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Primary ciliary dyskinesia (PCD) is a respiratory disease caused by dysfunction of the cilia with currently no approved treatments. This predominantly autosomal recessive disease is caused by mutations in any one of over 50 genes involved in cilia function; DNAI1 is one of the more frequently mutated genes, accounting for approximately 5-10% of diagnosed PCD cases. A codon-optimized mRNA encoding DNAI1 and encapsulated in a lipid nanoparticle (LNP) was administered to mice via aerosolized inhalation resulting in the expression human DNAI1 in the multiciliated cells of the pseudostratified columnar epithelia. The spatial localization of DNAI1 expression in the bronchioles indicate that delivery of the DNAI1 mRNA transpires the lower airways. In a PCD disease model, exposure to the LNP-encapsulated DNAI1 mRNA resulted in increased ciliary beat frequency using high speed videomicroscopy showing the potential for an mRNA therapeutic to correct cilia function in patients with PCD due to DNAI1 mutations., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

After lung injury, damage-associated transient progenitors (DATPs) emerge, representing a transitional state between injured epithelial cells and newly regenerated alveoli. DATPs express profibrotic genes, suggesting that they might promote idiopathic pulmonary fibrosis (IPF). However, the molecular pathways that induce and/or maintain DATPs are incompletely understood. Here we show that the bifunctional kinase/RNase-IRE1α-a central mediator of the unfolded protein response (UPR) to endoplasmic reticulum (ER) stress is a critical promoter of DATP abundance and function. Administration of a nanomolar-potent, monoselective kinase inhibitor of IRE1α (KIRA8)-or conditional epithelial IRE1α gene knockout-both reduce DATP cell number and fibrosis in the bleomycin model, indicating that IRE1α cell-autonomously promotes transition into the DATP state. IRE1α enhances the profibrotic phenotype of DATPs since KIRA8 decreases expression of integrin αvβ6, a key activator of transforming growth factor β (TGF-β) in pulmonary fibrosis, corresponding to decreased TGF-β-induced gene expression in the epithelium and decreased collagen accumulation around DATPs. Furthermore, IRE1α regulates DNA damage response (DDR) signaling, previously shown to promote the DATP phenotype, as IRE1α loss-of-function decreases H2AX phosphorylation, Cdkn1a (p21) expression, and DDR-associated secretory gene expression. Finally, KIRA8 treatment increases the differentiation of Krt19 CreERT2 -lineage-traced DATPs into type 1 alveolar epithelial cells after bleomycin injury, indicating that relief from IRE1α signaling enables DATPs to exit the transitional state. Thus, IRE1α coordinates a network of stress pathways that conspire to entrap injured cells in the DATP state. Pharmacological blockade of IRE1α signaling helps resolve the DATP state, thereby ameliorating fibrosis and promoting salutary lung regeneration.

Skin is the largest organ of the body and serves as the principle barrier to the environment. Composed of multiple cell types arranged in stratified layers with highly specialized appendages, it serves sensory and immune surveillance roles in addition to its primary mechanical function. Several complex in vitro models of skin (i.e. microphysiological systems (MPS) including but not limited to 3D tissues, organ-on-a-chip, organoids), have been developed and assays validated for regulatory purposes. As such, skin is arguably the most advanced organ with respect to model development and adoption across industries including chemical, cosmetic, and to a somewhat lesser extent, pharmaceutical. Early adoption of complex skin models and associated assays for assessment of irritation and corrosion spurred research into other areas such as sensitization, absorption, phototoxicity, and genotoxicity. Despite such considerable advancements, opportunities remain for immune capabilities, inclusion of appendages such as hair follicles, fluidics, and innervation, among others. Herein, we provide an overview of current complex skin model capabilities and limitations within the drug development scheme, and recommendations for future model development and assay qualification and/or validation with the intent to facilitate wider adoption of use within the pharmaceutical industry.

Master regulators of the unfolded protein response (UPR), IRE1α and PERK, promote adaptation or apoptosis depending on the level of endoplasmic reticulum (ER) stress. Although the UPR is activated in many cancers, its effects on tumor growth remain unclear. Derived from endocrine cells, pancreatic neuroendocrine tumors (PanNET) universally hypersecrete one or more peptide hormones, likely sensitizing these cells to high ER protein-folding stress. To assess whether targeting the UPR is a viable therapeutic strategy, we analyzed human PanNET samples and found evidence of elevated ER stress and UPR activation. Genetic and pharmacologic modulation of IRE1α and PERK in cultured cells, xenograft, and spontaneous genetic (RIP-Tag2) mouse models of PanNETs revealed that UPR signaling was optimized for adaptation and that inhibiting either IRE1α or PERK led to hyperactivation and apoptotic signaling through the reciprocal arm, thereby halting tumor growth and survival. These results provide a strong rationale for therapeutically targeting the UPR in PanNETs and other cancers with elevated ER stress. SIGNIFICANCE: The UPR is upregulated in pancreatic neuroendocrine tumors and its inhibition significantly reduces tumor growth in preclinical models, providing strong rationale for targeting the UPR in these cancers., (©2019 American Association for Cancer Research.)

