共查询到20条相似文献,搜索用时 31 毫秒
1.
Tjakko J. van Ham Karen L. Thijssen Rainer Breitling Robert M. W. Hofstra Ronald H. A. Plasterk Ellen A. A. Nollen 《PLoS genetics》2008,4(3)
Inclusions in the brain containing α-synuclein are the pathological hallmark of Parkinson''s disease, but how these inclusions are formed and how this links to disease is poorly understood. We have developed a C. elegans model that makes it possible to monitor, in living animals, the formation of α-synuclein inclusions. In worms of old age, inclusions contain aggregated α- synuclein, resembling a critical pathological feature. We used genome-wide RNA interference to identify processes involved in inclusion formation, and identified 80 genes that, when knocked down, resulted in a premature increase in the number of inclusions. Quality control and vesicle-trafficking genes expressed in the ER/Golgi complex and vesicular compartments were overrepresented, indicating a specific role for these processes in α-synuclein inclusion formation. Suppressors include aging-associated genes, such as sir-2.1/SIRT1 and lagr-1/LASS2. Altogether, our data suggest a link between α-synuclein inclusion formation and cellular aging, likely through an endomembrane-related mechanism. The processes and genes identified here present a framework for further study of the disease mechanism and provide candidate susceptibility genes and drug targets for Parkinson''s disease and other α-synuclein related disorders. 相似文献
2.
3.
Wellens A Garofalo C Nguyen H Van Gerven N Slättegård R Hernalsteens JP Wyns L Oscarson S De Greve H Hultgren S Bouckaert J 《PloS one》2008,3(4):e2040
Background
Escherichia coli strains adhere to the normally sterile human uroepithelium using type 1 pili, that are long, hairy surface organelles exposing a mannose-binding FimH adhesin at the tip. A small percentage of adhered bacteria can successfully invade bladder cells, presumably via pathways mediated by the high-mannosylated uroplakin-Ia and α3β1 integrins found throughout the uroepithelium. Invaded bacteria replicate and mature into dense, biofilm-like inclusions in preparation of fluxing and of infection of neighbouring cells, being the major cause of the troublesome recurrent urinary tract infections.Methodology/Principal Findings
We demonstrate that α-d-mannose based inhibitors of FimH not only block bacterial adhesion on uroepithelial cells but also antagonize invasion and biofilm formation. Heptyl α-d-mannose prevents binding of type 1-piliated E. coli to the human bladder cell line 5637 and reduces both adhesion and invasion of the UTI89 cystitis isolate instilled in mouse bladder via catheterization. Heptyl α-d-mannose also specifically inhibited biofilm formation at micromolar concentrations. The structural basis of the great inhibitory potential of alkyl and aryl α-d-mannosides was elucidated in the crystal structure of the FimH receptor-binding domain in complex with oligomannose-3. FimH interacts with Manα1,3Manβ1,4GlcNAcβ1,4GlcNAc in an extended binding site. The interactions along the α1,3 glycosidic bond and the first β1,4 linkage to the chitobiose unit are conserved with those of FimH with butyl α-d-mannose. The strong stacking of the central mannose with the aromatic ring of Tyr48 is congruent with the high affinity found for synthetic inhibitors in which this mannose is substituted for by an aromatic group.Conclusions/Significance
The potential of ligand-based design of antagonists of urinary tract infections is ruled by the structural mimicry of natural epitopes and extends into blocking of bacterial invasion, intracellular growth and capacity to fluxing and of recurrence of the infection. 相似文献4.
Büttner S Delay C Franssens V Bammens T Ruli D Zaunschirm S de Oliveira RM Outeiro TF Madeo F Buée L Galas MC Winderickx J 《PloS one》2010,5(10):e13700
Background
Parkinson''s disease is characterized by the presence of cytoplasmic inclusions, known as Lewy bodies, containing both aggregated α-synuclein and its interaction partner, synphilin-1. While synphilin-1 is known to accelerate inclusion formation by α-synuclein in mammalian cells, its effect on cytotoxicity remains elusive.Methodology/Principal Findings
We expressed wild-type synphilin-1 or its R621C mutant either alone or in combination with α-synuclein in the yeast Saccharomyces cerevisiae and monitored the intracellular localization and inclusion formation of the proteins as well as the repercussions on growth, oxidative stress and cell death. We found that wild-type and mutant synphilin-1 formed inclusions and accelerated inclusion formation by α-synuclein in yeast cells, the latter being correlated to enhanced phosphorylation of serine-129. Synphilin-1 inclusions co-localized with lipid droplets and endomembranes. Consistently, we found that wild-type and mutant synphilin-1 interacts with detergent-resistant membrane domains, known as lipid rafts. The expression of synphilin-1 did not incite a marked growth defect in exponential cultures, which is likely due to the formation of aggresomes and the retrograde transport of inclusions from the daughter cells back to the mother cells. However, when the cultures approached stationary phase and during subsequent ageing of the yeast cells, both wild-type and mutant synphilin-1 reduced survival and triggered apoptotic and necrotic cell death, albeit to a different extent. Most interestingly, synphilin-1 did not trigger cytotoxicity in ageing cells lacking the sirtuin Sir2. This indicates that the expression of synphilin-1 in wild-type cells causes the deregulation of Sir2-dependent processes, such as the maintenance of the autophagic flux in response to nutrient starvation.Conclusions/Significance
Our findings demonstrate that wild-type and mutant synphilin-1 are lipid raft interacting proteins that form inclusions and accelerate inclusion formation of α-synuclein when expressed in yeast. Synphilin-1 thereby induces cytotoxicity, an effect most pronounced for the wild-type protein and mediated via Sir2-dependent processes. 相似文献5.
