LRH-1/NR5A2 targets mitochondrial dynamics to reprogram type 1 diabetes macrophages and dendritic cells into an immune tolerance phenotype.

Background: The complex aetiology of type 1 diabetes (T1D), characterised by a detrimental cross-talk between the immune system and insulin-producing beta cells, has hindered the development of effective disease-modifying therapies. The discovery that the pharmacological activation of LRH-1/NR5A2 can reverse hyperglycaemia in mouse models of T1D by attenuating the autoimmune attack coupled to beta cell survival/regeneration prompted us to investigate whether immune tolerisation could be translated to individuals with T1D by LRH-1/NR5A2 activation and improve islet survival.

Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from individuals with and without T1D and derived into various immune cells, including macrophages and dendritic cells.

Functional analyses of a human vascular tumor FOS variant identify a novel degradation mechanism and a link to tumorigenesis.

Epithelioid hemangioma is a locally aggressive vascular neoplasm, found in bones and soft tissue, whose cause is currently unknown, but may involve oncogene activation. FOS is one of the earliest viral oncogenes to be characterized, and normal cellular FOS forms part of the activator protein 1 (AP-1) transcription factor complex, which plays a pivotal role in cell growth, differentiation, and survival as well as the DNA damage response. Despite this, a causal link between aberrant FOS function and naturally occurring tumors has not yet been established.

Site-specific mapping of the human SUMO proteome reveals co-modification with phosphorylation.

Small ubiquitin-like modifiers (SUMOs) are post-translational modifications (PTMs) that regulate nuclear cellular processes. Here we used an augmented K0-SUMO proteomics strategy to identify 40,765 SUMO acceptor sites and quantify their fractional contribution for 6,747 human proteins. Structural-predictive analyses revealed that lysines residing in disordered regions are preferentially targeted by SUMO, in notable contrast to other widespread lysine modifications.

Label-Free Identification and Quantification of SUMO Target Proteins.

Mass spectrometry-based approaches are utilized with increasing frequency to facilitate identification of novel SUMO target proteins and to elucidate the dynamics of SUMOylation in response to cellular stresses. Here, we describe a robust method for the identification of SUMO target proteins, and the relative quantification of SUMOylation dynamics, using a label-free approach. The method relies on a decahistidine (His10)-tagged SUMO, which is expressed at a low level in a mammalian cell line or model organism.

A high-yield double-purification proteomics strategy for the identification of SUMO sites.

The small ubiquitin-like modifier (SUMO) is a protein modifier that is post-translationally coupled to thousands of lysines in more than a thousand proteins. An understanding of which lysines are modified by SUMO is critical in unraveling its function as a master regulator of all nuclear processes, as well as its involvement in diseases such as cancer. Here we describe a protocol for the lysine-deficient (K0) method for efficient identification of SUMOylated lysines by mass spectrometry (MS).

A comprehensive compilation of SUMO proteomics.

Small ubiquitin-like modifiers (SUMOs) are essential for the regulation of several cellular processes and are potential therapeutic targets owing to their involvement in diseases such as cancer and Alzheimer disease. In the past decade, we have witnessed a rapid expansion of proteomic approaches for identifying sumoylated proteins, with recent advances in detecting site-specific sumoylation. In this Analysis, we combined all human SUMO proteomics data currently available into one cohesive database.

Ubiquitin-dependent and independent roles of SUMO in proteostasis.

Cellular proteomes are continuously undergoing alterations as a result of new production of proteins, protein folding, and degradation of proteins. The proper equilibrium of these processes is known as proteostasis, implying that proteomes are in homeostasis. Stress conditions can affect proteostasis due to the accumulation of misfolded proteins as a result of overloading the degradation machinery.

A cascading activity-based probe sequentially targets E1-E2-E3 ubiquitin enzymes.

Post-translational modifications of proteins with ubiquitin (Ub) and ubiquitin-like modifiers (Ubls), orchestrated by a cascade of specialized E1, E2 and E3 enzymes, control a wide range of cellular processes. To monitor catalysis along these complex reaction pathways, we developed a cascading activity-based probe, UbDha. Similarly to the native Ub, upon ATP-dependent activation by the E1, UbDha can travel downstream to the E2 (and subsequently E3) enzymes through sequential trans-thioesterifications.

Mapping the SUMOylated landscape.

SUMOylation is a post-translational modification that regulates a multitude of cellular processes, including replication, cell-cycle progression, protein transport and the DNA damage response. Similar to ubiquitin, SUMO (small ubiquitin-like modifier) is covalently attached to target proteins in a reversible process via an enzymatic cascade. SUMOylation is essential for nearly all eukaryotic organisms, and deregulation of the SUMO system is associated with human diseases such as cancer and neurodegenerative diseases.

System-wide identification of wild-type SUMO-2 conjugation sites.

SUMOylation is a reversible post-translational modification (PTM) regulating all nuclear processes. Identification of SUMOylation sites by mass spectrometry (MS) has been hampered by bulky tryptic fragments, which thus far necessitated the use of mutated SUMO. Here we present a SUMO-specific protease-based methodology which circumvents this problem, dubbed Protease-Reliant Identification of SUMO Modification (PRISM).

c-Myc is targeted to the proteasome for degradation in a SUMOylation-dependent manner, regulated by PIAS1, SENP7 and RNF4.

c-Myc is the most frequently overexpressed oncogene in tumors, including breast cancer, colon cancer and lung cancer. Post-translational modifications comprising phosphorylation, acetylation and ubiquitylation regulate the activity of c-Myc. Recently, it was shown that c-Myc-driven tumors are strongly dependent on the SUMO pathway.

