Cytoplasmic sumoylation by PIAS-type Siz1-SUMO ligase

SUMO (small ubiquitin-related modifier), a 12 kDa protein with distant similarity to ubiquitin, covalently binds to many proteins in eukaryotic cells. In contrast to ubiquitination, which mainly regulates proteasome-dependent degradation and protein sorting, sumoylation is known to regulate assembly and disassembly of protein complexes, protein localization and stability, and so on. SUMO is primarily localized to the nucleus, and many SUMO substrates are nuclear proteins involved in DNA transaction. However, certain roles of SUMO conjugates have been shown outside the nucleus. Particularly in budding yeast, SUMO is also localized to the bud-neck in a cell cycle-dependent manner. The first and prominent SUMO substrates are septins, evolutionally conserved proteins required for cytokinesis in yeast. Recent analysis of human septin structure would greatly facilitate the study of the functions of these SUMO conjugates. SUMO modification of septins is regulated by cell cycle-dependent nuclear transport of PIAS-type Siz1 (SUMO E3) and Ulp1 desumoylation enzyme in yeast. Domains outside the SUMO-ligase core (SP-RING) of Siz1 ensure its regulations. Furthermore, newly discovered ubiquitin ligases that specifically recognize poly-SUMO conjugates could lead to degradation of SUMO conjugates. Thus, protein modifications seem to be regulated in an unexpectedly complex manner. In this review, we focus on various regulations in yeast septin sumoylation and discuss its possible functions.     

SUMO protein modification

SUMO (small ubiquitin-related modifier) family proteins are not only structurally but also mechanistically related to ubiquitin in that they are posttranslationally attached to other proteins. As ubiquitin, SUMO is covalently linked to its substrates via amide (isopeptide) bonds formed between its C-terminal glycine residue and the epsilon-amino group of internal lysine residues. The enzymes involved in the reversible conjugation of SUMO are similar to those mediating the ubiquitin conjugation. Since its discovery in 1996, SUMO has received a high degree of attention because of its intriguing and essential functions, and because its substrates include a variety of biomedically important proteins such as tumor suppressor p53, c-jun, PML and huntingtin. SUMO modification appears to play important roles in diverse processes such as chromosome segregation and cell division, DNA replication and repair, nuclear protein import, protein targeting to and formation of certain subnuclear structures, and the regulation of a variety of processes including the inflammatory response in mammals and the regulation of flowering time in plants.     

An improved SUMmOn‐based methodology for the identification of ubiquitin and ubiquitin‐like protein conjugation sites identifies novel ubiquitin‐like protein chain linkages

Ubiquitin (Ub) and the ubiquitin‐like proteins (Ubls) comprise a remarkable assortment of polypeptides that are covalently conjugated to target proteins (or other biomolecules) to modulate their intracellular localization, half‐life, and/or activity. Identification of Ub/Ubl conjugation sites on a protein of interest can thus be extremely important for understanding how it is regulated. While MS has become a powerful tool for the study of many classes of PTMs, the identification of Ub/Ubl conjugation sites presents a number of unique challenges. Here, we present an improved Ub/Ubl conjugation site identification strategy, utilizing SUMmOn analysis and an additional protease (lysyl endopeptidase C), as a complement to standard approaches. As compared with standard trypsin proteolysis‐database search protocols alone, the addition of SUMmOn analysis can (i) identify Ubl conjugation sites that are not detected by standard database searching methods, (ii) better preserve Ub/Ubl conjugate identity, and (iii) increase the number of identifications of Ub/Ubl modifications in lysine‐rich protein regions. Using this methodology, we characterize for the first time a number of novel Ubl linkages and conjugation sites, including alternative yeast (K54) and mammalian small ubiquitin‐related modifier (SUMO) chain (SUMO‐2 K42, SUMO‐3 K41) assemblies, as well as previously unreported NEDD8 chain (K27, K33, and K54) topologies.     