Post-translational modifications of α-synuclein occur in the brain of patients affected by Parkinson''s disease and other α-synucleinopathies, as indicated by the accumulation of Lewy inclusions containing phosphorylated (at serine 129) and nitrated α-synuclein. Here we found that phospho-Ser 129 and nitrated α-synuclein are also formed within dopaminergic neurons of the monkey substantia nigra as a result of normal aging. Dopaminergic cell bodies immunoreactive for phospho-Ser 129 and nitrated α-synuclein were rarely seen in adult mature animals but became significantly more frequent in the substantia nigra of old primates. Dual labeling with antibodies against phospho-Ser 129 and nitrated α-synuclein revealed only limited colocalization and mostly stained distinct sub-populations of dopaminergic neurons. Age-related elevations of modified protein paralleled an increase in the number of neurons immunoreactive for unmodified α-synuclein, supporting a relationship between higher levels of normal protein and enhanced phosphorylation/nitration. Other mechanisms were also identified that likely contribute to α-synuclein modifications. In particular, increased expression of Polo-like kinase 2 within neurons of older animals could contribute to phospho-Ser 129 α-synuclein production. Data also indicate that a pro-oxidant environment characterizes older neurons and favors α-synuclein nitration. Aging is an unequivocal risk factor for human α-synucleinopathies. These findings are consistent with a mechanistic link between aging, α-synuclein abnormalities and enhanced vulnerability to neurodegenerative processes. 相似文献
6.
Tomoya Isaji Yuya Sato Tomohiko Fukuda Jianguo Gu 《The Journal of biological chemistry》2009,284(18):12207-12216
N-Glycosylation of integrin α5β1 plays a crucial role
in cell spreading, cell migration, ligand binding, and dimer formation, but
the detailed mechanisms by which N-glycosylation mediates these
functions remain unclear. In a previous study, we showed that three potential
N-glycosylation sites (α5S3–5) on the β-propeller of
the α5 subunit are essential to the functional expression of the
subunit. In particular, site 5 (α5S5) is the most important for its
expression on the cell surface. In this study, the function of the
N-glycans on the integrin β1 subunit was investigated using
sequential site-directed mutagenesis to remove the combined putative
N-glycosylation sites. Removal of the N-glycosylation sites
on the I-like domain of the β1 subunit (i.e. the Δ4-6
mutant) decreased both the level of expression and heterodimeric formation,
resulting in inhibition of cell spreading. Interestingly, cell spreading was
observed only when the β1 subunit possessed these three
N-glycosylation sites (i.e. the S4-6 mutant). Furthermore,
the S4-6 mutant could form heterodimers with either α5S3-5 or α5S5
mutant of the α5 subunit. Taken together, the results of the present
study reveal for the first time that N-glycosylation of the I-like
domain of the β1 subunit is essential to both the heterodimer formation
and biological function of the subunit. Moreover, because the
α5S3-5/β1S4-6 mutant represents the minimal
N-glycosylation required for functional expression of the β1
subunit, it might also be useful for the study of molecular structures.Integrin is a heterodimeric glycoprotein that consists of both an α
and a β subunit (1). The
interaction between integrin and the extracellular matrix is essential to both
physiologic and pathologic events, such as cell migration, development, cell
viability, immune homeostasis, and tumorigenesis
(2,
3). Among the integrin
superfamily, β1 integrin can combine with 12 distinct α subunits
(α1–11, αv) to form heterodimers, thereby acquiring a wide
variety of ligand specificity
(1,
4). Integrins are thought to be
regulated by inside-out signaling mechanisms that provoke conformational
changes, which modulate the affinity of integrin for the ligand
(5). However, an increasing
body of evidence suggests that cell-surface carbohydrates mediate a variety of
interactions between integrin and its extracellular environment, thereby
affecting integrin activity and possibly tumor metastasis as well
(6–8).Guo et al. (9)
reported that an increase in β1–6-GlcNAc sugar chains on the
integrin β1 subunit stimulated cell migration. In addition, elevated
sialylation of the β1 subunit, because of Ras-induced STGal-I transferase
activity, also induced cell migration
(10,
11). Conversely, cell
migration and spreading were reduced by the addition of a bisecting GlcNAc,
which is a product of N-acetylglucosaminyltransferase III
(GnT-III),2 to the
α5β1 and α3β1 integrins
(12,
13). Alterations of
N-glycans on integrins might also regulate their cis interactions
with membrane-associated proteins, including the epidermal growth factor
receptor, the galectin family, and the tetraspanin family of proteins
(14–19).In addition to the positive and negative regulatory effects of
N-glycan, several research groups have reported that
N-glycans must be present on integrin α5β1 for the
αβ heterodimer formation and proper integrin-matrix interactions.
