Chemical protein synthesis combined with protein cell delivery reveals new insights on the maturation process of SUMO2.

The Small Ubiquitin-like Modifier (SUMO) is a crucial post-translational modifier of proteins, playing a key role in various cellular functions. All SUMOs are synthesized as precursor proteins that must be proteolytically processed. However, the maturation process of cleaving the extending C-terminal tail, preceding SUMOylation of substrates, remains poorly understood, especially within cellular environments.

Analysis of a degron-containing reporter protein GFP-CL1 reveals a role for SUMO1 in cytosolic protein quality control.

Misfolded proteins are recognized and degraded through protein quality control (PQC) pathways, which are essential for maintaining proteostasis and normal cellular functions. Defects in PQC can result in disease, including cancer, cardiovascular disease, and neurodegeneration. The small ubiquitin-related modifiers (SUMOs) were previously implicated in the degradation of nuclear misfolded proteins, but their functions in cytoplasmic PQC are unclear.

RNF4 Regulates the BLM Helicase in Recovery From Replication Fork Collapse.

Sumoylation is an important enhancer of responses to DNA replication stress and the SUMO-targeted ubiquitin E3 ligase RNF4 regulates these responses by ubiquitylation of sumoylated DNA damage response factors. The specific targets and functional consequences of RNF4 regulation in response to replication stress, however, have not been fully characterized. Here we demonstrated that RNF4 is required for the restart of DNA replication following prolonged hydroxyurea (HU)-induced replication stress.

SUMO paralogue-specific functions revealed through systematic analysis of human knockout cell lines and gene expression data.

The small ubiquitin-related modifiers (SUMOs) regulate nearly every aspect of cellular function, from gene expression in the nucleus to ion transport at the plasma membrane. In humans, the SUMO pathway has five SUMO paralogues with sequence homologies that range from 45% to 97%. SUMO1 and SUMO2 are the most distantly related paralogues and also the best studied.

SUMOylation of mitofusins: A potential mechanism for perinuclear mitochondrial congression in cells treated with mitochondrial stressors.

Depolarized/damaged mitochondria aggregate at the perinuclear region prior to mitophagy in cells treated with mitochondrial stressors. However, the cellular mechanism(s) by which damaged mitochondria are transported and remain aggregated at the perinuclear region is unknown. Here, we demonstrate that mitofusins (Mfn1/2) are post-translationally modified by SUMO2 (Small Ubiquitin-related Modifier 2) in Human embryonic kidney 293 (Hek293) cells treated with protonophore CCCP and proteasome inhibitor MG132, both known mitochondrial stressors.

Keratin 17 regulates nuclear morphology and chromatin organization.

Keratin 17 (; K17), a non-lamin intermediate filament protein, was recently found to occur in the nucleus. We report here on K17-dependent differences in nuclear morphology, chromatin organization, and cell proliferation. Human tumor keratinocyte cell lines lacking K17 exhibit flatter nuclei relative to normal.

A cellular and bioinformatics analysis of the SENP1 SUMO isopeptidase in pancreatic cancer.

Sumoylation is an important post-translational modification that involves the conjugation of the Small Ubiquitin-related Modifier (SUMO) onto target proteins. This modification is reversed through the catalytic activity of SUMO isopeptidases, known as SENPs. One of these SENPs, SENP1, was reported to be overexpressed in human pancreatic cancer cells and patient tissues.

A Method for SUMO Modification of Proteins .

The Small Ubiquitin-related Modifier (SUMO) is a protein that is post-translationally added to and reversibly removed from other proteins in eukaryotic cells. SUMO and enzymes of the SUMO pathway are well conserved from yeast to humans and SUMO modification regulates a variety of essential cellular processes including transcription, chromatin remodeling, DNA damage repair, and cell cycle progression. One of the challenges in studying SUMO modification is the relatively low steady-state level of a SUMO-modified protein due in part to the activity of SUMO deconjugating enzymes known as SUMO Isopeptidases or SENPs.

The SUMO-specific isopeptidase SENP2 is targeted to intracellular membranes via a predicted N-terminal amphipathic α-helix.

Sumoylation regulates a wide range of essential cellular functions, many of which are associated with activities in the nucleus. Although there is also emerging evidence for the involvement of the small ubiquitin-related modifier (SUMO) at intracellular membranes, the mechanisms by which sumoylation is regulated at membranes is largely unexplored. In this study, we report that the SUMO-specific isopeptidase, SENP2, uniquely associates with intracellular membranes.

Sumoylation promotes optimal APC/C Activation and Timely Anaphase.

The Anaphase Promoting Complex/Cyclosome (APC/C) is a ubiquitin E3 ligase that functions as the gatekeeper to mitotic exit. APC/C activity is controlled by an interplay of multiple pathways during mitosis, including the spindle assembly checkpoint (SAC), that are not yet fully understood. Here, we show that sumoylation of the APC4 subunit of the APC/C peaks during mitosis and is critical for timely APC/C activation and anaphase onset.

Global Identification of Small Ubiquitin-related Modifier (SUMO) Substrates Reveals Crosstalk between SUMOylation and Phosphorylation Promotes Cell Migration.

Proteomics studies have revealed that SUMOylation is a widely used post-translational modification (PTM) in eukaryotes. However, how SUMO E1/2/3 complexes use different SUMO isoforms and recognize substrates remains largely unknown. Using a human proteome microarray-based activity screen, we identified over 2500 proteins that undergo SUMO E3-dependent SUMOylation.

RAP80, ubiquitin and SUMO in the DNA damage response.

