Loss of Anti-Bax Function in Gerstmann-Sträussler-Scheinker Syndrome-Associated Prion Protein Mutants

Abstract

Previously, we have shown the loss of anti-Bax function in Creutzfeldt Jakob disease (CJD)-associated prion protein (PrP) mutants that are unable to generate cytosolic PrP (CyPrP). To determine if the anti-Bax function of PrP modulates the manifestation of prion diseases, we further investigated the anti-Bax function of eight familial Gerstmann-Sträussler-Scheinker Syndrome (GSS)-associated PrP mutants. These PrP mutants contained their respective methionine (M) or valine (V) at codon 129. All of the mutants lost their ability to prevent Bax-mediated chromatin condensation or DNA fragmentation in primary human neurons. In the breast carcinoma MCF-7 cells, the F198SV, D202NV, P102LV and Q217RV retained, whereas the P102LM, P105LV, Y145stopM and Q212PM PrP mutants lost their ability to inhibit Bax-mediated condensed chromatin. The inhibition of Bax-mediated condensed chromatin depended on the ability of the mutants to generate cytosolic PrP. However, except for the P102LV, none of the mutants significantly inhibited Bax-mediated caspase activation. These results show that the cytosolic PrP generated from the GSS mutants is not as efficient as wild type PrP in inhibiting Bax-mediated cell death. Furthermore, these results indicate that the anti-Bax function is also disrupted in GSS-associated PrP mutants and is not associated with the difference between CJD and GSS.

Human GBP1 binds LPS to initiate assembly of a caspase-4 activating platform on cytosolic bacteria

“…The pAIP vectors expressing HA-GBP1 or HA-GBP2 were a kind gift from T. Henry (CIRI, Lyon) and were used to generate HA-tagged GBPs, by replacing GBP1 by the GBP3 or −4 coding sequences at the EcoRI sites. The bicistronic plasmids encoding FLAG-GBP3 + HA-GBP4 were generated by inserting GBP3 or GBP4 at the NotI/PmeI sites of the pBud-EGFP vector (addgene 23027) 44 . Doxycyclineinducible eGFP-GBP1, −2, −3, −4 were generated by amplifying eGFP-GBPs generated above by PCR and inserting the coding sequences at the BamHI site of the pLVX-Puro vector (Clontech).…”

Luman contributes to brefeldin A-induced prion protein gene expression by interacting with the ERSE26 element

“…The DNA sequence coding for HA-Luman was amplified from MCF-7 cDNA with high fidelity Pfu polymerase (Agilent Technologies, Santa Clara CA) using the forward 5′-CCG CTA AAG CTT ACC ATG GCA TAC CCA TAC GAC GTC CCA GAC TAC GCT GAG CTG GAA TTG GAT GCT GGT GA-3′ and the reverse 5′-ACG CGA GTC GAC TAG CCT GAG TAT CTG TCC TGC-3′ primers, while amplification of the sequence coding for Luman amino acid 1 to 215 was accomplished using the forward 5′-CCG CTA AAG CTT AGC ATG GAG CTG GAA TTG GAT GC-3′ and reverse 5′-ACG CGA GTC GAC TAG GCC TGG AGT TTC CTC AGT TG-3′ primers. Both pairs introduced flanking HindIII and SalI sites used for cloning into the pBud-eGFP vector 55 . pBud-eGFP-ΔLuman-Myc was generated by mutating the stop codon by QuikChange Site-Directed Mutagenesis (Stratagene, LaJolla, CA), using the forward primer 5′-CTG AGG AAA CTC CAG GCC TCG TCG ACA TCG ATC TTA AGC-3′ and its reverse complement.…”

Presenilin 2 overexpression is associated with apoptosis in Neuro2a cells

“…Interestingly, the high level of chromatin condensation observed can be correlated with an upregulation of Bax, which activates caspases. This leads to increased chromatin condensation and cellular apoptosis [28]. Moreover, Bax-mediated p53-independent apoptotic activation of caspase 3 has also been reported in several studies [29, 30].…”