Mucus polymer concentration and in vivo adaptation converge to define the antibiotic response of Pseudomonas aeruginosa during chronic lung infection.

The airway milieu of individuals with muco-obstructive airway diseases (MADs) is defined by the accumulation of dehydrated mucus due to hyperabsorption of airway surface liquid and defective mucociliary clearance. Pathological mucus becomes progressively more viscous with age and disease severity due to the concentration and overproduction of mucin and accumulation of host-derived extracellular DNA (eDNA). Respiratory mucus of MADs provides a niche for recurrent and persistent colonization by respiratory pathogens, including Pseudomonas aeruginosa, which is responsible for the majority of morbidity and mortality in MADs. Despite high concentration inhaled antibiotic therapies and the absence of antibiotic resistance, antipseudomonal treatment failure in MADs remains a significant clinical challenge. Understanding the drivers of antibiotic tolerance is essential for developing more effective treatments that eradicate persistent infections. The complex and dynamic environment of diseased airways makes it difficult to model antibiotic efficacy in vitro. We aimed to understand how mucin and eDNA concentrations, the two dominant polymers in respiratory mucus, alter the antibiotic tolerance of P. aeruginosa. Our results demonstrate that polymer concentration and molecular weight affect P. aeruginosa survival post antibiotic challenge. Polymer-driven antibiotic tolerance was not explicitly associated with reduced antibiotic diffusion. Lastly, we established a robust and standardized in vitro model for recapitulating the ex vivo antibiotic tolerance of P. aeruginosa observed in expectorated sputum across age, underlying MAD etiology, and disease severity, which revealed the inherent variability in intrinsic antibiotic tolerance of host-evolved P. aeruginosa populations. IMPORTANCE: Antibiotic treatment failure in Pseudomonas aeruginosa chronic lung infections is associated with increased morbidity and mortality, illustrating the clinical challenge of bacterial infection control. Understanding the underlying infection environment, as well as the host and bacterial factors driving antibiotic tolerance and the ability to accurately recapitulate these factors in vitro, is crucial for improving antibiotic treatment outcomes. Here, we demonstrate that increasing concentration and molecular weight of mucin and host eDNA drive increased antibiotic tolerance to tobramycin. Through systematic testing and modeling, we identified a biologically relevant in vitro condition that recapitulates antibiotic tolerance observed in ex vivo treated sputum. Ultimately, this study revealed a dominant effect of in vivo evolved bacterial populations in defining inter-subject ex vivo antibiotic tolerance and establishes a robust and translatable in vitro model for therapeutic development.

The alpha-1A adrenergic receptor regulates mitochondrial oxidative metabolism in the mouse heart.

AIMS: The sympathetic nervous system regulates numerous critical aspects of mitochondrial function in the heart through activation of adrenergic receptors (ARs) on cardiomyocytes. Mounting evidence suggests that α1-ARs, particularly the α1A subtype, are cardioprotective and may mitigate the deleterious effects of chronic ß-AR activation by shared ligands. The mechanisms underlying these adaptive effects remain unclear. Here, we tested the hypothesis that α1A-ARs adaptively regulate cardiomyocyte oxidative metabolism in both the uninjured and infarcted heart. METHODS: We used high resolution respirometry, fatty acid oxidation (FAO) enzyme assays, substrate-specific electron transport chain (ETC) enzyme assays, transmission electron microscopy (TEM) and proteomics to characterize mitochondrial function comprehensively in the uninjured hearts of wild type and α1A-AR knockout mice and defined the effects of chronic ß-AR activation and myocardial infarction on selected mitochondrial functions. RESULTS: We found that isolated cardiac mitochondria from α1A-KO mice had deficits in fatty acid-dependent respiration, FAO, and ETC enzyme activity. TEM revealed abnormalities of mitochondrial morphology characteristic of these functional deficits. The selective α1A-AR agonist A61603 enhanced fatty-acid dependent respiration, fatty acid oxidation, and ETC enzyme activity in isolated cardiac mitochondria. The ß-AR agonist isoproterenol enhanced oxidative stress in vitro and this adverse effect was mitigated by A61603. A61603 enhanced ETC Complex I activity and protected contractile function following myocardial infarction. CONCLUSIONS: Collectively, these novel findings position α1A-ARs as critical regulators of cardiomyocyte metabolism in the basal state and suggest that metabolic mechanisms may underlie the protective effects of α1A-AR activation in the failing heart.