A decade has passed since the first reported connection between RAP80 and BRCA1 in DNA double-strand break repair. Despite the initial identification of RAP80 as a factor localizing BRCA1 to DNA double-strand breaks and potentially promoting homologous recombination, there is increasing evidence that RAP80 instead suppresses homologous recombination to fine-tune the balance of competing DNA repair processes during the S/G phase of the cell cycle. RAP80 opposes homologous recombination by inhibiting DNA end-resection and sequestering BRCA1 into the BRCA1-A complex.

Global Analysis of SUMO-Binding Proteins Identifies SUMOylation as a Key Regulator of the INO80 Chromatin Remodeling Complex.

SUMOylation is a critical regulator of a broad range of cellular processes, and is thought to do so in part by modulation of protein interaction. To comprehensively identify human proteins whose interaction is modulated by SUMOylation, we developed an binding assay using human proteome microarrays to identify targets of SUMO1 and SUMO2. We then integrated these results with protein SUMOylation and protein-protein interaction data to perform network motif analysis.

A high throughput mutagenic analysis of yeast sumo structure and function.

Sumoylation regulates a wide range of essential cellular functions through diverse mechanisms that remain to be fully understood. Using S. cerevisiae, a model organism with a single essential SUMO gene (SMT3), we developed a library of >250 mutant strains with single or multiple amino acid substitutions of surface or core residues in the Smt3 protein.

Detection of SUMOylation in Plasmodium falciparum.

Reversible protein modification by small ubiquitin-related modifiers (SUMOs) regulates many cellular processes, including transcription, protein quality control, cell division, and oxidative stress. SUMOylation is therefore essential for normal cell function and represents a potentially valuable target for the development of inhibitors of pathogenic eukaryotic organisms, including the malaria parasite, Plasmodium falciparum (Pf). The specific and essential functions of SUMOylation in Pf, however, remain largely uncharacterized.

Concepts and Methodologies to Study Protein SUMOylation: An Overview.

Protein modification by the small ubiquitin-related modifier (SUMO) was simultaneously discovered by several groups at the middle of the 1990s. Although distinct names were proposed including Sentrin, GMP1, PIC1, or SMT3, SUMO became the most popular. Early studies on the functions of SUMOylation focused on activities in the nucleus, including transcription activation, chromatin structure, and DNA repair.

Characterizing Requirements for Small Ubiquitin-like Modifier (SUMO) Modification and Binding on Base Excision Repair Activity of Thymine-DNA Glycosylase in Vivo.

Thymine-DNA glycosylase (TDG) plays critical roles in DNA base excision repair and DNA demethylation. It has been proposed, based on structural studies and in vitro biochemistry, that sumoylation is required for efficient TDG enzymatic turnover following base excision. However, whether sumoylation is required for TDG activity in vivo has not previously been tested.

Characterization and Structural Insights into Selective E1-E2 Interactions in the Human and Plasmodium falciparum SUMO Conjugation Systems.

Protein modification by small ubiquitin-related modifiers (SUMOs) is essential and conserved in the malaria parasite, Plasmodium falciparum. We have previously shown that interactions between the SUMO E1-activating and E2-conjugating enzyme in P. falciparum are distinct compared with human, suggesting a potential target for development of parasite-specific inhibitors of SUMOylation.

The C9orf72 repeat expansion disrupts nucleocytoplasmic transport.

The hexanucleotide repeat expansion (HRE) GGGGCC (G4C2) in C9orf72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recent studies support an HRE RNA gain-of-function mechanism of neurotoxicity, and we previously identified protein interactors for the G4C2 RNA including RanGAP1. A candidate-based genetic screen in Drosophila expressing 30 G4C2 repeats identified RanGAP (Drosophila orthologue of human RanGAP1), a key regulator of nucleocytoplasmic transport, as a potent suppressor of neurodegeneration.

Identification of SUMO E3 ligase-specific substrates using the HuProt human proteome microarray.

The functional protein microarray is a powerful and versatile systems biology and proteomics tool that allows the rapid activity profiling of thousands of proteins in parallel. We have recently developed a human proteome array, the HuProt array, which includes ~80 % of all the full-length proteins of the human proteome. In one recent application of the HuProt array, we identified numerous SUMO E3 ligase-dependent SUMOylation substrates.

Identification of SUMO-2/3-modified proteins associated with mitotic chromosomes.

Sumoylation is essential for progression through mitosis, but the specific protein targets and functions remain poorly understood. In this study, we used chromosome spreads to more precisely define the localization of SUMO-2/3 (small ubiquitin-related modifier) to the inner centromere and protein scaffold of mitotic chromosomes. We also developed methods to immunopurify proteins modified by endogenous, untagged SUMO-2/3 from mitotic chromosomes.

Characterization of the SUMO-binding activity of the myeloproliferative and mental retardation (MYM)-type zinc fingers in ZNF261 and ZNF198.

SUMO-binding proteins interact with SUMO modified proteins to mediate a wide range of functional consequences. Here, we report the identification of a new SUMO-binding protein, ZNF261. Four human proteins including ZNF261, ZNF198, ZNF262, and ZNF258 contain a stretch of tandem zinc fingers called myeloproliferative and mental retardation (MYM)-type zinc fingers.

E2-mediated small ubiquitin-like modifier (SUMO) modification of thymine DNA glycosylase is efficient but not selective for the enzyme-product complex.

Thymine DNA glycosylase (TDG) initiates the repair of G·T mismatches that arise by deamination of 5-methylcytosine (mC), and it excises 5-formylcytosine and 5-carboxylcytosine, oxidized forms of mC. TDG functions in active DNA demethylation and is essential for embryonic development. TDG forms a tight enzyme-product complex with abasic DNA, which severely impedes enzymatic turnover.