Han- Jia Lin Ph.D National Taiwan Ocean University

1. Functional and structural diversification of spermidine/spermine N1-acetyltransferase in zebrafish Han-Jia Lin Ph.D National Taiwan Ocean University

2. Our polyamine research team in Taiwan

4. Polyamines are very important! • Present in almost all organism • Essential for cell survival Putrescine (Put) Spermidine (Spd) Spermine (Spm) Wallace, 2003

5. Homeostasis of polyamine is also very important! In human, SSAT1 is the key enzyme to reduce cellular [Spm] and [Spd]! de novo synthesis Transport ODC: ornithinedecarboxylase MAT: methionineadenosyltransferase SAMDC: S-AdoMetdecarboxylase PAO: polyamine oxidase catabolism Wallace et al., 2003

6. Background of SSAT1 • Spermidine/spermine N1-acetyltransferase 1 • Belongs to GCN5 related acetyltransferase (GNAT) superfamily • Homodimer: ~171 amino acids • Enzyme activity: • Adding acetyl groups to the aminopropyl end of spermidine or spermine

7. Multiple levels of activity regulation for SSAT1 Pegg et al., 2008

8. Multiple functions of SSAT1 Via protein-protein interaction Pegg et al., 2008

9. SSAT-related genes in human Thialysine

10. Functional convergency of SSAT1 and SSAT2? • They are all involved in HIF-1α regulation, though the mechanisms are different… Baek, J.H. et al., 2007

11. What is the evolutionary path of SSAT1 and polyamine interconversion pathway?

12. We need both “dry lab” and “wet lab” works • Use zebrafish as a model to study…. • The evolutionary path of SSAT1’s multiple functions and regulatory mechanisms • Key factors related to the functional and structural diversification of SSAT1 and SSAT2

13. Result Part I • Bioinformatics approaches Han-Jia Lin Ph.D Taiwan Ocean University Hanjia’s Metabolomic Biochemistry Laboratory

14. Phylogenetic Analysis of SSAT related enzymes in Chordates • Although there are 4 Ssat-related enzymes in amphioxus, none of them are group with Ssat1!

15. Identification of SSAT1 locus • PRDX4 － ACOT9－SAT1－APOO region is conserved • SSAT1 gene of human, mouse and zebrafish maybe come form the same ancient SSAT gene. • Zebrafish have experienced an extra duplication of whole genome, therefore two SSAT1 locus are identified on Ch5 and Ch24. • zSSAT1b and zSSAT1c seems to be the products of gene repeat

16. Result Part II • Expression patterns and regulations Han-Jia Lin Ph.D Taiwan Ocean University Hanjia’s Metabolomic Biochemistry Laboratory

17. Zebrafishssat-related genes used in this study

18. The spatial expression profile of ssat-related genes in adult zebrafish • Ubiquitous! • The expression of ssat1c and ssat2b is more abundant in most tissues! • Co-expression!

19. The temporal expression profiles of ssat-related genes during zebrafish embryogenesis • Ssat1: ssat1c is the most abundant! • Ssat2: only ssat2b was expressed during zebrafish embryogenesis. • The RNA expression of ssat2b mRNA did not induced by polyamine (DENSPM is a polyamine analog).

20. Cross-species promoter analysis of ssat1 genes • Polyamine-responsive element (PRE) is located at ~ -1.5 kb of human SSAT1 promoter.Wang, Y. et. al (1998) JBC 273, p34623 • PRE is not found in the promoter of zebrafishssat1isogenes

21. Cross-species analysis of the alternative spliced ssat-X sequence in ssat1 genes • Zebrafishssat1b is the only fish’s gene which has sequence similar to X-intron in intron 3. However, such kind of alternative splicing did not observed by RT-PCR Fishes

22. Translational regulation of Ssat1 • Both human and zebrafish use the same translational regulatory mechanism? • Translation of zebrafish Ssat1a is less regulated, why? ZebrafishZF4 cells HumanHEK293T

23. Search for the key region responsible for the translational regulation • Crucial regions: • 3’ of ssat1b (332-513) • 5’ of ssat1b (1-389) • Both 5’ and 3’ regions are important?

24. Protein stability of zebrafish Ssat1 isoenzymes • By increasing 2x transfected plasmid and 10x protein loading  basal protein expression of Ssat1b and Ssat1c could be observed without induction! • Without addition of Spd, only Ssat1b turns over rapidly in HEK293 cells Incubation time 0h 6h 2h 4h 6h 2h 4h 6h

25. Search for the key region responsible for the rapid degradation • The last 70 residues of Ssat1b is important for the rapid degradation! • There are 14 variants between the last 70 residues of Ssat1a and Ssat1b.

