共查询到20条相似文献,搜索用时 15 毫秒
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Bharathi Suresh Suresh Ramakrishna Yong-Soo Kim Sun-Myoung Kim Myung-Sun Kim Kwang-Hyun Baek 《The Journal of biological chemistry》2010,285(46):35340-35349
The evolutionarily conserved lethal giant larvae (Lgl) tumor suppressor gene has an essential role in establishing apical-basal cell polarity, cell proliferation, differentiation, and tissue organization. However, the precise molecular mechanism by which the Lgl carries out its function remains obscure. In the current study, we have identified Ran-binding protein M (RanBPM) as a novel binding partner of Mgl-1, a mammalian homolog of Drosophila tumor suppressor protein lethal (2) giant larvae (L(2)gl) by yeast two-hybrid screening. RanBPM seems to act as a scaffolding protein with a modulatory function with respect to Mgl-1. The Mgl-1 and RanBPM association was confirmed by co-immunoprecipitation and GST pull-down experiments. Additionally, expression of RanBPM resulted in inhibition of Mgl-1 degradation, and thereby extended the half-life of Mgl-1. Furthermore, the ability of Mgl-1 activity in cell migration and colony formation assay was enhanced by RanBPM. Taken together, our findings reveal that RanBPM plays a novel role in regulating Mgl-1 stability and contributes to its biological function as a tumor suppressor. 相似文献
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Studies investigating the subcellular localization of periplasmic proteins have been hampered by problems with the export of green fluorescent protein (GFP). Here we show that a superfolding variant of GFP (sfGFP) is fluorescent following Sec-mediated transport and works best when the cotranslational branch of the pathway is employed. 相似文献
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Anshika Bajaj Nicole E. LaPlante Victoria E. Cotero Kenneth M. Fish Roger M. Bjerke Tiberiu Siclovan Cristina A. Tan Hehir 《The journal of histochemistry and cytochemistry》2013,61(1):19-30
The ability to visualize myelin is important in the diagnosis of demyelinating disordersand the detection of myelin-containing nerves during surgery. The development ofmyelin-selective imaging agents requires that a defined target for these agents beidentified and that a robust assay against the target be developed to allow for assessmentof structure-activity relationships. We describe an immunohistochemical analysis and afluorescence polarization binding assay using purified myelin basic protein (MBP) thatprovides quantitative evidence that MBP is the molecular binding partner of previouslydescribed myelin-selective fluorescent dyes such as BMB, GE3082, and GE3111. 相似文献
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Cell swelling induced by hypo-osmotic stress results in activation of volume-regulated anion channels (VRAC) that drive a compensatory regulatory volume decrease. We have previously shown that the Best1 gene in Drosophila encodes a VRAC that is also activated by increases in intracellular Ca2+. The role of Best1 as a VRAC has recently been independently confirmed by the Clapham lab in an unbiased RNAi screen. Although dBest1 is clearly a volume-regulated channel, its mechanisms of regulation remain unknown. Here we investigate Drosophila Best1 (dBest1) regulation using the Drosophila S2 cell model system. Because dBest1 activates slowly after establishing whole-cell recording, we tested the hypothesis that the channel is activated by phosphorylation. Two experiments indicate that phosphorylation is required for dBest1 activation: nonspecific protein kinase inhibitors or intracellular perfusion with the non-hydrolyzable ATP analog AMP-PNP dramatically reduce the amplitude of dBest1 currents. Furthermore, intracellular perfusion with ATP-γ-S augments channel activation. The kinase responsible for dBest1 activation is likely Ca2+/calmodulin dependent kinase II (CaMKII), because specific inhibitors of this kinase dramatically inhibit dBest1 current activation. Neither specific PKA inhibitors nor inactive control inhibitors have effects on dBest1currents. Our results demonstrate that dBest1 currents are regulated by phosphorylation via a CaMKII dependent mechanism. 相似文献
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Sean R. Stowell Moonjae Cho Christa L. Feasley Connie M. Arthur Xuezheng Song Jennifer K. Colucci Sougata Karmakar Padmaja Mehta Marcelo Dias-Baruffi Rodger P. McEver Richard D. Cummings 《The Journal of biological chemistry》2009,284(8):4989-4999
Galectin-1 (Gal-1) regulates leukocyte turnover by inducing the cell
surface exposure of phosphatidylserine (PS), a ligand that targets cells for
phagocytic removal, in the absence of apoptosis. Gal-1 monomer-dimer
equilibrium appears to modulate Gal-1-induced PS exposure, although the
mechanism underlying this regulation remains unclear. Here we show that
monomer-dimer equilibrium regulates Gal-1 sensitivity to oxidation. A mutant
form of Gal-1, containing C2S and V5D mutations (mGal-1), exhibits impaired
dimerization and fails to induce cell surface PS exposure while retaining the
ability to recognize carbohydrates and signal Ca2+ flux in
leukocytes. mGal-1 also displayed enhanced sensitivity to oxidation, whereas
ligand, which partially protected Gal-1 from oxidation, enhanced Gal-1
dimerization. Continual incubation of leukocytes with Gal-1 resulted in
gradual oxidative inactivation with concomitant loss of cell surface PS,
whereas rapid oxidation prevented mGal-1 from inducing PS exposure.
