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Supplementary Table S5. is a List of Intact glycopeptides of purified FAM198B identified by Orbitrap Velos MS analysis and Byonic processing.

Purpose: The comprehensive understanding of mechanisms involved in the tumor metastasis is urgently needed for discovering novel metastasis-related genes for developing effective diagnoses and treatments for lung cancer.Experimental Design: FAM198B was identified from an isogenic lung cancer metastasis cell model by microarray analysis. To investigate the clinical relevance of FAM198B, the FAM198B expression of 95 Taiwan lung adenocarcinoma patients was analyzed by quantitative real-time PCR and correlated to patients' survivals. The impact of FAM198B on cell invasion, metastasis, and tumor growth was examined by in vitro cellular assays and in vivo mouse models. In addition, the N-glycosylation–defective FAM198B mutants generated by site-directed mutagenesis were used to study protein stability and subcellular localization of FAM198B. Finally, the microarray and pathway analyses were used to elucidate the underlying mechanisms of FAM198B-mediated tumor suppression.Results: We found that the high expression of FAM198B was associated with favorable survival in Taiwan lung adenocarcinoma patients and in a lung cancer public database. Enforced expression of FAM198B inhibited cell invasion, migration, mobility, proliferation, and anchorage-independent growth, and FAM198B silencing exhibited opposite activities in vitro. FAM198B also attenuated tumor growth and metastasis in vivo. We further identified MMP-1 as a critical downstream target of FAM198B. The FAM198B-mediated MMP-1 downregulation was via inhibition of the phosphorylation of ERK. Interestingly deglycosylation nearly eliminated the metastasis suppression activity of FAM198B due to a decrease of protein stability.Conclusions: Our results implicate FAM198B as a potential tumor suppressor and to be a prognostic marker in lung adenocarcinoma. Clin Cancer Res; 24(4); 916–26. ©2017 AACR.

The Lon AAA+ protease (LonA) is a ubiquitous ATP-dependent proteolytic machine, which selectively degrades damaged proteins or native proteins carrying exposed motifs (degrons). Here we characterize the structural basis for substrate recognition and discrimination by the N-terminal domain (NTD) of LonA. The results reveal that the six NTDs are attached to the hexameric LonA chamber by flexible linkers such that the formers tumble independently of the latter. Further spectral analyses show that the NTD selectively interacts with unfolded proteins, protein aggregates, and degron-tagged proteins by two hydrophobic patches of its N-lobe, but not intrinsically disordered substrate, α-casein. Moreover, the NTD selectively binds to protein substrates when they are thermally induced to adopt unfolded conformations. Collectively, our findings demonstrate that NTDs enable LonA to perform protein quality control to selectively degrade proteins in damaged states and suggest that substrate discrimination and selective degradation by LonA are mediated by multiple NTD interactions., eLife digest There are many different types of protein which each have different roles in biology. Most proteins are surrounded by water and are folded so that their water-attracting regions are on the outside and more fat-like regions, which repel water, are on the inside. When a protein becomes damaged or is assembled incorrectly, some of the fat-like regions end up on the outside of the protein and become exposed to water. This can prevent the protein from performing its role and harm the cell instead. LonA proteases are responsible for dismantling and recycling these harmful proteins, as well as proteins that have been labelled for destruction. They do this by unfolding the unwanted protein and transporting it into an enclosed chamber made of six LonA molecules. Once inside the chamber, the target protein is broken down into smaller fragments that can be used to build other structures. LonA proteases contain a region called the N-terminal domain, or NTD for short, which is thought to be responsible for identifying which proteins need degrading. Yet it remained unclear how the NTD recognizes and binds to these target proteins. To answer this question, Tzeng et al. studied the detailed structure of a LonA protease that had been purified from bacteria cells. This revealed that the NTD of LonA contains two water-repelling regions which bind to fat-like segments on the surface of proteins that have become unfolded or tagged for destruction. Further experiments showed that the NTD is bound to the main body of LonA via a ‘flexible linker’. This led Tzeng et al. to propose that the NTD sways around loosely at the end of LonA searching for proteins with exposed water-repelling regions. Once an NTD identifies and attaches to a target, the NTDs of the other LonA molecules then bind to the protein and help insert it into the chamber. Proteases are a vital component of all biological systems. Controlling protein destruction and recycling is a key factor in how cells divide and respond to a changing environment. This study provides new insights into how LonA operates in bacteria, which may apply to proteases more widely. This contributes to our knowledge of fundamental biology and may also be relevant in a range of diseases where protein recycling is defective or inefficient.