Cardiomyocyte Alpha-1A Adrenergic Receptors Mitigate Postinfarct Remodeling and Mortality by Constraining Necroptosis.

Clinical studies have shown that α1-adrenergic receptor antagonists (α-blockers) are associated with increased heart failure risk. The mechanism underlying that hazard and whether it arises from direct inhibition of cardiomyocyte α1-ARs or from systemic effects remain unclear. To address these issues, we created a mouse with cardiomyocyte-specific deletion of the α1A-AR subtype and found that it experienced 70% mortality within 7 days of myocardial infarction driven, in part, by excessive activation of necroptosis. We also found that patients taking α-blockers at our center were at increased risk of death after myocardial infarction, providing clinical correlation for our translational animal models.

Prefusion-stabilized SARS-CoV-2 S2-only antigen provides protection against SARS-CoV-2 challenge.

Ever-evolving SARS-CoV-2 variants of concern (VOCs) have diminished the effectiveness of therapeutic antibodies and vaccines. Developing a coronavirus vaccine that offers a greater breadth of protection against current and future VOCs would eliminate the need to reformulate COVID-19 vaccines. Here, we rationally engineer the sequence-conserved S2 subunit of the SARS-CoV-2 spike protein and characterize the resulting S2-only antigens. Structural studies demonstrate that the introduction of interprotomer disulfide bonds can lock S2 in prefusion trimers, although the apex samples a continuum of conformations between open and closed states. Immunization with prefusion-stabilized S2 constructs elicits broadly neutralizing responses against several sarbecoviruses and protects female BALB/c mice from mouse-adapted SARS-CoV-2 lethal challenge and partially protects female BALB/c mice from mouse-adapted SARS-CoV lethal challenge. These engineering and immunogenicity results should inform the development of next-generation pan-coronavirus therapeutics and vaccines.

Viral afterlife: SARS-CoV-2 as a reservoir of immunomimetic peptides that reassemble into proinflammatory supramolecular complexes.

It is unclear how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leads to the strong but ineffective inflammatory response that characterizes severe Coronavirus disease 2019 (COVID-19), with amplified immune activation in diverse cell types, including cells without angiotensin-converting enzyme 2 receptors necessary for infection. Proteolytic degradation of SARS-CoV-2 virions is a milestone in host viral clearance, but the impact of remnant viral peptide fragments from high viral loads is not known. Here, we examine the inflammatory capacity of fragmented viral components from the perspective of supramolecular self-organization in the infected host environment. Interestingly, a machine learning analysis to SARS-CoV-2 proteome reveals sequence motifs that mimic host antimicrobial peptides (xenoAMPs), especially highly cationic human cathelicidin LL-37 capable of augmenting inflammation. Such xenoAMPs are strongly enriched in SARS-CoV-2 relative to low-pathogenicity coronaviruses. Moreover, xenoAMPs from SARS-CoV-2 but not low-pathogenicity homologs assemble double-stranded RNA (dsRNA) into nanocrystalline complexes with lattice constants commensurate with the steric size of Toll-like receptor (TLR)-3 and therefore capable of multivalent binding. Such complexes amplify cytokine secretion in diverse uninfected cell types in culture (epithelial cells, endothelial cells, keratinocytes, monocytes, and macrophages), similar to cathelicidin's role in rheumatoid arthritis and lupus. The induced transcriptome matches well with the global gene expression pattern in COVID-19, despite using <0.3% of the viral proteome. Delivery of these complexes to uninfected mice boosts plasma interleukin-6 and CXCL1 levels as observed in COVID-19 patients.

A comprehensive SARS-CoV-2 and COVID-19 review, Part 2: host extracellular to systemic effects of SARS-CoV-2 infection.

COVID-19, the disease caused by SARS-CoV-2, has caused significant morbidity and mortality worldwide. The betacoronavirus continues to evolve with global health implications as we race to learn more to curb its transmission, evolution, and sequelae. The focus of this review, the second of a three-part series, is on the biological effects of the SARS-CoV-2 virus on post-acute disease in the context of tissue and organ adaptations and damage. We highlight the current knowledge and describe how virological, animal, and clinical studies have shed light on the mechanisms driving the varied clinical diagnoses and observations of COVID-19 patients. Moreover, we describe how investigations into SARS-CoV-2 effects have informed the understanding of viral pathogenesis and provide innovative pathways for future research on the mechanisms of viral diseases.

