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Sumoylation is an essential post-translational modification that is catalysed by a small number of modifying enzymes but regulates thousands of target proteins in a dynamic manner. Small ubiquitin-like modifiers (SUMOs) can be attached to target proteins as one or more monomers or in the form of polymers of different types. Non-covalent readers recognize SUMO-modified proteins via SUMO interaction motifs. SUMO simultaneously modifies groups of functionally related proteins to regulate predominantly nuclear processes, including gene expression, the DNA damage response, RNA processing, cell cycle progression and proteostasis. Recent progress has increased our understanding of the cellular and pathophysiological roles of SUMO modifications, extending their functions to the regulation of immunity, pluripotency and nuclear body assembly in response to oxidative stress, which partly occurs through the recently characterized mechanism of liquid–liquid phase separation. Such progress in understanding the roles and regulation of sumoylation opens new avenues for the targeting of SUMO to treat disease, and indeed the first drug blocking sumoylation is currently under investigation in clinical trials as a possible anticancer agent.Sumoylation regulates thousands of proteins, many of which are nuclear. Recent studies have implicated sumoylation in liquid–liquid phase separation and assembly of nuclear bodies, and have uncovered its roles in immunity and pluripotency and links to disease, thereby opening new therapeutic avenues.

Key Points Advances in mass spectrometry-based proteomics have enabled the global identification and characterization of thousands of acetylation sites, ushering in the age of acetylomics. Acetylation intersects with cellular metabolism at multiple levels: metabolic intermediates regulate acetyltransferases and deacetylases, acetylation can affect metabolic pathways, and metabolites may directly acetylate proteins through non-enzymatic mechanisms. Acetylation regulates protein function by affecting protein interactions with nucleic acids and with other proteins, the catalytic activity of proteins, and protein localization. Emerging data indicate that other types of acylations, such as succinylation and glutarylation, are also linked to metabolic activity and are regulated by sirtuin-class deacylases. Recent technical advances are expanding our understanding of how lysine acetylation, as well as other metabolite-sensitive acylations, regulates various cellular processes. Emerging findings point to new functions for different acylations and deacylating enzymes, and clarify the intricate link between lysine acetylation and cellular metabolism. Lysine acetylation is a conserved protein post-translational modification that links acetyl-coenzyme A metabolism and cellular signalling. Recent advances in the identification and quantification of lysine acetylation by mass spectrometry have increased our understanding of lysine acetylation, implicating it in many biological processes through the regulation of protein interactions, activity and localization. In addition, proteins are frequently modified by other types of acylations, such as formylation, butyrylation, propionylation, succinylation, malonylation, myristoylation, glutarylation and crotonylation. The intricate link between lysine acylation and cellular metabolism has been clarified by the occurrence of several such metabolite-sensitive acylations and their selective removal by sirtuin deacylases. These emerging findings point to new functions for different lysine acylations and deacylating enzymes and also highlight the mechanisms by which acetylation regulates various cellular processes.

Protein aggregates and damaged organelles are tagged with ubiquitin chains to trigger selective autophagy. To initiate mitophagy, the ubiquitin kinase PINK1 phosphorylates ubiquitin to activate the ubiquitin ligase parkin, which builds ubiquitin chains on mitochondrial outer membrane proteins, where they act to recruit autophagy receptors. Using genome editing to knockout five autophagy receptors in HeLa cells, here we show that two receptors previously linked to xenophagy, NDP52 and optineurin, are the primary receptors for PINK1- and parkin-mediated mitophagy. PINK1 recruits NDP52 and optineurin, but not p62, to mitochondria to activate mitophagy directly, independently of parkin. Once recruited to mitochondria, NDP52 and optineurin recruit the autophagy factors ULK1, DFCP1 and WIPI1 to focal spots proximal to mitochondria, revealing a function for these autophagy receptors upstream of LC3. This supports a new model in which PINK1-generated phospho-ubiquitin serves as the autophagy signal on mitochondria, and parkin then acts to amplify this signal. This work also suggests direct and broader roles for ubiquitin phosphorylation in other autophagy pathways. The PINK1 ubiquitin kinase is shown to recruit the two autophagy receptors NDP52 and OPTN to mitochondria to activate mitophagy directly, independently of the ubiquitin ligase parkin; once recruited to mitochondria, NDP52 and OPTN recruit autophagy initiation components, and parkin may amplify the phospho-ubiquitin signal generated by PINK1, resulting in robust autophagy induction. The role of parkin in mitophagy As in other forms of selective autophagy, during mitophagy the damaged cargo — the mitochondrion — is tagged with ubiquitin chains for recognition and subsequent degradation. Specifically, the enzyme PINK1 phosphorylates ubiquitin as part of the process to activate the ubiquitin ligase enzyme parkin. Consequently, parkin can build ubiquitin chains on mitochondrial outer membrane proteins to recruit autophagy receptors. Richard Youle and colleagues report an additional layer of regulatory complexity in this pathway, with a cellular role for phosphorylated ubiquitin. Using genome editing to knockout multiple autophagy receptors, the authors find that PINK1 recruits only two such receptors, NDP52 and optineurin, to mitochondria to directly activate mitophagy, independent of parkin. NDP52 and optineurin then recruit other autophagy components. These observations call for a revision of the current model of the role of parkin in mitophagy, suggesting that it amplifies the phospho-ubiquitin signal generated by PINK1 to signal autophagy.

