Cell Stress and Chaperones

Glutathione is a redox buffer, best known for preventing oxidative stress and ferroptosis, but rarely studied in stem cells. We studied glutathione function in hematopoietic stem cells (HSCs) by deleting the glutamate-cysteine ligase catalytic subunit (GCLC), which synthesizes glutathione, or glutathione peroxidase 4 (GPX4), which employs glutathione to prevent ferroptosis. GCLC deficiency eliminated glutathione from hematopoietic cells, severely impairing HSC function and hematopoiesis. In contrast, GPX4 deficiency did not affect HSC function and had little effect on hematopoiesis, even after vitamin E depletion, suggesting glutathione has ferroptosis-independent functions in HSCs. Consistent with this, GCLC deficiency increased reactive oxygen species levels in hematopoietic cells. But treatment with antioxidants or ferroptosis inhibitors gave little or no rescue of HSC function despite reducing ROS levels. Rather, GCLC-deficient, but not GPX4-deficient, HSCs exhibited unfolded protein accumulation, proteostatic stress, and cell death marked by elevated p53 levels and Annexin V staining. Deficiency for p53 prolonged the function of GCLC-deficient HSCs after transplantation. Glutathione is thus necessary in HSCs for proteostasis but may be dispensable for ferroptosis inhibition in vivo.

Palmitate-associated lipotoxic stress activates the endoplasmic-reticulum stress sensor protein IRE1, but how IRE1 signaling influences genotoxic-stress responses in triple-negative breast cancer remains unclear. Here, we investigated IRE1 and XBP1 signaling in MDA-MB-231 cells exposed to palmitate and etoposide. Under combined lipotoxic and genotoxic stress, XBP1 depletion increased DNA-damage accumulation in IRE1 wild-type cells but not in IRE1-deficient cells, whereas IRE1 re-expression reduced DNA damage. Pharmacologic IRE1 RNase inhibition also increased etoposide-associated DNA damage, supporting a protective role for IRE1 RNase-dependent signaling. IRE1 status and XBP1 manipulation differentially affected H2A.X and {gamma}H2A.X abundance, indicating context-dependent regulation of chromatin-associated DNA-damage signaling. At the RNA level, H2A.X was enriched in IRE1 immunoprecipitates, and the H2A.X-linked DNA-damage-response transcripts HUWE1 and BRCA2 also associated with IRE1 under combined stress. These findings link palmitate-activated IRE1-XBP1 signaling to genome-protective responses during genotoxic stress and identify a network of IRE1-associated DNA-damage-response transcripts in triple-negative breast cancer cells. Direct IRE1-mediated cleavage of these transcripts remains to be established.

Introduction: With global warming, heatwaves are increasing in severity, affecting species worldwide. Recent studies show associations between heat exposure in pregnancy and adverse birth outcomes such as preterm birth, low birthweight and stillbirth across multiple species. This suggests potentially conserved mechanisms such as maternal inflammation, dehydration and changes in placental physiology in response to heat exposure. Moreover, the gestational timing of antenatal heat likely impacts the type and severity of outcome, yet this is not fully understood. Methods: We conducted a scoping review of studies on heat exposure and pregnancy outcomes in humans and other animals, to identify common mechanisms and temporal dynamics of effects. Temperature-, trimester- and species- specific patterns were reviewed across 197 studies and ten species (human, cow, sheep, pig, goat, rat, mouse, guinea pig, lizard and rabbit). Results: Qualitative trends indicate that high temperatures are associated with adverse birth outcomes. In particular, third trimester exposure increases risk of preterm birth and low birthweight. Many studies emphasise placental involvement in adverse outcomes and point to potential aberrations in placental physiology or cortisol signaling pathways. Conclusions: Overall, literature presents that heat exposure increases risk of adverse birth outcomes across studies, and taxa. Understanding the relationship between heat exposure and adverse birth outcomes, as well as the mechanisms and timing, is vital for informing public health policies and creating mitigating strategies for expecting mothers.

