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1.

Background

Ionizing irradiation causes not only growth arrest and cell death, but also release of growth factors or signal transmitters, which promote cancer malignancy. Extracellular ATP controls cancer growth through activation of purinoceptors. However, there is no report of radiation-induced ATP release from cancer cells. Here, we examined γ-irradiation-induced ATP release and its mechanism in B16 melanoma.

Methods

Extracellular ATP was measured by luciferin–luciferase assay. To investigate mechanism of radiation-induced ATP release, we pharmacologically inhibited the ATP release and established stable P2X7 receptor-knockdown B16 melanoma cells using two short hairpin RNAs targeting P2X7 receptor.

Results

Cells were exposed to 0.5–8 Gy of γ-rays. Extracellular ATP was increased, peaking at 5 min after 0.5 Gy irradiation. A selective P2X7 receptor channel antagonist, but not anion transporter inhibitors, blocked the release of ATP. Further, radiation-induced ATP release was significantly decreased in P2X7 receptor-knockdown cells. Our results indicate that γ-irradiation evokes ATP release from melanoma cells, and P2X7 receptor channel plays a significant role in mediating the ATP release.

General Significance

We suggest that extracellular ATP could be a novel intercellular signaling molecule released from cancer cells when cells are exposed to ionizing radiation.  相似文献   

2.

Background

Extracellular matrix (ECM) components and intracellular pH (pHi) may serve as regulators of cell migration in various cell types.

Methods

The Oris migration assay was used to assess the effect of fibronectin (FN) on cell motility. The Na+/H+ exchanger (NHE)-1 activity was evaluated by measuring pHi and [22Na+] uptake. To examine activated signaling molecules, western blot analysis and immunoprecipitation was performed.

Results

ECM components (FN, laminin, fibrinogen, and collagen type I) increased [22Na+] uptake, pHi, and cell migration. In addition, FN-induced increase of cell migration was inhibited by NHE-1 inhibitor amiloride or NHE-1-specific siRNA. FN selectively increased the mRNA and protein expression of NHE-1, but not that of NHE-2 or NHE-3. FN binds integrin β1 and subsequently stimulates caveolin-1 phosphorylation and Ca2 + influx. Then, NHE-1 is phosphorylated by RhoA and Rho kinases, and Ca2 +/calmodulin (CaM) signaling elicits complex formation with NHE-1, which is enriched in lipid raft/caveolae microdomains of the plasma membrane. Activation of NHE-1 continuously induces an increase of [22Na+] uptake and pHi. Finally, NHE-1-dependent extracellular signal-regulated kinase (ERK) 1/2 phosphorylation enhanced matrix metalloproteinase-2 (MMP-2) and filamentous-actin (F-actin) expression, partially contributing to the regulation of embryonic stem cells (ESCs) migration.

Conclusions

FN stimulated mESCs migration and proliferation through NHE-1 activation, which were mediated by lipid raft-associated caveolin-1, RhoA/ROCK, and Ca2 +/CaM signaling pathways.

General significance

The precise role of NHE in the modulation of ECM-related physiological functions such as proliferation and migration remains poorly understood. Thus, this study analyzed the relationship between FN and NHE in regulating the migration of mouse ESCs and their related signaling pathways.  相似文献   

3.
Extracellular nucleotides and their metabolites activate ionotropic P2X and metabotropic P2Y receptors on the surface of various types of cells. Here, we investigated the involvement of P2X and P2Y receptor-mediated signaling in TCR-dependent T cell activation. Murine T cells were activated by stimulation of TCR, and both CD25 expression and interleukin (IL)-2 production were observed in activated T cells. Ecto-nucleotidase apyrase and P2Y6 antagonist MRS2578 significantly blocked the increases of both CD25 expression and IL-2 production, and P2X7 antagonists A438079 and oxidized ATP inhibited IL-2 production rather than CD25 expression, suggesting the involvement of P2Y6 and P2X7 receptors in different processes of T cell activation. MRS2578 also blocked TCR-dependent elevation of cytosolic Ca2+ in T cells. The P2X7 and P2Y6 receptors were expressed in murine CD4 T cells. In conclusion, our results indicate that activation of P2Y6 and P2X7 receptors contributes to T cell activation via TCR.  相似文献   

4.

