HACE1 is a HECT family E3 ubiquitin–protein ligase with broad but incompletely understood tumor suppressor activity. Here, we report a previously unrecognized link between HACE1 and signaling ...complexes containing mammalian target of rapamycin (mTOR). HACE1 blocks mTORC1 and mTORC2 activities by reducing mTOR stability in an E3 ligase‐dependent manner. Mechanistically, HACE1 binds to and ubiquitylates Ras‐related C3 botulinum toxin substrate 1 (RAC1) when RAC1 is associated with mTOR complexes, including at focal adhesions, leading to proteasomal degradation of RAC1. This in turn decreases the stability of mTOR to reduce mTORC1 and mTORC2 activity. HACE1 deficient cells show enhanced mTORC1/2 activity, which is reversed by chemical or genetic RAC1 inactivation but not in cells expressing the HACE1‐insensitive mutant, RAC1K147R. In vivo, Rac1 deletion reverses enhanced mTOR expression in KRasG12D‐driven lung tumors of Hace1−/− mice. HACE1 co‐localizes with mTOR and RAC1, resulting in RAC1‐dependent loss of mTOR protein stability. Together, our data demonstrate that HACE1 destabilizes mTOR by targeting RAC1 within mTOR‐associated complexes, revealing a unique ubiquitin‐dependent process to control the activity of mTOR signaling complexes.
Synopsis
HACE1 E3 ligase mediated degradation of RAC1 within mTORC1 and mTORC2 signaling complexes controls the signalling activity of both complexes.
HACE1 regulates mTORC1 and mTORC2 activity in an E3 ligase dependent manner.
HACE1 targets RAC1 for ubiquitylation when it is associated with mTOR signaling complexes.
RAC1 inactivation reduces activity of mTOR signaling complexes.
HACE1 E3 ligase mediated degradation of RAC1 within mTORC1 and mTORC2 signaling complexes controls the signalling activity of both complexes.
DNA methylation-based biomarkers of aging are highly correlated with actual age. Departures of methylation-estimated age from actual age can be used to define epigenetic measures of child development ...or age acceleration (AA) in adults. Very little is known about genetic or environmental determinants of these epigenetic measures of aging. We obtained DNA methylation profiles using Infinium HumanMethylation450 BeadChips across five time-points in 1018 mother-child pairs from the Avon Longitudinal Study of Parents and Children. Using the Horvath age estimation method, we calculated epigenetic age for these samples. AA was defined as the residuals from regressing epigenetic age on actual age. AA was tested for associations with cross-sectional clinical variables in children. We identified associations between AA and sex, birth weight, birth by caesarean section and several maternal characteristics in pregnancy, namely smoking, weight, BMI, selenium and cholesterol level. Offspring of non-drinkers had higher AA on average but this difference appeared to resolve during childhood. The associations between sex, birth weight and AA found in ARIES were replicated in an independent cohort (GOYA). In children, epigenetic AA measures are associated with several clinically relevant variables, and early life exposures appear to be associated with changes in AA during adolescence. Further research into epigenetic aging, including the use of causal inference methods, is required to better our understanding of aging.
Ewing sarcoma is the second most common bone sarcoma in children, with 1 case per 1.5 million in the United States. Although the survival rate of patients diagnosed with localized disease is ...approximately 70%, this decreases to approximately 30% for patients with metastatic disease and only approximately 10% for treatment-refractory disease, which have not changed for decades. Therefore, new therapeutic strategies are urgently needed for metastatic and refractory Ewing sarcoma.
This study analyzed 19 unique Ewing sarcoma patient- or cell line-derived xenografts (from 14 primary and 5 metastatic specimens) using proteomics to identify surface proteins for potential immunotherapeutic targeting. Plasma membranes were enriched using density gradient ultracentrifugation and compared with a reference standard of 12 immortalized non-Ewing sarcoma cell lines prepared in a similar manner. In parallel, global proteome analysis was carried out on each model to complement the surfaceome data. All models were analyzed by Tandem Mass Tags-based mass spectrometry to quantify identified proteins.
