Investigations on Cellular Localization of Coiled-Coil Domain-Containing Protein 43 (CCDC43) Under Stress Conditions

Öz

Coiled-coil domain containing (CCDC) family proteins regulate multiple biological functions in the cell. These domain structures have physical properties favorable for liquid-liquid phase separation (LLPS) phenomena, which play important roles in cell biology dynamics. LLPS is a fundamental mechanism in forming membrane-less organelles, including stress granules (SG) and P-body, nucleolus, PML nuclear body, Cajal bodies, nuclear speckles, and centrosomes. One of the new members of the CCDC protein family is CCDC43. In this study, sequence-based analyses of CCDC43, performed as a first step to understanding the role of CCDC43 in the cellular network, revealed the LLPS potential of the protein. We report that CCDC43, which has an evolutionarily conserved protein sequence, structurally possesses RNA-binding sites and its amino acid composition analysis may show an aggregation propensity and LLPS behavior under cellular conditions. CCDC43 is known to show a diffuse localization in the cytoplasm. We investigated whether this cellular localization is present in SGs whose formation is based on LLPS. We found that CCDC43 does not aggregate at SGs under given stress conditions. These data provide the first insights into the intracellular behavior and roles of CCDC43 in a disease-independent manner.

Anahtar Kelimeler

• CCDC43
• liquid-liquid phase separation
• stress granules

Sarılı-sarmal domain içeren 43 proteininin (CCDC43) stres koşulları altında hücresel lokalizasyonu üzerine araştırmalar

Öz

ÖZET Sarılı-sarmal domain ailesi proteinleri hücrede çoklu biyolojik fonksiyonları düzenler. Bu domain yapıları, hücre biyolojisi dinamiklerinde önemli roller oynayan sıvı-sıvı faz ayrımı (LLPS) olayları için uygun fiziksel özelliklere sahiptir. LLPS, stres granülleri (SG) ve P-cisimciği, nükleolus, PML nükleer cisimciği, Cajal cisimcikleri, nükleer benekler ve sentrozomlar dahil olmak üzere zarsız organellerin oluşturulmasında temel bir mekanizmadır. CCDC protein ailesinin daha az çalışılmış üyelerinden biri CCDC43'tür. Bu çalışmada, CCDC43'ün hücresel ağdaki rolünü anlamak için ilk adım olarak gerçekleştirilen CCDC43'ün sekans bazlı analizleri, proteinin LLPS potansiyelini ortaya koymuştur. Evrimsel olarak korunmuş bir protein dizisine sahip olan CCDC43'ün yapısal olarak RNA bağlanma bölgelerine sahip olduğunu ve amino asit kompozisyon analizinin hücresel koşullar altında bir agregasyon eğilimi ve LLPS davranışı gösterebileceğini bildiriyoruz. CCDC43'ün sitoplazmada yaygın bir lokalizasyon gösterdiği bilinmektedir. Bu hücresel lokalizasyonun, oluşumu LLPS'ye dayanan SG'lerde mevcut olup olmadığını araştırdık. CCDC43'ün verilen stres koşulları altında SG'lerde toplanmadığını bulduk. Bu veriler, hastalıktan bağımsız bir şekilde CCDC43'ün hücre içi davranışı ve rolleri hakkında ilk bilgileri sağlamaktadır.

Anahtar Kelimeler

• CCDC43
• sıvı-sıvı faz ayrımı
• stres granül

Kaynakça

1. [1]. Ford, LK, Fioriti, L. 2020. Coiled-Coil Motifs of RNA-Binding Proteins: Dynamicity in RNA Regulation. Front Cell Dev Biol; 19;8:607947.
2. [2]. Liu, Z, Yan, W, Liu, S, Liu, Z, Xu, P, Fang, W. 2023. Regulatory network and targeted interventions for CCDC family in tumor pathogenesis. Cancer letters; 565, 216225.
3. [3]. Priyanka, PP, Yenugu, S. 2021. Coiled-Coil Domain-Containing (CCDC) Proteins: Functional Roles in General and Male Reproductive Physiology. Reprod. Sci; 28, 2725–2734.
4. [4]. Wang, Y, Wang, Y, Zhou, J, Ying, P, Wang, Z, Wu, Y, Hao, M, Qiu, S, Jin, H, Wang, X. 2023. A novel coiled-coil domain containing-related gene signature for predicting prognosis and treatment effect of breast cancer. J Cancer Res Clin Oncol; 149(15):14205-14225.
5. [5]. Huang, H, Arighi, CN, Ross, KE, Ren, J, Li, G, Chen, SC, Wang, Q, Cowart, J, Vijay-Shanker, K, Wu, CH. 2018. iPTMnet: an integrated resource for protein post-translational modification network discovery. Nucleic Acids Res; 4;46(D1):D542-D550.
6. [6]. Tanikawa, C, Ueda, K, Suzuki, A, Iida, A, Nakamura, R, Atsuta, N, Tohnai, G, Sobue, G, Saichi N, Momozawa, Y, Kamatani, Y, Kubo, M, Yamamoto, K, Nakamura, Y, Matsuda, K. 2018. Citrullination of RGG Motifs in FET Proteins by PAD4 Regulates Protein Aggregation and ALS Susceptibility. Cell Rep; 6;22(6):1473-1483.
7. [7]. Kumar, M, Thakur, V, Raghava, GP. 2008. COPid: Composition based protein identification. In Silico Biol; 8(2):121-8.
8. [8]. Livi, CM, Klus, P, Delli, Ponti, R, Tartaglia, GG. 2016. catRAPID signature: identification of ribonucleoproteins and RNA-binding regions. Bioinformatics; 1;32(5):773-785.

