Journal of Biological Chemistry

doi: 10.1016/s0021-9258(20)48547-3pmid: N/A

doi: 10.1016/s0021-9258(20)48613-2pmid: N/A

Wanaguru, Madushi;Crosnier, Cécile;Johnson, Steven;Rayner, Julian C.;Wright, Gavin J.

doi: 10.1074/jbc.m113.484840pmid: 24043627

<p><i>Pf</i>EBA175 has an important role in the invasion of human erythrocytes by <i>Plasmodium falciparum</i> and is therefore considered a high priority blood-stage malaria vaccine candidate. <i>Pf</i>EBA175 mediates adhesion to erythrocytes through binding of the Duffy-binding-like (DBL) domains in its extracellular domain to Neu5Acα2–3Gal displayed on the <i>O</i>-linked glycans of glycophorin-A (GYPA). Because of the difficulties in expressing active full-length (FL) <i>P. falciparum</i> proteins in a recombinant form, previous analyses of the <i>Pf</i>EBA175-GYPA interaction have largely focused on the DBL domains alone, and therefore they have not been performed in the context of the native protein sequence. Here, we express the entire ectodomain of <i>Pf</i>EBA175 (<i>Pf</i>EBA175 FL) in soluble form, allowing us to compare the biochemical and immunological properties with a fragment containing only the tandem DBL domains ("region II," <i>Pf</i>EBA175 RII). Recombinant <i>Pf</i>EBA175 FL bound human erythrocytes in a trypsin and neuraminidase-sensitive manner and recognized Neu5Acα2–3Gal-containing glycans, confirming its biochemical activity. A quantitative binding analysis showed that <i>Pf</i>EBA175 FL interacted with native GYPA with a <i>K_D</i> ∼0.26 μm and is capable of self-association. By comparison, the RII fragment alone bound GYPA with a lower affinity demonstrating that regions outside of the DBL domains are important for interactions with GYPA; antibodies directed to these other regions also contributed to the inhibition of parasite invasion. These data demonstrate the importance of <i>Pf</i>EBA175 regions other than the DBL domains in the interaction with GYPA and merit their inclusion in an EBA175-based vaccine.</p><p><b>Background:</b> The GYPA-<i>Pf</i>EBA175 interaction is important for erythrocyte invasion by the malaria parasite.</p><p><b>Results:</b> The entire ectodomain of EBA175 interacted with GYPA with different biochemical parameters to the previously determined GYPA-binding fragment containing two DBL domains.</p><p><b>Conclusion:</b> Regions outside of the tandem DBL domains contribute to GYPA binding by EBA175.</p><p><b>Significance:</b> These findings may assist the design of an EBA175-based malaria vaccine.</p>

doi: 10.1074/jbc.p113.484840pmid: N/A

♦ See referenced article, J. Biol. Chem. 2013, 288, 32106–32117

Lawson, Kevin A.;Teteak, Colin J.;Zou, Junhui;Hacquebord, Jacques;Ghatan, Andrew;Zielinska-Kwiatkowska, Anna;Fernandes, Russell J.;Chansky, Howard A.;Yang, Liu

