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1.
Shotaro Saita Michiko Shirane Tohru Natume Shun-ichiro Iemura Keiichi I. Nakayama 《The Journal of biological chemistry》2009,284(20):13766-13777
Protrudin is a protein that contains a Rab11-binding domain and a FYVE
(lipid-binding) domain and that functions to promote neurite formation through
interaction with the GDP-bound form of Rab11. Protrudin also contains a short
sequence motif designated FFAT (two phenylalanines in an acidic tract), which
in other proteins has been shown to mediate binding to vesicle-associated
membrane protein-associated protein (VAP). We now show that protrudin
associates and colocalizes with VAP-A, an isoform of VAP expressed in the
endoplasmic reticulum. Both the interaction between protrudin and VAP-A as
well as the induction of process formation by protrudin were markedly
inhibited by mutation of the FFAT motif. Furthermore, depletion of VAP-A by
RNA interference resulted in mislocalization of protrudin as well as in
inhibition of neurite outgrowth induced by nerve growth factor in rat
pheochromocytoma PC12 cells. These defects resulting from depletion of
endogenous rat VAP-A in PC12 cells were corrected by forced expression of (RNA
interference-resistant) human VAP-A but not by VAP-A mutants that have lost
the ability to interact with protrudin. These results suggest that VAP-A is an
important regulator both of the subcellular localization of protrudin and of
its ability to stimulate neurite outgrowth.The molecular mechanisms that underlie neurite formation include both
cytoskeletal remodeling and membrane trafficking. Membrane components are
transported in a directional manner within the cell by a membrane recycling
system, resulting in expansion of the surface area of the neurite. The small
GTPase Rab11 regulates membrane recycling and constitutive exocytosis
(1), and it is thought to
contribute to neurite formation through regulation of directional membrane
transport.We have recently identified protrudin as a key regulator of Rab11-dependent
membrane trafficking during neurite extension. Protrudin interacts with FKBP38
(also known as FKBP8) (2),
which is a member of the immunophilin family of proteins that bind the
immunosuppressant drug FK506
(3). FKBPs are multifunctional
proteins that regulate the folding or export of other proteins as a result of
their peptidyl-prolyl cis-trans-isomerase activity
(4). Protrudin was found to
interact with FKBP38, but not with other FKBP proteins such as FKBP12 or
FKBP52 (5). Protrudin is
hyperphosphorylated in Fkbp38-/- mice, which manifest
abnormal extension of nerve fibers
(5).Protrudin contains a Rab11-binding domain (RBD11), two transmembrane
domains (TM1 and
TM2),2 an FFAT (two
phenylalanines in an acidic tract) motif
(6), a coiled-coil domain, and
a FYVE domain (7). These
structural characteristics suggested that protrudin might function in membrane
trafficking, particularly in membrane recycling. The gene encoding ZFYVE27 (a
synonym of human protrudin) was recently found to be mutated in a German
family with an autosomal dominant form of hereditary spastic paraplegia
(AD-HSP), which is characterized by selective degeneration of axons
(8). The phenotype of the
affected individuals is similar to that of patients with AD-HSP caused by
mutation of spastin, a protein implicated in neuronal vesicular trafficking
(9), and protrudin was shown to
interact with spastin (8).
These findings support the notion that protrudin plays a key role in
Rab11-mediated directional membrane transport during neurite formation.The subcellular localization of protrudin is dynamic. Whereas it is
localized to the endoplasmic reticulum (ER) under basal conditions, nerve
growth factor (NGF) triggers the translocation of protrudin from the ER, via
recycling endosomes, to the tip of membrane protrusions in neuronal cells.
