共查询到20条相似文献,搜索用时 15 毫秒
1.
Simina Bogatan Duygu Cevik Valentin Demidov Jessica Vanderploeg Abdullah Panchbhaya Alex Vitkin J. Roger Jacobs 《PloS one》2015,10(6)
Mechanotransduction of tension can govern the remodeling of cardiomyocytes during growth or cardiomyopathy. Tension is signaled through the integrin adhesion complexes found at muscle insertions and costameres but the relative importance of signalling during cardiomyocyte growth versus remodelling has not been assessed. Employing the Drosophila cardiomyocyte as a genetically amenable model, we depleted the levels of Talin, a central component of the integrin adhesion complex, at different stages of heart growth and remodeling. We demonstrate a continuous requirement for Talin during heart growth to maintain the one-to-one apposition of myofibril ends between cardiomyocytes. Retracted myofibrils cannot regenerate appositions to adjacent cells after restoration of normal Talin expression, and the resulting deficit reduces heart contraction and lifespan. Reduction of Talin during heart remodeling after hatching or during metamorphosis results in pervasive degeneration of cell contacts, myofibril length and number, for which restored Talin expression is insufficient for regeneration. Resultant dilated cardiomyopathy results in a fibrillating heart with poor rhythmicity. Cardiomyocytes have poor capacity to regenerate deficits in myofibril orientation and insertion, despite an ongoing capacity to remodel integrin based adhesions. 相似文献
2.
The melanization reaction is a major immune response in Arthropods and involves the rapid synthesis of melanin at the site of infection and injury. A key enzyme in the melanization process is phenoloxidase (PO), which catalyzes the oxidation of phenols to quinones, which subsequently polymerize into melanin. The Drosophila genome encodes three POs, which are primarily produced as zymogens or prophenoloxidases (PPO). Two of them, PPO1 and PPO2, are produced by crystal cells. Here we have generated flies carrying deletions in PPO1 and PPO2. By analyzing these mutations alone and in combination, we clarify the functions of both PPOs in humoral melanization. Our study shows that PPO1 and PPO2 are responsible for all the PO activity in the hemolymph. While PPO1 is involved in the rapid early delivery of PO activity, PPO2 is accumulated in the crystals of crystal cells and provides a storage form that can be deployed in a later phase. Our study also reveals an important role for PPO1 and PPO2 in the survival to infection with Gram-positive bacteria and fungi, underlining the importance of melanization in insect host defense. 相似文献
3.
4.
5.
Multivesicular endosome (MVE) sorting depends on proteins of the Endosomal Sorting Complex Required for Transport (ESCRT) family. These are organized in four complexes (ESCRT-0, -I, -II, -III) that act in a sequential fashion to deliver ubiquitylated cargoes into the internal luminal vesicles (ILVs) of the MVE. Drosophila genes encoding ESCRT-I, -II, -III components function in sorting signaling receptors, including Notch and the JAK/STAT signaling receptor Domeless. Loss of ESCRT-I, -II, -III in Drosophila epithelia causes altered signaling and cell polarity, suggesting that ESCRTs genes are tumor suppressors. However, the nature of the tumor suppressive function of ESCRTs, and whether tumor suppression is linked to receptor sorting is unclear. Unexpectedly, a null mutant in Hrs, encoding one of the components of the ESCRT-0 complex, which acts upstream of ESCRT-I, -II, -III in MVE sorting is dispensable for tumor suppression. Here, we report that two Drosophila epithelia lacking activity of Stam, the other known components of the ESCRT-0 complex, or of both Hrs and Stam, accumulate the signaling receptors Notch and Dome in endosomes. However, mutant tissue surprisingly maintains normal apico-basal polarity and proliferation control and does not display ectopic Notch signaling activation, unlike cells that lack ESCRT-I, -II, -III activity. Overall, our in vivo data confirm previous evidence indicating that the ESCRT-0 complex plays no crucial role in regulation of tumor suppression, and suggest re-evaluation of the relationship of signaling modulation in endosomes and tumorigenesis. 相似文献
6.
