Heterotrimeric G proteins have been implicated in the regulation of membrane trafficking, but the mechanisms involved are not well understood. Here, we report that overexpression of the stimulatory G protein subunit (Gαs) promotes ligand-dependent degradation of epidermal growth factor (EGF) receptors and Texas Red EGF, and knock-down of Gαs expression by RNA interference (RNAi) delays receptor degradation. We also show that Gαs and its GTPase activating protein (GAP), RGS-PX1, interact with hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), a critical component of the endosomal sorting machinery. Gαs coimmunoprecipitates with Hrs and binds Hrs in pull-down assays. By immunofluorescence, exogenously expressed Gαs colocalizes with myc-Hrs and GFP-RGS-PX1 on early endosomes, and expression of either Hrs or RGS-PX1 increases the localization of Gαs on endosomes. Furthermore, knock-down of both Hrs and Gαs by double RNAi causes greater inhibition of EGF receptor degradation than knock-down of either protein alone, suggesting that Gαs and Hrs have cooperative effects on regulating EGF receptor degradation. These observations define a novel regulatory role for Gαs in EGF receptor degradation and provide mechanistic insights into the function of Gαs in endocytic sorting

RGS-PX1 (also known as sorting nexin 13) is a member of both the regulator of G protein signaling (RGS) and sorting nexin (SNX) protein families. Biochemical and cell culture studies have shown that RGS-PX1͞SNX13 attenuates G␣s-mediated signaling through its RGS domain and regulates endocytic trafficking and degradation of the epidermal growth factor receptor. To understand the functions of RGS-PX1͞SNX13 in vivo, we generated mice carrying targeted mutations of Snx13 and found that systemic Snx13-null mice were embryonic lethal around midgestation. Snx13-null embryos had significant overall growth retardation and defects in neural tube closure, blood vessel formation, and the formation of the placental labyrinthine layer. Moreover, the Snx13-null visceral yolk sac endoderm cells showed dramatic changes in the organization of endocytic compartments, abundant autophagic vacuoles, and abnormal localization of several endocytic markers, including megalin, a receptor for nutrients and proteins; ARH, a coat protein that binds megalin; LAMP2; and LC3. These changes suggest that Snx13-null embryos are defective in nutrient uptake and transport, which may contribute to the other developmental abnormalities observed. Taken together, our findings demonstrate an essential role for RGS-PX1͞SNX13 in mouse development and provide previously undescribed insights into its cellular function in the regulation of endocytosis dynamics.megalin ͉ receptor endocytosis ͉ regulator of G protein signaling ͉ visceral yolk sac endoderm R eceptor-mediated endocytosis is the process by which plasma membrane components and extracellular materials such as nutrients, hormones, antigens, and other macromolecules are selectively internalized into cytoplasmic vesicles, delivered to early and late endosomes and degraded in lysosomes or recycled back to the plasma membrane through recycling endosomes (1-3). Trafficking between these endocytic compartments is highly regulated and often involves critical sorting steps. Recently, a large family of sorting nexin (SNX) proteins characterized by the presence of Phox (PX) domains has been identified and implicated in the regulation of different steps of the endocytic pathway (4, 5). For example, the founding member of the SNX family, SNX1, was identified as an interacting partner of the epidermal growth factor receptor, and overexpression of SNX1 was found to enhance lysosomal degradation of epidermal growth factor receptor (6). More recently, knock-down of SNX1 expression by RNA interference was shown to perturb the steady state distribution of the cation-independent mannose-6-phosphate receptor (CI-MPR) from the trans-Golgi network to endosomes and to increase the degradation rate of CI-MPR, establishing a role for SNX1 in endosome-to-trans-Golgi network transport (7).RGS-PX1 (also known as SNX 13, the designation by the human gene nomenclature committee) was identified originally as a member of the regulator of G protein signaling (RGS) protein family through bioinformatics analysis (8). RGS-PX1 contains...