Given that the FFAT motif is thought to serve as an ER targeting signal
(6), this motif might be
expected to contribute both to the localization of protrudin to the ER and to
the regulation of neurite formation by this protein. The FFAT motif (consensus
amino acid sequence of EFFDAXE, where X is any amino acid)
is present in several lipid-binding proteins that are implicated in the
transfer of lipids between the ER and other organelles such as the Golgi
apparatus (10,
11). Vesicle-associated
membrane protein-associated protein (VAP) interacts with these lipid-binding
proteins through their FFAT motifs
(6,
11,
12). The VAP-A and VAP-B
isoforms of mammalian VAP are ER-resident type II membrane proteins
(13) that are encoded by
different genes (14); VAP-C is
a splicing variant of VAP-B that lacks the membrane-spanning domain. VAP-A and
VAP-B share ∼60% amino acid sequence identity, form homo- or heterodimers,
and are expressed in many tissues
(14-16).
In addition to their localization to the ER
(16), VAP-A and VAP-B are
present in a wide range of intracellular membranes or membrane structures,
including the Golgi, the ER-Golgi intermediate compartment
(17), tight junctions
(18), neuromuscular junctions
(19), recycling endosomes, and
the plasma membrane (20).We have now identified VAP-A and VAP-B as proteins that interact with
protrudin. Protrudin preferentially interacts with VAP-A via its FFAT motif,
and this motif was found to be required for the protrudin-dependent formation
of membrane protrusions in HeLa cells. In addition, depletion of VAP-A by RNA
interference resulted in inhibition of NGF-induced neurite outgrowth in the
PC12 rat pheochromocytoma cell line. This inhibition of neurite outgrowth was
reversed by expression of human VAP-A but not by that of VAP-A mutants that
have lost the ability to bind to protrudin. These results suggest that
interaction of protrudin with VAP-A is important both for its ER retention and
for its ability to stimulate neurite formation. 相似文献
2.
Lizama C Rojas-Benitez D Antonelli M Ludwig A Moreno RD 《Journal of cellular physiology》2012,227(2):829-838
Germ cell apoptosis is important to regulate sperm production in the mammalian testis, but the molecular mechanisms underlying apoptosis are still poorly understood. We have recently shown that in vitro, etoposide induces upregulation of TACE/ADAM17 and ADAM10, two membrane-bound extracellular metalloproteases. Here we show that in vivo these enzymes are involved in etoposide-, but not in heat shock-, induced apoptosis in rat spermatogenesis. Germ cell apoptosis induced by DNA damage was associated with an increase in protein levels and cell surface localization of TACE/ADAM17 and ADAM10. On the contrary, apoptosis of germ cells induced by heat stress, another cell death stimulus, did not change levels or localization of these proteins. Pharmacological in vivo inhibition of TACE/ADAM17 and ADAM10 prevents etoposide-induced germ cell apoptosis. Finally, Gleevec (STI571) a pharmacological inhibitor of p73, a master gene controlling apoptosis induced by etoposide, prevented the increase of TACE/ADAM17 levels. Our results strongly suggest that TACE/ADAM17 participates in in vivo apoptosis of male germ cells induced by DNA damage. 相似文献
3.
4.