Stabilization of Gal-1 or mGal-1 with iodoacetamide fully protected Gal-1 and
mGal-1 from oxidation. Alkylation-induced stabilization allowed Gal-1 to
signal sustained PS exposure in leukocytes and mGal-1 to signal both
Ca2+ flux and PS exposure. Taken together, these results
demonstrate that monomer-dimer equilibrium regulates Gal-1 sensitivity to
oxidative inactivation and provides a mechanism whereby ligand partially
protects Gal-1 from oxidation.Immunological homeostasis relies on efficient contraction of activated
leukocytes following an inflammatory episode. Several factors, including
members of the galectin and tumor necrosis factor families
(1,
2), regulate leukocyte turnover
by inducing apoptotic cell death. In contrast, several galectin family
members, in particular galectin-1
(Gal-1),2 uniquely
regulate neutrophil turnover by inducing phosphatidylserine (PS) exposure,
which normally sensitizes apoptotic cells to phagocytic removal
(3,
4), independent of apoptosis, a
process recently termed preaparesis
(5).Previous studies suggested that dimerization may be required for
Gal-1-induced PS exposure, as a mutant form of Gal-1 (mGal-1) containing two
point mutations within the dimer interface, C2S and V5D (C2S,V5D), displays
impaired Gal-1 dimerization and fails to induce PS exposure
(6). However, the manner in
which monomer-dimer equilibrium regulates Gal-1 signaling remains unclear.
Previous studies suggest that dimerization may be required for efficient
cross-linking of functional receptors or the formation of signaling lattices
(7–9).
Consistent with this, monomeric mutants of several other galectins fail to
induce PS exposure or signal leukocytes
(4,
8). Gal-1 signaling of PS
exposure requires initial signaling events, such as mobilization of
intracellular Ca2+ followed by sustained receptor engagement
(10). Although mGal-1 fails to
induce PS exposure (6), whether
mGal-1 can induce these initial signaling events remains unknown
(10).In addition to directly regulating signaling, monomer-dimer equilibrium may
also regulate other aspects of Gal-1 function. Unlike many other proteins
involved in the regulation of immunity, Gal-1 displays unique sensitivity to
oxidative inactivation
(11–15).
Although engagement of ligand partially protects Gal-1 from oxidation
(15), the impact of Gal-1
oxidation on signaling remains enigmatic. During oxidation, Gal-1 forms three
distinct intramolecular disulfide bridges that facilitate profound
conformational changes that preclude ligand binding and Gal-1 dimerization
(12–14),
suggesting that monomerdimer equilibrium may also regulate Gal-1 sensitivity
to oxidative inactivation.Previous studies utilized dithiothreitol (DTT) in treatment conditions to
protect Gal-1 from oxidative inactivation
(16,
17). Indeed, failure to
include DTT precluded Gal-1-induced death in T cells
(3,
18), suggesting that Gal-1
undergoes rapid oxidation in vivo in the absence of reducing
conditions. However, DTT itself can induce apoptosis in leukocytes
(19), leaving questions
regarding the impact of Gal-1 oxidation on these signaling events. In
contrast, recent studies utilizing iodoacetamide-alkylated Gal-1 (iGal-1),
previously shown to protect Gal-1 from oxidative inactivation
(20–29),
demonstrated that DTT actually primes cells to become sensitive to
Gal-1-induced apoptosis regardless of Gal-1 sensitivity to oxidation
(5).As the engagement of leukocyte ligands requires glycan recognition and
oxidation precludes this binding
(11,
15), understanding the impact
of oxidation on Gal-1 signals will facilitate a greater appreciation of the
factors that govern Gal-1 oxidation and therefore function. Our results
demonstrate that Gal-1 monomer-dimer equilibrium provides a key regulatory
point controlling both Gal-1 sensitivity to oxidation and its ability to
signal PS exposure in leukocytes. These results provide novel insights into
Gal-1 function and explain at a biochemical level the mechanisms regulating
Gal-1 oxidative inactivation and signaling. 相似文献
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Jens Waak Stephanie S. Weber Karin G?rner Christoph Schall Hidenori Ichijo Thilo Stehle Philipp J. Kahle 《The Journal of biological chemistry》2009,284(21):14245-14257
Parkinson disease (PD)-associated genomic deletions and the destabilizing
L166P point mutation lead to loss of the cytoprotective DJ-1 protein. The
effects of other PD-associated point mutations are less clear. Here we
demonstrate that the M26I mutation reduces DJ-1 expression, particularly in a
null background (knockout mouse embryonic fibroblasts). Thus, homozygous M26I
mutation causes loss of DJ-1 protein. To determine the cellular consequences,
we measured suppression of apoptosis signal-regulating kinase 1 (ASK1) and
cytotoxicity for [M26I]DJ-1, and systematically all other DJ-1 methionine and
cysteine mutants. C106A mutation of the central redox site specifically
abolished binding to ASK1 and the cytoprotective activity of DJ-1. DJ-1 was
apparently recruited into the ASK1 signalosome via Cys-106-linked mixed
disulfides. The designed higher order oxidation mimicking [C106DD]DJ-1
non-covalently bound to ASK1 even in the absence of hydrogen peroxide and
conferred partial cytoprotection. Interestingly, mutations of peripheral redox
sites (C46A and C53A) and M26I also led to constitutive ASK1 binding.