Subtle variations in the activity and signaling of PERK, an endoplasmic reticulum kinase, can shift stress response outcomes. PERK has three commonly occurring exonic single-nucleotide variants frequently inherited together as part of the PERK haplotype B (PERK B), which occurs in 28% of the global population and is associated with several chronic neurodegenerative diseases, although its impact on PERK activity remains controversial. We hypothesized that PERK-B was associated with increased activity, altering stress response and stress tolerance, and that minorly but consistently elevated PERK activity, in combination with additional environmental or genetic risk factors, would lead to adverse outcomes. Comparison of stress response between PERK A, the major PERK haplotype, and PERK B in neuronal cultures using novel knock-in PERK-B mice alongside wild-type PERK A mice revealed differential, transient changes. Following ER stress, astrocyte-containing neuronal cultures, but not neuron-only cultures expressing PERK B exhibited similar levels of eIF2 phosphorylation, the canonical PERK substrate at PERK peak activation point (2h), but showed higher levels at 4 to 6 h, consistent with altered kinetics of PERK signaling. PERK-B neuronal cultures pretreated with a low dose PERK-specific activator exhibited increased vulnerability to N-methyl-D-aspartate-induced death compared to PERK-A neuronal cultures, indicating impaired acquired stress tolerance. Additionally, neuron-astrocyte coculture experiments revealed that the observed difference in acquired stress tolerance was dependent on the astrocyte genotype. The association of PERK-B with transient changes in stress response and reduced stress tolerance with higher vulnerability to cellular stress, albeit transient and small/moderate in size, has clinical relevance based on its potential contribution to the overall genetic vulnerability in neurodegenerative diseases.

Oxidative phosphorylation (OxPhos) and substrate-level phosphorylation (SLP) are the primary biochemical mechanisms for ATP generation in mammalian cells. Increased dependency on SLP can arise from either transient (e.g., Crabtree effect) or chronic (e.g., Warburg effect) suppression of OxPhos. We evaluated the steady-state bioenergetics of quiescent and proliferating mouse cells matched in genetic background and cell biology, including primary thioglycollate-elicited peritoneal macrophages, malignant VM-M3 glioma with macrophage characteristics, and malignant CT-2A astrocytoma. Data are presented showing: 1) OxPhos insufficiency was the primary bioenergetic hallmark of proliferating malignant cells. 2) Dysregulated proliferation was associated with lower metabolic flexibility under nutrient stress than was growth arrest or regulated proliferation. 3) The Crabtree effect was present in macrophages and involved a glucose- and time-dependent suppression of OxPhos coupled to ATP production through SLP and resistance to mitochondrial inhibitors; lowering glucose concentration or substituting glucose with respiratory fuels increased OxPhos and restored sensitivity to mitochondrial inhibitors. 4) The Warburg effect in tumor cells involved a persistent SLP dependency and resistance to mitochondrial inhibitors under equivalent glucose-replete conditions. 5) OxPhos could sustain the regulated proliferation of non-tumor cells in the absence of a Warburg effect. Collectively, our findings emphasize that comparisons between normal and cancer cell metabolism require long-term experimental endpoints that ensure steady-state ATP sufficiency.

The allorecognition system of the hydroid Hydractinia symbiolongicarpus plays an essential role in mediating self vs. non-self recognition among cnidarian colonies, analogous to the role of the major histocompatibility complex (MHC) in bilaterian immune systems. Both complexes have contiguous stretches of similar, highly duplicated genes, suggesting a homologous relationship. Protein structural prediction methods such as AlphaFold2 also indicate structural similarity between the proteins encoded within the Hydractinia ARC and the MHC. We have determined the structure of the ARC in the related hydrozoan Podocoryna americana to determine the degree of conservation of the cnidarian allorecognition complex. Protein structural prediction methods have revealed a number of candidate allorecognition proteins that form a long contiguous complex. Phylogenies suggest homology between cnidarian allorecognition genes, as well as syntenic conservation of this gene complex across hydrozoans. Ongoing comparative genomic analyses across bilaterian species aim to clarify the relationship between the ARC and the MHC, thereby informing our understanding of the evolutionary origins of bilaterian adaptive immunity.