The surfaceome and global proteome analyses identified 1,131 and 1,030 annotated surface proteins, respectively. Among surface proteins identified, both approaches identified known Ewing sarcoma-associated proteins, including IL1RAP, CD99, STEAP1, and ADGRG2, and many new cell surface targets, including ENPP1 and CDH11. Robust staining of ENPP1 was demonstrated in Ewing sarcoma tumors compared with other childhood sarcomas and normal tissues.
Our comprehensive proteomic characterization of the Ewing sarcoma surfaceome provides a rich resource of surface-expressed proteins in Ewing sarcoma. This dataset provides the preclinical justification for exploration of targets such as ENPP1 for potential immunotherapeutic application in Ewing sarcoma. See related commentary by Bailey, p. 934.
Health literacy is an important health promotion concern and recently children and adolescents have been the focus of increased academic attention. To assess the health literacy of this population, ...researchers have been focussing on developing instruments to measure their health literacy. Compared to the wider availability of instruments for adults, only a few tools are known for younger age groups. The objective of this study is to systematically review the field of generic child and adolescent health literacy measurement instruments that are currently available.
A systematic literature search was undertaken in five databases (PubMed, CINAHL, PsycNET, ERIC, and FIS) on articles published between January 1990 and July 2015, addressing children and adolescents ≤18 years old. Eligible articles were analysed, data was extracted, and synthesised according to review objectives.
Fifteen generic health literacy measurement instruments for children and adolescents were identified. All, except two, are self-administered instruments. Seven are objective measures (performance-based tests), seven are subjective measures (self-reporting), and one uses a mixed-method measurement. Most instruments applied a broad and multidimensional understanding of health literacy. The instruments were developed in eight different countries, with most tools originating in the United States (n = 6). Among the instruments, 31 different components related to health literacy were identified. Accordingly, the studies exhibit a variety of implicit or explicit conceptual and operational definitions, and most instruments have been used in schools and other educational contexts. While the youngest age group studied was 7-year-old children within a parent-child study, there is only one instrument specifically designed for primary school children and none for early years.
Despite the reported paucity of health literacy research involving children and adolescents, an unexpected number of health literacy measurement studies in children's populations was found. Most instruments tend to measure their own specific understanding of health literacy and not all provide sufficient conceptual information. To advance health literacy instruments, a much more standardised approach is necessary including improved reporting on the development and validation processes. Further research is required to improve health literacy instruments for children and adolescents and to provide knowledge to inform effective interventions.
Ewing sarcoma (EwS) is an aggressive pediatric cancer of bone and soft tissues characterized by scant T cell infiltration and predominance of immunosuppressive myeloid cells. Given the important ...roles of extracellular vesicles (EVs) in cancer-host crosstalk, we hypothesized that EVs secreted by EwS tumors target myeloid cells and promote immunosuppressive phenotypes. Here, EVs were purified from EwS and fibroblast cell lines and exhibited characteristics of small EVs, including size (100–170 nm) and exosome markers CD63, CD81, and TSG101. Treatment of healthy donor-derived CD33+ and CD14+ myeloid cells with EwS EVs but not with fibroblast EVs induced pro-inflammatory cytokine release, including IL-6, IL-8, and TNF. Furthermore, EwS EVs impaired differentiation of these cells towards monocytic-derived dendritic cells (moDCs), as evidenced by reduced expression of co-stimulatory molecules CD80, CD86 and HLA-DR. Whole transcriptome analysis revealed activation of gene expression programs associated with immunosuppressive phenotypes and pro-inflammatory responses. Functionally, moDCs differentiated in the presence of EwS EVs inhibited CD4+ and CD8+ T cell proliferation as well as IFNγ release, while inducing secretion of IL-10 and IL-6. Therefore, EwS EVs may promote a local and systemic pro-inflammatory environment and weaken adaptive immunity by impairing the differentiation and function of antigen-presenting cells.
