Multi-omics of a pre-clinical model of diabetic cardiomyopathy reveals increased fatty acid supply impacts mitochondrial metabolic selectivity.

The incidence of type 2 diabetes (T2D) is increasing globally, with long-term implications for human health and longevity. Heart disease is the leading cause of death in T2D patients, who display an elevated risk of an acute cardiovascular event and worse outcomes following such an insult. The underlying mechanisms that predispose the diabetic heart to this poor prognosis remain to be defined. This study developed a pre-clinical model (Rattus norvegicus) that complemented caloric excess from a high-fat diet (HFD) and pancreatic ß-cell dysfunction from streptozotocin (STZ) to produce hyperglycaemia, peripheral insulin resistance, hyperlipidaemia and elevated fat mass to mimic the clinical features of T2D. Ex vivo cardiac function was assessed using Langendorff perfusion with systolic and diastolic contractile depression observed in T2D hearts. Cohorts representing untreated, individual HFD- or STZ-treatments and the combined HFD + STZ approach were used to generate ventricular samples (n = 9 per cohort) for sequential and integrated analysis of the proteome, lipidome and metabolome by liquid chromatography-tandem mass spectrometry. This study found that in T2D hearts, HFD treatment primed the metabolome, while STZ treatment was the major driver for changes in the proteome. Both treatments equally impacted the lipidome. Our data suggest that increases in ß-oxidation and early TCA cycle intermediates promoted rerouting via 2-oxaloacetate to glutamate, γ-aminobutyric acid and glutathione. Furthermore, we suggest that the T2D heart activates networks to redistribute excess acetyl-CoA towards ketogenesis and incomplete ß-oxidation through the formation of short-chain acylcarnitine species. Multi-omics provided a global and comprehensive molecular view of the diabetic heart, which distributes substrates and products from excess ß-oxidation, reduces metabolic flexibility and impairs capacity to restore high energy reservoirs needed to respond to and prevent subsequent acute cardiovascular events.

Affordable housing through the low-income housing tax credit program and intimate partner violence-related homicide.

The most severe outcome of intimate partner violence (IPV) is IPV-related homicide. Access to affordable housing may both facilitate exit from abusive relationships and reduce financial stress in intimate relationships, potentially preventing IPV-related homicide. We examined the association of the availability of rental housing through the Low-Income Housing Tax Credit (LIHTC) program, a federal program providing tax incentives to support the development of affordable housing, with IPV-related homicide and assessed whether this association differed by eviction rates at the state-level. We used 2005-2016 National Violent Death Reporting System, LIHTC Property, and Eviction Lab data for 13 states and compared the rate of IPV-related homicide in state-years with ≥30 to state-years with <30 LIHTC units per 100,000 population, overall and stratified by eviction rates. We conducted analyses in fall 2020. Adjusting for potential state-level confounders, the rate of IPV-related homicide in state-years with ≥30 LIHTC units per 100,000 population was lower than in state-years with <30 LIHTC units per 100,000 population (RR = 0.89, 95% CI 0.81, 0.98). The reduction in the rate of IPV-related homicide was slightly larger in state-years with higher eviction rates (≥3500 evictions per 100,000 renter population; RR = 0.83, 95% CI 0.74, 0.93) compared to state-years with lower eviction rates (<3500 evictions per 100,000 renter population; RR = 0.91, 95% CI 0.81, 1.03). Overall, at the state-level, increased availability of affordable housing through the LIHTC program was associated with lower rates of IPV-related homicide. Increasing the availability of affordable housing may be one tool for preventing IPV-related homicide.

Improving Outcome of Superior Mediastinal Lymph Node Dissection During Esophagectomy: A Novel Approach Combining Continuous and Intermittent Recurrent Laryngeal Nerve Monitoring.