UBE1DC1, an ubiquitin-activating enzyme, activates two different ubiquitin-like proteins

Ubiquitin and ubiquitin-like proteins are known to be covalently conjugated to a variety of cellular substrates via a three-step enzymatic pathway. These modifications lead to the degradation of substrates or change its functional status. The ubiquitin-activating enzyme (E1) plays a key role in the first step of ubiquitination pathway to activate ubiquitin or ubiquitin-like proteins. Ubiquitin-activating enzyme E1-domain containing 1 (UBE1DC1) had been proved to activate an ubiquitin-like protein, ubiquitin-fold modifier 1 (Ufm1), by forming a high-energy thioester bond. In this report, UBE1DC1 is proved to activate another ubiquitin-like protein, SUMO2, besides Ufm1, both in vitro and in vivo by immunological analysis. It indicated that UBE1DC1 could activate two different ubiquitin-like proteins, SUMO2 and Ufm1, which have no significant similarity with each other. Subcellular localization in AD293 cells revealed that UBE1DC1 was especially distributed in the cytoplasm; whereas UBE1DC1 was mainly distributed in the nucleus when was cotransfected with SUMO2. It presumed that UBE1DC1 greatly activated SUMO2 in the nucleus or transferred activated-SUMO2 to nucleus after it conjugated SUMO2 in the cytoplasm.     

Regulation of the p53 pathway by ubiquitin and related proteins

The p53 tumour suppressor protein is subject to many levels of control, including modification with ubiquitin and related proteins such as SUMO and NEDD8. These modifications regulate p53 at a number of levels, including control of protein turnover, alterations in sub-cellular localization and changes in the ability to regulate gene expression. Numerous E3 ligases that can mediate these modifications of p53 have been described, some of which promote conjugation with more than one ubiquitin-like protein. Understanding the complexity of this mechanism of p53 regulation will help in the development of therapeutic drugs that function to modulate these events.     

The binding interface between an E2 (UBC9) and a ubiquitin homologue (UBL1).

Human UBC9 is a member of the E2 (ubiquitin conjugation enzyme) family of proteins. Instead of conjugating to ubiquitin, it conjugates with a ubiquitin homologue UBL1 (also known as SUMO-1, GMP1, SMTP3, PIC1, and sentrin). UBC9 has been shown to be involved in cell cycle regulation, DNA repair, and p53-dependent processes. The binding interfaces of the UBC9 and UBL1 complex have been determined by chemical shift perturbation using nuclear magnetic resonance spectroscopy. The binding site of UBL1 resides on the ubiquitin domain, and the binding site of UBC9 is located on a structurally conserved region of E2. Because the UBC9-UBL1 system shares many similarities with the ubiquitin system in structures and in conjugation with each other and with target proteins, the observed binding interfaces may be conserved in E2-ubiquitin interactions in general.     

MAPL is a new mitochondrial SUMO E3 ligase that regulates mitochondrial fission

The modification of proteins by the small ubiquitin‐like modifier (SUMO) is known to regulate an increasing array of cellular processes. SUMOylation of the mitochondrial fission GTPase dynamin‐related protein 1 (DRP1) stimulates mitochondrial fission, suggesting that SUMOylation has an important function in mitochondrial dynamics. The conjugation of SUMO to its substrates requires a regulatory SUMO E3 ligase; however, so far, none has been functionally associated with the mitochondria. By using biochemical assays, overexpression and RNA interference experiments, we characterized the mitochondrial‐anchored protein ligase (MAPL) as the first mitochondrial‐anchored SUMO E3 ligase. Furthermore, we show that DRP1 is a substrate for MAPL, providing a direct link between MAPL and the fission machinery. Importantly, the large number of unidentified mitochondrial SUMO targets suggests a global role for SUMOylation in mitochondrial function, placing MAPL as a crucial component in the regulation of multiple conjugation events.     

SUMO1‐conjugation is altered during normal aging but not by increased amyloid burden

A proper equilibrium of post‐translational protein modifications is essential for normal cell physiology, and alteration in these processes is key in neurodegenerative disorders such as Alzheimer's disease. Recently, for instance, alteration in protein SUMOylation has been linked to amyloid pathology. In this work, we aimed to elucidate the role of protein SUMOylation during aging and increased amyloid burden in vivo using a His₆‐HA‐SUMO1 knock‐in mouse in the 5XFAD model of Alzheimer's disease. Interestingly, we did not observe any alteration in the levels of SUMO1‐conjugation related to Alzheimer's disease. SUMO1 conjugates remained localized to neuronal nuclei upon increased amyloid burden and during aging and were not detected in amyloid plaques. Surprisingly however, we observed age‐related alterations in global levels of SUMO1 conjugation and at the level of individual substrates using quantitative proteomic analysis. The identified SUMO1 candidate substrates are dominantly nuclear proteins, mainly involved in RNA processing. Our findings open novel directions of research for studying a functional link between SUMOylation and its role in guarding nuclear functions during aging.     

