Degradation of CFTR-F508del By the Ubiquitin E2 Conjugating Enzyme UBE 2L6 and the E3 Ligase RNF19B CURRENT POSTED

Cystic Fibrosis (CF) is the most common, lethal autosomal-recessive disorder, and is caused by mutations in the cystic fibrosis transmembrane conductance regulator protein (CFTR), an anion channel that is found in epithelial cells lining multiple organs. The most common mutation of CFTR is deletion of phenylalanine at position 508 (CFTR-F508del), which produces a misfolded protein. This misfolded protein is confined in the endoplasmic reticulum (ER), ubiquitinated and signaled for degradation via the cytosolic proteasome. Previous studies demonstrating experimental restoration of CFTR-F508del (F508del) trafficking to the plasma membrane showed partial function of the chloride channel, raising therapeutic speculations. However, many mechanisms that underly its degradation is still unclear. We discovered that the E2 conjugating enzyme, UBE 2L6, aids in the degradation of F508del. We also show that UBE 2L6’s interacting E3 ligase, RNF19B, also assists in F508del degradation. We used siRNA-mediated silencing of endogenous UBE 2L6 and RNF19B in the CF human bronchial epithelial (HBE) cell line CFBE-F508del to demonstrate that there is an increase in F508del-CFTR expression. We also co-expressed UBE 2L6 and RNF 19B with F508del in HEK293 cells that demonstrated a decrease in F508del compared to control. Cycloheximide-chase (CHX) experiments using HEK 293 cells overexpressing RNF19B and F508del showed that there was a decrease in F508del half-life. Lastly, using siRNA-mediated silencing of endogenous UBE 2L6 and RNF19B in CFBE-F508del cells increased forskolin-stimulated short-circuit currents. Interestingly, there seemed to be cooperatively larger increases in short-circuit currents in CFBE-F508del cells that were silenced with UBE 2L6 or RNF19B were treated for These data suggest that there is an additional mechanism where the E2 conjugating enzyme, UBE 2L6, and the E3 ubiquitin ligase, RNF19B, is responsible for the ubiquitination and subsequent proteasomal degradation of the misfolded F508del. This study presents a novel therapeutic strategy to synergistically restore the function of F508del. membrane of the cells. Our results provide a clearer landscape of the molecular mechanism that underlies the proteasomal degradation of F508del through UBE 2L6 and RNF19B.


Conclusion
These data suggest that there is an additional mechanism where the E2 conjugating enzyme, UBE 2L6, and the E3 ubiquitin ligase, RNF19B, is responsible for the ubiquitination and subsequent proteasomal degradation of the misfolded F508del. This study presents a novel therapeutic strategy to synergistically restore the function of F508del.

