Identification of the Proteins Required for Biosynthesis of Diphthamide, the Target of Bacterial ADP-Ribosylating Toxins on

Translation Elongation Factor 2

Shihui Liu, G. Todd Milne, Jeffrey G. Kuremsky, Gerald R. Fink, Stephen H. Leppla

Molecular and Cellular Biology, Nov 2004: 9487-9497

Diphthamide

Ethyl methane

sulfonate

Grow 6 generations Spheroblasts

Incubate with diphtheria fragment

Isolate resistant yeast

Random Yeast Mutagenesis

-Mutated yeasts isolated and sporolated, analyzed for ADPR-EF2 production (as opposed to other survival factors)

-Isolates crossed with Toxin-sensitive mating strain

-Genetic crossing analysis performed

- Resistance segregated genetically as if single-copy

- Over 31 sets crossed pair-wise

- All fell into one of 5 complimentation sets

- dph1, dph2, dph3, dph4, dph5

Dph5

-enzymatic action identified as late process

-Dph5 mutant produced EF2 can be converted to ADPRTable EF2 using dph5 based lysate

-Dph5 determined later to be responsible for the trimethylation

-Gene identified: 300 residue

-AdoMet transferase conserved function

Dph2

• Gene identified 534 residue

• No conserved domains, function suggestion

CHO-Cells-Similar work done previously with CHO cells and mutants

-4 complimenting strains identified as toxin-resistant

-CG-1, CG-2, CG-3, CG-4

-CG-2 recently identified 82 residue protein, with a functional homolog in yeast responsible for zymocin toxin sensitivity

This research..

• Borrowed the dph# yeast mutants from Bodley• Borrowed the CG-# CHO strains• Created a large series of mating strains for

crossing• Created strains using recently identified putative

genes as cross-testing candidates

Mutagenesis-Cell lines mutagenized by transformation of transposon-mutagenized genomic library

-Also used a pool of spontaneous mutations

-Cells transfected to express DT catalytic fragment on demand, to screen for resistivity and sensitivity

-Complimentation crossings done with dph# strains

-Of 484 recessive mutants identified, all were one of dpn1, dph2, dph4 or dph5 -- none in dpn3

Dph1/dph4 discovery

-Given that dph5 and dph2 genes had been previously identified, focus was on the unidentified genes

-Genetic-crossing tests consistant with single-gene mutations

-Transposon marker always followed the DTR marker

-Transposon insertion sites were sequenced

-Genes identified, and deletion-mutant strains prepared for each

Dph1: YIL103w ORF Dph4: YJR097w ORF

Dph3-Not obtained from transposon-based mutagenesis method

-Screened only from DT-expression/selection of 10^8 cells

-363 recessive DTR mutants divided into one of all 5 dph groups

-Very elaborate screening to isolate dph3 mutant strains

-Overlapping genetic-fragment analysis used to identify gene

-Deletion mutant of dph3 created

Dph3: YBL071w-A ORF

CHO-Genes-CHO based mutants made by fusion analysis

-This mutation system revealed a new CG-4 compliment group

-CG-2 mutation previously identified: DESR1

- is a homolog of TBL071w-A (dph3)

-CG-genes identified by sequence-similarity to yeast genes

-Corresponding mouse genes put into protein expression system

-Transformed into the various CG strains

-Reversal of DTR to confirm gene function

Dph1/2-These genes have 16% identity between each other

-Previously, human dph1 has been called dph2L due to misidentification because of homology

-yeast/mammalian sequences:

Dph1 – 49% identity (CG-4)

Dph2 – 22% identity (CG-3)

-Non-interchangible products, they ARE functionally distinct

Biosynthetic Intermediates-EF2 products and ribosylated products run on nondenaturing PAGE

-Net 0 charge – HIS precursor, “intermediate” and diphthine

-Net +1 charge – diphthamide

-Net –1 charge – ADPR-diphthamide

-Different bands observed on gels, but ALL could run as ADPR-Dip when cells transfected with missing gene

-dph1, dph2 involved first-step approximately equally

-dph5 later (known)

Dph1/dph2 Interact-Dph1, dph2 AND dph3 appear to be involved in early steps

-Similarity of dph1 and dph2, and small (82 res) size dph3 suggests the possibility of a catalytic complex

-All gene products produced with his/myc tags

- Each product restores activity in defective organism (no tag probs)