System-wide Analysis of SUMOylation Dynamics in Response to Replication Stress Reveals Novel Small Ubiquitin-like Modified Target Proteins and Acceptor Lysines Relevant for Genome Stability.

Genotoxic agents can cause replication fork stalling in dividing cells because of DNA lesions, eventually leading to replication fork collapse when the damage is not repaired. Small Ubiquitin-like Modifiers (SUMOs) are known to counteract replication stress, nevertheless, only a small number of relevant SUMO target proteins are known. To address this, we have purified and identified SUMO-2 target proteins regulated by replication stress in human cells.

SUMOylation and PARylation cooperate to recruit and stabilize SLX4 at DNA damage sites.

SUMOylation plays important roles in the DNA damage response. However, whether it is important for interstrand crosslink repair remains unknown. We report that the SLX4 nuclease scaffold protein is regulated by SUMOylation.

Uncovering global SUMOylation signaling networks in a site-specific manner.

SUMOylation is a reversible post-translational modification essential for genome stability. Using high-resolution MS, we have studied global SUMOylation in human cells in a site-specific manner, identifying a total of >4,300 SUMOylation sites in >1,600 proteins. To our knowledge, this is the first time that >1,000 SUMOylation sites have been identified under standard growth conditions.

Uncovering SUMOylation dynamics during cell-cycle progression reveals FoxM1 as a key mitotic SUMO target protein.

Loss of small ubiquitin-like modification (SUMOylation) in mice causes genomic instability due to the missegregation of chromosomes. Currently, little is known about the identity of relevant SUMO target proteins that are involved in this process and about global SUMOylation dynamics during cell-cycle progression. We performed a large-scale quantitative proteomics screen to address this and identified 593 proteins to be SUMO-2 modified, including the Forkhead box transcription factor M1 (FoxM1), a key regulator of cell-cycle progression and chromosome segregation.

Remodeling and spacing factor 1 (RSF1) deposits centromere proteins at DNA double-strand breaks to promote non-homologous end-joining.

The cellular response to ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) in native chromatin requires a tight coordination between the activities of DNA repair machineries and factors that modulate chromatin structure. SMARCA5 is an ATPase of the SNF2 family of chromatin remodeling factors that has recently been implicated in the DSB response. It forms distinct chromatin remodeling complexes with several non-canonical subunits, including the remodeling and spacing factor 1 (RSF1) protein.

RNF12 controls embryonic stem cell fate and morphogenesis in zebrafish embryos by targeting Smad7 for degradation.

TGF-β members are of key importance during embryogenesis and tissue homeostasis. Smad7 is a potent antagonist of TGF-β family/Smad-mediated responses, but the regulation of Smad7 activity is not well understood. We identified the RING domain-containing E3 ligase RNF12 as a critical component of TGF-β signaling.

Positively charged amino acids flanking a sumoylation consensus tetramer on the 110kDa tri-snRNP component SART1 enhance sumoylation efficiency.

Covalent attachment of Small Ubiquitin-like MOdifiers (SUMOs) to the epsilon-amino group of lysine residues in target proteins regulates many cellular processes. Previously, we have identified the 110kDa U4/U6.U5 tri-snRNP component SART1 as a target protein for SUMO-1 and SUMO-2.

SUMO chains: polymeric signals.

Ubiquitin and ubiquitin-like proteins are conjugated to a wide variety of target proteins that play roles in all biological processes. Target proteins are conjugated to ubiquitin monomers or to ubiquitin polymers that form via all seven internal lysine residues of ubiquitin. The fate of these target proteins is controlled in a chain architecture-dependent manner.

RNF4 and VHL regulate the proteasomal degradation of SUMO-conjugated Hypoxia-Inducible Factor-2alpha.

Hypoxia-inducible factors (HIFs) are critical transcription factors that mediate cell survival during reduced oxygen conditions (hypoxia). At regular oxygen conditions (normoxia), HIF-1alpha and HIF-2alpha are continuously synthesized in cells and degraded via the ubiquitin-proteasome pathway. During hypoxia, these proteins are stabilized and translocate to the nucleus to activate transcription of target genes that enable cell survival at reduced oxygen levels.

Telomeric DNA mediates de novo PML body formation.

The cell nucleus harbors a variety of different bodies that vary in number, composition, and size. Although these bodies coordinate important nuclear processes, little is known about how they are formed. Among the most intensively studied bodies in recent years is the PML body.

Identification of SUMO target proteins by quantitative proteomics.

The identification of target proteins for small ubiquitin-like modifiers (SUMOs) is a critical step towards a detailed understanding of the cellular functions of SUMOs. Substrate protein identification for SUMOs is hampered by the low abundance of SUMO targets, the finding that only a small fraction of these target proteins is sumoylated, and the high activity of deconjugating enzymes. Quantitative proteomics is a powerful tool to overcome these challenges, because it allows discrimination between contaminating proteins in SUMO-enriched preparations and true target proteins.