Han-Jia Lin Ph.D
National Taiwan Ocean University
Our polyamine research team in
Taiwan
Polyamines are very
important!
 Present in almost all organism
Putrescine (Put)
 Essential for cell survival
Spermidine (Spd)
Spermine (Spm)
Wallace, 2003
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:
ornithine decarboxylase
MAT:
methionine adenosyltransferase
SAMDC:
S-AdoMet decarboxylase
PAO:
polyamine oxidase
catabolism
Wallace et al., 2003
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
Multiple levels of activity
regulation for SSAT1
Pegg et al., 2008
Multiple functions of SSAT1
Via protein-protein interaction
Pegg et al., 2008
SSAT-related genes in human
SSAT1
SSAT2
SSAT-like 1
(SATL1)
NP_002961
NP_597998
NP_001012998
Chromosome
X
17
X
Length (a. a.)
171
170
632
Active form
Homodimer
Homodimer
?
Substrates
Spm/Spd
Thialysine
?
Polyamine
catabolism
Thialysine
catabolism?
?
Accession #
Enzyme function
Thialysine
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
What is the evolutionary path of SSAT1
and polyamine interconversion pathway?
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
Han-Jia Lin Ph.D
Taiwan Ocean University
Hanjia’s
Metabolomic Biochemistry
Laboratory
Phylogenetic Analysis of SSAT
related enzymes in Chordates
 Although there
are 4 Ssatrelated enzymes
in amphioxus,
none of them are
group with Ssat1!
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
Han-Jia Lin Ph.D
Taiwan Ocean University
Hanjia’s
Metabolomic Biochemistry
Laboratory
Zebrafish ssat-related genes used
in this study
Ssat1a
Ssat1b
Ssat1c
Ssat2a
Ssat2b
Accession #
NM_001093748
NM_001030199
NM_001002169
NM_001002554
NM_001008598
Chromsome
24
5
5
7
5
Amino acids
171
171
170
171
171
21
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!
22
The temporal expression profiles of ssatrelated 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).
23
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
Cross-species analysis of the alternative
spliced ssat-X sequence in ssat1 genes
GTTACAGTCTCTAGCTTCGCCATGTACATG----GCCCTT
GTTACAGTCTCTAGCTTCGCCATGTACATG----GCCCTT
GTTACAGTCTCTAGCTTCGCCATGTACATG----GCCCTT
GTTACAGTCTCTAGCTTCGCCATGTACATG----GCCCTT
GTTACAGTCTCTAGCTTCGCCATGTACATG----GCCCTT
GTTACAGTCTCTAGCTTCGCCATGTACATG----GCCCTT