doi: 10.1074/jbc.m113.473827pmid: 24056368

<p>The exact molecular mechanisms governing articular chondrocytes remain unknown in skeletal biology. In this study, we have found that ESET (an ERG-associated protein with a SET domain, also called SETDB1) histone methyltransferase is expressed in articular cartilage. To test whether ESET regulates articular chondrocytes, we carried out mesenchyme-specific deletion of the ESET gene in mice. ESET knock-out did not affect generation of articular chondrocytes during embryonic development. Two weeks after birth, there was minimal qualitative difference at the knee joints between wild-type and ESET knock-out animals. At 1 month, ectopic hypertrophy, proliferation, and apoptosis of articular chondrocytes were seen in the articular cartilage of ESET-null animals. At 3 months, additional signs of terminal differentiation such as increased alkaline phosphatase activity and an elevated level of matrix metalloproteinase (MMP)-13 were found in ESET-null cartilage. Staining for type II collagen and proteoglycan revealed that cartilage degeneration became progressively worse from 2 weeks to 12 months at the knee joints of ESET knock-out mutants. Analysis of over 14 pairs of age- and sex-matched wild-type and knock-out mice indicated that the articular chondrocyte phenotype in ESET-null mutants is 100% penetrant. Our results demonstrate that expression of ESET plays an essential role in the maintenance of articular cartilage by preventing articular chondrocytes from terminal differentiation and may have implications in joint diseases such as osteoarthritis.</p><p><b>Background:</b> Articular chondrocytes are responsible for producing articular cartilage and do not normally enter into terminal differentiation.</p><p><b>Results:</b> Conditional knock-out of ESET histone methyltransferase results in hypertrophy, apoptosis, and terminal differentiation of articular chondrocytes.</p><p><b>Conclusion:</b> ESET is essential for the normal maintenance of articular cartilage and joint function in adult animals.</p><p><b>Significance:</b> Learning regulatory mechanisms of articular chondrocytes is critical to the understanding of joint diseases.</p>

Zhang, Wenliang;Zhao, Jiawei;Lee, Jen-Fu;Gartung, Allison;Jawadi, Hiba;Lambiv, Wanyu Louis;Honn, Kenneth V.;Lee, Menq-Jer

doi: 10.1074/jbc.m113.495218pmid: 24064218

<p>Sphingosine-1-phosphate (S1P)-regulated chemotaxis plays critical roles in various physiological and pathophysiological conditions. S1P-regulated chemotaxis is mediated by the S1P family of G-protein-coupled receptors. However, molecular details of the S1P-regulated chemotaxis are incompletely understood. Cultured human lung adenocarcinoma cell lines abundantly express S1P receptor subtype 3 (S1P₃), thus providing a tractable <i>in vitro</i> system to characterize molecular mechanism(s) underlying the S1P₃ receptor-regulated chemotactic response. S1P treatment enhances CD44 expression and induces membrane localization of CD44 polypeptides via the S1P₃/Rho kinase (ROCK) signaling pathway. Knockdown of CD44 completely diminishes the S1P-stimulated chemotaxis. Promoter analysis suggests that the CD44 promoter contains binding sites of the ETS-1 (v-<i>ets</i> erythroblastosis virus E26 oncogene homolog 1) transcriptional factor. ChIP assay confirms that S1P treatment stimulates the binding of ETS-1 to the CD44 promoter region. Moreover, S1P induces the expression and nuclear translocation of ETS-1. Knockdown of S1P₃ or inhibition of ROCK abrogates the S1P-induced ETS-1 expression. Furthermore, knockdown of ETS-1 inhibits the S1P-induced CD44 expression and cell migration. In addition, we showed that S1P₃/ROCK signaling up-regulates ETS-1 via the activity of JNK. Collectively, we characterized a novel signaling axis, <i>i.e.</i>, ROCK-JNK-ETS-1-CD44 pathway, which plays an essential role in the S1P₃-regulated chemotactic response.</p><p><b>Background:</b> S1P₃-mediated chemotaxis plays a pivotal role in various physiological and pathophysiological activities.</p><p><b>Results:</b> S1P/S1P₃ signaling activates ROCK/JNK/ETS-1/CD44 pathway, and inhibition of this pathway abrogates S1P₃-stimulated chemotaxis.</p><p><b>Conclusion:</b> ETS-1/CD44 signaling mediates S1P/S1P₃-regulated chemotaxis.</p><p><b>Significance:</b> Therapeutically manipulating S1P₃-mediated chemotaxis requires a molecular understanding of its regulated signaling pathway.</p>

