Loss of TRIM29 mitigates viral myocarditis by attenuating PERK-driven ER stress response in male mice.

Viral myocarditis, an inflammatory disease of the myocardium, is a significant cause of sudden death in children and young adults. The current coronavirus disease 19 pandemic emphasizes the need to understand the pathogenesis mechanisms and potential treatment strategies for viral myocarditis. Here, we found that TRIM29 was highly induced by cardiotropic viruses and promoted protein kinase RNA-like endoplasmic reticulum kinase (PERK)-mediated endoplasmic reticulum (ER) stress, apoptosis, and reactive oxygen species (ROS) responses that promote viral replication in cardiomyocytes in vitro. TRIM29 deficiency protected mice from viral myocarditis by promoting cardiac antiviral functions and reducing PERK-mediated inflammation and immunosuppressive monocytic myeloid-derived suppressor cells (mMDSC) in vivo. Mechanistically, TRIM29 interacted with PERK to promote SUMOylation of PERK to maintain its stability, thereby promoting PERK-mediated signaling pathways. Finally, we demonstrated that the PERK inhibitor GSK2656157 mitigated viral myocarditis by disrupting the TRIM29-PERK connection, thereby bolstering cardiac function, enhancing cardiac antiviral responses, and curbing inflammation and immunosuppressive mMDSC in vivo. Our findings offer insight into how cardiotropic viruses exploit TRIM29-regulated PERK signaling pathways to instigate viral myocarditis, suggesting that targeting the TRIM29-PERK axis could mitigate disease severity.

Wall-mounted folding chairs to promote resident physician sitting at the hospital bedside.

BACKGROUND: Sitting at the bedside may improve patient-clinician communication; however, many clinicians do not regularly sit during inpatient encounters. OBJECTIVE: To determine the impact of adding wall-mounted folding chairs inside patient rooms, beyond any impact from a resident education campaign, on the patient-reported frequency of sitting at the bedside by internal medicine resident physicians. DESIGN, SETTING, AND PARTICIPANTS: Prospective, controlled pre-post trial between 2019 and 2022 (data collection paused 2020-2021 due to COVID-19) at an academic hospital in Baltimore, Maryland. Folding chairs were installed in two of four internal medicine units and educational activities were delivered equally across all units. MAIN OUTCOME AND MEASURES: Patient-reported frequency of sitting at bedside, assessed as means on Likert-type items with 1 being "never" and 5 being "every single time." We also examined the frequency of other patient-reported communication behaviors. RESULTS: Two hundred fifty six and 206 patients enrolled in the pre and post-intervention periods, respectively. The mean frequency of patient-reported sitting by resident physicians increased from 1.8 (SD 1.2) to 2.3 (1.2) on education-only units (absolute difference 0.48 [95% CI: 0.21-0.75]) and from 2.0 (1.3) to 3.2 (1.4) on units receiving chairs (1.16, [0.87-1.45]). Comparing differences between groups using ordered logistic regression adjusting for clustering within residents, units with added chairs had greater increases in sitting (odds ratio 2.05 [1.10-3.82]), spending enough time at the bedside (2.43 [1.32-4.49]), and checking for understanding (3.04 [1.44-6.39]). Improvements in sitting and other behaviors were sustained on both types of units. CONCLUSIONS: Adding wall-mounted folding chairs may help promote effective patient-clinician communication.

Chagas Disease Diagnostic Practices at Four Major Hospital Systems in California and Texas.

BACKGROUND: Chagas disease (CD) is a parasitic disease that affects ∼300 000 people living in the United States. CD leads to cardiac and/or gastrointestinal disease in up to 30% of untreated people. However, end-organ damage can be prevented with early diagnosis and antiparasitic therapy. METHODS: We reviewed electronic health records of patients who underwent testing for CD at four hospital systems in California and Texas between 2016 and 2020. Descriptive analyses were performed as a needs assessment for improving CD diagnosis. RESULTS: In total, 470 patients were tested for CD. Cardiac indications made up more than half (60%) of all testing, and the most frequently cited cardiac condition was heart failure. Fewer than 1% of tests were ordered by obstetric and gynecologic services. Fewer than half (47%) of patients had confirmatory testing performed at the Centers for Disease Control and Prevention. DISCUSSION: Four major hospitals systems in California and Texas demonstrated low overall rates of CD diagnostic testing, testing primarily among older patients with end-organ damage, and incomplete confirmatory testing. This suggests missed opportunities to diagnose CD in at-risk individuals early in the course of infection when antiparasitic treatment can reduce the risk of disease progression and prevent vertical transmission.

