This technique is groundbreaking in Brazil and permitted the discovery of how molecules are organized within the cell; the target was glutaminase, which is essential in the energy production cycle in the mitochondria as well as biomass production, and is related to some types of cancer
The discovery that glutaminase is arranged in a quinary structure as helicoid filaments which in turn form long bundles within the mitochondria could shift the direction of various studies. This is the first time that the structure of a metabolic enzyme has been revealed within the cellular environment.
Staff at the Brazilian Center for Research in Energy and Materials (CNPEM) were responsible for an article published in the high-impact journal Nature Structural & Molecular Biology, in collaboration with researchers from the Department of Physics at USP–São Carlos, UNICAMP, and the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany. The study utilized cutting-edge techniques including cryoelectron tomography (cryo-ET) and also demonstrated some of the functions of the filaments of these proteins, such as making the mitochondria longer and more resistant to degradation that results from the cellular recycling process.
The researcher who coordinated the project, Sandra Dias, and the lead author of the article, Douglas Adamoski, are both researchers at CNPEM’s Brazilian Biosciences National Laboratory (LNBio), and explain that revealing molecular structures in situ (within the cellular environment) will make it easier to understand how proteins interact with each other inside the cell and when and in which physiological situations these interactions occur.
The study also contributes to the understanding of disease mechanisms and creates possibilities for developing pharmaceuticals in a more precise and contextualized manner. “The purpose of this type of technique is to close the gap between structural biology and cellular biology, showing the structure of the molecules where they actually occur in biological settings,” says Adamoski.
Prostate cancer tumor cells: cell mitochondria (red), filament bundles of the glutaminase enzyme (green), cell cytoplasm (cyan), and nucleic DNA (dark blue). Yellow indicates overlaps between green and red, where the glutaminase filaments are located (Outreach/CNPEM)
The mitochondria are responsible for producing the energy used for all the functions that take place within animal organisms: each muscle movement, blink of an eye, and thought produced by the nervous system, and even to maintain the cells themselves. Various molecules are “burned” to unleash the cycle of chemical reactions within the mitochondria, with glucose and glutamine acting as the main “fuels,” especially in cells that divide constantly.
Glutaminase, which has been studied for many years at CNPEM, is the enzyme that breaks down glutamine, an amino acid, into glutamate, another amino acid. In previous findings, the group showed how this mechanism is dysregulated in various types of tumor cells. The new research casts light on what may be a new area of investigation on the physiology of this fundamental organelle, and its potential impacts on neurological and cancerous diseases.
EIllustration showing the function of glutamine and glucose molecules in energy production within the mitochondria. The glutaminase enzyme acts in the energy cycle and also generates various other molecules that comprise the basic building blocks of cells: proteins, nucleic acids, and lipids. In cancerous cells, glucose processing is dysregulated, and this is also often the case for glutamine
According to Dias, other groups of researchers are currently also working to resolve the molecular structures of proteins within the cell. “Previously, in order to define the structure of a protein we often had to get the gene sequence, put it in a bacterium, produce this protein in large quantities, and study it separately from its system. We did this with glutaminase some time ago, and during the process, we observed that it formed these strange, long structures, these polymers, which was unexpected,” she notes, explaining that the use of the advanced technology was essential to be able to prove to the scientific community that the filaments are also within the mitochondria, inside the cell, and have a function that extends beyond what is known for this enzyme.
Filaments were previously known
An interesting fact noted by Dias and Adamoski is that glutaminase filaments had already been observed in vitro in 1970 by Bjørn Olsen and Trond Eskeland, during electron microscopy involving purified pig liver protein. Although Olsen and Eskeland described them, they had no idea how they were structurally organized or what molecules were involved in their formation, much less their physiological significance.
Decades later and completely independently, Sandra Dias also observed the filaments with an electron microscope, and like Olsen & Eskeland, when isolating the protein in vitro. But only today, with the study published in Nature and cryo-ET technology, are the functions of this structure starting to be revealed.
Several techniques were needed to obtain these results. The researchers used cryogenic electron microscopy (cryo-EM) to study the purified protein in vitro, using the state-of-the-art Titan Krios microscope. “It is important to note that Krios is a unique electron microscope, and that CNPEM makes it available for the entire academic community to use,” adds Dias. The Brazilian Nanotechnology National Laboratory (LNNano), which operates the facility, is a pioneer in revealing protein structures via cryo-EM in Latin America.
Another stage took place in Germany at the European Molecular Biology Laboratory for additional procedures to define the structure, which is absolutely unprecedented in the country. This involved creating narrow cell slices which are essential to determine the structures within the cells. The technique, known as in situ cryo-ET, is at its zenith in global academic research and only recently has been acquired by some Brazilian institutions. “We already have broad know-how for the analyses. Right now, we are implementing the cryo-ET technique to study the ultrastructure of cells, and we soon hope to also have the necessary infrastructure for in situ cryo-ET analyses,” states Rodrigo Portugal, coordinator of the Electron Cryomicroscopy Laboratory at LNNano.
These multiple strategies allowed the researchers to determine that glutaminase is also present in the form of filaments in its natural environment, in other words, within the mitochondria in cell cultures. The advantage emerges because substances act differently within a test tube than under normal physiological conditions within the cell. This difference impacts the quality of deductions and scientific conclusions, as seen in many studies in the area of health that obtain excellent results in the laboratory but frustrating clinical outcomes. The more closely experiments resemble the holistic function of an organism, the better.
The study also concluded that the glutaminase filaments may have been formed in different types of cells, but only under specific conditions, and that this formation capacity is maintained in cancer, for example. The researchers noted that these structures change the morphology of the mitochondria, making them longer, as well as some functions, making these organelles more likely to survive and resist the “cleaning” that cells conduct within the mitochondria from time to time.
Gaining an enzymatic function in the mitochondria due to a higher-order structural organization beyond the enzymatic role was not expected in the scientific literature. Adamoski emphasizes the fact that much still remains to be discovered. “Now it is a fact that the filaments change the morphology of the mitochondria. But whether this is something positive or harmful for the organism, and in which organs they are found, are some of the group’s future research questions. It is impressive because even today we do not entirely understand how the mitochondria, which are so important to the function of all our organs, function within the cell,” he notes.
The research group also hypothesizes that a series of other enzymes may also have a similar capacity, which is being investigated as part of a thematic project funded by FAPESP and the Ministry of Science, Technology and Innovation (MCTI), to which CNPEM is linked.
About CNPEM
A sophisticated and effervescent environment for research and development, unique in Brazil and present in few scientific centers in the world, the Brazilian Center for Research in Energy and Materials (CNPEM) is a private non-profit organization, under the supervision of the Ministry of Science, Technology and Innovation (MCTI). The Center operates four National Laboratories and is the birthplace of the most complex project in Brazilian science – Sirius – one of the most advanced synchrotron light sources in the world. CNPEM brings together highly specialized multi-thematic teams, globally competitive laboratory infrastructures open to the scientific community, strategic lines of investigation, innovative projects in partnership with the productive sector and training of researchers and students. The Center is an environment driven by the search for solutions with impact in the areas of Health, Energy and Renewable Materials, Agro-environment, and Quantum Technologies. As of 2022, with the support of the Ministry of Education (MEC), CNPEM expanded its activities with the opening of the Ilum School of Science. The interdisciplinary higher course in Science, Technology and Innovation adopts innovative proposals with the aim of offering excellent, free, full-time training with immersion in the CNPEM research environment. Through the CNPEM 360 Platform, it is possible to explore, in a virtual and immersive way, the main environments and activities of the Center, visit: https://pages.cnpem.br/cnpem360/.
