A bacterial binary toxin system that kills both insects and aquatic crustaceans: Photorhabdus insect-related toxins A and B.

Photorhabdus insect-related toxins A and B (PirA and PirB) were first recognized as insecticidal toxins from Photorhabdus luminescens. However, subsequent studies showed that their homologs from Vibrio parahaemolyticus also play critical roles in the pathogenesis of acute hepatopancreatic necrosis disease (AHPND) in shrimps. Based on the structural features of the PirA/PirB toxins, it was suggested that they might function in the same way as a Bacillus thuringiensis Cry pore-forming toxin. However, unlike Cry toxins, studies on the PirA/PirB toxins are still scarce, and their cytotoxic mechanism remains to be clarified. In this review, based on our studies of V. parahaemolyticus PirAvp/PirBvp, we summarize the current understanding of the gene locations, expression control, activation, and cytotoxic mechanism of this type of toxin. Given the important role these toxins play in aquatic disease and their potential use in pest control applications, we also suggest further topics for research. We hope the information presented here will be helpful for future PirA/PirB studies.

Expression of the AHPND Toxins PirAvp and PirBvp Is Regulated by Components of the Vibrio parahaemolyticus Quorum Sensing (QS) System.

Acute hepatopancreatic necrosis disease (AHPND) in shrimp is caused by Vibrio strains that harbor a pVA1-like plasmid containing the pirA and pirB genes. It is also known that the production of the PirA and PirB proteins, which are the key factors that drive the observed symptoms of AHPND, can be influenced by environmental conditions and that this leads to changes in the virulence of the bacteria. However, to our knowledge, the mechanisms involved in regulating the expression of the pirA/pirB genes have not previously been investigated. In this study, we show that in the AHPND-causing Vibrio parahaemolyticus 3HP strain, the pirAvp and pirBvp genes are highly expressed in the early log phase of the growth curve. Subsequently, the expression of the PirAvp and PirBvp proteins continues throughout the log phase. When we compared mutant strains with a deletion or substitution in two of the quorum sensing (QS) master regulators, luxO and/or opaR (luxOD47E, ΔopaR, ΔluxO, and ΔopaRΔluxO), our results suggested that expression of the pirAvp and pirBvp genes was related to the QS system, with luxO acting as a negative regulator of pirAvp and pirBvp without any mediation by opaRvp. In the promoter region of the pirAvp/pirBvp operon, we also identified a putative consensus binding site for the QS transcriptional regulator AphB. Real-time PCR further showed that aphBvp was negatively controlled by LuxOvp, and that its expression paralleled the expression patterns of pirAvp and pirBvp. An electrophoretic mobility shift assay (EMSA) showed that AphBvp could bind to this predicted region, even though another QS transcriptional regulator, AphAvp, could not. Taken together, these findings suggest that the QS system may regulate pirAvp/pirBvp expression through AphBvp.

Structural insights into the interaction between phytoplasmal effector causing phyllody 1 and MADS transcription factors.

Phytoplasmas are bacterial plant pathogens which can induce severe symptoms including dwarfism, phyllody and virescence in an infected plant. Because phytoplasmas infect many important crops such as peanut and papaya they have caused serious agricultural losses. The phytoplasmal effector causing phyllody 1 (PHYL1) is an important phytoplasmal pathogenic factor which affects the biological function of MADS transcription factors by interacting with their K (keratin-like) domain, thus resulting in abnormal plant developments such as phyllody. Until now, lack of information on the structure of PHYL1 has prevented a detailed understanding of the binding mechanism between PHYL1 and the MADS transcription factors. Here, we present the crystal structure of PHYL1 from peanut witches'-broom phytoplasma (PHYL1PnWB ). This protein was found to fold into a unique α-helical hairpin with exposed hydrophobic residues on its surface that may play an important role in its biological function. Using proteomics approaches, we propose a binding mode of PHYL1PnWB with the K domain of the MADS transcription factor SEPALLATA3 (SEP3_K) and identify the residues of PHYL1PnWB that are important for this interaction. Furthermore, using surface plasmon resonance we measure the binding strength of PHYL1PnWB proteins to SEP3_K. Lastly, based on confocal images, we found that α-helix 2 of PHYL1PnWB plays an important role in PHYL1-mediated degradation of SEP3. Taken together, these results provide a structural understanding of the specific binding mechanism between PHYL1PnWB and SEP3_K.

