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Han-Jia Lin Ph.D National Taiwan Ocean University

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Our polyamine research team in Taiwan

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

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

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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

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

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

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SSAT-related genes in human SSAT1SSAT2 SSAT-like 1 (SATL1) Accession #NP_002961NP_597998NP_001012998 ChromosomeX17X Length (a. a.)171170632 Active formHomodimer ? SubstratesSpm/SpdThialysine? Enzyme function Polyamine catabolism Thialysine catabolism? ? Thialysine

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Bioinformatics analysis of SSAT related genes 58 species, 145 SSAT related genes

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MammaliaMammalia AvesAves Reptilia & Amphibia FishesFishes Data from Prof. Tun-Wen Pai Degree of variation of each SSAT related genes SSAT1 was only found in vertebrates, and its homology is ranging from 85~70% SSAT2 was not found in some Aves, and its homology is ranging from 80~41% SATL1 was not found in fishes, and its homology is ranging from 64~18%

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Degree of variation between SSAT1 and SSAT related genes in each species AvesAves Reptilia & Amphibia FishesFishes MammaliaMammalia Data from Prof. Tun-Wen Pai The sequence homology between SSAT2 and SSAT1 is remained at ~35% Some important features between SSAT2 and SSAT1 have been conserved? The sequence homology between SATL1 and SSAT1 is increased from 14% to 58% SATL1 is a developing gene?

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

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

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We need both dry lab and wet lab works Use zebrafish as a model to study. The evolutionary path of SSAT1s multiple functions and regulatory mechanisms Key factors related to the functional and structural diversification of SSAT1 and SSAT2

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Han-Jia Lin Ph.D Taiwan Ocean University Hanjias Metabolomic Biochemistry Laboratory

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

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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

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Synteny of SSAT1 and ACOT9 In th

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Han-Jia Lin Ph.D Taiwan Ocean University Hanjias Metabolomic Biochemistry Laboratory

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21 Zebrafish ssat-related genes used in this study Ssat1aSsat1bSsat1cSsat2aSsat2b Accession # NM_001093748NM_001030199NM_001002169NM_001002554NM_001008598 Chromsome245575 Amino acids171 170171

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22 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!

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23 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).

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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 zebrafish ssat1 isogenes

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Cross-species analysis of the alternative spliced ssat-X sequence in ssat1 genes Zebrafish ssat1b is the only fishs gene which has sequence similar to X-intron in intron 3. However, such kind of alternative splicing did not observed by RT-PCR Fishes

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Translational regulation of Ssat1 Both human and zebrafish use the same translational regulatory mechanism? Translation of zebrafish Ssat1a is less regulated, why? Zebrafish ZF4 cells Human HEK293T

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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?

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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 0h 6h 2h 4h 6h 2h 4h 6h Incubation time

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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.

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Han-Jia Lin Ph.D Taiwan Ocean University Hanjias Metabolomic Biochemistry Laboratory

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Enzymatic properties of zSsat1a

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

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

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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!

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

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Ssat2 is a kind of thialysine acetyltransferase (TLAT) 36 FEBS Letters 579 (2005) 53475352 81 Uni-cell protozoa and parasite

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Ser 81 plays a key role in the activity of Ssat2 (TLAT)? 37 C D A B 81 A.B.C.D motifs are GNAT superfamily conserved regions.

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

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Enzymatic activities of Ssat2 mutants 39 Both Ssat2a_N81S and Ssat2b_S81G can use thialysine as substrate. As Ssat2a, no obvious activity of Ssat2ab was found. Ssat2ba can use thialysine and putrescine as substrate. 2a_N81S 2b_S81G

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40 Enzyme Kinetics of Ssat2 isozymes (with thialysine) R 2 =0.999 Ssat2b R 2 =0.999 Ssat2a_N81S R 2 =0.988 Ssat2b_S81G R 2 =0.996 Ssat2ba Km (mM)Kcat (S -1 )Vmax (min) kcat / Km (M -1 S -1 ) Ssat2b0.78 0.071421.750.1327388.01 Ssat2a_N81S12.79 2.0041.320.06103.31 Ssat2b_S81G3.18 0.87473.420.181176.34 Ssat2ba50.93 3.21791.560.1930.6

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41 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

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Resolution range ()27.01 - 2.652 (2.746 - 2.652) Space groupP 4 3 2 1 2 Unit cell92.118 92.118 144.156 90 90 90 Total reflections Unique reflections18428 (1831) Completeness (%)98.87 (100.00) Mean I/sigma(I)22.85 (5.25) Wilson B-factor57.72 R-sym R-factor0.1990 (0.3015) R-free0.2727 (0.4020) Protein residues503 RMS(bonds)0.012 RMS(angles)1.64 Ramachandran favored (%)97 Ramachandran outliers (%)0 Crystal structure of Ssat2ba

