Mitotic SUMOylation: from the mechanism to functions

ABSTRACT Protein modification by conjugation of SUMO molecules to target proteins is an essential process for both genomic stability and cell viability. In vertebrates, the SUMOylation process involves three SUMO paralogues, SUMO 1, 2, and 3. During cell division, certain chromosome-associated protein are modified by SUMO2/3; however, the regulatory mechanisms that monitor and control the SUMOylation process are poorly defined. Previous studies have revealed that DNA Topoisomerase IIα (TopoIIα) is a mitotic target of SUMO2/3, and that defects in TopoIIα SUMOylation are linked to chromosomal missegregation, suggesting a relationship between TopoIIα SUMOylation and the regulation of mitotic progression. Using an in vitro SUMOylation and decatenation assay with recombinant TopoIIα protein, we demonstrated that SUMO conjugation inhibits the intrinsic activity of TopoIIα. By mass spectrometry and biochemical analysis, we identified Lys 660 of TopoIIα as a SUMOylation site in both Xenopus egg extracts (XEE) and in vitro assays. Lys 660 is located within the catalytic domain of TopoIIα and elimination of Lys 660 SUMOylation by mutation abolished the SUMOylation-mediated inhibition of TopoIIα activity that is normally observed in wildtype (WT), suggesting that Lys 660 SUMOylation is responsible for regulating TopoIIα activity. In addition, biochemical analysis has shown that SUMO conjugation of Lys 660 requires the presence of DNA, suggesting that catalytically active TopoIIα is a target of SUMO conjugation during mitosis. Subsequent investigations of mitotic SUMOylation, lead to the isolation of SUMOylated forms of another protein from mitotic chromosomes. Using mass spectrometry, we identified this protein, poly (ADP-ribose) polymerase 1 (PARP1) as another SUMO2/3 target during mitosis. Similar to the conjugation pattern seen with TopoIIα, SUMO conjugation of PARP1 first appears during prometaphase, shows the strongest intensity during metaphase, and disappears with the onset of anaphase. Interruption of SUMOylation increased PARP1-dependant PARylation, implicating SUMO2/3 conjugation in the regulation of mitotic PARylation. We also investigated the role of PIASy, a SUMO E3 enzyme of the SIZ/PIAS family that is essential for the completion of SUMO2/3 modification during mitosis. Analysis of PIAS family proteins has shown that only PIASy is able to bind to chromosomes during mitosis. Domain analysis has suggested that the N-terminus of PIASy is responsible for chromosome binding. In our studies, we analyzed the functional importance of the N-terminus of PIASy using recombinant PIAS N-terminal truncations. We demonstrated that the most N-terminal region of PIASy is not involved in either substrate binding or in SUMO modification; however, it is required for governing chromosome interaction. Furthermore, an ~130 amino acid polypeptide located at the N-terminus of PIASy was capable of accumulating at the centromere, the site where most mitotic SUMO2/3 conjugation takes place. Mass spectrometry following pull-down assays have shown that Rod/Zw10, a critical component of spindle checkpoints, specifically interacts with the N-terminus of PIASy, but not with other PIAS family members. We demonstrated that elimination of Rod proteins by immunodepletion in XEE causes mislocalization of PIASy on mitotic chromosomes followed by abnormal SUMOylation. In summary, we have investigated how SUMOylation is regulated in a spatial and temporal manner by the SUMO E3 ligase, PIASy. We have also analyzed the function of SUMO conjugation during mitosis using two SUMO target proteins, TopoIIα and PARP1. Our in vitro reconstitution assay has enabled us to closely examine the nature of mitotic SUMOylation and has demonstrated potential roles for SUMOylation during mitosis. Together, these results contribute to the understanding of mitotic SUMO conjugations and raise specific questions for future studies

SUMOylation of Psmd1 Controls Adrm1 Interaction with the Proteasome

SummarySUMOylation is the covalent conjugation of SUMO polypeptides to cellular target proteins. Psmd1 is a subunit of the proteasomal 19S regulatory particle that acts as a docking site for Adrm1, another proteasome subunit that recruits ubiquitinated substrates for proteolysis. Here, we show that the SUMO deconjugating enzyme xSENP1 specifically interacts with Psmd1 and that disruption of xSENP1 targeting delays mitotic exit. Psmd1 becomes SUMOylated through the action of the SUMO E3 enzyme PIASy. We mapped SUMOylation sites within Psmd1 and found that SUMOylation of a critical lysine immediately adjacent to the Adrm1-binding domain regulates the association of Adrm1 with Psmd1. Together, our findings suggest that the interaction of Psmd1 with Adrm1 is controlled by SUMOylation in a manner that may alter proteasome composition and function. These findings demonstrate a mechanism for regulation of ubiquitin-mediated protein degradation by ubiquitin-like proteins of the SUMO family

