Marsters, S. A. | bioRxiv Science

bioRxiv2024

TRAIL induces caspase-dependent cleavage of specific components of the clathrin-mediated endocytosis machinery

It has been reported that caspase activation by the proapoptotic ligand Apo2L/TRAIL disrupts the clathrin-mediated endocytosis machinery (CMEM). To confirm whether TRAIL induces caspase-mediated cleavage of specific CMEM components, we examined the temporal and functional relationship between TRAIL-induced processing of the apoptosis-initiating protease, caspase-8, and the apoptosis-executing protease, caspase-3, versus cleavage of specific adaptin (AP) and clathrin heavy chain (CHC) proteins. TRAIL induced time-dependent proteolytic processing of caspase-8 and caspase-3, which coincided with cleavage of AP2 and CHC. The pan caspase inhibitor zVAD-FMK, which blocked caspase processing in response to TRAIL, also prevented the cleavage AP2. Whereas FADD-deficient or caspase-8-deficient cells showed little or no TRAIL-driven cleavage of either AP2 or CHC, Bax-deficient or caspase-3-deficient cell lines retained AP2 cleavage but failed to cleave CHC in response to TRAIL. The DNA-damaging agent doxorubicin also led to processing of caspase-8 and caspase-3 in conjunction ith cleavage of AP2 and CHC. Together, these results confirm that TRAIL induces caspase-dependent cleavage of AP2 and CHC. Whereas TRAIL-driven cleavage of both AP2 and CHC requires caspase-8, cleavage of CHC, but not of AP2, requires caspase-3. MATERIALS AND METHODS Cell lines and cell cultureAll cell lines were obtained from ATCC or as kind gifts from Dr. Bert Vogelstein (HCT116 Bax-/-) and Dr. John Blenis (Jurkat I9.2 and E1). Cell lines were cultured with standard media as previously described (1). Immunoblot analysisCells were lysed in RIPA lysis buffer (20-188, Millipore) supplemented with Halt protease and phosphatase inhibitor cocktail (ThermoFisher Scientific) and kept on ice for 30 min. Lysates were cleared by centrifugation at 13,600 x g for 15 min at 4 {degrees}C, and protein amount was determined by BCA protein assay (ThermoFisher Scientific). Protein was denatured by adding NuPAGE LDS buffer and DTT reducing buffer (Invitrogen) and incubating the samples at 95 {degrees}C for 5 min. Equal amounts of denatured protein were loaded into each well of NuPAGE pre-cast gels (Invitrogen), resolved by SDS-PAGE, and electro-transferred to nitrocellulose membranes using the iBLOT2 system (Invitrogen). Membranes were blocked in a 5% nonfat milk solution for 1 hr at room temperature and probed with the corresponding primary antibody at 1:1,000 dilution overnight at 4 {degrees}C. This was followed by incubation with the corresponding horseradish peroxidase (HRP)- conjugated secondary antibody at 1:10,000 dilution during 1 hr at room temperature. All secondary antibodies were from Jackson Laboratories. The primary antibodies and secondary HRP-conjugated antibodies are listed respectively in Table 1 and Table 2. {beta}-actin IB was used to verify uniformity of protein loading and electro-transfer. O_TBL View this table: org.highwire.dtl.DTLVardef@ed5a65org.highwire.dtl.DTLVardef@c9aecdorg.highwire.dtl.DTLVardef@f31b4aorg.highwire.dtl.DTLVardef@849deaorg.highwire.dtl.DTLVardef@9b847b_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 1.C_FLOATNO O_TABLECAPTIONPrimary antibodies used for IB analysis C_TABLECAPTION C_TBL O_TBL View this table: org.highwire.dtl.DTLVardef@16c33forg.highwire.dtl.DTLVardef@6c6b81org.highwire.dtl.DTLVardef@5efb4org.highwire.dtl.DTLVardef@1bc354forg.highwire.dtl.DTLVardef@15007aa_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 2.C_FLOATNO O_TABLECAPTIONHorseradish peroxidase conjugated secondary antibodies used for IB analysis C_TABLECAPTION C_TBL Additional reagentsRecombinant soluble non-tagged human TRAIL (Apo2L.0) and Flag-tagged TRAIL were prepared at Genentech. Flag-tagged TRAIL was crosslinked with anti-Flag M2 antibody at a ligand-to-antibody molar ratio of 1:2. zVAD-FMK was purchased from R & D Systems, FMK001. Doxorubicin was purchased from Sigma Aldrich, D1515.

