To all the author's family members for their support and encouragement to apply for the 2022 Amgen Scholars Program at Kyoto University and for their guidance that the author was able to reach Japan and return to Indonesia;. Jun Suzuki as a field supervisor to introduce the author to the Amgen Scholars Program 2022 at Kyoto University, and support and guide the author during his stay in Japan until the writing of the report and his feedback and suggestions for the poster and presentation as part of the Amgen Scholars Program assignment;. Amgen Scholars Program to the Kyoto University Committee for organizing this program, accepting the author as one of the awardees, and helping the author adapt to Japanese culture through many cultural activities, including pottery and calligraphy;.
Other awardees of the Amgen Scholars Program 2022 at Kyoto University for their emotional support; and. Method for gating and analysis of the raw data for flow cytometry from Lipid Scrambling. The phosphorylation status of the XRCC4/C terminus in active Xkr4 can also be observed using mass spectrometry.
The Amgen Scholars Program at Kyoto University is part of the Amgen Scholars Asia Program, where 22 selected undergraduate students from around the world are invited for 8 weeks of intensive hands-on research over the summer, sponsored by the Amgen Foundation. Each of the Amgen Scholars will be assigned to one of several departments or interdisciplinary areas of Kyoto University that support traditional science and biotechnology research, including the field of biochemistry. Kyoto University (京都大学, Kyōto daigaku) is one of the most prestigious public research universities in Kyoto, Japan.
The practice was carried out in one of the groups from the field of cell biology led by Prof.
PROJECT DESCRIPTION Project background
This strategy involves generating a single amino acid mutation of the XRCC4/C terminus from Serine/Threonine to Alanine to eliminate the phosphorylation site(s). The result of this project will contribute to establishing a complete understanding of the Xkr4 activation mechanism. After generating the mutants, Lipid Scrambling Assay was then performed to observe its Xkr4 activity during apoptosis.
Generation of the cDNA mutant was done by amplification of the XRCC4 cDNA template using Polymerase Chain Reaction (PCR) with PrimeStar DNA polymerase (Takara Bio) and primers containing the desired mutation (Eurofins Genomics; Merck). However, the location of the PacI and EcoRI restriction sites in plasmid overlap (Figure 2.2). The mixture was then incubated at 37°C for 1 hour, followed by the addition of the EcoRI enzyme (NEB) to the mixture and ended with overnight incubation at 37°C.
Plasmid construction was performed using the In-Fusion® HD cloning kit (Takara Bio). Once the plasmid was fully constructed, confirmation of the correct sequence of the XRCC4/C-terminal cDNA mutant was performed by double restriction enzyme and insert sequencing. However, due to large differences between the size of the insert and the plasmid vector, the enzyme KpnI (NEB) was used for double digestion with PacI instead of EcoRI (Figure 2.2).
The digestion product was then subjected to 1% agarose gel electrophoresis (135 V; 20 minutes) to detect the presence of the inserted band. After the presence of the insert was detected, the concentration of the remaining plasmid isolates was measured using NanoDrop. After confirming the presence of the correct XRCC4/C-terminus cDNA mutant, the rest of the transformed E .
After amplification, the plasmid construct was then used to induce stable expression of the XRCC4/C-terminal mutant cDNA in PLB cells using lentivirus. To determine the activity of the Xkr4 mutant, a positive control (wild type) and a negative control (PLB cells without XRCC4/C-terminus expression) should also be subjected to a lipid coding assay in each experiment. The NBD-PC fluorescence level of each cell in the third cell population was then analyzed (Figure 2.3E and Figure 2.3F).
Method for gating and analyzing the Lipid Scrambling Assay flow cytometry raw data. The "Red Fluorescence" level indicates the presence of tagRFP from the expression of the XRCC4/C terminus, and was measured using the Phycoerythrin (PE) channel with the maximum excitation of 565 nm (yellow-green) and an emission peak at 573 nm (yellow).
FINDINGS Result
The sequence results from Eurofins Genomics further confirm the successful insertion of XRCC4/C-terminus cDNA mutant. A band with a size of approximately 2,000 bp (red arrow) represents the presence of the XRCC4/C-terminus cDNA inserts. To determine the Xkr4 activity during apoptosis, each XRCC4/C-terminus mutant(s) undergoes Lipid Scrambling Assay together with the WT XRCC4/C-terminus (positive control) and non-transfected cell (negative control).