GTTACAGTCTCTAGCTTCGCCATGTACATG----GCCCTT
GTTACAGTCTCTAGCTTCGCCATGTACATG----GCCCTT
GTTACAGTCTCTAGCTTCGCCATGTACATG----GCCCTT
GTTACAGTCTCTAGCTTCGCCATGTACATG----GCCCTT
GTTACAGTCTCTAGCTTCGCCATGTACATG----GCCCTT
GTTACAGTCTCTAGCTTCGCCATGTACATG----GCCCTT
GTTACAGTCTCTAGCTTCGCCATGTACATG----GCCCTT
GCCCTT
GCCCTT
GCCCTT
GCCCTT
GCCCTT
GCCCTT
GCCCTT
GCCCTT
GCCCTT
GCCCTT
GCCCTT
GCCCTT
GCCCTT
CCGTGTACATGGATGGGCGGGGA
GGTAACTAAAAGATCCTTT--ACACAA-TAAAGTAGATGATCATGATAAA TGAG----CCGTGTACATGGATGGGCGGGGA
GGTAACTAAAAGATCCTTT--ACACAA-TAAAGTAGATGATCATGATAAA TGAG----CCGTGTACATGGATGGGCGGGGA
GGTAACTAAAAGATCCTTT--ACACAA-TAAAGTAGATGATCATGATAAA TGAG----CCGTGTACATGGATGGGCGGGGA
GGTAACTAAAAGATCCTTT--ACACAA-TAAAGTAGATGATCATGATAAA TGAG----CCGTGTACATGGATGGGCGGGGA
GGTAACTAAAAGATCCTTT--ACACAA-TAAAGTAGATGATCATGATAAA TGAG----CCGTGTACATGGATGGGCGGGGA
GGTAACTAAAAGATCCTTT--ACACAA-TAAAGTAGATGATCATGATAAA TGAG----CCGTGTACATGGATGGGCGGGGA
GGTAACTAAAAGATCCTTT--ACACAA-TAAAGTAGATGATCATGATAAA TGAG----CCGTGTACATGGATGGGCGGGGA
GGTAACTAAAAGATCCTTT--ACACAA-TAAAGTAGATGATCATGATAAA TGAG----CCGTGTACATGGATGGGCGGGGA
GGTAACTAAAAGATCCTTT--ACACAA-TAAAGTAGATGATCATGATAAA TGAG----CCGTGTACATGGATGGGCGGGGA
GGTAACTAAAAGATCCTTT--ACACAA-TAAAGTAGATGATCATGATAAA TGAG----CCGTGTACATGGATGGGCGGGGA
GGTAACTAAAAGATCCTTT--ACACAA-TAAACTAGATGATCATGATAAA TGAG----CTGTGTACATGGATGGGCGGGGA
GGTAACTAAAAGACCCTTT--ACACAA-TAAAGTAGATGATCATGATAAA TGAG----CTGTGTACATGGATGGNCGGGAG
GGTAACTAAAAGATCC-TT--ACACAAATAAAGTAGATGATCATGATAAA TGAG-----
GTTACAGTCTCTAGCTTCGCCATGTACATG----GCCCTT
GTTACAGTCTCTAGCTTCGCCATGTACATG----GCCCTT
GTTACAGTCTCTAGCTTCGCCATGTACATG----GCCCTT
GTTACAGTCTCTAGCTTCGCCATGTACATG----GCCCTT
GTTACAGTCTCTAGCTTCGCCATGTACATG----GCCCTT
GTTACAGTCTCTAGCTTCGCCATGTACATG----GCCCTT
GTTACAGTCTCTAGCTTCGCCATGTACATG----GCCCTT
GTTACAGTCTCTAGCTTCGCCATGTACATG----GCCCTT
GTTACAGTCTCTAGCTTCGCCATGTACATG----GCCCTT
GTTACAGTCTCTAGCTTCGCCATGTACATG----GCCCTT
GTTACAGTCTCTAGCTTCGCCATGTACATG----GCCCTT
GTTACAGTCTCTAGCTTCGCCATGTACATG----GCCCTT
GTTACAGTCTCTAGCTTCGCCATGTACATG----GCCCTT
GTTACAGTCTCTAGCTTCGCCATGTACATG----GCCCTT
GCCCTT
GCCCTT
GCCCTT
GCCCTT
GCCCTT
GCCCTT
GCCCTT
GCCCTT
GCCCTT
GCCCTT
GCCCTT
GCCCTT
GCCCTT
GCCCTT
CCGTGTACATGGATGGGCGGGGA
GGTAACTAAAAGATCCTTT--ACACAA-TAAAGTAGATGATCATGATAAA TGAG----CCGTGTACATGGATGGGCGGGGA