Cytoprotective [wt]DJ-1 bound to the ASK1 N terminus (which is known to bind
another negative regulator, thioredoxin 1), whereas [M26I]DJ-1 bound to
aberrant C-terminal site(s). Consequently, the peripheral cysteine mutants
retained cytoprotective activity, whereas the PD-associated mutant [M26I]DJ-1
failed to suppress ASK1 activity and nuclear export of the death
domain-associated protein Daxx and did not promote cytoprotection. Thus,
cytoprotective binding of DJ-1 to ASK1 depends on the central redox-sensitive
Cys-106 and may be modulated by peripheral cysteine residues. We suggest that
impairments in oxidative conformation changes of DJ-1 might contribute to PD
neurodegeneration.Loss-of-function mutations in the DJ-1 gene (PARK7) cause
autosomal-recessive hereditary Parkinson disease
(PD)2
(1). The most dramatic
PD-associated mutation L166P impairs DJ-1 dimer formation and dramatically
destabilizes the protein
(2–7).
Other mutations such as M26I
(8) and E64D
(9) have more subtle defects
with unclear cellular consequences
(4,
7,
10,
11). In addition to this
genetic association, DJ-1 is neuropathologically linked to PD. DJ-1 is
up-regulated in reactive astrocytes, and it is oxidatively modified in brains
of sporadic PD patients
(12–14).DJ-1 protects against oxidative stress and mitochondrial toxins in cell
culture
(15–17)
as well as in diverse animal models
(18–21).
The cytoprotective effects of DJ-1 may be stimulated by oxidation and mediated
by molecular chaperoning (22,
23), and/or facilitation of
the pro-survival Akt and suppression of apoptosis signal-regulating kinase 1
(ASK1) pathways (6,
24,
25). The cytoprotective
activity of DJ-1 against oxidative stress depends on its cysteine residues
(15,
17,
26). Among the three cysteine
residues of DJ-1, the most prominent one is the easiest oxidizable Cys-106
(27) that is in a constrained
conformation (28), but the
other cysteine residues Cys-46 and Cys-53 have been implicated with DJ-1
activity as well (22).
However, the molecular basis of oxidation-mediated cytoprotective activity of
DJ-1 is not clear. Moreover, the roles of PD-mutated and in vivo
oxidized methionines are not known.Here we have mutagenized all oxidizable residues within DJ-1 and studied
the effects on protein stability and function. The PD-associated mutation M26I
within the DJ-1 dimer interface selectively reduced protein expression as well
as ASK1 suppression and cytoprotective activity in oxidatively stressed cells.
These cell culture results support a pathogenic effect of the clinical M26I
mutation (8). Furthermore,
oxidation-defective C106A mutation abolished binding to ASK1 and
cytoprotective activity of DJ-1, whereas the designed higher order oxidation
mimicking mutant [C106DD]DJ-1 bound to ASK1 even in the absence of
H2O2 and conferred partial cytoprotection. The
peripheral cysteine mutants [C46A]DJ-1 and [C53A]DJ-1 were also cytoprotective
and were incorporated into the ASK1 signalosome even in the basal state. Thus,
DJ-1 may be activated by a complex mechanism, which depends on the redox
center Cys-106 and is modulated by the peripheral cysteine residues.
