Yoshinori Satomi

A ubiquitin-like system mediates protein lipidation

Autophagy is a dynamic membrane phenomenon for bulk protein degradation in the lysosome/vacuole. Apg8/Aut7 is an essential factor for autophagy in yeast. We previously found that the carboxy-terminal arginine of nascent Apg8 is removed by Apg4/Aut2 protease, leaving a glycine residue at the C terminus. Apg8 is then converted to a form (Apg8-X) that is tightly bound to the membrane. Here we report a new mode of protein lipidation. Apg8 is covalently conjugated to phosphatidylethanolamine through an amide bond between the C-terminal glycine and the amino group of phosphatidylethanolamine. This lipidation is mediated by a ubiquitination-like system. Apg8 is a ubiquitin-like protein that is activated by an E1 protein, Apg7 (refs 7, 8), and is transferred subsequently to the E2 enzymes Apg3/Aut1 (ref. 9). Apg7 activates two different ubiquitin-like proteins, Apg12 (ref. 10) and Apg8, and assigns them to specific E2 enzymes, Apg10 (ref. 11) and Apg3, respectively. These reactions are necessary for the formation of Apg8-phosphatidylethanolamine. This lipidation has an essential role in membrane dynamics during autophagy.

The Atg12-Atg5 Conjugate Has a Novel E3-like Activity for Protein Lipidation in Autophagy

Autophagy is a bulk degradation process in eukaryotic cells; autophagosomes enclose cytoplasmic components for degradation in the lysosome/vacuole. Autophagosome formation requires two ubiquitin-like conjugation systems, the Atg12 and Atg8 systems, which are tightly associated with expansion of autophagosomal membrane. Previous studies have suggested that there is a hierarchy between these systems; the Atg12 system is located upstream of the Atg8 system in the context of Atg protein organization. However, the concrete molecular relationship is unclear. Here, we show using an in vitro Atg8 conjugation system that the Atg12-Atg5 conjugate, but not unconjugated Atg12 or Atg5, strongly enhances the formation of the other conjugate, Atg8-PE. The Atg12-Atg5 conjugate promotes the transfer of Atg8 from Atg3 to the substrate, phosphatidylethanolamine (PE), by stimulating the activity of Atg3. We also show that the Atg12-Atg5 conjugate interacts with both Atg3 and PE-containing liposomes. These results indicate that the Atg12-Atg5 conjugate is a ubiquitin-protein ligase (E3)-like enzyme for Atg8-PE conjugation reaction, distinctively promoting protein-lipid conjugation.

Involvement of linear polyubiquitylation of NEMO in NF-|[kappa]|B activation

Nuclear factor-κB (NF-κB) is a key transcription factor in inflammatory, anti-apoptotic and immune processes. The ubiquitin pathway is crucial in regulating the NF-κB pathway. We have found that the LUBAC ligase complex, composed of the two RING finger proteins HOIL-1L and HOIP, conjugates a head-to-tail-linked linear polyubiquitin chain to substrates. Here, we demonstrate that LUBAC activates the canonical NF-κB pathway by binding to NEMO (NF-κB essential modulator, also called IKKγ) and conjugates linear polyubiquitin chains onto specific Lys residues in the CC2–LZ domain of NEMO in a Ubc13-independent manner. Moreover, in HOIL-1 knockout mice and cells derived from these mice, NF-κB signalling induced by pro-inflammatory cytokines such as TNF-α and IL-1β was suppressed, resulting in enhanced TNF-α–induced apoptosis in hepatocytes of HOIL-1 knockout mice. These results indicate that LUBAC is involved in the physiological regulation of the canonical NF-κB activation pathway through linear polyubiquitylation of NEMO.

Transmembrane phosphoprotein Cbp regulates the activities of Src-family tyrosine kinases.

