SPRY Domain-containing SOCS Box Protein Research Articles

On the human health benefits of microalgal phytohormones: An explorative in silico analysis

Phytohormones represent a group of secondary metabolites with different chemical structures, in which belong auxins, cytokinins, gibberellins, or brassinosteroids. In higher plants, they cover active roles in growth or defense function, while their potential benefits for human health protection were noted for some phytohormones and little explored for many others. In this study, we developed a target fishing strategy on fifty-three selected naturally occurring phytohormones covering different families towards proteins involved in key cellular functions related to human metabolism and health protection/disease. This in silico analysis strategy aims to screen the potential human health-driven bioactivity of more than fifty phytohormones through the analysis of their interactions with specific targets. From this analysis, twenty-eight human targets were recovered. Some targets e.g., the proteins mitochondrial glutamate dehydrogenase (GLUD1) or nerve growth factor (NGF) bound many phytohormones, highlighting their involvement in amino acid metabolism and/or in the maintenance or survival of neurons. Conversely, some phytohormones specifically interacted with some proteins, e.g., SPRY domain-containing SOCS box protein 2 (SPSB2) or Inosine-5′-monophosphate dehydrogenase 1 (IMPDH1), both involved in human immune response. They were then investigated with a molecular docking analysis approach. Our bioprospecting study indicated that many phytohormones may endow human health benefits, with potential functional role in multiple cellular processes including immune response and cell cycle progression.

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.

Macrocyclic Modalities Combining Peptide Epitopes and Natural Product Fragments.

“Hot loop” protein segments have variable structure and conformation and contribute crucially to protein–protein interactions. We describe a new hot loop mimicking modality, termed PepNats, in which natural product (NP)-inspired structures are incorporated as conformation-determining and -restricting structural elements into macrocyclic hot loop-derived peptides. Macrocyclic PepNats representing hot loops of inducible nitric oxide synthase (iNOS) and human agouti-related protein (AGRP) were synthesized on solid support employing macrocyclization by imine formation and subsequent stereoselective 1,3-dipolar cycloaddition as key steps. PepNats derived from the iNOS DINNN hot loop and the AGRP RFF hot spot sequence yielded novel and potent ligands of the SPRY domain-containing SOCS box protein 2 (SPSB2) that binds to iNOS, and selective ligands for AGRP-binding melanocortin (MC) receptors. NP-inspired fragment absolute configuration determines the conformation of the peptide part responsible for binding. These results demonstrate that combination of NP-inspired scaffolds with peptidic epitopes enables identification of novel hot loop mimics with conformationally constrained and biologically relevant structure.

Abstract GMM-035: TARGETING SPSB1 SENSITIZES PLATINUM-RESISTANT OVARIAN CANCER CELLS TO CISPLATIN

Abstract OBJECTIVE: Emergence of chemoresistance remains a critical clinical issue responsible for treatment failure in patients with ovarian cancer. The precise mechanisms underlying the development of chemoresistance are not fully understood. In our previous study using methylomic and transcriptomic analyses, SPRY domain-containing SOCS box protein 1 (SPSB1) was found to be differentially expressed between cisplatin-sensitive and –resistant ovarian cancer cells. Here, we investigated the roles of SPSB1 as a new therapeutic target for ovarian cancer cells resistant to cisplatin. METHODS: Knockdown of SPSB1 gene was performed by transfection of SPSB1 siRNA. Ovarian cancer cell lines were cultured for 5 days and 12 days after transfection for MTT assay and colony forming assay, respectively. Protein level was analyzed by western blot analysis. Cell migration assay was performed using Boyden chamber method. RESULTS: Knockdown of SPSB1 using RNAi method dramatically inhibited cell viability and migratory capability of ovarian cancer cells. Also, SPSB1 knockdown significantly induced CDK inhibitors, p21 and p27 and inhibited anti-apoptotic Bcl-2, indicating that SPSB1 inhibition induces cell cycle arrest and apoptosis in ovarian cancer cells. CONCLUSION: Our results suggest that SPSB1 might be a potential therapeutic target for cisplatin-resistant ovarian cancer. Citation Format: Mi-Kyung Kim, Hyun-Jung Kim, Yun Hwan Kim, Seung Cheol Kim, Woong Ju. TARGETING SPSB1 SENSITIZES PLATINUM-RESISTANT OVARIAN CANCER CELLS TO CISPLATIN [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-035.

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.

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.

