Part:BBa_K4016009
2XCDK4 αCHelix
This part is used as the targeting module to bind with cyclinD1. Considering other structures may disrupts the position of the specific helix(Morgan, 1997) or produce other impacts, the CDK4 is truncated to maintain its αCHelix to exclude the effects of other factors as far as possible.
Usage and Biology
External growth factors usually influence the cell’s commitment to division in G1 of cell proliferation in vertebrates. The activation of CDK4, a member of growth-dependent cyclin-dependent kinase (CDK) family, is a key response to growth factors in many cell types. (Morgan, 1997) The main outcome of CDK4 activation is the phosphorylation and, thus, inhibition of the retinoblastoma protein leading to G1–S cell-cycle transition. In addition, CDK4 directly phosphorylates other proteins that promote cell-cycle progression and inhibit both cell senescence and apoptosis.
During G1 phase, CDK activity promotes DNA replication and initiates G1-S transition. CDK activation initiates a positive feedback loop that further increases CDK activity, and this commits the cell to division by inducing genome-wide transcriptional changes. G1–S transcripts encode proteins that regulate downstream cell cycle events. (Bertoli et al., 1997) CDK4 and its close homolog CDK6 are serine/threonine kinases that form heterodimers with D-type cyclins and are central regulators of G1–S transition of the cell cycle. (Sheppard et al., 2013)
The enzymatic activities of CDK4 and CDK6 in G1 of the cell cycle are governed by D-type cyclins expressed in response to various extracellular signals, including stimulatory mitogens, inhibitory cytokines, differentiation inducers, cell–cell contacts, and other spatial cues, (Sherr et al., 2016) thus providing a number of cancer treatments by developing drugs, which can inhibit their activities.
Based on the former researches of CDK4, it is particularly actionable in tumors that a pure CDK4 inhibitor would elicit a single phenotype as a direct reflection of the engagement of RB to suppress gene expression and proliferation. Apart from the ER+ breast, the first indication in which CDK4/CDK6 inhibitor received full approval from FDA in 2015, more are being actively explored in MCL, NSCLC, GBM, melanoma, liposarcoma and other cancer subtypes. (Sherr et al., 2016, Asghar et al., 2015, VanArsdale et al., 2015,)
Characterization
This part was measured through three ways:PCR, enzyme digestion and sequencing.
PCR
The PCR is performed with Green Taq Mix by Vazyme.
F-Prime: 5’-CTAGCGTTTAAACTTAAGCTTGCCACCATGGAGGTTCAACTGGAGGAGTCTG-3’
R-Prime: 5’- TGGATATCTGCAGAATTCTTACACGTTCACGCCGCCGTCGAT-3’
The PCR protocol is selected based on the Users Manuel. The Electrophoresis was performed on a 1% Agarose gel.
Enzyme Digestion
After the assembly ,the plasmid was transferred into the Competent E. coli DH5α). After culturing overnight in LB, we minipreped the plasmid for cutting. The preparation of the plasmid was performed with TIANprep Mini Plasmid Kit from TIANGEN. The cutting procedure was performed with XbaI and KpnI restriction endonuclease bought from TAKARA.
The plasmid was cutted in a 20μL system at 37 ℃ for 2 hours. The Electrophoresis was performed on a 1％ Agarose glu.
Sequecing
The plasmid was sequenced correct.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Functional Validation
To validate our basic part 2x CDK4 αCHelix (BBa_K4016009), which can target to CyclinD thus regulating the cell cycle, we designed part 2x CDK4 αCHelix-Coh2 (Part:BBa_K4016028) and part Trim21-Docs (Part:BBa_K3396005) since Docs and Coh2 can interact with each other therefore initiate the degradation process.
Result
The result showed a significant decrease of 450nm absorbance compared to the control group, indacating that in the experimental group, the growth of cells was inhibited. The result successfully proved our system can work as we expected—target and degrade CyclinD, indicating our basic part CDK4 αCHelix can bind with CyclinD as we expected.
Reference
[1]Asghar U, Witkiewicz AK, Turner NC, Knudsen ES. The history and future of targeting cyclin-dependent kinases in cancer therapy. Nat Rev Drug Discov. 2015 Feb;14(2):130-46.
[2]Bertoli C, Skotheim JM, de Bruin RA. Control of cell cycle transcription during G1 and S phases. Nat Rev Mol Cell Biol. 2013 Aug;14(8):518-28. doi: 10.1038/nrm3629.
[3]Morgan DO. Cyclin-dependent kinases: engines, clocks, and microprocessors. Annu Rev Cell Dev Biol. 1997;13:261-91. Sheppard KE, McArthur GA. The cell-cycle regulator CDK4: an emerging therapeutic target in melanoma. Clin Cancer Res. 2013 Oct 1;19(19):5320-8.
[4]Sherr CJ, Beach D, Shapiro GI. Targeting CDK4 and CDK6: From Discovery to Therapy. Cancer Discov. 2016 Apr;6(4):353-67.
[5]VanArsdale T, Boshoff C, Arndt KT, Abraham RT. Molecular Pathways: Targeting the Cyclin D-CDK4/6 Axis for Cancer Treatment. Clin Cancer Res. 2015 Jul 1;21(13):2905-10.
| None |