Pseudomonas aeruginosa inhibits endocytic recycling of CFTR in polarized human airway epithelial cells

The most common mutation in the CFTR gene in individuals with cystic fibrosis (CF), F508, leads to the absence of CFTR Cl^– channels in the apical plasma membrane, which in turn results in impairment of mucociliary clearance, the first line of defense against inhaled bacteria. Pseudomonas aeruginosa is particularly successful at colonizing and chronically infecting the lungs and is responsible for the majority of morbidity and mortality in patients with CF. Rescue of F508-CFTR by reduced temperature or chemical means reveals that the protein is at least partially functional as a Cl^– channel. Thus current research efforts have focused on identification of drugs that restore the presence of CFTR in the apical membrane to alleviate the symptoms of CF. Because little is known about the effects of P. aeruginosa on CFTR in the apical membrane, whether P. aeruginosa will affect the efficacy of new drugs designed to restore the plasma membrane expression of CFTR is unknown. Accordingly, the objective of the present study was to determine whether P. aeruginosa affects CFTR-mediated Cl^– secretion in polarized human airway epithelial cells. We report herein that a cell-free filtrate of P. aeruginosa reduced CFTR-mediated transepithelial Cl^– secretion by inhibiting the endocytic recycling of CFTR and thus the number of WT-CFTR and F508-CFTR Cl^– channels in the apical membrane in polarized human airway epithelial cells. These data suggest that chronic infection with P. aeruginosa may interfere with therapeutic strategies aimed at increasing the apical membrane expression of F508-CFTR. cystic fibrosis     

The deubiquitinating enzyme USP1 regulates the Fanconi anemia pathway

Protein ubiquitination and deubiquitination are dynamic processes implicated in the regulation of numerous cellular pathways. Monoubiquitination of the Fanconi anemia (FA) protein FANCD2 appears to be critical in the repair of DNA damage because many of the proteins that are mutated in FA are required for FANCD2 ubiquitination. By screening a gene family RNAi library, we identify the deubiquitinating enzyme USP1 as a novel component of the Fanconi anemia pathway. Inhibition of USP1 leads to hyperaccumulation of monoubiquitinated FANCD2. Furthermore, USP1 physically associates with FANCD2, and the proteins colocalize in chromatin after DNA damage. Finally, analysis of crosslinker-induced chromosomal aberrations in USP1 knockdown cells suggests a role in DNA repair. We propose that USP1 deubiquitinates FANCD2 when cells exit S phase or recommence cycling after a DNA damage insult and may play a critical role in the FA pathway by recycling FANCD2.     

The deubiquitinating enzyme UCH-L3 regulates the apical membrane recycling of the epithelial sodium channel

The epithelial sodium channel (ENaC) is ubiquitinated by the E3 ligase Nedd4-2 at the apical membranes of polarized cortical collecting duct (CCD) epithelial cells. This leads to ENaC endocytosis and possible degradation. Because ENaC is known to recycle at the apical membranes of CCD cells, deubiquitinating enzymes (DUBs) are likely involved in regulating ENaC surface density by facilitating ENaC recycling as opposed to degradation. Using a chemical probe approach to tag active DUBs, we identified ubiquitin C-terminal hydrolase (UCH) isoform L3 as the predominant DUB in endosomal compartments of CCD cells. Blocking UCH-L3 activity or reducing its expression by selective knockdown increased ENaC ubiquitination and resulted in its removal from the apical membranes of CCD cells. Functionally this caused a rapid reduction in transepithelial Na(+) currents across the CCD epithelia. Surface biotinylation demonstrated the loss of ENaC from the apical surface when UCH-L3 was inhibited. Whole cell or apical surface immunoprecipitation demonstrated increased ENaC ubiquitination with UCH-L3 inhibition. This constitutes a novel function for UCH in epithelia and in the regulation of ion channels and demonstrates the dynamic regulation of apically located ENaC by recycling, which is facilitated by this DUB.     