Background
Cystic fibrosis (CF) is the most common, lethal autosomal recessive disease caused by a mutation in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) [1,2]. CFTR is a cAMP regulated chloride channel that is a member of the ABC (ATPbinding cassette) superfamily. The channel is found at the apical membranes of epithelial cells lining the airway and other organs [3]. The most common mutation amongst CF patients is the deletion of the phenylalanine residue at position 508 (CFTR-F508del). CFTR-F508del (F508del) is unable to achieve a native, folded state that is required for the protein to export from the endoplasmic reticulum (ER) to the apical membrane of epithelial cells [4]. More than 90% of CF patients have at least one copy of the F508del allele. F508del is synthesized in the ER, and it is trapped in the ER because it is unable to obtain its native state [5][6][7][8]. This trapped F508del is then recognized by the ubiquitinproteasome system (UPS), where it is polyubiquitinated, translocated to the cytosol and is eventually degraded by the 26S proteasome [8][9][10]. This network is generally called ERassociated degradation pathway (ERAD) [11]. Studies have previously shown that F508del has a folding defect that can be adjusted by chemicals to promote proper folding and introducing channel function at the plasma membrane [8,10,12]. The ER folding pathway of CFTR is tightly coordinated with the ERAD pathway whereby misfolded CFTR are targeted to the cytosolic proteasome [13]. This is extremely important because 4 pharmacological agents that may help with F508del folding or the blocking of its degradation may help CF patients.
A small amount of F508del can return to its proper folding pathway with the help of correctors, such as VX-809 [14,15]. Experimental restoration of F508del trafficking to the plasma membrane results in partial function of the chloride channel, raising therapeutic speculations [10,12]. The UPS is essential in the degradation of F508del. Ubiquitin is covalently attached to F508del by an isopeptide linkage between the c-terminal glycine of ubiquitin and amino group of lysine of the substrate (F508del). This is done through a cascade system, which consists of E1 activating enzyme, E2 conjugating enzymes, and E3 ligase enzymes [16]. Ubiquitination of F508del occurs when the E3 ligase binds to the F508del and E2 enzyme that is thioesterifed with ubiquitin [17,18], and brings both to close proximity so that ubiquitin is transferred from the E2 to the substrate. This process occurs several times to the point a polyubiquitin tail is formed on F508del, which is then a signal for the protein to be degraded by the proteasome [19]. Ubiquitin E3 ligases are important enzymes that transfer ubiquitin from an E2 ubiquitin-conjugating enzyme to the mutant CFTR. E3 ligases are important for they show substrate specificity for ubiquitin transfer to the target proteins. To date, there are little studies in mammalian cells that identify E2 conjugating enzymes and ER-localized E3 ubiquitin ligases that function in the degradation of F508del.
About 60% of newly synthesized wild type-CFTR (WT-CFTR) and almost 99% of F508del is degraded by the UPS [20]. Therefore, there seems to be specific checkpoints set in place within the ER that allows F508del to stray apart from its WT counterpart during early stages of CFTR biogenesis. We believe that ER resident E3 ligases and there corresponding E2 conjugating enzymes are the initial checkpoint proteins that may be responsible for the divergence of WT-CFTR and F508del during CFTR biogenesis.
Previous studies have identified the E3 RMA1 and the E2, UBC6e cooperate to degrade both CFTR and F508del. [21,22] However, there seems to be other mechanisms that facilitate F508del degradation because these studies still illustrate a significant amount of F508del that is being degraded, even when the above-mentioned proteins were silenced.
The E2 UBCH5 forms an E3 complex with CHIP and the E3 ligase Hdj2 to ubiquitinate F508del [5,22,23]. When this complex is destroyed, F508del is still not able to traffic out of the ER, but instead is degraded. Therefore, other E2/E3 complexes may exist in F508del degradation and other mechanisms need to be sorted out to determine other potential therapeutic approaches to help prevent F508del degradation.
Through a literature search of E2 conjugating enzymes that seem to interact with ER membrane-bound E3 ligases, we discovered the E2 conjugating enzyme UBE 2L6, whom has its role in the ubiquitin conjugating pathway [24][25][26] This E2 interacts with RING E3 ligases that are bound to the ER such as RNF122, RNF19B, and also RNF19A [27]. This is important because it has already been shown that certain E3 ligases, such as RMA1, acts in the ER membrane to identify the misfolded F508del [21]. We were able to determine that the E2 UBE2L6 was a key player in F508del degradation, and its interacting E3 ligase, RNF19B, also mediates F508del. In this study, we explore the possible roles of UBE 2L6 and the E3 ligase RNF19B in the proteasomal degradation of F508del. We determined that both proteins degraded F508del when overexpressed. Also, when both proteins were knocked down through siRNA-mediated silencing in HBE cells, there was an increase in expression of F508del, which was further enhanced when treated with VX-809. The same cells treated with VX-809 also had more chloride channel activity at apical membrane of the cells. Our results provide a clearer landscape of the molecular mechanism that underlies the proteasomal degradation of F508del through UBE 2L6 and RNF19B.
HA-ubiquitin was purchased from Addgene.

Immunoblotting
Cells were lysed by sonication in lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1% NP40, 10% glycerol and protease inhibitors cocktail). Specifically, protein samples were 7 resolved by SDS-PAGE and transferred to PVDF membranes, which were blocked at room temperature for 1 h with 5% (w/v) non-fat milk in TBST (10 mM Tris (pH 8.0), 150 mM NaCl, 0.05% Tween 20). The blots were incubated with primary antibodies in TBST with 10% fetal bovine serum at room temperature for 2-3 h. The blots were then washed three times with TBST and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h, followed by three washes with TBST. The reactive bands were visualized by incubation with enhanced chemiluminescence substrates (PerkinElmer Products) and exposure to Xray film (Eastman Kodak Co).
Immunocomplexes were resuspended in SDS sample buffer and subjected to SDS-PAGE gel and immunoblotting.

Cycloheximide (CHX) chase analysis
After 24 h treatment with designated reagent, cells were continued to be cultured in medium supplemented with 50 μg/ml CHX and harvested at designated time points. Cell extracts were subjected to the immunoblotting analysis with appropriate antibodies.