GGTAACTAAAAGATCCTTT--ACACAA-TAAACTAGATGATCATGATAAA TGAG----CCGTGTACATGGATGGGCGGGGA
GGTAACTAAAAGATCCTTT--ACACAA-TAAAGTAGATGATCATGATAAA TGAG----CCGTGTACATGGATGGGCGGGGN
NNNNNNNNNNNNNNNNNNN--NNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNN------CCGTGTACATGGATGGGCGGGGA
GGTAACTAAAAGATCCTTT--ACACAA-TAAAGTAGATGATCATGATAAA TGAG----CCGTGTACATGGATGGGCGGGGA
GGTAACTAAAAGATCCTTT--ACACAA-TAAAGTAGATGATCATGATAAA TGAG----CCGTGTACATGGATGGGCGGGGA
GGTAACTAAAAGATCCTTT--ACACAA-TAAAGTAGATGATCATGATAAA TGAG----CCGTGTACATGGATGGGCGGGGA
GGTAACTAAAAGATCCTTT--ACACAA-TAAAGTAGATGATCATGATAAA TGAG----CCGTGTACATGGATGGGCGGGGA
GGTAACTAAAAGATCCTTT--ACACAA-TAAAGTAGATGATCATGATAAA TGAG----CCGTGTACATGGATGGGCGGGGA
GGTAACTAAAAGATCCTTT--ACACAA-TAAAGTAGATGATCATGATAAA TGAG----CCGTGTACATGGATGGGCGGGGA
GGTAACTAAAAGATCCTTT--ACACAA-TAAAGTAGATGATCATGATAAA TGAG----CCGTGTACATGGATGGGCGGGGA
GGTAACTAAAAGATCCTTT--ACACAA-TAAAGTAGATGATCATGATAAA TGAG----CCGTGTACATGGATGGGCGGGGA
GGTAACTAAAAGATCCTTT--ACACAA-TAAAGTAGATGATCATGATAAAATGAG---CCGTGTACATGGATGGGCGGGGA
GGTAACTAAAAGATCCTTT--ACCCAA-TAAAGTAGATGATCATGATAAA---------
GTTACAGTCTCTAGCTTCGCCATGTACATG----GCCCTT
GTTACAGTCTCTAGCTTCGCCATGTACATG----GCCCTT
GTTACAGTCTCTAGCTTCGCCATGTACATG----GCCCTT
GTTACAGTCTCTAGCTTCGCCATGTACATG----GCCCTT
GCCCTT
GCCCTT
GCCCTT
GCCCTT
CCGTGTACATGGATGGGCGGGGA
GGTAACTAAAAGATCCTTT--ACACAA-TAAAGTAGATGATCATGATAAA
CCGTGTACATGGATGGGCGGGGA
GGTAACTAAAAGATCCTTT--ACACAA-TAAAGTAGATGATCATGATAAA
CTGTGTACATGGATGGGCGGGGA
GGTAACTAAAAGATCCTTT--ACACAA-TAAAGTAGATGATCATGATAAA
CCGTGTACATGGATGGGCGGGGA
GGTAACTAAAAGATCCTTT--ACACAA-TAAAGTAGATGATCATGATAAA
GTTACAGTCTCTAGCTTCGCCATGTACATG----GCCCTT
GCCCTT
CCGTGTACATGGATGGGCGGGGA
GGTAACTAAAAGATCCGTT--ACTCAA-TAAAGTAGATGATCATGATAAA TGAG-----
GTTACAGTCTCTAGCTTCGCCATGTACATG----GCCCTT
GTTACAGTCTCTAGCTTCGCCATGTACATG----GCCCTT
GTTACAGTCTCTAGCTTCGCCATGTACATG----GCCCTT
GTTACAGTCTCTAGCTTCGCCATGTACATG----GCCCTT
GTTACAGTCTCTAGCTTCGCCATGTACATG----GCCCTT
GTTACAGTCTCTAGCTTCGCCATGTACATG----GCCCTT
GTTACAGTCTCTAGCTTCGCCATGTACATG---GTTACAGTCTCTAGCTTCGCCATGTACATG----G
AT
GTTACAGTCTCTAGCTTCGCCATGTACATG---GTTACAGTCTCTAGCTTCGCCATGTACATG----
GCCCTT
GCCCTT
GCCCTT
GCCCTT
GCCCTT
GCCCTT
GCCCTT
GGCCCTT
GCCCTT--A
GCCCTT--A
CCGTGTACATGGATGGGCGGGGA
GGTAACTAAAAGATCCGTT--ACTCAA-TAAAGTAGATGATCATGATAAA TGAG----CCGTGTACATGGATGGGCGGGGA
