C9orf10/Ossa regulates the bone metastasis of established lung adenocarcinoma cell subline H322L-BO4 in a mouse model.

Lung cancer frequently metastasizes to the bones. An in vivo model is urgently required to identify potential therapeutic targets for the prevention and treatment of lung cancer with bone metastasis. We established a lung adenocarcinoma cell subline (H322L-BO4) that specifically showed metastasis to the leg bones and adrenal glands. This was achieved by repeated isolation of metastatic cells from the leg bones of mice. The cells were intracardially injected into nude mice. Survival was prolonged for mice that received H322L-BO4 cells versus original cells (H322L). H322L-BO4 cells did not exhibit obvious changes in general in vitro properties associated with the metastatic potential (e.g., cell growth, migration, and invasion) compared with H322L cells. However, the phosphorylation of chromosome 9 open reading frame 10/oxidative stress-associated Src activator (C9orf10/Ossa) was increased in H322L-BO4 cells. This result confirmed the increased anchorage independence through C9orf10/Ossa-mediated activation of Src family tyrosine kinase. Reduction of C9orf10/Ossa by shRNA reduced cells' metastasis to the leg bone and prolonged survival in mice. These findings indicate that H322L-BO4 cells can be used to evaluate the effect of candidate therapeutic targets against bone metastatic lung cancer cells. Moreover, C9orf10/Ossa may be a useful target for treatment of lung cancer with bone metastasis.

An ionic liquid-assisted sample preparation method for sensitive integral-membrane proteome analysis.

Many ion channels and receptor proteins are potential targets for new drugs. However, standard methods for profiling these integral membrane proteins (IMPs) have not been fully established, especially when applied to rare and quantity-limited biological samples. We previously demonstrated that a mixture containing 1-butyl-3-methylimidazolium cyanate, an ionic liquid (IL), and NaOH (termed i-soln) is an excellent solubilizer for insoluble aggregates. In this study, we present a combined i-soln-assisted proteomic sample preparation platform (termed pTRUST), which is compatible with starting materials in the sub-microgram range, using our previously reported i-soln-based sample preparation strategy (iBOPs) and an in-StageTip technique. This novel and straightforward approach allows for the rapid solubilization and processing of a variety of IMPs from human samples to support highly sensitive mass spectrometry analysis. We also demonstrated that the performance of this technology surpasses that of conventional methods such as filter-aided sample preparation methods, FASP and i-FASP. The convenience and availability of pTRUST technology using the IL system have great potential for proteomic identification and characterization of novel drug targets and disease biology in research and clinical settings.

Ubiquitination-coupled liquid phase separation regulates the accumulation of the TRIM family of ubiquitin ligases into cytoplasmic bodies.

Many members of the tripartite motif (TRIM) family of ubiquitin ligases localize in spherical, membrane-free structures collectively referred to as cytoplasmic bodies (CBs) in a concentration-dependent manner. These CBs may function as aggresome precursors or storage compartments that segregate potentially harmful excess TRIM molecules from the cytosolic milieu. However, the manner in which TRIM proteins accumulate into CBs is unclear. In the present study, using TRIM32, TRIM5α and TRIM63 as examples, we demonstrated that CBs are in a liquid droplet state, resulting from liquid-liquid phase separation (LLPS). This finding is based on criteria that defines phase-separated structures, such as recovery after photobleaching, sensitivity to hexanediol, and the ability to undergo fusion. CB droplets, which contain cyan fluorescent protein (CFP)-fused TRIM32, were purified from HEK293 cells using a fluorescence-activated cell sorter and analyzed by LC-MS/MS. We found that in addition to TRIM32, these droplets contain a variety of endogenous proteins and enzymes including ubiquitin. Localization of ubiquitin within CBs was further verified by fluorescence microscopy. We also found that the activation of the intracellular ubiquitination cascade promotes the assembly of TRIM32 molecules into CBs, whereas inhibition causes suppression. Regulation is dependent on the intrinsic E3 ligase activity of TRIM32. Similar regulation by ubiquitination on the TRIM assembly was also observed with TRIM5α and TRIM63. Our findings provide a novel mechanical basis for the organization of CBs that couples compartmentalization through LLPS with ubiquitination.

