Our lab studies a number of proteins and regulatory pathways that promote chromosomal stability through the various stages of the mitotic cell division process. We investigate these players and events in budding yeast (Saccharomyces cerevisiae) and human cells (healthy and cancer cells). Particular interest lies in the activities occurring at the centromeres (including the kinetochore complex that assembles on them), rDNA, and telomeres. Dysregulated activities at these regions trigger accelerated aging, genomic imbalance, and a variety of diseases, including cancer and aneuploidy syndromes. Studying chromosome activities through the cell cycle will allow us to better understand the aetiology of these diseases but also to develop drugs that inhibit or rescue them.
Our research implements genetics, biochemistry and proteomics, molecular biology techniques, high-resolution live-cell (video)microscopy, bioinformatics, systems network biology, structural biology, computational modelling of drug-protein interactions, and chemosynthetic drug development.
Regulation of centromere transcription and kinetochore activity
Following DNA replication, kinetochores hierarchically assemble from 60 (yeast) or 100 proteins (humans) onto the centromere sequences to bi-orient the sister chromatids to the mitotic spindle, and to meditate their faithful transmission into both daughter cells. Using S. cerevisiae and human cells, we study how kinase/ATPase RioK1 promotes the timely activity of kinetochores through the cell cycle.
Kinetochore-inhibiting anti-cancer drugs
Many tumours driven by a variety of well-known mutations are susceptible to reduced kinetochore activity. By using the structure of the human Ndc80 kinetochore subcomplex, in combination with in-house developed biochemical assays, we have identified a number of small molecules that inhibit the interaction between the outer kinetochore and microtubule tips. Via additional chemosynthetic optimization, biological analyses, and mouse cancer models we aspire to convert these compounds both into broad-spectrum anti-cancer drugs and experimental tools that will allow us to investigate still uncharacterized mitotic activities.
Defining RioK1 activity
Using S. cerevisiae and human cells we study the activity of RioK1 at the DNA, RNA and protein levels. We have shown that yeast RioK1 is an upstream signalling kinase that manages a response network comprising hundreds of genes and proteins. Specifically, RioK1 activates growth and proliferation under positive conditions, while restraining them under negative (stress) circumstances. By mapping its activities during cell cycle progression both at a global and single gene/RNA/protein level we wish to describe its biology. This knowledge will help to explain how overexpression of this enzyme, which characterizes up to 20% of all malignancies, dysregulates cell biology and triggers chromosomal instability, culminating in cellular transformation, cancer progression, and metastasis.
Development of RioK1 inhibitors
Using state-of-the-art computational techniques, and a unique intracellular human RioK1 activity assay we are evaluating candidate inhibitors of RioK1. We aspire to convert them into potent medicines to eradicate hitherto incurable tumours. Small compound inhibitors will also prove highly valuable as new research tools to study RioK1 biology.
Activity study and drug-based rescue of PRUNE/PPX1-driven microcephaly
Using S. cerevisiae as a genetic model we study how a conserved, dominant-negative heterozygous mutation in human exopolyphosphatase PRUNE1 (PPX1 in yeast) alters cell cycle progression resulting in the microcephaly syndrome. Using yeast we have also developed a real-time assay that we use to identify compounds that inactivate the PRUNE1/PPX1 mutation, opening a path towards the future treatment of this syndrome.
- Peter De Wulf, PI
- Michela Damizia, post-doctoral researcher
- Ksenia Smurova, post-doctoral researcher
- Valeria Facchini, research assistant
- Anna Bresciani, thesis student
- Chiara Morandini, thesis student
- Riccardo Tonelli, thesis student
- Alessandra Vivian, thesis student
- Nikolas Walteros, thesis student
- Prof. Zoran Culig (Medical University Innsbruck, Austria)
- Prof. Erik Dassi (Univ. Trento)
- Prof. Saverio Minucci (IEO Milano)
- Prof. Romano Silvestri (Univ. Roma LaSapienza)
- Prof. Massimo Zollo (Univ. Napoli)
Damizia M., Moretta G.M., De Wulf P. (2023). Kinase/ATPase RioK1 integrates the oncogenic pathways that determine p53 activity. Submitted.
Handle F., Puhr M., Gruber M., Ph.D.; Andolfi C.; Schäfer G., Klocker H.; Haybaeck J., De Wulf P., Culig Z. (2023). The oncogenic protein kinase/ATPase RIOK1 is up-regulated via the MYC/E2F transcription factor axis in prostate cancer. American Journal of Pathology. In press.
