Summary The unfolded protein response (UPR) maintains endoplasmic reticulum (ER) proteostasis through the activation of transcription factors such as XBP1s and ATF6. The functional consequences of these transcription factors for ER proteostasis remain poorly defined. Here, we describe methodology that enables orthogonal, small-molecule-mediated activation of the UPR-associated transcription factors XBP1s and/or ATF6 in the same cell independent of stress. We employ transcriptomics and quantitative proteomics to evaluate ER proteostasis network remodeling owing to the XBP1s and/or ATF6 transcriptional programs. Furthermore, we demonstrate that the three ER proteostasis environments accessible by activating XBP1s and/or ATF6 differentially influence the folding, trafficking, and degradation of destabilized ER client proteins without globally affecting the endogenous proteome. Our data reveal how the ER proteostasis network is remodeled by the XBP1s and/or ATF6 transcriptional programs at the molecular level and demonstrate the potential for selective restoration of aberrant ER proteostasis of pathologic, destabilized proteins through arm-selective UPR activation.

The discovery of molecules that bind tightly and selectively to desired proteins continues to drive innovation at the interface of chemistry and biology. This paper describes the binding of human insulin by the synthetic receptor cucurbit[7]uril (Q7) in vitro. Isothermal titration calorimetry and fluorescence spectroscopy experiments show that Q7 binds to insulin with an equilibrium association constant of 1.5 × 106 M-1 and with 50-100-fold selectivity versus proteins that are much larger but lack an N-terminal aromatic residue, and >1000-fold selectivity versus an insulin variant lacking the N-terminal phenylalanine (Phe) residue. The crystal structure of the Q7•insulin complex shows that binding occurs at the N-terminal Phe residue and that the N-terminus unfolds to enable binding. These findings suggest that site-selective recognition is based on the properties inherent to a protein terminus, including the unique chemical epitope presented by the terminal residue and the greater freedom of the terminus to unfold, like the end of a ball of string, to accommodate binding. Insulin recognition was predicted accurately from studies on short peptides and exemplifies an approach to protein recognition by targeting the terminus.

Light-chain amyloidosis (AL) is a degenerative disease characterized by the extracellular aggregation of a destabilized amyloidogenic Ig light chain (LC) secreted from a clonally expanded plasma cell. Current treatments for AL revolve around ablating the cancer plasma cell population using chemotherapy regimens. Unfortunately, this approach is limited to the ∼70% of patients who do not exhibit significant organ proteotoxicity and can tolerate chemotherapy. Thus, identifying new therapeutic strategies to alleviate LC organ proteotoxicity should allow AL patients with significant cardiac and/or renal involvement to subsequently tolerate established chemotherapy treatments. Using a small-molecule screening approach, the unfolded protein response (UPR) was identified as a cellular signaling pathway whose activation selectively attenuates secretion of amyloidogenic LC, while not affecting secretion of a nonamyloidogenic LC. Activation of the UPR-associated transcription factors XBP1s and/or ATF6 in the absence of stress recapitulates the selective decrease in amyloidogenic LC secretion by remodeling the endoplasmic reticulum proteostasis network. Stress-independent activation of XBP1s, or especially ATF6, also attenuates extracellular aggregation of amyloidogenic LC into soluble aggregates. Collectively, our results show that stress-independent activation of these adaptive UPR transcription factors offers a therapeutic strategy to reduce proteotoxicity associated with LC aggregation.ER proteostasis | amyloid L ight-chain amyloidosis (AL) afflicts 8-10 people per million per year, making it the most prominent systemic amyloid disease (1). AL is a gain-of-toxic function disease driven by a clonally expanded plasma cell that secretes amyloidogenic Ig light chains (LCs). These amyloidogenic LCs undergo extracellular misfolding and aggregation into proteotoxic soluble oligomers and amyloid fibrils that interact with distal tissues such as the kidney, heart, and gastrointestinal tract, leading to organ dysfunction and ultimately death by unknown proteotoxicity mechanism(s) (2).The majority of AL patients must combat both a cancer (i.e., the clonally expanded plasma cell) and LC aggregation-associated proteotoxicity. The standard treatment for AL patients is chemotherapy (often combined with stem cell transplant) to eliminate the cancerous plasma cell population (3, 4). The proteasome inhibitor bortezomib, which takes advantage of the stress sensitivity of aggressively proliferating plasma cells, has transformed chemotherapy effectiveness (5-7). Regardless, ∼30% of AL patients with substantial cardiac or renal LC proteotoxicity are too ill at diagnosis to tolerate chemotherapeutics (8-10). Thus, new strategies to reduce LC organ proteotoxicity must be developed to allow more AL patients to take advantage of chemotherapy.LC aggregation requires conformational changes and a sufficient concentration of misfolded LC in plasma, which determine the rate and extent of its concentration-dependent aggregation. Over 500 distinct LC s...

The Unfolded Protein Response (UPR) indirectly regulates extracellular proteostasis through transcriptional remodeling of endoplasmic reticulum (ER) proteostasis pathways. This remodeling attenuates secretion of misfolded, aggregation-prone proteins during ER stress. Through these activities, the UPR has a critical role in preventing the extracellular protein aggregation associated with numerous human diseases. Here, we demonstrate that UPR activation also directly influences extracellular proteostasis through the upregulation and secretion of the ER HSP40 ERdj3/ DNAJB11. Secreted ERdj3 binds misfolded proteins in the extracellular space, substoichiometrically inhibits protein aggregation, and attenuates proteotoxicity of disease-associated toxic prion protein.Moreover, ERdj3 can co-secrete with destabilized, aggregationprone proteins in a stable complex under conditions where ER chaperoning capacity is overwhelmed, preemptively providing extracellular chaperoning of proteotoxic misfolded proteins that evade ER quality control. This regulated co-secretion of ERdj3 with misfolded clients directly links ER and extracellular proteostasis during conditions of ER stress. ERdj3 is, to our knowledge, the first metazoan chaperone whose secretion into the extracellular space is regulated by the UPR, revealing a new mechanism by which UPR activation regulates extracellular proteostasis.

The heat shock response is an evolutionarily conserved, stress-responsive signaling pathway that adapts cellular proteostasis in response to pathologic insult. In metazoans, the heat shock response primarily functions through the posttranslational activation of heat shock factor 1 (HSF1), a stress-responsive transcription factor that induces the expression of cytosolic proteostasis factors including chaperones, cochaperones, and folding enzymes. HSF1 is a potentially attractive therapeutic target to ameliorate pathologic imbalances in cellular proteostasis associated with human disease, although the underlying impact of stress-independent HSF1 activation on cellular proteome composition remains to be defined. Here, we employ a highly controllable, ligand-regulated HSF1 that activates HSF1 to levels compatible with those that could be achieved using selective small molecule HSF1 activators. Using a combination of RNAseq and quantitative proteomics, we define the impact of stress-independent HSF1 activation on the composition of the cellular proteome. We show that stress-independent HSF1 activation selectively remodels cytosolic proteostasis pathways without globally influencing the composition of the cellular proteome. Furthermore, we show that stress-independent HSF1 activation decreases intracellular aggregation of a model polyglutamine-containing protein and reduces the cellular toxicity of environmental toxins like arsenite that disrupt cytosolic proteostasis. Collectively, our results reveal a proteome-level view of stress-independent HSF1 activation, providing a framework to establish therapeutic approaches to correct pathologic imbalances in cellular proteostasis through the selective targeting of HSF1.