GGTAACTAAAAGATCCTTT--ACACAA-TAAAGTAGATGATCATGATAAA TGAG----CCGTGTACATGGATGGGCGGGGA
GGTAACTAAAAGATCCGTT--ACACAA-TAAAGTAGATGATCATGTGATCCGAGGTAAG
CTGTGTACATGGATGGGCGGGGA
GGTAACTAAAAGATCCGTT--ACACAA-TAAAGTAGATGATCAT--------------CTGTGTACATGGATGGGCGGGGA
GGTAACTAAAAGATCCGTT--ACACAA-TAAAGTAGATGATCAT--------------CTGTGTACATGGATGGGCGGGGA
GGTAACTAAAAGATCCGTT--ACACAA-TAAAGTAGATGATCAT--------------CCGTGTACATGGATGGGCGGGGA
GGTAACTAAAAGATCCGTT--ACACAA-TAAAGTAGATGATCATGATATA TGAG----CCGTGTACATGGGTGGGCGGGGA
GGTAACTAAAAGATCCGTT--ACACAA-TAAAGTAGATGATCATGA--AACGAG----GCGTGTACATGGATGGGCGGGGA
GGTAACTGAAAG
-----TT--ACACAA-TAAACTAGATGATCATGA------------GCGTGTACATGGATGGGCGGGGA
GGTAACTGAAAG
-----TT--ACACAA-TAAACTAGATGATCATGA-------------
TGAG----TGAG----TGAG----TGAG-----
Fishes
GCTACAGTCTCTTGCTTCGCCATGTACATT----GCTCAT
GCTACAGTCTCTTGCTTCGCCATGTACATT----GCTCAT
G
G
GCGTGTACATGGATGGGCGGGGA
GGTAACTGAAAGAAAGATCCGGTACAA-TAGTGTA-ACATTTACAGTAAAGCAGAG--GCGTGTACATGGATGGGCGGGGA
GGTAACTGAAAGAAAGATCCGGTACAA-TAGTGTA-ACATTTACAGTAAAGCAGAG---
 Zebrafish ssat1b 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
Translational regulation of Ssat1
Zebrafish
ZF4 cells
Human
HEK293T
 Both human and zebrafish
use the same translational
regulatory mechanism?
 Translation of zebrafish Ssat1a
is less regulated, why?
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?
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
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.
Han-Jia Lin Ph.D
Taiwan Ocean University
Hanjia’s
Metabolomic Biochemistry
Laboratory
Enzymatic properties of zSsat1a
Enzymatic properties of zSsat1b
(A) Temperature
(B) pH
Enzymatic properties of zSsat1c
(A) Temperature
(B) pH
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!
Enzyme activity of zebrafish Ssat2
isoenzymes
35
Ssat2 enzyme activity
0.7
• Ssat2a did not
react with any
substrates.
0.6
Ssat2a
Ssat2b
0.5
Activity (OD412)
• Ssat2b could
only react with
thialysine but
not with
polyamines
0.4
0.3
0.2
0.1
0.0
put
spd
spm
Substrate
thia
35
Ssat2 is a kind of thialysine
acetyltransferase (TLAT)
81
Uni-cell protozoa and
parasite
FEBS Letters 579 (2005) 5347–5352
36
Ser81 plays a key role in the activity of
Ssat2 (TLAT)?
C
81
D
A
B
• A.B.C.D motifs are GNAT superfamily
conserved regions.
