1. Study of psymberin’s mode of action using forward genetics
Vincent Tsao, Cheng-Yang Wu, and Dr. Michael Roth*
UT Southwestern Medical Center, Biochemistry Department, Dallas, TX
Background
Psmyberin is a member of the pederin family and inhibits translation in
vivo and in vitro. However, the inhibition by psymberin is 40 to 100
fold more effective than cycloheximide, a well-known translational
inhibitor. Looking at the half maximum values of translation inibition
and cytotoxicity of psymberin and cycloheximide reveal that
translation inhibition may not be the only method by which psymberin
induces cell death. In cycloheximide, the these values are very similar.
However, psymberin’s half maximum concentration of translation is
almost 20 times greater than it’s concentration of cytotoxicity.
Furthermore, in 8 hours where cells were treated by both psymberin
and cycloheximide separately, there are more cells detached from the
plate with psymberin treatment. This suggests that the two processes
are uncoupled.
Abstract
Psymberin, also known as Irciniastatin A, is an extremely potent
cytotoxin found in marine sponges Psammocinia and Ircinia ramose1
.
Many cancer cell lines, including breast cancer, melanoma, and colon
cancer are very sensitive to psymberin2
. Like other members of the
natural product family pederin, psymberin acts as an inhibitor of
protein synthesis. However, previous studies suggest that psymberin
may have additional bioactivity to induce cell death. These studies
suggest that psymberin might be a good candidate for cancer therapy
because it disturbs multiple cell pathways. In order to understand the
mode of activity of psymberin in cells, we use forward genetics in C
elegans. In our lab, 3 psymberin-resistant mutants were generated by
EMS mutagenesis and selected with psymberin treatment. Using
single nucleotide polymorphism mapping to locate the mutation, we
have found the mutation of a psymberin-resistant mutant to be on
chromosome II. We have mapped the mutation to be in the region
between +3.1 cM and +3.5 cM of chromosome II, and sequenced the
mutant gene, rpl-413
.
Hypothesis/Aim
Psymberin may induce cell death by pathways other than translation
inhibition. Our goal is to find the mode of action of psymberin in cells.
Future Directions
Acknowledgements
I would like to thank Dr. Nancy Street, Ms. Vanessa Powell, and all of
the people associated with the SURF program for giving me the
amazing opportunity to work and learn on the UT-Southwestern
campus. I would also to thank Dr. Michael Roth, Cheng-Yang Wu, and
the rest of the lab for welcoming me into the lab, assisting me in my
research, and making my experience rich and rewarding.
Strategy/Tool
• Forward genetics screening in C elegans to select drug-resistant
mutants.
• Single Nucleotide Polymorphism Mapping in semi-dominant
psymberin-resistant mutants (SNP mapping):
Summary
Wildtype: G Q T KG Q T K PP I F R KI F R K
GGA CAA ACC AAG CCA ATC TTC AGA AAG
16: G Q T KG Q T K LL I F R KI F R K
GGA CAA ACC AAG TCA ATC TTC AGA AAG
RPL-41 homologues:
C elegans RPL41 MVNVPKARRTFCDGKCRKHTNHKVTQYKKGKESKFAQGRRRYDRKQSGFGGQTKPIFRKK
Drosophila RPL36a MVNVPKARRTFCDGKCRKHTNHKVTQYKKGKESKFAQGRRRYDRKQSGFGGQTKPIFRKK
Human RPL36a MVNVPKTRRTFCK-KCGKHQPHKVTQYKKGKDSLYAQGKRRYDRKQSGYGGQTKPIFRKK
Human RPL36al MVNVPKTRRTFCK-KCGKHQPHKVTQYKKGKDSLYAQGRRRYDRKQSGYGGQTKPIFRKK
Mouse RPL36a MVNVPKTRRTFCK-KCGKHQPHKVTQYKKGKDSLYAQGKRRYDRKQSGYGGQTKPIFRKK
C elegans RPL41 AKTTKKIVLRMECTE--CKHKKQLPIKRCKHFELGGQKKSRGQVIQF
Drosophila RPL36a AKTTKKIVLRMECTE--CKHKKQLPIKRCKHFELGGQKKSRGQVIQF
Human RPL36a AKTTKKIVLRLECVEPNCRSKRMLAIKRCKHFELGGDKKRKGQVIQF
Human RPL36al AKTTKKIVLRLECVEPNCRSKRMLAIKRCKHFELGGDKKRKGQVIQF
Mouse RPL36a AKTTKKIVLRLECVEPNCRSKRMLAIKRCKHFELGGDKKRKGQVIQF
Through SNP mapping, we were able to narrow down the
region of the mutation in a pysmberin-resistant mutant 16 to
be between +3.1 cM and +3.5 cM on chromosome II. We
were able to pinpoint the mutation in the rpl-41 gene. The
mutation is a C to T mutation, which changes the amino acid
sequence by substituting proline with luecine. The effect of
this change will be further studied. rpl36a, which is the
human homologue of the rpl-41 gene in C elegans, encodes
105 amino acids and is a member of the RPL44E family.
