Quantitative Interactome Proteomics Reveals a Molecular Basis for ATF6-Dependent Regulation of a Destabilized Amyloidogenic Protein

Plate, Lars; Rius, Bibiana; Nguyen, Bianca; Genereux, Joseph C.; Wiseman, R. Luke

doi:10.1016/j.chembiol.2019.04.001

Cited by 31 publications

(40 citation statements)

References 65 publications

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“…To identify protein–protein interactions implicated in the aberrant processing of CH-associated mutations, we implemented an affinity purification–mass spectrometry method coupled with tandem mass tag (TMT) labeling to allow for multiplexed identification and quantification of interacting proteins ( Fig. 2 A ) ( 45 ). Transient interactions between Tg and PN components were captured using the cell permeable and reversable cross-linker dithiobis(succinimidyl propionate) (DSP) ( 46 ).…”

Section: Resultsmentioning

confidence: 99%

“…By using quantitative multiplexed interactome proteomics, we identified specific PN components that may act as therapeutic targets for rescuing Tg secretion. A similar method has been used to investigate the molecular basis of activating transcription factor 6 (ATF6)-dependent regulation of immunoglobulin light-chain secretion in amyloidosis ( 45 ). Methods to pharmacologically target the UPR may be further applicable to rescuing Tg secretion.…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, pharmacologic modulation of UPR activity to regulate the abundance of ER proteostasis factors in a coordinated manner could act to restore mutant Tg secretion. In the case of amyloidogenic light-chain proteins, overexpression of UPR-regulated chaperones, in particular BiP and GRP94, was able reduce the secretion of an aggregation-prone protein variant ( 24 , 45 ). Similar effects were also observed for a model aggregation-prone polyglutamine protein as cytosolic heat shock activation-attenuated intracellular aggregation and cellular toxicity ( 80 ).…”

Section: Discussionmentioning

confidence: 99%

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Thyroglobulin Interactome Profiling Defines Altered Proteostasis Topology Associated With Thyroid Dyshormonogenesis

Wright

Kouba

Plate

2021

Molecular & Cellular Proteomics

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Thyroglobulin (Tg) is a secreted iodoglycoprotein serving as the precursor for T3 and T4 hormones. Many characterized Tg gene mutations produce secretion-defective variants resulting in congenital hypothyroidism (CH). Tg processing and secretion is controlled by extensive interactions with chaperone, trafficking, and degradation factors comprising the secretory proteostasis network. While dependencies on individual proteostasis network components are known, the integration of proteostasis pathways mediating Tg protein quality control and the molecular basis of mutant Tg misprocessing remain poorly understood. We employ a multiplexed quantitative affinity purification–mass spectrometry approach to define the Tg proteostasis interactome and changes between WT and several CH-variants. Mutant Tg processing is associated with common imbalances in proteostasis engagement including increased chaperoning, oxidative folding, and engagement by targeting factors for ER-associated degradation (ERAD). Furthermore, we reveal mutation-specific changes in engagement with N-glycosylation components, suggesting distinct requirements for one Tg variant on dual engagement of both oligosaccharyltransferase complex isoforms for degradation. Modulating dysregulated proteostasis components and pathways may serve as a therapeutic strategy to restore Tg secretion and thyroid hormone biosynthesis.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Thyroglobulin Interactome Profiling Defines Altered Proteostasis Topology Associated With Thyroid Dyshormonogenesis

Wright

Kouba

Plate

2021

Molecular & Cellular Proteomics

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“…TMT-AP-MS allows direct quantitative comparisons of prey recovery across multiple replicates in a single run, simplifying evaluation of whether potential interactors are preferentially recovered with the bait 22,28 . However, variation in bait levels between conditions, particularly when using transient transfection, leads to variability in recovered prey levels.…”