The opportunistic marine pathogen Vibrio parahaemolyticus becomes virulent by acquiring a plasmid that expresses a deadly toxin.

Acute hepatopancreatic necrosis disease (AHPND) is a severe, newly emergent penaeid shrimp disease caused by Vibrio parahaemolyticus that has already led to tremendous losses in the cultured shrimp industry. Until now, its disease-causing mechanism has remained unclear. Here we show that an AHPND-causing strain of V. parahaemolyticus contains a 70-kbp plasmid (pVA1) with a postsegregational killing system, and that the ability to cause disease is abolished by the natural absence or experimental deletion of the plasmid-encoded homologs of the Photorhabdus insect-related (Pir) toxins PirA and PirB. We determined the crystal structure of the V. parahaemolyticus PirA and PirB (PirA(vp) and PirB(vp)) proteins and found that the overall structural topology of PirA(vp)/PirB(vp) is very similar to that of the Bacillus Cry insecticidal toxin-like proteins, despite the low sequence identity (<10%). This structural similarity suggests that the putative PirAB(vp) heterodimer might emulate the functional domains of the Cry protein, and in particular its pore-forming activity. The gene organization of pVA1 further suggested that pirAB(vp) may be lost or acquired by horizontal gene transfer via transposition or homologous recombination.

Structural Insights into the Cytotoxic Mechanism of Vibrio parahaemolyticus PirAvp and PirBvp Toxins.

In aquaculture, shrimp farming is a popular field. The benefits of shrimp farming include a relatively short grow-out time, high sale price, and good cost recovery. However, outbreaks of serious diseases inflict serious losses, and acute hepatopancreatic necrosis disease (AHPND) is an emerging challenge to this industry. In South American white shrimp (Penaeus vannamei) and grass shrimp (Penaeus monodon), this disease has a 70-100% mortality. The pathogenic agent of AHPND is a specific strain of Vibrio parahaemolyticus which contains PirAvp and PirBvp toxins encoded in the pVA1 plasmid. PirAvp and PirBvp have been shown to cause the typical histological symptoms of AHPND in infected shrimps, and in this review, we will focus on our structural understanding of these toxins. By analyzing their structures, a possible cytotoxic mechanism, as well as strategies for anti-AHPND drug design, is proposed.

White spot syndrome virus protein kinase 1 defeats the host cell's iron-withholding defense mechanism by interacting with host ferritin.

UNLABELLED: Iron is an essential nutrient for nearly all living organisms, including both hosts and invaders. Proteins such as ferritin regulate the iron levels in a cell, and in the event of a pathogenic invasion, the host can use an iron-withholding mechanism to restrict the availability of this essential nutrient to the invading pathogens. However, pathogens use various strategies to overcome this host defense. In this study, we demonstrated that white spot syndrome virus (WSSV) protein kinase 1 (PK1) interacted with shrimp ferritin in the yeast two-hybrid system. A pulldown assay and 27-MHz quartz crystal microbalance (QCM) analysis confirmed the interaction between PK1 and both ferritin and apoferritin. PK1 did not promote the release of iron ions from ferritin, but it prevented apoferritin from binding ferrous ions. When PK1 was overexpressed in Sf9 cells, the cellular labile iron pool (LIP) levels were elevated significantly. Immunoprecipitation and atomic absorption spectrophotometry (AAS) further showed that the number of iron ions bound by ferritin decreased significantly at 24 h post-WSSV infection. Taken together, these results suggest that PK1 prevents apoferritin from iron loading, and thus stabilizes the cellular LIP levels, and that WSSV uses this novel mechanism to counteract the host cell's iron-withholding defense mechanism. IMPORTANCE: We show here that white spot syndrome virus (WSSV) ensures the availability of iron by using a previously unreported mechanism to defeat the host cell's iron-withholding defense mechanism. This defense is often implemented by ferritin, which can bind up to 4,500 iron atoms and acts to sequester free iron within the cell. WSSV's novel counterstrategy is mediated by a direct protein-protein interaction between viral protein kinase 1 (PK1) and host ferritin. PK1 interacts with both ferritin and apoferritin, suppresses apoferritin's ability to sequester free iron ions, and maintains the intracellular labile iron pool (LIP), and thus the availability of free iron is increased within cells.

