Lyn Kinase Suppresses the Transcriptional Activity of IRF5 in the TLR-MyD88 Pathway to Restrain the Development of Autoimmunity.

Interferon regulatory factor-5 (IRF5), a transcription factor critical for the induction of innate immune responses, contributes to the pathogenesis of the autoimmune disease systemic lupus erythematosus (SLE) in humans and mice. Lyn, a Src family kinase, is also implicated in human SLE, and Lyn-deficient mice develop an SLE-like disease. Here, we found that Lyn physically interacted with IRF5 to inhibit ubiquitination and phosphorylation of IRF5 in the TLR-MyD88 pathway, thereby suppressing the transcriptional activity of IRF5 in a manner independent of Lyn's kinase activity. Conversely, Lyn did not inhibit NF-κB signaling, another major branch downstream of MyD88. Monoallelic deletion of Irf5 alleviated the hyperproduction of cytokines in TLR-stimulated Lyn(-/-) dendritic cells and the development of SLE-like symptoms in Lyn(-/-) mice. Our results reveal a role for Lyn as a specific suppressor of the TLR-MyD88-IRF5 pathway and illustrate the importance of fine-tuning IRF5 activity for the maintenance of immune homeostasis.

Identification of gravity-responsive serum proteins in spaceflight mice using a quantitative proteomic approach with data-independent acquisition mass spectrometry.

Physical inactivity associated with gravity unloading, such as microgravity during spaceflight and hindlimb unloading (HU), can cause various physiological changes. In this study, we attempted to identify serum proteins whose levels fluctuated in response to gravity unloading. First, we quantitatively assessed changes in the serum proteome profiles of spaceflight mice using mass spectrometry with data-independent acquisition. The serum levels of several proteins involved in the responses to estrogen and glucocorticoid, blood vessel maturation, osteoblast differentiation, and ossification were changed by microgravity exposure. Furthermore, a collective evaluation of serum proteomic data from spaceflight and HU mice identified 30 serum proteins, including Mmp2, Igfbp2, Tnc, Cdh5, and Pmel, whose levels varied to a similar extent in both gravity unloading models. These changes in serum levels could be involved in the physiological changes induced by gravity unloading. A collective evaluation of serum, femur, and soleus muscle proteome data of spaceflight mice also showed 24 serum proteins, including Igfbp5, Igfbp3, and Postn, whose levels could be associated with biological changes induced by microgravity. This study examined serum proteome profiles in response to gravity unloading, and may help deepen our understanding of microgravity adaptation mechanisms during prolonged spaceflight missions.

Changes in the astronaut serum proteome during prolonged spaceflight.

The molecular mechanisms associated with spaceflight-induced biological adaptations that may affect many healthy tissue functions remain poorly understood. In this study, we analyzed temporal changes in the serum proteome of six astronauts during prolonged spaceflight missions using quantitative comprehensive proteome analysis performed with the data-independent acquisition method of mass spectrometry (DIA-MS). All six astronauts participated in a spaceflight mission for approximately 6 months and showed a decreasing trend in T-scores at almost all sites where dual-energy X-ray absorptiometry scans were performed. DIA-MS successfully identified 624 nonredundant proteins in sera and further quantitative analysis for each sampling point provided information on serum protein profiles closely related to several time points before (pre-), during (in-), and after (post-) spaceflight. Changes in serum protein levels between spaceflight and on the ground suggest that abnormalities in bone metabolism are induced in astronauts during spaceflight. Furthermore, changes in the proteomic profile occurring during spaceflight suggest that serum levels of bone metabolism-related proteins, namely ALPL, COL1A1, SPP1, and POSTN, could serve as highly responsive indicators of bone metabolism status in spaceflight missions. This study will allow us to accelerate research to improve our understanding of the molecular mechanisms of biological adaptations associated with prolonged spaceflight.

Cancer cell-derived CD69 induced under lipid and oxygen starvation promotes ovarian cancer progression through fibronectin.

