What are Lon proteases? What is their attraction to scientists?

Lon proteases are large protein complexes. Scientists first discovered them in fecal coliform bacteria, and later came to understand that they are present in all bacteria, and in the cells of all higher life forms (e.g. in human mitochondria).

A Lon protease, which is ATP-dependent (i.e., it requires ATP as an energy source to function), takes damaged proteins and breaks them down in its central chamber. Says Chang: “These properties are quite interesting, which is why scientists have wanted to solve the structure of Lon proteases.”

Cellular “street sweeper”

Lon proteases enable cells to function normally in harsh environments, such as the 50–60°C hot spring water of Wu­lai. Many pathogenic cells and cancerous cells need Lon proteases, which play a vitally important role by “taking out the trash,” so to speak.

Figure I: Chang Chung-i (left) and grad student Lin Jianzhu use a probe to pick up a bit of crystallized protein, which they will examine using X-ray-crystallography.

Wu explains that Lon proteases repair or break down damaged proteins, as appropriate. “If it’s repairable, they repair it. If not, they break it down.” Protein breakdown is a very important function. Alzheimer’s disease, for example, occurs precisely from the buildup of too many useless proteins.

Chang adds that scientists have long known of the importance of Lon proteases, but for years had been unable to use them to develop medicines because they couldn’t figure out their structure.

Eluding observation

Protein structure is usually resolved through the use of X-ray crystallography, but it’s not doable unless the protein is in crystalline form. And the crystal must measure at least 0.05 millimeters, explains ­Chang, for X-ray crystallography to work.

The problem, however, is that not all proteins can be induced to crystallize. For over 30 years, academic studies on Lon proteases in fecal coliform bacteria got nowhere because researchers could not get Lon proteases to crystallize. Lon proteases were found to be present in human mitochondria in the 1990s, but the problem of crystallization stymied researchers.

The research team at Academia Sinica isolated a Lon protease from Meiothermus taiwanensis and named it LonC protease. It is far more stable than the LonA protease used in previous studies, and the team was luckily able to get it to crystallize, which made it possible for the first time to get a look at its structure.

After completing his BS at the National Taiwan University (NTU) School of Pharmacy and an MS at the NTU Graduate Institute of Biochemistry and Molecular Biology, ­Chang went on to receive a PhD from the University of Texas Southwestern Medical Center at Dallas, and worked for a few years at Array BioPharma in the United States before Academia Sinica lured him back to Taiwan in 2009. He more or less stumbled onto the LonC protease topic, and after three years achieved some preliminary success with it.

“This is the least scientific sort of guesswork in all of science,” says Chang, explaining that protein crystallization is like art. It doesn’t occur in nature. “It’s up to the fates whether you succeed with it or not.”

The structure of LonC protease is in some respects a bit different from what the team had expected. It has a central chamber and six tentacle-like receptors. These six activation domains are like scissors that slice up any proteins sent their way.

“We knew beforehand that it would be shaped like a cage.” After resolving the structure, they further ascertained its hollow hexagonal shape, as opposed to the four-sided or seven-sided structure that scientists had conjectured about.

Hot springs hide a big secret

Professor Tsay Shan-shan of the NTU Department of Botany, now retired, first isolated the thermophilic Wulai hot springs bacterium in 2002, and named it Meio­thermus tai­wan­en­sis.

Wu Shih-­hsiung, who had been a student of Professor Tsay, followed up on Tsay’s research at the Institute of Biological Chemistry by genetically sequencing the filamentous bacterium and discovering the presence of LonC protease.

Figure II: Under the-microscope, a thermophilic bacterium discovered in the hot springs of Wulai, south of Taipei.Its discoverers have named it Meiothermus taiwanensis (courtesy of Wu Shih-hsiung).

Now 60 years old, Wu holds a BS in botany and an MS in biochemistry from NTU, and a PhD in pharmacy from the University of Wisconsin in Madison. Since returning to Taiwan, he has spent over a decade studying M. tai­wan­en­sis. Wu explains that this bacterium has a genome of over 3 million bases (as opposed to 3 billion in a human being), and over 3,000 enzymes, of which LonC protease is just one.

However, unlike previously discovered Lon proteases, LonC is ATP-independent and thus requires no energy source to take proteins into its interior chamber to be broken down. The Academia Sinica research team named the newly discovered protease LonC, and reported on it last year in the international scientific journal PLOS ONE.

The proteins in hot springs bacteria, explains Wu, have better heat stability, and the resistance of their enzymes to heat and acidity makes them very useful industrially. There is one enzyme, for example, with an especially good capacity for absorbing and breaking down proteins.

When added to feed for chicken and pigs, it improves the animals’ digestion and ability to absorb the nutrients. Overseas feed makers have already traveled to Taiwan to discuss technical cooperation.

“The power of microorganisms is limitless,” enthuses Wu, adding that bacteria, when used properly, can be put to work for us.

Resolving the structure of the LonC protease is just the start of something much bigger.

Cancer applications

Lon proteases have attracted interest for their possible application in the fight against cancer.

The effects of some targeted drugs, says Chang, have not yet been narrowed to cancerous cells, but progress might be made by suppressing the ability of cancerous cells to dispose of their refuse.

In theory, if a drug can bond with the six activation domains in the LonC molecule’s central chamber and suppress its capacity to dispose of useless proteins, then the cancerous cell will not be able to proliferate. Current research indicates that Lon proteases are very important to the survival of some kinds of cancerous cells, lymphoma being one example. And some pathogenic bacteria, such as typhoid, also need Lon proteases in order survive within the host.

Some drugs are effective, but the reasons for their efficacy are unknown. Now that the structure of LonC protease has been resolved, however, it is possible to determine how it could be incorporated into a small-molecule drug. This would make it possible to improve drugs by making them even more precisely targeted. A targeted cancer drug called Velcade, for example, was originally used clinically to suppress the breakdown of proteins, but ­Chang discovered that it also suppresses the activity of mitochondrial Lon proteases.

The discovery inspired two new directions in his research. First he began working to improve Velcade by eliminating its suppressant effect upon mitochondrial Lon proteases, so as to narrow the drug’s effect and reduce side effects. Then, building on this work, he set about designing a suppressant for Lon proteases.

“Doing research on a local bacteria is like a dream come true,” exclaims ­Chang, adding that one of the best things about scientific research is that one study leads to another.

For example, LonC protease is ATP-independent, while the LonA protease in human mitochondria and yeasts is ATP-dependent. ­Chang surmises that perhaps the pore to the chamber in LonA protease is relatively small and therefore needs energy from ATP to get proteins into the chamber, while the pore in LonC protease is larger and therefore needs no ATP.

However, he stresses, “the difference between them remains to be proven.”

So, it turns out, the “beauty springs” of Wu­lai do a lot more than just invigorate the circulation and beautify the skin. Now we find that bacteria there harbor secrets awaiting scientific discovery. And some scientists from Taiwan have already begun to crack the code!

< photos by Chin Hung-hao/tr. by David Smith >