NINJ1 mediates plasma membrane rupture during lytic cell death.

Plasma membrane rupture (PMR) is the final cataclysmic event in lytic cell death. PMR releases intracellular molecules known as damage-associated molecular patterns (DAMPs) that propagate the inflammatory response1-3. The underlying mechanism of PMR, however, is unknown. Here we show that the cell-surface NINJ1 protein4-8, which contains two transmembrane regions, has an essential role in the induction of PMR. A forward-genetic screen of randomly mutagenized mice linked NINJ1 to PMR. Ninj1-/- macrophages exhibited impaired PMR in response to diverse inducers of pyroptotic, necrotic and apoptotic cell death, and were unable to release numerous intracellular proteins including HMGB1 (a known DAMP) and LDH (a standard measure of PMR). Ninj1-/- macrophages died, but with a distinctive and persistent ballooned morphology, attributable to defective disintegration of bubble-like herniations. Ninj1-/- mice were more susceptible than wild-type mice to infection with Citrobacter rodentium, which suggests a role for PMR in anti-bacterial host defence. Mechanistically, NINJ1 used an evolutionarily conserved extracellular domain for oligomerization and subsequent PMR. The discovery of NINJ1 as a mediator of PMR overturns the long-held idea that cell death-related PMR is a passive event.

IRF2 transcriptionally induces GSDMD expression for pyroptosis.

Gasdermin-D (GSDMD) is cleaved by caspase-1, caspase-4, and caspase-11 in response to canonical and noncanonical inflammasome activation. Upon cleavage, GSDMD oligomerizes and forms plasma membrane pores, resulting in interleukin-1ß (IL-1ß) secretion, pyroptotic cell death, and inflammatory pathologies, including periodic fever syndromes and septic shock-a plague on modern medicine. Here, we showed that IRF2, a member of the interferon regulatory factor (IRF) family of transcription factors, was essential for the transcriptional activation of GSDMD. A forward genetic screen with N-ethyl-N-nitrosourea (ENU)-mutagenized mice linked IRF2 to inflammasome signaling. GSDMD expression was substantially attenuated in IRF2-deficient macrophages, endothelial cells, and multiple tissues, which corresponded with reduced IL-1ß secretion and inhibited pyroptosis. Mechanistically, IRF2 bound to a previously uncharacterized but unique site within the GSDMD promoter to directly drive GSDMD transcription for the execution of pyroptosis. Disruption of this single IRF2-binding site abolished signaling by both the canonical and noncanonical inflammasomes. Together, our data illuminate a key transcriptional mechanism for expression of the gene encoding GSDMD, a critical mediator of inflammatory pathologies.

Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling.

Intracellular lipopolysaccharide from Gram-negative bacteria including Escherichia coli, Salmonella typhimurium, Shigella flexneri, and Burkholderia thailandensis activates mouse caspase-11, causing pyroptotic cell death, interleukin-1ß processing, and lethal septic shock. How caspase-11 executes these downstream signalling events is largely unknown. Here we show that gasdermin D is essential for caspase-11-dependent pyroptosis and interleukin-1ß maturation. A forward genetic screen with ethyl-N-nitrosourea-mutagenized mice links Gsdmd to the intracellular lipopolysaccharide response. Macrophages from Gsdmd(-/-) mice generated by gene targeting also exhibit defective pyroptosis and interleukin-1ß secretion induced by cytoplasmic lipopolysaccharide or Gram-negative bacteria. In addition, Gsdmd(-/-) mice are protected from a lethal dose of lipopolysaccharide. Mechanistically, caspase-11 cleaves gasdermin D, and the resulting amino-terminal fragment promotes both pyroptosis and NLRP3-dependent activation of caspase-1 in a cell-intrinsic manner. Our data identify gasdermin D as a critical target of caspase-11 and a key mediator of the host response against Gram-negative bacteria.

Notes from the field: the evaluation of Maine Nutrition and Physical Activity Self-Assessment for Child Care (NAPSACC) experience.

More than half of all children in the United States aged 3 to 6 years are enrolled in child care centers. Maine received funds from the U.S. Department of Health and Human Services' Communities Putting Prevention to Work to promote the adoption of Nutrition and Physical Activity Self-Assessment for Child Care (NAPSACC), an evidence-based program for the child care setting. We evaluated the rollout and adoption of NAP SACC in Maine using multiple methods. Our findings suggest that the NAP SACC program has been successfully adopted in Maine. Nutrition and physical activity policies and offerings have improved, especially with regard to purchasing healthier options in the child care setting.

Location of food stores near schools does not predict the weight status of Maine high school students.