Activation of Both MAP Kinase and Phosphatidylinositide 3-Kinase by Ras Is Required for Hepatocyte Growth Factor/Scatter Factor–induced Adherens Junction Disassembly
下载免费PDF全文

Hepatocyte growth factor/scatter factor (HGF/SF) stimulates the motility of epithelial cells, initially inducing centrifugal spreading of colonies followed by disruption of cell–cell junctions and subsequent cell scattering. In Madin–Darby canine kidney cells, HGF/SF-induced motility involves actin reorganization mediated by Ras, but whether Ras and downstream signals regulate the breakdown of intercellular adhesions has not been established. Both HGF/SF and V12Ras induced the loss of the adherens junction proteins E-cadherin and β-catenin from intercellular junctions during cell spreading, and the HGF/SF response was blocked by dominant-negative N17Ras. Desmosomes and tight junctions were regulated separately from adherens junctions, because they were not disrupted by V12Ras. MAP kinase, phosphatidylinositide 3-kinase (PI 3-kinase), and Rac were required downstream of Ras, because loss of adherens junctions was blocked by the inhibitors PD098059 and LY294002 or by dominant-inhibitory mutants of MAP kinase kinase 1 or Rac1. All of these inhibitors also prevented HGF/SF-induced cell scattering. Interestingly, activated Raf or the activated p110α subunit of PI 3-kinase alone did not induce disruption of adherens junctions. These results indicate that activation of both MAP kinase and PI 3-kinase by Ras is required for adherens junction disassembly and that this is essential for the motile response to HGF/SF. 相似文献
7.
Mark Le Rachelle Naridze Jasmine Morrison Leah C. Biggs Lindsey Rhea Brian C. Schutte Vesa Kaartinen Martine Dunnwald 《PloS one》2012,7(10)
Wound healing is a complex process that relies on proper levels of cytokines and growth factors to successfully repair the tissue. Of particular interest are the members of the transforming growth factor family. There are three TGF-ß isoforms–TGF- ß 1, 2, and 3, each isoform showing a unique expression pattern, suggesting that they each play a distinct function during development and repair. Previous studies reported an exclusive role for TGF-ß 3 in orofacial development and a potent anti-scarring effect. However, the role of TGF- ß 3 in excisional wound healing and keratinocyte migration remains poorly understood. We tested the effect of TGF-ß 3 levels on excisional cutaneous wounds in the adult mouse by directly injecting recombinant TGF-ß 3 or neutralizing antibody against TGF-ß 3 (NAB) in the wounds. Our results demonstrate that TGF-ß 3 does not promote epithelialization. However, TGF-ß 3 is necessary for wound closure as wounds injected with neutralizing antibody against TGF-ß 3 showed increased epidermal volume and proliferation in conjunction with a delay in keratinocyte migration. Wild type keratinocytes treated with NAB and Tgfb3-deficient keratinocytes closed an in vitro scratch wound with no delay, suggesting that our in vivo observations likely result from a paracrine effect. 相似文献
8.
Gennadiy Tenin Christopher Clowes Kathryn Wolton Eliska Krejci Jayne A. Wright Simon C. Lovell David Sedmera Kathryn E. Hentges 《PloS one》2014,9(9)
The heart is the first organ required to function during embryonic development and is absolutely necessary for embryo survival. Cardiac activity is dependent on both the sinoatrial node (SAN), which is the pacemaker of heart''s electrical activity, and the cardiac conduction system which transduces the electrical signal though the heart tissue, leading to heart muscle contractions. Defects in the development of cardiac electrical function may lead to severe heart disorders. The Erbb2 (Epidermal Growth Factor Receptor 2) gene encodes a member of the EGF receptor family of receptor tyrosine kinases. The Erbb2 receptor lacks ligand-binding activity but forms heterodimers with other EGF receptors, stabilising their ligand binding and enhancing kinase-mediated activation of downstream signalling pathways. Erbb2 is absolutely necessary in normal embryonic development and homozygous mouse knock-out Erbb2 embryos die at embryonic day (E)10.5 due to severe cardiac defects. We have isolated a mouse line, l11Jus8, from a random chemical mutagenesis screen, which carries a hypomorphic missense mutation in the Erbb2 gene. Homozygous mutant embryos exhibit embryonic lethality by E12.5-13. The l11Jus8 mutants display cardiac haemorrhage and a failure of atrial function due to defects in atrial electrical signal propagation, leading to an atrial-specific conduction block, which does not affect ventricular conduction. The l11Jus8 mutant phenotype is distinct from those reported for Erbb2 knockout mouse mutants. Thus, the l11Jus8 mouse reveals a novel function of Erbb2 during atrial conduction system development, which when disrupted causes death at mid-gestation. 相似文献
9.
