Journal articles on the topic 'Inactive surface mutants'
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1
Honegger, A. M., A. Schmidt, A. Ullrich, and J. Schlessinger. "Separate endocytic pathways of kinase-defective and -active EGF receptor mutants expressed in same cells." Journal of Cell Biology 110, no. 5 (May 1, 1990): 1541–48. http://dx.doi.org/10.1083/jcb.110.5.1541.
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Ligand binding to the membrane receptor for EGF induces its clustering and internalization. Both receptor and ligand are then degraded by lysosomal enzymes. A kinase defective point mutant (K721A) of EGF receptor undergoes internalization similarly to the wild-type receptor. However, while internalized EGF molecules bound to either the wild-type or mutant receptors are degraded, the K721A mutant receptor molecules recycle to the cell surface for reutilization. To investigate the mechanism of receptor trafficking, we have established transfected NIH-3T3 cells coexpressing the kinase-negative mutant (K721A) together with a mutant EGF receptor (CD63) with active kinase. CD63 was chosen because it behaves like wild-type EGF receptor with respect to biological responsiveness and cellular routing but afforded immunological distinction between kinase active and inactive mutants. Although expressed in the same cells, the two receptor mutants followed their separate endocytic itineraries. Like wild-type receptor, the CD63 mutant was downregulated and degraded in response to EFG while the kinase-negative mutant K721A returned to the cell surface for reutilization. Intracellular trafficking of EGF receptor must be determined by a sorting mechanism that specifically recognizes EGF receptor molecules according to their intrinsic kinase activity.
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Mo, Xiu-Lei, Rui Yang, and Ya-Xiong Tao. "Functions of transmembrane domain 3 of human melanocortin-4 receptor." Journal of Molecular Endocrinology 49, no. 3 (September 26, 2012): 221–35. http://dx.doi.org/10.1530/jme-12-0162.
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The melanocortin-4 receptor (MC4R) is a G protein-coupled receptor critical for maintaining energy homeostasis. Transmembrane domain 3 (TM3) of MC4R contains residues that were suggested to be essential in ligand binding and signaling. SeveralMC4Rmutations in TM3 are associated with human obesity. To gain a better understanding of the functions of TM3, we analyzed the functions of 26 residues in TM3 using alanine-scanning mutagenesis. We showed that all mutants had normal cell-surface expression. Four mutants were defective in ligand binding and signaling and six mutants had normal ligand binding but impaired cAMP production. L140A had increased basal cAMP level. To further characterize the function of L140, we generated 17 additional L140 mutants. Fifteen L140 mutants had significantly decreased cell-surface expression, with L140R and L140V expressed normally. Ten L140 mutants had increased basal cAMP activities. Four L140 mutants were defective in ligand-stimulated cAMP generation. Interestingly, with the ERK1/2 pathway, we showed that nine constitutively active mutants had similar levels of basal pERK1/2 as that of WT, and two signaling defective mutants had similar levels of pERK1/2 as that of WT upon agonist stimulation, different from their cAMP signaling properties, suggesting biased signaling in these mutant receptors. In summary, we identified 13 residues in TM3 that were essential for ligand binding and/or signaling. Moreover, L140 was critical for locking MC4R in inactive conformation and several mutants showed biased signaling in cAMP and ERK1/2 signaling pathways.
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Kinne, Anita, Stephan Roth, Heike Biebermann, Josef Köhrle, Annette Grüters, and Ulrich Schweizer. "Surface translocation and tri-iodothyronine uptake of mutant MCT8 proteins are cell type-dependent." Journal of Molecular Endocrinology 43, no. 6 (July 31, 2009): 263–71. http://dx.doi.org/10.1677/jme-09-0043.
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Mutations in the gene encoding the thyroid hormone transporter, monocarboxylate transporter 8 (MCT8), underlie severe mental retardation. We wanted to understand the functional consequences of a series of missense mutations in MCT8 in order to identify therapeutic options for affected patients. We established cell lines stably expressing 12 MCT8 variants in JEG1 and MDCK1 cells. The cell lines were characterized according to MCT8 mRNA and protein expression, tri-iodothyronine (T3) transport activity, substrate KM characteristics, surface expression, and responsiveness to T3 preincubation and chemical chaperones. Functional activities of ins235V and L568P MCT8 mutants depend on the cell type in which they are expressed. These mutants and R271H exhibited considerable transport activity when present at the cell surface as verified by surface biotinylation and kinetic analysis. Most mutants, however, were inactive in T3 transport even when present at the cell surface (e.g. S194F, A224V, ΔF230, L512P). Preincubation of G558D with T3 increased T3 uptake in MDCK1 cells to a small, but significant, extent. Chemical chaperones were ineffective. The finding that the cell type determines surface expression and T3 transport activities of missense mutants in MCT8 may be important to understand phenotypic variability among carriers of different mutations. In particular, the clinical observation that the severity of derangements of thyroid hormone levels does not correlate with mental impairments of the patients may be based on different residual activity of mutant MCT8 in different cell types.
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Filippa, Nathalie, Carol L. Sable, Brian A. Hemmings, and Emmanuel Van Obberghen. "Effect of Phosphoinositide-Dependent Kinase 1 on Protein Kinase B Translocation and Its Subsequent Activation." Molecular and Cellular Biology 20, no. 15 (August 1, 2000): 5712–21. http://dx.doi.org/10.1128/mcb.20.15.5712-5721.2000.
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ABSTRACT In this report we investigated the function of phosphoinositide-dependent protein kinase 1 (PDK1) in protein kinase B (PKB) activation and translocation to the cell surface. Wild-type and PDK1 mutants were transfected into HeLa cells, and their subcellular localization was analyzed. PDK1 was found to translocate to the plasma membrane in response to insulin, and this process did not require a functional catalytic activity, since a catalytically inactive kinase mutant (Kd) of PDK1 was capable of translocating. The PDK1 presence at the cell surface was shown to be linked to phospholipids and therefore to serum-dependent phosphatidylinositol 3-kinase activity. Using confocal microscopy in HeLa cells we found that PDK1 colocalizes with PKB at the plasma membrane. Further, after cotransfection of PKB and a PDK1 mutant (Mut) unable to translocate to the plasma membrane, PKB was prevented from moving to the cell periphery after insulin stimulation. In response to insulin, a PKB mutant with its PH domain deleted (ΔPH-PKB) retained the ability to translocate to the plasma membrane when coexpressed with PDK1. Finally, we found that ΔPH-PKB was highly active independent of insulin stimulation when cotransfected with PDK1 mutants defective in their PH domain. These findings suggest that PDK1 brings PKB to the plasma membrane upon exposure of cells to insulin and that the PH domain of PDK1 acts as a negative regulator of its enzyme activity.
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Zorreguieta, Angeles, Christine Finnie, and J. Allan Downie. "Extracellular Glycanases of Rhizobium leguminosarum Are Activated on the Cell Surface by an Exopolysaccharide-Related Component." Journal of Bacteriology 182, no. 5 (March 1, 2000): 1304–12. http://dx.doi.org/10.1128/jb.182.5.1304-1312.2000.
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ABSTRACT Rhizobium leguminosarum secretes two extracellular glycanases, PlyA and PlyB, that can degrade exopolysaccharide (EPS) and carboxymethyl cellulose (CMC), which is used as a model substrate of plant cell wall cellulose polymers. When grown on agar medium, CMC degradation occurred only directly below colonies of R. leguminosarum, suggesting that the enzymes remain attached to the bacteria. Unexpectedly, when a PlyA-PlyB-secreting colony was grown in close proximity to mutants unable to produce or secrete PlyA and PlyB, CMC degradation occurred below that part of the mutant colonies closest to the wild type. There was no CMC degradation in the region between the colonies. By growing PlyB-secreting colonies on a lawn of CMC-nondegrading mutants, we could observe a halo of CMC degradation around the colony. Using various mutant strains, we demonstrate that PlyB diffuses beyond the edge of the colony but does not degrade CMC unless it is in contact with the appropriate colony surface. PlyA appears to remain attached to the cells since no such diffusion of PlyA activity was observed. EPS defective mutants could secrete both PlyA and PlyB, but these enzymes were inactive unless they came into contact with an EPS+ strain, indicating that EPS is required for activation of PlyA and PlyB. However, we were unable to activate CMC degradation with a crude EPS fraction, indicating that activation of CMC degradation may require an intermediate in EPS biosynthesis. Transfer of PlyB to Agrobacterium tumefaciens enabled it to degrade CMC, but this was only observed if it was grown on a lawn ofR. leguminosarum. This indicates that the surface ofA. tumefaciens is inappropriate to activate CMC degradation by PlyB. Analysis of CMC degradation by other rhizobia suggests that activation of secreted glycanases by surface components may occur in other species.
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Yamanouchi, Jun, Takaaki Hato, Hiroshi Fujiwara, Yoshihiro Yakushijin, and Masaki Yasukawa. "A Glycan Wedge Between βTD and βI Domains Activates Integrin αIIbβ3,." Blood 118, no. 21 (November 18, 2011): 3259. http://dx.doi.org/10.1182/blood.v118.21.3259.3259.
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Abstract Abstract 3259 Integrin αIIbβ3 undergoes allosteric conformational changes in its extracellular domains, resulting in integrin activation that allows high affinity binding with soluble ligands. The crystal structure of the integrin β subunit revealed an interaction of the β-tail domain (βTD) with the βI domain containing ligand-binding sites, suggesting that βTD may be involved in allosteric mechanism for integrin activation. However, previous studies have shown conflicting results on the functional role of βTD in integrin activation. In this study, we conducted site-directed mutagenesis in the βTD domain and tested ligand binding to αIIbβ3 mutants. We produced αIIbβ3 mutants in which the β3TD loop residues (DSSG) were substituted with the corresponding β1 (NGNN) or β2TD residues (DGMD). The αIIbβ3 mutants were expressed on the surface of CHO cells by cotransfection of mutant β3 and wild-type αIIb cDNAs, and were tested for binding of PAC1, a ligand-mimetic anti-αIIbβ3 antibody. The NGNN, but not DGMD mutant bound significant PAC1 binding without any stimulation, indicating a constitutively active state. To identify the residue(s) responsible for αIIbβ3 activation in the βTD, we produced αIIbβ3 mutants in which the individual residues in the β3TD loop were substituted with the corresponding β1TD residues. Among them, only G675N bound significant PAC1 binding without any stimulation. Since G675N mutation creates a sequence known to be a consensus sequence for glycosylation (Asn-X-Ser/Thr), it is possible that the insertion of glycans into the βTD loop induces conformational changes in αIIbβ3 which allow ligand binding. To test this hypothesis, we added substitution of S677 with Thr, Ala or Asp to the G675N mutation. The resultant G675N/S677T double mutant, in which the N-glycosylation site was preserved, was constitutively active. In contrast, G675N/S677A and G675N/S677D, in which the N-glycosylation site was disrupted, were in an inactive state. These results suggest that an artificial glycan wedge between βTD and βI domains activates αIIbβ3. However, our study does not provide evidence that the βTD domain constrains wild type αIIbβ3 inactive although the separation of βTD and βI domains may be able to activate integrins. Disclosures: No relevant conflicts of interest to declare.
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Moon, Andrea F., Philippe Gaudu, and Lars C. Pedersen. "Structural characterization of the virulence factor nuclease A fromStreptococcus agalactiae." Acta Crystallographica Section D Biological Crystallography 70, no. 11 (October 23, 2014): 2937–49. http://dx.doi.org/10.1107/s1399004714019725.
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The group B pathogenStreptococcus agalactiaecommonly populates the human gut and urogenital tract, and is a major cause of infection-based mortality in neonatal infants and in elderly or immunocompromised adults. Nuclease A (GBS_NucA), a secreted DNA/RNA nuclease, serves as a virulence factor forS. agalactiae, facilitating bacterial evasion of the human innate immune response. GBS_NucA efficiently degrades the DNA matrix component of neutrophil extracellular traps (NETs), which attempt to kill and clear invading bacteria during the early stages of infection. In order to better understand the mechanisms of DNA substrate binding and catalysis of GBS_NucA, the high-resolution structure of a catalytically inactive mutant (H148G) was solved by X-ray crystallography. Several mutants on the surface of GBS_NucA which might influence DNA substrate binding and catalysis were generated and evaluated using an imidazole chemical rescue technique. While several of these mutants severely inhibited nuclease activity, two mutants (K146R and Q183A) exhibited significantly increased activity. These structural and biochemical studies have greatly increased our understanding of the mechanism of action of GBS_NucA in bacterial virulence and may serve as a foundation for the structure-based drug design of antibacterial compounds targeted toS. agalactiae.
