Shigenori Ogata

The interaction of two tethering factors, p115 and COG complex, is required for Golgi integrity.

The vesicle‐tethering protein p115 functions in endoplasmic reticulum–Golgi trafficking. We explored the function of homologous region 2 (HR2) of the p115 head domain that is highly homologous with the yeast counterpart, Uso1p. By expression of p115 mutants in p115 knockdown (KD) cells, we found that deletion of HR2 caused an irregular assembly of the Golgi, which consisted of a cluster of mini‐stacked Golgi fragments, and gathered around microtubule‐organizing center in a microtubule‐dependent manner. Protein interaction analyses revealed that p115 HR2 interacted with Cog2, a subunit of the conserved oligomeric Golgi (COG) complex that is known another putative cis‐Golgi vesicle‐tethering factor. The interaction between p115 and Cog2 was found to be essential for Golgi ribbon reformation after the disruption of the ribbon by p115 KD or brefeldin A treatment and recovery by re‐expression of p115 or drug wash out, respectively. The interaction occurred only in interphase cells and not in mitotic cells. These results strongly suggested that p115 plays an important role in the biogenesis and maintenance of the Golgi by interacting with the COG complex on the cis‐Golgi in vesicular trafficking.

Interaction of Golgin-84 with the COG Complex Mediates the Intra-Golgi Retrograde Transport

The coiled‐coil Golgi membrane protein golgin‐84 functions as a tethering factor for coat protein I (COPI) vesicles. Protein interaction analyses have revealed that golgin‐84 interacts with another tether, the conserved oligomeric Golgi (COG) complex, through its subunit Cog7. Therefore, we explored the function of golgin‐84 as the tether for COPI vesicles of intra‐Golgi retrograde traffic. First, glycosylic maturation of both plasma membrane (CD44) and lysosomal (lamp1) glycoproteins was distorted in golgin‐84 knockdown (KD) cells. The depletion of golgin‐84 caused fragmentation of the Golgi with the mislocalization of Golgi resident proteins, resulting in the accumulation of vesicles carrying intra‐Golgi soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs) and cis‐Golgi membrane protein GPP130. Similar observations were obtained by diminution of the COG complex, suggesting a strong correlation between the two tethers. Indeed, COG complex‐dependent (CCD) vesicles that accumulate in Cog3 or Cog7 KD cells carried golgin‐84. Surprisingly, the interaction between golgin‐84 and another candidate tethering partner CASP (CDP/cut alternatively spliced product) decreased in Cog3 KD cells. These results indicate that golgin‐84 on COPI vesicles interact with the COG complex before SNARE assembly, suggesting that the interaction of golgin‐84 with COG plays an important role in the tethering process of intra‐Golgi retrograde vesicle traffic.

Multiple Forms of Rat-Serum α1-Protease Inhibitor

alpha 1-Protease inhibitor was purified from rat serum by two different methods, of which an immunoaffinity method should be preferentially used to obtain all of the multiple forms. The protein thus obtained showed a single protein band in dodecylsulphate/polyacrylamide gel electrophoresis corresponding to a molecular weight of 54000, and contained 13.2% carbohydrate by weight. By column isoelectric focusing in a pH 3.5-5.0 gradient the purified alpha 1-protease inhibitor was separated into five forms with pI values from 4.3-4.7. The amino acid composition of each form was identical, while sialic acid content was significantly different from each other. The most acidic form contained 6.7 residues/molecule, the most basic form, 5.1 residues/molecule, and three forms between them showed proportionally intermediate values between the two. When alpha 1-protease inhibitor was treated with neuraminidase, the five forms were converted finally into three major forms with pI values of 5.3-5.7. In addition, the major form (band 3) of the inhibitor was also converted into three forms after complete removal of sialic acid. These results suggest that alpha 1-protease inhibitor originally exists as three forms with different charges, possibly due to modification of amino acids which might not be detectable by the amino acid composition analysis in the present study. A possible explanation was presented for involvement of sialic acid in appearance of multiple forms originating from three parental forms.