Cancer tissues generally have molecular oxygen and serum component deficiencies because of poor vascularization. Recently, we revealed that ICAM1 is strongly activated through lipophagy in ovarian clear cell carcinoma (CCC) cells in response to starvation of long-chain fatty acids and oxygen and confers resistance to apoptosis caused by these harsh conditions. CD69 is a glycoprotein that is synthesized in immune cells and is associated with their activation through cellular signaling pathways. However, the expression and function of CD69 in nonhematological cells is unclear. Here, we report that CD69 is induced in CCC cells as in ICAM1. Mass spectrometry analysis of phosphorylated peptides followed by pathway analysis revealed that CD69 augments CCC cell binding to fibronectin (FN) in association with the phosphorylation of multiple cellular signaling molecules including the focal adhesion pathway. Furthermore, CD69 synthesized in CCC cells could facilitate cell survival because the CD69-FN axis can induce epithelial-mesenchymal transition. Experiments with surgically removed tumor samples revealed that CD69 is predominantly expressed in CCC tumor cells compared with other histological subtypes of epithelial ovarian cancer. Overall, our data suggest that cancer cell-derived CD69 can contribute to CCC progression through FN.

Evaluation of four phosphopeptide enrichment strategies for mass spectrometry-based proteomic analysis.

Information about phosphorylation status can be used to prioritize and characterize biological processes in the cell. Various analytical strategies have been proposed to address the complexity of phosphorylation status and comprehensively identify phosphopeptides. In this study, we evaluated four strategies for phosphopeptide enrichment, using titanium dioxide (TiO2 ) and Phos-tag ligand particles from in-gel or in-solution digests prior to mass spectrometry-based analysis. Using TiO2 and Phos-tag magnetic beads, it was possible to enrich phosphopeptides from in-gel digests of phosphorylated ovalbumin separated by Phos-tag SDS-PAGE or in-solution serum digests, while minimizing non-specific adsorption. The tip-column strategy with TiO2 particles enabled enrichment of phosphopeptides from in-solution digests of whole-cell lysates with high efficiency and selectivity. However, the tip-column strategy with Phos-tag agarose beads yielded the greatest number of identified phosphopeptides. The strategies using both types of tip columns had a high degree of overlap, although there were differences in selectivity between the identified phosphopeptides. Together, our results indicate that multi-enrichment strategies using TiO2 particles and Phos-tag agarose beads are useful for comprehensive phosphoproteomic analysis.

Recent developments in Phos-tag electrophoresis for the analysis of phosphoproteins in proteomics.

INTRODUCTION: Phosphate-binding tag (Phos-tag) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is an important development capable of analyzing the phosphorylation state of proteins. Conventionally, proteins were separated via SDS-PAGE and Phos-tag SDS-PAGE that use different gels to identify phosphorylated proteins. However, it was often difficult to compare the electrophoretic mobility of the proteins in the different gels used. The recently developed Phos-tag diagonal electrophoresis has been able to solve this problem. It can indicate the SDS-PAGE and Phos-tag SDS-PAGE patterns on a single gel; therefore, phosphorylated proteins can be distinguished easily from non-phosphorylated proteins. AREAS COVERED: This review assesses the importance of Phos-tag electrophoresis, which enables the analysis of protein phosphorylation states, in the field of proteomics. Additionally, this review describes the significance and actual experimental technique of Phos-tag diagonal electrophoresis, which was recently developed to overcome the drawbacks of Phos-tag SDS-PAGE. EXPERT OPINION: Although shotgun analysis of proteins allows detecting many phosphorylation sites, it is challenging to clarify the differences in the phosphorylation states of protein molecules using this technique. Therefore, Phos-tag SDS-PAGE is frequently used to determine the phosphorylation state of proteins. This technique has become more powerful with the recent development of Phos-tag diagonal electrophoresis.Abbreviations: BIS, N,N'-methylenebis(acrylamide); CBB, Coomassie brilliant blue R250; ESI, electrospray ionization; hnRNP, heterogeneous ribonucleoprotein K; LTQ-Orbitrap, Linear trap quadrupole-Orbitrap; LC, liquid chromatography; MS, mass spectrometry; MALDI, matrix-assisted laser desorption ionization; Phos-tag, phosphate-binding tag [1,3-bis [bis (pyridine-2-ylmethyl) amino] propane-2-olate]; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TOF, time of flight; 2D-DIGE, fluorescence-labeled two-dimensional difference gel electrophoresis; 2-DE, two-dimensional gel electrophoresis.