Aims

We previously reported that fluvoxamine, a selective serotonin reuptake inhibitor with high affinity for the σ1-receptor (σ1R), ameliorates cardiac hypertrophy and dysfunction via σ1R stimulation. Although σ1R on non-cardiomyocytes interacts with the IP3 receptor (IP3R) to promote mitochondrial Ca2 + transport, little is known about its physiological and pathological relevance in cardiomyocytes.

Main methods

Here we performed Ca2 + imaging and measured ATP production to define the role of σ1Rs in regulating sarcoplasmic reticulum (SR)-mitochondrial Ca2 + transport in neonatal rat ventricular cardiomyocytes treated with angiotensin II to promote hypertrophy.

Key finding

These cardiomyocytes exhibited imbalances in expression levels of σ1R and IP3R and impairments in both phenylephrine-induced mitochondrial Ca2 + mobilization from the SR and ATP production. Interestingly, σ1R stimulation with fluvoxamine rescued impaired mitochondrial Ca2 + mobilization and ATP production, an effect abolished by treatment of cells with the σ1R antagonist, NE-100. Under physiological conditions, fluvoxamine stimulation of σ1Rs suppressed intracellular Ca2 + mobilization through IP3Rs and ryanodine receptors (RyRs). In vivo, chronic administration of fluvoxamine to TAC mice also rescued impaired ATP production.

Significance

These results suggest that σ1R stimulation with fluvoxamine promotes SR-mitochondrial Ca2 + transport and mitochondrial ATP production, whereas σ1R stimulation suppresses intracellular Ca2 + overload through IP3Rs and RyRs. These mechanisms likely underlie in part the anti-hypertrophic and cardioprotective action of the σ1R agonists including fluvoxamine.  相似文献   

5.
This work shows that ATP activates JNK1, but not JNK2, in rat osteoblasts and ROS-A 17/2.8 osteoblast-like cells. In ROS-A 17/2.8 cells ATP induced JNK1 phosphorylation in a dose- and time-dependent manner. JNK1 phosphorylation also increased after osteoblast stimulation with ATPγS and UTP, but not with ADPβS. RT-PCR studies supported the expression of P2Y2 receptor subtype. ATP-induced JNK1 activation was reduced by PI-PLC, IP3 receptor, PKC and Src inhibitors and by gadolinium, nifedipine and verapamil or a Ca2+-free medium. ERK 1/2 or p38 MAPK inhibitors diminished JNK1 activation by ATP, suggesting a cross-talk between these pathways. ATP stimulated osteoblast-like cell proliferation consistent with the participation of P2Y2 receptors. These results show that P2Y2 receptor stimulation by ATP induces JNK1 phosphorylation in ROS-A 17/2.8 cells in a way dependent on PI-PLC/IP3/intracellular Ca2+ release and Ca2+ influx through stress activated and L-type voltage-dependent calcium channels and involves PKC and Src kinases.  相似文献   

6.
The presynaptic P2X7 receptor (P2X7R) plays an important role in the modulation of transmitter release. We recently demonstrated that, in nerve terminals of the adult rat cerebral cortex, P2X7R activation induced Ca2+-dependent vesicular glutamate release and significant Ca2+-independent glutamate efflux through the P2X7R itself. In the present study, we investigated the effect of the new selective P2X7R competitive antagonist 3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methyl pyridine (A-438079) on cerebrocortical terminal intracellular calcium (intrasynaptosomal calcium concentration;[Ca2+]i signals and glutamate release, and evaluated whether P2X7R immunoreactivity was consistent with these functional tests. A-438079 inhibited functional responses. P2X7R immunoreactivity was found in about 45% of cerebrocortical terminals, including glutamatergic and non-glutamatergic terminals. This percentage was similar to that of synaptosomes showing P2X7R-mediated [Ca2+]i signals. These findings provide compelling evidence of functional presynaptic P2X7R in cortical nerve terminals.  相似文献   

7.

