植物SIZ1 SUMO E3连接酶的研究进展

SUMO化修饰是一种重要的翻译后修饰,对蛋白的翻译后调控起到重要作用。植物SIZ1是一种SUMOE3连接酶,在SUMO化的过程中起着关键作用。本文概述了SIZ1的基本结构和功能,阐述了其在植物响应非生物胁迫如高温、低温、干旱、盐和离子胁迫时所发挥的调节功能,并展望了植物SIZ1研究中有待解决的问题。     

Heterologous expression of OsSIZ1, a rice SUMO E3 ligase,enhances broad abiotic stress tolerance in transgenic creeping bentgrass

Sumoylation is a posttranslational regulatory process in higher eukaryotes modifying substrate proteins through conjugation of small ubiquitin‐related modifiers (SUMOs). Sumoylation modulates protein stability, subcellular localization and activity; thus, it regulates most cellular functions including response to environmental stress in plants. To study the feasibility of manipulating SUMO E3 ligase, one of the important components in the sumoylation pathway in transgenic (TG) crop plants for improving overall plant performance under adverse environmental conditions, we have analysed TG creeping bentgrass (Agrostis stolonifera L.) plants constitutively expressing OsSIZ1, a rice SUMO E3 ligase. Overexpression of OsSIZ1 led to increased photosynthesis and overall plant growth. When subjected to water deficiency and heat stress, OsSIZ1 plants exhibited drastically enhanced performance associated with more robust root growth, higher water retention and cell membrane integrity than wild‐type (WT) controls. OsSIZ1 plants also displayed significantly better growth than WT controls under phosphate‐starvation conditions, which was associated with a higher uptake of phosphate (Pi) and other minerals, such as potassium and zinc. Further analysis revealed that overexpression of OsSIZ1 enhanced stress‐induced SUMO conjugation to substrate in TG plants, which was associated with modified expression of stress‐related genes. This strongly supports a role sumoylation plays in regulating multiple molecular pathways involved in plant stress response, establishing a direct link between sumoylation and plant response to environmental adversities. Our results demonstrate the great potential of genetic manipulation of sumoylation process in TG crop species for improved resistance to broad abiotic stresses.     

The SUMO‐E3 ligase PIAS3 targets pyruvate kinase M2

Pyruvate kinase M2 (M2‐PK) controls the rate‐limiting step at the end of the glycolytic pathway in normal proliferating and tumor cells. Other functions of M2‐PK in addition to its role in glycolysis are little understood. The aim of this study was to identify new cellular interaction partners of M2‐PK in order to discover novel links between M2‐PK and cellular functions. Here we show that the SUMO‐E3 ligase protein PIAS3 (inhibitor of activated STAT3) physically interacts with M2‐PK and its isoenzyme M1‐PK. Moreover, we demonstrate that endogenous SUMO‐1‐M2‐PK conjugates exist in mammalian cells. Furthermore, we show that transient expression of PIAS3 but not the RING domain mutant PIAS3 (C299S, H301A) is consistent with nuclear localization of M2‐PK and PIAS3 and M2‐PK partially co‐localize in the nucleus of these cells. This study suggests a link between PIAS3 and nuclear pyruvate kinase. J. Cell. Biochem. 107: 293–302, 2009. © 2009 Wiley‐Liss, Inc.     

SUMO E3 Ligases GmSIZ1a and GmSIZ1b regulate vegetative growth in soybean

SIZ1 is a small ubiquitin‐related modifier (SUMO) E3 ligase that mediates post‐translational SUMO modification of target proteins and thereby regulates developmental processes and hormonal and environmental stress responses in Arabidopsis. However, the role of SUMO E3 ligases in crop plants is largely unknown. Here, we identified and characterized two Glycine max (soybean) SUMO E3 ligases, GmSIZ1a and GmSIZ1b. Expression of GmSIZ1a and GmSIZ1b was induced in response to salicylic acid (SA), heat, and dehydration treatment, but not in response to cold, abscisic acid (ABA), and NaCl treatment. Although GmSIZ1a was expressed at higher levels than GmSIZ1b, both genes encoded proteins with SUMO E3 ligase activity in vivo. Heterologous expression of GmSIZ1a or GmSIZ1b rescued the mutant phenotype of Arabidopsis siz1‐2, including dwarfism, constitutively activated expression of pathogen‐related genes, and ABA‐sensitive seed germination. Simultaneous downregulation of GmSIZ1a and GmSIZ1b (GmSIZ1a/b) using RNA interference (RNAi)‐mediated gene silencing decreased heat shock‐induced SUMO conjugation in soybean. Moreover, GmSIZ1RNAi plants exhibited reduced plant height and leaf size. However, unlike Arabidopsis siz1‐2 mutant plants, flowering time and SA levels were not significantly altered in GmSIZ1RNAi plants. Taken together, our results indicate that GmSIZ1a and GmSIZ1b mediate SUMO modification and positively regulate vegetative growth in soybean.     