Receptor-mediated selective autophagy degrades the endoplasmic reticulum and the nucleus.

Macroautophagy (hereafter referred to as autophagy) degrades various intracellular constituents to regulate a wide range of cellular functions, and is also closely linked to several human diseases. In selective autophagy, receptor proteins recognize degradation targets and direct their sequestration by double-membrane vesicles called autophagosomes, which transport them into lysosomes or vacuoles. Although recent studies have shown that selective autophagy is involved in quality/quantity control of some organelles, including mitochondria and peroxisomes, it remains unclear how extensively it contributes to cellular organelle homeostasis. Here we describe selective autophagy of the endoplasmic reticulum (ER) and nucleus in the yeast Saccharomyces cerevisiae. We identify two novel proteins, Atg39 and Atg40, as receptors specific to these pathways. Atg39 localizes to the perinuclear ER (or the nuclear envelope) and induces autophagic sequestration of part of the nucleus. Atg40 is enriched in the cortical and cytoplasmic ER, and loads these ER subdomains into autophagosomes. Atg39-dependent autophagy of the perinuclear ER/nucleus is required for cell survival under nitrogen-deprivation conditions. Atg40 is probably the functional counterpart of FAM134B, an autophagy receptor for the ER in mammals that has been implicated in sensory neuropathy. Our results provide fundamental insight into the pathophysiological roles and mechanisms of 'ER-phagy' and 'nucleophagy' in other organisms.

Detailed Structure and Pathophysiological Roles of the IgA-Albumin Complex in Multiple Myeloma.

Immunoglobulin A (IgA)-albumin complexes may be associated with pathophysiology of multiple myeloma, although the etiology is not clear. Detailed structural analyses of these protein-protein complexes may contribute to our understanding of the pathophysiology of this disease. We analyzed the structure of the IgA-albumin complex using various electrophoresis, mass spectrometry, and in silico techniques. The data based on the electrophoresis and mass spectrometry showed that IgA in the sera of patients was dimeric, linked via the J chain. Only dimeric IgA can bind to albumin molecules leading to IgA-albumin complexes, although both monomeric and dimeric forms of IgA were present in the sera. Molecular interaction analyses in silico implied that dimeric IgA and albumin interacted not only via disulfide bond formation, but also via noncovalent bonds. Disulfide bonds were predicted between Cys34 of albumin and Cys311 of IgA, resulting in an oxidized form of albumin. Furthermore, complex formation prolongs the half-life of IgA molecules in the IgA-albumin complex, leading to excessive glycation of IgA molecules and affects the accumulation of IgA in serum. These findings may demonstrate why complications such as hyperviscosity syndrome occur more often in patients with IgA dimer producing multiple myeloma.

Linagliptin Ameliorates Hepatic Steatosis via Non-Canonical Mechanisms in Mice Treated with a Dual Inhibitor of Insulin Receptor and IGF-1 Receptor.

Abnormal hepatic insulin signaling is a cause or consequence of hepatic steatosis. DPP-4 inhibitors might be protective against fatty liver. We previously reported that the systemic inhibition of insulin receptor (IR) and IGF-1 receptor (IGF1R) by the administration of OSI-906 (linsitinib), a dual IR/IGF1R inhibitor, induced glucose intolerance, hepatic steatosis, and lipoatrophy in mice. In the present study, we investigated the effects of a DPP-4 inhibitor, linagliptin, on hepatic steatosis in OSI-906-treated mice. Unlike high-fat diet-induced hepatic steatosis, OSI-906-induced hepatic steatosis is not characterized by elevations in inflammatory responses or oxidative stress levels. Linagliptin improved OSI-906-induced hepatic steatosis via an insulin-signaling-independent pathway, without altering glucose levels, free fatty acid levels, gluconeogenic gene expressions in the liver, or visceral fat atrophy. Hepatic quantitative proteomic and phosphoproteomic analyses revealed that perilipin-2 (PLIN2), major urinary protein 20 (MUP20), cytochrome P450 2b10 (CYP2B10), and nicotinamide N-methyltransferase (NNMT) are possibly involved in the process of the amelioration of hepatic steatosis by linagliptin. Thus, linagliptin improved hepatic steatosis induced by IR and IGF1R inhibition via a previously unknown mechanism that did not involve gluconeogenesis, lipogenesis, or inflammation, suggesting the non-canonical actions of DPP-4 inhibitors in the treatment of hepatic steatosis under insulin-resistant conditions.

