caspase-activated DNase

{{Infobox protein family

| Symbol = DFF40

| Name = DNA fragmentation factor 40 kDa

| image = PDB 1v0d EBI.jpg

| width =

| caption = Crystal structure of caspase-activated DNAse (CAD)

| Pfam = PF09230

| Pfam_clan =

| InterPro = IPR015311

| SMART =

| PROSITE =

| MEROPS =

| SCOP = 1v0d

| TCDB =

| OPM family =

| OPM protein =

| CAZy =

| CDD =

}}

Caspase-activated DNase (CAD) or DNA fragmentation factor subunit beta is a protein that in humans is encoded by the DFFB gene.{{cite journal | vauthors = Liu X, Zou H, Slaughter C, Wang X | title = DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis | journal = Cell | volume = 89 | issue = 2 | pages = 175–84 | date = April 1997 | pmid = 9108473 | doi = 10.1016/S0092-8674(00)80197-X | s2cid = 14800864 | doi-access = free }}{{cite journal | vauthors = Halenbeck R, MacDonald H, Roulston A, Chen TT, Conroy L, Williams LT | title = CPAN, a human nuclease regulated by the caspase-sensitive inhibitor DFF45 | journal = Current Biology | volume = 8 | issue = 9 | pages = 537–40 | date = April 1998 | pmid = 9560346 | doi = 10.1016/S0960-9822(98)79298-X | s2cid = 9837862 | doi-access = free | bibcode = 1998CBio....8..537H }}{{cite web | title = Entrez Gene: DFFB DNA fragmentation factor, 40kDa, beta polypeptide (caspase-activated DNase)| url = https://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=1677}} It breaks up the DNA during apoptosis and promotes cell differentiation. It is usually an inactive monomer inhibited by ICAD. This is cleaved before dimerization.

Function

Apoptosis is a cell self-destruct process that removes toxic and/or useless cells during mammalian development and other life processes. The apoptotic process is accompanied by the shrinkage and fragmentation of cells and nuclei, as well as the degradation of the chromosomal DNA into nucleosomal units. DNA fragmentation factor (DFF) is a heterodimeric protein composed of 40-kD (DFFB) and 45-kD (DFFA) subunits. DFFA is the substrate for caspase-3 and triggers DNA fragmentation during apoptosis. DFF becomes activated when DFFA is cleaved by caspase-3. The cleaved fragments of DFFA dissociate from DFFB, the active component of DFF. DFFB has been found to trigger both DNA fragmentation and chromatin condensation during apoptosis. Multiple alternatively spliced transcript variants encoding distinct isoforms have been identified for this gene; however, the biological validity of some variants has not been determined.

Image:CAD and ICAD forms.png

Despite this gene being present in every cell, the protein it encodes is only expressed in various tissues and cell types, including the pancreas, heart, colon, leukocytes, prostate, ovary, placenta, kidney, spleen and thymus.{{cite web|last1=Davidson College|url=http://www.bio.davidson.edu/courses/immunology/students/spring2006/ryan/yfip.html |title = Caspase Activated Deoxyribonuclease (CAD) |access-date=21 Jan 2016}}

It is also known as caspase activated nuclease (CPAN), dna fragmentation factor 40 (DFF-40), DFF2 and DFFB. Additionally, other nomenclatures have resulted from combining these previous ones.{{cite journal | vauthors = Yuste VJ, Sánchez-López I, Solé C, Moubarak RS, Bayascas JR, Dolcet X, Encinas M, Susin SA, Comella JX | title = The contribution of apoptosis-inducing factor, caspase-activated DNase, and inhibitor of caspase-activated DNase to the nuclear phenotype and DNA degradation during apoptosis | journal = The Journal of Biological Chemistry | volume = 280 | issue = 42 | pages = 35670–83 | date = October 2005 | pmid = 16049016 | doi = 10.1074/jbc.M504015200 | doi-access = free }}{{cite journal | vauthors = Sakahira H, Iwamatsu A, Nagata S | title = Specific chaperone-like activity of inhibitor of caspase-activated DNase for caspase-activated DNase | journal = The Journal of Biological Chemistry | volume = 275 | issue = 11 | pages = 8091–6 | date = March 2000 | pmid = 10713130 | doi = 10.1074/jbc.275.11.8091 | doi-access = free }}{{cite journal | vauthors = Sakahira H, Enari M, Nagata S | title = Functional differences of two forms of the inhibitor of caspase-activated DNase, ICAD-L, and ICAD-S | journal = The Journal of Biological Chemistry | volume = 274 | issue = 22 | pages = 15740–4 | date = May 1999 | pmid = 10336474 | doi = 10.1074/jbc.274.22.15740 | doi-access = free }}