Consistent with this hypothesis, in the presence of the glycosylation
inhibitor, tunicamycin, normal integrin-substrate binding and transport to the
cell surface are inhibited
(20). Moreover, treatment of
purified integrin with N-glycosidase F blocked both the inherent
association of the subunits and the interaction between integrin and
fibronectin (FN) (21). These
results suggest that N-glycosylation is essential to the functional
expression of α5β1. However, because integrin α5β1
contains 26 potential N-linked glycosylation sites, 14 in the α
subunit and 12 in the β subunit, identification of the sites that are
essential to its biological functions is key to understanding the molecular
mechanisms by which N-glycans alter integrin function. Recently, our
group determined that N-glycosylation of the β-propeller domain
on the α5 subunit is essential to both heterodimerization and biological
functions of the subunit. Furthermore, we determined that sites 3–5 are
the most important sites for α5 subunit-mediated cell spreading and
migration on FN (22). The
purpose of this study was to clarify the roles of N-glycosylation of
the β1 subunit. Therefore, we performed combined substitutions in the
putative N-glycosylation sites by replacement of asparagine residues
with glutamine residues. We subsequently introduced these mutated genes into
β1-deficient epithelial cells (GE11). The results of these mutation
experiments revealed that the N-glycosylation sites on the I-like
domain of the β1 subunit, sites number 4–6 (S4-6), are essential to
both heterodimer formation and biological functions, such as cell
spreading. 相似文献
7.
Shouryadeep Srivastava Ayman K. Hamouda Akash Pandhare Phaneendra K. Duddempudi Mitesh Sanghvi Jonathan B. Cohen Michael P. Blanton 《The Journal of biological chemistry》2009,284(37):24939-24947
Nicotinic acetylcholine receptor (nAChR) agonists, such as epibatidine and its molecular derivatives, are potential therapeutic agents for a variety of neurological disorders. In order to identify determinants for subtype-selective agonist binding, it is important to determine whether an agonist binds in a common orientation in different nAChR subtypes. To compare the mode of binding of epibatidine in a muscle and a neuronal nAChR, we photolabeled Torpedo α2βγδ and expressed human α4β2 nAChRs with [3H]epibatidine and identified by Edman degradation the photolabeled amino acids. Irradiation at 254 nm resulted in photolabeling of αTyr198 in agonist binding site Segment C of the principal (+) face in both α subunits and of γLeu109 and γTyr117 in Segment E of the complementary (−) face, with no labeling detected in the δ subunit. For affinity-purified α4β2 nAChRs, [3H]epibatidine photolabeled α4Tyr195 (equivalent to Torpedo αTyr190) in Segment C as well as β2Val111 and β2Ser113 in Segment E (equivalent to Torpedo γLeu109 and γTyr111, respectively). Consideration of the location of the photolabeled amino acids in homology models of the nAChRs based upon the acetylcholine-binding protein structure and the results of ligand docking simulations suggests that epibatidine binds in a single preferred orientation within the α-γ transmitter binding site, whereas it binds in two distinct orientations in the α4β2 nAChR.Nicotinic acetylcholine receptors (nAChRs)3 are prototypical members of the Cys loop superfamily of neurotransmitter-gated ion channels that mediate the actions of the neurotransmitter acetylcholine (1). nAChRs from vertebrate skeletal muscle and the electric organs of Torpedo rays are heteropentamers of homologous subunits with a stoichiometry of 2α:β:γ(ϵ):δ that are arranged pseudosymmetrically around central cation-selective ion channels (1, 2). There are 12 mammalian neuronal nAChR subunit genes: nine neuronal α subunits (α2–α10) and three neuronal β subunits (β2–β4). The α4β2 nAChR is the most abundant and widely distributed nAChR subtype expressed in the brain and is a major target for potential therapeutic agents for neurological diseases and conditions, including nicotine dependence and Alzheimer and Parkinson diseases (3, 4). Although the ratio of α4 to β2 subunit in vivo is uncertain, expressed receptors containing either three α4 or three β2 subunits have distinct pharmacological properties (5, 6).The agonist binding sites (ABS) of nAChRs are located within the amino-terminal extracellular domain at the interface of adjacent subunits (α-γ and α-δ in the Torpedo nAChR), and different nAChR subunit combinations form ABS with distinct physical and pharmacological properties (3, 7). Affinity labeling studies with Torpedo nAChR and site-directed mutational analyses of muscle and neuronal nAChRs identified key amino acids delineating the ABS from three noncontiguous stretches of the α subunit (Segments A-C, the principal component (+ face)) and three noncontiguous regions of the non-α subunit (Segments D–F, the complementary component (− face)) (8, 9). The three-dimensional structure of the ABS in the absence