The gap junction channel protein connexin 43 is covalently modified and regulated by SUMOylation

SUMOylation is a posttranslational modification in which a member of the small ubiquitin-like modifier (SUMO) family of proteins is conjugated to lysine residues in specific target proteins. Most known SUMOylation target proteins are located in the nucleus, but there is increasing evidence that SUMO may also be a key determinant of many extranuclear processes. Gap junctions consist of arrays of intercellular channels that provide direct transfer of ions and small molecules between adjacent cells. Gap junction channels are formed by integral membrane proteins called connexins, of which the best-studied isoform is connexin 43 (Cx43). Here we show that Cx43 is posttranslationally modified by SUMOylation. The data suggest that the SUMO system regulates the Cx43 protein level and the level of functional Cx43 gap junctions at the plasma membrane. Cx43 was found to be modified by SUMO-1, -2, and -3. Evidence is provided that the membrane-proximal lysines at positions 144 and 237, located in the Cx43 intracellular loop and C-terminal tail, respectively, act as SUMO conjugation sites. Mutations of lysine 144 or lysine 237 resulted in reduced Cx43 SUMOylation and reduced Cx43 protein and gap junction levels. Altogether, these data identify Cx43 as a SUMOylation target protein and represent the first evidence that gap junctions are regulated by the SUMO system.     

The fate of metaphase kinetochores is weighed in the balance of SUMOylation during S phase

Genetic evidence suggests that conjugation of Small Ubiquitin-like Modifier proteins (SUMOs) plays an important role in kinetochore function, although the mechanism underlying these observations are poorly defined. We found that depletion of the SUMO protease SENP6 from HeLa cells causes chromosome misalignment, prolonged mitotic arrest and chromosome missegregation. Many inner kinetochore proteins (IKPs) were mis-localized in SENP6-depleted cells. This gross mislocalization of IKPs is due to proteolytic degradation of CENP-I and CENP-H via the SUMO targeted Ubiquitin Ligase (STUbL) pathway. Our findings show that SENP6 is a key regulator of inner kinetochore assembly that antagonizes the cellular STUbL pathway to protect IKPs from degradation during S phase. Here, we will briefly review the implications of our findings and present new data on how SUMOylation during S phase can control chromosome alignment in the subsequent metaphase.     

The fate of metaphase kinetochores is weighed in the balance of SUMOylation during S phase

Genetic evidence suggests that conjugation of Small Ubiquitin-like Modifier proteins (SUMOs) plays an important role in kinetochore function, although the mechanism underlying these observations are poorly defined. we found that depletion of the SUMO protease SENP6 from HeLa cells causes chromosome misalignment, prolonged mitotic arrest and chromosome missegregation. Many inner kinetochore proteins (IKPs) were mis-localized in SENP6-depleted cells. This gross mislocalization of IKPs is due to proteolytic degradation of CENP-I and CENP-H via the SUMO targeted Ubiquitin Ligase (STUbL) pathway. Our findings show that SENP6 is a key regulator of inner kinetochore assembly that antagonizes the cellular STUbL pathway to protect IKPs from degradation during S phase. Here, we will briefly review the implications of our findings and present new data on how SUMOylation during S phase can control chromosome alignment in the subsequent metaphase.Key words: SUMO, kinetochore, mitosis, SENP6, CENP-H, CENP-I     

Rod/Zw10 Complex Is Required for PIASy-dependent Centromeric SUMOylation

SUMO conjugation of cellular proteins is essential for proper progression of mitosis. PIASy, a SUMO E3 ligase, is required for mitotic SUMOylation of chromosomal proteins, yet the regulatory mechanism behind the PIASy-dependent SUMOylation during mitosis has not been determined. Using a series of truncated PIASy proteins, we have found that the N terminus of PIASy is not required for SUMO modification in vitro but is essential for mitotic SUMOylation in Xenopus egg extracts. We demonstrate that swapping the N terminus of PIASy protein with the corresponding region of other PIAS family members abolishes chromosomal binding and mitotic SUMOylation. We further show that the N-terminal domain of PIASy is sufficient for centromeric localization. We identified that the N-terminal domain of PIASy interacts with the Rod/Zw10 complex, and immunofluorescence further reveals that PIASy colocalizes with Rod/Zw10 in the centromeric region. We show that the Rod/Zw10 complex interacts with the first 47 residues of PIASy which were particularly important for mitotic SUMOylation. Finally, we show that depletion of Rod compromises the centromeric localization of PIASy and SUMO2/3 in mitosis. Together, we demonstrate a fundamental mechanism of PIASy to localize in the centromeric region of chromosome to execute centromeric SUMOylation during mitosis.     

