Keiji Kito

Fluorescent differential display: Arbitrarily primed RT-PCR fingerprinting on an automated DNA sequencer

We established robust, reliable protocols for ‘Differential Display (DD),’ an RNA fingerprinting method originally developed by Liang and Pardee [(1992) Science 257, 967–971] using RT‐PCR with arbitrary primers. Our protocols are optimized so that reliable DD analysis can be performed on a fluorescent DNA sequencer to ensure high throughput as well as improved operational safety, compared with the original one using radioactive compounds. Such ‘Fluorescent Differential Display (FDD)’ techniques will accelerate the identification of differentially expressed as well as polymorphic transcripts to address various biological questions.

Mass Spectrometry-Based Approaches Toward Absolute Quantitative Proteomics

Mass spectrometry has served as a major tool for the discipline of proteomics to catalogue proteins in an unprecedented scale. With chemical and metabolic techniques for stable isotope labeling developed over the past decade, it is now routinely used as a method for relative quantification to provide valuable information on alteration of protein abundance in a proteome-wide scale. More recently, absolute or stoichiometric quantification of proteome is becoming feasible, in particular, with the development of strategies with isotope-labeled standards composed of concatenated peptides. On the other hand, remarkable progress has been also made in label-free quantification methods based on the number of identified peptides. Here we review these mass spectrometry-based approaches for absolute quantification of proteome and discuss their implications.

A proteomic screen reveals the mitochondrial outer membrane protein Mdm34p as an essential target of the F-box protein Mdm30p

Ubiquitination plays various critical roles in eukaryotic cellular regulation and is medated by a cascade of enzymes including ubiquitin protein ligase (E3). The Skp1–Cullin–F‐box protein complex comprises the largest E3 family, in each member of which a unique F‐box protein binds its targets to define substrate specificity. Although genome sequencing uncovers a growing number of F‐box proteins, most of them have remained as “orphans” because of the difficulties in identification of their substrates. To address this issue, we tested a quantitative proteomic approach by combining the stable isotope labeling by amino acids in cell culture (SILAC), parallel affinity purification (PAP) that we had developed for efficient enrichment of ubiquitinated proteins, and mass spectrometry (MS). We applied this SILAC‐PAP‐MS approach to compare ubiquitinated proteins between yeast cells with and without over‐expressed Mdm30p, an F‐box protein implicated in mitochondrial morphology. Consequently, we identified the mitochondrial outer membrane protein Mdm34p as a target of Mdm30p. Furthermore, we found that mitochondrial defects induced by deletion of MDM30 are not only recapitulated by a mutant Mdm34p defective in interaction with Mdm30p but alleviated by ubiquitination‐mimicking forms of Mdm34p. These results indicate that Mdm34p is a physiologically important target of Mdm30p.

Fluorescent differential display analysis of gene expression in differentiating neuroblastoma cells

Identification of differentially-expressed genes provides an important step toward the elucidation of molecular mechanisms underlying a variety of biological processes. A novel PCR-based approach to detect and clone such transcripts is the so-called differential display (DD). We established an improved DD protocol that can be performed on an automated fluorescent DNA sequencer to ensure high throughput as well as operational safety. Using this fluorescent DD (FDD) technique, we analyzed the gene expression profile in the retinoic acid-induced differentiation of a human neuroblastoma cell line SH-SY5Y. Screening with 102 primer combinations at eight different time points revealed 66 cDNA bands with variously different behaviors out of approximately 6000 bands displayed. Subsequent analyses with 15 cloned species confirmed the differential expression of corresponding transcripts in all the cases, thereby demonstrating the high reliability of FDD analysis. These clones were composed of seven novel and eight known genes, the latter of which included those that had never been described in the context of neuronal differentiation. These results indicate that FDD is an effective approach to obtain not only novel genes but also clues to possible novel functions of known genes involved in various biological phenomena.

The yeast eIF4E-associated protein Eap1p attenuates GCN4 translation upon TOR-inactivation.

Amino acid‐starved yeast activates the eIF2α kinase Gcn2p to suppress general translation and to selectively derepress the transcription factor Gcn4p, which induces various biosynthetic genes to elicit general amino acid control (GAAC). Well‐fed yeast activates the target of rapamycin (TOR) to stimulate translation via the eIF4F complex. A crosstalk was demonstrated between the pathways for GAAC and TOR signaling: the TOR‐specific inhibitor rapamycin activates Gcn2p. Here we demonstrate that, upon TOR‐inactivation, the putative TOR‐regulated eIF4E‐associated protein Eap1p likely functions downstream of Gcn2p to attenuate GCN4 translation via a mechanism independent of eIF4E‐binding, thereby constituting another interface between the two pathways.

