Yuuki Hashimoto

Absolute Quantitation of Low Abundance Plasma APL1β peptides at Sub-fmol/mL Level by SRM/MRM without Immunoaffinity Enrichment

Selected/multiple reaction monitoring (SRM/MRM) has been widely used for the quantification of specific proteins/peptides, although it is still challenging to quantitate low abundant proteins/peptides in complex samples such as plasma/serum. To overcome this problem, enrichment of target proteins/peptides is needed, such as immunoprecipitation; however, this is labor-intense and generation of antibodies is highly expensive. In this study, we attempted to quantify plasma low abundant APLP1-derived Aβ-like peptides (APL1β), a surrogate marker for Alzheimers disease, by SRM/MRM using stable isotope-labeled reference peptides without immunoaffinity enrichment. A combination of Cibacron Blue dye mediated albumin removal and acetonitrile extraction followed by C18-strong cation exchange multi-StageTip purification was used to deplete plasma proteins and unnecessary peptides. Optimal and validated precursor ions to fragment ion transitions of APL1β were developed on a triple quadruple mass spectrometer, and the nanoliquid chromatography gradient for peptide separation was optimized to minimize the biological interference of plasma. Using the stable isotope-labeled (SI) peptide as an internal control, absolute concentrations of plasma APL1β peptide could be quantified as several hundred amol/mL. To our knowledge, this is the lowest detection level of endogenous plasma peptide quantified by SRM/MRM.

Formation of long and winding nuclear F-actin bundles by nuclear c-Abl tyrosine kinase

The non-receptor-type tyrosine kinase c-Abl is involved in actin dynamics in the cytoplasm. Having three nuclear localization signals (NLSs) and one nuclear export signal, c-Abl shuttles between the nucleus and the cytoplasm. Although monomeric actin and filamentous actin (F-actin) are present in the nucleus, little is known about the relationship between c-Abl and nuclear actin dynamics. Here, we show that nuclear-localized c-Abl induces nuclear F-actin formation. Adriamycin-induced DNA damage together with leptomycin B treatment accumulates c-Abl into the nucleus and increases the levels of nuclear F-actin. Treatment of c-Abl-knockdown cells with Adriamycin and leptomycin B barely increases the nuclear F-actin levels. Expression of nuclear-targeted c-Abl (NLS-c-Abl) increases the levels of nuclear F-actin even without Adriamycin, and the increased levels of nuclear F-actin are not inhibited by inactivation of Abl kinase activity. Intriguingly, expression of NLS-c-Abl induces the formation of long and winding bundles of F-actin within the nucleus in a c-Abl kinase activity-dependent manner. Furthermore, NLS-c-AblΔC, which lacks the actin-binding domain but has the full tyrosine kinase activity, is incapable of forming nuclear F-actin and in particular long and winding nuclear F-actin bundles. These results suggest that nuclear c-Abl plays critical roles in actin dynamics within the nucleus.

Proteome-Wide Discovery of Unknown ATP-Binding Proteins and Kinase Inhibitor Target Proteins Using an ATP Probe

ATP-binding proteins, including protein kinases, play essential roles in many biological and pathological processes and thus these proteins are attractive as drug targets. Acyl-ATP probes have been developed as efficient probes for kinase enrichment, and these probes have also been used to enrich other ATP-binding proteins. However, a robust method to identify ATP-binding proteins with systematic elimination of nonspecific binding proteins has yet to be established. Here, we describe an ATP competition assay that permitted establishment of a rigorous ATP-binding protein list with virtual elimination of nonspecific proteins. A total of 539 ATP-binding protein candidates were identified, including 178 novel candidates. In informatics analysis, ribosomal proteins were overrepresented in the list of novel candidates. We also found multiple ATP-competitive sites for several kinases, including epidermal growth factor receptor, serine/threonine-protein kinase PRP4 homologue, cyclin-dependent kinase 12, eukaryotic elongation factor 2 kinase, ribosomal protein S6 kinase alpha-1, and SRSF protein kinase 1. Using our cataloged ATP-binding protein list, a selectivity profiling method that covers the kinome and ATPome was established to identify off-target binding sites of ATP-competitive kinase inhibitors, staurosporine and crizotinib.

c-Abl induces stabilization of histone deacetylase 1 (HDAC1) in a kinase activity-dependent manner.