The gene encodes a ribosomal protein that is a 60S
ribosome large subunit. RPL36a is also up-regulated in
different cancer cell lines. Overexpression of RPL36a in
hepatocellular carcinoma correlates to cell proliferation4
. The
location of RPL36a in ribosome is distant from peptide chain
synthesis core. These observations suggest RPL36a is a
potential cancer therapeutic target.
Figure 2. Results of gel electrophoresis after
digestion by the restriction endonuclease Apo1 for
samples 17,19,20,21,25-42,N2, and CB. Apo1 recognizes
the 6-base sequence 5’RAATTY’3 and cleaves after the first base
pair between bases R and A. This sequence is found in the N2
strain while altered by a SNP in the CB strain. Thus, a double band
would signify heterozygous chromosomes, a higher single band
would represent uncut homozygous CB chromosomes, and a lower
band would indicate homozygous N2 chromosomes.
+6.75
Seg 13 Seg 13 Seg 14 Seg 15 Seg 15 Seg 16 Seg 16 Seg 16 Seg 17
ChrII +1.38 +1.77 +3.1 +3.35 +3.5 +4.04 +4.11 +5.9 +6.75
loc cM cM cM cM cM cM cM cM cM
6 H H H N2 N2
62 H H H N2 N2
77 H H N2 N2 N2
96 H H H N2 N2
105 H H H N2 N2
N2
1 N2 N2 N2 CB
2 N2 H H H H H
6 N2 H H CB
7 N2 N2 CB CB CB CB
16 N2 N2 N2 CB CB CB
Figure 3. Sequencing data for samples 6, 58, 62, 77,
105, N2. The SNP site is indicated above. Each nucleotide (A,C,G,T)
is represented by a specific color and curve in the data. Samples 58,77,
and N2 exhibit the nucleotide C at the SNP site which corresponds to
the N2 wild type sequence. However, samples 6,62, and 105 exhibit
heterozygous chromosomes due to the presence of two curves and a
non-specific nucleotide listing.
Table I. Data from sequencing and digestion organized as
being homozygous N2, homozygous CB, or heterozygous
(H) at various positions along Chr II. The data from Figure 2
corresponds to the data listed for samples 6,62,77,96, and 105 on the table
at +3.1 cM. Data for samples 1,2,6,7, and 16 at +3.35 were collected in the
same procedure. Data for the samples at various other locations along ChrII
were collected via digestion (Figure 1) or sequencing (Figure 2).
Figure 4. Sequencing data for the point mutation in the rpl-41
gene. The mutation site is indicated above. The correct sequence for the rpl-
41 gene is also highlighted. The wild type nucleotide for rpl-41 is C, but these
samples have a T at this position. This mutation in the rpl-41 sequence for the
16 mutant could correspond to a change in the amino acid sequence.
Figure 5. Amino acid change from the point mutation in the rpl-
41 gene. In wild type C elegans, the amino acid produced from the ‘CCA’
sequence is proline. However, in the 16 mutant, the C to T mutation produces
leucine.
Figure 7. RPL-41 homologues including C elegans, Drosophila, Humans, and Mice. The amino acids are
highly conserved throughout these organisms. The sequences in blue show conserved residues. The mutation site (red) is in the
conserved region.
• Complement experiments in worm and human cells
• Explore how RPL36a affects ribosomes assembly and
translation.
• Explore how RPL36a contributes to psymberin-induced cell
death.
Figure 8. The structure of the RPL36a gene and the location in
60S ribosome. The structure of RPL36a is in green. The structure of 60S
ribosome is in gray. The location of the mutation in RPL36a is in yellow.
O
N
H
OMe
OH
OOMeMe
Me
OHO
O
HO
OH
Me
OH
Me
Me
4 8
Figure 1. The structure of the translational inhibitor
psymberin. The addition of the dihydroisocoumarin side
chain to the structure may contribute a secondary
bioactivity in psymberin to induce cell death.
N2 CB
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