Protein degradation in all prokaryotic and eukaryotic cells is tightly regulated by ATP-dependent compartmental proteases. These enzymes of the AAA+ family use ATP hydrolysis in a hexameric ATPase motor to drive the mechanical unfolding of protein substrates and their translocation into the sequestered chamber of an associated peptidase for degradation. The major ATP-dependent protease in eukaryotic cells is the 26S proteasome, a 35-subunit complex that degrades proteins marked with poly-ubiquitin chains and thereby controls protein homeostasis as well as numerous vital processes, including transcription, cell division, differentiation, signal transduction, and apoptosis. Despite the great importance of the 26S proteasome for cell viability, its detailed mechanisms for substrate processing and regulation still remain largely elusive. Over the past five years, we were already able to significantly advance our understanding of proteasome structure and function. We established heterologous E. coli-expression systems for the yeast proteasome lid and base subcomplexes, which together with the in-vitro reconstitution of partially recombinant 26S holoenzymes revolutionized mutational, mechanistic, and structural studies of the proteasome. Using cryo-EM, we revealed the complete subunit architecture of the proteasome and discovered major substrate-induced conformational changes that allow deubiquitination, unfolding, and processive translocation. Furthermore, we were able to uncover that the individual ATPase subunits differentially contribute to the activities of the heterohexameric AAA+ motor, and we provided novel biophysical insights into forceful protein unfolding and the mechanochemistry of ATP-dependent proteases by performing single-molecule optical-tweezers measurements on the related bacterial protease ClpXP. Our established biochemical tools, recombinant systems, and site-specific fluorescence-labeling strategies put us into a unique position to tackle the numerous outstanding questions about ubiquitin-mediated protein turnover, the molecular mechanisms of the 26S proteasome and other AAA+ motors, as well as the regulation of pathways connected to the ubiquitin- proteasome system. We will employ a multidisciplinary approach that includes in-vitro biochemical, single- molecule, and atomic-resolution structural studies. Our mechanistic dissection of proteasome function and regulation, together with the characterization of important determinants for cellular substrate selection, will open numerous future opportunities for extending our research to other crucial pathways that feed into or are regulated by the ubiquitin-proteasome system. We are expanding our research to the AAA+ translocase Pex1/Pex6, which is essential for peroxisome biogenesis and delivers ubiquitinated proteins to the proteasome for degradation. Due to the important regulatory functions of the proteasome and its role in cancer biology, our research also has substantial medical relevance and offers great potential for the development of new small- molecule drugs.
The 26S proteasome is the main proteolytic molecular machine in eukaryotic cells that regulates numerous vital processes. This proposal aims to understand in molecular detail the biochemistry, mechanics, and regulation of substrate recognition, ATP-dependent unfolding, and processing by the 26S proteasome and related protein translocases of the AAA+ family, including the peroxisomal Pex1/Pex6 motor and the ClpXP protease from E. coli.
|de la Peña, Andres H; Goodall, Ellen A; Gates, Stephanie N et al. (2018) Substrate-engaged 26S proteasome structures reveal mechanisms for ATP-hydrolysis-driven translocation. Science 362:|
|Gardner, Brooke M; Castanzo, Dominic T; Chowdhury, Saikat et al. (2018) The peroxisomal AAA-ATPase Pex1/Pex6 unfolds substrates by processive threading. Nat Commun 9:135|
|San Martín, Álvaro; Rodriguez-Aliaga, Piere; Molina, José Alejandro et al. (2017) Knots can impair protein degradation by ATP-dependent proteases. Proc Natl Acad Sci U S A 114:9864-9869|
|Rodriguez-Aliaga, Piere; Ramirez, Luis; Kim, Frank et al. (2016) Substrate-translocating loops regulate mechanochemical coupling and power production in AAA+ protease ClpXP. Nat Struct Mol Biol 23:974-981|
|Dambacher, Corey M; Worden, Evan J; Herzik, Mark A et al. (2016) Atomic structure of the 26S proteasome lid reveals the mechanism of deubiquitinase inhibition. Elife 5:e13027|
|Bashore, Charlene; Dambacher, Corey M; Goodall, Ellen A et al. (2015) Ubp6 deubiquitinase controls conformational dynamics and substrate degradation of the 26S proteasome. Nat Struct Mol Biol 22:712-9|
|Yang, Bei; Stjepanovic, Goran; Shen, Qingtao et al. (2015) Vps4 disassembles an ESCRT-III filament by global unfolding and processive translocation. Nat Struct Mol Biol 22:492-8|
|Gardner, Brooke M; Chowdhury, Saikat; Lander, Gabriel C et al. (2015) The Pex1/Pex6 complex is a heterohexameric AAA+ motor with alternating and highly coordinated subunits. J Mol Biol 427:1375-1388|
|Nyquist, Kristofor; Martin, Andreas (2014) Marching to the beat of the ring: polypeptide translocation by AAA+ proteases. Trends Biochem Sci 39:53-60|
|Sen, Maya; Maillard, Rodrigo A; Nyquist, Kristofor et al. (2013) The ClpXP protease unfolds substrates using a constant rate of pulling but different gears. Cell 155:636-646|
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