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1.
Haipeng Cheng Kulandaivelu S. Vetrivel Renaldo C. Drisdel Xavier Meckler Ping Gong Jae Yoon Leem Tong Li Meghan Carter Ying Chen Phuong Nguyen Takeshi Iwatsubo Taisuke Tomita Philip C. Wong William N. Green Maria Z. Kounnas Gopal Thinakaran 《The Journal of biological chemistry》2009,284(3):1373-1384
Proteolytic processing of amyloid precursor protein (APP) by β- and
γ-secretases generates β-amyloid (Aβ) peptides, which
accumulate in the brains of individuals affected by Alzheimer disease.
Detergent-resistant membrane microdomains (DRM) rich in cholesterol and
sphingolipid, termed lipid rafts, have been implicated in Aβ production.
Previously, we and others reported that the four integral subunits of the
γ-secretase associate with DRM. In this study we investigated the
mechanisms underlying DRM association of γ-secretase subunits. We report
that in cultured cells and in brain the γ-secretase subunits nicastrin
and APH-1 undergo S-palmitoylation, the post-translational covalent
attachment of the long chain fatty acid palmitate common in lipid
raft-associated proteins. By mutagenesis we show that nicastrin is
S-palmitoylated at Cys689, and APH-1 is
S-palmitoylated at Cys182 and Cys245.
S-Palmitoylation-defective nicastrin and APH-1 form stable
γ-secretase complexes when expressed in knock-out fibroblasts lacking
wild type subunits, suggesting that S-palmitoylation is not essential
for γ-secretase assembly. Nevertheless, fractionation studies show that
S-palmitoylation contributes to DRM association of nicastrin and
APH-1. Moreover, pulse-chase analyses reveal that S-palmitoylation is
important for nascent polypeptide stability of both proteins. Co-expression of
S-palmitoylation-deficient nicastrin and APH-1 in cultured cells
neither affects Aβ40, Aβ42, and AICD production, nor intramembrane
processing of Notch and N-cadherin. Our findings suggest that
S-palmitoylation plays a role in stability and raft localization of
nicastrin and APH-1, but does not directly modulate γ-secretase
processing of APP and other substrates.Alzheimer disease is the most common among neurodegenerative diseases that
cause dementia. This debilitating disorder is pathologically characterized by
the cerebral deposition of 39–42 amino acid peptides termed Aβ,
which are generated by proteolytic processing of amyloid precursor protein
(APP)2 by β- and
γ-secretases (1,
2). The β-site APP
cleavage enzyme 1 cleaves full-length APP within its luminal domain to
generate a secreted ectodomain leaving behind a C-terminal fragment
(β-CTF). γ-Secretase cleaves β-CTF within the transmembrane
domain to release Aβ and APP intracellular
C-terminal domain (AICD). γ-Secretase is a
multiprotein complex, comprising at least four subunits: presenilins (PS1 and
PS2), nicastrin, APH-1, and PEN-2 for its activity
(3). PS1 is synthesized as a
42–43-kDa polypeptide and undergoes highly regulated endoproteolytic
processing within the large cytoplasmic loop domain connecting putative
transmembrane segments 6 and 7 to generate stable N-terminal (NTF) and
C-terminal fragments (CTF) by an uncharacterized proteolytic activity
(4). This endoproteolytic event
has been identified as the activation step in the process of PS1 maturation as
it assembles with other γ-secretase subunits
(3). Nicastrin is a heavily
glycosylated type I membrane protein with a large ectodomain that has been
proposed to function in substrate recognition and binding
(5), but this putative function
has not been confirmed by others
(6). APH-1 is a
seven-transmembrane protein encoded by two human or three rodent genes that
are alternatively spliced (7).
Although PS1 (or PS2), nicastrin, APH-1, and PEN-2 are sufficient for
γ-secretase processing of APP, a type I membrane protein, termed p23
(also referred toTMP21), was recently identified as a γ-secretase
component that modulates γ-secretase activity and regulates secretory
trafficking of APP (8,
9).A growing number of type I integral membrane proteins has been identified
as γ-secretase substrates within the last few years, including Notch1
homologues, Notch ligands, Delta and Jagged, cell adhesion receptors N- and
E-cadherins, low density lipoprotein receptor-related protein, ErbB-4, netrin
receptor DCC, and others (10).