and presence of nAChR agonists or competitive antagonists has been determined for snail acetylcholine-binding proteins (AChBPs) that are soluble homopentamers homologous to the extracellular (amino-terminal) domain of a nAChR (10–12). In the AChBP, four aromatic amino acids from Segments A–C that are conserved within α subunits, along with a conserved Trp in Segment D, form a core aromatic “pocket” with a dimension optimal for accommodation of a trimethylammonium group. The other amino acids in the non-α subunits closest to the aromatic pocket, which are generally not conserved among γ, δ, or neuronal β subunits, are on three antiparallel β strands. The AChBP structure was used to refine the structure of the Torpedo nAChR in the absence of agonist to 4 Å resolution (13). In this structure, there is a reorientation of Segments A–C, resulting in the absence of a well defined core aromatic binding pocket.Analysis of agonist interactions with mutant nAChRs containing fluorine-substituted core aromatic residues provides evidence that cation-π interactions, particularly with αTrp149 in Segment B, are important determinants of agonist binding affinity (14) and for the higher affinity binding of nicotine to α4β2 nAChRs compared with α2βγδ nAChRs (15). Mutational analyses and molecular docking calculations have also provided evidence that two molecules of very similar structure may actually bind to a single receptor in very different orientations, as seen for two high affinity antagonists, d-tubocurarine and its quaternary ammonium analog metocurine, binding to the AChBP and to the muscle nAChR (16, 17).Photoaffinity labeling provides an alternative means to identify amino acids contributing to a drug binding site (18, 19) and has been used to determine the orientation of drugs bound in the ABS of Torpedo nAChR (20). Epibatidine binds with very high affinity (∼10 pm) to heteromeric neuronal nAChRs (e.g. α4β2) and with nanomolar affinity to α7 and muscle-type/Torpedo nAChRs (3). Utilizing a photoreactive analogue of epibatidine (azidoepibatidine; Fig. 1) and mass spectrometry, Tomizawa et al. (21) identified photolabeled amino acids in the Aplysia AChBP (Tyr195 in Segment C and Met116 in Segment E), establishing an orientation for bound azidoepibatidine consistent with the orientation of epibatidine in an AChBP crystal structure (12).Open in a separate windowFIGURE 1.Structure of [3H]epibatidine (top) and azidoepibatidine (bottom).In this report, we use [3H]epibatidine as a photoaffinity reagent to identify the amino acids photolabeled in an expressed α4β2 nAChR and in the Torpedo α2βγδ nAChR. Comparisons of the labeled amino acids seen in the Torpedo nAChR α-γ binding site and in the α4β2 nAChR, in conjunction with the results of docking calculations for epibatidine binding to homology models of the α2βγδ and α4β2 nAChRs, suggests that epibatidine binds in a single orientation in the α-γ site but in two orientations in the α4β2 ABS. 相似文献
8.
Anthony Ludlam Joseph Brunzelle Thomas Pribyl Xingjue Xu Domenico L. Gatti Sharon H. Ackerman 《The Journal of biological chemistry》2009,284(25):17138-17146
Mitochondrial F1-ATPase contains a hexamer of alternating α and β subunits. The assembly of this structure requires two specialized chaperones, Atp11p and Atp12p, that bind transiently to β and α. In the absence of Atp11p and Atp12p, the hexamer is not formed, and α and β precipitate as large insoluble aggregates. An early model for the mechanism of chaperone-mediated F1 assembly (Wang, Z. G., Sheluho, D., Gatti, D. L., and Ackerman, S. H. (2000) EMBO J. 19, 1486–1493) hypothesized that the chaperones themselves look very much like the α and β subunits, and proposed an exchange of Atp11p for α and of Atp12p for β; the driving force for the exchange was expected to be a higher affinity of α and β for each other than for the respective chaperone partners. One important feature of this model was the prediction that as long as Atp11p is bound to β and Atp12p is bound to α, the two F1 subunits cannot interact at either the catalytic site or the noncatalytic site interface. Here we present the structures of Atp11p from Candida glabrata and Atp12p from Paracoccus denitrificans, and we show that some features of the Wang model are correct, namely that binding of the chaperones to α and β prevents further interactions between these F1 subunits. However, Atp11p and Atp12p do not resemble α or β, and it is instead the F1 γ subunit that initiates the release of the chaperones from α and β and their further assembly into the mature complex.Mitochondrial F1-ATPase consists of three α and three β subunits occupying alternate positions in a hexamer that surrounds a rod-like element containing one each of γ, δ, and ϵ subunits (1–3). Three nucleotide-binding catalytic sites (CS)4 and three noncatalytic sites (NCS) alternate at the six α/β interfaces. Early work with respiratory-deficient strains of Saccharomyces cerevisiae (4) revealed that two