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How cells recover from stress. Cancer cells need cysteine to proliferate. Method to make small membrane proteins. Read about papers on these topics recently published in the Journal of Biological Chemistry.

How cells recover from stress

Ribosome ubiquitination during oxidative stress pauses translation and helps cells regulate the proteome, but translation must resume when the stress dissipates. The yeast deubiquitinating enzyme Ubp2 removes polyubiquitin chains. Scientists want to elucidate details of Ubp2’s function and regulation to gain a better understanding of how this enzyme may reverse aspects of the cellular response to stress.

Illustration of ubiquitin chains attached to a protein. Deubiquitinating enzymes, like Ubp2, remove these chains to regulate proteostasis.

Clara Santos and a team of researchers, led by Gustavo Silva, from Duke University recently published their in the Journal of Biological Chemistry outlining factors that regulate Ubp2 function. They used enzyme activity assays to show that Ubp2 activity was inhibited by hydrogen peroxide, a type of reactive oxygen species that can accumulate during cellular oxidative stress. Importantly, they found that reducing agents can reverse Ubp2 inhibition, indicating that changing redox conditions during cellular oxidative stress recovery may contribute to Ubp2’s regulation.

The authors also investigated how domains of Ubp2 regulate its activity, focusing on the Ubp2 catalytic domain and three repeated domains. They tested truncated Ubp2 variants’ ability to reverse polyubiquitin chain formation and observed less polyubiquitin chain accumulation in assays with the Ubp2 variant missing the first repeated domain. They speculated that the first repeated domain could negatively regulate Ubp2’s overall function.

Finally, the researchers used a green fluorescent protein, or GFP, reporter assay in yeast cells to assess Ubp2’s role in translation restart after a period of oxidative stress. They found that GFP only accumulated in the normal strain and not in a strain lacking Ubp2, suggesting that Ubp2 is important for translation to resume during the recovery process. A proteomic analysis of translation restoration in these two strains indicated that loss of Ubp2 may affect specific cellular pathways related to cell division, and future experiments will elucidate more details of Ubp2’s exact role in these processes.

Cancer cells need cysteine to proliferate

Cancer cells ramp up the intake of key nutrients to sustain metabolic functions. Cysteine accumulation helps cancer cells produce glutathione and ward off oxidative stress. Scientists have also shown that some cancer cells dampen glutathione production even while increasing cysteine intake, prompting a search for other roles of cysteine in cancer cell growth and survival.

In a recent Journal of Biological Chemistry , Yumi Okano and Tomoaki Yamauchi from Kyushu University and a team of researchers in Japan determined that cysteine deprivation slows the growth of four cancer cell lines derived from mice. Limiting cysteine in their control group of nonmalignant mouse hepatocytes did not affect growth, which suggests that cysteine could play a unique role in cancer cell proliferation. The authors performed a flow cytometric analysis of hepatocarcinoma cells and determined that diminished cysteine leads to a buildup of cells in the resting G0/G1 phase of the cell cycle. Further analysis indicated that lower levels of cysteine correlated with decreased expression of D-type cyclins, proteins that regulate progression from the G0/G1 phase to the S phase.

The reliance on accumulated cysteine for cancer cell growth emphasizes alterations in these cells’ metabolism and pinpoints vulnerabilities; the authors suggest that targeting cysteine uptake pathways in combination with cell cycle inhibitors could form the basis of future therapeutics for cancer patients.

Method to make small membrane proteins

Recent advances in bioinformatics and ribosomal profiling have identified many small membrane proteins that may participate in cell signaling and protein complex regulation. However, producing these proteins in the lab is difficult and requires overcoming challenges of hydrophobicity and toxicity in overexpression hosts, such as E. coli. Shan Jiang from the Max Planck Institute for Terrestrial Microbiology and a team of researchers in Germany successfully produced small membrane proteins in the lab and published their in the Journal of Biological Chemistry. They combined a minimal cell-free system that provides translation machinery with lipid sponge droplets, which contain a glycolipid and nonionic detergents to form a large membrane surface for protein insertion.

The authors tested their method by producing the small E. coli membrane protein AcrZ, which regulates a drug efflux pump, and the human protein sarcolipin, which regulates adenosine triphosphate synthase activity in muscle cells, highlighting the versatility of the platform. The researchers showed that their purified AcrZ functions normally in a coimmunoprecipitation assay that identified known interactors and other membrane proteins, indicating that their system produces proteins compatible with downstream target discovery experiments.

The authors indicated that their method will improve investigations of small membrane proteins and enable the characterization of proteins with unknown functions. Further use of this system will help determine its compatibility with a variety of functional assays.