A collaborative team of scientists including biochemist Peter Chien at the University of Massachusetts Amherst has reconstructed how bacteria tightly control their growth and division, a process known as the cell cycle, by specifically destroying key proteins through regulated protein degradation.
Regulated protein degradation uses specific enzymes called energy dependent proteases to selective destroy certain targets. Because regulated protein degradation is critical for bacterial virulence and invasion, understanding how these proteases function should help to uncover pathways that can be targeted by new antibiotics.
All organisms use controlled degradation of specific proteins to alter cellular behavior in response to internal or external cues, says Chien, an assistant professor of biochemistry and molecular biology. And, a process that has to happen as reliably and stably as cell division also has to be flexible enough to allow the organism to grow and respond to its ever-changing environment. But little has been known about the molecular mechanics of how cells meet these challenges.
This work, done in collaboration with Kathleen Ryan and colleagues at the University of California, Berkeley, was supported by the NIH's National Institute for General Medical Sciences. Results appeared this week in an early online edition of Proceedings of the National Academy of Sciences.
Energy dependent proteases can be thought of as tiny molecular-level machines, says Chien. By selectively cutting and destroying key proteins at precise time points during cell division, they take charge of when, and at what rate, a cell grows and divides. They are found in all kingdoms of life, but are especially important in bacteria where they help cells overcome stressful conditions such as an attack by antibiotic treatment.
"When the environment becomes damaging, these proteases selectively target particular proteins to stop cell division so the bacteria can turn to focus instead on repair until the stress is over," Chien explains. "Understanding how bacteria use these machines at the cellular and molecular level could reveal avenues for discovery of new drugs to treat infectious diseases."
The researchers focused on the bacterium Caulobacter crescentus. The cell cycle for this bacterium is controlled by the destruction of key proteins such as the essential transcription factor known as CtrA, but until now it has been unclear how this actually worked at the molecular level. Researchers have known for more than 20 years that one of the factors important for this protein destruction is an energy dependent protease ClpXP.
But ClpXP is always present through the bacterial cell cycle, not always actively breaking down CtrA, suggesting that more complex regulation was going on. Further, more recent work showed that CtrA degradation requires changes in second messengers, small molecules that help different cell pathways communicate with each other. CtrA degradation also needs dephosphorylation of proteins known as adaptors, Chien notes.
His graduate student Kamal Joshi found that these additional proteins were needed to create a scaffold that linked the CtrA substrate to the ClpXP protease. Importantly, this scaffold had to bind the small molecule second messengers in order to hold CtrA and had to contain properly dephosphorylated adaptors in order to hold the ClpXP protease.
"By requiring both these inputs, the cell ensures that degradation of CtrA only occurs at a very specific time," Chien summarizes. "We show that three proteins work together as a multi-component adaptor to stimulate the degradation of CtrA in Caulobacter crescentus. The adaptor only functions when one of the components, CpdR, is unphosphorylated and when another component, PopA, is bound to the signaling molecule, cyclic diguanylate. All this ensures that CtrA is only broken down during a specific window in the bacterium's cell-division cycle."
Chien recently received a five-year, $1.4 million grant from NIH to further explore how bacteria deal with stress by destroying their own proteins. His future work should reveal new pathways that could be targeted to block bacterial virulence or to prevent bacteria from resisting the stresses produced by antibiotics now in use.
- S. C. Smith, K. K. Joshi, J. J. Zik, K. Trinh, A. Kamajaya, P. Chien, K. R. Ryan. Cell cycle-dependent adaptor complex for ClpXP-mediated proteolysis directly integrates phosphorylation and second messenger signals. Proceedings of the National Academy of Sciences, 2014; DOI: 10.1073/pnas.1407862111
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