Presentation on theme: "Characterization of an ERAD Pathway for Nonglycosylated BiP Substrates, which Require Herp Yuki Okuda-Shimizu and Linda M. Hendershot Molecular Cell 28:544-554,"— Presentation transcript:
Characterization of an ERAD Pathway for Nonglycosylated BiP Substrates, which Require Herp Yuki Okuda-Shimizu and Linda M. Hendershot Molecular Cell 28:544-554, November 30, 2007 PTRM Student presentation November 4, 2008 Chang-Hyun Kim
ER-associated Degradation (ERAD) 1.Recognition of a substrate (misfolded or unfolded) within ER. 2.Transported across the ER membrane through the retrotranslocon. 3.In cytosol, Polyubiquitination and Degradation by 26S proteasome.
Functions of the ER 1. Translocating nascent secretory pathway proteins into the ER 3. Maintaining intracellular Ca 2+ stores, oxidizing environment 2. Folding and assembly of nascent proteins 5. Monitoring conditions for proper folding and activating the UPR 4. Identifying and targeting incompletely or improperly folded proteins for degradation Proteosome
ERAD subpathways in Yeast ERAD-L pathway 1.Misfold on ER-luminal domain 2.Hrd1p/Hrd3p ubiquitin ligase form complex with Der1p via the linker protein Usa1p. ERAD-M pathway: Misfold on transmembrane domain ERAD-C pathway: Misfold on cytosolic domain
Carvalho et al, 2006 Cell 126:361-373 The scheme shows the ubiquitin-ligase complexes involved in the ERAD-L, -M, and –C pathways. Components in orange and green belong to the Hrd1p core and Cdc48p ATPase complexes, respectively. Stars show the location of the misfolded domain of a substrate. Ub is ubiquitin.
Mammalian homologs or functional equivalents of the components of the yeast ubiquitin ligase complexes. Question marks (?) indicate uncertainty. Carvalho et al, 2006 Cell 126:361-373
Der1p in Yeast Four transmembrane domains Constitute part of the channel for retrotranslocation of substrate with misfold on ER-domain Mammalian equivalents: Derlin-1, Derlin-2, Derlin-3
Derlin-1 Mammalian equivalent of Der1p in yeast. Derlin-1 is in a complex with Herp, AAA ATPase Cdc48/p97, and ubiquitin ligase Hrd1.
Figure 6 Model for US11-mediated retro-translocation of MHC class I heavy chains. US11 recognizes HC in the ER lumen and targets it to Derlin-1, a proposed component of the retro-translocation channel. The p97 ATPase complex is recruited to Derlin-1 by VIMP. HC emerging into the cytosol is bound by p97. Poly-ubiquitin chains (Poly-Ub, red) are attached and recognized by both the N- domain (N) of p97 and the cofactor Ufd1/Npl4 (U/N). ATP hydrolysis by p97 moves HC into the cytosol. The retro-translocation of misfolded ER proteins may occur similarly, with US11 being replaced by other targeting components. Ye et al, 2004. Nature 429:841-847
FIG. 9. Herp on the ER membrane. The majority of the Herp molecule on the ER membrane faces the cytoplasm. Hydropathic profile of the amino acid sequence of human Herp was obtained according to the algorithm of Kyte and Doolittle (41). Positive values represent increased hydrophobicity. (Kokame et al, (2000 ) J. Biol. Chem. 275:32846-32853)
ERAD for misfolded glycoprotein in mammalian cells Most glycoproteins interact with calnexin/calreticulin chaperone family during folding. The chaperones monitor cyclic processing of N-linked glycans ER-degradation-enhancing alpha-mannosidase-like protein (EDEM) recognize the protein of which glycan is trimed too far by mannosidases ERAD e.g. misfolded 1-antitrypsin Null Hong Kong (AAT NHK) glycoprotein is a substrate of calnexin/calreticulin and requires either Derlin-2 or -3 for its degradation
Study question It is much less clear how unfolded, nonglycosylated proteins that utilize BiP are recognized and targeted for degradation. How are unfold, nonglycosylated proteins that utilize BiP recognized and targeted for degradation?