Mounting evidence suggests that APP processing occurs within cholesterol- and
sphingolipid-enriched lipid rafts, which are biochemically defined as
detergentresistant membrane microdomains (DRM)
(11,
12). Previously we reported
that each of the γ-secretase subunits localizes in lipid rafts in
post-Golgi and endosome membranes enriched in syntaxin 6
(13). Moreover, loss of
γ-secretase activity by gene deletion or exposure to γ-secretase
inhibitors results in the accumulation of APP CTFs in lipid rafts indicating
that cleavage of APP CTFs likely occurs in raft microdomains
(14). In contrast, CTFs
derived from Notch1, Jagged2, N-cadherin, and DCC are processed by
γ-secretase in non-raft membranes
(14). The mechanisms
underlying association of γ-secretase subunits with lipid rafts need
further clarification to elucidate spatial segregation of amyloidogenic
processing of APP in membrane microdomains.Post-translational S-palmitoylation is increasingly recognized as
a potential mechanism for regulating raft association, stability,
intracellular trafficking, and function of several cytosolic and transmembrane
proteins
(15–17).
S-palmitoylation refers to the addition of 16-carbon palmitoyl moiety
to certain cysteine residues through thioester linkage. Cysteines close to
transmembrane domains or membrane-associated domains in non-integral membrane
proteins are preferred S-palmitoylation sites, although no conserved
motif has been identified
(18). Palmitoylation modifies
numerous neuronal proteins, including postsynaptic density protein PSD-95
(19),
a-amino-3-hydroxyl-5-methyl-4-isoxazole propionic acid receptors
(20), nicotinic α7
receptors (21), neuronal
t-SNAREs SNAP-25, synaptobrevin 2 and synaptogagmin
(22,
23), neuronal
growth-associated protein GAP-43
(24), protein kinase CLICK-III
(CL3)/CaMKIγ (25),
β-secretase (26), and
Huntingtin (27). Although
palmitoylation can occur in vitro without the involvement of an
enzyme, a family of palmitoyltransferases that specifically catalyze
S-palmitoylation has been identified
(28,
29).In this study, we have identified S-palmitoylation of
γ-secretase subunits nicastrin and APH-1, and characterized its role on
DRM association, protein stability, and γ-secretase enzyme activities.
We show that nicastrin is S-palmitoylated at Cys689, and
APH-1 at Cys182 and Cys245. Mutagenesis of
palmitoylation sites results in increased degradation of nascent nicastrin and
APH-1 polypeptides and reduced association with DRM. Nevertheless, in cultured
cells overexpression of S-palmitoylation-deficient nicastrin and
APH-1 does not modulate γ-secretase processing of APP or other
substrates. 相似文献
2.
Yi Qian Intaek Lee Wang-Sik Lee Meiqian Qian Mariko Kudo William M. Canfield Peter Lobel Stuart Kornfeld 《The Journal of biological chemistry》2010,285(5):3360-3370
UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase is an α2β2γ2 hexamer that mediates the first step in the synthesis of the mannose 6-phosphate recognition marker on lysosomal acid hydrolases. Using a multifaceted approach, including analysis of acid hydrolase phosphorylation in mice and fibroblasts lacking the γ subunit along with kinetic studies of recombinant α2β2γ2 and α2β2 forms of the transferase, we have explored the function of the α/β and γ subunits. The findings demonstrate that the α/β subunits recognize the protein determinant of acid hydrolases in addition to mediating the catalytic function of the transferase. In mouse brain, the α/β subunits phosphorylate about one-third of the acid hydrolases at close to wild-type levels but require the γ subunit for optimal phosphorylation of the rest of the acid hydrolases. In addition to enhancing the activity of the α/β subunits toward a subset of the acid hydrolases, the γ subunit facilitates the addition of the second GlcNAc-P to high mannose oligosaccharides of these substrates. We postulate that the mannose 6-phosphate receptor homology domain of the γ subunit binds and presents the high mannose glycans of the acceptor to the α/β catalytic site in a favorable manner. 相似文献
3.
4.
β Subunits of Voltage-Gated Calcium Channels 总被引:12,自引:0,他引:12
Dolphin AC 《Journal of bioenergetics and biomembranes》2003,35(6):599-620
Calcium channel beta subunits have marked effects on the trafficking and on several of the biophysical properties of all high voltage activated calcium channels. In this article I shall review information on the different genes, on the structure of the beta subunits, and on their differential expression and post-translational modification. Their role in trafficking and assembly of the calcium channel heteromultimer will be described, and I will then review their effects on voltage-dependent and kinetic properties, stressing the differences between palmitoylated beta2a and the other beta subunits. Evidence for effects on calcium channel pharmacology will also be examined. I shall discuss the hypothesis that beta subunits can bind reversibly to calcium channels, and examine their role in the G protein modulation of calcium channels. Finally, I shall describe the consequences of knock-out of different beta subunit genes, and describe evidence for the involvement of beta subunits in disease. 相似文献
5.
