Mammalian Ddi2 is a shuttling factor containing a retroviral protease domain that influences binding of ubiquitylated proteins and proteasomal degradation.

Although several proteasome subunits have been shown to bind ubiquitin (Ub) chains, many ubiquitylated substrates also associate with 26S proteasomes via "shuttling factors." Unlike the well-studied yeast shuttling factors Rad23 and Dsk2, vertebrate homologs Ddi2 and Ddi1 lack a Ub-associated domain; therefore, it is unclear how they bind Ub. Here, we show that deletion of Ddi2 leads to the accumulation of Ub conjugates with K11/K48 branched chains. We found using affinity copurifications that Ddi2 binds Ub conjugates through its Ub-like domain, which is also required for Ddi2 binding to proteasomes. Furthermore, in cell extracts, adding Ub conjugates increased the amount of Ddi2 associated with proteasomes, and adding Ddi2 increased the binding of Ub conjugates to purified proteasomes. In addition, Ddi2 also contains a retroviral protease domain with undefined cellular roles. We show that blocking the endoprotease activity of Ddi2 either genetically or with the HIV protease inhibitor nelfinavir increased its binding to Ub conjugates but decreased its binding to proteasomes and reduced subsequent protein degradation by proteasomes leading to further accumulation of Ub conjugates. Finally, nelfinavir treatment required Ddi2 to induce the unfolded protein response. Thus, Ddi2 appears to function as a shuttling factor in endoplasmic reticulum-associated protein degradation and delivers K11/K48-ubiquitylated proteins to the proteasome. We conclude that the protease activity of Ddi2 influences this shuttling factor activity, promotes protein turnover, and helps prevent endoplasmic reticulum stress, which may explain nelfinavir's ability to enhance cell killing by proteasome inhibitors.

Mechanisms That Activate 26S Proteasomes and Enhance Protein Degradation.

Although ubiquitination is widely assumed to be the only regulated step in the ubiquitin-proteasome pathway, recent studies have demonstrated several important mechanisms that regulate the activities of the 26S proteasome. Most proteasomes in cells are inactive but, upon binding a ubiquitinated substrate, become activated by a two-step mechanism requiring an association of the ubiquitin chain with Usp14 and then a loosely folded protein domain with the ATPases. The initial activation step is signaled by Usp14's UBL domain, and many UBL-domain-containing proteins (e.g., Rad23, Parkin) also activate the proteasome. ZFAND5 is a distinct type of activator that binds ubiquitin conjugates and the proteasome and stimulates proteolysis during muscle atrophy. The proteasome's activities are also regulated through subunit phosphorylation. Agents that raise cAMP and activate PKA stimulate within minutes Rpn6 phosphorylation and enhance the selective degradation of short-lived proteins. Likewise, hormones, fasting, and exercise, which raise cAMP, activate proteasomes and proteolysis in target tissues. Agents that raise cGMP and activate PKG also stimulate 26S activities but modify different subunit(s) and stimulate also the degradation of long-lived cell proteins. Both kinases enhance the selective degradation of aggregation-prone proteins that cause neurodegenerative diseases. These new mechanisms regulating proteolysis thus have clear physiological importance and therapeutic potential.

Methods to Rapidly Prepare Mammalian 26S Proteasomes for Biochemical Analysis.

Rapid, gentle isolation of 26S proteasomes from cells or tissues is an essential step for studies of the changes in proteasome activity and composition that can occur under different physiological or pathological conditions and in response to pharmacological agents. We present here three different approaches to affinity purify or to prepare proteasome-rich cell fractions. The first method uses affinity tags fused to proteasome subunits and has been useful in several cell lines for studies of proteasome structure by cryo-electron microscopy and composition by mass spectrometry. A second method uses the proteasome's affinity for a ubiquitin-like (UBL) domain and can be used to purify these particles from any cell or tissue. This method does not require expression of a tagged subunit and has proven to be very useful to investigate how proteasomal activity changes in different physiological states (e.g., fasting or aging), with neurodegenerative diseases, and with drugs or hormones that cause subunit phosphorylation. A third, simple method that is based on the 26S proteasome's high molecular weight (about 2.5 MDa) concentrates these particles greatly by differential centrifugation. This method maintains the association of proteasomes with ubiquitin (Ub) conjugates and many other loosely associated regulatory proteins and is useful to study changes in proteasome composition under different conditions.

Measurement of the Multiple Activities of 26S Proteasomes.

Because proteasomes catalyze most of the protein degradation in mammalian cells, and their functioning is essential for cellular homeostasis, proteasome structure, biochemical mechanisms, and regulation in normal and disease states are now widely studied and are of major importance. In addition, inhibitors of the proteasome's peptidase activity have proven to be very valuable as research tools and in the treatment of hematologic malignancies, and a number of newer pharmacological agents that alter proteasome function are being developed. The rapid degradation of ubiquitinated proteins by the 26S proteasome involves multiple enzymatic and non-enzymatic steps, including the binding of ubiquitinated substrates to the 19S particle (Subheading 3.2), opening the gated substrate entry channel into the 20S particle (Subheading 3.3), disassembly of the Ub chain (Subheading 3.4), ATP hydrolysis (Subheading 3.5), substrate unfolding and translocation, and proteolysis within the 20S particle (Subheadings 3.3 and 3.7). Assaying each of these processes is important if we are to fully understand the physiological regulation of proteasome function and the effects of disease or drugs. Here, we describe several methods that we have found useful to measure many of these individual activities using purified proteasomes. Studies using these approaches have already provided valuable new insights into the effects of post-synthetic modifications to 26S subunits, the physiological regulation of the ubiquitin-proteasome system, and the impairment of proteasome activity in neurodegenerative disease. These advances would not have been possible if only the standard assays of peptidase activity were used.

The Logic of the 26S Proteasome.

The ubiquitin proteasome pathway is responsible for most of the protein degradation in mammalian cells. Rates of degradation by this pathway have generally been assumed to be determined by rates of ubiquitylation. However, recent studies indicate that proteasome function is also tightly regulated and determines whether a ubiquitylated protein is destroyed or deubiquitylated and survives longer. This article reviews recent advances in our understanding of the proteasome's multistep ATP-dependent mechanism, its biochemical and structural features that ensure efficient proteolysis and ubiquitin recycling while preventing nonselective proteolysis, and the regulation of proteasome activity by interacting proteins and subunit modifications, especially phosphorylation.