Regulation of degradation of deltaF508 CFTR in CF

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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 This suggest molecular sensor role for Hdj-2 {Sun, F. unpublished data, The 21st Annual North American CF conference Anaheim Convention Center, Anaheim, California, October 3-6, 2007}.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].

References:

  1. Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok S, Plavsic N, Chou JL
    Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science (New York, N.Y.) 1989 Sep 8;245(4922):1066-73
  2. Gelman MS, Kannegaard ES, Kopito RR
    A principal role for the proteasome in endoplasmic reticulum-associated degradation of misfolded intracellular cystic fibrosis transmembrane conductance regulator. The Journal of biological chemistry 2002 Apr 5;277(14):11709-14
  3. Kopito RR
    Biosynthesis and degradation of CFTR. Physiological reviews 1999 Jan;79(1 Suppl):S167-73
  4. Zhang Y, Nijbroek G, Sullivan ML, McCracken AA, Watkins SC, Michaelis S, Brodsky JL
    Hsp70 molecular chaperone facilitates endoplasmic reticulum-associated protein degradation of cystic fibrosis transmembrane conductance regulator in yeast. Molecular biology of the cell 2001 May;12(5):1303-14
  5. Rowe SM, Miller S, Sorscher EJ
    Cystic fibrosis. The New England journal of medicine 2005 May 12;352(19):1992-2001
  6. Meacham GC, Lu Z, King S, Sorscher E, Tousson A, Cyr DM
    The Hdj-2/Hsc70 chaperone pair facilitates early steps in CFTR biogenesis. The EMBO journal 1999 Mar 15;18(6):1492-505
  7. Rubenstein RC, Zeitlin PL
    Sodium 4-phenylbutyrate downregulates Hsc70: implications for intracellular trafficking of DeltaF508-CFTR. American journal of physiology. Cell physiology 2000 Feb;278(2):C259-67
  8. Meacham GC, Patterson C, Zhang W, Younger JM, Cyr DM
    The Hsc70 co-chaperone CHIP targets immature CFTR for proteasomal degradation. Nature cell biology 2001 Jan;3(1):100-5
  9. Ahner A, Nakatsukasa K, Zhang H, Frizzell RA, Brodsky JL
    Small heat-shock proteins select deltaF508-CFTR for endoplasmic reticulum-associated degradation. Molecular biology of the cell 2007 Mar;18(3):806-14
  10. Ward CL, Omura S, Kopito RR
    Degradation of CFTR by the ubiquitin-proteasome pathway. Cell 1995 Oct 6;83(1):121-7
  11. Jensen TJ, Loo MA, Pind S, Williams DB, Goldberg AL, Riordan JR
    Multiple proteolytic systems, including the proteasome, contribute to CFTR processing. Cell 1995 Oct 6;83(1):129-35
  12. Goldstein RF, Niraj A, Sanderson TP, Wilson LS, Rab A, Kim H, Bebok Z, Collawn JF