Salicylic acid-mediated innate immunity in Arabidopsis is regulated by SIZ1 SUMO E3 ligase

Reversible modifications of target proteins by small ubiquitin-like modifier (SUMO) proteins are involved in many cellular processes in yeast and animals. Yet little is known about the function of sumoylation in plants. Here, we show that the SIZ1 gene, which encodes an Arabidopsis SUMO E3 ligase, regulates innate immunity. Mutant siz1 plants exhibit constitutive systemic-acquired resistance (SAR) characterized by elevated accumulation of salicylic acid (SA), increased expression of pathogenesis-related (PR) genes, and increased resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. Transfer of the NahG gene to siz1 plants results in reversal of these phenotypes back to wild-type. Analyses of the double mutants, npr1 siz1, pad4 siz1 and ndr1 siz1 revealed that SIZ1 controls SA signalling. SIZ1 interacts epistatically with PAD4 to regulate PR expression and disease resistance. Consistent with these observations, siz1 plants exhibited enhanced resistance to Pst DC3000 expressing avrRps4, a bacterial avirulence determinant that responds to the EDS1/PAD4-dependent TIR-NBS-type R gene. In contrast, siz1 plants were not resistant to Pst DC3000 expressing avrRpm1, a bacterial avirulence determinant that responds to the NDR1-dependent CC-NBS-type R gene. Jasmonic acid (JA)-induced PDF1.2 expression and susceptibility to Botrytis cinerea were unaltered in siz1 plants. Taken together, these results demonstrate that SIZ1 is required for SA and PAD4-mediated R gene signalling, which in turn confers innate immunity in Arabidopsis.     

Solution structures and DNA binding properties of the N-terminal SAP domains of SUMO E3 ligases from Saccharomyces cerevisiae and Oryza sativa

SUMO E3 ligase of the Siz/PIAS family that promotes sumoylation of target proteins contains SAP motif in its N-terminal region. The SAP motif with a consensus sequence of 35 residues was first proposed to be as a new DNA binding motif found in diverse nuclear proteins involved in chromosomal organization. We have determined solution structures of the SAP domains of SUMO ligases Siz1 from yeast and rice by NMR spectroscopy, showing that the structure of the SAP domain (residues 2-105) of rice Siz1 is a four-helix bundle with an up-down-extended loop-down-up topology, whereas the SAP domain (residues 1-111) of yeast Siz1 is comprised of five helices where the fifth helix alpha5 causes a significant change in the alignment of the four-helix bundle characteristic to the SAP domains of the Siz/PIAS family. We have also demonstrated that both SAP domains have binding ability to an A/T-rich DNA, but that binding affinity of yeast Siz1 SAP is at least by an order of magnitude higher than that of rice Siz1 SAP. Our NMR titration experiments clearly showed that yeast Siz1 SAP uses alpha2-helix for DNA binding more effectively than rice Siz1 SAP, which would result from the dislocation of this helix due to the existence of the extra helix alpha5. In addition, based on the structures of the SAP domains determined here and registered in Protein Data Bank, general features of structures of the SAP domains are discussed in conjunction with equivocal nature of their DNA binding.     

A novel tomato SUMO E3 ligase,SlSIZ1, confers drought tolerance in transgenic tobacco

SUMOylation is an important post‐translational modification process that regulates different cellular functions in eukaryotes. SIZ/PIAS‐type SAP and Miz1 (SIZ1) proteins exhibit SUMO E3 ligase activity, which modulates SUMOylation. However, SIZ1 in tomato has been rarely investigated. In this study, a tomato SIZ1 gene (SlSIZ1) was isolated and its molecular characteristics and role in tolerance to drought stress are described. SlSIZ1 was up‐regulated by cold, sodium chloride (NaCl), polyethylene glycol (PEG), hydrogen peroxide (H₂O₂) and abscisic acid (ABA), and the corresponding proteins were localized in the nucleus. The expression of SlSIZ1 in Arabidopsis thaliana (Arabidopsis) siz1‐2 mutants partially complemented the phenotypes of dwarf, cold sensitivity and ABA hypersensitivity. SlSIZ1 also exhibited the activity of SUMO E3 ligase to promote the accumulation of SUMO conjugates. Under drought stress, the ectopic expression of SlSIZ1 in transgenic tobacco lines enhanced seed germination and reduced the accumulation of reactive oxygen species. SlSIZ1 overexpression conferred the plants with improved growth, high free proline content, minimal malondialdehyde accumulation and increased accumulation of SUMO conjugates. SlSIZ1 is a functional homolog of Arabidopsis SIZ1 with SUMO E3 ligase activity. Therefore, overexpression of SlSIZ1 enhanced the tolerance of transgenic tobacco to drought stress.     

