Molecular cages provide cryo-EM researchers with new insights into cancer proteins

Cryogenic electron microscope reconstruction of KIX (red) sandwiched between MBP outer shell (purple) and apoferritin inner shell (blue). Sandwiching technology has helped researchers get the best-ever look of KIX, a potential target for treating acute myeloid leukemia. Credits: Greg Stewart / SLAC National Accelerator Laboratory

The American Cancer Institute estimates that acute myeloid leukemia (AML) will affect more than 20,000 Americans this year, killing more than 11,000 of them. Many people who are treated with intensive chemotherapy or stem cell transplants have side effects such as infections, hair loss, and vomiting, in addition to long-term complications.

To remedy that situation, Professor Kathleen Sakamoto of Stanford University School of Medicine has been working on developing treatments for AML and other blood disorders. However, as her team is pursuing new ideas for how to treat AML, between the two techniques used to understand the structure and function of proteins (X-ray crystallography and cryo-electron microscopy). Their search has been hampered by the subtle gaps in. (Cryo EM) The other.

Researchers at Stanford University’s School of Medicine and the SLAC National Accelerator Laboratory at the Ministry of Energy have now found a way to fill the gap with a type of molecular cage to stabilize certain medium-sized proteins. Time with cryo-EM that can reveal near atomic level details. Kaiming Zhang, a postdoctoral researcher at Stanford University, and Naoki Horikoshi, a visiting assistant professor, were the first authors at the time of the study, and his colleagues announced the results on February 7. ACS Central Science..

At issue, according to SLAC and Professor Soichi Wakatsuki of Stanford University, is KIX, which is part of the CREB-binding protein (CBP) used by AML cancer cells to transcribe genes important for growth and survival. is. With a better understanding of its structure, researchers can design drugs that block KIX and prevent cancer cell replication. However, efforts to study proteins using X-ray crystallography have not been successful. According to crystallographic standards, the relatively large size of the molecule makes it difficult to crystallize, and even if it does, the details of the process make it difficult. Analyze the part of KIX that the drug designer wants to target.

At the same time, KIX is a bit too small on its own to be effectively studied with CryoEM. Wakatsuki explains that in order to get a good image of a protein in a cryo-EM, it is necessary to find many copies of the protein in the electron micrograph to understand how they are oriented. increase. Such as that.Just find and arrange many images of protein The cryo-EM method can produce high resolution structures. The relatively small size of KIX (according to the cryo-EM standard) makes it difficult. Another option, Nuclear magnetic resonanceHas been used to determine the structure of KIX when bound to other natural molecules, but this method requires extensive preparation and analysis and is ideal for rapid molecular structure determination. It is not targeted and therefore not ideal for studying the potential impact of KIX. -Inhibitor.

The solution came when Wakatsuki and Zhang, who worked at SLAC and Professor Wouchiu’s lab at Stanford University, had lunch in Tokyo and were working on another project. Outer molecular cage. This “double shell” is much larger than the individual KIX molecules, making it easier to detect and orient in cryo-EM images and to obtain high-resolution images of the KIX molecules themselves.

In addition to confirming the structure of KIX, Wakatsuki collaborated with Sakamoto and Ron Dror, a professor of computer science at Stanford University, to add other molecules to the mixture and KIX. He said he was able to see if it could bind to and inhibit the function of. Already, the team reports that they were able to strengthen the bond by about 200 times. This may help scientists develop low-dose, effective drugs. “The name of the game is to find compounds that inhibit KIX at low concentrations,” Wakatsuki said. “This isn’t enough yet, but we’ve made progress.”

Team results suggest that this method may also be useful for other medium-sized proteins (possibly including some viral proteins) that are difficult to study with either cryo-electron microscopy or X-ray crystallography. It also suggests that. “We are making progress to expand the scope of this approach,” Wakatsuki said.

Atom or noise?New methods help cryo-EM researchers see the difference

For more information:
Kaiming Zhang et al, Cryo-EM, protein engineering, and simulation will enable the development of peptide treatments for acute myeloid leukemia. ACS Central Science (2022). DOI: 10.1021 /acscentsci.1c01090

Quote: The molecular cage was obtained by cryo-EM researchers from https: // on February 11, 2022 (cancer protein). Provides new insights on (February 11, 2022) .html

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Molecular cages provide cryo-EM researchers with new insights into cancer proteins

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