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Gennady Ermak Karl J. Hench Kevin T. Chang Sean Sachdev Kelvin J. A. Davies 《The Journal of biological chemistry》2009,284(18):11845-11853
Our work suggests an important new link between the RCAN1 gene and
Huntington disease. Huntington disease is caused by expansion of glutamine
repeats in the huntingtin protein. How the huntingtin protein with expanded
polyglutamines (mutant huntingtin) causes the disease is still unclear, but
phosphorylation of huntingtin appears to be protective. Increased huntingtin
phosphorylation can be produced either by inhibition of the phosphatase
calcineurin or by activation of the Akt kinase. The RCAN1 gene
encodes regulators of calcineurin, and we now demonstrate, for the first time,
that RCAN1-1L is depressed in Huntington disease. We also show that RCAN1-1L
overexpression can protect against mutant huntingtin toxicity in an ST14A cell
culture model of Huntington disease and that increased phosphorylation of
huntingtin via calcineurin inhibition, rather than via Akt induction or
activation, is the likely mechanism by which RCAN1-1L may be protective
against mutant huntingtin. These findings suggest that RCAN1-1L
“deficiency” may actually play a role in the etiology of
Huntington disease. In addition, our results allow for the possibility that
controlled overexpression of RCAN1-1L in the striatal region of the brain
might be a viable avenue for therapeutic intervention in Huntington disease
patients (and perhaps other polyglutamine expansion disorders).Huntington disease is a genetic disorder characterized by abnormal body
movements and a reduction of various cognitive functions. It is caused by an
expansion of a trinucleotide (CAG) repeat encoding glutamine in the
IT15 gene, which encodes the huntingtin protein. The neuropathology
of Huntington disease is characterized by neuronal death specifically in the
striatal region, consisting of the caudate nucleus and the putamen. The
severity of the disease is proportional to the number of glutamine repeats in
the huntingtin protein. Aging
(1) and disorders such as
dentatorubropallidoluysian atrophy and spinobulbar muscular atrophy are also
associated with polyglutamine expansion. Because short polyglutamine repeats
are normally present in huntingtin and other proteins, we will call huntingtin
with abnormally long expanded polyglutamines that actually cause toxic effects
“mutant huntingtin.” The functions of normal huntingtin and the
exact mechanism by which mutant huntingtin protein actually causes Huntington
disease are still unclear. It has been found, however, that phosphorylation of
huntingtin is neuroprotective
(2,
3). Increased phosphorylation
has so far been produced either by chemical inhibition of calcineurin with
FK506 or by overexpression of the dominant interfering form of calcineurin
(3) or by activation of the
serine/threonine kinase Akt
(2). Both decreased huntingtin
dephosphorylation (by calcineurin inhibition) and increased huntingtin
phosphorylation (by Akt) are neuroprotective
(2,
3).It has been demonstrated in several laboratories that the RCAN1
gene (4,
5) encodes regulators of
calcineurin
(6–10);
thus, it may present us with a highly specific way to regulate/inhibit
calcineurin. The RCAN1 gene consists of seven exons, four of which
(exons 1–4) can be alternatively transcribed or spliced to produce a
number of different mRNA isoforms
(4,
5). In brain, RCAN1 is
expressed predominantly in neurons rather than in astrocytes or microglia
(11). We have demonstrated
that at least two RCAN1 mRNA isoforms are expressed in adult human
brain and that the mRNA levels of isoform 1 are much higher than those of
isoform 4 (11). We have also
tested whether the various potential RCAN1 proteins (generated by alternate