The theory of cancer immunoediting refers to mechanisms by which the immune system can suppress or promote tumour progression. A major challenge for the development of novel cancer immunotherapies is to find ways to exploit the immune system's antitumour activity while concomitantly reducing its protumour activity. Using the PyVmT model of mammary tumourigenesis, we show that lack of the Usp18 gene significantly inhibits tumour growth by creating a tumour-suppressive microenvironment. Generation of this antitumour environment is driven by elevated secretion of the potent T-cell chemoattractant Cxcl10 by Usp18 deficient mammary epithelial cells (MECs), which leads to recruitment of Th1 subtype CD4+ T cells. Furthermore, we show that Cxcl10 upregulation in MECs is promoted by interferon-λ and that Usp18 is a novel inhibitor of interferon-λ signalling. Knockdown of the interferon-λ specific receptor subunit IL-28R1 in Usp18 deficient MECs dramatically enhances tumour growth. Taken together, our data suggest that targeting Usp18 may be a viable approach to boost antitumour immunity while suppressing the protumour activity of the immune system.

Mitochondria are dynamic organelles that play key roles in metabolism, energy production, and apoptosis. Coordination of these processes is essential to maintain normal cellular functions. Here we characterized hNOA1, the human homologue of AtNOA1 (Arabidopsis thaliana nitric oxide-associated protein 1), a large mitochondrial GTPase. By immunofluorescence, immunoelectron microscopy, and mitochondrial subfractionation, endogenous hNOA1 is localized within mitochondria where it is peripherally associated with the inner mitochondrial membrane facing the mitochondrial matrix. Overexpression and knockdown of hNOA1 led to changes in mitochondrial shape implying effects on mitochondrial dynamics. To identify the interaction partners of hNOA1 and to further understand its cellular functions, we performed immunoprecipitation-mass spectrometry analysis of endogenous hNOA1 from enriched mitochondrial fractions and found that hNOA1 interacts with both Complex I of the electron transport chain and DAP3 (death-associated protein 3), a positive regulator of apoptosis. Knockdown of hNOA1 reduces mitochondrial O 2 consumption ϳ20% in a Complex I-dependent manner, supporting a functional link between hNOA1 and Complex I. Moreover, knockdown of hNOA1 renders cells more resistant to apoptotic stimuli such as ␥-interferon and staurosporine, supporting a role for hNOA1 in regulating apoptosis. Thus, based on its interactions with both Complex I and DAP3, hNOA1 may play a role in mitochondrial respiration and apoptosis.Emerging evidence indicates that mitochondrial metabolism, apoptosis, and dynamics (fission and fusion) are closely intertwined. Apoptosis and changes in metabolism are associated with morphological changes in mitochondria (1, 2). Conversely, when mitochondrial morphology is altered either by mutations or altered expression of mitochondrial fission or fusion proteins such as the dynamin like large G proteins Drp1 and Opa1, the cell's susceptibility to apoptotic agents (3) or ability to generate ATP (4, 5) is altered.Apoptosis is controlled by a diverse range of cell signals, which may originate either extracellularly (extrinsic inducers) or intracellularly (intrinsic inducers), and mitochondria play central roles in both pathways (6). The apoptotic pathways involve a growing list of mitochondria-associated proteins, such as Bad, cytochrome c, Smac, AIF, Bcl-2, and others, most of which are located either on the outer mitochondrial membrane (OMM) 3 or in the intermembrane space (IMS) (7). Recently, proteins of the mitochondrial matrix such as DAP3, have also been shown to be involved in apoptosis (8). DAP3 has been reported to be involved in both ␥-interferon-(9) and tumor necrosis factor-␣-induced (10) apoptosis as well as staurosporine-induced mitochondrial fragmentation (11), but the detailed mechanisms involved remain to be elucidated.Besides their role in apoptosis, much more is known about the functions of mitochondria in respiration and generation of ATP. The electron transport chain in the inner mitochondrial mem...