Core mitochondrial genes are down-regulated during SARS-CoV-2 infection of rodent and human hosts.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins bind to host mitochondrial proteins, likely inhibiting oxidative phosphorylation (OXPHOS) and stimulating glycolysis. We analyzed mitochondrial gene expression in nasopharyngeal and autopsy tissues from patients with coronavirus disease 2019 (COVID-19). In nasopharyngeal samples with declining viral titers, the virus blocked the transcription of a subset of nuclear DNA (nDNA)-encoded mitochondrial OXPHOS genes, induced the expression of microRNA 2392, activated HIF-1α to induce glycolysis, and activated host immune defenses including the integrated stress response. In autopsy tissues from patients with COVID-19, SARS-CoV-2 was no longer present, and mitochondrial gene transcription had recovered in the lungs. However, nDNA mitochondrial gene expression remained suppressed in autopsy tissue from the heart and, to a lesser extent, kidney, and liver, whereas mitochondrial DNA transcription was induced and host-immune defense pathways were activated. During early SARS-CoV-2 infection of hamsters with peak lung viral load, mitochondrial gene expression in the lung was minimally perturbed but was down-regulated in the cerebellum and up-regulated in the striatum even though no SARS-CoV-2 was detected in the brain. During the mid-phase SARS-CoV-2 infection of mice, mitochondrial gene expression was starting to recover in mouse lungs. These data suggest that when the viral titer first peaks, there is a systemic host response followed by viral suppression of mitochondrial gene transcription and induction of glycolysis leading to the deployment of antiviral immune defenses. Even when the virus was cleared and lung mitochondrial function had recovered, mitochondrial function in the heart, kidney, liver, and lymph nodes remained impaired, potentially leading to severe COVID-19 pathology.

Mouse genomic associations with in vitro sensitivity to simulated space radiation.

Exposure to ionizing radiation is considered by NASA to be a major health hazard for deep space exploration missions. Ionizing radiation sensitivity is modulated by both genomic and environmental factors. Understanding their contributions is crucial for designing experiments in model organisms, evaluating the risk of deep space (i.e. high-linear energy transfer, or LET, particle) radiation exposure in astronauts, and also selecting therapeutic irradiation regimes for cancer patients. We identified single nucleotide polymorphisms in 15 strains of mice, including 10 collaborative cross model strains and 5 founder strains, associated with spontaneous and ionizing radiation-induced in vitro DNA damage quantified based on immunofluorescent tumor protein p53 binding protein (53BP1) positive nuclear foci. Statistical analysis suggested an association with pathways primarily related to cellular signaling, metabolism, tumorigenesis and nervous system damage. We observed different genomic associations in early (4 and 8 h) responses to different LET radiation, while later (24 hour) DNA damage responses showed a stronger overlap across all LETs. Furthermore, a subset of pathways was associated with spontaneous DNA damage, suggesting 53BP1 positive foci as a potential biomarker for DNA integrity in mouse models. Our results suggest several mouse strains as new models to further study the impact of ionizing radiation and validate the identified genetic loci. We also highlight the importance of future human in vitro studies to refine the association of genes and pathways with the DNA damage response to ionizing radiation and identify targets for space travel countermeasures.

Mucus polymer concentration and in vivo adaptation converge to define the antibiotic response of Pseudomonas aeruginosa during chronic lung infection.

The airway milieu of individuals with muco-obstructive airway diseases (MADs) is defined by the accumulation of dehydrated mucus due to hyperabsorption of airway surface liquid and defective mucociliary clearance. Pathological mucus becomes progressively more viscous with age and disease severity due to the concentration and overproduction of mucin and accumulation of host-derived extracellular DNA (eDNA). Respiratory mucus of MADs provides a niche for recurrent and persistent colonization by respiratory pathogens, including Pseudomonas aeruginosa , which is responsible for the majority of morbidity and mortality in MADs. Despite high concentration inhaled antibiotic therapies and the absence of antibiotic resistance, antipseudomonal treatment failure in MADs remains a significant clinical challenge. Understanding the drivers of antibiotic recalcitrance is essential for developing more effective treatments that eradicate persistent infections. The complex and dynamic environment of diseased airways makes it difficult to model antibiotic efficacy in vitro . We aimed to understand how mucin and eDNA concentrations, the two dominant polymers in respiratory mucus, alter the antibiotic tolerance of P. aeruginosa . Our results demonstrate that polymer concentration and molecular weight affect P. aeruginosa survival post antibiotic challenge. Polymer-driven antibiotic tolerance was not explicitly associated with reduced antibiotic diffusion. Lastly, we established a robust and standardized in vitro model for recapitulating the ex vivo antibiotic tolerance of P. aeruginosa observed in expectorated sputum across age, underlying MAD etiology, and disease severity, which revealed the inherent variability in intrinsic antibiotic tolerance of host-evolved P. aeruginosa populations. Importance: Antibiotic treatment failure in Pseudomonas aeruginosa chronic lung infections is associated with increased morbidity and mortality, illustrating the clinical challenge of bacterial infection control. Understanding the underlying infection environment, as well as the host and bacterial factors driving antibiotic tolerance and the ability to accurately recapitulate these factors in vitro , is crucial for improving antibiotic treatment outcomes. Here, we demonstrate that increasing concentration and molecular weight of mucin and host eDNA drive increased antibiotic tolerance to tobramycin. Through systematic testing and modeling, we identified a biologically relevant in vitro condition that recapitulates antibiotic tolerance observed in ex vivo treated sputum. Ultimately, this study revealed a dominant effect of in vivo evolved bacterial populations in defining inter-subject ex vivo antibiotic tolerance and establishes a robust and translatable in vitro model for therapeutic development.

