As the endpoint for the ubiquitin-proteasome system, the 26S proteasome is the principal proteolytic machine responsible for regulated protein degradation in eukaryotic cells. The proteasome's cellular functions range from general protein homeostasis and stress response to the control of vital processes such as cell division and signal transduction. To reliably process all the proteins presented to it in the complex cellular environment, the proteasome must combine high promiscuity with exceptional substrate selectivity. Recent structural and biochemical studies have shed new light on the many steps involved in proteasomal substrate processing, including recognition, deubiquitination, and ATP-driven translocation and unfolding. In addition, these studies revealed a complex conformational landscape that ensures proper substrate selection before the proteasome commits to processive degradation. These advances in our understanding of the proteasome's intricate machinery set the stage for future studies on how the proteasome functions as a major regulator of the eukaryotic proteome.

Structure and Function of the 26S Proteasome.

To maintain protein homeostasis, AAA+ proteolytic machines degrade damaged and unneeded proteins in bacteria, archaea and eukaryotes. This process involves the ATP-dependent unfolding of a target protein and its subsequent translocation into a self-compartmentalized proteolytic chamber. Related AAA+ enzymes also disaggregate and remodel proteins. Recent structural and biochemical studies, in combination with direct visualization of unfolding and translocation in single-molecule experiments, have illuminated the molecular mechanisms behind these processes and suggest how remodelling of macromolecular complexes by AAA+ enzymes could occur without global denaturation. In this Review, we discuss the structural and mechanistic features of AAA+ proteases and remodelling machines, focusing on the bacterial ClpXP and ClpX as paradigms. We also consider the potential of these enzymes as antibacterial targets and outline future challenges for the field.

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Mechanistic insights into bacterial AAA+ proteases and protein-remodelling machines.

Inflammasomes are multiprotein platforms that initiate innate immunity by recruitment and activation of caspase-1. The NLRP1B inflammasome is activated upon direct cleavage by the anthrax lethal toxin protease. However, the mechanism by which cleavage results in NLRP1B activation is unknown. In this study, we find that cleavage results in proteasome-mediated degradation of the amino-terminal domains of NLRP1B, liberating a carboxyl-terminal fragment that is a potent caspase-1 activator. Proteasome-mediated degradation of NLRP1B is both necessary and sufficient for NLRP1B activation. Consistent with our functional degradation model, we identify IpaH7.8, a Shigella flexneri ubiquitin ligase secreted effector, as an enzyme that induces NLRP1B degradation and activation. Our results provide a unified mechanism for NLRP1B activation by diverse pathogen-encoded enzymatic activities.

https://science.sciencemag.org/content/sci/early/2019/03/13/science.aau1330.full.pdf

Functional degradation: A mechanism of NLRP1 inflammasome activation by diverse pathogen enzymes

The 26S proteasome is the primary eukaryotic degradation machine and thus is critically involved in numerous cellular processes. The heterohexameric adenosine triphosphatase (ATPase) motor of the proteasome unfolds and translocates targeted protein substrates into the open gate of a proteolytic core while a proteasomal deubiquitinase concomitantly removes substrate-attached ubiquitin chains. However, the mechanisms by which ATP hydrolysis drives the conformational changes responsible for these processes have remained elusive. Here we present the cryo-electron microscopy structures of four distinct conformational states of the actively ATP-hydrolyzing, substrate-engaged 26S proteasome. These structures reveal how mechanical substrate translocation accelerates deubiquitination and how ATP-binding, -hydrolysis, and phosphate-release events are coordinated within the AAA+ (ATPases associated with diverse cellular activities) motor to induce conformational changes and propel the substrate through the central pore.

/pdf/substrate-engaged-26sproteasome-structures-reveal-mechanisms-349zuu0liq.pdf

Substrate-engaged 26Sproteasome structures reveal mechanisms for ATP-hydrolysis-driven translocation.

The ubiquitin proteasome system (UPS) is the main ATP-dependent protein degradation pathway in the cytosol and nucleus of eukaryotic cells. At its centre is the 26S proteasome, which degrades regulatory proteins and misfolded or damaged proteins. In a major breakthrough, several groups have determined high-resolution structures of the entire 26S proteasome particle in different nucleotide conditions and with and without substrate using cryo-electron microscopy combined with other techniques. These structures provide some surprising insights into the functional mechanism of the proteasome and will give invaluable guidance for genetic and biochemical studies of this key regulatory system.

/pdf/regulated-protein-turnover-snapshots-of-the-proteasome-in-4mco39vbrg.pdf

Regulated protein turnover: snapshots of the proteasome in action.

The ClpXP protease unfolds substrates using a constant rate of pulling but different gears.

https://www.cell.com/trends/biochemical-sciences/pdf/S0968-0004(13)00185-0.pdf

Marching to the beat of the ring: polypeptide translocation by AAA+ proteases

ATP-dependent proteases exist in all cells and are crucial regulators of the pro-teome. These biological nanomachines contain a homohexameric molecular motorwhich generates mechanical force to unfold proteins and translocate unstructuredpolypeptides into a degradation chamber for proteolysis. Using E. coli ClpXP asa model system for investigating the function of ATP-dependent proteases, I usedsingle-molecule optical tweezers to explore how individual subunits within the hex-americ motor convert the chemical energy from ATP hydrolysis into the mechanicalwork required to perform protein unfolding and polypeptide translocation.These studies revealed key aspects of motor function that highlight how ATP-dependent proteases are tailored to serve their specific cellular tasks. My analysesindicated that ClpXP translocates in bursts resulting from highly coordinated con-formational changes in two to four ATPase subunits and that these bursts of translo-cation couple to the phosphate release step of the ATP hydrolysis cycle. I foundthat firing of four ATPase subunits is required to successfully unfold GFP, but thatpolypeptide translocation robustly occurs even when only two ATPase subunits fire.Because this work pushed the envelope of single-molecule data analysis techniques,I developed new algorithms to detect steps in passive-mode optical tweezers data,where the noise statistics vary in time.The results discussed in this dissertation improve our understanding of how ClpXPmechanically operates. Strong structural similarities to other ATP-dependent pro-teases, as well as other ring translocases, suggest that the operating principles I havediscerned and describe here may generalize to other biological molecular motors.

Mechanism of protein unfolding and polypeptide translocation by the AAA+ protease ClpXP

/pdf/theoretical-description-of-trapped-low-dimensional-dipoles-1b0mddye1j.pdf

Kristofor Nyquist

Papers

The ClpXP protease unfolds substrates using a constant rate of pulling but different gears.

Marching to the beat of the ring: polypeptide translocation by AAA+ proteases

Mechanism of protein unfolding and polypeptide translocation by the AAA+ protease ClpXP

Theoretical Description of Trapped Low-Dimensional Dipoles in the Strongly-Interacting Regime