Two methods for identifying protein isoforms that are concurrently phosphorylated and ubiquitylated are applied in yeast to identify phosphorylation sites that regulate ubiquitin proteasome–mediated proteome degradation. Cross-talk between different types of post-translational modifications on the same protein molecule adds specificity and combinatorial logic to signal processing, but it has not been characterized on a large-scale basis. We developed two methods to identify protein isoforms that are both phosphorylated and ubiquitylated in the yeast Saccharomyces cerevisiae , identifying 466 proteins with 2,100 phosphorylation sites co-occurring with 2,189 ubiquitylation sites. We applied these methods quantitatively to identify phosphorylation sites that regulate protein degradation via the ubiquitin-proteasome system. Our results demonstrate that distinct phosphorylation sites are often used in conjunction with ubiquitylation and that these sites are more highly conserved than the entire set of phosphorylation sites. Finally, we investigated how the phosphorylation machinery can be regulated by ubiquitylation. We found evidence for novel regulatory mechanisms of kinases and 14-3-3 scaffold proteins via proteasome-independent ubiquitylation.

Poly (ADP-ribose) polymerase (PARP) inhibitors elicit antitumour activity in homologous recombination-defective cancers by trapping PARP1 in a chromatin-bound state. How cells process trapped PARP1 remains unclear. Using wild-type and a trapping-deficient PARP1 mutant combined with rapid immunoprecipitation mass spectrometry of endogenous proteins and Apex2 proximity labelling, we delineated mass spectrometry-based interactomes of trapped and non-trapped PARP1. These analyses identified an interaction between trapped PARP1 and the ubiquitin-regulated p97 ATPase/segregase. We found that following trapping, PARP1 is SUMOylated by PIAS4 and subsequently ubiquitylated by the SUMO-targeted E3 ubiquitin ligase RNF4, events that promote recruitment of p97 and removal of trapped PARP1 from chromatin. Small-molecule p97-complex inhibitors, including a metabolite of the clinically used drug disulfiram (CuET), prolonged PARP1 trapping and enhanced PARP inhibitor-induced cytotoxicity in homologous recombination-defective tumour cells and patient-derived tumour organoids. Together, these results suggest that p97 ATPase plays a key role in the processing of trapped PARP1 and the response of tumour cells to PARP inhibitors. Krastev et al. report that trapped PARP1 undergoes SUMOylation, followed by ubiquitylation, resulting in the recruitment of the p97 ATPase to remove trapped PARP1 from chromatin and prevent PARP inhibitor-induced cytotoxicity.

Abnormal neddylation activation is frequently observed in human cancers and neddylation inhibition has been proposed as a therapy for cancer. Here, we report that MLN4924, a small-molecule inhibitor of neddylation activating enzyme, increases glutamine uptake in breast cancer cells by causing accumulation of glutamine transporter ASCT2/SLC1A5, via inactivation of CRL3-SPOP E3 ligase. We show the E3 ligase SPOP promotes ASCT2 ubiquitylation, whereas SPOP itself is auto-ubiquitylated upon glutamine deprivation. Thus, SPOP and ASCT2 inversely regulate glutamine uptake and metabolism. SPOP knockdown increases ASCT2 levels to promote growth which is rescued by ASCT2 knockdown. Adding ASCT2 inhibitor V-9302 enhances MLN4924 suppression of tumor growth. In human breast cancer specimens, SPOP and ASCT2 levels are inversely correlated, whereas lower SPOP with higher ASCT2 predicts a worse patient survival. Collectively, our study links neddylation to glutamine metabolism via the SPOP-ASCT2 axis and provides a rational drug combination for enhanced cancer therapy. Neddylation inhibition has been reported as a therapy for cancer. Here, the authors show that neddylation inhibition increases glutamine metabolism by stabilizing glutamine transporter ASCT2, therefore targeting ASCT2 improves the anti-cancer effect of neddylation inhibitors.

The serine/threonine kinase Akt plays a central role in cell proliferation, survival and metabolism, and its hyperactivation is linked to cancer progression. Here we report that Akt undergoes K64 methylation by SETDB1, which is crucial for cell membrane recruitment, phosphorylation and activation of Akt following growth factor stimulation. Furthermore, we reveal an adaptor function of histone demethylase JMJD2A, which is important for recognizing Akt K64 methylation and recruits E3 ligase TRAF6 and Skp2-SCF to the Akt complex, independently of its demethylase activity, thereby initiating K63-linked ubiquitination, cell membrane recruitment and activation of Akt. Notably, the cancer-associated Akt mutant E17K displays enhanced K64 methylation, leading to its hyper-phosphorylation and activation. SETDB1-mediated Akt K64 methylation is upregulated and correlated with Akt hyperactivation in non-small-cell lung carcinoma (NSCLC), promotes tumour development and predicts poor outcome. Collectively, these findings reveal complicated layers of Akt activation regulation coordinated by SETDB1-mediated Akt K64 methylation to drive tumorigenesis. Wang et al. show that Akt methylation by SETDB1 is recognized by demethylase JMJD2A, which then recruits E3 ligases to induce K63-linked Akt ubiquitination, leading to Akt activation and tumorigenesis.

