Summary In eukaryotic cells, ubiquitination of proteins leads to their degradation by the 26S proteasome. We tested if the ubiquitin (Ub) chain also regulates the proteasome’s capacity for proteolysis. After incubation with polyubiquitinated proteins, 26S proteasomes hydrolyzed peptides and proteins 2–7 fold faster. Ub conjugates enhanced peptide hydrolysis by stimulating gate opening in the 20S proteasome, since this stimulation was seen when this gate was closed or transiently open, but not maximally open. Gate opening requires conjugate association with Usp14/Ubp6, since it is also mediated by occupancy of the Ubp6 active site with Ub aldehyde. No stimulation was observed with 26S from Ubp6Δ mutants, but was restored by addition of Usp14/Ubp6 or an inactive Ubp6 mutant. The stimulation of gate-opening by Ub conjugates through Usp14/Ubp6 requires nucleotide binding to the gate-regulatory ATPases. This activation enhances the selectivity of the 26S proteasome for ubiquitinated proteins and links their deubiquitination to their degradation.

Summary Due to genome instability, most cancers exhibit loss of regions containing tumor suppressor genes and collateral loss of other genes. To identify cancer-specific vulnerabilities that are the result of copy-number losses, we performed integrated analyses of genome-wide copy-number and RNAi profiles and identified 56 genes for which gene suppression specifically inhibited the proliferation of cells harboring partial copy-number loss of that gene. These CYCLOPS (Copy-number alterations Yielding Cancer Liabilities Owing to Partial losS) genes are enriched for spliceosome, proteasome and ribosome components. One CYCLOPS gene, PSMC2, encodes an essential member of the 19S proteasome. Normal cells express excess PSMC2, which resides in a complex with PSMC1, PSMD2, and PSMD5 and acts as a reservoir protecting cells from PSMC2 suppression. Cells harboring partial PSMC2 copy-number loss lack this complex and die after PSMC2 suppression. These observations define a distinct class of cancer-specific liabilities resulting from genome instability.

Recent studies, mainly in yeast, have identified various cofactors that associate with the 26S proteasome and appear to influence its function. To identify these proteins in different cells and physiological states, we developed a method to gently and rapidly isolate 26S proteasomes and associated proteins without the need for genetic modifications of the proteasome. This method is based on the affinity of this complex for the ubiquitin-like (UBL) domain of hHR23B and elution with a competing polypeptide containing a ubiquitin-interacting motif. Associated with 26S proteasomes from rat muscle were a variety of known proteasome-interacting proteins, activators, and ubiquitin conjugates. In addition, we identified over 40 proteins not previously known to associate with the 26S proteasome, some of which were tightly associated with the proteasome in a substoichiometric fashion, e.g., the deubiquitinating enzymes USP5/isopeptidase T and USP7/HAUSP and the ubiquitin ligases ARF-BP1/HUWE1 and p600/UBR4. By altering buffer conditions, we also purified by this approach complexes of the ATPase p97/VCP associated with its adaptor proteins Ufd1-Npl4, p47, SAKS1, and FAF1, all of which contain ubiquitin-binding motifs. These complexes were isolated with ubiquitin conjugates bound and were not previously known to bind to the UBL domain of hHR23B. These various UBL-interacting proteins, dubbed the UBL interactome, represent a network of proteins that function together in ubiquitin-dependent proteolysis, and the UBL method offers many advantages for studies of the diversity, functions, and regulation of 26S proteasomes and p97 complexes under different conditions.

Among the hallmarks of aged organisms are an accumulation of misfolded proteins and a reduction in skeletal muscle mass ("sarcopenia"). We have examined the effects of aging and dietary restriction (which retards many age-related changes) on components of the ubiquitin proteasome system (UPS) in muscle. The hindlimb muscles of aged (30 months old) rats showed a marked loss of muscle mass and contained 2-3-fold higher levels of 26S proteasomes than those of adult (4 months old) controls. 26S proteasomes purified from muscles of aged and adult rats showed a similar capacity to degrade peptides, proteins, and an ubiquitylated substrate, but differed in levels of proteasome-associated proteins (e.g. the ubiquitin ligase E6AP and deubiquitylating enzyme USP14). Also, the activities of many other deubiquitylating enzymes were greatly enhanced in the aged muscles. Nevertheless, their content of polyubiquitylated proteins was higher than in adult animals. The aged muscles contained higher levels of the ubiquitin ligase CHIP, involved in eliminating misfolded proteins, and MuRF1, which ubiquitylates myofibrillar proteins. These muscles differed from ones rapidly atrophying due to disease, fasting, or disuse in that Atrogin-1/MAFbx expression was low and not inducible by glucocorticoids. Thus, the muscles of aged rats showed many adaptations indicating enhanced proteolysis by the UPS, which may enhance their capacity to eliminate misfolded proteins and seems to contribute to the sarcopenia. Accordingly, dietary restriction decreased or prevented the aging-associated increases in proteasomes and other UPS components and reduced muscle wasting.

Degradation rates of most proteins in eukaryotic cells are determined by their rates of ubiquitination. However, possible regulation of the proteasome's capacity to degrade ubiquitinated proteins has received little attention, although proteasome inhibitors are widely used in research and cancer treatment. We show here that mammalian 26S proteasomes have five associated ubiquitin ligases and that multiple proteasome subunits are ubiquitinated in cells, especially the ubiquitin receptor subunit, Rpn13. When proteolysis is even partially inhibited in cells or purified 26S proteasomes with various inhibitors, Rpn13 becomes extensively and selectively polyubiquitinated by the proteasome-associated ubiquitin ligase, Ube3c/Hul5. This modification also occurs in cells during heat-shock or arsenite treatment, when poly-ubiquitinated proteins accumulate. Rpn13 ubiquitination strongly decreases the proteasome's ability to bind and degrade ubiquitin-conjugated proteins, but not its activity against peptide substrates. This autoinhibitory mechanism presumably evolved to prevent binding of ubiquitin conjugates to defective or stalled proteasomes, but this modification may also be useful as a biomarker indicating the presence of proteotoxic stress and reduced proteasomal capacity in cells or patients.

The Lon protease from the archaeon Thermoplasma acidophilum (TaLon) is composed of an N-terminal ATPase associated with various cellular activities (AAA þ ) domain and a C-terminal Lon protease domain. Although related in sequence to the soluble Lon proteases, TaLon was shown to be membranebound in its native host and also when expressed in Escherichia coli. Recombinant TaLon was purified as a functional highmolecular weight complex displaying ATPase and proteolytic activity. Mutagenesis of conserved AAA þ residues revealed that the Walker A and B motifs, and the sensor 1 and sensor 2 0 residues were essential for the ATPase activity, while the sensor 2 and the arginine finger were involved in activation of the protease domain.

Assembly of complex structures such as the eukaryotic 26S proteasome requires intricate mechanisms that ensure precise subunit arrangements. Recent studies have shed light on the pathway for ordered assembly of the base of the 19S regulatory particle of the 26S proteasome by identifying new precursor complexes and four dedicated chaperones involved in its assembly.

Increasingly over the past decade, faculty in medical and graduate schools have received requests from digital millennial learners for concise faculty-made educational videos. At our institution, over the past couple of years alone, several hundred educational videos have been created by faculty who teach in a flipped-classroom setting of the pre-clinical medical school curriculum. Despite the appeal and potential learning benefits of digital chalk-talk videos first popularized by Khan Academy, we have observed that the conceptual and technological barriers for creating chalk-talk videos can be high for faculty. To this end, this tips article offers an easy-to-follow 12-step conceptual framework to guide at-home production of chalk-talk educational videos.