OBJECTIVE: This study aimed at demonstrating the effects and learning curve of utilizing combined intermittent and continuous recurrent laryngeal nerve (RLN) monitoring for lymphadenectomy during esophagectomy. BACKGROUND: RLN lymphadenectomy is oncologically important but is technically demanding. Vocal cord (VC) palsy as a result from RLN injury, carries significant morbidities. METHODS: This is a retrospective study of consecutive esophageal squamous cell carcinoma (ESCC) patients who underwent transthoracic esophagectomy from 2010 to 2020. Combined nerve monitoring (CNM) included: CNM which involved a periodic stimulating left vagal electrode and intermittent nerve monitoring which utilized a stimulating probe to identify the RLNs. The integrity of the RLNs was assessed both intermittently and continuously. This technique was introduced in 2014. Patients were divided into "before CNM" and "CNM" groups. The primary outcome was the difference in number of RLN lymph nodes harvested and VC palsy rate. Learning curves were demonstrated by cumulative sum (CUSUM) analysis. RESULTS: Two hundred and fifty-five patients were included with 157 patients in "CNM" group. The mean number of RLN lymph nodes harvested was significantly higher (4.31 vs 0.45, P < 0.0001) for the "CNM" group. VC palsy rates were significantly lower (17.8% vs 32.7%, P = 0.007). There was an initial increase in VC palsy rate, peaked at around 46 cases. The increase in lymph nodes harvested above the mean plateaued at around 96 cases. CONCLUSIONS: CNM helped improve bilateral RLN lymphadenectomy. Lymph node harvesting was increased with reduction of VC palsy after a learning curve.

The role of microorganisms in soy sauce production.

Soy sauce is a salty condiment commonly used in Eastern Asia that is made from soy beans with varying amounts of wheat or no wheat at all. It is known as shoyu in Japan, chiang-yu (or -yi) in China, kecup in Indonesia, kunjang in Korea, toyo in The Philippines, and see-ieu in Thailand (Beuchat, 1985; Djien, 1982; Fukushima, 1989). It provides flavor in an otherwise bland diet, and nutritionally it provides salt (NaCl) and predigested proteins in a diet that is traditionally protein poor. It has been made for centuries on a small scale in many towns and villages in Asia, but since 1950, particularly in Japan, the manufacturing process has been studied and modernized so that its manufacture is now concentrated in large factories using modern, controlled methods of production (Sasaki & Nunomura, 1993). In Japan, soy sauce fermentation is a major food manufacturing activity. More than 1.1 million kiloliters of soy sauce was produced in 1986 by 3000 producers, and the Kikkoman Company supplied 30% of the market (Fukushima, 1989). By 1990 there were 2871 manufacturers, 5 of which produced about 50% of the total production (Sasaki & Nunomura, 1993). While modern methods are used for most of the soy sauce produced in Japan, and factory production in other Asian countries is growing, soy sauce is still produced by methods involving no modern technological inputs (Röling, Prasetyo, Timotius, Stouthamer, & van Verseveld, 1994).

Structured and disordered regions cooperatively mediate DNA-binding autoinhibition of ETS factors ETV1, ETV4 and ETV5.

Autoinhibition enables spatial and temporal regulation of cellular processes by coupling protein activity to surrounding conditions, often via protein partnerships or signaling pathways. We report the molecular basis of DNA-binding autoinhibition of ETS transcription factors ETV1, ETV4 and ETV5, which are often overexpressed in prostate cancer. Inhibitory elements that cooperate to repress DNA binding were identified in regions N- and C-terminal of the ETS domain. Crystal structures of these three factors revealed an α-helix in the C-terminal inhibitory domain that packs against the ETS domain and perturbs the conformation of its DNA-recognition helix. Nuclear magnetic resonance spectroscopy demonstrated that the N-terminal inhibitory domain (NID) is intrinsically disordered, yet utilizes transient intramolecular interactions with the DNA-recognition helix of the ETS domain to mediate autoinhibition. Acetylation of selected lysines within the NID activates DNA binding. This investigation revealed a distinctive mechanism for DNA-binding autoinhibition in the ETV1/4/5 subfamily involving a network of intramolecular interactions not present in other ETS factors. These distinguishing inhibitory elements provide a platform through which cellular triggers, such as protein-protein interactions or post-translational modifications, may specifically regulate the function of these oncogenic proteins.