TR4 nuclear receptor promotes prostate cancer metastasis via upregulation of CCL2/CCR2 signaling.

Testicular nuclear receptor 4 (TR4) plays protective roles against oxidative stress and DNA damage and might contribute to aging. Our recent clinical tumor tissue staining results showed higher expression of TR4 in prostate cancer (PCa) patients with high Gleason scores compared to the tissues with the low Gleason scores. In vitro migration/invasion assays after manipulation of the TR4 expression in PCa cells showed that TR4 promoted PCa cells migration/invasion. Mechanism dissection found that the CCL2/CCR2 signal plays the key role in the mediation of TR4-promoted PCa cells migration/invasion. Chromatin immunoprecipitation and Luciferase assays further confirmed TR4 modulation of CCL2 at the transcriptional level and addition of the CCR2 antagonist led to interruption of the TR4-enhanced PCa cells migration/invasion. Finally, the orthotopic xenografted mice studies using the luciferase expressing CWR22Rv1 cells found that TR4 enhanced PCa metastasis and this increased metastasis was reversed when the CCR2 antagonist was injected into the mice. Together, these in vitro and in vivo results revealed a positive role of TR4 in PCa metastasis and demonstrated CCL2/CCR2 signaling as an important mediator in exerting TR4 action. This finding suggests that TR4 may represent a biomarker related to PCa metastasis and targeting the TR4-CCL2/CCR2 axis may become a new therapeutic approach to battle PCa metastasis.

TR4 nuclear receptor functions as a tumor suppressor for prostate tumorigenesis via modulation of DNA damage/repair system.

Testicular nuclear receptor 4 (TR4), a member of the nuclear receptor superfamily, plays important roles in metabolism, fertility and aging. The linkage of TR4 functions in cancer progression, however, remains unclear. Using three different mouse models, we found TR4 could prevent or delay prostate cancer (PCa)/prostatic intraepithelial neoplasia development. Knocking down TR4 in human RWPE1 and mouse mPrE normal prostate cells promoted tumorigenesis under carcinogen challenge, suggesting TR4 may play a suppressor role in PCa initiation. Mechanism dissection in both in vitro cell lines and in vivo mice studies found that knocking down TR4 led to increased DNA damage with altered DNA repair system that involved the modulation of ATM expression at the transcriptional level, and addition of ATM partially interrupted the TR4 small interfering RNA-induced tumorigenesis in cell transformation assays. Immunohistochemical staining in human PCa tissue microarrays revealed ATM expression is highly correlated with TR4 expression. Together, these results suggest TR4 may function as a tumor suppressor to prevent or delay prostate tumorigenesis via regulating ATM expression at the transcriptional level.

Increased chemosensitivity via targeting testicular nuclear receptor 4 (TR4)-Oct4-interleukin 1 receptor antagonist (IL1Ra) axis in prostate cancer CD133+ stem/progenitor cells to battle prostate cancer.

Prostate cancer (PCa) stem/progenitor cells are known to have higher chemoresistance than non-stem/progenitor cells, but the underlying molecular mechanism remains unclear. We found the expression of testicular nuclear receptor 4 (TR4) is significantly higher in PCa CD133(+) stem/progenitor cells compared with CD133(-) non-stem/progenitor cells. Knockdown of TR4 levels in the established PCa stem/progenitor cells and the CD133(+) population of the C4-2 PCa cell line with lentiviral TR4 siRNA led to increased drug sensitivity to the two commonly used chemotherapeutic drugs, docetaxel and etoposide, judging from significantly reduced IC50 values and increased apoptosis in the TR4 knockdown cells. Mechanism dissection studies found that suppression of TR4 in these stem/progenitor cells led to down-regulation of Oct4 expression, which, in turn, down-regulated the IL-1 receptor antagonist (IL1Ra) expression. Neutralization experiments via adding these molecules into the TR4 knockdown PCa stem/progenitor cells reversed the chemoresistance, suggesting that the TR4-Oct4-IL1Ra axis may play a critical role in the development of chemoresistance in the PCa stem/progenitor cells. Together, these studies suggest that targeting TR4 may alter chemoresistance of PCa stem/progenitor cells, and this finding provides the possibility of targeting TR4 as a new and better approach to overcome the chemoresistance problem in PCa therapeutics.

