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Purification of in vitro His-SUMO-1 target proteins. An in vitro reaction of HeLa nuclear extract was performed with HisSUMO-1, which was previously bound to Ni 2 beads. The reaction mixture was obtained by incubating Ubc9, inorganic pyrophosphatase, and ATP in sumoylation buffer with and in the absence of SUMO-1 as control. A, anti-SUMO-1 Western blot (WB) of purified His-SUMO-1-conjugated proteins shows an enrichment of conjugated species. B, SDS-PAGE of purified His-SUMO-1 proteins stained by silver shows a very specific signal in the reaction mixture compared with the control (ctrl). The gel was excised in 34 slices for nLC-MS/MS analysis.
Source publication
Many cellular processes are regulated by the coordination of several post-translational modifications that allow a very fine modulation of substrates. Recently it has been reported that there is a relationship between sumoylation and ubiquitination. Here we propose that the nucleolus is the key organelle in which SUMO-1 conjugates accumulate in res...
Contexts in source publication
Context 1
... inorganic pyrophosphatase, and ATP as the components of the sumoylation buffer. As a control, the reaction mixture was incu- bated in the absence of SUMO-1. His-SUMO-1-tagged proteins were affinity purified on Ni 2 beads. 10% of the eluted sumoy- lated targets were separated by SDS-PAGE and immunoblotted using anti-SUMO-1 antibody. As shown in Fig. 6A, the in vitro reaction sharply increased the level of sumoylated proteins. The remaining part of the purified His-SUMO-1 targets was separated by SDS-PAGE. Comparing the elution with the con- trol, the recovery of SUMO target proteins was highly specific (Fig. 6B). The top part of the silver-stained gel lane, which contains the ...
Context 2
... were separated by SDS-PAGE and immunoblotted using anti-SUMO-1 antibody. As shown in Fig. 6A, the in vitro reaction sharply increased the level of sumoylated proteins. The remaining part of the purified His-SUMO-1 targets was separated by SDS-PAGE. Comparing the elution with the con- trol, the recovery of SUMO target proteins was highly specific (Fig. 6B). The top part of the silver-stained gel lane, which contains the majority of the SUMO-1 conjugates, was cut in several slices, and proteins were in-gel digested with trypsin. The peptide mixtures were analyzed by nLC-MS/MS, identi- fied by database search with the Mascot search engine, and validated by Scaffold software. In Table I, in ...
Citations
... However, the SUMO1 pattern depends on the cell type, organ, and physiologic state. This SUMO1 signal at 150KDa concurs with previous studies showing similar SUMO1 patterns in HeLa cells (83). This pattern is specific to the cell types of the organ, and we didn't see SUMO1 signal at 150KDa in other organs such as the kidney or lungs. ...
Introduction
The recent discovery of TAK981(Subasumstat), the first-in-class selective inhibitor of SUMOylation, enables new immune treatments. TAK981 is already in clinical trials to potentiate immunotherapy in metastatic tumors and hematologic malignancies. Cancer patients have more than ten times higher risk of infections, but the effects of TAK981 in sepsis are unknown and previous studies on SUMO in infections are conflicting.
Methods
We used TAK981 in two sepsis models; polymicrobial peritonitis (CLP) and LPS endotoxemia. Splenectomy was done in both models to study the role of spleen. Western blotting of SUMO-conjugated proteins in spleen lysates was done. Global SUMO1 and SUMO3 knockout mice were used to study the specific SUMO regulation of inflammation in LPS endotoxemia. Splenocytes adoptive transfer was done from SUMO knockouts to wild type mice to study the role of spleen SUMOylation in experimental sepsis.
Results and discussion
Here, we report that inhibition of SUMOylation with TAK981 improved survival in mild polymicrobial peritonitis by enhancing innate immune responses and peritoneal bacterial clearance. Thus, we focused on the effects of TAK981 on the immune responses to bacterial endotoxin, showing that TAK981 enhanced early TNFα production but did not affect the resolution of inflammation. Splenectomy decreased serum TNFα levels by nearly 60% and TAK981-induced TNFα responses. In the spleen, endotoxemia induced a distinct temporal and substrate specificity for SUMO1 and SUMO2/3, and both were inhibited by TAK981. Global genetic depletion of SUMO1, but not SUMO3, enhanced TNFα production and metabolic acidosis. The transfer of SUMO1-null, but not wild-type, splenocytes into splenectomized wild-type mice exacerbated TNFα production and metabolic acidosis in endotoxemia.