Invited Commentary: Gaze Aversion and Unnoticed Phenomena.

Child abuse is a social phenomenon that has been underresearched and undersupported. The scale of the problem is large, with over 3 million US children reported for abuse or neglect each year. An estimated 15% of confirmed victims may be placed in out-of-home care to ensure their safety. Studies have not previously examined the impact of losing a child into foster care on maternal health and mortality. Family and maternal risk factors, such as teen pregnancy, intimate partner violence, or drug and alcohol abuse, have been well documented as risk factors for maltreatment. The findings of Wall-Wieler et al. (Am J Epidemiol. 2018;187(6):1182-1188) are not so much unexpected as they are surprising in that the impact of foster care on maternal health has heretofore been largely unexamined. The innovative use of maternal sisters with children not in foster care as controls provides a powerful control for family genetics, and family environment with likely common early life experiences, in the mortality of the mothers whose children were removed. With over 3 million children reported for suspected maltreatment each year, the related health and social outcomes for both children and their families require more careful examination using public health methods.

Development and Prospective Validation of Tools to Accurately Identify Neurosurgical and Critical Care Events in Children With Traumatic Brain Injury.

OBJECTIVE: To develop and validate case definitions (computable phenotypes) to accurately identify neurosurgical and critical care events in children with traumatic brain injury. DESIGN: Prospective observational cohort study, May 2013 to September 2015. SETTING: Two large U.S. children's hospitals with level 1 Pediatric Trauma Centers. PATIENTS: One hundred seventy-four children less than 18 years old admitted to an ICU after traumatic brain injury. MEASUREMENTS AND MAIN RESULTS: Prospective data were linked to database codes for each patient. The outcomes were prospectively identified acute traumatic brain injury, intracranial pressure monitor placement, craniotomy or craniectomy, vascular catheter placement, invasive mechanical ventilation, and new gastrostomy tube or tracheostomy placement. Candidate predictors were database codes present in administrative, billing, or trauma registry data. For each clinical event, we developed and validated penalized regression and Boolean classifiers (models to identify clinical events that take database codes as predictors). We externally validated the best model for each clinical event. The primary model performance measure was accuracy, the percent of test patients correctly classified. The cohort included 174 children who required ICU admission after traumatic brain injury. Simple Boolean classifiers were greater than or equal to 94% accurate for seven of nine clinical diagnoses and events. For central venous catheter placement, no classifier achieved 90% accuracy. Classifier accuracy was dependent on available data fields. Five of nine classifiers were acceptably accurate using only administrative data but three required trauma registry fields and two required billing data. CONCLUSIONS: In children with traumatic brain injury, computable phenotypes based on simple Boolean classifiers were highly accurate for most neurosurgical and critical care diagnoses and events. The computable phenotypes we developed and validated can be used in any observational study of children with traumatic brain injury and can reasonably be applied in studies of these interventions in other patient populations.

Functional Status Scale in Children With Traumatic Brain Injury: A Prospective Cohort Study.