The procofactor, factor VIII, is activated by thrombin or factor
Xa-catalyzed cleavage at three P1 residues: Arg-372, Arg-740, and Arg-1689.
The catalytic efficiency for thrombin cleavage at Arg-740 is greater than at
either Arg-1689 or Arg-372 and influences reaction rates at these sites.
Because cleavage at Arg-372 appears rate-limiting and dependent upon initial
cleavage at Arg-740, we investigated whether cleavage at Arg-1689 influences
catalysis at this step. Recombinant B-domainless factor VIII mutants, R1689H
and R1689Q were prepared and stably expressed to slow and eliminate cleavage,
respectively. Specific activity values for the His and Gln mutations were
∼50 and ∼10%, respectively, that of wild type. Thrombin activation of
the R1689H variant showed an ∼340-fold reduction in the rate of Arg-1689
cleavage, whereas the R1689Q variant was resistant to thrombin cleavage at
this site. Examination of heavy chain cleavages showed ∼4- and 11-fold
reductions in A2 subunit generation and ∼3- and 7-fold reductions in A1
subunit generation for the R1689H and R1689Q mutants, respectively. These
results suggest a linkage between light chain cleavage and cleavages in heavy
chain. Results obtained evaluating proteolysis of the factor VIII mutants by
factor Xa revealed modest rate reductions (<5-fold) in generating A2 and A1
subunits and in cleaving light chain at Arg-1721 from either variant,
suggesting little dependence upon prior cleavage at residue 1689 as compared
with thrombin. Overall, these results are consistent with a competition
between heavy and light chains for thrombin exosite binding and subsequent
proteolysis with binding of the former chain preferred.Factor VIII, a plasma protein missing or defective in individuals with
hemophilia A, is synthesized as an ∼300-kDa single chain polypeptide
corresponding to 2332 amino acids. Within the protein are six domains based on
internal homologies and ordered as NH2-A1-A2-B-A3-C1-C2-COOH
(1,
2). Bordering the A domains are
short segments containing high concentrations of acidic residues that follow
the A1 and A2 domains and precede the A3 domain and are designated a1
(residues 337–372), a2 (residues 711–740), and a3
(1649–1689). Factor VIII is processed by cleavage at the B-A3 junction
to generate a divalent metal ion-dependent heterodimeric protein composed of a
heavy chain (A1-a1-A2-a2-B domains) and a light chain (a3-A3-C1-C2 domains)
(3).The activated form of factor VIII, factor VIIIa, functions as a cofactor
for factor IXa, increasing its catalytic efficiency by several orders of
magnitude in the phospholipid- and Ca2+-dependent conversion of
factor X to factor Xa (4). The
factor VIII procofactor is converted to factor VIIIa through limited
proteolysis catalyzed by thrombin or factor Xa
(5,
6). Thrombin is believed to act
as the physiological activator of factor VIII, as association of factor VIII
with von Willebrand factor impairs the capacity for the membrane-dependent
factor Xa to efficiently activate the procofactor
(5,
7). Activation of factor VIII
occurs through proteolysis by either protease via cleavage of three P1
residues at Arg-740 (A2-B domain junction), Arg-372 (A1-A2 domain junction),
and Arg-1689 (a3-A3 junction)
(5). After factor VIII
activation, there is a weak electrostatic interaction between the A1 and A2
domains of factor VIIIa (8,
9) and spontaneous inactivation
of the cofactor occurs through A2 subunit dissociation from the A1/A3-C1-C2
dimer, consequently dampening factor Xase
(3).Thrombin cleavage of factor VIII appears to be an ordered pathway, with
relative rates at Arg-740 > Arg-1689 > Arg-372 and the initial
proteolysis at Arg-740 facilitating proteolysis at Arg-372 as well as Arg-1689
(10). This latter observation
was based upon results showing that mutations at Arg-740, impairing this
cleavage, significantly reduced cleavage rates at the two other P1 sites.