Zhaohuan Zhang Xiaohui Xu Yong Zhang Jianfeng Zhou Zhongwang Yu Cheng He 《The Journal of biological chemistry》2009,284(23):15717-15728
LINGO-1 is a component of the tripartite receptor complexes, which act as a convergent mediator of the intracellular signaling in response to myelin-associated inhibitors and lead to collapse of growth cone and inhibition of neurite extension. Although the function of LINGO-1 has been intensively studied, its downstream signaling remains elusive. In the present study, a novel interaction between LINGO-1 and a serine-threonine kinase WNK1 was identified by yeast two-hybrid screen. The interaction was further validated by fluorescence resonance energy transfer and co-immunoprecipitation, and this interaction was intensified by Nogo66 treatment. Morphological evidences showed that WNK1 and LINGO-1 were co-localized in cortical neurons. Furthermore, either suppressing WNK1 expression by RNA interference or overexpression of WNK1-(123–510) attenuated Nogo66-induced inhibition of neurite extension and inhibited the activation of RhoA. Moreover, WNK1 was identified to interact with Rho-GDI1, and this interaction was attenuated by Nogo66 treatment, further indicating its regulatory effect on RhoA activation. Taken together, our results suggest that WNK1 is a novel signaling molecule involved in regulation of LINGO-1 mediated inhibition of neurite extension.Axons of the adult mammalian central nervous system possess an extremely limited ability to regenerate after injury, largely because of inhibitory components of myelin preventing axon growth (1, 2). Several myelin-associated inhibitors have been identified, including myelin-associated glycoprotein (3–5), chondroitin sulfate proteoglycans (6), oligodendrocyte myelin glycoprotein (7), and Nogo (8–10). Myelin-associated glycoprotein, oligodendrocyte myelin glycoprotein, and Nogo bind to the Nogo-66 receptor (NgR)3 and exert their actions through a tripartite receptor complex NgR/LINGO-1/p75NTR (11) or NgR/LINGO-1/TROY (12, 13).LINGO-1 is a transmembrane protein that contains a leucine-rich repeat, an immunoglobulin domain, and a short intracellular tail (11). LINGO-1 functions as an essential component of the NgR complexes that mediate the activity of myelin inhibitors to regulate central nervous system axon growth (11, 14). In neurons, the NgR complexes activate RhoA in the presence of myelin inhibitors, which lead to growth cone collapse and neurite extension inhibition (11). Attenuation of LINGO-1 function is able to overcome the myelin inhibitory activity in the spinal cord that prevents axonal regeneration after lesion in rats (15). Besides, it has been reported that LINGO-1 is also expressed in oligodendrocytes, where it negatively regulates oligodendrocyte differentiation and axon myelination (16). Inhibition of LINGO-1 promotes spinal cord remyelination in an experimental model of autoimmune encephalitis (17). Moreover, inhibition of LINGO-1 has been shown to enhance survival, structure, and function of dopaminergic neurons in Parkinson disease models (18). Although the function of LINGO-1 has been intensively studied, much less is known about its downstream signaling.To gain insight into the mechanisms by which LINGO-1 functions, it is of considerable importance to identify new binding partners of LINGO-1. Therefore, using the intracellular domain of LINGO-1 as bait, we employed yeast two-hybrid screening on a brain cDNA library and identified several candidates that interact with LINGO-1, one of which is the protein kinase WNK1.WNKs (with no lysine [K]) are a distinct subfamily of serine-threonine kinases, which are characterized by a unique placement of the lysine that is involved in binding ATP and catalyzing phosphoryl transfer (19). Thus far, WNKs are known composed of four members, WNK1, WNK2, WNK3, and WNK4. Mutations in the serine-threonine kinases WNK1 and WNK4 cause a Mendelian disease PAHII, featuring hypertension and hyperkalemia (20, 21), and their roles in the regulation of electrolyte flux in the kidney have been well established (22). Recently, other important features of WNKs are beginning to be understood. WNKs have also been proposed functioning in a number of non-transport processes, including cell growth, differentiation, and apoptosis (23–26). Although WNK1 has been shown to be expressed in brain (27, 28), little is known about its function in the nervous system until recently; mutations of a nervous system-specific exon of the WNK1 gene were found to cause Hereditary sensory and autonomic neuropathy type II (HSANII) (29). In this study WNK1 was demonstrated to interact with LINGO-1 and regulate Nogo-induced inhibition of neurite extension. 相似文献
5.
6.
7.
8.