Method BiP is required for retrotranslocation of ERAD substrate. BiP-binding domain controls the rate of degradation of Ig LC mutants. Three BiP-binding proteins: 1.Nonsecreted Ig LC 2.Mutant Ig LC 3.Truncated Ig HC
Figure 1. Nonsecreted LCs in P3U.1 are degraded by the 26S Proteasome P3U.1 cells produces LC (no LC). In reduced condition, disulfide bonds get lost. Lactacystin is a selective inhibitor of proteasome.
Figure 1. Nonsecreted LCs in P3U.1 are degraded by the 26S Proteasome…….continued Lactacystin is a selective inhibitor of proteasome. NH 4 Cl is an inhibitor of lysosomal degradation.
Figure 2. The partially oxidized form of nonsecreted LC in P3U1 cells in ubiquitinated Cells were harvested after 6-hour treatment.
Figure 2. The partially oxidized form of nonsecreted LC in P3U1 cells in ubiquitinated…….continued
NS1 LC is degraded by the 26S proteasome Partially oxidized (Ox1) form of NS1 LC is ubiquitinated How is NS1 LC extracted from the ER lumen?
Figure 3. The partially oxidized form of nonsecreted LC, which is a BiP substrate, interact with Herp and Derlin-1 P3X ( +, + ), P3U.1 ( -, + ), Ag8(8) ( +, - ), Ag8.653 ( -, - ) ?
Figure 3. The partially oxidized form of nonsecreted LC, which is a BiP substrate, interact with Herp and Derlin-1…….continued 1.Immunoprecipitated with anti-Herp 2.boiled in the presence of SDS to release bound proteins from the beads. 3.divided into three portions for a second immunoprecipitation with either anti-Herp, anti-k, or Protein A-Sepharose alone
Figure 3. The partially oxidized form of nonsecreted LC, which is a BiP substrate, interact with Herp and Derlin-1…….continued 1.Herp and Derlin-1 form a complex with p97 and Hrd1 2.Cotransfect 293T with NS1 LC along with p97 or Hrd1
Figure 4. Overexpressed Herp-FLAG interacts with the BiP substrates, that is, nonsecreted LC mutant and unassembled Ig HC mutant, but not with the calnexin/calreticulin substrates, that is 1-antitrypsin variants Cotransfection 293T with FLAG tagged Herp along with BiP substrates
Figure 4. Overexpressed Herp-FLAG interacts with the BiP substrates, that is, nonsecreted LC mutant and unassembled Ig HC mutant, but not with the calnexin/calreticulin substrates, that is 1-antitrypsin variants…….continued
Tm=Tunicamycin: inhibits the synthesis of N-linked glycoprotein
Misfolded AAT NHK ( 1-antitrypsin Null Hong Kong ) Substrate for calnexin/calreticulin Requires Derlin-2 or -3 for its degradation
Figure 5. Herp interacts with the 26S proteasome and ubiquitinated substrates
Figure 5. Herp interacts with the 26S proteasome and ubiquitinated substrates…….continued
Figure 6. siRNA-mediated repression of Herp leads to the stabilization of nonsecreted LC, but not of 1-antitrypsin variants
1. ER Stress Signal BiP-associated unfolded proteins ER Nucleus 2. Signal Transducers Ire1, PERK, ATF6 3. Downstream Elements eIF2- phosphorylation p38 activation ATF6 cleavage CHOP induction NF B activation XBP1 cleavage ATF4 induction eIF-2 P P P P P Translation inhibition Cell cycle arrest ATF4 synthesis 5. Defeat Caspase 12 activation Apoptosis 4. Transcriptional Responses GRPs / XBP-1 CHOP ?? NF B targets XBP1 targets - -
Figure 6. siRNA-mediated repression of Herp leads to the stabilization of nonsecreted LC, but not of 1-antitrypsin variants…….continued