Ayuko Kimura

The amphioxus genome illuminates vertebrate origins and cephalochordate biology

Cephalochordates, urochordates, and vertebrates evolved from a common ancestor over 520 million years ago. To improve our understanding of chordate evolution and the origin of vertebrates, we intensively searched for particular genes, gene families, and conserved noncoding elements in the sequenced genome of the cephalochordate Branchiostoma floridae, commonly called amphioxus or lancelets. Special attention was given to homeobox genes, opsin genes, genes involved in neural crest development, nuclear receptor genes, genes encoding components of the endocrine and immune systems, and conserved cis-regulatory enhancers. The amphioxus genome contains a basic set of chordate genes involved in development and cell signaling, including a fifteenth Hox gene. This set includes many genes that were co-opted in vertebrates for new roles in neural crest development and adaptive immunity. However, where amphioxus has a single gene, vertebrates often have two, three, or four paralogs derived from two whole-genome duplication events. In addition, several transcriptional enhancers are conserved between amphioxus and vertebrates--a very wide phylogenetic distance. In contrast, urochordate genomes have lost many genes, including a diversity of homeobox families and genes involved in steroid hormone function. The amphioxus genome also exhibits derived features, including duplications of opsins and genes proposed to function in innate immunity and endocrine systems. Our results indicate that the amphioxus genome is elemental to an understanding of the biology and evolution of nonchordate deuterostomes, invertebrate chordates, and vertebrates.

N-Myristoylation of the Rpt2 Subunit Regulates Intracellular Localization of the Yeast 26S Proteasome

The 26S proteasome is a large, complex multisubunit protease involved in protein quality control and other critical processes in eukaryotes. More than 110 post-translational modification (PTM) sites have been identified by a mass spectrometry of the 26S proteasome of Saccharomyces cerevisiae and are predicted to be implicated in the dynamic regulation of proteasomal functions. Here, we report that the N-myristoylation of the Rpt2 subunit controls the intracellular localization of the 26S proteasome. While proteasomes were mainly localized in the nucleus in normal cells, mutation of the N-myristoylation site of Rpt2 caused diffusion of the nuclear proteasome into the cytoplasm, where it formed aggregates. In mutant cells, the level of accumulation of cytoplasmic proteasomes was significantly increased in the nonproliferating state. Although the molecular assembly and peptidase activity of the 26S proteasome were totally unchanged in the nonmyristoylated mutants of Rpt2, an increased level of accumulation of polyubiquitinated proteins and a severe growth defect were observed in mutant cells induced for protein misfolding. In addition, polyubiquitinated protein and the nuclear protein Gcn4 tended not to colocalize with the proteasome in normal and mutant cells. Our results suggest that N-myristoylation is involved in regulating the proper intracellular distribution of proteasome activity by controlling the nuclear localization of the 26S proteasome.

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 Lyns 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.

Molecular cloning of the terminal complement components C6 and C8β of cartilaginous fish

The terminal complement components (TCCs) of mammals, C6, C7, C8alpha, C8beta, and C9, are a group of serum proteins involved in the cytolytic killing of microbial pathogens. The mammalian TCCs share a unique core domain structure and were probably generated by the duplication of the ancestral TCC gene and subsequent addition and/or deletion of the N- and C-terminal domains. Proteins and genes for all the TCCs have been identified from bony fish. In contrast, no TCC gene has been identified from cyclostome lamprey using whole-genome shotgun-sequence analysis and liver EST analysis. To clarify the evolutionary origin of TCCs, we performed degenerate RT-PCR and RACE analyses of the cartilaginous fish liver and identified the C6 gene from a shark, Mustelus manazo, and the C8B gene from a chimaera, Chimaera phantasma. The presence of the C6 gene in shark suggests that one of the most crucial steps in the establishment of the cytolytic complement pathway, the addition of the FIM and CCP domains to the primitive TCC, occurred in a common ancestor of the jawed vertebrates. These results also indicate that the gene duplications among TCCs occurred at an early stage of the jawed vertebrate evolution.

Biological significance of co- and post-translational modifications of the yeast 26S proteasome.

UNLABELLED In yeast (Saccharomyces cerevisiae), co- and post-translational modifications of the 26S proteasome, a large protein complex, were comprehensively detected by proteomic techniques, and their functions were investigated. The presence, number, site, and state of co- and post-translational modifications of the 26S proteasome differ considerably among yeast, human, and mouse. The roles of phosphorylation, N(α)-acetylation, N(α)-myristoylation, N(α)-methylation, and N-terminal truncation in the yeast 26S proteasome were investigated. Although there is only one modification site for either N(α)-acetylation, N(α)-myristoylation, or N(α)-methylation, these modifications play an important role in the functions of the yeast proteasome. In contrast, there are many phosphorylation sites in the yeast 26S proteasome. However, the phosphorylation patterns might be a few, suggesting that tiny modifications exert considerable effects on the function of the proteasome. BIOLOGICAL SIGNIFICANCE Protein co- and post-translational modifications produce different protein species which often have different functions. The yeast 26S proteasome, a large protein complex, consisting of many subunits has a number of co- and post-translational modification sites. This review describes the effects of the modifications on the function of the protein complex. This article is part of a Special Issue entitled: Protein species. Guest Editors: Peter Jungblut, Hartmut Schlüter and Bernd Thiede.

