Structural and functional characterization of Tomato SUMO1 gene

Small ubiquitin-related modifier (SUMO) genes regulate various functions of target proteins through post-translational modification. The SUMO proteins have a similar 3-dimensional structure as that of ubiquitin proteins and occur through a cascade of enzymatic reactions. In the present study we have cloned a new SUMO gene from Tomato (Solanum lycopersicum L.), cv Saudi-1, named SlS-SUMO1 gene by PCR using specific primers. This gene has SUMO member's features such as C-terminal diglycine (GG) motif as processing site by ULP (ubiquitin-like SUMO protease) and has SUMO consensus ΨKXE/D sequence. Phylogenetic analysis showed that SlS-SUMO1 gene is highly conserved and homologous to Potatoes Ca-SUMO1 and Ca-SUMO2 genes based on sequence similarity. Expression protein of SlS-SUMO1 gene found to be localized in the nucleus, cytoplasm, and nuclear envelop or nuclear pore complex. SUMO conjugating enzyme SCE1a with SlS-SUMO1 protein co-expressed and co-localized in nucleus and formed nuclear subdomains. This study reported that the SlS-SUMO1 gene is a member of SUMO family and its SUMO protein processing using GG motif and activate and transport to nucleus through Sumoylation system in the plant cell.     

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。     

Combined Effect of Cameo2 and CBP on the Cellular Uptake of Lutein in the Silkworm,Bombyx mori

Formation of yellow-red color cocoons in the silkworm, Bombyx mori, occurs as the result of the selective delivery of carotenoids from the midgut to the silk gland via the hemolymph. This process of pigment transport is thought to be mediated by specific cellular carotenoids carrier proteins. Previous studies indicated that two proteins, Cameo2 and CBP, are associated with the selective transport of lutein from the midgut into the silk gland in Bombyx mori. However, the exact roles of Cameo2 and CBP during the uptake and transport of carotenoids are still unknown. In this study, we investigated the respective contributions of these two proteins to lutein and β-carotene transport in Bombyx mori as well as commercial cell-line. We found that tissues, expressed both Cameo2 and CBP, accumulate lutein. Cells, co-expressed Cameo2 and CBP, absorb 2 fold more lutein (P<0.01) than any other transfected cells, and the rate of cellular uptake of lutein was concentration-dependent and reached saturation. From immunofluorescence staining, confocal microscopy observation and western blot analysis, Cameo2 was localized at the membrane and CBP was expressed in the cytosol. What’s more, bimolecular fluorescence complementation analysis showed that these two proteins directly interacted at cellular level. Therefore, Cameo2 and CBP are necessarily expressed in midguts and silk glands for lutein uptake in Bombyx mori. Cameo2 and CBP, as the membrane protein and the cytosol protein, respectively, have the combined effect to facilitate the cellular uptake of lutein.     

Low METTL3 level in midgut of the Bombyx mori inhibit the proliferation of nucleopolyhedrovirus

N6-methyladeosine (m⁶A) plays an important role in virus infection and replication. Bombyx mori nuclear polyhedrosis is caused by Bombyx mori nucleopolyhedrovirus (BmNPV) infection. Expression levels of m⁶A-modification-related genes after the infection of BmNPV were detected at first. Then, expression levels of BmNPV nucleocapsid protein gene VP39 and envelope fusion protein gene GP64 after knockdown of METTL3in vitro were quantified to identify the effect of m⁶A modification on BmNPV. BmNPV firstly infects the larval midgut in case of oral infection. Subsequently, to clarify the relationship between m⁶A modification and resistance of the silkworm to BmNPV, we detected the expression levels of m⁶A-modification-related genes invivo before and after infection of BmNPV. The results indicated that low METTL3 level hindered the proliferation of BmNPV to some extent, and silkworm strain with low METTL3 level showed stronger resistance against BmNPV. This study will accumulate new experimental data for elucidating the resistance mechanism of silkworm against BmNPV.     