The deubiquitinating enzyme USP2a regulates the p53 pathway by targeting Mdm2

Mdm2 is an E3 ubiquitin ligase that promotes its own ubiquitination and also ubiquitination of the p53 tumour suppressor. In a bacterial two-hybrid screen, using Mdm2 as bait, we identified an Mdm2-interacting peptide that bears sequence similarity to the deubiquitinating enzyme USP2a. We have established that full-length USP2a associates with Mdm2 in cells where it can deubiquitinate Mdm2 while demonstrating no deubiquitinating activity towards p53. Ectopic expression of USP2a causes accumulation of Mdm2 in a dose-dependent manner and consequently promotes Mdm2-mediated p53 degradation. This differs from the behaviour of HAUSP, which deubiquitinates p53 in addition to Mdm2 and thus protects p53 from Mdm2-mediated degradation. We further demonstrate that suppression of endogenous USP2a destabilises Mdm2 and causes accumulation of p53 protein and activation of p53. Our data identify the deubiquitinating enzyme USP2a as a novel regulator of the p53 pathway that acts through its ability to selectively target Mdm2.     

Structure and mechanisms of the proteasome-associated deubiquitinating enzyme USP14

The ubiquitin-specific processing protease (UBP) family of deubiquitinating enzymes plays an essential role in numerous cellular processes. Mammalian USP14 (Ubp6 in yeast) is unique among known UBP enzymes in that it is activated catalytically upon specific association with the 26S proteasome. Here, we report the crystal structures of the 45-kDa catalytic domain of USP14 in isolation and in a complex with ubiquitin aldehyde, which reveal distinct structural features. In the absence of ubiquitin binding, the catalytic cleft leading to the active site of USP14 is blocked by two surface loops. Binding by ubiquitin induces a significant conformational change that translocates the two surface loops thereby allowing access of the ubiquitin C-terminus to the active site. These structural observations, in conjunction with biochemical characterization, identify important regulatory mechanisms for USP14.     

The deubiquitinating enzyme USP20 stabilizes ULK1 and promotes autophagy initiation

Autophagy begins with the formation of autophagosomes, a process that depends on the activity of the serine/threonine kinase ULK1 (hATG1). Although earlier studies indicated that ULK1 activity is regulated by dynamic polyubiquitination, the deubiquitinase involved in the regulation of ULK1 remained unknown. In this study, we demonstrate that ubiquitin‐specific protease 20 (USP20) acts as a positive regulator of autophagy initiation through stabilizing ULK1. At basal state, USP20 binds to and stabilizes ULK1 by removing the ubiquitin moiety, thereby interfering with the lysosomal degradation of ULK1. The stabilization of basal ULK1 protein levels is required for the initiation of starvation‐induced autophagy, since the depletion of USP20 by RNA interference inhibits LC3 puncta formation, a marker of autophagic flux. At later stages of autophagy, USP20 dissociates from ULK1, resulting in enhanced ULK1 degradation and apoptosis. Taken together, our findings provide the first evidence that USP20 plays a crucial role in autophagy initiation by maintaining the basal expression level of ULK1.     

The deubiquitinating enzyme USP36 controls selective autophagy activation by ubiquitinated proteins

Initially described as a nonspecific degradation process induced upon starvation, autophagy is now known also to be involved in the degradation of specific ubiquitinated substrates such as mitochondria, bacteria and aggregated proteins, ensuring crucial functions in cell physiology and immunity. We report here that the deubiquitinating enzyme USP36 controls selective autophagy activation in Drosophila and in human cells. We show that dUsp36 loss of function autonomously inhibits cell growth while activating autophagy. Despite the phenotypic similarity, dUSP36 is not part of the TOR signaling pathway. Autophagy induced by dUsp36 loss of function depends on p62/SQSTM1, an adaptor for delivering cargo marked by polyubiquitin to autophagosomes. Consistent with p62 requirement, dUsp36 mutant cells display nuclear aggregates of ubiquitinated proteins, including Histone H2B, and cytoplasmic ubiquitinated proteins; the latter are eliminated by autophagy. Importantly, USP36 function in p62-dependent selective autophagy is conserved in human cells. Our work identifies a novel, crucial role for a deubiquitinating enzyme in selective autophagy.     