Different post-translational modifications of Ca channel subunits have been identified. Recent studies have characterized the palmitoylation of the Ca channel 2a subunit, as well as one effect of this modification on channel function. The potential importance of palmitoylation on other channel properties is discussed. Other studies have addressed the role of phosphorylation of subunits in the regulation of voltage-dependent Ca channels. Phosphorylation of subunits by second messenger-activated protein kinases, as well as by unidentified protein kinases, may affect interactions between channel subunits and other aspects of channel function. The differential modification of Ca channel subunit isoforms by post-translational events likely results in diversely regulated channels with unique properties. 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.
Lutz Birnbaumer Ning Qin Riccardo Olcese Erwin Tareilus Daniela Platano Jim Costantin Enrico Stefani 《Journal of bioenergetics and biomembranes》1998,30(4):357-375
Calcium channel subunits have profound effects on how 1 subunits perform. In this article we summarize our present knowledge of the primary structures of subunits as deduced from cDNAs and illustrate their different properties. Upon co-expression with 1 subunits, the effects of subunits vary somewhat between L-type and non-L-type channels mostly because the two types of channels have different responses to voltage which are affected by subunits, such as long-lasting prepulse facilitation of 1C (absent in 1E) and inhibition by G protein dimer of 1E, absent in 1C. One subunit, a brain 2a splice variant that is palmitoylated, has several effects not seen with any of the others, and these are due to palmitoylation. We also illustrate the finding that functional expression of 1 in oocytes requires a subunit even if the final channel shows no evidence for its presence. We propose two structural models for Ca2+ channels to account for 1 alone channels seen in cells with limited subunit expression. In one model, dissociates from the mature 1 after proper folding and membrane insertion. Regulated channels seen upon co-expression of high levels of would then have subunit composition 1. In the other model, the chaperoning remains associated with the mature channel and 1 alone channels would in fact be 1 channels. Upon co-expression of high levels of the regulated channels would have composition [1]. 相似文献
8.
Wen-yi Lo Andre H. Lagrange Ciria C. Hernandez Katharine N. Gurba Robert L. Macdonald 《Neurochemical research》2014,39(6):1088-1103
GABAA receptors, the major mediators of fast inhibitory neuronal transmission, are heteropentameric glycoproteins assembled from a panel of subunits, usually including α and β subunits with or without a γ2 subunit. The α1β2γ2 receptor is the most abundant GABAA receptor in brain. Co-expression of γ2 with α1 and β2 subunits causes conformational changes, increases GABAA receptor channel conductance, and prolongs channel open times. We reported previously that glycosylation of the three β2 subunit glycosylation sites, N32, N104 and N173, was important for α1β2 receptor channel gating. Here, we examined the hypothesis that steric effects or conformational changes caused by γ2 subunit co-expression alter the glycosylation of partnering β2 subunits. We found that co-expression of γ2 subunits hindered processing of β2 subunit N104 N-glycans in HEK293T cells. This γ2 subunit-dependent effect was strong enough that a decrease of γ2 subunit expression in heterozygous GABRG2 knockout (γ2+/?) mice led to appreciable changes in the endoglycosidase H digestion pattern of neuronal β2 subunits. Interestingly, as measured by flow cytometry, γ2 subunit surface levels were decreased by mutating each of the β2 subunit glycosylation sites. The β2 subunit mutation N104Q also decreased GABA potency to evoke macroscopic currents and reduced conductance, mean open time and open probability of single channel currents. Collectively, our data suggested that γ2 subunits interacted with β2 subunit N-glycans and/or subdomains containing the glycosylation sites, and that γ2 subunit co-expression-dependent alterations in the processing of the β2 subunit N104 N-glycans were involved in altering the function of surface GABAA receptors. 相似文献
9.
Elizabeth Scotto-Lavino Miguel Garcia-Diaz Guangwei Du Michael A. Frohman 《The Journal of biological chemistry》2010,285(9):6419-6424
Myosin II association with actin, which triggers contraction, is regulated by orchestrated waves of phosphorylation/dephosphorylation of the myosin regulatory light chain. Blocking myosin regulatory light chain phosphorylation with small molecule inhibitors alters the shape, adhesion, and migration of many types of smooth muscle and cancer cells. Dephosphorylation is mediated by myosin phosphatase (MP), a complex that consists of a catalytic subunit (protein phosphatase 1c, PP1c), a large subunit (myosin phosphatase targeting subunit, MYPT), and a small subunit of unknown function. MYPT functions by targeting PP1c onto its substrate, phosphorylated myosin II. Using RNA interference, we show here that stability of PP1c β and MYPT1 is interdependent; knocking down one of the subunits decreases the expression level of the other. Associated changes in cell shape also occur, characterized by flattening and spreading accompanied by increased cortical actin, and cell numbers decrease secondary to apoptosis. Of the three highly conserved isoforms of PP1c, we show that MYPT1 binding is restricted to PP1c β, and, using chimeric analysis and site-directed mutations, that the central region of PP1c β confers the isoform-specific binding. This finding was unexpected because the MP crystal structure has been solved and was reported to identify the variable, C-terminal domain of PP1c β as being the region key for isoform-specific interaction with MYPT1. These findings suggest a potential screening strategy for cardiovascular and cancer therapeutic agents based on destabilizing MP complex formation and function. 相似文献
10.
