Gennaro Napolitano

Publications (7)65.52 Total impact

• Article: Munc13-4 regulates the oxidative response and is essential for phagosomal maturation and bacterial killing in neutrophils.

  Jlenia Monfregola, Jennifer Linda Johnson, Michael M Meijler, Gennaro Napolitano, Sergio Daniel Catz
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  ABSTRACT: Neutrophils use diverse mechanisms to kill pathogens including phagocytosis, exocytosis, the generation of reactive oxygen species (ROS) and neutrophil extracellular traps (NETs). These mechanisms rely on their ability to mobilize intracellular organelles and to deliver granular cargoes to specific cellular compartments or into the extracellular milieu, but the molecular mechanisms regulating vesicular trafficking in neutrophils are not well understood. Munc13-4 is a Rab27a effector that coordinates exocytosis in hematopoietic cells and its deficiency is associated with the human immunodeficiency familial hemophagocytic lymphohistiocytosis type 3. In this work, we have established an essential role for Munc13-4 in selective vesicular trafficking, phagosomal maturation and intracellular bacterial killing in neutrophils. Using neutrophils from Munc13-4-knockout (KO) mice, we show that Munc13-4 is necessary for the regulation of p22phox-expressing granule trafficking to the plasma membrane and regulates extracellular ROS production. Munc13-4 was also essential for the regulation of intracellular ROS production induced by Pseudomona aeruginosa despite normal trafficking of p22phox-expressing vesicles towards the phagosome. Importantly, in the absence of Munc13-4, phagosomal maturation was impaired as observed by the defective delivery of azurophilic granules and multivesicular bodies to the phagosome. Significantly, this mechanism was intact in Rab27a KO neutrophils. Intracellular bacterial killing was markedly impaired in Munc13-4 KO neutrophils. Munc13-4-deficient cells showed a significant increase in NET formation but were unable to compensate for the impaired bacterial killing. Altogether, these findings characterize novel functions of Munc13-4 in the neutrophil innate immune response and have direct implications for the understanding of immunodeficiencies in patients with Munc13-4 deficiency.
  Journal of Biological Chemistry 10/2012; · 4.77 Impact Factor
• Article: Vesicular trafficking through cortical actin during exocytosis is regulated by the Rab27a effector JFC1/Slp1 and the RhoA-GTPase-activating protein Gem-interacting protein.

  Jennifer L Johnson, Jlenia Monfregola, Gennaro Napolitano, William B Kiosses, Sergio D Catz
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  ABSTRACT: Cytoskeleton remodeling is important for the regulation of vesicular transport associated with exocytosis, but a direct association between granular secretory proteins and actin-remodeling molecules has not been shown, and this mechanism remains obscure. Using a proteomic approach, we identified the RhoA-GTPase-activating protein Gem-interacting protein (GMIP) as a factor that associates with the Rab27a effector JFC1 and modulates vesicular transport and exocytosis. GMIP down-regulation induced RhoA activation and actin polymerization. Importantly, GMIP-down-regulated cells showed impaired vesicular transport and exocytosis, while inhibition of the RhoA-signaling pathway induced actin depolymerization and facilitated exocytosis. We show that RhoA activity polarizes around JFC1-containing secretory granules, suggesting that it may control directionality of granule movement. Using quantitative live-cell microscopy, we show that JFC1-containing secretory organelles move in areas near the plasma membrane deprived of polymerized actin and that dynamic vesicles maintain an actin-free environment in their surroundings. Supporting a role for JFC1 in RhoA inactivation and actin remodeling during exocytosis, JFC1 knockout neutrophils showed increased RhoA activity, and azurophilic granules were unable to traverse cortical actin in cells lacking JFC1. We propose that during exocytosis, actin depolymerization commences near the secretory organelle, not the plasma membrane, and that secretory granules use a JFC1- and GMIP-dependent molecular mechanism to traverse cortical actin.
  Molecular biology of the cell 03/2012; 23(10):1902-16. · 5.98 Impact Factor
• Article: Crystal structure of inhibitor of κB kinase β.