A real-world observational study assessing relationships between excessive daytime sleepiness and patient satisfaction in obstructive sleep apnea.

OBJECTIVES/BACKGROUND: To estimate prevalence and severity of excessive daytime sleepiness among patients with obstructive sleep apnea (OSA) who were prescribed treatment; assess perception and satisfaction of OSA-related care; describe relationships between excessive daytime sleepiness, treatment adherence, and patient satisfaction. PATIENTS/METHODS: A national population-based cross-sectional sample of US adults with clinician-diagnosed OSA was surveyed in January 2021 via Evidation Health's Achievement App. Patients completed the Epworth Sleepiness Scale, rated satisfaction with healthcare provider and overall OSA care, and reported treatment adherence. Covariates affecting excessive daytime sleepiness (average weekly sleep duration, treatment adherence, sleepiness-inducing medications, age, sex, body mass index, nasal congestion, smoking status, and comorbidities) were adjusted in multivariate regression models. RESULTS: In 2289 participants (50.3 % women; 44.8 ± 11.1 years), EDS was highly prevalent (42 %), and was experienced by 36 % of patients with high positive airway pressure (PAP) therapy adherence. Each additional hour of nightly PAP use was associated with improved sleepiness (a 0.28-point lower Epworth score; p < 0.001). Excessive daytime sleepiness was associated with lower patient satisfaction with healthcare providers and overall care (OR [95 % CI] 0.62 [0.48-0.80] and 0.50 [0.39-0.64], respectively; p < 0.0001), whereas PAP adherence was associated with higher patient satisfaction (OR [95 % CI] 2.37 [1.64-3.43] and 2.91 [2.03-4.17]; p < 0.0001), after adjusting for confounders. CONCLUSIONS: In a real-world population-based study of patients with OSA, excessive daytime sleepiness was highly prevalent and associated with poor patient satisfaction ratings. Better patient-centered care among patients with OSA may require interventions aimed at addressing excessive daytime sleepiness and treatment adherence.

Long-term effects of vaginal surgery and endogenous ovarian hormones on the vagina and bladder.

Background: Surgery is a common treatment for pelvic organ prolapse (POP); however, risk of recurrence and reoperation is high, resulting in a negative impact on quality of life and sexual function. Aim: To examine the long-term effects of POP surgery and endogenous circulating ovarian hormones on the vagina and bladder. Methods: Our animal model simulated surgical injury of the vagina and bladder during POP surgery. Female Rowett nude rats were divided into 4 groups: intact control (IC), vaginal surgery only (V), ovariectomy only (O), and ovariectomy + vaginal surgery (OV). Rats were euthanized 10 weeks postsurgery. Proximal vagina and bladder dome/trigone underwent (1) organ bath myography to assess smooth muscle contractility; (2) real-time quantitative polymerase chain reaction to quantify mRNA expression of elastin, collagen I and III, and PGP9.5 (protein gene product 9.5); (3) enzyme-linked immunosorbent assay for protein quantification of elastin and collagen I and III; and (4) hematoxylin-eosin/immunohistochemistry staining. Outcomes: The primary outcome was tissue contractility as measured by organ bath myography. Secondary outcomes included gene and protein expression of collagen I and III and elastin. Results: O and OV showed reduced vaginal wall contractility vs IC and V (P < .002). Bladder dome and trigone displayed different contractile patterns, with significant differences between O and OV (P < .05), suggesting a negative effect from surgery rather than ovariectomy. OV demonstrated consistent reductions in contractility and elastin/collagen protein expression for the vagina and bladder vs IC. V had similar contractility and increased collagen I expression vs IC, suggesting a protective effect of ovarian hormones. Vaginal epithelium thinning was confirmed in the ovariectomized groups (P = .001), although there was no statistical significance in muscularis thinning with surgery or ovariectomy. O, V, and OV showed significant downregulation of PGP9.5 mRNA expression vs IC. Clinical Translation: These data allow researchers to gain insights into the long-term effects of surgery and deprivation of ovarian hormones. Future studies can use this animal model to investigate other mechanisms that may affect long-term tissue changes due to surgical intervention. Strengths and Limitations: Major strengths are long-term data on the effects of POP surgery and development of an animal model for future studies. However, the animal model limits our ability to extrapolate to humans, where tissue healing is modulated by many factors. Conclusion: Our animal model provides evidence that ovarian hormone deprivation and POP surgery result in negative long-term effects on tissue function and extracellular matrix.

