Akiko Okayama

Co- and post-translational modifications of the 26S proteasome in yeast

The yeast (Saccharomyces cerevisiae) 26S proteasome consists of the 19S regulatory particle (19S RP) and 20S proteasome subunits. We detected comprehensively co‐ and post‐translational modifications of these subunits using proteomic techniques. First, using MS/MS, we investigated the N‐terminal modifications of three 19S RP subunits, Rpt1, Rpn13, and Rpn15, which had been unclear, and found that the N‐terminus of Rpt1 is not modified, whereas that of Rpn13 and Rpn15 is acetylated. Second, we identified a total of 33 Ser/Thr phosphorylation sites in 15 subunits of the proteasome. The data obtained by us and other groups reveal that the 26S proteasome contains at least 88 phospho‐amino acids including 63 pSer, 23 pThr, and 2 pTyr residues. Dephosphorylation treatment of the 19S RP with λ phosphatase resulted in a 30% decrease in ATPase activity, demonstrating that phosphorylation is involved in the regulation of ATPase activity in the proteasome. Third, we tried to detect glycosylated subunits of the 26S proteasome. However, we identified neither N‐ and O‐linked oligosaccharides nor O‐linked β‐N‐acetylglucosamine in the 19S RP and 20S proteasome subunits. To date, a total of 110 co‐ and post‐translational modifications, including Nα‐acetylation, Nα‐myristoylation, and phosphorylation, in the yeast 26S proteasome have been identified.

A distinct role for Pin1 in the induction and maintenance of pluripotency

The prominent characteristics of pluripotent stem cells are their unique capacity to self-renew and pluripotency. Although pluripotent stem cell proliferation is maintained by specific intracellular phosphorylation signaling events, it has not been well characterized how the resulting phosphorylated proteins are subsequently regulated. We here report that the peptidylprolyl isomerase Pin1 is indispensable for the self-renewal and maintenance of pluripotent stem cells via the regulation of phosphorylated Oct4 and other substrates. Pin1 expression was found to be up-regulated upon the induction of induced pluripotent stem (iPS) cells, and the forced expression of Pin1 with defined reprogramming factors was observed to further enhance the frequency of iPS cell generation. The inhibition of Pin1 activity significantly suppressed colony formation and induced the aberrant differentiation of human iPS cells as well as murine ES cells. We further found that Pin1 interacts with the phosphorylated Ser12-Pro motif of Oct4 and that this in turn facilitates the stability and transcriptional activity functions of Oct4. Our current findings thus uncover an atypical role for Pin1 as a putative regulator of the induction and maintenance of pluripotency via the control of phosphorylation signaling. These data suggest that the manipulation of Pin1 function could be a potential strategy for the stable induction and proliferation of human iPS cells.

The phosphorylation of HIV-1 Gag by atypical protein kinase C facilitates viral infectivity by promoting Vpr incorporation into virions

BackgroundHuman immunodeficiency virus type 1 (HIV-1) Gag is the main structural protein that mediates the assembly and release of virus-like particles (VLPs) from an infected cell membrane. The Gag C-terminal p6 domain contains short sequence motifs that facilitate virus release from the plasma membrane and mediate incorporation of the viral Vpr protein. Gag p6 has also been found to be phosphorylated during HIV-1 infection and this event may affect virus replication. However, the kinase that directs the phosphorylation of Gag p6 toward virus replication remains to be identified. In our present study, we identified this kinase using a proteomic approach and further delineate its role in HIV-1 replication.ResultsA proteomic approach was designed to systematically identify human protein kinases that potently interact with HIV-1 Gag and successfully identified 22 candidates. Among this panel, atypical protein kinase C (aPKC) was found to phosphorylate HIV-1 Gag p6. Subsequent LC-MS/MS and immunoblotting analysis with a phospho-specific antibody confirmed both in vitro and in vivo that aPKC phosphorylates HIV-1 Gag at Ser487. Computer-assisted structural modeling and a subsequent cell-based assay revealed that this phosphorylation event is necessary for the interaction between Gag and Vpr and results in the incorporation of Vpr into virions. Moreover, the inhibition of aPKC activity reduced the Vpr levels in virions and impaired HIV-1 infectivity of human primary macrophages.ConclusionOur current results indicate for the first time that HIV-1 Gag phosphorylation on Ser487 is mediated by aPKC and that this kinase may regulate the incorporation of Vpr into HIV-1 virions and thereby supports virus infectivity. Furthermore, aPKC inhibition efficiently suppresses HIV-1 infectivity in macrophages. aPKC may therefore be an intriguing therapeutic target for HIV-1 infection.