splicing and alternate translation start sites) are expressed in adult human
brain, and we found at least three isoforms transcribed in adults:
RCAN1-1L, RCAN1–1S, and RCAN1–4
(5,
12). These isoforms have
different expression patterns and different cellular distribution patterns and
may also have somewhat different functions and different mechanisms of
regulation. Nevertheless, all three isoforms can potentially inhibit
calcineurin. Therefore, all three physiologically relevant RCAN1 proteins may
have therapeutic potential for Huntington disease, and we have analyzed this
possibility in our studies. 相似文献
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Lifu Wang John C. Lawrence Jr. Thomas W. Sturgill Thurl E. Harris 《The Journal of biological chemistry》2009,284(22):14693-14697
mTORC1 contains multiple proteins and plays a central role in cell growth
and metabolism. Raptor (regulatory-associated protein of mammalian target of
rapamycin (mTOR)), a constitutively binding protein of mTORC1, is essential
for mTORC1 activity and critical for the regulation of mTORC1 activity in
response to insulin signaling and nutrient and energy sufficiency. Herein we
demonstrate that mTOR phosphorylates raptor in vitro and in
vivo. The phosphorylated residues were identified by using phosphopeptide
mapping and mutagenesis. The phosphorylation of raptor is stimulated by
insulin and inhibited by rapamycin. Importantly, the site-directed mutation of
raptor at one phosphorylation site, Ser863, reduced mTORC1 activity
both in vitro and in vivo. Moreover, the Ser863
mutant prevented small GTP-binding protein Rheb from enhancing the
phosphorylation of S6 kinase (S6K) in cells. Therefore, our findings indicate
that mTOR-mediated raptor phosphorylation plays an important role on
activation of mTORC1.Mammalian target of rapamycin
(mTOR)2 has been shown
to function as a critical controller in cellular growth, survival, metabolism,
and development (1). mTOR, a
highly conserved Ser-Thr phosphatidylinositol 3-kinase-related protein kinase,
structurally forms two distinct complexes, mTOR complex 1 (mTORC1) and mTOR
complex 2 (mTORC2), each of which catalyzes the phosphorylation of different
substrates (1). The best
characterized substrates for mTORC1 are eIF4E-binding protein (4E-BP, also
known as PHAS) and p70 S6 kinase (S6K)
(1), whereas mTORC2
phosphorylates the hydrophobic and turn motifs of protein kinase B
(Akt/protein kinase B) (2) and
protein kinase C (3,
4). mTORC1 constitutively
consists of mTOR, raptor, and mLst8/GβL
(1), whereas the proline-rich
Akt substrate of 40 kDa (PRAS40) is a regulatory component of mTORC1 that
disassociates after growth factor stimulation
(5,
6). Raptor is essential for
mTORC1 activity by providing a substrate binding function
(7) but also plays a regulatory
role on mTORC1 with stimuli of growth factors and nutrients
(8). In response to insulin,
raptor binding to substrates is elevated through the release of the
competitive inhibitor PRAS40 from mTORC1
(9,
10) because PRAS40 and the
substrates of mTORC1 (4E-BP and S6K) appear to bind raptor through a consensus
sequence, the TOR signaling (TOS) motif
(10–14).
In response to amino acid sufficiency, raptor directly interacts with a
heterodimer of Rag GTPases and promotes mTORC1 localization to the
Rheb-containing vesicular compartment
(15).mTORC1 integrates signaling pathways from growth factors, nutrients,
energy, and stress, all of which generally converge on the tuberous sclerosis
complex (TSC1-TSC2) through the phosphorylation of TSC2
(1). Growth factors inhibit the
GTPase-activating protein activity of TSC2 toward the small GTPase Rheb via
the PI3K/Akt pathway (16,
17), whereas energy depletion
activates TSC2 GTPase-activating protein activity by stimulating AMP-activated
protein kinase (AMPK) (18).
