Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phagosomes are key organelles for the innate ability of macrophages to participate in tissue remodeling, clear apoptotic cells, and restrict the spread of intracellular pathogens. To understand the functions of phagosomes, we initiated the systematic identification of their proteins. Using a proteomic approach, we identified >140 proteins associated with latex bead-containing phagosomes. Among these were hydrolases, proton pump ATPase subunits, and proteins of the fusion machinery, validating our approach. A series of unexpected proteins not previously described along the endocytic/phagocytic pathways were also identified, including the apoptotic proteins galectin3, Alix, and TRAIL, the anti-apoptotic protein 14-3-3, the lipid raft-enriched flotillin-1, the anti-microbial molecule lactadherin, and the small GTPase rab14. In addition, 24 spots from which the peptide masses could not be matched to entries in any database potentially represent new phagosomal proteins. The elaboration of a two-dimensional gel database of >160 identified spots allowed us to analyze how phagosome composition is modulated during phagolysosome biogenesis. Remarkably, during this process, hydrolases are not delivered in bulk to phagosomes, but are instead acquired sequentially. The systematic characterization of phagosome proteins provided new insights into phagosome functions and the protein or groups of proteins involved in and regulating these functions.
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PMID:The phagosome proteome: insight into phagosome functions. 1114 29

Apert (Ap) syndrome is a craniofacial malformation characterized by premature fusion of cranial sutures (craniosynostosis). We previously showed that the Ser252Trp fibroblast growth factor receptor 2 (FGFR-2) mutation in Ap syndrome increases osteoblast differentiation and subperiosteal bone matrix formation, leading to premature calvaria ossification. In this study, we used the emerging technology of complementary DNA (cDNA) microarray to identify genes that are involved in osteoblast abnormalities induced by the Ser252Trp FGFR-2 mutation. To identify the signaling pathways involved in this syndrome, we used radioactively labeled cDNAs derived from two sources of cellular messenger RNAs (mRNAs) for hybridization: control (Co) and mutant Ap immortalized osteoblastic cells. Among genes that were differentially expressed, protein kinase Ca (PKC-alpha), interleukin-1alpha (IL-1alpha), and the small guanosine-5'-triphosphatase (GTPase) RhoA were increased in FGFR-2 mutant Ap cells compared with Co cells. The validity of the hybridization array was confirmed by Northern blot analysis using mRNAs derived from different cultures. Furthermore, immunochemical and Western blot analyses showed that mutant Ap cells displayed increased PKC-alpha, IL-1alpha, and RhoA protein levels compared with Co cells. Treatment of Co and Ap cells with the PKC inhibitor calphostin C decreased IL-1alpha and RhoA mRNA and protein levels in Ap cells, indicating that PKC is upstream of IL-1alpha and RhoA. Moreover, SB203580, a specific inhibitor of p38 mitogen-activated protein kinase (MAPK), and PD-98059, a specific inhibitor of MAPK kinase (MEKK), also reduced IL-1alpha and RhoA expression in Ap cells. These data show that the Ser252Trp FGFR-2 mutation in Ap syndrome induces constitutive overexpression of PKC-alpha, IL-1alpha, and small GTPase RhoA, suggesting a role for these effectors in osteoblast alterations induced by the mutation. The cDNA microarray technology appears to be a useful tool to gain information on abnormal gene expression and molecular pathways induced by genetic mutations in bone cells.
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PMID:Increased expression of protein kinase Calpha, interleukin-1alpha, and RhoA guanosine 5'-triphosphatase in osteoblasts expressing the Ser252Trp fibroblast growth factor 2 receptor Apert mutation: identification by analysis of complementary DNA microarray. 1131 98