Conclusion
These results suggest that specific regulation of splenic SUMO1 can modulate immune and metabolic responses to bacterial infection.
... A number of nucleolar ribosome biogenesis-associated proteins are modified by SUMO, such as NPM (41,42), nucleolin (43), Las1L (44,45), Pelp1 (46) and snoRNP complex components Nop58, Nop56, Nhp2 and DKC1 (33,47,48). Proteomic studies also found that ribosome biogenesis-related proteins are one of the major groups of SUMOylated proteins (49)(50)(51)(52)(53). Thus, USP36 acting as a SUMO E3 reveals an additional mechanism underlying its crucial role in ribosome biogenesis (33). ...
The RNA exosome is an essential 3' to 5' exoribonuclease complex that mediates degradation, processing and quality control of virtually all eukaryotic RNAs. The nucleolar RNA exosome, consisting of a nine-subunit core and a distributive 3' to 5' exonuclease EXOSC10, plays a critical role in processing and degrading nucleolar RNAs, including pre-rRNA. However, how the RNA exosome is regulated in the nucleolus is poorly understood. Here, we report that the nucleolar ubiquitin-specific protease USP36 is a novel regulator of the nucleolar RNA exosome. USP36 binds to the RNA exosome through direct interaction with EXOSC10 in the nucleolus. Interestingly, USP36 does not significantly regulate the levels of EXOSC10 and other tested exosome subunits. Instead, it mediates EXOSC10 SUMOylation at lysine (K) 583. Mutating K583 impaired the binding of EXOSC10 to pre-rRNAs, and the K583R mutant failed to rescue the defects in rRNA processing and cell growth inhibition caused by knockdown of endogenous EXOSC10. Furthermore, EXOSC10 SUMOylation is markedly reduced in cells in response to perturbation of ribosomal biogenesis. Together, these results suggest that USP36 acts as a SUMO ligase to promote EXOSC10 SUMOylation critical for the RNA exosome function in ribosome biogenesis.
... Histones H2A S1/3 Transcriptional repression or chromatin compaction Shiio and Eisenman (2003), Chen et al. (2013) H2B S1/3 (Shiio and Eisenman, 2003) (Ryu et al., 2020) H3 S1/3 K18 Shiio and Eisenman (2003) H4 S1/3 K12 Inhibits chromatin compaction Shiio and Eisenman (2003), Chen et al. (2013), Dhall et al. (2014), Dhall et al. (2017) H2A.X S1 K5, K9, K13, K15, K118, K119, K127, K133, K134 Chen et al. (2013) H1 S1 Chromatin condensation Matafora et al. (2009) HDACs HDAC1 S1/2 K444, K476 Enhances histone deacetylase activity David et al. (2002), Cheng et al. (2004), Citro et al. (2013) HDAC4 S1/2 K559 Enhances histone deacetylase activity Kirsh et al. (2002) HDAC2 S1 K462 Recruits to promoters Brandl et al. (2012) HATs EP300 S1/2/3 CRD Domain Recruits HDAC6 Girdwood et al. (2003), Park et al. (2017) CBP S1 K999, K1034, K1057 Recruits DAXX and HDAC2 Kuo et al. (2005), Park et al. (2017) Frontiers in Molecular Biosciences frontiersin.org proteins in response to a variety of signaling (Yu et al., 2004), and are regarded as key drivers of tumorigenesis (Hogg et al., 2021). ...
... The evidence linking SUMOylation with the process of RNA capping, splicing and tailing is majorly based upon proteomic analysis (Richard et al., 2017). In the past decade, the affinity purification strategy based on tagged-SUMO peptides has led to the development of mass spectrometry (MS) identifying large-scale SUMOylated proteins, many of which are involved in RNA processing events such as capping, splicing, polyadenylation and mRNA export in mammals Manza et al., 2004;Gocke et al., 2005;Guo et al., 2005;Rosas-Acosta et al., 2005;Vertegaal et al., 2006;Blomster et al., 2009;Golebiowski et al., 2009;Matafora et al., 2009;Matic et al., 2010;Tatham et al., 2011;Becker et al., 2013;Schimmel et al., 2014;Tammsalu et al., 2014;Yang and Paschen, 2015). In yeast, a significant proportion (17%) of the SUMO-modified proteins identified is found to be involved in RNA-related processes (Denison et al., 2005). ...