Impairments of oxidative DJ-1 activation might contribute to the pathogenesis
of PD. 相似文献
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Maria Adelaida Gomez Laleh Alisaraie Marina Tiemi Shio Albert M. Berghuis Colette Lebrun Isabelle Gautier-Luneau Martin Olivier 《The Journal of biological chemistry》2010,285(32):24620-24628
The involvement of macrophages (Mφs) as host, accessory, and effector cells in the development of infectious diseases, together with their central role in iron homeostasis, place these immune cells as key players in the interface between iron and infection. Having previously shown that the functional expression of NRAMP-1 results in increased protein phosphorylation mediated in part by an iron-dependent inhibition of Mφ protein-tyrosine phosphatase (PTP) activity, we sought to study the mechanism(s) underlying this specific event. Herein we have identified the mononuclear dicitrate iron complex [Fe(cit)2H4-x](1+x)− as the species responsible for the specific inhibition of Mφ PTP activity. By using biochemical and computational approaches, we show that [Fe(cit)2]5− targets the catalytic pocket of the PTP SHP-1, competitively inhibiting its interaction with an incoming phosphosubstrate. In vitro and in vivo inhibition of PTP activity by iron-citrate results in protein hyperphosphorylation and enhanced MAPK signaling in response to LPS stimulation. We propose that iron-citrate-mediated PTP inhibition represents a novel and biologically relevant regulatory mechanism of signal transduction. 相似文献
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We used in situ hybridization to investigate Kunitz trypsin inhibitor gene expression programs at the cell level in soybean embryos and in transformed tobacco seeds. The major Kunitz trypsin inhibitor mRNA, designated as KTi3, is first detectable in a specific globular stage embryo region, and then becomes localized within the axis of heart, cotyledon, and maturation stage embryos. By contrast, a related Kunitz trypsin inhibitor mRNA class, designated as KTi1/2, is not detectable during early embryogenesis. Nor is the KTi1/2 mRNA detectable in the axis at later developmental stages. Outer perimeter cells of each cotyledon accumulate both KTi1/2 and KTi3 mRNAs early in maturation. These mRNAs accumulate progressively from the outside to inside of each cotyledon in a "wave-like" pattern as embryogenesis proceeds. A similar KTi3 mRNA localization pattern is observed in soybean somatic embryos and in transformed tobacco seeds. An unrelated mRNA, encoding [beta]-conglycinin storage protein, also accumulates in a wave-like pattern during soybean embryogenesis. Our results indicate that cell-specific differences in seed protein gene expression programs are established early in development, and that seed protein mRNAs accumulate in a precise cellular pattern during seed maturation. We also show that seed protein gene expression patterns are conserved at the cell level in embryos of distantly related plants, and that these patterns are established in the absence of non-embryonic tissues. 相似文献
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We have investigated the regulation and localization of mitogen-activated protein kinase (MAPK) and mitogen-activated protein kinase kinase (MAPKK) in both cytosolic and nuclear fractions of glomerular mesangial cells. p42 MAPK was localized by both immunoblot and kinase activity in both cytosol and nucleus and was rapidly activated, in both fractions, by fetal bovine serum and TPA. Downregulation of protein kinase C (PKC) by TPA inhibited stimulation of cytosolic p42 MAPK, but unexpectedly had no effect on stimulated p42 MAPK in the nucleus. Next we studied the upstream kinase p45 MAPKK by indirect immunofluorescence microscopy, Western blot analysis, and kinase specific activity. Unlike MAPK, p45 MAPKK is almost exclusively cytosolic in resting cells and kinase activity stimulated by TPA is restricted to the cytosol. Interestingly, PKC downregulation for 24 h with TPA dramatically enhanced nuclear MAPKK as assessed by all three techniques. Cytosolic stimulated MAPKK was attenuated in PKC downregulation. Collectively these results show that in mesangial cells: (i) p42 MAPK and p45 MAPKK localize in both the cytosol and the nucleus, and (ii) PKC exerts a negative effect on nuclear MAPKK activity as documented by PKC downregulation, which augments p45 MAPKK nuclear mass and activity. These results indicate that the dual regulation of these two kinases is under differential control in the cytosol and the nucleus. 相似文献
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A Gene Encoding Proline Dehydrogenase Is Not Only Induced by
Proline and Hypoosmolarity, but Is Also Developmentally Regulated in
the Reproductive
Organs of Arabidopsis 总被引:8,自引:0,他引:8 下载免费PDF全文
Kazuo Nakashima Rie Satoh Tomohiro Kiyosue Kazuko Yamaguchi-Shinozaki Kazuo Shinozaki 《Plant physiology》1998,118(4):1233-1241
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C. Nick Pace Gerald R. Grimsley J. Martin Scholtz 《The Journal of biological chemistry》2009,284(20):13285-13289
The structure, stability, solubility, and function of proteins depend on
their net charge and on the ionization state of the individual residues.
Consequently, biochemists are interested in the pK values of the
ionizable groups in proteins and how these pK values depend on their
environment. We review what has been learned about pK values of
ionizable groups in proteins from experimental studies and discuss the
important contributions they make to protein stability and solubility. 相似文献
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