Architecture and assembly of poly-SUMO chains on PCNA in Saccharomyces cerevisiae

Posttranslational modifications of proliferating cell nuclear antigen (PCNA), the eukaryotic processivity clamp for DNA polymerases, regulate the pathways by which replication problems are resolved. In the budding yeast Saccharomyces cerevisiae, ubiquitylation of PCNA in response to DNA damage facilitates the replicative bypass of lesions, whereas conjugation of the ubiquitin-related modifier (SUMO) prevents unscheduled crossover events during S phase. We have analyzed the SUMO modification pattern of budding yeast PCNA in vivo and in vitro and found that most aspects of our in vitro sumoylation reactions reflect the situation under physiological conditions. We show that two oligomeric SUMO chains of two to three moieties each, linked via internal sumoylation consensus motifs within the SUMO sequence, are assembled on PCNA. The SUMO-specific ligase Siz1 both stimulates the overall efficiency of sumoylation and selects the modification site on PCNA. Furthermore, ubiquitin and SUMO chains are assembled independently, and we found evidence that both modifiers can coexist in vivo on a common PCNA subunit. Our results demonstrate for the first time the in vivo assembly of polymeric SUMO chains of defined linkage on a physiological substrate in yeast, but they also indicate that SUMO-SUMO polymers are dispensable for PCNA^SUMO function in replication and recombination.     

SUMO modification through rapamycin-mediated heterodimerization reveals a dual role for Ubc9 in targeting RanGAP1 to nuclear pore complexes

SUMOs (small ubiquitin-related modifiers) are eukaryotic proteins that are covalently conjugated to other proteins and thereby regulate a wide range of important cellular processes. The molecular mechanisms by which SUMO modification influences the functions of most target proteins and cellular processes, however, remain poorly defined. A major obstacle to investigating the effects of SUMO modification is the availability of a system for selectively inducing the modification or demodification of an individual protein. To address this problem, we have developed a procedure using the rapamycin heterodimerizer system. This procedure involves co-expression of rapamycin-binding domain fusion proteins of SUMO and candidate SUMO substrates in living cells. Treating cells with rapamycin induces a tight association between SUMO and a single SUMO substrate, thereby allowing specific downstream effects to be analyzed. Using RanGAP1 as a model SUMO substrate, the heterodimerizer system was used to investigate the molecular mechanism by which SUMO modification targets RanGAP1 from the cytoplasm to nuclear pore complexes (NPCs). Our results revealed a dual role for Ubc9 in targeting RanGAP1 to NPCs: In addition to conjugating SUMO-1 to RanGAP1, Ubc9 is also required to form a stable ternary complex with SUMO-1 modified RanGAP1 and Nup358. As illustrated by our studies, the rapamycin heterodimerizer system represents a novel tool for studying the molecular effects of SUMO modification.     

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。     

Identification of a Bombyx mori gene encoding small heat shock protein BmHsp27.4 expressed in response to high-temperature stress

Elucidating the mechanisms underlying the response and resistance to high-temperature stress in the Lepidoptera is essential for understanding the effect of high-temperature on the regulation of gene expression. A tag (CATGAACGTGAAGAGATTCAG) matching the predicted gene BGIBMGA005823-TA in SilkDB identified the most significant response to high-temperature stress in a screen of the heat-treated digital gene expression library of Bombyx mori (B. mori) (Unpublished data). BLAST and RACE showed that the gene is located on chromosome 5 and has an open reading frame (ORF) of 741 bp. Phylogenetic analysis found that B. mori small heat shock protein 27.4 (BmHSP27.4) is in an evolutionary branch separate from other small heat shock proteins. Expression analysis showed that BmHsp27.4 is highly expressed in brain, eyes and fat bodies in B. mori. Its mRNA level was elevated at high-temperature and this increase was greater in females. The ORF without the signal peptide sequence was cloned into vector pET-28a(+), transformed and over-expressed in Escherichia coli Rosetta (DE3). Western blotting and immunofluorescence analysis with a polyclonal antibody, confirmed that the level of protein BmHSP27.4 increased at a high-temperature, in accordance with its increased mRNA level. In this study, BmHsp27.4 was identified as a novel B. mori gene with an important role in response to high-temperature stress.     