植物SUMO化修饰及其生物学功能

SUMO化修饰是细胞内蛋白质功能调节的重要方式之一。植物中的SUMO化修饰途径由SUMO分子和SUMO化酶系组成。SUMO化修饰是一个可逆的动态过程。SUMO前体蛋白在SUMO特异性蛋白酶的作用下成熟,随后通过SUMO活化酶、SUMO结合酶和SUMO连接酶将靶蛋白SUMO化,最后SUMO特异性蛋白酶将SUMO与靶蛋白分离,重新进入SUMO化循环。初步研究表明,植物SUMO化修饰参与植物花期调控、激素信号转导、抗病防御以及逆境应答等生理过程。     

In situ SUMOylation analysis reveals a modulatory role of RanBP2 in the nuclear rim and PML bodies

SUMO modification plays a critical role in a number of cellular functions including nucleocytoplasmic transport, gene expression, cell cycle and formation of subnuclear structures such as promyelocytic leukemia (PML) bodies. In order to identify the sites where SUMOylation takes place in the cell, we developed an in situ SUMOylation assay using a semi-intact cell system and subsequently combined it with siRNA-based knockdown of nucleoporin RanBP2, also known as Nup358, which is one of the known SUMO E3 proteins. With the in situ SUMOylation assay, we found that both nuclear rim and PML bodies, besides mitotic apparatuses, are major targets for active SUMOylation. The ability to analyze possible SUMO conjugation sites would be a valuable tool to investigate where SUMO E3-like activities and/or SUMO substrates exist in the cell. Specific knockdown of RanBP2 completely abolished SUMOylation along the nuclear rim and dislocated RanGAP1 from the nuclear pore complexes. Interestingly, the loss of RanBP2 markedly reduced the number of PML bodies, in contrast to other, normal-appearing nuclear compartments including the nuclear lamina, nucleolus and chromatin, suggesting a novel link between RanBP2 and PML bodies. SUMOylation facilitated by RanBP2 at the nuclear rim may be a key step for the formation of a particular subnuclear organization. Our data imply that SUMO E3 proteins like RanBP2 facilitate spatio-temporal SUMOylation for certain nuclear structure and function.     

植物SUMO化修饰及其生物学功能

SUMO化修饰是细胞内蛋白质功能调节的重要方式之一。植物中的SUMO化修饰途径由SUMO分子和SUMO化酶系组成。SUMO化修饰是一个可逆的动态过程。SUMO前体蛋白在SUMO特异性蛋白酶的作用下成熟, 随后通过SUMO活化酶、SUMO结合酶和SUMO连接酶将靶蛋白SUMO化, 最后SUMO特异性蛋白酶将SUMO与靶蛋白分离, 重新进入SUMO化循环。初步研究表明, 植物SUMO化修饰参与植物花期调控、激素信号转导、抗病防御以及逆境应答等生理过程。     

A differential requirement for SUMOylation in proliferating and non-proliferating cells during Drosophila development

SUMOylation is a highly conserved post-translational modification shown to modulate target protein activity in a wide variety of cellular processes. Although the requirement for SUMO modification of specific substrates has received significant attention in vivo and in vitro, the developmental requirements for SUMOylation at the cell and tissue level remain poorly understood. Here, we show that in Drosophila melanogaster, both heterodimeric components of the SUMO E1-activating enzyme are zygotically required for mitotic progression but are dispensable for cell viability, homeostasis and DNA synthesis in non-dividing cells. Explaining the lack of more pleiotropic effects following a global block of SUMO conjugation, we further demonstrate that low levels of global substrate SUMOylation are detected in mutants lacking either or both E1 subunits. These results not only suggest that minimal SUMOylation persists in the absence of Aos1/Uba2, but also show that the process of cell division is selectively sensitive to reductions in global SUMOylation. Supporting this view, knockdown of SUMO or its E1 and E2 enzymes robustly disrupts proliferating cells in the developing eye, without any detectable effects on the development or differentiation of neighboring post-mitotic cells.     

Battling Alzheimer’s Disease: Targeting SUMOylation-Mediated Pathways

SUMO (small ubiquitin-like modifier) conjugation is a critically important control process in all eukaryotic cells, because it acts as a biochemical switch and regulates the function of hundreds of proteins in many different pathways. Although the diverse functional consequences and molecular targets of SUMOylation remain largely unknown, SUMOylation is becoming increasingly implicated in the pathophysiology of Alzheimer’s disease (AD). Apart from the central SUMO-modified disease-associated proteins, such as amyloid precursor protein, amyloid β, and tau, SUMOylation also regulates several other processes underlying AD. These are involved in inflammation, mitochondrial dynamics, synaptic transmission and plasticity, as well as in protective responses to cell stress. Herein, we review current reports on the involvement of SUMOylation in AD, and present an overview of potential SUMO targets and pathways underlying AD pathogenesis.     