Neural cell differentiation during development is controlled by multiple signaling pathways, in which protein phosphorylation and dephosphorylation play an important role. In this study, we examined the role of pyrophosphatase1 (PPA1) in neuronal differentiation using the loss and gain of function analysis. Neuronal differentiation induced by external factors was studied using a mouse neuroblastoma cell line (N1E115). The neuronal like differentiation in N1E115 cells was determined by morphological analysis based on neurite growth length. In order to analyze the loss of the PPA1 function in N1E115, si-RNA specifically targeting PPA1 was generated. To study the effect of PPA1 overexpression, an adenoviral gene vector containing the PPA1 gene was utilized to infect N1E115 cells. To address the need for pyrophosphatase activity in PPA1, D117A PPA1, which has inactive pyrophosphatase, was overexpressed in N1E115 cells. We used valproic acid (VPA) as a neuronal differentiator to examine the effect of PPA1 in actively differentiated N1E115 cells. Si-PPA1 treatment reduced the PPA1 protein level and led to enhanced neurite growth in N1E115 cells. In contrast, PPA1 overexpression suppressed neurite growth in N1E115 cells treated with VPA, whereas this effect was abolished in D117A PPA1. PPA1 knockdown enhanced the JNK phosphorylation level, and PPA1 overexpression suppressed it in N1E115 cells. It seems that recombinant PPA1 can dephosphorylate JNK while no alteration of JNK phosphorylation level was seen after treatment with recombinant PPA1 D117A. Enhanced neurite growth by PPA1 knockdown was also observed in rat cortical neurons. Thus, PPA1 may play a role in neuronal differentiation via JNK dephosphorylation. 相似文献
9.
10.
The sigma-1 receptor is a molecular chaperone protein highly enriched in the brain. Recent studies linked it to many diseases, such as drug addition, Alzheimer’s disease, stroke, depression, and even cancer. Sigma-1 receptor is enriched in lipid rafts, which are membrane microdomains essential in signaling processes. One of those signaling processes is ADAM17- and ADAM10-dependent ectodomain shedding. By using an alkaline phosphatase tagged substrate reporter system, we have shown that ADAM10-dependent BTC shedding was very sensitive to both membrane lipid component change and sigma-1 receptor agonist DHEAS treatment while ADAM17-dependent HB-EGF shedding was not; and overexpression of sigma-1 receptor diminished ADAM17- and ADAM10-dependent shedding. Our results indicate that sigma-1 receptor plays an important role in modifying the function of transmembrane proteases. 相似文献
11.
Yong Zhang Yong-Gang Wang Qi Zhang Xiu-Jie Liu Xuan Liu Li Jiao Wei Zhu Zhao-Huan Zhang Xiao-Lin Zhao Cheng He 《The Journal of biological chemistry》2009,284(18):12469-12479
TrkA receptor signaling is essential for nerve growth factor (NGF)-induced
survival and differentiation of sensory neurons. To identify possible
effectors or regulators of TrkA signaling, yeast two-hybrid screening was
performed using the intracellular domain of TrkA as bait. We identified
muc18-1-interacting protein 2 (Mint2) as a novel TrkA-binding protein and
found that the phosphotyrosine binding domain of Mint2 interacted with TrkA in
a phosphorylation- and ligand-independent fashion. Coimmunoprecipitation
assays showed that endogenous TrkA interacted with Mint2 in rat tissue
homogenates, and immunohistochemical evidence revealed that Mint2 and TrkA
colocalized in rat dorsal root ganglion neurons. Furthermore, Mint2
overexpression inhibited NGF-induced neurite outgrowth in both PC12 and
cultured dorsal root ganglion neurons, whereas inhibition of Mint2 expression
by RNA interference facilitated NGF-induced neurite outgrowth. Moreover, Mint2
was found to promote the retention of TrkA in the Golgi apparatus and inhibit
its surface sorting. Taken together, our data provide evidence that Mint2 is a
novel TrkA-regulating protein that affects NGF-induced neurite outgrowth,
possibly through a mechanism involving retention of TrkA in the Golgi
apparatus.The neurotrophin family member nerve growth factor
(NGF)3 is
essential for proper development, patterning, and maintenance of nervous
systems (1,
2). NGF has two known
receptors; TrkA, a single-pass transmembrane receptor-tyrosine kinase that
binds selectively to NGF, and p75, a transmembrane glycoprotein that binds all
members of the neurotrophin family
(3,
4). NGF binding activates the
kinase domain of TrkA, leading to autophosphorylation
(5). The resulting
phosphotyrosines become docking sites for adaptor proteins involved in signal
transduction pathways that lead to the activation of Ras, Rac,
phosphatidylinositol 3-kinase, phospholipase Cγ, and other effectors
(2,
6). Many of these
TrkA-interacting adaptor proteins have been identified and include, Grb2, APS,
SH2B, fibroblast growth factor receptor substrate 2 (FRS-2), Shc, and human
tumor imaginal disc 1 (TID1)
(7-10).