Mass Spectrometric Analysis of the Phosphorylation Levels of the SWI/SNF Chromatin Remodeling/Tumor Suppressor Proteins ARID1A and Brg1 in Ovarian Clear Cell Adenocarcinoma Cell Lines

Protein phosphorylation is one of the major factors involved in tumor progression and malignancy. We performed exploratory studies aimed at identifying phosphoproteins characteristic to cell lines derived from ovarian clear cell adenocarcinoma (CCA), a highly malignant type of ovarian cancer. Comparative phosphoproteome analysis revealed that the phosphopeptides of five SWI/SNF chromatin remodeling/tumor suppressor components, including ARID1A and BRG1, were significantly down-regulated in CCA cells. We then quantitatively determined the phosphorylation levels of ARID1A and BRG1 by immunoprecipitation-multiple reaction monitoring (IP-MRM) that we used for analysis of the cognate phospho- and nonphosphopeptides of low-abundance proteins. The phosphorylation level of Brg1 at Ser1452 was down-regulated in CCA cells, whereas the phosphorylation level of ARID1A at Ser696 did not significantly differ between CCA and non-CCA cells. These results were consistent with the results of immunoblotting showing that Brg1 levels were comparable, but ARID1A levels were lower, in CCA cells relative to non-CCA cells. This is the first report to demonstrate reduced phosphorylation of Brg1 in CCA-derived cells. Our data also indicated that the IP-MRM/MS method we used is a powerful tool for validation of the phosphoproteins detected by shotgun analysis of phosphopeptides.

N-Myristoylation of the Rpt2 subunit of the yeast 26S proteasome is implicated in the subcellular compartment-specific protein quality control system.

Ubiquitination is the posttranslational modification of a protein by covalent attachment of ubiquitin. Controlled proteolysis via the ubiquitin-proteasome system (\UPS) alleviates cellular stress by clearing misfolded proteins. In budding yeast, UPS within the nucleus degrades the nuclear proteins as well as proteins imported from the cytoplasm. While the predominantly nuclear localization of the yeast proteasome is maintained by the importin-mediated transport, N-myristoylation of the proteasome subunit Rpt2 was indicated to cause dynamic nucleo-cytoplasmic localization of proteasomes. Here, we quantitatively analyzed the ubiquitinated peptides using anti-K-ε-GG antibody in yeast cell lines with or without a mutation in the N-myristoylation site of Rpt2 and detected upregulated ubiquitination of proteins with nucleo-cytoplasmic localizations in the mutant strains. Moreover, both the protein and ubiquitinated peptide levels of two Hsp70 family chaperones involved in the nuclear import of misfolded proteins, Ssa and Sse1, were elevated in the mutant strains, whereas levels of an Hsp70 family chaperone involved in the nuclear export, Ssb, were reduced. Taken together, our results indicate that N-myristoylation of Rpt2 is involved in controlled proteolysis via regulation of the nucleo-cytoplasmic localization of the yeast proteasome.

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.

Serum Quantitative Proteomic Analysis Reveals Soluble EGFR To Be a Marker of Insulin Resistance in Male Mice and Humans

To identify circulating factors as candidates involved in type 2 diabetes mellitus (T2DM), we conducted two different quantitative proteomic analyses: (1) db/db mouse sera were compared with db/+ mouse sera obtained at 4, 8, 12, and 24 weeks of age, and (2) db/db mouse sera from animals treated with liraglutide were compared with sera from animals without liraglutide treatment. Twenty proteins were differentially expressed in db/db mouse sera in the first experiment and eight proteins were differentially expressed in db/db mouse sera after liraglutide treatment in the second experiment. Soluble epidermal growth factor receptor (sEGFR) was identified as a common factor, and its protein level was significantly affected in both experiments. An enzyme-linked immunosorbent assay confirmed that the relatively low serum sEGFR levels in db/db mice were restored by liraglutide treatment. The serum sEGFR levels were elevated in diabetic mice with impaired insulin secretion and decreased in high-fat diet-fed mice and ob/ob mice. The serum sEGFR levels increased after the administration of a dual inhibitor of IGF-1/insulin receptor or streptozotocin. In humans with normal glucose tolerance or T2DM, the serum sEGFR levels were correlated with the fasting blood glucose, fasting serum insulin, homeostatic model assessment of insulin resistance, HbA1c, total cholesterol, low-density lipoprotein cholesterol, and triglycerides levels. These findings suggest that sEGFR might be a biomarker for evaluating insulin resistance or a therapeutic target of liraglutide.