Structural insights into the molecular mechanism of phytoplasma immunodominant membrane protein.

Immunodominant membrane protein (IMP) is a prevalent membrane protein in phytoplasma and has been confirmed to be an F-actin-binding protein. However, the intricate molecular mechanisms that govern the function of IMP require further elucidation. In this study, the X-ray crystallographic structure of IMP was determined and insights into its interaction with plant actin are provided. A comparative analysis with other proteins demonstrates that IMP shares structural homology with talin rod domain-containing protein 1 (TLNRD1), which also functions as an F-actin-binding protein. Subsequent molecular-docking studies of IMP and F-actin reveal that they possess complementary surfaces, suggesting a stable interaction. The low potential energy and high confidence score of the IMP-F-actin binding model indicate stable binding. Additionally, by employing immunoprecipitation and mass spectrometry, it was discovered that IMP serves as an interaction partner for the phytoplasmal effector causing phyllody 1 (PHYL1). It was then shown that both IMP and PHYL1 are highly expressed in the S2 stage of peanut witches' broom phytoplasma-infected Catharanthus roseus. The association between IMP and PHYL1 is substantiated through in vivo immunoprecipitation, an in vitro cross-linking assay and molecular-docking analysis. Collectively, these findings expand the current understanding of IMP interactions and enhance the comprehension of the interaction of IMP with plant F-actin. They also unveil a novel interaction pathway that may influence phytoplasma pathogenicity and host plant responses related to PHYL1. This discovery could pave the way for the development of new strategies to overcome phytoplasma-related plant diseases.

A model for apoptotic interaction between white spot syndrome virus and shrimp.

White spot syndrome virus (WSSV) is an enveloped, large dsDNA virus that mainly infects penaeid shrimp, causing serious damage to the shrimp aquaculture industry. Like other animal viruses, WSSV infection induces apoptosis. Although this occurs even in by-stander cells that are free of WSSV virions, apoptosis is generally regarded as a kind of antiviral immune response. To counter this response, WSSV has evolved several different strategies. From the presently available literature, we construct a model of how the host and virus both attempt to regulate apoptosis to their respective advantage. The basic sequence of events is as follows: first, when a WSSV infection occurs, cellular sensors detect the invading virus, and activate signaling pathways that lead to (1) the expression of pro-apoptosis proteins, including PmCasp (an effecter caspase), MjCaspase (an initiator caspase) and voltage-dependent anion channel (VDAC); and (2) mitochondrial changes, including the induction of mitochondrial membrane permeabilization and increased oxidative stress. These events initiate the apoptosis program. Meanwhile, WSSV begins to express its genes, including two anti-apoptosis proteins: AAP-1, which is a direct caspase inhibitor, and WSV222, which is an E3 ubiquitin ligase that blocks apoptosis through the ubiquitin-mediated degradation of shrimp TSL protein (an apoptosis inducer). WSSV also induces the expression of a shrimp anti-apoptosis protein, Pm-fortilin, which can act on Bax to inhibit mitochondria-triggered apoptosis. This is a life and death struggle because the virus needs to prevent apoptosis in order to replicate. If WSSV succeeds in replicating in sufficient numbers, this will result in the death of the infected penaeid shrimp host.

Reduced osteoblast activity in the mice lacking TR4 nuclear receptor leads to osteoporosis.