cell biology↗

bioRxiv2021

Antigen-derived peptides directly engage the unfolded-protein sensor IRE1α to curb cross-presentation by dendritic cells

Dendritic cells (DCs) promote adaptive immunity by cross-presenting antigen-based epitopes to CD8+ T cells. DCs process internalized protein antigens into peptides that enter the endoplasmic reticulum (ER) and upload onto major histocompatibility type I (MHC-I) protein complexes for cell-surface transport and cross-presentation. Perplexingly, DCs often exhibit activation of the ER-stress sensor IRE1 in the absence of classical ER stress--leaving the underlying mechanism unexplained. Here we show that antigen-derived hydrophobic peptides directly engage ER-resident IRE1 by masquerading as unfolded proteins. Furthermore, IRE1 activation depletes MHC-I heavy-chain mRNAs through regulated IRE1-dependent decay (RIDD), thereby curtailing antigen cross-presentation. In tumor-bearing mice, IRE1 disruption increased MHC-I expression on tumor-infiltrating DCs, and enhanced recruitment and activation of CD8+ T cells. Moreover, IRE1 inhibition synergized with anti-PD-L1 antibody treatment to cause tumor regression. Our findings elucidate the mechanism and consequence of antigen-driven IRE1 activation in DCs, yielding a promising combination strategy for cancer immunotherapy.

cell biology↗

bioRxiv2018

Disruption of IRE1α through its Kinase Domain Attenuates Multiple Myeloma

Multiple myeloma (MM) arises from malignant immunoglobulin-secreting plasma cells and remains an incurable, often lethal disease despite recent therapeutic advances. The unfolded-protein response sensor IRE1 supports protein secretion by deploying a kinase-endoribonuclease module to activate the transcription factor XBP1s. MM cells may coopt the IRE1-XBP1s pathway; however, the validity of IRE1 as a potential MM therapeutic target is controversial. Here we show that genetic disruption of IRE1 or XBP1s, or pharmacologic IRE1 kinase inhibition, attenuated subcutaneous or orthometastatic growth of MM tumors in mice, and augmented efficacy of two well-established frontline antimyeloma agents, bortezomib or lenalidomide. Mechanistically, IRE1 perturbation inhibited expression of key components of the ER-associated degradation machinery, as well as cytokines and chemokines known to promote MM growth. Selective IRE1 kinase inhibition reduced viability of CD138+ plasma cells while sparing CD138- cells from bone marrow of newly diagnosed MM patients or patients whose disease relapsed after 1 - 4 lines of treatment in both US- and EU-based cohorts. IRE1 inhibition preserved survival and glucose-induced insulin secretion by pancreatic microislets. Together, these results establish a strong therapeutic rationale for targeting IRE1 with kinase-based small-molecule inhibitors in MM.\n\nSignificance statementMultiple myeloma (MM) is a lethal malignancy of plasma cells. MM cells have an expanded endoplasmic reticulum (ER) that is constantly under stress due to immunoglobulin hyperproduction. The ER-resident sensor IRE1 mitigates ER stress by expanding the ERs protein-folding capacity while supporting proteasomal degradation of misfolded ER proteins. IRE1 elaborates these functions by deploying its cytoplasmic kinase-RNase module to activate the transcription factor XBP1s. The validity of IRE1 as a potential therapeutic target in MM has been questioned. Using genetic and pharmacologic disruption in vitro and in vivo, we demonstrate that the IRE1-XBP1s pathway plays a critical role in MM growth. We further show that IRE1s kinase domain is an effective and safe potential small-molecule target for MM therapy.

cancer biology↗