Robust Enhancement of Lentivirus Production by Promoter Activation

Lentiviral vectors are a valuable tool to deliver exogenous genes for stable expression in cells. While much progress has been made in processing lentiviral vector-containing culture medium, it remains to be explored how the production of lentiviral vector from producer cells can be increased. We initially found that co-expression of the SPRY domain-containing SOCS box protein 1 (SPSB1) promotes the production of human immunodeficiency virus type 1 (HIV-1) and lentiviral vector with increased expression of the Gag and envelope proteins and activation of the HIV-1 LTR and CMV promoter. The presence of AP-1, NF-κB and CREB/ATF recognition sites in these promoters prompted us to utilize human T-lymphotropic virus type 1 (HTLV-1) Tax for lentiviral vector production because Tax activates all these transcription factors. Co-expression of a small amount of Tax markedly increased both the expression of viral structural proteins in producer cells and release of lentiviral vector particles, resulting in a more than 10-fold enhancement of transduction efficiency. Of note, the Tax protein was not detected in the lentiviral vector particles concentrated by ultracentrifugation, supporting the safety of this preparation. Collectively, these results indicate that promoter activation in producer cells represents a promising approach to preparing high-titer lentiviral vectors.

Hsp90 inhibition renders iNOS aggregation and the clearance of iNOS aggregates by proteasomes requires SPSB2.

Inducible nitric oxide synthase (iNOS) plays important roles in cell injury and host defense. Our early study demonstrated that heat shock protein 90 (Hsp90) interacts with iNOS and this interaction enhances iNOS function. Recently, we reported that Hsp90 is also essential for iNOS gene transactivation. In the present study, we investigate the role of Hsp90 in controlling iNOS protein stability. In mouse macrophages, Hsp90 inhibition dissociated Hsp90 from iNOS and the latter subsequently formed aggregates. Aggregation deactivated iNOS. iNOS aggregates were cleared by the ubiquitin-proteasome system (UPS) inside cells. CHIP, an Hsp90-dependent E3 ligase, was previously implicated in iNOS turnover. However, CHIP knockdown had little effect on iNOS degradation in Hsp90-inhibited cells, indicating that other E3 ligases accounted for the clearance of iNOS aggregates. Further studies revealed that the SPRY domain-containing SOCS box protein 2 (SPSB2), an E3 ligase-recruiting protein, was essential for the ubiquitination of iNOS aggregates. SPSB2 knockdown or deleting the SPSB2-interacting domain on iNOS prevented the clearance of iNOS aggregates in Hsp90-inhibited cells. Thus, besides modulating iNOS function and gene transcription, Hsp90 is also essential for the protein stability of iNOS. Hsp90 blockade induces iNOS aggregation and SPSB2 is required for UPS degradation of iNOS aggregates.

Crystal structure of SPSB2 in complex with a rational designed RGD-containing cyclic peptide inhibitor of SPSB2-iNOS interaction

SPRY domain-containing SOCS box protein 2 (SPSB2) is a negative regulator of inducible nitric oxide synthase (iNOS) that modulates the lifetime of iNOS and thus the levels of nitric oxide (NO) production. Inhibitors that can disrupt the endogenous SPSB2-iNOS interaction and augment NO production have potential as novel antimicrobial and anticancer drugs. In this study, we have designed a cyclic peptide (cR8), containing an RGD motif and the SPSB2 binding motif (DINNNV). ITC and chemical shift perturbation showed that cR8 binds to the iNOS binding site on SPSB2 with a Kd of 671 nM, and saturation transfer difference NMR showed that cR8 binds to αvβ3 integrin-expressing cells. Moreover, we determined the crystal structure of SPSB2 in complex with cR8, at a resolution of 1.34 Å. cR8 forms extensive hydrogen bonding with SPSB2 residues, but loss of an intramolecular hydrogen bond that is present in SPSB2-bound iNOS peptide may destabilize the bound conformation of cR8 and lead to a gentle reduction in SPSB2 binding affinity. These results serve as a useful basis for designing site-directed SPSB2 inhibitors in the future.

SPSB1-mediated HnRNP A1 ubiquitylation regulates alternative splicing and cell migration in EGF signaling.

