Protein posttranslational modifications by small ubiquitin-like modifiers (SUMOs) are critical for regulating a variety of cellular processes. Dysregulation of SUMOylation is increasingly... Show moreProtein posttranslational modifications by small ubiquitin-like modifiers (SUMOs) are critical for regulating a variety of cellular processes. Dysregulation of SUMOylation is increasingly associated with cancer and inhibitors that block SUMOylation are currently being explored as cancer treatment. We aimed to gain a better understanding of how the SUMO system governs the integrated network of nuclear signaling pathways, with a specific focus on genome maintenance. For this, we combined unbiased global mass spectrometry-based proteomics approaches with mechanistic follow-up studies. We found that the SUMO protease SENP6 maintains genome stability by ensuring the timely removal of SUMO chains on proteins involved in the response to DNA damage, affecting their localization, kinetics and nuclear condensation state. In addition, we found that noncovalent protein interactions with SUMOs mediated by SUMO-interaction motifs (SIMs) are also critical in maintaining genome stability, by facilitating the recruitment of DNA damage response proteins to the sites of damage. Lastly, we offer valuable new insights on noncovalent SUMO interactions by providing a comprehensive dataset of proteins that bind SUMO independently of the classical SUMO-SIM interaction. This suggests that the relevance of alternate modes of SUMO interactions is currently underestimated by the field and opens up new avenues for future research. Show less
The SUMO protease SENP6 maintains genomic stability, but mechanistic understanding of this process remains limited. We find that SENP6 deconjugates SUMO2/3 polymers on a group of DNA damage... Show moreThe SUMO protease SENP6 maintains genomic stability, but mechanistic understanding of this process remains limited. We find that SENP6 deconjugates SUMO2/3 polymers on a group of DNA damage response proteins, including BRCA1-BARD1, 53BP1, BLM and ERCC1-XPF. SENP6 maintains these proteins in a hypo-SUMOylated state under unstressed conditions and counteracts their polySUMOylation after hydroxyurea-induced stress. Co-depletion of RNF4 leads to a further increase in SUMOylation of BRCA1, BARD1 and BLM, suggesting that SENP6 antagonizes targeting of these proteins by RNF4. Functionally, depletion of SENP6 results in uncoordinated recruitment and persistence of SUMO2/3 at UVA laser and ionizing radiation induced DNA damage sites. Additionally, SUMO2/3 and DNA damage response proteins accumulate in nuclear bodies, in a PML-independent manner driven by multivalent SUMO-SIM interactions. These data illustrate coordinated regulation of SUMOylated DNA damage response proteins by SENP6, governing their timely localization at DNA damage sites and nuclear condensation state. Show less
BackgroundSUMOylation involves the attachment of small ubiquitin-like modifier (SUMO) proteins to specific lysine residues on thousands of substrates with target-specific effects on protein... Show moreBackgroundSUMOylation involves the attachment of small ubiquitin-like modifier (SUMO) proteins to specific lysine residues on thousands of substrates with target-specific effects on protein function. Sentrin-specific proteases (SENPs) are proteins involved in the maturation and deconjugation of SUMO. Specifically, SENP7 is responsible for processing polySUMO chains on targeted substrates including the heterochromatin protein 1 & alpha; (HP1 & alpha;). MethodsWe performed exome sequencing and segregation studies in a family with several infants presenting with an unidentified syndrome. RNA and protein expression studies were performed in fibroblasts available from one subject. ResultsWe identified a kindred with four affected subjects presenting with a spectrum of findings including congenital arthrogryposis, no achievement of developmental milestones, early respiratory failure, neutropenia and recurrent infections. All died within four months after birth. Exome sequencing identified a homozygous stop gain variant in SENP7 c.1474C>T; p.(Gln492*) as the probable aetiology. The proband's fibroblasts demonstrated decreased mRNA expression. Protein expression studies showed significant protein dysregulation in total cell lysates and in the chromatin fraction. We found that HP1 & alpha; levels as well as different histones and H3K9me3 were reduced in patient fibroblasts. These results support previous studies showing interaction between SENP7 and HP1 & alpha;, and suggest loss of SENP7 leads to reduced heterochromatin condensation and subsequent aberrant gene expression. ConclusionOur results suggest a critical role for SENP7 in nervous system development, haematopoiesis and immune function in humans. Show less
In contrast to our extensive knowledge on covalent small ubiquitin-like modifier (SUMO) target proteins, we are limited in our understanding of non-covalent SUMO-binding proteins. We identify... Show moreIn contrast to our extensive knowledge on covalent small ubiquitin-like modifier (SUMO) target proteins, we are limited in our understanding of non-covalent SUMO-binding proteins. We identify interactors of different SUMO isoforms-monomeric SUMO1, monomeric SUMO2, or linear trimeric SUMO2 chains-using a mass spectrometry-based proteomics approach. We identify 379 proteins that bind to different SUMO isoforms, mainly in a preferential manner. Interestingly, XRCC4 is the only DNA repair protein in our screen with a preference for SUMO2 trimers over mono-SUMO2, as well as the only protein in our screen that belongs to the non-homologous end joining (NHEJ) DNA double-strand break repair pathway. A SUMO interaction motif (SIM) in XRCC4 regulates its recruitment to sites of DNA damage and phosphorylation of S320 by DNA-PKcs. Our data highlight the importance of non-covalent and covalent sumoylation for DNA double-strand break repair via the NHEJ pathway and provide a resource of SUMO isoform interactors. Show less
Jia, Y.Q.; Claessens, L.A.; Vertegaal, A.C.O.; Ovaa, H. 2019
