Endoplasmic Reticulum-associated Degradation of Niemann-Pick C1

Background: Mutant Niemann-Pick C1 (NPC1) is degraded by the proteasome. Results: NPC1 is one of the clients of HSP/CHIP (heat shock protein/carboxyl terminus of Hsp70-interacting protein) complexes and accepts ubiquitin at multiple lysine residues. Conclusion: HSPs are critical in the quality control of NPC1. Significance: Unraveling the mechanisms of NPC1 degradation is crucial for development of a chaperone therapy. Most cases with Niemann-Pick disease type C carry mutations in NPC1. Some of the mutations, including the most frequent I1061T, give rise to unstable proteins selected for endoplasmic reticulum-associated degradation. The purpose of the current study was to shed mechanistic insights into the degradation process. A proteasome inhibitor MG132 prolonged the life span of the wild-type NPC1 expressed in COS cells. The expressed protein associated with multiple chaperones including heat shock protein 90 (Hsp90), Hsp70, heat shock cognate protein 70 (Hsc70), and calnexin. Accordingly, expression of an E3 ligase CHIP (carboxyl terminus of Hsp70-interacting protein) enhanced MG132-induced accumulation of ubiquitylated NPC1. Co-expression and RNAi knockdown experiments in HEK cells indicated that Hsp70/Hsp90 stabilized NPC1, whereas Hsc70 destabilized it. In human fibroblasts carrying the I1061T mutation, adenovirus-mediated expression of Hsp70 or treatment with an HSP-inducer geranylgeranylacetone (GGA) increased the level of the mutant protein. In GGA-treated cells, the rescued protein was localized in the late endosome and ameliorated cholesterol accumulation. MALDI-TOF mass spectrometry revealed three lysine residues at amino acids 318, 792, and 1180 as potential ubiquitin-conjugation sites. Substitutions of the three residues with alanine yielded a mutant protein with a steady-state level more than three times higher than that of the wild-type. Introduction of the same substitutions to the I1061T mutant resulted in an increase in its protein level and functional restoration. These findings indicated the role of HSPs in quality control of NPC1 and revealed the role of three lysine residues as ubiquitin-conjugation sites.

[1]  W. Skach,et al.  Endoplasmic Reticulum Protein Quality Control is Determined by Cooperative Interactions Between Hsp / c 70 and the CHIP E 3 Ligase , 2013 .

[2]  L. Suaud,et al.  Differential effects of Hsc70 and Hsp70 on the intracellular trafficking and functional expression of epithelial sodium channels. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[3]  T. Yoshimori,et al.  Cholesterol depletion facilitates ubiquitylation of NPC1 and its association with SKD1/Vps4 , 2006, Journal of Cell Science.

[4]  J. Brodsky,et al.  How early studies on secreted and membrane protein quality control gave rise to the ER associated degradation (ERAD) pathway: the early history of ERAD. , 2013, Biochimica et biophysica acta.

[5]  Yiannis A. Ioannou,et al.  Topological Analysis of Niemann-Pick C1 Protein Reveals That the Membrane Orientation of the Putative Sterol-sensing Domain Is Identical to Those of 3-Hydroxy-3-methylglutaryl-CoA Reductase and Sterol Regulatory Element Binding Protein Cleavage-activating Protein* , 2000, The Journal of Biological Chemistry.

[6]  W. Nastainczyk,et al.  BAG-2 acts as an inhibitor of the chaperone-associated ubiquitin ligase CHIP. , 2005, Molecular biology of the cell.

[7]  Paul Helquist,et al.  Histone deacetylase inhibitor treatment dramatically reduces cholesterol accumulation in Niemann-Pick type C1 mutant human fibroblasts , 2011, Proceedings of the National Academy of Sciences.

[8]  Martin Bard,et al.  An “Exacerbate-reverse” Strategy in Yeast Identifies Histone Deacetylase Inhibition as a Correction for Cholesterol and Sphingolipid Transport Defects in Human Niemann-Pick Type C Disease*♦ , 2011, The Journal of Biological Chemistry.

[9]  R. Cotter,et al.  Approach for determining protein ubiquitination sites by MALDI-TOF mass spectrometry. , 2005, Analytical chemistry.

[10]  I. Hisatome,et al.  Hsp90 prevents interaction between CHIP and HERG proteins to facilitate maturation of wild-type and mutant HERG proteins. , 2013, Cardiovascular research.

[11]  K. Yamakawa,et al.  Genotype-phenotype relationship of Niemann-Pick disease type C: a possible correlation between clinical onset and levels of NPC1 protein in isolated skin fibroblasts , 2000, Journal of medical genetics.

[12]  B. Bembi,et al.  Treatment of Human Fibroblasts Carrying NPC1 Missense Mutations with MG132 Leads to an Improvement of Intracellular Cholesterol Trafficking. , 2012, JIMD reports.

