction and are located at the NBD1-NBD2 interface or on the surface of the dimer. In particular, a cluster of aromatic amino acids, which includes F508 and straddles the two NBDs, might be directly involved in the interaction of the NBD1/NBD2 heterodimer with the channel-forming membrane-spanning domains.Received 24 May 2005; received after revision 13 June 2005; accepted 18 June 2005 相似文献
17.
Seyyed Abolghasem Ghadami Francesco Bemporad Benedetta Maria Sala Guido Tiana Stefano Ricagno Fabrizio Chiti 《Cellular and molecular life sciences : CMLS》2017,74(19):3577-3598
Transthyretin (TTR) is an extracellular protein able to deposit into well-defined protein aggregates called amyloid, in pathological conditions known as senile systemic amyloidosis, familial amyloid polyneuropathy, familial amyloid cardiomyopathy and leptomeningeal amyloidosis. At least three distinct partially folded states have been described for TTR, including the widely studied amyloidogenic state at mildly acidic pH. Here, we have used fluorescence resonance energy transfer (FRET) experiments in a monomeric variant of TTR (M-TTR) and in its W41F and W79F mutants, taking advantage of the presence of a unique, solvent-exposed, cysteine residue at position 10, that we have labelled with a coumarin derivative (DACM, acceptor), and of the two natural tryptophan residues at positions 41 and 79 (donors). Trp41 is located in an ideal position as it is one of the residues of β-strand C, whose degree of unfolding is debated. We found that the amyloidogenic state at low pH has the same FRET efficiency as the folded state at neutral pH in both M-TTR and W79F-M-TTR, indicating an unmodified Cys10–Trp41 distance. The partially folded state populated at low denaturant concentrations also has a similar FRET efficiency, but other spectroscopic probes indicate that it is distinct from the amyloidogenic state at acidic pH. By contrast, the off-pathway state accumulating transiently during refolding has a higher FRET efficiency, indicating non-native interactions that reduce the Cys10–Trp41 spatial distance, revealing a third distinct conformational state. Overall, our results clarify a negligible degree of unfolding of β-strand C in the formation of the amyloidogenic state and establish the concept that TTR is a highly plastic protein able to populate at least three distinct conformational states. 相似文献
18.
It is well documented that activation of calpain, a calcium-sensitive cysteine protease, marks the pathology of naturally and experimentally occuring neurodegenerative conditions. Calpain-mediated proteolysis of major membrane-skeletal protein, αII-spectrin, results in the appearance of two unique and highly stable breakdown products, which is an early event in neural cell pathology. This review focuses on spectrin degradation by calpain within neurons induced by diverse conditions, emphasizing a current picture of multi-pattern neuronal death and a recent success in the development of spectrin-based biomarkers. The issue is presented in the context of the major structural and functional properties of the two proteins.Received 7 March 2005; received after revision 22 April 2005; accepted 13 May 2005 相似文献
19.
20.
The morphological development of the embryonic gonads is very similar in birds and mammals, and recent evidence suggests
that the genes involved in this process are conserved between these classes of vertebrates. The genetic mechanism by which
sex is determined in birds remains to be elucidated, although recent studies have reinforced the contention that steroids
may play an important role in the structural development of the testes and ovaries in birds. So far, few genes have been assigned
to the avian sex chromosomes, but it is known that the Z and W chromosomes do not share significant homology with the mammalian
X and Y chromosomes. The commercial importance of poultry breeding has motivated considerable investment in developing physical
and genetic maps of the chicken genome. These efforts, in combination with modern molecular approaches to analyzing gene expression,
should help to elucidate the sex-determining mechanism in birds in the near future. 相似文献