Xiao-Qian Zhao Jian-Fei Hu Jun Yu 《基因组蛋白质组与生物信息学报(英文版)》2006,4(4):203-211
DNA polymerase Ⅲ is one of the five eubacterial DNA polymerases that is responsible for the replication of DNA duplex. Among the ten subunits of the DNA polymerase Ⅲ core enzyme, the alpha subunit catalyzes the reaction for polymerizing both DNA strands. In this study, we extracted genomic sequences of the alpha subunit from 159 sequenced eubacterial genomes, and carried out sequence- based phylogenetic and structural analyses. We found that all eubacterial genomes have one or more alpha subunits, which form either homodimers or heterodimers. Phylogenetic and domain structural analyses as well as copy number variations of the alpha subunit in each bacterium indicate the classification of alpha subunit into four basic groups: polC, dnaE1, dnaE2, and dnaE3. This classification is of essence in genome composition analysis. We also consolidated the naming convention to avoid further confusion in gene annotations. 相似文献
11.
12.
The Functional Role of β Subunits in Oligomeric P-Type ATPases 总被引:5,自引:0,他引:5
Käthi Geering 《Journal of bioenergetics and biomembranes》2001,33(5):425-438
Na,K-ATPase and gastric and nongastric H,K-ATPases are the only P-type ATPases of higher organisms that are oligomeric and are associated with a subunit, which is obligatory for expression and function of enzymes. Topogenesis studies suggest that subunits have a fundamental and unique role in K+-transporting P-type ATPases in that they facilitate the correct membrane integration and packing of the catalytic subunit of these P-type ATPases, which is necessary for their resistance to cellular degradation, their acquisition of functional properties, and their routing to the cell surface. In addition to this chaperone function, subunits also participate in the determination of intrinsic transport properties of Na,K- and H,K-ATPases. Increasing experimental evidence suggests that assembly is a highly ordered, isoform-specific process, which is mediated by multiple interaction sites that contribute in a coordinate, multistep process to the structural and functional maturation of Na,K- and H,K-ATPases. 相似文献
13.
Can Zhang Andrew Browne Daniel Child Jason R. DiVito Jesse A. Stevenson Rudolph E. Tanzi 《The Journal of biological chemistry》2010,285(12):8515-8526
Alzheimer disease (AD) is a devastating neurodegenerative disease with complex and strong genetic inheritance. Four genes have been established to either cause familial early onset AD (APP, PSEN1, and PSEN2) or to increase susceptibility for late onset AD (APOE). To date ∼80% of the late onset AD genetic variance remains elusive. Recently our genome-wide association screen identified four novel late onset AD candidate genes. Ataxin 1 (ATXN1) is one of these four AD candidate genes and has been indicated to be the disease gene for spinocerebellar ataxia type 1, which is also a neurodegenerative disease. Mounting evidence suggests that the excessive accumulation of Aβ, the proteolytic product of β-amyloid precursor protein (APP), is the primary AD pathological event. In this study, we ask whether ATXN1 may lead to AD pathogenesis by affecting Aβ and APP processing utilizing RNA interference in a human neuronal cell model and mouse primary cortical neurons. We show that knock-down of ATXN1 significantly increases the levels of both Aβ40 and Aβ42. This effect could be rescued with concurrent overexpression of ATXN1. Moreover, overexpression of ATXN1 decreased Aβ levels. Regarding the underlying molecular mechanism, we show that the effect of ATXN1 expression on Aβ levels is modulated via β-secretase cleavage of APP. Taken together, ATXN1 functions as a genetic risk modifier that contributes to AD pathogenesis through a loss-of-function mechanism by regulating β-secretase cleavage of APP and Aβ levels. 相似文献
14.