Section: Introductionmentioning

confidence: 99%

Bait Correlation Improves Interactor Identification by Tandem Mass Tag-Affinity Purification-Mass Spectrometry

et al. 2020

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The quantitative multiplexing capacity of isobaric Tandem Mass Tags (TMT) has increased the throughput of affinity purification mass spectrometry (AP-MS) to characterize protein interaction networks of immunoprecipitated bait proteins. However, variable bait levels between replicates can convolute interactor identification. We compared the Student's t-test and Pearson's R correlation as methods to generate t-statistics and assessed the significance of interactors following TMT-AP-MS. Using a simple linear model of protein recovery in immunoprecipitates to simulate reporter ion ratio distributions, we found that correlation-derived t-statistics protect against bait variance while robustly controlling Type I errors (false positives). We experimentally determined the performance of these two approaches for determining t-statistics under two experimental conditions: irreversible prey association to the Hsp40 mutant DNAJB8 H31Q followed by stringent washing, and reversible association to 14-3-3z with gentle washing. Correlation-derived t-statistics performed at least as well as Student's t-statistics for each sample, and with substantial improvement in performance for experiments with high bait level variance. Deliberately varying bait levels over a large range fails to improve selectivity but does increase robustness between runs. The use of correlation-derived t-statistics should improve identification of interactors using TMT-AP-MS. Data are available via ProteomeXchange with identifier PXD016613.

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“…ATF6 induces the expression of 73 many ER proteostasis factors including the ATP-dependent ER HSP70 BiP and multiple protein disulfide 74 isomerases (PDIs) (Haze, Yoshida et al, 1999, Shoulders, Ryno et al, 2013. Genetic activation of ATF6 using 75 a ligand-regulated system preferentially reduces secretion and subsequent aggregation of a destabilized, 76 amyloidogenic LC from HEK293T cells, without impacting the secretion of a non-amyloidogenic LC, fully-77 assembled IgGs, or the endogenous secretory proteome (Cooley, Ryno et al, 2014, Plate, Rius et al, 2019, 78 Shoulders et al, 2013. Interestingly, the ATF6-dependent reduction in amyloidogenic LC secretion results 79 from increased interactions with ER chaperones and PDIs, which preferentially retain the amyloidogenic LC 80 within the ER and prevent its secretion to downstream secretory environments .…”

Section: Impact Statement 36mentioning

confidence: 99%

ER Proteostasis Regulators Reduce Amyloidogenic Light Chain Secretion Through an On-Target, ATF6-Independent Mechanism

Rius

Mesgarzadeh

Romine

et al. 2020

Preprint

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SUMMARYThe plasma cell secretion and toxic aggregation of amyloidogenic immunoglobulin light chains (LCs) causes proteotoxicity in Light Chain Amyloidosis (AL). We recently identified endoplasmic reticulum (ER) proteostasis regulators such as compound 147 that reduce secretion and aggregation of LCs implicated in AL (Plate, Cooley et al., 2016). Compound 147 promotes adaptive ER proteostasis remodeling through a mechanism involving covalent modification of multiple protein disulfide isomerases (PDIs) and subsequent activation of the ATF6 unfolded protein response (UPR) -associated transcriptional signaling pathway (Paxman, Plate et al., 2018). Here, we show that the 147-dependent reduction in amyloidogenic LC secretion from AL patient plasma cells is independent of ATF6 activation, but instead requires on-target PDI modification. Our results reveal pharmacologic targeting of PDIs as a potential strategy to ameliorate AL-associated proteotoxicity and demonstrate that 147 can influence ER proteostasis through multiple on-target mechanisms including ATF6 activation and PDI modification.IMPACT STATEMENTThis study demonstrates the broad potential for endoplasmic reticulum proteostasis regulator compounds such as 147 to influence secretory proteostasis of disease-associated proteins through multiple on target mechanisms.

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Quantitative Interactome Proteomics Reveals a Molecular Basis for ATF6-Dependent Regulation of a Destabilized Amyloidogenic Protein

Cited by 31 publications

References 65 publications

Thyroglobulin Interactome Profiling Defines Altered Proteostasis Topology Associated With Thyroid Dyshormonogenesis

Thyroglobulin Interactome Profiling Defines Altered Proteostasis Topology Associated With Thyroid Dyshormonogenesis

Bait Correlation Improves Interactor Identification by Tandem Mass Tag-Affinity Purification-Mass Spectrometry

ER Proteostasis Regulators Reduce Amyloidogenic Light Chain Secretion Through an On-Target, ATF6-Independent Mechanism

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