[24] Membrane filter assay for detection of amyloid-like polyglutamine-containing protein aggregates

Wanker, Erich E.; Scherzinger, Eberhard; Heiser, Volker; Sittler, Annie; Eickhoff, Holger; Lehrach, Hans

doi:10.1016/s0076-6879(99)09026-6

Cited by 224 publications

(253 citation statements)

References 23 publications

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“…HEKT-cell lines stably expressing HTT-exon 1 with either 20, 51 or 83 CAG repeats were transfected either with MID1-or non-silencing control siRNA oligonucleotides. Cells were analysed for HTT-exon 1 protein expression either by western blotting or, for aggregate formation, in a filter-aggregation assay, which was developed by Wanker et al 33 . While the knockdown of MID1 (knockdown control see Supplementary Fig.…”

Section: Mid1mentioning

confidence: 99%

Translation of HTT mRNA with expanded CAG repeats is regulated by the MID1–PP2A protein complex

et al. 2013

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Expansion of CAG repeats is a common feature of various neurodegenerative disorders, including Huntington's disease. Here we show that expanded CAG repeats bind to a translation regulatory protein complex containing MID1, protein phosphatase 2A and 40S ribosomal S6 kinase. Binding of the MID1-protein phosphatase 2A protein complex increases with CAG repeat size and stimulates translation of the CAG repeat expansion containing messenger RNA in a MID1-, protein phosphatase 2A-and mammalian target of rapamycindependent manner. Our data indicate that pathological CAG repeat expansions upregulate protein translation leading to an overproduction of aberrant protein and suggest that the MID1-complex may serve as a therapeutic target for the treatment of CAG repeat expansion disorders.

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Section: Mid1mentioning

confidence: 99%

Translation of HTT mRNA with expanded CAG repeats is regulated by the MID1–PP2A protein complex

et al. 2013

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“…40 Briefly, protein lysates were solubilized in 1% SDS and directly spotted onto nitrocellulose membrane. Vacuum blotting was performed for 30 min by gel dryer (Bio-Rad, Munich, Germany).…”

Section: Determination Of Protein Aggregatesmentioning

confidence: 99%

BAG3 induction is required to mitigate proteotoxicity via selective autophagy following inhibition of constitutive protein degradation pathways

2013

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Simultaneous inhibition of the two major constitutive protein quality control (PQC) pathways, that is, the ubiquitin-proteasome system (UPS) and the aggresome-autophagy system, has been suggested as a promising strategy to trigger cell death in cancer cells. However, we observed that one third of rhabdomyosarcoma (RMS) cells survives parallel inhibition of the UPS by Bortezomib and the aggresome-autophagy pathway by the cytoplasmic histone deacetylase 6 inhibitor ST80, and is able to regrow upon drug removal, thus pointing to the induction of compensatory pathways. Here, we identify Bcl-2-associated athanogene 3 (BAG3) as a critical mediator of inducible resistance in surviving cells after concomitant blockage of constitutive PQC pathways by mitigating ST80/Bortezomib-triggered proteotoxicity via selective autophagy. ST80/Bortezomib cotreatment upregulates BAG3 mRNA and protein levels in surviving cells in addition to triggering the accumulation of insoluble protein aggregates. Intriguingly, knockdown of BAG3 by RNA interference severely impairs clearance of protein aggregates, significantly increases cell death and reduces longterm survival and clonogenic growth during recovery after ST80/Bortezomib cotreatment. Similarly, inhibition of autophagy by inducible autophagy-related protein 7 knockdown prevents removal of protein aggregates and cell regrowth during recovery after ST80/Bortezomib cotreatment. Also, the inhibition of lysosomal degradation using the V-ATPase pump inhibitor Bafilomycin A1 enhances accumulation of protein aggregates, and completely abolishes regrowth after Bortezomib/ST80-induced proteotoxic stress. By identifying BAG3 as a key mediator of inducible resistance by mitigating proteotoxicity via selective autophagy after inhibition of constitutive PQC systems, our study provides new insights into the regulation of PQC pathways in cancer cells and identifies new targets for therapeutic intervention.