Characterization of a novel chromodomain-containing gene from the silkworm, Bombyx mori

Heterochromatin protein 1 (HP1) is an evolutionarily conserved protein across different eukaryotic species, and is crucial in the establishment and maintenance of heterochromatin. HP1 proteins have two distinct functional domains, an N-terminal chromodomain (CD) and a C-terminal chromoshadow domain (CSD), which are required for the selective binding of HP1 proteins to modified histones. During our screen for HP1-like proteins in the Bombyx mori genome, we found a novel silkworm gene, Bombyx mori chromodomain protein 1 (BmCdp1), encoding a putative chromobox protein with only two CDs. The BmCdp1 family proteins are closely related to the HP1 proteins, and most of them belong to insect lineages. qRT-PCR analysis indicated that BmCdp1 mRNA was most abundantly expressed in early embryos, and relatively higher expression was observed in larval testes, hemocytes, and pupal ovaries. Western blot and immunostaining experiments showed that BmCdp1 was localized mainly in the nucleus of BmN4 cells. We searched BmCdp1-bound loci in the Bombyx genome by ChIP-seq analysis using Flag-tagged BmCdp1-expressing BmN4 cells. Combined with ChIP-qPCR experiments, we identified two reliable BmCdp1-bound loci in the genome. siRNA-mediated knockdown of BmCdp1 in BmN4 cells and early embryos did not affect the expression of the gene located close to the BmCdp1-bound locus.     

Nonvirus encoded proteins could be embedded into Bombyx mori cypovirus polyhedra

To explore whether the nonvirus encoded protein could be embedded into Bombyx mori cypovirus (BmCPV) polyhedra. The stable transformants of BmN cells expressing a polyhedrin (Polh) gene of BmCPV were constructed by transfection with a non-transposon derived vector containing a polh gene. The polyhedra were purified from the midguts of BmCPV-infected silkworms and the transformed BmN cells, respectively. The proteins embedded into polyhedra were determined by mass spectrometry analysis. Host derived proteins were detected in the purified polyhedra. Analysis of structure and hydrophilicity of embedded proteins indicated that the hydrophilic proteins, in structure, were similar to the left-handed structure of polyhedrin or the N-terminal domain of BmCPV structural protein VP3, which were easily embedded into the BmCPV polyhedra. The lysate of polyhedra purified from the infected transformation of BmN cells with modified B. mori baculovirus BmPAK6 could infect BmN cells, indicating that B. mori baculovirus could be embedded into BmCPV polyhedra. Both the purified polyhedra and its lysate could be coloured by X-gal, indicating that the β-galactosidase expressed by BmPAK6 could be incorporated into BmCPV polyhedra. These results suggested that some heterologous proteins and baculovirus could be embedded into polyhedra in an unknown manner.     

SWP25, A Novel Protein Associated with the Nosema bombycis Endospore1

ABSTRACT. Microsporidia are eukaryotic, obligate intracellular, spore-forming parasites. The resistant spores, which harbor a rigid cell wall, are critical for their host-to-host transmission and persistence in the environment. The spore wall comprises two major layers: the exospore and the endospore. In Nosema bombycis, two spore wall proteins have been characterized—an endosporal protein, SWP30, and an exosporal protein, SWP32. Here, we report the identification of the third spore wall protein of N. bombycis, SWP25, the gene of which has no known homologue. SWP25 is predicted to posses a signal peptide and a heparin-binding motif. Immunoelectron microscopy analysis showed that this protein is localized to the endospore. This characterization of a new spore wall protein of N. bombycis may facilitate our investigation of the relationship between N. bombycis and its host, Bombyx mori.     

Homology modeling,virtual screening and dynamics study of proteins involved in Pebrine - Serine protease inhibitor 106 and spore wall protein 26

Abstract

Pebrine is a microsporidian disease caused by Nosema bombycis in Bombyx mori (silk worm) which results in brown/black spots. The affected larvae either spin cocoons which are flimsy with low silk content or not spin a cocoon. It has been hypothesised that Serine Protease Inhibitor 106 (SPN106) is responsible for evasion of host immune system by inhibiting the melanization process in silkworms. Also, Spore Wall Protein 26 (SWP26) has been observed to bind with Ig- like protein Bombyx mori turtle-like protein (Bm-TLP) facilitating the attachment of the microsporidian to the host and contributing to infectivity. Till date, there is no crystal structure of the proteins SPN106, SWP26 and Bm-TLP available. In this study, we performed homology modeling of the three structures using Modeller v9.18 and the binding pockets were identified. Virtual screening was conducted using AutoDock Vina on a ligand library consisting of 28,870 lead-like molecules. The protein stability, compactness, fluctuations and protein-ligand interactions were investigated through Molecular Dynamics (MD) simulations studies using Desmond Maestro 11.3 and a potential lead molecule was identified.