Host transcriptional responses in nasal swabs identify potential SARS-CoV-2 infection in PCR negative patients.

We analyzed RNA sequencing data from nasal swabs used for SARS-CoV-2 testing. 13% of 317 PCR-negative samples contained over 100 reads aligned to multiple regions of the SARS-CoV-2 genome. Differential gene expression analysis compares the host gene expression in potential false-negative (FN: PCR negative, sequencing positive) samples to subjects with multiple SARS-CoV-2 viral loads. The host transcriptional response in FN samples was distinct from true negative samples (PCR & sequencing negative) and similar to low viral load samples. Gene Ontology analysis shows viral load-dependent changes in gene expression are functionally distinct; 23 common pathways include responses to viral infections and associated immune responses. GO analysis reveals FN samples had a high overlap with high viral load samples. Deconvolution of RNA-seq data shows similar cell content across viral loads. Hence, transcriptome analysis of nasal swabs provides an additional level of identifying SARS-CoV-2 infection.

The interplay between lncRNAs, RNA-binding proteins and viral genome during SARS-CoV-2 infection reveals strong connections with regulatory events involved in RNA metabolism and immune response.

Rationale: Viral infections are complex processes based on an intricate network of molecular interactions. The infectious agent hijacks components of the cellular machinery for its profit, circumventing the natural defense mechanisms triggered by the infected cell. The successful completion of the replicative viral cycle within a cell depends on the function of viral components versus the cellular defenses. Non-coding RNAs (ncRNAs) are important cellular modulators, either promoting or preventing the progression of viral infections. Among these ncRNAs, the long non-coding RNA (lncRNA) family is especially relevant due to their intrinsic functional properties and ubiquitous biological roles. Specific lncRNAs have been recently characterized as modulators of the cellular response during infection of human host cells by single stranded RNA viruses. However, the role of host lncRNAs in the infection by human RNA coronaviruses such as SARS-CoV-2 remains uncharacterized. Methods: In the present work, we have performed a transcriptomic study of a cohort of patients with different SARS-CoV-2 viral load and analyzed the involvement of lncRNAs in supporting regulatory networks based on their interaction with RNA-binding proteins (RBPs). Results: Our results revealed the existence of a SARS-CoV-2 infection-dependent pattern of transcriptional up-regulation in which specific lncRNAs are an integral component. To determine the role of these lncRNAs, we performed a functional correlation analysis complemented with the study of the validated interactions between lncRNAs and RBPs. This combination of in silico functional association studies and experimental evidence allowed us to identify a lncRNA signature composed of six elements - NRIR, BISPR, MIR155HG, FMR1-IT1, USP30-AS1, and U62317.2 - associated with the regulation of SARS-CoV-2 infection. Conclusions: We propose a competition mechanism between the viral RNA genome and the regulatory lncRNAs in the sequestering of specific RBPs that modulates the interferon response and the regulation of RNA surveillance by nonsense-mediated decay (NMD).

Ubiquitin ligase STUB1 destabilizes IFNγ-receptor complex to suppress tumor IFNγ signaling.