Rheb binds directly to mTOR, albeit with very low affinity
(19), and upon charging with
GTP, Rheb functions as an mTORC1 activator
(6). mTORC1 complexes isolated
from growth factor-stimulated cells show increased kinase activity yet do not
contain detectable levels of associated Rheb. Therefore, how Rheb-GTP binding
to mTOR leads to an increase in mTORC1 activity toward substrates, and what
the role of raptor is in this activation is currently unknown. More recently,
the AMPK and p90 ribosomal S6 kinase (RSK) have been reported to directly
phosphorylate raptor and regulate mTORC1 activity. The phosphorylation of
raptor directly by AMPK reduced mTORC1 activity, suggesting an alternative
regulation mechanism independent of TSC2 in response to energy supply
(20). RSK-mediated raptor
phosphorylation enhances mTORC1 activity and provides a mechanism whereby
stress may activate mTORC1 independent of the PI3K/Akt pathway
(21). Therefore, the
phosphorylation status of raptor can be critical for the regulation of mTORC1
activity.In this study, we investigated phosphorylation sites in raptor catalyzed by
mTOR. Using two-dimensional phosphopeptide mapping, we found that
Ser863 and Ser859 in raptor were phosphorylated by mTOR
both in vivo and in vitro. mTORC1 activity in vitro
and in vivo is associated with the phosphorylation of
Ser863 in raptor. 相似文献
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Farah H. Bardai Valerie Price Marcus Zaayman Lulu Wang Santosh R. D'Mello 《The Journal of biological chemistry》2012,287(42):35444-35453
Both neuroprotective and neurotoxic roles have previously been described for histone deacetylase-1 (HDAC1). Here we report that HDAC1 expression is elevated in vulnerable brain regions of two mouse models of neurodegeneration, the R6/2 model of Huntington disease and the Ca2+/calmodulin-dependent protein kinase (CaMK)/p25 double-transgenic model of tauopathic degeneration, suggesting a role in promoting neuronal death. Indeed, elevating HDAC1 expression by ectopic expression promotes the death of otherwise healthy cerebellar granule neurons and cortical neurons in culture. The neurotoxic effect of HDAC1 requires interaction and cooperation with HDAC3, which has previously been shown to selectively induce the death of neurons. HDAC1-HDAC3 interaction is greatly elevated under conditions of neurodegeneration both in vitro and in vivo. Furthermore, the knockdown of HDAC3 suppresses HDAC1-induced neurotoxicity, and the knockdown of HDAC1 suppresses HDAC3 neurotoxicity. As described previously for HDAC3, the neurotoxic effect of HDAC1 is inhibited by treatment with IGF-1, the expression of Akt, or the inhibition of glycogen synthase kinase 3β (GSK3β). In addition to HDAC3, HDAC1 has been shown to interact with histone deacetylase-related protein (HDRP), a truncated form of HDAC9, whose expression is down-regulated during neuronal death. In contrast to HDAC3, the interaction between HDRP and HDAC1 protects neurons from death, an effect involving acquisition of the deacetylase activity of HDAC1 by HDRP. We find that elevated HDRP inhibits HDAC1-HDAC3 interaction and prevents the neurotoxic effect of either of these two proteins. Together, our results suggest that HDAC1 is a molecular switch between neuronal survival and death. Its interaction with HDRP promotes neuronal survival, whereas interaction with HDAC3 results in neuronal death. 相似文献
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Ting Wu Yuanyue Li Deli Huang Felicia Han Ying-Ying Zhang Duan-Wu Zhang Jiahuai Han 《PloS one》2014,9(4)
Autophagy has diverse biological functions and is involved in many biological processes. The L929 cell death induced by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethyl ketone (zVAD) was shown to be an autophagy-mediated death for which RIP1 and RIP3 were both required. It was also reported that zVAD can induce a small amount of TNF production, which was shown to be required for zVAD-induced L929 cell death, arguing for the contribution of autophagy in the zVAD-induced L929 cell death. In an effort to study RIP3 mediated cell death, we identified regulator of G-protein signaling 19 (RGS19) as a RIP3 interacting protein. We showed that RGS19 and its partner Gα-inhibiting activity polypeptide 3 (GNAI3) are involved in zVAD-, but not TNF-, induced cell death. The role of RGS19 and GNAI3 in zVAD-induced cell death is that they are involved in zVAD-induced autophagy. By the use of small hairpin RNAs and chemical inhibitors, we further demonstrated that zVAD-induced autophagy requires not only RIP1, RIP3, PI3KC3 and Beclin-1, but also RGS19 and GNAI3, and this autophagy is required for zVAD-induced TNF production. Collectively, our data suggest that zVAD-induced L929 cell death is a synergistic result of autophagy, caspase inhibition and autocrine effect of TNF. 相似文献