OBJECTIVE: To examine the relationship between stores selling calorie-dense food near schools and student obesity risk, with the hypothesis that high availability predicts increased risk. METHODS: Mail surveys determined height, weight, and calorie-dense food consumption for 552 students at 11 Maine high schools. Driving distance from all food stores within 2 km (1.24 miles) of schools (or the closest store) was computed, and the impact of food store density and proximity to schools on student body mass index was determined by logistic regression. RESULTS: Ten schools had ≥ 1 store selling soda, and 8 schools had ≥1 fast-food restaurant within 1 km (0.62 miles). There were no significant relationships between the proximity or density of food stores around schools and student obesity risk. Students obtained sugar-sweetened beverages in many locations including at school. CONCLUSIONS AND IMPLICATIONS: Unhealthful food choices are ubiquitous. Consequently, stores selling these food items near schools have no significant affect on student obesity.

COP1 is a tumour suppressor that causes degradation of ETS transcription factors.

The proto-oncogenes ETV1, ETV4 and ETV5 encode transcription factors in the E26 transformation-specific (ETS) family, which includes the most frequently rearranged and overexpressed genes in prostate cancer. Despite being critical regulators of development, little is known about their post-translational regulation. Here we identify the ubiquitin ligase COP1 (also known as RFWD2) as a tumour suppressor that negatively regulates ETV1, ETV4 and ETV5. ETV1, which is mutated in prostate cancer more often, was degraded after being ubiquitinated by COP1. Truncated ETV1 encoded by prostate cancer translocation TMPRSS2:ETV1 lacks the critical COP1 binding motifs and was 50-fold more stable than wild-type ETV1. Almost all patient translocations render ETV1 insensitive to COP1, implying that this confers a selective advantage to prostate epithelial cells. Indeed, COP1 deficiency in mouse prostate elevated ETV1 and produced increased cell proliferation, hyperplasia, and early prostate intraepithelial neoplasia. Combined loss of COP1 and PTEN enhanced the invasiveness of mouse prostate adenocarcinomas. Finally, rare human prostate cancer samples showed hemizygous loss of the COP1 gene, loss of COP1 protein, and elevated ETV1 protein while lacking a translocation event. These findings identify COP1 as a tumour suppressor whose downregulation promotes prostatic epithelial cell proliferation and tumorigenesis.

Sensitivity to antitubulin chemotherapeutics is regulated by MCL1 and FBW7.

Microtubules have pivotal roles in fundamental cellular processes and are targets of antitubulin chemotherapeutics. Microtubule-targeted agents such as Taxol and vincristine are prescribed widely for various malignancies, including ovarian and breast adenocarcinomas, non-small-cell lung cancer, leukaemias and lymphomas. These agents arrest cells in mitosis and subsequently induce cell death through poorly defined mechanisms. The strategies that resistant tumour cells use to evade death induced by antitubulin agents are also unclear. Here we show that the pro-survival protein MCL1 (ref. 3) is a crucial regulator of apoptosis triggered by antitubulin chemotherapeutics. During mitotic arrest, MCL1 protein levels decline markedly, through a post-translational mechanism, potentiating cell death. Phosphorylation of MCL1 directs its interaction with the tumour-suppressor protein FBW7, which is the substrate-binding component of a ubiquitin ligase complex. The polyubiquitylation of MCL1 then targets it for proteasomal degradation. The degradation of MCL1 was blocked in patient-derived tumour cells that lacked FBW7 or had loss-of-function mutations in FBW7, conferring resistance to antitubulin agents and promoting chemotherapeutic-induced polyploidy. Additionally, primary tumour samples were enriched for FBW7 inactivation and elevated MCL1 levels, underscoring the prominent roles of these proteins in oncogenesis. Our findings suggest that profiling the FBW7 and MCL1 status of tumours, in terms of protein levels, messenger RNA levels and genetic status, could be useful to predict the response of patients to antitubulin chemotherapeutics.

Impact of Maine's statewide nutrition policy on high school food environments.