The cytokine IFNγ differentially impacts on tumors upon immune checkpoint blockade (ICB). Despite our understanding of downstream signaling events, less is known about regulation of its receptor (IFNγ-R1). With an unbiased genome-wide CRISPR/Cas9 screen for critical regulators of IFNγ-R1 cell surface abundance, we identify STUB1 as an E3 ubiquitin ligase for IFNγ-R1 in complex with its signal-relaying kinase JAK1. STUB1 mediates ubiquitination-dependent proteasomal degradation of IFNγ-R1/JAK1 complex through IFNγ-R1K285 and JAK1K249. Conversely, STUB1 inactivation amplifies IFNγ signaling, sensitizing tumor cells to cytotoxic T cells in vitro. This is corroborated by an anticorrelation between STUB1 expression and IFNγ response in ICB-treated patients. Consistent with the context-dependent effects of IFNγ in vivo, anti-PD-1 response is increased in heterogenous tumors comprising both wildtype and STUB1-deficient cells, but not full STUB1 knockout tumors. These results uncover STUB1 as a critical regulator of IFNγ-R1, and highlight the context-dependency of STUB1-regulated IFNγ signaling for ICB outcome.

TARGETED DOWN REGULATION OF CORE MITOCHONDRIAL GENES DURING SARS-COV-2 INFECTION.

Defects in mitochondrial oxidative phosphorylation (OXPHOS) have been reported in COVID-19 patients, but the timing and organs affected vary among reports. Here, we reveal the dynamics of COVID-19 through transcription profiles in nasopharyngeal and autopsy samples from patients and infected rodent models. While mitochondrial bioenergetics is repressed in the viral nasopharyngeal portal of entry, it is up regulated in autopsy lung tissues from deceased patients. In most disease stages and organs, discrete OXPHOS functions are blocked by the virus, and this is countered by the host broadly up regulating unblocked OXPHOS functions. No such rebound is seen in autopsy heart, results in severe repression of genes across all OXPHOS modules. Hence, targeted enhancement of mitochondrial gene expression may mitigate the pathogenesis of COVID-19.

With or without You: Co-Chaperones Mediate Health and Disease by Modifying Chaperone Function and Protein Triage.

Heat shock proteins (HSPs) are a family of molecular chaperones that regulate essential protein refolding and triage decisions to maintain protein homeostasis. Numerous co-chaperone proteins directly interact and modify the function of HSPs, and these interactions impact the outcome of protein triage, impacting everything from structural proteins to cell signaling mediators. The chaperone/co-chaperone machinery protects against various stressors to ensure cellular function in the face of stress. However, coding mutations, expression changes, and post-translational modifications of the chaperone/co-chaperone machinery can alter the cellular stress response. Importantly, these dysfunctions appear to contribute to numerous human diseases. Therapeutic targeting of chaperones is an attractive but challenging approach due to the vast functions of HSPs, likely contributing to the off-target effects of these therapies. Current efforts focus on targeting co-chaperones to develop precise treatments for numerous diseases caused by defects in protein quality control. This review focuses on the recent developments regarding selected HSP70/HSP90 co-chaperones, with a concentration on cardioprotection, neuroprotection, cancer, and autoimmune diseases. We also discuss therapeutic approaches that highlight both the utility and challenges of targeting co-chaperones.

Role of miR-2392 in driving SARS-CoV-2 infection.

MicroRNAs (miRNAs) are small non-coding RNAs involved in post-transcriptional gene regulation that have a major impact on many diseases and provide an exciting avenue toward antiviral therapeutics. From patient transcriptomic data, we determined that a circulating miRNA, miR-2392, is directly involved with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) machinery during host infection. Specifically, we show that miR-2392 is key in driving downstream suppression of mitochondrial gene expression, increasing inflammation, glycolysis, and hypoxia, as well as promoting many symptoms associated with coronavirus disease 2019 (COVID-19) infection. We demonstrate that miR-2392 is present in the blood and urine of patients positive for COVID-19 but is not present in patients negative for COVID-19. These findings indicate the potential for developing a minimally invasive COVID-19 detection method. Lastly, using in vitro human and in vivo hamster models, we design a miRNA-based antiviral therapeutic that targets miR-2392, significantly reduces SARS-CoV-2 viability in hamsters, and may potentially inhibit a COVID-19 disease state in humans.

CHIP protects against MPP+/MPTP-induced damage by regulating Drp1 in two models of Parkinson's disease.

