Tag Archives: #yeast

Sloan Kettering Institute Scientists Solve a 100-year-old Mystery About Cancer (Medicine)

A long-standing mystery is why fast-growing cells, like cancer cells and immune cells, rely on a seemingly inefficient form of metabolizing glucose to power their activities. In a new study, scientists at the Sloan Kettering Institute offer a compelling solution.

The year 2021 marks the 100th anniversary of a fundamental discovery that’s taught in every biochemistry textbook. In 1921, German physician Otto Warburg observed that cancer cells harvest energy from glucose sugar in a strangely inefficient manner: rather than “burn” it using oxygen, cancer cells do what yeast do — they ferment it. This oxygen-independent process occurs quickly, but leaves much of the energy in glucose untapped.

MSK immunologist Ming Li

Various hypotheses to explain the Warburg effect have been proposed over the years, including the idea that cancer cells have defective mitochondria — their “energy factories” — and therefore cannot perform the controlled burning of glucose. But none of these explanations has withstood the test of time. (Cancer cells’ mitochondria work just fine, for example.)

Now a research team at the Sloan Kettering Institute led by immunologist Ming Li offers a new answer, based on a hefty set of genetic and biochemical experiments and published January 21 in the journal Science.

It comes down to a previously unappreciated link between Warburg metabolism and the activity of a powerhouse enzyme in the cell called PI3 kinase.

“PI3 kinase is a key signaling molecule that functions almost like a commander-in-chief of cell metabolism,” Dr. Li says. “Most of the energy-costly cellular events in cells, including cell division, occur only when PI3 kinase gives the cue.”

As cells shift to Warburg metabolism, the activity of PI3 kinase is increased, and in turn, the cells’ commitment to divide is strengthened. It’s a bit like giving the commander-in-chief a megaphone.

The findings revise the commonly accepted view among biochemists that sees metabolism as secondary to cell signaling. They also suggest that targeting metabolism could be an effective way to thwart cancer growth.

Challenging the Textbook View

Dr. Li and his team, including graduate student Ke Xu, studied Warburg metabolism in immune cells, which also rely on this seemingly inefficient form of metabolism. When immune cells are alerted to the presence of an infection, a certain type called T cells shift from the typical oxygen-burning form of metabolism to Warburg metabolism as they grow in number and ramp up infection-fighting machinery.

The key switch that controls this shift is an enzyme called lactate dehydrogenase A (LDHA), which is made in response to PI3 kinase signaling. As a result of this switch, glucose remains only partially broken down and the cell’s energy currency, called ATP, is quickly generated in the cell’s cytosol. (In contrast, when cells use oxygen to burn glucose, the partially broken down molecules travel to the mitochondria and are further broken down there to make ATP on a delay.)

Dr. Li and his team found that in mice, T cells lacking LDHA could not sustain their PI3 kinase activity, and as a result could not effectively fight infections. To Dr. Li and his team, this implied that this metabolic enzyme was controlling a cell’s signaling activity.

“The field has worked under the assumption that metabolism is secondary to growth factor signaling,” Dr. Li says. “In other words, growth factor signaling drives metabolism, and metabolism supports cell growth and proliferation. So the observation that a metabolic enzyme like LDHA could impact growth factor signaling through PI3 kinase really caught our attention.”

Like other kinases, PI3 kinase relies on ATP to do its work. Since ATP is the net product of Warburg metabolism, a positive feedback loop is set up between Warburg metabolism and PI3 kinase activity, securing PI3 kinase’s continued activity — and therefore cell division.

As for why activated immune cells would preferentially resort to this form of metabolism, Dr. Li suspects it has to do with the cells’ need to produce ATP quickly to ramp up their cell division and infection-fighting machinery. The positive feedback loop ensures that once this program is engaged, it will be sustained until the infection is eradicated.

The Cancer Connection

Though the team made their discoveries in immune cells, there are clear parallels to cancer.

“PI3 kinase is a very, very critical kinase in the context of cancer,” Dr. Li says. “It’s what sends the growth signal for cancer cells to divide, and is one of the most overly active signaling pathways in cancer.”

