Quantitative Measurement of Secretory Protein Mistargeting by Proximity Labeling and Parallel Reaction Monitoring.

Proximity labeling is a powerful approach for characterizing subcellular proteomes. We recently demonstrated that proximity labeling can be used to identify mistrafficking of secretory proteins, such as occurs during pre-emptive quality control (pre-QC) following endoplasmic reticulum (ER) stress. This assay depends on protein quantification by immunoblotting and densitometry, which is only semi-quantitative and suffers from poor sensitivity. Here, we integrate parallel reaction monitoring mass spectrometry to enable a more quantitative platform for ER import. PRM as opposed to densitometry improves quantification of transthyretin mistargeting while also achieving at least a ten-fold gain in sensitivity. The multiplexing of PRM also enabled us to evaluate a series of normalization approaches, revealing that normalization to auto-labeled APEX2 peroxidase is necessary to account for drug treatment-dependent changes in labeling efficiency. We apply this approach to systematically characterize the relationship between chemical ER stressors and ER pre-QC induction in HEK293T cells. Using dual-FLAG-tagged transthyretin (FLAGTTR) as a model secretory protein, we find that Brefeldin A treatment as well as ER calcium depletion cause pre-QC, while tunicamycin and dithiothreitol do not, indicating ER stress alone is not sufficient. This finding contrasts with the canonical model of pre-QC induction, and establishes the utility of our platform.

Cellular Exposure to Chloroacetanilide Herbicides Induces Distinct Protein Destabilization Profiles.

Herbicides in the widely used chloroacetanilide class harbor a potent electrophilic moiety, which can damage proteins through nucleophilic substitution. In general, damaged proteins are subject to misfolding. Accumulation of misfolded proteins compromises cellular integrity by disrupting cellular proteostasis networks, which can further destabilize the cellular proteome. While direct conjugation targets can be discovered through affinity-based protein profiling, there are few approaches to probe how cellular exposure to toxicants impacts the stability of the proteome. We apply a quantitative proteomics methodology to identify chloroacetanilide-destabilized proteins in HEK293T cells based on their binding to the H31Q mutant of the human Hsp40 chaperone DNAJB8. We find that a brief cellular exposure to the chloroacetanilides acetochlor, alachlor, and propachlor induces misfolding of dozens of cellular proteins. These herbicides feature distinct but overlapping profiles of protein destabilization, highly concentrated in proteins with reactive cysteine residues. Consistent with the recent literature from the pharmacology field, reactivity is driven by neither inherent nucleophilic nor electrophilic reactivity but is idiosyncratic. We discover that propachlor induces a general increase in protein aggregation and selectively targets GAPDH and PARK7, leading to a decrease in their cellular activities. Hsp40 affinity profiling identifies a majority of propachlor targets identified by competitive activity-based protein profiling (ABPP), but ABPP can only identify about 10% of protein targets identified by Hsp40 affinity profiling. GAPDH is primarily modified by the direct conjugation of propachlor at a catalytic cysteine residue, leading to global destabilization of the protein. The Hsp40 affinity strategy is an effective technique to profile cellular proteins that are destabilized by cellular toxin exposure. Raw proteomics data is available through the PRIDE Archive at PXD030635.

Profiling protein targets of cellular toxicant exposure.

Environmental agents of exposure can damage proteins, affecting protein function and cellular protein homeostasis. Specific residues are inherently chemically susceptible to damage from individual types of exposure. Amino acid content is not completely predictive of protein susceptibility, as secondary, tertiary, and quaternary structures of proteins strongly influence the reactivity of the proteome to individual exposures. Because we cannot readily predict which proteins will be affected by which chemical exposures, mass spectrometry-based proteomic strategies are necessary to determine the protein targets of environmental toxins and toxicants. This review describes the mechanisms by which environmental exposure to toxins and toxicants can damage proteins and affect their function, and emerging omic methodologies that can be used to identify the protein targets of a given agent. These methods include target identification strategies that have recently revolutionized the drug discovery field, such as activity-based protein profiling, protein footprinting, and protein stability profiling technologies. In particular, we highlight the necessity of multiple, complementary approaches to fully interrogate how protein integrity is challenged by individual exposures.

Quantitative Measurement of Transthyretin Mistargeting by Proximity Labeling and Parallel Reaction Monitoring.

