RNF4‐Dependent Hybrid SUMO‐Ubiquitin Chains are Signals for RAP80 and thereby Mediate the Recruitment of BRCA1 to Sites of DNA Damage

Guzzo, Catherine M.; Berndsen, Christopher E.; Zhu, Jianmei; Gupta, Vibhor; Datta, Aritreyee; Greenberg, Roger A.; Wolberger, Cynthia; Matunis, Michael J.

doi:10.1096/fasebj.27.1_supplement.782.7

Cited by 3 publications

(4 citation statements)

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“…Besides ubiquitination, around 10 different Ub‐like protein (UbL) families have been discovered, like interferon (IFN)‐stimulated gene 15 (ISG15), small Ub‐related modifier (SUMO) and the neural precursor cell expressed, developmentally downregulated 8 (NEDD8), each equipped with dedicated E1‐E2‐E3‐like conjugation machineries and specific biological functions [19]. Extensive cross‐modifications of Ub, UbL and additional post‐translational modifications have been reported [20], for example, the Really Interesting New Gene (RING)‐type E3 Ub ligase RNF4 mediates poly‐ubiquitination of SUMO chains [21] and hybrid Ub‐NEDD8 chains protect against proteotoxicity [22].…”

Section: Ubiquitination and Regulation By Deubiquitinating Enzymesmentioning

confidence: 99%

Surviving death: emerging concepts of RIPK3 and MLKL ubiquitination in the regulation of necroptosis

Karlowitz

Wijk

2021

The FEBS Journal

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Lytic forms of programmed cell death, like necroptosis, are characterised by cell rupture and the release of cellular contents, often provoking inflammatory responses. In the recent years, necroptosis has been shown to play important roles in human diseases like cancer, infections and ischaemia/ reperfusion injury. Coordinated interactions between RIPK1, RIPK3 and MLKL lead to the formation of a dedicated death complex called the necrosome that triggers MLKL-mediated membrane rupture and necroptotic cell death. Necroptotic cell death is tightly controlled by posttranslational modifications, among which especially phosphorylation has been characterised in great detail. Although selective ubiquitination is relatively well-explored in the early initiation stages of necroptosis, the mechanisms and functional consequences of RIPK3 and MLKL ubiquitination for necrosome function and necroptosis are only starting to emerge. This review provides an overview on how site-specific ubiquitination of RIPK3 and MLKL regulates, fine-tunes and reverses the execution of necroptotic cell death.

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Section: Ubiquitination and Regulation By Deubiquitinating Enzymesmentioning

confidence: 99%

Surviving death: emerging concepts of RIPK3 and MLKL ubiquitination in the regulation of necroptosis

Karlowitz

Wijk

2021

The FEBS Journal

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“…This conjugate binds 80-fold more tightly to the BRCA1-A complex subunit RAP80 than SUMO-2 or K63-di-Ub alone. 63 In order to shed light on this process, there is a great demand for hybrid chains where SUMO is linked through its different Lys residues to the Ub chain. Notably, only Ub fused to the N-terminus of SUMO could be prepared enzymatically.…”

Section: Total Chemical Synthesis Of 245 Amino Acidmentioning

confidence: 99%

The Journey for the Total Chemical Synthesis of a 53 kDa Protein

Sun

Brik

2019

Acc. Chem. Res.

