Regulation of degradation of deltaF508 CFTR in CF

Regulation of degradation of deltaF508 CFTR

Cystic fibrosis (CF), the most common life-threatening autosomal-recessive genetic disease of Caucasians, is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) [1]. Over 1500 mutations have been identified in the CFTR gene; the most common of them is loss of a Phe residue at position 508 (DeltaF508 CFTR).

DeltaF508 CFTR potentially retains transporter functionality, but it fails to fold into its native conformations and is selected for endoplasmic reticulum (ER)-associated degradation (ERAD) by molecular chaperones and associated proteins [2].

Two chaperones, cytoplasmic 70-kDa heat shock protein from HSP70 family, and transmembrane ER chaperone calnexin, form transient complexes with nascent, newly synthesized core-glycosylated forms of immature CFTR molecules, one on each side of the ER membrane. Both chaperons interact with DeltaF50 8CFTR, and complexes of DeltaF508 CFTR with Hsp70 are more stable than those with wt-CFTR [3]. It was shown that Hsp70 facilitates endoplasmic reticulum-associated protein degradation of CFTR in yeast [4], [5].

Cytoplasmic heat shock protein from HSP70 family, Hsc70 and its co-chaperone Hdj-2, interact with immature form of CFTR. A function of the Hsc70/Hdj-2 pair was suggested to be the co-translational stabilization of NBD1 and the promotion of intramolecular assembly between it and the R-domain of CFTR. Normally, wt-CFTR is released from chaperones Hsc70 (Hdj-2/Hsp70) as it achieves its native conformation, but Hdj-2/Hsp70 remains attached to misfolded DeltaF508 CFTR [6], [7], [8].

Hdj-2 is capable of associating with both immature and ubiquitinated CFTR. It was proposed that Hdj-2 discriminates between wt-CFTR and DeltaF508 CFTR and inducing degradation of the latter. This suggest molecular sensor role for Hdj-2 {Sun, F. unpublished data, The 21^st Annual North American CF conference Anaheim Convention Center, Anaheim, California, October 3-6, 2007}.

Other characterized chaperones are small heat-shock proteins alpha A-crystallin and HSP27. alpha A-crystallin and HSP27 distinguish terminally misfolded forms of DeltaF50 8 CFTR from the wild-type protein and interact preferentially with DeltaF50 8CFTR. Overexpression of these proteins selectively accelerates degradation of DeltaF508 CFTR, leaving the biogenesis of wild-type CFTR unchanged [9], (Ahner A. et al., unpublished data (The 21st Annual North American CF conference Anaheim Convention Center, Anaheim, California, October 3-6, 2007)).

For DeltaF508 CFTR in mammalian cells ubiquitin-proteasome-mediated degradation is the dominant pathway [2], [10], [11], [12].

Two ubiquitin ligase complexes mark DeltaF508 CFTR for degradation - ER ubiquitin ligase complex and cytosolic ubiquitin ligase complex.

The first complex - ER membrane-associated ubiquitin ligase complex -contains the E3 RMA1 (RNF5), the E2 Ubc6e (UNE2J1), and Derlin-1 [13], [14], [15].

The second complex - cytosolic ubiquitin ligase complex -contains E3 CHIP [13], and UBCH5a. Complex acts upon Hsc70-bound DeltaF508 CFTR and its action is dependent upon Hdj2 [8], [15], [16], [17].

Cochaperone HspBP1 is an inhibitor of CHIP. HspBP1 attenuates the ubiquitin ligase activity of CHIP when complexed with Hsc70. As a consequence, HspBP1 interferes with the CHIP-induced degradation of immature forms and may modulate the function of the Hsc70/CHIP complex [18].

DnaJ homolog subfamily C member 5 (Csp), blocks ER exit of CFTR. Additionally, Csp associates with CHIP and facilitates degradation of immature CFTR (Schmidt, B. unpublished data, The 21^st Annual North American CF conference Anaheim Convention Center, Anaheim, California, October 3-6, 2007}

RNF5 is capable of recognizing folding defects in DeltaF508 CFTR coincident with translation, whereas the CHIP E3 appears to act posttranslationally. RNF5 and CHIP E3 ubiquitin ligases act sequentially in ER membrane and cytosol to monitor the folding status of CFTR and DeltaF508 CFTR, and their triage.

In addition, it is shown that a multi-ubiquitin chain assembly factor (E4) Autocrine motility factor receptor (AMFR) is also involved in DeltaF508 CFTR ubiquitination. RNF5 functiones as an E3 enzyme upstream of AMFR [19].

Ubiquitylated DeltaF508-CFTR is transported through the Sec61 trimeric complex back to the cytosol, escorted by the beta subunit of Sec61 [20].

VCP/p97, a Type II AAA ATPase component of the retrotranslocation machinery, forms a complex with substrate-recruiting cofactors Ufd1/Npl4. VCP binds polyubiquinated DeltaF508 CFTR while Ufd1/Npl4 interacts to the ubiquitin chains on the substrate [12], [21]. VCP activity may be regulated by Ataxin-3 [22].

In situations where 26S proteasome are compromised or overwhelmed, ubiquitinated DeltaF508-CFTR is transported to a perinuclear location near the microtubule-organizing center to form aggresomes [15]. Ataxin-3, Histone deacetylase 6 (HDAC6) and Dynein participate in DeltaF508-CFTR aggresome formation [23].

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