The identification of these binding partners has contributed greatly to our
understanding of the mechanisms underlying the functional diversity of
NGF-TrkA signaling.Studies have indicated that the transmission of NGF signaling in neurons
involves retrograde transport of NGF-TrkA complexes from the neurite tip to
the cell body
(11-14).
TrkA associates with components of cytoplasmic dynein, and it is thought that
vesicular trafficking of neurotrophins occurs via direct interaction of Trk
receptors with the dynein motor machinery
(14). Furthermore, the
atypical protein kinase C-interacting protein, p62, associates with TrkA and
plays a novel role in connecting receptor signals with the endosomal signaling
network required for mediating TrkA-induced differentiation
(15). Recently, the
membrane-trafficking protein Pincher has been found to mediate
macroendocytosis underlying retrograde signaling by TrkA
(16). Despite the progress
made to date in understanding Trk complex internalization and trafficking, the
mechanisms remain poorly understood.Mint2 (muc18-1-interacting protein 2) belongs to the Mint protein family,
which consists of three members, Mint1, Mint2, and Mint3. Mint proteins were
first identified as interacting proteins of the synaptic vesicle-docking
protein Munc18-1 (17,
18). Mint1 is also sometimes
referred to as mLIN-10, as it is the mammalian orthologue of the
Caenorhabditis elegans LIN-10
(19). Additionally, Mint1,
Mint2, and Mint3 are also referred to as X11α or X11, X11β or X11L
(X11-like), and X11γ or X11L2 (X11-like 2), respectively
(20). All Mint proteins
contain a conserved central phosphotyrosine binding (PTB) domain and two
contiguous C-terminal PDZ domains (repeated sequences in the brain-specific
protein PSD-95, the Drosophila septate junction protein Discs large,
and the epithelial tight junction protein ZO-1)
(17,
18,
21). Mint1 and Mint2 are
expressed only in neuronal tissue
(17), whereas Mint3 is
ubiquitously expressed (18).
Although the function of Mints proteins is not fully clear, their interactions
with the docking and exocytosis factors Mun18 -1 and CASK, ADP-ribosylation
factor (Arf) GTPases involved in vesicle budding
(22), and other synaptic
adaptor proteins, such as neurabin-II/spinophilin
(23), tamalin
(24), and kalirin-7
(25), all suggest possible
roles for Mints in synaptic vesicle docking and exocytosis. Mint proteins have
also been implicated in the trafficking and/or processing of β-amyloid
precursor protein (β-APP). Through their PTB domains, all three Mints
bind to a motif within the cytoplasmic domain of β-APP
(21,
26-29),
and Mint1 and Mint2 can stabilize β-APP, affect β-APP processing,
and inhibit the production and secretion of Aβ
(28,
30-32).
Although the mechanisms by which Mints inhibit β-APP processing are not
yet well known, Mints and their binding partners have emerged as potential
therapeutic targets for the treatment of Alzheimer disease.To uncover new TrkA-interacting factors and gain insight into the
mechanisms that guide TrkA intracellular trafficking and other aspects of TrkA
signaling, we conducted a yeast two-hybrid screen of a brain cDNA library
using the intracellular domain of TrkA as bait. The screen identified several
candidate TrkA-interacting proteins, one of which was Mint2. Follow-up binding
assays showed that the PTB domain of Mint2 alone was necessary and sufficient
for mediating the interaction with TrkA. Endogenous Mint2 was also
coimmunoprecipitated and colocalized with TrkA in rat DRG tissue.
Overexpression and knockdown studies showed that Mint2 could significantly
inhibit NGF-induced neurite outgrowth in both TrkA-expressing PC12 cells and
DRG neurons. Moreover, Mint2 was found to induce the retention of TrkA in the
Golgi apparatus and inhibit its surface sorting. Our results suggest that
Mint2 is a novel regulator of TrkA receptor signaling. 相似文献
12.