Thrombin-catalyzed activation of factor VIII is dependent upon interactions
involving the anion binding exosites of the proteinase
(11,
12). Exosite binding is
believed to determine substrate affinity, whereas subsequent active site
docking primarily affects Vmax
(13). Furthermore, the complex
interactions involving multiple cleavages within a single substrate may
utilize a ratcheting mechanism
(14) where presentation of the
scissile bond is facilitated by a prior cleavage event.Cleavage at Arg-372 is a critical step in thrombin activation of factor
VIII as it exposes a cryptic functional factor IXa-interactive site in the A2
domain (15), whereas cleavage
at Arg-1689 liberates factor VIII from von Willebrand factor
(16) and contributes to factor
VIIIa specific activity (17,
18). Although cleavage at
Arg-740 represents a fast step relative to cleavages at other P1 residues in
the activation of factor VIII
(19), the influence of
Arg-1689 cleavage on cleavages in the heavy chain remains unknown. In the
present study cleavage at Arg-1689 is examined using recombinant factor VIII
variants possessing single point mutations of R1689Q and R1689H. Results
indicating reduced rates of A1 and A2 subunit generation, which are dependent
upon the residue at position 1689, suggest that cleavage at Arg-1689 affects
rates of proteolysis at Arg-740 and Arg-372. These observations are consistent
with a mechanism whereby heavy chain and light chain compete for a binding
thrombin exosite(s), with heavy chain preferred over light chain. In this
competition mechanism, cleavage at Arg-740 is favored over Arg-1689.
Subsequent cleavage at Arg-372 in heavy chain may involve a ratcheting
mechanism after initial cleavage at Arg-740. On the other hand, the mechanism
for factor Xa-catalyzed activation of factor VIII appears to be less dependent
on cleavage at the Arg-1689 site as compared with thrombin. 相似文献
10.
11.
Jennifer L. Macdonald-Obermann Linda J. Pike 《The Journal of biological chemistry》2009,284(20):13570-13576
We have previously shown that the binding of epidermal growth factor (EGF)
to its receptor can best be described by a model that involves negative
cooperativity in an aggregating system (Macdonald, J. L., and Pike, L. J.
(2008) Proc. Natl. Acad. Sci. U. S. A. 105, 112–117). However,
despite the fact that biochemical analyses indicate that EGF induces
dimerization of its receptor, the binding data provided no evidence for
positive linkage between EGF binding and dimer assembly. By analyzing the
binding of EGF to a number of receptor mutants, we now report that in naive,
unphosphorylated EGF receptors, ligand binding is positively linked to
receptor dimerization but the linkage is abolished upon autophosphorylation of
the receptor. Both phosphorylated and unphosphorylated EGF receptors exhibit
negative cooperativity, indicating that mechanistically, cooperativity is
distinct from the phenomenon of linkage. Nonetheless, both the positive
linkage and the negative cooperativity observed in EGF binding require the
presence of the intracellular juxtamembrane domain. This indicates the
existence of inside-out signaling in the EGF receptor system. The
intracellular juxtamembrane domain has previously been shown to be required
for the activation of the EGF receptor tyrosine kinase (Thiel, K. W., and
Carpenter, G. (2007) Proc. Natl. Acad. Sci. U. S. A. 104,
19238–19243). Our experiments expand the role of this domain to include
the allosteric control of ligand binding by the extracellular domain.The EGF2 receptor
is a tyrosine kinase composed of an ∼620-amino-acid extracellular domain
that recognizes and binds EGF, a single pass α-helical transmembrane
domain, and an intracellular tyrosine kinase domain, encompassing roughly
residues 685–950 (1). In
addition, the receptor contains an ∼230-amino-acid-long C-terminal tail
that contains the bulk of the sites of receptor autophosphorylation
(2–4).
An intracellular juxtamembrane domain of about 40 residues connects the
transmembrane domain to the kinase domain and has been shown to be crucial in
the allosteric activation of the EGF receptor kinase
(5,
6).In the membrane, the EGF receptor exists as a monomer, but a wealth of data
indicate that the binding of EGF induces the formation of EGF receptor dimers
(7–10).
