protein dynamics and control of proteostasis
Cells must keep millions of protein molecules in a properly working condition and avoid accumulating damaged proteins. This tendency is known as proteostasis. Proteostasis is achieved via balance of different activities including translation of new proteins, chaperone-mediated folding, post-translational modifications and elimination of non-functional proteins. Distortion of this balance is detrimental. For example, some of the most socially impactful disorders - Alzheimer’s disease and Parkinson’s disease - result from the proteostasis failure.
My lab is interested in how cells control the functional state of proteins to maintain proteostasis. To answer these questions we are using biochemical and genetic techniques to study protein dynamics - a “life record” of proteins within cells including when and how proteins fold, assemble into complexes, get modified and eliminated. The analysis of protein dynamics in normal cells and in conditions when the protein’s quality is deteriorated helps us to understand the mechanisms that control proteostasis.
We are conducting our research in budding yeast. Although yeast is a unicellular organism, it is a well-established model system to study proteostasis and is extremely well-suited for biochemical and genetic work. The evolutionary conservation of the proteostasis control machinery also ensures that our results are relevant for a wide research community and for biomedical applications.
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. Proceedings of the National Academy of Sciences of the United States of America 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. Proceedings of the National Academy of Sciences of the United States of America.
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. Current opinion in cell biology 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. The Journal of cell biology 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. Journal of cell science 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. Experimental cell research 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. Molecular biology of the 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.
high-throughput analysis of protein dynamics
Protein dynamics could be thought of as a chain of events that cellular proteins encounter from the moment of biosynthesis and until elimination. The examples of such events are synthesis on ribosomes, folding, interaction with other proteins to form multiprotein complexes or any other changes in the protein structure or interactions. The abnormal flow of protein dynamic events leads to the loss of the protein’s quality and deterrorates cellular function.
In spite of the paramount importance (especially for understanding the control of proteostasis) protein dynamics is a very understudied topic. For example, the assembly pathways (the way proteins interact with one another to form complexes) are well known only for a handful of ~4000 protein complexes identified in mammals alone.
To foster elucidation of protein dynamics, my lab is developing kinetic analysis of incorporation rates in macromolecular assemblies (KARMA) - a high-throughput approach to analyze the protein dynamic events in live cells. The idea behind KARMA is that every newly made protein needs time to transition to a certain dynamic state within a cell, representing one of the protein’s dynamic events. In KARMA, we aim to measure such transition times for thousands of newly-made proteins in a single experiment by taking advantage of biochemical isolation techniques and quantitative mass-spectrometry. These measurements are then integrated together across multiple KARMA experiments to draw the full picture of the protein’s dynamics.
The development of KARMA is an effort at the intersection of biochemistry, quantitative mass-spectrometry and metabolomics. Our work on KARMA is expected to allow researchers to explore a completely uncharted territory in biology and address questions ranging from the control of protein quality to the mechanisms of viral infection.
Onishchenko, Evgeny and Noor, Elad and Fischer, Jonas and Gillet, Ludovic and Wojtynek, Matthias and Vallotton, Pascal and Weis, Karsten, Maturation Kinetics of a Multiprotein Complex Revealed by Metabolic Labeling. CELL-D-20-01041. Available at SSRN: https://ssrn.com/abstract=3596618 or http://dx.doi.org/10.2139/ssrn.3596618