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Ferjoux, Geraldine, Frederic Lopez, Jean-Pierre Esteve, Audrey Ferrand, Eric Vivier, Frederic Vely, Nathalie Saint-Laurent, Lucien Pradayrol, Louis Buscail, and Christiane Susini. "Critical Role of Src and SHP-2 in sst2 Somatostatin Receptor-mediated Activation of SHP-1 and Inhibition of Cell Proliferation." Molecular Biology of the Cell 14, no. 9 (September 2003): 3911–28. http://dx.doi.org/10.1091/mbc.e03-02-0069.
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The G protein-coupled sst2 somatostatin receptor acts as a negative cell growth regulator. Sst2 transmits antimitogenic signaling by recruiting and activating the tyrosine phosphatase SHP-1. We now identified Src and SHP-2 as sst2-associated molecules and demonstrated their role in sst2 signaling. Surface plasmon resonance and mutation analyses revealed that SHP-2 directly associated with phosphorylated tyrosine 228 and 312, which are located in sst2 ITIMs (immunoreceptor tyrosine-based inhibitory motifs). This interaction was required for somatostatin-induced SHP-1 recruitment and activation and consequent inhibition of cell proliferation. Src interacted with sst2 and somatostatin promoted a transient Gβγ-dependent Src activation concomitant with sst2 tyrosine hyperphosphorylation and SHP-2 activation. These steps were abrogated with catalytically inactive Src. Both catalytically inactive Src and SHP-2 mutants abolished somatostatin-induced SHP-1 activation and cell growth inhibition. Sst2–Src–SHP-2 complex formation was dynamic. Somatostatin further induced sst2 tyrosine dephosphorylation and complex dissociation accompanied by Src and SHP-2 inhibition. These steps were defective in cells expressing a catalytically inactive Src mutant. All these data suggest that Src acts upstream of SHP-2 in sst2 signaling and provide evidence for a functional role for Src and SHP-2 downstream of an inhibitory G protein-coupled receptor.
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Ebanks, R. O., and D. E. Isenman. "Evidence for the involvement of arginine 462 and the flanking sequence of human C4 beta-chain in mediating C5 binding to the C4b subcomponent of the classical complement pathway C5 convertase." Journal of Immunology 154, no. 6 (March 15, 1995): 2808–20. http://dx.doi.org/10.4049/jimmunol.154.6.2808.
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Abstract Replacement of human C4 beta-chain residue arginine 458 by tryptophan, a substitution that occurs naturally in the hemolytically inactive A6 allotype of C4, totally abrogates the molecule's ability to act as a C5 binding subunit of the classical pathway C5 convertase. Hydropathy plots predict R458 to be within a hydrophilic segment extending from residue 455 to 469 and having the sequence SIERPDSRPPRVGDT. To further assess the potential involvement of this segment in the C5 binding function of C4, we have engineered "ala-scan" mutants through this segment, concentrating predominantly on charged residues, and analyzed their functional profiles. C4B isotype mutant proteins S455A (0.7), E457A (1.1), R458A (0.3), D460A (0.2), R462A (0.0), R465A (0.6), and D468A (0.3) displayed the relative to wild-type hemolytic activities indicated in the parentheses. In all cases, the hemolytic defect was accounted for solely at the C5 convertase stage. The total absence of C5 binding activity in the R462A mutant suggests a requirement for the guanidinium group per se, because mutants with a charge-conservative lysine or a relatively isosteric methionine at this position were also completely inactive. In contrast, the inactivity of the C4A6-like R458W mutant is probably caused by the intolerance of tryptophan in a hydrophilic segment, as substitution of R458 by alanine or methionine yielded recombinant molecules that retained 30% and 60% of wild-type hemolytic activity, respectively. Taken together, the mutagenesis results strongly imply that residues in the 455-469 segment contribute to the C5 binding site in C4; however, the conformational context of the segment appears to be crucial, as a synthetic peptide corresponding to this segment displayed no ability to inhibit C5 binding to surface-bound C4b.
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Ohradanova, Anna, Daniela Vullo, Juraj Kopacek, Claudia Temperini, Tatiana Betakova, Silvia Pastorekova, Jaromir Pastorek, and Claudiu T. Supuran. "Reconstitution of carbonic anhydrase activity of the cell-surface-binding protein of vaccinia virus." Biochemical Journal 407, no. 1 (September 12, 2007): 61–67. http://dx.doi.org/10.1042/bj20070816.
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The N-terminal region of a 32 kDa cell-surface-binding protein, encoded by the D8L gene of vaccinia virus, shows sequence homology to CAs (carbonic anhydrases; EC 4.2.1.1). The active CAs catalyse the reversible hydration of CO2 to bicarbonate participating in many physiological processes. The CA-like domain of vaccinia protein [vaccCA (vaccinia virus CA-like protein)] contains one of the three conserved histidine residues required for co-ordination to the catalytic zinc ion and for enzyme activity. In the present study, we report the engineering of catalytically active vaccCA mutants by introduction of the missing histidine residues into the wild-type protein. The wild-type vaccCA was inactive as a catalyst and does not bind sulfonamide CA inhibitors. Its position on a phylogram with other hCAs (human CAs) shows a relationship with the acatalytic isoforms CA X and XI, suggesting that the corresponding viral gene was acquired from the human genome by horizontal gene transfer. The single mutants (vaccCA N92H/Y69H) showed low enzyme activity and low affinity for acetazolamide, a classical sulfonamide CA inhibitor. The activity of the double mutant, vaccCA N92H/Y69H, was much higher, of the same order of magnitude as that of some human isoforms, namely CA VA and CA XII. Moreover, its affinity for acetazolamide was high, comparable with that of the most efficient human isoenzyme, CA II (in the low nanomolar range). Multiplication of vaccinia virus in HeLa cells transfected with the vaccCA N92H/Y69H double mutant was approx. 2-fold more efficient than in wild-type vaccCA transfectants, suggesting that the reconstitution of the enzyme activity improved the virus life cycle.
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Wright, J. H., P. Drueckes, J. Bartoe, Z. Zhao, S. H. Shen, and E. G. Krebs. "A role for the SHP-2 tyrosine phosphatase in nerve growth-induced PC12 cell differentiation." Molecular Biology of the Cell 8, no. 8 (August 1997): 1575–85. http://dx.doi.org/10.1091/mbc.8.8.1575.
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SHP-1 and SHP-2 are intracellular protein tyrosine phosphatases containing two adjacent src homology 2 domains that target these phosphatases to cell surface receptor signaling complexes and play a role in receptor signal transduction. In this report the PC12 cell system was used to investigate the potential roles of SHP-1 and SHP-2 in the induction of neuronal differentiation by nerve growth factor (NGF). By using neurite outgrowth as a marker for differentiation, the effects of transfected constructs of SHP-1 and SHP-2 were assessed. Overexpression of a catalytically inactive SHP-2, but not a catalytically inactive SHP-1, blocked NGF-stimulated neurite outgrowth. The mitogen-activated protein kinase (MAPK) signaling cascade is important for the morphological differentiation in PC12 cells, and both SHP-1 and SHP-2 have been implicated to act upstream of MAPK in other receptor signaling systems. A positive role for SHP-2 but not SHP-1 in the activation of MAPK by NGF was demonstrated by introduction of the SHP-2 phosphatase mutants along with hemagglutinin-tagged MAPK. Coexpression studies with the SHP-2 mutant along with mutant forms of MAPK kinase suggested that SHP-2 functions upstream of MAPK kinase and MAPK in NGF-induced neurite outgrowth.
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Yamanouchi, Jun, Takaaki Hato, Yoshihiro Yakushijin, Ikuya Sakai, and Masaki Yasukawa. "Identification of Critical Residues for Affinity Regulation of β3 Integrins in the β6-α7 Loop of the I-Like Domain." Blood 108, no. 11 (November 16, 2006): 1526. http://dx.doi.org/10.1182/blood.v108.11.1526.1526.
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Abstract Integrin αIIbβ3 exists in a low-affinity state and requires activation for high-affinity binding with soluble ligands. αIIbβ3 activation is linked to rearrangements of the β3 I-like domain structure and the largest movement in the β3 I-like domain following ligand binding occurs in the β6-α7 loop. Although this loop does not comprise a ligand binding site, a recent mutational study has shown that introduction of disulfide bonds into the β6/α7 region to lock the β3 I-like domain in the open or closed conformation renders αIIbβ3 constitutively active or inactive, suggesting the regulatory role of this region in integrin activation. However, it remains to be determined which residues of the β6-α7 loop in the β I-like domain are critical for integrin activation. We therefore conducted alanine-scanning mutagenesis of the β6-α7 loop residues in the β3 I-like domain and tested for ligand binding to mutant β3 integrins. The β6-α7 loop is composed of residues from S334 to N339, and the region between L333 and V340 was targeted for mutagenesis. The mutant β3 cDNA was transfected into CHO cells together with wild-type αIIb cDNA. The expression of αIIbβ3 mutants on the cell surface was 56–116% that of the wild-type αIIbβ3. Then binding of an activation-dependent antibody. PAC1, to the αIIbβ3 mutants was examined in the presence or absence of the αIIbβ3-activating antibody PT25-2. As expected, wild-type αIIbβ3 showed PAC1 binding only after activation of αIIbβ3 by PT25-2. The L333A, S334A, M335A and V340A mutations had no effect on PAC1 binding. In contrast, the S337A and N339A mutations induced significant PAC1 binding in the absence of PT25-2, indicating a constitutively active state. Although the D336A and S338A mutations retained αIIbβ3 in an inactive state in the absence of PT25-2, they induced 3- and 4.5-fold as much PAC1 binding as wild-type αIIbβ3 in response to PT25-2, respectively. The S337A and N339A mutations, which rendered αIIbβ3 constitutively active, induced further PAC1 binding in the presence of PT25-2 (2.7~5.1-fold as much PAC1 binding as wild-type αIIbβ3). When soluble fibrinogen was used instead of PAC1, similar results were obtained. None of the mutations tested had any effect on PT25-2 binding. We next quantified adhesion of CHO cells stably expressing mutant or wild-type αIIbβ3 to immobilized fibrinogen. The S337A and N339A mutations enhanced cell adhesion to fibrinogen and the extent of adhesion of the mutants was comparable to that of wild-type αIIbβ3 activated by Mn2+. To determine whether the mutations of the β3 subunit responsible for αIIbβ3 activation also induce constitutive activation of αVβ3, CHO cells expressing αVβ3 mutants were tested for soluble fibrinogen binding. The surface expression of mutant αVβ3 was comparable to that of wild-type αVβ3. The αVβ3S337A and N339A mutants bound significantly more fibrinogen than wild-type αVβ3 without any stimulation, indicating that the S337A and N339A mutations also rendered another β3 integrin, αVβ3, constitutively active. These results suggest that the S337 and N339 residues in the β3 I-like domain are essential for constraining β3 integrins in a default low-affinity state and that structural rearrangement in the 336DSSN sequence of the β6-α7 loop alters the affinity state of αIIbβ3. Since the DSSN sequence is highly conserved among integrin β subunits, this motif may be a common regulatory component of integrin activation.
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Schwake, Michael, Thomas Friedrich, and Thomas J. Jentsch. "An Internalization Signal in ClC-5, an Endosomal Cl−Channel Mutated in Dent's Disease." Journal of Biological Chemistry 276, no. 15 (December 14, 2000): 12049–54. http://dx.doi.org/10.1074/jbc.m010642200.