Gärtner, Florian;Seidel, Thorsten;Schulz, Uwe;Gummert, Jan;Milting, Hendrik

doi: 10.1074/jbc.m113.461566pmid: 24064210

<p>Endothelin receptor A (ET_A), a G protein-coupled receptor, mediates endothelin signaling, which is regulated by GRK2. Three Ser and seven Thr residues recently proven to be phosphoacceptor sites are located in the C-terminal extremity (CTE) of the receptor following its palmitoylation site. We created various phosphorylation-deficient ET_A mutants. The phospholipase C activity of mutant receptors in HEK-293 cells was analyzed during continuous endothelin stimulation to investigate the impact of phosphorylation sites on ET_A desensitization. Total deletion of phosphoacceptor sites in the CTE affected proper receptor regulation. However, proximal and distal phosphoacceptor sites both turned out to be sufficient to induce WT-like desensitization. Overexpression of the Gα_q coupling-deficient mutant GRK2-D110A suppressed ET_A-WT signaling but failed to decrease phospholipase C activity mediated by the phosphorylation-deficient mutant ET_A-6PD. In contrast, GRK2-WT acted on both receptors, whereas the kinase-inactive mutant GRK2-D110A/K220R failed to inhibit signaling of ET_A-WT and ET_A-6PD. This demonstrates that ET_A desensitization involves at least two autonomous GRK2-mediated components: 1) a phosphorylation-independent signal decrease mediated by blocking of Gα_q and 2) a mechanism involving phosphorylation of Ser and Thr residues in the CTE of the receptor in a redundant fashion, able to incorporate either proximal or distal phosphoacceptor sites. High level transfection of GRK2 variants influenced signaling of ET_A-WT and ET_A-6PD and hints at an additional phosphorylation-independent regulatory mechanism. Furthermore, internalization of mRuby-tagged receptors was observed with ET_A-WT and the phosphorylation-deficient mutant ET_A-14PD (lacking 14 phosphoacceptor sites) and turned out to be based on a phosphorylation-independent mechanism.</p><p><b>Background:</b> The involvement of phosphorylation of endothelin receptor A (ET_A) in its desensitization and internalization is unclear.</p><p><b>Results:</b> Phosphorylation-deficient ET_A mutants display defective regulation, whereas receptor internalization is not affected.</p><p><b>Conclusion:</b> GRK2-mediated phosphorylation contributes to ET_A desensitization, but not to ET_A internalization.</p><p><b>Significance:</b> This work contributes to the understanding of the complex GRK2-mediated ET_A regulation involving multiple mechanisms.</p>

McCarthy, Donald A.;Clark, Ryan R.;Bartling, Toni R.;Trebak, Mohamed;Melendez, J. Andres

doi: 10.1074/jbc.m113.493841pmid: 24062309

<p>Senescent cells accumulate in aged tissue and are causally linked to age-associated tissue degeneration. These non-dividing, metabolically active cells are highly secretory and alter tissue homeostasis, creating an environment conducive to metastatic disease progression. IL-1α is a key senescence-associated (SA) proinflammatory cytokine that acts as a critical upstream regulator of the SA secretory phenotype (SASP). We established that SA shifts in steady-state H₂O₂ and intracellular Ca²⁺ levels caused an increase in IL-1α expression and processing. The increase in intracellular Ca²⁺ promoted calpain activation and increased the proteolytic cleavage of IL-1α. Antioxidants and low oxygen tension prevented SA IL-1α expression and restricted expression of SASP components IL-6 and IL-8. Ca²⁺ chelation or calpain inhibition prevented SA processing of IL-1α and its ability to induce downstream cytokine expression. Conditioned medium from senescent cells treated with antioxidants or Ca²⁺ chelators or cultured in low oxygen markedly reduced the invasive capacity of proximal metastatic cancer cells. In this paracrine fashion, senescent cells promoted invasion by inducing an epithelial-mesenchymal transition, actin reorganization, and cellular polarization of neighboring cancer cells. Collectively, these findings demonstrate how SA alterations in the redox state and Ca²⁺ homeostasis modulate the inflammatory phenotype through the regulation of the SASP initiator IL-1α, creating a microenvironment permissive to tumor invasion.</p><p><b>Background:</b> The senescent microenvironment is permissive to disease progression, and the role of oxidants in this process remains uncharacterized.</p><p><b>Results:</b> Senescent fibroblasts promote tumor invasion through redox/calcium regulation of the cytokine IL-1α.</p><p><b>Conclusion:</b> Senescence-associated oxidants and calcium drive the secretory phenotype, altering the microenvironment.</p><p><b>Significance:</b> Targeting senescent cells with antioxidant-based therapeutics may restrict inflammation and combat age-related disease progression.</p>

