Gene Section Review

Atlas of Genetics and Cytogenetics in Oncology and Haematology

OPEN ACCESS JOURNAL INIST-CNRS Gene Section Review

BOK (BCL2 family apoptosis regulator BOK) Pinelopi I. Artemaki, Andreas Scorilas, Christos K. Kontos Department of Biochemistry and Molecular Biology, National and Kapodistrian University of Athens, Athens, Greece / [email protected]

Published in Atlas Database: April 2019 Online updated version : http://AtlasGeneticsOncology.org/Genes/BOKID824ch2q37.html Printable original version : http://documents.irevues.inist.fr/bitstream/handle/2042/70575/04-2019-BOKID824ch2q37.pdf DOI: 10.4267/2042/70575 This article is an update of : Yakovlev AG. BOK (BCL2-related ovarian killer). Atlas Genet Cytogenet Oncol Haematol 2007;11(2)

distinct fifth exons with a length of 164, 50 and 19 nt Abstract have been identified. Review on BOK, with data on DNA, on the protein Regarding the two possible first exons, the first is encoded, and where the gene is implicated. 249-nt long and the second one has a length of 171 Keywords nt. However, the length of the second possible exon is BOK; Apoptosis calculated based on in silico analysis. The 5' untranslated region (UTR) has not been extensively Identity studied, and therefore an uncertainty exists regarding Other names: Mtd, BOKL, BCL2-like 9, BCL2L9, the 5' end. The exon 3 consists of 193 nt, the exon 4 MGC4631 of 129 nt, and the exon 5 of 164 nt. The exon 3 is an Local order outcome of computational analysis of expressed sequence tags (ESTs) and has not been validated yet. MIR3133, STK25, BOK, THAP4, ATG4B Furthermore, there is evidence of an extension in the HGNC (Hugo): BOK second exon and a truncated version of the sixth exon Location: 2q37.3 lacking an internal segment in human, based on our preliminary results and their existence in other DNA/RNA primates, such as in chimpanzee. Description Transcription The BCL2 ovarian killer gene (BOK) is located on The aforementioned transcript NM_032515.4 is the chromosome 2q and comprises 6 exons and 5 considered as mRNA and encodes the protein intervening introns. isoform NP_115904.1. In silico analysis revealed The number of the verified transcripts is nine and that 3 (MK328044.1, MK328045.1, MK328047.1) eight of them have recently been discovered and out of the 9 transcripts have an open reading frame submitted to GenBank (NM_032515.4, and could hence be translated to a functional protein MK354020.1, MK354019.1, MK354018.1, isoform. MK354017.1, MK328047.1, MK328046.1, Moreover, there is a predicted transcript, derived MK328045.1, MK328044.1). from computational analysis, with indications of So far, 2 alternative, untranslated first exons, 5 being a coding mRNA. The presence of three alternative second exons with a length of 249, 119, different stop codons is well-worth to be mentioned. 138, 100, and 97 nucleotides (nt) respectively, and 3 The rest of the transcripts do not seem to have an open reading frame and, therefore, are considered as candidates for non-sense mediated mRNA decay.

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Figure 1. Schematic illustration of BOK transcripts structure with their accession number. The boxes illustrate the exons, the lines illustrate the introns, while the numbers inside the boxes and above the lines represent the length of the exons and the introns, respectively. The presence of the two parallel lines (//) indicates that these shapes are not constructed based on a scale. The grey-colored regions of the boxes represent the coding regions of the mRNA, while the uncolored ones depict the non- coding regions. The question marks (?) symbolize an undetermined untranslated region (UTR), while the arrows and the asterisks indicate the position of the translation start (ATG) codon and the translation stop (TGA) codon, respectively.