Dimerization appears to be mediated in large part by the extracellular domain
of the receptor, which is comprised of four subdomains, designated I through
IV. X-ray crystallography data suggest that in the absence of ligand, the
extracellular domain is held in a closed configuration through the interaction
of loops or arms that extend from the backs of subdomains II and IV
(11). Upon binding of EGF,
this intramolecular tether is released, allowing the receptor to adopt an open
conformation in which EGF is tightly bound between subdomains I and III. In
this configuration, the “dimerization arm” that was previously
involved in tethering the receptor closed mediates the formation of a
back-to-back EGF receptor dimer
(12,
13).Analyses of the binding of 125I-EGF to its receptor have
invariably resulted in concave up Scatchard plots that have been interpreted
as indicating the presence of two classes of EGF binding sites. However, we
have recently used global analysis of the binding of 125I-EGF to
cells expressing increasing levels of EGF receptors to show that EGF binding
is best described by a model involving negative cooperativity in an
aggregating system (14) (see
Fig. 6). Ligand binding is
negatively cooperative if the binding of ligand to the first site on a dimer
reduces the affinity of the ligand for binding to the second site on the
dimer.Open in a separate windowFIGURE 6.Model for the binding of EGF to its receptor. Circles
represent receptor subunits. E represents a molecule of EGF. The
equilibrium association constants are written above or beside the reaction to
which they apply.The concept of cooperativity only applies to existing dimers. It does not
relate to the effect of ligand on the assembly or disassembly of those dimers.
The effect of ligand on the formation of receptor dimers is captured in the
concept of linkage (15,
16). If ligand binding is
positively linked to dimer formation, then ligand promotes the assembly of
receptor dimers. In a monomer-dimer equilibrium, positive linkage arises when
a ligand binds with higher affinity to the first site on the dimer than to the
monomer. Under these circumstances, the ligand will preferentially bind to the
dimer, shifting the equilibrium in favor of the dimeric species. In the case
of the EGF receptor, biochemical data suggest that EGF induces receptor
dimerization; however, evidence for positive linkage in binding studies has
been lacking.By analyzing the binding of 125I-EGF to cells expressing various
EGF receptor mutants, we now report that in naive, unphosphorylated EGF
receptors, ligand binding is, in fact, positively linked to receptor
dimerization. Autophosphorylation of the EGF receptor abolishes the positive
linkage that is present during the initial phase of the ligand binding
reaction. Negative cooperativity is present in both the phosphorylated and the
non-phosphorylated states of the receptor. Structure-function analyses
demonstrate that both cooperativity and linkage are lost when the EGF receptor
is truncated immediately after the transmembrane domain. However, both forms
of regulation are restored in receptors that include the additional 40 amino
acids that correspond to the intracellular juxtamembrane domain. These data
expand the role of the intracellular juxtamembrane domain to include the
allosteric regulation of EGF binding by the extracellular domain and
demonstrate the presence of inside-out signaling in the EGF receptor
system. 相似文献
12.
Loss-of-function mutations in TRPML1 (transient receptor potential mucolipin 1) cause the lysosomal storage disorder, mucolipidosis type IV (MLIV). Here, we report that flies lacking the TRPML1 homolog displayed incomplete autophagy and reduced viability during the pupal period-a phase when animals rely on autophagy for nutrients. We show that TRPML was required for fusion of amphisomes with lysosomes, and its absence led to accumulation of vesicles of significantly larger volume and higher luminal Ca(2+). We also found that trpml(1) mutant cells showed decreased TORC1 (target of rapamycin complex 1) signaling and a concomitant upregulation of autophagy induction. Both of these defects in the mutants were reversed by genetically activating TORC1 or by feeding the larvae a high-protein diet. The high-protein diet?also reduced the pupal lethality and the increased volume of acidic vesicles. Conversely, further inhibition of TORC1 activity by rapamycin exacerbated the mutant phenotypes. Finally, TORC1 exerted reciprocal control on TRPML function. A high-protein diet caused cortical localization of TRPML, and this effect was blocked by rapamycin. Our findings delineate the interrelationship between the TRPML and TORC1 pathways and raise the intriguing possibility that a high-protein diet might reduce the severity of MLIV. 相似文献
13.
14.
Sriram Devanathan Alexander Erban Rodolfo Perez-Torres Jr Joachim Kopka Christopher A. Makaroff 《PloS one》2014,9(4)
The glyoxalase pathway, which consists of the two enzymes, GLYOXALASE 1 (GLX 1) (E.C.: 4.4.1.5) and 2 (E.C.3.1.2.6), has a vital role in chemical detoxification. In Arabidopsis thaliana there are at least four different isoforms of glyoxalase 2, two of which, GLX2-1 and GLX2-4 have not been characterized in detail. Here, the functional role of Arabidopsis thaliana GLX2-1 is investigated. Glx2-1 loss-of-function mutants and plants that constitutively over-express GLX2-1 resemble wild-type plants under normal growth conditions. Insilico analysis of publicly available microarray datasets with ATTEDII, Mapman and Genevestigator indicate potential role(s) in stress response and acclimation. Results presented here demonstrate that GLX2-1 gene expression is up-regulated in wild type Arabidopsis thaliana by salt and anoxia stress, and by excess L-Threonine. Additionally, a mutation in GLX2-1 inhibits growth and survival during abiotic stresses. Metabolic profiling studies show alterations in the levels of sugars and amino acids during threonine stress in the plants. Elevated levels of polyamines, which are known stress markers, are also observed. Overall our results suggest that Arabidopsis thaliana GLX2-1 is not essential during normal plant life, but is required during specific stress conditions. 相似文献
15.
