Haplotype variations of sucrose phosphate synthase B gene among sugarcane accessions with different sucrose content.

BACKGROUND: Sucrose phosphate synthase B (SPSB) gene encoding an important rate-limiting enzyme for sucrose synthesis in sugarcane is mainly expressed on leaves, where its alleles control sucrose synthesis. In this study, genetic variation of SPSB gene represented by different haplotypes in sugarcane was investigated in hybrid clones with high and low sugar content and various accessory species. RESULTS: A total of 39 haplotypes of SPSB gene with 2, 824 bp in size were identified from 18 sugarcane accessions. These haplotypes mainly distributed on Chr3B, Chr3C, and Chr3D according to the AP85-441 reference genome. Single nucleotide polymorphisms (SNPs) and insertion/deletion (InDels) were very dense (42 bp/sequence variation) including 44 transitional and 23 transversional SNPs among the 39 haplotypes. The sequence diversity related Hd, Eta, and Pi values were all lower in clones of high sucrose content (HS) than those in clones of low sucrose content (LS). The evolutionary network analysis showed that about half SPSB haplotypes (19 out of 39) were clustered into one group, including 6 (HAP4, HAP6, HAP7, HAP9, HAP17 and HAP20) haplotypes under positive selection in comparison to HAP26 identified in Badila (S. officinarum), an ancestry noble cane species and under purification selection (except HAP19 under neutral selection) in comparison to HAP18 identified from India1 (S. spontaneum), an ancestry species with low sugar content but high stress tolerance. The average number of haplotypes under positive selection in HS clones was twice as that in LS. Most of the SNPs and InDels sequence variation sites were positively correlated with sucrose and fiber content and negatively correlated with reducing sugar. CONCLUSIONS: A total of 39 haplotypes of SPSB gene were identified in this study. Haplotypes potentially associated with high sucrose synthesis efficiency were identified. The mutations of SPSB haplotypes in HS were favorable and tended to be selected and fixed. The results of this study are informative and beneficial to the molecular assisted breeding of sucrose synthesis in sugarcane in the future.

Enhanced nitric oxide production by macrophages treated with a cell-penetrating peptide conjugate.

The SPRY domain-containing SOCS box protein-2 (SPSB2) plays a critical role in the degradation of inducible nitric oxide synthase (iNOS) in macrophages. In this study, we have conjugated a peptide inhibitor of the iNOS-SPSB2 interaction with a cell-penetrating peptide (CPP) for delivery into macrophages, and confirmed its binding to SPSB2. We have assessed the uptake of a fluorophore-tagged analogue by RAW 264.7 and immortalised bone marrow derived macrophage (iBMDM) cell lines, and shown that the CPP-peptide conjugate enhanced NO production. The findings of this study will be useful in further refinement of CPP-peptide conjugates as leads in the development of new antibiotics that target the host innate immune response.

Structural basis for the regulation of inducible nitric oxide synthase by the SPRY domain-containing SOCS box protein SPSB2, an E3 ubiquitin ligase.

Relatively high concentration of nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) in response to a variety of stimuli is a source of reactive nitrogen species, an important weapon of host innate immune defense. The SPRY domain-containing SOCS box protein 2 (SPSB2) is an E3 ubiquitin ligase that regulates the lifetime of iNOS. SPSB2 interacts with the N-terminal region of iNOS via a binding site on the SPRY domain of SPSB2, and recruits an E3 ubiquitin ligase complex to polyubiquitinate iNOS, leading to its proteasomal degradation. Although critical residues for the SPSB2-iNOS interaction have been identified, structural basis for the interaction remains to be explicitly determined. In this study, we have determined a crystal structure of the N-terminal region of iNOS in complex with the SPRY domain of SPSB2 at 1.24 Å resolution. We have resolved the roles of some flanking residues, whose contribution to the SPSB2-iNOS interaction was structurally unclear previously. Furthermore, we have evaluated the effects of SPSB2 inhibitors on NO production using transient transfection and cell-penetrating peptide approaches, and found that such inhibitors can elevate NO production in RAW264.7 macrophages. These results thus provide a useful basis for the development of potent SPSB2 inhibitors as well as recruiting ligands for proteolysis targeting chimera (PROTAC) design.

SPSB1 enhances ovarian cancer cell survival by destabilizing p21.