Endoplasmic reticulum stress (ER stress) has been implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF), a disease of progressive fibrosis and respiratory failure. ER stress activates a signaling pathway called the unfolded protein response (UPR) that either restores homeostasis or promotes apoptosis. The bifunctional kinase/RNase IRE1α is a UPR sensor/effector that promotes apoptosis if ER stress remains high and irremediable (i.e., a "terminal" UPR). Using multiple small molecule inhibitors against IRE1α, we show that ER stress-induced apoptosis of murine alveolar epithelial cells can be mitigated in vitro. In vivo, we show that bleomycin exposure to murine lungs causes early ER stress to activate IRE1α and the terminal UPR prior to development of pulmonary fibrosis. Small-molecule IRE1α kinase-inhibiting RNase attenuators (KIRAs) that we developed were used to evaluate the contribution of IRE1α activation to bleomycin-induced pulmonary fibrosis. One such KIRA-KIRA7-provided systemically to mice at the time of bleomycin exposure decreases terminal UPR signaling and prevents lung fibrosis. Administration of KIRA7 14 days after bleomycin exposure even promoted the reversal of established fibrosis. Finally, we show that KIRA8, a nanomolar-potent, monoselective KIRA compound derived from a completely different scaffold than KIRA7, likewise promoted reversal of established fibrosis. These results demonstrate that IRE1α may be a promising target in pulmonary fibrosis and that kinase inhibitors of IRE1α may eventually be developed into efficacious anti-fibrotic drugs., Competing Interests: The authors have declared that no competing interests exist.

By employing a genetic selection that forces the cell to fold an unstable, aggregation-prone test protein in order to survive, we have generated bacterial strains with enhanced periplasmic folding capacity. These strains enhance the soluble steady-state level of the test protein. Most of the bacterial variants we isolated were found to overexpress one or more periplasmic proteins including OsmY, Ivy, DppA, OppA, and HdeB. Of these proteins, only HdeB has convincingly been previously shown to function as chaperone in vivo. By giving bacteria the stark choice between death and stabilizing a poorly folded protein, we have now generated designer bacteria selected for their ability to stabilize specific proteins., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Unbiased genetic studies have uncovered surprising molecular mechanisms in human cellular immunity and autoimmunity. We performed whole-exome sequencing and targeted sequencing in five families with an apparent mendelian syndrome of autoimmunity characterized by high-titer autoantibodies, inflammatory arthritis and interstitial lung disease. We identified four unique deleterious variants in the COPA gene (encoding coatomer subunit α) affecting the same functional domain. Hypothesizing that mutant COPA leads to defective intracellular transport via coat protein complex I (COPI), we show that COPA variants impair binding to proteins targeted for retrograde Golgi-to-ER transport. Additionally, expression of mutant COPA results in ER stress and the upregulation of cytokines priming for a T helper type 17 (TH17) response. Patient-derived CD4(+) T cells also demonstrate significant skewing toward a TH17 phenotype that is implicated in autoimmunity. Our findings uncover an unexpected molecular link between a vesicular transport protein and a syndrome of autoimmunity manifested by lung and joint disease.

The era in which ROS (reactive oxygen species) were simply the 'bad boys of biology' is clearly over. High levels of ROS are still rightfully considered to be toxic to many cellular processes and, as such, contribute to disease conditions and cell death. However, the high toxicity of ROS is also extremely beneficial, particularly as it is used to kill invading micro-organisms during mammalian host defence. Moreover, a transient, often more localized, increase in ROS levels appears to play a major role in signal transduction processes and positively affects cell growth, development and differentiation. At the heart of all these processes are redox-regulated proteins, which use oxidation-sensitive cysteine residues to control their function and by extension the function of the pathways that they are part of. Our work has contributed to changing the view about ROS through: (i) our characterization of Hsp33 (heat-shock protein 33), one of the first redox-regulated proteins identified, whose function is specifically activated by ROS, (ii) the development of quantitative tools that reveal extensive redox-sensitive processes in bacteria and eukaryotes, and (iii) the discovery of a link between early exposure to oxidants and aging. Our future research programme aims to generate an integrated and system-wide view of the beneficial and deleterious effects of ROS with the central goal to develop more effective antioxidant strategies and more powerful antimicrobial agents.