An Ionic Liquid-Based Sample Preparation Method for Next-Stage Aggregate Proteomic Analysis.

A wide variety of proteomic methods have been applied for protein profiling of insoluble aggregates or inclusion bodies deposited in various cells or tissues. However, these are essentially optimized or modified classical protein chemistry techniques using conventional denaturing agents such as formic acid, urea, and sodium dodecyl sulfate (SDS). The use of these denaturants has several shortcomings, including limited solubilization, contamination, and restrictions on absolute sample quantity and throughput. Here, we describe an alternative proteomic sample preparation platform for widespread aggregation analysis. This approach combines two techniques, (1) the use of ionic liquid for protein solubilization and (2) the recently published microbead-based and organic-media-assisted proteolysis strategy (BOPs), into a single-tube workflow. We demonstrate that the combined approach (iBOPs) enabled the successful solubilization of heat-aggregated hen egg whites within 10 min and supported sensitive mass spectrometry (MS) analysis. The performance of the iBOPs system surpassed those of conventional detergents and chaotropes. Moreover, this technology enabled ultrasensitive proteomic characterization of protein aggregates deposited in individual Caenorhabditis elegans nematodes. We identified ubiquitin and other molecules as candidate stochastic factors whose accumulation levels varied among aging nematode individuals. The sensitivity and applicability of the present iBOPs make it especially attractive for next-stage aggregate proteomic analysis of various biological processes.

Homophilic complex formation of CDCP1 via the extracellular CUB2 domain facilitates SFK activation and promotes cancer cell migration.

CUB domain­containing protein 1 (CDCP1) is phosphorylated by Src family kinases (SFK), and is thought to serve an important role in tumor metastasis through downstream signaling subsequent to its interaction with protein kinase C δ. The present study investigated the mechanisms of activation for CDCP1 signaling, and demonstrated that CDCP1 is able to activate SFK via a homophilic complex of the extracellular complement C1r/C1s, urchin embryonic growth factor, bone morphogenetic protein 1 (CUB) 2 domain. Deletion of the extracellular CDCP1 region abolished homophilic complex formation of CDCP1 and the ability to promote cancer cell migration. When the culture medium was supplemented with recombinant CUB2 domain protein fused with maltose binding protein (rMBP­CUB2), CDCP1 homophilic complex formation was effectively inhibited. rMBP­CUB2 also inhibited SFK activation and the migratory capacity of invasive human lung adenocarcinoma A549 cells, and human pancreatic BxPC3 cells. These findings demonstrated a novel function for the extracellular CUB2 domain of CDCP1, promoting cancer cell migration via SFK activation on the plasma membrane. It was also indicated that the region blocking the homophilic binding site may be a potential therapeutic target against CDCP1­dependent tumor invasion.

A Sensitive Microbead-Based Organic Media-Assisted Method for Proteomics Sample Preparation from Dilute and Denaturing Solutions.

We developed a robust and sensitive sample preparation method for proteomics termed microbead-based and organic-media-assisted proteolysis strategy (BOPs). BOPs combines two advantages of current techniques, (1) unbiased binding of reversed-phase polymeric microbeads to any type of protein and (2) enhanced trypsin digestion efficiency in CH3CN-aqueous solvent systems, into a single-tube workflow. Compared with conventional techniques, this method effectively concentrates proteins and improves proteolytic digestion, and can be used with submicromolar protein samples in dilute or denaturing solutions, such as 70% formic acid, 8 M urea, or 7 M guanidine hydrochloride without any sample pretreatment. Proteome analysis of single Caenorhabditis elegans organisms demonstrates that BOPs has the sensitivity, reproducibility, and unbiasedness required to characterize worm proteins at a single organism level. We also show that, by simply incorporating an acetone washing step for detergent removal, BOPs is applicable to low concentration samples contaminated with a variety of detergents, including sodium dodecyl sulfate, with negligible protein loss. Moreover, the utility of this modification has also been demonstrated through proteomic characterization of 2000 human (HEK293T) cells lysed using 1% Triton X-100. The simplicity and availability of the present BOPs make it especially attractive for next-stage proteomics of rare and sample-limited systems.

TRIM32-Cytoplasmic-Body Formation Is an ATP-Consuming Process Stimulated by HSP70 in Cells.