The multifunctional regulator VelB physically interacts with other velvet regulators and the resulting complexes govern development and secondary metabolism in the filamentous fungus Aspergillus nidulans. Here, we further characterize VelB’s role in governing asexual development and conidiogenesis in A. nidulans. In asexual spore formation, velB deletion strains show reduced number of conidia, and decreased and delayed mRNA accumulation of the key asexual regulatory genes brlA, abaA, and vosA. Overexpression of velB induces a two-fold increase of asexual spore production compared to wild type. Furthermore, the velB deletion mutant exhibits increased conidial germination rates in the presence of glucose, and rapid germination of conidia in the absence of external carbon sources. In vivo immuno-pull-down analyses reveal that VelB primarily interacts with VosA in both asexual and sexual spores, and VelB and VosA play an inter-dependent role in spore viability, focal trehalose biogenesis and control of conidial germination. Genetic and in vitro studies reveal that AbaA positively regulates velB and vosA mRNA expression during sporogenesis, and directly binds to the promoters of velB and vosA. In summary, VelB acts as a positive regulator of asexual development and regulates spore maturation, focal trehalose biogenesis and germination by interacting with VosA in A. nidulans. 相似文献
13.
Neurite Extension Occurs in the Absence of Regulated Exocytosis in PC12 Subclones 总被引:5,自引:0,他引:5 下载免费PDF全文
Chiara Leoni Andrea Menegon Fabio Benfenati Daniela Toniolo Maria Pennuto Flavia Valtorta 《Molecular biology of the cell》1999,10(9):2919-2931
We have investigated the process leading to differentiation of PC12 cells. This process is known to include extension of neurites and changes in the expression of subsets of proteins involved in cytoskeletal rearrangements or in neurosecretion. To this aim, we have studied a PC12 clone (trk-PC12) stably transfected with the nerve growth factor receptor TrkA. These cells are able to undergo both spontaneous and neurotrophin-induced morphological differentiation. However, both undifferentiated and nerve growth factor-differentiated trk-PC12 cells appear to be completely defective in the expression of proteins of the secretory apparatus, including proteins of synaptic vesicles and large dense-core granules, neurotransmitter transporters, and neurotransmitter-synthesizing enzymes. These results indicate that neurite extension can occur independently of the presence of the neurosecretory machinery, including the proteins that constitute the fusion machine, suggesting the existence of differential activation pathways for the two processes during neuronal differentiation. These findings have been confirmed in independent clones obtained from PC12-27, a previously characterized PC12 variant clone globally incompetent for regulated secretion. In contrast, the integrity of the Rab cycle appears to be necessary for neurite extension, because antisense oligonucleotides against the neurospecific isoform of Rab-guanosine diphosphate-dissociation inhibitor significantly interfere with process formation. 相似文献
14.
15.
Shigeki Okumura Osamu Muraoka Yasuhiro Tsukamoto Hidekazu Tanaka Keiko Kohama Naomasa Miki Eiichi Taira 《Experimental cell research》2001,271(2):269-276
Gicerin is a cell adhesion molecule belonging to the immunoglobulin superfamily. To study the functional differences between l- and s-gicerin, we first examined the distribution of endogenous gicerin in B16 cells and found that l-gicerin was densely localized in microvilli. To clarify the relationship between gicerin and the microvilli, we established independent stable cell lines expressing l- and s-gicerin in L cells and found that l-gicerin localized to the microvilli. Scanning electron microscopic analysis revealed that the microvilli of l-gicerin-transfected cells were longer than those of s-gicerin and control transfectants. This suggested that l-gicerin might participate in the elongation of the microvilli. When cells were double-stained with antibodies to gicerin and moesin, a microvilli-specific protein, the staining of l-gicerin corresponded to that of moesin in the elongated microvilli. Moesin was coprecipitated with glutathione S-transferase-fusion proteins of the l-gicerin cytoplasmic domain but not with the s-gicerin cytoplasmic domain. To determine the region involved in the extension of microvilli, we generated transfectants of two truncated forms of l-gicerin cytoplasmic domain, and we found that only the transfectants of the longer mutant had the longer microvilli, while the shorter mutant exhibited short microvilli. These results suggested that l-gicerin-specific amino acid residues, especially amino acids 16-39, within the cytoplasmic domain of l-gicerin might be involved in the extension of microvilli. 相似文献
16.