Structure

This heterodimer is an endonuclease{{cite journal | vauthors = Jog NR, Frisoni L, Shi Q, Monestier M, Hernandez S, Craft J, Prak ET, Caricchio R | title = Caspase-activated DNase is required for maintenance of tolerance to lupus nuclear autoantigens | journal = Arthritis and Rheumatism | volume = 64 | issue = 4 | pages = 1247–56 | date = April 2012 | pmid = 22127758 | pmc = 3292632 | doi = 10.1002/art.33448 }}{{cite journal | vauthors = Widlak P, Lanuszewska J, Cary RB, Garrard WT | title = Subunit structures and stoichiometries of human DNA fragmentation factor proteins before and after induction of apoptosis | journal = The Journal of Biological Chemistry | volume = 278 | issue = 29 | pages = 26915–22 | date = July 2003 | pmid = 12748178 | doi = 10.1074/jbc.M303807200 | doi-access = free }} with a high content of cysteine residues. It remains inactive in growing cells when associated with its inhibitor (ICAD, DNA fragmentation factor 45 kDa subunit, DFFA or DFF45), resulting in a complex ICAD-CAD.{{cite journal | vauthors = Reh S, Korn C, Gimadutdinow O, Meiss G | title = Structural basis for stable DNA complex formation by the caspase-activated DNase | journal = The Journal of Biological Chemistry | volume = 280 | issue = 50 | pages = 41707–15 | date = December 2005 | pmid = 16236713 | doi = 10.1074/jbc.m509133200 | doi-access = free }}{{cite journal | vauthors = Widlak P, Li P, Wang X, Garrard WT | title = Cleavage preferences of the apoptotic endonuclease DFF40 (caspase-activated DNase or nuclease) on naked DNA and chromatin substrates | journal = The Journal of Biological Chemistry | volume = 275 | issue = 11 | pages = 8226–32 | date = March 2000 | pmid = 10713148 | doi = 10.1074/jbc.275.11.8226 | doi-access = free }}{{cite journal | vauthors = Sharif-Askari E, Alam A, Rhéaume E, Beresford PJ, Scotto C, Sharma K, Lee D, DeWolf WE, Nuttall ME, Lieberman J, Sékaly RP | title = Direct cleavage of the human DNA fragmentation factor-45 by granzyme B induces caspase-activated DNase release and DNA fragmentation | journal = The EMBO Journal | volume = 20 | issue = 12 | pages = 3101–13 | date = June 2001 | pmid = 11406587 | pmc = 150191 | doi = 10.1093/emboj/20.12.3101 }}{{cite journal | vauthors = Liu X, Zou H, Widlak P, Garrard W, Wang X | title = Activation of the apoptotic endonuclease DFF40 (caspase-activated DNase or nuclease). Oligomerization and direct interaction with histone H1 | journal = The Journal of Biological Chemistry | volume = 274 | issue = 20 | pages = 13836–40 | date = May 1999 | pmid = 10318789 | doi = 10.1074/jbc.274.20.13836 | doi-access = free }} Their dissociation allows DFF40 to oligomerize to form a large functional complex, which is, by itself, an active DNase.

= DFF40 subunit or CAD =

It weighs 40 kDa. Moreover, it contains three domains making up a CAD monomer: C1 or N-terminal CAD; C2, which consists of three separate α chains; and lastly, C3, which is the largest and functionally the most important. What is more, combining C3’s amino acids leads to 5 α helices, 4 β lamina and a loop at the catalytic C-terminal which interacts with each other. Therefore, a cavity (active site) where DNA can fit is produced, even though there is another binding region responsible for a stable DNA complex during its fragmentation.{{cite journal | vauthors = Uegaki K, Otomo T, Sakahira H, Shimizu M, Yumoto N, Kyogoku Y, Nagata S, Yamazaki T | title = Structure of the CAD domain of caspase-activated DNase and interaction with the CAD domain of its inhibitor | journal = Journal of Molecular Biology | volume = 297 | issue = 5 | pages = 1121–8 | date = April 2000 | pmid = 10764577 | doi = 10.1006/jmbi.2000.3643 }}

= DFF45 subunit or ICAD =

DFFA is encoded by an alternatively encrypted mRNAs originating two distinct forms: short (ICAD-S) and long (ICAD-L), which act like a specific chaperone ensuring the correct CAD's folding Besides, it contains two aspartic acid residues (Asp117 and Asp224) where CAD is identified and, consequently, it stays bounded until Caspase-3 splits this union.

