Daniel A. Kraut

ATP-dependent Proteases Differ Substantially in Their Ability to Unfold Globular Proteins

ATP-dependent proteases control the concentrations of hundreds of regulatory proteins and remove damaged or misfolded proteins from cells. They select their substrates primarily by recognizing sequence motifs or covalent modifications. Once a substrate is bound to the protease, it has to be unfolded and translocated into the proteolytic chamber to be degraded. Some proteases appear to be promiscuous, degrading substrates with poorly defined targeting signals, which suggests that selectivity may be controlled at additional levels. Here we compare the abilities of representatives from all classes of ATP-dependent proteases to unfold a model substrate protein and find that the unfolding abilities range over more than 2 orders of magnitude. We propose that these differences in unfolding abilities contribute to the fates of substrate proteins and may act as a further layer of selectivity during protein destruction.

Disordered proteinaceous machines.

Monika Fuxreiter,† Agnes Toth-Petroczy,‡ Daniel A. Kraut, Andreas T. Matouschek, Roderick Y. H. Lim, Bin Xue, Lukasz Kurgan, and Vladimir N. Uversky* †MTA-DE Momentum Laboratory of Protein Dynamics, Department of Biochemistry and Molecular Biology, University of Debrecen, Nagyerdei krt. 98, H-4032 Debrecen, Hungary ‡Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 7610001, Israel Department of Chemistry, Villanova University, 800 East Lancaster Avenue, Villanova, Pennsylvania 19085, United States Section of Molecular Genetics and Microbiology, Institute for Cellular & Molecular Biology, The University of Texas at Austin, 2506 Speedway, Austin, Texas 78712, United States Biozentrum and the Swiss Nanoscience Institute, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Department of Cell Biology, Microbiology and Molecular Biology, College of Fine Arts and Sciences, and Department of Molecular Medicine and USF Health Byrd Alzheimer’s Research Institute, Morsani College of Medicine, University of South Florida, Tampa, Florida 33612, United States Department of Electrical and Computer Engineering, University of Alberta, Edmonton, AB T6G 2R3, Canada Institute for Biological Instrumentation, Russian Academy of Sciences, 142290 Pushchino, Moscow Region 119991, Russia

Sequence- and Species-Dependence of Proteasomal Processivity

The proteasome is the degradation machine at the center of the ubiquitin-proteasome system and controls the concentrations of many proteins in eukaryotes. It is highly processive so that substrates are degraded completely into small peptides, avoiding the formation of potentially toxic fragments. Nonetheless, some proteins are incompletely degraded, indicating the existence of factors that influence proteasomal processivity. We have quantified proteasomal processivity and determined the underlying rates of substrate degradation and release. We find that processivity increases with species complexity over a 5-fold range between yeast and mammalian proteasome, and the effect is due to slower but more persistent degradation by proteasomes from more complex organisms. A sequence stretch that has been implicated in causing incomplete degradation, the glycine-rich region of the NFκB subunit p105, reduces the proteasomes ability to unfold its substrate, and polyglutamine repeats such as found in Huntingtons disease reduce the processivity of the proteasome in a length-dependent manner.

Proteasomal degradation from internal sites favors partial proteolysis via remote domain stabilization.

The ubiquitin-proteasome system controls the concentrations of hundreds of regulatory proteins and removes misfolded and damaged proteins in eukaryotic cells. The proteasome recognizes ubiquitinated proteins and then engages its substrates at unstructured initiation regions. After initiation, it proceeds along the polypeptide chain, unraveling folded domains sequentially and degrading the protein completely. In vivo the proteasome can, and likely often does, initiate degradation at internal sites within its substrates, but it is not known how this affects the outcome of the degradation reaction. Here we find that domains flanking the initiation region can protect each other against degradation without interacting directly. The magnitude of this effect is related to the stability of both domains and can be tuned from complete degradation to complete protection of one domain. Partial proteasomal degradation has been observed in the cell in three signaling pathways and is associated with internal initiation. Thus, the basic biochemical mechanism of remote stabilization of protein domains is important in proteasome biology.