Extracellular signals have been shown to impact on alternative pre-mRNA splicing; however, the molecular mechanisms and biological significance of signal-induced splicing regulation remain largely unknown. Here, we report that epidermal growth factor (EGF) induces splicing changes through ubiquitylation of a well-known splicing regulator, hnRNP A1. EGF signaling upregulates an E3 ubiquitin (Ub) ligase adaptor, SPRY domain-containing SOCS box protein 1 (SPSB1), which recruits Elongin B/C-Cullin complexes to conjugate lysine 29-linked polyUb chains onto hnRNP A1. Importantly, SPSB1 and ubiquitylation of hnRNP A1 have a critical role in EGF-driven cell migration. Mechanistically, EGF-induced ubiquitylation of hnRNP A1 together with the activation of SR protein kinases (SRPKs) results in the upregulation of a Rac1 splicing isoform, Rac1b, to promote cell motility. These findings unravel a novel crosstalk between protein ubiquitylation and alternative splicing in EGF/EGF receptor signaling, and identify a new EGF/SPSB1/hnRNP A1/Rac1 axis in modulating cell migration, which may have important implications for cancer treatment.

Molecular Insights into the Interaction Between the SPRY Domain-Containing SOCS Box Protein SPSB2 and Peptides Based on the Binding Motif from iNOS

SPRY domain-containing SOCS box proteins SPSB1, 2, and 4 mediate the proteasomal degradation of inducible nitric oxide synthase (iNOS) and thereby modulate the amount of NO available for combating infectious organisms. A highly conserved Asp-Ile-Asn-Asn-Asn (DINNN) motif found at the N-terminus of iNOS binds to SPSB2 with nanomolar affinity. The design of specific and potent inhibitors of iNOS–SPSB interactions will be aided by a better understanding of the interactions of this DINNN sequence with SPSB2. Although crystal structures of SPSB complexes with DINNN peptides are available, aspects of the interaction between peptide and protein are still not fully understood. Here, our results from surface plasmon resonance and NMR spectroscopy indicate that residues flanking the DINNN motif, which make no direct contact with SPSB2 in the available crystal structures, nonetheless play an important role in enhancing the binding affinity to SPSB2, by up to 80-fold. Mutational analysis of the DINNN sequence showed that mutation of the Asp or the first Asn residue to Ala reduced the binding affinity by 200- or 600-fold respectively, whereas mutation of the third Asn made binding undetectable. Ala substitution of the second Asn residue caused a 30-fold drop in binding affinity. Substitution of the Ile had very little effect on the binding affinity and substitutions with bulky residues were tolerated. This provides an opportunity for further modification for therapeutic applications. These results highlight the complex interplay of peptide sequence and protein binding and inform efforts to design peptide therapeutics to disrupt the iNOS–SPSB interaction.

SPSB1, a Novel Negative Regulator of the Transforming Growth Factor-β 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-β (TGF-β) 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-β 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-β signaling pathway at the receptor level is relatively low, and there are no known direct modulators for the TGF-β type II receptor (TβRII). Here we identify SPSB1 (a Spry domain-containing Socs box protein) as a novel regulator of the TGF-β signaling pathway. SPSB1 negatively regulates the TGF-β signaling pathway through its interaction with both endogenous and overexpressed TβRII (and not TβRI) via its Spry domain. As such, TβRII and SPSB1 co-localize on the cell membrane. SPSB1 maintains TβRII at a low level by enhancing the ubiquitination levels and degradation rates of TβRII through its Socs box. More importantly, silencing SPSB1 by siRNA results in enhanced TGF-β signaling and migration and invasion of tumor cells.

Effects of Titanium Dioxide Nanoparticles on the Synthesis of Fibroin in Silkworm (Bombyx mori).