additional mitochondrial proteins, Atp11p and Atp12p, which are not integral subunits of the enzyme, are nonetheless necessary for the assembly of F1-ATPase. Besides their failure to assemble F1, a particularly interesting feature of atp11 and atp12 mutants is that they accumulate α and β subunits as high molecular weight aggregates (4) that can be recognized as densely stained inclusion bodies in the mitochondrial matrix (5). Subsequent studies in yeast have shown that Atp12p binds to F1 α (6) and that Atp11p binds to β (7); these interactions include binding determinants in the nucleotide binding domains (NBD) of the two F1 subunits. On this basis, it is now recognized that Atp11p and Atp12p are members of two new families of molecular chaperones, pfam06644 and pfam07542 (8), which are required for the assembly of mitochondrial ATP synthase in all eukaryotes. In fact, the first nuclear genetic lesion associated to a defect of mitochondrial ATP synthase in humans was identified in the locus ATPAF2 for Atp12p and was responsible for the death of a 14-month-old infant (9). Atp12p is also present in the α subdivision of Proteobacteria, consistent with the proposed origin of mitochondria from this ancestral line (10).The nature of the interactions between the F1 subunits and Atp11p and Atp12p has remained elusive because of the lack of structural information for these chaperones. As α and β aggregate in the absence of Atp11p and Atp12p, it is usually assumed that the F1 subunits are themselves poorly soluble, and that the two chaperones maintain them in a dispersed state until they are incorporated in the mature enzyme. Based on the analysis of the distribution of hydrophilic and hydrophobic areas on the surface of the α and β subunits of F1, and on the interaction energies between these subunits at the interfaces that provide the CS and NCS sites, Wang et al. (6) have proposed a model of F1 assembly in which Atp11p binds at the region of the β subunit that contributes to the CS site, and Atp12p binds at the region of the α subunit that contributes to the NCS site. One consequence of this particular binding of Atp11p and Atp12p to the F1 subunits is that as long as Atp11p is bound to β and Atp12p is bound to α, the two F1 subunits cannot interact at either the CS or the NCS interface. Since no other modulators of chaperone release are known, the Wang model requires an exchange of Atp11p for α and of Atp12p for β. Implied in this model is that the chaperones must themselves look very much like the α and β subunits, and that the driving force for the exchange must simply be a higher affinity of α and β for each other than for the respective chaperone partners. Here we present the structures of Atp11p from Candida glabrata and Atp12p from Paracoccus denitrificans, and we show that some features of the Wang model are correct, namely that binding of the chaperones to α and β prevents further interactions between these F1 subunits. However, Atp11p and Atp12p do not resemble α or β, and it is instead the F1 γ subunit that initiates the release of the chaperones from α and β and their further assembly into mature complex. 相似文献
9.
10.
11.
Cytoplasmic Sequestration of the Polyomavirus Enhancer Binding Protein 2 (PEBP2)/Core Binding Factor α (CBFα) Subunit by the Leukemia-Related PEBP2/CBFβ-SMMHC Fusion Protein Inhibits PEBP2/CBF-Mediated Transactivation 下载免费PDF全文
Yuka Kanno Tomohiko Kanno Chohei Sakakura Suk-Chul Bae Yoshiaki Ito 《Molecular and cellular biology》1998,18(7):4252-4261
12.
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
13.
14.
15.
Yuya Sato Toshihiko Uemura Keisuke Morimitsu Ryoko Sato-Nishiuchi Ri-ichiroh Manabe Junichi Takagi Masashi Yamada Kiyotoshi Sekiguchi 《The Journal of biological chemistry》2009,284(21):14524-14536
Integrin α8β1 interacts with a variety of Arg-Gly-Asp
(RGD)-containing ligands in the extracellular matrix. Here, we examined the
binding activities of α8β1 integrin toward a panel of
RGD-containing ligands. Integrin α8β1 bound specifically to
nephronectin with an apparent dissociation constant of 0.28 ± 0.01
nm, but showed only marginal affinities for fibronectin and other
RGD-containing ligands. The high-affinity binding to α8β1 integrin
was fully reproduced with a recombinant nephronectin fragment derived from the
RGD-containing central “linker” segment. A series of deletion
mutants of the recombinant fragment identified the LFEIFEIER sequence on the
C-terminal side of the RGD motif as an auxiliary site required for
high-affinity binding to α8β1 integrin. Alanine scanning
mutagenesis within the LFEIFEIER sequence defined the EIE sequence as a
critical motif ensuring the high-affinity integrin-ligand interaction.
Although a synthetic LFEIFEIER peptide failed to inhibit the binding of
α8β1 integrin to nephronectin, a longer peptide containing both the
RGD motif and the LFEIFEIER sequence was strongly inhibitory, and was
∼2,000-fold more potent than a peptide containing only the RGD motif.