BACKGROUND: Early studies suggested that TR4 nuclear receptor might play important roles in the skeletal development, yet its detailed mechanism remains unclear. METHODS: We generated TR4 knockout mice and compared skeletal development with their wild type littermates. Primary bone marrow cells were cultured and we assayed bone differentiation by alkaline phosphatase and alizarin red staining. Primary calvaria were cultured and osteoblastic marker genes were detected by quantitative PCR. Luciferase reporter assays, chromatin immunoprecipitation (ChIP) assays, and electrophoretic mobility shift assays (EMSA) were performed to demonstrate TR4 can directly regulate bone differentiation marker osteocalcin. RESULTS: We first found mice lacking TR4 might develop osteoporosis. We then found that osteoblast progenitor cells isolated from bone marrow of TR4 knockout mice displayed reduced osteoblast differentiation capacity and calcification. Osteoblast primary cultures from TR4 knockout mice calvaria also showed higher proliferation rates indicating lower osteoblast differentiation ability in mice after loss of TR4. Mechanism dissection found the expression of osteoblast markers genes, such as ALP, type I collagen alpha 1, osteocalcin, PTH, and PTHR was dramatically reduced in osteoblasts from TR4 knockout mice as compared to those from TR4 wild type mice. In vitro cell line studies with luciferase reporter assay, ChIP assay, and EMSA further demonstrated TR4 could bind directly to the promoter region of osteocalcin gene and induce its gene expression at the transcriptional level in a dose dependent manner. CONCLUSIONS: Together, these results demonstrate TR4 may function as a novel transcriptional factor to play pathophysiological roles in maintaining normal osteoblast activity during the bone development and remodeling, and disruption of TR4 function may result in multiple skeletal abnormalities.

Deficiency in TR4 nuclear receptor abrogates Gadd45a expression and increases cytotoxicity induced by ionizing radiation.

The testicular receptor 4 (TR4) is a member of the nuclear receptor superfamily that controls various biological activities. A protective role of TR4 against oxidative stress has recently been discovered. We here examined the protective role of TR4 against ionizing radiation (IR) and found that small hairpin RNA mediated TR4 knockdown cells were highly sensitive to IR-induced cell death. IR exposure increased the expression of TR4 in scramble control small hairpin RNA expressing cells but not in TR4 knockdown cells. Examination of IR-responsive molecules found that the expression of Gadd45a, the growth arrest and DNA damage response gene, was dramatically decreased in Tr4 deficient (TR4KO) mice tissues and could not respond to IR stimulation in TR4KO mouse embryonic fibroblast cells. This TR4 regulation of GADD45A was at the transcriptional level. Promoter analysis identified four potential TR4 response elements located in intron 3 and exon 4 of the GADD45A gene. Reporter and chromatin immunoprecipitation (ChIP) assays provided evidence indicating that TR4 regulated the GADD45A expression through TR4 response elements located in intron 3 of the GADD45A gene. Together, we find that TR4 is essential in protecting cells from IR stress. Upon IR challenges, TR4 expression is increased, thereafter inducing GADD45A through transcriptional regulation. As GADD45A is directly involved in the DNA repair pathway, this suggests that TR4 senses genotoxic stress and up-regulates GADD45A expression to protect cells from IR-induced genotoxicity.

TR4 nuclear receptor functions as a fatty acid sensor to modulate CD36 expression and foam cell formation.

Testicular orphan nuclear receptor 4 (TR4) is an orphan member of the nuclear receptor superfamily with diverse physiological functions. Using TR4 knockout (TR4(-/-)) mice to study its function in cardiovascular diseases, we found reduced cluster of differentiation (CD)36 expression with reduced foam cell formation in TR4(-/-) mice. Mechanistic dissection suggests that TR4 induces CD36 protein and mRNA expression via a transcriptional regulation. Interestingly, we found this TR4-mediated CD36 transactivation can be further enhanced by polyunsaturated fatty acids (PUFAs), such as omega-3 and -6 fatty acids, and their metabolites such as 15-hydroxyeico-satetraonic acid (15-HETE) and 13-hydroxy octa-deca dieonic acid (13-HODE) and thiazolidinedione (TZD)-rosiglitazone. Both electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) assays demonstrate that TR4 binds to the TR4 response element located on the CD36 5'-promoter region for the induction of CD36 expression. Stably transfected TR4-siRNA or functional TR4 cDNA in the RAW264.7 macrophage cells resulted in either decreased or increased CD36 expression with decreased or increased foam cell formation. Restoring functional CD36 cDNA in the TR4 knockdown macrophage cells reversed the decreased foam cell formation. Together, these results reveal an important signaling pathway controlling CD36-mediated foam cell formation/cardiovascular diseases, and findings that TR4 transactivation can be activated via its ligands/activators, such as PUFA metabolites and TZD, may provide a platform to screen new drug(s) to battle the metabolism syndrome, diabetes, and cardiovascular diseases.