As with immune cells, cancer cells may employ Warburg metabolism as a way to sustain the activity of this signaling pathway and therefore ensure their continued growth and division. The results raise the intriguing possibility that doctors could curb cancer growth by blocking the activity of LDHA — the Warburg “switch.”

This study received financial support from the National Institutes of Health (grant R01 AI 102888), the Howard Hughes Medical Institute, and the Memorial Sloan Kettering Cancer Center Support Grant/Core Grant P30 CA08748. The study authors declare no competing interests.

Reference: Ke Xu, Na Yin, Min Peng, Efstathios G. Stamatiades, Amy Shyu, Peng Li, Xian Zhang, Mytrang H. Do, Zhaoquan Wang, Kristelle J. Capistrano, Chun Chou, Andrew G. Levine, Alexander Y. Rudensky, Ming O. Li, “Glycolysis fuels phosphoinositide 3-kinase signaling to bolster T cell immunity”, Science  22 Jan 2021: Vol. 371, Issue 6527, pp. 405-410 DOI: 10.1126/science.abb2683 https://science.sciencemag.org/content/371/6527/405

Provided by MSKCC

Researchers Develop New Method to Revamp and Minimize Yeast Genome (Biology)

Researchers from the Shenzhen Institutes of Advanced Technology (SIAT) of the Chinese Academy of Sciences developed a method termed SCRaMbLE-based genome compaction (SGC) to revamp and minimize the yeast genome.

Schematic illustration of the SCRaMbLE-based genome compaction (SGC) method. (Image by SIAT)  

They showed that a synthetic chromosome arm (synXIIL) could be efficiently reduced by this method. Their study was published in Genome Biology on Jan 4. 

Redundancy is a common feature of genomes, presumably to ensure robust growth under different and changing conditions. Genome compaction removes sequences nonessential for given conditions. 

The synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) system is a unique feature implanted in the synthetic yeast genome (Sc2.0), which has been proposed as an effective tool for genome minimization. 

As the Sc2.0 project is about to be completed, the researchers have begun to explore the application of the SCRaMbLE system in genome compaction. 

“With SGC, all the strains we identified harbor a reduced synthetic chromosome,” said Associate Professor LUO Zhouqing from SIAT, first author of the study. “The nonessential genes located approximate to the essential one could not be removed by SGC directly, if there is no loxP site in between.”

The researchers constructed an episomal essential gene array and introduced it prior to activate SCRaMbLE, which enhanced the deletion ability of SGC, not only by removing nonessential genes located close to the essential ones, but also by deleting more chromosomal sequences in a single SGC process. 

Further compaction was achieved through iterative SGC, revealing that at least 39 out of 65 nonessential genes in synXIIL can be removed collectively without affecting cell viability at 30°C in rich medium. 

“We developed iterative SGC with the aid of eArray as a generic yet effective tool to compact the synthetic yeast genome,” said Dr. DAI Junbiao from SIAT, the co-corresponding author of the study. 

Reference: Luo, Z., Yu, K., Xie, S. et al. Compacting a synthetic yeast chromosome arm. Genome Biol 22, 5 (2021). https://genomebiology.biomedcentral.com/articles/10.1186/s13059-020-02232-8 https://doi.org/10.1186/s13059-020-02232-8

Provided by Chinese Academy of Sciences

Scientists Resolve Solution Structure of Yeast Tim23 Channel in Complex with Peptide (Biology)

Recently, a research team led by Prof. WANG Junfeng and Dr. ZHOU Shu from the High Magnetic Field Laboratory of the Hefei Institutes of Physical Science (HFIPS) resolved the three-dimensional structure of voltage-gated Tim23 channel.