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45 o Substrate preference of Ssat isozymes may be explained from their crystal structures

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Han-Jia Lin Ph.D Taiwan Ocean University Hanjias Metabolomic Biochemistry Laboratory

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

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Heterodimerization between zSsat1 isozymes pCDNA3.1a myczSSAT1 transfection Harvest cells and protein extraction Pull-down by GST or GST-SSAT and western blotting

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

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Summary of protein-protein interactions between Ssat1 and Ssat2 isoenzymes 48 Ssat2a could form heterdimer with Ssat1c. Ssat2b could form heterdimer with Ssat1a and Ssat1b and Ssat1c. Ssat1a Ssat1b Ssat1c Ssat2a Ssat2b

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Protein-protein interaction between Ssat1 and HIF-1 Hypoxia inducible factor 1 alpha subunit Under normoxia, HIF-1 is constitutively synthesized and degraded. Under hypoxia, HIF-1 is stabilized and became a transcription factor which activates genes responsible for hypoxia condition. Human SSAT1 could enhance the degradation of HIF-1 under hypoxia condition. 49 Baek, J.H. et al., 2007

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Interaction of zebrafish Hif-1 and Ssat1 isozymes Only Ssat1b and Ssat1c could interacted with Hif-1 PAS-B domain

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Protein-protein interaction between Ssat1 and Integrin 9 Integrins are cell surface proteins that mediate cell- cell communication and cell morphology. Integrin 9 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 9.

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Protein-protein interaction between Ssat1 and Integrin 9 By using GST-pull down experiments, we confirmed that zebrafish integrin 9 interacts with Ssat1b and Ssat1c, but not Ssat1a.

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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 9 thus regulates the migration signaling.

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Han-Jia Lin Ph.D Taiwan Ocean University Hanjias Metabolomic Biochemistry Laboratory

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Characteristics of zebrafish Ssat2 isoenzymes Reacted with Spd/Spm Reacted with thialysine Reacted with 5-hydroxylysine Interacted with SSAT1 Interacted with HIF-1 Data source S. pombe TLAT x N.D. Biochem. J. (2004) 384, 129 137 D. major TLAT x N.D. FEBS Letters (2005) 579 5347 5352 C. elegans D2023.4 x N.D. Biochem. J. (2004) 384, 129 137 Amphioxus XM_002595182 x N.D. This work Zebrafish Ssat2a x xx zSSAT1c? This work Zebrafish Ssat2b x ? This work Human SSAT2 x N.D. Biochem. J. (2004) 384, 139 148

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Physiological significant of Ssat2 Ssat2 was found in most eukaryotic species. The activity of thialysine and hydroxylysine acetylation are conserved. Other unidentified substrates for Ssat2? Remained ~35% sequence identity with Ssat1 in most vertebrates Functional convergency with Ssat1? (Ex: regulation of hypoxia signaling pathway) Thialysine 5-Hydroxylysine

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Characteristics of zebrafish Ssat1 isoenzymes zSsat1azSsat1bzSsat1chSSAT1 Acetylation of thialysinexxxx Acetylation of Spd/Spm Synteny with Acot9 Transcriptional regulationxxx Alternative splicingx x Translational regulation Protein stability regulationx x Interaction with HIF-1 x Interaction with Integrin 9 x

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Evolutionary path of Ssat1 3 fates of duplicated genes: nonfunctionalization, neofunctionalization and subfunctionalization 2 rounds of gene duplication before vertebrate evolved; one extra round in the ray-finned fish lineage Ssat1 was derived from Ssat2 during the evolution of vertebrates as well as Integrin 9 and Hif-1 (neofunctionaliztion?) 3 zebrafish Ssat1 isoenzymes (subfunctionalization) Transltional and protein stability regulation were also found the common ancestor of human and fish? Ssat1 is a polyamine sensor?

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Heterodimerization of Ssat1 and Ssat2 Ssat2b + Ssat2a lost enzyme activity? Dominant negative regulation? Ssat2b + Ssat1a/b/c What kinds of activity? Use spermidine or thialysine or others as substrates? Ssat1 + Ssat2 better regulation of Hypoxia and integrin 9 signaling pathway? 59

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Han-Jia Lin Ph.D Taiwan Ocean University Hanjias Metabolomic Biochemistry Laboratory Lab members: Lien, Yi-Chin Lin, Yu-Tzu Ou, Ting-Yu Kuo, Po-Chi Collaboration: Hsu, Chun-Hua Ph.D National Taiwan University sa olun