OBJECTIVES: In children with traumatic brain injury, 1) to describe the hospital discharge functional outcome and change from baseline function using the Functional Status Scale and 2) to determine any associations between discharge Functional Status Scale and age, injury mechanism, neurologic examination, imaging, and other predictors of outcome. DESIGN: Prospective observational cohort study, May 2013 to November 2015. SETTING: Two U.S. children's hospitals designated as American College of Surgeons level 1 pediatric trauma centers. PATIENTS: Children less than 18 years old admitted to an ICU with acute traumatic brain injury and either a surgical or critical care intervention within the first 24 hours or in-hospital mortality. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The primary outcome was hospital discharge Functional Status Scale. Most, 133 of 196 (68%), had severe traumatic brain injury (admission Glasgow Coma Scale, 3-8). Overall hospital mortality was 14%; 20% among those with severe traumatic brain injury. Hospital discharge Functional Status Scale had an inverse relationship with Glasgow Coma Scale: for each increase in admission Glasgow Coma Scale by 1, the discharge Functional Status Scale decreased by 0.5 (95% CI, 0.7-0.3). Baseline Functional Status Scale was collected at one site (n = 75). At that site, nearly all (61/62) of the survivors had normal or near-normal (≤ 7) preinjury Functional Status Scale. More than one-third, 23 of 62 (37%), of survivors had new morbidity at hospital discharge (increase in Functional Status Scale, ≥ 3). Among children with severe traumatic brain injury who had baseline Functional Status Scale collected, 21 of 41 survivors (51%) had new morbidity at hospital discharge. The mean change in Functional Status Scale from baseline to hospital discharge was 3.9 ± 4.9 overall and 5.2 ± 5.4 in children with severe traumatic brain injury. CONCLUSIONS: More than one-third of survivors, and approximately half of survivors with severe traumatic brain injury, will have new morbidity. Hospital discharge Functional Status Scale, change from baseline Functional Status Scale, and new morbidity acquisition can be used as outcome measures for hospital-based care process improvement initiatives and interventional studies of children with traumatic brain injury.

Variation in Acceptable Child Discipline Practices by Child Age: Perceptions of Community Norms by Medical and Legal Professionals.

Mandated child abuse reporters may judge specific disciplinary practices as unacceptable for young children, whereas child law professionals arbitrating allegations may be less inclusive. Do the views of these groups diverge, by child age, regarding discipline? Judgments of community norms across a wide range of children's ages were obtained from 380 medical and legal professionals. Because the Parent-Child Conflict Tactics Scale (PC-CTS) can be used to assess the epidemiology of child disciplinary behaviors and as a proxy to examine the incidence or prevalence of child abuse, the disciplinary practices described on the PC-CTS were presented as triggers for questions. Significant child age effects were found for disciplinary practices classified as "harsh." The consistencies between legal and medical professionals were striking. Both groups reflected changes in United States norms, as non-physical approaches were the most approved. We conclude that instruments estimating the prevalence of child maltreatment by parent-report should consider modifying how specific disciplinary practices are classified. Copyright © 2016 John Wiley & Sons, Ltd.

Pathogenic T cells persist after reversal of autoimmune disease by immunosuppression with regulatory T cells.

Autoimmune disease can be prevented with immunosuppressive agents; however, the effectiveness of these treatments in advanced stage of disease and the fate of pathogenic T cells following such treatments are not clear. In this study we demonstrate that a single dose of in vitro-induced Treg cells (iTreg cells) resulted in the functional repair and restitution of stomach tissue that had been severely damaged in advanced autoimmune gastritis. iTreg cells caused depletion or inactivation of autoreactive naïve T cells that were antigen inexperienced, however, autoreactive effector/memory T cells persisted in treated mice, resulting in residual cellular infiltrates within the repaired stomach tissue. The persisting autoreactive T cells were able to rapidly cause autoimmune disease if iTreg cells were removed. Similar data were obtained from mice treated continuously with corticosteroid, in that there was substantial restitution of the gastric mucosa; however, effector T cells persisted and rapidly caused pathology following drug removal. Therefore, iTreg cells or corticosteroid can suppress pathogenic autoreactive cells in advanced autoimmune disease, reversing tissue damage and improving tissue function. However, the persistence of pathogenic T cells represents a disease risk.

Leakage Mechanism and Cycling Behavior of Ferroelectric Al0.7Sc0.3N.