The MHD Kelvin-Helmholtz Instability III: The Role of Sheared Magnetic Field in Planar Flows

We have carried out simulations of the nonlinear evolution of the magnetohydrodynamic (MHD) Kelvin-Helmholtz (KH) instability for compressible fluids in $2\frac{1}{2}$-dimensions, extending our previous work by Frank et al (1996) and Jones \etal (1997). In the present work we have simulated flows in the x-y plane in which a ``sheared'' magnetic field of uniform strength ``smoothly'' rotates across a thin velocity shear layer from the z direction to the x direction, aligned with the flow field. We focus on dynamical evolution of fluid features, kinetic energy dissipation, and mixing of the fluid between the two layers, considering their dependence on magnetic field strength for this geometry. The introduction of magnetic shear can allow a Cat's Eye-like vortex to form, even when the field is stronger than the nominal linear instability limit given above. For strong fields that vortex is asymmetric with respect to the preliminary shear layer, however, so the subsequent dissipation is enhanced over the uniform field cases of comparable field strength. In fact, so long as the magnetic field achieves some level of dynamical importance during an eddy turnover time, the asymmetries introduced through the magnetic shear will increase flow complexity, and, with that, dissipation and mixing. The degree of the fluid mixing between the two layers is strongly influenced by the magnetic field strength. Mixing of the fluid is most effective when the vortex is disrupted by magnetic tension during transient reconnection, through local chaotic behavior that follows.Comment: 14 pages including 9 figures (4 figures in degraded jpg format), full paper with original quality figures available via anonymous ftp at ftp://canopus.chungnam.ac.kr/ryu/mhdkh2d.uu, to appear in The Astrophysical Journa

Evaluating a theory-based intervention for improving eHealth literacy in older adults: a single group, pretest–posttest design

All authors wish to express their gratitude to our participants for their enthusiastic participation in the intervention over a long five-week period. Also, all authors are deeply grateful to The National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning for their support (Grant No. NRF-2017R1C1B5017768).Background The Internet is considered an important channel for providing health information to older adults. We developed an intervention to improve eHealth literacy in older adults according to the information-motivation-behavioral skills (IMB) theory and Intervention Mapping. This study aimed to analyze the effect of a developed intervention on information, motivation, behavioral skills, and behaviors related to eHealth information in older adults. Methods Forty-six older adults over the age of 65 were recruited from two senior welfare centers in a city in South Korea. We divided the participants into four groups and conducted one intervention per group from March to December 2019. One intervention consisted of 5 sessions and was performed once a week (2 h/1 time) for 5 weeks, culminating in a total lecture time of 10 h. One lecture instructor and two assistant instructors supported the participants in the computer practices. Results Participants’ computer/web knowledge, perceived ease of use, perceived enjoyment, and attitude toward eHealth information showed statistically significant increases. The eHealth literacy efficacy score, searching performance score, and understanding score were also significantly increased. However, there was no significant difference in perceived usefulness. Conclusion The application of the current theory-based methodology can improve the quality of research in eHealth literacy. Additionally, various interventions should be developed and continuously applied to improve eHealth literacy among older adults.This work was supported by The National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning for their support (Grant No. NRF-2017R1C1B5017768)

Phospholipase A2β mediates light-induced stomatal opening in Arabidopsis

Phospholipase A2 (PLA2) catalyses the hydrolysis of phospholipids into lysophospholipids and free fatty acids. Physiological studies have indicated that PLA2 is involved in stomatal movement. However, genetic evidence of a role of PLA2 in guard cell signalling has not yet been reported. To identify PLA2 gene(s) that is (are) involved in light-induced stomatal opening, stomatal movement was examined in Arabidopsis thaliana plants in which the expression of PLA2 isoforms was reduced or knocked-out. Light-induced stomatal opening in PLA2α knockout plants did not differ from wild-type plants. Plants in which PLA2β was silenced by RNA interference exhibited delayed light-induced stomatal opening, and this phenotype was reversed by exogenous lysophospholipids, which are products of PLA2. Stomatal opening in transgenic plants that over-expressed PLA2β was faster than wild-type plants. The expression of PLA2β was localized to the endoplasmic reticulum of guard cells, and increased in response to light in the mature leaf. Aristolochic acid, which inhibits light-induced stomatal opening, inhibited the activity of purified PLA2β. Collectively, these results provide evidence that PLA2β is involved in light-induced stomatal opening in Arabidopsis