Depending on endoplasmic reticulum (ER) stress levels, the ER transmembrane multidomain protein IRE1α promotes either adaptation or apoptosis. Unfolded ER proteins cause IRE1α lumenal domain homo-oligomerization, inducing trans autophosphorylation that further drives homo-oligomerization of its cytosolic kinase/endoribonuclease (RNase) domains to activate mRNA splicing of adaptive XBP1 transcription factor. However, under high/chronic ER stress, IRE1α surpasses an oligomerization threshold that expands RNase substrate repertoire to many ER-localized mRNAs, leading to apoptosis. To modulate these effects, we developed ATP-competitive IRE1α Kinase-Inhibiting RNase Attenuators-KIRAs-that allosterically inhibit IRE1α's RNase by breaking oligomers. One optimized KIRA, KIRA6, inhibits IRE1α in vivo and promotes cell survival under ER stress. Intravitreally, KIRA6 preserves photoreceptor functional viability in rat models of ER stress-induced retinal degeneration. Systemically, KIRA6 preserves pancreatic β cells, increases insulin, and reduces hyperglycemia in Akita diabetic mice. Thus, IRE1α powerfully controls cell fate but can itself be controlled with small molecules to reduce cell degeneration., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Obstacles in elucidating the role of oxidative stress in aging include difficulties in (1) tracking in vivo oxidants, in (2) identifying affected proteins, and in (3) correlating changes in oxidant levels with life span. Here, we used quantitative redox proteomics to determine the onset and the cellular targets of oxidative stress during Caenorhabditis elegans' life span. In parallel, we used genetically encoded sensor proteins to determine peroxide levels in live animals in real time. We discovered that C. elegans encounters significant levels of oxidants as early as during larval development. Oxidant levels drop rapidly as animals mature, and reducing conditions prevail throughout the reproductive age, after which age-accompanied protein oxidation sets in. Long-lived daf-2 mutants transition faster to reducing conditions, whereas short-lived daf-16 mutants retain higher oxidant levels throughout their mature life. These results suggest that animals with improved capacity to recover from early oxidative stress have significant advantages later in life., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Accumulation of reactive oxygen species has been implicated in various diseases and aging. However, the precise physiological effects of accumulating oxidants are still largely undefined. Here, we applied a short-term peroxide stress treatment to young Caenorhabditis elegans and measured behavioral, physiological, and cellular consequences. We discovered that exposure to peroxide stress causes a number of immediate changes, including loss in mobility, decreased growth rate, and decreased cellular adenosine triphosphate levels. Many of these alterations, which are highly reminiscent of changes in aging animals, are reversible, suggesting the presence of effective antioxidant systems in young C. elegans. One of these antioxidant systems involves the highly abundant protein peroxiredoxin 2 (PRDX-2), whose gene deletion causes phenotypes symptomatic of chronic peroxide stress and shortens lifespan. Applying the quantitative redox proteomic technique OxICAT to oxidatively stressed wild-type and prdx-2 deletion worms, we identified oxidation-sensitive cysteines in 40 different proteins, including proteins involved in mobility and feeding (e.g., MYO-2 and LET-75), protein translation and homeostasis (e.g., elongation factor 1 [EFT-1] and heat shock protein 1), and adenosine triphosphate regeneration (e.g., nucleoside diphosphate kinase). The oxidative modification of some of these redox-sensitive cysteines may contribute to the physiological and behavioral changes observed in oxidatively stressed animals.

Eukaryotic peroxiredoxins are highly susceptible to sulfinic acid formation. This overoxidation, which is thought to convert peroxiredoxins into chaperones, can be reversed by sulfiredoxins. Several organisms, including Caenorhabditis elegans, lack sulfiredoxins but encode sestrins, proteins proposed to be functionally equivalent. We induced peroxiredoxin overoxidation in C. elegans with a short peroxide pulse. We found that reduction of overoxidized peroxiredoxin 2 (PRDX-2) was extremely slow and sestrin-independent, strongly implying that worms lack an efficient repair system. Analysis of PRDX-2's overoxidation status during C. elegans lifespan revealed no accumulation of overoxidized PRDX-2 at any point, questioning whether PRDX-2 overoxidation in worms is physiologically relevant.

Redox-regulated proteins play fundamentally important roles not only during the defense of organisms against oxidative stress conditions but also as targets of cellular signaling events. This realization has spurred the development of proteomic techniques geared towards characterizing the redoxome; proteins with highly reactive cysteine residues, whose thiol oxidation state controls the function of the proteins, and by extension, the pathways they are part of. We will here summarize the most recent advances made in the field of redox proteomic analysis, aimed to elucidate the cellular redox networks that appear to control prokaryotic and eukaryotic organisms., (Copyright © 2010. Published by Elsevier Ltd.)