Laszlo L. P. Hosszu Clare R. Trevitt Samantha Jones Mark Batchelor David J. Scott Graham S. Jackson John Collinge Jonathan P. Waltho Anthony R. Clarke 《The Journal of biological chemistry》2009,284(33):21981-21990
Prion propagation involves a conformational transition of the cellular form of prion protein (PrPC) to a disease-specific isomer (PrPSc), shifting from a predominantly α-helical conformation to one dominated by β-sheet structure. This conformational transition is of critical importance in understanding the molecular basis for prion disease. Here, we elucidate the conformational properties of a disulfide-reduced fragment of human PrP spanning residues 91–231 under acidic conditions, using a combination of heteronuclear NMR, analytical ultracentrifugation, and circular dichroism. We find that this form of the protein, which similarly to PrPSc, is a potent inhibitor of the 26 S proteasome, assembles into soluble oligomers that have significant β-sheet content. The monomeric precursor to these oligomers exhibits many of the characteristics of a molten globule intermediate with some helical character in regions that form helices I and III in the PrPC conformation, whereas helix II exhibits little evidence for adopting a helical conformation, suggesting that this region is a likely source of interaction within the initial phases of the transformation to a β-rich conformation. This precursor state is almost as compact as the folded PrPC structure and, as it assembles, only residues 126–227 are immobilized within the oligomeric structure, leaving the remainder in a mobile, random-coil state.Prion diseases, such as Creutzfeldt-Jacob and Gerstmann-Sträussler-Scheinker in humans, scrapie in sheep, and bovine spongiform encephalopathy in cattle, are fatal neurological disorders associated with the deposition of an abnormally folded form of a host-encoded glycoprotein, prion (PrP)2 (1). These diseases may be inherited, arise sporadically, or be acquired through the transmission of an infectious agent (2, 3). The disease-associated form of the protein, termed the scrapie form or PrPSc, differs from the normal cellular form (PrPC) through a conformational change, resulting in a significant increase in the β-sheet content and protease resistance of the protein (3, 4). PrPC, in contrast, consists of a predominantly α-helical structured domain and an unstructured N-terminal domain, which is capable of binding a number of divalent metals (5–12). A single disulfide bond links two of the main α-helices and forms an integral part of the core of the structured domain (13, 14).According to the protein-only hypothesis (15), the infectious agent is composed of a conformational isomer of PrP (16) that is able to convert other isoforms to the infectious isomer in an autocatalytic manner. Despite numerous studies, little is known about the mechanism of conversion of PrPC to PrPSc. The most coherent and general model proposed thus far is that PrPC fluctuates between the dominant native state and minor conformations, one or a set of which can self-associate in an ordered manner to produce a stable supramolecular structure composed of misfolded PrP monomers (3, 17). This stable, oligomeric species can then bind to, and stabilize, rare non-native monomer conformations that are structurally complementary. In this manner, new monomeric chains are recruited and the system can propagate.In view of the above model, considerable effort has been devoted to generating and characterizing alternative, possibly PrPSc-like, conformations in the hope of identifying common properties or features that facilitate the formation of amyloid oligomers. This has been accomplished either through PrPSc-dependent conversion reactions (18–20) or through conversion of PrPC in the absence of a PrPSc template (21–25). The latter approach, using mainly disulfide-oxidized recombinant PrP, has generated a wide range of novel conformations formed under non-physiological conditions where the native state is relatively destabilized. These conformations have ranged from near-native (14, 26, 27), to those that display significant β-sheet content (21, 23, 28–33). The majority of these latter species have shown a high propensity for aggregation, although not all are on-pathway to the formation of amyloid. Many of these non-native states also display some of the characteristics of PrPSc, such as increased β-sheet content, protease resistance, and a propensity for oligomerization (28, 29, 31) and some have been claimed to be associated with the disease process (34).One such PrP folding intermediate, termed β-PrP, differs from the majority of studied PrP intermediate states in that it is formed by refolding the PrP molecule from the native α-helical conformation (here termed α-PrP), at acidic pH in a reduced state, with the disulfide bond broken (22, 35). Although no covalent differences between the PrPC and PrPSc have been consistently identified to date, the role of the disulfide bond in prion propagation remains disputed (25, 36–39). β-PrP is rich in β-sheet structure (22, 35), and displays many of the characteristics of a PrPSc-like precursor molecule, such as partial resistance to proteinase K digestion, and the ability to form amyloid fibrils in the presence of physiological concentrations of salts (40).The β-PrP species previously characterized, spanning residues 91–231 of PrP, was soluble at low ionic strength buffers and monomeric, according to elution volume on gel filtration (22). NMR analysis showed that it displayed radically