• Biotechnology and Bioprocess Engineering:BBE
• /
• 제5권1호
• /
• pp.61-64
• /
• 2000

A series of 5-oxaproline peptide derivatives was synthesized and evaluated for its ability to inhibit the prolyl 4-hydroxylase in vitro. Structure-activity studies show that the 5-oxaproline sequences, prepared by the 1,3-dipolar cycloaddition of the C-methoxycarbonyl-N-mannosyl nitrone in the presence of the ethylene, are more active than the corresponding proline derivatives. Prolyl 4-hydroxylase belongs to a family of $Fe^{2+}-dependent$ dioxygenase, which catalyzes the formation of 4-hydroxyproline in collagens by the hydroxylation of proline residues in -Gly-Xaa-Pro-Gly- of procollagen chains. In this paper we discover the more selective N-Cbz-Gly-Phe-Pro-Gly-OEt $(K_m\;=\;520\;{\mu}M)$ sequences which are showed stronger binding than others in vitro. Therefore, we set out to investigate constrained tetrapeptide that was designed to mimic the proline structure of pep tides for the development of prolyl 4-hydroxylase inhibitor. From this result, we found that the most potent inhibitor is N-Dansyl-Gly-Phe-5-oxaPro-Gly-OEt $(K_i\;=\;1.6\;{\mu}M)$. This has prompted attempts to develop drugs which inhibit collagen synthesis. Prolyl 4-hydroxylase would seem a particularly suitable target for antifibrotic therapy.

• The Korean Journal of Physiology and Pharmacology
• /
• 제17권2호
• /
• pp.111-120
• /
• 2013

Prolyl 4 hydroxylases (P4H) are iron- and 2-oxoglutamate-dependent dioxygenase enzymes and hypoxia-inducible transcription factor (HIF)-P4Hs play a critical role in the regulating oxygen homeostasis in the local tissues as well in the systemic circulation. Over a period of time, a number of prolyl hydroxylase inhibitors and activators have been developed. By employing the pharmacological tools and transgenic knock out animals, the critical role of these enzymes has been established in the pathophysiology of number of diseases including myocardial infarction, congestive heart failure, stroke, neurodegeneration, inflammatory disease, respiratory diseases, retinopathy and others. The present review discusses the different aspects of these enzymes including their pathophysiological role in disease development.

• Biomolecules & Therapeutics
• /
• 제15권4호
• /
• pp.261-265
• /
• 2007

Hydroxyproline (HYP) is a post-translational product of proline hydroxylation catalyzed by an enzyme prolyl 4-hydroxylase (P4H) which plays a crucial role in the synthesis of all collagens. Considering the role of collagen and its significance in many clinically important diseases such as liver fibrosis, a great deal of attention has been directed toward the development of an assay at cell-based system. The reason is that cell-based assay system is more efficient than enzyme-based in vitro system and takes much less time than in vivo system. Several assay procedures developed for P4H are laborious, time-consuming and not feasible for the massive-screening. Here, we report the cell-based assay method of prolyl 4-hydroxylase in immortalized rat hepatic stellate HSC-T6 cells. To optimize the cell culture condition to assay for HYP content, various concentrations of reagents were treated for different times in HSC-T6 cells. Our data showed that the treatment with ascorbate in a hypoxic condition for 24 h resulted in the maximal increase of HYP by 1.8 fold. Alternatively, cobalt chloride ($5\;{\mu}M$) and ascorbate ($50\;{\mu}M$) in normoxic states exhibited similar effect on the production of HYP as in a hypoxic condition. Therefore, cobalt chloride can be substituted for a hypoxic condition when an anaerobic chamber is not available. Rosiglitazone and HOE077, known as inhibitors of collagen, synthesis decreased P4H enzyme activity by 32.3% and 15%, respectively, which coincided with previous reports from liver tissues. The level of the smooth muscle ${\alpha}$-actin, a marker of activated stellate cells, was significantly increased under hypoxia, suggesting that our experimental condition could work for screening the anti-fibrotic compounds. The assay procedure took only 3 days after treatment with agents, while assays from the primary stellate cells or liver tissues have taken several weeks. Considering the time and expenses, this assay method could be useful to screen the compounds for the inhibitor of prolyl 4-hydroxylase.

• Bulletin of the Korean Chemical Society
• /
• 제27권6호
• /
• pp.909-917
• /
• 2006

2,2-Dimethyl-3,4-dialkoxy-substituted 6-aminobenzopyran analogues (eg., 7 and 8) were identified as prolyl 4-hydroxylase inhibitors via a screening process using HSC-T6 and LI 90 cells that express an immortalized rat hepatic stellate cell line and as part of a test of the type I collagen contents employing the ELISA method. A subsequent lead optimization effort based on solid-phase parallel synthesis led to the identification of 2,2-dimethyl-3,4-dialkoxy-substituted 6-aminobenzopyrans as potent inhibitors of prolyl 4-hydroxylase.