A Review of the Functional Annotations of Important Genes in the AHPND-Causing pVA1 Plasmid.

Acute hepatopancreatic necrosis disease (AHPND) is a lethal shrimp disease. The pathogenic agent of this disease is a special Vibrio parahaemolyticus strain that contains a pVA1 plasmid. The protein products of two toxin genes in pVA1, pirAvp and pirBvp, targeted the shrimp's hepatopancreatic cells and were identified as the major virulence factors. However, in addition to pirAvp and pirBvp, pVA1 also contains about ~90 other open-reading frames (ORFs), which may encode functional proteins. NCBI BLASTp annotations of the functional roles of 40 pVA1 genes reveal transposases, conjugation factors, and antirestriction proteins that are involved in horizontal gene transfer, plasmid transmission, and maintenance, as well as components of type II and III secretion systems that may facilitate the toxic effects of pVA1-containing Vibrio spp. There is also evidence of a post-segregational killing (PSK) system that would ensure that only pVA1 plasmid-containing bacteria could survive after segregation. Here, in this review, we assess the functional importance of these pVA1 genes and consider those which might be worthy of further study.

Structural insight into the differential interactions between the DNA mimic protein SAUGI and two gamma herpesvirus uracil-DNA glycosylases.

Uracil-DNA glycosylases (UDGs) are conserved DNA-repair enzymes that can be found in many species, including herpesviruses. Since they play crucial roles for efficient viral DNA replication in herpesviruses, they have been considered as potential antiviral targets. In our previous work, Staphylococcus aureus SAUGI was identified as a DNA mimic protein that targets UDGs from S. aureus, human, Herpes simplex virus (HSV) and Epstein-Barr virus (EBV). Interestingly, SAUGI has the strongest inhibitory effects with EBVUDG. Here, we determined complex structures of SAUGI with EBVUDG and another γ-herpesvirus UDG from Kaposi's sarcoma-associated herpesvirus (KSHVUDG), which SAUGI fails to effectively inhibit. Structural analysis of the SAUGI/EBVUDG complex suggests that the additional interaction between SAUGI and the leucine loop may explain why SAUGI shows the highest binding capacity with EBVUDG. In contrast, SAUGI appears to make only partial contacts with the key components responsible for the compression and stabilization of the DNA backbone in the leucine loop extension of KSHVUDG. The findings in this study provide a molecular explanation for the differential inhibitory effects and binding strengths that SAUGI has on these two UDGs, and the structural basis of the differences should be helpful in developing inhibitors that would interfere with viral DNA replication.

A shrimp glycosylase protein, PmENGase, interacts with WSSV envelope protein VP41B and is involved in WSSV pathogenesis.

Viral glycoproteins are expressed by many viruses, and during infection they usually play very important roles, such as receptor attachment or membrane fusion. The mature virion of the white spot syndrome virus (WSSV) is unusual in that it contains no glycosylated proteins, and there are currently no reports of any glycosylation mechanisms in the pathogenesis of this virus. In this study, we cloned a glycosylase, mannosyl-glycoprotein endo-ß-N-acetylglucosaminidase (ENGase, EC 3.2.1.96), from Penaeus monodon and found that it was significantly up-regulated in WSSV-infected shrimp. A yeast two-hybrid assay showed that PmENGase interacted with both structural and non-structural proteins, and GST-pull down and co-immunoprecipitation (Co-IP) assays confirmed its interaction with the envelope protein VP41B. In the WSSV challenge tests, the cumulative mortality and viral copy number were significantly decreased in the PmEngase-silenced shrimp, from which we conclude that shrimp glycosylase interacts with WSSV in a way that benefits the virus. Lastly, we speculate that the deglycosylation activity of PmENGase might account for the absence of glycosylated proteins in the WSSV virion.

Structural Insights to the Heterotetrameric Interaction between the Vibrio parahaemolyticus PirAvp and PirBvp Toxins and Activation of the Cry-Like Pore-Forming Domain.