However, the effect of XRCC4/C-terminus phosphorylation on Xkr4 activation during apoptosis has never been examined. Therefore, this project sought to examine the effect of XRCC4/C-terminus phosphorylation on Xkr4 activation. In this project, the effect of XRCC4/C-terminus phosphorylation on Xkr4 activation was investigated using the XRCC4/C-terminus Alanine Mutation Screening strategy.
Therefore, the alanine mutation resulted in the elimination of the phosphorylation site in the XRCC4/C terminus. This indicates that the direct interaction of the XRCC4/C-terminus mutant with Xkr4 and the activation of Xkr4 will be prevented if the XRCC4/C-terminus phosphorylation has an effect on Xkr4 activation. The direct interaction with Xkr4 and Xkr4 activation would not occur if the XRCC4/C-terminus phosphorylation affects Xkr4 activity.
Several modifications were made to the XRCC4/C-terminus cDNA sequence before it was mutated and/or inserted for stable expression in PLB cells (Figure 3.8). To ensure that tagRFP does not interfere with XRCC4/C-terminus activity, the (GGGGS)2 linker sequence was also included directly downstream of the XRCC4/C-terminus cDNA sequence (WT and mutant) as part of the insert. After these modifications, the amino acid sequence of the wild-type XRCC4/C terminus (which will be mutated as part of the alanine mutation screening) used as an insert for this project can be seen in Figure 3.8.
The RFP tag was translated directly next to the (GGGGS)2 linker (blue font) within the same XRCC4/C-terminal peptide molecule. Therefore, loss of one or two phosphorylations at the XRCC4/C terminus does not alter Xkr4 activity to expose PS and incorporate PC. According to Normanno et al. 2017 ), XRCC4/C-terminus phosphorylation occurs after DNA double-strand break binding.
Therefore, it can be suggested that phosphorylation of the XRCC4/C terminus may not be sufficient to have an effect on the Xkr4 activation. The phosphorylation status of the wild-type XRCC4/C-terminus in active Xkr4 can also be observed by mass spectrometry to further confirm the effect of XRCC4/C-terminus phosphorylation in Xkr4 activity.
CONCLUSION AND RECOMMENDATION
SELF REFLECTION
Red fluorescent indicates expression of the RFP-tagged XRCC4/C-terminus mutant due to lentivirus transfection of the plasmid construct. These 288 base pairs of nucleotide sequence were used as a template for PCR primer design to generate a site-directed XRCC4/C-terminus mutant. The stop codon was removed to ensure translation of the tagRFP sequence into the same XRCC4/C-terminal DNA amino acid sequence.
Nucleotide in green box will be translated to DVTD which is the cleavage site of caspase. Nucleotide in red box will be translated to serine or threonine which was changed or mutated for the alanine mutation screening strategy. XRCC4 represents the full-length XRCC4 cDNA sequence and is used as a template to generate the XRCC4/C-terminus cDNA mutant.
Photo of the author and other members of the Laboratory for Medical Biochemistry and Cell Membrane Biology. Photo by the author during a molecular biology experiment (left) and a cell culture experiment (right). Photo of the author in front of a poster of this project during a symposium with other awardees of the 2022 Amgen Scholars Program at Kyoto University.
The symposium was held in person on August 1, 2022 as part of the 2022 Amgen Scholars Program at Kyoto University. The screenshot of the author's poster in the symposium with other award winners of the Amgen Scholars Asia Program 2022. The symposium was held virtually from 04 – 05 August 2022 as part of the Amgen Scholars Program 2022 at Kyoto University.
The photo of the author with other awardees of the Amgen Scholars Program 2022 at Kyoto University, whose work under the Institute for Integrated Cell-Material Sciences (iCeMS). The photo of the author and other awardees of the Amgen Scholars Program 2022 at Kyoto University, whose work under the Institute for Integrated Cell-Material Sciences (iCeMS), with each of their Kyoto University faculty mentors. Mutational phospho-mimicry reveals a regulatory role for XRCC4 and XLF C-terminal tails in modulating DNA bridging during classical non-homologous end joining.