Activation process

As usual in non-apoptotic growing cells, caspase activated dnase is held in check and inactivated in the cytoplasm thanks to its association with the inhibitor, inhibitor of caspase-activated DNase (ICAD) also known as DNA fragmentation factor 45 kDa (DFF45).

ICAD is encoded by alternatively spliced mRNAs, which generate long (ICAD-L) and short (ICAD-S) forms of ICAD. Therefore, ICAD has a dual function; it acts as a CAD inhibitor and also as a chaperone for CAD synthesis, assisting in the correct assembly of the protein.{{PDB|1V0D}}; {{cite journal | vauthors = Woo EJ, Kim YG, Kim MS, Han WD, Shin S, Robinson H, Park SY, Oh BH | title = Structural mechanism for inactivation and activation of CAD/DFF40 in the apoptotic pathway | journal = Molecular Cell | volume = 14 | issue = 4 | pages = 531–9 | date = May 2004 | pmid = 15149602 | doi = 10.1016/S1097-2765(04)00258-8 | doi-access = free }}

ICAD has two caspase recognition sites at Asp117 and Asp224. CAD release from ICAD inhibition is achieved by cleavage of ICAD at these Asp residues by caspase-3.{{Cite web | url=https://www.ncbi.nlm.nih.gov/gene/836 |title = CASP3 caspase 3 [Homo sapiens (human)] - Gene - NCBI}}

Caspase-3 is activated in the apoptotic cell. Caspase-3 activation is a cell requirement during the early stages of skeletal myoblast differentiation. Its catalytic site involves the sulfohydryl group of Cys-285 and the imidazole ring of His-237. The caspase-3 His-237 stabilizes the target Aspartate, causing the break of the association of ICAD and CAD, leaving the endonuclease CAD active, allowing it to degrade chromosomal DNA.

Once the inhibitor is released, and for the CAD monomers to function properly, they must come together to form a functional dimer with vertical symmetry.

Interactions

DFFB has been shown to interact with DFFA.{{cite journal | vauthors = Ewing RM, Chu P, Elisma F, Li H, Taylor P, Climie S, McBroom-Cerajewski L, Robinson MD, O'Connor L, Li M, Taylor R, Dharsee M, Ho Y, Heilbut A, Moore L, Zhang S, Ornatsky O, Bukhman YV, Ethier M, Sheng Y, Vasilescu J, Abu-Farha M, Lambert JP, Duewel HS, Stewart II, Kuehl B, Hogue K, Colwill K, Gladwish K, Muskat B, Kinach R, Adams SL, Moran MF, Morin GB, Topaloglou T, Figeys D | title = Large-scale mapping of human protein-protein interactions by mass spectrometry | journal = Molecular Systems Biology | volume = 3 | issue = 1 | pages = 89 | year = 2007 | pmid = 17353931 | pmc = 1847948 | doi = 10.1038/msb4100134 }}{{cite journal | vauthors = McCarty JS, Toh SY, Li P | title = Study of DFF45 in its role of chaperone and inhibitor: two independent inhibitory domains of DFF40 nuclease activity | journal = Biochemical and Biophysical Research Communications | volume = 264 | issue = 1 | pages = 176–80 | date = October 1999 | pmid = 10527860 | doi = 10.1006/bbrc.1999.1497 }}

Cell differentiation

Caspase 3 is responsible for cellular differentiation, although it is unclear how this kind of protein can promote cell apoptosis. Caspase signals resulting from the activation of nuclease CAD indicate that the cell differentiation is due to a CAD modification in chromatin structure.

CAD leads to the initiation of DNA strand breakage, which occurs during terminal differentiation of some cells, such as skeletal muscle cells. The targeting of the p21 promoter is responsible for inducing cell differentiation, which is promoted by modifying the nuclear DNA microenvironment. {{cite journal | vauthors = Larsen BD, Rampalli S, Burns LE, Brunette S, Dilworth FJ, Megeney LA | title = Caspase 3/caspase-activated DNase promote cell differentiation by inducing DNA strand breaks | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 107 | issue = 9 | pages = 4230–5 | date = March 2010 | pmid = 20160104 | pmc = 2840077 | doi = 10.1073/pnas.0913089107 | bibcode = 2010PNAS..107.4230L | doi-access = free }}

Cell diversity originates from cell differentiation, which is attributed to the activation of specific transcription factors. It also depends on the activity of a protein or a common signal. The factor that seems to induce more cell differentiation is caspase-3 protease.{{cite journal | vauthors = Fernando P, Megeney LA | title = Is caspase-dependent apoptosis only cell differentiation taken to the extreme? | journal = FASEB Journal | volume = 21 | issue = 1 | pages = 8–17 | date = January 2007 | pmid = 17093139 | doi = 10.1096/fj.06-5912hyp | doi-access = free | s2cid = 11933880 }} This was identified as the penultimate stage of the apoptosis pathway.