[13]  D. Cyr,et al.  The Hsc70 co-chaperone CHIP targets immature CFTR for proteasomal degradation , 2000, Nature Cell Biology.

[14]  John R Yates,et al.  Reduced histone deacetylase 7 activity restores function to misfolded CFTR in cystic fibrosis. , 2010, Nature chemical biology.

[15]  I. Hisatome,et al.  Transcriptional activation of the anchoring protein SAP97 by heat shock factor (HSF)-1 stabilizes Kv1.5 channels in HL-1 cells , 2011, British journal of pharmacology.

[16]  Y. Ioannou,et al.  Niemann-Pick C1 is a late endosome-resident protein that transiently associates with lysosomes and the trans-Golgi network. , 1999, Molecular genetics and metabolism.

[17]  N. Rosen,et al.  An acetylation site in the middle domain of Hsp90 regulates chaperone function. , 2007, Molecular cell.

[18]  C. Tomasetto,et al.  Niemann-Pick C1 disease: correlations between NPC1 mutations, levels of NPC1 protein, and phenotypes emphasize the functional significance of the putative sterol-sensing domain and of the cysteine-rich luminal loop. , 2001, American journal of human genetics.

[19]  M. Horie,et al.  Reciprocal Control of hERG Stability by Hsp70 and Hsc70 With Implication for Restoration of LQT2 Mutant Stability , 2011, Circulation research.

[20]  A. Lieberman,et al.  Ryanodine receptor antagonists adapt NPC1 proteostasis to ameliorate lipid storage in Niemann-Pick type C disease fibroblasts. , 2012, Human molecular genetics.

[21]  Katsumi Higaki,et al.  Increased NPC1 mRNA in skin fibroblasts from Niemann-Pick disease type C patients , 2004, Brain & development (Tokyo. 1979).

[22]  C. Borchers,et al.  Regulation of the Cytoplasmic Quality Control Protein Degradation Pathway by BAG2* , 2005, Journal of Biological Chemistry.

[23]  R. Wattiaux,et al.  Identification of HE1 as the second gene of Niemann-Pick C disease. , 2000, Science.

[24]  G. Sobue,et al.  Pharmacological induction of heat-shock proteins alleviates polyglutamine-mediated motor neuron disease. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[25]  Seiichiro Ogawa,et al.  N-Octyl-β-valienamine up-regulates activity of F213I mutant β-glucosidase in cultured cells: a potential chemical chaperone therapy for Gaucher disease , 2004 .

[26]  W. Skach,et al.  Endoplasmic Reticulum Protein Quality Control Is Determined by Cooperative Interactions between Hsp/c70 Protein and the CHIP E3 Ligase* , 2013, The Journal of Biological Chemistry.

[27]  N. Baumann,et al.  The adult form of Niemann-Pick disease type C. , 2006, Brain : a journal of neurology.

[28]  S. Gale,et al.  Niemann-Pick Type C1 I1061T Mutant Encodes a Functional Protein That Is Selected for Endoplasmic Reticulum-associated Degradation Due to Protein Misfolding* , 2008, Journal of Biological Chemistry.

[29]  K. G. Coleman,et al.  Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis. , 1997, Science.

[30]  G. Millat,et al.  Structure and function of the NPC2 protein. , 2004, Biochimica et biophysica acta.

[31]  J. Brodsky.  Cleaning Up: ER-Associated Degradation to the Rescue , 2012, Cell.

[32]  Keiji Tanaka,et al.  CHIP is a chaperone‐dependent E3 ligase that ubiquitylates unfolded protein , 2001, EMBO reports.

[33]  B. Song,et al.  Gp78, a membrane-anchored ubiquitin ligase, associates with Insig-1 and couples sterol-regulated ubiquitination to degradation of HMG CoA reductase. , 2005, Molecular cell.

[34]  Daniel Kaganovich,et al.  Protein quality control: chaperones culling corrupt conformations , 2005, Nature Cell Biology.

[35]  T. Sommer,et al.  ERAD: the long road to destruction , 2005, Nature Cell Biology.

[36]  M. Patterson,et al.  PART 16 : LYSOSOMAL DISORDERS Chapter 145 : Niemann-Pick Disease Type C : A Lipid Trafficking Disorder , 2007 .

[37]  Kenji Ohgane,et al.  Discovery of oxysterol-derived pharmacological chaperones for NPC1: implication for the existence of second sterol-binding site. , 2013, Chemistry & biology.

[38]  K. Higaki,et al.  Accumulation of cholera toxin and GM1 ganglioside in the early endosome of Niemann–Pick C1-deficient cells , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[39]  Y. Kitaura,et al.  Active HSF1 Significantly Suppresses Polyglutamine Aggregate Formation in Cellular and Mouse Models* , 2005, Journal of Biological Chemistry.