Furthermore, trans-complementation assays using recombinant fragments
containing either the RGD motif or LFEIFEIER sequence revealed a clear
synergism in the binding to α8β1 integrin. Taken together, these
results indicate that the specific high-affinity binding of nephronectin to
α8β1 integrin is achieved by bipartite interaction of the integrin
with the RGD motif and LFEIFEIER sequence, with the latter serving as a
synergy site that greatly potentiates the RGD-driven integrin-ligand
interaction but has only marginal activity to secure the interaction by
itself.Integrins are a family of adhesion receptors that interact with a variety
of extracellular ligands, typically cell-adhesive proteins in the
extracellular matrix
(ECM).2 They play
mandatory roles in embryonic development and the maintenance of tissue
architectures by providing essential links between cells and the ECM
(1). Integrins are composed of
two non-covalently associated subunits, termed α and β. In mammals,
18 α and 8 β subunits have been identified, and combinations of
these subunits give rise to at least 24 distinct integrin heterodimers. Based
on their ligand-binding specificities, ECM-binding integrins are classified
into three groups, namely laminin-, collagen- and RGD-binding integrins
(2,
3), of which the RGD-binding
integrins have been most extensively investigated. The RGD-binding integrins
include α5β1, α8β1, αIIbβ3, and
αV-containing integrins, and have been shown to interact with a variety
of ECM ligands, such as fibronectin and vitronectin, with distinct binding
specificities.The α8 integrin subunit was originally identified in chick nerves
(4). Integrin α8β1
is expressed in the metanephric mesenchyme and plays a crucial role in
epithelial-mesenchymal interactions during the early stages of kidney
morphogenesis. Disruption of the α8 gene in mice was found to be
associated with severe defects in kidney morphogenesis
(5) and stereocilia development
(6). To date, α8β1
integrin has been shown to bind to fibronectin, vitronectin, osteopontin,
latency-associated peptide of transforming growth factor-β1, tenascin-W,
and nephronectin (also named POEM)
(7–13),
among which nephronectin is believed to be an α8β1 integrin ligand
involved in kidney development
(10).Nephronectin is one of the basement membrane proteins whose expression and
localization patterns are restricted in a tissue-specific and developmentally
regulated manner (10,
11). Nephronectin consists of
five epidermal growth factor-like repeats, a linker segment containing the RGD
cell-adhesive motif (designated RGD-linker) and a meprin-A5 protein-receptor
protein-tyrosine phosphatase μ (MAM) domain (see
Fig. 3A). Although the
physiological functions of nephronectin remain only poorly understood, it is
thought to play a role in epithelial-mesenchymal interactions through binding
to α8β1 integrin, thereby transmitting signals from the epithelium
to the mesenchyme across the basement membrane
(10). Recently, mice deficient
in nephronectin expression were produced by homologous recombination
(14). These
nephronectin-deficient mice frequently displayed kidney agenesis, a phenotype
reminiscent of α8 integrin knock-out mice
(14), despite the fact that
other RGD-containing ligands, including fibronectin and osteopontin, were
expressed in the embryonic kidneys
(9,
15). The failure of the other
RGD-containing ligands to compensate for the deficiency of nephronectin in the
developing kidneys suggests that nephronectin is an indispensable
α8β1 ligand that plays a mandatory role in epithelial-mesenchymal
interactions during kidney development.Open in a separate windowFIGURE 3.Binding activities of α8β1 integrin to nephronectin and its
fragments. A, schematic diagrams of full-length nephronectin
(NN) and its fragments. RGD-linker and RGD-linker
(GST), the central RGD-containing linker segments expressed in
mammalian and bacterial expression systems, respectively; PRGDV, a
short RGD-containing peptide modeled after nephronectin and expressed as a GST
fusion protein (see Fig.
4A for the peptide sequence). The arrowheads
indicate the positions of the RGD motif. B, purified recombinant
proteins were analyzed by SDS-PAGE in 7–15% gradient (left and
center panels) and 12% (right panels) gels, followed by
Coomassie Brilliant Blue (CBB) staining, immunoblotting with an
anti-FLAG mAb, or lectin blotting with PNA. The quantities of proteins loaded
were: 0.5 μg (for Coomassie Brilliant Blue staining) and 0.1 μg (for
blotting with anti-FLAG and PNA) in the left and center
panels;1 μg in the right panel. C, recombinant proteins (10
nm) were coated on microtiter plates and assessed for their binding
activities toward α8β1 integrin (10 nm) in the presence
of 1 mm Mn2+. The backgrounds were subtracted as
described in the legend to Fig.
2. The results represent the mean ± S.D. of triplicate
determinations. D, titration curves of α8β1 integrin bound
to full-length nephronectin (NN, closed squares), the RGD-linker
segments expressed in 293F cells (RGD-linker, closed triangles) and
E. coli (RGD-linker (GST), open
triangles), the MAM domain (MAM, closed diamonds), and the PRGDV
peptide expressed as a GST fusion protein in E. coli (PRGDV
(GST), open circles). The assays were performed as described
in the legend to Fig.
2B. The results represent the means of duplicate
determinations.Although ligand recognition by RGD-binding integrins is primarily
determined by the RGD motif in the ligands, it is the residues outside the RGD
motif that define the binding specificities and affinities toward individual
integrins (16,
17). For example,
α5β1 integrin specifically binds to fibronectin among the many
RGD-containing ligands, and requires not only the RGD motif in the 10th type
III repeat but also the so-called “synergy site” within the
preceding 9th type III repeat for fibronectin recognition
(18). Recently, DiCara et
al. (19) demonstrated
that the high-affinity binding of αVβ6 integrin to its natural
ligands, e.g. foot-and-mouth disease virus, requires the RGD motif
immediately followed by a Leu-Xaa-Xaa-Leu/Ile sequence, which forms a helix to
align the two conserved hydrophobic residues along the length of the helix.
