TRIM21 Ubiquitylates SQSTM1/p62 and Suppresses Protein Sequestration to Regulate Redox Homeostasis.

TRIM21 is a RING finger domain-containing ubiquitin E3 ligase whose expression is elevated in autoimmune disease. While TRIM21 plays an important role in immune activation during pathogen infection, little is known about its inherent cellular function. Here we show that TRIM21 plays an essential role in redox regulation by directly interacting with SQSTM1/p62 and ubiquitylating p62 at lysine 7 (K7) via K63-linkage. As p62 oligomerizes and sequesters client proteins in inclusions, the TRIM21-mediated p62 ubiquitylation abrogates p62 oligomerization and sequestration of proteins including Keap1, a negative regulator of antioxidant response. TRIM21-deficient cells display an enhanced antioxidant response and reduced cell death in response to oxidative stress. Genetic ablation of TRIM21 in mice confers protection from oxidative damages caused by arsenic-induced liver insult and pressure overload heart injury. Therefore, TRIM21 plays an essential role in p62-regulated redox homeostasis and may be a viable target for treating pathological conditions resulting from oxidative damage.

Class IA PI3K p110ß subunit promotes autophagy through Rab5 small GTPase in response to growth factor limitation.

Autophagy is an evolutionarily conserved membrane trafficking process. Induction of autophagy in response to nutrient limitation or cellular stress occurs by similar mechanisms in organisms from yeast to mammals. Unlike yeast, metazoan cells rely more on growth factor signaling for a wide variety of cellular activities including nutrient uptake. How growth factor availability regulates autophagy is poorly understood. Here we show that, upon growth factor limitation, the p110ß catalytic subunit of the class IA phosphoinositide 3-kinases (PI3Ks) dissociates from growth factor receptor complexes and increases its interaction with the small GTPase Rab5. This p110ß-Rab5 association maintains Rab5 in its guanosine triphosphate (GTP)-bound state and enhances the Rab5-Vps34 interaction that promotes autophagy. p110ß mutants that fail to interact with Rab5 are defective in autophagy promotion. Hence, in mammalian cells, p110ß acts as a molecular sensor for growth factor availability and induces autophagy by activating a Rab5-mediated signaling cascade.

Contribution of Asparagine Catabolism to Salmonella Virulence.

Salmonellae are pathogenic bacteria that cause significant morbidity and mortality in humans worldwide. Salmonellae establish infection and avoid clearance by the immune system by mechanisms that are not well understood. We previously showed that l-asparaginase II produced by Salmonella enterica serovar Typhimurium (S Typhimurium) inhibits T cell responses and mediates virulence. In addition, we previously showed that asparagine deprivation such as that mediated by l-asparaginase II of S Typhimurium causes suppression of activation-induced T cell metabolic reprogramming. Here, we report that STM3997, which encodes a homolog of disulfide bond protein A (dsbA) of Escherichia coli, is required for l-asparaginase II stability and function. Furthermore, we report that l-asparaginase II localizes primarily to the periplasm and acts together with l-asparaginase I to provide S Typhimurium the ability to catabolize asparagine and assimilate nitrogen. Importantly, we determined that, in a murine model of infection, S Typhimurium lacking both l-asparaginase I and II genes competes poorly with wild-type S Typhimurium for colonization of target tissues. Collectively, these results indicate that asparagine catabolism contributes to S Typhimurium virulence, providing new insights into the competition for nutrients at the host-pathogen interface.

Class III PI3K Vps34 plays an essential role in autophagy and in heart and liver function.

