Rintaro Saito

A travel guide to Cytoscape plugins

Cytoscape is open-source software for integration, visualization and analysis of biological networks. It can be extended through Cytoscape plugins, enabling a broad community of scientists to contribute useful features. This growth has occurred organically through the independent efforts of diverse authors, yielding a powerful but heterogeneous set of tools. We present a travel guide to the world of plugins, covering the 152 publicly available plugins for Cytoscape 2.5–2.8. We also describe ongoing efforts to distribute, organize and maintain the quality of the collection.

Metabolomics Reveals Signature of Mitochondrial Dysfunction in Diabetic Kidney Disease

Diabetic kidney disease is the leading cause of ESRD, but few biomarkers of diabetic kidney disease are available. This study used gas chromatography-mass spectrometry to quantify 94 urine metabolites in screening and validation cohorts of patients with diabetes mellitus (DM) and CKD(DM+CKD), in patients with DM without CKD (DM-CKD), and in healthy controls. Compared with levels in healthy controls, 13 metabolites were significantly reduced in the DM+CKD cohorts (P≤0.001), and 12 of the 13 remained significant when compared with the DM-CKD cohort. Many of the differentially expressed metabolites were water-soluble organic anions. Notably, organic anion transporter-1 (OAT1) knockout mice expressed a similar pattern of reduced levels of urinary organic acids, and human kidney tissue from patients with diabetic nephropathy demonstrated lower gene expression of OAT1 and OAT3. Analysis of bioinformatics data indicated that 12 of the 13 differentially expressed metabolites are linked to mitochondrial metabolism and suggested global suppression of mitochondrial activity in diabetic kidney disease. Supporting this analysis, human diabetic kidney sections expressed less mitochondrial protein, urine exosomes from patients with diabetes and CKD had less mitochondrial DNA, and kidney tissues from patients with diabetic kidney disease had lower gene expression of PGC1α (a master regulator of mitochondrial biogenesis). We conclude that urine metabolomics is a reliable source for biomarkers of diabetic complications, and our data suggest that renal organic ion transport and mitochondrial function are dysregulated in diabetic kidney disease.

A new role for expressed pseudogenes as ncRNA: regulation of mRNA stability of its homologous coding gene

AbstractWe have earlier generated a mutant mouse in a course of making a transgenic line that exhibited interesting heterozygote phenotypes, which exhibited failure to thrive, severe bone deformities, and polycystic kidneys. This mutant mouse provided a clue to uncover a unique role of expressed pseudogenes. In this mutant the transgene was integrated into the vicinity of the expressing pseudogene of Makorin1 called Makorin1-p1. This insertion reduced transcription of the Makorin1-p1, resulting in destabilization of the Makorin1 mRNA in trans via a cis-acting RNA decay element within the 5′ region of Makorin1 that is homologous between Makorin1 and Makorin1-p1. These findings demonstrate a novel and specific regulatory role of an expressed pseudogene as well as functional significance for noncoding RNAs. Next, we developed an original algorithm to determine how many pseudogenes are expressed. Based on our examination 2–3% of human processed pseudogenes are expressed using the most strict criteria. Interestingly, the mouse has a much smaller proportion of expressed pseudogenes (0.5–1%). Pseudogenes are functionally less constrained, and have accumulated more mutations than translated genes. If they have some functions in gene regulation, this property would allow more rapid functional diversification than protein-coding genes. In addition, some genetic phenomena that exhibit incomplete penetrance might be attributed to “mutation” or “variation” of pseudogenes.

Bioinformatic analysis of post-transcriptional regulation by uORF in human and mouse

RNA decay is thought to exert an important influence on gene expression by maintaining a steady‐state level of transcripts and/or by eliminating aberrant transcripts. However, the sequence elements which control such processes have not been determined. Upstream open reading frames (uORFs) in the transcripts of several genes are reported to control translational initiation by stalling ribosomes and thereby promote RNA decay. We therefore performed bioinformatic analysis of the tissue‐wide expression profiles and mRNA half‐life of transcripts containing uORFs in humans and mice to assess the relationship between RNA decay and the presence of uORFs in transcripts. The expression levels of transcripts containing uORF were markedly lower than those not containing uORF. Moreover, the half‐life of the uORF‐containing transcripts was also shorter. These results suggest that uORFs are sequence elements that down‐regulate RNA transcripts via RNA decay mechanisms.