different spectra to those of α-PrP, with considerably fewer observable peaks and markedly reduced chemical shift dispersion. Data from circular dichroism experiments showed that fixed side chain (tertiary) interactions were lost, in contrast to the well defined β-sheet secondary structure, and thus in conjunction with the NMR data, indicated that β-PrP possessed a number of characteristics associated with a “molten globule” folding intermediate (22). Such states have been proposed to be important in amyloid and fibril formation (41). Indeed, antibodies raised against β-PrP (e.g. ICSM33) are capable of recognizing native PrPSc (but not PrPC) (42–44). Subsequently, a related study examining the role of the disulfide bond in PrP folding confirmed that a monomeric molten globule-like form of PrP was formed on refolding the disulfide-reduced protein at acidic pH, but reported that, under their conditions, the circular dichroism response interpreted as β-sheet structure was associated with protein oligomerization (45). Indeed, atomic force microscopy on oligomeric full-length β-PrP (residues 23–231) shows small, round particles, showing that it is capable of formation of oligomers without forming fibrils (35). Notably, however, salt-induced oligomeric β-PrP has been shown to be a potent inhibitor of the 26 S proteasome, in a similar manner to PrPSc (46). Impairment of the ubiquitin-proteasome system in vivo has been linked to prion neuropathology in prion-infected mice (46).Although the global properties of several PrP intermediate states have been determined (30–32, 35), no information on their conformational properties on a sequence-specific basis has been obtained. Their conformational properties are considered important, as the elucidation of the chain conformation may provide information on the way in which these chains pack in the assembly process, and also potentially provide clues on the mechanism of amyloid assembly and the phenomenon of prion strains. As the conformational fluctuations and heterogeneity of molten globule states give rise to broad NMR spectra that preclude direct observation of their conformational properties by NMR (47–50), here we use denaturant titration experiments to determine the conformational properties of β-PrP, through the population of the unfolded state that is visible by NMR. In addition, we use circular dichroism and analytical ultracentrifugation to examine the global structural properties, and the distribution of multimeric species that are formed from β-PrP. 相似文献
15.
Nikolaos Giagtzoglou Cindy V. Ly Hugo J. Bellen 《Cold Spring Harbor perspectives in biology》2009,1(4)
Synapses are asymmetric intercellular junctions that mediate neuronal communication. The number, type, and connectivity patterns of synapses determine the formation, maintenance, and function of neural circuitries. The complexity and specificity of synaptogenesis relies upon modulation of adhesive properties, which regulate contact initiation, synapse formation, maturation, and functional plasticity. Disruption of adhesion may result in structural and functional imbalance that may lead to neurodevelopmental diseases, such as autism, or neurodegeneration, such as Alzheimer''s disease. Therefore, understanding the roles of different adhesion protein families in synapse formation is crucial for unraveling the biology of neuronal circuit formation, as well as the pathogenesis of some brain disorders. The present review summarizes some of the knowledge that has been acquired in vertebrate and invertebrate genetic model organisms.Synapses are asymmetric, intercellular junctions that are the basic structural units of neuronal transmission. The correct development of synaptic specializations and the establishment of appropriate connectivity patterns are crucial for the assembly of functional neuronal circuits. Improper synapse formation and function may cause neurodevelopmental disorders, such as mental retardation (MsR) and autism spectrum disorders (ASD) (McAllister 2007; Sudhof 2008), and likely play a role in neurodegenerative disorders, such as Alzheimer''s disease (AD) (Haass and Selkoe 2007).At chemical synapses (reviewed in Sudhof 2004; Zhai and Bellen 2004; Waites et al. 2005; McAllister 2007; Jin and Garner 2008), the presynaptic compartment contains synaptic vesicles (SV), organized in functionally distinct subcellular pools. A subset of SVs docks to the presynaptic membrane around protein-dense release sites, named active zones (AZ). Upon the arrival of an action potential at the terminal, the docked and “primed” SVs fuse with the plasma membrane and release neurotransmitter molecules into the synaptic cleft. Depending on the type of synapse (i.e., excitatory vs. inhibitory synapses), neurotransmitters ultimately activate an appropriate set of postsynaptic receptors that are accurately apposed to the AZ.Synapse formation occurs in several steps (Fig. 1) (reviewed in Eaton and Davis 2003; Goda and Davis 2003; Waites et al. 2005; Garner et al. 2006; Gerrow and El-Husseini 2006; McAllister 2007). Spatiotemporal signals guide axons through heterogeneous cellular environments to contact appropriate postsynaptic targets. At their destination, axonal growth cones initiate synaptogenesis through adhesive interactions with target cells. In the mammalian central nervous system (CNS), immature postsynaptic dendritic spines initially protrude as thin, actin-rich filopodia on the surface of dendrites. Similarly, at the Drosophila neuromuscular junction (NMJ), myopodia develop from the muscles (Ritzenthaler et al. 2000). The stabilization of intercellular contacts and their elaboration into mature, functional synapses involves cytoskeletal arrangements and recruitment of pre- and postsynaptic components to contact sites in spines and boutons. Conversely, retraction of contacts results in synaptic elimination. Both stabilization and retraction sculpt a functional neuronal circuitry.Open in a separate windowFigure 1.