Ferroelectric scandium-doped aluminum nitride (Al1-xScxN) is of considerable research interest because of its superior ferroelectricity. Studies indicate that Al1-xScxN may suffer from a high leakage current, which can hinder further thickness scaling and long-term reliability. In this work, we systematically investigate the origin of the leakage current in Al0.7Sc0.3N films via experiments and theoretical calculations. The results reveal that the leakage may originate from the nitrogen vacancies with positively charged states and fits well with the trap-assisted Poole-Frenkel (P-F) emission. Moreover, we examine the cycling behavior of ferroelectric Al0.7Sc0.3N-based FeRAM devices. We observe that the leakage current substantially increases when the device undergoes bipolar cycling with a pulse amplitude larger than the coercive electric field. Our analysis shows that the increased leakage current in bipolar cycling is caused by the monotonously reduced trap energy level by monitoring the direct current (DC) leakage under different temperatures and the P-F emission fitting.

Affordable housing through the Low-Income Housing Tax Credit program and opioid overdose emergency department visits.

INTRODUCTION: The United States continues to experience an opioid overdose crisis. As a key social determinant of health, housing insecurity may contribute to initiation of substance use and can threaten outcomes for those with substance use disorders by increasing stress, risky substance use, discontinuity of treatment, and return to use, all of which may increase the risk of overdose. The Low-Income Housing Tax Credit (LIHTC) program supports access to rental housing for low-income populations. By facilitating access to affordable housing, this program may improve housing security, thereby reducing overdose risk. METHODS: We used data from LIHTC Property Data and the State Emergency Department Database (SEDD) to identify the number of LIHTC units available and opioid overdoses discharged from the emergency department (ED) in 13 states between 2005 and 2014. RESULTS: Between 2005 and 2014, mean opioid overdose ED visits were higher in states with fewer LIHTC units (<28 LIHTC units per 100,000 population) at 26.5 per 100,000 population as compared to states with higher LIHTC units (≥28 LIHTC units per 100,000 population) at 21.1 per 100,000. We find that greater availability of LIHTC units was associated with decreased rates of opioid overdose ED visits (RR 0.94; CI 0.90, 1.00). CONCLUSIONS: Given the importance of housing as a key social determinant of health, the provision of affordable housing may mitigate substance misuse and prevent nonfatal opioid overdose.

Both IFN-γ and IL-17 are required for the development of severe autoimmune gastritis.

IL-17, produced by a distinct lineage of CD4(+) helper T (Th) cells termed Th17 cells, induces the production of pro-inflammatory cytokines from resident cells and it has been demonstrated that over-expression of IL-17 plays a crucial role in the onset of several auto-immune diseases. Here we examined the role of IL-17 in the pathogenesis of autoimmune gastritis, a disease that was previously believed to be mediated by IFN-γ. Significantly higher levels of IL-17 and IFN-γ were found in the stomachs and stomach-draining lymph nodes of mice with severe autoimmune gastritis. Unlike IL-17, which was produced solely by CD4(+) T cells in gastritic mice, the majority of IFN-γ-producing cells were CD8(+) T cells. However, CD8(+) T cells alone were not able to induce autoimmune gastritis. T cells that were deficient in IL-17 or IFN-γ production were able to induce autoimmune gastritis but to a much lower extent compared with the disease induced by wild-type T cells. These data demonstrate that production of neither IL-17 nor IFN-γ by effector T cells is essential for the initiation of autoimmune gastritis, but suggest that both are required for the disease to progress to the late pathogenic stage that includes significant tissue disruption.

Arl5b is a Golgi-localised small G protein involved in the regulation of retrograde transport.