Purpose: The comprehensive understanding of mechanisms involved in the tumor metastasis is urgently needed for discovering novel metastasis-related genes for developing effective diagnoses and treatments for lung cancer. Experimental Design: FAM198B was identified from an isogenic lung cancer metastasis cell model by microarray analysis. To investigate the clinical relevance of FAM198B, the FAM198B expression of 95 Taiwan lung adenocarcinoma patients was analyzed by quantitative real-time PCR and correlated to patients' survivals. The impact of FAM198B on cell invasion, metastasis, and tumor growth was examined by in vitro cellular assays and in vivo mouse models. In addition, the N-glycosylation–defective FAM198B mutants generated by site-directed mutagenesis were used to study protein stability and subcellular localization of FAM198B. Finally, the microarray and pathway analyses were used to elucidate the underlying mechanisms of FAM198B-mediated tumor suppression. Results: We found that the high expression of FAM198B was associated with favorable survival in Taiwan lung adenocarcinoma patients and in a lung cancer public database. Enforced expression of FAM198B inhibited cell invasion, migration, mobility, proliferation, and anchorage-independent growth, and FAM198B silencing exhibited opposite activities in vitro. FAM198B also attenuated tumor growth and metastasis in vivo. We further identified MMP-1 as a critical downstream target of FAM198B. The FAM198B-mediated MMP-1 downregulation was via inhibition of the phosphorylation of ERK. Interestingly deglycosylation nearly eliminated the metastasis suppression activity of FAM198B due to a decrease of protein stability. Conclusions: Our results implicate FAM198B as a potential tumor suppressor and to be a prognostic marker in lung adenocarcinoma. Clin Cancer Res; 24(4); 916–26. ©2017 AACR.

// Yi-Jing Hsiao 1 , Kang-Yi Su 1, 2, 3 , Yi-Chiung Hsu 4 , Gee-Chen Chang 5, 6 , Jin-Shing Chen 7 , Hsuan-Yu Chen 2, 4 , Qi-Sheng Hong 1 , Shih-Chun Hsu 1 , Po-Hsiang Kang 1 , Chia-Ying Hsu 1 , Bing-Ching Ho 1 , Tsung-Hui Yang 1 , Chia-Yu Wang 1 , Yuh-Shan Jou 8 , Pan-Chyr Yang 2, 8, 9 , Sung-Liang Yu 1, 2, 3, 10, 11, 12 1 Department of Clinical and Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, Taipei, Taiwan 2 Center of Genomic Medicine, National Taiwan University College of Medicine, Taipei, Taiwan 3 Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan 4 Institute of Statistical Science, Academia Sinica, Taipei, Taiwan 5 Faculty of Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan 6 Division of Chest Medicine, Department of Internal Medicine, Taichung Veterans General Hospital, Taichung, Taiwan 7 Division of Thoracic Surgery and Department of Surgery, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan 8 Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan 9 Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan 10 Department of Pathology and Graduate Institute of Pathology, National Taiwan University College of Medicine, Taipei, Taiwan 11 Center for Optoelectronic Biomedicine, National Taiwan University College of Medicine, Taipei, Taiwan 12 Institute of Medical Device and Imaging, National Taiwan University College of Medicine, Taipei, Taiwan Correspondence to: Sung-Liang Yu, email: slyu@ntu.edu.tw Keywords: SPANX family, AP-1, SLUG, E-cadherin, metastasis Received: January 07, 2016 Accepted: June 01, 2016 Published: June 15, 2016 ABSTRACT SPANXA (Sperm Protein Associated with the Nucleus on the X-chromosome, family members A1/A2) acts as a cancer-testis antigen expressed in normal testes, but dysregulated in various tumors. We found that SPANXA is highly expressed in low-invasive CL1-0 cells compared with isogenous high-invasive CL1-5 cells. SPANXA was preferably expressed in tumor tissues and associated with the prolonged survival of lung adenocarcinomas. SPANXA suppressed the invasion and metastasis of lung cancer cells in vitro and in vivo . By the expression microarray and pathway analysis, we found that the SPANXA-altered genes were enriched in the epithelial–mesenchymal transition (EMT) pathway. SPANXA reduced SNAI2 expression resulted in up-regulating E-cadherin . c-JUN acts as the positive-regulator of EMT. Silencing SPANXA increased c-JUN mRNA expression and blockage of c-JUN led to SNAI2 down-regulation. Our results clearly characterized SPANXA as an EMT inhibitor by suppressing c-JUN-SNAI2 axis in lung adenocarcinoma.

