Structural insight into glucose dehydrogenase from the thermoacidophilic archaeon Thermoplasma volcanium.

Glucose dehydrogenase from the thermoacidophilic archaeon Thermoplasma volcanium (tvGlcDH) is highly active towards D-glucose and D-galactose, but does not utilize aldopentoses such as D-xylose as substrates. In the present study, the crystal structures of substrate/cofactor-free tvGlcDH and of a tvGlcDH T277F mutant in a binary complex with NADP and in a ternary complex with D-glucose and nicotinic acid adenine dinucleotide phosphate, an NADP analogue, were determined at resolutions of 2.6, 2.25 and 2.33 Å, respectively. The overall structure of each monomer showed notable similarity to that of the enzyme from Sulfolobus solfataricus (ssGlcDH-1), which accepts a broad range of C5 and C6 sugars as substrates. However, the amino-acid residues of tvGlcDH involved in substrate binding markedly differed from those of ssGlcDH-1. Structural comparison revealed that a decreased number of interactions between the C3-hydroxyl group of the sugar and the enzyme are likely to be responsible for the lack of reactivity of tvGlcDH towards D-xylose.

Intracellular retention of the extracellular domain of the (pro)renin receptor in mammalian cells.

As a component of the renin-angiotensin system, the (pro)renin receptor [(P)RR] activates prorenin along with intracellular signaling pathways. In this study, the glutathione S-transferase-fused extracellular domain of (P)RR expressed in mammalian cells was recovered in the detergent phase in detergent-based two-phase separation experiments, and intracellular localization was observed by immunocytochemistry, suggesting retention inside the cell through stable membrane association.

[A case of Stage IV sigmoid colon cancer that achieved long-term survival with oral anticancer drugs].

An 80-year-old man presenting with abdominal distension was admitted to our hospital. He was diagnosed with sigmoid cancer with multiple liver and lung metastases. We first performed a sigmoidectomy to avoid obstruction, and then initiated chemotherapy with S-1(120mg/day). The tumor showed a complete clinical response after 10 courses, but we had to change the regimen after 18 courses because of growth of the lung metastases. After 10 courses of capecitabine(4,200mg/ day)treatment, we again observed growth of the lung metastases; a new nodule, which was also considered to be a metastasis, appeared on the abdominal wall. We then decided to administer mFOLFOX6(5-fluorouracil+Leucovorin+oxaliplatin) after the patient had received oral anticancer drugs for 3 years 4 month. In conclusion, oral chemotherapy drugs may prevent tumor growth over a long period and improve quality of life(QOL)in elderly patients with Stage IV colon cancer.

[A case of complete response(CR)to S-1 and paclitaxel(PTX)combination therapy in a patient with unresectable gastric cancer].

We report a case of a patient with unresectable gastric cancer who showed complete response(CR)to S-1 and paclitaxel (PTX)combination therapy. The patient(a 67-year-old woman)was diagnosed with unresectable advanced gastric cancer with metastases in the Virchow's lymph nodes and para-aortic lymph nodes. Systemic chemotherapy with 70mg/m2 S-1 (days 1-14)and 70mg/m2 PTX(day 1)was administered every 3 weeks. At the end of 7 courses of chemotherapy, the primary lesion and swollen lymph nodes became markedly smaller. After 7 courses, an additional 39 courses were administered over 2.5 years. No notable adverse events were seen, and the patient's performance status(PS)was 0. CR was monitored by imaging studies. No cancer cells were detected on cytological examination of the primary lesion. Monotherapy with 70mg/m2 S-1(days 1-28, 2-week drug holiday)has been administered for the past 3 years. The patient is currently treated as an outpatient and maintains CR and a PS of 0.

The coexistence of Ser84 in renin and His13 in angiotensinogen brings a pH profile of two separate peaks to the reaction of human renin and sheep angiotensinogen.

The pH dependence and kinetics parameters of renin-angiotensinogen reactions were determined using wild-type and S84G mutant human renins and wild-type and H13Y mutant sheep angiotensinogens. It is explained in this report that (i) renin catalyzes acidic and basic reactions of which the optimum pHs are 5.5 and 7.5-8.2 respectively, both of which produce angiotensin I; (ii) Ser84 specific to human renin accelerates the acidic reaction by 75-110% through elevation of V(max), and shifts the optimum pH of the basic reaction from 7.5 to 8.0-8.2; and (iii) His13 specific to sheep angiotensinogen accelerates the acidic and basic reactions by 25-42% through reduction of K(m). It is concluded from these results that the coexistence of Ser84 in renin and His13 in angiotensinogen brings a pH profile of two separate peaks at pHs 5.5 and 8.2 to the reaction of human renin and sheep angiotensinogen.