SPRY domain-containing SOCS box protein 1 (SPSB1) is an E3 ligase adaptor protein with unknown functions in cancer cells. In this study, we found that SPSB1 knockdown markedly decreased the viability and migration of ovarian cancer cells, while ectopic SPSB1 overexpression in IL-3-dependent Ba/F3 cells significantly increased their proliferation rate compared with empty vector-transfected cells. SPSB1 knockdown significantly elevated p21 protein and mRNA levels and induced apoptosis in ovarian cancer cells, as evidenced by increased levels of cleaved PARP and decreased levels of Bcl-2. Notably, mechanistic investigations revealed that SPSB1 accelerated p21 destabilization by directly interacting with p21 and promoting its ubiquitin-mediated proteasomal degradation. Taken together, our findings provide novel insights into the role of SPSB1 in ovarian cancer cells.

Ras enhances TGF-ß signaling by decreasing cellular protein levels of its type II receptor negative regulator SPSB1.

BACKGROUND: Transformation by oncogene Ras overcomes TGF-ß mediated growth inhibition in epithelial cells. However, it cooperates with each other to mediate epithelial to mesenchymal transition (EMT). The mechanism of how these two pathways interact with each other is controversial. METHODS: Molecular techniques were used to engineer expression plasmids for Ras, SPRY, TGF-ß receptors, type I and II and ubiquitin. Immunoprecipitation and western blots were employed to determine protein-protein interactions, preotein levels, protein phosphorylation while immunofluorecesent staining for molecular co-localization. TGF-ß signalling activities is also determined by its luciferase reporter assay. Trans-well assays were used to measure cell migration and invasion. RESULTS: Ras interacts with the SPSB1's SPRY domain to enhance TGF-ß signaling. Ras interacts and colocalizes with the TGF-ß type II receptor's (TßRII) negative regulator SPSB1 on the cell membrane, consequently promoting SPSB1 protein degradation via enhanced mono- and di-ubiquitination. Reduced SPSB1 levels result in the stablization of TßRII, in turn the increase of receptor levels significantly enhance Smad2/3 phosphorylation and signaling. Importantly, forced expression of SPSB1 in Ras transformed cells suppresses TGF-ß signaling and its mediated migration and invasion. CONCLUSION: Ras positively cooperates with TGF-ß signaling by reducing the cellular protein levels of TßRII negative regualtor SPSB1.

SPSB1, a Novel Negative Regulator of the Transforming Growth Factor-ß Signaling Pathway Targeting the Type II Receptor.

Appropriate cellular signaling is essential to control cell proliferation, differentiation, and cell death. Aberrant signaling can have devastating consequences and lead to disease states, including cancer. The transforming growth factor-ß (TGF-ß) signaling pathway is a prominent signaling pathway that has been tightly regulated in normal cells, whereas its deregulation strongly correlates with the progression of human cancers. The regulation of the TGF-ß signaling pathway involves a variety of physiological regulators. Many of these molecules act to alter the activity of Smad proteins. In contrast, the number of molecules known to affect the TGF-ß signaling pathway at the receptor level is relatively low, and there are no known direct modulators for the TGF-ß type II receptor (TßRII). Here we identify SPSB1 (a Spry domain-containing Socs box protein) as a novel regulator of the TGF-ß signaling pathway. SPSB1 negatively regulates the TGF-ß signaling pathway through its interaction with both endogenous and overexpressed TßRII (and not TßRI) via its Spry domain. As such, TßRII and SPSB1 co-localize on the cell membrane. SPSB1 maintains TßRII at a low level by enhancing the ubiquitination levels and degradation rates of TßRII through its Socs box. More importantly, silencing SPSB1 by siRNA results in enhanced TGF-ß signaling and migration and invasion of tumor cells.

Dynamic Nature of Staphylococcus aureus Type I Signal Peptidases.

Molecular dynamics simulations are used to interrogate the dynamic nature of Staphylococcus aureus Type I signal peptidases, SpsA and SpsB, including the impact of the P29S mutation of SpsB. Fluctuations and plasticity- rigidity characteristics vary among the proteins, particularly in the extracellular domain. Intriguingly, the P29S mutation, which influences susceptibility to arylomycin antibiotics, affect the mechanically coupled motions in SpsB. The integrity of the active site is crucial for catalytic competency, and variations in sampled structural conformations among the proteins are consistent with diverse peptidase capabilities. We also explored the intricate interactions between the proteins and the model S. aureus membrane. It was observed that certain membrane-inserted residues in the loop around residue 50 (50s) and C-terminal loops, beyond the transmembrane domain, give rise to direct interactions with lipids in the bilayer membrane. Our findings are discussed in the context of functional knowledge about these signal peptidases, offering additional understanding of dynamic aspects relevant to some cellular processes with potential implications for drug targeting strategies.

SPSB1-mediated inhibition of TGF-ß receptor-II impairs myogenesis in inflammation.

