Gennaro Napolitano

TFEB at a glance

ABSTRACT The transcription factor EB (TFEB) plays a pivotal role in the regulation of basic cellular processes, such as lysosomal biogenesis and autophagy. The subcellular localization and activity of TFEB are regulated by mechanistic target of rapamycin (mTOR)-mediated phosphorylation, which occurs at the lysosomal surface. Phosphorylated TFEB is retained in the cytoplasm, whereas dephosphorylated TFEB translocates to the nucleus to induce the transcription of target genes. Thus, a lysosome-to-nucleus signaling pathway regulates cellular energy metabolism through TFEB. Recently, in vivo studies have revealed that TFEB is also involved in physiological processes, such as lipid catabolism. TFEB has attracted a lot of attention owing to its ability to induce the intracellular clearance of pathogenic factors in a variety of murine models of disease, such as Parkinsons and Alzheimers, suggesting that novel therapeutic strategies could be based on the modulation of TFEB activity. In this Cell Science at a Glance article and accompanying poster, we present an overview of the latest research on TFEB function and its implication in human diseases. Summary: The article discusses the roles of TFEB as a regulator of lysosomal biogenesis and intracellular clearance, and its involvement in human diseases.

Vesicular trafficking through cortical actin during exocytosis is regulated by the Rab27a effector JFC1/Slp1 and the RhoA-GTPase-activating protein Gem-interacting protein.

The mechanism of cytoskeleton remodeling during exocytosis is not well defined. A combination of vesicular dynamics and functional studies shows that the Rab27a effector JFC1 and the RhoA-GTPase–activating protein Gem-interacting protein are necessary for RhoA regulation, actin depolymerization, and vesicular transport through the actin cortex during exocytosis.

Vesicular trafficking through cortical actin during exocytosis is regulated by the Rab27a effector JFC1/Slp1 and the RhoA-GAP GMIP

The mechanism of cytoskeleton remodeling during exocytosis is not well defined. A combination of vesicular dynamics and functional studies shows that the Rab27a effector JFC1 and the RhoA-GTPase–activating protein Gem-interacting protein are necessary for RhoA regulation, actin depolymerization, and vesicular transport through the actin cortex during exocytosis.

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 γ-Tubulin

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 γ-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.

Impairment of chaperone-mediated autophagy leads to selective lysosomal degradation defects in the lysosomal storage disease cystinosis

Metabolite accumulation in lysosomal storage disorders (LSDs) results in impaired cell function and multi‐systemic disease. Although substrate reduction and lysosomal overload‐decreasing therapies can ameliorate disease progression, the significance of lysosomal overload‐independent mechanisms in the development of cellular dysfunction is unknown for most LSDs. Here, we identify a mechanism of impaired chaperone‐mediated autophagy (CMA) in cystinosis, a LSD caused by defects in the cystine transporter cystinosin (CTNS) and characterized by cystine lysosomal accumulation. We show that, different from other LSDs, autophagosome number is increased, but macroautophagic flux is not impaired in cystinosis while mTOR activity is not affected. Conversely, the expression and localization of the CMA receptor LAMP2A are abnormal in CTNS‐deficient cells and degradation of the CMA substrate GAPDH is defective in Ctns−/− mice. Importantly, cysteamine treatment, despite decreasing lysosomal overload, did not correct defective CMA in Ctns−/− mice or LAMP2A mislocalization in cystinotic cells, which was rescued by CTNS expression instead, suggesting that cystinosin is important for CMA activity. In conclusion, CMA impairment contributes to cell malfunction in cystinosis, highlighting the need for treatments complementary to current therapies that are based on decreasing lysosomal overload.

Upregulation of the Rab27a-Dependent Trafficking and Secretory Mechanisms Improves Lysosomal Transport, Alleviates Endoplasmic Reticulum Stress, and Reduces Lysosome Overload in Cystinosis