A critical regulator of autophagy is the Class III PI3K Vps34 (also called PIK3C3). Although Vps34 is known to play an essential role in autophagy in yeast, its role in mammals remains elusive. To elucidate the physiological function of Vps34 and to determine its precise role in autophagy, we have generated Vps34(f/f) mice, in which expression of Cre recombinase results in a deletion of exon 4 of Vps34 and a frame shift causing a deletion of 755 of the 887 amino acids of Vps34. Acute ablation of Vps34 in MEFs upon adenoviral Cre infection results in a diminishment of localized generation of phosphatidylinositol 3-phosphate and blockade of both endocytic and autophagic degradation. Starvation-induced autophagosome formation is blocked in both Vps34-null MEFs and liver. Liver-specific Albumin-Cre;Vps34(f/f) mice developed hepatomegaly and hepatic steatosis, and impaired protein turnover. Ablation of Vps34 in the heart of muscle creatine kinase-Cre;Vps34(f/f) mice led to cardiomegaly and decreased contractility. In addition, while amino acid-stimulated mTOR activation was suppressed in the absence of Vps34, the steady-state level of mTOR signaling was not affected in Vps34-null MEFs, liver, or cardiomyocytes. Taken together, our results indicate that Vps34 plays an essential role in regulating functional autophagy and is indispensable for normal liver and heart function.

Identification of a stem-like cell population by exposing metastatic breast cancer cell lines to repetitive cycles of hypoxia and reoxygenation.

INTRODUCTION: The irregular vasculature of solid tumors creates hypoxic regions, which are characterized by cyclic periods of hypoxia and reoxygenation. Accumulated evidence suggests that chronic and repetitive exposure to hypoxia and reoxygenation seem to provide an advantage to tumor growth. Although the development of hypoxia tolerance in tumors predicts poor prognosis, mechanisms contributing to hypoxia tolerance remain to be elucidated. Recent studies have described a subpopulation of cancer stem cells (CSC) within tumors, which have stem-like properties such as self-renewal and the ability to differentiate into multiple cell types. The cancer stem cell theory suggests CSCs persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumors. Since hypoxia is considered to be one of the critical niche factors to promote invasive growth of tumors, we hypothesize that repetitive cycles of hypoxia/reoxygenation also play a role in the enrichment of breast CSCs. METHODS: Two metastatic human breast cancer cell lines (MDA-MB 231 and BCM2) were used to optimize the conditions of hypoxia and reoxygenation cycles. The percentage of CSCs in the cycling hypoxia selected subpopulation was analyzed based on the CD44, CD24, ESA, and E-cadherin expression by three-color flow cytometry. Colony formation assays were used to assess the ability of this subpopulation to self-renew. Limiting dilution assays were performed to evaluate the tumor-initiating and metastatic ability of this subpopulation. Induction of EMT was examined by the expression of EMT-associated markers and EMT-associated microRNAs. RESULTS: Using an optimized hypoxia and reoxygenation regimen, we identified a novel cycling hypoxia-selected subpopulation from human breast cancer cell lines and demonstrated that a stem-like breast cancer cell subpopulation could be expanded through repetitive hypoxia/reoxygenation cycles without genetic manipulation. We also found that cells derived from this novel subpopulation form colonies readily, are highly tumorigenic in immune-deficient mice, and exhibit both stem-like and EMT phenotypes. CONCLUSIONS: These results provide the validity to the newly developed hypoxia/reoxygenation culture system for examining the regulation of CSCs in breast cancer cell lines by niche factors in the tumor microenvironment and developing differential targeting strategies to eradicate breast CSCs.

Endothelial JAK2V617F mutation leads to thrombosis, vasculopathy, and cardiomyopathy in a murine model of myeloproliferative neoplasm.

OBJECTIVE: Cardiovascular complications are the leading cause of morbidity and mortality in patients with myeloproliferative neoplasms (MPNs). The acquired kinase mutation JAK2V617F plays a central role in these disorders. Mechanisms responsible for cardiovascular dysfunction in MPNs are not fully understood, limiting the effectiveness of current treatment. Vascular endothelial cells (ECs) carrying the JAK2V617F mutation can be detected in patients with MPNs. The goal of this study was to test the hypothesis that the JAK2V617F mutation alters endothelial function to promote cardiovascular complications in patients with MPNs. APPROACH AND RESULTS: We employed murine models of MPN in which the JAK2V617F mutation is expressed in specific cell lineages. When JAK2V617F is expressed in both blood cells and vascular ECs, the mice developed MPN and spontaneous, age-related dilated cardiomyopathy with an increased risk of sudden death as well as a prothrombotic and vasculopathy phenotype on histology evaluation. In contrast, despite having significantly higher leukocyte and platelet counts than controls, mice with JAK2V617F-mutant blood cells alone did not demonstrate any cardiac dysfunction, suggesting that JAK2V617F-mutant ECs are required for this cardiovascular disease phenotype. Furthermore, we demonstrated that the JAK2V617F mutation promotes a pro-adhesive, pro-inflammatory, and vasculopathy EC phenotype, and mutant ECs respond to flow shear differently than wild-type ECs. CONCLUSIONS: These findings suggest that the JAK2V617F mutation can alter vascular endothelial function to promote cardiovascular complications in MPNs. Therefore, targeting the MPN vasculature represents a promising new therapeutic strategy for patients with MPNs.