Ugolev, Yelena;Segal, Tali;Yaffe, Dana;Gros, Yael;Schuldiner, Shimon

doi: 10.1074/jbc.m113.502971pmid: 24062308

<p>Vesicular monoamine transporter 2 (VMAT2) transports monoamines into storage vesicles in a process that involves exchange of the charged monoamine with two protons. VMAT2 is a member of the DHA12 family of multidrug transporters that belongs to the major facilitator superfamily of secondary transporters. Tetrabenazine (TBZ) is a non-competitive inhibitor of VMAT2 that is used in the treatment of hyperkinetic disorders associated with Huntington disease and Tourette syndrome. Previous biochemical studies suggested that the recognition site for TBZ and monoamines is different. However, the precise mechanism of TBZ interaction with VMAT2 remains unknown. Here we used a random mutagenesis approach and selected TBZ-resistant mutants. The mutations clustered around the lumenal opening of the transporter and mapped to either conserved proline or glycine, or to residues immediately adjacent to conserved proline and glycine. Directed mutagenesis provides further support for the essential role of the latter residues. Our data strongly suggest that the conserved α-helix breaking residues identified in this work play an important role in conformational rearrangements required for TBZ binding and substrate transport. Our results provide a novel insight into the mechanism of transport and TBZ binding by VMAT2.</p><p><b>Background:</b> Transport of monoamines into storage vesicles, mediated by the vesicular monoamine transporter 2 (VMAT2), is inhibited by tetrabenazine via an unknown mechanism.</p><p><b>Results:</b> We identified residues essential for conformational rearrangements required for tetrabenazine binding and substrate transport.</p><p><b>Conclusion:</b> Conformational rearrangements are required for binding of the inhibitor.</p><p><b>Significance:</b> The results provide a novel insight into the mechanism of transport.</p>

Ermert, David;Weckel, Antonin;Agarwal, Vaibhav;Frick, Inga-Maria;Björck, Lars;Blom, Anna M.

doi: 10.1074/jbc.m113.502955pmid: 24064215

<p><i>Streptococcus pyogenes</i> AP1, a strain of the highly virulent M1 serotype, uses exclusively protein H to bind the complement inhibitor C4b-binding protein (C4BP). We found a strong correlation between the ability of AP1 and its isogenic mutants lacking protein H to inhibit opsonization with complement C3b and binding of C4BP. C4BP bound to immobilized protein H or AP1 bacteria retained its cofactor activity for degradation of ¹²⁵I-C4b. Furthermore, C4b deposited from serum onto AP1 bacterial surfaces was processed into C4c/C4d fragments, which did not occur on strains unable to bind C4BP. Recombinant C4BP mutants, which (i) lack certain CCP domains or (ii) have mutations in single aa as well as (iii) mutants with additional aa between different CCP domains were used to determine that the binding is mainly mediated by a patch of positively charged amino acid residues at the interface of domains CCP1 and CCP2. Using recombinant protein H fragments, we narrowed down the binding site to the N-terminal domain A. With a peptide microarray, we identified one single 18-amino acid-long peptide comprising residues 92–109, which specifically bound C4BP. Biacore was used to determine <i>K_D</i> = 6 × 10⁻⁷ m between protein H and a single subunit of C4BP. C4BP binding also correlated with elevated levels of adhesion and invasion to endothelial cells. Taken together, we identified the molecular basis of C4BP-protein H interaction and found that it is not only important for decreased opsonization but also for invasion of endothelial cells by <i>S. pyogenes</i>.</p><p><b>Background:</b> Pathogens such as <i>Streptococcus pyogenes</i> acquire protection by binding complement inhibitors to their surface.</p><p><b>Results:</b> Domain A of protein H binds to C4BP CCP1–2, increasing invasiveness.</p><p><b>Conclusion:</b> Protein H but not M1 protein is the major virulence factor mediating invasion and preventing C3b deposition.</p><p><b>Significance:</b> Understanding molecular details of host pathogen interactions is crucial for development of novel therapeutics.</p>