Uncovering the mechanisms that underpin how tumor cells adapt to microenvironmental stress is essential to better understand cancer progression. The HACE1 (HECT domain and ankyrin repeat-containing ...E3 ubiquitin-protein ligase) gene is a tumor suppressor that inhibits the growth, invasive capacity, and metastasis of cancer cells. However, the direct regulatory pathways whereby HACE1 confers this tumor-suppressive effect remain to be fully elucidated. In this report, we establish a link between HACE1 and the major stress factor, hypoxia-inducible factor 1 alpha (HIF1α). We find that HACE1 blocks the accumulation of HIF1α during cellular hypoxia through decreased protein stability. This property is dependent on HACE1 E3 ligase activity and loss of Ras-related C3 botulinum toxin substrate 1 (RAC1), an established target of HACE1 mediated ubiquitinylation and degradation. In vivo, genetic deletion of Rac1 reversed the increased HIF1α expression observed in Hace1
mice in murine KRas
-driven lung tumors. An inverse relationship was observed between HACE1 and HIF1α levels in tumors compared to patient-matched normal kidney tissues, highlighting the potential pathophysiological significance of our findings. Together, our data uncover a previously unrecognized function for the HACE1 tumor suppressor in blocking HIF1α accumulation under hypoxia in a RAC1-dependent manner.
Whether interactions between synaptotagmin-1 (syt-1) and the soluble NSF attachment protein receptors (SNAREs) are required during neurotransmission is debated. We examined five SNAP-25 mutations ...designed to interfere with syt-1 interactions. One mutation, D51/E52/E55A, targeted negative charges within region II of the primary interface (Zhou et al., 2015); two mutations targeted region I (D166A and D166/E170A) and one mutation targeted both (D51/E52/E55/D166A). The final mutation (D186/D193A) targeted C-terminal residues not expected to interact with syt-1. An in vitro assay showed that the region I, region II, and region I+II (D51/E52/E55/D166A) mutants markedly reduced the attachment between syt-1 and t-SNARE-carrying vesicles in the absence of phosphatidylinositol 4,5-bisphosphate PI(4,5)P
. In the presence of PI(4,5)P
, vesicle attachment was unaffected by mutation. When expressed in Snap-25-null mouse autaptic neurons, region I mutations reduced the size of the readily releasable pool of vesicles, whereas the region II mutation reduced vesicular release probability. Combining both in the D51/E52/E55/D166A mutation abrogated evoked release. These data point to a division of labor between region I (vesicle priming) and region II (evoked release). Spontaneous release was disinhibited by region I mutations and found to correlate with defective complexin (Cpx) clamping in an in vitro fusion assay, pointing to an interdependent role of synaptotagmin and Cpx in release clamping. Mutation in region II (D51/E52/E55A) also unclamped release, but this effect could be overcome by synaptotagmin overexpression, arguing against an obligatory role in clamping. We conclude that three synaptic release functions of syt-1, vesicle priming, spontaneous release clamping, and evoked release triggering, depend on direct SNARE complex interaction.
The function of synaptotagmin-1 (syt-1):soluble NSF attachment protein receptor (SNARE) interactions during neurotransmission remains unclear. We mutated SNAP-25 within the recently identified region I and region II of the primary synaptotagmin:SNARE interface. Using in vitro assays and rescue experiments in autaptic neurons, we show that interactions within region II of the primary interface are necessary for synchronized calcium-triggered release, whereas region I is involved in vesicle priming. Spontaneous release was disinhibited by region I mutation and found to correlate with defective complexin (Cpx) clamping in vitro, pointing to an interdependent role of synaptotagmin and Cpx in release clamping. Therefore, vesicle priming, clamping spontaneous release, and eliciting evoked release are three different functions of syt-1 that involve different interaction modes with the SNARE complex.
We carried out a trans-ancestry genome-wide association and replication study of blood pressure phenotypes among up to 320,251 individuals of East Asian, European and South Asian ancestry. We find ...genetic variants at 12 new loci to be associated with blood pressure (P = 3.9 × 10(-11) to 5.0 × 10(-21)). The sentinel blood pressure SNPs are enriched for association with DNA methylation at multiple nearby CpG sites, suggesting that, at some of the loci identified, DNA methylation may lie on the regulatory pathway linking sequence variation to blood pressure. The sentinel SNPs at the 12 new loci point to genes involved in vascular smooth muscle (IGFBP3, KCNK3, PDE3A and PRDM6) and renal (ARHGAP24, OSR1, SLC22A7 and TBX2) function. The new and known genetic variants predict increased left ventricular mass, circulating levels of NT-proBNP, and cardiovascular and all-cause mortality (P = 0.04 to 8.6 × 10(-6)). Our results provide new evidence for the role of DNA methylation in blood pressure regulation.