Hundreds of E3 ligases play a critical role in recognizing specific substrates for modification by ubiquitin (Ub). Separating genuine targets of E3s from E3-interactors remains a challenge. We present BioE3, a powerful approach for matching substrates to Ub E3 ligases of interest. Using BirA-E3 ligase fusions and bioUb, site-specific biotinylation of Ub-modified substrates of particular E3s facilitates proteomic identification. We show that BioE3 identifies both known and new targets of two RING-type E3 ligases: RNF4 (DNA damage response, PML bodies), and MIB1 (endocytosis, autophagy, centrosome dynamics). Versatile BioE3 identifies targets of an organelle-specific E3 (MARCH5) and a relatively uncharacterized E3 (RNF214). Furthermore, BioE3 works with NEDD4, a HECT-type E3, identifying new targets linked to vesicular trafficking. BioE3 detects altered specificity in response to chemicals, opening avenues for targeted protein degradation, and may be applicable for other Ub-likes (UbLs, e.g., SUMO) and E3 types. BioE3 applications shed light on cellular regulation by the complex UbL network. Here, the authors describe BioE3, a biotin-based method to discriminate direct substrates of ubiquitin E3 ligases of interest from mere interactors using proximity proteomics. BioE3 responds to chemical treatments, and works with RING- and HECT-type E3s, as well as ubiquitin-likes (e.g., SUMO).

Signalling is known to regulate metabolism, and it is becoming clear that this regulation is reciprocal, with signalling pathways being regulated by the availability of nutrient-sensitive modifications, such as acetylation and glycosylation. This tight link between signalling and metabolism allows cells to modulate their activities according to metabolic status. It is becoming increasingly clear that cellular signalling and metabolism are not just separate entities but rather are tightly linked. Although nutrient metabolism is known to be regulated by signal transduction, an emerging paradigm is that signalling and transcriptional networks can be modulated by nutrient-sensitive protein modifications, such as acetylation and glycosylation, which depend on the availability of acetyl-CoA and sugar donors such as UDP- N -acetylglucosamine (UDP-GlcNAc), respectively. The integration of metabolic and signalling cues allows cells to modulate activities such as metabolism, cell survival and proliferation according to their intracellular metabolic resources.

Key Points Post-translation modification of proteins by ubiquitin and ubiquitin-like proteins, such as small ubiquitin-related modifier (SUMO), requires the sequential activities of E1, E2 and E3 enzymes. SUMO modification regulates a wide array of cellular processes that include transcription, replication, chromosome segregation, DNA repair and response to environmental stress. The SUMO pathway relies on a single E1 and E2 enzyme and just a few E3 enzymes to regulate substrate specificity. This is achieved in part because the SUMO E2 can specifically recognize and conjugate SUMO to substrates in the absence of an E3 by recognition of a ψKX(D/E) consensus motif in the substrate, where ψ is a large hydrophobic residue. Longer consensus motifs for SUMO interaction with the E2 also exist. SUMO-interacting motifs (SIMs) mediate non-covalent interactions between SUMO and SIM-containing proteins. SIMs are characterized by a short stretch of hydrophobic amino acids that are often flanked by acidic residues. SIMs are present in SUMO enzymes, SUMO substrates and SUMO-binding proteins. SUMO E3 ligases catalyse SUMO transfer through at least two distinct mechanisms. They can bind an E2∼SUMO thioester complex and hold it in a productive orientation for catalysis in complexes in which the E2 mediates substrate specificity, or they can interact directly with both the substrate and E2∼SUMO to facilitate SUMO transfer to the substrate Lys acceptor. Phosphorylation of SUMO enzymes and SUMO substrates has been shown to contribute to the regulation of the SUMO pathway. Examples exist that illustrate both positive and negative regulation of SUMO modification by phosphorylation. Substrate Lys residues that are modified by SUMO are sometimes sites for other post-translational modifications, such as ubiquitylation or acetylation. Switching between these various modifications can influence downstream signalling. Conjugation of the ubiquitin-like protein SUMO to proteins regulates many biological processes. Insights are emerging into mechanisms that regulate the SUMO modification pathway, including other post-translational modifications. Many substrates also harbour additional characteristics that facilitate their modification. Proteins of the small ubiquitin-related modifier (SUMO) family are conjugated to proteins to regulate such cellular processes as nuclear transport, transcription, chromosome segregation and DNA repair. Recently, numerous insights into regulatory mechanisms of the SUMO modification pathway have emerged. Although SUMO-conjugating enzymes can discriminate between SUMO targets, many substrates possess characteristics that facilitate their modification. Other post-translational modifications also regulate SUMO conjugation, suggesting that SUMO signalling is integrated with other signal transduction pathways. A better understanding of SUMO regulatory mechanisms will lead to improved approaches for analysing the function of SUMO and substrate conjugation in distinct cellular pathways.