An unlikely assembly plant

If the ribosome makes the body’s proteins, what makes it? Oddly enough, a giant.

Imagine that you’re out for a stroll in your neighborhood, passing the same familiar landmarks you see every day, when you suddenly come upon an enormous factory. Now imagine how astonished you’d be to learn that the behemoth had been there all along, but no one had been able to see it until now. That’s about how surprised Susan J. Baserga, M.D. ’88, Ph.D. ’88, was to find the cellular equivalent of an assembly plant hiding in plain sight.

Baserga and colleagues discovered and purified a new cellular entity, dubbed the SSU processome, which plays a key role in the making of ribosomes, the cellular machinery responsible for manufacturing all proteins. Surprisingly, the SSU processome, undetected until now, is nearly as large as a ribosome, about 80 Svedberg units.

“We were surprised to discover that it takes a complex as big as a ribosome to make a ribosome,” said Baserga, associate professor of molecular biophysics and biochemistry, therapeutic radiology and genetics. The SSU processome—a complex of RNA and many proteins—wasn’t detected until now because previous techniques used to look for RNA-protein complexes filtered out the larger ones, leaving only smaller material. “We were able to find it because we made our extracts—the starting material for purifying RNA-protein complexes—differently,” said Baserga. The mass spectrometry work of collaborator Donald F. Hunt, Ph.D., of the University of Virginia, also figured prominently.

The researchers, who reported their work last June in Nature, named the complex a processome “because it’s essential for processing the RNA that becomes part of the ribosome,” said Baserga. The “SSU” in the name stands for small subunit, because the complex is required for processing small ribosomal subunits, but not large ones. Though they’re not sure exactly how it functions, Baserga and co-workers believe the SSU processome and its proteins help fold ribosomal RNA into the proper configuration. It’s already known that the RNA portion of the ribosome is “the business end” that facilitates the ribosome’s protein-producing work, so properly processed ribosomal RNA is essential to the ribosome’s function, noted Baserga. The SSU processome appears to be so critical to cell growth and health, in fact, that Baserga suspects that defective processomes may be at the root of some diseases for which the causes are not well understood.

Researchers also are interested in the basic science behind RNA processing in all types of cells, Baserga added. “There’s pretty good reason to think that RNA was the first molecule, and that DNA and all the other molecules came from RNA. So studying anything that affects RNA metabolism brings you closer to understanding basic cellular processes.”

Next, the research team wants to explore how the SSU processome assembles into the huge complex it eventually becomes. “We think there’s a definite order of assembly,” says Baserga. Working with helicases—enzymes that fold and unfold protein and RNA—one of her students hopes to “freeze” the process in mid-course, providing a snapshot of assembly in progress. Baserga’s group will continue to tease out the exact details of how the SSU processome coaxes ribosomal RNA into the proper form. “That,” she said, “is going to be the next 10 years or so of work.”

—Nancy Ross-Flanigan

Nature studies offer a new view of the immune response, from a dendritic perspective

When the body is under pathogenic attack, it is the long-armed dendritic cells in the skin that identify the foreign invaders and instruct the body’s killer cells to fight them. Understanding how these multitalented sentinels operate could be central to producing the next generation of vaccines to combat diseases such as cancer and HIV, according to immunologists at Yale and Harvard who published their findings last August in Nature.

Discovered in 1868 by the German scientist Paul Langerhans, dendritic cells became of interest to immunologists in the 1960s but have only recently revealed their operational secrets. It took a novel imaging approach to observe the role the cells play in precipitating the body’s immune response.

By tagging the relevant molecules with green fluorescent dye, the groups from Yale and Harvard used video imaging of cultured cells to chart the pathway of an antigen, a protein that stimulates an immune response. For the first time, scientists observed molecules moving in a live dendritic cell.

Dendritic cells reside in the skin, constantly feasting on the proteins that surround them. If a foreign antigen is present, it is consumed and transported to an acidic compartment deep within the dendritic cell called a lysosome. Aided by enzymes, the lysosome chops the proteins up into more manageable chunks called peptides. Meanwhile the dendritic cell travels to the killer T cells in the lymph, which ultimately deal with invaders.

But how do peptides get from the enclosed compartment, the lysosome, at the cell’s core—so impenetrable that Yale’s Ira Mellman, Ph.D., chair and professor of cell biology, calls it “Dante’s seventh level”—to its surface, where they can interact with T cells?

Under Mellman’s supervision, graduate student Amy Chow and associate research scientist Derek Toomre, Ph.D., watched the growth of long thin tubules that became the peptides’ escape routes. They begin emanating from the lysosome shortly after a foreign invader is detected, and finally fuse with the cell’s surface. Chow saw the green-glowing carrier molecules drag the freshly chopped peptides along the tubules.

At the cell’s surface, the carrier molecules display their cargo of peptides, flagging those that represent dangerous invaders differently from those that came from the body’s own harmless proteins. T cells respond by self-destructing if the peptide is benign and by propagating if they must unleash their arsenal upon it.

“Dendritic cells sit at a critical nexus, deciding whether to respond or ignore a protein. Most immunologists so far have been T-cell-centric, but T cells can’t do anything unless a dendritic cell instructs them,” said Mellman. “Dendritic cells are like cellular psychiatrists. They bring out the deep-seated problems, wait for them to come to the surface and then interpret them.”

Jacques Banchereau, Ph.D., director of the Baylor Institute for Immunology Research in Dallas, said the unprecedented inside view of how dendritic cells operate offered by the Nature studies could pave the way to a more rational approach to vaccine design.

—Celeste Biever

Et Cetera

A promising target

Yale researchers have shown that an artificial gene switch can induce the growth of new blood vessels in a mouse model, a new approach to gene therapy that has implications for heart disease and cancer. The technique uses an engineered transcription factor to switch on inactive genes that are already present in the mouse and has the potential to spur the growth of new vessels in tissue that has a diminished blood supply. The principle could also be extended to control overactive genes that need switching off, such as those for cancerous tumor growth.

In previous gene therapy trials, patients have been injected with the genes that encode the growth factors that drive angiogenesis, the process of vessel formation. But Yale’s Frank J. Giordano, M.D., and colleagues at Sangamo BioSciences used a viral vector to deliver the genes that code for the switch, a zinc finger protein transcription factor dubbed Vegfa-ZFP. The mouse then produced the transcription factor, which switched on the growth genes. Healthy blood vessels formed and wound healing was augmented, according to the paper, published online in Nature Medicine on November 4.

—Celeste Biever

From the mouths of ticks

An anti-coagulant protein in the saliva of the deer tick allows it to suck blood from a single wound for days, according to Yale scientists. The identification of the protein, called Salp14, could lead to therapies for clotting disorders or vaccines against tick-borne diseases.

Mosquitoes and tsetse flies can feed for only a few seconds before clots form. Exactly how ticks bypass this natural defense had been a mystery. “Tick saliva has an array of potent pharmacologic functions,” said Erol Fikrig, M.D., principal investigator for the study, published in the December issue of Insect Molecular Biology. Fikrig said Salp14 blocks the actions of prothrombin, a key participant in the cascade of reactions that lead to blood clots. “If we study it in more detail, it could be used to combat any disease where clot formation is a problem,” said Fikrig. “But this is just the tip of the iceberg.”

—Celeste Biever


			Originally published in Yale Medicine, Winter 2003. Copyright © 2003 Yale University School of Medicine. All rights reserved.