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The dynamic and reversible post-translational modification of proteins and protein complexes with the ubiquitin-related SUMO modifier regulates a wide variety of nuclear functions, such as transcription, replication and DNA repair. SUMO can be attached as a monomer to its targets, but can also form polymeric SUMO chains. While monoSUMOylation is generally involved in the assembly of protein complexes, multi- or polySUMOylation may have very different consequences. The evolutionary conserved paradigmatic signaling process initiated by multi- or polySUMOylation is the SUMO-targeted Ubiquitin ligase (StUbL) pathway, where the presence of multiple SUMO moieties primes ubiquitylation by the mammalian E3 ubiquitin ligases RNF4 or RNF111, or the yeast Slx5/8 heterodimer. The mammalian SUMO chain-specific isopeptidases SENP6 or SENP7, or yeast Ulp2, counterbalance chain formation thereby limiting StUbL activity. Many facets of SUMO chain signaling are still incompletely understood, mainly because only a limited number of polySUMOylated substrates have been identified. Here we summarize recent work that revealed a highly interconnected network of candidate polySUMO modified proteins functioning in DNA damage response and chromatin organization. Based on these datasets and published work on distinct polySUMO-regulated processes we discuss overarching concepts in SUMO chain function. We propose an evolutionary conserved role of polySUMOylation in orchestrating chromatin dynamics and genome stability networks by balancing chromatin-residency of protein complexes. This concept will be exemplified in processes, such as centromere/kinetochore organization, sister chromatid cohesion, DNA repair and replication.
Throughout their life cells of eukaryotic organisms can be confronted with a variety of proteotoxic stresses and in order to survive, corresponding resistance mechanisms had to evolve. Proteotoxic stresses can cause misfolding of proteins and accumulation of toxic protein aggregates. Failure to remove aggregates of misfolded proteins compromises cellular function and can ultimately cause cell death and disease. To deal with this challenge, cells utilize a complex network of protein quality control pathways, including chaperones, the ubiquitin-proteasome system and the autophagy system.
Another mechanism to cope with proteotoxic stresses is the stalling of translation initiation in order to save valuable resources and prevent faulty translation. Upon stress, intrinsically disordered RNA-binding proteins such as TIA-1 or G3BP1/2 are recruited to stalled preinitiation complexes and a network of multivalent interactions between RNAs and proteins is formed. These mRNP networks can merge with each other and phase separate into membraneless liquid-like structures called stress granules (SGs). Once stress is released, SGs are quickly resolved and translation continues. Yet, chronic stress or mutations of SG-associated proteins can cause persistent SGs, which can sequester misfolded proteins and have been linked to neurodegenerative diseases such as amyotrophic lateral sclerosis or frontotemporal dementia.
In mammalian cells, three isoforms of the small ubiquitin-related modifier (SUMO), SUMO1, SUMO2 and SUMO3 are covalently attached to lysine residues of target proteins. SUMO conjugation is catalyzed via an enzymatic cascade of an heteromeric E1 activating enzyme, the E2 conjugating enzyme Ubc9 and in some cases one of a limited number of E3 SUMO ligases. SUMOylation is a dynamic modification and can be reversed by SUMO isopeptidases, the best characterized of which belong to the SENP family. Cellular stresses such as heat or oxidative stress strongly induce SUMOylation resulting in increased numbers of poly-SUMOylation (formation of SUMO2/3 chains) on nuclear proteins.
The SUMO-targeted ubiquitin ligase (STUbL) RNF4 harbors four SUMO interaction motifs in its N-terminal domain. This feature allows RNF4 to specifically bind poly-SUMOylated proteins and catalyze their proteolytic or non-proteolytic ubiquitylation.
A variety of substrate proteins have been shown to undergo SUMO-primed ubiquitylation by RNF4 in response to stress or DNA damage. RNF4-mediated ubiquitylation is often a signal for proteolytic degradation of these substrates.
In this work we aimed by identify novel RNF4 targets, in heat-stressed cells in order to gain a wider understanding of the nuclear proteotoxic stress response. Analysis by mass spectrometry revealed that a large fraction of RNF4-interacting proteins in heatstressed cells are nuclear RNA-binding proteins, many of which shuttle outside the nucleus and associate with SGs upon stress. We validated, that nuclear RNA-binding proteins, such as TDP-43 and hnRNP M are indeed heat-induced targets of SUMOprimed ubiquitylation by RNF4.
These initial results led us to further investigate the links between the SUMO/RNF4-mediated, nuclear protein quality control and the dynamics of cytosolic heat- or arsenite-induced SGs. SUMO2/3 and RNF4 are mainly nuclear proteins and we confirmed that they do not associate with SGs. Yet, we could demonstrate that depletion of SUMO2/3, the E3 SUMO ligase PML or RNF4 as well as chemical inhibition of SUMOylation strongly delayed SG clearance upon stress release, indicating that a functional STUbL pathway is essential for the timely clearance of SGs.
Next, we investigated how stress-induced poly-SUMOylation is regulated. Our data shows that SENP levels and activities are reduced in response to heat and arsenite stress, which allows the buildup of poly-SUMO chains on nuclear proteins. Limitation of poly-SUMOylation by overexpression of the SUMO chain-specific isopeptidases SENP6 and SENP7 induced SG formation. In contrast, poly-SUMO-priming by chemical depletion of SENP6 with the drug hinokiflavone drastically limited SG formation upon stress treatment. These results indicate a clear role of chain-specific SENPs in the regulation of stress-induced poly-SUMOylation and SG dynamics.
Last, we investigated whether the STUbL pathway could affect the phase separation of FUSP525 (an ALS-linked mutant of the SG-associated protein FUS) and observed that perturbations of the STUbL pathway lead to an increased phase separation of FUSP525L.
Thus, our work connects the SUMO/RNF4 protein quality control mechanism to the dynamics of SGs supporting the hypothesis that release of proteotoxic stress in the nucleus facilitates the clearance of cytosolic SGs. Thereby, we discovered a previously unknown link between the nuclear and cytosolic axis of proteotoxic stress response.
SUMO : glue or solvent for phase-separated ribonucleoprotein complexes and molecular condensates?
(2021)
Spatial organization of cellular processes in membranous or membrane-less organelles (MLOs, alias molecular condensates) is a key concept for compartmentalizing biochemical pathways. Prime examples of MLOs are the nucleolus, PML nuclear bodies, nuclear splicing speckles or cytosolic stress granules. They all represent distinct sub- cellular structures typically enriched in intrinsically disordered proteins and/or RNA and are formed in a process driven by liquid-liquid phase separation. Several MLOs are critically involved in proteostasis and their formation, disassembly and composition are highly sensitive to proteotoxic insults. Changes in the dynamics of MLOs are a major driver of cell dysfunction and disease. There is growing evidence that post-translational modifications are critically involved in controlling the dynamics and composition of MLOs and recent evidence supports an important role of the ubiquitin-like SUMO system in regulating both the assembly and disassembly of these structures. Here we will review our current understanding of SUMO function in MLO dynamics under both normal and pathological conditions.