# CAD

## Overview
The CAD gene encodes a multifunctional enzyme complex that is pivotal in the de novo pyrimidine biosynthesis pathway, which is essential for the synthesis of pyrimidine nucleotides such as uridine monophosphate (UMP). This enzyme complex, known as carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase, is categorized as a multifunctional enzyme due to its integration of three distinct enzymatic activities. These activities catalyze the initial steps of pyrimidine biosynthesis, which are crucial for DNA and RNA synthesis in human cells (del2021Deciphering; Li2021Pyrimidine). The CAD protein is organized into a hexameric structure, enhancing its metabolic efficiency and regulatory capacity through feedback inhibition and allosteric activation. It is also involved in other cellular processes, such as protein glycosylation and phospholipid biosynthesis, underscoring its significance in cellular physiology and growth (del2021Deciphering; Li2021Pyrimidine). Mutations in the CAD gene are linked to developmental and epileptic encephalopathy 50 (DEE-50), a severe neurometabolic disorder, which can be treated with uridine supplementation, highlighting the gene's clinical importance (Zhou2020Case; Duan2024Novel).

## Structure
The CAD protein is a multifunctional enzyme involved in de novo pyrimidine biosynthesis, comprising three enzymatic activities: carbamoyl-phosphate synthetase (CPS), aspartate transcarbamoylase (ATC), and dihydroorotase (DHO) (del2021Deciphering). The primary structure of CAD includes distinct domains for each enzymatic function, organized in the order GLN-SYN-DHO-ATC (del2019CAD). The CPS domain is responsible for carbamoyl phosphate synthesis and regulation, while the ATC domain is located at the carboxyl end of the polypeptide chain (Grayson1985Immunochemical).

The DHO domain is a Zn metalloenzyme with a 'TIM' barrel fold, featuring eight parallel beta strands flanked by alpha-helices. It contains two catalytic Zn2+ ions and a third Zn2+ ion that may stabilize the structure (del2019CAD). The ATC domain forms a homotrimer, with each subunit having an N-terminal domain for carbamoyl phosphate binding and a C-terminal domain for aspartate binding (del2021Deciphering).

CAD self-assembles into hexamers, primarily through interactions between the ATC domains, forming a 'dimer of trimers' structure (del2019CAD). The protein's quaternary structure is flexible, allowing for conformational changes necessary for catalysis (del2019CAD). The linker between the DHO and ATC domains is phosphorylated, enhancing oligomerization and pyrimidine synthesis (del2019CAD).

## Function
The CAD gene encodes a multifunctional enzyme complex that plays a critical role in the de novo pyrimidine biosynthesis pathway, essential for synthesizing pyrimidine nucleotides like UMP, which are vital for DNA and RNA synthesis in healthy human cells (del2021Deciphering; Li2021Pyrimidine). This enzyme complex is composed of three enzymatic activities: carbamoyl-phosphate synthetase 2 (CPS-2), aspartate transcarbamoylase (ATC), and dihydroorotase (DHO), which catalyze the initial steps of pyrimidine biosynthesis (del2021Deciphering; Lin2023Complexed).

The CAD protein is structured as a hexamer, enhancing metabolic efficiency by ensuring coordinated production and co-localization within the cell, minimizing diffusion of intermediates, and reducing interference with other pathways (del2021Deciphering). It is regulated by feedback inhibition and allosteric activation, primarily through UTP and PRPP, and is subject to posttranslational modifications such as phosphorylation, which modulate its activity and localization (Li2021Pyrimidine).

In addition to its role in nucleotide synthesis, CAD is involved in protein glycosylation and phospholipid biosynthesis, contributing to cellular and organismal physiology (Li2021Pyrimidine). The enzyme's activity is crucial for cell growth and proliferation, as it provides the necessary precursors for nucleic acid synthesis (del2021Deciphering).

## Clinical Significance
Mutations in the CAD gene are primarily associated with developmental and epileptic encephalopathy 50 (DEE-50), an autosomal recessive disorder. DEE-50 is characterized by drug-refractory epilepsy, psychomotor retardation, anemia, and progressive brain atrophy. Some patients may experience developmental delay without seizures. The disorder is progressive and has a poor prognosis but can be effectively treated with uridine, which compensates for the pyrimidine deficiency caused by CAD mutations (Zhou2020Case; Duan2024Novel).

CAD deficiency, also known as early infantile epileptic encephalopathy-50 (EIEE-50), results from biallelic variants in the CAD gene. This condition is marked by progressive epileptic encephalopathy, recurrent status epilepticus, loss of skills, and dyserythropoietic anemia. It can also present with milder symptoms such as isolated developmental delay or intellectual disability. Uridine supplementation has been shown to improve clinical outcomes significantly (Rymen2020Expanding).

The CAD gene's role in pyrimidine biosynthesis is crucial for nucleotide homeostasis, and its dysfunction can lead to severe neurometabolic disorders. These disorders are treatable with uridine supplements, highlighting the importance of early genetic diagnosis and treatment (del2020Cellbased; Koch2016CADmutations).

