EXPLORING THE REGULATION OF NRD-CONVERTASE EXPRESSION BY RETINOIC ACID

KRISHNA MOORTHYSUPERVISOR: DR. ADRIAN PIEROTTI

INTRODUCTION

• NRD-CONVERTASE (NRDc) is a cytosolic and cell-surface metallopeptidase enzyme.

• It is classified under Clan ME in Family M16, Subfamily A, M16.005 (merops).

• This enzyme is present in abundant amount in endocrine tissues, in rat testis at late stages of spermatogenesis and brain cortex.

INTRODUCTION CONTINUED….

• The 90 amino acid acidic domain inserted close to NRDc active site differentiates it from other members of the M16 family of metalloendopeptidases.

• NRDc acts by cleaving arginine residues on N-terminus of basic amino acid or lysine in basic pairs.

IMPORTANCE OF ENZYME

• Role in Alzheimer Disease, Brain Diseases, Down Syndrome, Leukemia Neoplasms and Neuroblastoma

PREVIOUS STUDIES:• Draoui M et al (1997): When treated with retinoic

acid for 2 days, SK-N-BE(2) cell lines showed an increase in the expression of NRD Convertase in the cytoplasm.

• BE(2)-NA cell lines showed 9-fold increase in the expression of NRD Convertase within few hours to a day treatment.

• Interestingly, NRD Convertase expression was down regulated in BE(2)-M(17) cells after 5 days treatment of Retinoic acid.

• The authors used immunochemistry and northern blotting to do this study.

• Real time-PCR is used in this study to get more accurate results.

OBJECTIVES:

• To investigate the possible modulation of NRD-convertase expression in the presence of Retinoic acid (1 µM, 5 µM), Tamibarotene (1 µM) at different time points.

METHODS

Figure 1. Samples of SK-N-BE cells of Actin and NRDc on gel electrophoresis

Figure 2. Restriction digest of Actin and NRDc on gel electrophoresis

STD OF ACTINConc Log

[conc]Actin Only (Ct values)

Average

10pg 7 12.29 13.52 12.91

100fg 5 19.59 18.83 19.21

1fg 3 25.43 25.72 25.58

10ag 1 29.17 30.14 29.66

CONCENTRATION OF ACTIN

STD OF NRDConc Log [ag] NRD only

(Ct values)Average

10pg 7 13.23 13.49 13.36

100fg 5 19.46 20.51 19.99

1fg 3 27.08 25.95 26.52

10ag 1 30.56 29.68 30.12

CONCENTRATION OF NRD

ACTIN IN COMBO WITH NRDlog [ag] Actin+NRD Actin only

1 33.51 32.05

3 28.91 28.04

5 22.7 21.75

7 16.69 17.05

CONCENTRATION OF ACTIN

NRD in comboLog[ag] NRD only NRD + ACTIN

1 32.23 32.4

3 28.89

5 21.75 22.74

7 16.01 16.71

CONCENTRATION OF NRD

CELL TISSUE CULTURE• TREATMENTS 1µM TAMBIROTENE 1µM RETINOIC ACID 5µM RETINOIC ACID

• TIMES 6 HOURS 24 HOURS 48 HOURS

• MEASURE Ct NRD Ct ACT in same tube Calculate ∆Ct = CtNRD-CtACT Calculate ∆∆Ct = ∆CtControl-∆CtTreatment ∆∆Ct = 1 = 2 fold change

∆∆Ct of NRDc level

∆∆Ct for

Treatment 6 hrs treatment 24 hrs treatment 48 hrs treatment

Retinoic acid 1µM 0.537333 -0.395 -0.335

Retinoic acid 5µM 0.401667 -0.238333333 -0.56166667

Tamibarotene 1µM 0.3425 -0.708333333 -0.155

control 0 0 0

FOLD CHANGE OF NRDc LEVEL AFTER DRUG TREATMENT

FOLD CHANGE OF NRDc LEVEL AFTER

BAR CHART REPRESENTATION OF FOLD CHANGE OF NRDc LEVEL

Conclusion:

• As presence of retinoic acid causes changes in expression of NRD levels in human SK B NE cells, it can be concluded that retinoic acid plays an important role in pathogenesis of brain diseases.

• The results is not similar with previous works. This may be due to small number of samples treated. To get more accurate result this experiment must be repeated with more number of treatment.