The Src family of protein tyrosine kinases (Src-PTKs) is important in the regulation of growth and differentiation of eukaryotic cells. The activity of Src-PTKs in cells of different types is negatively controlled by Csk, which specifically phosphorylates a conserved regulatory tyrosine residue at the carboxy-terminal tail of the Src-PTKs. Csk is mainly cytoplasmic and Src-PTKs are predominantly membrane-associated. This raises a question about the mechanism of interaction between these enzymes. Here we present Cbp—a transmembrane phosphoprotein that is ubiquitously expressed and binds specifically to the SH2 domain of Csk. Cbp is involved in the membrane localization of Csk and in the Csk-mediated inhibition of c-Src. In the plasma membrane Cbp is exclusively localized in the GM1 ganglioside-enriched detergent-insoluble membrane domain, which is important in receptor-mediated signalling. These findings reveal Cbp as a new component of the regulatory mechanism controlling the activity of membrane-associated Src-PTKs.

A sequential program of dual phosphorylation of KaiC as a basis for circadian rhythm in cyanobacteria

The circadian phosphorylation cycle of the cyanobacterial clock protein KaiC has been reconstituted in vitro. The phosphorylation profiles of two phosphorylation sites in KaiC, serine 431 (S431) and threonine 432 (T432), revealed that the phosphorylation cycle contained four steps: (i) T432 phosphorylation; (ii) S431 phosphorylation to generate the double‐phosphorylated form of KaiC; (iii) T432 dephosphorylation; and (iv) S431 dephosphorylation. We then examined the effects of mutations introduced at one KaiC phosphorylation site on the intact phosphorylation site. We found that the product of each step in the phosphorylation cycle regulated the reaction in the next step, and that double phosphorylation converted KaiC from an autokinase to an autophosphatase, whereas complete dephosphorylation had the opposite effect. These mechanisms serve as the basis for cyanobacterial circadian rhythm generation. We also found that associations among KaiA, KaiB, and KaiC result from S431 phosphorylation, and these interactions would maintain the amplitude of the rhythm.

Roles of CLOCK Phosphorylation in Suppression of E-Box-Dependent Transcription

ABSTRACT In mammalian circadian clockwork, the CLOCK-BMAL1 heterodimer activates E-box-dependent transcription, while its activity is suppressed by circadian binding with negative regulators, such as CRYs. Here, we found that the CLOCK protein is kept mostly in the phosphorylated form throughout the day and is partly hyperphosphorylated in the suppression phase of E-box-dependent transcription in the mouse liver and NIH 3T3 cells. Coexpression of CRY2 in NIH 3T3 cells inhibited the phosphorylation of CLOCK, whereas CIPC coexpression markedly stimulated phosphorylation, indicating that CLOCK phosphorylation is regulated by a combination of the negative regulators in the suppression phase. CLOCK-BMAL1 purified from the mouse liver was subjected to tandem mass spectrometry analysis, which identified Ser38, Ser42, and Ser427 as in vivo phosphorylation sites of CLOCK. Ser38Asp and Ser42Asp mutations of CLOCK additively and markedly weakened the transactivation activity of CLOCK-BMAL1, with downregulation of the nuclear amount of CLOCK and the DNA-binding activity. On the other hand, CLOCKΔ19, lacking the CIPC-binding domain, was far less phosphorylated and much more stabilized than wild-type CLOCK in vivo. Calyculin A treatment of cultured NIH 3T3 cells promoted CLOCK phosphorylation and facilitated its proteasomal degradation. Together, these results show that CLOCK phosphorylation contributes to the suppression of CLOCK-BMAL1-mediated transactivation through dual regulation: inhibition of CLOCK activity and promotion of its degradation.

Peptidomic Identification and Biological Validation of Neuroendocrine Regulatory Peptide-1 and -2