Role of "handle" region of prorenin prosegment in the non-proteolytic activation of prorenin by binding to membrane anchored (pro)renin receptor.

A role of the "handle" region in the prorenin prosegment sequence was investigated to demonstrate the crucial non-proteolytic activation of prorenin by binding to the recombinant (pro)renin receptor on the COS-7 cell membrane. The plasmid DNA containing either rat or human (pro)renin receptor was transfected into the COS-7 cells. The highest amount of receptor was observed on the COS-7 cell membrane after 18 h transfection. Of the total rat and human prorenin, 90% and 50% were bound to each of the respective receptors, respectively. The Kd values were 0.89 and 1.8 nM, respectively. Rat prorenin was activated non-proteolytically by the receptor. The Km was determined 1.0 microM when sheep angiotensinogen was used as the substrate. Human prorenin was also activated by the receptor. The Km was 0.71 microM. Additionally, decapeptides (10P-19P) known as "decoy" peptide and pentapeptides (11P-15P) named "handle" region peptide, were observed to inhibit the binding of both prorenins to receptors, respectively. The Ki were similar around 7 nM for both the peptides. Other two region peptides in the prosegment did not interfere the binding. These results show that the "handle" region probably plays a crucial role in prorenin binding to the receptor and in its enzymic activity by non-proteolytic activation.

Efficient production of recombinant human (pro)renin utilizing a decahistidine tag attached at the C-terminus.

Human prorenin attached by a decahistidine tag at the C-terminus was produced in Chinese hamster ovary cells. The tagged protein secreted into the culture medium was in the inactive prorenin form, and was activated to mature renin by proteolytic removal of its prosegment by trypsin in the same manner as native prorenin. The tagged (pro)renin was efficiently purified by metal-chelate affinity chromatography. The enzymatic properties of mature renin carrying the tag were similar to native renin. These results indicate that the introduction of a decahistidine tag at the C-terminus does not interfere with either the correct folding of prorenin or the catalytic activity of mature renin.

The His-Pro-Phe motif of angiotensinogen is a crucial determinant of the substrate specificity of renin.

The amino acid sequence His-Pro-Phe as N-terminal residues 6-8 of the natural renin substrate, angiotensinogen, is conserved among species. We investigated whether this His-Pro-Phe motif functions as the determinant of the substrate specificity of renin. Mutant angiotensinogens in which the Ile-His-Pro-Phe-His-Leu sequence at positions 5-10 of wild-type angiotensinogen was replaced by either His-Pro-Phe-His-Leu-Leu or Ala-Ile-His-Pro-Phe-His were cleaved by renin at the C-terminal side of residues 9 and 11, respectively, while wild-type angiotensinogen was cleaved at residue 10. A triple Ala substitution for the His-Pro-Phe motif of angiotensinogen prevented its cleavage by renin. In contrast, triple Ala substitution for residues 9-11, including the natural site of cleavage by renin, allowed cleavage between the two Ala residues at positions 10 and 11. Furthermore, the 33-residue C-terminal peptide of human megsin, which carries a naturally occurring His-Pro-Phe sequence, was cleaved by renin at the C-terminal side of the His-Pro-Phe-Leu-Phe sequence. These results indicate that the His-Pro-Phe motif of angiotensinogen is a crucial determinant of the substrate specificity of renin. By binding to a corresponding pocket on renin, the His-Pro-Phe motif may act as a molecular anchor to recruit the scissile peptide bond to a favorable site for catalysis.

Ser84 of human renin contributes to the biphasic pH dependence of the renin-angiotensinogen reaction.

The pH dependence of the reaction of various renins was investigated using sheep angiotensinogen as a substrate. Human renin showed two separate peaks, but rat and mouse Ren1 renins showed one peak with a shoulder. A comparison of the predicted subsite residues of human renin with those of rat and mouse Ren1 renins revealed that Arg82, Ser84, Thr85, Ala229, and Thr312 are unique in the human sequence. We examined the possible importance of these residues in the unique pH profile of the human renin reaction by replacing these residues with the corresponding residues of rat renin. The replacement of Ser84 of human renin with Gly changed the pH dependence of the reaction to one peak, similarly to rat and mouse Ren1 renins. Other mutant human renins kept two separate peaks, similarly to wild-type human renin. These results indicate that Ser84 of human renin contributes to the biphasic pH dependence of the renin-angiotensinogen reaction.