Whether sexual selection facilitates or hinders adaptation to novel environments is a question of fundamental importance due to current rapid environmental change. Using phylogenetic comparative analysis in 42 Drosophila species, we found that sperm length - a sexually selected ornament - positively associates with the ability of males to survive high temperatures. However, the ability to remain fertile does not increase with sperm length to the same extent, meaning that longer sperm species experience a larger gap between temperatures that kill and temperatures that sterilize males. The findings are consistent with the idea that strong sexual selection on condition-dependent exaggerated traits may facilitate somatic resilience to environmental stressors, but that susceptibility to heat-induced infertility may be a cost of extreme sperm ornamentation. Overall, the data suggests multifaceted links between postcopulatory sexual selection and climate change.

Early embryos must mount stress defenses without compromising the developmental programs that drive morphogenesis, but whether genetic background alters this balance remains unclear. We compared cellularizing Drosophila melanogaster embryos from inbred and outcrossed backgrounds under control conditions and after acute or chronic heat-stress. Both backgrounds developed successfully and showed modest transcriptomic differences at 18{degrees}C. Heat stress at 32{degrees}C, however, sharply separated their phenotypic and transcriptional responses. Inbred embryos showed poor larval hatching after acute and chronic heat, and this deficit persisted through adult eclosion. Their transcriptomes exhibited a greater stress-response burden, with enrichment of proteostasis, DNA repair, mitochondrial maintenance, RNA processing, and metabolic remodeling programs. Outcrossed embryos retained substantially greater developmental survival and higher abundance of transcripts associated with development, adhesion, epithelial organization, signaling, transport, sensory responses, and morphogenesis. Background-dependent divergence was greatest during acute heat. Under chronic heat, background-dependent differences were fewer, with most overlapping those seen during acute heat and a smaller set detected only under chronic heat. Transcript-origin analysis further showed that acute outcrossed-biased genes were predominantly maternally derived and showed no enrichment for purely zygotic transcripts, whereas chronic outcrossed-biased genes contained a larger zygotic component. In summary, the less viable inbred background mounted the broader molecular stress signature, whereas the more viable outcrossed background retained greater abundance of development-associated transcripts. These findings identify genetic background as a major determinant of how embryonic heat responses are distributed between molecular stress defense and developmental gene expression.

Stressful events are associated with long-term adverse health impacts, but the causal mechanisms linking these exposures to subsequent disease remain poorly understood. One plausible mechanism is persistent changes to hormonal signaling that regulate metabolic homeostasis. Here, we examine the long-term impacts of stress associated with exposure to the 2004 Indian Ocean tsunami on levels of free triiodothyronine (FT3), a circulating measure of biologically active thyroid hormone, using data from the Study of Tsunami Aftermath and Recovery. We measure biomarkers 20 years after the tsunami in a population-representative sample age 35y+ in 2024 who, at the time of the tsunami, were living along the coast of Aceh, Indonesia, the most affected part of the Indian Ocean Basin. Because respondents were first interviewed before the tsunami and subsequently tracked regardless of migration, the sample avoids a major source of selection bias common in long-term disaster studies. We identify the causal effect of tsunami exposure using variation in community-level tsunami mortality, comparing communities within the same sub-district. We find that greater tsunami exposure reduced levels of FT3 20 years later. Exposure also altered relationships between FT3, body composition, and cortisol and increased cardiometabolic risk. These findings identify persistent alteration of thyroid hormone physiology as a potential pathway linking severe stressful exposures to long-term cardiometabolic disease risk.