Mitochondrial dysfunction has been implicated in the pathogenesis of Parkinson's disease (PD). Carboxyl terminus of Hsp70-interacting protein (CHIP) is a key regulator of mitochondrial dynamics, and mutations in CHIP or deficits in its expression have been associated with various neurological diseases. This study explores the protective role of CHIP in cells and murine PD models. In SH-SY5Y cell line, overexpression of CHIP improved the cell viability and increased the ATP levels upon treatment with 1-methyl-4-phenylpyridinium (MPP+). To achieve CHIP overexpression in animal models, we intravenously injected mice with AAV/BBB, a new serotype of adeno-associated virus that features an enhanced capacity to cross the blood-brain barrier. We also generated gene knock-in mice that overexpressed CHIP in neural tissue. Our results demonstrated that CHIP overexpression in mice suppressed 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced damage, including movement impairments, motor coordination, and spontaneous locomotor activity, as well as loss of dopaminergic neurons. In vitro and in vivo experiments showed that overexpression of CHIP inhibited the pathological increase in Drp1 observed in the PD models, suggesting that CHIP regulates Drp1 degradation to attenuate MPP+/MPTP-induced injury. We conclude that CHIP plays a protective role in MPP+/MPTP-induced PD models. Our experiments further revealed that CHIP maintains the integrity of mitochondria.

Circulating miRNA Spaceflight Signature Reveals Targets for Countermeasure Development.

We have identified and validated a spaceflight-associated microRNA (miRNA) signature that is shared by rodents and humans in response to simulated, short-duration and long-duration spaceflight. Previous studies have identified miRNAs that regulate rodent responses to spaceflight in low-Earth orbit, and we have confirmed the expression of these proposed spaceflight-associated miRNAs in rodents reacting to simulated spaceflight conditions. Moreover, astronaut samples from the NASA Twins Study confirmed these expression signatures in miRNA sequencing, single-cell RNA sequencing (scRNA-seq), and single-cell assay for transposase accessible chromatin (scATAC-seq) data. Additionally, a subset of these miRNAs (miR-125, miR-16, and let-7a) was found to regulate vascular damage caused by simulated deep space radiation. To demonstrate the physiological relevance of key spaceflight-associated miRNAs, we utilized antagomirs to inhibit their expression and successfully rescue simulated deep-space-radiation-mediated damage in human 3D vascular constructs.

Comprehensive Multi-omics Analysis Reveals Mitochondrial Stress as a Central Biological Hub for Spaceflight Impact.

Spaceflight is known to impose changes on human physiology with unknown molecular etiologies. To reveal these causes, we used a multi-omics, systems biology analytical approach using biomedical profiles from fifty-nine astronauts and data from NASA's GeneLab derived from hundreds of samples flown in space to determine transcriptomic, proteomic, metabolomic, and epigenetic responses to spaceflight. Overall pathway analyses on the multi-omics datasets showed significant enrichment for mitochondrial processes, as well as innate immunity, chronic inflammation, cell cycle, circadian rhythm, and olfactory functions. Importantly, NASA's Twin Study provided a platform to confirm several of our principal findings. Evidence of altered mitochondrial function and DNA damage was also found in the urine and blood metabolic data compiled from the astronaut cohort and NASA Twin Study data, indicating mitochondrial stress as a consistent phenotype of spaceflight.

CHIP phosphorylation by protein kinase G enhances protein quality control and attenuates cardiac ischemic injury.

Proteotoxicity from insufficient clearance of misfolded/damaged proteins underlies many diseases. Carboxyl terminus of Hsc70-interacting protein (CHIP) is an important regulator of proteostasis in many cells, having E3-ligase and chaperone functions and often directing damaged proteins towards proteasome recycling. While enhancing CHIP functionality has broad therapeutic potential, prior efforts have all relied on genetic upregulation. Here we report that CHIP-mediated protein turnover is markedly post-translationally enhanced by direct protein kinase G (PKG) phosphorylation at S20 (mouse, S19 human). This increases CHIP binding affinity to Hsc70, CHIP protein half-life, and consequent clearance of stress-induced ubiquitinated-insoluble proteins. PKG-mediated CHIP-pS20 or expressing CHIP-S20E (phosphomimetic) reduces ischemic proteo- and cytotoxicity, whereas a phospho-silenced CHIP-S20A amplifies both. In vivo, depressing PKG activity lowers CHIP-S20 phosphorylation and protein, exacerbating proteotoxicity and heart dysfunction after ischemic injury. CHIP-S20E knock-in mice better clear ubiquitinated proteins and are cardio-protected. PKG activation provides post-translational enhancement of protein quality control via CHIP.