Sunkyu Choi

Eukaryotic Translation Initiation Factor 5A (EIF5A) Regulates Pancreatic Cancer Metastasis by Modulating RhoA and Rho-associated Kinase (ROCK) Protein Expression Levels

Background: Eukaryotic translation initiation factor 5A (eIF5A) regulates pancreatic cancer pathogenesis. Results: eIF5A was shown to control the expression of a set of key signaling molecules including RhoA and ROCK2, and to promote the invasive potential of pancreatic cancer cells. Conclusion: Hypusine/eIF5A/RhoA/ROCK cascade promotes pancreatic cancer cell metastasis. Significance: eIF5A may be a novel druggable target to treat metastatic pancreatic cancer. Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with an overall survival rate of less than 5%. The poor patient outcome in PDAC is largely due to the high prevalence of systemic metastasis at the time of diagnosis and lack of effective therapeutics that target disseminated cells. The fact that the underlying mechanisms driving PDAC cell migration and dissemination are poorly understood have hindered drug development and compounded the lack of clinical success in this disease. Recent evidence indicates that mutational activation of K-Ras up-regulates eIF5A, a component of the cellular translational machinery that is critical for PDAC progression. However, the role of eIF5A in PDAC cell migration and metastasis has not been investigated. We report here that pharmacological inhibition or genetic knockdown of eIF5A reduces PDAC cell migration, invasion, and metastasis in vitro and in vivo. Proteomic profiling and bioinformatic analyses revealed that eIF5A controls an integrated network of cytoskeleton-regulatory proteins involved in cell migration. Functional interrogation of this network uncovered a critical RhoA/ROCK signaling node that operates downstream of eIF5A in invasive PDAC cells. Importantly, eIF5A mediates PDAC cell migration and invasion by modulating RhoA/ROCK protein expression levels. Together our findings implicate eIF5A as a cytoskeletal rheostat controlling RhoA/ROCK protein expression during PDAC cell migration and metastasis. Our findings also implicate the eIF5A/RhoA/ROCK module as a potential new therapeutic target to treat metastatic PDAC cells.

Proteomic analysis of tumor necrosis factor-alpha (TNF-α)-induced L6 myotube secretome reveals novel TNF-α-dependent myokines in diabetic skeletal muscle.

There is a strong possibility that skeletal muscle can respond to irregular metabolic states by secreting specific cytokines. Obesity-related chronic inflammation, mediated by pro-inflammatory cytokines, is believed to be one of the causes of insulin resistance that results in type 2 diabetes. Here, we attempted to identify and characterize the members of the skeletal muscle secretome in response to tumor necrosis factor-alpha (TNF-α)-induced insulin resistance. To conduct this study, we comparatively analyzed the media levels of proteins released from L6 skeletal muscle cells. We found 28 TNF-α modulated secretory proteins by using separate filtering methods: Gene Ontology, SignalP, and SecretomeP, as well as the normalized Spectral Index for label-free quantification. Ten of these secretory proteins were increased and 18 secretory proteins were decreased by TNF-α treatment. Using microarray analysis of Zuker diabetic rat skeletal muscle combined with bioinformatics and Q-PCR, we found a correlation between TNF-α-mediated insulin resistance and type 2 diabetes. This novel approach combining analysis of the conditioned secretome and transcriptome has identified several previously unknown, TNF-α-dependent secretory proteins, which establish a foothold for research on the different causes of insulin resistance and their relationships with each other.

Afamin secreted from nonresorbing osteoclasts acts as a chemokine for preosteoblasts via the Akt-signaling pathway

Although it is well known that osteoclastic bone resorption is followed by osteoblastic bone formation, questions remain as to when coupling factors are produced during bone resorption and which stages of bone formation are affected by these factors. To clarify these mechanisms, we established an in vitro system to investigate the coupling phenomenon. We obtained conditioned media (CM) from osteoclasts in the early and late stages of differentiation and from bone resorption stages. The collected CM was used to treat primary mouse calvarial osteoblasts and preosteoblastic MC3T3-E1 cells and to evaluate its influence on the migration, viability, proliferation, and differentiation of osteoblasts. We found that CM from osteoclasts in the early stage of differentiation predominantly stimulated the migration of osteoblastic lineages. By further performing fractional analyses of the CM with liquid chromatography-tandem mass spectrometry, we identified afamin, which has binding activity with vitamin E, as a possible coupling factor. The CM collected from afamin siRNA-transfected osteoclasts significantly suppressed preosteoblast migration. Afamin activated Akt in preosteoblasts, and pretreatment with Akt inhibitor significantly blocked afamin-stimulated preosteoblast migration. In conclusion, these results indicate that osteoclasts themselves play a central role in the coupling of bone resorption and formation by stimulating preosteoblast migration. In addition, we identified afamin as one of osteoclast-derived chemokines that affect preosteoblasts through the activation of the Akt-signaling pathway.

