Biology subjects

Schisler, J. C.

Publications and source records attributed to Schisler, J. C..

11 recordsLinked to original sources

bioRxiv2025

Domain-specific decoupling of co-chaperone and ligase functions in STUB1 underlies biochemical and clinical heterogeneity in SCA48

AO_SCPLOWBSTRACTC_SCPLOWThe carboxyl terminus of HSC70-interacting protein (CHIP, encoded by STUB1) integrates co-chaperone and E3 ubiquitin ligase activities to maintain proteostasis. Heterozygous STUB1 mutations cause the dominant cerebellar ataxia SCA48 through incompletely understood mechanisms. We characterized 13 SCA48-associated variants in the TPR and U-box domains using recombinant protein and cellular assays. TPR mutations retained intrinsic ligase activity but lost HSC70 binding, substrate ubiquitination efficiency, and protein stability. Conversely, U-box mutations abolished ligase function, induced aberrant high-molecular-weight oligomerization, and frequently elevated steady-state CHIP levels while only partially impairing co-chaperone activity. Many variants exhibited temperature-sensitive defects and stress-induced nuclear mislocalization. Principal component analysis revealed robust domain-specific biochemical clustering. RNA-seq following STUB1 knockdown demonstrated preserved HSF1-dependent transactivation but a loss of CHIP-dependent amplification of ubiquitination, chaperone, and transcriptional pathways under heat stress. Meta-analysis of 87 SCA48 patients linked TPR-like biochemical profiles to upper motor neuron involvement and U-box profiles to prominent dysarthria. Collectively, SCA48 mutations decouple CHIPs dual functions in a domain-dependent manner, exerting dominant-negative or gain-of-toxic effects that drive the observed clinical heterogeneity. These findings establish a direct biochemical-clinical correlation in SCA48 and provide a framework for domain-targeted therapeutic strategies exploiting residual ligase or chaperone activity.

neuroscience↗

bioRxiv2025

The MEK inhibitor trametinib incurs mitochondrial injury and induces innate immune responses in the mouse heart

Trametinib (Trm) is a highly selective MEK inhibitor that potently and persistently abrogates ERK1/2 activation. Trm initially was used to treat BRAF V600E-mutated melanoma but its FDA-approved indications are expanding rapidly. Trm generally is well tolerated but it can cause dose-limiting cardiomyopathy and heart failure. Here we characterize a mouse model of Trm cardiotoxicity using complementary in vitro approaches to show that Trm induces mitochondrial dysfunction in cardiomyocytes and some cancer cell types. In vivo, Trm caused contractile dysfunction within 3 days and heart failure within 2 weeks. High resolution respirometry using isolated cardiac mitochondria revealed that Trm compromises oxidative metabolism, in part through blunted activity of Electron Transport System Complexes. Trm-mediated mitochondrial injury led to the release of mitochondrial Damage-Associated Molecular Patterns including mitochondrial DNA in both mice and humans, triggering activation of canonical innate immune pathways including cGAS-STING. In multiple rodent and human cardiomyocyte platforms, Trm diminished mitochondrial respiratory capacity at nanomolar concentrations but this lesion was reversed by expression of a phosphomimetic STAT3-S727 construct. We also found that Trm induced mitochondrial dysfunction in some but not all cancer cell lines, identifying a previously unrecognized effect that could contribute to Trms anti-cancer efficacy.

physiology↗

bioRxiv2025

Phosphorylation-State Modulated Binding of HSP70: Structural Insights and Compensatory Protein Engineering

Protein quality control is crucial for cellular homeostasis, involving the heat shock response, the ubiquitin-proteasome system, and the autophagy-lysosome pathway. Central to these systems are the chaperone homologs heat shock protein 70 (HSP70) and heat shock cognate 70 (HSC70), which manage protein folding and degradation. This study investigated the impact of the C-terminal phosphorylation of HSP70 on its interaction with the co-chaperone CHIP (C-terminus of HSC70 interacting protein), an E3 ligase that ubiquitinates protein substrates for degradation. Using both cell-free and cell-based approaches, including X-ray crystallography, biolayer interferometry, and live cell biocomplementation assays, we demonstrate that phosphorylation at HSP70 T636 reduces CHIPs binding affinity, shifting the preference toward other co-chaperones like HOP. Structural analysis reveals that phosphorylation disrupts key hydrogen bonds, altering binding dynamics. We engineered a CHIP variant (CHIP-G132N) to restore binding affinity to phosphorylated HSP70. While CHIP-G132N effectively restored binding without additional functional domains, its effectiveness was diminished in full-length phosphomimetic constructs in cell-free and in-cell assays, suggesting that additional interactions may influence binding. Functional assays indicate that phosphorylation of HSP70 affects its stability and degradation, with implications for diseases such as cancer and neurodegeneration. Our findings highlight the complexity of chaperone-co-chaperone interactions and underscore the importance of post-translational modifications in regulating protein quality control mechanisms. By elucidating the molecular details of HSP70 and CHIP interactions, our study provides a foundation for developing therapeutic interventions for diseases characterized by proteostasis imbalance.