The spontaneous and energy-releasing reaction of protein aggregation is typically prevented by cellular quality control machinery (QC). TRIM32 is a member of the TRIM (tripartite motif-containing) ubiquitin E3 ligases, and when overexpressed in cultured cells, readily forms spherical inclusions designated as cytoplasmic bodies (CBs) even without proteasome inhibition. Here, we show that HSP70, a central QC component, is a primary binding factor of overexpressed TRIM32. Contrary to expectation, however, we find that this molecular chaperone facilitates and stabilizes CB assembly depending on intrinsic ATPase activity, rather than preventing CB formation. We also show that the HSP70-TRIM32 complex is biochemically distinct from the previously characterized 14-3-3-TRIM32 phospho-complex. Moreover, the two complexes have opposing roles, with HSP70 stimulating CB formation and 14-3-3 retaining TRIM32 in a diffuse form throughout the cytosol. Our results suggest that CB inclusion formation is actively controlled by cellular QC and requires ATP, similar to protein folding and degradation reactions.

14-3-3 proteins sequester a pool of soluble TRIM32 ubiquitin ligase to repress autoubiquitylation and cytoplasmic body formation.

Deregulated expression of tripartite motif-containing protein 32 (TRIM32, an E3 ubiquitin-protein ligase) contributes to various diseases. Here we report, using quantitative proteomics and biochemistry, that 14-3-3 proteins bind to phosphorylated TRIM32 and prevent TRIM32 autoubiquitylation and the formation of TRIM32-containing cytoplasmic bodies, which are potential autoregulatory mechanisms that can reduce the concentration of soluble free TRIM32. The 14-3-3-TRIM32 interaction is dependent on protein-kinase-A-catalyzed phosphorylation of TRIM32 at Ser651. We found that the inhibitory effect of 14-3-3 is, in part, a consequence of disrupting the propensity of TRIM32 to undergo higher-order self-association without affecting its dimerization. Consequently, dimerized TRIM32 bound to 14-3-3 was sequestered in a distinct cytoplasmic pool away from the microtubule network, whereas a TRIM32 mutant that cannot bind 14-3-3 underwent multimerization and was unavailable to facilitate cell growth. Our results reveal a novel connection between ubiquitylation and phosphorylation pathways, which could modulate a variety of cell events by stimulating the formation of the 14-3-3-TRIM32 signaling complex.

Interaction of nucleosome assembly proteins abolishes nuclear localization of DGKζ by attenuating its association with importins.

Diacylglycerol kinase (DGK) is involved in the regulation of lipid-mediated signal transduction through the metabolism of a second messenger diacylglycerol. Of the DGK family, DGKζ, which contains a nuclear localization signal, localizes mainly to the nucleus but translocates to the cytoplasm under pathological conditions. However, the detailed mechanism of translocation and its functional significance remain unclear. To elucidate these issues, we used a proteomic approach to search for protein targets that interact with DGKζ. Results show that nucleosome assembly protein (NAP) 1-like 1 (NAP1L1) and NAP1-like 4 (NAP1L4) are identified as novel DGKζ binding partners. NAP1Ls constitutively shuttle between the nucleus and the cytoplasm in transfected HEK293 cells. The molecular interaction of DGKζ and NAP1Ls prohibits nuclear import of DGKζ because binding of NAP1Ls to DGKζ blocks import carrier proteins, Qip1 and NPI1, to interact with DGKζ, leading to cytoplasmic tethering of DGKζ. In addition, overexpression of NAP1Ls exerts a protective effect against doxorubicin-induced cytotoxicity. These findings suggest that NAP1Ls are involved in a novel molecular basis for the regulation of nucleocytoplasmic shuttling of DGKζ and provide a clue to examine functional significance of its translocation under pathological conditions.

E6AP ubiquitin ligase mediates ubiquitin-dependent degradation of peroxiredoxin 1.

