Researchers at the Salk Institute for Biological Studies in collaboration with scientists at the University of California, San Diego (UCSD) took a high resolution "action shot" of a protein switch that plays a crucial role in the development of the nervous system. Their findings, published in the Dec. 8 issue of the journal Molecular Cell, provide a template for the design of small molecule inhibitors to control that switch, a protein called Scp1, at will.
"Scp1 is an important brake that regulates the transition from neuronal precursor to mature neuron," explains senior author Joseph Noel, Ph.D, a Howard Hughes Medical Institute investigator at Salk. "Loosening the brake with an inhibitor would allow us to influence the timing of neuronal differentiation," he adds.
A finely tuned network of molecular "on" and "off" switches orchestrates the differentiation of embryonic stem cells into different tissue types. Being able to manipulate individual switches would allow scientists to nudge embryonic stem cells into becoming specific cell types, a plus for both basic research and potential therapies.
"At the moment, the differentiation of stem cells into neurons in a Petri dish is a little bit like a black box and not very efficient," explains co-author Samuel Pfaff, Ph.D., a professor in the Salk's Gene Expression Laboratory, who together with co-author Gordon Gill, Ph.D., of the Departments of Medicine and Cellular and Molecular Medicine at UCSD, found that Scp1 silences neuron-specific genes in non-neuronal cells last year. "Having a specific inhibitor would give us a lot of insight into the development of the fetal nervous system and would allow us to chemically push embryonic stem cells to acquire a neuronal fate in an informed way," adds Pfaff.
Scp1 belongs to group of proteins called small carboxyl-terminal phosphatases (SCPs) that are expressed in almost all tissues of the body. When active, Scp1 prevents the enzyme RNA polymerase II from reading and switching on neuronal genes in tissues where they shouldn't be expressed, such as skin, muscle and liver. In the nervous system Scp1 is switched off, enabling RNA polymerase II to efficiently transcribe information encoded by neuronal genes and driving the maturation of neural stem cells into specialized neurons.
"Scp1 is an interesting twist on how genes can be regulated during development," says Pfaff. "In the past there has been a lot of emphasis on chromatin modifications and physical access to genes, but Scp1 regulates the activity of the enzyme that transcribes genes directly," he adds.
Scp1 is not the only protein that directly influences the activity of RNA polymerase II. A constantly fluctuating brigade of enzymatic foot soldiers regulates RNA polymerase II's activity by chemically modifying the long cord-like tail that hangs from its globular structure, like a chain on a light fixture.
Enzymes called kinases turn the "light" on by adding small phosphate chemical groups - giving RNA polymerase the go-ahead to transcribe genes - while removal of those phosphates by phosphatases like Scp1 turns out the light, effectively stopping RNA polymerase in its tracks.
Noel and postdoctoral fellow Yan Zhang, Ph.D, analyzed the crystal structure of Scp1 and RNA polymerase together and obtained a 3-dimensional image showing how Scp1 hangs onto the seven amino acid residues reiterated in the polymerase tail. "We captured Scp1 bound to a single seven amino-acid long repeat containing specific phosphates," explains Zhang, the paper's first author. "It turns out that only three amino acids are important for Scp1's ability to know how to remove phosphates from RNA polymerase."
She adds that knowing how enzymes like Scp1 precisely recognize that seven amino acid stretch is exactly the kind of "unambiguous information relevant for the design of a chemical inhibitor by a process known as structure-based drug design."
Capitalizing on information gleaned from their structural studies, Noel's lab has already started that structure-based program. "We have designed the first generation of inhibitors and now it is a matter of chemically synthesizing them, testing them in test tubes and cells, and imaging them bound to Scp1 in 3D," says Noel. This will set the stage for the rational fine-tuning of their efficacy using an established process Noel likens to molecular dentistry, because his group is striving to shape inhibitory molecules to fit the groove in Scp1 much the same way that a dentist molds a filling to fit the cavity in a patient's tooth.
Researchers who contributed to the work include Nicolas Genoud, Ph.D., in the Gene Expression Laboratory at the Salk Institute, Jack E. Dixon, Ph.D., and Youngjun Kim, Ph.D., in the Departments of Pharmacology and Cellular and Molecular Medicine at UCSD, and Jianmin Gao, Ph.D., and Jeffery W. Kelly, Ph.D., at the Skaggs Institute for Chemical Biology of The Scripps Research Institute, La Jolla.
The Salk Institute for Biological Studies in La Jolla, California, is an independent nonprofit organization dedicated to fundamental discoveries in the life sciences, the improvement of human health and the training of future generations of researchers. Jonas Salk, M.D., whose polio vaccine all but eradicated the crippling disease poliomyelitis in 1955, opened the Institute in 1965 with a gift of land from the City of San Diego and the financial support of the March of Dimes.
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