The deubiquitinating enzyme USP36 controls selective autophagy activation by ubiquitinated proteins

Initially described as a nonspecific degradation process induced upon starvation, autophagy is now known also to be involved in the degradation of specific ubiquitinated substrates such as mitochondria, bacteria and aggregated proteins, ensuring crucial functions in cell physiology and immunity. We report here that the deubiquitinating enzyme USP36 controls selective autophagy activation in Drosophila and in human cells. We show that dUsp36 loss of function autonomously inhibits cell growth while activating autophagy. Despite the phenotypic similarity, dUSP36 is not part of the TOR signaling pathway. Autophagy induced by dUsp36 loss of function depends on p62/SQSTM1, an adaptor for delivering cargo marked by polyubiquitin to autophagosomes. Consistent with p62 requirement, dUsp36 mutant cells display nuclear aggregates of ubiquitinated proteins, including Histone H2B, and cytoplasmic ubiquitinated proteins; the latter are eliminated by autophagy. Importantly, USP36 function in p62-dependent selective autophagy is conserved in human cells. Our work identifies a novel, crucial role for a deubiquitinating enzyme in selective autophagy.     

Rme-1 regulates the distribution and function of the endocytic recycling compartment in mammalian cells

RME-1 is an Eps15-homology (EH)-domain protein that was identified in a genetic screen for endocytosis genes in Caenorhabditis elegans. When expressed in a CHO cell line, the worm RME-1 protein and a mouse homologue are both associated with the endocytic recycling compartment. Here we show that expression of a dominant-negative construct with a point mutation near the EH domain results in redistribution of the endocytic recycling compartment and slowing down of transferrin receptor recycling. The delivery of a TGN38 chimaeric protein to the trans-Golgi network is also slowed down. The function of Rme-1 in endocytic recycling is evolutionarily conserved in metazoans as shown by the protein's properties in C. elegans.     

The deubiquitinating protein USP24 interacts with DDB2 and regulates DDB2 stability

Damage-specific DNA-binding protein 2 (DDB2) was first isolated as a subunit of the UV-DDB heterodimeric complex that is involved in DNA damage recognition in the nucleotide excision repair pathway (NER). DDB2 is required for efficient repair of CPDs in chromatin and is a component of the CRL4^DDB2 E3 ligase that targets XPC, histones and DDB2 itself for ubiquitination. In this study, a yeast two-hybrid screening of a human cDNA library was performed to identify potential DDB2 cellular partners. We identified a deubiquitinating enzyme, USP24, as a likely DDB2-interacting partner. Interaction between DDB2 and USP24 was confirmed by co-precipitation. Importantly, knockdown of USP24 in two human cell lines decreased the steady-state levels of DDB2, indicating that USP24-mediated DDB2 deubiquitination prevents DDB2 degradation. In addition, we demonstrated that USP24 can cleave an ubiquitinated form of DDB2 in vitro. Taken together, our results suggest that the ubiquitin-specific protease USP24 is a novel regulator of DDB2 stability.     

CED-10/Rac1 regulates endocytic recycling through the RAB-5 GAP TBC-2

Rac1 is a founding member of the Rho-GTPase family and a key regulator of membrane remodeling. In the context of apoptotic cell corpse engulfment, CED-10/Rac1 acts with its bipartite guanine nucleotide exchange factor, CED-5/Dock180-CED-12/ELMO, in an evolutionarily conserved pathway to promote phagocytosis. Here we show that in the context of the Caenorhabditis elegans intestinal epithelium CED-10/Rac1, CED-5/Dock180, and CED-12/ELMO promote basolateral recycling. Furthermore, we show that CED-10 binds to the RAB-5 GTPase activating protein TBC-2, that CED-10 contributes to recruitment of TBC-2 to endosomes, and that recycling cargo is trapped in recycling endosomes in ced-12, ced-10, and tbc-2 mutants. Expression of GTPase defective RAB-5(Q78L) also traps recycling cargo. Our results indicate that down-regulation of early endosome regulator RAB-5/Rab5 by a CED-5, CED-12, CED-10, TBC-2 cascade is an important step in the transport of cargo through the basolateral recycling endosome for delivery to the plasma membrane.     

