Han- Jia Lin Ph.D National Taiwan Ocean University

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Functional and structural diversification of spermidine/spermine N1-acetyltransferase in zebrafishHan-Jia Lin Ph.DNational Taiwan Ocean UniversityOur polyamine research team in Taiwan

Polyamines are very important! Present in almost all organismEssential for cell survival

Putrescine (Put)Spermidine (Spd)Spermine (Spm)

Wallace, 20034Homeostasis of polyamine is also very important!

de novosynthesiscatabolismTransportWallace et al., 2003ODC: ornithine decarboxylaseMAT: methionine adenosyltransferaseSAMDC: S-AdoMet decarboxylasePAO: polyamine oxidase

In human, SSAT1 is the key enzyme to reduce cellular [Spm] and [Spd]!Homeostasis of polyamine is also very important.

Background of SSAT1Spermidine/spermine N1-acetyltransferase 1Belongs to GCN5 related acetyltransferase (GNAT) superfamilyHomodimer: ~171 amino acidsEnzyme activity:Adding acetyl groups to the aminopropyl end of spermidine or spermine

Multiple levels of activity regulation for SSAT1

Pegg et al., 2008Multiple functions of SSAT1

Pegg et al., 2008Via protein-protein interactionFrom mouse and human models, we could tell that the incensement of SSAT1 activity may....8SSAT-related genes in humanSSAT1SSAT2SSAT-like 1 (SATL1)Accession #NP_002961NP_597998NP_001012998ChromosomeX17XLength (a. a.)171 170632Active formHomodimerHomodimer?SubstratesSpm/SpdThialysine?Enzyme functionPolyamine catabolismThialysine catabolism??

ThialysineIn human, actually, there are 3 ssat-related genes.

However, only SSAT1 is well studied!9Bioinformatics analysis of SSAT related genes58 species, 145 SSAT related genes

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MammaliaAvesReptilia & AmphibiaFishesData from Prof. Tun-Wen PaiDegree of variation of each SSAT related genesSSAT1 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% Here, we used human SSAT1 as a template. The sequence identity of cognate protein in each species is label in blue dots and their linear regression is represented by blue line.

Further, we also used human SSAT2 as a template~ Green

In general, SSAT1 is more conserved in all species, while SSAT2 and SATL1 may undergo divergency along with animal evolution.11Degree of variation between SSAT1 and SSAT related genes in each species

AvesReptilia & AmphibiaFishesMammaliaData from Prof. Tun-Wen PaiThe 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? Next, we used SSAT1 in each species as a template and compared with the sequence of SSAT2 or SATL1 protein in the same organism. The red dots represent the sequence identity of SSAT2 and SSAT1. The linear regression indicates~

On the other hand... 12Functional convergency of SSAT1 and SSAT2?They are all involved in HIF-1 regulation, though the mechanisms are different

Baek, J.H. et al., 200713What is the evolutionary path of SSAT1 and polyamine interconversion pathway?

14We need both dry lab and wet lab worksUse zebrafish as a model to study.The evolutionary path of SSAT1s multiple functions and regulatory mechanismsKey factors related to the functional and structural diversification of SSAT1 and SSAT2 Result Part I

Han-Jia Lin Ph.DTaiwan Ocean University

Hanjias Metabolomic BiochemistryLaboratory

Bioinformatics approachesPhylogenetic Analysis of SSAT related enzymes in ChordatesAlthough there are 4 Ssat-related enzymes in amphioxus, none of them are group with Ssat1!

Identification of SSAT1 locusPRDX4 ACOT9SAT1APOO region is conservedSSAT1 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

18Synteny of SSAT1 and ACOT9In th

Result Part II



Expression patterns and regulations21Zebrafish ssat-related genes used in this studySsat1aSsat1bSsat1cSsat2aSsat2bAccession #NM_001093748NM_001030199NM_001002169NM_001002554NM_001008598Chromsome245575Amino acids17117117017117122The spatial expression profile of ssat-related genes in adult zebrafishUbiquitous!

The expression of ssat1c and ssat2b is more abundant in most tissues!

Co-expression!

23The temporal expression profiles of ssat-related genes during zebrafish embryogenesisSsat1: 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).

Cross-species promoter analysis of ssat1 genesPolyamine-responsive element (PRE) is located at ~ -1.5 kb of human SSAT1 promoter. Wang, Y. et. al (1998) JBC 273, p34623PRE is not found in the promoter of zebrafish ssat1 isogenes

Cross-species analysis of the alternative spliced ssat-X sequence in ssat1 genesZebrafish 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

FishesTranslational regulation of Ssat1Both human and zebrafish use the same translational regulatory mechanism?Translation of zebrafish Ssat1a is less regulated, why?

Zebrafish ZF4 cellsHuman HEK293TSearch for the key region responsible for the translational regulationCrucial regions:3 of ssat1b (332-513)5 of ssat1b (1-389)Both 5 and 3 regions are important?

Protein stability of zebrafish Ssat1 isoenzymesBy 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 6hIncubation timeSearch for the key region responsible for the rapid degradationThe last 70 residues of Ssat1b is important for the rapid degradation!There are 14 variants between the last 70 residues of Ssat1a and Ssat1b.

Result Part III



Enzyme activitiesEnzymatic properties of zSsat1a

Enzymatic properties of zSsat1b

(A) Temperature(B) pHEnzymatic properties of zSsat1c

(A) Temperature(B) pHThe activities of zebrafish Ssat1 isoenzymesPutrescine is not a substrate for all Ssat1 isoenzyme!Ssat1a has better catalytic efficiencySsat1a and Ssat1b have better catalytic efficiency toward SpdSsat1c has almost equal catalytic efficiency toward Spd and Spm!