Metronomic doxifluridine chemotherapy combined with the anti-angiogenic agent TNP-470 inhibits the growth of human uterine carcinosarcoma xenografts

Uterine carcinosarcoma is a highly aggressive gynecological neoplasm that responds poorly to conventional chemotherapy and radiotherapy. Metronomic chemotherapy is accepted as a new approach for cancer treatment, and its underlying mechanism seems to involve the suppression of angiogenesis. However, the efficacy of metronomic and anti‐angiogenic therapies against uterine carcinosarcoma is unknown. The anti‐angiogenic effect of doxifluridine was assessed in vitro using human umbilical vein endothelial cells (HUVEC) co‐cultured with FU‐MMT‐1 human uterine carcinosarcoma cells. The antitumor and anti‐angiogenic effects of metronomic doxifluridine (delivered via oral gavage) in combination with TNP‐470 were evaluated in vivo. Tumor vascularity was assessed by contrast‐enhanced color Doppler ultrasound, laser Doppler and microvessel density staining. Doxifluridine suppressed tube formation of HUVEC and vascular endothelial growth factor production by FU‐MMT‐1 cells. Metronomic doxifluridine alone significantly suppressed tumor growth compared with the untreated (control) group, while metronomic doxifluridine in combination with TNP‐470 significantly inhibited tumor growth compared with each treatment alone. A significant reduction of intratumoral vascularity was observed in FU‐MMT‐1 xenografts following treatment with metronomic doxifluridine in combination with TNP‐470, as compared with each treatment alone. Intestinal bleeding was only observed when the maximum tolerated dose of doxifluridine was administered in combination with TNP‐470. Metronomic doxifluridine chemotherapy in combination with TNP‐470 might be effective for uterine carcinosarcoma without marked toxicity, possibly acting via its potent anti‐angiogenic effects. Clinical studies are needed to evaluate the safety and efficacy of this treatment in humans. (Cancer Sci 2011; 102: 1545–1552)

Isolation and characterization of human apolipoprotein A-I Fukuoka (110 Glu → Lys). A novel apolipoprotein variant

A novel genetic variant of apolipoprotein(apo) A-I Fukuoka, has been identified in a Japanese family. This variant has a relative charge of +2 compared to normal apolipoprotein A-I (A-I4), on the isoelectric focusing gels and the same molecular mass and immunologic characteristics as normal apolipoprotein A-I. This variant, transmitted as an autosomal co-dominant inheritance was purified by preparative Immobiline isoelectric focusing. Sequence analysis after cleavage with lysyl endopeptidase and CNBr, followed by high-performance liquid chromatography revealed a single substitution of lysine at position 110, instead of the usual glutamic acid. This mutant apolipoprotein A-I has much the same potential as to activate lecithin-cholesterol acyltransferase.

YIPF5 and YIF1A recycle between the ER and the Golgi apparatus and are involved in the maintenance of the Golgi structure.

Yip1p/Yif1p family proteins are five-span transmembrane proteins localized in the Golgi apparatus and the ER. There are nine family members in humans, and YIPF5 and YIF1A are the human orthologs of budding yeast Yip1p and Yif1p, respectively. We raised antisera against YIPF5 and YIF1A and examined the localization of endogenous proteins in HeLa cells. Immunofluorescence, immunoelectron microscopy and subcellular fractionation analysis suggested that YIPF5 and YIF1A are not restricted to ER exit sites but also localized in the ER-Golgi intermediate compartment (ERGIC) and some in the cis-Golgi at steady state. Along with ERGIC53, YIPF5 and YIF1A remained in the cytoplasmic punctate structures after brefeldin A treatment, accumulated in the ERGIC and the cis-Golgi after treatment with AlF4- and accumulated in the ER when ER to Golgi transport was inhibited by Sar1(H79G). These results supported the localization of YIPF5 and YIF1A in the ERGIC and the cis-Golgi, and strongly suggested that they are recycling between the ER and the Golgi apparatus. Analysis by blue native PAGE and co-immunoprecipitation showed that YIPF5 and YIF1A form stable complexes of three different sizes. Interestingly, the knockdown of YIPF5 or YIF1A caused partial disassembly of the Golgi apparatus suggesting that YIPF5 and YIF1A are involved in the maintenance of the Golgi structure.

[16] Dipeptidyl-peptidase IV from rat liver

Publisher Summary This chapter describes assay, purification, and properties of dipeptidyl-peptidase IV (DPPIV) from rat liver. DPPIV is a serine protease that cleaves N-terminal dipeptides from oligo- and polypeptides with a penultimate prolyl residue. It is a membrane-bound glycoprotein localized on the cell surface (ectoenzyme), in contrast to other DPPs localized in the lysosome and in the cytoplasm. The release of p -nitroaniline from dipeptidyl- p -nitroanilides is photometrically determined at 385 nm after the indicated incubation period. Gly-Pro- p -nitroanilide tosylate is used as a routine substrate, but any Xaa-Pro- p -nitroanilide can be used for the assay. Two forms of DPPIV, papain-cleaved soluble form and Triton X-100- solubilized membrane form, are purified from plasma membranes of rat liver. DPPIV is known to survive autolysis for up to 24 hr at pH 4 and 37°, with a recovery of more than 50% of the activity. DPPIV also exhibits a strong preference for substrates having a penultimate prolyl residue, which can be replaced only by alanine and hydroxyproline with much lower rates of hydrolysis.