Biotinylation and pulldown of CFTR on streptavidin beads
After treatment with specified reagent of 24 hours, cells were washed 3 times with PBS and exposed to 0.5mg/ml in PBS EZLink Sulfo-NHS-LC--biotin (Thermo Scientific) for 1 h on ice. Cells were washed 3 time with PBS, quenched with 100mM glycine in PBS, and rinsed 2 time again. Then cells were solubilized by sonication in RIPA buffer (150 mM NaCl, 1 mM Tris/HCl, 0.5% (w/v) deoxycholic acid, 1% (w/v) NP-40, 0.1% SDS, 2 mM EDTA, 50 mM NaF 8 and protease inhibitors). The resulting lysate was centrifuged at 21100 g for 10 min at 4 0 C, and supernatant protein content was determined using protein assay dye reagent (Bio Rad). The supernatant was incubated with streptavidin beads for overnight at 4 0 C.
After a brief centrifugation the supernatant was removed, the beads were washed 4 times with lysis buffer. Pull-downed proteins were resolved by SDS-PAGE, transferred to PVDF membranes and performed an immunoblot as described above.

Confocal Microscopy
Immunofluorescence staining of filter-grown cells was performed as described previously [35]. Briefly, CFBE-F508del cells were fixed in 4% paraformaldehyde and permeabilized with a mixture of 4% paraformaldehyde and 0.1% Triton X-100. The cells were then washed three times with buffer A (0.5% BSA and 0.15% glycine at pH 7.4 in phosphatebuffered saline). After blocking with purified goat serum, the monolayers were incubated in the appropriate primary antibodies (anti-Myc 1: 300, and anti-CFTR217 1:300) for 1 hour followed by three washes in buffer A and subsequent incubation with fluorescein isothiocyanate (green) or rhodamine (red)-labeled secondary antibodies (1:1000, Molecular Probes) for another hour. After washing with buffer A, the filters were mounted on glass coverslips using synthetic resin and subjected to confocal microscopy. Collected images were exported to ImageSpace (Molecular Dynamics) for subsequent reconstruction and processing.

Quantitative real-time reverse transcription PCR analysis
Total cellular RNA was extracted using TRIzol reagent and reverse-transcribed to cDNA using a random primer. The real-time PCR reaction mixture containing cDNA template, The immunoblot images were scanned at 600 dpi for densitometry analysis using Image J software. The Values are means ± SEM. Statistical significance of differences was determined using paired two-tailed Student's t-test.

Overexpressing UBE 2L6 degrades F508del
Through a literature search, we were able to discover UBE 2L6 interacts with many E3 ligases that were found in the ER [27]. This interaction with E3 ligases in the ER may mediate F508del degradation; therefore we wanted to observe what happens to CFTR when we overexpress UBE 2L6. We used HEK 293 cells and co-transfected with F508del and UBE 2L6 for 48 hours. 48-hours post-transfection, total protein lysates were harvested and probed for F508del levels by immunoblot analysis. Overexpressing UBE 2L6 decreased F508del expression by about 2.5-fold (Fig. 1A,1C).
To further confirm UBE 2L6 effects on degrading F508del, we developed a dominantnegative mutant of UBE 2L6 where a cysteine residue at position 86 had been mutated to a serine residue (UBE 2L6 DN). This mutation will destroy the catalytic activity of UBE 2L6 and therefore the activated ubiquitin is not able to transfer onto the E2. Hence, theoretically the ubiquitin is not able to transfer to the misfolded protein. We transiently transfected HEK 293 cells with F508del and with UBE 2L6 or UBE2L6 DN or a control (pCMV) (Fig. 1B). Consistent to pervious finding, overexpressing UBE 2L6 decreases F508del. When UBE 2L6 DN is overexpressed, F508del expression increases 1.9-fold (Fig.   1B, 1C). These data suggest that UBE 2L6 facilitates F508del degradation by ubiquitination.

Silencing UBE 2L6 in HBE cells with VX-809 rescues F508del
We next wanted to determine if we can rescue F508del if we silenced UBE 2L6 in human bronchiole epithelial (HBE) cells. We used cystic fibrosis HBE cells that have the F508 mutation (CFBE-F508del) and knockdown UBE 2L6 with siRNA. 48 hours post-transfection, the cells were treated with VX-809 or DMSO as control for 24 hours. VX-809 is a drug used in the CF field that folds F508del so that the protein is to traffic to the plasma membrane [14]. 24 hours post-treatment, the cells were harvested and probed for CFTR and actin.
When UBE 2L6 was knocked down with siRNA, there is a 2.8-fold increase in F508del expression, which is illustrated as Band-B (Fig. 2A, 2C). Band B is the misfolded CFTR that is trapped in the ER where it will be eventually degraded by the proteasome in the cytosol. Interestingly, with VX-809 treatment, there seems to be a 2.7-fold increase in Cband expression of CFTR compared to control ( Fig.2A, 2D). This is critical in that this shows that with UBE 2L6 knocked down, there seems to be a greater pool of B-band CFTR.
With this increased pool of B-band, VX-809 helps more F508del to traffic to the plasma membrane.

