Noncanonical structural requirements of neurofibromin SUMOylation reveal a folding-deficiency of several pathogenic mutants

Neurofibromin (Nf1) is a large multidomain protein encoded by the tumour-suppressor gene NF1. NF1 is mutated in a frequently occurring genetic disease, neurofibromatosis type I, and in various cancers. The best described function of Nf1 is its Ras-GTPase activity, carried out by its GAP-related domain (GRD). SecPH, another structurally well-characterized domain of Nf1, is immediately adjacent to the GRD and interacts with lipids and proteins, thus connecting Nf1 to diverse signalling pathways. Here, we demonstrate, for the first time, that Nf1 and SecPH are substrates of the SUMO pathway. We identified a well-defined SUMOylation profile of SecPH and a main SUMOylation event on Lys1731 that appears to play a role in Ras-GAP activity. Our data allowed us to characterize a new set of pathogenic Nf1 missense mutants that exhibits a disrupted SUMOylation profile that may correlate with their unfolding. Accordingly, Lys1731 SUMOylation is mediated by a noncanonical structural motif, therefore allowing a read-out of SecPH conformation and folding status.

Tissue-specific inhibition of protein sumoylation uncovers diverse SUMO functions during C. elegans vulval development

The sumoylation (SUMO) pathway is involved in a variety of processes during C. elegans development, such as gonadal and vulval fate specification, cell cycle progression and maintenance of chromosome structure. The ubiquitous expression of the sumoylation machinery and its involvement in many essential processes has made it difficult to dissect the tissue-specific roles of protein sumoylation and identify the specific target proteins. To overcome these challenges, we have established tools to block protein sumoylation and degrade sumoylated target proteins in a tissue-specific and temporally controlled manner. We employed the auxin-inducible protein degradation system (AID) to down-regulate AID-tagged SUMO E3 ligase GEI-17 or the SUMO ortholog SMO-1, either in the vulval precursor cells (VPCs) or in the gonadal anchor cell (AC). Tissue-specific inhibition of GEI-17 and SMO-1 revealed diverse roles of the SUMO pathway during vulval development, such as AC positioning, basement membrane (BM) breaching, vulval cell fate specification and epithelial morphogenesis. Inhibition of sumoylation in the VPCs resulted in an abnormal shape of the vulval toroids and ectopic cell fusions. Sumoylation of the ETS transcription factor LIN-1 at K169 mediates a subset of these SUMO functions, especially the proper contraction of the ventral vulA toroids. Thus, the SUMO pathway plays diverse roles throughout vulval development.

Therapeutic Potential of Targeting the SUMO Pathway in Cancer

SUMOylation is a dynamic and reversible post-translational modification, characterized more than 20 years ago, that regulates protein function at multiple levels. Key oncoproteins and tumor suppressors are SUMO substrates. In addition to alterations in SUMO pathway activity due to conditions typically present in cancer, such as hypoxia, the SUMO machinery components are deregulated at the genomic level in cancer. The delicate balance between SUMOylation and deSUMOylation is regulated by SENP enzymes possessing SUMO-deconjugation activity. Dysregulation of SUMO machinery components can disrupt the balance of SUMOylation, contributing to the tumorigenesis and drug resistance of various cancers in a context-dependent manner. Many molecular mechanisms relevant to the pathogenesis of specific cancers involve SUMO, highlighting the potential relevance of SUMO machinery components as therapeutic targets. Recent advances in the development of inhibitors targeting SUMOylation and deSUMOylation permit evaluation of the therapeutic potential of targeting the SUMO pathway in cancer. Finally, the first drug inhibiting SUMO pathway, TAK-981, is currently also being evaluated in clinical trials in cancer patients. Intriguingly, the inhibition of SUMOylation may also have the potential to activate the anti-tumor immune response. Here, we comprehensively and systematically review the recent developments in understanding the role of SUMOylation in cancer and specifically focus on elaborating the scientific rationale of targeting the SUMO pathway in different cancers.

SUMO and SUMOylation Pathway at the Forefront of Host Immune Response

Pathogens pose a continuous challenge for the survival of the host species. In response to the pathogens, the host immune system mounts orchestrated defense responses initiating various mechanisms both at the cellular and molecular levels, including multiple post-translational modifications (PTMs) leading to the initiation of signaling pathways. The network of such pathways results in the recruitment of various innate immune components and cells at the site of infection and activation of the adaptive immune cells, which work in synergy to combat the pathogens. Ubiquitination is one of the most commonly used PTMs. Host cells utilize ubiquitination for both temporal and spatial regulation of immune response pathways. Over the last decade, ubiquitin family proteins, particularly small ubiquitin-related modifiers (SUMO), have been widely implicated in host immune response. SUMOs are ubiquitin-like (Ubl) proteins transiently conjugated to a wide variety of proteins through SUMOylation. SUMOs primarily exert their effect on target proteins by covalently modifying them. However, SUMO also engages in a non-covalent interaction with the SUMO-interacting motif (SIM) in target proteins. Unlike ubiquitination, SUMOylation alters localization, interactions, functions, or stability of target proteins. This review provides an overview of the interplay of SUMOylation and immune signaling and development pathways in general. Additionally, we discuss in detail the regulation exerted by covalent SUMO modifications of target proteins, and SIM mediated non-covalent interactions with several effector proteins. In addition, we provide a comprehensive review of the literature on the importance of the SUMO pathway in the development and maintenance of a robust immune system network of the host. We also summarize how pathogens modulate the host SUMO cycle to sustain infectability. Studies dealing mainly with SUMO pathway proteins in the immune system are still in infancy. We anticipate that the field will see a thorough and more directed analysis of the SUMO pathway in regulating different cells and pathways of the immune system. Our current understanding of the importance of the SUMO pathway in the immune system necessitates an urgent need to synthesize specific inhibitors, bioactive regulatory molecules, as novel therapeutic targets.