Non-small cell lung cancer (NSCLC) is featured with complex genomic alterations. Molecular profiling of large cohort of NSCLC patients is thus a prerequisite for precision medicine. We first validated the detection performance of a next-generation sequencing (NGS) cancer hotspot panel, OncoAim, on formalin-fixed paraffin-embedded (FFPE) samples. We then utilized OncoAim to delineate the genomic aberrations in Chinese NSCLC patients. Overall detection performance was powerful for mutations with allele frequency (MAF) ≥ 5% at >500 × coverage depth, with >99% sensitivity, high specificity (positive predictive value > 99%), 94% accuracy and 96% repeatability. Profiling 422 NSCLC FFPE samples revealed that patient characteristics, including gender, age, lymphatic spread, histologic grade and histologic subtype were significantly associated with the mutation incidence of EGFR and TP53. Moreover, RTK signaling pathway activation was enriched in adenocarcinoma, while PI(3)K pathway activation, oxidative stress pathway activation, and TP53 pathway inhibition were more prevalent in squamous cell carcinoma. Additionally, novel co-existence (e.g., variants in BRAF and PTEN) and mutual-exclusiveness (e.g., alterations in EGFR and NFE2L2) were found. Finally, we revealed distinct mutation spectrum in TP53, as well as a previously undervalued PTEN aberration. Our findings could aid in improving diagnosis, prognosis and personalized therapeutic decisions of Chinese NSCLC patients.Lung cancer is the leading cause of cancer-associated mortality worldwide. Up to 90% lung cancer is non-small cell lung cancer (NSCLC) 1 . NSCLC is usually diagnosed at the advanced stage with limited therapeutic options (e.g., surgery, radiotherapy and chemotherapy) 1 .Conventional molecular detection techniques, such as fluorescence in situ hybridization and Sanger sequencing, only detect a limited number of biomarkers 2-4 . The advent of next-generation sequencing (NGS) has broadened the landscape of genetic aberrations 5-7 , making it possible to implement targeted treatment tailored for specific mutations in individual patients 1,8,9 . Lung cancer is characterized with complex genomic aberrations 10 . The most frequently mutated genes in NSCLC include EGFR (Epidermal growth factor receptor), TP53 (Tumor Protein p53), KRAS (Kirsten rat sarcoma viral oncogene) and PIK3CA (Phosphatidylinositol 3-kinase). In addition, Genetic mutations detected in NSCLC are complicated by patient demographic, racial, clinical and pathological characteristics 1,8,9,11 . For instance, the mutational frequency of EGFR can vary from 10% in Western populations to 30% in Asians, and KRAS mutation incidence in Western populations is approximately 18-26% versus 3.8-8% in Asians 12 , and within

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Next-generation sequencing (NGS) has become a promising approach for tumor somatic mutation detection. However, stringent validation is required for its application on clinical specimens, especially for low-quality formalin-fixed paraffin-embedded (FFPE) tissues. Here, we validated the performance of an amplicon-based targeted NGS assay, OncoAim™ DNA panel, on both commercial reference FFPE samples and clinical FFPE samples of Chinese colorectal cancer (CRC) patients. Then we profiled the mutation spectrum of 648 Chinese CRC patients in a multicenter study to explore its clinical utility. This NGS assay achieved 100% test specificity and 95-100% test sensitivity for variants with mutant allele frequency (MAF) ≥ 5% when median read depth ≥ 500×. The orthogonal methods including amplification refractory mutation system (ARMS)-PCR and Sanger sequencing validated that NGS generated three false negatives (FNs) but no false positives (FPs) among 516 clinical samples for KRAS aberration detection. Genomic profiling of Chinese CRC patients with this assay revealed that 63.3% of the tumors harbored clinically actionable alterations. Besides the commonly mutated genes including TP53 (52.82%), KRAS (46.68%), APC (24.09%), PIK3CA (18.94%), SMAD4 (9.47%), BRAF (6.15%), FBXW7 (5.32%), and NRAS (4.15%), other less frequently mutated genes were also identified. Statistically significant association of specific mutated genes with certain clinicopathological features was detected, e.g., both BRAF and PIK3CA were more prevalent in right-side CRC (p < 0.001 and p = 0.002, respectively). We concluded this targeted NGS assay is qualified for clinical practice, and our findings could help the diagnosis and prognosis of Chinese CRC patients.

Mutations of