How NSAIDs, like aspirin, affect tissue membranes
Researchers have found that three commonly used NSAIDs (nonsteroidal anti-inflammatory drugs), work by altering enzymes in cell membranes.
The finding suggests that if taken at higher-than-approved doses or even over long periods of time, prescription-level NSAIDs (and related drugs that affect cell membranes) can produce wide-ranging and unwanted side effects.
A summary of their results is published online in the journal Cell Reports.
"When drug designers think about possible side effects, they tend to think about proteins similar to those proteins being targeting by the new drug.
"But our group has found that drugs that affect cell membranes can alter proteins totally unrelated to the drug target."
Sinisa Urban, Ph.D., associate professor of molecular biology and genetics at the Johns Hopkins University School of Medicine, and a Howard Hughes Medical Institute investigator.
Working with Syed Moin PhD, Urban's project began as an investigation into the "cellular scissors" embedded within a cell membrane. Cellular scissors known as rhomboid proteases. Rhomboid proteases cut proteins which are then released from the membrane. Half a protein might exit the cell and act as a signal to another cell or both halves might end up leaking into the cell and being completely degraded without any function. It all depends on the protein and where it is cut.
Why is this important? Proteins are known to play roles in everything from malaria to Parkinson's disease.
Researchers had already found out that rhomboid proteases tend to cut proteins whose structures are unstable. While some proteins are inherently more stable than others, the cell membrane itself can physically support unstable proteins embedded within it. So, Urban went about changing the physical properties of a cell membrane "habitat" to see if such changes would allow unintended proteins to be cut up by the proteases.
Urban treated human cells with two chemicals to see if either made the membranes more flexible or distorted. When these types of distortion did occur — rhomboid proteases began cutting proteins not normally cut.
Amyloid-beta precursor protein (APP) and Delta, a signaling protein were two such proteins cut in addition to "client" or expected protein targets of the proteases.
If gamma-secretase cuts APP at the "wrong" site on the protein, the piece of protein that is generated clumps in the brain causing Alzheimer's.
Many drugs end up in the cell membrane. Certain prescription-only NSAIDs repair the function of gamma-secretase, an enzyme with more than 100 different protein clients the most famous one being APP. Some prescription-level NSAIDs, like flurbiprofen (which treats serious arthritis) make gamma-secretase less likely to cut the APP protein at the wrong site.
For over-the-counter NSAIDS like aspirin, ibuprofen and naproxen, the range of client proteins cut by rhomboid proteases increases only slightly, if at all.
In order to find out how NSAIDs actually are affecting cell membranes, Urban used an instrument that measures the melting temperature of fat.
Membranes are composed primarily of fat molecules, and heat can make membranes as fluid as melted butter. The same way solid shortening becomes liquid when heated, so can membrane fat molecules "melt."
A lower melting temperature produces a more flexible membrane. Researchers found that prescription-level NSAIDs that lower a membrane's melting temperature cause rhomboid proteases to cut nonclient proteins.
One of the benefits of this study is that the same methods used to measure the melting temperature of membrane molecules (which makes them more flexible) can be used to test new drugs. Adds Urban: "Now we can use rhomboid proteases as predictors of a drug's possible effects on a membrane and its enzymes."
•Rhomboid enzymes catalyze proteolysis of specific protein targets in the membrane
•Perturbing cell membranes pharmacologically leads rhomboid to cleave nonsubstrates
•Several γ-secretase-modulating drugs also provoke rhomboid cleavage of nonsubstrates
•Membranes guide rhomboid specificity but are vulnerable to off-target drug effects
Rhomboid proteases are integral membrane enzymes that regulate cell signaling, adhesion, and organelle homeostasis pathways, making substrate specificity a key feature of their function. Interestingly, we found that perturbing the membrane pharmacologically in living cells had little effect on substrate processing but induced inappropriate cleavage of nonsubstrates by rhomboid proteases. A subclass of drugs known to modulate γ-secretase activity acted on the membrane directly and induced nonsubstrate cleavage by rhomboid proteases but left true substrate cleavage sites unaltered. These observations highlight an active role for the membrane in guiding rhomboid selectivity and caution that membrane-targeted drugs should be evaluated for cross-activity against membrane-resident enzymes that are otherwise unrelated to the intended drug target. Furthermore, some γ-secretase-modulating activity or toxicity could partly result from global membrane effects.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
On the Web
Link to article (live after embargo lifts): http://dx.doi.org/10.1016/j.celrep.2014.07.039
This work was supported by grants from the Howard Hughes Medical Institute, the David and Lucile Packard Foundation, and the National Institute of Allergy and Infectious Diseases (AI066025).
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