Improving imputation quality in Samoans through the integration of population-specific sequences into existing reference panels.

Genotype imputation is fundamental to association studies, and yet even gold standard panels like TOPMed are limited in the populations for which they yield good imputation. Specifically, Pacific Islanders are poorly represented in extant panels. To address this, we constructed an imputation reference panel using 1,285 Samoan individuals with whole-genome sequencing, combined with 1000 Genomes (1000G) samples, to create a reference panel that better represents Pacific Islander, specifically Samoan, genetic variation. We compared this panel to 1000G and TOPMed panels based on imputed variants using genotyping array data for 1,834 Samoan participants who were not part of the panels. The 1000G + 1285 Samoan panel yielded up to 2.25-2.76 times more well-imputed (r 2 ≥ 0.80) variants than TOPMed and 1000G. There was improved imputation accuracy across the minor allele frequency (MAF) spectrum, although it was more pronounced for variants with 0.01 ≤ MAF ≤ 0.05. Imputation accuracy (r 2 ) was greater for population-specific variants (high fixation index, F ST ) and those from larger haplotypes (high LD score). The gain in imputation accuracy over TOPMed was largest for small haplotypes (low LD score), reflecting the Samoan panel's ability to capture population-specific variation not well tagged by other panels. We also augmented the 1000G reference panel with varying numbers of Samoan samples and found that panels with 48 or more Samoans included outperformed TOPMed for all variants with MAF ≥ 0.001. This study identifies variants with improved imputation using population-specific reference panels and provides a framework for constructing other population-specific reference panels.

Designing the pressure-dependent shear modulus using tessellated granular metamaterials.

Jammed packings of granular materials display complex mechanical response. For example, the ensemble-averaged shear modulus 〈G〉 increases as a power law in pressure p for static packings of soft spherical particles that can rearrange during compression. We seek to design granular materials with shear moduli that can either increase or decrease with pressure without particle rearrangements even in the large-system limit. To do this, we construct tessellated granular metamaterials by joining multiple particle-filled cells together. We focus on cells that contain a small number of bidisperse disks in two dimensions. We first study the mechanical properties of individual disk-filled cells with three types of boundaries: periodic boundary conditions (PBC), fixed-length walls (FXW), and flexible walls (FLW). Hypostatic jammed packings are found for cells with FLW, but not in cells with PBC and FXW, and they are stabilized by quartic modes of the dynamical matrix. The shear modulus of a single cell depends linearly on p. We find that the slope of the shear modulus with pressure λ_{c}<0 for all packings in single cells with PBC where the number of particles per cell N≥6. In contrast, single cells with FXW and FLW can possess λ_{c}>0, as well as λ_{c}<0, for N≤16. We show that we can force the mechanical properties of multicell granular metamaterials to possess those of single cells by constraining the end points of the outer walls and enforcing an affine shear response. These studies demonstrate that tessellated granular metamaterials provide a platform for the design of soft materials with specified mechanical properties.

Local and global measures of the shear moduli of jammed disk packings.