F508del function is increased with UBE 2L6 knockdown and VX-809 treatment
To investigate how UBE 2L6 affect CFTR function, we used the Ussing chamber to measure there is a slight, but significant increase in CFTR function by about 2.9 µA/cm 2 when stimulated by forskolin and IBMX (Fig. 3A, 3B). When CFBE-F508del cells have UBE 2L6 knocked down and treated with VX-809, there is an increase in CFTR function by about 5.4 µA/cm 2 compared to cell transfected with scramble siRNA and treated with VX-809 (Fig.   3A, 3B). The quantification of three experiments illustrates that knockdown of UBE 2L6 and VX-809 treatment had a significantly greater increase in F508del function compared to controls (Fig. 3B).

RNF19B is a candidate in F508del degradation
To identify which E3 Ligase may cooperate with UBE 2L6 to promote F508del degradation, we performed an siRNA screen on ER resident E3 ligases on HBE cells. We determined that RNF19B may be a candidate in F508del degradation and it shown that RNF19B is a partner of UBE 2L6 [28]. RNF19B is a Ring finger protein family of E3 ligases, which interacts with E2 conjugating enzyme(s) and substrates, facilitating the transfer of an activated ubiquitin to the substrate [29,30]. RING finger E3 ligases have a RING structure that is involved in ubiquitinating its substrate. To show that this site is involved in ubiquitinating F508del, we generated a catalytically non-functioning RNF19B RING mutant where we mutated a cysteine residue at position 119 to a serine residue (c119s) to disable the RING domain.
24 hours post-transfection, HA-ubiquitin was transfected into these cells. The cells were than harvested and CFTR was pulled down (Fig. 4C). The left panel demonstrates that there is more HA-ubiquitin immunoprecipitated with F508del when RNF19B was overexpressed compared to control. The right panel demonstrates that total CFTR was the same between both conditions under MG-132 treatment. If RNF19B regulates F508del, they should be located in the same compartment. To determine if these proteins are localized within the same area of the cell, CFBE-F508del cells were transfected with Myc-13 RNF19B and then immunostained with Myc (green) and F508del (red) (Fig. 4D). The immunostaining shows that Myc-RNF19B is localized with F508del in the perinuclear area, which is indicative of the ER. To determine how RNF19B affects the half-life of F508del, we performed a cycloheximide-chase where HEK 293 cells were co-transfected with F508del and with the control or with Flag-RNF19B for 48 hours. The cells were than treated with 50µg/ml cycloheximide for the different indicated time periods. (Fig. 5A,   5B). Overexpressing RNF19B decreased the half-life of F508del from 60 minutes to about 20 minutes, suggesting RNF19B participates F508del degradation.

F508del function is enhanced by RNF19B knockdown in the presence of VX-809 treatment
To further determine if RNF19B is involved F508del degradation and its function, we 14 needed to determine if CFTR trafficking to the plasma membrane is affected by RNF19B.
We performed biotinylation experiments to determine if more F508del traffics to the plasma membrane when RNF19B is silenced (Fig. 7A). Knockdown of RNF19B promotes F508del C-band to the plasma membrane to act as a chloride channel. When RNF19B is knocked down with VX-809 treatment, there is a 1.8 fold increase in C-Band (Fig 7A, 7B).
To determine if there is enhanced F508del function when RNF19B is knocked down, we performed Ussing Chamber experiments. We transfected CFBE-F508del cells with siRNA against RNF19B or a scramble siRNA as a control. 48 hours post-transfection, we treated the cells with either 3 μM VX-809 or with DMSO for 24 hours. 24 hours post-treatment, cells were set up in the Ussing chamber to measure short-circuit currents. Short-circuit currents demonstrated that compared to control, RNF19B knocked down in CFBE-F508del cells increased short-circuit currents by about 3.25 µA/cm 2 compared to scramble controls when stimulated by forskolin and IBMX (Fig. 7C, 7D). There is an increase of about 7.7 µA/cm 2 in CFTR function when CFBE-F508del cells have RNF19B knocked down and treated with VX-809 compared to scramble control (Fig. 7C,7D). This set of data indicates that we are able to rescue F508del protein and function when RNF19B is silenced in HBE cells.