Proteomic Analysis of Proteins Related to Prognosis of Lung Adenocarcinoma

We attempted to identify prognosis-related proteins expressed in early resection lung adenocarcinomas that had higher metastatic potential. Early resection of lung adenocarcinoma tissues were collected from patients who experienced recurrence within 5 years after surgery; these patients are defined here as the poor prognosis group. From these samples, we prepared frozen tissue sections and then isolated cancerous areas by laser capture microdissection to allow extraction of cancer tissue-derived soluble proteins. Shotgun LC-MS/MS analysis detected and identified a total of 875 proteins in these cancer tissues. Relative quantitative analysis revealed that 17 proteins were preferentially expressed in the poor prognosis group relative to the good prognosis group, which consisted of patients who did not exhibit recurrence. Among them, 14-3-3 beta/alpha and calnexin were reported to be potentially involved in tumor recurrence and the malignant properties of lung cancer. Here immunological analyses confirmed disease-associated expression of these proteins. In a cell-culture model using A549, targeted depletion of either 14-3-3 beta/alpha or calnexin reduced proliferation, invasion, and migration, suggesting that both proteins are involved in determining the malignant properties of lung cancer that contribute to poor prognosis.

Mass spectrometric identification of glycosylphosphatidylinositol-anchored peptides.

Glycosylphosphatidylinositol (GPI) anchoring is a post-translational modification widely observed among eukaryotic membrane proteins. GPI anchors are attached to proteins via the carboxy-terminus in the outer leaflet of the cell membrane, where GPI-anchored proteins (GPI-APs) perform important functions as coreceptors and enzymes. Precursors of GPI-APs (Pre-GPI-APs) contain a C-terminal hydrophobic sequence that is involved in cleavage of the signal sequence from the protein and addition of the GPI anchor by the transamidase complex. In order to confirm that a given protein contains a GPI anchor, it is essential to identify the C-terminal peptide containing the GPI-anchor modification site (ω-site). Previously, efficient identification of GPI-anchored C-terminal peptides by mass spectrometry has been difficult, in part because of complex structure of the GPI-anchor moiety. We developed a method to experimentally identify GPI-APs and their ω-sites. In this method, a part of GPI-anchor moieties are removed from GPI-anchored peptides using phosphatidylinositol-specific phospholipase C (PI-PLC) and aqueous hydrogen fluoride (HF), and peptide sequence is then determined by mass spectrometry. Using this method, we successfully identified 10 GPI-APs and 12 ω-sites in the cultured ovarian adenocarcinoma cells, demonstrating that this method is useful for identifying efficiently GPI-APs.

N-Terminal methylation of proteasome subunit Rpt1 in yeast.

The 26S proteasome is a multicatalytic protease complex that degrades ubiquitinated proteins in eukaryotic cells. It consists of a proteolytic core (the 20S proteasome) as well as regulatory particles, which contain six ATPase (Rpt) subunits involved in unfolding and translocation of substrates to the catalytic chamber of the 20S proteasome. In this study, we used MS to analyze the N‐terminal modifications of the yeast Rpt1 subunit, which contains the N‐terminal recognition sequence for N‐methyltransferase. Our results revealed that following the removal of the initiation Met residue of yeast Rpt1, the N‐terminal Pro residue is either unmodified, mono‐methylated, or di‐methylated, and that this N‐methylation has not been conserved throughout evolution. In order to gain a better understanding of the possible function(s) of the Pro‐Lys (PK) sequence at positions 3 and 4 of yeast Rpt1, we generated mutant strains expressing an Rpt1 allele that lacks this sequence. The absence of the PK sequence abolished N‐methylation, decreased cell growth, and increased sensitivity to stress. Our data suggest that N‐methylation of Rpt1 and/or its PK sequence might be important in cell growth or stress tolerance in yeast.

Relationship between phosphorylation of sperm-specific antigen and prognosis of lung adenocarcinoma.