Roasting treatment increased levels of unsaturated fatty acids (linoleic, oleic and elaidic acids) as well as saturated fatty acids (palmitic and stearic acids) in almond (Prunus dulcis) kernel oils with temperature (150 or 180 °C) and duration (5, 10 or 20 min). Nonetheless, higher temperature (200 °C) and longer duration (10 or 20 min) roasting might result in breakdown of fatty acids especially for unsaturated fatty acids. Phenolic components (total phenols, flavonoids, condensed tannins and phenolic acids) of almond kernels substantially lost in the initial phase; afterward these components gradually increased with roasting temperature and duration. Similar results also observed for their antioxidant activities (scavenging DPPH and ABTS(+) radicals and ferric reducing power). The changes of phenolic acid and flavonoid compositions were also determined by HPLC. Maillard reaction products (estimated with non-enzymatic browning index) also increased with roasting temperature and duration; they might also contribute to enhancing the antioxidant attributes.

SPANX (Sperm protein associated with the nucleus, X-linked) gene family has been found higher expression in many cancers, such as testicular germ tumor, melanoma, glioblastoma and hematologic malignancies. However, in our previous study, the gene expression array analysis showed that the expression of SPANX family member A1 (SPANXA1) was higher in lower invasive lung cancer cells, CL 1-0, than in higher invasive cells, CL 1-5. Real-time PCR was used to confirm that mRNA expression of SPANXA1 was 8 times higher in CL 1-0 than CL 1-5. In vitro assays showed that migration and invasion abilities of CL 1-5 stable transfectants were decreased in a dose-depended manner. Interestingly, cell morphology dramatically changed from mesenchymal-like to epithelial-like in CL 1-5 SPANXA1 stable transfectants, which was consistent to its decreased invasive ability in vitro. Therefore, we demonstrated metastasis ability of CL 1-5 SPANXA1 stable transfectants would be down-regulated in vivo, which was performed in mouse metastasis model by tail-vein injection. Next, we set up CL 1-0 lentiviral-base shRNA knockdown clones, and the cell mobility and invasion ability were increased. Because SPANXA1 could induce the cell morphology change, we evaluate the effect of SPANXA1 on the expression of EMT markers. We found up-regulation of E-cadherin, an epithelial marker, and down-regulation of vimentin and N-cadherin, which were known as mesenchymal markers in presence of SPANXA1. Finally, to identify the underlying mechanism of SPANXA1, we carried out gene expression array and 3 of top 10 pathways are EMT-related. Furthermore, quantitative RT-PCR validation showed WNT pathway is involved in SPANXA1-repressed metastasis indicated that SPANXA1 might provide a new treatment in lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3990. doi:1538-7445.AM2012-3990