Post-translational modifications of proteins play very important roles in regulating RNA metabolism and affect many biological pathways. Here we mainly summarize the crucial functions of small ubiquitin-like modifier (SUMO) modification in RNA metabolism including transcription, splicing, tailing, stability and modification, as well as its impact on the biogenesis and function of microRNA (miRNA) in particular. This review also highlights the current knowledge about SUMOylation regulation in RNA metabolism involved in many cellular processes such as cell proliferation and apoptosis, which is closely related to tumorigenesis and cancer progression.
... Wariant 1 wykazuje ok. 47% identyczności sekwencji z wariantami 2 i 3, które z kolei różnią się między sobą tylko kilkoma pozycjami AA na N-końcu [84]. Wariant 1 zazwyczaj dołączany jest do białek pojedynczo, zaś 2 oraz 3 tworzą łańcuchy [84]. ...
... 47% identyczności sekwencji z wariantami 2 i 3, które z kolei różnią się między sobą tylko kilkoma pozycjami AA na N-końcu [84]. Wariant 1 zazwyczaj dołączany jest do białek pojedynczo, zaś 2 oraz 3 tworzą łańcuchy [84]. Udowodniono, że do histonów łącznikowych dołączany jest wariant 1 [84]. ...
... Wariant 1 zazwyczaj dołączany jest do białek pojedynczo, zaś 2 oraz 3 tworzą łańcuchy [84]. Udowodniono, że do histonów łącznikowych dołączany jest wariant 1 [84]. ...
Histony łącznikowe (H1) są białkami występującymi w jądrze komórkowym większości eukariontów. Pełnią ważną rolę w ich życiu i rozwoju, uczestnicząc m.in. w kondensacji chromatyny, regulacji transkrypcji genów czy indukcji apoptozy. Mimo powszechnego występowania oraz istotnych funkcji H1, na wiele pytań nie znaleziono dotąd odpowiedzi. Zagadką pozostaje mechanizm odpowiedzialny za tryb wiązania tego histonu do nukleosomu oraz wpływ każdego z trybów na strukturę chromatyny. Pojawiają się również doniesienia na temat uczestnictwa H1 w procesach fizjologicznych nie związanych bezpośrednio z organizacją materiału genetycznego. Postuluje się, że histon ten bierze udział w powstawaniu wrzeciona podziałowego u roślin, a jego fragmenty – w obronie ryb przed patogenami. Artykuł ten ma na celu przyjrzenie się histonowi H1 pod wieloma aspektami, zgromadzenie i uporządkowanie zdobytej już wiedzy oraz nakreślenie pytań, na które odpowiedzi należy szukać w przyszłości.
... RNA-binding proteins involved in translation is relevant in the assembly/disassembly of stress granules (SG), ribonucleoprotein compartments that are involved in the protection of mRNAs during the stress period [55]. Among others, oxidative stress plays an important role in the regulation of protein translation by ubiquitination. ...
mRNAs translation to proteins constitutes an important step of cellular gene expression that is highly regulated in response to different extracellular stimuli and stress situations. The fine control of protein synthesis is carried out both qualitatively and quantitatively, depending on the cellular demand at each moment. Post-translational modifications, in turn regulated by intracellular signaling pathways, play a key role in translation regulation. Among them, ubiquitination, whose role is becoming increasingly important in the control of translation, determines a correct balance between protein synthesis and degradation. In this review we focus on the role of ubiquitination (both degradative K48-linkage type and non-degradative K63-linkage type and monoubiquitination) in eukaryotic translation, both at the pre-translational level during the biogenesis/degradation of the components of translational machinery as well as at the co-translational level under stressful conditions. We also discuss other ubiquitin-dependent regulatory mechanisms of mRNA protection and resumption of translation after stress removal, where the ubiquitination of ribosomal proteins and associated regulatory proteins play an important role in the global rhythm of translation.