17.
《Biophysical journal》2020,118(8):1914-1920
The densely packed microtubule (MT) array found in neuronal cell projections (neurites) serves two fundamental functions simultaneously: it provides a mechanically stable track for molecular motor-based transport and produces forces that drive neurite growth. The local pattern of MT polarity along the neurite shaft has been found to differ between axons and dendrites. In axons, the neurons’ dominating long projections, roughly 90% of the MTs orient with their rapidly growing plus end away from the cell body, whereas in vertebrate dendrites, their orientations are locally mixed. Molecular motors are known to be responsible for cytoskeletal ordering and force generation, but their collective function in the dense MT cytoskeleton of neurites remains elusive. We here hypothesized that both the polarity pattern of MTs along the neurite shaft and the shaft’s global extension are simultaneously driven by molecular motor forces and should thus be regulated by the mechanical load acting on the MT array as a whole. To investigate this, we simulated cylindrical bundles of MTs that are cross-linked and powered by molecular motors by iteratively solving a set of force-balance equations. The bundles were subjected to a fixed load arising from actively generated tension in the actomyosin cortex enveloping the MTs. The magnitude of the load and the level of motor-induced connectivity between the MTs have been varied systematically. With an increasing load and decreasing motor-induced connectivity between MTs, the bundles became wider in cross section and extended more slowly, and the local MT orientational order was reduced. These results reveal two, to our knowledge, novel mechanical factors that may underlie the distinctive development of the MT cytoskeleton in axons and dendrites: the cross-linking level of MTs by motors and the load acting on this cytoskeleton during growth. 相似文献
18.
19.
20.
Akiko Maeda Tadao Maeda Marcin Golczak Steven Chou Amar Desai Charles L. Hoppel Shigemi Matsuyama Krzysztof Palczewski 《The Journal of biological chemistry》2009,284(22):15173-15183
Exposure to bright light can cause visual dysfunction and retinal
photoreceptor damage in humans and experimental animals, but the mechanism(s)
remain unclear. We investigated whether the retinoid cycle (i.e. the
series of biochemical reactions required for vision through continuous
generation of 11-cis-retinal and clearance of
all-trans-retinal, respectively) might be involved. Previously, we
reported that mice lacking two enzymes responsible for clearing
all-trans-retinal, namely photoreceptor-specific ABCA4 (ATP-binding
cassette transporter 4) and RDH8 (retinol dehydrogenase 8), manifested retinal
abnormalities exacerbated by light and associated with accumulation of
diretinoid-pyridinium-ethanolamine (A2E), a condensation product of
all-trans-retinal and a surrogate marker for toxic retinoids. Now we
show that these mice develop an acute, light-induced retinopathy. However,
cross-breeding these animals with lecithin:retinol acyltransferase knock-out
mice lacking retinoids within the eye produced progeny that did not exhibit
such light-induced retinopathy until gavaged with the artificial chromophore,
9-cis-retinal. No significant ocular accumulation of A2E occurred
under these conditions. These results indicate that this acute light-induced
retinopathy requires the presence of free all-trans-retinal and not,
as generally believed, A2E or other retinoid condensation products. Evidence
is presented that the mechanism of toxicity may include plasma membrane
permeability and mitochondrial poisoning that lead to caspase activation and
mitochondria-associated cell death. These findings further understanding of
the mechanisms involved in light-induced retinal degeneration.The retinoid cycle is a fundamental metabolic process in the vertebrate
retina responsible for continuous generation of 11-cis-retinal from
its all-trans-isomer
(1-3).