(A–C) Different stages of synapse formation. (A) Target selection, (B) Synapse assembly, (C) Synapse maturation and stabilization. (D–F) The role of cell adhesion molecules in synapse formation is exemplified by the paradigm of N-cadherin and catenins in regulation of the morphology and strength of dendritic spine heads. (D) At an early stage the dendritic spines are elongated from motile structures “seeking” their synaptic partners. (E) The contacts between the presynaptic and postsynaptic compartments are stabilized by recruitment of additional cell adhesion molecules. Adhesional interactions activate downstream pathways that remodel the cytoskeleton and organize pre- and postsynaptic apparatuses. (F) Cell adhesion complexes, stabilized by increased synaptic activity, promote the expansion of the dendritic spine head and the maturation/ stabilization of the synapse. Retraction and expansion is dependent on synaptic plasticity.In addition to the plastic nature of synapse formation, the vast heterogeneity of synapses (in terms of target selection, morphology, and type of neurotransmitter released) greatly enhances the complexity of synaptogenesis (reviewed in Craig and Boudin 2001; Craig et al. 2006; Gerrow and El-Husseini 2006). The complexity and specificity of synaptogenesis relies upon the modulation of adhesion between the pre- and postsynaptic components (reviewed in Craig et al. 2006; Gerrow and El-Husseini 2006; Piechotta et al. 2006; Dalva et al. 2007; Shapiro et al. 2007; Yamada and Nelson 2007; Gottmann 2008). Cell adhesive interactions enable cell–cell recognition via extracellular domains and also mediate intracellular signaling cascades that affect synapse morphology and organize scaffolding complexes. Thus, cell adhesion molecules (CAMs) coordinate multiple synaptogenic steps.However, in vitro and in vivo studies of vertebrate CAMs are often at odds with each other. Indeed, there are no examples of mutants for synaptic CAMs that exhibit prominent defects in synapse formation. This apparent “resilience” of synapses is probably caused by functional redundancy or compensatory effects among different CAMs (Piechotta et al. 2006). Hence, studies using simpler organisms less riddled by redundancy, such as Caenorhabditis elegans and Drosophila, have aided in our understanding of the role that these molecules play in organizing synapses.In this survey, we discuss the roles of the best characterized CAM families of proteins involved in synaptogenesis. Our focus is to highlight the complex principles that govern the molecular basis of synapse formation and function from a comparative perspective. We will present results from cell culture studies as well as in vivo analyses in vertebrate systems and refer to invertebrate studies, mainly performed in Drosophila and C. elegans, when they have provided important insights into the role of particular CAM protein families. However, we do not discuss secreted factors, for which we refer the reader to numerous excellent reviews (as for example Washbourne et al. 2004; Salinas 2005; Piechotta et al. 2006; Shapiro et al. 2006; Dalva 2007; Yamada and Nelson 2007; Biederer and Stagi 2008; Salinas and Zou 2008). 相似文献
16.
Jennie Garcia-Olivares Delany Torres-Salazar William A. Owens Tracy Baust David P. Siderovski Susan G. Amara Jun Zhu Lynette C. Daws Gonzalo E. Torres 《PloS one》2013,8(3)
Uptake through the Dopamine Transporter (DAT) is the primary mechanism of terminating dopamine signaling within the brain, thus playing an essential role in neuronal homeostasis. Deregulation of DAT function has been linked to several neurological and psychiatric disorders including ADHD, schizophrenia, Parkinson’s disease, and drug addiction. Over the last 15 years, several studies have revealed a plethora of mechanisms influencing the activity and cellular distribution of DAT; suggesting that fine-tuning of dopamine homeostasis occurs via an elaborate interplay of multiple pathways. Here, we show for the first time that the βγ subunits of G proteins regulate DAT activity. In heterologous cells and brain tissue, a physical association between Gβγ subunits and DAT was demonstrated by co-immunoprecipitation. Furthermore, in vitro pull-down assays using purified proteins established that this association occurs via a direct interaction between the intracellular carboxy-terminus of DAT and Gβγ. Functional assays performed in the presence of the non-hydrolyzable GTP analog GTP-γ-S, Gβγ subunit overexpression, or the Gβγ activator mSIRK all resulted in rapid inhibition of DAT activity in heterologous systems. Gβγ activation by mSIRK also inhibited dopamine uptake in brain synaptosomes and dopamine clearance from mouse striatum as measured by high-speed chronoamperometry in vivo. Gβγ subunits are intracellular signaling molecules that regulate a multitude of physiological processes through interactions with enzymes and ion channels. Our findings add neurotransmitter transporters to the growing list of molecules regulated by G-proteins and suggest a novel role for Gβγ signaling in the control of dopamine homeostasis. 相似文献
17.
