Characterizing Pathologically Relevant Mutations in Human Plastin 3 (PLS3)
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Date
2020-05
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The Ohio State University
Abstract
Plastins are a conserved family of proteins that non-covalently crosslink actin filaments into bundled networks aiding in cellular adhesion, division, and motility. Plastins contain a Ca2+-binding head-piece domain, and two actin-binding domains (ABD1 & ABD2). Ca2+-binding to the head-piece domain of plastin inhibits the bundling of F-actin. Of the three human plastin isoforms, plastin-3 (PLS3) is expressed in most solid tissues, and has been linked to diseases such as cancer, spinal muscular atrophy, X-linked osteogenesis imperfecta (OI), and congenital diaphragmatic hernias (CDH). Recently, 8 different mutations resulting in full length PLS3 in patients has been linked to OI (A368D, E249_A250insI-L, A253_L254insN, N446S, & L478P) and CDH (E270K, W499C, & M592V).
OI, more generally known as osteoporosis, is characterized by improper bone formation and low bone mineral density resulting in numerous peripheral or vertebral fractures from moderate or no impact. Specific therapies are lacking for the treatment of OI. CDH is a developmental disorder that is characterized by improper formation of the diaphragm, resulting in contents of the abdominal cavity moving up into the thoracic cavity. CDH is often fixed through surgical repair, and is associated with a high level of infant morbidity and mortality related to pulmonary issues. The role of PLS3 in these diseases is not well understood. Since the previously mentioned mutations are present in patients with these diseases, we know that PLS3 plays some underlying role. The learning goals of this project includes characterizing how each mutation affects PLS3 functionality in vitro, PLS3 behavior in cellulo, and further exploring the underlying link between PLS3 and OI/CDH.
Each mutant was cloned and purified for in vitro characterization. Differential scanning fluorimetry (DSF) was done to obtain each protein's melting point, which can be used to assess each protein's stability. It was observed that in the osteoporosis-related mutants, there was a slight destabilization observed to PLS3 WT, which was apparent with a decrease in melting temperature across varying degrees (2.6-7.6°C). In the CDH-related mutants, there was no significant change in each protein's melting temperature with respect to PLS3 WT. Since the transition point for all of the proteins was well above physiological temperatures, this suggests that each mutant maintains its tertiary structure in the patients, and that the disease pathologies for OI and CDH may be a result of altering PLS3's binding and bundling capabilities.
Next, each protein's binding and bundling capabilities were assessed by performing high-speed and low-speed co-sedimentation assays, respectively. None of the mutations affected PLS3's ability to bind to F-actin. However, the mutations have various effects on PLS3's ability to bundle F-actin. The CDH-related mutants bundled actin similarly to PLS3 WT in the absence of Ca2+, but these experiments must be repeated to confirm these results. The osteoporosis-related mutants could be categorized into 3 groups based on their bundling abilities; bundling incompetent (L478P), Ca2+-hypersensitive (N446S and A253_L254insN), and Ca2+-hyposensitive (A368D and E249_A250insI-L). Each OI-related mutant's sensitivity to Ca2+ regulation was confirmed through light-scattering combined with Ca2+ titrations.
The osteoporosis-related mutants were expressed into fibroblasts and osteoblasts, which confirmed the biochemical results from the in vitro assays. The Ca2+-hypersensitive mutants and -hyposensitive mutants were localized primarily to the lamellipodia or focal adhesions respectively, while PLS3 WT was distributed between both regions. L478P did not strongly associate with any actin structures and was diffuse throughout the cytoplasm. The importance of Ca2+ in PLS3 localization was confirmed by observing the redistribution of WT and hypersensitive mutants from the leading edge to focal adhesions upon depletion of intracellular Ca2+. The CDH-related mutants were also expressed into cells and illustrate a different association to actin structures compared to PLS3 WT. All of these findings illustrate how mutations perturbing the fine regulation of PLS3 may be the underlying link in these human diseases. This presents PLS3 as a valuable research and therapeutic target in the biomedical field for disease pathologies.
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Keywords
actin, actin cytoskeleton, osteoporosis, hernia, plastin