Background

The secretory activity of Sertoli cells (SC) is dependent on ion channel functions and protein synthesis and is critical to ongoing spermatogenesis. The aim of this study was to investigate the mechanism of action associated with a non-metabolizable amino acid [14C]-MeAIB (α-(methyl-amino)isobutyric acid) accumulation stimulated by T4 and the role of the integrin receptor in this event, and also to clarify whether the T4 effect on MeAIB accumulation and on Ca2+ influx culminates in cell secretion.

Methods

We have studied the rapid and plasma membrane initiated effects of T4 by using 45Ca2+ uptake and [45C]-MeAIB accumulation assays, respectively. Thymidine incorporation into DNA was used to monitor nuclear activity and quinacrine to analyze the secretory activity on SC.

Results

The stimulation of MeAIB accumulation by T4 appears to be mediated by the integrin receptor in the plasma membrane since tetrac and RGD peptide were able to nullify the effect of this hormone. In addition, T4 increases extracellular Ca2+ uptake and Ca2+ from intracellular stocks to enhance nuclear activity, but this genomic action seems not to influence SC secretion mediated by T4. Also, the cytoskeleton and ClC-3 chloride channel contribute to the membrane-associated responses of SC.

Conclusions

T4 integrin receptor activation ultimately determines the plasma membrane responses on amino acid transport in SC, but it is not involved in calcium influx, cell secretion or the nuclear effect of the hormone.

General significance

The integrin receptor activation by T4 may take a role in plasma membrane processes involved in the male reproductive system.  相似文献   

8.
Purinergic receptors have been shown to be involved in neuronal development, but the functions of specific subtypes of P2 receptors during neuronal development remain elusive. In this study we investigate the distribution of P2X7 receptors (P2X7Rs) in the embryonic rat brain using in situ hybridization. At E15.5, P2X7R mRNA was observed in the ventricular zone and subventricular zone, and colocalized with nestin, indicating that P2X7R might be expressed in neural progenitor cells (NPCs). P2X7R mRNA was also detected in the subgranular zone and dentate gyrus of the E18.5 and P4 brain. To investigate the roles of P2X7R and elucidate its mechanism, we established NPC cultures from the E15.5 rat brain. Stimulation of P2X7Rs induced Ca2+ influx, inhibited proliferation, altered cell cycle progression and enhanced the expression of neuronal markers, such as TUJ1 and MAP2. Similarly, knockdown of P2X7R by shRNA nearly abolished the agonist-stimulated increases in intracellular Ca2+ concentration and the expression of TUJ1 and NeuN. Furthermore, stimulation of P2X7R induced activation of ERK1/2, which was inhibited by the removal of extracellular Ca2+ and treatment with blockers for P2X7R and PKC activity. Stimulation of P2X7R also induced translocation of PKCα and PKCγ, but not of PKCβ, whereas knockdown of either PKCα or PKCγ inhibited ERK1/2 activation. Inhibition of PKC or p-ERK1/2 also caused a decrease in the number of TUJ1-positive cells and a concomitant increase in the number of GFAP-positive cells. Taken together, the activation of P2X7R in NPCs induced neuronal differentiation through a PKC-ERK1/2 signaling pathway.  相似文献   

9.

Back ground

Stress-induced phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF2α), involved in translation, promotes cell suicide or survival. Since multiple signaling pathways are implicated in cell death, the present study has analyzed the importance of PKC activation in the stress-induced eIF2α phosphorylation, caspase activation and cell death in the ovarian cells of Spodoptera frugiperda (Sf9) and in their extracts.

Methods

Cell death is analyzed by flow cytometry. Caspase activation is measured by Ac-DEVD-AFC hydrolysis and also by the cleavage of purified recombinant PERK, an endoplasmic reticulum-resident eIF2α kinase. Status of eIF2α phosphorylation and cytochrome c levels are analyzed by western blots.