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“…41 While polyQ-containing mutant HTT formed tight protein aggregates resistant to harsh chemicals such as 6 M urea (Fig. 8A), we did not detect such aggregates in cells expressing GST-BHMT with or without MG132 treatment.…”

Section: Proteasome Inhibition-induced Gst-bhmt Fragmentation Requirementioning

confidence: 57%

“…41 Briefly, cells transiently expressed with GST-BHMT or GFP-HTT-exon1-Q72 were lysed in phosphate-buffered saline by sonication. Bradford method was used to calculate protein concentration.…”

Section: Filter Trap Assaymentioning

confidence: 99%

The GST-BHMT assay reveals a distinct mechanism underlying proteasome inhibition-induced macroautophagy in mammalian cells

Rui

Chen

et al. 2015

Autophagy

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#These authors equally contributed to this work.Keywords: BHMT, cargo receptors SQSTM1/p62 and NBR1, MTOR, PRKAA/AMPK, proteasome inhibition, selective macroautophagyAbbreviations: ACACA/B, acetyl-CoA carboxylase a/b; ACTB, actin, b; ATF4, activating transcription factor 4; ATF6, activating transcription factor 6; ATG7, autophagy-related 7; Baf A1, bafilomycin A 1 ; BCL2, B-cell CLL/lymphoma 2; BECN1, Beclin 1, autophagy-related; BHMT, betaine-homocysteine S-methyltransferase; CTNNB1, catenin (cadherin-associated protein), b 1, 88kDa; Cvt, cytoplasm-to-vacuole-targeting; DDIT3, DNA-damage-inducible transcript 3; EBSS, Earle's Balanced Salt Solution; EIF2AK3, eukaryotic translation initiation factor 2-a, kinase 3; EIF4EBP1, eukaryotic translation initiation factor 4E binding protein 1; ER, endoplasmic reticulum; ERN1, endoplasmic reticulum to nucleus signaling 1; GST, glutathionine S-transferase; GST-BHMT, a fusion protein of glutathionine S-transferase N-terminal to betaine-homocysteine S-methyltransferase; GST-BHMT (FRAG) , an autophagy-mediated cleavage product of the GST-BHMT reporter; HA, hemagglutinin; HSPA5, heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa); LSCS, linker-specific cleavage site; MAP1LC3, microtubule-associated protein 1 light chain 3; MAP2K7, mitogen-activated protein kinase kinase 7; MAPK8, mitogen-activated protein kinase 8; MTOR, mechanistic target of rapamycin (serine/threonine kinase); MTORC1, MTOR complex 1; NBR1, neighbor of BRCA1 gene 1; P4HB, prolyl 4-hydroxylase, b polypeptide; PRKAA, protein kinase, AMP-activated, a catalytic subunit; RHEB, Ras homolog enriched in brain; RM, rich medium; RPS6KB1, ribosomal protein S6 kinase, 70kDa, polypeptide 1; SQSTM1, sequestosome 1; TSC1/2, tuberous sclerosis 1/2; ULK1, unc-51 like autophagy activating kinase 1; UPR, unfolded protein response; UPS, ubiquitin proteasome system; XBP1, X-box binding protein 1By monitoring the fragmentation of a GST-BHMT (a protein fusion of glutathionine S-transferase N-terminal to betaine-homocysteine S-methyltransferase) reporter in lysosomes, the GST-BHMT assay has previously been established as an endpoint, cargo-based assay for starvation-induced autophagy that is largely nonselective. Here, we demonstrate that under nutrient-rich conditions, proteasome inhibition by either pharmaceutical or genetic manipulations induces similar autophagy-dependent GST-BHMT processing. However, mechanistically this proteasome inhibition-induced autophagy is different from that induced by starvation as it does not rely on regulation by MTOR (mechanistic target of rapamycin [serine/threonine kinase]) and PRKAA/AMPK (protein kinase, AMP-activated, a catalytic subunit), the upstream central sensors of cellular nutrition and energy status, but requires the presence of the cargo receptors SQSTM1/p62 (sequestosome 1) and NBR1 (neighbor of BRCA1 gene 1) that are normally involved in the selective autophagy pathway. Further, it depends on ER (endoplasmic reticulum) stress signaling, in particular ERN1/IRE1 (endoplasmic ...

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[24] Membrane filter assay for detection of amyloid-like polyglutamine-containing protein aggregates

Cited by 224 publications

References 23 publications

Translation of HTT mRNA with expanded CAG repeats is regulated by the MID1–PP2A protein complex

Translation of HTT mRNA with expanded CAG repeats is regulated by the MID1–PP2A protein complex

BAG3 induction is required to mitigate proteotoxicity via selective autophagy following inhibition of constitutive protein degradation pathways

The GST-BHMT assay reveals a distinct mechanism underlying proteasome inhibition-induced macroautophagy in mammalian cells

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