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Sanhita Roy Mausita Karmakar Eric Pearlman 《The Journal of biological chemistry》2014,289(2):1174-1182
In the current study, we examined the role of CD14 in regulating LPS activation of corneal epithelial cells and Pseudomonas aeruginosa corneal infection. Our findings demonstrate that LPS induces Toll-like receptor 4 (TLR4) internalization in corneal epithelial cells and that blocking with anti-CD14 selectively inhibits TLR4 endocytosis, spleen tyrosine kinase (Syk) and IRF3 phosphorylation, and production of CCL5/RANTES and IFN-β, but not IL-8. Using a murine model of P. aeruginosa corneal infection, we show that although infected CD14−/− corneas produce less CCL5, they exhibit significantly increased CXC chemokine production, neutrophil recruitment to the corneal stroma, and bacterial clearance than C57BL/6 mice. We conclude that CD14 has a critical role in mediating TLR4 signaling through IRF3 in resident corneal epithelial cells and macrophages and thereby modulates TLR4 cell surface activation of the MyD88/NF-κB/AP-1 pathway and production of CXC chemokines and neutrophil infiltration to infected tissues. 相似文献
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Ku-Chi Tsao Cheng-Fen Tu Shyh-Jye Lee Ruey-Bing Yang 《The Journal of biological chemistry》2013,288(7):5017-5026
scube1 (signal
peptide-CUB (complement protein C1r/C1s, Uegf, and
Bmp1)-EGF domain-containing protein
1), the founding member of a novel secreted and cell
surface SCUBE protein family, is expressed predominantly in various developing
tissues in mice. However, its function in primitive hematopoiesis remains
unknown. In this study, we identified and characterized zebrafish
scube1 and analyzed its function by injecting antisense
morpholino-oligonucleotide into embryos. Whole-mount in situ
hybridization revealed that zebrafish scube1 mRNA is maternally
expressed and widely distributed during early embryonic development. Knockdown
of scube1 by morpholino-oligonucleotide down-regulated the
expression of marker genes associated with early primitive hematopoietic
precursors (scl) and erythroid (gata1 and
hbbe1), as well as early (pu.1) and late
(mpo and l-plastin) myelomonocytic
lineages. However, the expression of an early endothelial marker
fli1a and vascular morphogenesis appeared normal in
scube1 morphants. Overexpression of bone morphogenetic
protein (bmp) rescued the expression of scl in
the posterior lateral mesoderm during early primitive hematopoiesis in
scube1 morphants. Biochemical and molecular analysis
revealed that Scube1 could be a BMP co-receptor to augment BMP signaling. Our
results suggest that scube1 is critical for and functions at
the top of the regulatory hierarchy of primitive hematopoiesis by modulating BMP
activity during zebrafish embryogenesis. 相似文献
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Andrea Taferner Haymo Pircher Rafal Koziel Susanne von Grafenstein Giorgia Baraldo Konstantinos Palikaras Klaus R. Liedl Nektarios Tavernarakis Pidder Jansen-Dürr 《PloS one》2015,10(8)
The fumarylacetoacetate hydrolase (FAH) protein superfamily of metabolic enzymes comprises a diverse set of enzymatic functions, including ß-diketone hydrolases, decarboxylases, and isomerases. Of note, the FAH superfamily includes many prokaryotic members with very distinct functions that lack homologs in eukaryotes. A prokaryotic member of the FAH superfamily, referred to as Cg1458, was shown to encode a soluble oxaloacetate decarboxylase (ODx). Based on sequence homologies to Cg1458, we recently identified human FAH domain containing protein-1 (FAHD1) as the first eukaryotic oxaloacetate decarboxylase. The physiological functions of ODx in eukaryotes remain unclear. Here we have probed the function of fahd-1, the nematode homolog of FAHD1, in the context of an