Metabolomics Reveals a Key Role for Fumarate in Mediating the Effects of NADPH Oxidase 4 in Diabetic Kidney Disease

The NADPH oxidase (NOX) isoform NOX4 has been linked with diabetic kidney disease (DKD). However, a mechanistic understanding of the downstream effects of NOX4 remains to be established. We report that podocyte-specific induction of NOX4 in vivo was sufficient to recapitulate the characteristic glomerular changes noted with DKD, including glomerular hypertrophy, mesangial matrix accumulation, glomerular basement membrane thickening, albuminuria, and podocyte dropout. Intervention with a NOX1/NOX4 inhibitor reduced albuminuria, glomerular hypertrophy, and mesangial matrix accumulation in the F1 Akita model of DKD. Metabolomic analyses from these mouse studies revealed that tricarboxylic acid (TCA) cycle-related urinary metabolites were increased in DKD, but fumarate levels were uniquely reduced by the NOX1/NOX4 inhibitor. Expression of fumarate hydratase (FH), which regulates urine fumarate accumulation, was reduced in the diabetic kidney (in mouse and human tissue), and administration of the NOX1/NOX4 inhibitor increased glomerular FH levels in diabetic mice. Induction of Nox4 in vitro and in the podocyte-specific NOX4 transgenic mouse led to reduced FH levels. In vitro, fumarate stimulated endoplasmic reticulum stress, matrix gene expression, and expression of hypoxia-inducible factor-1α (HIF-1α) and TGF-β. Similar upregulation of renal HIF-1α and TGF-β expression was observed in NOX4 transgenic mice and diabetic mice and was attenuated by NOX1/NOX4 inhibition in diabetic mice. In conclusion, NOX4 is a major mediator of diabetes-associated glomerular dysfunction through targeting of renal FH, which increases fumarate levels. Fumarate is therefore a key link connecting metabolic pathways to DKD pathogenesis, and measuring urinary fumarate levels may have application for monitoring renal NOX4 activity.

Comparative expression analysis uncovers novel features of endogenous antisense transcription

Increasing numbers of sense-antisense transcripts (SATs), which are transcribed from the same chromosomal location but in opposite directions, have been identified in various eukaryotic species, but the biological meanings of most SATs remain unclear. To improve understanding of natural sense-antisense transcription, we performed comparative expression profiling of SATs conserved among humans and mice. Using custom oligo-arrays loaded with probes that represented SATs with both protein-coding and non-protein-coding transcripts, we showed that 33% of the 291 conserved SATs displayed identical expression patterns in the two species. Among these SATs, expressional balance inversion of sense-antisense genes was mostly observed in testis at a tissue-specific manner. Northern analyses of the individual conserved SAT loci revealed that: (i) a smeary hybridization pattern was present in mice, but not in humans, and (2) small RNAs (about 60 to 80 nt) were detected from the exon-overlapping regions of SAT loci. In addition, further analyses showed marked alteration of sense-antisense expression balance throughout spermatogenesis in testis. These results suggest that conserved SAT loci are rich in potential regulatory roles that will help us understand this new class of transcripts underlying the mammalian genome.

A Comprehensive Resource of Interacting Protein Regions for Refining Human Transcription Factor Networks

Large-scale data sets of protein-protein interactions (PPIs) are a valuable resource for mapping and analysis of the topological and dynamic features of interactome networks. The currently available large-scale PPI data sets only contain information on interaction partners. The data presented in this study also include the sequences involved in the interactions (i.e., the interacting regions, IRs) suggested to correspond to functional and structural domains. Here we present the first large-scale IR data set obtained using mRNA display for 50 human transcription factors (TFs), including 12 transcription-related proteins. The core data set (966 IRs; 943 PPIs) displays a verification rate of 70%. Analysis of the IR data set revealed the existence of IRs that interact with multiple partners. Furthermore, these IRs were preferentially associated with intrinsic disorder. This finding supports the hypothesis that intrinsically disordered regions play a major role in the dynamics and diversity of TF networks through their ability to structurally adapt to and bind with multiple partners. Accordingly, this domain-based interaction resource represents an important step in refining protein interactions and networks at the domain level and in associating network analysis with biological structure and function.

Integrative features of the yeast phosphoproteome and protein-protein interaction map.