37
Search for the key region responsible
Constructs of ssat2 isozymes
for TLAT activity
•Site-directed mutants:
ssat2a_N81S
N
S
ssat2b_S81G
S
81
G
81
•Chimeric mutants:
ssat2a
ssat2b
92 96
chimeric
＋
gene
92 96
ssat2ab
ssat2ba
overlappig
38
Enzymatic activities of Ssat2 mutants
2D Graph 2
Chimeric enzyme activity
0.6
0.7
0.6
2b_S81G
Ssat2a_N81S
Ssat2b_S81G
Ssat2b
2a_N81S
0.5
Activity (OD412)
Activity (OD412)
0.5
0.4
0.3
Ssat2ab
Ssat2ba
Ssat2b
0.4
0.3
0.2
0.2
0.1
0.1
0.0
0.0
put
spd
spm
thia
Substrate
• Both Ssat2a_N81S and
Ssat2b_S81G can use thialysine
as substrate.
put
spd
spm
thia
Substrate
• As Ssat2a, no obvious activity
of Ssat2ab was found.
• Ssat2ba can use thialysine and
putrescine as substrate.
39
6
Enzyme Kinetics of Ssat2
isozymes (with thialysine)
Chimeric zSSAT2_2b2a kinetic
zSSAT2b_S81G kientic
Ssat2b_S81G
Mut_zSSAT2a_N81S
Ssat2a_N81S
Ssat2b
Ssat2ba
35
50
R2=0.999
R2=0.999
20
40
R2=0.996
R2=0.988
30
40
20
1/V (min mM-1)
10
1/v (min mM-1)
1/V (min mM-1)
1/V (min mM-1)
25
30
15
30
20
20
15
10
5
10
10
5
-4
-2
0
2
1/[Thialysine] (mM-1)
4
6
-0.10 8
-0.05
0.00
0.05
0.10
0.15
-2
-1
0
1
2
3
-0.10
-0.05
0.00
0.05
0.10
1/[Thialysine] (mM-1)
1/ [Thialysine] (mM-1)
1/[Thialysine] (mM-1)
kcat / Km
(M-1 S-1)
Km (mM)
Kcat (S-1)
Vmax (min)
Ssat2b
0.78 ± 0.0714
21.75
0.13
27388.01
Ssat2a_N81S
12.79 ± 2.004
1.32
0.06
103.31
Ssat2b_S81G
3.18 ± 0.8747
3.42
0.18
1176.34
Ssat2ba
50.93 ± 3.2179
1.56
0.19
30.6
40
0.1
Can Ssat2 isozymes use substrates
other than thialysine?
2D Graph 2
• Hydroxylysine
(sturcture similar to
thialysine)
1.0
Ssat2a
Ssat2b
Ssat2a_N81S
Ssat2b_S81G
Ssat2ba
Amphixous
• Structure similar to
putrescine (4C):
• 1,3-diaminopropane
(3C)
• Cadaverine (5C)
• 1,8-diaminoctane (8C)
• Monoamine 
serotonin
Activity (OD412)
0.8
0.6
0.4
0.2
0.0
e
ne
lysin
ropa
y
p
x
o
o
in
dr
diam
5-hy
1,3-
ne
ine
octa
aver
n
i
d
a
C
diam
1,8-
n
t on i
s er o
41
Crystal structure of Ssat2ba
Resolution range (Ã…)
27.01 - 2.652
(2.746 - 2.652)
Space group
Unit cell
P 43 21 2
92.118 92.118
144.156 90 90 90
Total reflections
Unique reflections
18428 (1831)
Completeness (%)
98.87 (100.00)
Mean I/sigma(I)
22.85 (5.25)
Wilson B-factor
57.72
R-sym
R-factor
0.1990 (0.3015)
R-free
0.2727 (0.4020)
Protein residues
503
RMS(bonds)
0.012
RMS(angles)
1.64
Ramachandran favored (%)
97
Ramachandran outliers (%)
0
Substrate preference of Ssat isozymes may
be explained from their crystal structures
45o
Han-Jia Lin Ph.D
Taiwan Ocean University
Hanjia’s
Metabolomic Biochemistry
Laboratory
Heterodimer formation between
zSSAT1 isoenzymes?