Proximity labeling is a powerful approach for characterizing subcellular proteomes. We recently demonstrated that proximity labeling can be used to identify mistrafficking of secretory proteins, such as occurs during pre-emptive quality control (pre-QC) following endoplasmic reticulum (ER) stress. This assay depends on protein quantification by immunoblotting and densitometry, which sometimes suffers from poor sensitivity. Here, we integrate parallel reaction monitoring (PRM) mass spectrometry to enable a more quantitative platform, and assess how chemical ER stressors impact pre-QC of the model secretory protein transthyretin in HEK293T cells. We find that some drug treatments affect labeling efficiency, which can be controlled for by normalizing to APEX2 auto-labeling. While some chemical ER stress inducers including Brefeldin A and thapsigargin induce pre-QC, tunicamycin and dithiothreitol do not, indicating ER stress alone is not sufficient. This finding contrasts with the canonical model of pre-QC induction, and establishes the utility of our platform.

Heat shock protein Hspa13 regulates endoplasmic reticulum and cytosolic proteostasis through modulation of protein translocation.

Most eukaryotic secretory proteins are cotranslationally translocated through Sec61 into the endoplasmic reticulum (ER). Because these proteins have evolved to fold in the ER, their mistargeting is associated with toxicity. Genetic experiments have implicated the ER heat shock protein 70 (Hsp70) Hspa13/STCH as involved in processing of nascent secretory proteins. Herein, we evaluate the role of Hspa13 in protein import and the maintenance of cellular proteostasis in human cells, primarily using the human embryonic kidney 293T cell line. We find that Hspa13 interacts primarily with the Sec61 translocon and its associated factors. Hspa13 overexpression inhibits translocation of the secreted protein transthyretin, leading to accumulation and aggregation of immature transthyretin in the cytosol. ATPase-inactive mutants of Hspa13 further inhibit translocation and maturation of secretory proteins. While Hspa13 overexpression inhibits cell growth and ER quality control, we demonstrate that HSPA13 knockout destabilizes proteostasis and increases sensitivity to ER disruption. Thus, we propose that Hspa13 regulates import through the translocon to maintain both ER and cytosolic protein homeostasis. The raw mass spectrometry data associated with this article have been deposited in the PRIDE archive and can be accessed at PXD033498.

Monitoring Protein Import into the Endoplasmic Reticulum in Living Cells with Proximity Labeling.

The proper trafficking of eukaryotic proteins is essential to cellular function. Genetic, environmental, and other stresses can induce protein mistargeting and, in turn, threaten cellular protein homeostasis. Current methods for measuring protein mistargeting are difficult to translate to living cells, and thus the role of cellular signaling networks in stress-dependent protein mistargeting processes, such as ER pre-emptive quality control (ER pQC), is difficult to parse. Herein, we use genetically encoded peroxidases to characterize protein import into the endoplasmic reticulum (ER). We show that the ERHRP/cytAPEX pair provides good selectivity and sensitivity for both multiplexed protein labeling and for identifying protein mistargeting, using the known ER pQC substrate transthyretin (TTR). Although ERHRP labeling induces formation of detergent-resistant TTR aggregates, this is minimized by using low ERHRP expression, without loss of labeling efficiency. cytAPEX labeling recovers TTR that is mistargeted as a consequence of Sec61 inhibition or ER stress-induced ER pQC. Furthermore, we discover that stress-free activation of the ER stress-associated transcription factor ATF6 recapitulates the TTR import deficiency of ER pQC. Hence, proximity labeling is an effective strategy for characterizing factors that influence ER protein import in living cells.

ATF6 Activation Reduces Amyloidogenic Transthyretin Secretion through Increased Interactions with Endoplasmic Reticulum Proteostasis Factors.

