| Project/Area Number |
23K19715
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| Research Category |
Grant-in-Aid for Research Activity Start-up
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| Allocation Type | Multi-year Fund |
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| Review Section |
0907:Oral science and related fields
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| Research Institution | Niigata University |
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Principal Investigator |
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| Project Period (FY) |
2023-08-31 – 2025-03-31
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| Project Status |
Granted (Fiscal Year 2023)
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| Budget Amount *help |
¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000)
Fiscal Year 2024: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
Fiscal Year 2023: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
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| Keywords | isforms / splice variants / protein interactions / protein modification / Protein / DEL-1 / Isoform |
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| Outline of Research at the Start |
Developmental endothelial locus-1 (DEL-1), a multifunctional secreted protein, has been shown to play an integral role in resolving inflammation and promoting bone growth following injury caused by chronic inflammation. We will use molecular, genetic, and biophysical methods to identify cell-type specific DEL-1 isoforms and post-translational modifications and determine their role in the function of DEL-1. We will determine the full-length structure of DEL-1 protein.
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| Outline of Annual Research Achievements |
Objective 1: Discovery of del1 splice variants and isoforms. We created primers that spanned exon-exon junctions to determine if any exons were missing, greater than or less than the size expected, or the expected size. We confirmed that del1 has two distinct isoforms with exon 3 removed in osteoclasts and osteoblasts and found at least one other potential splice variant that is being investigated further. These different isoforms may lead to the discovery of different isoform-specific functions of DEL-1 that may be linked to specific cellular functions. For objectives 3 and 4, looking at DEL-1 structure and protein interaction, we are creating the final reagents required to begin these studies. We will be starting studies using these reagents this year. Understanding the structure of DEL-1 will help us understand DEL-1 functions and potentially cell-type-specific functions. By evaluating DEL-1's interactions with the alphaV beta 3 integrin receptor, we can better understand this interaction and find modulators for this interaction. For objective 2, evaluating DEL-1 post-translation modifications, protein samples have been obtained from osteoclasts. We are collecting protein samples from osteoblasts and will send both samples for mass spectrometry. This is important as understanding how DEL-1 is modified may help us better understand its functions in target cells.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
One known splice variant has been confirmed, and at least one other potential splice variant has been discovered. This second splice variant is being evaluated to verify its validity before creating an expression vector. Due to the difficulty of constructing the reagents for DEL-1 protein structure and protein interaction projects, the reagents are progressing with only minor delays. Finally, the collection of protein samples from target cell types from mice and humans is progressing as planned and samples will be sent for mass spectrometry analysis.
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| Strategy for Future Research Activity |
We will validate the potential splice variants found through qPCR and sequencing. We will create expression vectors to determine their function in target-cell populations. We will continue making reagents for protein structure and protein interaction experiments and begin with structural studies and protein interaction studies within the year. We will finish collecting protein samples from target cells in mice and humans, send them for mass spectrometry, and create expression vectors that reflect the post-translation modifications discovered to determine their role in DEL-1 function.
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