Torpor is a physiological strategy used by which some species conserve energy by lowering metabolic rates and body temperatures. While mammalian hibernation is well-studied, the molecular mechanisms governing the rapid entry into avian torpor remain poorly understood. This study represents the first multi-tissue whole-transcriptomic analysis of torpor entry in an avian system. By combining real-time thermal imaging and respirometry with transcriptomic profiling across seven tissues, we identified the key molecular pathways orchestrating this transition in Anna's hummingbirds (Calypte anna). Our results reveal that avian torpor entry is characterised by a subtle transcriptomic change (<5% of the genome differentially expressed across tissues) compared to mammalian hibernation (10%-60%). Most of these genes are differentially expressed before torpor entry is complete, rather than tracking body temperature linearly. This indicates that active transcriptional reprogramming precedes and potentially drives the transition. Thus, hummingbird torpor entry is not a passive shutdown, but a highly regulated, tissue-specific process. Vital organs, such as the heart and lungs, remain transcriptionally stable to maintain essential functions, while the liver and gut exhibit extensive metabolic rewiring. We found evidence of global transcriptional and translational suppression, and cell-cycle arrest: the three major contributors to cellular energy budgets. We hypothesise that avian torpor entry is orchestrated through an arrest of apoptosis, a metabolic switch from carbohydrate to lipid utilisation, coupled with alterations in alternative splicing and circadian rhythm regulation. Finally, the counterintuitive upregulation of genes involved in mitochondrial metabolism suggests a preparation for rapid arousal despite the depressed metabolic state.

Forms I&prime, I&Prime, and I RuBisCOs were recently identified in bacterial metagenome-assembled genomes (MAGs) and are significant for evolutionarily bridging form I, which is responsible for the major part of carbon fixation on Earth, and other forms. Pioneering studies focused mainly on the biochemical and structural characteristics of "form I-related" RuBisCOs and deduced the evolution of this protein family over phylogenetic relationships among form I and form I-related sequences. We here surveyed form I-related sequences in public databases and conducted phylogenetic analyses incorporating the updated diversity of form I-related RuBisCOs. This study yielded two major outcomes regarding RuBisCO evolution. First, the analyses presented here suggest uncertainties overlooked in the previously published studies. It appeared to be difficult to conclude (i) the phylogenetic relationship among forms I, I&prime, and I&Prime, (ii) the monophyly of form I&prime, or (iii) the monophyly of form I. Second, we identified a gene encoding form I RuBisCO in an archaeal MAG, and this protein is likely involved in the pentose bisphosphate pathway. The results presented here suggest that the evolutionary route leading to form I in RuBisCO evolution remains unsettled.

The teleost whole-genome duplication (TGD) contributed to functional diversification of opsins. Some TGD paralogs, including those of parapinopsin (PP), Vertebrate Ancient (VA) opsin, and long wavelength-sensitive (LWS) opsin, show different absorption spectra and/or expression patterns. However, our knowledge of detailed evolutionary processes and mechanisms by which TGD contributed to opsin diversification is still limited. Here, we report that TGD paralogs of parietopsin (PT1 and PT2) are retained in extant species. While most species, including the zebrafish, have only PT1, several characins, including the red piranha (Pygocentrus nattereri), retain both PT1 and PT2. The Mexican tetra (Astyanax mexicanus) and catfishes have only PT2. To assess the degree of functional diversification between PT1 and PT2, we characterized spectral properties and expression patterns. Maximum absorption spectra differ slightly among PTs. Those of red piranha PT1, PT2, Mexican tetra PT2, and Japanese catfish (Silurus asotus) PT2 were located at 517 nm, 528 nm, 517 nm, and ~535 nm, respectively. Fluorescence in situ hybridization showed that (1) piranha PT1 and PT2 are expressed in the same pineal cells, and (2) they are also co-expressed with PP1. Based on these findings, functional diversification of opsins after gene duplication is discussed.