intact organism. We found that mutation of fahd-1 resulted in reduced brood size, a deregulation of the egg laying process and a severe locomotion deficit, characterized by a reduced frequency of body bends, reduced exploratory movements and reduced performance in an endurance exercise test. Notably, mitochondrial function was altered in the fahd-1(tm5005) mutant strain, as shown by a reduction of mitochondrial membrane potential and a reduced oxygen consumption of fahd-1(tm5005) animals. Mitochondrial dysfunction was accompanied by lifespan extension in worms grown at elevated temperature; however, unlike in mutant worms with a defect in the electron transport chain, the mitochondrial unfolded protein response was not upregulated in worms upon inactivation of fahd-1. Together these data establish a role of fahd-1 to maintain mitochondrial function and consequently physical activity in nematodes. 相似文献
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The human methyltransferases (MTases) METTL21A and VCP-KMT (METTL21D) were recently shown to methylate single lysine residues in Hsp70 proteins and in VCP, respectively. The yet uncharacterized MTase encoded by the YNL024C gene in Saccharomyces cerevisiae shows high sequence similarity to METTL21A and VCP-KMT, as well as to their uncharacterized paralogues METTL21B and METTL21C. Despite being most similar to METTL21A, the Ynl024c protein does not methylate yeast Hsp70 proteins, which were found to be unmethylated on the relevant lysine residue. Eukaryotic translation elongation factor eEF1A in yeast has been reported to contain four methylated lysine residues (Lys30, Lys79, Lys318 and Lys390), and we here show that the YNL024C gene is required for methylation of eEF1A at Lys390, the only of these methylations for which the responsible MTase has not yet been identified. Lys390 was found in a partially monomethylated state in wild-type yeast cells but was exclusively unmethylated in a ynl024cΔ strain, and over-expression of Ynl024c caused a dramatic increase in Lys390 methylation, with trimethylation becoming the predominant state. Our results demonstrate that Ynl024c is the enzyme responsible for methylation of eEF1A at Lys390, and in accordance with prior naming of similar enzymes, we suggest that Ynl024c is renamed to Efm6 (Elongation factor MTase 6). 相似文献
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Ichiro Kikkawa Shinji Saito Kaoru Tominaga Yuichi Hoshino Yoshio Ooi Masayasu Nakano 《Microbiology and immunology》1998,42(9):591-598
Osteoclasts (OCL) resorb bone. They are essential for the development of normal bones and the repair of impaired bones. The function of OCL is presumed to be supported by cytokines and other biological mediators, including tumor necrosis factor (TNF)-α and nitric oxide (NO). Bacterial lipopolysaccharide (LPS) is a potent inducer of TNF-α and inducible nitric oxide synthase (iNOS), which is the specific enzyme for synthesizing NO from L-arginine. To obtain direct evidence on LPS-induced TNF-α production and iNOS expression by OCL, OCL-enriched cultures were prepared by 7-day cocultures of bone marrow cells of adult BALB/c mice and osteoblastic cells (OBs) derived from calvaria of newborn BALB/c mice, and the generation of TNF-α and iNOS in OCL stimulated with LPS was examined immunocytochemically. When the cultured cells were stimulated with 100 ng/ml of LPS, OCL clearly showed TNF-α and iNOS expression. Without LPS-stimulation, no expression was observed. TNF activity in the culture supernatants of the OCL-enriched cultures in the presence of LPS was also detected by cytotoxic assay that used TNF-sensitive L929 cells. The dentin resorption activity of OCL was estimated by area and number of pits formed on dentin slices, which were covered by the OCL fraction and cultured in the presence or absence of LPS, sodium nitroprusside (SNP; a NO generating compound), NG-monomethyl L-arginine acetate (L-NMMA; a competitive inhibitor of NO synthase (NOS)), or LPS plus L-NMMA. Pit formation was obviously inhibited in the presence of SNP and slightly inhibited in the presence of L-NMMA, but it was not affected in the presence of LPS or LPS plus L-NMMA. These findings indicate that OCL produces TNF and expresses iNOS in response to LPS, but the LPS-activation of OCL scarcely affects pit formation by them. 相似文献