Polyamines are very important! Present in almost all organism
Essential for cell survival Putrescine (Put) Spermidine (Spd)
Spermine (Spm) Wallace, 2003
Slide 5
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]!
Slide 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
Slide 7
Multiple levels of activity regulation for SSAT1 Pegg et al.,
2008
Slide 8
Multiple functions of SSAT1 Pegg et al., 2008 Via
protein-protein interaction
Slide 9
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
Slide 10
Bioinformatics analysis of SSAT related genes 58 species, 145
SSAT related genes
Slide 11
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%
Slide 12
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?
Slide 13
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
Slide 14
What is the evolutionary path of SSAT1 and polyamine
interconversion pathway?
Slide 15
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
Slide 16
Han-Jia Lin Ph.D Taiwan Ocean University Hanjias Metabolomic
Biochemistry Laboratory
Slide 17
Phylogenetic Analysis of SSAT related enzymes in Chordates
Although there are 4 Ssat- related enzymes in amphioxus, none of
them are group with Ssat1!
Slide 18
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
Slide 19
Synteny of SSAT1 and ACOT9 In th
Slide 20
Han-Jia Lin Ph.D Taiwan Ocean University Hanjias Metabolomic
Biochemistry Laboratory
Slide 21
21 Zebrafish ssat-related genes used in this study
Ssat1aSsat1bSsat1cSsat2aSsat2b Accession #
NM_001093748NM_001030199NM_001002169NM_001002554NM_001008598
Chromsome245575 Amino acids171 170171
Slide 22
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!
Slide 23
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).
Slide 24
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
Slide 25
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
Slide 26
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
Slide 27
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?
Slide 28
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
Slide 29
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.
Slide 30
Han-Jia Lin Ph.D Taiwan Ocean University Hanjias Metabolomic
Biochemistry Laboratory
Slide 31
Enzymatic properties of zSsat1a
Slide 32
Enzymatic properties of zSsat1b (A) Temperature (B) pH
Slide 33
Enzymatic properties of zSsat1c (A) Temperature(B) pH
Slide 34
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!
Slide 35
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.
Slide 36
Ssat2 is a kind of thialysine acetyltransferase (TLAT) 36 FEBS
Letters 579 (2005) 53475352 81 Uni-cell protozoa and parasite
Slide 37
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.
Slide 38
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
Slide 39
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
Slide 40
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
Slide 41
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
Slide 42
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
Slide 43
45 o Substrate preference of Ssat isozymes may be explained
from their crystal structures
Slide 44
Han-Jia Lin Ph.D Taiwan Ocean University Hanjias Metabolomic
Biochemistry Laboratory
Heterodimerization between zSsat1 isozymes pCDNA3.1a myczSSAT1
transfection Harvest cells and protein extraction Pull-down by GST
or GST-SSAT and western blotting
Slide 47
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
Slide 48
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
Slide 49
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
Slide 50
Interaction of zebrafish Hif-1 and Ssat1 isozymes Only Ssat1b
and Ssat1c could interacted with Hif-1 PAS-B domain
Slide 51
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.
Slide 52
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.
Slide 53
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.
Slide 54
Han-Jia Lin Ph.D Taiwan Ocean University Hanjias Metabolomic
Biochemistry Laboratory
Slide 55
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
Slide 56
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
Slide 57
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
Slide 58
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?
Slide 59
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
Slide 60
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