Targeted label-free quantitative analysis of secretory proteins from adipocytes in response to oxidative stress

Adipocytes are well known to release regulation factors associated with metabolic disorders. In particular, increased oxidative stress in adipocytes contributes to dysregulation of adipokine production. In this study, we applied relative quantitative proteomic analysis based on label-free multiple reaction monitoring (MRM) to discover biological changes of adipokines under oxidative stress. Among a total of 194 identified proteins, 8 proteins were selected and quantified between control and hydrogen peroxide (H(2)O(2))-treated groups by label-free MRM quantification. The secretion levels of matrix metalloproteinase-2 (MMP-2), stromal cell-derived factor-1 (SDF-1, CXCL12), resistin, and complement factor D (CFD, adipsin) decreased, whereas the secretion levels of tissue inhibitor of metalloproteinase-2 (TIMP-2) and aldolase A increased. Here we suggest that our study with label-free quantitative analysis will contribute to the efficient quantitative analysis of targeted proteins in complex mixtures and specifically to a better understanding of changes of adipokines under oxidative stress.

Osmotic Stress Regulates Mammalian Target of Rapamycin (mTOR) Complex 1 via c-Jun N-terminal Kinase (JNK)-mediated Raptor Protein Phosphorylation

Background: mTORC1 integrates diverse signals including stress to control cell growth. Results: JNK phosphorylates Raptor, a component of mTORC1, and activates mTORC1 kinase upon osmotic stress. Conclusion: mTORC1 is regulated by JNK during osmotic stress. Significance: Our findings provide the JNK-Raptor relationship as a potential mechanism by which stress activates mTORC1 signaling pathway. mTOR complex 1 (mTORC1) is a multiprotein complex that integrates diverse signals including growth factors, nutrients, and stress to control cell growth. Raptor is an essential component of mTORC1 that functions to recruit specific substrates. Recently, Raptor was suggested to be a key target of regulation of mTORC1. Here, we show that Raptor is phosphorylated by JNK upon osmotic stress. We identified that osmotic stress induces the phosphorylation of Raptor at Ser-696, Thr-706, and Ser-863 using liquid chromatography-tandem mass spectrometry. We found that JNK is responsible for the phosphorylation. The inhibition of JNK abolishes the phosphorylation of Raptor induced by osmotic stress in cells. Furthermore, JNK physically associates with Raptor and phosphorylates Raptor in vitro, implying that JNK is responsible for the phosphorylation of Raptor. Finally, we found that osmotic stress activates mTORC1 kinase activity in a JNK-dependent manner. Our findings suggest that the molecular link between JNK and Raptor is a potential mechanism by which stress regulates the mTORC1 signaling pathway.

Comparative proteome analysis using amine-reactive isobaric tagging reagents coupled with 2D LC/MS/MS in 3T3-L1 adipocytes following hypoxia or normoxia.

Hypoxia during the expansion of adipocytes is known to contribute both to the secretion of multiple inflammation-related adipokines as well as to obesity. We therefore investigated the nature of protein changes occurring in adipocytes during hypoxia by observation of the intracellular proteins that are expressed in 3T3-L1 adipocytes. Lysates were utilized for quantitative proteome analysis using isobaric tags for relative and absolute quantitation (iTRAQ) combined with peptide separation by multi-dimensional liquid chromatography. Antioxidants and elongation factors, as well as glycolytic enzymes were increased in hypoxic adipocytes. These changes were supported by similar changes suggested by real-time PCR. The proteins showing changes are all potential targets for revering the mechanism behind the phenomenon of induction of obese adipocytes by hypoxia. This study can therefore aid in defining the proteomic changes that occur in adipocytes in response to oxygen stress, and can further characterize adipocyte metabolism and adaptation to low oxygen conditions.

eIF5A-PEAK1 Signaling Regulates YAP1/TAZ Protein Expression and Pancreatic Cancer Cell Growth

In pancreatic ductal adenocarcinoma (PDAC), mutant KRAS stimulates the translation initiation factor eIF5A and upregulates the focal adhesion kinase PEAK1, which transmits integrin and growth factor signals mediated by the tumor microenvironment. Although eIF5A-PEAK1 signaling contributes to multiple aggressive cancer cell phenotypes, the downstream signaling processes that mediate these responses are uncharacterized. Through proteomics and informatic analyses of PEAK1-depleted PDAC cells, we defined protein translation, cytoskeleton organization, and cell-cycle regulatory pathways as major pathways controlled by PEAK1. Biochemical and functional studies revealed that the transcription factors YAP1 and TAZ are key targets of eIF5A-PEAK1 signaling. YAP1/TAZ coimmunoprecipitated with PEAK1. Interfering with eIF5A-PEAK1 signaling in PDAC cells inhibited YAP/TAZ protein expression, decreasing expression of stem cell-associated transcription factors (STF) including Oct4, Nanog, c-Myc, and TEAD, thereby decreasing three-dimensional (3D) tumor sphere growth. Conversely, amplified eIF5A-PEAK1 signaling increased YAP1/TAZ expression, increasing expression of STF and enhancing 3D tumor sphere growth. Informatic interrogation of mRNA sequence databases revealed upregulation of the eIF5A-PEAK1-YAP1-TEAD signaling module in PDAC patients. Taken together, our findings indicate that eIF5A-PEAK1-YAP signaling contributes to PDAC development by regulating an STF program associated with increased tumorigenicity. Cancer Res; 77(8); 1997-2007. ©2017 AACR.

