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{Sun, F. unpublished data, The 21st Annual North American CF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wild-type CFTR unchanged [9], (Ahner A. et al., unpublished data (The 21st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 21st 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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