Abstract Background: Chronic subdural hematoma (cSDH) remains a significant and increasingly prevalent cause of morbidity in older adults. Disease progression is sustained by de novo formed highly vascularized pseudomembranes and a persistent cycle of fluid accumulation and hematoma expansion. Although inflammatory and angiogenic pathways have been implicated in cSDH pathogenesis, the endothelial mechanisms underlying persistent vascular leak remain poorly understood. We investigated the cSDH vascular microenvironment in human patients and examined the role of vascular endothelial cadherin (VECAD) remodeling in cSDH endothelial dysfunction. Methods: Human cSDH membranes and fluid were evaluated by multiplex profiling, histology, immunofluorescence, immunoprecipitation, western blotting and exploratory bulk RNA sequencing. A human brain endothelial cell (HBEC) line was exposed to patient-derived cSDH fluid to assess VECAD remodeling, endothelial barrier permeability, and proliferation. Results: Multiplex profiling of patient-derived cSDH fluid detected mediators associated with endothelial cell (EC) activation, extracellular matrix remodeling, inflammation, and angiogenesis. In patient pseudomembranes, discontinuous VECAD coincided with erythrocyte extravasation. Two hyperdense (high-risk) specimens showed more extensive disruption than the hypodense (low-risk) specimen while vessel counts appeared similar. Bulk RNA sequencing identified an angiogenic environment and enrichment in cadherin regulation. A soluble VECAD (sVECAD) isoform was detected in cSDH fluid. Exposure of HBECs to patient-derived cSDH fluid increased EC proliferation, cytoplasmic VECAD (cVECAD) accumulation, and EC barrier permeability. Concurrent dexamethasone or minocycline treatment attenuated VECAD cleavage. Conclusions: VECAD disruption is a prominent feature of cSDH and is associated with EC barrier dysfunction. These findings support further evaluation of soluble VECAD as a candidate biomarker and of junction-preserving pathways as potential therapeutic targets.

Background. Cancer cachexia is a multifactorial syndrome characterized by substantial metabolic disturbances including systemic inflammation, and muscle atrophy, which may affect the regulation of iron metabolism in patients. This study aimed to characterize this regulation in two murine models of colorectal and pancreatic cancer, both associated with a high prevalence of cachexia. Methods. Histological, biochemical and molecular analyses were performed in serum, skeletal muscle, liver, spleen and tumor from UN-KC-6141 pancreatic tumor bearing mice and from ApcMin/+ mice (colorectal cancer model), both models exhibiting variable cachexia severity. Results. Both models induce cachexia, but diverge markedly in their regulation of iron metabolism. UNKC mice display reduced plasma iron availability (-9.6% transferrin saturation, p<0.001) without concomitant anemia, while hepatic hepcidin synthesis is upregulated (+100.6%, p<0.001) by inflammation, promoting ferroportin (FPN) downregulation (-42.4%, p<0.001) and iron sequestration in the spleen (+134% total iron content, p<0.001). In the atrophied gastrocnemius, iron concentration is preserved through increased ferritin (+86.6%, p=0.010) and hemoproteins (i.e., myoglobin, CYSC, COXIV) expression. Conversely, ApcMin/+ mice exhibit anemia together with hepatic and splenic iron depletion (-48.4%, p<0.001 and -84.9%, p<0.001, respectively). Hepcidin synthesis is downregulated (-99.9%, p<0.001) despite persistent inflammation, indicating that hypoxia and/or iron-deficiency signals probably predominate over the inflammatory pathway. FPN is markedly decreased (-94.2%, p<0.001) in the iron-depleted spleen despite low hepcidin. In the atrophied quadriceps of ApcMin/+ mice, iron deficiency is associated with decreased FPN and ferritin (-87.9%, p<0.001, and -38.4%, p=0.016, respectively), and increased TfR1 expression (+305%, p=0.107). Finally, tumor iron import may play a significant role in cachexia progression, as suggested by the association between tumor weight, tumor TfR1 protein levels and body weight loss in UNKC mice. Conclusion. These findings reveal cancer type-specific signatures of iron regulation in cachexia, which appear to be more driven by cancer type specific features than by cachexia severity itself, with skeletal muscle iron metabolism impaired mainly when cachexia is associated with anemia. This suggests that the clinical management of iron metabolism in cachectic patients should be guided by the underlying cancer type.

