As the name suggests, ubiquitination involves the participation of ubiquitin. The Ubiquitin-Proteasome System (UPS), as one might guess, involves both ubiquitin and proteasomes. First, let's take a quick review of these key terms with me.

• Ubiquitin: A small protein composed of 76 amino acids.
• Ubiquitylation: A post-translational modification involving the covalent attachment of ubiquitin to target proteins, catalyzed by a series of enzymes (E1/E2/E3/E4).
• Proteasome: A large, tubular protein complex present in eukaryotic cells. Its main function is to degrade unneeded or damaged proteins through proteolysis, an enzymatic process also known as protein hydrolysis.

Eukaryotic cells have two main degradation systems: the Ubiquitin-Proteasome System and the Lysosome Degradation System. In eukaryotes, over 80% of protein degradation relies on the "Ubiquitin-Proteasome System."

This degradation process can be divided into two stages: (1) "tagging" discarded proteins - ubiquitin interacts with substrate proteins, effectively tagging them with ubiquitin; (2) "waste management" of tagged proteins - proteins modified by ubiquitination are recognized by specific proteasomes, leading to their degradation^[1].

Apart from the classic "Ubiquitin-Proteasome System" for protein degradation, proteasomes can also degrade proteins through non-ubiquitin modifications. For instance, studies have shown that the Fos family can be degraded through both ubiquitin-dependent and independent mechanisms^[2]. However, the mechanism for the rapid targeted degradation of IEG proteins (Immediate-early genes) has remained unclear, until now...

Recently, a breakthrough research was published in the top journal Science by Professor Michael E. Greenberg's team from Harvard Medical School.

Through genome-wide CRISPR-Cas9 screenings for genes that regulate IEG protein stability, Greenberg's team discovered Midnolin, a protein in mammals that has hardly been characterized before. Midnolin can facilitate the proteasome degradation of various IEG proteins. This led to the discovery of a completely new protein degradation mechanism — the Midnolin-Proteasome pathway.

Midnolin is induced by multiple factors, including growth factors and neuronal stimuli. Within cell nuclei, Midnolin binds to the proteasome via its C-terminal α-helix, utilizes its Catch domain for selectivity, and employs its N-terminal ubiquitin-like domain (Ubl) to facilitate substrate degradation (Figure 2). Research has found that the Midnolin-Proteasome pathway bypasses the typical ubiquitination system to achieve selective degradation of numerous nuclear proteins^[2].

Midnolin, the midbrain nucleolar protein, encoded by the MIDN gene, is a transcription factor that controls development by regulating mRNA transport^[3].

• Midnolin was initially discovered in embryonic stem cells by T Sukahara and colleagues in 2000^[4].

(1) Using a gene trap method and selection of in vitro embryonic stem cells, they identified a new gene, Midnolin, which is involved in the regulation of nucleolar neurogenesis.

(2) Midnolin encodes a protein containing 508 amino acids (aa), which includes a ubiquitin-like domain.

(3) The distribution of Midnolin within cells was studied using a Midnolin-Green Fluorescent Protein (GFP) fusion protein. Midnolin was found to be located in the cell nucleus and nucleolus but not in the cytoplasm. The nucleolar localization signal (NLS) is a peptide consisting of 28 amino acids located in the C-terminal region of Midnolin (440-QQKRLRRKARRDARGPYHWTPSRKAGRS-467).

Midnolin is a modulator of IEG protein degradation

To study the mechanism of IEG protein degradation, the authors focused on two common transcription factors from different protein families - EGR1 and Fos. By constructing stable HEK-293T cell lines expressing EGR1 or FosB GPS (The global protein stability) reporter genes, and performing genome-wide CRISPR-Cas9 screening, they identified MIDN as a hotspot gene negatively regulating the stability of EGR1 and FosB. They further discovered that knocking out the MIDN gene increases the stability of EGR1 and FosB.

In addition, overexpression of Midnolin in NIH/3T3 fibroblasts (Figure 3D-E) and primary cortical neurons (Figure 3F-G) reduced the protein levels of multiple IGEs, such as EGR1, FosB, c-Fos, and NR4A1, and Knocking out Midnolin resulted in the opposite effect. The above results indicate that these IEGs proteins may undergo targeted degradation through Midnolin. Further studies revealed that Midnolin may also be involved in the degradation of hundreds of other transcription factors in the nucleus in addition to Fos and EGR1.

Midnolin degrades non-ubiquitinated proteins by binding to its substrates and the 26S proteasome

The authors tagged endogenous Midnolin with 3xHA for proteomic analysis, discovering that Midnolin can immunoprecipitate both 19S regulatory particles and 20S core particles (Figure 4A). Simultaneously, using MG132 (a proteasome inhibitor) to inhibit substrate protein degradation, they found that Midnolin interacts with substrates c-Fos, FosB, EGR1, as well as PSMD2 and PSMA2 (which are components of the 19S and 20S proteasomes respectively) (Figure 4B).

In addition, MG132 can increase Midnolin protein levels, but TAK-243 (ubiquitin E1 enzyme inhibitor) does not affect Midnolin protein levels (Figure 4C), and TAK-243 does not affect the degradation of substrate proteins by Midnolin, thus proving Midnolin degrades proteins independently of ubiquitination.

NOTE: The 26S proteasome consists of a cylindrical 20S core particle in the middle and one or two 19S regulatory particles covering the ends.

Using the artificial intelligence platform AlphaFold for structure prediction, the authors showed that the Midnolin protein contains three stable structural domains: an N-terminal ubiquitin-like domain (Ubl), a "catch" domain with two pseudosymmetric parts, and a long α-helix at the C-terminus (αHelix-C) (Figure 5A).

Next, the authors used immunoprecipitation experiments to identify which structural domains are necessary for Midnolin to interact with substrates and/or proteasomes. The results showed that point mutations/deletions/inductions in the Ubl domain have no effect on the interaction between Midnolin and EGR1 or proteasomes. However, deletion of the Catch domain prevents substrate proteins (like EGR1) from being precipitated (Figure 5C Left), and deletion of the αHelix-C or NLS domains prevents the capture of proteasomes (like PSMD2) (Figure 5C Right).

That means, Midnolin interacts with target proteins through the Catch domain and binds to the 26S proteasome through the α Helix-C domain. At the same time, Midnolin can bind to unstructured regions within the substrate through its Catch domain and form β-strands. Although the Midnolin-proteasome pathway can bypass the ubiquitination system to achieve selective degradation of many nuclear proteins, how to achieve specific target protein degradation remains to be further studied.