A role for the deubiquitinating enzyme USP28 in control of the DNA-damage response

The Chk2-p53-PUMA pathway is a major regulator of DNA-damage-induced apoptosis in response to double-strand breaks in vivo. Through analysis of 53BP1 complexes we have discovered a new ubiquitin protease, USP28, which regulates this pathway. Using a human cell line that faithfully recapitulated the Chk2-p53-PUMA pathway, we show that USP28 is required to stabilize Chk2 and 53BP1 in response to DNA damage. In this cell line, both USP28 and Chk2 are required for DNA-damage-induced apoptosis, and they accomplish this in part through regulation of the p53 induction of proapoptotic genes like PUMA. Our studies implicate DNA-damage-induced ubiquitination and deubiquitination as a major regulator of the DNA-damage response for Chk2, 53BP1, and a number of other proteins in the DNA-damage checkpoint pathway, including several mediators, such as Mdc1, Claspin, and TopBP1.     

The deubiquitinating protein USP24 interacts with DDB2 and regulates DDB2 stability

Damage-specific DNA-binding protein 2 (DDB2) was first isolated as a subunit of the UV-DDB heterodimeric complex that is involved in DNA damage recognition in the nucleotide excision repair pathway (NER). DDB2 is required for efficient repair of CPDs in chromatin and is a component of the CRL4^DDB2 E3 ligase that targets XPC, histones and DDB2 itself for ubiquitination. In this study, a yeast two-hybrid screening of a human cDNA library was performed to identify potential DDB2 cellular partners. We identified a deubiquitinating enzyme, USP24, as a likely DDB2-interacting partner. Interaction between DDB2 and USP24 was confirmed by co-precipitation. Importantly, knockdown of USP24 in two human cell lines decreased the steady-state levels of DDB2, indicating that USP24-mediated DDB2 deubiquitination prevents DDB2 degradation. In addition, we demonstrated that USP24 can cleave an ubiquitinated form of DDB2 in vitro. Taken together, our results suggest that the ubiquitin-specific protease USP24 is a novel regulator of DDB2 stability.     

The deubiquitinating enzyme Ubp1 affects sorting of the ATP-binding cassette-transporter Ste6 in the endocytic pathway

Deubiquitinating enzymes (Dubs) are potential regulators of ubiquitination-dependent processes. Here, we focus on a member of the yeast ubiquitin-specific processing protease (Ubp) family, the Ubp1 protein. We could show that Ubp1 exists in two forms: a longer membrane-anchored form (mUbp1) and a shorter soluble form (sUbp1) that seem to be independently expressed from the same gene. The membrane-associated mUbp1 variant could be localized to the endoplasmic reticulum (ER) membrane by sucrose density gradient centrifugation and by immunofluorescence microscopy. Overexpression of the soluble Ubp1 variant stabilizes the ATP-binding cassette-transporter Ste6, which is transported to the lysosome-like vacuole for degradation, and whose transport is regulated by ubiquitination. Ste6 stabilization was not the result of a general increase in deubiquitination activity, because overexpression of Ubp1 had no effect on the degradation of the ER-associated degradation substrate carboxypeptidase Y^* and most importantly on Ste6 ubiquitination itself. Also, overexpression of another yeast Dub, Ubp3, had no effect on Ste6 turnover. This suggests that the Ubp1 target is a component of the protein transport machinery. On Ubp1 overexpression, Ste6 accumulates at the cell surface, which is consistent with a role of Ubp1 at the internalization step of endocytosis or with enhanced recycling to the cell surface from an internal compartment.     