Retinal neurons rely on continuous oxygen supply to maintain function, yet some species, like goldfish, withstand prolonged hypoxia due to adaptations that remain unclear. Horizontal cells (HCs) are inhibitory interneurons of the retina that express ionotropic glutamate receptors (iGluRs) and secrete gama-aminobutyric acid (GABA), allowing them to shape visual signalling by feeding back to photoreceptors. We investigated whether iGluRs in dissociated goldfish HCs are suppressed during hypoxia, and whether this process is mediated by oxygen-sensitive activation of mitochondrial ATP-sensitive K+ (mKATP) channels. Through perforated patch-clamp electrophysiology, we determined that 20 min of hypoxia reduced peak inward current density associated with glutamate application by ~25% (P = 0.0059). When mKATP channels were selectively blocked with 100 microM glibenclamide, hypoxic suppression of iGluRs was abolished. Inhibiting mitochondrial Ca2+ uptake via the mitochondrial Ca2+ uniporter with ruthenium red, or blocking Ca2+ release through ryanodine receptors in the endoplasmic reticulum, also abolished the hypoxia-induced suppression. Similarly, inhibition of calmodulin with calmidazolium, or of protein phosphatase 1 and 2A (PP1/PP2A) activity with calyculin A, prevented the hypoxia-induced suppression of iGluRs. Taken together, these results indicate that mKATP activation is required to mediate an intracellular Ca2+ signal that downregulates iGluR activity during hypoxia. Through the reduction of iGluR activity, HCs may lower their metabolic demand, allowing them to maintain cell function during hypoxia. Additionally, GABA release from HCs, as measured by mass spectrometry, was not affected by hypoxia, and we propose that downregulation of iGluR may account for hypoxic suppression of the retina in hypoxia-tolerant organisms.

Endoplasmic reticulum (ER) stress is associated with many human diseases, but current understanding of how ER stress responses reshape the stressed proteome over time is still emerging. Specifically, how organellar protein quality control and clearance pathways coordinate to maintain proteostasis in early and prolonged ER stress is unclear. Here we describe a spatiotemporal proteomic strategy termed simultaneous proteome localization and turnover analysis with time resolution (SPLAT-TR) to interrogate the synthesis, degradation, and localization changes of over 4,000 proteins in early (1-4 hours) and prolonged (16-24 hours) ER stress. As ER stress progresses, each time point is distinguished by distinct protein translocation and clearance regulations, coupled to differential organellar proteostasis and usage of protein degradation pathways. Mitochondria show a bimodal response, with a shift from early activation of respiratory protein towards protein quality control pathways in prolonged stress, coinciding with energetics decline. In the Golgi, the increased clearance of collagen proteins is paradoxically coupled to an increased synthesis of secretory pathway components, suggesting removal by secretion. In the ER, the proteome remodels under prolonged stress via increased synthesis of UFMylation and ER-phagy-related proteins coupled to the selective degradation of ER membrane and microdomain proteins. Inhibition of UFMylation alters ER-phagy receptor usage and synergistically induces stress-induced cell death. These results present a systematic delineation of cell compartment proteostasis under unfolded protein response and highlight the utility of SPLAT-TR to investigate time-dependent cellular events.

Acute respiratory distress syndrome (ARDS) is marked by severe pulmonary edema and concomitant hypoxia, affecting hundreds of thousands of people a year, especially those in critical care conditions or suffering from septic shock. Previous studies have implicated that the ketogenic diet, a high-fat and low-carbohydrate diet, modulates inflammatory responses. However, the impact of the ketogenic diet on septic ARDS outcomes is unknown. Here, we demonstrated that mice on a ketogenic diet showed strikingly reduced lung injury and inflammation compared to those on a control diet during a murine model of endotoxin-induced lung injury, induced by intratracheal lipopolysaccharide (LPS) injection. Immune mass cytometry studies on lung tissue indicated that the ketogenic diet reduces immune cell infiltration. Treating mice with beta-hydroxybutyrate (BHB), the primary metabolite of ketogenesis, after the onset of ARDS reduced pulmonary edema and lung inflammation, as well as NF-kB activity, suggesting strong therapeutic potential. By multiplex analysis in bronchial alveolar lavage fluid, we observed that the ketogenic diet or BHB administration attenuates the chemotaxis and activation of immune cells. Altogether, our findings reveal that the ketogenic diet provides lung protection during endotoxin-induced lung injury through BHB.