INTRODUCTION: We assessed the effect on the food environments of public high schools of Maine's statewide nutrition policy (Chapter 51), which banned "foods of minimal nutritional value" (FMNV) in public high schools that participated in federally funded meal programs. We documented allowable exceptions to the policy and describe the school food environments. METHODS: We mailed surveys to 89 high school food-service directors to assess availability pre-Chapter 51 and post-Chapter 51 of soda, other sugar-sweetened beverages, and junk food. Frequency data were tabulated pre-Chapter 51 and post-Chapter 51, and Fisher exact test was used to assess significance in changes. We conducted food and beverage inventories at 11 high schools. RESULTS: The survey return rate was 61% (N = 54). Availability of soda in student vending significantly decreased pre-Chapter 51 versus post-Chapter 51 (P = .04). No significant changes were found for other sugar-sweetened beverages and junk food. Exceptions to Chapter 51 were permitted to staff (67%), to the public (86%), and in career and technical education programs (31%). Inventories in a subset of schools found no availability of soda for students, whereas other sugar-sweetened beverages and junk food were widely available in à la carte, vending machines, and school stores. Candy, considered a FMNV, was freely available. Soda advertisement on school grounds was common. CONCLUSION: Student vending choices improved after the implementation of Chapter 51; however, use of FMNV as the policy standard may be limiting, as availability of other sugar-sweetened beverages and junk food was pervasive. School environments were not necessarily supportive of the policy, as advertisement of soda was common and some FMNV were available. Furthermore, local exceptions to Chapter 51 likely reduced the overall effect of the policy.

Absent in melanoma 2 is required for innate immune recognition of Francisella tularensis.

Macrophages respond to cytosolic nucleic acids by activating cysteine protease caspase-1 within a complex called the inflammasome. Subsequent cleavage and secretion of proinflammatory cytokines IL-1beta and IL-18 are critical for innate immunity. Here, we show that macrophages from mice lacking absent in melanoma 2 (AIM2) cannot sense cytosolic double-stranded DNA and fail to trigger inflammasome assembly. Caspase-1 activation in response to intracellular pathogen Francisella tularensis also required AIM2. Immunofluorescence microscopy of macrophages infected with F. tularensis revealed striking colocalization of bacterial DNA with endogenous AIM2 and inflammasome adaptor ASC. By contrast, type I IFN (IFN-alpha and -beta) secretion in response to F. tularensis did not require AIM2. IFN-I did, however, boost AIM2-dependent caspase-1 activation by increasing AIM2 protein levels. Thus, inflammasome activation was reduced in infected macrophages lacking either the IFN-I receptor or stimulator of interferon genes (STING). Finally, AIM2-deficient mice displayed increased susceptibility to F. tularensis infection compared with wild-type mice. Their increased bacterial burden in vivo confirmed that AIM2 is essential for an effective innate immune response.

Ubiquitin hydrolase Dub3 promotes oncogenic transformation by stabilizing Cdc25A.

The dual specificity (Tyr/Thr) phosphatase Cdc25A activates cyclin-dependent kinases (Cdks) to promote cell-cycle progression and has significant oncogenic potential. Cdc25A protein levels are regulated tightly in normal tissues, but many human cancers overexpress Cdc25A. The underlying mechanism for overexpression has been enigmatic. Here we show that Cdc25A is stabilized by the ubiquitin hydrolase Dub3. Upon binding Cdc25A, Dub3 removes the polyubiquitin modifications that mark Cdc25A for proteasomal degradation. Dub3 knockdown in cells increased Cdc25A ubiquitylation and degradation, resulting in reduced Cdk/Cyclin activity and arrest at G1/S and G2/M phases of the cell cycle. In contrast, acute Dub3 overexpression produced a signature response to oncogene induction: cells accumulated in S and G2 because of replication stress, and activated a DNA damage response. Dub3 also transformed NIH-3T3 cells and cooperated with activated H-Ras to promote growth in soft agar. Importantly, we show that Dub3 overexpression is responsible for an abnormally high level of Cdc25A in a subset of human breast cancers. Moreover, Dub3 knockdown significantly retarded the growth of breast tumour xenografts in nude mice. As a major regulator of Cdc25A, Dub3 is an example of a transforming ubiquitin hydrolase that subverts a key component of the cell cycle machinery.

Deubiquitinase USP9X stabilizes MCL1 and promotes tumour cell survival.

MCL1 is essential for the survival of stem and progenitor cells of multiple lineages, and is unique among pro-survival BCL2 family members in that it is rapidly turned over through the action of ubiquitin ligases. B- and mantle-cell lymphomas, chronic myeloid leukaemia, and multiple myeloma, however, express abnormally high levels of MCL1, contributing to chemoresistance and disease relapse. The mechanism of MCL1 overexpression in cancer is not well understood. Here we show that the deubiquitinase USP9X stabilizes MCL1 and thereby promotes cell survival. USP9X binds MCL1 and removes the Lys 48-linked polyubiquitin chains that normally mark MCL1 for proteasomal degradation. Increased USP9X expression correlates with increased MCL1 protein in human follicular lymphomas and diffuse large B-cell lymphomas. Moreover, patients with multiple myeloma overexpressing USP9X have a poor prognosis. Knockdown of USP9X increases MCL1 polyubiquitination, which enhances MCL1 turnover and cell killing by the BH3 mimetic ABT-737. These results identify USP9X as a prognostic and therapeutic target, and they show that deubiquitinases may stabilize labile oncoproteins in human malignancies.