The extracellular aggregation of destabilized transthyretin (TTR) variants is implicated in the onset and pathogenesis of familial TTR-related amyloid diseases. One strategy to reduce the toxic, extracellular aggregation of TTR is to decrease the population of aggregation-prone proteins secreted from mammalian cells. The stress-independent activation of the unfolded protein response (UPR)-associated transcription factor ATF6 preferentially decreases the secretion and subsequent aggregation of destabilized, aggregation-prone TTR variants. However, the mechanism of this reduced secretion was previously undefined. Here, we implement a mass-spectrometry-based interactomics approach to identify endoplasmic reticulum (ER) proteostasis factors involved in ATF6-dependent reductions in destabilized TTR secretion. We show that ATF6 activation reduces amyloidogenic TTR secretion and subsequent aggregation through a mechanism involving ER retention that is mediated by increased interactions with ATF6-regulated ER proteostasis factors including BiP and PDIA4. Intriguingly, the PDIA4-dependent retention of TTR is independent of both the single TTR cysteine residue and the redox activity of PDIA4, indicating that PDIA4 retains destabilized TTR in the ER through a redox-independent mechanism. Our results define a mechanistic basis to explain the ATF6 activation-dependent reduction in destabilized, amyloidogenic TTR secretion that could be therapeutically accessed to improve treatments of TTR-related amyloid diseases.

Chemical Diversity of Citrus Leaf Essential Oils.

The essential oils from leaves of 20 commercial citrus accessions maintained by the University of California, Riverside Givaudan Citrus Variety Collection and selected on the basis of their odor profile were analyzed by GCMS/FID. The main components were quantified while the semi-quantitative percentage composition data was compiled with data from other publications for sample visualization, classification and comparison with leaf oils from other citrus accessions. Some compositional clusters aligned closely with the taxonomic clades of sweet orange, bitter orange, and C. hystrix while other clades like the mandarins and lemons showed distinct chemical sub-groups. Characteristic compounds for the clusters included linalyl acetate and linalool (bitter orange leaf), sabinene (sweet orange leaf), methyl N-methyl anthranilate (mandarin leaf), γ-terpinene (yuzu leaf), citronellal (C. hystrix), limonene, citronellal and citral (lemons and citrons). A chemometric approach combined with t-SNE cluster plots can be more informative than taxonomic assignments when considering flavor and fragrance characteristics.

Hsp40 Affinity to Identify Proteins Destabilized by Cellular Toxicant Exposure.

Environmental toxins and toxicants can damage proteins and threaten cellular proteostasis. Most current methodologies to identify misfolded proteins in cells survey the entire proteome for sites of changed reactivity. We describe and apply a quantitative proteomics methodology to identify destabilized proteins based on their binding to the human Hsp40 chaperone DNAJB8. These protein targets are validated by an orthogonal limited proteolysis assay using parallel reaction monitoring. We find that a brief exposure of HEK293T cells to meta-arsenite increases the affinity of two dozen proteins to DNAJB8, including known arsenite-sensitive proteins. In particular, arsenite treatment destabilizes both the pyruvate dehydrogenase complex E1 subunit and several RNA-binding proteins. This platform can be used to explore how environmental toxins impact cellular proteostasis and to identify the susceptible proteome.

Methodologies for Measuring Protein Trafficking across Cellular Membranes.

Nearly all proteins are synthesized in the cytosol. The majority of this proteome must be trafficked elsewhere, such as to membranes, to subcellular compartments, or outside of the cell. Proper trafficking of nascent protein is necessary for protein folding, maturation, quality control and cellular and organismal health. To better understand cellular biology, molecular and chemical technologies to properly characterize protein trafficking (and mistrafficking) have been developed and applied. Herein, we take a biochemical perspective to review technologies that enable spatial and temporal measurement of protein distribution, focusing on both the most widely adopted methodologies and exciting emerging approaches.

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

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 DNAJB8H31Q followed by stringent washing, and reversible association to 14-3-3ζ 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 the 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.

Protein profiling and pseudo-parallel reaction monitoring to monitor a fusion-associated conformational change in hemagglutinin.

Influenza infection requires viral escape from early endosomes into the cytosol, which is enabled by an acid-induced irreversible conformational transformation in the viral protein hemagglutinin. Despite the direct relationship between this conformational change and infectivity, label-free methods for characterizing this and other protein conformational changes in biological mixtures are limited. While the chemical reactivity of the protein backbone and side-chain residues is a proxy for protein conformation, coupling this reactivity to quantitative mass spectrometry is a challenge in complex environments. Herein, we evaluate whether electrophilic amidination coupled with pseudo-parallel reaction monitoring is an effective label-free approach to detect the fusion-associated conformational transformation in recombinant hemagglutinin (rHA). We identified rHA peptides that are differentially amidinated between the pre- and post-fusion states, and validated that this difference relies upon the fusion-associated conformational switch. We further demonstrate that we can distinguish the fusion profile in a matrix of digested cellular lysate. This fusion assay can be used to evaluate fusion competence for modified HA. Graphical abstract.