Sumoylation of sodium/calcium exchanger in brain ischemia and ischemic preconditioning

The small ubiquitin-like modifier (SUMO) conjugation (or SUMOylation) is a post-translational protein modification mechanism activated by different stress conditions that has been recently investigated in experimental models of cerebral ischemia. The expression of SUMOylation enzymes and substrates is not restricted to the nucleus, since they are present also in the cytoplasm and on plasma membrane and are involved in several physiological and pathological conditions.In the last decades, convincing evidence have supported the idea that the increased levels of SUMOylated proteins may induce tolerance to ischemic stress. In particular, it has been established that protein SUMOylation may confer neuroprotection during ischemic preconditioning.Considering the increasing evidence that SUMO can modify stability and expression of ion channels and transporters and the relevance of controlling ionic homeostasis in ischemic conditions, the present review will resume the main aspects of SUMO pathways related to the key molecules involved in maintenance of ionic homeostasis during cerebral ischemia and ischemic preconditioning, with a particular focus on the on Na⁺/Ca²⁺ exchangers.     

The small ubiquitin-like modifier (SUMO)-conjugating system of Toxoplasma gondii

SUMOylation, the reversible covalent attachment of small ubiquitin-like modifier (SUMO) peptides has emerged as an important regulator of target protein function. Here we show, by characterization of the Toxoplasma gondii SUMO pathway, that the SUMO conjugation system operates in apicomplexan parasites. A gene encoding the SUMO tag was discovered as were genes encoding the various enzymes required for SUMO processing, ligation and release. Various SUMO conjugates were immuno-detected and by means of a global proteomic-based approach, we identified several T. gondii SUMOylated proteins that reveal many diverse cellular processes in which the modification plays a role. More specifically, SUMO conjugates were seen at the tachyzoite surface in response to signaling generated by host cell contact at the time of invasion. Also, under tissue culture conditions that stimulate bradyzoite differentiation (alkaline pH), we observed the conjugates at the parasitophorous vacuole membrane. The labeling was also at the surface of the mature cysts isolated from parasite-infected mouse brain. Overall, the SUMO conjugation system appears to be a complex and functionally heterogeneous pathway for protein modification in T. gondii with initial data indicating that it is likely to play a putative role in host cell invasion and cyst genesis.     

MAPL is a new mitochondrial SUMO E3 ligase that regulates mitochondrial fission

The modification of proteins by the small ubiquitin‐like modifier (SUMO) is known to regulate an increasing array of cellular processes. SUMOylation of the mitochondrial fission GTPase dynamin‐related protein 1 (DRP1) stimulates mitochondrial fission, suggesting that SUMOylation has an important function in mitochondrial dynamics. The conjugation of SUMO to its substrates requires a regulatory SUMO E3 ligase; however, so far, none has been functionally associated with the mitochondria. By using biochemical assays, overexpression and RNA interference experiments, we characterized the mitochondrial‐anchored protein ligase (MAPL) as the first mitochondrial‐anchored SUMO E3 ligase. Furthermore, we show that DRP1 is a substrate for MAPL, providing a direct link between MAPL and the fission machinery. Importantly, the large number of unidentified mitochondrial SUMO targets suggests a global role for SUMOylation in mitochondrial function, placing MAPL as a crucial component in the regulation of multiple conjugation events.     

SENP3‐mediated deSUMOylation of dynamin‐related protein 1 promotes cell death following ischaemia

Global increases in small ubiquitin‐like modifier (SUMO)‐2/3 conjugation are a neuroprotective response to severe stress but the mechanisms and specific target proteins that determine cell survival have not been identified. Here, we demonstrate that the SUMO‐2/3‐specific protease SENP3 is degraded during oxygen/glucose deprivation (OGD), an in vitro model of ischaemia, via a pathway involving the unfolded protein response (UPR) kinase PERK and the lysosomal enzyme cathepsin B. A key target for SENP3‐mediated deSUMOylation is the GTPase Drp1, which plays a major role in regulating mitochondrial fission. We show that depletion of SENP3 prolongs Drp1 SUMOylation, which suppresses Drp1‐mediated cytochrome c release and caspase‐mediated cell death. SENP3 levels recover following reoxygenation after OGD allowing deSUMOylation of Drp1, which facilitates Drp1 localization at mitochondria and promotes fragmentation and cytochrome c release. RNAi knockdown of SENP3 protects cells from reoxygenation‐induced cell death via a mechanism that requires Drp1 SUMOylation. Thus, we identify a novel adaptive pathway to extreme cell stress in which dynamic changes in SENP3 stability and regulation of Drp1 SUMOylation are crucial determinants of cell fate.     

The activity-dependent stimuli increase SUMO modification in SHSY5Y cells

Post-translational modification of proteins by the small ubiquitin-like modifiers (SUMOs) has emerged as an important regulatory mechanism for alteration of protein activity, stability, and cellular localization. It has been reported that SUMOylation plays an important role in some activities of neuronal cells. However, the link between SUMOylation and activity-dependent stimuli of neurons remains to be elucidated. Here we showed that KCl-induced depolarization increased SUMO conjugation in SHSY5Y cell line in a time-dependent manner. The increase of SUMOylation was largely dependent on calcium influx and intracellular calcium signals. Our study demonstrates the link between the activity-dependent stimuli and global SUMOs conjugation, which may play an important role in activity-dependent signals of neurons.