Impact of a primary care intervention on physician practice and patient and family behavior: keep ME Healthy---the Maine Youth Overweight Collaborative.

OBJECTIVE: To evaluate the effect of a pediatric primary care-based intervention, on improved clinical decision support and family management of risk behaviors for childhood overweight. METHODS: An experimental field trial was conducted with 12 intervention sites in urban and rural areas of Maine and nonrandomized control sites. Change was assessed by using clinical and parent measures from 9 intervention and 10 control sites before and during the Maine Youth Overweight Collaborative intervention. Longitudinal information was collected from chart audits of patients aged 5-18 years (n = 600), systematic samples of parents collected before (n = 346) and during (n = 386) the intervention in 12 sites, and systematic samples of parents in 9 intervention (n = 235) and 10 control (n = 304) sites collected during the intervention. Surveys of health care providers (n = 14 and 17) before and during the intervention were also collected. Teams worked over 18 months to implement improvements in clinical decision support, including tracking BMI percentiles, identification of overweight patients, appropriate laboratory tests, counseling of families and patients use of a behavioral screening tool, and other improvements following the chronic-care model targeting patients aged 5 to 18 and their families. RESULTS: Large changes occurred in clinical practice from before to during the Maine Youth Overweight Collaborative: increases in assessment of BMI (38%-94%), BMI percentile for age and gender (25%-89%), use of the 5-2-1-0 behavioral screening tool (0%-82%), and weight classification (19%-75%). Parent surveys indicated improvements in providers' behavior and rates of counseling. Intervention providers reported improvements in knowledge, attitudes, self-efficacy, and practice. CONCLUSIONS: The Maine Youth Overweight Collaborative intervention improved clinical decision support and family management of risk behaviors, indicating a promising primary care-based approach to address overweight risk among children and youth.

Association of C-terminal ubiquitin hydrolase BRCA1-associated protein 1 with cell cycle regulator host cell factor 1.

Protein ubiquitination provides an efficient and reversible mechanism to regulate cell cycle progression and checkpoint control. Numerous regulatory proteins direct the addition of ubiquitin to lysine residues on target proteins, and these are countered by an army of deubiquitinating enzymes (DUBs). BRCA1-associated protein-1 (Bap1) is a ubiquitin carboxy-terminal hydrolase and is frequently mutated in lung and sporadic breast tumors. Bap1 can suppress growth of lung cancer cells in athymic nude mice and this requires its DUB activity. We show here that Bap1 interacts with host cell factor 1 (HCF-1), a transcriptional cofactor found in a number of important regulatory complexes. Bap1 binds to the HCF-1 beta-propeller using a variant of the HCF-binding motif found in herpes simplex virus VP16 and other HCF-interacting proteins. HCF-1 is K48 and K63 ubiquitinated, with a major site of linkage at lysines 1807 and 1808 in the HCF-1(C) subunit. Expression of a catalytically inactive version of Bap1 results in the selective accumulation of K48 ubiquitinated polypeptides. Depletion of Bap1 using small interfering RNA results in a modest accumulation of HCF-1(C), suggesting that Bap1 helps to control cell proliferation by regulating HCF-1 protein levels and by associating with genes involved in the G(1)-S transition.

DUBA: a deubiquitinase that regulates type I interferon production.

Production of type I interferon (IFN-I) is a critical host defense triggered by pattern-recognition receptors (PRRs) of the innate immune system. Deubiquitinating enzyme A (DUBA), an ovarian tumor domain-containing deubiquitinating enzyme, was discovered in a small interfering RNA-based screen as a regulator of IFN-I production. Reduction of DUBA augmented the PRR-induced IFN-I response, whereas ectopic expression of DUBA had the converse effect. DUBA bound tumor necrosis factor receptor-associated factor 3 (TRAF3), an adaptor protein essential for the IFN-I response. TRAF3 is an E3 ubiquitin ligase that preferentially assembled lysine-63-linked polyubiquitin chains. DUBA selectively cleaved the lysine-63-linked polyubiquitin chains on TRAF3, resulting in its dissociation from the downstream signaling complex containing TANK-binding kinase 1. A discrete ubiquitin interaction motif within DUBA was required for efficient deubiquitination of TRAF3 and optimal suppression of IFN-I. Our data identify DUBA as a negative regulator of innate immune responses.