Results

PMA, an activator of PKC, does not promote cell death or affect eIF2α phosphorylation. However, PMA enhances late stages of UV-irradiation or cycloheximide-induced caspase activation, eIF2α phosphorylation and apoptosis in Sf9 cells. PMA also enhances cytochrome c-induced caspase activation and eIF2α phosphorylation in cell extracts. These changes are mitigated more efficiently by caspase inhibitor, z-VAD-fmk, than by calphostin, an inhibitor of PKC. In contrast, tunicamycin-induced eIF2α phosphorylation that does not lead to caspase activation or cell death is unaffected by PMA, z-VAD-fmk or by calphostin.

Conclusions

While caspase activation is a cause and consequence of eIF2α phosphorylation, PKC activation that follows caspase activation further enhances caspase activation, eIF2α phosphorylation, and cell death in Sf9 cells.

General significance

Caspases can activate multiple signaling pathways to enhance cell death.  相似文献   

10.

Background

Hydroxy-1-aryl-isochromans (HAIC) are newly emerging natural polyphenolic antioxidants, enriched in extravirgin olive oil, whose antioxidative potency was only scarcely characterized using cell-free systems and cells.

Methods

We characterized the activity of HAIC to inactivate reactive oxygen species (ROS) generated by the xanthine/xanthine oxidase system, mitochondria (rat brain) and neural cells. ROS levels were estimated using ROS-sensitive probes, such as Amplex Red, MitoSOXRED.

Results

HAIC (with 2, 3 or 4 hydroxyl substituents) effectively scavenge ROS released from mitochondria. EC50 values estimated with mitochondria and submitochondrial particles were around 20 μM. Moreover, in PC12 and cultured neural primary cells, HAIC buffered cytosolic ROS. Although HAIC permeate biological membranes, HAIC fail to buffer matrix ROS in isolated mitochondria. We show that hydrogen peroxide was effectively abolished by HAIC, whereas the production of superoxide was not affected.

Conclusion

HAIC exert high antioxidative activity to reduce hydrogen peroxide. The antioxidative activity of HAIC is comparable with that of the stilbene-like, polyphenolic resveratrol, but much higher than that of trolox, N-acetylcysteine or melatonin.

General significance

Unlike resveratrol, HAIC do not impair mitochondrial ATP synthesis or Ca2+ retention by mitochondria. Thus, HAIC have the decisive advantage to be potent antioxidants with no detrimental side effects on mitochondrial functions.  相似文献   

11.

Background

ATP exerts diverse effects on various cell types via specific purinergic P2Y receptors. Intracellular signaling cascades are the main routes of communication between P2Y receptors and regulatory targets in the cell.

Methods and results

We examined the role of ATP in the modulation of ERK1/2, JNK1/2, and p38 MAP kinases (MAPKs) in human colon cancer Caco-2 cells. Immunoblot analysis showed that ATP induces the phosphorylation of MAPKs in a time- and dose-dependent manner, peaking at 5 min at 10 µM ATP. Moreover, ATPγS, UTP, and UDP but not ADP or ADPβS increased phosphorylation of MAPKs, indicating the involvement of, at least, P2Y2/P2Y4 and P2Y6 receptor subtypes. RT–PCR studies and PCR product sequencing supported the expression of P2Y2 and P2Y4 receptors in this cell line. Spectrofluorimetric measurements showed that cell stimulation with ATP induced transient elevations in intracellular calcium concentration. In addition, ATP-induced phosphorylation of MAPKs in Caco-2 cells was dependent on Src family tyrosine kinases, calcium influx, and intracellular Ca2+ release and was partially dependent on the cAMP/PKA and PKC pathways and the EGFR.

General significance

These findings provide new molecular basis for further understanding the mechanisms involved in ATP functions, as a signal transducer and activator of MAP kinase cascades, in colon adenocarcinoma Caco-2 cells.  相似文献   

12.

Background

Activation of ATP-gated P2X7 receptors (P2X7R) in macrophages leads to production of reactive oxygen species (ROS) by a mechanism that is partially characterized. Here we used J774 cells to identify the signaling cascade that couples ROS production to receptor stimulation.

Methods

J774 cells and mP2X7-transfected HEK293 cells were stimulated with Bz-ATP in the presence and absence of extracellular calcium. Protein inhibitors were used to evaluate the physiological role of various kinases in ROS production. In addition, phospho-antibodies against ERK1/2 and Pyk2 were used to determine activation of these two kinases.