Parmil K. Bansal Amanda Nourse Rashid Abdulle Katsumi Kitagawa 《The Journal of biological chemistry》2009,284(6):3586-3592
The kinetochore, which consists of DNA sequence elements and structural
proteins, is essential for high-fidelity chromosome transmission during cell
division. In budding yeast, Sgt1 and Hsp90 help assemble the core kinetochore
complex CBF3 by activating the CBF3 components Skp1 and Ctf13. In this study,
we show that Sgt1 forms homodimers by performing in vitro and in
vivo immunoprecipitation and analytical ultracentrifugation analyses.
Analyses of the dimerization of Sgt1 deletion proteins showed that the
Skp1-binding domain (amino acids 1–211) contains the Sgt1
homodimerization domain. Also, the Sgt1 mutant proteins that were unable to
dimerize also did not bind Skp1, suggesting that Sgt1 dimerization is
important for Sgt1-Skp1 binding. Restoring dimerization activity of a
dimerization-deficient sgt1 mutant (sgt1-L31P) by using the
CENP-B (centromere protein-B) dimerization
domain suppressed the temperature sensitivity, the benomyl sensitivity, and
the chromosome missegregation phenotype of sgt1-L31P. These results
strongly suggest that Sgt1 dimerization is required for kinetochore
assembly.Spindle microtubules are coupled to the centromeric region of the
chromosome by a structural protein complex called the kinetochore
(1,
2). The kinetochore is thought
to generate a signal that arrests cells during mitosis when it is not properly
attached to microtubules, thereby preventing aberrant chromosome transmission
to the daughter cells, which can lead to tumorigenesis
(3,
4). The kinetochore of the
budding yeast Saccharomyces cerevisiae has been characterized
thoroughly, genetically and biochemically; thus, its molecular structure is
the most well detailed to date. More than 70 different proteins comprise the
budding yeast kinetochore, and several of those are conserved in mammals
(2).The budding yeast centromere DNA is a 125-bp region that contains three
conserved regions, CDEI, CDEII, and CDEIII
(5,
6). CDEI is bound by Cbf1
(7–9).
CDEIII (25 bp) is essential for centromere function
(10) and is the site where
CBF3 binds to centromeric DNA. CBF3 contains four proteins: Ndc10, Cep3, Ctf13
(11–18),
and Skp1 (17,
18), all of which are
essential for viability. Mutations in any of the four CBF3 proteins abolish
the ability of CDEIII to bind to CBF3
(19,
20). All of the described
kinetochore proteins, except the CDEI-binding Cbf1, localize to kinetochores
dependent on the CBF3 complex
(2). Therefore, the CBF3
complex is the fundamental structure of the kinetochore, and the mechanism of
CBF3 assembly is of major interest.We previously isolated SGT1, the skp1-4
kinetochore-defective mutant dosage suppressor
(21). Sgt1 and Skp1 activate
Ctf13; thus, they are required for assembly of the CBF3 complex
(21). The molecular chaperone
Hsp90 is also required for the formation of the Skp1-Ctf13 complex
(22). Sgt1 has two highly
conserved motifs that are required for protein-protein interaction, the
tetratricopeptide repeat
(TPR)2
(21) and the CS
(CHORD protein- and Sgt1-specific) motif. We and others
(23–26)
have found that both domains are important for the interaction with Hsp90. The
Sgt1-Hsp90 interaction is required for the assembly of the core kinetochore
complex; this interaction is an initial step in kinetochore assembly
(24,
26,
27) that is conserved between
yeast and humans (28,
29).In this study, we further characterized the molecular mechanism of this
assembly process. We found that Sgt1 forms dimers in vivo, and our
results strongly suggest that Sgt1 dimerization is required for kinetochore
assembly in budding yeast. 相似文献
16.