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The ClC-5 chloride channel resides mainly in vesicles of the endocytotic pathway and contributes to their acidification. Its disruption in mice entails a broad defect in renal endocytosis and causes secondary changes in calciotropic hormone levels. Inactivating mutations in Dent's disease lead to proteinuria and kidney stones. Possibly by recycling, a small fraction of ClC-5 also reaches the plasma membrane. Here we identify a carboxyl-terminal internalization motif in ClC-5. It resembles the PY motif, which is crucial for the endocytosis and degradation of epithelial Na+channels. Mutating this motif increases surface expression and currents about 2-fold. This is probably because of interactions with WW domains, because dominant negative mutants of the ubiquitin-protein ligase WWP2 increased surface expression and currents of ClC-5 only when its PY motif was intact. Stimulating endocytosis by expressing rab5 or its GTPase-deficient Q79L mutant decreased WT ClC-5 currents but did not affect channels with mutated motifs. Similarly, decreasing endocytosis by expressing the inactive S34N mutant of rab5 increased ClC-5 currents only if its PY-like motif was intact. Thus, the endocytosis of ClC-5, which itself is crucial for the endocytosis of other proteins, depends on the interaction of a carboxyl-terminal internalization signal with ubiquitin-protein ligases containing WW domains.
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Fang, Eric G. C., and Ralph A. Dean. "Site-Directed Mutagenesis of the magB Gene Affects Growth and Development in Magnaporthe grisea." Molecular Plant-Microbe Interactions® 13, no. 11 (November 2000): 1214–27. http://dx.doi.org/10.1094/mpmi.2000.13.11.1214.
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G protein signaling is commonly involved in regulating growth and differentiation of eukaryotic cells. We previously identified MAGB, encoding a Gα subunit, from Magnaporthe grisea, and disruption of MAGB led to defects in a number of cellular responses, including appres-sorium formation, conidiation, sexual development, mycelial growth, and surface sensing. In this study, site-directed mutagenesis was used to further dissect the pleiotropic effects controlled by MAGB. Conversion of glycine 42 to arginine was predicted to abolish GTPase activity, which in turn would constitutively activate G protein signaling in magBG42R. This dominant mutation caused autolysis of aged colonies, misscheduled melanization, reduction in both sexual and asexual reproduction, and reduced virulence. Furthermore, magBG42R mutants were able to produce appressoria on both hydrophobic and hydrophilic surfaces, although development on the hydrophilic surface was delayed. A second dominant mutation, magBG203R (glycine 203 converted to arginine), was expected to block dissociation of the Gβγ from the Gα subunit, thus producing a constitutively inactive G protein complex. This mutation did not cause drastic phenotypic changes in the wild-type genetic background, other than increased sensitivity to repression of conidiation by osmotic stress. However, magBG203R is able to complement phenotypic defects in magB mutants. Comparative analyses of the phenotypical effects of different magB mutations are consistent with the involvement of the Gβγ subunit in the signaling pathways regulating cellular development in M. grisea.
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Walters, Jad, Joshua L. Schipper, Paul Swartz, Carla Mattos, and A. Clay Clark. "Allosteric modulation of caspase 3 through mutagenesis." Bioscience Reports 32, no. 4 (June 28, 2012): 401–11. http://dx.doi.org/10.1042/bsr20120037.
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A mutation in the allosteric site of the caspase 3 dimer interface of Val266 to histidine abolishes activity of the enzyme, and models predict that the mutation mimics the action of small molecule allosteric inhibitors by preventing formation of the active site. Mutations were coupled to His266 at two sites in the interface, E124A and Y197C. We present results from X-ray crystallography, enzymatic activity and molecular dynamics simulations for seven proteins, consisting of single, double and triple mutants. The results demonstrate that considering allosteric inhibition of caspase 3 as a shift between discrete ‘off-state’ or ‘on-state’ conformations is insufficient. Although His266 is accommodated in the interface, the structural defects are propagated to the active site through a helix on the protein surface. A more comprehensive view of allosteric regulation of caspase 3 requires the representation of an ensemble of inactive states and shows that subtle structural changes lead to the population of the inactive ensemble.
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De la Vieja, Antonio, Christopher S. Ginter, and Nancy Carrasco. "Molecular Analysis of a Congenital Iodide Transport Defect: G543E Impairs Maturation and Trafficking of the Na+/I− Symporter." Molecular Endocrinology 19, no. 11 (November 1, 2005): 2847–58. http://dx.doi.org/10.1210/me.2005-0162.
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Abstract The Na+/I− symporter (NIS) is a key membrane glycoprotein that mediates active I− transport in the thyroid and other tissues. Upon isolation of the cDNA encoding NIS, 10 NIS mutations that cause congenital iodide transport defect have been identified. Three of these mutations (T354P, G395R, and Q267E) have been thoroughly characterized at the molecular level. All three NIS mutant proteins are correctly targeted to the plasma membrane; however, whereas Q267E displays minimal activity, T354P and G395R are inactive. Here, we show that in contrast to these mutants, G543E NIS matures only partially and is retained intracellularly; thus, it is not targeted properly to the cell surface, apparently because of faulty folding. These findings indicate that the G543 residue plays significant roles in NIS maturation and trafficking. Remarkably, NIS activity was rescued by small neutral amino acid substitutions (volume < 129 Å3) at this position, suggesting that G543 is in a tightly packed region of NIS.
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Kittanakom, Saranya, Emmanuelle Cordat, and Reinhart A. F. Reithmeier. "Dominant-negative effect of Southeast Asian ovalocytosis anion exchanger 1 in compound heterozygous distal renal tubular acidosis." Biochemical Journal 410, no. 2 (February 12, 2008): 271–81. http://dx.doi.org/10.1042/bj20070615.
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The human chloride/bicarbonate AE1 (anion exchanger) is a dimeric glycoprotein expressed in the red blood cell membrane, and expressed as an N-terminal (Δ1–65) truncated form, kAE1 (kidney AE1), in the basolateral membrane of α-intercalated cells in the distal nephron. Mutations in AE1 can cause SAO (Southeast Asian ovalocytosis) or dRTA (distal renal tubular acidosis), an inherited kidney disease resulting in impaired acid secretion. The dominant SAO mutation (Δ400–408) that results in an inactive transporter and altered eythrocyte shape occurs in many dRTA families, but does not itself result in dRTA. Compound heterozygotes of four dRTA mutations (R602H, G701D, ΔV850 and A858D) with SAO exhibit dRTA and abnormal red blood cell properties. Co-expression of kAE1 and kAE1 SAO with the dRTA mutants was studied in polarized epithelial MDCK (Madin–Darby canine kidney) cells. Like SAO, the G701D and ΔV850 mutants were predominantly retained intracellularly, whereas the R602H and A858D mutants could traffic to the basolateral membrane. When co-expressed in transfected cells, kAE1 WT (wild-type) and kAE1 SAO could interact with the dRTA mutants. MDCK cells co-expressing kAE1 SAO with kAE1 WT, kAE1 R602H or kAE1 A858D showed a decrease in cell-surface expression of the co-expressed proteins. When co-expressed, kAE1 WT co-localized with the kAE1 R602H, kAE1 G701D, kAE1 ΔV850 and kAE1 A858D mutants at the basolateral membrane, whereas kAE1 SAO co-localized with kAE1 WT, kAE1 R602H, kAE1 G701D, kAE1 ΔV850 and kAE1 A858D in MDCK cells. The decrease in cell-surface expression of the dRTA mutants as a result of the interaction with kAE1 SAO would account for the impaired expression of functional kAE1 at the basolateral membrane of α-intercalated cells, resulting in dRTA in compound heterozygous patients.
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Ing, Bryant, Alina Shteiman-Kotler, MaryLisa Castelli, Pauline Henry, Youngshil Pak, Bryan Stewart, Gabrielle L. Boulianne, and Daniela Rotin. "Regulation of Commissureless by the Ubiquitin Ligase DNedd4 Is Required for Neuromuscular Synaptogenesis in Drosophila melanogaster." Molecular and Cellular Biology 27, no. 2 (October 30, 2006): 481–96. http://dx.doi.org/10.1128/mcb.00463-06.
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ABSTRACT Muscle synaptogenesis in Drosophila melanogaster requires endocytosis of Commissureless (Comm), a binding partner for the ubiquitin ligase dNedd4. We investigated whether dNedd4 and ubiquitination mediate this process. Here we show that Comm is expressed in intracellular vesicles in the muscle, whereas Comm bearing mutations in the two PY motifs (L/PPXY) responsible for dNedd4 binding [Comm(2PY→AY)], or bearing Lys→Arg mutations in all Lys residues that serve as ubiquitin acceptor sites [Comm(10K→R)], localize to the muscle surface, suggesting they cannot endocytose. Accordingly, aberrant muscle innervation is observed in the Comm(2PY→AY) and Comm(10K→R) mutants expressed early in muscle development. Similar muscle surface accumulation of Comm and innervation defects are observed when dNedd4 is knocked down by double-stranded RNA interference in the muscle, in dNedd4 heterozygote larvae, or in muscles overexpressing catalytically inactive dNedd4. Expression of the Comm mutants fused to a single ubiquitin that cannot be polyubiquitinated and mimics monoubiquitination [Comm(2PY→AY)-monoUb or Comm(10K→R)-monoUb] prevents the defects in both Comm endocytosis and synaptogenesis, suggesting that monoubiquitination is sufficient for Comm endocytosis in muscles. Expression of the Comm mutants later in muscle development, after synaptic innervation, has no effect. These results demonstrate that dNedd4 and ubiquitination are required for Commissureless endocytosis and proper neuromuscular synaptogenesis.
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Strittmatter, S. M., D. Valenzuela, and M. C. Fishman. "An amino-terminal domain of the growth-associated protein GAP-43 mediates its effects on filopodial formation and cell spreading." Journal of Cell Science 107, no. 1 (January 1, 1994): 195–204. http://dx.doi.org/10.1242/jcs.107.1.195.
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GAP-43 is a neuronal protein that is believed to be important to neuronal growth and nerve terminal plasticity. It is enriched on the inner surface of growth cone membranes, a localization that may depend upon palmitoylation of Cys3 and Cys4. It is a major substrate for protein kinase C, which phosphorylates Ser41. Isolated GAP-43 can bind to actin and to calmodulin, and can activate the heterotrimeric GTP-binding proteins, G(o) and Gi. A peptide consisting of the GAP-43 sequence 39–55 binds calmodulin, and an amino-terminal GAP-43 (1–10) peptide activates G(o), suggesting that these stretches may be functional domains of the intact protein. When expressed in non-neuronal cells, GAP-43 enhances filopodial extension and has effects upon cell spreading. We have examined the effects of various GAP-43 domains upon this assay, by expression of GAP-43, GAP-43 mutant proteins, and GAP-43-CAT fusion proteins in COS-7 cells. We find that the amino terminus (Met-Leu-Cys-Cys-Met-Arg-Arg-Thr-Lys-Gln) is an important contributor to these effects on cell shape. A GAP-43 protein mutant in Cys3 and Cys4 does not bind to the membrane, and is inactive. Mutants in Arg6 or Lys9 also are inactive, although they remain localized to particulate fractions; Arg7 mutants are active. A chimeric gene consisting of GAP-43 (1–10) fused to chloramphenicol acetyl transferase (CAT) also causes cell shape changes. As for GAP-43, the effects of this fusion protein are abolished by mutations of Cys3, Cys4, Arg6 or Lys9, but not by mutation of Arg7. Therefore, the cell surface activity of transfected GAP-43 depends upon its amino terminus, although other domains may regulate it in this regard. Since the amino-terminal domain includes the peptide stretch known to be capable of activating G(o) and Gi, we examined the effect of GAP-43 on a Gi-regulated second messenger system, the inhibition of cAMP production in A431 cells. A431 cells stably transfected with GAP-43 spread less well than do controls. In addition, they evidence decreased levels of forskolin-stimulated cAMP, consistent with chronic stimulation of Gi. Stimulation of adenylate cyclase by isoproterenol reverses the GAP-43-induced changes in cell shape. This suggests that G protein stimulation is involved in GAP-43 effects upon cell shape.
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Lavillette, Dimitri, and David Kabat. "Porcine Endogenous Retroviruses Infect Cells Lacking Cognate Receptors by an Alternative Pathway: Implications for Retrovirus Evolution and Xenotransplantation." Journal of Virology 78, no. 16 (August 15, 2004): 8868–77. http://dx.doi.org/10.1128/jvi.78.16.8868-8877.2004.