Some studies have shown that this differentiation is due to the presence of many CAD kinase substrates. Referring to the example of skeletal cells, their differentiation is associated with the cleavage of the kinase MST1.{{cite journal | vauthors = Fernando P, Kelly JF, Balazsi K, Slack RS, Megeney LA | title = Caspase 3 activity is required for skeletal muscle differentiation | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 99 | issue = 17 | pages = 11025–30 | date = August 2002 | pmid = 12177420 | doi = 10.1073/pnas.162172899 | bibcode = 2002PNAS...9911025F | pmc=123204| doi-access = free }}

Moreover, it has been observed that CAD participates in the formation of the genome, whose DNA breaks occur during the early stages of cell differentiation. Besides, Caspase 3 induces DNA breaks in the promoter of the p21 factor, and this strand breakup is related to p21 gene expression.

Cell apoptotic death

The protein caspase DNase is an endonuclease involved in the cell apoptotic process that facilitates the DNA breakup.{{cite journal|author-link2=Wong Chi-huey|author-link3=Yuan-Pern Lee | vauthors = Lai SK, Wong CH, Lee YP, Li HY | title = Caspase-3-mediated degradation of condensin Cap-H regulates mitotic cell death | journal = Cell Death and Differentiation | volume = 18 | issue = 6 | pages = 996–1004 | date = June 2011 | pmid = 21151026 | doi = 10.1038/cdd.2010.165 | pmc=3131938}} Cell apoptotic death is a process executed by cysteine proteases{{cite journal | vauthors = Marsden VS, O'Connor L, O'Reilly LA, Silke J, Metcalf D, Ekert PG, Huang DC, Cecconi F, Kuida K, Tomaselli KJ, Roy S, Nicholson DW, Vaux DL, Bouillet P, Adams JM, Strasser A | title = Apoptosis initiated by Bcl-2-regulated caspase activation independently of the cytochrome c/Apaf-1/caspase-9 apoptosome | journal = Nature | volume = 419 | issue = 6907 | pages = 634–7 | date = October 2002 | pmid = 12374983 | doi = 10.1038/nature01101 | bibcode = 2002Natur.419..634M | s2cid = 4415828 }} that allows animals to maintain their homeostasis, also regulated by other mechanisms such as growth and cell differentiation. This biological response is characterized by the chromosomal DNA’s degradation into tiny fragments within the nucleus of the cell.{{cite journal | vauthors = Enari M, Sakahira H, Yokoyama H, Okawa K, Iwamatsu A, Nagata S | title = A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD | journal = Nature | volume = 391 | issue = 6662 | pages = 43–50 | date = January 1998 | pmid = 9422506 | doi = 10.1038/34112 | bibcode = 1998Natur.391...43E | s2cid = 4407426 }} After many investigations and research, it was possible to ensure that Caspase-activated DNase is mainly responsible for this destruction due to a long list of stimuli.

One of the experiments conducted by the investigators to validate the theory involved introducing a mutated form of the protein into both TF-1 human cells and Jurkat cells, which had previously responded to the usual (non-mutated) form of the endonuclease and had died of apoptosis. As a result, these cells died, taking into account this genetic modification, but they did not show DNA breakup. This was the key evidence to prove that the CAD form is implicated in this part of the process, because without its contribution, the fragmentation would not have occurred.{{cite journal | vauthors = McIlroy D, Sakahira H, Talanian RV, Nagata S | title = Involvement of caspase 3-activated DNase in internucleosomal DNA cleavage induced by diverse apoptotic stimuli | journal = Oncogene | volume = 18 | issue = 31 | pages = 4401–8 | date = August 1999 | pmid = 10442630 | doi = 10.1038/sj.onc.1202868 | doi-access = free }}

Later, it was found that the way this protein induces the DNA breakup is explained by its forms CAD and ICAD, which facilitate both the entry and exit from the nucleus of the cell.