  Guozhou Xu, Yu-Chih Lo, Qiubai Li, Gennaro Napolitano, Xuefeng Wu, Xuliang Jiang, Michel Dreano, Michael Karin, Hao Wu
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  ABSTRACT: Inhibitor of κB (IκB) kinase (IKK) phosphorylates IκB proteins, leading to their degradation and the liberation of nuclear factor κB for gene transcription. Here we report the crystal structure of IKKβ in complex with an inhibitor, at a resolution of 3.6 Å. The structure reveals a trimodular architecture comprising the kinase domain, a ubiquitin-like domain (ULD) and an elongated, α-helical scaffold/dimerization domain (SDD). Unexpectedly, the predicted leucine zipper and helix-loop-helix motifs do not form these structures but are part of the SDD. The ULD and SDD mediate a critical interaction with IκBα that restricts substrate specificity, and the ULD is also required for catalytic activity. The SDD mediates IKKβ dimerization, but dimerization per se is not important for maintaining IKKβ activity and instead is required for IKKβ activation. Other IKK family members, IKKα, TBK1 and IKK-i, may have a similar trimodular architecture and function.
  Nature 03/2011; 472(7343):325-30. · 36.28 Impact Factor
• Article: Functional characterization of Wiskott-Aldrich syndrome protein and scar homolog (WASH), a bi-modular nucleation-promoting factor able to interact with biogenesis of lysosome-related organelle subunit 2 (BLOS2) and gamma-tubulin.

  Jlenia Monfregola, Gennaro Napolitano, Michele D'Urso, Pekka Lappalainen, Matilde Valeria Ursini
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  ABSTRACT: The Arp2/3 complex is essential for actin filament nucleation in a variety of cellular processes. The activation of the Arp2/3 complex is mediated by nucleation-promoting factors, such as the Wiskott-Aldrich syndrome family proteins, which share a WCA (WH2 domain, central region, acidic region) catalytic module at the C-terminal region, required for Arp2/3 activation, but diverge at the N-terminal region, required for binding to specific activators. Here, we report the characterization of WASH, a new member of the WAS family that has nucleation-promoting factor activity and recently has been demonstrated to play a role in endosomal sorting. We found that overexpression of the WASH-WCA domain induced disruption of the actin cytoskeleton, whereas overexpression of full-length WASH in mammalian cells did not affect stress fiber organization. Furthermore, our analysis has revealed that nerve growth factor treatment of PC12 cells overexpressing full-length WASH leads to disruption of the actin cytoskeleton. We have also found that WASH interacts through its N-terminal region with BLOS2, a centrosomal protein belonging to the BLOC-1 complex that functions as a scaffolding factor in the biogenesis of lysosome-related organelles. In addition to BLOS2, WASH also interacts with centrosomal gamma-tubulin and with pallidin, an additional component of the BLOC-1 complex. Collectively, our data propose that WASH is a bimodular protein in which the C terminus is involved in Arp2/3-mediated actin nucleation, whereas the N-terminal portion is required for its regulation and localization in the cells. Moreover, our data suggest that WASH is also a component of the BLOC-1 complex that is associated with the centrosomes.
  Journal of Biological Chemistry 03/2010; 285(22):16951-7. · 4.77 Impact Factor
• Article: Sphingolipids: the oil on the TRAFire that promotes inflammation.

