Research
proteome dynamics and control of proteostasis
Each cell must keep millions of protein molecules properly functional and avoid accumulating damaged proteins. This condition, known as proteostasis, is achieved via balance of different activities like translation of new proteins, chaperone-mediated folding, post-translational modifications, elimination of non-functional proteins etc.. Distortion of this balance is detrimental. For example, some of the most socially impactful disorders - Alzheimer’s disease and Parkinson’s disease - stem from the proteostasis failure.
My group is interested in how cells control the functional state of proteins and maintain proteostasis. To answer these questions we are analysing protein dynamics - a “life record” of proteins within cells including when and how proteins fold, assemble into complexes, get modified and eliminated. By observing dynamics of proteins and protein complexes in normal cells and under conditions of proteostasis failure we aim to understand the mechanisms that govern proteostasis.
We are conducting our research primarily in budding yeast and applying a broad range of techniques including biochemistry, genetics and mathematical modeling. Although budding yeast is a unicellular organism, it is a well-established system to study proteins and protein complexes, and is extremely well-suited for biochemical and genetic work. The evolutionary conservation of the proteostasis control machinery also reassures that our results are relevant for a wider research community and for biomedical applications.
In the News
Teaching
Publications
Academic literature review
See a complete overview of publications in Cristin.
Dultz, E., Wojtynek, M., Medalia, O., and Onischenko, E. (2022). The Nuclear Pore Complex: Birth, Life, and Death of a Cellular Behemoth. Cells 11.
Kralt, A., Wojtynek, M., Fischer, J.S., Agote-Aran, A., Mancini, R., Dultz, E., Noor, E., Uliana, F., Tatarek-Nossol, M., Antonin, W., Onischenko E., Medalia, O. and Weis, K. (2022) An amphipathic helix in Brl1 is required for nuclear pore complex biogenesis in S. cerevisiae eLife 11:e78385
Onischenko, E.*, Noor, E.*, Fischer, J.*, Gillet, L., Wojtynek, M., Vallotton, P., and Weis, K. (2020). Maturation Kinetics of a Multiprotein Complex Revealed by Metabolic Labeling. Cell
Vallotton, P., Rajoo, S., Wojtynek, M., Onischenko, E., Kralt, A., Derrer, C.P., and Weis, K. (2019). Mapping the native organization of the yeast nuclear pore complex using nuclear radial intensity measurements. Proc Nat Acad Sci USA 116, 14606-14613.
Rajoo, S., Vallotton, P., Onischenko, E., and Weis, K. (2018). Stoichiometry and compositional plasticity of the yeast nuclear pore complex revealed by quantitative fluorescence microscopy. Proc Nat Acad Sci USA 115, E3969-E3977.
Onischenko, E., Tang, J.H., Andersen, K.R., Knockenhauer, K.E., Vallotton, P., Derrer, C.P., Kralt, A., Mugler, C.F., Chan, L.Y., Schwartz, T.U., et al. (2017). Natively Unfolded FG Repeats Stabilize the Structure of the Nuclear Pore Complex. Cell 171, 904-917 e919.
Andersen, K.R.*, Onischenko, E.*, Tang, J.H., Kumar, P., Chen, J.Z., Ulrich, A., Liphardt, J.T., Weis, K., and Schwartz, T.U. (2013). Scaffold nucleoporins Nup188 and Nup192 share structural and functional properties with nuclear transport receptors. eLife 2, e00745.
Onischenko, E., and Weis, K. (2011). Nuclear pore complex-a coat specifically tailored for the nuclear envelope. Curr Opin Cell Biol 23, 293-301.
Onischenko, E., Stanton, L.H., Madrid, A.S., Kieselbach, T., and Weis, K. (2009). Role of the Ndc1 interaction network in yeast nuclear pore complex assembly and maintenance. J Cell Biol 185, 475-491.
Buch, C., Lindberg, R., Figueroa, R., Gudise, S., Onischenko, E., and Hallberg, E. (2009). An integral protein of the inner nuclear membrane localizes to the mitotic spindle in mammalian cells. J Cell Sci 122, 2100-2107.
Onischenko, E.A., Crafoord, E., and Hallberg, E. (2007). Phosphomimetic mutation of the mitotically phosphorylated serine 1880 compromises the interaction of the transmembrane nucleoporin gp210 with the nuclear pore complex. Exp Cell Res 313, 2744-2751.
