Study of psymberin’s mode of action using forward geneticsVincent 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

AcknowledgementsI 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 MVNVPKARRTFCDGKCRKHTNHKVTQYKKGKESKFAQGRRRYDRKQSGFGGQTKPIFRKKDrosophila RPL36a MVNVPKARRTFCDGKCRKHTNHKVTQYKKGKESKFAQGRRRYDRKQSGFGGQTKPIFRKKHuman RPL36a MVNVPKTRRTFCK-KCGKHQPHKVTQYKKGKDSLYAQGKRRYDRKQSGYGGQTKPIFRKKHuman RPL36al MVNVPKTRRTFCK-KCGKHQPHKVTQYKKGKDSLYAQGRRRYDRKQSGYGGQTKPIFRKKMouse RPL36a MVNVPKTRRTFCK-KCGKHQPHKVTQYKKGKDSLYAQGKRRYDRKQSGYGGQTKPIFRKKC elegans RPL41 AKTTKKIVLRMECTE--CKHKKQLPIKRCKHFELGGQKKSRGQVIQFDrosophila RPL36a AKTTKKIVLRMECTE--CKHKKQLPIKRCKHFELGGQKKSRGQVIQFHuman RPL36a AKTTKKIVLRLECVEPNCRSKRMLAIKRCKHFELGGDKKRKGQVIQFHuman RPL36al AKTTKKIVLRLECVEPNCRSKRMLAIKRCKHFELGGDKKRKGQVIQFMouse 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 +6.75loc cM cM cM cM cM cM cM cM cM

6 H H H N2 N2

62 H H H N2 N277 H H N2 N2 N2

96 H H H N2 N2105 H H H N2 N2

N21 N2 N2 N2 CB

2 N2 H H H H H6 N2 H H CB

7 N2 N2 CB CB CB CB16 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 leuc

ine.

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

NH

OMe

OH

OOMeMe

Me

OHO

O

HO

OH

Me

OH

MeMe

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

References

1. Cichewicz, R.H., Valeriote, F.A., Crews, P. (2004). Org Lett. 6, 1951.

2. Jiang, X., Williams, N., De Brabander, J. K. (2007). Org Lett. 9, 227.

3. Wu, C.-Y., Cardenas, E. R. and Roth, M. G. unpublished result.

4. a. Kim, J.-H. et al. (2004). Hepatology,39(1). b. Hoebeke, I. et al. (2007). Lukemia, 21, 311. c. Uechi. T. et al. (2002). Nuc. Aci. Res. 30(24), 5369. d. Amsterdam, A. et al. (2004). PLoS Bio., 2(5), 690.

5. Chandramouli et al. 2008. Structure,16(4), 535.