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ABSTRACT A PHQ motif near the amino termini of gammaretroviral envelope glycoprotein surface (SU) subunits is important for infectivity but not for incorporation into virions or binding to cognate receptors. The H residue of this motif is most critical, with all substitutions we tested being inactive. Interestingly, porcine endogenous retroviruses (PERVs) of all three host-range groups, A, B, and C, lack full PHQ motifs, but most members have an H residue at position 10. H10A PERV mutants are noninfectious but were efficiently transactivated by adding to the assays a PHQ-containing SU or receptor-binding subdomain (RBD) derived from a gibbon ape leukemia virus (GALV). A requirement of this transactivation was a functional GALV receptor on the cells. In contrast to this heterologous transactivation, PERV RBDs and SUs were inactive in all tested cells, including porcine ST-IOWA cells. Surprisingly, transactivation by GALV RBD enabled wild-type or H10A mutant PERVs of all three host-range groups to efficiently infect cells from humans and rodents that lack functional PERV receptors and it substantially enhanced infectivities of wild-type PERVs, even for cells with PERV receptors. Thus, PERVs can suboptimally infect cells that contain cognate receptors or they can employ a transactivation pathway to more efficiently infect all cells. This ability to infect cells lacking cognate receptors was previously demonstrated only for nontransmissible variant gammaretroviruses with recombinant and mutant envelope glycoproteins. We conclude that some endogenously inherited mammalian retroviruses also have a receptor-independent means for overcoming host-range and interference barriers, implying a need for caution in xenotransplantation, especially of porcine tissues.
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Hands, Sarah L., Lisa E. Holland, Mireille Vankemmelbeke, Lauren Fraser, Colin J. Macdonald, Geoffrey R. Moore, Richard James, and Christopher N. Penfold. "Interactions of TolB with the Translocation Domain of Colicin E9 Require an Extended TolB Box." Journal of Bacteriology 187, no. 19 (October 1, 2005): 6733–41. http://dx.doi.org/10.1128/jb.187.19.6733-6741.2005.
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ABSTRACT The mechanism by which enzymatic E colicins such as colicin E3 (ColE3) and ColE9 cross the outer membrane, periplasm, and cytoplasmic membrane to reach the cytoplasm and thus kill Escherichia coli cells is unique in prokaryotic biology but is poorly understood. This requires an interaction between TolB in the periplasm and three essential residues, D35, S37, and W39, of a pentapeptide sequence called the TolB box located in the N-terminal translocation domain of the enzymatic E colicins. Here we used site-directed mutagenesis to demonstrate that the TolB box sequence in ColE9 is actually larger than the pentapeptide and extends from residues 34 to 46. The affinity of the TolB box mutants for TolB was determined by surface plasmon resonance to confirm that the loss of biological activity in all except one (N44A) of the extended TolB box mutants correlates with a reduced affinity of binding to TolB. We used a PCR mutagenesis protocol to isolate residues that restored activity to the inactive ColE9 D35A, S37A, and W39A mutants. A serine residue at position 35, a threonine residue at position 37, and phenylalanine or tyrosine residues at position 39 restored biological activity of the mutant ColE9. The average area predicted to be buried upon folding (AABUF) was correlated with the activity of the variants at positions 35, 37, and 39 of the TolB box. All active variants had AABUF profiles that were similar to the wild-type residues at those positions and provided information on the size, stereochemistry, and potential folding pattern of the residues of the TolB Box.
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Briken, V., D. Lankar, and C. Bonnerot. "New evidence for two MHC class II-restricted antigen presentation pathways by overexpression of a small G protein." Journal of Immunology 159, no. 10 (November 15, 1997): 4653–58. http://dx.doi.org/10.4049/jimmunol.159.10.4653.
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Abstract Exogenous Ags may be presented by MHC class II molecules through two distinct pathways distinguished by their sensitivity to drugs that inhibit the protein synthesis. Using this approach, we previously showed that the subunits Ig-alpha and Ig-beta, associated to B cell Ag receptor, targeted Ags either to newly synthesized or to preexisting pools of MHC class II molecules, respectively. To further characterize these two Ag presentation pathways, we altered the intra-Golgi transport of newly synthesized MHC class II by stably overexpressing, in B cells, mutants of a small G protein involved in the intra-Golgi transport, Rab6. Overexpression of GTP-bound rab6 (Q72L) mutant proteins reduced the cell surface arrival of MHC class II molecules and consequently slowed down Ag presentation dependent upon newly synthesized class II molecules. In contrast, this mutant had no effect on Ag presentation dependent upon preexisting pools of class II molecules, and the overexpression of an inactive GDP-bound form of rab6 (T27N) did not affect any Ag presentation pathway. MHC class II-restricted Ag presentation pathways can therefore be distinguished by their sensitivity to the overexpression of proteins modifying the intracellular transport of newly synthesized class II molecules.
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Enz, Sabine, Heidi Brand, Claudia Orellana, Susanne Mahren, and Volkmar Braun. "Sites of Interaction between the FecA and FecR Signal Transduction Proteins of Ferric Citrate Transport in Escherichia coli K-12." Journal of Bacteriology 185, no. 13 (July 1, 2003): 3745–52. http://dx.doi.org/10.1128/jb.185.13.3745-3752.2003.
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ABSTRACT Transcription of the fecABCDE ferric citrate transport genes of Escherichia coli K-12 is initiated by a signaling cascade from the cell surface into the cytoplasm. FecR receives the signal in the periplasm from the outer membrane protein FecA loaded with ferric citrate, transmits the signal across the cytoplasmic membrane, and converts FecI in the cytoplasm to an active sigma factor. In this study, it was shown through the use of a bacterial two-hybrid system that, in the periplasm, the C-terminal FecR237-317 fragment interacts with the N-terminal FecA1-79 fragment. In the same C-terminal region, amino acid residues important for the interaction of FecR with FecA were identified by random and site-directed mutagenesis. They were preferentially located in and around a leucine motif (residues 247 to 268) which was found to be highly conserved in FecR-like proteins. The degree of residual binding of FecR mutant proteins to FecA was correlated with the degree of transcription initiation in response to ferric citrate in the culture medium. Three randomly generated inactive FecR mutants, FecR(L254E), FecR(L269G), and FecR(F284L), were suppressed to different degrees by the mutants FecA(G39R) and FecR(D43E). One FecR mutant, FecR (D138E, V197A), induced fecA promoter-directed transcription constitutively in the absence of ferric citrate and bound more strongly than wild-type FecR to FecA. The data showed that FecR interacts in the periplasm with FecA to confer ferric citrate-induced transcription of the fec transport genes and identified sites in FecR and FecA that are important for signal transduction.
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Qin, Haixia, Michael A. Frohman, and Wendy B. Bollag. "Phospholipase D2 Mediates Acute Aldosterone Secretion in Response to Angiotensin II in Adrenal Glomerulosa Cells." Endocrinology 151, no. 5 (March 10, 2010): 2162–70. http://dx.doi.org/10.1210/en.2009-1159.
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In primary bovine adrenal glomerulosa cells, the signaling enzyme phospholipase D (PLD) is suggested to mediate priming, the enhancement of aldosterone secretion after pretreatment with and removal of angiotensin II (AngII), via the formation of persistently elevated diacylglycerol (DAG). To further explore PLD’s role in priming, glomerulosa cells were pretreated with an exogenous bacterial PLD. Using this approach, phosphatidic acid (PA) is generated on the outer, rather than the inner, leaflet of the plasma membrane. Although PA is not readily internalized, the PA is nonetheless rapidly hydrolyzed by cell-surface PA phosphatases to DAG, which efficiently flips to the inner leaflet and accesses the cell interior. Pretreatment with bacterial PLD resulted in priming upon subsequent AngII exposure, supporting a role of DAG in this process, because the increase in DAG persisted after exogenous PLD removal. To determine the PLD isoform mediating aldosterone secretion, and presumably priming, primary glomerulosa cells were infected with adenoviruses expressing GFP, PLD1, PLD2, or lipase-inactive mutants. Overexpressed PLD2 increased aldosterone secretion by approximately 3-fold over the GFP-infected control under basal conditions, with a significant enhancement to about 16-fold over the basal value upon AngII stimulation. PLD activity was also increased basally and upon stimulation with AngII. In contrast, PLD1 overexpression had little effect on aldosterone secretion, despite the fact that PLD activity was enhanced. In both cases, the lipase-inactive PLD mutants showed essentially no effect on PLD activity or aldosterone secretion. Our results suggest that PLD2 is the isoform that mediates aldosterone secretion and likely priming.
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Shetty, Sreerama, Malathesha Ganachari, Ming-Cheh Liu, Ali Azghani, Harish Muniyappa, and Steven Idell. "Regulation of urokinase receptor expression by phosphoglycerate kinase is independent of its catalytic activity." American Journal of Physiology-Lung Cellular and Molecular Physiology 289, no. 4 (October 2005): L591—L598. http://dx.doi.org/10.1152/ajplung.00319.2004.
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Posttranscriptional regulation of urokinase-type plasminogen activator receptor (uPAR) mRNA involves the interaction of a uPAR mRNA coding region sequence with phosphoglycerate kinase (PGK), a 50-kDa uPAR mRNA binding protein. PGK catalyzes a reversible transfer of a phosphoryl group from 1,3-biphosphoglycerate to ADP in the glycolytic pathway. Our previous studies showed that overexpression of PGK in uPAR-overproducing H157 lung carcinoma cells results in decreased cytoplasmic uPAR mRNA and cell surface uPAR protein expression through destabilization of the mRNA. In order to determine the role of PGK enzymatic activity on uPAR mRNA stability we mutated PGK by changing amino acid P204H and amino acid D219A. The mutant proteins were expressed in Epicurian coli BL21 cells, and the purified proteins were analyzed for PGK activity. We found that mutation of amino acid P204H and D219A reduced PGK activity by 99 and 83%, respectively. By gel mobility shift and Northwestern assay, we found that the mutant proteins were able to bind to uPAR mRNA as effectively as wild-type PGK. Overexpression of mutant, inactive PGK in H157 cells reduced cell surface uPAR protein as well as uPAR mRNA expression. Run-on transcription analysis indicated that overexpression of mutant PGKs fails to alter the rate of synthesis of uPAR mRNA, whereas transcription chase experiments demonstrated that both mutants and wild-type PGK reduce the stability of the uPAR mRNA transcripts to a similar extent. Overexpression of mutant PGK also inhibited the rate of DNA synthesis and the invasion-migration ratio. These results demonstrate that uPAR mRNA binding activity as well as PGK-mediated regulation of uPAR mRNA are independent of PGK enzymatic activity.
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Cain, Natalie E., and Chris A. Kaiser. "Transport activity–dependent intracellular sorting of the yeast general amino acid permease." Molecular Biology of the Cell 22, no. 11 (June 2011): 1919–29. http://dx.doi.org/10.1091/mbc.e10-10-0800.
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Intracellular trafficking of the general amino acid permease, Gap1p, of Saccharomyces cerevisiae is regulated by amino acid abundance. When amino acids are scarce Gap1p is sorted to the plasma membrane, whereas when amino acids are abundant Gap1p is sorted from the trans-Golgi through the multivesicular endosome (MVE) and to the vacuole. Here we test the hypothesis that Gap1p itself is the sensor of amino acid abundance by examining the trafficking of Gap1p mutants with altered substrate specificity and transport activity. We show that trafficking of mutant Gap1pA297V, which does not transport basic amino acids, is also not regulated by these amino acids. Furthermore, we have identified a catalytically inactive mutant that does not respond to complex amino acid mixtures and constitutively sorts Gap1p to the plasma membrane. Previously we showed that amino acids govern the propensity of Gap1p to recycle from the MVE to the plasma membrane. Here we propose that in the presence of substrate the steady-state conformation of Gap1p shifts to a state that is unable to be recycled from the MVE. These results indicate a parsimonious regulatory mechanism by which Gap1p senses its transport substrates to set an appropriate level of transporter activity at the cell surface.
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McCallum, Nadine, Markus Bischoff, Hideki Maki, Akihito Wada, and Brigitte Berger-Bächi. "TcaR, a Putative MarR-Like Regulator of sarS Expression." Journal of Bacteriology 186, no. 10 (May 15, 2004): 2966–72. http://dx.doi.org/10.1128/jb.186.10.2966-2972.2004.
