The Path Less Traveled

A freshly demonstrated theory on how peptides enter cells sparks ongoing debate.

The uptake of fluorescein-labeled peptides into cells via endocytosis at 5 microM peptide (top) and independent of endocytosis at 20 microM peptide (bottom)
Courtesy of Roland Brock

Describing how peptides and proteins traverse cell membranes is huge in the field of cell biology. Peptides, short chains of amino acids, could one day be used as molecular ferries transporting therapeutic genes or proteins across lipid bilayers and directly into the cytosol, where many crucial biochemical pathways start. But the journey from outside a cell to the cytoplasm is no small feat for bulky peptides—researchers have been trying to map the various routes for decades.

Since the early 1990s, researchers studying how cell-penetrating peptides, which include some transcription factors and parts of viruses, gain access to the inside of cells have focused on different modes of...

In 2007, a paper published by European researchers seemed to quell the debate over which mode of uptake was most important for peptides entering cells. Roland Brock, a cell biologist at the Nijmegen Centre of Molecular Life Sciences in the Netherlands and senior author on the paper, showed three different peptides entering human cells by simultaneously using three separate pathways: macropinocytosis and two modes that depend on membrane proteins and/or lipids.

“[The paper] in a way released the field” from arguing about which mode of endocytic uptake was most important, says Brock. “Everyone was very happy because everyone felt represented in this paper.”

“It’s obviously one of the most comprehensive studies in this field,” says Stockholm University biochemist Ülo Langel, who was not involved with the study. “The data are very trustable.”

But Brock and his colleagues noted something even more interesting: at higher extracellular peptide concentrations, the macromolecules made their way into cells via an endocytic-independent pathway. It appeared that the peptides were permeating directly into the cell’s cytoplasm. “For us, this was actually the most exciting finding,” Brock recalls. And one with potential clinical significance, since it would make it easier to deliver drug molecules directly to cytoplasm if they didn’t have to escape from vesicles that are formed during endocytosis.

This finding has not been as universally well received, and continues to stoke debate 2 years after it was published. To this day, some scientists claim that peptides didn’t enter cells independent of endocytosis and any finding that suggests otherwise was an artifact, while others are accepting the finding and trying to pinpoint the molecular mechanism behind it.

Not so fast

Some researchers maintain that Brock’s finding of endocytosis-independent entry represents an artifact of extremely high peptide concentrations outside the cell, rather than some novel mechanism of cellular uptake. Steve Dowdy, a cell biologist at the University of California, San Diego, says Brock and his colleagues were likely seeing peptides fill the cytoplasm of cells because at high concentrations—20 micromolar—molecules were in essence “blowing out a hole” in the membrane and allowing the peptides to rush in. This, Dowdy notes, makes this mode of peptide entry unacceptable for potential therapeutic use. “It’s literally destroying the membrane,” he says. “You can’t punch holes in membranes and not have some consequences for the cell.”

“We were certainly confronted with this concern during the submission process,” Brock admits. “We never claimed that this direct membrane permeation may be therapeutically relevant. We just said it was interesting.”

Other labs, working independently of Brock’s, observed similar phenomena around the same time. Arwyn Jones, cell biologist at Cardiff University’s Welsh School of Pharmacy, and his colleagues exposed the cells to peptide concentrations up to 12.5 micromolar, and observed endocytosis-independent uptake without any apparent damage to the cells (Biochem J, 403:335–42, 2007). “The cells look very healthy, and they live, and they divide, and there is no sign that there is a problem with them,” says Jones.

M. Cristina Cardoso, a cell biologist at the Max Delbrück Center for Molecular Medicine in Berlin, agrees that Brock’s observation of the nonendocytic uptake was valid. Cardoso’s lab published a recent paper corroborating the nonendocytic uptake pathway in mouse cells where modes of endocytic uptake were blocked by chemical inhibitors (J Biol Chem, 284:3370–78, 2009). “I truly believe that the data on nonendocytic uptake is absolutely correct,” she says.

Towards therapy

Even if the endocytosis-independent uptake noted by Brock and others is a valid biological phenomenon, using cell-penetrating peptides in a therapeutic context is far from becoming a reality. Brock’s study, like many others that support his findings, use peptides carrying fluorescent tags, which lets them track movement. But in a clinical setting, peptides would have to carry drugs or genetic molecules, which are much bigger. “If you want to deliver things with these cell-penetrating peptides, then the cargo has to be significantly larger,” Jones says.

A bevy of labs is trying to pinpoint the molecular mechanism that is at play in endocytosis-independent entry. Jones suggests that a membrane potential gradient might be “pulling” peptides into cells at high concentrations. Others suggest the existence of transient pores that are opened in specific extracellular conditions. Brock claims that his group has created a knockout mouse that does not show the nonendocytic uptake, suggesting a single enzyme mediates this peptide transduction, but declines to provide more information before publishing.

Jones says that researchers should continue studying all peptide uptake pathways for their potential use in delivering therapies. “What we need to do,” Jones says, “is learn more from these systems.”

Data derived from the Science Watch/Hot Papers database and the Web of Science (Thomson ISI) show that Hot Papers are cited 50 to 100 times more often than the average paper of the same type and age.
F. Duchardt et al., “A comprehensive model for the cellular uptake of cationic cell-penetrating peptides,” Traffic, 8: 848–66, 2007. (Cited in 80 papers)

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