The distribution of transmembrane proteins is considered to be crucial for their activities because these proteins mediate the information coming from outside of cells. A small GTPase Rho participates in many cellular functions through its downstream effectors. In this study, we examined the effects of RhoA on the distribution of Na(+),K(+)-ATPase, one of the transmembrane proteins. In polarized renal epithelium, Na(+),K(+)-ATPase is known to be localized at the basolateral membrane. By microinjection of the constitutively active mutant of RhoA (RhoA(Val14)) into cultured renal epithelial cells, Na(+),K(+)-ATPase was translocated to the spike-like protrusions over the apical surfaces. Microinjection of the constitutively active mutant of other Rho family GTPases, Rac1 or Cdcd42, did not induce the translocation. The translocation induced by RhoA(Val14) was inhibited by treatment with Y-27632, a Rho-kinase specific inhibitor, or by coinjection of the dominant negative mutant of Rho-kinase. These results indicate that Rho and Rho-kinase are involved in the regulation of the localization of Na(+),K(+)-ATPase. We also found that Na(+),K(+)-ATPase seemed to be colocalized with ERM proteins phosphorylated at T567 (ezrin), T564 (radixin), and T558 (moesin) in cells microinjected with RhoA(Val14).
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PMID:Translocation of Na(+),K(+)-ATPase is induced by Rho small GTPase in renal epithelial cells. 1237 19

Subosteoclastic bone resorption is a result of HCl and proteinase secretion through a late endosome-like bone facing membrane domain called ruffled border. As bone matrix is degraded, it enters osteoclasts' transcytotic vesicles for further processing and is then finally exocytosed to the intercellular space. The present study clarifies the spatial relationship between these vesicle fusion and matrix uptake processes at the ruffled border. Our results show the presence of vacuolar H+-ATPase, small GTPase rab7 as well as dense aggregates of F-actin at the peripheral ruffled border, where basolaterally endocytosed transferrin and cathepsin K are delivered. On the contrary, rhodamine-labeled bone matrix enters transcytotic vesicles at the central ruffled border, where the vesicle budding proteins such as clathrin, AP-2 and dynamin II are also localized. We present a model for the mechanism of ruffled border turnover and suggest that, due to its late endosomal characteristics, the ruffled border serves as a valuable model for studying the dynamic organization of other endosomal compartments as well.
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PMID:Osteoclast ruffled border has distinct subdomains for secretion and degraded matrix uptake. 1255 37

Cell-substratum interactions trigger key signaling pathways that modulate growth control and tissue-specific gene expression. We have previously shown that abolishing adhesive interactions by suspension culture results in G(0) arrest of myoblasts. We report that blocking intracellular transmission of adhesion-dependent signals in adherent cells mimics the absence of adhesive contacts. We investigated the effects of pharmacological inhibitors of acto-myosin contractility on growth and differentiation of C2C12 myogenic cells. ML7 (5-iodonaphthalene-1-sulfonyl homopiperazine) and BDM (2,3, butanedione monoxime) are specific inhibitors of myosin light chain kinase, and myosin heavy chain ATPase, respectively. ML7 and BDM affected cell shape by reducing focal adhesions and stress fibers. Both inhibitors rapidly blocked DNA synthesis in a dose-dependent, reversible fashion. Furthermore, both ML7 and BDM suppressed expression of MyoD and myogenin, induced p27(kip1) but not p21(cip1), and inhibited differentiation. Thus, as with suspension-arrest, inhibition of acto-myosin contractility in adherent cells led to arrest uncoupled from differentiation. Over-expression of inhibitors of the small GTPase RhoA (dominant negative RhoA and C3 transferase) mimicked the effects of myosin inhibitors. By contrast, wild-type RhoA induced arrest, maintained MyoD and activated myogenin and p21 expression. The Rho effector kinase ROCK did not appear to mediate Rho's effects on MyoD. Thus, ROCK and MLCK play different roles in the myogenic program. Signals regulated by MLCK are critical, since inhibition of MLCK suppressed MyoD expression but inhibition of ROCK did not. Inhibition of contractility suppressed MyoD but did not reduce actin polymer levels. However, actin depolymerization with latrunculin B inhibited MyoD expression. Taken together, our observations indicate that actin polymer status and contractility regulate MyoD expression. We suggest that in myoblasts, the Rho pathway and regulation of acto-myosin contractility may define a control point for conditional uncoupling of differentiation and the cell cycle.
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PMID:Modulation of acto-myosin contractility in skeletal muscle myoblasts uncouples growth arrest from differentiation. 1525 13