    VCP/p97 AAA-ATPase does not interact with the endogenous wild-type cystic fibrosis transmembrane conductance regulator. American journal of respiratory cell and molecular biology 2007 Jun;36(6):706-14
  13. Younger JM, Chen L, Ren HY, Rosser MF, Turnbull EL, Fan CY, Patterson C, Cyr DM
    Sequential quality-control checkpoints triage misfolded cystic fibrosis transmembrane conductance regulator. Cell 2006 Aug 11;126(3):571-82
  14. Sun F, Zhang R, Gong X, Geng X, Drain PF, Frizzell RA
    Derlin-1 promotes the efficient degradation of the cystic fibrosis transmembrane conductance regulator (CFTR) and CFTR folding mutants. The Journal of biological chemistry 2006 Dec 1;281(48):36856-63
  15. Turnbull EL, Rosser MF, Cyr DM
    The role of the UPS in cystic fibrosis. BMC biochemistry 2007 Nov 22;8 Suppl 1:S11
  16. Jiang J, Ballinger CA, Wu Y, Dai Q, Cyr DM, Hohfeld J, Patterson C
    CHIP is a U-box-dependent E3 ubiquitin ligase: identification of Hsc70 as a target for ubiquitylation. The Journal of biological chemistry 2001 Nov 16;276(46):42938-44
  17. Younger JM, Ren HY, Chen L, Fan CY, Fields A, Patterson C, Cyr DM
    A foldable CFTR{Delta}F508 biogenic intermediate accumulates upon inhibition of the Hsc70-CHIP E3 ubiquitin ligase. The Journal of cell biology 2004 Dec 20;167(6):1075-85
  18. Alberti S, Bohse K, Arndt V, Schmitz A, Hohfeld J
    The cochaperone HspBP1 inhibits the CHIP ubiquitin ligase and stimulates the maturation of the cystic fibrosis transmembrane conductance regulator. Molecular biology of the cell 2004 Sep;15(9):4003-10
  19. Morito D, Hirao K, Oda Y, Hosokawa N, Tokunaga F, Cyr DM, Tanaka K, Iwai K, Nagata K
    Gp78 cooperates with RMA1 in endoplasmic reticulum-associated degradation of CFTRDeltaF508. Molecular biology of the cell 2008 Apr;19(4):1328-36
  20. Bebok Z, Mazzochi C, King SA, Hong JS, Sorscher EJ
    The mechanism underlying cystic fibrosis transmembrane conductance regulator transport from the endoplasmic reticulum to the proteasome includes Sec61beta and a cytosolic, deglycosylated intermediary. The Journal of biological chemistry 1998 Nov 6;273(45):29873-8
  21. Gnann A, Riordan JR, Wolf DH
    Cystic fibrosis transmembrane conductance regulator degradation depends on the lectins Htm1p/EDEM and the Cdc48 protein complex in yeast. Molecular biology of the cell 2004 Sep;15(9):4125-35
  22. Zhong X, Pittman RN
    Ataxin-3 binds VCP/p97 and regulates retrotranslocation of ERAD substrates. Human molecular genetics 2006 Aug 15;15(16):2409-20
  23. Burnett BG, Pittman RN
    The polyglutamine neurodegenerative protein ataxin 3 regulates aggresome formation. Proceedings of the National Academy of Sciences of the United States of America 2005 Mar 22;102(12):4330-5