-Various combinations of constructs transfected into CHO cells

-immunoprecipitation of dph1 brings out dph2, and vice-versa

-dph5 expressed at high levels, but no cross-precipitation

-dph3 not expressed at detectable levels for precipitations

-Two-hybrid studies elsewhere of dph1/2 locate interaction regions,

- These two proteins likely form a heterodimer/multimer

Details – dph1Yeast 425 residue, 49% identity with mouse dph1

Identified in CG-4 cells

Previously identified as OVCA1, dphl1

-dphl1: identified as tumor suppressor gene in humans

-Located in highly conserved region on Ch17 which shows loss of heterozygosity in ovarian/breast cancer cells

-Knockout single dph1 in mice Increased tumerogenesis

-Knockout both dph1 in mice embroyonic lethality

-Forced expression suppressed cell proliferation in ovarian cell line

Details – dph2Yeast 534 residue protein, 22% indentity to mouse gene

16% identity to dph1, identified as CG-3

Interacts with dph1, likely heterodimer

Mutation in either 1 or 2 no synthesis, thus functionally different from dph1 despite similarities

Like dph1 gene product, dph2 gene product is localized in the cytoplasm (where dip-synthesis expected)

Details – dph3Yeast 82 residue protein, small in others too

Previously identified DESR1 gene

High percentage of negatively charged residues

Highly conserved in residues 1-60, differing C-termini

Conserved region: CSL Zinc-finger domain (cys-chelating)

*An identified allele sequence results in subbing tyr for cys

Dph3 localized in both cytoplasm and nucleus

continued….

Details – dph3

Dph3 previously identified as KTI11 zymocin resistance in Yeast

KTI11shown to associate with dph1, dph2 and EF2 via tandem-purifications. Also associates with Elongator core complex, Elp1, Elp2, Elp3

Dph1 AND dph2 mutants are slightly resistant to zymocin, suggesting a large complex is involved at some stage

*While link appears between dip-synthesis and elongator-complex, its not reciprocal: mutants of elp1, elp2, elp3 are not defective in dipthamide biosynthesis

•dph3 is the only dph# mutant which doesn’t have subtle phenotype changes

• posess pleiotropic growth defects

• primarily slow growth and drug / temperature hypersensitivity

Details – dph4Yeast YJR097w gene, encodes DNAJ-like protein, 172 residues

Has no corresponding CHO compliment mutant set

First half of gene= typical J-domain

- DnaJ-like proteins cochaperones for HSP70 proteins

- assist in folding of newly synthesized proteins

•May ensure proper folding required for other dph genes?

•Second half of gene = typical CSL zinc-finger domain

•Mammalian homologs show same arrangement as yeast

•Mouse homolog shown to be expressed ubiquitously, is a cochaperone, and stimulates ATPase activity of several HSP70 proteins

•Localized in both cytoplasm and nucleus

Details – dph5Previously identified 300-residue SAM-methyltransferase

Highly homologous to mammalian homologs (50% to 55%)

Belong to larger class of SAM-methyltransferases

No functional redundancy found elsewhere in yeast or CHO

* Unique methylation step

Dph5 protein localized in cytoplasm

Biosynthesis of Diphthamide

1) Dph1, 2 and 3 = multimer complex to stick on carbon chain

2) Dph4 stabilizes the complex

3) Dph5 Unique methylation step

** Unknown, does it do the first one, or all of them

4) Unidentified product ATP-driven amidation

Role of Diphthamide?Targeted by two externally produced bacterial toxins

Found in eukaryotes in all environments, as well as the extremeophile archeabacteria (volcanic caves, acid pools, thermal vents, metallic brines)

Produced by multiple enzymes, multiple enzyme steps – important!

Likely it plays a STRUCTURAL or REGULATORY role

His699all 19 other aas, non of them adp-ribosylatable

13 of them expressed temperature sensitive growth

6 of them were non-functional

* Strongly indicative of a STRUCTURAL role

Role of Diphthamide?However, the ADP-Ribosylation of diphthamide suggests regulatory role

Mono-ADPRT in bacterial systems as posttranslational regulation mechanism

Similar possibilities for many monoADPRTs in other eukaryotic systems

•There most likely does appear to exist an intrinsic ADPRTase for EF2

Take-home power-observation linker:

Dph1 gene is previously identified having a role as a tumor suppressor and thus suggests Diphthamide might have a role in cell growth regulation