Onischenko, E.A., Gubanova, N.V., Kiseleva, E.V., and Hallberg, E. (2005). Cdk1 and okadaic acid-sensitive phosphatases control assembly of nuclear pore complexes in Drosophila embryos. Mol Biol Cell 16, 5152-5162.
Onischenko, E.A., Gubanova, N.V., Kieselbach, T., Kiseleva, E.V., and Hallberg, E. (2004). Annulate lamellae play only a minor role in the storage of excess nucleoporins in Drosophila embryos. Traffic 5, 152-164.
Projects
Understanding Nuclear Pore Complex biogenesis
In eukaryotic cells genome is confined within membrane-enclosed cell nucleus requiring intense macromolecular communication across the nuclear border. It is estimated that in all cells in our body around 1kg of macromolecules cross the nuclear membrane every minute. This traffic is accomplished by multiprotein Nuclear Pore Complex (NPC) channels embedded in the nuclear membrane. The are ~ 2000 NPCs in the human cell nucleus and ~ 150 in the yeast nucleus.
The NPCs are gigantic mucltiprotein channels measuring around 100 nm in diameter and consisting of 500-1000 nucleoporin proteins depending on the species (see image on the side). Some or the numerous nucleoporins form NPC scaffold while and others fill up the NPC transport channel thanks to special naively disordered Phenyalanine-Glycine (FG) repeat segments. Wheres FG segments prevent free diffusion of macromolecules above ~ 40 kDa, specific Nuclear Transport Receptors (NTRs) mediate directional nucleoytoplamic transport of macromoleculer cargos that could be as large as the viral particles comparable in dimensions to the NPC itself.
To make a new NPC cells must seamlessly insert the gigantic complex into the nuclear membrane and if the NPC is out of order they must be somehow eliminated or repaired to maintain them properly functional. It is especially intriguing how this is accomplished in cells that never divide (like neurones) and therefore must control their NPCs while keeping the nuclear membrane intact. In fact NPC dysfunction in the non-proliferating cell types recently attracted attention as the hotspot of age-related disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's and Parkinson's diseases and various myodystrophies.
Our group is interested in understanding cellular control over the events of the NPC lifecycle and the challenges posed by the intact nuclear membrane. We address these topics by investigating NPC lifecycle from assembly to elimination in budding yeast, which is an excellent genetic and biochemical model system. Although yeast is a unicellular organism, the yeast NPCs always stay in the intact nuclear membrane, keeping many parallels with the NPCs with non-dividing human cells. Thanks to 90 minute generation time, any genetic manipulations in yeast take only couple of days thus allowing to test various hypotheses.
High-throughput analysis of protein dynamics
Protein dynamics can be thought of as a chain of events that cellular proteins undergo from the moment of biosynthesis and until elimination. Examples of such events are synthesis on ribosomes, protein folding, interaction with other proteins, proteasomal degradation etc... In spite of the paramount importance, protein dynamics is a highly understudied topic. For example, the assembly pathways (the way proteins interact with one another to form complexes) are known only for a handful of ~4000 human protein complexes.
This project focuses on the development of tools to explore dynamics of cellular protein complexes.Our approach to tackle protein dynamics is based on the idea that biogenesis of protein complexes is seen as the flow of metabolic reactions except that the "metabolites" are not typical small molecules but cellular proteins and their assemblies. Based on this idea we currently developed an approach entitled kinetic analysis of incorporation rates in macromolecular assemblies (KARMA) that utilises a combination of isotope metabolic labelling and quantitative mass spectrometry to dissect in vivo dynamics of protein complexes. This project amis at development of the analytical techniques to address protein dynamics of various cellular protein complexes, host-pathogen interactions or other kinds of biomolecules.
Dynamic profiling of proteostasis network
Quality of cellular proteins is controlled by a sophisticated machinery - proteostasis network - including folding chaperones, disaggregates, autophagy machinery, proteasome and numerous other factors. It is estimated that more than 10% of cellular proteome is devoted to the protein quality control. What is the functional specialisation of the PN elements? At which stage of the protein's lifecycle the PN interrogates its clients? We are seeking answers to these questions by profiling in a time-resolved manner the interactions of PN with the cellular proteome using quantitative mass-spectrometry and mathematical models based on proteomic data.