Discussion
In this paper we have identified an E2 conjugating enzyme, UBE 2L6 that mediates the proteasomal degradation of F508del. We've found that when UBE 2L6 is knocked down in human airway epithelial cells and treated with VX-809, there is an increase in expression of CFTR C-band, the functional form of CFTR. UBE 2L6 works as a conjugating enzyme, acting as a transient holder of activated ubiquitin to be ready to tag a substrate [31], in this case misfolded F508del. We have shown that by mutating UBE 2L6 so that it cannot carry that activated ubiquitin, there is an increase is expression of F508del, therefore demonstrating that UBE 2L6 does indeed mediate the degradation of F508del through the ubiquitin proteasome system. Also, this shows that UBE 2L6 is an important player in the F508del degradation because the mutation to UBE 2L6 rescues F508del to similar levels as seen in control. Therefore, there is a greater pool of F508del that can be used on by VX-809 to allow for greater trafficking of the misfolded protein to the plasma membrane.
Future studies will be focusing on developing and discovering small inhibitors to act as a drug to inhibit UBE 2L6 function so that there is a greater pool of F508del protein for VX-809 to act on. VX-809 will help fold F508del so that is can traffic to the plasma membrane.
With the increase in F508del pool, we also showed that there is an increase in CFTR function when UBE 2L6 in knocked down. Using human airway epithelial cells, we knocked down UBE 2L6 and determined that there was an increase CFTR function when forskolin and IBMX was added to the cells. Importantly, when the same cells that had UBE 2L6 knocked down were treated with VX-809, there was a greater CFTR function compared to control. These experiments further support the idea that when UBE 2L6 is knocked down there is a greater pool CFTR protein that VX-809 can work on.
Our data also illustrates an interacting partner of UBE 2L6 that mediates the degradation of F508del. We have shown that RNF19B, an E3 ligase that interacts with UBE 2L6, mediates the degradation of F508del. Interestingly, RNF19B is localized in the endoplasmic reticulum [32,33], where F508del is localized. Our data also demonstrates that VX-809 treatment RNF19B knocked down in HBE cells, there is an increase in F508del function at the plasma membrane. This is similar to what was seen when performing similar experiments with UBE 2L6. This is important in that not only does UBE 2L6 aids in the degradation of F508del, but that it interacts with and ER localized E3 ligases to assist in this degradation. Therefore, this is a potential early checkpoint in CFTR degradation because these two proteins that are involved in the ubiquitin proteasome system prevent F508del from trafficking to the plasma membrane to act as a chloride channel. If we are able to use small inhibitors to prevent these proteins from ubiquitinating F508del, we will be able to provide the cell with greater pool of F508del that is not degraded by the proteasome but allow VX-809 to act on more F508del so that it is able to traffic to the plasma membrane. Synergy between the knockdown of UBE 2L6 and RNF19B with VX-809 treatment, which enhanced F508del chloride secretion, indicates that these two proteins may offer a therapeutic approach for CF. This allows for more chloride channels to the plasma membrane that will be extremely beneficial to CF patients.
Previous studies have shown that RMA1 and Ubc6e are involved in the proteasomal degradation of F508del [21], however we have also identified that RNF19B and its interacting partner, UBE 2L6, mediates F508del degradation. Based on the data presented, we propose the following mechanism that is involved by F508del degradation. Quality control checkpoints within the ER senses the misfolded F508del due to the F508del mutation. The misfolded F508del is brought into association with RNF19B and UBE 2L6 where both the E3 and E2 proteins cooperate to ubiquitinate F508del. The ubiquitinated F508del is than signaled to proceed to the proteasome for degradation.
F508del the most common mutation amongst CF patients, where these patients lack the functional form of CFTR to act as an anion channel [34]. With this mutation, therapeutic strategies that create the mature form of CFTR partially restores the function of CFTR at the apical membrane of cells. Therefore, understanding the early quality control checkpoints in CFTR degradation in important is revealing possible avenues in trying to rescue CFTR protein in CF patients.

Conclusions
This study reveals additional proteins that are involved in F508del degradation, and possible targets for therapeutic intervention. The E2 conjugating enzyme UBE 2L6, and its' interacting E3 ligase, RNF19B, both mediate the proteasomal degradation of F508del.
Also, our data demonstrates that VX-809, an already established processing drug treatment, operates to enhance F508del function at the plasma membrane when UBE 2L6 and RNF19B are also silenced. Therefore, modulation of UBE 2L6/RNF19B mediated degradation of F508del offers a promising area for therapeutic intervention.