UNLABELLED Lung cancer is generally considered as a highly malignant cancer. A major challenge for the management of lung adenocarcinoma patients is to predict the clinical course of the disease after resection. We analyzed the different levels of phosphorylation of proteins in lung adenocarcinoma tissues between a poor prognosis (PP) group, in which six patients exhibited recurrence within five years after surgery, and a good prognosis (GP) group, in which seven patients did not exhibit recurrence within five years after surgery. We found that phosphorylation at Ser92 of the sperm-specific antigen 2 (SSFA2) [phospho-SSFA2(pS92)] was stimulated in the PP group. Using samples from a total of 46 patients, we investigated the utility of phospho-SSFA2(pS92) to discriminate patients of GP and PP groups, with multiple reaction monitoring (MRM) mass spectrometry. Consequently, we confirmed that the PP group had significantly elevated phospho-SSFA2(pS92) levels. Additionally, no expression of SSFA2 recognized in the normal lung tissues. From these results, we demonstrate that phospho-SSFA2 (pS92) is related to the prognosis of early resected lung adenocarcinomas. Therefore, we suggest that phosphorylation of this protein indicates its role as a potential biomarker and new therapeutic target. BIOLOGICAL SIGNIFICANCE Lung adenocarcinoma patients often experience a high rate of recurrence after surgery. It is important to discover biomarkers for prognostic prediction and therapeutic targets for treatment of early-stage lung adenocarcinoma. In this study, using tissue samples obtained from patients with lung adenocarcinoma that had been stored for five years at -80°C, we identified 13 unique phosphorylated peptides, which were differentially expressed between poor and good prognosis groups. We confirmed that phosphorylation at Ser92 of the sperm-specific antigen 2 (SSFA2)[phospho-SSFA2 (pS92)], was related to poor prognosis. Our study demonstrates that prognostic prediction of early-stage lung adenocarcinoma is possible, and suggests new therapeutic targets for its treatment.

Identification of Tyrosine-Phosphorylated Proteins Upregulated during Epithelial–Mesenchymal Transition Induced with TGF-β

The epithelial-to-mesenchymal transition (EMT) is a unique process for the phenotypic changes of tumor cells characterized by a transition from polarized rigid epithelial cells to migrant mesenchymal cells, thus conferring the ability of tumor invasion and metastasis. A major challenge in the treatment of lung adenocarcinoma is to identify early stage patients at a high risk of recurrence or metastasis, thereby permitting the best therapeutic strategy and prognosis. In this study, we used a transforming growth factor-β (TGF-β)-induced EMT model to quantitatively identify protein tyrosine phosphorylation during the course of EMT in relation to malignant characteristics of lung adenocarcinoma cells. We performed relative quantitation analysis of tyrosine-phosphorylated peptides in TGF-β-treated and -untreated lung adenocarcinoma cells and identified tyrosine-phosphorylated proteins that were upregulated in TGF-β-treated cells. These include tensin-1 (TNS1) phosphorylated on Y1404, hepatocyte growth factor receptor (c-Met) phosphorylated on Y1234, and NT-3 growth factor receptor (TrkC) phosphorylated on Y516. We also found that these protein phosphorylation profiles were specifically observed in tissue samples of patients with poor prognostic lung adenocarcinoma. Tyrosine phosphorylations of these proteins represent possible candidates of prognostic prediction markers for lung adenocarcinoma.

Multiplex detection and identification of proteins on a PVDF membrane blocked with a synthetic polymer-based reagent

2‐DE is one of the most powerful methods for analyzing proteins expressed in cells and tissues. Immunodetection of proteins blotted on a polymer membrane is the method of choice for detecting specific proteins in 2‐D gels. To precisely locate spots of immunoreactive proteins in 2‐D gels, both dye staining and immunodetection were performed on the same PVDF membrane. Prior to immunodetection, nonspecific adsorption of the antibodies to the membrane was blocked with a synthetic polymer‐based reagent (N‐102) after protein transfer. The protein was then stained with colloidal gold or CBB followed by protein spot identification by LC‐MS. Described herein is a method for multiplex analysis of proteins transferred to a PVDF membrane. Proteins that were phosphorylated at tyrosine in the phosphoproteome of rice callus or human ovarian cancer cells were detected by immunoblotting and subsequently identified with high precision.

Identification of candidate diagnostic serum biomarkers for Kawasaki disease using proteomic analysis

Kawasaki disease (KD) is a systemic vasculitis and childhood febrile disease that can lead to cardiovascular complications. The diagnosis of KD depends on its clinical features, and thus it is sometimes difficult to make a definitive diagnosis. In order to identify diagnostic serum biomarkers for KD, we explored serum KD-related proteins, which differentially expressed during the acute and recovery phases of two patients by mass spectrometry (MS). We identified a total of 1,879 proteins by MS-based proteomic analysis. The levels of three of these proteins, namely lipopolysaccharide-binding protein (LBP), leucine-rich alpha-2-glycoprotein (LRG1), and angiotensinogen (AGT), were higher in acute phase patients. In contrast, the level of retinol-binding protein 4 (RBP4) was decreased. To confirm the usefulness of these proteins as biomarkers, we analyzed a total of 270 samples, including those collected from 55 patients with acute phase KD, by using western blot analysis and microarray enzyme-linked immunosorbent assays (ELISAs). Over the course of this experiment, we determined that the expression level of these proteins changes specifically in the acute phase of KD, rather than the recovery phase of KD or other febrile illness. Thus, LRG1 could be used as biomarkers to facilitate KD diagnosis based on clinical features.