c‐Abl is a non‐receptor‐type tyrosine kinase that regulates various cellular events, including cell proliferation, differentiation, and apoptosis, through phosphorylation of cytoplasmic and nuclear targets. Although we showed that c‐Abl induces histone deacetylation, the molecular mechanisms of this phenomenon are largely unknown. Here, we analyzed the effect of c‐Abl on the expression of histone deacetylase 1 (HDAC1), because c‐Abl was shown to be involved in maintenance of nuclear protein levels of HDAC1. Co‐transfection of HDAC1 with c‐Abl increased the levels of HDAC1 protein in a kinase activity‐dependent manner without affecting its mRNA levels. Treatment with the proteasome inhibitor MG132 increased protein levels of HDAC1 in cells transfected with HDAC1 but not in cells co‐transfected with HDAC1 and c‐Abl. Among class I HDACs, knockdown of endogenous c‐Abl preferentially suppressed endogenous protein levels of HDAC1, suggesting that c‐Abl stabilizes HDAC1 protein by inhibiting its proteasomal degradation. Subcellular fractionation showed that the stabilization of HDAC1 by c‐Abl occurred in the nucleus. Despite the fact that HDAC1 was phosphorylated by co‐expression with c‐Abl, stabilization of HDAC1 by c‐Abl was not affected by mutations in its sites phosphorylated by c‐Abl. Co‐expression with HDAC1 and nuclear‐targeted c‐Abl did not affect HDAC1 stabilization. Therefore, these results suggest that c‐Abl induces HDAC1 stabilization possibly through phosphorylation of a cytoplasmic target that is involved in proteasomal degradation of HDAC1.

Tyrosine Phosphorylation of the Pioneer Transcription Factor FoxA1 Promotes Activation of Estrogen Signaling

The pioneer transcription factor FoxA1 plays an important role in estrogen signaling by opening closed chromatin and promoting recruitment of the estrogen receptor to its target regions in DNA. In this study, we analyzed tyrosine phosphorylation of FoxA1 by the non‐receptor‐type tyrosine kinase c‐Abl. c‐Abl was shown to phosphorylate FoxA1 at multiple sites, especially in the N‐ and C‐terminal regions. Tyr429 and Tyr464 were identified as the major phosphorylation sites in the FoxA1 C‐terminal region. The phosphomimetic and nonphosphorylatable FoxA1 mutants were generated by glutamic acid and phenylalanine substitutions at these tyrosine residues, respectively. The phosphomimetic FoxA1 promoted the activation of estrogen signaling, whereas the nonphosphorylatable FoxA1 suppressed its activation. Stimulation with the epidermal growth factor, which activates c‐Abl, enhanced the activation of estrogen signaling. In contrast, the c‐Abl inhibitor imatinib reduced its activation. The phosphomimetic FoxA1 mutant showed a higher affinity toward histone H3 than the wild‐type. These results suggest that c‐Abl‐mediated phosphorylation of FoxA1 promotes the activation of estrogen signaling by inducing its binding to histones. J. Cell. Biochem. 118: 1453–1461, 2017.

Differential Proteome Analysis Identifies TGF-β-Related Pro-Metastatic Proteins in a 4T1 Murine Breast Cancer Model

Transforming growth factor-β (TGF-β) has a dual role in tumorigenesis, acting as either a tumor suppressor or as a pro-oncogenic factor in a context-dependent manner. Although TGF-β antagonists have been proposed as anti-metastatic therapies for patients with advanced stage cancer, how TGF-β mediates metastasis-promoting effects is poorly understood. Establishment of TGF-β-related protein expression signatures at the metastatic site could provide new mechanistic information and potentially allow identification of novel biomarkers for clinical intervention to discriminate TGF-β oncogenic effects from tumor suppressive effects. In the present study, we found that systemic administration of the TGF-β receptor kinase inhibitor, SB-431542, significantly inhibited lung metastasis from transplanted 4T1 mammary tumors in Balb/c mice. The differentially expressed proteins in the comparison of lung metastases from SB-431542 treated and control vehicle-treated groups were analyzed by a quantitative LTQ Orbitrap Velos system coupled with stable isotope dimethyl labeling. A total of 36,239 peptides from 6,694 proteins were identified, out of which 4,531 proteins were characterized as differentially expressed. A subset of upregulated proteins in the control group was validated by western blotting and immunohistochemistry. The eukaryotic initiation factor (eIF) family members constituted the most enriched protein pathway in vehicle-treated compared with SB-43512-treated lung metastases, suggesting that increased protein expression of specific eIF family members, especially eIF4A1 and eEF2, is related to the metastatic phenotype of advanced breast cancer and can be down-regulated by TGF-β pathway inhibitors. Thus our proteomic approach identified eIF pathway proteins as novel potential mediators of TGF-β tumor-promoting activity.