Communicated by Ramaswamy H. Sarma     

Small Peptide Binding Stiffens the Ubiquitin-like Protein SUMO1

Posttranslational modification by small ubiquitin-like modifiers (SUMOs), known as SUMOylation, is a key regulatory event in many eukaryotic cellular processes in which SUMOs interact with a large number of target proteins. SUMO binding motifs (SBMs) are small peptides derived from these target proteins that interact noncovalently with SUMOs and induce conformational changes. To determine the effect of SBMs on the mechanical properties of SUMO1 (the first member of the human SUMO family), we performed single-molecule force spectroscopy experiments on SUMO1/SBM complexes. The unfolding force of SUMO1 (at a pulling speed of 400 nm/s) increased from ∼130 pN to ∼170 pN upon binding to SBMs, indicating mechanical stabilization upon complexation. Pulling-speed-dependent experiments and Monte Carlo simulations measured a large decrease in distance to the unfolding transition state for SUMO1 upon SBM binding, which is by far the largest change measured for any ligand binding protein. The stiffness of SUMO1 (measured as a spring constant for the deformation response along the line joining the N- and C-termini) increased upon SBM binding from ∼1 N/m to ∼3.5 N/m. The relatively higher flexibility of ligand-free SUMO1 might play a role in accessing various conformations before binding to a target.     

SUMO conjugation in plants

Covalent attachment of small proteins to substrates can regulate protein activity in eukaryotes. SUMO, the small ubiquitin-related modifier, can be covalently linked to a broad spectrum of substrates. An understanding of SUMOs role in plant biology is still in its infancy. In this review, we briefly summarize the enzymology of SUMO conjugation (sumoylation), and the current knowledge of SUMO modification in Arabidopsis thaliana (L.) Heynh. and other plants, in comparison to animals and fungi. Furthermore, we assemble a list of potential pathway components in the genome of A. thaliana that have either been functionally defined, or are suggested by similarity to pathway components from other organisms.     

Single-Molecule Studies on PolySUMO Proteins Reveal Their Mechanical Flexibility

Proteins with β-sandwich and β-grasp topologies are resistant to mechanical unfolding as shown by single-molecule force spectroscopy studies. Their high mechanical stability has generally been associated with the mechanical clamp geometry present at the termini. However, there is also evidence for the importance of interactions other than the mechanical clamp in providing mechanical stability, which needs to be tested thoroughly. Here, we report the mechanical unfolding properties of ubiquitin-like proteins (SUMO1 and SUMO2) and their comparison with those of ubiquitin. Although ubiquitin and SUMOs have similar size and structural topology, they differ in their sequences and structural contacts, making them ideal candidates to understand the variations in the mechanical stability of a given protein topology. We observe a two-state unfolding pathway for SUMO1 and SUMO2, similar to that of ubiquitin. Nevertheless, the unfolding forces of SUMO1 (∼130 pN) and SUMO2 (∼120 pN) are lower than that of ubiquitin (∼190 pN) at a pulling speed of 400 nm/s, indicating their lower mechanical stability. The mechanical stabilities of SUMO proteins and ubiquitin are well correlated with the number of interresidue contacts present in their structures. From pulling speed-dependent mechanical unfolding experiments and Monte Carlo simulations, we find that the unfolding potential widths of SUMO1 (∼0.51 nm) and SUMO2 (∼0.33 nm) are much larger than that of ubiquitin (∼0.19 nm), indicating that SUMO1 is six times and SUMO2 is three times mechanically more flexible than ubiquitin. These findings might also be important in understanding the functional differences between ubiquitin and SUMOs.     

Modification in reverse: the SUMO proteases

SUMOs (small ubiquitin-like modifiers) are ubiquitin-related proteins that become covalently conjugated to cellular target proteins that are involved in a variety of processes. Frequently, this modification has a key role in regulating the activities of those targets and, thus, their cellular functions. SUMO conjugation is a highly dynamic process that can be rapidly reversed by the action of members of the Ubl (ubiquitin-like protein)-specific protease (Ulp) family. The same family of enzymes is also responsible for maturation of newly synthesized SUMOs prior to their initial conjugation. Recent advances in structural, biochemical and cell biological analysis of Ulp/SENPs reveal their high degree of specificity towards SUMO paralogs, in addition to discrimination between processing, deconjugation and chain-editing reactions. The dissimilar sub-nuclear localization patterns of Ulp/SENPs and phenotypes of Ulp/SENP mutants further indicate that different Ulp/SENPs have distinct and non-redundant roles.