A Pro-Cathepsin L Mutant Is a Luminal Substrate for Endoplasmic-Reticulum-Associated Degradation in C. elegans

Miedel, Mark T.; Graf, Nathan J.; Stephen, Kate E.; Long, Olivia S.; Pak, Stephen C.; Perlmutter, David H.; Silverman, Gary A.; Luke, Cliff J.

doi:10.1371/journal.pone.0040145

Cited by 41 publications

(42 citation statements)

References 53 publications

(69 reference statements)

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“…3E ). In support of these observations, 35 S-methionine/cysteine labeling showed that basal-B lines (Hs578T, BT549, MDA-MB-157, SUM159, MDA-MB-231, and 4T1) also exhibited, on average, a more than 40-fold increase in protein secretion relative to luminal lines (MCF7, T47D, BT474, and ZR-75-3; Fig. 3F ).…”

Section: Cells That Undergo An Emt Are Highly Secretorysupporting

confidence: 68%

“…Relative expression To determine whether increased ECM secretion is a general feature of EMT, we examined the expression of ECM genes identifi ed to be upregulated upon EMT ( Fig. 3A ) in basal-B and luminal breast cancer lines ( 35 ). Basal-B cancer lines ( n = 9) expressed many EMT ECM genes-including FN1 , COL1A1 , COL1A2 , COL4A1 , COL5A1 , POSTN , FBN1 , and COL6A1 -at signifi cantly higher levels than luminal breast cancer lines ( n = 13; Fig.…”

Section: Cells That Undergo An Emt Are Highly Secretorymentioning

confidence: 99%

“…If this were indeed the case, then reducing ECM levels would attenuate UPR activation in response to ER stressors. To examine this, we analyzed the proteins secreted by two nontumorigenic EMT lines (HMLE_shEcad and HMLE_Twist) that, by 35 S-methionine/ cysteine labeling, strongly upregulated secretion of a limited number of proteins ( Fig. 3C ).…”

Section: Upregulated Ecm Secretion Following Emt Sensitizes Cells To mentioning

confidence: 99%

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Epithelial-to-Mesenchymal Transition Activates PERK–eIF2α and Sensitizes Cells to Endoplasmic Reticulum Stress

et al. 2014

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Epithelial-to-mesenchymal transition (EMT) promotes both tumor progression and drug resistance, yet few vulnerabilities of this state have been identifi ed. Using selective small molecules as cellular probes, we show that induction of EMT greatly sensitizes cells to agents that perturb endoplasmic reticulum (ER) function. This sensitivity to ER perturbations is caused by the synthesis and secretion of large quantities of extracellular matrix (ECM) proteins by EMT cells. Consistent with their increased secretory output, EMT cells display a branched ER morphology and constitutively activate the PERK-eIF2α axis of the unfolded protein response (UPR). Protein kinase RNA-like ER kinase (PERK) activation is also required for EMT cells to invade and metastasize. In human tumor tissues, EMT gene expression correlates strongly with both ECM and PERK-eIF2α genes, but not with other branches of the UPR. Taken together, our fi ndings identify a novel vulnerability of EMT cells, and demonstrate that the PERK branch of the UPR is required for their malignancy. SIGNIFICANCE:EMT drives tumor metastasis and drug resistance, highlighting the need for therapies that target this malignant subpopulation. Our fi ndings identify a previously unrecognized vulnerability of cancer cells that have undergone an EMT: sensitivity to ER stress. We also fi nd that PERK-eIF2α signaling, which is required to maintain ER homeostasis, is also indispensable for EMT cells to invade and metastasize. Cancer Discov; 4(6);

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Section: Cells That Undergo An Emt Are Highly Secretorysupporting

confidence: 68%

Section: Cells That Undergo An Emt Are Highly Secretorymentioning

confidence: 99%

Section: Upregulated Ecm Secretion Following Emt Sensitizes Cells To mentioning

confidence: 99%

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Epithelial-to-Mesenchymal Transition Activates PERK–eIF2α and Sensitizes Cells to Endoplasmic Reticulum Stress

et al. 2014

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“…Subsequently the amount of aggregates in the head region of 10 worms was counted. As a control for efficient inhibition of the autophagy pathway, autophagosome formation of nhx-2:: mCherry::lgg-1; myo-2::GFP worms was visualized upon RNAi treatment following 4 hr starvation as previously described (Miedel et al 2012).…”