E6-associated protein (E6AP) is a cellular ubiquitin protein ligase that mediates ubiquitylation and degradation of tumor suppressor p53 in conjunction with the high-risk human papillomavirus E6 protein. We previously reported that E6AP targets annexin A1 protein for ubiquitin-dependent proteasomal degradation. To gain a better understanding of the physiological function of E6AP, we have been seeking to identify novel substrates of E6AP. Here, we identified peroxiredoxin 1 (Prx1) as a novel E6AP-binding protein using a tandem affinity purification procedure coupled with mass spectrometry. Prx1 is a 25-kDa member of the Prx family, a ubiquitous family of antioxidant peroxidases that regulate many cellular processes through intracellular oxidative signal transduction pathways. Immunoprecipitation analysis showed that E6AP binds Prx1 in vivo. Pull-down experiments showed that E6AP binds Prx1 in vitro. Ectopic expression of E6AP enhanced the degradation of Prx1 in vivo. In vivo and in vitro ubiquitylation assays revealed that E6AP promoted polyubiquitylation of Prx1. RNAi-mediated downregulation of endogenous E6AP increased the level of endogenous Prx1 protein. Taken together, our data suggest that E6AP mediates the ubiquitin-dependent proteasomal degradation of Prx1. Our findings raise a possibility that E6AP may play a role in regulating Prx1-dependent intracellular oxidative signal transduction pathways.

Identification of a suppressive mechanism for Hedgehog signaling through a novel interaction of Gli with 14-3-3.

Gli transcription factors are central effectors of Hedgehog signaling in development and tumorigenesis. Using a tandem affinity purification (TAP) strategy and mass spectrometry, we have found that Gli1 interacts with 14-3-3epsilon, and that Gli2 and Gli3 also bind to 14-3-3epsilon through homologous sites. This interaction depends on their phosphorylation, and cAMP-dependent protein kinase (PKA), a known negative regulator of Hedgehog signaling serves as a responsible kinase. A Gli2 mutant engineered to eliminate this interaction exhibited increased transcriptional activity (2 approximately 3x). Transcriptional repression by 14-3-3 binding was also observed with Gli3, when its N-terminal repressor domain was deleted. The phosphorylation sites responsible for the binding to 14-3-3 are distinct from those required for proteolysis, the known mechanism for PKA-induced repression of Hh signaling. Our data propose a novel mechanism in which PKA down-regulates Hedgehog signaling by promoting the interaction between Gli and 14-3-3 as well as proteolysis. Given the certain neuronal or malignant disorders in human caused by the abnormality of 17p13 encompassing 14-3-3epsilon overlap with increased Hh signaling, the Gli-14-3-3 interaction may have pathological significance for those human diseases.

Two Beclin 1-binding proteins, Atg14L and Rubicon, reciprocally regulate autophagy at different stages.

Beclin 1, a protein essential for autophagy, binds to hVps34/Class III phosphatidylinositol-3-kinase and UVRAG. Here, we have identified two Beclin 1 associated proteins, Atg14L and Rubicon. Atg14L and UVRAG bind to Beclin 1 in a mutually exclusive manner, whereas Rubicon binds only to a subpopulation of UVRAG complexes; thus, three different Beclin 1 complexes exist. GFP-Atg14L localized to the isolation membrane and autophagosome, as well as to the ER and unknown puncta. Knockout of Atg14L in mouse ES cells caused a defect in autophagosome formation. GFP-Rubicon was localized at the endosome/lysosome. Knockdown of Rubicon caused enhancement of autophagy, especially at the maturation step, as well as enhancement of endocytic trafficking. These data suggest that the Beclin 1-hVps34 complex functions in two different steps of autophagy by altering the subunit composition.

Single-step purification of pepsin-derived monoclonal antibody fragments from crude murine ascitic fluids by ceramic hydroxyapatite high-performance liquid chromatography.

Ceramic hydroxyapatite (CHT) high-performance liquid chromatography (HPLC) is used to purify a variety of classes of monoclonal antibodies (mAbs) from crude murine ascites fluids. We report here that this method is also applicable for simple and efficient purification of many mAb fragments that are generated by pepsin treatment of crude ascites. F(ab')(2) fragments were quantitatively generated from IgG(1) mAbs in ascitic fluids by incubation with pepsin for 6 h at pH 3.9-4.1. Under the same conditions, pepsin also cleaved unwanted ascites components, such as albumin and transferrin to very low molecular weight polypeptides. The F(ab')(2) fragments, but not the low molecular weight products, selectively bound to and were eluted from the CHT column using a linear gradient of phosphate ion concentration over 15 min. The recovery of the F(ab')(2) fragments by CHT-HPLC was >90%. This method also allowed single-step purification of mAb fragments from distinct IgG subclasses (IgG(2a) and IgG(2b)) and IgM directly from crude digested ascitic samples. This CHT-HPLC method combined with direct pepsinolysis of murine ascites is a useful strategy for rapid purification and characterization of many types of mAb fragments.