Results

ROS generation in either J774 or HEK293 cells (expressing P2X7, NOX2, Rac1, p47phox and p67phox) was strictly dependent on calcium entry via P2X7R. Stimulation of P2X7R activated Pyk2 but not calmodulin. Inhibitors of MEK1/2 and c-Src abolished ERK1/2 activation and ROS production but inhibitors of PI3K and p38 MAPK had no effect on ROS generation. PKC inhibitors abolished ERK1/2 activation but barely reduced the amount of ROS produced by Bz-ATP. In agreement, the amount of ROS produced by PMA was about half of that produced by Bz-ATP.

Conclusions

Purinergic stimulation resulted in calcium entry via P2X7R and subsequent activation of the PKC/c-Src/Pyk2/ERK1/2 pathway to produce ROS. This signaling mechanism did not require PI3K, p38 MAPK or calmodulin.

General significance

ROS is generated in order to kill invading pathogens, thus elucidating the mechanism of ROS production in macrophages and other immune cells allow us to understand how our body copes with microbial infections.  相似文献   

13.

Background

Sleep is a physiological event that directly influences health by affecting the immune system, in which calcium (Ca2 +) plays a critical signaling role. We performed live cell measurements of cytosolic Ca2 + mobilization to understand the changes in Ca2 + signaling that occur in splenic immune cells after various periods of sleep deprivation (SD).

Methods

Adult male mice were subjected to sleep deprivation by platform technique for different periods (from 12 to 72 h) and Ca2 + intracellular fluctuations were evaluated in splenocytes by confocal microscopy. We also performed spleen cell evaluation by flow cytometry and analyzed intracellular Ca2 + mobilization in endoplasmic reticulum and mitochondria. Additionally, Ca2 + channel gene expression was evaluated

Results

Splenocytes showed a progressive loss of intracellular Ca2 + maintenance from endoplasmic reticulum (ER) stores. Transient Ca2 + buffering by the mitochondria was further compromised. These findings were confirmed by changes in mitochondrial integrity and in the performance of the store operated calcium entry (SOCE) and stromal interaction molecule 1 (STIM1) Ca2 + channels.

Conclusions and general significance

These novel data suggest that SD impairs Ca2 + signaling, most likely as a result of ER stress, leading to an insufficient Ca2 + supply for signaling events. Our results support the previously described immunosuppressive effects of sleep loss and provide additional information on the cellular and molecular mechanisms involved in sleep function.  相似文献   

14.
The expression of protein kinase C (PKC) isoforms and the modulation of Ca2+ mobilization by PKC were investigated in the human submandibular duct cell line A253. Three new PKC (nPKC) isoforms (, , and ) and one atypical PKC (aPKC) isoform () are expressed in this cell line. No classical PKC (cPKC) isoforms were present. The effects of the PKC activator phorbol 12-myristate-13-acetate (PMA) and of the PKC inhibitors calphostin C (CC) and bisindolymaleimide I (BSM) on inositol 1,4,5-trisphosphate (IP3) and Ca2+ responses to ATP and to thapsigargin (TG) were investigated. Pre-exposure to PMA inhibited IP3 formation, Ca2+ release and Ca2+ influx in response to ATP. Pre-exposure to CC or BSM slightly enhanced IP3 formation but inhibited the Ca2+ release and the Ca2+ influx induced by ATP. In contrast, pre-exposure to PMA did not modify the Ca2+ release induced by TG, but reduced the influx of Ca2+ seen in the presence of this Ca2+-ATPase inhibitor. These results suggest that PKC modulates elements of the IP3/Ca2+ signal transduction pathway in A253 cells by (1) inhibiting phosphatidylinositol turnover and altering the sensitivity of the Ca2+ channels to IP3, (2) altering the activity, the sensitivity to inhibitors, or the distribution of the TG-sensitive Ca2+ ATPase, and (3) modulating Ca2+ entry pathways.  相似文献   

15.