Implementation outcomes serve as progress and success indicators of the implementation process. They are also key antecedents to achieving the more traditional clinical outcomes typically associated ...with a service. Despite their importance, there are few implementation outcomes measures with appropriate psychometric properties, none of which have yet been adapted for medication optimization services.
This study aims to develop and validate the Implementation Outcomes Questionnaire (IOQ) to assess implementation of medication optimization services, starting with Comprehensive Medication Management (CMM). The resulting IOQ is a 40-item self-report instrument for six implementation outcomes, including adoption, acceptability, feasibility, appropriateness, penetration, and sustainability.
A three-phase approach was used to develop and validate the IOQ. Development of the instrument, Phase I, was informed by a targeted search of existing implementation outcomes measures in other fields, a review of suitableoptions options by an expert panel, and item adaptation. To assess content validity, Phase II, an internal vetting process was conducted using an adapted version of Rubio and colleagues’ methodology. Evidence of reliability and construct validity, Phase III, was obtained through a pilot test with 167 pharmacists within 78 different care settings.
Overall, the results supported the reliability and validity (both content and construct) of the IOQ, with further psychometric testing needed for adoption. The items' relevance, clarity, and alignment with each implementation concept were high, except for Penetration. As a result, the Penetration items were refined for further use. Best-fit models were identified for each outcome based on the MCFA analyses, thereby providing insights into the factor structures and interpretation for each measure. Cronbach’ alphas indicated good internal consistency.
This questionnaire is the first of its kind tailored to medication optimization services, starting with CMM. Access to this survey should facilitate measurement of implementation outcomes, thereby increasing the likelihood of achieving the desired clinical outcomes.
•First study to validate an implementation outcomes questionnaire for pharmacy practice.•Provides evidence of questionnaire reliability, content validity, and construct validity.•Results in the 40-item self-report Implementation Outcomes Questionnaire (IOQ).•Evaluates adoption, acceptability, feasibility, appropriateness, penetration, and sustainability.•Will facilitate assessment of the implementation process and thus clinical outcomes.
High infant weight increases the risk of childhood overweight, while breastfeeding may reduce the risk. However, some infants have a very high weight gain even though they are exclusively breastfed. ...We examined the risk of a high body mass index (BMI) and overweight in childhood for infants ≥2.5 SD above the median weight‐for‐age (WAZ) at age 5 months according to duration of exclusive breastfeeding (≤2, >2 to <4 or ≥4 months). The study is based on 13,401 7‐year‐old and 9,819 11‐year‐old children enrolled into the Danish National Birth Cohort (born 1997–2003). Linear and logistic regression analyses were used to examine the associations while adjusting for presumed confounders including birth weight. The results showed that infants ≥2.5 SD at 5 months, breastfed exclusively ≤2, >2 to <4 or ≥4 months had adjusted odds ratios (ORs) for overweight at age 7 at 3.67 (95% confidence interval CI 2.10, 6.43), 3.42 (95% CI 2.32, 5.04) and 3.19 (95% CI 1.90, 5.36) respectively, when compared with infants <2.5 SD WAZ exclusively breastfed ≥4 months. The corresponding results for BMI z‐scores were 0.82 (95% CI 0.60, 1.04), 0.63 (95% CI 0.48, 0.78) and 0.57 (95% CI 0.38, 0.77). For the ≥2.5 SD infants, the differences in risk of overweight and BMI according to duration of exclusive breastfeeding were neither significantly different among the 7‐year nor among the 11‐year‐old children. A high infant weight increases the odds of overweight and is associated with a higher BMI in childhood. Whereas the odds and BMI z‐scores tended to be lower for those exclusively breastfed longer, the differences were not statistically significant.
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