Slippery substrates impair ATP-dependent protease function by slowing unfolding

Background: ATP-dependent proteases cannot fully degrade so-called “slippery” substrates. Results: Reanalysis indicates ClpXP has a reduced unfolding rate for slippery substrates, not a faster substrate release rate. Conclusion: The proteasome and ClpXP share a common mechanism of inhibition by slippery substrates. Significance: Comparison of the mechanisms of ATP-dependent proteases across species help explain how these unfolding machines work and evolve. ATP-dependent proteases are responsible for most energy-dependent protein degradation across all species. Proteases initially bind an unstructured region on a substrate and then translocate along the polypeptide chain, unfolding and degrading protein domains as they are encountered. Although this process is normally processive, resulting in the complete degradation of substrate proteins to small peptides, some substrates are released prematurely. Regions of low sequence complexity within the substrate such as the glycine-rich region (GRR) from p105 or glycine-alanine repeats (GAr) from the EBNA1 (Epstein-Barr virus nuclear antigen-1) protein, can trigger partial degradation and fragment release. Loss of processivity could be due to inability to hold on to the substrate (faster release) or inability to unfold and degrade a substrate domain (slower unfolding). I previously showed that the GRR slows domain unfolding by the proteasome (Kraut, D. A., Israeli, E., Schrader, E. K., Patil, A., Nakai, K., Nanavati, D., Inobe, T., and Matouschek, A. (2012) ACS Chem. Biol. 7, 1444–1453). In contrast, a recently published study concluded that GArs increase the rate of substrate release from ClpXP, a bacterial ATP-dependent protease (Too, P. H., Erales, J., Simen, J. D., Marjanovic, A., and Coffino, P. (2013) J. Biol. Chem. 288, 13243–13257). Here, I show that these apparently contradictory results can be reconciled through a reanalysis of the ClpXP GAr data. This reanalysis shows that, as with the proteasome, low complexity sequences in substrates slow their unfolding and degradation by ClpXP, with little effect on release rates. Thus, despite their evolutionary distance and limited sequence identity, both ClpXP and the proteasome share a common mechanism by which substrate sequences regulate the processivity of degradation.

How to pick a protein and pull at it

In this issue of Nature Structural & Molecular Biology, work on the bacterial AAA+ machine ClpX provides insight into how the ATPase subunits exert a translocating force on their substrates.

Slipping up: Partial substrate degradation by ATP‐dependent proteases

ATP‐dependent proteases are present in all organisms, where they are responsible for much of intracellular protein degradation. Most proteins are processively unfolded and degraded into small peptides; however, in a few so‐called slippery substrates, the protease stalls at a folded domain and releases a large protein fragment. In this review, we describe the properties of physiological slippery substrates that are processed in this manner by ATP‐dependent proteases and the recent advances that have been made in understanding the mechanism underlying their partial degradation.

Substrate Ubiquitination Controls the Unfolding Ability of the Proteasome

In eukaryotic cells, proteins are targeted to the proteasome for degradation by polyubiquitination. These proteins bind to ubiquitin receptors, are engaged and unfolded by proteasomal ATPases, and are processively degraded. The factors determining to what extent the proteasome can successfully unfold and degrade a substrate are still poorly understood. We find that the architecture of polyubiquitin chains attached to a substrate affects the ability of the proteasome to unfold and degrade the substrate, with K48- or mixed-linkage chains leading to greater processivity than K63-linked chains. Ubiquitin-independent targeting of substrates to the proteasome gave substantially lower processivity of degradation than ubiquitin-dependent targeting. Thus, even though ubiquitin chains are removed early in degradation, during substrate engagement, remarkably they dramatically affect the later unfolding of a protein domain. Our work supports a model in which a polyubiquitin chain associated with a substrate switches the proteasome into an activated state that persists throughout the degradation process.

Pup grows up: in vitro characterization of the degradation of pupylated proteins.

By reconstituting the recently discovered prokaryotic ubiquitin-like protein (Pup)--proteasome degradation system in vitro, Weber-Ban and colleagues (Striebel et al, 2010) elucidate its mechanism and describe a surprising variation on the established principles of protease targeting. Nevertheless, their findings suggest that the bacterial and eukaryotic systems follow the same overall principles even if the details differ.

Correction to Disordered Proteinaceous Machines

A.M. acknowledges support from the National Institutes of Health (U54GM105816) and the Welch Foundation (F-1817). A.M.’s name appeared as Andreas T. Matouschek. His preferred name for publication is as above, Andreas Matouschek.