Given the presence of many naturally occurring RGD-containing ligands, it is
conceivable that the specificities of the RGD-binding integrins are dictated
by the sequences flanking the RGD motif or those in neighboring domains that
come into close proximity with the RGD motif in the intact ligand proteins.
However, the preferences of α8β1 integrin for RGD-containing
ligands and how it secures its high-affinity binding toward its preferred
ligands remain unknown.In the present study, we investigated the binding specificities of
α8β1 integrin toward a panel of RGD-containing cell-adhesive
proteins. Our data reveal that nephronectin is a preferred ligand for
α8β1 integrin, and that a LFEIFEIER sequence on the C-terminal side
of its RGD motif serves as a synergy site to ensure the specific high-affinity
binding of nephronectin to α8β1 integrin. 相似文献
16.
Yuya Sato Tomoya Isaji Michiko Tajiri Shumi Yoshida-Yamamoto Tsuyoshi Yoshinaka Toshiaki Somehara Tomohiko Fukuda Yoshinao Wada Jianguo Gu 《The Journal of biological chemistry》2009,284(18):11873-11881
Recently we reported that N-glycans on the β-propeller domain
of the integrin α5 subunit (S-3,4,5) are essential for α5β1
heterodimerization, expression, and cell adhesion. Herein to further
investigate which N-glycosylation site is the most important for the
biological function and regulation, we characterized the S-3,4,5 mutants in
detail. We found that site-4 is a key site that can be specifically modified
by N-acetylglucosaminyltransferase III (GnT-III). The introduction of
bisecting GlcNAc into the S-3,4,5 mutant catalyzed by GnT-III decreased cell
adhesion and migration on fibronectin, whereas overexpression of
N-acetylglucosaminyltransferase V (GnT-V) promoted cell migration.
The phenomenon is similar to previous observations that the functions of the
wild-type α5 subunit were positively and negatively regulated by GnT-V
and GnT-III, respectively, suggesting that the α5 subunit could be
duplicated by the S-3,4,5 mutant. Interestingly GnT-III specifically modified
the S-4,5 mutant but not the S-3,5 mutant. This result was confirmed by
erythroagglutinating phytohemagglutinin lectin blot analysis. The reduction in
cell adhesion was consistently observed in the S-4,5 mutant but not in the
S-3,5 mutant cells. Furthermore mutation of site-4 alone resulted in a
substantial decrease in erythroagglutinating phytohemagglutinin lectin
staining and suppression of cell spread induced by GnT-III compared with that
of either the site-3 single mutant or wild-type α5. These results, taken
together, strongly suggest that N-glycosylation of site-4 on the
α5 subunit is the most important site for its biological functions. To
our knowledge, this is the first demonstration that site-specific modification
of N-glycans by a glycosyltransferase results in functional
regulation.Glycosylation is a crucial post-translational modification of most secreted
and cell surface proteins (1).
Glycosylation is involved in a variety of physiological and pathological
events, including cell growth, migration, differentiation, and tumor invasion.
It is well known that glycans play important roles in cell-cell communication,
intracellular signal transduction, protein folding, and stability
(2,
3).Integrins comprise a family of receptors that are important for cell
adhesion. The major function of integrins is to connect cells to the
extracellular matrix, activate intracellular signaling pathways, and regulate
cytoskeletal formation (4).
Integrin α5β1 is well known as a fibronectin
(FN)3 receptor. The
interaction between integrin α5 and FN is essential for cell migration,
cell survival, and development
(5–8).
In addition, integrins are N-glycan carrier proteins. For example,
α5β1 integrin contains 14 and 12 putative N-glycosylation
sites on the α5 and β1 subunits, respectively. Several studies
suggest that N-glycosylation is essential for functional integrin
α5β1. When human fibroblasts were cultured in the presence of
1-deoxymannojirimycin, which prevents N-linked oligosaccharide
processing, immature α5β1 integrin appeared on the cell surface,
and FN-dependent adhesion was greatly reduced
(9). Treatment of purified
integrin α5β1 with N-glycosidase F, which cleaves between
the innermost N-acetylglucosamine (GlcNAc) and asparagine
N-glycan residues of N-linked glycoproteins, prevented the
inherent association between subunits and blocked α5β1 binding to
FN (10).A growing body of evidence indicates that the presence of the appropriate
oligosaccharide can modulate integrin activation.
N-Acetylglucosaminyltransferase III (GnT-III) catalyzes the addition
of GlcNAc to mannose that is β1,4-linked to an underlying
N-acetylglucosamine, producing what is known as a
“bisecting” GlcNAc linkage as shown in
Fig. 1B. GnT-III is
generally regarded as a key glycosyltransferase in N-glycan
biosynthetic pathways and contributes to inhibition of metastasis. The
introduction of a bisecting GlcNAc catalyzed by GnT-III suppresses additional
processing and elongation of N-glycans. These reactions, which are
catalyzed in vitro by other glycosyltransferases, such as
N-acetylglucosaminyltransferase V (GnT-V), which catalyzes the
formation of β1,6 GlcNAc branching structures
(Fig. 1B) and plays
important roles in tumor metastasis, do not proceed because the enzymes cannot
utilize the bisected N-glycans as a substrate. Introduction of the
bisecting GlcNAc to integrin α5 by overexpression of GnT-III resulted in
decreased in ligand binding and down-regulation of cell adhesion and migration
(11–13).
