Structural Biochemistry/Brooke-Spiegler syndrome

Brooke-Spiegler syndrome is an illness involving skin tumors developed from skin appendages, for example, hair follicles and sweat glands. It is caused by the mutation of CYLD, a tumor suppressor. CYLD is a human gene and function as a deubiquitinating enzyme.

Deubiquitinating enzymes
Deubiquitinating enzymes (DUBs) belong to a large group of proteases. They regulate the ubiquitin-mediated pathways. They also function with proteasome by direct or indirect association. They are involved in many stages of biological functions: cell growth,cell differentiation, cell development, oncogenesis, neuronal diseases, and transcriptional regulation.

DUBs is used as a catalyst in the removal of ubiquitin from C-terminal extension peptides and linear poly-ubiquitin fusion. DUBs usually contains esterase and amidase activites in vitro. Ubiquitin-esters and ubiquitin-amidocoumarins are used as substrates for activity assays.

DUBs are divided into two divisions. One of them is ubiquitin C-terminal hydrolases (UCHs). They are enzyme with small size and they act as a catalyst in the removal of peptides and small molecules from the C-terminus of ubiquitin. UCHs usually cannot generate monomeric ubiquitin from protein conjugates or disassemble poly-ubiquitin chains.

Another group is ubiquitin-specific proteases (USPs/UBPs). They are bigger size compared to UCHs. They usually contain N-terminal extensions which can used in substrate regonition, subcellular localization and protein-protein interactions. They also can remove ubiquitin from protein conjugates and disassemble ubiquitin chains. An example of USP is Isopeptidase T (IsoT). It is capable of binding ubiquitin and disassembles free poly-ubiquitin chains

CYLD
CYLD is one of the deubiquitylating enzymes and the regulate NFKB activity negatively. NFKB (nuclear factor kappa beta; or nuclear factor KB) is a transcriptional regulator. It is important in the immune system and they regulate the expression of lots of genes that are critical for the regulation of tumorigenesis, autoimmune diseases, cancer, apoptosis, and inflammation. CYLD gives instruction for making a protein to help regulating NFKB. The protein makes the cells response properly to signals to destroy themselves when the cells become abnormal. CYLD, as a tumor suppressor, inhibits the uncontrolled growth and division of cells. The mutations usually occur in the C-terminal of CYLD which contains a DUB domain. Its function is related to the signal transduction involved in the activation of NFkB 12, 13, 14. The DUB catalytic activity is disrupted by the mutations of CYLD and causes the resistance of cell death and tumorigenesis.

The tumor-suppressive activity of CYLD depends on the ability to inhibit the anti-apoptotic activity of NFkB. The stimulants of cell to survive apoptotic would increase when CYLD is depleted by RNA interference.

Patients with Brooke-Spiegler syndrome have 20 CYLD gene mutations. Patients have mutation in one of the copies of CYLD gene in each cell when they are born, leading to the inhibition of the production of CYLD protein. Also, there is another mutation in another copy of CYLD in some cells which causes genetic changes. If there are mutations in both copies of CYLD gene, there would be no instruction regarding the apoptosis of abnormal cells. The regulation of NFkB will be inhibited. Therefore, the cells will undergo division rapidly and lead to the formation of tumor. There will be formation of multiple noncancerous tumors that develop in skin appendages. There are different kinds of possible developed skin appendages due to the mutation of CYLD, and they are spiradenomas – tumors of sweat glands, trichoepitheliomas – tumors of hair follicles, and cylindromas – tumors of hair follicles.

If patients have 22 mutations in CYLD gene, they will develop multiple familial trichoepithelioma, and they will have a lot of developed trichoepitheliomas. If patients have30 CYLD gene mutations, they will develop familial cylindromatosis. Patients will have a much larger number of cylindroma. All patients are born with a mutated CYLD gene and have another mutation in another copy of CYLD gene (similar to Brooke-Spiegler syndrome).

Image of the genomic location of CYLD: http://www.genecards.org/cgi-bin/carddisp.pl?gene=CYLD

NFkB
As mentioned, CYLD negatively regulates NFKB activity. NFKB is a protein composed of different members of the Rel family of transcription factors. They control genes that regulate a lot of biological processes by acting as dimeric transcription factors. NFkB is hidden in the cytoplasm and is bound by inhibitor proteins IkBa, IkBb, IkBg, and IkBe. When NFkB is activated, phosphorylation of IkB will occur, and its ubiquitination and degradation will occur next. This leads to the exposure of nuclear localization signals on NFkB subunits and the translocation to the nucleus. In the nucleus, NFkB binds to a gene with specific sequence (5'GGGACTTTCC3') and activates their transcription. IkB is phosphorylated by IkB kinase complex which contains IKK1/IKKa, IKK2/IKKb and IKK3/IKKg.They phosphorylate IkB and lead to its ubiquitination and degradation. The activator tumor necrosis factor binds to its receptor and recruits TNF receptor death domain (TRADD). TRADD binds to TNF receptor associated factor (TRAF2). TRAF2 recruits NFkB inducible kinase. IKK1 and IKK2 can be phosphorylated by MAP kinase NIK/MEKK1 and they can phosphorylate IkBa and IkBb. TRAF2 also has the ability to interact with A20. A20 is a zinc finger protein in which the expression is induced by agents that activate NFkB. It is used to inhibit TRAF2-mediated NFkB activation. It is also used as inhibitor of TNF and IL-1 induced activation of NFkB. Therefore, A20 is considered an inhibitor of NFkB activation. There are two pathways:

1. Canonical pathway:

NFkB/Rel proteins are bound and inhibited by IkB proteins. The IKK complex is activated by proinflammatory cytokines, LPS, growth factors, and antigen receptors. IKK complex phosphorylates IkB proteins, leading the ubiquitination and proteasomal degradation and the NFkB/Rel complexes will be freed. NFkB/Rel complex are also activated by phosphorylation and translocate to the nucleus, inducing the target gene expression.

2. Noncanonical pathway:

NFkB2 p100/RelB complex remains inactive in the cytoplasm. The IKKα complexes are activated by the signaling through a subset of receptors. The activated IKKα phosphorylates C-terminal residues in NFkB p100 which leads to the ubiquitination and proteasomal processing to NFkB2 p52. The NFkB p52/RelB complexes translocate to the nucles, inducing target gene expression.