Sumoylation of the GTPase Ran by the RanBP2 SUMO E3 Ligase Complex

The SUMO E3 ligase complex RanBP2/RanGAP1*SUMO1/Ubc9 localizes at cytoplasmic nuclear pore complex (NPC) filaments and is a docking site in nucleocytoplasmic transport. RanBP2 has four Ran binding domains (RBDs), two of which flank RanBP2''s E3 ligase region. We thus wondered whether the small GTPase Ran is a target for RanBP2-dependent sumoylation. Indeed, Ran is sumoylated both by a reconstituted and the endogenous RanBP2 complex in semi-permeabilized cells. Generic inhibition of SUMO isopeptidases or depletion of the SUMO isopeptidase SENP1 enhances sumoylation of Ran in semi-permeabilized cells. As Ran is typically associated with transport receptors, we tested the influence of Crm1, Imp β, Transportin, and NTF2 on Ran sumoylation. Surprisingly, all inhibited Ran sumoylation. Mapping Ran sumoylation sites revealed that transport receptors may simply block access of the E2-conjugating enzyme Ubc9, however the acceptor lysines are perfectly accessible in Ran/NTF2 complexes. Isothermal titration calorimetry revealed that NTF2 prevents sumoylation by reducing RanGDP''s affinity to RanBP2''s RBDs to undetectable levels. Taken together, our findings indicate that RanGDP and not RanGTP is the physiological target for the RanBP2 SUMO E3 ligase complex. Recognition requires interaction of Ran with RanBP2''s RBDs, which is prevented by the transport factor NTF2.     

Determinants of small ubiquitin-like modifier 1 (SUMO1) protein specificity, E3 ligase, and SUMO-RanGAP1 binding activities of nucleoporin RanBP2

The RanBP2 nucleoporin contains an internal repeat domain (IR1-M-IR2) that catalyzes E3 ligase activity and forms a stable complex with SUMO-modified RanGAP1 and UBC9 at the nuclear pore complex. RanBP2 exhibits specificity for SUMO1 as RanGAP1-SUMO1/UBC9 forms a more stable complex with RanBP2 compared with RanGAP1-SUMO2 that results in greater protection of RanGAP-SUMO1 from proteases. The IR1-M-IR2 SUMO E3 ligase activity also shows a similar preference for SUMO1. We utilized deletions and domain swap constructs in protease protection assays and automodification assays to define RanBP2 domains responsible for RanGAP1-SUMO1 protection and SUMO1-specific E3 ligase activity. Our data suggest that elements in both IR1 and IR2 exhibit specificity for SUMO1. IR1 protects RanGAP1-SUMO1/UBC9 and functions as the primary E3 ligase of RanBP2, whereas IR2 retains the ability to interact with SUMO1 to promote SUMO1-specific E3 ligase activity. To determine the structural basis for SUMO1 specificity, a hybrid IR1 construct and IR1 were used to determine three new structures for complexes containing UBC9 with RanGAP1-SUMO1/2. These structures show more extensive contacts among SUMO, UBC9, and RanBP2 in complexes containing SUMO1 compared with SUMO2 and suggest that differences in SUMO specificity may be achieved through these subtle conformational differences.     

Role of the fission yeast SUMO E3 ligase Pli1p in centromere and telomere maintenance

Sumoylation represents a conserved mechanism of post-translational protein modification. We report that Pli1p, the unique fission yeast member of the SP-RING family, is a SUMO E3 ligase in vivo and in vitro. pli1Delta cells display no obvious mitotic growth defects, but are sensitive to the microtubule-destabilizing drug TBZ and exhibit enhanced minichromosome loss. The weakened centromeric function of pli1Delta cells may be related to the defective heterochromatin structure at the central core, as shown by the reduced silencing of an ura4 variegation reporter gene inserted at cnt and imr. Interestingly, pli1Delta cells also exhibit enhanced loss of the ura4 reporter at these loci, likely by gene conversion using homologous sequences as information donors. Moreover, pli1Delta cells exhibit consistent telomere length increase, possibly achieved by a similar process. Point mutations within the RING finger of Pli1p totally or partially reproduce the pli1 deletion phenotypes, thus correlating with their sumoylation activity. Altogether, these results strongly suggest that Pli1p, and by extension sumoylation, is involved in mechanisms that regulate recombination in particular heterochromatic repeated sequences.     