## Interactions
The CAD protein, encoded by the CAD gene, participates in several significant interactions with other proteins. In non-apoptotic cells, CAD, also known as DFF40, forms a heterodimer with its inhibitor DFF45 (ICAD), which acts as a chaperone to ensure proper folding and prevent premature activation. During apoptosis, caspase-3 or -7 cleaves DFF45, releasing active CAD to execute DNA fragmentation (Widlak2005Discovery). CAD's nuclease activity is enhanced by interactions with chromosomal proteins such as histone H1, HMGB1/2, and topoisomerase II, which influence its substrate preference (Widlak2005Discovery).

CAD also interacts with mLST8, a component of the mTOR complexes, through multiple regions except the ATC domain. This interaction is distinct from mLST8's interaction with mTOR and is involved in regulating CAD's enzymatic activity (Nakashima2013Association). Additionally, CAD interacts with the androgen receptor (AR) in prostate cancer cells, affecting AR transcriptional activity and facilitating its nuclear translocation (Morin2011Identification). These interactions highlight CAD's multifunctional role in cellular processes, including apoptosis and pyrimidine synthesis.


## References


[1. (del2021Deciphering) Francisco del Caño‐Ochoa and Santiago Ramón‐Maiques. Deciphering <scp>cad</scp>: structure and function of a mega‐enzymatic pyrimidine factory in health and disease. Protein Science, 30(10):1995–2008, July 2021. URL: http://dx.doi.org/10.1002/pro.4158, doi:10.1002/pro.4158. This article has 25 citations and is from a peer-reviewed journal.](https://doi.org/10.1002/pro.4158)

[2. (Zhou2020Case) Ling Zhou, Jie Deng, Sarah L. Stenton, Ji Zhou, Hua Li, Chunhong Chen, Holger Prokisch, and Fang Fang. Case report: rapid treatment of uridine-responsive epileptic encephalopathy caused by cad deficiency. Frontiers in Pharmacology, December 2020. URL: http://dx.doi.org/10.3389/fphar.2020.608737, doi:10.3389/fphar.2020.608737. This article has 9 citations and is from a peer-reviewed journal.](https://doi.org/10.3389/fphar.2020.608737)

[3. (del2019CAD) Francisco del Caño-Ochoa, María Moreno-Morcillo, and Santiago Ramón-Maiques. CAD, A Multienzymatic Protein at the Head of de Novo Pyrimidine Biosynthesis, pages 505–538. Springer International Publishing, 2019. URL: http://dx.doi.org/10.1007/978-3-030-28151-9_17, doi:10.1007/978-3-030-28151-9_17. This article has 30 citations.](https://doi.org/10.1007/978-3-030-28151-9_17)

[4. (Morin2011Identification) Aurélie Morin, Lauriane Fritsch, Jacques R. R. Mathieu, Criste’le Gilbert, Basma Guarmit, Virginie Firlej, Catherine Gallou‐Kabani, Annick Vieillefond, Nicolas Barry Delongchamps, and Florence Cabon. Identification of cad as an androgen receptor interactant and an early marker of prostate tumor recurrence. The FASEB Journal, 26(1):460–467, October 2011. URL: http://dx.doi.org/10.1096/fj.11-191296, doi:10.1096/fj.11-191296. This article has 15 citations.](https://doi.org/10.1096/fj.11-191296)

[5. (Lin2023Complexed) En-Shyh Lin, Yen-Hua Huang, Po-Chun Yang, Wei-Feng Peng, and Cheng-Yang Huang. Complexed crystal structure of the dihydroorotase domain of human cad protein with the anticancer drug 5-fluorouracil. Biomolecules, 13(1):149, January 2023. URL: http://dx.doi.org/10.3390/biom13010149, doi:10.3390/biom13010149. This article has 5 citations and is from a peer-reviewed journal.](https://doi.org/10.3390/biom13010149)

[6. (Rymen2020Expanding) Daisy Rymen, Martijn Lindhout, Maria Spanou, Farah Ashrafzadeh, Ira Benkel, Cornelia Betzler, Christine Coubes, Hans Hartmann, Julie D. Kaplan, Diana Ballhausen, Johannes Koch, Jan Lotte, Mohammad Hasan Mohammadi, Marianne Rohrbach, Argirios Dinopoulos, Marieke Wermuth, Daniel Willis, Karin Brugger, Ron A. Wevers, Eugen Boltshauser, Jörgen Bierau, Johannes A. Mayr, and Saskia B. Wortmann. Expanding the clinical and genetic spectrum of cad deficiency: an epileptic encephalopathy treatable with uridine supplementation. Genetics in Medicine, 22(10):1589–1597, October 2020. URL: http://dx.doi.org/10.1038/s41436-020-0933-z, doi:10.1038/s41436-020-0933-z. This article has 22 citations and is from a highest quality peer-reviewed journal.](https://doi.org/10.1038/s41436-020-0933-z)