Smurova K., Damizia M., Irene C., Stancari S., Berto G., Perticari G., Iacovella M.G., D’Ambrosio I., Giubettini M., Philippe, R., Baggio C., Callegaro E., Casagranda A., Corsini A., Gentile Polese V., Ricci A., Dassi E., De Wulf P. (2023). Rio1 downregulates centromeric RNA levels to promote the timely assembly of structurally fit kinetochores. Nature Communications. In press. DOI 10.1038/s41467-023-38920-9.
Jurková K., De Wulf P., Cusanelli E. (2021). Nuclear periphery and telomere maintenance: TERRA sets the stage. Trends in Genetics, 37:608-611.
Berto G., Ferreira-Cerca S., De Wulf P. (2019). The Rio1 protein kinases/ATPases: conserved regulators of growth, division, and genomic stability. Current Genetics, 65:457-466.
Iacovella M.G., Bremang M., Basha O., Giaco L., Carotenuto W., Golfieri C., Szakal B., Dal MaschioM., Infantino V., Beznoussenko G.V., Chinnu R.J., Visintin C., Mironov A.A., Visintin R., Branzei D., Ferreia-Cerca S., Yeger-Lotem E., De Wulf P. (2018). Integrating Rio1 activities discloses its nutrient-activated network in Saccharomyces cerevisiae. Nucleic Acids Research, 46: 7586-7611.
Smurova K., De Wulf P. (2018). Centromere and pericentromere transcription: roles and regulation... in sickness and in health. Frontiers in Genetics, 9:674.
Iacovella M.G., Golfieri C., Massari L.F., Busnelli S., Pagliuca C., Dal Maschio M., Infantino V., Visintin R., Mechtler K., Ferreira-Cerca S., De Wulf P. (2015). Rio1 promotes rDNA stability and downregulates RNA polymerase I to ensure rDNA segregation. Nature Communications, 6:6643.
Thapa K.S., Oldani A., Pagliuca C., De Wulf P., Hazbun T.R. (2015). The Mps1 kinase modulates the recruitment and activity of Cnn1CENP-T at Saccharomyces cerevisiae kinetochores. Genetics, 200:79-90.
Bock L.J., Pagliuca C., Kobayashi N., Grove R.A., Oku Y., Shrestha K., Alfieri C., Golfieri C., Oldani A., Dal Maschio M., Bermejo R., Hazbun T.R., Tanaka T.U., De Wulf P. (2012). Cnn1 inhibits the interactions between the KMN complexes of the yeast kinetochore. Nature Cell Biology, 14:614-624.
Nguyen T.L., Cera M.T., Pinto A., Lo Presti L., Hamel E., Conti P., Gussio R., De Wulf P. (2012). Evading Pgp activity in drug-resistant cancer cells: a structural and functional study of antitubulin furan metotica compounds. Molecular Cancer Therapeutics, 11:1103-1111.
Screpanti E., Santaguida S., Nguyen T.L., Silvestri R., Gussio R., Musacchio A., Hamel E., De Wulf P. (2010). A screen for kinetochore-microtubule interaction inhibitors identifies novel antitubulin compounds. PLoS One, 5:e11603.
De Wulf P., Montani F., Visintin R. (2009) Protein phosphatases take the mitotic stage. Current Opinion in Cell Biology, 21:806-815.
Pagliuca C., Draviam V.M., Marco E., Sorger P.K., De Wulf P. (2009). Roles for the conserved Spc105p/Kre28p complex in kinetochore-microtubule binding and the spindle assembly checkpoint. PLoS One, 4:e7640.
Fukagawa T., De Wulf P. (2009). Kinetochore composition, formation and organization. In: "The Kinetochore: from Molecular Discoveries to Cancer Therapy". Eds. De Wulf P., Earnshaw W.C. Springer Publ., New York City, p. 133-191.
Ciferri C., Pasqualato S., Screpanti E., Maiolica A., Polka J., DeLuca J.B., De Wulf P., Salek M., Rappsilber J., Moores C.A., Salmon E.D., Musacchio A. (2008). Implications for kinetochore-microtubule attachment from the structure of an engineered Ndc80 complex. Cell, 133:427-439.
Cohen R.L., Espelin C.W., De Wulf P., Sorger P.K., Harrison S.C., Simons K.T. (2008). Structural and functional dissection of Mif2p, a conserved DNA-binding kinetochore protein. Molecular Biology of the Cell, 19:4480-4491.
De Wulf P., Visintin R. (2008). Cdc14B and APC/C tackle DNA damage. Cell, 134:210-212.
Miranda JJ.M., De Wulf P., Sorger P.K., Harrison S.C. (2005). The yeast DASH complex forms closed rings on microtubules. Nature Structural and Molecular Biology, 12:138-143.
De Wulf P., McAinsh A.D., Sorger P.K. (2003). Hierarchical assembly of the budding yeast kinetochore from multiple subcomplexes. Genes and Development, 17:2902-2921.