Studart-Guimarães C Gibon Y Frankel N Wood CC Zanor MI Fernie AR Carrari F 《Plant molecular biology》2005,59(5):781-791
Despite the central importance of the TCA cycle in plant metabolism not all of the genes encoding its constituent enzymes
have been functionally identified. In yeast, the heterodimeric protein succinyl CoA ligase is encoded for by two single-copy
genes. Here we report the isolation of two tomato cDNAs coding for α- and one coding for the β-subunit of succinyl CoA ligase.
These three cDNAs were used to complement the respective Saccharomyces cerevisiae mutants deficient in the α- and β-subunit, demonstrating that they encode functionally active polypeptides. The genes encoding
for the subunits were expressed in all tissues, but most strongly in floral and leaf tissues, with equivalent expression of
the two α-subunit genes being expressed to equivalent levels in all tissues. In all instances GFP fusion expression studies
confirmed an expected mitochondrial location of the proteins encoded. Following the development of a novel assay to measure
succinyl CoA ligase activity, in the direction of succinate formation, the evaluation of the maximal catalytic activities
of the enzyme in a range of tissues revealed that these paralleled those of mRNA levels. We also utilized this assay to perform
a preliminary characterisation of the regulatory properties of the enzyme suggesting allosteric control of this enzyme which
may regulate flux through the TCA cycle in a manner consistent with its position therein. 相似文献
18.
Mukesh Saxena Samarendra Singh Shamsu Zzaman Deepak Bastia 《The Journal of biological chemistry》2010,285(8):5695-5704
A typical plasmid replicon of Escherichia coli, such as ori γ of R6K, contains tandem iterons (iterated initiator protein binding sites), an AT-rich region that melts upon initiator-iteron interaction, two binding sites for the bacterial initiator protein DnaA, and a binding site for the DNA-bending protein IHF. R6K also contains two structurally atypical origins called α and β that are located on either side of γ and contain a single and a half-iteron, respectively. Individually, these sites do not bind to initiator protein π but access it by DNA looping-mediated interaction with the seven π-bound γ iterons. The π protein exists in 2 interconvertible forms: inert dimers and active monomers. Initiator dimers generally function as negative regulators of replication by promoting iteron pairing (“handcuffing”) between pairs of replicons that turn off both origins. Contrary to this existing paradigm, here we show that both the dimeric and the monomeric π are necessary for ori α-driven plasmid maintenance. Furthermore, efficient looping interaction between α and γ or between 2 γ iterons in vitro also required both forms of π. Why does α-γ iteron pairing promote α activation rather than repression? We show that a weak, transitory α-γ interaction at the iteron pairs was essential for α-driven plasmid maintenance. Swapping the α iteron with one of γ without changing the original sequence context that caused enhanced looping in vitro caused a significant inhibition of α-mediated plasmid maintenance. Therefore, the affinity of α iteron for π-bound γ and not the sequence context determined whether the origin was activated or repressed. 相似文献
19.
Evgeny Kobrinsky Parwiz Abrahimi Son Q. Duong Sam Thomas Jo Beth Harry Chirag Patel Qi Zong Lao Nikolai M. Soldatov 《PloS one》2009,4(5)
Background
Voltage-gated Cav1.2 calcium channels play a crucial role in Ca2+ signaling. The pore-forming α1C subunit is regulated by accessory Cavβ subunits, cytoplasmic proteins of various size encoded by four different genes (Cavβ1 - β4) and expressed in a tissue-specific manner.Methods and Results
Here we investigated the effect of three major Cavβ types, β1b, β2d and β3, on the structure of Cav1.2 in the plasma membrane of live cells. Total internal reflection fluorescence microscopy showed that the tendency of Cav1.2 to form clusters depends on the type of the Cavβ subunit present. The highest density of Cav1.2 clusters in the plasma membrane and the smallest cluster size were observed with neuronal/cardiac β1b present. Cav1.2 channels containing β3, the predominant Cavβ subunit of vascular smooth muscle cells, were organized in a significantly smaller number of larger clusters. The inter- and intramolecular distances between α1C and Cavβ in the plasma membrane of live cells were measured by three-color FRET microscopy. The results confirm that the proximity of Cav1.2 channels in the plasma membrane depends on the Cavβ type. The presence of different Cavβ subunits does not result in significant differences in the intramolecular distance between the termini of α1C, but significantly affects the distance between the termini of neighbor α1C subunits, which varies from 67 Å with β1b to 79 Å with β3.Conclusions
Thus, our results show that the structural organization of Cav1.2 channels in the plasma membrane depends on the type of Cavβ subunits present. 相似文献20.
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