Background

In recent years, as our understanding of the various roles played by Ca2 + signaling in development and differentiation has expanded, the challenge of imaging Ca2 + dynamics within living cells, tissues, and whole animal systems has been extended to include specific signaling activity in organelles and non-membrane bound sub-cellular domains.

Scope of review

In this review we outline how recent advances in genetics and molecular biology have contributed to improving and developing current bioluminescence-based Ca2 + imaging techniques. Reporters can now be targeted to specific cell types, or indeed organelles or domains within a particular cell.

Major conclusions

These advances have contributed to our current understanding of the specificity and heterogeneity of developmental Ca2 + signaling. The improvement in the spatial resolution that results from specifically targeting a Ca2 + reporter has helped to reveal how a ubiquitous signaling messenger like Ca2 + can regulate coincidental but different signaling events within an individual cell; a Ca2 + signaling paradox that until now has been hard to explain.

General significance

Techniques used to target specific reporters via genetic means will have applications beyond those of the Ca2 + signaling field, and these will, therefore, make a significant contribution in extending our understanding of the signaling networks that regulate animal development. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signalling.  相似文献   

16.

Background

Phospholipid scramblases are a group of four homologous proteins conserved from C. elegans to human. In human, two members of the scramblase family, hPLSCR1 and hPLSCR3 are known to bring about Ca2+ dependent translocation of phosphatidylserine and cardiolipin respectively during apoptotic processes. However, affinities of Ca2+/Mg2+ binding to human scramblases and conformational changes taking place in them remains unknown.

Methods

In the present study, we analyzed the Ca2+ and Mg2+ binding to the calcium binding motifs of hPLSCR1–4 and hPLSCR1 by spectroscopic methods and isothermal titration calorimetry.

Results

The results in this study show that (i) affinities of the peptides are in the order hPLSCR1  > hPLSCR3 > hPLSCR2 > hPLSCR4 for Ca2+ and in the order hPLSCR1 > hPLSCR2 > hPLSCR3 > hPLSCR4 for Mg2+, (ii) binding of ions brings about conformational change in the secondary structure of the peptides. The affinity of Ca2+ and Mg2+ binding to protein hPLSCR1 was similar to that of the peptide I. A sequence comparison shows the existence of scramblase-like motifs among other protein families.

Conclusions

Based on the above results, we hypothesize that the Ca2+ binding motif of hPLSCR1 is a novel type of Ca2+ binding motif.

General significance

Our findings will be relevant in understanding the calcium dependent scrambling activity of hPLSCRs and their biological function.  相似文献   

17.

Background

Transplanted mesenchymal stem cells (MSC) can differentiate into cardiac cells that have the potential to contribute to heart repair following ischemic injury. Overexpression of GATA-4 can significantly increase differentiation of MSC into cardiomyocytes (CM). However, the specific impact of GATA-4 overexpression on the electrophysiological properties of MSC-derived CM has not been well documented.

Methods

Adult rat bone marrow MSC were retrovirally transduced with GATA-4 (MSCGATA-4) and GFP (MSCNull) and subsequently co-cultured with neonatal rat ventricular cardiomyocytes (CM). Electrophysiological properties and mRNA levels of ion channels were assessed in MSC using patch-clamp technology and real-time PCR.

Results

MSCGATA-4 exhibited higher levels of the TTX-sensitive Na+ current (INa.TTX), L-type calcium current (ICa.L), transient outward K+ current (Ito), delayed rectifier K+ current (IKDR) and inwardly rectifying K+ current (IK1) channel activities reflective of electrophysiological characteristics of CM. Real-time PCR analyses showed that MSCGATA-4 exhibited upregulated mRNA levels of Kv1.2, Kv2.1, SCN2a1, CCHL2a, KV1.4 and Kir1.1 channels versus MSCNull. Interestingly, MSCGATA-4 treated with IGF-1 neutralizing antibodies resulted in a significant decrease in Kir1.1, Kv2.1, KV1.4, CCHL2a and SCN2a1 channel mRNA expression. Similarly, MSCGATA-4 treated with VEGF neutralizing antibodies also resulted in an attenuated expression of Kv2.1, Kv1.2, Kv1.4, Kir1.1, CCHL2a and SCN2a1 channel mRNAs.