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ABSTRACT TcaR, which shares sequence homology with MarR-like transcriptional regulators, has been identified as a novel Staphylococcus aureus regulator affecting the expression of the global regulatory element SarS (SarH1), as well as that of the cell surface-associated protein SasF (N315-SA2439). Microarray analysis, confirmatory Northern blots, and genetic complementation experiments showed that TcaR upregulates sarS and thus spa transcription. In addition, it attenuates whole-length transcription of sasF, thereby producing a truncated transcript lacking the 3′ terminus, which codes for the cell wall anchor motif. Hence, in strains containing an intact tcaR gene, TcaR is likely to decrease the amount of the surface-associated protein SasF and to increase that of the surface-associated protein A. The widely used laboratory strains derived from NCTC8325 were found to be natural, truncated mutants of tcaR, harboring an inactive TcaR and therefore expressing very low levels of sarS. The data presented here identified TcaR as a further activator of sarS, and a modulator of sasF expression that has to be taken into account in studies of virulence gene expression in S. aureus.
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Burns, Michael L., Thomas M. Malott, Kevin J. Metcalf, Benjamin J. Hackel, Jonah R. Chan, and Eric V. Shusta. "Directed Evolution of Brain-Derived Neurotrophic Factor for Improved Folding and Expression in Saccharomyces cerevisiae." Applied and Environmental Microbiology 80, no. 18 (July 11, 2014): 5732–42. http://dx.doi.org/10.1128/aem.01466-14.
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ABSTRACTBrain-derived neurotrophic factor (BDNF) plays an important role in nervous system function and has therapeutic potential. Microbial production of BDNF has resulted in a low-fidelity protein product, often in the form of large, insoluble aggregates incapable of binding to cognate TrkB or p75 receptors. In this study, employingSaccharomyces cerevisiaedisplay and secretion systems, it was found that BDNF was poorly expressed and partially inactive on the yeast surface and that BDNF was secreted at low levels in the form of disulfide-bonded aggregates. Thus, for the purpose of increasing the compatibility of yeast as an expression host for BDNF, directed-evolution approaches were employed to improve BDNF folding and expression levels. Yeast surface display was combined with two rounds of directed evolution employing random mutagenesis and shuffling to identify BDNF mutants that had 5-fold improvements in expression, 4-fold increases in specific TrkB binding activity, and restored p75 binding activity, both as displayed proteins and as secreted proteins. Secreted BDNF mutants were found largely in the form of soluble homodimers that could stimulate TrkB phosphorylation in transfected PC12 cells. Site-directed mutagenesis studies indicated that a particularly important mutational class involved the introduction of cysteines proximal to the native cysteines that participate in the BDNF cysteine knot architecture. Taken together, these findings show that yeast is now a viable alternative for both the production and the engineering of BDNF.
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Yu, Xiaochun, Kailash D. Sharma, Tsuyoshi Takahashi, Ryo Iwamoto, and Eisuke Mekada. "Ligand-independent Dimer Formation of Epidermal Growth Factor Receptor (EGFR) Is a Step Separable from Ligand-induced EGFR Signaling." Molecular Biology of the Cell 13, no. 7 (July 2002): 2547–57. http://dx.doi.org/10.1091/mbc.01-08-0411.
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Dimerization and phosphorylation of the epidermal growth factor (EGF) receptor (EGFR) are the initial and essential events of EGF-induced signal transduction. However, the mechanism by which EGFR ligands induce dimerization and phosphorylation is not fully understood. Here, we demonstrate that EGFRs can form dimers on the cell surface independent of ligand binding. However, a chimeric receptor, comprising the extracellular and transmembrane domains of EGFR and the cytoplasmic domain of the erythropoietin receptor (EpoR), did not form a dimer in the absence of ligands, suggesting that the cytoplasmic domain of EGFR is important for predimer formation. Analysis of deletion mutants of EGFR showed that the region between835Ala and918Asp of the EGFR cytoplasmic domain is required for EGFR predimer formation. In contrast to wild-type EGFR ligands, a mutant form of heparin-binding EGF-like growth factor (HB2) did not induce dimerization of the EGFR-EpoR chimeric receptor and therefore failed to activate the chimeric receptor. However, when the dimerization was induced by a monoclonal antibody to EGFR, HB2 could activate the chimeric receptor. These results indicate that EGFR can form a ligand-independent inactive dimer and that receptor dimerization and activation are mechanistically distinct and separable events.
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SERPA, Jacinta, Nuno MENDES, Celso A. REIS, Luis F. SANTOS SILVA, Raquel ALMEIDA, Jacques LE PENDU, and Leonor DAVID. "Two new FUT2 (fucosyltransferase 2 gene) missense polymorphisms, 739G→A and 839T→C, are partly responsible for non-secretor status in a Caucasian population from Northern Portugal." Biochemical Journal 383, no. 3 (October 26, 2004): 469–74. http://dx.doi.org/10.1042/bj20040803.
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Secretor status is defined by the expression of H type 1 antigen on gastric surface epithelium and external secretions. The H type 1 structure, and other fucosylated carbohydrates (Lea, sialyl-Lea, Leb, Lex, sialyl-Lex and Ley), can serve as ligands for several pathogens, including Helicobacter pylori, and are cancer-associated antigens. Secretor individuals are more susceptible to some bacterial and viral infections of the genito-urinary and digestive tracts. The aim of the present study was to examine FUT2 (fucosyltransferase 2 gene) polymorphisms in a Caucasian population of non-secretor individuals (n=36) from northern Portugal and to evaluate the activity of the mutant FUT2 enzymes. The secretor status was determined by UEAI [Ulex europaeus (gorse) lectin] histochemistry in gastric mucosa, and FUT2 polymorphisms were studied by restriction-fragment-length polymorphism and direct sequencing. The majority of non-secretors (88.9%) were homozygous for 428G→A polymorphism; 5.6% were homozygous for 571C→T and 5.6% were homozygous for two new missense polymorphisms, 739G→A (2.8%) and 839T→C (2.8%). By kinetic studies it was demonstrated that the two new FUT2 mutants (739G→A and 839T→C) are almost inactive and are responsible for some non-secretor cases.
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KEYNAN, Shoshana, Nigel M. HOOPER, and Anthony J. TURNER. "Identification by site-directed mutagenesis of three essential histidine residues in membrane dipeptidase, a novel mammalian zinc peptidase." Biochemical Journal 326, no. 1 (August 15, 1997): 47–51. http://dx.doi.org/10.1042/bj3260047.
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Membrane dipeptidase (EC 3.4.13.19) is a plasma membrane zinc peptidase that is involved in the renal metabolism of glutathione and its conjugates, such as leukotriene D4. The enzyme lacks the classical signatures of other zinc-dependent hydrolases and shows no homology with any other mammalian protein. We have used site-directed mutagenesis to explore the roles of five histidine residues in pig membrane dipeptidase that are conserved among mammalian species. When expressed in COS-1 cells, the mutants H49K and H128L exhibited a specific activity and Km for the substrate Gly-D-Phe comparable with those of the wild-type enzyme. However, the mutants H20L, H152L and H198K were inactive, but were expressed at the cell surface at equivalent levels to the wild-type, as assessed by immunoblotting and immunofluorescence. These three mutants were compared with regard to their ability to bind to the competitive inhibitor cilastatin, which binds with equal efficacy to native and EDTA-treated pig kidney membrane dipeptidase. Expressed wild-type enzyme and mutants H20L and H198K were efficiently bound by cilastatin–Sepharose, but H152L failed to bind. Thus His-152 appears to be involved in the binding of substrate or inhibitor, whereas His-20 and His-198 appear to be involved in catalysis. Membrane dipeptidase shares some similarity with a dipeptidase recently cloned from Acinetobacter calcoaceticus. In particular, His-20 and His-198 of membrane dipeptidase are conserved in the bacterial enzyme, as are Glu-125 and His-219, previously shown to be required for catalytic activity.
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Shipitsin, Michail, and Larry A. Feig. "RalA but Not RalB Enhances Polarized Delivery of Membrane Proteins to the Basolateral Surface of Epithelial Cells." Molecular and Cellular Biology 24, no. 13 (July 1, 2004): 5746–56. http://dx.doi.org/10.1128/mcb.24.13.5746-5756.2004.
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ABSTRACT RalA and RalB constitute a family of highly similar (85% identity) Ras-related GTPases. Recently, active forms of both RalA and RalB have been shown to bind to the exocyst complex, implicating them in the regulation of cellular secretion. However, we show here that only active RalA enhances the rate of delivery of E-cadherin and other proteins to their site in the basolateral membrane of MDCK cells, consistent with RalA being a regulator of exocyst function. One reason for this difference is that RalA binds more effectively to the exocyst complex than active RalB does both in vivo and in vitro. Another reason is that active RalA localizes to perinuclear recycling endosomes, where regulation of vesicle sorting is thought to take place, while active RalB does not. Strikingly, analysis of chimeras made between RalA and RalB reveals that high-affinity exocyst binding by RalA is due to unique amino acid sequences in RalA that are distal to the common effector-binding domains shared by RalA and RalB. Moreover, these chimeras show that the perinuclear localization of active RalA is due in part to its unique variable domain near the C terminus. This distinct localization appears to be important for RalA effects on secretion because all RalA mutants tested that failed to localize to the perinuclear region also failed to promote basolateral delivery of E-cadherin. Interestingly, one of these inactive mutants maintained binding to the exocyst complex, suggesting that RalA binding to the exocyst is necessary but not sufficient for RalA to promote basolateral delivery of membrane proteins.
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Jané-Valbuena, Judit, Laura A. Breun, Leslie A. Schiff, and Max L. Nibert. "Sites and Determinants of Early Cleavages in the Proteolytic Processing Pathway of Reovirus Surface Protein σ3." Journal of Virology 76, no. 10 (May 15, 2002): 5184–97. http://dx.doi.org/10.1128/jvi.76.10.5184-5197.2002.
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ABSTRACT Entry of mammalian reovirus virions into target cells requires proteolytic processing of surface protein σ3. In the virion, σ3 mostly covers the membrane-penetration protein μ1, appearing to keep it in an inactive form and to prevent it from interacting with the cellular membrane until the proper time in infection. The molecular mechanism by which σ3 maintains μ1 in this inactive state and the structural changes that accompany σ3 processing and μ1 activation, however, are not well understood. In this study we characterized the early steps in σ3 processing and determined their effects on μ1 function and particle infectivity. We identified two regions of high protease sensitivity, “hypersensitive” regions located at residues 208 to 214 and 238 to 244, within which all proteases tested selectively cleaved σ3 as an early step in processing. Further processing of σ3 was required for infection, consistent with the fact that the fragments resulting from these early cleavages remained bound to the particles. Reovirus type 1 Lang (T1L), type 3 Dearing (T3D), and T1L × T3D reassortant virions differed in the sites of early σ3 cleavage, with T1L σ3 being cleaved mainly at residues 238 to 244 and T3D σ3 being cleaved mainly at residues 208 to 214. These virions also differed in the rates at which the early cleavages occurred, with cleavage of T1L σ3 occurring faster than cleavage of T3D σ3. Analyses using chimeric and site-directed mutants of recombinant σ3 identified carboxy-proximal residues 344, 347, and 353 as the primary determinants of these strain differences. The spatial relationships between these more carboxy-proximal residues and the hypersensitive regions were discerned from the σ3 crystal structure. The results indicate that proteolytic processing of σ3 during reovirus disassembly is a multistep pathway with a number of molecular determinants.
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Wood, Jeremy P., Jay R. Silveira, Nicole M. Maille, Laura M. Haynes, and Paula B. Tracy. "Prothrombin activation on the activated platelet surface optimizes expression of procoagulant activity." Blood 117, no. 5 (February 3, 2011): 1710–18. http://dx.doi.org/10.1182/blood-2010-09-311035.