The expression of ERCC1, a member of the nucleotide excision repair (NER) family, is enhanced in cells transfected with insulin-like growth factor 1 (IGF-1) receptors. Of interest, an excellent concordance between ERCC1 expression and NER-mediated cell survival has been demonstrated. The two aims of the present study were to determine the signaling pathways used by IGF-1 to confer protection against apoptotic cell death in Chinese hamster ovary (CHO) cells and to assess the role of NER in this IGF-1 action. Experiments with pharmacological inhibitors indicated that phosphatidylinositol 3-kinase (PI 3-kinase) but not mitogen-activated protein kinase (ERK1/ERK2) mediates IGF-1 antiapoptotic activity. Using two series of CHO cells that have altered expression of ERCC1 or XPB/ERCC3, we examined IGF-1's ability to delay apoptotic death and reduction of mitochondrial oxidative function mediated by growth factor withdrawal. IGF-1 effectively blocked apoptosis, concomitant with increased MTT activity, in a pair of CHO cell lines expressing inactive ERCC1 (43-3B cells) and the transfected line of the mutant carrying the expressed human ERCC1 gene (83-G5 cells). Similarly, repair-deficient UV24 cells, which lack XPB/ERCC3, and their parental line AA8 were also responsive to the IGF-1's antiapoptotic capacity. In the presence of IGF-1, these cell lines became resistant to the cleavage of poly(ADP-ribose) polymerase, a key player in DNA damage recognition and DNA repair. These results suggest that PI 3-kinase activation plays a determinant role in the antiapoptotic function of IGF-1, but that functional NER does not play a critical part in mediating this IGF-1 response. 相似文献
17.
Pregnancy-associated plasma protein-A (PAPPA) has been reported to regulate the activity of insulin-like growth factor (IGF) signal pathway through proteolytic degradation of IGF binding proteins (IGFBPs) thereby increasing the local concentration of free IGFs available to receptors. In this study we found that PAPPA is secreted from two out of seven lung cancer cell lines examined. None of immortalized normal bronchial epithelial cells (HBE) tested secrets PAPPA. There is no correlation between expression level and secretion of PAPPA in these cells. A cell line over-expressing PAPPA accompanied with secretion shows no notable changes in proliferation under cell culture conditions in vitro, but displays significantly augmentation of tumor growth in vivo in a xenograft model. In contrast, a cell line over-expressing PAPPA without secretion exhibits reduction of tumor growth both in vitro and in vivo. Down-regulation of PAPPA expression and secretion by RNAi knockdown decreases tumor growth after implanted in vivo. The tumor promoting activity of PAPPA appears to be mediated mainly through augmentation of the IGF signaling pathway as indicated by notable increases in downstream Akt kinase phosphorylation in tumor samples. Our results indicate that PAPPA secretion may play an important role in lung cancer growth and progression. 相似文献
18.
Xiurong Wu Wan-Ting He Shuye Tian Dan Meng Yuanyue Li Wanze Chen Lisheng Li Lili Tian Chuan-Qi Zhong Felicia Han Jianming Chen Jiahuai Han 《PLoS pathogens》2014,10(4)
Viruses hijack host factors for their high speed protein synthesis, but information about these factors is largely unknown. In searching for genes that are involved in viral replication, we carried out a forward genetic screen for Drosophila mutants that are more resistant or sensitive to Drosophila C virus (DCV) infection-caused death, and found a virus-resistant line in which the expression of pelo gene was deficient. Our mechanistic studies excluded the viral resistance of pelo deficient flies resulting from the known Drosophila anti-viral pathways, and revealed that pelo deficiency limits the high level synthesis of the DCV capsid proteins but has no or very little effect on the expression of some other viral proteins, bulk cellular proteins, and transfected exogenous genes. The restriction of replication of other types of viruses in pelo deficient flies was also observed, suggesting pelo is required for high level production of capsids of all kinds of viruses. We show that both pelo deficiency and high level DCV protein synthesis increase aberrant 80S ribosomes, and propose that the preferential requirement of pelo for high level synthesis of viral capsids is at least partly due to the role of pelo in dissociation of stalled 80S ribosomes and clearance of aberrant viral RNA and proteins. Our data demonstrated that pelo is a host factor that is required for high efficiency translation of viral capsids and targeting pelo could be a strategy for general inhibition of viral infection. 相似文献
19.