Characterization of an ERAD Pathway forNonglycosylated BiP Substrates, which

Require Herp

Yuki Okuda-Shimizu and Linda M. HendershotMolecular Cell 28:544-554, November 30, 2007

PTRM Student presentationNovember 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 Ca2+ 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 pathway1.Misfold on ER-luminal domain2.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 NullHong 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 LC2.Mutant Ig LC3.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.NH4Cl 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-Herp2. 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 Hrd12. 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

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 NullHong 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 SignalBiP-associated unfolded proteins

ER

Nucleus

2. Signal TransducersIre1, PERK, ATF6

3. Downstream ElementseIF2- phosphorylationp38 activationATF6 cleavageCHOP inductionNFB activationXBP1 cleavageATF4 induction

eIF-2 P

P

P

PP

Translation inhibition Cell cycle arrest ATF4 synthesis

5. DefeatCaspase 12 activationApoptosis

4. Transcriptional Responses

GRPs / XBP-1

CHOP

??NFB targets

XBP1 targets

--

Figure 6. siRNA-mediated repression of Herp leads to the stabilization of nonsecreted LC, but not of

1-antitrypsin variants…….continued

Figure 6. siRNA-mediated repression of Herp leads to the stabilization of nonsecreted LC, but not of

1-antitrypsin variants…….continued

Figure 7. Model for degradation of NS1 LC