ComBio: Shadoo not a shadow of itself

The functions of the prion protein (PrP) are still a puzzle. Many have been suggested, but it appears to be a bit of a Jekyll and Hyde character. It has many “good” functions, such as acting as a protector against neural insults like stroke by preventing cerebellar degeneration, or as a copper-dependent antioxidant. There is also evidence for a role in the maintenance of long-term memory.

But on its bad days, in its misfolded form, PrP is associated with the lethal brain pathology of the transmissible spongiform encephalopathies that affect cattle, sheep, deer and humans.

Exactly how a misfolded protein becomes an infectious agent – or an essential part of it – is unknown, despite a great deal of work having taken place on prions, particularly since mad cow disease (BSE) was found in 1995 to be transmitted to humans through meat consumption. Increased interest in PrP and prions led researchers to look for related proteins.

The second member of the PrP family to be found is Doppel (Dpl), meaning “double”. Dpl is mainly expressed in the testes and spermatazoa and has an essential role in male fertility. But unlike PrP and the third family member, Shadoo (Sho), Dpl is expressed at only low levels in the central nervous system.

Shadoo was discovered by an ANU group in 2002, in a serendipitous collaboration with an Italian group led by Professor Tatjana Simonic from the University of Milan. The Italian group had been trying to identify PrP-analogue genes in fish. As a result of the BSE outbreak and variant Creutzfeldt-Jakob disease in humans, there was concern of a similar human disease from farmed fish fed meat-based meal.

Simonic’s PhD student, Lorenzo Sangiogio, had identified a PrP-like gene in zebrafish but had not been able to find homologous protein sequences in public databases. The Italians shared this lead sequence with the ANU group of PhD student Marko Premzl, Professor Jill Gready and Professor Jenny Graves, who were successful in finding mammalian homologues in the sequence databases.

The ANU group was able to rapidly flesh out a picture of the gene and its protein and where it was expressed in the body. Tissue expression indicating high expression in the brain was confirmed experimentally.

The protein was called Shadoo, Japanese for shadow, and the gene SPRN. In silico analysis suggested the SPRN gene was highly conserved through fish and mammals but at a different chromosomal location than the PrP and Dpl genes.

Subsequent work at ANU, using a combined in silico and experimental comparative genomics approach, has filled in the picture of the evolution of the gene environment and protein sequence in the intermediate vertebrate branches – frog, bird, platypus and marsupial. A striking feature of the Sho sequence is the conservation of a middle hydrophobic region of about 20 residues which is very similar to that in PrPs but is not observed in any other protein, including Dpl.

Last year, a Canadian research team working on prion proteins, led by Professor David Westaway, found that Shadoo also has neuro-protective qualities similar to PrP, is linked to the outer cell membrane by a glycosylphosphatidylinositol (GPI)-anchor, just like PrP and Dpl and, intriguingly, is dramatically reduced in prion disease-infected mice.

Sue Corley, a PhD student at ANU, will present new findings on Shadoo in the protein structure prediction session at ComBio next week.

---PB--- Sho and its RGG box

By comparing all the accumulated vertebrate sequences, Corley has now found that Sho has an RGG box, a series of arginine-glycine-glycine repeats that is a known RNA-binding motif, and which may also have something to do with mediating protein-protein interactions.

“These repeats of RGG are a motif that was first identified in 1992 by Gideon Dreyfus, who is the expert on RGG motifs,” Corley says. “He found this motif in one of the heterogenous RNPs (heterogeneous nuclear ribonucleoparticles or hnRNPs), which are generally found in the nucleus. This protein, hnRNP U, has no other RNA binding motif and through various experiments it was found that the RGG area was competent to bind RNA.

“There hadn’t been any hint of binding or a suggestion of RGG boxes in Sho, but I started with some comparative genomics, and something stood out for me. That was an area at the N-terminus which was conserved from fish through to human exactly, a GGRGG repeat, which is an arginine methylation site.

“I did key word searches in the literature to see if it meant anything and found that methylation often occurs in RGG box proteins. Then I looked again at my sequences and found that there was not only this GGRGG but there are actually three RGGs. It looks like an RGG box protein.”

“We hypothesise that the normal function of Sho may involve binding of RNA or other ligands to the RGG box region.”

Corley then did more literature searches and comparative genomics to find out about other RGG box proteins. She found about 45 other proteins with RGG boxes and Sho certainly seemed to fit all of the parameters.

One protein that stood out for its similarity was the Fragile X Mental Retardation protein (FMRP), which has a tendency to bind to RNA that forms a tertiary structure called a G quadruplex. This is a structure in which a string of RNA with a number of guanines folds into planes, which then stack on top of each other.

“They are stabilised by positively charged cations such as potassium,” Corley says. “These ions sit between these planes – so if you imagine a sandwich, they are the cherry tomato between the two slices of bread. They have quite a stable structure and are found at the end of DNA in the telomeres and so have been looked at quite carefully in terms of cancer research.”

PrP also seems to have an affinity with G quadruplexes, she says. PrP is certainly competent to bind to both RNA and DNA, but Corley says there are no studies as yet at to whether this is important functionally for either PrP or Sho.

“There is a question mark over that but if this line of research is continued it might shed some light on PrP as well.”