The Doa4 deubiquitinating enzyme is functionally linked to the vacuolar protein-sorting and endocytic pathways

The Saccharomyces cerevisiae DOA4 gene encodes a deubiquitinating enzyme that is required for rapid degradation of ubiquitin-proteasome pathway substrates. Both genetic and biochemical data suggest that Doa4 acts in this pathway by facilitating ubiquitin recycling from ubiquitinated intermediates targeted to the proteasome. Here we describe the isolation of 12 spontaneous extragenic suppressors of the doa4-1 mutation; these involve seven different genes, six of which were cloned. Surprisingly, all of the cloned DID (Doa4-independent degradation) genes encode components of the vacuolar protein-sorting (Vps) pathway. In particular, all are class E Vps factors, which function in the maturation of a late endosome/prevacuolar compartment into multivesicular bodies that then fuse with the vacuole. Four of the six Did proteins are structurally related, suggesting an overlap in function. In wild-type and several vps strains, Doa4-green fluorescent protein displays a cytoplasmic/nuclear distribution. However, in cells lacking the Vps4/Did6 ATPase, a large fraction of Doa4-green fluorescent protein, like several other Vps factors, concentrates at the late endosome-like class E compartment adjacent to the vacuole. These results suggest an unanticipated connection between protein deubiquitination and endomembrane protein trafficking in which Doa4 acts at the late endosome/prevacuolar compartment to recover ubiquitin from ubiquitinated membrane proteins en route to the vacuole.     

Basal chloride currents in murine airway epithelial cells: modulation by CFTR

We have isolatedciliated respiratory cells from the nasal epithelium of wild-type andcystic fibrosis (CF) null mice and used the patch-clamp technique toinvestigate their basal conductances. Current-clamp experiments onunstimulated cells indicated the presence ofK⁺ andCl conductances and, undercertain conditions, a small Na⁺conductance. Voltage-clamp experiments revealed three distinct Cl conductances.I_tv-indep wastime and voltage independent with a linear current-voltage(I-V)plot; I_v-actexhibited activation at potentials greater than ±50 mV, giving anS-shapedI-Vplot; andI_hyp-act wasactivated by hyperpolarizing potentials and had an inwardly rectifiedI-Vplot. The current density sequence was I_hyp-act = I_v-act  I_tv-indep. Theseconductances hadCl-to-N-methyl-D-glucaminecation permeability ratios of between 2.8 and 10.3 and were unaffectedby tamoxifen, flufenamate, glibenclamide, DIDS, and5-nitro-2-(3-phenylpropylamino) benzoic acid but were inhibited byZn²⁺ andGd³⁺.I_tv-indep andI_v-act werepresent in wild-type and CF cells at equal density and frequency.However, I_hyp-actwas detected in only 3% of CF cells compared with 26% of wild-typecells, suggesting that this conductance may be modulated by cysticfibrosis transmembrane conductance regulator (CFTR).

     

CFTR targeting in epithelial cells

We used polarized and nonpolarized colonic cell lines (HT-29) to correlate CFTR function and expression with epithelial cell morphogenesis. Unpolarized cells express levels of CFTR mRNA and protein that are equivalent to those observed in polarized cells, and the extent of CFTR glycosylation is also similar. Despite these similarities in CFTR expression, the polarized cells secreted Cl in response tocAMP, but there was nocAMP-stimulated Cl conductance response in the unpolarized cells. In the polarized cells, CFTR is localized in the apical membrane domain, but in unpolarized cells the protein is retained at a perinuclear location. These findings indicate that a peripheral targeting mechanism, distal to the Golgi cisternae, controls the progression of N-linked glycoproteins like CFTR to the apical membrane. This targeting process does not become active until epithelial cells polarize. It may determine whether mutant forms of CFTR are targeted to the apical membrane.