• Toxicological Research
• /
• 제24권2호
• /
• pp.101-107
• /
• 2008

Studies on hypoxia-signaling pathways have revealed novel Fe(II) and $\alpha$-ketoglutarate-dependent dioxygenases that hydroxylate prolyl or asparaginyl residues of a transactivator, Hypoxia-Inducible $Factor-\alpha(HIF-\alpha)$ protein. The recognition of these unprecedented dioxygenases has led to open a new paradigm that the hydroxylation mediates an instant post-translational modification of a protein in response to the changes in cellular concentrations of oxygen, reducing agents, or $\alpha$-ketoglutarate. Activity of $HIF-\alpha$ is repressed by two hydroxylases. One is $HIF-\alpha$ specific prolyl-hydroxylases, referred as prolyl-hydroxylase domain(PHD). The other is $HIF-\alpha$ specific asparaginyl-hydroxylase, referred as factor-inhibiting HIF-1(FIH-1). The facts (i) that many dioxygenases commonly use molecular oxygen and reducing agents during detoxification of xenobiotics, (ii) that detoxification reaction produces radicals and reactive oxygen species, and (iii) that activities of both PHD and FIH-1 are regulated by the changes in the balance between oxygen species and reducing agents, imply the possibility that the activity of $HIF-\alpha$ can be increased during detoxification process. The importance of $HIF-\alpha$ in cancer and ischemic diseases has been emphasized since its target genes mediate various hypoxic responses including angiogenesis, erythropoiesis, glycolysis, pH balance, metastasis, invasion and cell survival. Therefore, activators of PHDs and FIH-1 can be potential anticancer drugs which could reduce the activity of HIF, whereas inhibitors, for preventing ischemic diseases. This review highlights these novel dioxygenases, PHDs and FIH-1 as specific target against not only cancers but also ischemic diseases.

• 한국독성학회:학술대회논문집
• /
• 한국독성학회 2002년도 Molecular and Cellular Response to Toxic Substances
• /
• pp.161-161
• /
• 2002

The abnormal accumulation of collagen is progressive and often results in impairment of liver function, i.e. liver cirrhosis. Collagen synthesis requires several posttranslational events. Prolyl 4-hydroxylase is the key enzyme in collagen synthesis that catalyzes the hydroxylation of peptide-bound proline residues to 4-hydroxyroline.(omitted)

• Asian Pacific Journal of Cancer Prevention
• /
• 제15권14호
• /
• pp.5819-5823
• /
• 2014

Purpose: To investigate the expression of hypoxia-inducible factor prolyl hydroxylase 3 (HIFPH3) in non-small cell lung cancer (NSCLC) and explore the correlation of HIFPH3 expression with lymph node metastasis and microvessel density (MVD). Materials and Methods: A total of 73 cases of NSCLC specimens, 24 cases of para-cancerous tissues, and 20 normal pulmonary tissues were collected for HIFPH3 and CD31 immunohistochmical (IHC) study. Microvessel density (MVD) of the NSCLC tissues was also determined based on the expression of CD31. Results: The expression of HIFPH3 in carcinoma tissue was statistically higher than para-cancerous and normal pulmonary tissues (${\chi}^2=48.806$, p<0.05). Compared withthe negative lymph node metastasis group, the lymph node metastasis group showed significantly higher HIFPH3 expression (${\chi}^2=6.300$, p<0.05). The strong HIFPH3+group displayed a significantly higher MVD than weak HIFPH3+ and HIFPH3- groups (p<0.05). No differences in positive HIFPH3 expression were noted regarding the tumor diameter, age, smoking status, gender of NSCLC patients, tumor size, histopathology, or differentiation. Conclusions: HIFPH3 expression in human NSCLC lesions is significantly higher than that in para-cancerous and normal lung tissues and is positively associated with lymph node metastasis and MVD.

• Asian-Australasian Journal of Animal Sciences
• /
• 제20권11호
• /
• pp.1655-1661
• /
• 2007