Intracellular targeting of proteins by sumoylation.

A novel host cell posttranslational modification system, termed sumoylation, has recently been characterized. Sumoylation is an enzymatic process that is biochemically analogous to, but functionally distinct from, ubiquitinylation. As in ubiquitinylation, sumoylation involves the covalent attachment of a small protein moiety, SUMO, to substrate proteins. However, conjugation of SUMO does not typically lead to degradation of the substrate and instead has a more diverse array of effects on substrate function. As the list of sumoylation substrates has expanded, a common theme is that many substrates exhibit sumoylation-dependent subcellular distribution. While the molecular mechanisms by which sumoylation targets protein localization are still poorly understood, it is clear that this modification system is an important regulator of intracellular protein localization, particularly involving nuclear uptake and punctate intranuclear accumulation.     

Identification of a substrate recognition site on Ubc9

Human Ubc9 is homologous to ubiquitin-conjugating enzymes. However, instead of conjugating ubiquitin, it conjugates a ubiquitin homologue, small ubiquitin-like modifier 1 (SUMO-1), also known as UBL1, GMP1, SMTP3, PIC1, and sentrin. The SUMO-1 conjugation pathway is very similar to that of ubiquitin with regard to the primary sequences of the ubiquitin-activating enzymes (E1), the three-dimensional structures of the ubiquitin-conjugating enzymes (E2), and the chemistry of the overall conjugation pathway. The interaction of substrates with Ubc9 has been studied using NMR spectroscopy. Peptides with sequences that correspond to those of the SUMO-1 conjugation sites from p53 and c-Jun both bind to a surface adjacent to the active site Cys93 of human Ubc9, which has been previously shown to include residues that demonstrate the most significant dynamics on the microsecond to millisecond time scale. Mutations in this region, Q126A, Q130A, A131D, E132A, Y134A, and T135A, were constructed to evaluate the role of these residues in SUMO-1 conjugation. These alterations have significant effects on the conjugation of SUMO-1 with the target proteins p53, E1B, and promyelocytic leukemia protein and define a substrate binding site on Ubc9. Furthermore, the SUMO-1 conjugation site of p53 does not form any defined secondary structure when either free or bound to Ubc9. This suggests that a defined secondary structure at SUMO-1 conjugation sites in target proteins is not necessary for recognition and conjugation by the SUMO-1 pathway.     

Mutual interactions between the SUMO and ubiquitin systems: a plea of no contest

Posttranslational modification by ubiquitin and SUMO is recognized as an effective means of controlling the stability, localization or activity of intracellular proteins, thereby contributing to the regulation of many biological processes. Over the past few years, it has become apparent that the two modification systems often communicate and jointly affect the properties of common substrate proteins, in some cases by being targeted to the same site. However, although SUMO and ubiquitin might have very different effects on a given target, their actions can rarely be explained by simple competition. This article gives an overview of target proteins that can serve as substrates for both SUMO and ubiquitin to highlight the diversity of regulatory strategies that result from the crosstalk between the two modification systems.     

Ubiquitin-dependent proteolytic control of SUMO conjugates

Posttranslational protein modification with small ubiquitin-related modifier (SUMO) is an important regulatory mechanism implicated in many cellular processes, including several of biomedical relevance. We report that inhibition of the proteasome leads to accumulation of proteins that are simultaneously conjugated to both SUMO and ubiquitin in yeast and in human cells. A similar accumulation of such conjugates was detected in Saccharomyces cerevisiae ubc4 ubc5 cells as well as in mutants lacking two RING finger proteins, Ris1 and Hex3/Slx5-Slx8, that bind to SUMO as well as to the ubiquitin-conjugating enzyme Ubc4. In vitro, Hex3-Slx8 complexes promote Ubc4-dependent ubiquitylation. Together these data identify a previously unrecognized pathway that mediates the proteolytic down-regulation of sumoylated proteins. Formation of substrate-linked SUMO chains promotes targeting of SUMO-modified substrates for ubiquitin-mediated proteolysis. Genetic and biochemical evidence indicates that SUMO conjugation can ultimately lead to inactivation of sumoylated substrates by polysumoylation and/or ubiquitin-dependent degradation. Simultaneous inhibition of both mechanisms leads to severe phenotypic defects.