The midnolin-proteasome pathway catches proteins for ubiquitination-independent degradation

Cells use ubiquitin to mark proteins for proteasomal degradation. Although the proteasome also eliminates proteins that are not ubiquitinated, how this occurs mechanistically is unclear. Here, we found that midnolin promoted the destruction of many nuclear proteins, including transcription factors encoded by the immediate-early genes. Diverse stimuli induced midnolin, and its overexpression was sufficient to cause the degradation of its targets by a mechanism that did not require ubiquitination. Instead, midnolin associated with the proteasome via an α helix, used its Catch domain to bind a region within substrates that can form a β strand, and used a ubiquitin-like domain to promote substrate destruction. Thus, midnolin contains three regions that function in concert to target a large set of nuclear proteins to the proteasome for degradation. Description Editor’s summary Eukaryotic cells contain a macromolecular protease called the proteasome that degrades proteins modified by ubiquitin. The proteasome can also degrade proteins that are not ubiquitinated, but how this occurs mechanistically has remained mysterious. Gu et al. identified midnolin, an inducible protein that localizes within the nucleus to promote the proteasomal degradation of numerous transcriptional regulators independently of ubiquitination (see the Perspective by Schilling and Weber-Ban). Midnolin stably associates with the proteasome and uses a structural domain that incorporates a free β strand to “catch” substrates for destruction. Thus, the midnolin-proteasome pathway bypasses the canonical ubiquitination system to achieve selective degradation of many nuclear proteins. —Stella M. Hurtley Midnolin associates with the proteasome to promote degradation of nuclear proteins through a ubiquitination-independent mechanism. INTRODUCTION In mammals, the transcriptional response to growth factor, neuronal, and immune stimuli is mediated by a group of genes called immediate-early genes (IEGs), which encode transcription factors of the Fos, EGR, and NR4A families. IEG proteins are activated stereotypically in virtually all mammalian cells but promote the transcription of late-response genes (LRGs) that are cell-type specific and crucial for the appropriate response to the initial stimulus. The physiological importance of IEGs is underscored by the fact that misregulation of their expression can lead to cancer, immune deficiencies, and neurological disorders. The IEG mRNAs accumulate within minutes after the initial stimulus and, once translated, their proteins are rapidly degraded to allow for a transient burst of protein expression. Although the mechanisms that regulate IEG transcription are well characterized, how IEG proteins are swiftly targeted for destruction has remained mysterious for many years. RATIONALE Eukaryotic cells rely on a macromolecular protease called the proteasome that canonically degrades proteins marked with ubiquitin. It has been suggested that the Fos family is targeted to the proteasome by both ubiquitination-dependent and -independent mechanisms, but the molecular events that orchestrate these processes have remained elusive. We hypothesized that there exists a cellular pathway dedicated to the rapid destruction of c-Fos and other IEG proteins. By harnessing the power of forward genetic screens, we sought to identify the machinery that controls the degradation of these proteins. RESULTS We performed genome-wide CRISPR-Cas9 screens to search for genes that regulate the stability of IEG proteins. We found that midnolin, a largely uncharacterized protein in mammals, promoted the proteasomal destruction of IEG proteins from structurally distinct families including c-Fos, FosB, EGR1, and NR4A1. These results prompted us to search for additional midnolin targets. We used the global protein stability (GPS) assay with a human open reading frame library (ORFeome) to assess changes in protein stability for ~12,000 human proteins simultaneously. In addition to IEG proteins, midnolin promoted the degradation of IRF4, NeuroD1, PAX8, GATA1, and many other cell-type–specific transcriptional regulators in the nucleus, where midnolin itself resides. Diverse stimuli that activate IEGs also induced midnolin, and midnolin overexpression was sufficient to cause the destruction of its targets by a mechanism that does not require ubiquitination. Multiple lines of evidence support this ubiquitination-independent mechanism of protein degradation. Midnolin still bound to and promoted the degradation of many targets that had been mutated to lack lysine residues. Moreover, inhibition of the proteasome, but not E1 ubiquitin–activating enzymes, abrogated midnolin function. Additionally, midnolin does not contain RING or HECT domains that are characteristic of E3 ubiquitin ligases or ubiquitin-binding domains found in proteasomal processivity factors such as Rad23. Instead, midnolin engaged substrates using its “Catch” domain, which was necessary and sufficient to interact with unstructured regions within substrates that have the potential to form a β strand upon binding midnolin. These unstructured regions with the propensity to form a β strand were also necessary and sufficient to bind the Catch domain, thus functioning as a midnolin degron. In addition, midnolin stably associated with the proteasome through a C-terminal α helix and promoted the degradation of Catch-bound targets using its N-terminal ubiquitin-like domain. Thus, midnolin contains three conserved structural domains that function in concert to directly target a large set of nuclear proteins to the proteasome for ubiquitination-independent degradation. CONCLUSION Our study suggests that the midnolin-proteasome pathway may represent a general mechanism by which the proteasome bypasses the canonical ubiquitination system to achieve selective degradation of nuclear proteins, many of which are crucial for transcription. Within substrates, midnolin recognizes relatively degenerate amphipathic regions with the potential to form β strands, so the midnolin degron may be a common structural component of numerous proteins. How the midnolin-proteasome pathway is regulated by various cues in diverse cell types to control transcriptional programs will be an important subject of future exploration. The midnolin-proteasome pathway degrades many nuclear proteins independently of ubiquitination. Midnolin is induced by diverse cues, including growth factors and neuronal stimuli. Within the nucleus, midnolin associates with the proteasome through a C-terminal α helix (αHelix-C) and promotes the degradation of bound substrates using an N-terminal ubiquitin-like domain (Ubl). Midnolin achieves selectivity using its Catch domain, which binds an unstructured region within substrates that can form a β strand. Structures shown are AlphaFold models. Figure was created with BioRender.com. NLS, nuclear localization sequence.