... Global proteomic studies of SUMOylated proteins indicate that, while SUMOylated proteins can be identified in nearly all cellular compartments, SUMOylation is a predominantly nuclear event and occurs primarily in transcription factors, chromatin-associated proteins and RNA-binding proteins (8)(9)(10)(11)(12). In addition, histones, the central components of chromatin, are subjected to SUMOylation both in yeast and mammalian cells and histone SUMOylation is typically associated with transcriptional repression (13)(14)(15)(16)(17). Extensive studies on transcription factor and cofactor SUMOylation have established SUMOylation as a major mechanism for transcriptional regulation, most likely due to the potential marked impact of SUMOylation on protein-protein interaction, conformation and subcellular localization of substrate proteins (1,5,16,(18)(19)(20). Given the functional significance of SUMOylation in transcription, several studies have investigated the genome-wide distribution of SUMOylated proteins as well as SUMOmodification enzymes under both regular and stressed conditions. ...
As a conserved post-translational modification, SUMOylation has been shown to play important roles in chromatin-related biological processes including transcription. However, how the SUMOylation machinery associates with chromatin is not clear. Here, we present evidence that multiple SUMOylation machinery components, including SUMO E1 proteins SAE1 and SAE2 and the PIAS (protein inhibitor of activated STAT) family SUMO E3 ligases, are primarily associated with the nuclear matrix rather than with chromatin. We show using nuclease digestion that all PIAS family proteins maintain nuclear matrix association in the absence of chromatin. Importantly, we identify multiple histones including H3 and H2A.Z as directly interacting with PIAS1, and demonstrate that this interaction requires the PIAS1 SAP (SAF-A/B, Acinus, and PIAS) domain. We demonstrate that PIAS1 promotes SUMOylation of histones H3 and H2B in both a SAP domain- and an E3 ligase activity-dependent manner. Furthermore, we show that PIAS1 binds to heat shock-induced genes and represses their expression, and that this function also requires the SAP domain. Altogether, our study reveals for the first time the nuclear matrix as the compartment most enriched in SUMO E1 and PIAS family E3 ligases. Our finding that PIAS1 interacts directly with histone proteins also suggests a molecular mechanism as to how nuclear matrix-associated PIAS1 is able to regulate transcription and other chromatin-related processes.
... Primary sumoylation of human histone H4 as well as weak sumoylation signals from H2A, H2B and H3 were first observed in 2003 (12), and subsequent studies identified sumoylation at K12 of H4 (13) and K18 of H3 (14). Sumoylation of histone variant H2A.X (15) and H1 (16) were also reported in human cells (Table 1). In S. cerevisiae, SUMO can be conjugated to all four core histones (17), as well as the H2A variant H2A.Z (17,18) and H3 variant Cse4 (19). ...
Chromatin structure and gene expression are dynamically controlled by post-translational modifications (PTMs) on histone proteins, including ubiquitylation, methylation, acetylation and small ubiquitin-like modifier (SUMO) conjugation. It was initially thought that histone sumoylation exclusively suppressed gene transcription, but recent advances in proteomics and genomics have uncovered its diverse functions in cotranscriptional processes, including chromatin remodeling, transcript elongation, and blocking cryptic initiation. Histone sumoylation is integral to complex signaling codes that prime additional histone PTMs as well as modifications of the RNA polymerase II carboxy-terminal domain (RNAPII-CTD) during transcription. In addition, sumoylation of histone variants is critical for the DNA double-strand break (DSB) response and for chromosome segregation during mitosis. This review describes recent findings on histone sumoylation and its coordination with other histone and RNAPII-CTD modifications in the regulation of chromatin dynamics.
... In human, a number of nucleolar proteins involved in ribosome biogenesis, including nucleophosmin (NPM) (Tago et al, 2005;Liu et al, 2007), nucleolin (Zhang et al, 2015), and Las1L (Finkbeiner et al, 2011;Castle et al, 2012), are modified by SUMOylation. Proteomic studies found that ribosome biogenesis-related proteins are one of the major classes of SUMOylated proteins in cells (Vertegaal et al, 2004;Matafora et al, 2009;Amente et al, 2012;Hendriks et al, 2014). Likewise, deSUMOylation is also important for ribosome biogenesis. ...