SUMO E3连接酶在植物生长发育中的功能研究进展

SUMO化是真核生物中一种重要的蛋白质翻译后修饰。SUMO E3连接酶具有对底物特异的识别功能, 可以促进SUMO化反应, 是SUMO化修饰过程中的重要组成部分。目前, 在植物中已经鉴定出多种SUMO E3连接酶, 它们参与植物重要器官的发育调控。该文对植物SUMO E3连接酶在根系发育、开花途径、配子发育和光形态建成中的作用及其调节机制进行综述。     

A rice really interesting new gene H2‐type E3 ligase,OsSIRH2‐14, enhances salinity tolerance via ubiquitin/26S proteasome‐mediated degradation of salt‐related proteins

Salinity is a deleterious abiotic stress factor that affects growth, productivity, and physiology of crop plants. Strategies for improving salinity tolerance in plants are critical for crop breeding programmes. Here, we characterized the rice (Oryza sativa) really interesting new gene (RING) H2‐type E3 ligase, OsSIRH2‐14 (previously named OsRFPH2‐14), which plays a positive role in salinity tolerance by regulating salt‐related proteins including an HKT‐type Na⁺ transporter (OsHKT2;1). OsSIRH2‐14 expression was induced in root and shoot tissues treated with NaCl. The OsSIRH2‐14‐EYFP fusion protein was predominately expressed in the cytoplasm, Golgi, and plasma membrane of rice protoplasts. In vitro pull‐down assays and bimolecular fluorescence complementation assays revealed that OsSIRH2‐14 interacts with salt‐related proteins, including OsHKT2;1. OsSIRH2‐14 E3 ligase regulates OsHKT2;1 via the 26S proteasome system under high NaCl concentrations but not under normal conditions. Compared with wild type plants, OsSIRH2‐14‐overexpressing rice plants showed significantly enhanced salinity tolerance and reduced Na⁺ accumulation in the aerial shoot and root tissues. These results suggest that the OsSIRH2‐14 RING E3 ligase positively regulates the salinity stress response by modulating the stability of salt‐related proteins.     

The SUMO2/3 specific E3 ligase ZNF451-1 regulates PML stability

The small ubiquitin related modifier SUMO regulates protein functions to maintain cell homeostasis. SUMO attachment is executed by the hierarchical action of E1, E2 and E3 enzymes of which E3 ligases ensure substrate specificity. We recently identified the ZNF451 family as novel class of SUMO2/3 specific E3 ligases and characterized their function in SUMO chain formation. The founding member, ZNF451 isoform1 (ZNF451-1) partially resides in PML bodies, nuclear structures organized by the promyelocytic leukemia gene product PML. As PML and diverse PML components are well known SUMO substrates the question arises whether ZNF451-1 is involved in their sumoylation. Here, we show that ZNF451-1 indeed functions as SUMO2/3 specific E3 ligase for PML and selected PML components in vitro. Mutational analysis indicates that substrate sumoylation employs an identical biochemical mechanism as we described for SUMO chain formation. In vivo, ZNF451-1 RNAi depletion leads to PML stabilization and an increased number of PML bodies. By contrast, PML degradation upon arsenic trioxide treatment is not ZNF451-1 dependent. Our data suggest a regulatory role of ZNF451-1 in fine-tuning physiological PML levels in a RNF4 cooperative manner in the mouse neuroblastoma N2a cell-line.     

DNA activates the Nse2/Mms21 SUMO E3 ligase in the Smc5/6 complex

Modification of chromosomal proteins by conjugation to SUMO is a key step to cope with DNA damage and to maintain the integrity of the genome. The recruitment of SUMO E3 ligases to chromatin may represent one layer of control on protein sumoylation. However, we currently do not understand how cells upregulate the activity of E3 ligases on chromatin. Here we show that the Nse2 SUMO E3 in the Smc5/6 complex, a critical player during recombinational DNA repair, is directly stimulated by binding to DNA. Activation of sumoylation requires the electrostatic interaction between DNA and a positively charged patch in the ARM domain of Smc5, which acts as a DNA sensor that subsequently promotes a stimulatory activation of the E3 activity in Nse2. Specific disruption of the interaction between the ARM of Smc5 and DNA sensitizes cells to DNA damage, indicating that this mechanism contributes to DNA repair. These results reveal a mechanism to enhance a SUMO E3 ligase activity by direct DNA binding and to restrict sumoylation in the vicinity of those Smc5/6‐Nse2 molecules engaged on DNA.