  1. Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok S, Plavsic N, Chou JL
    Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science (New York, N.Y.) 1989 Sep 8;245(4922):1066-73
  2. Gelman MS, Kannegaard ES, Kopito RR
    A principal role for the proteasome in endoplasmic reticulum-associated degradation of misfolded intracellular cystic fibrosis transmembrane conductance regulator. The Journal of biological chemistry 2002 Apr 5;277(14):11709-14
  3. Kopito RR
    Biosynthesis and degradation of CFTR. Physiological reviews 1999 Jan;79(1 Suppl):S167-73
  4. Zhang Y, Nijbroek G, Sullivan ML, McCracken AA, Watkins SC, Michaelis S, Brodsky JL
    Hsp70 molecular chaperone facilitates endoplasmic reticulum-associated protein degradation of cystic fibrosis transmembrane conductance regulator in yeast. Molecular biology of the cell 2001 May;12(5):1303-14
  5. Rowe SM, Miller S, Sorscher EJ
    Cystic fibrosis. The New England journal of medicine 2005 May 12;352(19):1992-2001
  6. Meacham GC, Lu Z, King S, Sorscher E, Tousson A, Cyr DM
    The Hdj-2/Hsc70 chaperone pair facilitates early steps in CFTR biogenesis. The EMBO journal 1999 Mar 15;18(6):1492-505
  7. Rubenstein RC, Zeitlin PL
    Sodium 4-phenylbutyrate downregulates Hsc70: implications for intracellular trafficking of DeltaF508-CFTR. American journal of physiology. Cell physiology 2000 Feb;278(2):C259-67
  8. Meacham GC, Patterson C, Zhang W, Younger JM, Cyr DM
    The Hsc70 co-chaperone CHIP targets immature CFTR for proteasomal degradation. Nature cell biology 2001 Jan;3(1):100-5
  9. Ahner A, Nakatsukasa K, Zhang H, Frizzell RA, Brodsky JL
    Small heat-shock proteins select deltaF508-CFTR for endoplasmic reticulum-associated degradation. Molecular biology of the cell 2007 Mar;18(3):806-14
  10. Ward CL, Omura S, Kopito RR
    Degradation of CFTR by the ubiquitin-proteasome pathway. Cell 1995 Oct 6;83(1):121-7
  11. Jensen TJ, Loo MA, Pind S, Williams DB, Goldberg AL, Riordan JR
    Multiple proteolytic systems, including the proteasome, contribute to CFTR processing. Cell 1995 Oct 6;83(1):129-35
  12. Goldstein RF, Niraj A, Sanderson TP, Wilson LS, Rab A, Kim H, Bebok Z, Collawn JF
    VCP/p97 AAA-ATPase does not interact with the endogenous wild-type cystic fibrosis transmembrane conductance regulator. American journal of respiratory cell and molecular biology 2007 Jun;36(6):706-14
  13. Younger JM, Chen L, Ren HY, Rosser MF, Turnbull EL, Fan CY, Patterson C, Cyr DM
    Sequential quality-control checkpoints triage misfolded cystic fibrosis transmembrane conductance regulator. Cell 2006 Aug 11;126(3):571-82
  14. Sun F, Zhang R, Gong X, Geng X, Drain PF, Frizzell RA
    Derlin-1 promotes the efficient degradation of the cystic fibrosis transmembrane conductance regulator (CFTR) and CFTR folding mutants. The Journal of biological chemistry 2006 Dec 1;281(48):36856-63
  15. Turnbull EL, Rosser MF, Cyr DM
    The role of the UPS in cystic fibrosis. BMC biochemistry 2007 Nov 22;8 Suppl 1:S11
  16. Jiang J, Ballinger CA, Wu Y, Dai Q, Cyr DM, Hohfeld J, Patterson C
    CHIP is a U-box-dependent E3 ubiquitin ligase: identification of Hsc70 as a target for ubiquitylation. The Journal of biological chemistry 2001 Nov 16;276(46):42938-44
  17. Younger JM, Ren HY, Chen L, Fan CY, Fields A, Patterson C, Cyr DM
    A foldable CFTR{Delta}F508 biogenic intermediate accumulates upon inhibition of the Hsc70-CHIP E3 ubiquitin ligase. The Journal of cell biology 2004 Dec 20;167(6):1075-85
  18. Alberti S, Bohse K, Arndt V, Schmitz A, Hohfeld J
    The cochaperone HspBP1 inhibits the CHIP ubiquitin ligase and stimulates the maturation of the cystic fibrosis transmembrane conductance regulator. Molecular biology of the cell 2004 Sep;15(9):4003-10
  19. Morito D, Hirao K, Oda Y, Hosokawa N, Tokunaga F, Cyr DM, Tanaka K, Iwai K, Nagata K
    Gp78 cooperates with RMA1 in endoplasmic reticulum-associated degradation of CFTRDeltaF508. Molecular biology of the cell 2008 Apr;19(4):1328-36
  20. Bebok Z, Mazzochi C, King SA, Hong JS, Sorscher EJ
    The mechanism underlying cystic fibrosis transmembrane conductance regulator transport from the endoplasmic reticulum to the proteasome includes Sec61beta and a cytosolic, deglycosylated intermediary. The Journal of biological chemistry 1998 Nov 6;273(45):29873-8
  21. Gnann A, Riordan JR, Wolf DH
    Cystic fibrosis transmembrane conductance regulator degradation depends on the lectins Htm1p/EDEM and the Cdc48 protein complex in yeast. Molecular biology of the cell 2004 Sep;15(9):4125-35
  22. Zhong X, Pittman RN
    Ataxin-3 binds VCP/p97 and regulates retrotranslocation of ERAD substrates. Human molecular genetics 2006 Aug 15;15(16):2409-20
  23. Burnett BG, Pittman RN
    The polyglutamine neurodegenerative protein ataxin 3 regulates aggresome formation. Proceedings of the National Academy of Sciences of the United States of America 2005 Mar 22;102(12):4330-5

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