A degron-based strategy reveals new insights into Aurora B function in C. elegans

The widely conserved kinase Aurora B regulates important events during cell division. Surprisingly, recent work has uncovered a few functions of Aurora-family kinases that do not require kinase activity. Thus, understanding this important class of cell cycle regulators will require strategies to distinguish kinase-dependent from independent functions. Here, we address this need in C. elegans by combining germline-specific, auxin-induced Aurora B (AIR-2) degradation with the transgenic expression of kinase-inactive AIR-2. Through this approach, we find that kinase activity is essential for AIR-2’s major meiotic functions and also for mitotic chromosome segregation. Moreover, our analysis revealed insight into the assembly of the ring complex (RC), a structure that is essential for chromosome congression in C. elegans oocytes. AIR-2 localizes to chromosomes and recruits other components to form the RC. However, we found that while kinase-dead AIR-2 could load onto chromosomes, other components were not recruited. This failure in RC assembly appeared to be due to a loss of RC SUMOylation, suggesting that there is crosstalk between SUMOylation and phosphorylation in building the RC and implicating AIR-2 in regulating the SUMO pathway in oocytes. Similar conditional depletion approaches may reveal new insights into other cell cycle regulators.

PIE-1 SUMOylation promotes germline fates and piRNA-dependent silencing in C. elegans

Germlines shape and balance heredity, integrating and regulating information from both parental and foreign sources. Insights into how germlines handle information have come from the study of factors that specify or maintain the germline fate. In early Caenorhabditis elegans embryos, the CCCH zinc finger protein PIE-1 localizes to the germline where it prevents somatic differentiation programs. Here, we show that PIE-1 also functions in the meiotic ovary where it becomes SUMOylated and engages the small ubiquitin-like modifier (SUMO)-conjugating machinery. Using whole-SUMO-proteome mass spectrometry, we identify HDAC SUMOylation as a target of PIE-1. Our analyses of genetic interactions between pie-1 and SUMO pathway mutants suggest that PIE-1 engages the SUMO machinery both to preserve the germline fate in the embryo and to promote Argonaute-mediated surveillance in the adult germline.

Repression of interrupted and intact rDNA by the SUMO pathway in Drosophila melanogaster

Ribosomal RNAs (rRNAs) are essential components of the ribosome and are among the most abundant macromolecules in the cell. To ensure high rRNA level, eukaryotic genomes contain dozens to hundreds of rDNA genes, however, only a fraction of the rRNA genes seems to be active, while others are transcriptionally silent. We found that individual rDNA genes have high level of cell-to-cell heterogeneity in their expression in Drosophila melanogaster. Insertion of heterologous sequences into rDNA leads to repression associated with reduced expression in individual cells and decreased number of cells expressing rDNA with insertions. We found that SUMO (Small Ubiquitin-like Modifier) and SUMO ligase Ubc9 are required for efficient repression of interrupted rDNA units and variable expression of intact rDNA. Disruption of the SUMO pathway abolishes discrimination of interrupted and intact rDNAs and removes cell-to-cell heterogeneity leading to uniformly high expression of individual rDNA in single cells. Our results suggest that the SUMO pathway is responsible for both repression of interrupted units and control of intact rDNA expression.

A conserved SUMO pathway repairs topoisomerase DNA-protein cross-links by engaging ubiquitin-mediated proteasomal degradation

Topoisomerases form transient covalent DNA cleavage complexes to perform their reactions. Topoisomerase I cleavage complexes (TOP1ccs) are trapped by camptothecin and TOP2ccs by etoposide. Proteolysis of the trapped topoisomerase DNA-protein cross-links (TOP-DPCs) is a key step for some pathways to repair these lesions. We describe a pathway that features a prominent role of the small ubiquitin-like modifier (SUMO) modification for both TOP1- and TOP2-DPC repair. Both undergo rapid and sequential SUMO-2/3 and SUMO-1 modifications in human cells. The SUMO ligase PIAS4 is required for these modifications. RNF4, a SUMO-targeted ubiquitin ligase (STUbL), then ubiquitylates the TOP-DPCs for their subsequent degradation by the proteasome. This pathway is conserved in yeast with Siz1 and Slx5-Slx8, the orthologs of human PIAS4 and RNF4.