MCP-1 receptor binding affinity is up-regulated by pre-stimulation with MCP-1 in an actin polymerization-dependent manner

Monocyte chemoattractant protein‐1 (MCP‐1) induces monocyte chemotaxisvia interaction with the MCP‐1 receptor CCR2. We found that MCP‐1binding to monocytic THP‐1 cells was increased by pre‐treatment withMCP‐1. The amount of CCR2 mRNA and the cell‐surface expression of CCR2were not affected by MCP‐1 stimuli. In contrast, the MCP‐1‐treatedTHP‐1 cells showed a sixfold increase in MCP‐1 binding affinitycompared with untreated cells. MCP‐1 binding to CCR2B‐transfectedHEK‐293 cells was also enhanced by pre‐treatment with MCP‐1, and MCP‐1binding affinity increased by sixfold. In both cell lines, theenhancement of MCP‐1 binding by stimulation with MCP‐1 was blocked bycytochalasin D, an inhibitor of actin polymerization. This effect ofpre‐treatment with MCP‐1 is insensitive to pertussis toxin andpartially blocked by U73122, an inhibitor of phospholipase C. Theseresults demonstrate that the MCP‐1 receptor binding affinity isup‐regulated by MCP‐1 stimuli in an actin polymerization‐dependentmanner.

Discrimination between stable and dynamic components of protein complexes by means of quantitative proteomics.

To discriminate between stable and dynamic protein–protein interactions, we propose a strategy in which cells with and without tagged bait are differentially labeled with stable isotope and combined prior to complex purification. Mass‐spectrometric analysis of the purified complexes identifies stable and dynamic components as those derived exclusively from the tagged cells and those from both cells, respectively. We successfully applied this strategy to analyze two yeast protein complexes, eIF2B–eIF2 and cyclin–Cdc28.

A parallel affinity purification method for selective isolation of polyubiquitinated proteins.

We developed a parallel affinity purification (PAP) procedure, in which ubiquitinated proteins are purified from the cells that coexpress two affinity‐tagged ubiquitins by sequential use of affinity chromatography specific to each tag. In contrast with previous procedures using a single affinity‐tagged ubiquitin, the PAP eliminates highly abundant ubiquitin monomers and monoubiquitinated proteins to selectively enrich proteins bearing both affinity‐tags, or poly‐ and multiubiquitinated proteins. Accordingly, it would serve as a powerful method to facilitate mass‐spectrometric identification of ubiquitinated proteins.

Proteome analysis of shell matrix proteins in the brachiopod Laqueus rubellus

BackgroundThe calcitic brachipod shells contain proteins that play pivotal roles in shell formation and are important in understanding the evolution of biomineralization. Here, we performed a large-scale exploration of shell matrix proteins in the brachiopod Laqueus rubellus.ResultsA total of 40 proteins from the shell were identified. Apart from five proteins, i.e., ICP-1, MSP130, a cysteine protease, a superoxide dismutase, and actin, all other proteins identified had no homologues in public databases. Among these unknown proteins, one shell matrix protein was identified with a domain architecture that includes a NAD(P) binding domain, an ABC-type transport system, a transmembrane region, and an aspartic acid rich region, which has not been detected in other biominerals. We also identified pectin lyase-like, trypsin inhibitor, and saposin B functional domains in the amino acid sequences of the shell matrix proteins. The repertoire of brachiopod shell matrix proteins also contains two basic amino acid-rich proteins and proteins that have a variety of repeat sequences.ConclusionsOur study suggests an independent origin and unique mechanisms for brachiopod shell formation.

MafB protein stability is regulated by the JNK and ubiquitin-proteasome pathways.

MafB is a basic leucine zipper transcription factor that plays important roles in development and differentiation processes. During osteoclastogenesis, its expression is downregulated at the transcriptional level via the JNK and p38 MAP kinase pathways. In the present study, we demonstrated that MafB protein stability is regulated by JNK and identified a phosphorylation site, Thr62. The expression of a constitutively active form of JNK (a fusion protein MKK7alpha1-JNK1beta1) promoted the degradation of MafB in COS7 cells, and a T62A substitution significantly reduced the instability of MafB. The introduction of a fourfold (T58A/T62A/S70A/S74A) substitution in an acidic transcription-activating domain almost protected the instability resulting from the activation of JNK. Furthermore, treatment with proteasome inhibitors increased the MafB level, and a high-molecular-weight smear, characteristic of polyubiquitination, was observed in lysates from cells in which MafB, ubiquitin, and MKK7alpha1-JNK1beta1 were co-expressed. These results suggest that phosphorylation of MafB by JNK confers susceptibility to proteasomal degradation.