134
hSSAT1
zSSAT1a
zSSAT1b
zSSAT1c
140
155
hSSAT1
zSSAT1a
zSSAT1b
zSSAT1c
163
Heterodimerization between zSsat1
isozymes
zSSAT1
myc
pCDNA3.1a
transfection
Harvest cells and protein extraction
Pull-down by GST or GST-SSAT
and western blotting
Heterodimerization of Ssat2
isoenzymes
Pull down
GST
WB
GST_Ssat2a
10% 2b_myc
Anti-Ssat2b_myc
Pull down
GST
Ssat2a_myc
1a
1b
WB
1c
10% 2a_myc
Anti-Ssat2a_myc
Ssat2b_myc
Pull down
GST
1a
1b
WB
1c
10% 2b_myc
Anti-Ssat2b_myc
47
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.
48
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
49
Interaction of zebrafish Hif-1α and
Ssat1 isozymes
 Only Ssat1b and Ssat1c
could interacted with
Hif-1α PAS-B domain
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.
Protein-protein interaction between
Ssat1 and Integrin 9
 By using GST-pull down
experiments, we confirmed
that zebrafish integrin α9
interacts with Ssat1b and
Ssat1c, but not Ssat1a.
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.
Han-Jia Lin Ph.D
Taiwan Ocean University
Hanjia’s
Metabolomic Biochemistry
Laboratory
Characteristics of zebrafish Ssat2
isoenzymes
Reacted with
Spd/Spm
S. pombe
TLAT
D. major
TLAT
C. elegans
D2023.4
x
x
Reacted with
thialysine
√
√
Reacted with
5-hydroxylysine
√
√
Interacted
with SSAT1
N.D.
N.D.
Interacted
with HIF-1α
Data source
N.D.
Biochem. J.
(2004) 384, 129–
137
N.D.
FEBS Letters
(2005) 579 5347–
5352
x
√
√
N.D.
N.D.
Biochem. J.
(2004) 384, 129–
137
x
√
√
N.D.
N.D.
This work
Zebrafish
Ssat2a
x
x
x
zSSAT1c
?
This work
Zebrafish
Ssat2b
x
√
√
√
?
This work
√
Biochem. J.
(2004) 384, 139–
148
Amphioxus
XM_002595182
Human
SSAT2
x
√
√
N.D.
Physiological significant of Ssat2
 Ssat2 was found in most eukaryotic species.
 The activity of thialysine and hydroxylysine acetylation
are conserved.
Thialysine
5-Hydroxylysine
 Other unidentified substrates for Ssat2?
 Remained ~35% sequence identity with Ssat1 in most
vertebrates

Functional convergency with Ssat1?
(Ex: regulation of hypoxia signaling pathway)
Characteristics of zebrafish Ssat1
isoenzymes
zSsat1a
zSsat1b
zSsat1c
hSSAT1
Acetylation of thialysine
x
x
x
x
Acetylation of Spd/Spm
√
√
√
√
Synteny with Acot9
√
∆
∆
√
Transcriptional regulation
x
x
x
√
Alternative splicing
x
∆
x
√
Translational regulation
∆
√
√
√
Protein stability regulation
x
√
x
√
Interaction with HIF-1α
x
√
√
√
Interaction with Integrin α9
x
√
√
√
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 α9 and Hif-1α
(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 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 α9 signaling pathway?
59
Lab members:
Lien, Yi-Chin
Lin, Yu-Tzu
Ou, Ting-Yu
Kuo, Po-Chi
sağ olun
Collaboration:
Hsu, Chun-Hua Ph.D
National Taiwan University
Han-Jia Lin Ph.D
Taiwan Ocean University
Hanjia’s
Metabolomic Biochemistry
Laboratory