BACKGROUND: Sepsis-induced intensive care unit-acquired weakness (ICUAW) features profound muscle atrophy and attenuated muscle regeneration related to malfunctioning satellite cells. Transforming growth factor beta (TGF-ß) is involved in both processes. We uncovered an increased expression of the TGF-ß receptor II (TßRII)-inhibitor SPRY domain-containing and SOCS-box protein 1 (SPSB1) in skeletal muscle of septic mice. We hypothesized that SPSB1-mediated inhibition of TßRII signalling impairs myogenic differentiation in response to inflammation. METHODS: We performed gene expression analyses in skeletal muscle of cecal ligation and puncture- (CLP) and sham-operated mice, as well as vastus lateralis of critically ill and control patients. Pro-inflammatory cytokines and specific pathway inhibitors were used to quantitate Spsb1 expression in myocytes. Retroviral expression plasmids were used to investigate the effects of SPSB1 on TGF-ß/TßRII signalling and myogenesis in primary and immortalized myoblasts and differentiated myotubes. For mechanistical analyses we used coimmunoprecipitation, ubiquitination, protein half-life, and protein synthesis assays. Differentiation and fusion indices were determined by immunocytochemistry, and differentiation factors were quantified by qRT-PCR and Western blot analyses. RESULTS: SPSB1 expression was increased in skeletal muscle of ICUAW patients and septic mice. Tumour necrosis factor (TNF), interleukin-1ß (IL-1ß), and IL-6 increased the Spsb1 expression in C2C12 myotubes. TNF- and IL-1ß-induced Spsb1 expression was mediated by NF-κB, whereas IL-6 increased the Spsb1 expression via the glycoprotein 130/JAK2/STAT3 pathway. All cytokines reduced myogenic differentiation. SPSB1 avidly interacted with TßRII, resulting in TßRII ubiquitination and destabilization. SPSB1 impaired TßRII-Akt-Myogenin signalling and diminished protein synthesis in myocytes. Overexpression of SPSB1 decreased the expression of early (Myog, Mymk, Mymx) and late (Myh1, 3, 7) differentiation-markers. As a result, myoblast fusion and myogenic differentiation were impaired. These effects were mediated by the SPRY- and SOCS-box domains of SPSB1. Co-expression of SPSB1 with Akt or Myogenin reversed the inhibitory effects of SPSB1 on protein synthesis and myogenic differentiation. Downregulation of Spsb1 by AAV9-mediated shRNA attenuated muscle weight loss and atrophy gene expression in skeletal muscle of septic mice. CONCLUSIONS: Inflammatory cytokines via their respective signalling pathways cause an increase in SPSB1 expression in myocytes and attenuate myogenic differentiation. SPSB1-mediated inhibition of TßRII-Akt-Myogenin signalling and protein synthesis contributes to a disturbed myocyte homeostasis and myogenic differentiation that occurs during inflammation.

A strategy to assess the cellular activity of E3 ligase components against neo-substrates using electrophilic probes.

While there are hundreds of predicted E3 ligases, characterizing their applications for targeted protein degradation has proved challenging. Here, we report a chemical biology approach to evaluate the ability of modified recombinant E3 ligase components to support neo-substrate degradation. Bypassing the need for specific E3 ligase binders, we use maleimide-thiol chemistry for covalent functionalization followed by E3 electroporation (COFFEE) in live cells. We demonstrate that electroporated recombinant von Hippel-Lindau (VHL) protein, covalently functionalized at its ligandable cysteine with JQ1 or dasatinib, induces degradation of BRD4 or tyrosine kinases, respectively. Furthermore, by applying COFFEE to SPSB2, a Cullin-RING ligase 5 receptor, as well as to SKP1, the adaptor protein for Cullin-RING ligase 1 F box (SCF) complexes, we validate this method as a powerful approach to define the activity of previously uncharacterized ubiquitin ligase components, and provide further evidence that not only E3 ligase receptors but also adaptors can be directly hijacked for neo-substrate degradation.

Integrative RNA profiling of TBEV-infected neurons and astrocytes reveals potential pathogenic effectors.