biochemistry↗

bioRxiv2023

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. ImportanceAntibiotic 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.

microbiology↗

bioRxiv2022

Cardiomyocyte alpha-1A adrenergic receptors mitigate post-infarct remodeling and mortality by constraining necroptosis

Activation of alpha-1-adrenergic receptors (1-ARs), particularly the 1A subtype, protects the murine heart against injury, whereas human studies show that 1-AR antagonists (-blockers) may increase the risk of heart failure. We created a cardiomyocyte-specific 1A-AR knockout mouse (cmAKO) to define the mechanisms underlying these effects and to elucidate whether they arise from cardiomyocyte 1A-ARs or systemic factors. Myocardial infarction (MI) resulted in 70% 7-day mortality in cmAKO compared to 10% in wild type (WT) mice. cmAKO mice exhibited exaggerated ventricular remodeling and increased cell death compared to WT mice 3 days post-MI, coupled to upregulation of canonical mediators of necroptosis: receptor-interacting protein (RIP) kinases RIP1 and RIP3 and mixed lineage kinase domain-like protein. An 1A-AR agonist mitigated ischemia-induced cardiomyocyte death and necroptotic signaling in vitro. A RIP1 antagonist abrogated the protective effects of 1A activation in vivo and in vitro. We found that patients at our center who were taking -blockers at the time of MI experienced a higher risk of mortality (hazard ratio 1.53, p=0.029) during 5-year follow-up, providing clinical correlation for our experimental data. Collectively our findings indicate that cardiomyocyte 1A-ARs constrain ischemia-induced necroptosis and suggest caution in the use of -blockers in patients at risk for MI.

pathology↗

bioRxiv2022

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.

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. In the present work, we have performed a transcriptomic study of a cohort of patients with different SARS-CoV-2 viral load. 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 RNA-binding proteins (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. 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). O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/485903v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@4368c9org.highwire.dtl.DTLVardef@1948201org.highwire.dtl.DTLVardef@e31fd9org.highwire.dtl.DTLVardef@1400805_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical abstractModel of interactions among lncRNA and cognate RNA-binding proteins in SARS-CoV-2 infection. According to our model, the viral genome can establish direct interactions with three core proteins (DDX3X, UPF1 and IGF2BP2) involved in mRNA metabolism and regulation of the interferon response, which are also components of a SARS-CoV-2 lncRNA-centered regulatory network. The competition between viral RNA and lncRNAs could act as a counteracting factor for the normal function of homeostatic lncRNA-centered regulatory networks, contributing to viral progression and replication. Black arrows depict physical interactions between network components; red arrows represent functional relationships.

bioinformatics↗

bioRxiv2022

Mouse Genomic Associations with Ex Vivo 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 ex vivo DNA damage quantified based on immunofluorescent 53BP1+ 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 hour) 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+ foci as a potential biomarker for DNA integrity in mouse models. Based on our results, we 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 ex vivo studies to refine the association of genes and pathways with the DNA damage response to ionizing radiation and identify targets for space travel countermeasures.

genomics↗

bioRxiv2022

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. One-Sentence SummaryCovid-19 is associated with targeted inhibition of mitochondrial gene transcription.

molecular biology↗

bioRxiv2019

Modeling clinical phenotypes with protein function to explain the disease spectrum in patients with CHIP mutations