Acute hepatopancreatic necrosis disease (AHPND) is a newly emergent penaeid shrimp disease which can cause 70-100% mortality in Penaeus vannamei and Penaeus monodon, and has resulted in enormous economic losses since its appearance. AHPND is caused by the specific strains of Vibrio parahaemolyticus that harbor the pVA1 plasmid and express PirAvp and PirBvp toxins. These two toxins have been reported to form a binary complex. When both are present, they lead to the death of shrimp epithelial cells in the hepatopancreas and cause the typical histological symptoms of AHPND. However, the binding mode of PirAvp and PirBvp has not yet been determined. Here, we used isothermal titration calorimetry (ITC) to measure the binding affinity of PirAvp and PirBvp. Since the dissociation constant (Kd = 7.33 ± 1.20 µM) was considered too low to form a sufficiently stable complex for X-ray crystallographic analysis, we used alternative methods to investigate PirAvp-PirBvp interaction, first by using gel filtration to evaluate the molecular weight of the PirAvp/PirBvp complex, and then by using cross-linking and hydrogen-deuterium exchange (HDX) mass spectrometry to further understand the interaction interface between PirAvp and PirBvp. Based on these results, we propose a heterotetrameric interaction model of this binary toxin complex. This model provides insight of how conformational changes might activate the PirBvp N-terminal pore-forming domain and should be helpful for devising effective anti-AHPND strategies in the future.

TR2 and TR4 Orphan Nuclear Receptors: An Overview.

Testicular nuclear receptors 2 and 4 (TR2, TR4), also known as NR2C1 and NR2C2, belong to the nuclear receptor superfamily and were first cloned in 1989 and 1994, respectively. Although classified as orphan receptors, several natural molecules, their metabolites, and synthetic compounds including polyunsaturated fatty acids (PUFAs), PUFA metabolites 13-hydroxyoctadecadienoic acid, 15-hydroxyeicosatetraenoic acid, and the antidiabetic drug thiazolidinediones can transactivate TR4. Importantly, many of these ligands/activators can also transactivate peroxisome proliferator-activated receptor gamma (PPARγ), also known as NR1C3 nuclear receptor. Both TR4 and PPARγ can bind to similar hormone response elements (HREs) located in the promoter of their common downstream target genes. However, these two nuclear receptors, even with shared ligands/activators and shared binding ability for similar HREs, have some distinct functions in many diseases they influence. In cancer, PPARγ inhibits thyroid, lung, colon, and prostate cancers but enhances bladder cancer. In contrast, TR4 inhibits liver and prostate cancer initiation but enhances pituitary corticotroph, liver, and prostate cancer progression. In type 2 diabetes, PPARγ increases insulin sensitivity but TR4 decreases insulin sensitivity. In cardiovascular disease, PPARγ inhibits atherosclerosis but TR4 enhances atherosclerosis through increasing foam cell formation. In bone physiology, PPARγ inhibits bone formation but TR4 increases bone formation. Together, the contrasting impact of TR4 and PPARγ on different diseases may raise a critical issue about drug used to target any one of these nuclear receptors.

The monomeric form of Neisseria DNA mimic protein DMP19 prevents DNA from binding to the histone-like HU protein.

DNA mimicry is a direct and effective strategy by which the mimic competes with DNA for the DNA binding sites on other proteins. Until now, only about a dozen proteins have been shown to function via this strategy, including the DNA mimic protein DMP19 from Neisseria meningitides. We have shown previously that DMP19 dimer prevents the operator DNA from binding to the transcription factor NHTF. Here, we provide new evidence that DMP19 monomer can also interact with the Neisseria nucleoid-associated protein HU. Using BS3 crosslinking, gel filtration and isothermal titration calorimetry assays, we found that DMP19 uses its monomeric form to interact with the Neisseria HU dimer. Crosslinking conjugated mass spectrometry was used to investigate the binding mode of DMP19 monomer and HU dimer. Finally, an electrophoretic mobility shift assay (EMSA) confirmed that the DNA binding affinity of HU is affected by DMP19. These results showed that DMP19 is bifunctional in the gene regulation of Neisseria through its variable oligomeric forms.