Regulation of membrane transport is controlled by small G proteins, which include members of the Rab and Arf families. Whereas the role of the classic Arf family members are well characterized, many of the Arf-like proteins (Arls) remain poorly defined. Here we show that Arl5a and Arl5b are localised to the trans-Golgi in mammalian cells, and furthermore have identified a role for Arl5b in the regulation of retrograde membrane transport from endosomes to the trans-Golgi network (TGN). The constitutively active Arl5b (Q70L)-GFP mutant was localised efficiently to the Golgi in HeLa cells whereas the dominant-negative Arl5b (T30N)-GFP mutant was dispersed throughout the cytoplasm and resulted in perturbation of the Golgi apparatus. Stable HeLa cells expressing GFP-tagged Arl5b (Q70L) showed an increased rate of endosome-to-Golgi transport of the membrane cargo TGN38 compared with control HeLa cells. Depletion of Arl5b by RNAi resulted in an alteration in the intracellular distribution of mannose-6-phosphate receptor, and significantly reduced the endosome-to-TGN transport of the membrane cargo TGN38 and of Shiga toxin, but had no affect on the anterograde transport of the cargo E-cadherin. Collectively these results suggest that Arl5b is a TGN-localised small G protein that plays a key role in regulating transport along the endosome-TGN pathway.

Ras signaling requires dynamic properties of Ets1 for phosphorylation-enhanced binding to coactivator CBP.

Ras/MAPK signaling is often aberrantly activated in human cancers. The downstream effectors are transcription factors, including those encoded by the ETS gene family. Using cell-based assays and biophysical measurements, we have determined the mechanism by which Ras/MAPK signaling affects the function of Ets1 via phosphorylation of Thr38 and Ser41. These ERK2 phosphoacceptors lie within the unstructured N-terminal region of Ets1, immediately adjacent to the PNT domain. NMR spectroscopic analyses demonstrated that the PNT domain is a four-helix bundle (H2-H5), resembling the SAM domain, appended with two additional helices (H0-H1). Phosphorylation shifted a conformational equilibrium, displacing the dynamic helix H0 from the core bundle. The affinity of Ets1 for the TAZ1 (or CH1) domain of the coactivator CBP was enhanced 34-fold by phosphorylation, and this binding was sensitive to ionic strength. NMR-monitored titration experiments mapped the interaction surfaces of the TAZ1 domain and Ets1, the latter encompassing both the phosphoacceptors and PNT domain. Charge complementarity of these surfaces indicate that electrostatic forces act in concert with a conformational equilibrium to mediate phosphorylation effects. We conclude that the dynamic helical elements of Ets1, appended to a conserved structural core, constitute a phospho-switch that directs Ras/MAPK signaling to downstream changes in gene expression. This detailed structural and mechanistic information will guide strategies for targeting ETS proteins in human disease.

Correlates of parent-youth discordance about youth-witnessed violence: a brief report.

Studies have consistently demonstrated a lack of agreement between youth and parent reports regarding youth-witnessed violence (YWV). However, little empirical investigation has been conducted on the correlates of disagreement. Concordance between youth and parents about YWV was examined in 766 parent-youth dyads from the Longitudinal Studies of Child Abuse and Neglect (LONGSCAN). Results showed that significantly more youth (42%) than parents (15%) reported YWV. Among the dyads in which at least one informant reported YWV (N = 344), we assessed whether youth delinquency, parental monitoring, parent-child relationship quality, history of child maltreatment, income, and parental depression were predictive of parent-youth concordance. Findings indicated that youth engagement in delinquent activities was higher in the groups in which the youth reported violence exposure. More empirical study is needed to assess correlates of agreement in high-risk youth to better inform associations found between exposures and outcomes as well as practice and policy for violence exposed youth.

Towards a robust test to detect gene doping for anabolic enhancement in human athletes.