Common Repository of FBS Proteins (cRFP) To Be Added to a Search Database for Mass Spectrometric Analysis of Cell Secretome.

We propose to use cRFP (common Repository of FBS Proteins) in the MS (mass spectrometry) raw data search of cell secretomes. cRFP is a small supplementary sequence list of highly abundant fetal bovine serum proteins added to the reference database in use. The aim behind using cRFP is to prevent the contaminant FBS proteins from being misidentified as other proteins in the reference database, just as we would use cRAP (common Repository of Adventitious Proteins) to prevent contaminant proteins present either by accident or through unavoidable contacts from being misidentified as other proteins. We expect it to be widely used in experiments where the proteins are obtained from serum-free media after thorough washing of the cells, or from a complex media such as SILAC, or from extracellular vesicles directly.

Network-guided analysis of hippocampal proteome identifies novel proteins that colocalize with Aß in a mice model of early-stage Alzheimer's disease.

Alzheimer's disease (AD) is an incurable neurodegenerative disease characterized by memory loss and neurotoxic amyloid beta (Aß) plaques accumulation. Numerous pharmacological interventions targeting Aß plaques accumulation have failed to alleviate AD. Also, the pathological alterations in AD start years before the onset of clinical symptoms. To identify proteins at play during the early stage of AD, we conducted proteomic analysis of the hippocampus of young AppNL-F mice model of AD at the preclinical phase of the disease. This was followed by interactome ranking of the proteome into hubs that were further validated in vivo using immunoblot analysis. We also performed double-immunolabeling of these hub proteins and Aß to quantify colocalization. Behavioral analysis revealed no significant difference in memory performance between 8-month-old AppNL-F and control mice. The upregulation and downregulation of several proteins were observed in the AppNL-F mice compared to control. These proteins corresponded to pathways and processes related to Aß clearance, inflammatory-immune response, transport, mitochondrial metabolism, and glial cell proliferation. Interactome analysis revealed several proteins including DLGP5, DDX49, CCDC85A, ADCY6, HEPACAM, HCN3, PPT1 and TNPO1 as essential proteins in the AppNL-F interactome. Validation by immunoblot confirmed the over-expression of these proteins except HCN3 in the early-stage AD mice hippocampus. Immunolabeling revealed a significant increase in ADCY6/Aß and HEPACAM/Aß colocalized puncta in AppNL-F mice compared to WT. These data suggest that these proteins may be involved in the early stage of AD. Our work suggests new targets and biomarkers for AD diagnosis and therapeutic intervention.

Matrix metalloproteinase-7 induces homotypic tumor cell aggregation via proteolytic cleavage of the membrane-bound Kunitz-type inhibitor HAI-1.

Matrix metalloproteinase-7 (MMP-7) plays important roles in tumor progression and metastasis. Our previous studies have demonstrated that MMP-7 binds to colon cancer cells via cell surface-bound cholesterol sulfate and induces significant cell aggregation by cleaving cell-surface protein(s). These aggregated cells exhibit a dramatically enhanced metastatic potential. However, the molecular mechanism inducing this cell-cell adhesion through the proteolytic action of MMP-7 remained to be clarified. Here, we explored MMP-7 substrates on the cell surface; the proteins on the cell surface were first biotinylated, and a labeled protein fragment specifically released from the cells after MMP-7 treatment was analyzed using LC-MS/MS. We found that hepatocyte growth factor activator inhibitor type 1 (HAI-1), a membrane-bound Kunitz-type serine protease inhibitor, is an MMP-7 substrate. We also found that the cell-bound MMP-7 cleaves HAI-1 mainly between Gly451 and Leu452 and thereby releases the extracellular region as soluble HAI-1 (sHAI-1). We further demonstrated that this sHAI-1 can induce cancer cell aggregation and determined that the HAI-1 region corresponding to amino acids 141-249, which does not include the serine protease inhibitor domain, has the cell aggregation-inducing activity. Interestingly, a cell-surface cholesterol sulfate-independent proteolytic action of MMP-7 is critical for the sHAI-1-mediated induction of cell aggregation, whereas cholesterol sulfate is needed for the MMP-7-catalyzed generation of sHAI-1. Considering that MMP-7-induced cancer cell aggregation is an important mechanism in cancer metastasis, we propose that sHAI-1 is an essential component of MMP-7-induced stimulation of cancer metastasis and may therefore represent a suitable target for antimetastatic therapeutic strategies.