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CONSPECTUS: Chemical protein synthesis has been proved as an efficient way to afford medium-sized proteins with high homogeneity in workable quantities for various biochemical, structural, and functional studies. In particular, chemical protein synthesis has enabled access to proteins that are difficult or impossible to prepare by molecular biology approaches, such as those with post-translational modifications and mirror-image proteins. One prominent example is related to ubiquitination, a well-known modification that mediates a variety of cellular processes (e.g., proteasomal degradation). Ubiquitination is considered as a modification that is difficult to introduce into proteins in a test tube to generate ubiquitin (Ub) conjugates with high homogeneity with respect to the chain length and the anchored Lys residue in workable quantities to perform the biochemical and biophysical studies. Chemical protein synthesis has emerged as a powerful approach to prepare Ub conjugates for studies aiming to understand ubiquitination in great detail and decipher its roles in cell processes. Nevertheless, in order to answer more challenging questions in this field, it has been clear that researchers must also prepare Ub conjugates with increased size and complexity. Employing solid-phase peptide synthesis and chemoselective ligation, chemical protein synthesis offers a powerful way to furnish polypeptides composed of 100−200 residues. However, to synthesize larger proteins such as Ub conjugates, longer and more segments are required. This on the other hand leads to difficulties related to solubility, purification, ligation, and late-stage modifications. These challenges have encouraged us to explore more practical synthetic tools to facilitate the synthesis of complex Ub conjugates. In this Account, we summarize the synthetic tools that we have developed to achieve these goals. These include (1) δ-mercaptolysine-mediated isopeptide chemical ligation, (2) chemical synthesis of Ub building blocks, (3) palladium-mediated deprotection of key side chains during protein synthesis, (4) one-pot ligation and desulfurization, and (5) improving the solubility of peptide segments. The developed chemical toolbox has been a key for our successes in the synthesis of diverse and complex Ub conjugates. In this Account, we describe our approaches for generating various Ub conjugates, including (1) the K48 tetra-Ub chain composed of 304 amino acids, (2) the ubiquitinated histones and their analogues made of >200 amino acids, (3) the di-Ub-SUMO-2 hybrid chain composed of 245 amino acids, and (4) the 53 kDa tetra-Ub-α-globin composed of 472 amino acids, which represents the largest protein composed of natural amino acids ever made using chemical protein synthesis. The last target, Flag-Ub-Ub-Ub-Myc-Ub-(HA-α-globin), was prepared in the labeled form where the proximal Ub and distal Ub in the chain were labeled with Myc and Flag tags, respectively, while the α-globin was labeled with the HA tag. Applying the tetra-Ub-α-globin in proteasomal degrada...

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“…Many proteins involved in DSB repair signalling are SUMOylated in basal and stressed conditions, but for many, the role modifications have is not clear (Garvin and Morris, 2017, Hendriks and Vertegaal, 2016, Kumar et al, 2017). Multiple DSB repair factors interact with SUMO1-3 through SUMO Interacting Motifs (SIMs) (Pfeiffer et al, 2017, Guzzo et al, 2012, Hu et al, 2012) or other SUMO binding domains such as ZZ domain (Danielsen et al, 2012), or MYM type zinc fingers (Guzzo et al, 2014, Lee et al, 2022). These interactions are consistent with the proposed role of SUMO as a “molecular glue” wherein high local concentrations of SUMOylated factors - such as chromatin surrounding a DSB, act as binding platforms (Psakhye and Jentsch, 2012, Gonzalez-Prieto et al, 2021).…”

Section: Introductionmentioning

confidence: 99%

“…RAP80 contains tandem SIM-ubiquitin interaction motifs that interact with both SUMO and K63-Ub (Guzzo et al, 2012, Hu et al, 2012). To determine if RAP80 is affected by SUMO4 loss, we measured RAP80 foci kinetics in siSUMO4 cells.…”

Section: Introductionmentioning

confidence: 99%

SUMO4 regulates DNA double-strand break repair independently of conjugation

Garvin

Lanz

Densham

et al. 2022

Preprint

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The small ubiquitin-like modifier (SUMO) family is composed of five members, SUMO1, the highly similar SUMO2/SUMO3, SUMO4 and the tissue-specific SUMO5 (SUMO1P1). Sequence variation in SUMO4 is thought to prevent its maturation, resulting in an un-conjugatable SUMO isoform, and consequently, its functions are poorly understood. Here we show for the first time that SUMO4 promotes DNA double-strand break signalling in a manner distinct from SUMO1 or SUMO2/3. We show that SUMO4 function depends on interaction with partner proteins through SUMO interacting motifs and, on its inability, to be conjugated. We show that SUMO4 promotes the activity of the SUMO protease SENP1. In the absence of SUMO4, reduced SENP1 catalytic activity results in hyperSUMOylation that unbalances the recruitment of several DSB repair factors, including RAP80. These data reveal that SUMO4 acts as a buffer for the SUMOylation system.

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RNF4‐Dependent Hybrid SUMO‐Ubiquitin Chains are Signals for RAP80 and thereby Mediate the Recruitment of BRCA1 to Sites of DNA Damage

Cited by 3 publications

References 0 publications

Surviving death: emerging concepts of RIPK3 and MLKL ubiquitination in the regulation of necroptosis

Surviving death: emerging concepts of RIPK3 and MLKL ubiquitination in the regulation of necroptosis

The Journey for the Total Chemical Synthesis of a 53 kDa Protein

SUMO4 regulates DNA double-strand break repair independently of conjugation

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