Extensive expression sequence tag (EST) analysis possible regulating properties as far as the BOK- led to the discovery of many more transcripts, induced apoptosis is concerned. Its presence has only resulting from alternative splicing. been validated in testis and in particular cancer The alternative splicing of BOK pre-mRNA tissues, but not in the majority of normal ones producing several distinct transcripts is expected to (Zhang et al., 2009). result in protein isoforms with a different pattern of BCL2 homology (BH) domains or to transcripts with Protein regulatory function. For instance, the presence of a potential transcript, derived from EST analysis, Description lacking exons 2, 3, and 4 is an example of a possible The BOK gene encodes a main protein isoform of alternative BH motif. the BCL2 family, named BOK-L, with a length of These results underline the need of further research 212 amino acid residues and a mass of 23.3 kDa. It in the field of alternative splicing in BOK, as it will is characterized by the presence of the BH1, BH2, shed light on the enigmatic cellular role of BOK. BH3, and BH4 domains, a C-terminal Regarding its expression levels, high RNA levels transmembrane domain, and a nuclear export signal. have been observed in fat, liver, lung, kidney and The existence of the BH4 domain is considered as prostate tissue. However, its expression in particular typical feature for BCL2 family proteins with anti- cancerous tissues, as it defined by distinct research apoptotic function. Therefore, this finding creates an studies, seems to be downregulated. ambiguity regarding the cellular role of this Pseudogene molecule (Soleymanlou et al., 2005). None. Even though pseudogenes are absent, an Except of the full-length protein, an additional antisense gene has been characterized, known as isoform has been detected with a truncated BH3 BOKAS. Both genes are located in the same region, domain (170 aa) in mouse, named Bok-S. It is but in different orientations. There is a restricted hypothesized that this isoform derives from the overlap between these two genes in the region of the deletion of 43 aa residues from the main isoform and first exon of BOK. The BOKAS comprises two leads to a fusion of the N-terminal-half of its BH3 exons and is considered as a non-coding RNA with domain to the C-terminal half of the BH1 domain.

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Figure 2. Schematic illustration of the two curated amino acid sequences of BOK protein isoforms in Homo sapiens, accompanied by the location of their BH domains.

An alternative splice isoform has been discovered in recent valid research study demonstrate the ability of human tissues (BOK-P, also known as Mtd-P) with BOK to induce cytochrome c release, and a length of 135 aa residues. subsequent apoptosis in BAK/BAX deficient cell This isoform is considered to result from the deletion model systems. Therefore, the ability of BOK to of exon 2 and part of the 5' UTR (249 bp deletion). induce apoptosis in a BAK- and BAX- independent Due to the 249-bp deletion, this isoform comprises way is proposed (Einsele-Scholz et al., 2016). only BH1 and BH2 domains, and perhaps a partial Moreover, BOK seems to play a role in multiple BH3 domain (Soleymanlou et al., 2005). Because of signaling pathways, some of which are mentioned the absence of BH4 domain in this isoform, the pro- below; it is involved in granulose cell apoptosis apoptotic function of BOK could be attributed to this through Caspase-3 ( CASP3) activation particular molecule. (Jaaskelainen et al., 2010) and in the intrinsic Expression apoptotic mechanism depending on the tumor protein p53 ( TP53) (Yakovlev et al., 2004). This It was initially discovered in ovarian tissue, hence mechanism is activated in response to DNA damage. the designation "ovarian killer". After further It is, also, capable of inducing apoptosis without the research, it has been detected in other tissue types as prerequisite of heterodimerization with other pro- well, including brain, liver, rectum, kidney, but its apoptotic members of BCL2 family, when the ER- main expression has been observed in reproductive associated protein degradation (ERAD) system is tissues (Jaaskelainen et al., 2010,Echeverry et al., inhibited. More specifically, BOK, independently of 2013,Llambi et al., 2016). BAX and BAK, participates in mitochondrial outer Localisation membrane permeabilization, leading to cytochrome c release (Einsele-Scholz et al., 2016). In the early BOK is localized in multiple intracellular stages of placental development, regulation of membranes, including ER, Golgi, and associated trophoblast cell proliferation could be achieved by membranes (Echeverry et al., 2013). A fraction of BOK through its effect on the G1/S transition. This BOK has also been detected in outer mitochondrial can be achieved by the regulation of cyclin E1 membrane or in contact sites between the (CCNE1) expression (Ray et al., 2010). Finally, mitochondria and the endoplasmic reticulum (ER) there are indications that BOK can disturb MCL1 (Llambi et al., 2016). The BOK-P isoform has also and Beclin-1 (BECN1) interaction and therefore has been detected in the cytoplasm (Ray et al., 2010). an effect on the induction of autophagy (Kalkat et al., Function 2013). Due to the homology of BOK with BAX and BAK1, BOK protein has been examined more thoroughly in its pro-apoptotic properties were proposed. Contrary the mouse. The results of these studies revealed to BAK and BAX proteins, BOK is not well-studied alternative functions of this molecule. In and its role remains enigmatic, probably because of circumstances of ER stress, BOK induces positive its low expression in the cell lines which are regulation of EIF2AK3 (PKR-like ER kinase, commonly used for experiments. At the beginning, PERK) to initiate a downstream unfolded protein BOK was considered to act similarly to BAX and response and a downstream regulation of BAX/BAK BAK because of the formation of clustered activation, thus leading to the mitochondrial structures in the outer mitochondrial membrane. apoptotic pathway (Carpio et al., 2015). Besides its These structures indicate a possible association with pro-apoptotic role, there are indications that BOK cytochrome c release. However, the results of a has anti-apoptotic properties during neuronal