Proteomic analysis of aortic smooth muscle cell secretions reveals an association of myosin heavy chain 11 with abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is a life-threatening disease, and no disease-specific circulating biomarkers for AAA screening are currently available. We have identified a smooth muscle cell (SMC)-specific biomarker for AAA. We cultured aneurysmal tunica media that were collected from eight patients undergoing elective open-repair surgeries. Secreted proteins in culture medium were subjected to liquid chromatography/tandem mass spectrometry. Myosin heavy chain 11 (myosin-11) was identified as a SMC-specific protein in the tunica media-derived secretions of all patients. We then examined myosin-11 protein concentrations by ELISA in plasma samples from patients with AAA ( n = 35) and age-matched healthy control subjects ( n = 34). Circulating myosin-11 levels were significantly higher in patients with AAA than control subjects. The area under the receiver-operating characteristic curve (AUC) of myosin-11 was 0.77, with a specificity of 65% at a sensitivity of 91%. Multivariate logistic regression analysis showed a significant association between the myosin-11 level and presence of AAA. When the myosin-11 level was combined with hypertension, it improved the prediction of AAA (AUC 0.88) more than hypertension per se. We then investigated the correlation between aortic diameter and circulating myosin-11 levels using AAA serum samples from patients undergoing endovascular aneurysm repair ( n = 20). Circulating myosin-11 levels were significantly correlated with maximum aortic diameter. Furthermore, changes in myosin-11 concentrations from the baseline 12 mo after endovascular aneurysm repair were associated with those in aortic diameter. These data suggest that circulating levels of myosin-11, which is a SMC-specific myosin isoform, may be useful as a biomarker for AAA. NEW & NOTEWORTHY Extensive studies have revealed that inflammation- or proteolysis-related proteins are proposed as biomarkers for abdominal aortic aneurysm (AAA). Changes in these protein concentrations are not specific for smooth muscle, which is a major part of AAA pathologies. Hence, no disease-specific circulating markers for AAA are currently available. We found, using secretome-based proteomic analysis on human AAA tunica media, that myosin heavy chain 11 was associated with AAA. Circulating myosin heavy chain 11 may be a new tissue-specific AAA marker.

Atg13 HORMA domain recruits Atg9 vesicles during autophagosome formation.

During autophagosome formation, autophagosome-related (Atg) proteins are recruited hierarchically to organize the preautophagosomal structure (PAS). Atg13, which plays a central role in the initial step of PAS formation, consists of two structural regions, the N-terminal HORMA (from Hop1, Rev7, and Mad2) domain and the C-terminal disordered region. The C-terminal disordered region of Atg13, which contains the binding sites for Atg1 and Atg17, is essential for the initiation step in which the Atg1 complex is formed to serve as a scaffold for the PAS. The N-terminal HORMA domain of Atg13 is also essential for autophagy, but its molecular function has not been established. In this study, we searched for interaction partners of the Atg13 HORMA domain and found that it binds Atg9, a multispanning membrane protein that exists on specific cytoplasmic vesicles (Atg9 vesicles). After the Atg1 complex is formed, Atg9 vesicles are recruited to the PAS and become part of the autophagosomal membrane. HORMA domain mutants, which are unable to interact with Atg9, impaired the PAS localization of Atg9 vesicles and exhibited severe defects in starvation-induced autophagy. Thus, Atg9 vesicles are recruited to the PAS via the interaction with the Atg13 HORMA domain. Based on these findings, we propose that the two distinct regions of Atg13 play crucial roles in distinct steps of autophagosome formation: In the first step, Atg13 forms a scaffold for the PAS via its C-terminal disordered region, and subsequently it recruits Atg9 vesicles via its N-terminal HORMA domain.

Comprehensive behavioral study and proteomic analyses of CRMP2-deficient mice.