Silkworm (Bombyx mori) is an economically important insect, and its silk production capacity largely depends on its ability to synthesize fibroin. While breeding of B. mori varieties has been a key strategy to improve silk production, little improvement of B. mori silk production has been achieved to date. As a result, the development of sericulture economy has not progressed well, pointing to the need of new ways for improvement of B. mori silk production. Titanium dioxide nanoparticles (TiO2 NPs), a food additive widely used for livestock, have been shown to promote animal growth and increase the protein synthesis in animals. However, no studies on effect of TiO2 NPs on fibroin synthesis in B. mori have been available. In this study, the differential expression profiles of genes and proteins in the silk gland of B. mori fed without or with TiO2 NPs (5 μg ml(-1)) were analyzed and compared using digital gene expression (DGE), reverse transcription quantitative polymerase chain reaction (RT-qPCR), semi-qPCR, and Western blot analysis. The effects of TiO2 NPs feeding on the activity of proteases in the midgut and the synthesis and transportation of amino acids in hemolymph were also investigated. DGE analyses showed that among a total of 4,741 genes detected, 306 genes were differentially expressed after the TiO2 NPs feeding, of which 137 genes were upregulated whereas 169 genes were downregulated. 106 genes were shown to be involved in fibroin synthesis, of which 97 genes, including those encoding cuticular protein glycine-rich 10, serine protease inhibitor 28, aspartate aminotransferase, lysyl-tRNA synthetase, and splicing factor arginine/serine-rich 6, and silk gland factor-1 (SGF-1), were upregulated with the maximum induction of 8.52-folds, whereas nine genes, including those encoding aspartylglucosaminidase, the cathepsin L in Tribolium castaneum, and similar to SPRY domain-containing SOCS box protein 3, were downregulated with the maximum reduction of 8.11-folds. Transcription levels of nine genes were further verified by RT-qPCR, and the results were consistent with those with DGE. Transcription and expression levels of fibroin light chain (Fib-L) gene were increased after TiO2 NPs feeding, indicating that TiO2 NPs improves fibroin synthesis. Compared with that of control, the mean protease activity was increased by 56.67% in the B. mori fed with TiO2 NPs, and the transport of four key amino acids used for fibroin synthesis in hemolymph was also increased. These findings indicated that TiO2 NPs feeding can improve the absorption and utilization of amino acids from the feed and could be a new way to increase the fibroin synthesis in B. mori.

19F NMR as a probe of ligand interactions with the iNOS binding site of SPRY domain-containing SOCS box protein 2.

SPRY domain-containing SOCS box protein 2 (SPSB2) regulates inducible nitric oxide synthase (iNOS) by targeting it for proteasomal degradation. Inhibiting this interaction prolongs the intracellular lifetime of iNOS, leading in turn to enhanced killing of infectious pathogens such as bacteria and parasites. SPSB2 recognizes a linear motif (DINNN) in the disordered N-terminus of iNOS, and ligands that target the DINNN binding site on SPSB2 are potentially novel anti-infective agents. We have explored (19)F NMR as a means of probing ligand binding to SPSB2. All six Trp residues in SPSB2 were replaced with 5-fluorotryptophan (5-F-Trp) by utilizing a Trp auxotroph strain of Escherichia coli. The labeled protein was well folded and bound a DINNN-containing peptide with similar affinity to native SPSB2. Six well-resolved 5-F-Trp resonances were observed in the (19)F NMR spectrum and were assigned using site-directed mutagenesis. The (19)F resonance of W207 was significantly perturbed upon binding to DINNN-containing peptides. Other resonances were perturbed to a lesser extent although in a way that was sensitive to the composition of the peptide. Analogues of compounds identified in a fragment screen also perturbed the W207 resonance, confirming their binding to the iNOS peptide-binding site on SPSB2. (19)F NMR promises to be a valuable approach in developing inhibitors that bind to the DINNN binding site.

Genetic Deletion of Murine SPRY Domain-Containing SOCS Box Protein 2 (SSB-2) Results in Very Mild Thrombocytopenia

The SSB family is comprised of four highly homologous proteins containing a C-terminal SOCS box motif and a central SPRY domain. No function has yet been ascribed to any member of this family in mammalian species despite a clear role for other SOCS proteins in negative regulation of cytokine signaling. To investigate its physiological role, the murine Ssb-2 gene was deleted by homologous recombination. SSB-2-deficient mice were shown to have a reduced rate of platelet production, resulting in very mild thrombocytopenia (25% decrease in circulating platelets). Tissue histology and other hematological parameters were normal, as was the majority of serum biochemistry, with the exception that blood urea nitrogen (BUN) levels were decreased in mice lacking SSB-2. Quantitative analysis of SSB mRNA levels indicated that SSB-1, -2, and -3 were ubiquitously expressed; however, SSB-4 was only expressed at very low levels. SSB-2 expression was observed in the kidney and in megakaryocytes, a finding consistent with the phenotype of mice lacking this gene. Deletion of SSB-2 thus perturbs the steady-state level of two tightly controlled homeostatic parameters and identifies a critical role for SSB-2 in regulating platelet production and BUN levels.

Letter to the Editor: Backbone 1H, 13C and 15N assignments of the 25 kDa SPRY domain-containing SOCS box protein 2 (SSB-2)

Letter to the Editor: Backbone 1H, 13C and 15N assignments of the 25 kDa SPRY domain-containing SOCS box protein 2 (SSB-2)