Cartoon representation of the Tim23-pCoxIV structure (a-g); electrophysiological analysis of the Tim23 wild type and Tim23 sequence variant channels in the absence or presence of pCoxIV (h-j); the proposed mechanism whereby the Tim23 channel transports precursor proteins into the mitochondrial matrix (l-n). (Image by ZHOU Shu)

Using the method of solution-state nuclear magnetic resonance (NMR), the team determined the structure in complex with a mitochondrial presequence peptide. “Tim23 channel structure was never reported before. This is the first time,” said Dr. ZHOU.

Most mitochondrial proteins are synthesized in the cytosol and transported into various mitochondrial subcompartments. Tim23 protein, the key component of the TIM23 complex forming a channel in the mitochondrial inner membrane, is believed to recognize and translocate precursor proteins into the mitochondrial matrix or to release them into the mitochondrial inner membrane. Therefore, in recent years, lots of research have been conducted on the function of Tim23 channel, but structure of it was still unknown.

In this research, scientists found presequence binds to a coiled-coil motif in the mitochondrial intermembrane space via electrostatic forces. A short helix containing salt bridges was used as a voltage sensor and aromatic rings at the membrane interface served as a lock to operate channel gating, which was validated by electrophysiological analysis on the structure-based sequence variants.

The study offers mechanistic insight that how a voltage-sensitive channel transfers precursor protein into the mitochondrial matrix.

This study was supported by grants from the Ministry of Science and Technology of China and the National Natural Science Foundation of China, and it was funded by the Horizon 2020 program of the European Commission.

The NMR experiment was supported by the High Magnetic Field Laboratory of Chinese Academy of Sciences and the Biomolecular Magnetic Resonance Center at University of Frankfurt.

Reference: Zhou, S., Ruan, M., Li, Y. et al. Solution structure of the voltage-gated Tim23 channel in complex with a mitochondrial presequence peptide. Cell Res (2020). https://www.nature.com/articles/s41422-020-00452-y https://doi.org/10.1038/s41422-020-00452-y

Provided by Chinese Academy of Sciences

Ribosome Assembly – The Final Trimming Step (Biology)

Ribosomes synthesize all the proteins in cells. Studies mainly done on yeast have revealed much about how ribosomes are put together, but an Ludwig-Maximilians-Universitaet (LMU) in Munich team now reports that ribosome assembly in human cells requires factors that have no counterparts in simpler model organisms.

From particle to fine structure: A cryo-electron micrograph of the 40S ribosomal subunit and the derived structural model. Source: M. Ameismeier

In every cell, hundreds of thousands of intricate molecular machines called ribosomes fabricate new proteins, extending each growing chain at a rate of a few amino acids per second. Not surprisingly therefore, the construction of these vital protein factories is itself a highly complex operation, in which more than 200 assembly factors are transiently involved. Mature ribosomes are made up of approximately 80 proteins and four ribosomal RNAs. But how these constituents are assembled in the correct order to yield a functional ribosome is still not fully understood. Moreover, most of our knowledge of the process comes from studies carried out on model organisms like bacteria and yeast, and may not necessarily be applicable to the cells of higher organisms. Researchers led by Professor Roland Beckmann (Gene Center, LMU Munich) have now uncovered new details of the crucial steps in the maturation of ribosomes in human cells.

Active ribosomes consist of two separately assembled particles, which differ in size and interact with each other only after the first steps in protein synthesis have taken place on the smaller of the two (in human cells, the ’40S subunit’). Beckmann’s team has used cryo-electron microscopy to determine the structures of several precursors of the 40S subunit isolated from human cells and follow the course of its maturation. “This study follows on from an earlier project, in which we obtained initial insights into the process,” says Michael Ameismeier. He is a doctoral student in Beckmann’s team and lead author of the new report, which is concerned with the final steps in the assembly of the small subunit.

At this late stage in the process, one end of the ribosomal RNA associated with the small particle protrudes from the body of the immature subunit. The last step in the maturation of the 18S subunit consists in the removal of this now superfluous segment. To ensure that this reaction does not occur prematurely, the enzyme responsible – NOB1 – is maintained in an inactive state until it is required. The new study shows that the activation of NOB1 is preceded by a conformational change that results in the detachment of a binding partner from the enzyme. This in turn triggers a structural rearrangement in NOB1 itself, which enables the enzyme to snip off the protruding rRNA segment. “The activation of NOB1 is coordinated by another enzyme,” Ameismeier explains. Together with a protein we have discovered – which is not found in yeast – the latter enzyme inserts like a wedge into the maturing 40S subunit, and this facilitates the decisive conformational change in NOB1.”