Strain-controlled isotropic compression gives rise to jammed packings of repulsive, frictionless disks with either positive or negative global shear moduli. We carry out computational studies to understand the contributions of the negative shear moduli to the mechanical response of jammed disk packings. We first decompose the ensemble-averaged, global shear modulus as 〈G〉=(1-F_{-})〈G_{+}〉+F_{-}〈G_{-}〉, where F_{-} is the fraction of jammed packings with negative shear moduli and 〈G_{+}〉 and 〈G_{-}〉 are the average values from packings with positive and negative moduli, respectively. We show that 〈G_{+}〉 and 〈|G_{-}|〉 obey different power-law scaling relations above and below pN^{2}∼1. For pN^{2}>1, both 〈G_{+}〉N and 〈|G_{-}|〉N∼(pN^{2})^{ß}, where ß∼0.5 for repulsive linear spring interactions. Despite this, 〈G〉N∼(pN^{2})^{ß^{'}} with ß^{'}≳0.5 due to the contributions from packings with negative shear moduli. We show further that the probability distribution of global shear moduli P(G) collapses at fixed pN^{2} and different values of p and N. We calculate analytically that P(G) is a Γ distribution in the pN^{2}≪1 limit. As pN^{2} increases, the skewness of P(G) decreases and P(G) becomes a skew-normal distribution with negative skewness in the pN^{2}≫1 limit. We also partition jammed disk packings into subsystems using Delaunay triangulation of the disk centers to calculate local shear moduli. We show that the local shear moduli defined from groups of adjacent triangles can be negative even when G>0. The spatial correlation function of local shear moduli C(r[over ⃗]) displays weak correlations for pn_{sub}^{2}<10^{-2}, where n_{sub} is the number of particles within each subsystem. However, C(r[over ⃗]) begins to develop long-ranged spatial correlations with fourfold angular symmetry for pn_{sub}^{2}≳10^{-2}.

A Polynesian-specific missense CETP variant alters the lipid profile.

Identifying population-specific genetic variants associated with disease and disease-predisposing traits is important to provide insights into the genetic determinants of health and disease between populations, as well as furthering genomic justice. Various common pan-population polymorphisms at CETP associate with serum lipid profiles and cardiovascular disease. Here, sequencing of CETP identified a missense variant rs1597000001 (p.Pro177Leu) specific to Maori and Pacific people that associates with higher HDL-C and lower LDL-C levels. Each copy of the minor allele associated with higher HDL-C by 0.236 mmol/L and lower LDL-C by 0.133 mmol/L. The rs1597000001 effect on HDL-C is comparable with CETP Mendelian loss-of-function mutations that result in CETP deficiency, consistent with our data, which shows that rs1597000001 lowers CETP activity by 27.9%. This study highlights the potential of population-specific genetic analyses for improving equity in genomics and health outcomes for population groups underrepresented in genomic studies.

A stop-gain variant in BTNL9 is associated with atherogenic lipid profiles.

Current understanding of lipid genetics has come mainly from studies in European-ancestry populations; limited effort has focused on Polynesian populations, whose unique population history and high prevalence of dyslipidemia may provide insight into the biological foundations of variation in lipid levels. Here, we performed an association study to fine map a suggestive association on 5q35 with high-density lipoprotein cholesterol (HDL-C) seen in Micronesian and Polynesian populations. Fine-mapping analyses in a cohort of 2,851 Samoan adults highlighted an association between a stop-gain variant (rs200884524; c.652C>T, p.R218∗; posterior probability = 0.9987) in BTNL9 and both lower HDL-C and greater triglycerides (TGs). Meta-analysis across this and several other cohorts of Polynesian ancestry from Samoa, American Samoa, and Aotearoa New Zealand confirmed the presence of this association (ßHDL-C = -1.60 mg/dL, p HDL-C = 7.63 × 10-10; ßTG = 12.00 mg/dL, p TG = 3.82 × 10-7). While this variant appears to be Polynesian specific, there is also evidence of association from other multiancestry analyses in this region. This work provides evidence of a previously unexplored contributor to the genetic architecture of lipid levels and underscores the importance of genetic analyses in understudied populations.

Multivariate analysis of a missense variant in CREBRF reveals associations with measures of adiposity in people of Polynesian ancestries.