ABSTRACT Cystinosis is a lysosomal storage disorder caused by the accumulation of the amino acid cystine due to genetic defects in the CTNS gene, which encodes cystinosin, the lysosomal cystine transporter. Although many cellular dysfunctions have been described in cystinosis, the mechanisms leading to these defects are not well understood. Here, we show that increased lysosomal overload induced by accumulated cystine leads to cellular abnormalities, including vesicular transport defects and increased endoplasmic reticulum (ER) stress, and that correction of lysosomal transport improves cellular function in cystinosis. We found that Rab27a was expressed in proximal tubular cells (PTCs) and partially colocalized with the lysosomal marker LAMP-1. The expression of Rab27a but not other small GTPases, including Rab3 and Rab7, was downregulated in kidneys from Ctns−/− mice and in human PTCs from cystinotic patients. Using total internal reflection fluorescence microscopy, we found that lysosomal transport is impaired in Ctns−/− cells. Ctns−/− cells showed significant ER expansion and a marked increase in the unfolded protein response-induced chaperones Grp78 and Grp94. Upregulation of the Rab27a-dependent vesicular trafficking mechanisms rescued the defective lysosomal transport phenotype and reduced ER stress in cystinotic cells. Importantly, reconstitution of lysosomal transport mediated by Rab27a led to decreased lysosomal overload, manifested as reduced cystine cellular content. Our data suggest that upregulation of the Rab27a-dependent lysosomal trafficking and secretory pathways contributes to the correction of some of the cellular defects induced by lysosomal overload in cystinosis, including ER stress.

MUNC13-4 Protein Regulates the Oxidative Response and Is Essential for Phagosomal Maturation and Bacterial Killing in Neutrophils

Background: MUNC13-4 regulates vesicular trafficking, and its deficiency causes immunodeficiency in humans. Results: MUNC13-4 regulates ROS production, phagosomal maturation, and bacterial killing in neutrophils. Conclusion: MUNC13-4 is essential for the neutrophil-dependent innate immune response. Significance: This study identifies MUNC13-4 as a potential target for therapeutic intervention during bacterial infections. Neutrophils use diverse mechanisms to kill pathogens including phagocytosis, exocytosis, generation of reactive oxygen species (ROS), and neutrophil extracellular traps. 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 knock-out (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 Pseudomonas aeruginosa despite normal trafficking of p22phox-expressing vesicles toward 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 neutrophil extracellular trap formation but were unable to compensate for the impaired bacterial killing. Altogether, these findings characterize novel functions of MUNC13-4 in the innate immune response of the neutrophil and have direct implications for the understanding of immunodeficiencies in patients with MUNC13-4 deficiency.

Munc13-4 interacts with syntaxin 7 and regulates late endosomal maturation, endosomal signaling, and TLR9-initiated cellular responses.

Munc13-4 regulates late endosome maturation and Toll-like receptor 9–dependent endosome-initiated signaling through a mechanism that involves interaction with the endocytic SNAREs syntaxin 7 and VAMP8. The results thus provide new mechanistic insight into endosomal maturation and function.

Cystinosin, the small GTPase Rab11, and the Rab7 effector RILP regulate intracellular trafficking of the chaperone-mediated autophagy receptor LAMP2A

The lysosomal storage disease cystinosis, caused by cystinosin deficiency, is characterized by cell malfunction, tissue failure, and progressive renal injury despite cystine-depletion therapies. Cystinosis is associated with defects in chaperone-mediated autophagy (CMA), but the molecular mechanisms are incompletely understood. Here, we show CMA substrate accumulation in cystinotic kidney proximal tubule cells. We also found mislocalization of the CMA lysosomal receptor LAMP2A and impaired substrate translocation into the lysosome caused by defective CMA in cystinosis. The impaired LAMP2A trafficking and localization were rescued either by the expression of wild-type cystinosin or by the disease-associated point mutant CTNS-K280R, which has no cystine transporter activity. Defective LAMP2A trafficking in cystinosis was found to associate with decreased expression of the small GTPase Rab11 and the Rab7 effector RILP. Defective Rab11 trafficking in cystinosis was rescued by treatment with small-molecule CMA activators. RILP expression was restored by up-regulation of the transcription factor EB (TFEB), which was down-regulated in cystinosis. Although LAMP2A expression is independent of TFEB, TFEB up-regulation corrected lysosome distribution and lysosomal LAMP2A localization in Ctns−/− cells but not Rab11 defects. The up-regulation of Rab11, Rab7, or RILP, but not its truncated form RILP-C33, rescued LAMP2A-defective trafficking in cystinosis, whereas dominant-negative Rab11 or Rab7 impaired LAMP2A trafficking. Treatment of cystinotic cells with a CMA activator increased LAMP2A localization at the lysosome and increased cell survival. Altogether, we show that LAMP2A trafficking is regulated by cystinosin, Rab11, and RILP and that CMA up-regulation is a potential clinically relevant mechanism to increase cell survival in cystinosis.

NESCA: A new NEMO/IKKgamma and TRAF6 interacting protein†

NEMO/IKKγ 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. J. Cell. Physiol. 220: 410–417, 2009.