Recent advances in peptidomics have enabled the identification of previously uncharacterized peptides. However, sequence information alone does not allow us to identify candidates for bioactive peptides. To increase an opportunity to discover bioactive peptides, we have focused on C-terminal amidation, a post-translational modification shared by many bioactive peptides. We analyzed peptides secreted from human medullary thyroid carcinoma TT cells that produce amidated peptides, and we identified two novel amidated peptides, designated neuroendocrine regulatory peptide (NERP)-1 and NERP-2. NERPs are derived from distinct regions of the neurosecretory protein that was originally identified as a product of a nerve growth factor-responsive gene in PC12 cells. Mass spectrometric analysis of the immunoprecipitate using specific antibodies as well as reversed phase-high performance liquid chromatography coupled with radioimmunoassay analysis of brain extract demonstrated the endogenous presence of NERP-1 and NERP-2 in the rat. NERPs are abundant in the paraventricular and supraoptic nuclei of the rat hypothalamus and colocalized frequently with vasopressin but rarely with oxytocin. NERPs dose-dependently suppressed vasopressin release induced by intracerebroventricular injection of hypertonic NaCl or angiotensin II in vivo. NERPs also suppressed basal and angiotensin II-induced vasopressin secretion from hypothalamic explants in vitro. Bioactivity of NERPs required C-terminal amidation. Anti-NERP IgGs canceled plasma vasopressin reduction in response to water loading, indicating that NERPs could be potent endogenous suppressors of vasopressin release. These findings suggest that NERPs are novel modulators in body fluid homeostasis.

N-glycosylation at Asn491 in the Asn-Xaa-Cys motif of human transferrin

Glycopeptides derived from human transferrin were exhaustively analyzed by matrix‐assisted laser desorption ionization and electrospray ionization mass spectrometry (MS). Both MS techniques clearly revealed the sequences of and the attachment sites of bi‐antennary complex‐type oligosaccharides, at both Asn432and Asn630, both of which are located in a well‐known motif for N‐glycosylation, Asn‐Xaa‐Ser/Thr, but also at Asn491in the Asn‐Xaa‐Cys motif. The latter has been reported to be a minor N‐glycosylation site in several glycoproteins. The relative abundance of this abnormal glycosylation was estimated to be approximately 2 mol% of the transferrin preparation used in this study.

Three Maize Leaf Ferredoxin:NADPH Oxidoreductases Vary in Subchloroplast Location, Expression, and Interaction with Ferredoxin

In higher plants, ferredoxin (Fd):NADPH oxidoreductase (FNR) catalyzes reduction of NADP+ in the final step of linear photosynthetic electron transport and is also implicated in cyclic electron flow. We have identified three leaf FNR isoenzymes (LFNR1, LFNR2, and LFNR3) in maize (Zea mays) chloroplasts at approximately equivalent concentrations. Fractionation of chloroplasts showed that, while LFNR3 is an exclusively soluble enzyme, LFNR1 is only found at the thylakoid membrane and LFNR2 has a dual location. LFNR1 and LFNR2 were found to associate with the cytochrome b6f complex following its partial purification. We cloned LFNR3 and produced all three isoenzymes as stable, soluble proteins. Measurement of Fd reduction ability showed no significant differences between these recombinant enzymes. Column chromatography revealed variation between the interaction mechanisms of LFNR1 and LFNR2 with Fd, as detected by differential dependence on specific intermolecular salt bridges and variable sensitivity of interactions to changes in pH. A comparison of LFNR transcripts in leaves of plants grown on variable nitrogen regimes revealed that LFNR1 and LFNR2 transcripts are relatively more abundant under conditions of high demand for NADPH. These results are discussed in terms of the functional differentiation of maize LFNR isoenzymes.

Automated interpretation of low-energy collision-induced dissociation spectra by SeqMS, a software aid for de novo sequencing by tandem mass spectrometry

SeqMS, a software aid for de novo sequencing by tandem mass spectrometry (MS/MS), which was initially developed for the automated interpretation of high‐energy collision‐induced dissociation (CID) MS/MS spectra of peptides, has been applied to the interpretation of low‐energy CID and post‐source decay (PSD) spectra of peptides. Based on peptide backbone fragmented ions and their related ions, which are the dominant ions observed in the latter two techniques, the types of ions and their propensities to be observed have been optimized for efficient interpretation of the spectra. In a typical example, the modified SeqMS allowed the complete sequencing of a 31‐amino acid synthetic peptide, except for the isobaric amino acids (Leu or Ile, and Lys or Gln), based on only the low‐energy CID‐MS/MS spectrum.