Tick-borne encephalitis virus (TBEV), the most medically relevant tick-transmitted flavivirus in Eurasia, targets the host central nervous system and frequently causes severe encephalitis. The severity of TBEV-induced neuropathogenesis is highly cell-type specific and the exact mechanism responsible for such differences has not been fully described yet. Thus, we performed a comprehensive analysis of alterations in host poly-(A)/miRNA/lncRNA expression upon TBEV infection in vitro in human primary neurons (high cytopathic effect) and astrocytes (low cytopathic effect). Infection with severe but not mild TBEV strain resulted in a high neuronal death rate. In comparison, infection with either of TBEV strains in human astrocytes did not. Differential expression and splicing analyses with an in silico prediction of miRNA/mRNA/lncRNA/vd-sRNA networks found significant changes in inflammatory and immune response pathways, nervous system development and regulation of mitosis in TBEV Hypr-infected neurons. Candidate mechanisms responsible for the aforementioned phenomena include specific regulation of host mRNA levels via differentially expressed miRNAs/lncRNAs or vd-sRNAs mimicking endogenous miRNAs and virus-driven modulation of host pre-mRNA splicing. We suggest that these factors are responsible for the observed differences in the virulence manifestation of both TBEV strains in different cell lines. This work brings the first complex overview of alterations in the transcriptome of human astrocytes and neurons during the infection by two TBEV strains of different virulence. The resulting data could serve as a starting point for further studies dealing with the mechanism of TBEV-host interactions and the related processes of TBEV pathogenesis.

The E3 Ligases Spsb1 and Spsb4 Regulate RevErbα Degradation and Circadian Period.

The time-dependent degradation of core circadian clock proteins is essential for the proper functioning of circadian timekeeping mechanisms that drive daily rhythms in gene expression and, ultimately, an organism's physiology. The ubiquitin proteasome system plays a critical role in regulating the stability of most proteins, including the core clock components. Our laboratory developed a cell-based functional screen to identify ubiquitin ligases that degrade any protein of interest and have started screening for those ligases that degrade circadian clock proteins. This screen identified Spsb4 as a putative novel E3 ligase for RevErbα. In this article, we further investigate the role of Spsb4 and its paralogs in RevErbα stability and circadian rhythmicity. Our results indicate that the paralogs Spsb1 and Spsb4, but not Spsb2 and Spsb3, can interact with and facilitate RevErbα ubiquitination and degradation and regulate circadian clock periodicity.

A SPRY domain-containing SOCS box protein 3 (SPSB3) involved in the regulation of cytokine production in granulocytes of Crassostrea gigas.

The sp1A/ryanodine receptor (SPRY) family members have been reported to involve in important biological pathways, including innate immune signaling, cytokine signaling suppression, development, cell growth, and retroviral restriction. In the present study, a SPRY domain-containing SOCS box protein (named as CgSPSB3) was identified and characterized from oyster Crassostrea gigas. The open reading frame of CgSPSB3 gene was of 699 bp, encoding a polypeptide of 232 amino acid residues with a central SPRY domain and a C-terminal SOCS box motif. CgSPSB3 mRNA transcripts could be detected in all the examined tissues with the highest level in hemocytes, which was about 82.72-fold (p < 0.05) of that in gonad. Furthermore, the expression level of CgSPSB3 mRNA in granulocytes was significantly higher than that in semi-granulocytes and agranulocytes, which was about 2.04-fold (p < 0.05) of the average level of hemocytes. Immunofluorescence assay further revealed that CgSPSB3 protein was mainly distributed in the cytoplasm of granulocytes. The mRNA expression of CgSPSB3 in hemocytes was up-regulated after lipopolysaccharide (LPS) and Vibrio splendidus stimulations. The mRNA expression of CgIFNLP, CgIL17-5 and CgTNF-1 decreased significantly (p < 0.05) at 24 h after the CgSPSB3 mRNA was knocked down by RNAi. These results collectively indicated that CgSPSB3 might play an important role in regulating cytokines production in granulocytes of C. gigas.

Cullin-5 Adaptor SPSB1 Controls NF-κB Activation Downstream of Multiple Signaling Pathways.