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

Activation of the unfolded protein response (UPR)-associated transcription factor ATF6 has emerged as a promising strategy to reduce the secretion and subsequent toxic aggregation of destabilized, amyloidogenic proteins implicated in systemic amyloid diseases. However, the molecular mechanism by which ATF6 activation reduces the secretion of amyloidogenic proteins remains poorly defined. We employ a quantitative interactomics platform to define how ATF6 activation reduces secretion of a destabilized, amyloidogenic immunoglobulin light chain (LC) associated with light-chain amyloidosis (AL). Using this platform, we show that ATF6 activation increases the targeting of this destabilized LC to a subset of pro-folding ER proteostasis factors that retains the amyloidogenic LC within the ER, preventing its secretion. Our results define a molecular basis for the ATF6-dependent reduction in destabilized LC secretion and highlight the advantage for targeting this UPR-associated transcription factor to reduce secretion of destabilized, amyloidogenic proteins implicated in AL and related systemic amyloid diseases.

Mass spectrometric approaches for profiling protein folding and stability.

Protein stability reports on protein homeostasis, function, and binding interactions, such as to other proteins, metabolites and drugs. As such, there is a pressing need for technologies that can report on protein stability. The ideal technique could be applied in vitro or in vivo systems, proteome-wide, independently of matrix, under native conditions, with residue-level resolution, and on protein at endogenous levels. Mass spectrometry has rapidly become a preferred technology for identifying and quantifying proteins. As such, it has been increasingly incorporated into methodologies for interrogating protein stability and folding. Although no single technology can satisfy all desired applications, several emerging approaches have shown outstanding success at providing biological insight into the stability of the proteome. This chapter outlines some of these recent emerging technologies.

XBP1s activation can globally remodel N-glycan structure distribution patterns.

Classically, the unfolded protein response (UPR) safeguards secretory pathway proteostasis. The most ancient arm of the UPR, the IRE1-activated spliced X-box binding protein 1 (XBP1s)-mediated response, has roles in secretory pathway maturation beyond resolving proteostatic stress. Understanding the consequences of XBP1s activation for cellular processes is critical for elucidating mechanistic connections between XBP1s and development, immunity, and disease. Here, we show that a key functional output of XBP1s activation is a cell type-dependent shift in the distribution of N-glycan structures on endogenous membrane and secreted proteomes. For example, XBP1s activity decreased levels of sialylation and bisecting GlcNAc in the HEK293 membrane proteome and secretome, while substantially increasing the population of oligomannose N-glycans only in the secretome. In HeLa cell membranes, stress-independent XBP1s activation increased the population of high-mannose and tetraantennary N-glycans, and also enhanced core fucosylation. mRNA profiling experiments suggest that XBP1s-mediated remodeling of the N-glycome is, at least in part, a consequence of coordinated transcriptional resculpting of N-glycan maturation pathways by XBP1s. The discovery of XBP1s-induced N-glycan structural remodeling on a glycome-wide scale suggests that XBP1s can act as a master regulator of N-glycan maturation. Moreover, because the sugars on cell-surface proteins or on proteins secreted from an XBP1s-activated cell can be molecularly distinct from those of an unactivated cell, these findings reveal a potential new mechanism for translating intracellular stress signaling into altered interactions with the extracellular environment.

Mapping wild-type and R345W fibulin-3 intracellular interactomes.

Fibulin-3 (F3) is an important, disulfide-rich, extracellular matrix glycoprotein that has been associated with a number of diseases ranging from cancer to retinal degeneration. An Arg345Trp (R345W) mutation in F3 causes the rare, autosomal dominant macular dystrophy, Malattia Leventinese. The purpose of this study was to identify and validate novel intracellular interacting partners of wild-type (WT) and R345W F3 in retinal pigment epithelium cells. We used stable isotope labeling by amino acids in cell culture (SILAC) to generate 'heavy' and 'light' isotopically labeled ARPE-19 cell populations which were subsequently infected with adenovirus encoding for FLAG-tagged WT or R345W F3. After immunoprecipitation, interacting proteins were identified by multidimensional protein identification technology (MudPIT). We identified sixteen new intracellular F3 interacting partners, the vast majority of which are involved in protein folding and/or degradation in the endoplasmic reticulum (ER). Eight of these interactions (ANXA5, ERdj5, PDIA4, P4HB, PDIA6, RCN1, SDF2L1, and TXNDC5) were verified at the western blotting level. These F3 interactome results can serve as the basis for pursuing targeted genetic or pharmacologic approaches in an effort to alter the fate of either WT or mutant F3.