Prolyl 4-hydroxylase (P4H) plays a central role in collagen synthesis by catalyzing the hydroxylation of the proline residue in the X-Pro-Gly amino acid sequence, and controls the biosynthesis of collagen that influences overall meat quality. In order to verify expression level of the catalytic ${\alpha}$ subunit of P4H, a 2.7 kb clone of the ${\alpha}$ subunit gene for P4H was selected from a cDNA library prepared from the muscular tissue of Sancheong berkshire pigs, and the whole gene sequence was determined. As expression level of the ${\alpha}$ subunit of P4H differed between tissues of pigs, we intended to assess more precisely the level of ${\alpha}$-subunit expression between tissues of Sancheong Berkshire pigs by using RT-PCR. Muscular and adipose tissues were taken from each pig grouped by growth stage (weighing 60, 80, and 110 kg) of Yorkshire and Sancheong Berkshire pigs, and the expression levels of the ${\alpha}$-subunit of P4H were examined. Since there were significant differences in the expression level with respect to variation in growth stage (p<0.01), an attempt was made to identify any influences of pig species and tissue variation. The muscular and adipose tissues of pigs weighing 110 kg showed higher expression levels than pigs weighing 60 kg and 80 kg. In general, significantly higher expression levels were found in muscular than in adipose tissue. The expression levels in Sancheong Berkshire were significantly higher than in Yorkshire pigs (p<0.01 or p<0.05). Since expression level of the ${\alpha}$-subunit of P4H affects the activity of P4H and is connected to the biosynthesis of collagen and increased collagen can improve meat texture, this finding may explain why meat quality of the Sancheong Berkshire pig is acclaimed in Korea. Given the higher expression levels of the ${\alpha}$-subunit gene in adipose than in muscular tissue, and also in the heavier pigs, more intensive studies are required to assess the correlation between expression level of the ${\alpha}$ subunit gene and overall meat quality.

• Journal of Nutrition and Health
• /
• 제42권6호
• /
• pp.505-515
• /
• 2009

본 연구에서는 비타민 C, silicon, 철분을 농도별로 피부섬유아 세포에 처리 후 콜라겐 합성 효소인 PH, LH의 mRNA와 protein 발현 및 분해 효소인 MMP-1와 저해제인 TIMP-1의 mRNA, protein 발현의 변화를 대조군과 비교 분석하였으며 그 결과를 요약하면 다음과 같다. 1) 피부 섬유아세포에서 비타민 C의 처리는 콜라겐 합성 효소인 PH의 mRNA 발현을 대조군에 비해 현저하게 증가시켰고 이와 병행하여, PH의 protein 발현도 대조군에 비해 유의적으로 증가하였다. 그러나 다른 콜라겐 합성 효소인 LH의 mRNA 발현에는 영향을 미치지 않았으나 protein의 발현을 증가시켰다. 또한, 콜라겐 분해 효소인 MMP-1의 mRNA 발현은 대조군에 비해 유의적으로 증가시켰으나 protein 발현에서는 대조군과 차이가 없었다. 2) 피부 섬유아세포에서 silicon의 혈중 내 농도 처리는 LH의 mRNA 발현의 현저한 증가와 더불어 protein 발현에도 긍정적인 영향을 미치는 것으로 나타났다. 콜라겐 분해 효소인 MMP-1과 저해제인 TIMP-1의 단백질 발현을 대조군에 비해 증가시켰다. 3) 피부 섬유아세포에서 철분의 혈중 내 농도 처리는 콜라겐 합성 및 분해 관련 효소의 mRNA 및 protein 발현에 영향을 미치지 않았다. 결론적으로, 피부섬유아세포에서 비타민 C 및 silicon의 처리는 콜라겐의 posttranslational modification 관련 효소의 mRNA의 발현 및 단백질의 발현을 증가시켜 궁극적으로 콜라겐 합성에 긍정적인 영향을 나타내었다.

• International Journal of Oral Biology
• /
• 제33권4호
• /
• pp.205-211
• /
• 2008

Gingival overgrowth can cause dental occlusion and seriously interfere with mastication, speech, and dental hygiene. It is observed in 25 to 81% of renal transplant patients treated with cyclosporine A (CsA). CsA-induced gingival overgrowth (CIGO) is caused by quantitative alteration of the extracellular matrix components, particularly collagen. However, the molecular mechanisms involved in the pathogenesis of CIGO remain poorly understood, despite intense clinical and laboratory investigations. The aim of the present work is to identify differentially expressed genes closely associated with CIGO. Human gingival fibroblasts were isolated by primary explant culture of gingival tissues from five healthy subjects (HGFs) and two patients with the CIGO (CIGO-HGFs). The proliferative activity of CsA-treated HGFs and CIGO-HGFs was examined using the MTT assay. The identification of differentially expressed genes in CsA-treated CIGO-HGF was performed by differential display reverse transcriptase-polymerase chain reaction (RT-PCR) followed by DNA sequencing. CsA significantly increased the proliferation of two HGFs and two CIGO-HGFs, whereas three HGFs were not affected. Seven genes, including the beta subunit of prolyl 4-hydroxylase (P4HB) and testican 1, were upregulated by CsA in a highly proliferative CIGO-HGF. The increased P4HB and testican-1 mRNA levels were confirmed in CsA-treated CIGO-HGFs by semiquantitative RT-PCR. Furthermore, CsA increased type I collagen mRNA levels and suppressed MMP-2 mRNA levels, which are regulated by P4HB and testican-1, respectively. These results suggest that CsA may induce gingival overgrowth through the upregulation of P4HB and testican-1, resulting in the accumulation of extracellular matrix components.