14-3-3 Proteins directly regulate Ca(2+)/calmodulin-dependent protein kinase kinase alpha through phosphorylation-dependent multisite binding.

Ca(2+)/calmodulin-dependent protein kinase kinase alpha (CaMKKalpha) plays critical roles in the modulation of neuronal cell survival as well as many other cellular activities. Here we show that 14-3-3 proteins directly regulate CaMKKalpha when the enzyme is phosphorylated by protein kinase A on either Ser74 or Ser475. Mutational analysis revealed that these two serines are both functional: the CaMKKalpha mutant with a mutation at either of these residues, but not the double mutant, was inhibited significantly by 14-3-3. The mode of regulation described herein differs the recently described mode of 14-3-3 regulation of CaMKKalpha.

Runx3 interacts with DNA repair protein Ku70.

Recent studies have suggested that Runt-related transcription factor 3 (Runx3) is associated with genesis and progression of gastric carcinoma. A proteomic approach was used to search for Runx3-interacting proteins to elucidate the molecular mechanisms of gastric carcinogenesis. Runx3 bound with myc and flag tags (MEF tags) is expressed in HEK293T cells, and the protein complex formed with Runx3 was purified and identified by mass spectrometry. Ku70 and Ku80, members of the DNA repair protein complex, were identified as Runx3-interacting proteins. Runx3, Ku70, and Ku80 associate in vivo, and in vitro interaction between Runx3 and Ku70 was confirmed via His-tag pull-down assay. The amino acids 241-322 of Runx3, which correspond to the transcriptional activation domain, and the amino acids 1-116 of Ku70 were necessary for binding with each other, and immunocytochemistry under confocal laser microscopy demonstrated that Runx3 and Ku70 localized throughout the nucleus excluding the nucleoli. Furthermore, Runx3 highly activated the transcription of p21, the target gene of Runx3, in Ku70 knockdown cells. These results suggest a possible link between a tumor suppressor function and DNA repair.

Proteomic analysis of in vivo 14-3-3 interactions in the yeast Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae produces two 14-3-3 proteins, Bmh1 and Bmh2, whose exact functions have remained unclear. Here, we performed a comprehensive proteomic analysis using multistep immunoaffinity purification and mass spectrometry and identified 271 yeast proteins that specifically bind to Bmh1 and -2 in a phosphorylation-dependent manner. The identified proteins have diverse biochemical functions and cellular roles, including cell signaling, metabolism, and cell cycle regulation. Importantly, there are a number of protein subsets that are involved in the regulation of yeast physiology through a variety of cell signaling pathways, including stress-induced transcription, cell division, and chitin synthesis at the cell wall. In fact, we found that a yeast mutant deficient in Bmh1 and -2 had defects in signal-dependent response of the MAPK (Hog1 and Mpk1) cascade and exhibited an abnormal accumulation of chitin at the bud neck. We propose that Bmh1 and -2 are common regulators of many cell signaling modules and pathways mediated by protein phosphorylation and regulate a variety of biological events by coordinately controlling the identified multiplex phosphoprotein components.

E6AP ubiquitin ligase mediates ubiquitylation and degradation of hepatitis C virus core protein.