Jun NH2-terminal kinase (JNK) prevents nuclear beta-catenin accumulation and regulates axis formation in Xenopus embryos.

Jun NH(2)-terminal kinases (JNKs) regulate convergent extension movements in Xenopus embryos through the noncanonical Wnt/planar cell polarity pathway. In addition, there is a high level of maternal JNK activity spanning from oocyte maturation until the onset of gastrulation that has no defined functions. Here, we show that maternal JNK activation requires Dishevelled and JNK is enriched in the nucleus of Xenopus embryos. Although JNK activity is not required for the glycogen synthase kinase-3-mediated degradation of beta-catenin, inhibition of the maternal JNK signaling by morpholino-antisense oligos causes hyperdorsalization of Xenopus embryos and ectopic expression of the Wnt/beta-catenin target genes. These effects are associated with an increased level of nuclear and nonmembrane-bound beta-catenin. Moreover, ventral injection of the constitutive-active Jnk mRNA blocks beta-catenin-induced axis duplication, and dorsal injection of active Jnk mRNA into Xenopus embryos decreases the dorsal marker gene expression. In mammalian cells, activation of JNK signaling reduces Wnt3A-induced and beta-catenin-mediated gene expression. Furthermore, activation of JNK signaling rapidly induces the nuclear export of beta-catenin. Taken together, these results suggest that JNK antagonizes the canonical Wnt pathway by regulating the nucleocytoplasmic transport of beta-catenin rather than its cytoplasmic stability. Thus, the high level of sustained maternal JNK activity in early Xenopus embryos may provide a timing mechanism for controlling the dorsal axis formation.

HIV-1 p55Gag encoded in the lysosome-associated membrane protein-1 as a DNA plasmid vaccine chimera is highly expressed, traffics to the major histocompatibility class II compartment, and elicits enhanced immune responses.

Several genetic vaccines encoding antigen chimeras containing the lysosome-associated membrane protein (LAMP) translocon, transmembrane, and cytoplasmic domain sequences have elicited strong mouse antigen-specific immune responses. The increased immune response is attributed to trafficking of the antigen chimera to the major histocompatibility class II (MHC II) compartment where LAMP is colocalized with MHC II. In this report, we describe a new form of an HIV-1 p55gag DNA vaccine, with the gag sequence incorporated into the complete LAMP cDNA sequence. Gag encoded with the translocon, transmembrane and cytoplasmic lysosomal membrane targeting sequences of LAMP, without the luminal domain, was poorly expressed, did not traffic to lysosomes or MHC II compartments of transfected cells, and elicited a limited immune response from DNA immunized mice. In contrast, addition of the LAMP luminal domain sequence to the construct resulted in a high level of expression of the LAMP/Gag protein chimera in transfected cells that was further increased by including the inverted terminal repeat sequences of the adeno-associated virus to the plasmid vector. This LAMP/Gag chimera with the complete LAMP protein colocalized with endogenous MHC II of transfected cells and elicited strong cellular and humoral immune responses of immunized mice as compared with the response to DNA-encoding native Gag, with a 10-fold increase in CD4+ responses, a 4- to 5-fold increase in CD8+ T-cell responses, and antibody titers of >100,000. These results reveal novel roles of the LAMP luminal domain as a determinant of Gag protein expression, lysosomal trafficking, and possibly of the immune response to Gag.