  Gennaro Napolitano, Michael Karin
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  ABSTRACT: Tumor necrosis factor receptor (TNFR)-associated factors (TRAFs) control inflammatory and immune responses by acting downstream of TNFRs and Toll-like receptors (TLRs). TRAF2 in particular has been extensively studied for its involvement in signaling by TNF-α, the classic inflammatory cytokine. Because it has a RING finger, it has been suggested that TRAF2 acts as an E3 ubiquitin ligase that catalyzes the noncanonical Lys-63 (K63)-linked polyubiquitination of receptor-induced protein 1 (RIP1), which is an essential event in the activation of the IκB kinase complex and consequently nuclear factor κB. Furthermore, TRAF2 itself is subject to K63-linked polyubiquitination, a modification that is rapidly induced upon receptor ligation and was interpreted to be the result of self-ubiquitination. However, formal evidence that TRAF2 is an active E3 ubiquitin ligase was lacking. New evidence shows that sphingosine-1-phosphate (S1P), a sphingolipid that is synthesized during inflammatory responses, is an essential cofactor for TRAF2 ubiquitin ligase activity. S1P binds to TRAF2 and promotes TRAF2-mediated K63-linked RIP1 polyubiquitination, providing direct evidence that TRAF2 is an active E3 ubiquitin ligase and also introducing lipid second messengers into the realm of TNFR and TLR signaling.
  Science Signaling 01/2010; 3(141):pe34. · 7.50 Impact Factor
• Article: NESCA: a new NEMO/IKKgamma and TRAF6 interacting protein.

  Gennaro Napolitano, Serena Mirra, Jlenia Monfregola, Alfonso Lavorgna, Antonio Leonardi, Matilde Valeria Ursini
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  ABSTRACT: NEMO/IKKgamma is the essential regulatory subunit of the IkB Kinase (IKK) complex, required for the activation of Nuclear Factor kB (NF-kB) in many physiological processes such as inflammation, immunity, apoptosis, or development. NEMO works at a converging point of the NF-kB pathway as it interacts with upstream signaling molecules to orchestrate its activation. Here we report on the identification of a novel NEMO-interacting protein, NESCA, an adapter molecule previously shown to be involved in the NGF-pathway via the TrkA receptor. We demonstrated that NESCA and NEMO interact by their N-terminal region. Beside to NEMO, we revealed that NESCA directly associates to the E3 ubiquitin ligase TRAF6, which in turn catalyzes NESCA polyubiquitination. Finally, we demonstrated that NESCA overexpression strongly inhibits TRAF6-mediated polyubiquitination of NEMO. In summary, our results highlight that NESCA represents a novel missing link in the NEMO-mediated NF-kB activation pathway.
  Journal of Cellular Physiology 05/2009; 220(2):410-7. · 3.87 Impact Factor
• Article: Functional characterization of the TMLH gene: promoter analysis, in situ hybridization, identification and mapping of alternative splicing variants.

  Jlenia Monfregola, Gennaro Napolitano, Ivan Conte, Armando Cevenini, Carmen Migliaccio, Michele D'Urso, Matilde Valeria Ursini
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  ABSTRACT: Carnitine is a molecule with well-documented pleiotropic functions whose biosynthesis involves four catalytic steps. Here, we report a detailed analysis of the expression and transcriptional control of TMLH gene, which codifies for the first enzyme of carnitine biosynthesis. TMLH maps at the extreme end of Xq28, a chromosomal region of high genomic instability. By 5' and 3' RACE, we identified and mapped two alternative 5' TMLH first exons and seven alternative 3'-splice variants, which are spread over a genomic region of about 250 kb. While the two alternative 5' exons have different expression profiles, all the 3' alternative forms are ubiquitously expressed. Reporter assays revealed that the 3'-UTRs of each TMLH isoform might influence its own expression at post-transcriptional level. In addition, we identified a highly conserved promoter region of TMLH. Functional analysis of this region showed the presence of a CpG island, whose methylation-status could control the level of TMLH transcription. Finally, by mRNA in situ hybridization, we found that TMLH expression is present at E12.5 dpc in the mouse liver, lung and brain, and is then maintained in the postnatal brain with a specific neuronal pattern. Collectively, our data highlight a tight transcriptional and post-transcriptional control of TMLH expression.
  Gene 07/2007; 395(1-2):86-97. · 2.34 Impact Factor