Joseph C. Genereux

Stress-Independent Activation of XBP1s and/or ATF6 Reveals Three Functionally Diverse ER Proteostasis Environments

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.

DNA-Mediated Charge Transport in Redox Sensing and Signaling

The transport of charge through the DNA base-pair stack offers a route to carry out redox chemistry at a distance. Here we describe characteristics of this chemistry that have been elucidated and how this chemistry may be utilized within the cell. The shallow distance dependence associated with these redox reactions permits DNA-mediated signaling over long molecular distances in the genome and facilitates the activation of redox-sensitive transcription factors globally in response to oxidative stress. The long-range funneling of oxidative damage to sites of low oxidation potential in the genome also may provide a means of protection within the cell. Furthermore, the sensitivity of DNA charge transport to perturbations in base-pair stacking, as may arise with base lesions and mismatches, may be used as a route to scan the genome for damage as a first step in DNA repair. Thus, the ability of double-helical DNA in mediating redox chemistry at a distance provides a natural mechanism for redox sensing and signaling in the genome.

Redox signaling between DNA repair proteins for efficient lesion detection

Base excision repair (BER) enzymes maintain the integrity of the genome, and in humans, BER mutations are associated with cancer. Given the remarkable sensitivity of DNA-mediated charge transport (CT) to mismatched and damaged base pairs, we have proposed that DNA repair glycosylases (EndoIII and MutY) containing a redox-active [4Fe4S] cluster could use DNA CT in signaling one another to search cooperatively for damage in the genome. Here, we examine this model, where we estimate that electron transfers over a few hundred base pairs are sufficient for rapid interrogation of the full genome. Using atomic force microscopy, we found a redistribution of repair proteins onto DNA strands containing a single base mismatch, consistent with our model for CT scanning. We also demonstrated in Escherichia coli a cooperativity between EndoIII and MutY that is predicted by the CT scanning model. This relationship does not require the enzymatic activity of the glycosylase. Y82A EndoIII, a mutation that renders the protein deficient in DNA-mediated CT, however, inhibits cooperativity between MutY and EndoIII. These results illustrate how repair proteins might efficiently locate DNA lesions and point to a biological role for DNA-mediated CT within the cell.

Stress-Regulated Translational Attenuation Adapts Mitochondrial Protein Import through Tim17A Degradation

Stress-regulated signaling pathways protect mitochondrial proteostasis and function from pathologic insults. Despite the importance of stress-regulated signaling pathways in mitochondrial proteome maintenance, the molecular mechanisms by which these pathways maintain mitochondrial proteostasis remain largely unknown. We identify Tim17A as a stress-regulated subunit of the translocase of the inner membrane 23 (TIM23) mitochondrial protein import complex. We show that Tim17A protein levels are decreased downstream of stress-regulated translational attenuation induced by eukaryotic initiation factor 2α (eIF2α) phosphorylation through a mechanism dependent on the mitochondrial protease YME1L. Furthermore, we demonstrate that decreasing Tim17A attenuates TIM23-dependent protein import, promotes the induction of mitochondrial unfolded protein response (UPR)-associated proteostasis genes, and confers stress resistance in C. elegans and mammalian cells. Thus, our results indicate that Tim17A degradation is a stress-responsive mechanism by which cells adapt mitochondrial protein import efficiency and promote mitochondrial proteostasis in response to the numerous pathologic insults that induce stress-regulated translation attenuation.

Single-Step Charge Transport through DNA over Long Distances

Quantum yields for charge transport across adenine tracts of increasing length have been measured by monitoring hole transport in synthetic oligonucleotides between photoexcited 2-aminopurine, a fluorescent analogue of adenine, and N(2)-cyclopropyl guanine. Using fluorescence quenching, a measure of hole injection, and hole trapping by the cyclopropyl guanine derivative, we separate the individual contributions of single- and multistep channels to DNA charge transport and find that with 7 or 8 intervening adenines the charge transport is a coherent, single-step process. Moreover, a transition occurs from multistep to single-step charge transport with increasing donor/acceptor separation, opposite to that generally observed in molecular wires. These results establish that coherent transport through DNA occurs preferentially across 10 base pairs, favored by delocalization over a full turn of the helix.

Ping-pong electron transfer through DNA.

Herein we describe a novel Ir system that is able to promote the reduction of pyrimidine bases from a distance without the presence of an external quencher. Instead, DNA-mediated ET is triggered by DNA-mediated HT. Thus, photoactivation of these Ir assemblies results in both a forward and a reverse pattern for charge migration, which we term ping-pong electron transfer through DNA (Scheme 1).

Unfolded protein response-induced ERdj3 secretion links ER stress to extracellular proteostasis

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, aggregation‐prone 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.

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. DOI: http://dx.doi.org/10.7554/eLife.15550.001

Induced Pluripotent Stem Cell Modeling of Multisystemic, Hereditary Transthyretin Amyloidosis

Summary Familial transthyretin amyloidosis (ATTR) is an autosomal-dominant protein-folding disorder caused by over 100 distinct mutations in the transthyretin (TTR) gene. In ATTR, protein secreted from the liver aggregates and forms fibrils in target organs, chiefly the heart and peripheral nervous system, highlighting the need for a model capable of recapitulating the multisystem complexity of this clinically variable disease. Here, we describe the directed differentiation of ATTR patient-specific iPSCs into hepatocytes that produce mutant TTR, and the cardiomyocytes and neurons normally targeted in the disease. We demonstrate that iPSC-derived neuronal and cardiac cells display oxidative stress and an increased level of cell death when exposed to mutant TTR produced by the patient-matched iPSC-derived hepatocytes, recapitulating essential aspects of the disease in vitro. Furthermore, small molecule stabilizers of TTR show efficacy in this model, validating this iPSC-based, patient-specific in vitro system as a platform for testing therapeutic strategies.

Back-electron transfer suppresses the periodic length dependence of DNA-mediated charge transport across adenine tracts.

DNA-mediated charge transport (CT) is exquisitely sensitive to the integrity of the bridging pi-stack and is characterized by a shallow distance dependence. These properties are obscured by poor coupling between the donor/acceptor pair and the DNA bridge, or by convolution with other processes. Previously, we found a surprising periodic length dependence for the rate of DNA-mediated CT across adenine tracts monitored by 2-aminopurine fluorescence. Here we report a similar periodicity by monitoring N 2-cyclopropylguanosine decomposition by rhodium and anthraquinone photooxidants. Furthermore, we find that this periodicity is attenuated by consequent back-electron transfer (BET), as observed by direct comparison between sequences that allow and suppress BET. Thus, the periodicity can be controlled by engineering the extent of BET across the bridge. The periodic length dependence is not consistent with a periodicity predicted by molecular wire theory but is consistent with a model where multiples of four to five base pairs form an ideal CT-active length of a bridging adenine domain.