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Abstract Effective hemostasis relies on the timely formation of α-thrombin via prothrombinase, a Ca2+-dependent complex of factors Va and Xa assembled on the activated platelet surface, which cleaves prothrombin at Arg271 and Arg320. Whereas initial cleavage at Arg271 generates the inactive intermediate prethrombin-2, initial cleavage at Arg320 generates the enzymatically active intermediate meizothrombin. To determine which of these intermediates is formed when prothrombin is processed on the activated platelet surface, the cleavage of prothrombin, and prothrombin mutants lacking either one of the cleavage sites, was monitored on the surface of either thrombin- or collagen-activated platelets. Regardless of the agonist used, prothrombin was initially cleaved at Arg271 generating prethrombin-2, with α-thrombin formation quickly after via cleavage at Arg320. The pathway used was independent of the source of factor Va (plasma- or platelet-derived) and was unaffected by soluble components of the platelet releasate. When both cleavage sites are presented within the same substrate molecule, Arg271 effectively competes against Arg320 (with an apparent IC50 = 0.3μM), such that more than 90% to 95% of the initial cleavage occurs at Arg271. We hypothesize that use of the prethrombin-2 pathway serves to optimize the procoagulant activity expressed by activated platelets, by limiting the anticoagulant functions of the alternate intermediate, meizothrombin.
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Burke, John E., Olga Perisic, Glenn R. Masson, Oscar Vadas, and Roger L. Williams. "Oncogenic mutations mimic and enhance dynamic events in the natural activation of phosphoinositide 3-kinase p110α (PIK3CA)." Proceedings of the National Academy of Sciences 109, no. 38 (September 4, 2012): 15259–64. http://dx.doi.org/10.1073/pnas.1205508109.
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The p110α catalytic subunit (PIK3CA) is one of the most frequently mutated genes in cancer. We have examined the activation of the wild-type p110α/p85α and a spectrum of oncogenic mutants using hydrogen/deuterium exchange mass spectrometry (HDX-MS). We find that for the wild-type enzyme, the natural transition from an inactive cytosolic conformation to an activated form on membranes entails four distinct events. Analysis of oncogenic mutations shows that all up-regulate the enzyme by enhancing one or more of these dynamic events. We provide the first insight into the activation mechanism by mutations in the linker between the adapter-binding domain (ABD) and the Ras-binding domain (RBD) (G106V and G118D). These mutations, which are common in endometrial cancers, enhance two of the natural activation events: movement of the ABD and ABD–RBD linker relative to the rest of the catalytic subunit and breaking the C2–iSH2 interface on binding membranes. C2 domain mutants (N345K and C420R) also mimic these events, even in the absence of membranes. A third event is breaking the nSH2–helical domain contact caused by phosphotyrosine-containing peptides binding to the enzyme, which is mimicked by a helical domain mutation (E545K). Interaction of the C lobe of the kinase domain with membranes is the fourth activation event, and is potentiated by kinase domain mutations (e.g., H1047R). All mutations increased lipid binding and basal activity, even mutants distant from the membrane surface. Our results elucidate a unifying mechanism in which diverse PIK3CA mutations stimulate lipid kinase activity by facilitating allosteric motions required for catalysis on membranes.
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Akimoto, Megumi, Makiko Ishihara, Kenji Kizawa, Hidenari Takahara, and Masaki Unno. "Two crystal forms of peptidylarginine deiminase typeIII (PAD3)." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C937. http://dx.doi.org/10.1107/s2053273314090627.
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Peptidylarginine deiminase (PAD) is a Ca2+-dependent enzyme that catalyzes the conversion of protein arginine residues to citrulline. Protein citrullination by PAD confers large structural and mechanical effects on the target proteins by altering intermolecular and intramolecular ionic or hydrophobic interactions. Five paralogous genes (PADI1 - 4 and 6) on human chromosome 1p35-36 encode the human PAD isozymes. Among the PADs, PAD3 shows the highest substrate specificity for synthetic and natural substrate. S100A3 is an EF-hand-type Ca2+-binding S100 protein family member that colocalizes with PAD3 in hair cuticular cells. PAD3 converts a symmetric pair of Arg51 residues on an S100A3 dimer surface to citrullines, causing assembly of a homotetramer, but does not convert other arginines. Although this specific citrullination is largely affected by the formation of two intramolecular disulfides in S100A3, it is not clear how the sheltered Arg51 residues is recognized by PAD3. We are aiming structural analysis of the substrate bound forms to elucidate structural factor that PAD3 recognize Arg51 residues only. Although X-ray crystal structures of the PAD4 isozyme and its complexes with substrate peptides have been reported, structural analysis of other PAD isozymes has not yet been conducted. To obtain the crystals of the substrate-complex, we prepared the C646A mutant and the other inactive mutants (D350A, H470A, D472A) of PAD3. We determined the crystal structures of wild-type PAD3, at first. Then, we have tried to determine the structure of the substrate complex with the mutants of PAD3. However, the solved structures did not contain the substrate at present stage. From our structural analysis, only the crystals of C646A were belonged to different crystal system from the others, and its difference didn't relate to their crystallization condition. In this conference, we discuss the origin(s) of the differences in the crystal system of C646 from the others.
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Frumence, Etienne, Sandrine Genetet, Pierre Ripoche, Achille Iolascon, Immacolata Andolfo, Caroline Le Van Kim, Yves Colin, Isabelle Mouro-Chanteloup, and Claude Lopez. "Rapid Cl−/HCO3−exchange kinetics of AE1 in HEK293 cells and hereditary stomatocytosis red blood cells." American Journal of Physiology-Cell Physiology 305, no. 6 (September 15, 2013): C654—C662. http://dx.doi.org/10.1152/ajpcell.00142.2013.
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Anion exchanger 1 (AE1) or band 3 is a membrane protein responsible for the rapid exchange of chloride for bicarbonate across the red blood cell membrane. Nine mutations leading to single amino-acid substitutions in the transmembrane domain of AE1 are associated with dominant hereditary stomatocytosis, monovalent cation leaks, and reduced anion exchange activity. We set up a stopped-flow spectrofluorometry assay coupled with flow cytometry to investigate the anion transport and membrane expression characteristics of wild-type recombinant AE1 in HEK293 cells, using an inducible expression system. Likewise, study of three stomatocytosis-associated mutations (R730C, E758K, and G796R), allowed the validation of our method. Measurement of the rapid and specific chloride/bicarbonate exchange by surface expressed AE1 showed that E758K mutant was fully active compared with wild-type (WT) AE1, whereas R730C and G796R mutants were inactive, reinforcing previously reported data on other experimental models. Stopped-flow analysis of AE1 transport activity in red blood cell ghost preparations revealed a 50% reduction of G796R compared with WT AE1 corresponding to a loss of function of the G796R mutated protein, in accordance with the heterozygous status of the AE1 variant patients. In conclusion, stopped-flow led to measurement of rapid transport kinetics using the natural substrate for AE1 and, conjugated with flow cytometry, allowed a reliable correlation of chloride/bicarbonate exchange to surface expression of AE1, both in recombinant cells and ghosts and therefore a fine comparison of function between different stomatocytosis samples. This technical approach thus provides significant improvements in anion exchange analysis in red blood cells.
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Yang, Li-Kun, and Ya-Xiong Tao. "Alanine Scanning Mutagenesis of the DRYxxI Motif and Intracellular Loop 2 of Human Melanocortin-4 Receptor." International Journal of Molecular Sciences 21, no. 20 (October 15, 2020): 7611. http://dx.doi.org/10.3390/ijms21207611.
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The melanocortin-4 receptor (MC4R) is a member of the G-protein-coupled receptor (GPCR) superfamily, which has been extensively studied in obesity pathogenesis due to its critical role in regulating energy homeostasis. Both the Gs-cAMP and ERK1/2 cascades are known as important intracellular signaling pathways initiated by the MC4R. The DRYxxI motif at the end of transmembrane domain 3 and the intracellular loop 2 (ICL2) are thought to be crucial for receptor function in several GPCRs. To study the functions of this domain in MC4R, we performed alanine-scanning mutagenesis on seventeen residues. We showed that one residue was critical for receptor cell surface expression. Eight residues were important for ligand binding. Mutations of three residues impaired Gs-cAMP signaling without changing the binding properties. Investigation on constitutive activities of all the mutants in the cAMP pathway revealed that six residues were involved in constraining the receptor in inactive states and five residues were important for receptor activation in the absence of an agonist. In addition, mutations of four residues impaired the ligand-stimulated ERK1/2 signaling pathway without affecting the binding properties. We also showed that some mutants were biased to the Gs-cAMP or ERK1/2 signaling pathway. In summary, we demonstrated that the DRYxxI motif and ICL2 were important for MC4R function.
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Owen-Schaub, L. B., W. Zhang, J. C. Cusack, L. S. Angelo, S. M. Santee, T. Fujiwara, J. A. Roth, A. B. Deisseroth, W. W. Zhang, and E. Kruzel. "Wild-type human p53 and a temperature-sensitive mutant induce Fas/APO-1 expression." Molecular and Cellular Biology 15, no. 6 (June 1995): 3032–40. http://dx.doi.org/10.1128/mcb.15.6.3032.
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Fas/APO-1 is a cell surface protein known to trigger apoptosis upon specific antibody engagement. Because wild-type p53 can activate transcription as well as induce apoptosis, we queried whether p53 might upregulate Fas/APO-1. To explore this possibility, we examined human p53-null (H358 non-small-cell lung adenocarcinoma and K562 erythroleukemia) and wild-type p53-containing (H460 non-small-cell lung adenocarcinoma) cell lines. When H358 or H460 cells were transduced with a replication-deficient adenovirus expression construct containing the human wild-type p53 gene but not with vector alone, a marked upregulation (approximately a three-to fourfold increase) of cell surface Fas/APO-1 was observed by flow cytometry. Similarly, K562, cells stably transfected with a plasmid vector containing the temperature-sensitive human p53 mutant Ala-143 demonstrated a four- to sixfold upregulation of Fas/APO-1 by flow-cytometric analysis at the permissive temperature of 32.5 degrees C. Temperature-sensitive upregulation of Fas/APO-1 in K562 Ala-143 cells was verified by immunoprecipitation and demonstrated to result from enhanced mRNA production by nuclear run-on and Northern (RNA) analyses. Stably transfected K562 cells expressing temperature-insensitive, transcriptionally inactive p53 mutants (His-175, Trp-248, His-273, or Gly-281) failed to upregulate Fas/APO-1 at either 32.5 degrees or 37.5 degrees C. The temperature-sensitive transcription of Fas/APO-1 occurred in the presence of cycloheximide, indicating that de novo protein synthesis was not required and suggested a direct involvement of p53. Collectively, these observations argue that Fas/APO-1 is a target gene for transcriptional activation by p53.
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Molinari, Antonio, Carla Giorgetti, Jacqueline Lansen, Fabrizio Vaghi, Gaetano Orsini, Elena Maria Faioni, and Pier Mannuccio Mannucci. "Thrombomodulin Is a Cofactor for Thrombin Degradation of Recombinant Single-Chain Urokinase Plasminogen Activator "In Vitro" and in a Perfused Rabbit Heart Model." Thrombosis and Haemostasis 67, no. 02 (1992): 226–32. http://dx.doi.org/10.1055/s-0038-1648417.
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SummaryThrombin cleaves single-chain urokinase-type plasminogen activator (scu-PA) to a two-chain derivative (tcu-PA) fibrinolyti-cally inactive. This reaction was accelerated in vitro by purified rabbit lung thrombomodulin in equimolar complex with thrombin. Polyclonal antibodies to rabbit thrombomodulin prevented this effect.We also observed that heparin and other sulfated polysaccharides had an accelerating effect on thrombin cleavage of recombinant scu-PA. Their effect was concentration-dependent and then reversed at high levels. The effect of heparin and heparan sulfate was independent and synergic with respect to thrombomodulin. All observations except the effect of heparin, could be confirmed in a Langendorff isolated rabbit heart model. From competition experiments carried out with scu-PA derivatives and mutants, we postulate that the amino-terminal sequence of rscu-PA, containing the epidermal growth factor (EGF)-like and the kringle domains is involved in the cofactor effect of thrombomodulin on scu-PA inactivation by thrombin. We conclude that a regulatory mechanism of scu-PA inactivation is present at the cell surface.
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Bach, Horacio, Yevgeny Berdichevsky, and David Gutnick. "An Exocellular Protein from the Oil-Degrading Microbe Acinetobacter venetianus RAG-1 Enhances the Emulsifying Activity of the Polymeric Bioemulsifier Emulsan." Applied and Environmental Microbiology 69, no. 5 (May 2003): 2608–15. http://dx.doi.org/10.1128/aem.69.5.2608-2615.2003.