The authors have also shown that yet another protein not found in yeast plays an (as yet) enigmatic role in the maturation of the 40S subunit. “This demonstrates the importance of considering the human system separately from other experimental models,” says Beckmann. Use of the evolutionarily simpler yeast system is sufficient for a basic understanding of the process. But certain pathological syndromes have been linked to errors in ribosomal biogenesis in humans, which provides an obvious rationale for the study of ribosomal assembly in human cell systems.

References: Ameismeier, M., Zemp, I., van den Heuvel, J. et al. Structural basis for the final steps of human 40S ribosome maturation. Nature (2020). https://www.nature.com/articles/s41586-020-2929-x https://doi.org/10.1038/s41586-020-2929-x

Provided by Ludwig-Maximilians-Universitat-Munchen

Genetic eraser: Newly Developed Technology Precisely and Rapidly Degrades Targeted Proteins (Biology)

Researchers can now more accurately and precisely target specific proteins in yeast, mammalian cells and mice to study how knocking down specific protein traits can influence physical manifestation in a cell or organism.

The Japan-based team published their results on November 11th in Nature Communications.

A degron-fused protein of interest is recognized for rapid degradation by a TIR1 mutant only in the presence of an auxin analog, which initiates the degradation process. The target protein is typically depleted in less than a few hours after treatment. ©Masato Kanemaki

“Conditional gene knockout and small interfering RNA (siRNA), which is used to silence proteins without knocking them out completely, has been employed in many studies,” said Masato T. Kanemaki, professor at the National Institute of Genetics in the Research Organization of Information and Systems (ROIS). “However, these technologies are not ideal for studying highly dynamic processes, such as cell cycle, differentiation, or neural activity, because of the slow rate of depletion of the protein of interest.”

Kanemaki and his team had previously developed an approach called the AID system, which uses a small protein tag, known as a degron, fused to proteins to induce degradation. To initiate the degradation process, the researchers administered auxin, a plant hormone that helps regulate plant growth. In previous conditional gene knockout and siRNA studies, according to Kanemaki, it typically takes two or three days for a target protein to deplete. The AID system allows for a general, more efficient approach by which the target protein can be depleted in less than a few hours.

“The original AID system has two major drawbacks: leaky degradation and the requirement for a high dose of auxin,” Kanemaki said. “These negative features make it difficult to control precisely the expression level of a protein of interest in living cells and apply this method to mice.”

The ability to knockout genes in mice is a critical step in genetic research and therapeutics. According to Kanemaki, an approach may work well in cultured cells, but it must work in a whole model system, such as a mouse. The “leaky degradation” of the AID system meant that a targeted protein would only degraded weakly without auxin, but the level of auxin required to induce full degradation appeared to have long-term negative effects on cell growth.

“In this paper, we describe the AID2 system, which overcomes all the drawbacks of the original AID system,” Kanemaki said, noting that they did not detect leaky degradation with the system, the degradation was quicker, and the required dose of auxin was much lower.

To establish the AID2 system, the researchers employed what is known as a “bump-and-hole” strategy to create an empty space in a mutant version of a plant protein (called TIR1) that recognizes and induces the degradation of degron-fused proteins. An auxin analog can bind directly to the TIR1 mutant and initiate the degradation process. Since the approach is very efficient, less auxin analog is needed. The researchers found that depletion could be induced at a concentration about 670 times lower than in the original system.

“With the AID 2 system, it is possible to rapidly deplete a protein of interest in cultured cells and mice,” Kanemaki said. “Next, we plan to use the system to find something new in chromosome biology, and to apply the AID2 system to other model organisms.”

Provided by Research Organization of Information and Systems