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apoptotic process. In the aforementioned process, it by its various associations with distinct protein regulates neuronal calcium homeostasis and molecules. More specifically, a strong association mitochondrial bioenergetics, resulting to was revealed between BOK and CALR downregulation of poly ADP-ribose polymerase (Calreticulin), but no significant correlation was (PARP1)-dependent cell death. This mechanism is observed between BOK and other ER-stress considered as a response to N-methyl-D-aspartate markers, including cleaved CASP3 (Carberry et al., excitation (D'Orsi et al., 2016). Finally, Bok seems 2018). to strongly interact with inositol 1,4,5-trisphosphate Non-small cell lung carcinoma (IP3) receptors via the BH4 domain. Regarding the outcome of a research study, this interaction, Prognosis possibly, enhances the structural stability of both IP3 Overexpression of BOK in lung adenocarcinoma- receptor tetramers and Bok, and leads to the derived A549 cells resulted in inhibition of TGFβ- limitation of overexpressed pro-apoptotic activity of induced migration and epithelial-to-mesenchymal BOK. One of the most interesting findings of this transition of these cells. Furthermore, after the study was the protection from degradation, which is comparison of BOK levels among patients with non- provided to BOK by this interaction. Based on the small cell lung carcinoma (NSCLC), tumors of grade similarity between the human and mouse protein 2 and 3 and/ or with nodal metastasis had lower BOK isoforms, we could hypothesize that human BOK expression levels than tumors of grade 1 without protein could be involved in the aforementioned nodal metastasis. These results support the favorable pathways and act in a similar manner. prognostic value of BOK in NSCLC patients. More specifically, there are indications that the BOK Homology overexpression in NSCLC patients is associated with BOK shares high amino acid sequence homology longer overall survival (Moravcikova et al., 2017). with the pro-apoptotic BCL2 family proteins BAK Preeclampsia and BAX (Hsu et al., 1997). Preeclampsia is a disorder that affects 5-8% of pregnancies worldwide, with frequent fatal Mutations consequences for the mother and the fetus, as well. Germinal Preeclampsia has been attributed to extensive trophoblast cell death, which seems to be related To the best of our knowledge, no germinal mutation with the presence of a group of bioactive has been discovered in vivo. sphingolipids, named ceramides. Somatic The aggregation of these structures affects BOK by To the best of our knowledge, none somatic mutation increasing its expression levels, resulting to has been discovered in vivo. However, an in-vitro extensive trophoblast autophagy and death. It is mutation in positions 71-73 in leucine-rich region important to clarify that preeclampsia is also led to significant accumulation of BOK in the correlated with increased phosphorylation of nucleus and to increased apoptotic activity. It is dynamin 1-like protein (DNM1L, also known as significant to mention that BOK is the only BCL2 DRP1). family member possessing this leucine-rich region Based on the results of a recent research study, a (Bartholomeusz et al., 2006). direct effect of BOK on DNM1L expression is highlighted on DNM1L expression, and hence its Implicated in pivotal role in mitochondrial energetics. These results enhance further the importance of BOK in Colorectal cancer this disease and underscore its potential drug Prognosis targeting for possible disease cure (Ausman et al., 2018). There are indications that BOK could act as a possible prognostic biomarker in colorectal cancer (CRC), because of the association of its high References expression levels with poor disease-free and overall Ausman J, Abbade J, Ermini L, Farrell A, Tagliaferro A, Post survival in CRC patients. However, in the M, Caniggia I. Ceramide-induced BOK promotes aforementioned research study, decreased mitochondrial fission in preeclampsia. Cell Death Dis. 2018 Feb 20;9(3):298 expression levels of BOK protein were measured in stage II and III CRC tumors compared to paired non- Bartholomeusz G, Wu Y, Ali Seyed M, Xia W, Kwong KY, Hortobagyi G, Hung MC. Nuclear translocation of the pro- cancerous tissue specimens. These seemingly apoptotic Bcl-2 family member Bok induces apoptosis. Mol contradictory results highlight the differential Carcinog. 2006 Feb;45(2):73-83 function of BOK and its involvement in multiple Carberry S, D'Orsi B, Monsefi N, Salvucci M, Bacon O, Fay activities. This assumption regarding the different J, Rehm M, McNamara D, Kay EW, Prehn JHM. The activities of BOK in CRC is additionally supported