Conclusions

GATA-4 overexpression increases Ito, IKDR, IK1, INa.TTX and ICa.L currents in MSC. Cytokine (VGEF and IGF-1) release from GATA-4 overexpressing MSC can partially account for the upregulated ion channel mRNA expression.

General significance

Our results highlight the ability of GATA4 to boost the cardiac electrophysiological potential of MSC.  相似文献   

18.

Background

To establish the physiological role of calpain, it is necessary to define how the protease can escape from the effect of its natural inhibitor calpastatin, since both proteins co-localize into the cell cytosol.

Methods

To answer this question, we have overexpressed four fluorescent calpastatin constructs, differing in the composition of their XL- and L-domains, and the intracellular trafficking of this protein inhibitor has been followed by single cell fluorescence imaging.

Results and conclusions

By the use of these calpastatin forms differing in the type of exon-derived sequences contained in the XL- and L-domains, we have demonstrated that the sequence coded by exon 6, containing multiple phosphorylation sites, is directly involved in determining the cell localization of calpastatin. In fact, exposure to cAMP promotes the recruitment into aggregates of those calpastatin forms containing the exon 6 sequence. These protein movements are directly related to the level of cytosolic inhibitory capacity and thereby to the extent of intracellular calpain activation.

General significance

The recruitment of calpastatin into aggregates allows the translocation and activation of the protease to the membranes; on the contrary, the presence of large amounts of calpastatin in the cytosol prevents both processes, protecting the cell from undesired proteolysis.  相似文献   

19.

Background

Redox signaling is an important emerging mechanism of cellular function. Dysfunctional redox signaling is increasingly implicated in numerous pathologies, including atherosclerosis, diabetes, and cancer. The molecular messengers in this type of signaling are reactive species which can mediate the post-translational modification of specific groups of proteins, thereby effecting functional changes in the modified proteins. Electrophilic compounds comprise one class of reactive species which can participate in redox signaling. Electrophiles modulate cell function via formation of covalent adducts with proteins, particularly cysteine residues.

Scope of review

This review will discuss the commonly used methods of detection for electrophile-sensitive proteins, and will highlight the importance of identifying these proteins for studying redox signaling and developing novel therapeutics.

Major conclusions

There are several methods which can be used to detect electrophile-sensitive proteins. These include the use of tagged model electrophiles, as well as derivatization of endogenous electrophile–protein adducts.

General significance

In order to understand the mechanisms by which electrophiles mediate redox signaling, it is necessary to identify electrophile-sensitive proteins and quantitatively assess adduct formation. Strengths and limitations of these methods will be discussed. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.  相似文献   

20.

Background

Hydrogen peroxide (H2O2) is an important signaling compound that has recently been identified as a new substrate for several members of the aquaporin superfamily in various organisms. Evidence is emerging about the physiological significance of aquaporin-facilitated H2O2 diffusion.

Scope of review

This review summarizes current knowledge about aquaporin-facilitated H2O2 diffusion across cellular membranes. It focuses on physicochemical and experimental evidence demonstrating the involvement of aquaporins in the transport of this redox signaling compound and discusses the regulation and structural prerequisites of these channels to transmit this signal. It also provides perspectives about the potential importance of aquaporin-facilitated H2O2 diffusion processes and places this knowledge in the context of the current understanding of transmembrane redox signaling processes.

Major conclusions

Specific aquaporin isoforms facilitate the passive diffusion of H2O2 across biological membranes and control H2O2 membrane permeability and signaling in living organisms.

General significance

Redox signaling is a very important process regulating the physiology of cells and organisms in a similar way to the well-characterized hormonal and calcium signaling pathways. Efficient transmembrane diffusion of H2O2, a key molecule in the redox signaling network, requires aquaporins and makes these channels important players in this signaling process. Channel-mediated membrane transport allows the fine adjustment of H2O2 levels in the cytoplasm, intracellular organelles, the apoplast, and the extracellular space, which are essential for it to function as a signal molecule. This article is part of a Special Issue entitled Aquaporins.  相似文献   

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