SUMOylation plays a crucial role in regulating diverse cellular processes including ribosome biogenesis. Proteomic analyses and experimental evidence showed that a number of nucleolar proteins involved in ribosome biogenesis are modified by SUMO. However, how these proteins are SUMOylated in cells is less understood. Here, we report that USP36, a nucleolar deubiquitinating enzyme (DUB), promotes nucleolar SUMOylation. Overexpression of USP36 enhances nucleolar SUMOylation, whereas its knockdown or genetic deletion reduces the levels of SUMOylation. USP36 interacts with SUMO2 and Ubc9 and directly mediates SUMOylation in cells and in vitro. We show that USP36 promotes the SUMOylation of the small nucleolar ribonucleoprotein (snoRNP) components Nop58 and Nhp2 in cells and in vitro and their binding to snoRNAs. It also promotes the SUMOylation of snoRNP components Nop56 and DKC1. Functionally, we show that knockdown of USP36 markedly impairs rRNA processing and translation. Thus, USP36 promotes snoRNP group SUMOylation and is critical for ribosome biogenesis and protein translation.
... MS was performed at IFOM Functional Proteomic Center (Milan, Italy). Bands of interest were cut from Coomassie-stained gels, reduced, alkylated, and finally digested overnight with trypsin (Roche) as previously described 59 . After acidification, peptide mixtures were concentrated and desalted on homemade Stagetips µC18 60 , dried in a Speed-Vac and resuspended in 10 µL of 0.1% formic acid. ...
Mutations in PRoline Rich Transmembrane protein 2 (PRRT2) cause pleiotropic syndromes including benign infantile epilepsy, paroxysmal kinesigenic dyskinesia, episodic ataxia, that share the paroxysmal character of the clinical manifestations. PRRT2 is a neuronal protein that plays multiple roles in the regulation of neuronal development, excitability, and neurotransmitter release. To better understand the physiopathology of these clinical phenotypes, we investigated PRRT2 interactome in mouse brain by a pulldown-based proteomic approach and identified α1 and α3 Na+/K+ ATPase (NKA) pumps as major PRRT2-binding proteins. We confirmed PRRT2 and NKA interaction by biochemical approaches and showed their colocalization at neuronal plasma membrane. The acute or constitutive inactivation of PRRT2 had a functional impact on NKA. While PRRT2-deficiency did not modify NKA expression and surface exposure, it caused an increased clustering of α3-NKA on the plasma membrane. Electrophysiological recordings showed that PRRT2-deficiency in primary neurons impaired NKA function during neuronal stimulation without affecting pump activity under resting conditions. Both phenotypes were fully normalized by re-expression of PRRT2 in PRRT2-deficient neurons. In addition, the NKA-dependent afterhyperpolarization that follows high-frequency firing was also reduced in PRRT2-silenced neurons. Taken together, these results demonstrate that PRRT2 is a physiological modulator of NKA function and suggest that an impaired NKA activity contributes to the hyperexcitability phenotype caused by PRRT2 deficiency.
... A gradient of eluent A (distilled water with 2% (v/v) acetonitrile, 0.5% (v/v) formic acid) and B (80% acetonitrile in distilled water with 0.1% (v/v) formic acid) was used to achieve separation from 4% buffer B to 50% buffer B in 320 min (0.2 µL/min flow rate). MS analysis was performed as reported previously [16]. We used exclusion list for predicted albumin peptides for all the MS runs. ...
Background
Hypertension is a complex disease and is the major cause of cardiovascular complications. In the vast majority of individuals, the aetiology of elevated blood pressure (BP) cannot be determined, thus impairing optimized therapies and prognosis for individual patients. A more precise understanding of the molecular pathogenesis of hypertension remains a pressing priority for both basic and translational research. Here we investigated the effect of salt on naive hypertensive patients in order to better understand the salt intake-blood pressure relationship.Methods
Patients underwent an acute saline infusion and were defined as salt-sensitive or salt-resistant according to mean blood pressure changes. Urinary proteome changes during the salt load test were analysed by a label-free quantitative proteomics approach.ResultsOur data show that salt-sensitive patients display equal sodium reabsorption as salt-resistant patients, as major sodium transporters show the same behaviour during the salt load. However, salt-sensitive patients regulate the renin angiotensin system (RAS) differently from salt-resistant patients, and upregulate proteins, as epidermal growth factor (EGF) and plasminogen activator, urokinase (PLAU), involved in the regulation of epithelial sodium channel ENaC activity.Conclusions
Salt-sensitive and salt-resistant subjects have similar response to a saline/volume infusion as detected by urinary proteome. However, we identified glutamyl aminopeptidase (ENPEP), PLAU, EGF and Xaa-Pro aminopeptidase 2 precursor XPNPEP2 as key molecules of salt-sensitivity, through modulation of ENaC-dependent sodium reabsorption along the distal tubule.Graphic abstract