Following recent advances in high-throughput mass spectrometry (MS)–based proteomics, the numbers of identified phosphoproteins and their phosphosites have greatly increased in a wide variety of organisms. Although a critical role of phosphorylation is control of protein signaling, our understanding of the phosphoproteome remains limited. Here, we report unexpected, large-scale connections revealed between the phosphoproteome and protein interactome by integrative data-mining of yeast multi-omics data. First, new phosphoproteome data on yeast cells were obtained by MS-based proteomics and unified with publicly available yeast phosphoproteome data. This revealed that nearly 60% of ∼6,000 yeast genes encode phosphoproteins. We mapped these unified phosphoproteome data on a yeast protein–protein interaction (PPI) network with other yeast multi-omics datasets containing information about proteome abundance, proteome disorders, literature-derived signaling reactomes, and in vitro substratomes of kinases. In the phospho-PPI, phosphoproteins had more interacting partners than nonphosphoproteins, implying that a large fraction of intracellular protein interaction patterns (including those of protein complex formation) is affected by reversible and alternative phosphorylation reactions. Although highly abundant or unstructured proteins have a high chance of both interacting with other proteins and being phosphorylated within cells, the difference between the number counts of interacting partners of phosphoproteins and nonphosphoproteins was significant independently of protein abundance and disorder level. Moreover, analysis of the phospho-PPI and yeast signaling reactome data suggested that co-phosphorylation of interacting proteins by single kinases is common within cells. These multi-omics analyses illuminate how wide-ranging intracellular phosphorylation events and the diversity of physical protein interactions are largely affected by each other.

Quantitative Proteomic and Functional Analysis of Liver Mitochondria from High Fat Diet (HFD) Diabetic Mice

Insulin resistance plays a major role in the development of type 2 diabetes and obesity and affects a number of biological processes such as mitochondrial biogenesis. Though mitochondrial dysfunction has been linked to the development of insulin resistance and pathogenesis of type 2 diabetes, the precise mechanism linking the two is not well understood. We used high fat diet (HFD)-induced obesity dependent diabetes mouse models to gain insight into the potential pathways altered with metabolic disease, and carried out quantitative proteomic analysis of liver mitochondria. As previously reported, proteins involved in fatty acid oxidation, branched chain amino acid degradation, tricarboxylic acid cycle, and oxidative phosphorylation were uniformly up-regulated in the liver of HFD fed mice compared with that of normal diet. Further, our studies revealed that retinol metabolism is distinctly down-regulated and the mitochondrial structural proteins—components of mitochondrial inter-membrane space bridging (MIB) complex (Mitofilin, Sam50, and ChChd3), and Tim proteins—essential for protein import, are significantly up-regulated in HFD fed mice. Structural and functional studies on HFD and normal diet liver mitochondria revealed remodeling of HFD mitochondria to a more condensed form with increased respiratory capacity and higher ATP levels compared with normal diet mitochondria. Thus, it is likely that the structural remodeling is essential to accommodate the increased protein content in presence of HFD: the mechanism could be through the MIB complex promoting contact site and crista junction formation and in turn facilitating the lipid and protein uptake.

In Silico Analysis of Phosphoproteome Data Suggests a Rich-get-richer Process of Phosphosite Accumulation over Evolution

Recent phosphoproteome analyses using mass spectrometry-based technologies have provided new insights into the extensive presence of protein phosphorylation in various species and have raised the interesting question of how this protein modification was gained evolutionarily on such a large scale. We investigated this issue by using human and mouse phosphoproteome data. We initially found that phosphoproteins followed a power-law distribution with regard to their number of phosphosites: most of the proteins included only a few phosphosites, but some included dozens of phosphosites. The power-law distribution, unlike more commonly observed distributions such as normal and log-normal distributions, is considered by the field of complex systems science to be produced by a specific rich-get-richer process called preferential attachment growth. Therefore, we explored the factors that may have promoted the rich-get-richer process during phosphosite evolution. We conducted a bioinformatics analysis to evaluate the relationship of amino acid sequences of phosphoproteins with the positions of phosphosites and found an overconcentration of phosphosites in specific regions of protein surfaces and implications that in many phosphoproteins these clusters of phosphosites are activated simultaneously. Multiple phosphosites concentrated in limited spaces on phosphoprotein surfaces may therefore function biologically as cooperative modules that are resistant to selective pressures during phosphoprotein evolution. We therefore proposed a hypothetical model by which the modularization of multiple phosphosites has been resistant to natural selection and has driven the rich-get-richer process of the evolutionary growth of phosphosite numbers.