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ABSTRACT The oil-degrading microorganism Acinetobacter venetianus RAG-1 produces an extracellular polyanionic, heteropolysaccharide bioemulsifier termed emulsan. Emulsan forms and stabilizes oil-water emulsions with a variety of hydrophobic substrates. Removal of the protein fraction yields a product, apoemulsan, which exhibits much lower emulsifying activity on hydrophobic substrates such as n-hexadecane. One of the key proteins associated with the emulsan complex is a cell surface esterase. The esterase (molecular mass, 34.5 kDa) was cloned and overexpressed in Escherichia coli BL21(DE3) behind the phage T7 promoter with the His tag system. After overexpression, about 80 to 90% of the protein was found in inclusion bodies. The overexpressed esterase was recovered from the inclusion bodies by solubilization with deoxycholate and, after slow dialysis, was purified by metal chelation affinity chromatography. Mixtures containing apoemulsan and either the catalytically active soluble form of the recombinant esterase isolated from cell extracts or the solubilized inactive form of the enzyme recovered from the inclusion bodies formed stable oil-water emulsions with very hydrophobic substrates such as hexadecane under conditions in which emulsan itself was ineffective. Similarly, a series of esterase-defective mutants were generated by site-directed mutagenesis, cloned, and overexpressed in E. coli. Mutant proteins defective in catalytic activity as well as others apparently affected in protein conformation were also active in enhancing the apoemulsan-mediated emulsifying activity. Other proteins, including a His-tagged overexpressed esterase from the related organism Acinetobacter calcoaceticus BD4, showed no enhancement.
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Kothari, Hema, Ramesh Nayak, L. Vijaya Mohan Rao, and Usha Pendurthi. "Cryptic Vs. Active Tissue Factor: Cystine186-Cystine 209 Disulfide Bond Switch Is Not the Answer." Blood 114, no. 22 (November 20, 2009): 333. http://dx.doi.org/10.1182/blood.v114.22.333.333.
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Abstract Abstract 333 Tissue factor (TF) plays the key role in initiation of blood coagulation by allosterically activating coagulation factor VIIa (FVIIa). TF is essential for hemostasis but aberrant expression would lead to thrombotic disorders. TF on cell surfaces resides mostly in a cryptic state. At present it is not entirely clear how cryptic TF differs from procoagulant active TF and how de-encryption occurs. Recent studies have suggested that cryptic and active TF may have different conformations, i.e., cryptic TF contains unpaired cysteine thiols at Cys 186 and Cys 209 in the membrane proximal domain whereas procoagulant active TF contains the oxidized Cys186-Cys209 disulfide bond. It has been suggested that the catalytic switching of this disulfide bond between reduced and oxidized states by protein disulfide isomerase (PDI) might modulate the activity status of TF. Although the validity of this hypothesis has been questioned, more recent reports have supported the contention that PDI-catalyzed disulfide bond formation of Cys186-Cys209 in TF plays a role in regulation of TF activity both in vitro and in vivo. To reconcile the conflicting data in the recent literature, we re-examined in the present study the central dogma on which the hypothesis was originated, i.e., a TF mutant lacking Cys186-Cys 209 disulfide bond is functionally inactive (cryptic) and that TF activation requires formation of the Cys186-Cys209 disulfide bond. CHO cells were transfected with wild type TF or TF mutants that selectively preclude Cys186-Cys209 disulfide bond formation, i.e., TFC186S, TFC209S, or TFC186S/C209S, and TF procoagulant and antigen expression levels at the cell surface were quantified accurately by measuring TF procoagulant activity in a factor × activation assay and TF antigen levels in radioligand binding studies utilizing two different TF mAb (10H10 and 5G9) and FVIIa. CHO cells transfected with TFC186S, TFC209S or TFC186S/C209S expressed very little procoagulant activity (less than 1% of CHO cells transfected with wild-type TF under identical experimental conditions). However, TF mAb and FVIIa binding studies revealed that CHO cells transfected with TFC186S, TFC209S or TFC186S/C209S expressed very little TF protein on the cell surface (∼ 1 to 2% of CHO cells transfected with wild-type TF). Similar results were obtained whether we mutated Cys 186 or Cys 209 to Ser or Ala or whether we used transient or stable transfections. To confirm that the lower expression of TFC186S, TFC209S or TFC186S/C209S is neither an artifact nor limited to CHO cell model system, we extended these studies to endothelial cells. Human umbilical vein endothelial cells (HUVECs) were transduced with adenovirus particles (25 moi/cell) encoding wild-type TF, TFC186S, TFC209S or TFC186S/C209S and TF antigen levels at the cell surface and in cell lysates were measured using multiple methods - confocal microscopy, FACS, TF antigen assay, immuno blot analysis, TF mAb binding and FVIIa binding. HUVEC transduced with adenoviral particles encoding TFC186S, TFC209S or TFC186S/C209S expressed very low levels of TF antigen both at the cell surface ( 1 to 5%) as well as in total (10 to 15%) (TFC186S > TFC209S >TFC186S/C209S) compared to HUVEC transduced with the same number of adenovirus particles expressing wild-type TF. Cell surface TF activity measured in the presence of saturating concentrations of FVIIa revealed that the TF procoagulant activity of the mutants correlate well with the amount of TF antigen present on the cell surface, i.e., there were no significant differences in the specific activity of TF mutants and wild-type TF (TF procoagulant activity/amount of TF antigen, pM FXa formed/femto mole TF: wild-type TF, 68 ± 6; TFC186S, 55 ± 10; TFC209S, 57 ± 17; and TFC186S/C209S, 64 ± 0.2). More importantly, treatment of HUVEC with the thiol-oxidizing agent HgCl2 or with ionomycin increased the cell surface TF activity to the same extent (200 to 400% increase over HUVEC treated with control vehicle) in HUVEC expressing TFC186S/C209S or wild-type TF. In summary the present data provide clear evidence for that TF lacking Cys186-Cys209 bond is coagulantly active and a lower TF procoagulant activity in cells expressing the mutant TF that precludes Cys186-Cys209 disulfide bond formation is due to severe impairment in TF protein synthesis/processing and not because the mutant TF assumes the cryptic conformation. Our data also show that TF de-encryption does not require the formation of Cys186-Cys209 disulfide bond. Disclosures: No relevant conflicts of interest to declare.
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Li, Wei, Douglas Metcalf, Roman Gorelik, Renhao Li, Neal Mitra, Vikas Nanda, Peter B. Law, James D. Lear, William F. DeGrado, and Joel S. Bennett. "Heteromeric and Homomeric Transmembrane Domain Associations Regulate αIIbβ3 Function." Blood 104, no. 11 (November 16, 2004): 329. http://dx.doi.org/10.1182/blood.v104.11.329.329.
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Abstract The integrin αIIbβ3 resides on the platelet surface in an equilibrium between inactive and active conformations that can be shifted in either direction by altering the distance between the stalks that anchor αIIbβ3 in the platelet membrane. Accordingly, the αIIb and β3 transmembrane (TM) domains, located near the ends of the stalks, are in proximity when αIIbβ3 is inactive and separate upon αIIbβ3 activation. Peptides corresponding to these domains undergo both homomeric and heteromeric interactions in biological membranes. Thus, it is possible that the shift between inactive and active αIIbβ3 conformations is accompanied by a shift from heteromeric to homomeric αIIb and β3 TM domain interactions. Indeed, we reported that introducing Asn, a residue known to strengthen homomeric TM helix interactions, into the β3 TM domain shifts αIIbβ3 to an active state. As a further test of this model of αIIbβ3 regulation, we studied the effects of mutations of the αIIb TM domain. First, we placed Asn at 10 consecutive positions in the αIIb TM domain, extending from residues V969 to L978, and co-expressed each mutant with WT β3 in CHO cells. Only one of the mutants, G972N, was constitutively active, binding ~ 8-fold more fibrinogen than WT αIIbβ3. Moreover, G972N was expressed in non-uniform patches on the CHO cell surface, consistent with the formation of αIIbβ3 clusters. G972 is the first residue of a GxxxG motif that is essential for dimerization of the αIIb TM domain. Using the TOXCAT assay to assess TM domain dimerization, we observed that G972N results in a 55% decrease in TOXCAT activity. This implies that the effect of G972N on the αIIbβ3 activation state was not a result of increased homo-dimerization of αIIb, but it is more likely that the mutation disrupted its heteromeric interaction with β3. To test this suggestion, we introduced mutations known to disrupt αIIb homo-dimerization (G972L, G976A, and G976L) into αIIbβ3 and measured their effect on αIIbβ3 function. Like G972N, each mutation induced constitutive αIIbβ3 activation and clustering. Lastly, we measured the effect of L980A, a mutation in the αIIb TM domain that unlike G972N, results in a 2.5-fold increase in TOXCAT activity. CHO cells expressing L980A constitutively bound ~ 6.5-fold more fibrinogen than did cells expressing WT αIIbβ3. Taken together, our results suggest a mechanism for αIIbβ3 regulation that involves both the heteromeric and homomeric association of the αIIb and β3 TM domains. Any process that destabilizes the heteromeric association of the αIIb and β3 TM domains would be expected to allow dissociation of these domains with concomitant αIIbβ3 activation. Hence, mutations that disrupt the heteromeric αIIb/β3 TM domain interface “push” αIIbβ3 toward activation. Conversely, intermolecular interactions that either require separation of the αIIb and β3 TM domains or are more favorable when they dissociate, such as homo-oligomerization of the αIIb and β3 TM domains, will “pull” the equilibrium toward the activated state.
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Leader, Avi, Ronit Mor-Cohen, Nurit Rosenberg, Ron Ram, Uri Seligsohn, Aida Inbal, and Judith Lahav. "The Role Of Protein Disulfide Isomerase In The Post-Ligation Phase Of β3 Integrin-Dependent Cell Adhesion." Blood 122, no. 21 (November 15, 2013): 2307. http://dx.doi.org/10.1182/blood.v122.21.2307.2307.
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Abstract Introduction Protein disulfide isomerase (PDI) is a member of the group of sulfhydryl isomerase enzymes which catalyze the formation, reduction and exchange of disulfide bonds. We and others have shown that PDI has a role in integrin-mediated platelet adhesion and aggregation, and among its proposed targets are integrins αIIbβ3 and αvβ3. There has been no clear evidence indicating whether disulfide bond exchange plays a role in the post-ligation phase of adhesion, which involves outside-in signaling. Objective We tested our hypothesis that free sulfhydryls and PDI play an essential role in the post-ligation phase of αIIbβ3 and αvβ3-dependent cell adhesion to fibrinogen. Methods Baby Hamster Kidney (BHK) cells were transfected with wild type (WT) αIIb and either WT β3 or β3 mutated at specific cysteines, resulting in surface expression of both αvβ3 and αIIbβ3. Single or double cysteine to serine mutations disrupting the Cys473-Cys503 and the Cys523-Cys544 disulfide bonds were generated. Evaluation of the expression and activity of WT or mutated integrins on the surface of BHK cells was performed by flow cytometry using P2 and PAC1 antibodies, respectively. Adhesion of BHK cells, expressing WT or mutated αIIbβ3, to fibrinogen-coated wells was studied in the presence or absence of bacitracin, a PDI inhibitor. Each experiment was performed with and without an αvβ3blocker (RO0655233-001). The adhered cells were stained and counted using light microscopy. Results Flow cytometry showed that BHK cells expressing the αIIbβ3 mutants bound both P2 and PAC1 while WT αIIbβ3-transfected cells bound only P2, indicating that the mutated αIIbβ3 receptors were constitutively active while WT αIIbβ3 was inactive as previously shown. The adhesion of BHK cells to fibrinogen was dependent on αIIbβ3 surface expression in both WT and mutants. Adhesion was reduced following treatment with the αvβ3 blocker, suggesting that both β3integrins support binding to immobilized fibrinogen (p < 0.05 for WT, C473S, C523S/C544S; p=0.053 for C544S). Without blocking of αvβ3, only cells expressing WT αIIbβ3 were inhibited by bacitracin (p=0.03, Figure 1A). Combination of the αvβ3 blocker and bacitracin, enabled a concentration-dependent inhibitory effect of bacitracin on adhesion of cells expressing either WT αIIbβ3or mutants disrupting the Cys523-Cys544 bond (C544S, C523S/C544S and C523S; p < 0.05 for each; Figure 1B). Statistical analysis failed to indicate a similar role for the Cys-473-Cys-503 bond (C473S and C503S; Figure 1B). Conclusion Cells expressing the constitutively active mutated αIIbβ3 were still dependent on PDI for adhesion, as shown by the inhibitory effect of the PDI inhibitor on their adhesion in the presence of an αvβ3 blocker, demonstrating that disulfide bond exchange plays an essential role in the post-ligation stage of αIIbβ3-mediated adhesion to fibrinogen. The difference between the inhibitory effect of bacitracin on the mutants disrupting the Cys523-Cys544 bond and the mutants disrupting the Cys473-Cys503 bond suggests that the role of disulfide bond exchange in the post-ligation phase of adhesion may differ between different disulfide bonds. In the absence of the αvβ3 blocker, bacitracin had no inhibitory effect on cells expressing the constitutively active mutants and the inhibitory effect of bacitracin on cells expressing the WT integrins was less prominent. Therefore, disulfide bond exchange mediated by PDI may have a pivotal role in the post-ligation phase of adhesion mediated by αIIbβ3 receptor, while the adhesion to fibrinogen mediated by αvβ3integrin depends on PDI to a lesser extent. Disclosures: No relevant conflicts of interest to declare.