[7. (Widlak2005Discovery) Piotr Widlak and William T. Garrard. Discovery, regulation, and action of the major apoptotic nucleases dff40/cad and endonuclease g. Journal of Cellular Biochemistry, 94(6):1078–1087, February 2005. URL: http://dx.doi.org/10.1002/jcb.20409, doi:10.1002/jcb.20409. This article has 171 citations and is from a peer-reviewed journal.](https://doi.org/10.1002/jcb.20409)

[8. (Grayson1985Immunochemical) D R Grayson, L Lee, and D R Evans. Immunochemical analysis of the domain structure of cad, the multifunctional protein that initiates pyrimidine biosynthesis in mammalian cells. Journal of Biological Chemistry, 260(29):15840–15849, December 1985. URL: http://dx.doi.org/10.1016/s0021-9258(17)36335-4, doi:10.1016/s0021-9258(17)36335-4. This article has 33 citations and is from a domain leading peer-reviewed journal.](https://doi.org/10.1016/s0021-9258(17)36335-4)

[9. (Duan2024Novel) Lifen Duan, Lei Ye, Runxiu Yin, Ying Sun, Wei Yu, Yi Zhang, Haiyan Zhong, Xinhua Bao, and Xin Tian. Novel cad gene mutations in a boy with developmental and epileptic encephalopathy 50 with dramatic response to uridine therapy: a case report and a review of the literature. BMC Pediatrics, March 2024. URL: http://dx.doi.org/10.1186/s12887-024-04593-6, doi:10.1186/s12887-024-04593-6. This article has 2 citations and is from a peer-reviewed journal.](https://doi.org/10.1186/s12887-024-04593-6)

[10. (Nakashima2013Association) Akio Nakashima, Ippei Kawanishi, Sumiko Eguchi, Eugene Hsin Yu, Satoshi Eguchi, Noriko Oshiro, Ken-ichi Yoshino, Ushio Kikkawa, and Kazuyoshi Yonezawa. Association of cad, a multifunctional protein involved in pyrimidine synthesis, with mlst8, a component of the mtor complexes. Journal of Biomedical Science, April 2013. URL: http://dx.doi.org/10.1186/1423-0127-20-24, doi:10.1186/1423-0127-20-24. This article has 17 citations and is from a domain leading peer-reviewed journal.](https://doi.org/10.1186/1423-0127-20-24)

[11. (Koch2016CADmutations) Johannes Koch, Johannes A. Mayr, Bader Alhaddad, Christian Rauscher, Jörgen Bierau, Reka Kovacs-Nagy, Karlien L. M. Coene, Ingrid Bader, Monika Holzhacker, Holger Prokisch, Hanka Venselaar, Ron A. Wevers, Felix Distelmaier, Tilman Polster, Steffen Leiz, Cornelia Betzler, Tim M. Strom, Wolfgang Sperl, Thomas Meitinger, Saskia B. Wortmann, and Tobias B. Haack. Cadmutations and uridine-responsive epileptic encephalopathy. Brain, 140(2):279–286, December 2016. URL: http://dx.doi.org/10.1093/brain/aww300, doi:10.1093/brain/aww300. This article has 100 citations and is from a highest quality peer-reviewed journal.](https://doi.org/10.1093/brain/aww300)

[12. (del2020Cellbased) Francisco del Caño-Ochoa, Bobby G. Ng, Malak Abedalthagafi, Mohammed Almannai, Ronald D. Cohn, Gregory Costain, Orly Elpeleg, Henry Houlden, Ehsan Ghayoor Karimiani, Pengfei Liu, M. Chiara Manzini, Reza Maroofian, Michael Muriello, Ali Al-Otaibi, Hema Patel, Edvardson Shimon, V. Reid Sutton, Mehran Beiraghi Toosi, Lynne A. Wolfe, Jill A. Rosenfeld, Hudson H. Freeze, and Santiago Ramón-Maiques. Cell-based analysis of cad variants identifies individuals likely to benefit from uridine therapy. Genetics in Medicine, 22(10):1598–1605, October 2020. URL: http://dx.doi.org/10.1038/s41436-020-0833-2, doi:10.1038/s41436-020-0833-2. This article has 17 citations and is from a highest quality peer-reviewed journal.](https://doi.org/10.1038/s41436-020-0833-2)

[13. (Li2021Pyrimidine) Guanya Li, Dunhui Li, Tao Wang, and Shanping He. Pyrimidine biosynthetic enzyme cad: its function, regulation, and diagnostic potential. International Journal of Molecular Sciences, 22(19):10253, September 2021. URL: http://dx.doi.org/10.3390/ijms221910253, doi:10.3390/ijms221910253. This article has 36 citations and is from a peer-reviewed journal.](https://doi.org/10.3390/ijms221910253)