Because 11-cis-retinal is the chromophore of rhodopsin and cone
visual pigments (4), disabling
mutations in genes encoding proteins of the retinoid cycle can cause a
spectrum of retinal diseases affecting sight
(3). Moreover, the efficiency
of the mammalian visual system and health of photoreceptors and retinal
pigment epithelium
(RPE)2 decrease
significantly with age. Even in the presence of a functional retinoid cycle,
A2E, retinal dimer (RALdi), and other toxic all-trans-retinal
condensation products
(5-7)
can accumulate as a consequence of aging
(8). Under experimental
conditions, these compounds can produce toxic effects on RPE cells
(9-11).
Patients affected by age-related macular degeneration, Stargardt disease, or
other retinal diseases associated with accumulation of surrogate markers, such
as A2E, all develop retinal degeneration
(12). Thus, elucidating the
fundamental causes of these age-dependent changes is of increasing importance.
Encouragingly, our understanding of both retinoid metabolism outside the eye
and production of 11-cis-retinal unique to the eye has accelerated
recently (Scheme 1)
(1-3),
and genetic mouse models are readily available to study these processes and
their potential aberrations in vivo
(13). Thus, a central question
can be addressed, namely what initiates the death of photoreceptor cells and
the underlining RPE?Open in a separate windowSCHEME 1.Retinoid flow and all-trans-retinal clearance in the visual
cycle. After diffusion from the RPE, the visual chromophore,
11-cis-retinal, combines with rhodopsin and then is photoisomerized
to all-trans-retinal. Most of the all-trans-retinal
dissociates from opsin into the cytoplasm, where it is reduced to
all-trans-retinol by RDHs, including RDH8. The fraction of
all-trans-retinal that dissociates into the disc lumen is transported
by ABCA4 into the cytoplasm
(23) before it is reduced.
All-trans-retinol then is translocated to the RPE, esterified by
LRAT, and recycled back to 11-cis-retinal. Mutations of ABCA4 are
associated with human macular degeneration, Stargardt disease, and age-related
macular degeneration (55,
56).Several mechanisms associated with retinoid metabolism may contribute to
different retinopathies (1).
For example, lack of retinoids in LRAT (lecithin:retinol acyltransferase) or
chromophore in retinoid isomerase knock-out (Rpe65-/-)
mice leads to rapid degeneration of cone photoreceptors and slowly progressive
death of rods (14). Such mice
do not produce toxic condensation products from all-trans-retinal.
Instead, their retinopathies have been attributed to continuous activation of
visual phototransduction (15)
due to either the basal activity of opsin
(16-18)
or disordered vectorial transport of cone visual pigments without bound
chromophore (19).
Paradoxically, an abnormally high flux of retinoids through the retinoid cycle
can also lead to retinopathy in other mouse models
(20,
21). Animal models featuring
anomalies in the retinoid cycle illustrate the importance of chromophore
regeneration and provide an approach to elucidating mechanisms involved in
human retinal dysfunction and disease.Recently, we showed that mice carrying a double knock-out of Rdh8
(retinol dehydrogenase 8), one of the main enzymes that reduces
all-trans-retinal in rod and cone outer segments
(22), and Abca4
(ATP-binding cassette transporter 4), which transports
all-trans-retinal from the inside to the outside of disc membranes
(23), rapidly accumulate
all-trans-retinal condensation products and exhibit accentuated
RPE/photoreceptor dystrophy at an early age
(24). Although these studies
suggest retinoid toxicity, it is still unclear if the elevated levels of
retinal and/or its condensation products, such as A2E, are the cause of this
retinopathy or merely a nonspecific reflection of impaired retinoid
metabolism. Here, we report that spent chromophore,
all-trans-retinal, is most likely responsible for photoreceptor
degeneration in Rdh8-/-Abca4-/- mice.
Toxic effects of all-trans-retinal include caspase activation and
mitochondria-associated cell death. 相似文献