Section: Autophagymentioning

confidence: 99%

A Novel Interaction Between Aging and ER Overload in a Protein Conformational Dementia

et al. 2013

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Intraneuronal deposition of aggregated proteins in tauopathies, Parkinson disease, or familial encephalopathy with neuroserpin inclusion bodies (FENIB) leads to impaired protein homeostasis (proteostasis). FENIB represents a conformational dementia, caused by intraneuronal polymerization of mutant variants of the serine protease inhibitor neuroserpin. In contrast to the aggregation process, the kinetic relationship between neuronal proteostasis and aggregation are poorly understood. To address aggregate formation dynamics, we studied FENIB in Caenorhabditis elegans and mice. Point mutations causing FENIB also result in aggregation of the neuroserpin homolog SRP-2 most likely within the ER lumen in worms, recapitulating morphological and biochemical features of the human disease. Intriguingly, we identified conserved protein quality control pathways to modulate protein aggregation both in worms and mice. Specifically, downregulation of the unfolded protein response (UPR) pathways in the worm favors mutant SRP-2 accumulation, while mice overexpressing a polymerizing mutant of neuroserpin undergo transient induction of the UPR in young but not in aged mice. Thus, we find that perturbations of proteostasis through impairment of the heat shock response or altered UPR signaling enhance neuroserpin accumulation in vivo. Moreover, accumulation of neuroserpin polymers in mice is associated with an age-related induction of the UPR suggesting a novel interaction between aging and ER overload. These data suggest that targets aimed at increasing UPR capacity in neurons are valuable tools for therapeutic intervention.

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“…Since depletion of mdt-15 had no effect on the degree of SRP-2 H302R ::YFP aggregation, they conclude that this mutation activates the UPR directly by altering ER membrane lipid content and not secondarily by impairing protein folding capability within the ER and stimulating the UPR by increasing the accumulation of misfolded species. Further, evidence for this direct effect is supported by no increase in a second ER folding sensor, the luminal ERAD substrate, CPL-1 W32A,Y35A :: YFP (Miedel et al 2012;Hou et al 2014). While the overall conclusions of this study are supported by the CPL-1 misfolding mutant, we are hesitant to draw this conclusion based on the experiments using SRP-2 H302R ::YFP per se.…”

Section: Discussionmentioning

confidence: 61%

The Aggregation-Prone Intracellular Serpin SRP-2 Fails to Transit the ER inCaenorhabditis elegans

Silverman¹,

Cummings²,

O’Reilly³

et al. 2015

Genetics

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Familial encephalopathy with neuroserpin inclusions bodies (FENIB) is a serpinopathy that induces a rare form of presenile dementia. Neuroserpin contains a classical signal peptide and like all extracellular serine proteinase inhibitors (serpins) is secreted via the endoplasmic reticulum (ER)-Golgi pathway. The disease phenotype is due to gain-of-function missense mutations that cause neuroserpin to misfold and aggregate within the ER. In a previous study, nematodes expressing a homologous mutation in the endogenous Caenorhabditis elegans serpin, srp-2, were reported to model the ER proteotoxicity induced by an allele of mutant neuroserpin. Our results suggest that SRP-2 lacks a classical N-terminal signal peptide and is a member of the intracellular serpin family. Using confocal imaging and an ER colocalization marker, we confirmed that GFP-tagged wild-type SRP-2 localized to the cytosol and not the ER. Similarly, the aggregationprone SRP-2 mutant formed intracellular inclusions that localized to the cytosol. Interestingly, wild-type SRP-2, targeted to the ER by fusion to a cleavable N-terminal signal peptide, failed to be secreted and accumulated within the ER lumen. This ER retention phenotype is typical of other obligate intracellular serpins forced to translocate across the ER membrane. Neuroserpin is a secreted protein that inhibits trypsinlike proteinase. SRP-2 is a cytosolic serpin that inhibits lysosomal cysteine peptidases. We concluded that SRP-2 is neither an ortholog nor a functional homolog of neuroserpin. Furthermore, animals expressing an aggregation-prone mutation in SRP-2 do not model the ER proteotoxicity associated with FENIB.

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A Pro-Cathepsin L Mutant Is a Luminal Substrate for Endoplasmic-Reticulum-Associated Degradation in C. elegans

Cited by 41 publications

References 53 publications

Epithelial-to-Mesenchymal Transition Activates PERK–eIF2α and Sensitizes Cells to Endoplasmic Reticulum Stress

Epithelial-to-Mesenchymal Transition Activates PERK–eIF2α and Sensitizes Cells to Endoplasmic Reticulum Stress

A Novel Interaction Between Aging and ER Overload in a Protein Conformational Dementia

The Aggregation-Prone Intracellular Serpin SRP-2 Fails to Transit the ER inCaenorhabditis elegans

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