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BAX/BAK-like protein BOK is a prognostic marker in Llambi F, Wang YM, Victor B, Yang M, Schneider DM, colorectal cancer. Cell Death Dis. 2018 Jan 26;9(2):125 Gingras S, Parsons MJ, Zheng JH, Brown SA, Pelletier S, Moldoveanu T, Chen T, Green DR. BOK Is a Non-canonical Carpio MA, Michaud M, Zhou W, Fisher JK, Walensky LD, BCL-2 Family Effector of Apoptosis Regulated by ER- Katz SG. BCL-2 family member BOK promotes apoptosis in Associated Degradation Cell 2016 Apr 7;165(2):421-33 response to endoplasmic reticulum stress Proc Natl Acad Sci U S A 2015 Jun 9;112(23):7201-6 Moravcikova E, Krepela E, Donnenberg VS, Donnenberg AD, Benkova K, Rabachini T, Fernandez-Marrero Y, D'Orsi B, Engel T, Pfeiffer S, Nandi S, Kaufmann T, Bachmann D, Kaufmann T. BOK displays cell death- Henshall DC, Prehn JH. Bok Is Not Pro-Apoptotic But independent tumor suppressor activity in non-small-cell Suppresses Poly ADP-Ribose Polymerase-Dependent Cell lung carcinoma Int J Cancer 2017 Nov 15;141(10):2050- Death Pathways and Protects against Excitotoxic and 2061 Seizure-Induced Neuronal Injury J Neurosci 2016 Apr 20;36(16):4564-78 Ray JE, Garcia J, Jurisicova A, Caniggia I. Mtd/Bok takes a swing: proapoptotic Mtd/Bok regulates trophoblast cell Echeverry N, Bachmann D, Ke F, Strasser A, Simon HU, proliferation during human placental development and in Kaufmann T. Intracellular localization of the BCL-2 family preeclampsia Cell Death Differ 2010 May;17(5):846-59 member BOK and functional implications Cell Death Differ 2013 Jun;20(6):785-99 Soleymanlou N, Wu Y, Wang JX, Todros T, Ietta F, Jurisicova A, Post M, Caniggia I. A novel Mtd splice isoform Einsele-Scholz S, Malmsheimer S, Bertram K, Stehle D, is responsible for trophoblast cell death in pre-eclampsia Johänning J, Manz M, Daniel PT, Gillissen BF, Schulze- Cell Death Differ 2005 May;12(5):441-52 Osthoff K, Essmann F. Bok is a genuine multi-BH-domain protein that triggers apoptosis in the absence of Bax and Yakovlev AG, Di Giovanni S, Wang G, Liu W, Stoica B, Bak J Cell Sci 2016 Aug 1;129(15):3054 Faden AI. BOK and NOXA are essential mediators of p53- dependent apoptosis J Biol Chem 2004 Jul Hsu SY, Kaipia A, McGee E, Lomeli M, Hsueh AJ. Bok is a 2;279(27):28367-74 pro-apoptotic Bcl-2 protein with restricted expression in reproductive tissues and heterodimerizes with selective Zhang H, Gao S, De Geyter C. A natural antisense anti-apoptotic Bcl-2 family members Proc Natl Acad Sci U S transcript, BOKAS, regulates the pro-apoptotic activity of A 1997 Nov 11;94(23):12401-6 human Bok Int J Oncol 2009 Apr;34(4):1135-8

Jæskeläinen M, Nieminen A, Pökkylä RM, Kauppinen M, This article should be referenced as such: Liakka A, Heikinheimo M, Vaskivuo TE, Klefström J, Tapanainen JS. Regulation of cell death in human fetal and Artemaki PI, Scorilas A, Kontos CK. BOK (BCL2 family adult ovaries--role of Bok and Bcl-X(L) Mol Cell Endocrinol apoptosis regulator BOK). Atlas Genet Cytogenet Oncol 2010 Dec 15;330(1-2):17-24 Haematol. 2019; 23(10):299-303. Kalkat M, Garcia J, Ebrahimi J, Melland-Smith M, Todros T, Post M, Caniggia I. Placental autophagy regulation by the BOK-MCL1 rheostat Autophagy 2013 Dec;9(12):2140-53

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