PIASy-dependent SUMOylation regulates DNA topoisomerase IIα activity

SUMOylation of TopoIIα inhibits its decatenation activity, preventing resolution of tangled DNA at centromeres before anaphase onset

Identification of dendritic cell precursor from the CD11c+ cells expressing high levels of MHC class II molecules in the culture of bone marrow with FLT3 ligand

Dendritic cells (DCs) are readily generated from the culture of mouse bone marrow (BM) treated with either granulocyte macrophage-colony stimulating factor (GM-CSF) or FMS-like tyrosine kinase 3 ligand (FLT3L). CD11c+MHCII+ or CD11c+MHCIIhi cells are routinely isolated from those BM cultures and generally used as in vitro-generated DCs for a variety of experiments and therapies. Here, we examined CD11c+ cells in the BM culture with GM-CSF or FLT3L by staining with a monoclonal antibody 2A1 that is known to recognize mature or activated DCs. Most of the cells within the CD11c+MHCIIhi DC gate were 2A1+ in the BM culture with GM-CSF (GM-BM culture). In the BM culture with FLT3L (FL-BM culture), almost of all the CD11c+MHCIIhi cells were within the classical DC2 (cDC2) gate. The analysis of FL-BM culture revealed that a majority of cDC2-gated CD11c+MHCIIhi cells exhibited a 2A1-CD83-CD115+CX3CR1+ phenotype, and the others consisted of 2A1+CD83+CD115-CX3CR1- and 2A1-CD83-CD115-CX3CR1- cells. According to the antigen uptake and presentation, morphologies, and gene expression profiles, 2A1-CD83-CD115-CX3CR1- cells were immature cDC2s and 2A1+CD83+CD115-CX3CR1- cells were mature cDC2s. Unexpectedly, however, 2A1-CD83-CD115+CX3CR1+ cells, the most abundant cDC2-gated MHCIIhi cell subset in FL-BM culture, were non-DCs. Adoptive cell transfer experiments in the FL-BM culture confirmed that the cDC2-gated MHCIIhi non-DCs were precursors to cDC2s, i.e., MHCIIhi pre-cDC2s. MHCIIhi pre-cDC2s also expressed the higher level of DC-specific transcription factor Zbtb46 as similarly as immature cDC2s. Besides, MHCIIhi pre-cDC2s were generated only from pre-cDCs and common DC progenitor (CDP) cells but not from monocytes and common monocyte progenitor (cMoP) cells, verifying that MHCIIhi pre-cDC2s are close lineage to cDCs. All in all, our study identified and characterized a new cDC precursor, exhibiting a CD11c+MHCIIhiCD115+CX3CR1+ phenotype, in FL-BM culture

The Effect of Pain Catastrophizing on Depression among Older Korean Adults with Chronic Pain: The Mediating Role of Chronic Pain Interference and Sleep Quality

Pain catastrophizing is a notable concept associated with change in chronic pain interference and depression. Sleep quality is also one of the important factors affecting geriatric depression. This study examined the mediating effects of chronic pain interference and sleep quality on the relationship between pain catastrophizing and depression. This study is a secondary data analysis that analyzed a total of 138 older Korean adults with chronic pain. The participants were selected from a single elderly daycare center in a city in South Korea. Also, the multiple regression analysis and PROCESS macro with bootstrapping were used. The results revealed that chronic pain interference and sleep quality mediated the relationship between pain catastrophizing and depression, respectively. Furthermore, chronic pain interference and sleep quality sequentially and dually mediated the effect of pain catastrophizing on depression. In the management of depression in the elderly, persistent complaints of pain should not be disregarded, irrespective of the intensity of their chronic pain. Psychological intervention is needed to alleviate negative thoughts about chronic pain and to increase the ability to cope with chronic pain. In addition, it is important to assess sleep patterns and to develop interventions to improve sleep quality, because depression in the elderly could appear as a symptom of a sleep problems