Collapsin response mediator protein 2 (CRMP2) plays a key role in axon guidance, dendritic morphogenesis and cell polarization. CRMP2 is implicated in various neurological and psychiatric disorders. However, in vivo functions of CRMP2 remain unknown. We generated CRMP2 gene-deficient (crmp2-/- ) mice and examined their behavioral phenotypes. During 24-h home cage monitoring, the activity level during the dark phase of crmp2-/- mice was significantly higher than that of wild-type (WT) mice. Moreover, the time during the open arm of an elevated plus maze was longer for crmp2-/- mice than for WT mice. The duration of social interaction was shorter for crmp2-/- mice than for WT mice. Crmp2-/- mice also showed mild impaired contextual learning. We then examined the methamphetamine-induced behavioral change of crmp2-/- mice. Crmp2-/- mice showed increased methamphetamine-induced ambulatory activity and serotonin release. Crmp2-/- mice also showed altered expression of proteins involved in GABAergic synapse, glutamatergic synapse and neurotrophin signaling pathways. In addition, SNAP25, RAB18, FABP5, ARF5 and LDHA, which are related genes to schizophrenia and methamphetamine sensitization, are also decreased in crmp2-/- mice. Our study implies that dysregulation of CRMP2 may be involved in pathophysiology of neuropsychiatric disorders.

A Phos-tag-based micropipette-tip method for rapid and selective enrichment of phosphopeptides.

Phosphorylated peptides are attractive targets in the study of the phosphoproteome. Here, we introduce a simple and convenient micropipette-tip method for the separation of phosphorylated and nonphosphorylated peptides by using a phosphate-binding zinc(II) complex of 1,3-bis(pyridin-2-ylmethylamino)propan-2-olate (Phos-tag). A 200-µL micropipette tip containing 10 µL of swollen agarose beads functionalized with Phos-tag moieties was prepared. All steps in the phosphate-affinity separation (binding, washing, and elution) were conducted by using aqueous buffers at neutral pH values. The entire separation protocol required less than 30 min per sample. By means of three independent separation experiments, followed by mass spectrometric (MS) analyses, we identified 1,649 non-redundant phosphopeptides from the lysates of human embryonic kidney cells (the peptides sample derived from 25 µg proteins per an MS analysis). The average ratio of identified phosphopeptides to total peptides in the respective experiments was >90%, showing a high selectivity. Furthermore, the high correlation between the triplicate analyses was confirmed by scatter plots based on the normalized abundance of each peptide, as calculated by a label-free peptide relative quantification analysis in Progenesis QI. This micropipette-tip method would be thus used preferentially as an alternative to existing tools for the reliable enrichment of phosphopeptides.

MZB1 in borderline resectable pancreatic cancer resected after neoadjuvant chemoradiotherapy.

BACKGROUND: A high accumulation of CD8+ tumor-infiltrating lymphocytes (TILs) induced by neoadjuvant chemoradiotherapy (NACRT) is associated with a favorable prognosis in patients with pancreatic ductal adenocarcinoma (PDAC). However, the correlation between a high accumulation of CD8+ TILs and a favorable prognosis has yet to be fully clarified. The aim of this study was to determine predictive markers of a high accumulation of CD8+ TILs, with a favorable prognosis, using proteomic analysis. MATERIALS AND METHODS: We studied 72 resected borderline resectable PDAC patients treated with NACRT between April 2009 and March 2014. Three matched pairs of high CD8+ TIL patients with a favorable prognosis and low CD8+ TIL patients with a poor prognosis were selected. Shotgun proteomics of the stroma and cancerous lesion was performed using formalin-fixed, paraffin-embedded tissue. Validation of the identified proteins was performed using immunohistochemical staining. Relationships between the identified proteins and TILs and clinical outcomes were assessed. RESULTS: Marginal zone B- and B1-cell-specific protein (MZB1) was detected in the tumor stroma. MZB1 expression was positively correlated with a high accumulation of CD8+ TILs. High stromal MZB1 expression also correlated with disease-free and overall survival. In a subgroup analysis of CD8+ expression, there was a significant association between stromal MZB1 expression and disease-free and overall survival in the high CD8+ TIL group. CONCLUSIONS: MZB1 is a potential marker of a high accumulation of CD8+ TILs in borderline resectable PDACs resected after NACRT. Combination of CD8+ TILs with MZB1 may be a new biomarker of resected cases after NACRT.