Contrary to the functions of GnT-III, overexpression of GnT-V promoted
integrin α5β1-mediated cell migration on FN
(14). These observations
clearly demonstrate that the alteration of N-glycan structure
affected the biological functions of integrin α5β1. Similarly
characterization of the carbohydrate moieties in integrin α3β1 from
non-metastatic and metastatic human melanoma cell lines showed that expression
of β1,6 GlcNAc branched structures was higher in metastatic cells
compared with non-metastatic cells, confirming the notion that the β1,6
GlcNAc branched structure confers invasive and metastatic properties to cancer
cells. In fact, Partridge et al.
(15) reported that
GnT-V-modified N-glycans containing
poly-N-acetyllactosamine, the preferred ligand for galectin-3, on
surface receptors oppose their constitutive endocytosis, promoting
intracellular signaling and consequently cell migration and tumor
metastasis.Open in a separate windowFIGURE 1.Potential N-glycosylation sites on the α5 subunit and its
modification by GnT-III and GnT-V. A, schematic diagram of
potential N-glycosylation sites on the α5 subunit. Putative
N-glycosylation sites are indicated by triangles, and point
mutations are indicated by crosses (N84Q, N182Q, N297Q, N307Q, N316Q,
N524Q, N530Q, N593Q, N609Q, N675Q, N712Q, N724Q, N773Q, and N868Q).
B, illustration of the reaction catalyzed by GnT-III and GnT-V.
Square, GlcNAc; circle, mannose. TM, transmembrane
domain.In addition, sialylation on the non-reducing terminus of N-glycans
of α5β1 integrin plays an important role in cell adhesion. Colon
adenocarcinomas express elevated levels of α2,6 sialylation and
increased activity of ST6GalI sialyltransferase. Elevated ST6GalI positively
correlated with metastasis and poor survival. Therefore, ST6GalI-mediated
hypersialylation likely plays a role in colorectal tumor invasion
(16,
17). In fact, oncogenic
ras up-regulated ST6GalI and, in turn, increased sialylation of
β1 integrin adhesion receptors in colon epithelial cells
(18). However, this is not
always the case. The expression of hyposialylated integrin α5β1 was
induced by phorbol esterstimulated differentiation in myeloid cells in which
the expression of the ST6GalI was down-regulated by the treatment, increasing
FN binding (19). A similar
phenomenon was also observed in hematopoietic or other epithelial cells. In
these cells, the increased sialylation of the β1 integrin subunit was
correlated with reduced adhesiveness and metastatic potential
(20–22).
In contrast, the enzymatic removal of α2,8-linked oligosialic acids from
the α5 integrin subunit inhibited cell adhesion to FN
(23). Collectively these
findings suggest that the interaction of integrin α5β1 with FN is
dependent on its N-glycosylation and the processing status of
N-glycans.Because integrin α5β1 contains multipotential
N-glycosylation sites, it is important to determine the sites that
are crucial for its biological function and regulation. Recently we found that
N-glycans on the β-propeller domain (sites 3, 4, and 5) of the
integrin α5 subunit are essential for α5β1
heterodimerization, cell surface expression, and biological function
(24). In this study, to
further investigate the underlying molecular mechanism of GnT-III-regulated
biological functions, we characterized the N-glycans on the α5
subunit in detail using genetic and biochemical approaches and found that
site-4 is a key site that can be specifically modified by GnT-III. 相似文献
17.
18.
Background
Mutation in αA-crystallin contributes to the development of congenital cataract in humans. Heterooligomerization of αA-crystallin and αB-crystallin is essential for maintaining transparency in the eye lens. The effect of congenital cataract causing mutants of αA-crystallin on subunit exchange and interaction with αB-crystallin is unknown. In the present study, interaction of the mutants of αA-crystallin with αB-crystallin was studied both in vitro and in situ by the fluorescence resonance energy transfer (FRET) technique.Methodology/Principal Findings
In vitro FRET technique was used to demonstrate the rates of subunit exchange of αB-wt with the following αA-crystallin mutants: R12C, R21L, R21W, R49C, R54C, and R116C. The subunit exchange rates (k values) of R21W and R116C with αB-wt decreased drastically as compared to αA-wt interacting with αB-wt. Moderately decreased k values were seen with R12C, R49C and R54C while R21L showed nearly normal k value. The interaction of αA- mutants with αB-wt was also assessed by in situ FRET. YFP-tagged αA mutants were co-expressed with CFP-tagged αB-wt in HeLa cells and the spectral signals were captured with a confocal microscope before and after acceptor laser photobleaching. The interaction of R21W and R116C with αB-wt was decreased nearly 50% as compared to αA-wt while the rest of the mutants showed slightly decreased interaction. Thus, there is good agreement between the in vitro and in situ FRET data.Conclusions/Significance
Structural changes occurring in these mutants, as reported earlier, could be the underlying cause for the decreased interaction with αB may contribute to development of congenital cataract. 相似文献19.