Hepatitis C virus (HCV) core protein is a major component of viral nucleocapsid and a multifunctional protein involved in viral pathogenesis and hepatocarcinogenesis. We previously showed that the HCV core protein is degraded through the ubiquitin-proteasome pathway. However, the molecular machinery for core ubiquitylation is unknown. Using tandem affinity purification, we identified the ubiquitin ligase E6AP as an HCV core-binding protein. E6AP was found to bind to the core protein in vitro and in vivo and promote its degradation in hepatic and nonhepatic cells. Knockdown of endogenous E6AP by RNA interference increased the HCV core protein level. In vitro and in vivo ubiquitylation assays showed that E6AP promotes ubiquitylation of the core protein. Exogenous expression of E6AP decreased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected Huh-7 cells. Furthermore, knockdown of endogenous E6AP by RNA interference increased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected cells. Taken together, our results provide evidence that E6AP mediates ubiquitylation and degradation of HCV core protein. We propose that the E6AP-mediated ubiquitin-proteasome pathway may affect the production of HCV particles through controlling the amounts of viral nucleocapsid protein.

Hepatitis C virus RNA replication is regulated by FKBP8 and Hsp90.

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a component of viral replicase and is well known to modulate the functions of several host proteins. Here, we show that NS5A specifically interacts with FKBP8, a member of the FK506-binding protein family, but not with other homologous immunophilins. Three sets of tetratricopeptide repeats in FKBP8 are responsible for interactions with NS5A. The siRNA-mediated knockdown of FKBP8 in a human hepatoma cell line harboring an HCV RNA replicon suppressed HCV RNA replication, and this reduction was reversed by the expression of an siRNA-resistant FKBP8 mutant. Furthermore, immunoprecipitation analyses revealed that FKBP8 forms a complex with Hsp90 and NS5A. Treatment of HCV replicon cells with geldanamycin, an inhibitor of Hsp90, suppressed RNA replication in a dose-dependent manner. These results suggest that the complex consisting of NS5A, FKBP8, and Hsp90 plays an important role in HCV RNA replication.

14-3-3 Mediates phosphorylation-dependent inhibition of the interaction between the ubiquitin E3 ligase Nedd4-2 and epithelial Na+ channels.

Although recent studies show that the 14-3-3 protein is a negative regulator of ubiquitin E3 protein ligases, the molecular mechanism remains largely unknown. We previously demonstrated that 14-3-3 specifically binds one of the E3 enzymes, Nedd4-2 (a human gene product of KIAA0439, termed hNedd4-2), which can be phosphorylated by serum glucocorticoid-inducible protein kinase 1 (SGK1); this binding protects the phosphorylated/inactive hNedd4-2 from phosphatase-catalyzed dephosphorylation [Ichimura, T., et al. (2005) J. Biol. Chem. 280, 13187-13194]. Here we report an additional mechanism of 14-3-3-mediated regulation of hNedd4-2. Using surface plasmon resonance spectrometry, we show that 14-3-3 inhibits the interaction between the WW domains of hNedd4-2 and the PY motif of the epithelial Na(+) channel, ENaC. The inhibition was dose-dependent and was dependent on SGK1-catalyzed phosphorylation of Ser468 located between the WW domains. Importantly, a mutant of hNedd4-2, which can be phosphorylated by SGK1 but cannot bind 14-3-3, reduced SGK1-mediated stimulation of the ENaC-induced current in Xenopus laevis oocytes. In addition, 14-3-3 had similar effects on hNedd4-2 that had been phosphorylated by cAMP-dependent protein kinase (PKA). Our results, together with the recent finding on 14-3-3/parkin interactions [Sato, S., et al. (2006) EMBO J. 25, 211-221], suggest that 14-3-3 suppresses ubiquitin E3 ligase activities by inhibiting the formation of the enzyme/substrate complex.

Nucleolar protein B23 interacts with Japanese encephalitis virus core protein and participates in viral replication.

Japanese encephalitis virus (JEV) core protein is detected not only in the cytoplasm but also in the nucleoli of infected cells. We previously showed that a mutant JEV lacking the nucleolar localization of the core protein impaired viral replication in mammalian cells. In this study, we identified a nucleolar phosphoprotein B23 as a protein binding with the core protein of JEV but not with that of dengue virus. The region binding with JEV core protein was mapped to amino acid residues 38 to 77 of B23. Upon JEV infection, some fraction of B23 was translocated from the nucleoli to the cytoplasm, and cytoplasmic B23 was colocalized with the core protein of wild-type JEV but not with that of the mutant JEV. Furthermore, overexpression of dominant negatives of B23 reduced JEV replication. These results suggest that B23 plays an important role in the intracellular localization of the core protein and replication of JEV.