Endoplasmic Reticulum Proteostasis Influences the Oligomeric State of an Amyloidogenic Protein Secreted from Mammalian Cells.

Transthyretin (TTR) is a tetrameric serum protein associated with multiple systemic amyloid diseases. In these disorders, TTR aggregates in extracellular environments through a mechanism involving rate-limiting dissociation of the tetramer to monomers, which then misfold and aggregate into soluble oligomers and amyloid fibrils that induce toxicity in distal tissues. Using an assay established herein, we show that highly destabilized, aggregation-prone TTR variants are secreted as both native tetramers and non-native conformations that accumulate as high-molecular-weight oligomers. Pharmacologic chaperones that promote endoplasmic reticulum (ER) proteostasis of destabilized TTR variants increase their fraction secreted as a tetramer and reduce extracellular aggregate populations. In contrast, disrupting ER proteostasis reduces the fraction of destabilized TTR secreted as a tetramer and increases extracellular aggregates. These results identify ER proteostasis as a factor that can affect conformational integrity and thus toxic aggregation of secreted amyloidogenic proteins associated with the pathology of protein aggregation diseases.

Small molecule proteostasis regulators that reprogram the ER to reduce extracellular protein aggregation.

Imbalances in endoplasmic reticulum (ER) proteostasis are associated with etiologically-diverse degenerative diseases linked to excessive extracellular protein misfolding and aggregation. Reprogramming of the ER proteostasis environment through genetic activation of the Unfolded Protein Response (UPR)-associated transcription factor ATF6 attenuates secretion and extracellular aggregation of amyloidogenic proteins. Here, we employed a screening approach that included complementary arm-specific UPR reporters and medium-throughput transcriptional profiling to identify non-toxic small molecules that phenocopy the ATF6-mediated reprogramming of the ER proteostasis environment. The ER reprogramming afforded by our molecules requires activation of endogenous ATF6 and occurs independent of global ER stress. Furthermore, our molecules phenocopy the ability of genetic ATF6 activation to selectively reduce secretion and extracellular aggregation of amyloidogenic proteins. These results show that small molecule-dependent ER reprogramming, achieved through preferential activation of the ATF6 transcriptional program, is a promising strategy to ameliorate imbalances in ER function associated with degenerative protein aggregation diseases.

A fluorogenic aryl fluorosulfate for intraorganellar transthyretin imaging in living cells and in Caenorhabditis elegans.

Fluorogenic probes, due to their often greater spatial and temporal sensitivity in comparison to permanently fluorescent small molecules, represent powerful tools to study protein localization and function in the context of living systems. Herein, we report fluorogenic probe 4, a 1,3,4-oxadiazole designed to bind selectively to transthyretin (TTR). Probe 4 comprises a fluorosulfate group not previously used in an environment-sensitive fluorophore. The fluorosulfate functional group does not react covalently with TTR on the time scale required for cellular imaging, but does red shift the emission maximum of probe 4 in comparison to its nonfluorosulfated analogue. We demonstrate that probe 4 is dark in aqueous buffers, whereas the TTR·4 complex exhibits a fluorescence emission maximum at 481 nm. The addition of probe 4 to living HEK293T cells allows efficient binding to and imaging of exogenous TTR within intracellular organelles, including the mitochondria and the endoplasmic reticulum. Furthermore, live Caenorhabditis elegans expressing human TTR transgenically and treated with probe 4 display TTR·4 fluorescence in macrophage-like coelomocytes. An analogue of fluorosulfate probe 4 does react selectively with TTR without labeling the remainder of the cellular proteome. Studies on this analogue suggest that certain aryl fluorosulfates, due to their cell and organelle permeability and activatable reactivity, could be considered for the development of protein-selective covalent probes.