SMG-8 and SMG-9, two novel subunits of the SMG-1 complex, regulate remodeling of the mRNA surveillance complex during nonsense-mediated mRNA decay.

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that detects and degrades mRNAs containing premature translation termination codons (PTCs). SMG-1 and Upf1 transiently form a surveillance complex termed "SURF" that includes eRF1 and eRF3 on post-spliced mRNAs during recognition of PTC. If an exon junction complex (EJC) exists downstream from the SURF complex, SMG-1 phosphorylates Upf1, the step that is a rate-limiting for NMD. We provide evidence of an association between the SURF complex and the ribosome in association with mRNPs, and we suggest that the SURF complex functions as a translation termination complex during NMD. We identified SMG-8 and SMG-9 as novel subunits of the SMG-1 complex. SMG-8 and SMG-9 suppress SMG-1 kinase activity in the isolated SMG-1 complex and are involved in NMD in both mammals and nematodes. SMG-8 recruits SMG-1 to the mRNA surveillance complex, and inactivation of SMG-8 induces accumulation of a ribosome:Upf1:eRF1:eRF3:EJC complex on mRNP, which physically bridges the ribosome and EJC through eRF1, eRF3, and Upf1. These results not only reveal the regulatory mechanism of SMG-1 kinase but also reveal the sequential remodeling of the ribosome:SURF complex to the predicted DECID (DECay InDucing) complex, a ribosome:SURF:EJC complex, as a mechanism of in vivo PTC discrimination.

Phosphoproteome analysis demonstrates the potential role of THRAP3 phosphorylation in androgen-independent prostate cancer cell growth.

Elucidating the androgen-independent growth mechanism is critical for developing effective treatment strategies to combat androgen-independent prostate cancer. We performed a comparative phosphoproteome analysis using a prostate cancer cell line, LNCaP, and an LNCaP-derived androgen-independent cell line, LNCaP-AI, to identify phosphoproteins involved in this mechanism. We performed quantitative comparisons of the phosphopeptide levels in tryptic digests of protein extracts from these cell lines using MS. We found that the levels of 69 phosphopeptides in 66 proteins significantly differed between LNCaP and LNCaP-AI. In particular, we focused on thyroid hormone receptor associated protein 3 (THRAP3), which is a known transcriptional coactivator of the androgen receptor. The phosphorylation level of THRAP3 was significantly lower at S248 and S253 in LNCaP-AI cells. Furthermore, pull-down assays showed that 32 proteins uniquely bound to the nonphosphorylatable mutant form of THRAP3, whereas 31 other proteins uniquely bound to the phosphorylation-mimic form. Many of the differentially interacting proteins were identified as being involved with RNA splicing and processing. These results suggest that the phosphorylation state of THRAP3 at S248 and S253 might be involved in the mechanism of androgen-independent prostate cancer cell growth by changing the interaction partners.

The δ2 glutamate receptor gates long-term depression by coordinating interactions between two AMPA receptor phosphorylation sites.

Long-term depression (LTD) commonly affects learning and memory in various brain regions. Although cerebellar LTD absolutely requires the δ2 glutamate receptor (GluD2) that is expressed in Purkinje cells, LTD in other brain regions does not; why and how cerebellar LTD is regulated by GluD2 remains unelucidated. Here, we show that the activity-dependent phosphorylation of serine 880 (S880) in GluA2 AMPA receptor subunit, which is an essential step for AMPA receptor endocytosis during LTD induction, was impaired in GluD2-null cerebellum. In contrast, the basal phosphorylation levels of tyrosine 876 (Y876) in GluA2 were increased in GluD2-null cerebellum. An in vitro phosphorylation assay revealed that Y876 phosphorylation inhibited subsequent S880 phosphorylation. Conversely, Y876 dephosphorylation was sufficient to restore S880 phosphorylation and LTD induction in GluD2-null Purkinje cells. Furthermore, megakaryocyte protein tyrosine phosphatase (PTPMEG), which binds to the C terminus of GluD2, directly dephosphorylated Y876. These data indicate that GluD2 gates LTD by coordinating interactions between the two phosphorylation sites of the GluA2.