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Elsen, Sylvie, Ophélie Duché, and Annette Colbeau. "Interaction between the H2Sensor HupUV and the Histidine Kinase HupT Controls HupSL HydrogenaseSynthesis in Rhodobactercapsulatus." Journal of Bacteriology 185, no. 24 (December 15, 2003): 7111–19. http://dx.doi.org/10.1128/jb.185.24.7111-7119.2003.
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ABSTRACT The photosynthetic bacterium Rhodobacter capsulatus contains two [NiFe]hydrogenases: an energy-generating hydrogenase, HupSL, and a regulatory hydrogenase, HupUV. The synthesis of HupSL is specifically activated by H2 through a signal transduction cascade comprising three proteins: the H2-sensing HupUV protein, the histidine kinase HupT, and the transcriptional regulator HupR. Whereas a phosphotransfer between HupT and HupR was previously demonstrated, interaction between HupUV and HupT was only hypothesized based on in vivo analyses of mutant phenotypes. To visualize the in vitro interaction between HupUV and HupT proteins, a six-His (His6)-HupU fusion protein and the HupV protein were coproduced by using a homologous expression system. The two proteins copurified as a His6-HupUHupV complex present in dimeric and tetrameric forms, both of which had H2 uptake activity. We demonstrated that HupT and HupUV interact and form stable complexes that could be separated on a native gel. Interaction was also monitored with surface plasmon resonance technology and was shown to be insensitive to salt concentration and pH changes, suggesting that the interactions involve hydrophobic residues. As expected, H2 affects the interaction between HupUV and HupT, leading to a weakening of the interaction, which is independent of the phosphate status of HupT. Several forms of HupT were tested for their ability to interact with HupUV and to complement hupT mutants. Strong interaction with HupUV was obtained with the isolated PAS domain of HupT and with inactive HupT mutated in the phosphorylable histidine residue, but only the wild-type HupT protein was able to restore normal H2 regulation.
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Ozawa, Masayuki, and Rolf Kemler. "The Membrane-proximal Region of the E-Cadherin Cytoplasmic Domain Prevents Dimerization and Negatively Regulates Adhesion Activity." Journal of Cell Biology 142, no. 6 (September 21, 1998): 1605–13. http://dx.doi.org/10.1083/jcb.142.6.1605.
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Cadherins are transmembrane glycoproteins involved in Ca2+-dependent cell–cell adhesion. Deletion of the COOH-terminal residues of the E-cadherin cytoplasmic domain has been shown to abolish its cell adhesive activity, which has been ascribed to the failure of the deletion mutants to associate with catenins. Based on our present results, this concept needs revision. As was reported previously, leukemia cells (K562) expressing E-cadherin with COOH-terminal deletion of 37 or 71 amino acid residues showed almost no aggregation. Cells expressing E-cadherin with further deletion of 144 or 151 amino acid residues, which eliminates the membrane-proximal region of the cytoplasmic domain, showed E-cadherin–dependent aggregation. Thus, deletion of the membrane-proximal region results in activation of the nonfunctional E-cadherin polypeptides. However, these cells did not show compaction. Chemical cross-linking revealed that the activated E-cadherin polypeptides can be cross-linked to a dimer on the surface of cells, whereas the inactive polypeptides, as well as the wild-type E-cadherin polypeptide containing the membrane-proximal region, can not. Therefore, the membrane-proximal region participates in regulation of the adhesive activity by preventing lateral dimerization of the extracellular domain.
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Nirmala, Jayaveeramuthu, Tom Drader, Xianming Chen, Brian Steffenson, and Andris Kleinhofs. "Stem Rust Spores Elicit Rapid RPG1 Phosphorylation." Molecular Plant-Microbe Interactions® 23, no. 12 (December 2010): 1635–42. http://dx.doi.org/10.1094/mpmi-06-10-0136.
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Stem rust threatens cereal production worldwide. Understanding the mechanism by which durable resistance genes, such as Rpg1, function is critical. We show that the RPG1 protein is phosphorylated within 5 min by exposure to spores from avirulent but not virulent races of stem rust. Transgenic mutants encoding an RPG1 protein with an in vitro inactive kinase domain fail to phosphorylate RPG1 in vivo and are susceptible to stem rust, demonstrating that phosphorylation is a prerequisite for disease resistance. Protein kinase inhibitors prevent RPG1 phosphorylation and result in susceptibility to stem rust, providing further evidence for the importance of phosphorylation in disease resistance. We conclude that phosphorylation of the RPG1 protein by the kinase activity of the pK2 domain induced by the interaction with an unknown pathogen spore product is required for resistance to the avirulent stem rust races. The pseudokinase pK1 domain is required for disease resistance but not phosphorylation. The very rapid phosphorylation of RPG1 suggests that an effector is already present in or on the stem rust urediniospores when they are placed on the leaf surface. However, spores must be alive, as determined by their ability to germinate, in order to elicit RPG1 phosphorylation.
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Shon, D. Judy, Stacy A. Malaker, Kayvon Pedram, Emily Yang, Venkatesh Krishnan, Oliver Dorigo, and Carolyn R. Bertozzi. "An enzymatic toolkit for selective proteolysis, detection, and visualization of mucin-domain glycoproteins." Proceedings of the National Academy of Sciences 117, no. 35 (August 17, 2020): 21299–307. http://dx.doi.org/10.1073/pnas.2012196117.
Full textAbstract:
Densely O-glycosylated mucin domains are found in a broad range of cell surface and secreted proteins, where they play key physiological roles. In addition, alterations in mucin expression and glycosylation are common in a variety of human diseases, such as cancer, cystic fibrosis, and inflammatory bowel diseases. These correlations have been challenging to uncover and establish because tools that specifically probe mucin domains are lacking. Here, we present a panel of bacterial proteases that cleave mucin domains via distinct peptide- and glycan-based motifs, generating a diverse enzymatic toolkit for mucin-selective proteolysis. By mutating catalytic residues of two such enzymes, we engineered mucin-selective binding agents with retained glycoform preferences. StcEE447Dis a pan-mucin stain derived from enterohemorrhagicEscherichia colithat is tolerant to a wide range of glycoforms. BT4244E575Aderived fromBacteroides thetaiotaomicronis selective for truncated, asialylated core 1 structures commonly associated with malignant and premalignant tissues. We demonstrated that these catalytically inactive point mutants enable robust detection and visualization of mucin-domain glycoproteins by flow cytometry, Western blot, and immunohistochemistry. Application of our enzymatic toolkit to ascites fluid and tissue slices from patients with ovarian cancer facilitated characterization of patients based on differences in mucin cleavage and expression patterns.
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Stewart, Andrew K., Prabhakar S. Kedar, Boris E. Shmukler, David H. Vandorpe, Ann Hsu, Bertil Glader, Alicia Rivera, Carlo Brugnara, and Seth L. Alper. "Functional characterization and modified rescue of novel AE1 mutation R730C associated with overhydrated cation leak stomatocytosis." American Journal of Physiology-Cell Physiology 300, no. 5 (May 2011): C1034—C1046. http://dx.doi.org/10.1152/ajpcell.00447.2010.
Full textAbstract:
We report the novel, heterozygous AE1 mutation R730C associated with dominant, overhydrated, cation leak stomatocytosis and well-compensated anemia. Parallel elevations of red blood cell cation leak and ouabain-sensitive Na+efflux (pump activity) were apparently unaccompanied by increased erythroid cation channel-like activity, and defined ouabain-insensitive Na+efflux pathways of nystatin-treated cells were reduced. Epitope-tagged AE1 R730C at the Xenopus laevis oocyte surface exhibited severely reduced Cl−transport insensitive to rescue by glycophorin A (GPA) coexpression or by methanethiosulfonate (MTS) treatment. AE1 mutant R730K preserved Cl−transport activity, but R730 substitution with I, E, or H inactivated Cl−transport. AE1 R730C expression substantially increased endogenous oocyte Na+-K+-ATPase-mediated86Rb+influx, but ouabain-insensitive flux was minimally increased and GPA-insensitive. The reduced AE1 R730C-mediated sulfate influx did not exhibit the wild-type pattern of stimulation by acidic extracellular pH (pHo) and, unexpectedly, was partially rescued by exposure to sodium 2-sulfonatoethyl methanethiosulfonate (MTSES) but not to 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA) or 2-(trimethylammonium)ethyl methanethiosulfonate bromide (MTSET). AE1 R730E correspondingly exhibited acid pHo-stimulated sulfate uptake at rates exceeding those of wild-type AE1 and AE1 R730K, whereas mutants R730I and R730H were inactive and pHoinsensitive. MTSES-treated oocytes expressing AE1 R730C and untreated oocytes expressing AE1 R730E also exhibited unprecedented stimulation of Cl−influx by acid pHo. Thus recombinant cation-leak stomatocytosis mutant AE1 R730C exhibits severely reduced anion transport unaccompanied by increased Rb+and Li+influxes. Selective rescue of acid pHo-stimulated sulfate uptake and conferral of acid pHo-stimulated Cl−influx, by AE1 R730E and MTSES-treated R730C, define residue R730 as critical to selectivity and regulation of anion transport by AE1.
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Powner, Dale J., Matthew N. Hodgkin, and Michael J. O. Wakelam. "Antigen-stimulated Activation of Phospholipase D1b by Rac1, ARF6, and PKCα in RBL-2H3 Cells." Molecular Biology of the Cell 13, no. 4 (April 2002): 1252–62. http://dx.doi.org/10.1091/mbc.01-05-0235.
Full textAbstract:
Phospholipase D (PLD) activity can be detected in response to many agonists in most cell types; however, the pathway from receptor occupation to enzyme activation remains unclear. In vitro PLD1b activity is phosphatidylinositol 4,5-bisphosphate dependent via an N-terminal PH domain and is stimulated by Rho, ARF, and PKC family proteins, combinations of which cooperatively increase this activity. Here we provide the first evidence for the in vivo regulation of PLD1b at the molecular level. Antigen stimulation of RBL-2H3 cells induces the colocalization of PLD1b with Rac1, ARF6, and PKCα at the plasma membrane in actin-rich structures, simultaneously with cooperatively increasing PLD activity. Activation is both specific and direct because dominant negative mutants of Rac1 and ARF6 inhibit stimulated PLD activity, and surface plasmon resonance reveals that the regulatory proteins bind directly and independently to PLD1b. This also indicates that PLD1b can concurrently interact with a member from each regulator family. Our results show that in contrast to PLD1b's translocation to the plasma membrane, PLD activation is phosphatidylinositol 3-kinase dependent. Therefore, because inactive, dominant negative GTPases do not activate PLD1b, we propose that activation results from phosphatidylinositol 3-kinase–dependent stimulation of Rac1, ARF6, and PKCα.