35Enzyme activity of zebrafish Ssat2 isoenzymes

35Ssat2b could only react with thialysine but not with polyamines

Ssat2a did not react with any substrates.

Ssat2 is a kind of thialysine acetyltransferase (TLAT)

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FEBS Letters 579 (2005) 5347535281

Uni-cell protozoa and parasiteSer81 plays a key role in the activity of Ssat2 (TLAT)?37

CDAB81A.B.C.D motifs are GNAT superfamily conserved regions. 38Chimeric mutants:chimeric genessat2assat2boverlappig92 96 92 96 ssat2abssat2baSite-directed mutants:ssat2a_N81Sssat2b_S81GN S81S G81Constructs of ssat2 isozymesSearch for the key region responsible for TLAT activityEnzymatic activities of Ssat2 mutants39

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_N81S2b_S81G

40Enzyme Kinetics of Ssat2 isozymes (with thialysine)

R2=0.999Ssat2bR2=0.999Ssat2a_N81S R2=0.988Ssat2b_S81G R2=0.996Ssat2baKm (mM)Kcat (S-1)Vmax (min)kcat / Km (M-1 S-1)Ssat2b0.78 0.0714 21.750.1327388.01Ssat2a_N81S12.79 2.004 1.320.06103.31Ssat2b_S81G3.18 0.87473.420.181176.34Ssat2ba50.93 3.21791.560.1930.6

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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

Resolution range ()27.01 - 2.652 (2.746 - 2.652)Space groupP 43 21 2Unit cell92.118 92.118 144.156 90 90 90Total reflectionsUnique reflections18428 (1831)Completeness (%)98.87 (100.00)Mean I/sigma(I)22.85 (5.25)Wilson B-factor57.72R-symR-factor0.1990 (0.3015)R-free0.2727 (0.4020)Protein residues503RMS(bonds)0.012RMS(angles)1.64Ramachandran favored (%)97Ramachandran outliers (%)0

Crystal structure of Ssat2ba 42

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Substrate preference of Ssat isozymes may be explained from their crystal structuresResult Part IV



Protein-protein interactionsHeterodimer formation between zSSAT1 isoenzymes?

134 140hSSAT1zSSAT1azSSAT1bzSSAT1c

hSSAT1zSSAT1azSSAT1bzSSAT1c155 163

Heterodimerization between zSsat1 isozymes

pCDNA3.1amyczSSAT1transfectionHarvest cells and protein extractionPull-down by GST or GST-SSAT and western blotting

47Heterodimerization of Ssat2 isoenzymes

Anti-Ssat2b_mycGST_Ssat2aGST

Pull downWB10% 2b_myc

GST 1a 1b 1cSsat2a_mycAnti-Ssat2a_myc

10% 2a_mycPull down

10% 2b_mycGST 1a 1b 1cSsat2b_mycPull downAnti-Ssat2b_mycWBWBSummary of protein-protein interactions between Ssat1 and Ssat2 isoenzymes48Ssat2a could form heterdimer with Ssat1c.Ssat2b could form heterdimer with Ssat1a and Ssat1b and Ssat1c.Ssat1aSsat1bSsat1cSsat2aSsat2bProtein-protein interaction between Ssat1 and HIF-1Hypoxia inducible factor 1 alpha subunitUnder 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.49Baek, J.H. et al., 2007

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

Protein-protein interaction between Ssat1 and Integrin 9Integrins are cell surface proteins that mediate cell-cell communication and cell morphology. Integrin 9A mammalian specific formStimulated by extracellular signals, such as tenascin C, osteopontin, and vascular cell adhesion molecules-1involved in embryogenesis, lymphangiogenesis, and wound healing. Over expression of human SSAT1 enhances cell migration mediated by integrin 9. Protein-protein interaction between Ssat1 and Integrin 9By using GST-pull down experiments, we confirmed that zebrafish integrin 9 interacts with Ssat1b and Ssat1c, but not Ssat1a.

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

Conclusions



DiscussionCharacteristics of zebrafish Ssat2 isoenzymesReacted with Spd/SpmReacted with thialysineReacted with 5-hydroxylysineInteracted with SSAT1Interacted with HIF-1Data sourceS. pombeTLATxN.D.N.D.Biochem. J. (2004) 384, 129137 D. majorTLATxN.D.N.D.FEBS Letters (2005) 579 53475352C. elegans D2023.4xN.D.N.D.Biochem. J. (2004) 384, 129137 Amphioxus XM_002595182xN.D.N.D.This workZebrafish Ssat2axxxzSSAT1c?This workZebrafish Ssat2bx?This workHuman SSAT2xN.D.Biochem. J. (2004) 384, 139148Physiological significant of Ssat2Ssat2 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 vertebratesFunctional convergency with Ssat1? (Ex: regulation of hypoxia signaling pathway)

Thialysine

5-Hydroxylysine Characteristics of zebrafish Ssat1 isoenzymeszSsat1azSsat1bzSsat1chSSAT1Acetylation of thialysinexxxxAcetylation of Spd/SpmSynteny with Acot9Transcriptional regulationxxxAlternative splicingxxTranslational regulation Protein stability regulationxxInteraction with HIF-1xInteraction with Integrin 9xEvolutionary path of Ssat1 3 fates of duplicated genes: nonfunctionalization, neofunctionalization and subfunctionalization2 rounds of gene duplication before vertebrate evolved; one extra round in the ray-finned fish lineageSsat1 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?

Heterodimerization of Ssat1 and Ssat2Ssat2b + 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?59Acknowledgements



Lab members:Lien, Yi-ChinLin, Yu-TzuOu, Ting-YuKuo, Po-Chi

Collaboration:Hsu, Chun-Hua Ph.DNational Taiwan University

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Han- Jia Lin Ph.D National Taiwan Ocean University

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