Quantitative proteomic analysis of cultured skin fibroblast cells derived from patients with triglyceride deposit cardiomyovasculopathy

BackgroundTriglyceride deposit cardiomyovasculopathy (TGCV) is a rare disease, characterized by the massive accumulation of triglyceride (TG) in multiple tissues, especially skeletal muscle, heart muscle and the coronary artery. TGCV is caused by mutation of adipose triglyceride lipase, which is an essential molecule for the hydrolysis of TG. TGCV is at high risk for skeletal myopathy and heart dysfunction, and therefore premature death. Development of therapeutic methods for TGCV is highly desirable. This study aims to discover specific molecules responsible for TGCV pathogenesis.MethodsTo identify differentially expressed proteins in TGCV patient cells, the stable isotope labeling with amino acids in cell culture (SILAC) method coupled with LC-MS/MS was performed using skin fibroblast cells derived from two TGCV patients and three healthy volunteers. Altered protein expression in TGCV cells was confirmed using the selected reaction monitoring (SRM) method. Microarray-based transcriptome analysis was simultaneously performed to identify changes in gene expression in TGCV cells.ResultsUsing SILAC proteomics, 4033 proteins were quantified, 53 of which showed significantly altered expression in both TGCV patient cells. Twenty altered proteins were chosen and confirmed using SRM. SRM analysis successfully quantified 14 proteins, 13 of which showed the same trend as SILAC proteomics. The altered protein expression data set was used in Ingenuity Pathway Analysis (IPA), and significant networks were identified. Several of these proteins have been previously implicated in lipid metabolism, while others represent new therapeutic targets or markers for TGCV. Microarray analysis quantified 20743 transcripts, and 252 genes showed significantly altered expression in both TGCV patient cells. Ten altered genes were chosen, 9 of which were successfully confirmed using quantitative RT-PCR. Biological networks of altered genes were analyzed using an IPA search.ConclusionsWe performed the SILAC- and SRM-based identification-through-confirmation study using skin fibroblast cells derived from TGCV patients, and first identified altered proteins specific for TGCV. Microarray analysis also identified changes in gene expression. The functional networks of the altered proteins and genes are discussed. Our findings will be exploited to elucidate the pathogenesis of TGCV and discover clinically relevant molecules for TGCV in the near future.

Enhancement of TGF-β-induced Smad3 activity by c-Abl-mediated tyrosine phosphorylation of its coactivator SKI-interacting protein (SKIP)

c-Abl is a non-receptor-type tyrosine kinase that plays an important role in cell proliferation, migration, apoptosis, and fibrosis. Furthermore, although c-Abl is involved in transforming growth factor-β (TGF-β) signaling, its molecular functions in TGF-β signaling are not fully understood. Here, we found that c-Abl phosphorylates SKI-interacting protein (SKIP), a nuclear cofactor of the transcription factor Smad3. The c-Abl inhibitor imatinib suppressed TGF-β-induced expression of Smad3 targets as well as SKIP/Smad3 interaction. TGF-β-stimulation induced tyrosine phosphorylation of SKIP, and this phosphorylation was suppressed by imatinib. Tyr292, Tyr430, and Tyr433 residues in SKIP were shown to be involved in c-Abl-mediated phosphorylation. Phosphomimetic glutamic acid substitution at Tyr292 in SKIP enhanced, whereas its phospho-dead phenylalanine substitution attenuated TGF-β-induced SKIP/Smad3 interaction. Moreover, the phosphomimetic mutant of SKIP augmented transcriptional activity of Smad3. Taken together, these results suggest that c-Abl phosphorylates SKIP mainly at Tyr292 and promotes SKIP/Smad3 interaction for the full activation of TGF-β/Smad3 signaling.

Data for proteomic analysis of ATP-binding proteins and kinase inhibitor target proteins using an ATP probe.

Interactions between ATP and ATP-binding proteins (ATPome) are common and are required for most cellular processes. Thus, it is clearly important to identify and quantify these interactions for understanding basic cellular mechanisms and the pathogenesis of various diseases. We used an ATP competition assay (competition between ATP and acyl-ATP probes) that enabled us to distinguish specific ATP-binding proteins from non-specific proteins (Adachi et al., 2014) [1]. As a result, we identified 539 proteins, including 178 novel ATP-binding protein candidates. We also established an ATPome selectivity profiling method for kinase inhibitors using our cataloged ATPome list. Normally only kinome selectivity is profiled in selectivity profiling of kinase inhibitors. In this data, we expand the profiled targets from the kinome to the ATPome through performance of ATPome selectivity profiling and obtained target profiles of staurosporine and (S)-crizotinib. The data accompanying the manuscript on this approach (Adachi et al., 2014) [1] have been deposited to the ProteomeXchange with identifier PXD001200.