The minor allele of rs373863828, a missense variant in CREB3 Regulatory Factor, is associated with several cardiometabolic phenotypes in Polynesian peoples. To better understand the variant, we tested the association of rs373863828 with a panel of correlated phenotypes (body mass index [BMI], weight, height, HDL cholesterol, triglycerides, and total cholesterol) using multivariate Bayesian association and network analyses in a Samoa cohort (n = 1632), Aotearoa New Zealand cohort (n = 1419), and combined cohort (n = 2976). An expanded set of phenotypes (adding estimated fat and fat-free mass, abdominal circumference, hip circumference, and abdominal-hip ratio) was tested in the Samoa cohort (n = 1496). In the Samoa cohort, we observed significant associations (log10 Bayes Factor [BF] ≥ 5.0) between rs373863828 and the overall phenotype panel (8.81), weight (8.30), and BMI (6.42). In the Aotearoa New Zealand cohort, we observed suggestive associations (1.5 < log10 BF < 5) between rs373863828 and the overall phenotype panel (4.60), weight (3.27), and BMI (1.80). In the combined cohort, we observed concordant signals with larger log10 BFs. In the Samoa-specific expanded phenotype analyses, we also observed significant associations between rs373863828 and fat mass (5.65), abdominal circumference (5.34), and hip circumference (5.09). Bayesian networks provided evidence for a direct association of rs373863828 with weight and indirect associations with height and BMI.

Early-stage structure-based drug discovery for small GTPases by NMR spectroscopy.

Small GTPase or Ras superfamily, including Ras, Rho, Rab, Ran and Arf, are fundamental in regulating a wide range of cellular processes such as growth, differentiation, migration and apoptosis. They share structural and functional similarities for binding guanine nucleotides and hydrolyzing GTP. Dysregulations of Ras proteins are involved in the pathophysiology of multiple human diseases, however there is still a stringent need for effective treatments targeting these proteins. For decades, small GTPases were recognized as 'undruggable' targets due to their complex regulatory mechanisms and lack of deep pockets for ligand binding. NMR has been critical in deciphering the structural and dynamic properties of the switch regions that are underpinning molecular switch functions of small GTPases, which pave the way for developing new effective inhibitors. The recent progress of drug or lead molecule development made for small GTPases profoundly delineated how modern NMR techniques reshape the field of drug discovery. In this review, we will summarize the progress of structural and dynamic studies of small GTPases, the NMR techniques developed for structure-based drug screening and their applications in early-stage drug discovery for small GTPases.

Surgical outcomes between temporal, extratemporal epilepsies and hypothalamic hamartoma: systematic review and meta-analysis of MRI-guided laser interstitial thermal therapy for drug-resistant epilepsy.

BACKGROUND: Approximately 1/3 of patients with epilepsy have drug-resistant epilepsy (DRE) and require surgical interventions. This meta-analysis aimed to review the effectiveness of MRI-guided laser interstitial thermal therapy (MRgLITT) in DRE. METHODS: The Population, Intervention, Comparator and Outcome approach and Preferred Reporting Items for Systematic Reviews and Meta-Analyses were followed. PubMed, MEDLINE and EMBASE databases were systematically searched for English language publications from 2012 to Nov 2020. Data on the prevalence outcome using the Engel Epilepsy Surgery Outcome Scale (Class I-IV), and postoperative complications were analysed with 95% CIs. RESULTS: Twenty-eight studies that included a total of 559 patients with DRE were identified. The overall prevalence of Engel class I outcome was 56% (95% CI 0.52% to 0.60%). Hypothalamic hamartomas (HH) patients had the highest seizure freedom rate of 67% (95% CI 0.57% to 0.76%) and outcome was overall comparable between mesial temporal lobe epilepsy (mTLE) (56%, 95% CI 0.50% to 0.61%) and extratemporal epilepsy (50% 95% CI 0.40% to 0.59%). The mTLE cases with mesial temporal sclerosis had better outcome vs non-lesional cases of mTLE. The prevalence of postoperative adverse events was 19% (95% CI 0.14% to 0.25%) and the most common adverse event was visual field deficits. The reoperation rate was 9% (95% CI 0.05% to 0.14%), which included repeat ablation and open resection. CONCLUSION: MRgLITT is an effective and safe intervention for DRE with different disease aetiologies. The seizure freedom outcome is overall comparable in between extratemporal and temporal lobe epilepsy; and highest with HH. TRAIL REGISTRATION NUMBER: The study protocol was registered with the National Institute for Health Research (CRD42019126365), which serves as a prospective register of systematic reviews. It is an international database of prospectively registered systematic reviews with a focus on health-related outcomes. Details about the protocol can be found at https://wwwcrdyorkacuk/PROSPERO/.