Functional interplay between Aurora B kinase and Ssu72 phosphatase regulates sister chromatid cohesion

Cohesins establish cohesion between replicated sister chromatids and are maintained as a multiprotein complex on chromosome arms until they are phosphorylated by mitotic kinases, such as Aurora B and Plk1. However, the mechanics of how the phosphorylation and dephosphorylation of cohesin subunits by kinases and phosphatases, respectively, leads to the dissociation of the cohesin complex from chromosomes remain unclear. Here we report that Aurora B kinase directly interacts with and phosphorylates Ssu72, a new cohesin-binding phosphatase, at Ser 19 in vitro and in vivo. The Aurora B-mediated phosphorylation of Ssu72 causes the structural modification of Ssu72 protein, downregulates phosphatase activity and triggers the ubiquitin-dependent degradation of Ssu72. Overexpression of the Aurora B-mediated phosphomimetic mutant of Ssu72 prevents maintainance chromosome arm cohesion. These results provide evidence that Aurora B kinase directly targets Ssu72 phosphatase for regulation of sister chromatid cohesion during early mitosis.

Sequential Fe3O4/TiO2 enrichment for phosphopeptide analysis by liquid chromatography/tandem mass spectrometry

Protein phosphorylation regulates a wide range of cellular functions and is associated with signaling pathways in cells. Various strategies for enrichment of phosphoproteins or phosphopeptides have been developed. Here, we developed a novel sequential phosphopeptide enrichment method, using magnetic iron oxide (Fe(3)O(4)) and titanium dioxide (TiO(2)) particles, to detect mono- and multi-phosphorylated peptides. In the first step, phosphopeptides were captured on Fe(3)O(4) particles. In a subsequent step, any residual phosphopeptides were captured on TiO(2) particles. The particles were eluted and rinsed to yield phosphopeptide-enriched fractions that were combined and analyzed using liquid chromatography/tandem mass spectrometry (LC/MS/MS). The validity of this sequential Fe(3)O(4)/TiO(2) enrichment strategy was demonstrated by the successful enrichment of bovine alpha-casein phosphopeptides. We then applied the sequential Fe(3)O(4)/TiO(2) enrichment method to the analysis of phosphopeptides in L6 muscle cell lysates and successfully identified mono- and multi-phosphorylated peptides.

Quantitative pattern analysis of the N-terminally processed isoforms of platelet factor-4 in serum

RATIONALE Platelet factor 4 (PF4) is a small cytokine belonging to the CXC chemokine family which has been shown to play a role in inflammation and in the regulation of angiogenesis. In general, chemokines are susceptible to proteolytic cleavage in amino and carboxy terminal regions, which usually results in dramatic changes to the chemokine bioactivity. The purpose of this study was to identify various platelet factor-4 (PF4) isoforms caused by proteolytic processing and to quantify their levels in normal serum. METHODS First, we identified the N-terminally truncated PF4 isoforms from a standard purified PF4 protein sample by using mass spectrometry (MS) and tandem mass spectrometry (MS/MS) analysis. Then, we used high-performance liquid chromatography (HPLC) to semi-purify PF4 from serum samples, and the levels of the four most abundant PF4 isoforms were quantitatively determined using selected reaction monitoring (SRM) assays on a nano-LC/triple-quadrupole mass spectrometer. RESULTS We have identified seven N-terminally processed PF4 isoforms and compared the levels of major PF4 isoforms from nine serum samples. Pro-p1 (EAEEDGDLQCLCVK-; average MW, 7765.2) is the major PF4 isoform in serum whereas the PF4 isoforms, designated Prot-p4 (FASAEAEEDGDLQCLCVK-;average MW, 8141.5), Prot-p3 (SAEAEEDGDLQCLCVK-; average MW, 7923.3), and Prot-p2 (AEEDGDLQCLCVK- ; average MW, 7836.3), are at about 16%, 3%, and 2% levels of the major one, respectively. CONCLUSIONS This study is the first report on the levels of N-terminally processed PF4 isoforms in serum. Also, this study shows the usefulness of SRM in determining concentrations of protein isoform variants, which can be often overlooked in immunoassay analysis.