Success in gene therapy in treating human disease makes this technology attractive to enhance athletic performance, creating the need for gene doping detection. In 2021, World Anti-Doping Agency (WADA) approved the first gene doping test. Here, we describe a new method to detect doping with four additional genes, follistatin, growth hormone 1, growth hormone-releasing hormone and insulin-like growth factor 1, that may improve performance by increasing muscle size and strength. The method utilises four hydrolysis probe-based polymerase chain reaction (PCR) assays that target the transgenes based on the coding sequence of the four endogenous genes. The assays are specific, reproducible and capable to detect five copies of transgene in the presence of very similar endogenous gene in 25,000 times excess. To underpin reliable and comparable routine method performance by doping testing laboratories, a synthetic reference material for the method was designed and generated following the ISO Guide 35. The complete method was validated in blood samples using plasma as extraction matrix and QIAamp DNA blood midi DNA extraction kit. All blood samples from different donors (n = 8) simulated to be negative or positive (1500 transgene copies spiked per millilitre of blood) for the transgenes were reported correctly. The new method that targets four additional genes will extend the capabilities of laboratories involved in doping control to protect athletes' health, fairness and equality.

Shape complementarity processes for ultrashort-burst sensitive M13-PEG-WS2-powered MCF-7 cancer cell sensors.

Biomarkers have the potential to be utilized in disease diagnosis, prediction and monitoring. The cancer cell type is a leading candidate for next-generation biomarkers. Although traditional digital biomolecular sensor (DBS) technology has shown to be effective in assessing cell-based interactions, low cell-population detection of cancer cell types is extremely challenging. Here, we controlled the electrical signature of a two-dimensional (2D) nanomaterial, tungsten disulfide (WS2), by utilizing a combination of the Phage-integrated Polymer and the Nanosheet (PPN), viz., the integration of the M13-conjugated polyethylene glycol (PEG) and the WS2, through shape-complementarity phenomena, and developed a sensor system, i.e., the Phage-based DBS (P-DBS), for the specific, rapid, sensitive detection of clinically-relevant MCF-7 cells. The P-DBS attains a detection limit of 12 cells per µL, as well as a contrast of 1.25 between the MCF-10A sample signal and the MCF-7 sample signal. A reading length of 200 µs was further achieved, along with a relative cell viability of ∼100% for both MCF-7 and MCF-10A cells and with the PNN. Atomistic simulations reveal the structural origin of the shape complementarity-facilitated decrease in the output impedance of the P-DBS. The combination of previously unreported exotic sensing materials and digital sensor design represents an approach to unlocking the ultra-sensitive detection of cancer cell types and provides a promising avenue for early cancer diagnosis, staging and monitoring.

Characterizing the N- and C-terminal Small ubiquitin-like modifier (SUMO)-interacting motifs of the scaffold protein DAXX.

DAXX is a scaffold protein with diverse roles that often depend upon binding SUMO via its N- and/or C-terminal SUMO-interacting motifs (SIM-N and SIM-C). Using NMR spectroscopy, we characterized the in vitro binding properties of peptide models of SIM-N and SIM-C to SUMO-1 and SUMO-2. In each case, binding was mediated by hydrophobic and electrostatic interactions and weakened with increasing ionic strength. Neither isolated SIM showed any significant paralog specificity, and the measured µM range K(D) values of SIM-N toward both SUMO-1 and SUMO-2 were ∼4-fold lower than those of SIM-C. Furthermore, SIM-N bound SUMO-1 predominantly in a parallel orientation, whereas SIM-C interconverted between parallel and antiparallel binding modes on an ms to µs time scale. The differences in affinities and binding modes are attributed to the differences in charged residues that flank the otherwise identical hydrophobic core sequences of the two SIMs. In addition, within its native context, SIM-N bound intramolecularly to the adjacent N-terminal helical bundle domain of DAXX, thus reducing its apparent affinity for SUMO. This behavior suggests a possible autoregulatory mechanism for DAXX. The interaction of a C-terminal fragment of DAXX with an N-terminal fragment of the sumoylated Ets1 transcription factor was mediated by SIM-C. Importantly, this interaction did not involve any direct contacts between DAXX and Ets1, but rather was derived from the non-covalent binding of SIM-C to SUMO-1, which in turn was covalently linked to the unstructured N-terminal segment of Ets1. These results provide insights into the binding mechanisms and hence biological roles of the DAXX SUMO-interacting motifs.