The endoplasmic reticulum (ER) is the major intracellular membrane system. The ER is essential for protein and lipid biosynthesis, transport of proteins along the secretory pathway, and calcium storage. Here, we describe our investigations into the dynamics and regulation of the ER in the early Caenorhabditis elegans embryo. Using a GFP fusion to the ER-resident signal peptidase SP12, we observed the morphological transitions of the ER through fertilization and the early cell-cycles in living embryos. These transitions were tightly coordinated with the division cycle: upon onset of mitosis, the ER formed structured sheets that redispersed at the initiation of cleavage. Although microtubules were not required for the transition of the ER between these different states, the actin cytoskeleton facilitated the dispersal of the ER at the end of mitosis. The ER had an asymmetric distribution in the early embryo, which was dependent on the establishment of polarity by the PAR proteins. The small GTPase ARF-1 played an essential role in the ER dynamics, although this function appeared to be unrelated to the role of ARF-1 in vesicular traffic. In addition, the ER-resident heat shock protein BiP and a homologue of the AAA ATPase Cdc48/p97 were found to be crucial for the ER transitions. Both proteins have been implicated in homotypic ER membrane fusion. We provide evidence that homotypic membrane fusion is required to form the sheet structure in the early embryo.
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PMID:Involvement of the actin cytoskeleton and homotypic membrane fusion in ER dynamics in Caenorhabditis elegans. 1571 56

The recruitment of the small GTPase Arf6 and ARNO from cytosol to endosomal membranes is driven by V-ATPase-dependent intra-endosomal acidification. The molecular mechanism that mediates this pH-sensitive recruitment and its role are unknown. Here, we demonstrate that Arf6 interacts with the c-subunit, and ARNO with the a2-isoform of V-ATPase. The a2-isoform is targeted to early endosomes, interacts with ARNO in an intra-endosomal acidification-dependent manner, and disruption of this interaction results in reversible inhibition of endocytosis. Inhibition of endosomal acidification abrogates protein trafficking between early and late endosomal compartments. These data demonstrate the crucial role of early endosomal acidification and V-ATPase/ARNO/Arf6 interactions in the regulation of the endocytic degradative pathway. They also indicate that V-ATPase could modulate membrane trafficking by recruiting and interacting with ARNO and Arf6; characteristics that are consistent with the role of V-ATPase as an essential component of the endosomal pH-sensing machinery.
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PMID:V-ATPase interacts with ARNO and Arf6 in early endosomes and regulates the protein degradative pathway. 1645 5

ATPase associated with various cellular activities (AAA) proteins are commonly distributed among eukaryotes, and are involved in a multitude of cellular functions. NtAAA1 is one such example, being involved in pathogen response in tobacco plants. When its activity was suppressed in RNAi transgenic tobacco plants, an elevated resistance to the pathogenic bacterium Pseudomonas syringae was observed in comparison with the wild type. As AAA proteins function through interaction with specific partners, NtAAA1-interacting proteins were screened by the yeast two-hybrid assay, and one particular gene encoding a small GTPase, an ADP ribosylation factor, was identified and designated as NtARF. Its specific binding to NtAAA1 was confirmed by in vitro pull-down assay, and their interaction was predominant between active forms of NtARF and NtAAA1, each bound to GTP and ATP, respectively. Their physical interaction in vivo around the plasma membrane was shown by fluorescence resonance energy transfer assays, suggesting their role in membrane trafficking. Transgenic tobacco plants constitutively expressing NtARF under the control of a cauliflower mosaic virus 35S promoter exhibited spontaneous and wound-induced lesion formation, and enhanced resistance to pathogen attack. Expression of NtAAA1 in leaves of NtARF transgenic plants attenuated lesion and suppressed pathogen resistance. In wild-type tobacco plants, transcripts of NtAAA1 and NtARF could be induced by ethylene and salicylic acid, respectively. These results suggest that NtAAA1 balances plant resistance through suppression of NtARF, and that the molecular basis for the known antagonistic actions of ethylene and salicylic acid in defense response could be partly attributable to these two proteins.
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PMID:Attenuation of the hypersensitive response by an ATPase associated with various cellular activities (AAA) protein through suppression of a small GTPase, ADP ribosylation factor, in tobacco plants. 1755 12