Balanced, Orientation-Dependent Dichoptic Masking in Cortex of Visually Normal Humans Measured Using Electroencephalography (EEG).

In the human visual system, cerebral cortex combines left- and right-eye retinal inputs, enabling single, comfortable binocular vision. In visual cortex, the signals from each eye inhibit one another (interocular suppression). While this mechanism may be disrupted by e.g. traumatic brain injury, clinical assessments of interocular suppression are subjective, qualitative, and lack reliability. EEG is a potentially useful clinical tool for objective, quantitative assessment of binocular vision. In a cohort of normal participants, we measured occipital, visual evoked potentials (VEPs) in response to dichoptically-presented vertical and/or horizontal sine-wave gratings. Response amplitudes to orthogonal gratings were greater than that of parallel gratings, which were in turn greater than that of monocular gratings. Our results indicate that interocular suppression is (normally) balanced, orientation-tuned, and that suppression per se is reduced for orthogonal gratings. This objective measure of suppression may have application in clinical settings.

Towards the Classification of Error-Related Potentials using Riemannian Geometry.

The error-related potential (ErrP) is an event-related potential (ERP) evoked by an experimental participant's recognition of an error during task performance. ErrPs, originally described by cognitive psychologists, have been adopted for use in brain-computer interfaces (BCIs) for the detection and correction of errors, and the online refinement of decoding algorithms. Riemannian geometry-based feature extraction and classification is a new approach to BCI which shows good performance in a range of experimental paradigms, but has yet to be applied to the classification of ErrPs. Here, we describe an experiment that elicited ErrPs in seven normal participants performing a visual discrimination task. Audio feedback was provided on each trial. We used multi-channel electroencephalogram (EEG) recordings to classify ErrPs (success/failure), comparing a Riemannian geometry-based method to a traditional approach that computes time-point features. Overall, the Riemannian approach outperformed the traditional approach (78.2% versus 75.9% accuracy, p <0.05); this difference was statistically significant (p <0.05) in three of seven participants. These results indicate that the Riemannian approach better captured the features from feedback-elicited ErrPs, and may have application in BCI for error detection and correction.

Mechanical response of packings of nonspherical particles: A case study of two-dimensional packings of circulo-lines.

We investigate the mechanical response of jammed packings of circulo-lines in two spatial dimensions, interacting via purely repulsive, linear spring forces, as a function of pressure P during athermal, quasistatic isotropic compression. The surface of a circulo-line is defined as the collection of points that is equidistant to a line; circulo-lines are composed of a rectangular central shaft with two semicircular end caps. Prior work has shown that the ensemble-averaged shear modulus for jammed disk packings scales as a power law, 〈G(P)〉∼P^{ß}, with ß∼0.5, over a wide range of pressure. For packings of circulo-lines, we also find robust power-law scaling of 〈G(P)〉 over the same range of pressure for aspect ratios R≳1.2. However, the power-law scaling exponent ß∼0.8-0.9 is much larger than that for jammed disk packings. To understand the origin of this behavior, we decompose 〈G〉 into separate contributions from geometrical families, G_{f}, and from changes in the interparticle contact network, G_{r}, such that 〈G〉=〈G_{f}〉+〈G_{r}〉. We show that the shear modulus for low-pressure geometrical families for jammed packings of circulo-lines can both increase and decrease with pressure, whereas the shear modulus for low-pressure geometrical families for jammed disk packings only decreases with pressure. For this reason, the geometrical family contribution 〈G_{f}〉 is much larger for jammed packings of circulo-lines than for jammed disk packings at finite pressure, causing the increase in the power-law scaling exponent for 〈G(P)〉.