Purification and characterization of a kinin- and angiotensin II-forming enzyme in the dog heart.

Objective To purify and characterize a kinin-forming enzyme in the dog heart and to examine the ability of this enzyme to generate angiotensin (Ang) II from Ang I. Methods The enzyme was isolated from heart homogenate using a diethylaminoethyl-Sepharose column, an aprotinin affinity column and a wheat germ lectin-Sepharose 6MB column. Kininogenase activity was assessed with a kinin radioimmunoassay after samples had been incubated with bovine low-molecular-mass kininogen at 37 °C for 1 h. Ang I-converting activity was assessed by the quantitation of Ang II formed by incubation of the sample with Ang I at 37 °C for 3 h, using high performance liquid chromatography. The enzyme was subjected to 12.5% sodium dodecyl sulphate-polyacrylamide gel electrophoresis, stained by Coomassie brilliant blue and transferred electrically to a membrane with glycoprotein staining. Results The purified enzyme is a glycoprotein with an apparent relative molecular mass of 65 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Its kininogenase activity was approximately 20 μg bradykinin/h per mg protein at an optimal pH of 8.0. The enzyme also converted Ang I to Ang II at an optimal pH of 6.5. Its specific activity was approximately 2 μg Ang II/h per mg protein. Both activities were inhibited by aprotinin, a tissue kallikrein inhibitor. Western blot analysis using polyclonal antibody against this enzyme demonstrated that this enzyme exists both in the myocardium and in the coronary artery. Conclusions The present study showed that the kininforming enzyme in the dog heart is a kallikrein-like enzyme that is different from cathepsin D, cathepsin G and chymase. It is also able to convert Ang I to Ang II. This enzyme might play a role in regulating myocardial perfusion, mainly by generating kinins and in part by forming Ang II.

Structure of a kallikrein-like enzyme and its tissue localization in the dog

We previously purified a kallikrein-like enzyme from the dog heart and demonstrated that it is not only able to form kinins but can also convert angiotensin (Ang) I to Ang II. The aim of the present study was to clarify the structure and tissue localization of this enzyme. Western blot analysis of various canine tissues was performed with antiserum against the purified dog heart enzyme. The purified enzyme was subjected to a determination of its amino acid composition and a sequence analysis. Western blotting indicated that this enzyme was present in the heart, aorta, kidney, pancreas, lung, liver, spleen, small intestine, and skeletal muscle. The amino acid composition of the enzyme was different from that of dog urinary kallikrein. Amino acid sequence analysis indicated that it is likely to be N-terminally blocked. The present study showed that this kallikrein-like enzyme is different from previously reported kallikrein and is distributed not only in the heart, but also in other tissues such as the aorta, kidney, lung, liver, spleen, small intestine, and skeletal muscle. This enzyme may exert local effects by generating kinins and Ang II.

Trans-Golgi protein p230/golgin-245 is involved in phagophore formation

p230/golgin-245 is a trans-Golgi coiled-coil protein that is known to participate in regulatory transport from the trans-Golgi network (TGN) to the cell surface. We investigated the role of p230 and its interacting protein, microtubule actin crosslinking protein 1 (MACF1), in amino acid starvation-induced membrane transport. p230 or MACF1 knock-down (KD) cells failed to increase the autophagic flow rate and the number of microtubule-associated protein 1 light chain 3 (LC3)-positive puncta under starvation conditions. Loss of p230 or MACF1 impaired mAtg9 recruitment to peripheral phagophores from the TGN, which was observed in the early step of autophagosome formation. Overexpression of the p230-binding domain of MACF1 resulted in the inhibition of mAtg9 trafficking in starvation conditions as in p230-KD or MACF1-KD cells. These results indicate that p230 and MACF1 cooperatively play an important role in the formation of phagophore through starvation-induced transport of mAtg9-containing membranes from the TGN. In addition, p230 itself was detected in autophagosomes/autolysosome with p62 or LC3 during autophagosome biogenesis. Thus, p230 is an important molecule in phagophore formation, although it remains unclear whether p230 has any role in late steps of autophagy.