9. [9]. Marcelo, A, Koppenol, R, de Almeida, LP. et al. 2021. Stress granules, RNA-binding proteins and polyglutamine diseases: too much aggregation? Cell Death Dis; 12, 592.
10. [10]. Chen, Y, Pei, M, Li, J, Wang, Z, Liu, S, Xiang, L, Zhang, J, Hong, L, Lin, J, Dai, W, Xiao, Y, Hu, H, Tang, W, Liu, G, Yang, Q, Lin, Z, Jiang, X, Wang, Y, Wu, X, Guo, Z, Wang, J. 2022. Disruption of the CCDC43-FHL1 interaction triggers apoptosis in gastric cancer cells. Exp Cell Res; Jun 1;415(1):113107.
11. [11]. Tao, M, Han, D, Wei, S, Gao, C. 2023. CCDC43 as a potential therapeutic target of Tian Yang Wan for the treatment of hepatocellular carcinoma by activating the hippo pathway. Front Oncol 8;13:1232190.
12. [12]. Wang, J, Wu, X, Dai, W, Li, J, Xiang, L, Tang, W, Lin, J, Zhang, W, Liu, G, Yang, Q, Lin, Z, Sun, Y, Zhang, Y, Chen, Y, Li, G, Li, A, Liu, S, Li, Y, Wang, J. 2020. The CCDC43-ADRM1 axis regulated by YY1, promotes proliferation and metastasis of gastric cancer. Cancer Lett; 10;482:90-101.
13. [13]. Wang, J, Liu, G, Liu, M, Xiang, L, Xiao, Y, Zhu, H, Wu, X, Peng, Y, Zhang, W, Jiang, P, Li, A, Nan, Q, Chen, Y, Chen, C, Cheng, T, Liu, S, Wang J. 2018. The FOXK1-CCDC43 Axis Promotes the Invasion and Metastasis of Colorectal Cancer Cells. Cell Physiol Biochem; 51(6):2547-2563.
14. [14]. Sambrook,J. Fritsch,E. F. and Maniatis,T., (1989)19901616061, English, Book, USA, 9780879693091, (Ed. 2), Cold Spring Harbor, NY, Molecular cloning: a laboratory manual., (xxxviii + 1546 pp.), Cold Spring Harbor Laboratory Press, Molecular cloning: a laboratory manual.
15. [15]. Hnasko, T. S., & Hnasko, R. M. 2015. The Western Blot. ELISA, 87–96.
16. [16]. Çakırca G, Öztürk MT, Telkoparan-Akillilar P, Güllülü Ö, Çetinkaya A, Tazebay UH. 2024. Proteomics analysis identifies the ribosome associated coiled-coil domain-containing protein-124 as a novel interaction partner of nucleophosmin-1. Biol Cell. Jan;116(1):e202300049.
17. [17]. Arimoto-Matsuzaki, K., Saito, H. & Takekawa, M. 2016. TIA1 oxidation inhibits stress granule assembly and sensitizes cells to stress-induced apoptosis. Nat Commun 7, 10252.
18. [18]. Jumper, J. et al. 2021. Highly accurate protein structure prediction with AlphaFold. Nature; 596, 583–589.
19. [19]. Hardenberg, M, Horvath, A, Ambrus, V, Fuxreiter, M, Vendruscolo, M. 2020. Widespread occurrence of the droplet state of proteins in the human proteome. Proc. Natl. Acad. Sci. U.S.A.;117 (52) 33254-33262.
20. [20]. Yariv, B, Yariv, E, Kessel, A, Masrati, G, Chorin, AB, Martz, E, Mayrose, I, Pupko, T, Ben-Tal, N. 2023. Using evolutionary data to make sense of macromolecules with a "face-lifted" ConSurf. Protein Sci; 32(3):e4582.
21. [21]. Landau, M, Mayrose, I, Rosenberg, Y, Glaser, F, Martz, E, Pupko, T, Ben-Tal N. 2005. ConSurf 2005: the projection of evolutionary conservation scores of residues on protein structures. Nucleic Acids Re; 1;33(Web Server issue):W299-302.
22. [22]. Gao, X, Jiang, L, Gong, Y, Chen, X, Ying, M, Zhu, H, He, Q, Yang, B, Cao, J. 2019. Stress granule: A promising target for cancer treatment. Br J Pharmacol; 176(23):4421-4433.
23. [23]. Ivanov, P, Kedersha, N, Anderson, P. 2019. Stress Granules and Processing Bodies in Translational Control. Cold Spring Harb Perspect Biol; 1;11(5):a032813.
24. [24]. Protter, DSW, Parker, R. 2016. Principles and Properties of Stress Granules. Trends Cell Biol; 26(9):668-679.