The controlled internalization of membrane receptors and lipids is crucial for cells to control signaling pathways and interact with their environment. During clathrin-mediated endocytosis, membrane constituents are transported via endocytic vesicles into early endosomes, from which they are further distributed within the cell. The small guanosine triphosphatase (GTPase) Rab5 is both required and sufficient for the formation of these early endosomes and can be used to experimentally address endocytic processes. Recent evidence shows that endocytic turnover of E-cadherin regulates the migration of mesendodermal cells during zebrafish gastrulation by modulating their adhesive interactions with neighboring cells. This in turn leads to effective and synchronized movement within the embryo. In this review, we discuss techniques to manipulate E-cadherin endocytosis by morpholino-mediated knockdown of rab5 during zebrafish gastrulation. We describe the use of antibodies specifically directed against zebrafish E-cadherin to detect its intracellular localization and of in situ hybridization and primary cell culture to reveal patterns of cell migration and adhesion, respectively.
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PMID:Probing E-cadherin endocytosis by morpholino-mediated Rab5 knockdown in zebrafish. 1836 59

We have previously shown that the V-ATPase a2-subunit isoform interacts specifically, and in an intra-endosomal acidification-dependent manner, with the Arf-GEF ARNO. In the present study, we examined the molecular mechanism of this interaction using synthetic peptides and purified recombinant proteins in protein-association assays. In these experiments, we revealed the involvement of multiple sites on the N-terminus of the V-ATPase a2-subunit (a2N) in the association with ARNO. While six a2N-derived peptides interact with wild-type ARNO, only two of them (named a2N-01 and a2N-03) bind to its catalytic Sec7-domain. However, of these, only the a2N-01 peptide (MGSLFRSESMCLAQLFL) showed specificity towards the Sec7-domain compared to other domains of the ARNO protein. Surface plasmon resonance kinetic analysis revealed a very strong binding affinity between this a2N-01 peptide and the Sec7-domain of ARNO, with dissociation constant KD=3.44x10(-7) M, similar to the KD=3.13x10(-7) M binding affinity between wild-type a2N and the full-length ARNO protein. In further pull-down experiments, we also revealed the involvement of multiple sites on ARNO itself in the association with a2N. However, while its catalytic Sec7-domain has the strongest interaction, the PH-, and PB-domains show much weaker binding to a2N. Interestingly, an interaction of the a2N to a peptide corresponding to ARNO's PB-domain was abolished by phosphorylation of ARNO residue Ser392. The 3D-structures of the non-phosphorylated and phosphorylated peptides were resolved by NMR spectroscopy, and we have identified rearrangements resulting from Ser392 phosphorylation. Homology modeling suggests that these alterations may modulate the access of the a2N to its interaction pocket on ARNO that is formed by the Sec7 and PB-domains. Overall, our data indicate that the interaction between the a2-subunit of V-ATPase and ARNO is a complex process involving various binding sites on both proteins. Importantly, the binding affinity between the a2-subunit and ARNO is in the same range as those previously reported for the intramolecular association of subunits within V-ATPase complex itself, indicating an important cell biological role for the interaction between the V-ATPase and small GTPase regulatory proteins.
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PMID:Specific motifs of the V-ATPase a2-subunit isoform interact with catalytic and regulatory domains of ARNO. 2015 92


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