A Method for SUMO Modification of Proteins in vitro

Lee, Christine C.; Li, Bing; Yu, Hongtao; Matunis, Michael J.

doi:10.21769/bioprotoc.3033

Cited by 4 publications

(3 citation statements)

References 21 publications

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“…Reactions were incubated at 30°C for the indicated times and stopped by addition of 2x SDS-sample buffer and analyzed by SDS-PAGE followed by autoradiography. In vitro sumoylation assays are described in more detail at Bio-protocol ( Lee et al, 2018 ).…”

Section: Methodsmentioning

confidence: 99%

Sumoylation promotes optimal APC/C activation and timely anaphase

Lee

et al. 2018

eLife

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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. We have also identified a functionally important SUMO interacting motif in the cullin-homology domain of APC2 located near the APC4 sumoylation sites and APC/C catalytic core. Our findings provide evidence of an important regulatory role for SUMO modification and binding in affecting APC/C activation and mitotic exit.

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Section: Methodsmentioning

confidence: 99%

Sumoylation promotes optimal APC/C activation and timely anaphase

Lee

et al. 2018

eLife

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“…As part of the complex PTM/UPS modification system, SUMO modification, similarly to ubiquitination, is an ATP-dependent enzymatic cascade involving activating, conjugating, and ligating E1, E2, and E3 enzymes, respectively [ 872 , 873 ]. Small ubiquitin-like modifiers (SUMO) recognize and conjugate many protein substrates that may also be targeted by ubiquitin [ 874 , 875 , 876 ], but often with different effects [ 877 ].…”

Section: Melatonin May Attenuate the Stress-induced Aggregation Of Pathological Mlos Via Post-translational Modification And Rna Modificamentioning

confidence: 99%

Melatonin: Regulation of Biomolecular Condensates in Neurodegenerative Disorders

Loh¹,

Reíter

2021

Antioxidants

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Biomolecular condensates are membraneless organelles (MLOs) that form dynamic, chemically distinct subcellular compartments organizing macromolecules such as proteins, RNA, and DNA in unicellular prokaryotic bacteria and complex eukaryotic cells. Separated from surrounding environments, MLOs in the nucleoplasm, cytoplasm, and mitochondria assemble by liquid–liquid phase separation (LLPS) into transient, non-static, liquid-like droplets that regulate essential molecular functions. LLPS is primarily controlled by post-translational modifications (PTMs) that fine-tune the balance between attractive and repulsive charge states and/or binding motifs of proteins. Aberrant phase separation due to dysregulated membrane lipid rafts and/or PTMs, as well as the absence of adequate hydrotropic small molecules such as ATP, or the presence of specific RNA proteins can cause pathological protein aggregation in neurodegenerative disorders. Melatonin may exert a dominant influence over phase separation in biomolecular condensates by optimizing membrane and MLO interdependent reactions through stabilizing lipid raft domains, reducing line tension, and maintaining negative membrane curvature and fluidity. As a potent antioxidant, melatonin protects cardiolipin and other membrane lipids from peroxidation cascades, supporting protein trafficking, signaling, ion channel activities, and ATPase functionality during condensate coacervation or dissolution. Melatonin may even control condensate LLPS through PTM and balance mRNA- and RNA-binding protein composition by regulating N6-methyladenosine (m6A) modifications. There is currently a lack of pharmaceuticals targeting neurodegenerative disorders via the regulation of phase separation. The potential of melatonin in the modulation of biomolecular condensate in the attenuation of aberrant condensate aggregation in neurodegenerative disorders is discussed in this review.

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“…SUMOylation increases the molecular mass of the target protein by ∼15 kDa, which can be detected as a mobility shift on polyacrylamide gels by staining or by western blot. The rate of the reaction is determined by measuring substrate modification at multiple time points (Eisenhardt, Ilic, Nagamalleswari, & Pichler, 2019;Lee, Li, Yu, & Matunis, 2018;Sarge, 2016).…”

Section: Introductionmentioning

confidence: 99%

A Fluorescence‐Based Assay to Monitor SUMOylation in Real‐Time

Tripathi

Das

2020

CP Protein Science

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The small ubiquitin‐like modifier (SUMO) is an important post‐translational modifier that regulates various cellular processes. Extensive investigations have been made to comprehend the enzymatic process and consequence of SUMOylation. In vitro SUMOylation assays are invaluable for understanding the fundamental mechanisms of SUMOylation. A majority of these assays monitor changes in the size of the substrate upon SUMO conjugation. Current methods typically detect the size difference through SDS‐PAGE and western blots, which makes these methods cumbersome, error‐prone, and time‐consuming. Here, we describe a fluorescence‐based assay for real‐time detection of SUMOylation. In the method, a fluorophore‐tagged substrate is used in the SUMOylation reaction. Upon SUMOylation, the size and correlation time (τc) of the substrate increases, and so does its anisotropy. The rate of change in anisotropy with time reflects the efficiency of the SUMOylation machinery. The real‐time SUMOylation assay protocol is elegant, time‐saving, and less prone to errors. © 2020 Wiley Periodicals LLC. Basic Protocol: Fluorescent anisotropy‐based in vitro SUMOylation assay

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A Method for SUMO Modification of Proteins in vitro

Cited by 4 publications

References 21 publications

Sumoylation promotes optimal APC/C activation and timely anaphase

Sumoylation promotes optimal APC/C activation and timely anaphase

Melatonin: Regulation of Biomolecular Condensates in Neurodegenerative Disorders

A Fluorescence‐Based Assay to Monitor SUMOylation in Real‐Time

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