Monogenetic disorders that cause cerebellar ataxia are characterized by defects in gait and atrophy of the cerebellum; however, patients often suffer from a spectrum of disease, complicating treatment options. Spinocerebellar ataxia autosomal recessive 16 (SCAR16) is caused by coding mutations in STUB1, a gene that encodes the multi-functional enzyme CHIP (C-terminus of HSC70-interacting protein). The spectrum of disease found in SCAR16 patients includes a wide range in the age of disease onset, cognitive dysfunction, increased tendon reflex, and hypogonadism. Although SCAR16 mutations span the multiple functional domains of CHIP, it is unclear if the location of the mutation contributes to the clinical spectrum of SCAR16 or with changes in the biochemical properties of CHIP. In this study, we examined the associations and relationships between the clinical phenotypes of SCAR16 patients and how they relate to changes in the biophysical, biochemical, and functional properties of the corresponding mutated protein. We found that the severity of ataxia did not correlate with age of onset; however, cognitive dysfunction, increased tendon reflex, and ancestry were able to predict 54% of the variation in ataxia severity. We further identified domain-specific relationships between biochemical changes in CHIP and clinical phenotypes, and specific biochemical activities that associate selectively to either increased tendon reflex or cognitive dysfunction, suggesting that specific changes to CHIP-HSC70 dynamics contributes to the clinical spectrum of SCAR16. Finally, linear models of SCAR16 as a function of the biochemical properties of CHIP support the concept that further inhibiting mutant CHIP activity lessens disease severity and may be useful in the design of patient-speciflc targeted approaches to treat SCAR16.

neuroscience↗

bioRxiv2018

TAS2R38 predisposition to bitter taste associated with differential changes in vegetable intake in response to a community-based dietary intervention

BackgroundAlthough vegetable consumption is associated with decreased risk for a variety of chronic diseases, few Americans meet the CDC recommendations for vegetable intake. The TAS2R38 gene encodes a taste receptor that confers bitter taste sensing from chemicals found in some vegetables. Common polymorphisms in TAS2R38, including rs713598, rs1726866, and rs10246939, lead to coding substitutions that alter receptor function and result in the loss of bitter taste perception.\n\nObjectiveOur study examines whether bitter taste perception TAS2R38 diplotypes were associated with vegetable consumption in participants enrolled in either an enhanced or a minimal nutrition counseling intervention within a community-based dietary intervention.\n\nMethodsDNA was isolated from the peripheral blood cells of study participants (N = 497) and analyzed for polymorphisms using genotyping arrays. The Block Fruit and Vegetable screener was used to determine frequency of vegetable consumption. Mixed effects models were used to test differences in frequency of vegetable consumption between intervention and genotype groups over time.\n\nResultsThere was no association between baseline vegetable consumption frequency and the bitter taste diplotype (p = 0.937), however after six months of the intervention, we observed an interaction between bitter taste diplotypes and time (p = 0.046). Participants in the enhanced intervention increased their vegetable consumption frequency (p = 0.020) and within this intervention group, the non-bitter and intermediate-bitter tasting participants had the largest increase in vegetable consumption. In contrast, in the minimal intervention group, the bitter tasting participants reported a decrease in vegetable consumption.\n\nConclusionsNon- and intermediate-bitter taste blind participants increased vegetable consumption in either intervention group more than those who perceive bitterness. Future applications of precision medicine could consider genetic variation in bitter taste perception genes when designing dietary interventions.\n\nAuthor summaryMost Americans under consume vegetables, despite clear associations between vegetable consumption and health benefits. Vegetables, such as broccoli, kale, and Brussels sprouts, contain bitter-tasting compounds, leading to taste aversion. Common polymorphisms on the TAS2R38 taste receptor gene (rs713598, rs1726866, and rs10246939) influence the perception of bitter taste. We tested whether genetic predisposition to bitter taste influenced vegetable intake in a dietary intervention and found that TAS2R38 diplotypes were related to vegetable consumption. Combining precision medicine approaches that identify taste profiles and personalizing dietary advice could help engage intervention participants and improve the impact of dietary interventions.

genomics↗

bioRxiv2017

Loss of CHIP Expression Perturbs Glucose Homeostasis and Leads to Type II Diabetes through Defects in Microtubule Polymerization and Glucose Transporter Localization

Recent evidence has implicated CHIP (carboxyl terminus of Hsc/Hsp70-interacting protein), a co-chaperone and ubiquitin ligase, in the functional support of several metabolism-related proteins, including AMPK and SirT6. In addition to previously reported aging and stress intolerance phenotypes, we find that CHIP -/- mice also demonstrate a Type II diabetes-like phenotype, including poor glucose tolerance, decreased sensitivity to insulin, and decreased insulin-stimulated glucose uptake in isolated skeletal muscle, characteristic of insulin resistance. In CHIP-deficient cells, glucose stimulation fails to induce translocation of Glut4 to the plasma membrane. This impairment in Glut4 translocation in CHIP-deficient cells is accompanied by decreased tubulin polymerization associated with decreased phosphorylation of stathmin, a microtubule-associated protein required for polymerization-dependent protein trafficking within the cell. Together, these data describe a novel role for CHIP in regulating microtubule polymerization that assists in glucose transporter translocation, promoting whole-body glucose homeostasis and sensitivity to insulin.

physiology↗