Use of national public health performance standards to assess Maine's diabetes system.

In the fall of 2003, Maine underwent a rigorous assessment of the diabetes public health system using a modified version of the State Public Health System Performance Instrument developed by the Centers for Disease Control and Prevention's National Public Health Performance Standards Program and other national partners. The assessment was intended to serve as the impetus for the development of a statewide improvement plan. This article details the assessment process and provides a case study highlighting the successful application of systems-based model standards for a categorical issue.

ATM engages autodegradation of the E3 ubiquitin ligase COP1 after DNA damage.

The ataxia telangiectasia mutated (ATM) protein kinase is a critical component of a DNA-damage response network configured to maintain genomic integrity. The abundance of an essential downstream effecter of this pathway, the tumor suppressor protein p53, is tightly regulated by controlled degradation through COP1 and other E3 ubiquitin ligases, such as MDM2 and Pirh2; however, the signal transduction pathway that regulates the COP1-p53 axis following DNA damage remains enigmatic. We observed that in response to DNA damage, ATM phosphorylated COP1 on Ser(387) and stimulated a rapid autodegradation mechanism. Ionizing radiation triggered an ATM-dependent movement of COP1 from the nucleus to the cytoplasm, and ATM-dependent phosphorylation of COP1 on Ser(387) was both necessary and sufficient to disrupt the COP1-p53 complex and subsequently to abrogate the ubiquitination and degradation of p53. Furthermore, phosphorylation of COP1 on Ser(387) was required to permit p53 to become stabilized and to exert its tumor suppressor properties in response to DNA damage.

Cryopyrin activates the inflammasome in response to toxins and ATP.

A crucial part of the innate immune response is the assembly of the inflammasome, a cytosolic complex of proteins that activates caspase-1 to process the proinflammatory cytokines interleukin (IL)-1beta and IL-18. The adaptor protein ASC is essential for inflammasome function, binding directly to caspase-1 (refs 3, 4), but the triggers of this interaction are less clear. ASC also interacts with the adaptor cryopyrin (also known as NALP3 or CIAS1). Activating mutations in cryopyrin are associated with familial cold autoinflammatory syndrome, Muckle-Wells syndrome and neonatal onset multisystem inflammatory disease, diseases that are characterized by excessive production of IL-1beta. Here we show that cryopyrin-deficient macrophages cannot activate caspase-1 in response to Toll-like receptor agonists plus ATP, the latter activating the P2X7 receptor to decrease intracellular K+ levels. The release of IL-1beta in response to nigericin, a potassium ionophore, and maitotoxin, a potent marine toxin, was also found to be dependent on cryopyrin. In contrast to Asc-/- macrophages, cells deficient in the gene encoding cryopyrin (Cias1-/-) activated caspase-1 and secreted normal levels of IL-1beta and IL-18 when infected with Gram-negative Salmonella typhimurium or Francisella tularensis. Macrophages exposed to Gram-positive Staphylococcus aureus or Listeria monocytogenes, however, required both ASC and cryopyrin to activate caspase-1 and secrete IL-1beta. Therefore, cryopyrin is essential for inflammasome activation in response to signalling pathways triggered specifically by ATP, nigericin, maitotoxin, S. aureus or L. monocytogenes.

De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-kappaB signalling.

NF-kappaB transcription factors mediate the effects of pro-inflammatory cytokines such as tumour necrosis factor-alpha and interleukin-1beta. Failure to downregulate NF-kappaB transcriptional activity results in chronic inflammation and cell death, as observed in A20-deficient mice. A20 is a potent inhibitor of NF-kappaB signalling, but its mechanism of action is unknown. Here we show that A20 downregulates NF-kappaB signalling through the cooperative activity of its two ubiquitin-editing domains. The amino-terminal domain of A20, which is a de-ubiquitinating (DUB) enzyme of the OTU (ovarian tumour) family, removes lysine-63 (K63)-linked ubiquitin chains from receptor interacting protein (RIP), an essential mediator of the proximal TNF receptor 1 (TNFR1) signalling complex. The carboxy-terminal domain of A20, composed of seven C2/C2 zinc fingers, then functions as a ubiquitin ligase by polyubiquitinating RIP with K48-linked ubiquitin chains, thereby targeting RIP for proteasomal degradation. Here we define a novel ubiquitin ligase domain and identify two sequential mechanisms by which A20 downregulates NF-kappaB signalling. We also provide an example of a protein containing separate ubiquitin ligase and DUB domains, both of which participate in mediating a distinct regulatory effect.