Our lab is trying to understand the workings of a giant protein machine, the proteasome, which selectively destroys proteins that are no longer needed by the cell or whose presence can cause harm. We want to find out how it finds, unfolds and ingests its substrates.

Proteasomes are the major neutral protease of eukaryotic cells. They constitute the effecter arm of the ubiquitin-proteasome system, which first tags proteins, usually by ubiquitin chain conjugation, and then delivers them to the proteasome, where they are recognized and cleaved to peptides. Proteasomes are ATP dependent proteases. A common feature of these taxonomically and structurally diverse molecular machines is the sequestration of proteolytic sites within a closed chamber. Substrate specificity is usually conferred by the ubiquitin tagging system, which determines which proteins can enter the chamber. Within that closed space, destruction is relatively independent of substrate sequence. Restricting entry to the chamber is critical to orderly cellular life, assuring that only misfolded or functionally redundant proteins will be destroyed. Access is through a narrow pore positioned axially at either end of the barrel-shaped chamber. Entry thus demands a sequence of events that (at the least) include tag recognition, unfolding and insertion. The unfolding step is critical for entry, as folded proteins are too bulky to traverse the narrow pore.

Significant progress has been made in understanding the initial and final steps of regulated protein degradation, substrate recognition and proteolysis, but the intermediate processes of unfolding and insertion have substantially eluded investigation. The functional proteasome, capable of degrading substrates in a native folded conformation, consists of the proteolytic 20S core particle capped at one or both ends by the 19S regulatory complex. The latter complex includes at least 18 distinct proteins, six of which are ATPases. In eukaryotes each of the six is distinct and in yeast all are essential. Hexameric ATPase rings, members of the very diverse AAA ATPase family, have the general capacity to change the conformation of their substrates. This energy-dependent work is fueled by a cycle of ATP binding, hydrolysis and release. It is therefore reasonable to conclude that the ATPases of the proteasome are the key mediators of substrate unfolding and insertion. How ATP-sourced energy is produce and consumed within the proteasome is important both for mechanistic understanding of its proteolytic function and in evaluating the overall energy budget of the cell.

Our lab has shown that ornithine decarboxylase (ODC) is a native substrate of the proteasome which does not require ubiquitin modification, but is instead recognized through its carboxy-terminal degradation tag. This 37 amino acid region of ODC is a molecular mimic of a ubiquitin chain, and can compete with polyubiquitin for proteasome recognition. It is a portable element that can be attached to other proteins, converting them to labile proteins which can associate with the proteasome and undergo unfolding and degradation. The tag is not only a recognition element but also the part of the substrate that first threads its way into the catalytic chamber of the proteasome. The structural simplicity and modular function of this tag has made it possible to design and use artificial substrates with engineered features useful for biochemical and genetic investigation of proteasome function.

One way to understand a system of energy production and delivery is to present it with loads that can drive it to failure, thereby defining the limits of its capacity. This is not easy to do with proteasomes. To perform adequately as general-purpose protein disposal machine they must be engineered, perhaps over-engineered, to deal with substrates of very diverse structure. One substrate that causes the proteasome to fail is the EBNA1 protein of Epstein-Barr virus. It contains an amino acid tract consisting entirely of glycine and alanine residues, termed the glycine-alanine repeat (GAr). The presence of that sequence acts in cis to impair EBNA1 degradation by the proteasome; a GAr is also cis-inhibitory when transferred to various other proteins. Our laboratory recently showed that the presence of a GAr does not prevent substrate recognition by the proteasome, nor does it impair initiation of degradation. It instead causes degradation to stall, rather than go to completion. Stalling can result in the production of partially processed intermediates that are trimmed at one end of the substrate, the end of the protein that first enters the proteasome. Our data were consistent with a model whereby stalling depends on a functional interaction between the GAr and a nearby folded domain of the substrate. We hypothesized that the GAr interacts ineffectively with the ATPases of the proteasome; this reduces the capacity of the ATPases to deliver energy. If a transient reduction of energy delivery associated with passage of the GAr through the ATPases coincides with a transient peak in the energy required for unfolding, this imbalance causes unfolding to fail. Insertion therefore pauses and proteolysis is limited to the portion of the substrate that has already entered the proteolytic 20S chamber. We have been testing various predictions of this model.

In other studies, we have shown that in budding yeast, Saccharomyces cerevisiae, yeast proteasomes act on mammalian ODC, as well as on the endogenous yeast ODC. This conservation of function between yeast and mammalian proteasomes implies that yeast genetics will also help us understand the mechanisms proteasomes use to degrade ODC and, by extension, the general mechanisms it uses for processing substrates. We have designed and implemented genetic screens to investigate the cellular components needed for proteasome function. We are especially interested in those that are functionally important but extrinsic to the proteasome as conventionally defined.