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Cellular biology

Action on the sly

Small proteins, previousy considered waste, help regulate cells

LORE KUTSCHERA/WIKIMEDIATwo varieties of a small protein called hemopressin may stimulate the appetite and give rise to sensations of pleasure in the same way as the components of narcotic plants, like marijuana, while another variety of hemopressin has the opposite effect. Research carried out in São Paulo and New York showed that these microproteíns, known as peptides, may do much more, are very varied, and unlike what even the specialists had thought, are not useless waste inside trillions of the organism’s cells.

In just one type of cell extracted from a human kidney, the teams of pharmacologists Emer Ferro at the University of São Paulo (USP), Lloyd Fricker at the Albert Einstein School of Medicine and Lakshmi Devi from the Mount Sinai School of Medicine, both in New York, identified and sequenced 116 microproteíns that help regulate cell functioning and facilitate the interaction of proteins – the group of hemopressins produced in neurons is, for the time being, the most widely studied.

“Besides regulating the internal functioning of the cell,  these peptides can modulate extracellular stimuli”, says Emer, based on studies published last year in the Journal of Biological Chemistry. As there have only been isolated reports of intracellular peptides with biological functions since 1983, this is possibly the first time these molecules have appeared in such large variety and are being jointly analyzed. Given what they can do – and apparently they must do a lot more in other cells – these molecules indicate that the functioning of cells and the organism do not depend only on giant molecules, like DNA, RNA and proteins, but also on this multitude of previously anonymous intermediaries, almost 50 times smaller than a protein like hemoglobin, which carries oxygen to the cells.

Why did no one pay any attention to these peptides before if there are so many and they are so abundant inside cells? There are two reasons, according to the researcher from USP. The first is that mass spectrometers, the equipment that identifies peptides, are relatively recent, as well as being costly. The second is that no one took them seriously. Emer says that in 2006 he himself had to convince Fricker that the nearly one thousand molecules that he, Fricker, had extracted from the brains of rats were not the so-called artifacts – an elegant word used in scientific circles to indicate any type of mistake – or pieces of other molecules. Produced continuously inside the cells as the result of the fragmentation of proteins they were indeed complete molecules that helped others – and the organism – to function.

Emer entered this line of research in 1989 when he saw that enzymes called oligopeptides were concentrated inside the cells, while the peptides on which they acted were outside. At the time, one of the cleaning mechanisms of cells was already known, the proteasomes that continually break down proteins with defects or that are too old to function properly. A protein with 700 amino acids, the blocks that form these molecules, may be broken down into at least 35 parts. Emer could not accept the possibility that 34 of these parts were inert raw material, waiting for other proteins that they could participate in, and only one of the parts had a function to adher to the surface of cells, thereby preventing the organism from identifying the protein of that specific type as something that should be destroyed. “It made no sense”, he thought. For Ferro the cell would not waste peptides; but as yet he had no way of proving what he was thinking.

In 1993 Emir attended a seminar at the Mount Sinai School of Medicine in which a specialist in protein recognition, Stephen Burley, presented a protein, the Tata box binding protein, which embraced the DNA at specific points as if it was a saddle on a horse, regulating other proteins that interact with the DNA. For Ferro this type of fit showed not only yet another refinement of the functioning of DNA, but also that small molecules, like peptides, could be very much more mobile than proteins or DNA itself and could help regulate the functioning of the cell.

Finally, the answers
 Ten years later in 2003, Vanessa Rioli, a biologist from the Federal University of São Paulo (Unifesp) who was a member of Emer’s team, identified one of these peptides, hemopressin (see Pesquisa FAPESP 84, February 2003). They immediately saw that hemopressin acted on proteins that controlled blood pressure, and later with the help of another biologist, Andrea Heimann (see Pesquisa FAPESP 143, January 2008) on the proteins that regulated hunger and pleasure. “Hemopressin was proof that peptides produced inside cells were there for a reason”, commemorated Emer.

At the time his group had already found some 30 peptides of the same size as hemopressin, with 5 to 17 amino acids and functions that still had to be demonstrated. Old doubts, like the one that had arisen 20 years before about oligopeptides, an enzyme that seemed to be far from the supposed target, got answers. “Everything began to fit together”, he says. “The enzyme and the peptides on which it acted are inside the cell, no longer separated, one inside and one outside.”

More recent studies have shown that hemopressin is not one, but at least four and with functions that are even opposite – two of these variations may increase and one reduce the activity of proteins on which the active principle of cannabis acts (the functions of the fourth form of hemopressin have not yet been studied). A doubt about this molecule that persisted for years was how  hemopressin could be generated in neurons from fragments of another molecule, hemoglobin, produced in the red blood cells? The reply was found this year in a study carried out at the University of California in Los Angeles (UCLA), showing that neurons could fabricate the alpha and beta chains, two of the blocks that form hemoglobin. So, in neurons these blocks form the hemopressin and the blood cells the hemoglobin.

Intracellular peptides have been well received. Since last year when the articles that described them began to appear, Emer has presented them to colleagues from the Biomedical Sciences Institute at USP, where he works, and to other researchers in congresses in the Netherlands, Japan and Israel. This May he is going to talk at a seminar at the National Institutes of Health (NIH), in the United States, alongside Fricker and Lakshmi, the coordinators of other groups with whom he is investigating intracellular peptides. The title of Fricker’s presentation (Non-classical bioactive peptides and micro-proteins: are they the next big small thing?) suggests there is still a lot to say about these small molecules.

The projects
Molecular cell biology of oligopeptidases (nº 04/04933-2); Modality
Thematic project; Coordinator Emer Ferro – ICB/USP; Investment
US$ 271,000.00 and R$ 270,000.00 (FAPESP)

Scientific articles
BERTI, D. A. et al. Analysis of intracellular substrates and products of Thimet oligopeptidase (EC in human embryonic kidney 293 cells. Journal of Biological Chemistry. 12 March 2009 (on-line).
CUNHA, F. M. et al. Intracellular peptides as natural regulators of cell signalingJournal of Biological Chemistry. 2008, 283 (36), 24.448–59.
GOMES, I. et al. Novel endogenous peptide agonists of cannabinoid receptorsFASEB Journal. 2 abr. 2009 (on-line).