26. Result Part III • Enzyme activities Han-Jia Lin Ph.D Taiwan Ocean University Hanjia’s Metabolomic Biochemistry Laboratory

27. Enzymatic properties of zSsat1a

28. Enzymatic properties of zSsat1b (A) Temperature (B) pH

29. Enzymatic properties of zSsat1c (A) Temperature (B) pH

30. The activities of zebrafish Ssat1 isoenzymes • Putrescine is not a substrate for all Ssat1 isoenzyme! • Ssat1a has better catalytic efficiency • Ssat1a and Ssat1b have better catalytic efficiency toward Spd • Ssat1c has almost equal catalytic efficiency toward Spd and Spm!

31. Enzyme activity of zebrafish Ssat2 isoenzymes 35 • Ssat2b could only react with thialysine but not with polyamines • Ssat2a did not react with any substrates.

32. Ssat2 is a kind of thialysineacetyltransferase (TLAT) 81 Uni-cell protozoa and parasite FEBS Letters 579 (2005) 5347–5352

33. Ser81 plays a key role in the activity of Ssat2 (TLAT)? C D B A 81 • A.B.C.D motifs are GNAT superfamily • conserved regions.

34. Search for the key region responsible for TLAT activity Constructs of ssat2 isozymes • Site-directed mutants: N S 81 • ssat2a_N81S S G 81 • ssat2b_S81G • Chimeric mutants: ssat2ab chimeric gene 92 96 ssat2a ＋ 92 96 ssat2ba ssat2b overlappig

35. Enzymatic activities of Ssat2 mutants 2b_S81G 2a_N81S • As Ssat2a, no obvious activity of Ssat2ab was found. • Ssat2ba can use thialysine and putrescineas substrate. • Both Ssat2a_N81S and Ssat2b_S81G can use thialysine as substrate.

36. Enzyme Kinetics of Ssat2 isozymes(with thialysine) Ssat2b_S81G Ssat2ba Ssat2a_N81S Ssat2b R2=0.999 R2=0.999 R2=0.996 R2=0.988

37. Can Ssat2 isozymes use substrates other than thialysine? • Hydroxylysine (sturcture similar to thialysine) • Structure similar to putrescine (4C): • 1,3-diaminopropane (3C) • Cadaverine (5C) • 1,8-diaminoctane (8C) • Monoamine  serotonin

38. Crystal structure of Ssat2ba

39. Substrate preference of Ssatisozymes may be explained from their crystal structures 45o

40. Result Part IV • Protein-protein interactions Han-Jia Lin Ph.D Taiwan Ocean University Hanjia’s Metabolomic Biochemistry Laboratory

41. Heterodimer formation between zSSAT1 isoenzymes? 134 140 155 163 hSSAT1 zSSAT1a zSSAT1b zSSAT1c hSSAT1 zSSAT1a zSSAT1b zSSAT1c

42. Heterodimerization between zSsat1 isozymes zSSAT1 myc pCDNA3.1a transfection Harvest cells and protein extraction Pull-down by GST or GST-SSAT and western blotting

43. Heterodimerization of Ssat2 isoenzymes Ssat2a_myc WB GST 1a 1b 1c 10% 2a_myc Pull down Anti-Ssat2a_myc Ssat2b_myc WB Pull down GST 1a 1b 1c 10% 2b_myc Anti-Ssat2b_myc WB Pull down GST 10% 2b_myc GST_Ssat2a Anti-Ssat2b_myc

44. Summary of protein-protein interactions between Ssat1 and Ssat2 isoenzymes Ssat1a Ssat2a Ssat1b Ssat2b Ssat1c • Ssat2a could form heterdimer with Ssat1c. • Ssat2b could form heterdimer with Ssat1a and Ssat1b and Ssat1c.

45. Protein-protein interaction between Ssat1 and HIF-1 • Hypoxia inducible factor 1 alpha subunit • Under normoxia, HIF-1α is constitutively synthesized and degraded. • Under hypoxia, HIF-1α is stabilized and became a transcription factor which activates genes responsible for hypoxia condition. • Human SSAT1 could enhance the degradation of HIF-1α under hypoxia condition. Baek, J.H. et al., 2007

46. Interaction of zebrafish Hif-1αand Ssat1 isozymes • Only Ssat1b and Ssat1c could interacted with Hif-1α PAS-B domain

47. Protein-protein interaction between Ssat1 and Integrin9 • Integrins are cell surface proteins that mediate cell-cell communication and cell morphology. • Integrin α9 • A mammalian specific form • Stimulated by extracellular signals, such as tenascin C, osteopontin, and vascular cell adhesion molecules-1 • involved in embryogenesis, lymphangiogenesis, and wound healing. • Over expression of human SSAT1 enhances cell migration mediated by integrin α9.

48. Protein-protein interaction between Ssat1 and Integrin9 • By using GST-pull down experiments, we confirmed that zebrafishintegrin α9 interacts with Ssat1b and Ssat1c, but not Ssat1a.

49. Protein-protein interaction between Ssat1 and Integrin9 • The first 20 amino acids of SSAT1 is crucial, since they could bind to the cytosolic domain of integrin α9 thus regulates the migration signaling.

50. Conclusions • Discussion Han-Jia Lin Ph.D Taiwan Ocean University Hanjia’s Metabolomic Biochemistry Laboratory