Synchrony between time-of-day and food intake is critical for maintaining homeostasis while desynchrony between these cues caused by repeated bouts of mistimed eating (ME) is profoundly disruptive to organismal health. However, it is unknown whether the same is true for a single bout of ME. While it is generally accepted that mammals recover quickly from acute alterations in feeding schedule and that chrono-disruption requires chronic, repeated changes, there have been no dedicated investigations of acute ME to suggest otherwise. Therefore, we exposed female and male C57Bl6/J mice to a single bout of ME or timed eating (TE) and continuously measured metabolic and behavioral responses for five days. A single bout of ME was sufficient to disrupt the daily rhythms of metabolic and behavioral outcomes for several days. Energy expenditure and wheel running activity were disrupted and light:dark variation in food intake and macronutrient utilization were altered for several days. Finally, ME changed the amount of time spent resting in for several days post-ME. The TE paradigm disrupted macronutrient utilization for two days but otherwise had no effect on metabolism or behavior. Our results demonstrate that one instance of ME alone has lasting impacts on daily rhythmicity and is an understudied source of circadian disruption.

Cisplatin remains as standard chemotherapy for patients with HNSCC, but rapid development of drug resistance has limited patient benefit. The p53 tumor suppressor plays a central role in the cellular response to DNA damage in cancer, triggering apoptosis to prevent propagation of damaged cells in tumor development. TP53 gene mutations occur in 65-86% of HNSCC. Small molecule PG3 induces the integrated stress response (ISR), leading to apoptosis via the HRI-eIF2-ATF4-PUMA axis. We hypothesized that a combination of PG3 plus cisplatin could increase apoptosis in TP53-mutated HNSCC cells through enhanced induction of the ISR and ATF4. PG3 synergized with cisplatin to inhibit cell viability, leading to potent apoptosis in TP53-deficient cells. The effect was regulated through the HRI-ATF4-NOXA pathway. Furthermore, we identified that cisplatin activates HRI and leads to the degradation of CReP (constitutive repressor of eIF2 phosphorylation) via E3 ligase {beta}-TrCP, contributing to the induction of the ISR. We noted decreased ATF4 levels after treatment with cisplatin, CPT, or PG3 and cisplatin. Thus, combined therapy of PG3 plus cisplatin likely results in adaptation and acquired resistance via degradation of ATF4. We targeted the degradation mechanism of ATF4 by inhibiting {beta}-TrCP1, CK1{delta}, or CK2, respectively. Each approach successfully blocked ATF4 degradation induced by cisplatin or PG3 plus cisplatin and enhanced apoptosis. Our results provide a rational strategy for triple treatments, involving an ISR inducer, a DNA damaging drug, and a {beta}-TrCP inhibitor/CK1{delta} inhibitor/CK2 inhibitor, to achieve potent and prolonged anti-tumor effects and overcome chemoresistance in HNSCC.

The human HSP70 chaperone network maintains cellular proteostasis through a diverse repertoire of HSP70s and co-chaperones. Here we examine alternative isoforms and co-chaperone hetero-complexes as additional sources of network complexity. To that end, we systematically mapped the isoform, tissue-expression, and interaction landscapes of the human HSP70 network, revealing a modular interactome containing known and novel DNAJ-DNAJ interactions. We found both tissue-expression isoform divergence as well as widespread alternative-canonical isoform co-expression, suggesting additional modes of functional diversification. Focusing on the uncharacterized DNAJA2-DNAJA4 hetero-complex, we identified the stress-inducible isoform DNAJA4-CTD-II. DNAJA4-CTD-II formed hetero-complexes with DNAJA2 and DNAJA4, with both interactions enhanced following sodium arsenite stress. Functionally, DNAJA4-CTD-II co-localized with TDP-43 aggregates and significantly suppressed their accumulation in a DNAJA2-dependent manner. Together, our data reveal extensive, uncharted isoform and interaction complexity within the HSP70 network, and uncover isoform-dependent hetero-complex remodeling as a new layer of chaperone network regulation.