The initiation of innate immune responses against pathogens relies on the activation of pattern-recognition receptors (PRRs) and corresponding intracellular signaling cascades. To avoid inappropriate or excessive activation of PRRs, these responses are tightly controlled. Cullin-RING E3 ubiquitin ligases (CRLs) have emerged as critical regulators of many cellular functions including innate immune activation and inflammation. CRLs form multiprotein complexes in which a Cullin protein acts as a scaffold and recruits specific adaptor proteins, which in turn recognize specific substrate proteins for ubiquitylation, hence providing selectivity. CRLs are divided into 5 main groups, each of which uses a specific group of adaptor proteins. Here, we systematically depleted all predicted substrate adaptors for the CRL5 family (the so-called SOCS-box proteins) and assessed the impact on the activation of the inflammatory transcription factor NF-κB. Depletion of SPSB1 resulted in a significant increase in NF-κB activation, indicating the importance of SPSB1 as an NF-κB negative regulator. In agreement, overexpression of SPSB1 suppressed NF-κB activity in a potent, dose-dependent manner in response to various agonists. Inhibition by SPSB1 was specific to NF-κB, because other transcription factors related to innate immunity and interferon (IFN) responses such as IRF-3, AP-1, and STATs remained unaffected by SPSB1. SPSB1 suppressed NF-κB activation induced via multiple pathways including Toll-like receptors and RNA and DNA sensing adaptors, and required the presence of its SOCS-box domain. To provide mechanistic insight, we examined phosphorylation and degradation of the inhibitor of κB (IκBα) and p65 translocation into the nucleus. Both remained unaffected by SPSB1, indicating that SPSB1 exerts its inhibitory activity downstream, or at the level, of the NF-κB heterodimer. In agreement with this, SPSB1 was found to co-precipitate with p65 after over-expression and at endogenous levels. Additionally, A549 cells stably expressing SPSB1 presented lower cytokine levels including type I IFN in response to cytokine stimulation and virus infection. Taken together, our results reveal novel regulatory mechanisms in innate immune signaling and identify the prominent role of SPSB1 in limiting NF-κB activation. Our work thus provides insights into inflammation and inflammatory diseases and new opportunities for the therapeutic targeting of NF-κB transcriptional activity.

Crystal structure of the SPRY domain of human SPSB2 in the apo state.

The SPRY domain-containing SOCS box protein 2 (SPSB2) is one of four mammalian SPSB proteins that are characterized by a C-terminal SOCS box and a central SPRY/B30.2 domain. SPSB2 interacts with inducible nitric oxide synthase (iNOS) via the SPRY domain and polyubiquitinates iNOS, resulting in its proteasomal degradation. Inhibitors that can disrupt SPSB2-iNOS interaction and augment NO production may serve as novel anti-infective and anticancer agents. The previously determined murine SPSB2 structure may not reflect the true apo conformation of the iNOS-binding site. Here, the crystal structure of human SPSB2 SPRY domain in the apo state is reported at a resolution of 1.9 Å. Comparison of the apo and ligand-bound structures reveals that the iNOS-binding site is highly preformed and that major conformational changes do not occur upon ligand binding. Moreover, the C-terminal His6 tag of the recombinant protein binds to a shallow pocket adjacent to the iNOS-binding site on a crystallographically related SPSB2 molecule. These findings may help in structure-based and fragment-based SPSB2 inhibitor design in the future.

Identification and expression analysis of a Spsb gene in planarian Dugesia japonica.

The SPSB family is comprised of four highly conserved proteins, each containing a C-terminal SOCS box motif and a central SPRY domain. Presently, Spsb genes have been found in mammals and in a few invertebrates, however, the specific functions of these genes are still unknown. In this study, we identified a Spsb gene from the planarian Dugesia japonica and termed it DjSpsb. The temporal and spatial expression patterns of DjSpsb were examined in both intact and regenerative animals, and expression levels were also quantified in response to various stressors. The results show that (1) DjSpsb is highly conserved in evolutionary history in metazoans and is at closer relationship to Spsb1, Spsb2 and Spsb4; (2) DjSpsb mRNA is mainly expressed in the head and also throughout head regeneration processes, particularly, its expression up-regulated observably on day 5 after amputation; (3) DjSpsb is also expressed in the testes and yolk glands; (4) DjSpsb expression is induced by high temperature and ethanol but inhibited by high doses of ionic liquids. The date suggests that the DjSpsb gene might be active in central nervous system (CNS) formation and functional recovery during head regeneration, and it is also involved in the development of germ cells and stress responses in the planarians.

Expression, purification and crystallization of a membrane-associated, catalytically active type I signal peptidase from Staphylococcus aureus.

Staphylococcus aureus infections are becoming increasingly difficult to treat as they rapidly develop resistance to existing antibiotics. Bacterial type I signal peptidases are membrane-associated, cell-surface serine proteases with a unique catalytic mechanism that differs from that of eukaryotic endoplasmic reticulum signal peptidases. They are thus potential antimicrobial targets. S. aureus has a catalytically active type I signal peptidase, SpsB, that is essential for cell viability. To elucidate its structure, the spsB gene from S. aureus Newman strain was cloned and overexpressed in Escherichia coli. After exploring many different protein-modification constructs, SpsB was expressed as a fusion protein with maltose-binding protein and crystallized by hanging-drop vapour diffusion. The crystals belonged to the monoclinic space group P2(1) and diffracted to 2.05 Šresolution. The crystal structure of SpsB is anticipated to provide structural insight into Gram-positive signal peptidases and to aid in the development of antibacterial agents that target type I signal peptidases.