捕捉神经干细胞分化之重要调控蛋白的3D影像

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捕捉神经干细胞分化之重要调控蛋白的3D影像 Salk研究所与加州大学圣地亚哥分校的研究人员合作，利用高分辨率的3D影像，捕捉神经系统发育过程中重要的蛋白质开关Scp1之作用。他们的研究结果发表于12月8日的MolecularCell中，为Scp1之小分子抑制剂的设计提供了一个模板。Scp1是调控神经细胞前体发育成为成熟神经元的重要开关。开启及抑制这个开关会影响神经细胞的分化时间。分子开关控制着胚胎干细胞分化成不同的组织类型。
Salk研究所与加州大学圣地亚哥分校的研究人员合作，利用高分辨率的3D影像，捕捉神经系统发育过程中重要的蛋白质开关Scp1之作用。他们的研究结果发表于12月8 日的Molecular Cell中，为Scp1之小分子抑制剂的设计提供了一个模板。  

    Scp1 是调控神经细胞前体发育成为成熟神经元的重要开关。开启及抑制这个开关会影响神经细胞的分化时间。  

    分子开关控制着胚胎干细胞分化成不同的组织类型。因此操控不同的开关将可以让科学家转换胚胎干细胞成为特殊的细胞类型。  

    研究人员于去年时发现，Scp1可以静默非神经细胞的神经元特殊基因。如果能利用特异性抑制剂，将有助于研究人员观察胎儿神经系统的发展，也可以让医学界将胚胎干细胞提升为具有特殊功能的神经细胞，而用于治疗上。  

    Scp1 属于一组名为小型羧端磷酸酶(SCPs)的蛋白质，这种蛋白质几乎在身体的所有组织中表现。当Scp1活化时，可以防止酵素RNA聚合酶 II在神经细胞基因不该表现的区域如皮肤、肌肉和肝脏组织中，读取和启动这些神经细胞基因。在神经系统中，Scp1被关闭，使RNA聚合酶 II可以有效地转录神经细胞的基因信息，并且使神经系统的干细胞成熟为特殊的神经元。  

    激酶可以为RNA聚合酶 II加上一个小的磷酸盐基团，使RNA聚合酶 II继续转录基因，而Scp1则会移除小的磷酸盐基团，使RNA聚合酶 II停工。在这项新研究中，研究人员分析了Scp1和RNA聚合酶 II，并且获得了Scp1如何附着于RNA聚合酶 II尾端重复的七个胺基酸残基的3D影像。  

    研究人员也发现到，这七个胺基酸中只有三个胺基酸，与Scp1去除与RNA聚合酶II结合的磷酸盐之能力有关。

英文原文：

Detailed 3-D image catches a key regulator of neural stem cell differentiation in action

La Jolla, CA —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.