Bromodomain and extraterminal domain (BET) protein inhibitors are emerging as promising anti-cancer therapies. The gene encoding the E3 ubiquitin ligase substrate-binding adaptor speckle-type POZ protein (SPOP) is most frequently mutated in prostate cancer. Here we demonstrate that wild-type SPOP binds to and induces ubiquitination and proteasomal degradation of BET proteins (BRD2, BRD3 and BRD4) by recognizing a common degron motif. In contrast, prostate cancer-associated SPOP mutants impair binding and proteasomal degradation of BET proteins, thus inducing their accumulation in prostate cancer cells and patient specimens. Transcriptome and BRD4 cistrome analyses reveal that SPOP mutation enhances BRD4-dependent expression of GTPase RAC1 and cholesterol biosynthesis genes and AKT-mTORC1 activation. SPOP mutant expression confers BET inhibitor resistance and this effect can be overcome by AKT inhibitors. Thus, SPOP mutations promote AKT-mTORC1 activation and intrinsic BET inhibitor resistance by stabilizing BET proteins, suggesting that SPOP mutation can be an effective biomarker to guide BET inhibitor-oriented therapy of prostate cancer.

Thioredoxin interacting protein (TXNIP) was originally characterized as an endogenous inhibitor of thioredoxin, a key regulator in cellular redox homeostasis. TXNIP is also known to play important roles in tumor growth and metastasis, glucose and lipid metabolism. TXNIP expression is induced by various stress stimuli. However, it has been unclear how TXNIP is down-regulated. Here, we report that TXNIP undergoes proteasomal degradation in cells. We identify Itch as the E3 ubiquitin ligase for TXNIP. We demonstrate that Itch mediates polyubiquitination of TXNIP both in vitro and in vivo. Overexpression of Itch leads to TXNIP proteasomal degradation. Knockdown of Itch by small interfering RNA causes an accumulation of the steady-state level of TXNIP. We also show that the PPXY motifs of TXNIP and the WW domains of Itch mediate their interaction. Furthermore, the Itch-TXNIP interaction regulates intracellular reactive oxygen species levels and apoptosis. These findings establish a new mechanism for the negative regulation of TXNIP by Itch and shed new light on the regulation of cellular redox homeostasis.

AMOT (angiomotin) is a membrane-associated protein that is expressed in ECs (endothelial cells) and controls migration, TJ (tight junction) formation, cell polarity and angiogenesis. Recent studies have revealed that AMOT and two AMOT-like proteins, AMOTL1 and AMOTL2, play critical roles in the Hippo pathway by regulating the subcellular localization of the co-activators YAP (Yes-associated protein) and TAZ (transcriptional co-activator with PDZ-binding motif). However, it has been unclear how AMOT is regulated. In the present study, we report that AMOT undergoes proteasomal degradation. We identify three members of Nedd4 (neural-precursor-cell-expressed developmentally down-regulated)-like ubiquitin E3 ligases, Nedd4, Nedd4-2 and Itch, as the ubiquitin E3 ligases for the long isoform of AMOT, AMOT/p130. We demonstrate that Nedd4, Nedd4-2 and Itch mediate poly-ubiquitination of AMOT/p130 in vivo. Overexpression of Nedd4, Nedd4-2 or Itch leads to AMOT/p130 proteasomal degradation. Knockdown of Nedd4, Nedd4-2 and Itch causes an accumulation of steady-state level of AMOT/p130. We also show that three L/P-PXY motifs of AMOT/p130 and the WW domains of Nedd4 mediate their interaction. Furthermore, Nedd4-like ubiquitin E3 ligases might compete with YAP for the binding to AMOT/p130, and subsequently targeting AMOT/p130 for ubiquitin-dependent degradation. Together, these observations reveal a novel post-translational regulatory mechanism of AMOT/p130.

SUMMARY Neutrophil degranulation plays an important role in acute innate immune responses and is tightly regulated because the granule contents can cause tissue damage. However, this regulation remains poorly understood. Here we identify the complex of STK24 and CCM3 as being an important regulator of neutrophil degranulation. Lack of either STK24 or CCM3 increases the release of a specific granule pool without affecting other neutrophil functions. STK24 appears to suppress exocytosis by interacting and competing with UNC13D C2B domain for lipid binding, whereas CCM3 has dual roles in exocytosis regulation. While CCM3 stabilizes STK24, it counteracts STK24-mediated inhibition of exocytosis by recruiting STK24 away from the C2B domain through its Ca2+-sensitive interaction with UNC13D C2A domain. This STK24/CCM3-regulated exocytosis plays an important role in protection of kidneys from ischemia-reperfusion injury. Together, these findings reveal a previously unknown function of the STK24 and CCM3 complex in the regulation of ligand-stimulated exocytosis.

Sepsis is a life-threatening condition caused by an immune response triggered by infection, and highly elevated cytokine/chemokine levels in the blood play crucial roles in the progression of sepsis. Serum exosomes are nanovesicles that have multiple biological functions, playing roles in antigen presentation, intercellular signal communication, inflammatory response and immune surveillance. However, the biological functions and related molecular bases remain to be elucidated. In this study, we investigated the profiles of cytokines/chemokines harbored in the exosomes of septic mice and explored the mechanisms of immunomodulation on T cells treated with exosomes harvested from septic mice. Blood cytokines/chemokines existed in both the soluble form and in the insoluble exosomal form; the profiles of the cytokines/chemokines in these two forms displayed different dynamics in the blood of mice challenged with LPS. Exosomes from septic mice induced the differentiation of Th1/Th2 cells, which was blocked by specific antibodies targeting IL-12 and IL-4. In addition, these exosomes significantly augmented the proliferation and migration of T lymphocytes. Furthermore, preadministration of exosomes by intravenous injection restrained the inflammatory response, attenuated lung and liver tissue damage, and prolonged the survival of cecal ligation and puncture (CLP) mice. Our results indicate that exosomes enriched with cytokines/chemokines play critical roles in T cell differentiation, proliferation and chemotaxis during the sepsis process and have a protective effect on cecal ligation and puncture (CLP) mice. Thus, these findings not only strengthen our understanding of the role of sepsis via exosomes but also provide potential targets for therapeutic applications.

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Osteoporosis is a chronic, progressive disease in which early diagnosis is very important. The neutrophil-to-lymphocyte ratio (NLR) and the platelet-to-lymphocyte ratio (PLR) have been reported as new predictors in inflammatory and immune diseases including osteoporosis. No studies have reported the relationship between monocyte-to-lymphocyte ratio (MLR) and osteoporosis patients. To investigated the ability of MLR to predict osteoporosis. Three hundred sixteen osteoporosis patients and 111 healthy control subjects were enrolled. Patients’ laboratory and clinical characteristics were recorded. MLR, NLR, and PLR levels were calculated. The differences were compared and the diagnostic values of MLR were analyzed. There were 76 male and 105 female patients included, with a mean age of 56.57 ± 9.95 years. The levels of MLR, NLR, and PLR in osteoporosis patients were all higher than those in healthy control subjects. The area under the curve of MLR was higher than those of NLR and PLR. Multivariate linear regression analysis showed that T-score was affected by age and MLR. MLR was positively correlated with C-reactive protein, erythrocyte sedimentation rate, red blood cell distribution width, age, sex, and inversely with hemoglobin. MLR and PLR levels were significantly higher in osteoporosis patients than in osteopenia patients ( P < .05). The present study shows that MLR had a higher diagnostic value for osteoporosis. MLR may be a reliable, inexpensive, and novel potential predictor of osteoporosis.