Investigation of structure and function of p63 in the quality control of oocytes
Final Report Abstract
The protein p63 is a homolog of the famous tumor suppressor p53. Knock-out mouse studies have, however, demonstrated that p63 is not a typical tumor suppressor but a transcription factor involved in developmental processes. So far two specific functions of p63 have been identified, both connected to a specific isoform of the protein. The ΔNp63α isoform gets highly expressed in the basal layer of epithelial tissue where it regulates proliferation and differentiation of keratinocytes. Mutations in p63 are causative of five different human syndromes that are characterized by development abnormalities of the skin and/or limbs. In particular the Hay-Wells syndrome is characterized by life-threatening skin lesions that result in chronic infections. Mutations in the SAM domain of p63 have been identified as the cause of this particular syndrome, a molecular mechanism, however, remained unclear. As part of this grant we have investigated the molecular mechanism of the Hay-Wells syndrome. We could show that mutations in the SAM domain destabilize this domain only moderately with melting temperatures still far above the body temperature. However, we could also show that unfolding of the mutant SAM domains is irreversible while the unfolding of the wild type domain is reversible. This led to the model that the Hay-Wells syndrome is based not on a loss of function but a gain of function mechanism by aggregation of the p63 SAM domain. In collaboration with the group of Caterina Missero, University of Naples, we could indeed confirm in a mouse model that p63 bearing Hay-Wells syndrome mutations lead to p63 aggregation in keratinocytes. Our results have for the first time identified the molecular mechanism of this disease. The paper has been mentioned in several online magazines, including FOCUS online. The other function of p63 is that of a quality control factor in oocytes. Females are born with a finite number of oocytes that are arrested in prophase of meiosis I. During this period, which can last up to 50 years in humans, oocytes express a high concentration of the TAp63α isoform of p63. We had shown that in resting oocytes, TAp63α is kept in an closed, autoinhibited and only dimeric conformation. Detection of DNA damage leads to the activation of a kinase cascade that results in the formation of the activated and tetrameric state of TAp63α which through expression of the two pro-apoptotic BH3-only proteins PUMA and NOXA induces apoptosis in the damaged oocytes. Chemotherapeutic drugs used to treat cancer patients as well as patients with some autoimmune diseases act by causing DNA damage with the aim to induce apoptosis in cancer cells. As DNA damage is also induced in oocytes the TAp63α based quality control program gets activated, resulting frequently in infertility and loss of ovarian endocrine function of female cancer patients. To prevent infertility and early start of menopause inhibition of oocyte loss with a fertoprotective therapy is of great importance. The development of such a therapy is particularly important for protecting fertility of children suffering from childhood cancers. The survival rate of children with childhood cancers has dramatically increased over the last decades and it is estimated that currently every 250th adult is a childhood cancer survivor. As part of this grant we could show that p63 gets activated to the full tetramer by consecutive phosphorylation by two different kinases. While Chk2 phosphorylates exactly one serine that does not lead to a change in the oligomeric state, this modification recruits the second kinase CK1 which adds four more phosphorylation sites. We could show that inhibition of CK1 with kinase inhibitors rescues oocytes in mouse ovary cultures from apoptosis induced by common chemotherapeutics such as Doxorubicin or Cisplatin. Our results have identified a new target for the future development of a fertoprotective therapy. Our paper was highlighted by a News&Views in NSMB and mentioned by many online journals resulting – according to the statistics of NSMB – in belonging to the 98th percentile (ranked 3,845th) of the 248,543 tracked articles of a similar age in all journals with respect to media attention.
Publications
- (2012). Loss of p63 and its microRNA-205 target results in enhanced cell migration and metastasis in prostate cancer. Proc Natl Acad Sci U S A 109, 15312-15317
Tucci P, Agostini M, Grespi F, Markert EK, Terrinoni A, Vousden KH, Muller PA, Dötsch V, Kehrloesser S, Sayan BS, Giaccone G, Lowe SW, Takahashi N, Vandenabeele P, Knight RA, Levine AJ, Melino G
(See online at https://doi.org/10.1073/pnas.1110977109) - (2013). Analysis of the oligomeric state and transactivation potential of TAp73α. Cell Death Differ 20, 1008-1016
Luh LM, Kehrloesser S, Deutsch GB, Gebel J, Coutandin D, Schäfer B, Agostini M, Melino G, Dötsch V
(See online at https://doi.org/10.1038/cdd.2013.23) - (2015). Functional interplay between MDM2, p63/p73 and mutant p53. Oncogene 34, 4300-10
Stindt MH, Muller PA, Ludwig RL, Kehrloesser S, Dötsch V, Vousden KH
(See online at https://doi.org/10.1038/onc.2014.359) - (2016). Intrinsic aggregation propensity of the p63 and p73 TI domains correlates with p53R175H interaction and suggests further significance of aggregation events in the p53 family. Cell Death Differ 23, 1952-1960
Kehrloesser S, Osterburg C, Tuppi M, Schäfer B, Vousden KH, Dötsch V
(See online at https://doi.org/10.1038/cdd.2016.75) - (2016). Mechanism of TAp73 inhibition by ΔNp63 and structural basis of p63/p73 hetero-tetramerization. Cell Death Differ 23, 1930-1940
Gebel J, Luh LM, Coutandin D, Osterburg C, Löhr F, Schäfer B, Frombach AS, Sumyk M, Buchner L, Krojer T, Salah E, Mathea S, Güntert P, Knapp S, Dötsch V
(See online at https://doi.org/10.1038/cdd.2016.83) - (2016). Quality control in oocytes by p63 is based on a spring-loaded activation mechanism on the molecular and cellular level. Elife pii: e13909
Coutandin D, Osterburg C, Srivastav RK, Sumyk M, Kehrloesser S, Gebel J, Tuppi M, Hannewald J, Schäfer B, Salah E, Mathea S, Müller-Kuller U, Doutch J, Grez M, Knapp S, Dötsch V
(See online at https://doi.org/10.7554/elife.13909) - (2017) Control mechanisms in germ cells mediated by p53 family proteins. J. Cell Science 130, 2663-2671
Gebel J, Tuppi M, Krauskopf K, Coutandin D, Pitzius S, Kehrloesser S, Osterburg C, Dötsch V
(See online at https://doi.org/10.1242/jcs.204859) - (2018) Oocyte DNA damage quality control requires consecutive interplay of CHK2 and CK1 to activate p63. Nat Struct Mol Biol, 25, 261-269
Tuppi M, Kehrloesser S, Coutandin DW, Rossi V, Luh LM, Strubel A, Hötte K, Hoffmeister M, Schäfer B, De Oliveira T, Greten F, Stelzer EHK, Knapp S, De Felici M, Behrends C, Klinger FG, Dötsch V
(See online at https://doi.org/10.1038/s41594-018-0035-7) - (2018) Protein aggregation of the p63 transcription factor underlies severe skin fragility in AEC syndrome. Proc Natl Acad Sci U S A, 115, E906-E915
Russo C, Osterburg C, Sirico A, Antonini D, Ambrosio R, Würz JM, Rinnenthal J, Ferniani M, Kehrloesser S, Schäfer B, Güntert P, Sinha S, Dötsch V, Missero C
(See online at https://doi.org/10.1073/pnas.1713773115)