Auranofin Resistance in Toxoplasma gondii Decreases the Accumulation of Reactive Oxygen Species but Does Not Target Parasite Thioredoxin Reductase.

Auranofin, a reprofiled FDA-approved drug originally designed to treat rheumatoid arthritis, has emerged as a promising anti-parasitic drug. It induces the accumulation of reactive oxygen species (ROS) in parasites, including Toxoplasma gondii. We generated auranofin resistant T. gondii lines through chemical mutagenesis to identify the molecular target of this drug. Resistant clones were confirmed with a competition assay using wild-type T. gondii expressing yellow fluorescence protein (YFP) as a reference strain. The predicted auranofin target, thioredoxin reductase, was not mutated in any of our resistant lines. Subsequent whole genomic sequencing analysis (WGS) did not reveal a consensus resistance locus, although many have point mutations in genes encoding redox-relevant proteins such as superoxide dismutase (TgSOD2) and ribonucleotide reductase. We investigated the SOD2 L201P mutation and found that it was not sufficient to confer resistance when introduced into wild-type parasites. Resistant clones accumulated less ROS than their wild type counterparts. Our results demonstrate that resistance to auranofin in T. gondii enhances its ability to abate oxidative stress through diverse mechanisms. This evidence supports a hypothesized mechanism of auranofin anti-parasitic activity as disruption of redox homeostasis.

Monoubiquitination of KRAS at Lysine104 and Lysine147 Modulates Its Dynamics and Interaction with Partner Proteins.

KRAS, a 21 kDa guanine nucleotide-binding protein that functions as a molecular switch, plays a key role in regulating cellular growth. Dysregulation of this key signaling node leads to uncontrolled cell growth, a hallmark of cancer cells. KRAS undergoes post-translational modification by monoubiquitination at various locations, including at lysine104 (K104) and lysine147 (K147). Previous studies have suggested that K104 stabilizes helix-2/helix-3 interactions and K147 is involved in nucleotide binding. However, the impact of monoubiquitination at these residues on the overall structure, dynamics, or function of KRAS is not fully understood. In this study, we examined KRAS monoubiquitination at these sites using data from extensive (12 µs aggregate time) molecular dynamics simulations complemented by nuclear magnetic resonance spectroscopy data. We found that ubiquitin forms dynamic nonspecific interactions with various regions of KRAS and that ubiquitination at both sites modulates conformational fluctuations. In both cases, ubiquitin samples a broad range of conformational space and does not form long-lasting noncovalent contacts with KRAS but it adopts several preferred orientations relative to KRAS. To examine the functional impact of these preferred orientations, we performed a systematic comparison of the dominant configurations of the ubiquitin/KRAS simulated complex with experimental structures of KRAS bound to regulatory and effector proteins as well as a model membrane. Results from these analyses suggest that conformational selection and population shift may minimize the deleterious effects of KRAS ubiquitination at K104 and K147 on binding to some but not all interaction partners. Our findings thus provide new insights into the steric effects of ubiquitin and suggest a potential avenue for therapeutic targeting.

KRAS Ubiquitination at Lysine 104 Retains Exchange Factor Regulation by Dynamically Modulating the Conformation of the Interface.

RAS proteins function as highly regulated molecular switches that control cellular growth. In addition to regulatory proteins, RAS undergoes a number of posttranslational modifications (PTMs) that regulate its activity. Lysine 104, a hot spot for multiple PTMs, is a highly conserved residue that forms key interactions that stabilize the RAS helix-2(H2)/helix-3(H3) interface. Mutation at 104 attenuates interaction with guanine nucleotide exchange factors (GEFs), whereas ubiquitination at lysine 104 retains GEF regulation. To elucidate how ubiquitination modulates RAS function, we generated monoubiquitinated KRAS at 104 using chemical biology approaches and conducted biochemical, NMR, and computational analyses. We find that ubiquitination promotes a new dynamic interaction network and alters RAS conformational dynamics to retain GEF function. These findings reveal a mechanism by which ubiquitination can regulate protein function.