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Kawasaki disease (KD) is the most common cause of pediatric cardiac disease in developed countries, and can lead to permanent coronary artery damage and long term sequelae such as coronary artery aneurysms. Given the prevalence and severity of KD, further research is warranted on its pathophysiology. It is known that endothelial cell damage and inflammation are two essential processes resulting in the coronary endothelial dysfunction in KD. However, detailed mechanisms are largely unknown. In this study, we investigated the role of pyroptosis in the setting of KD, and hypothesized that pyroptosis may play a central role in its pathophysiology. In vivo experiments of patients with KD demonstrated that serum levels of pyroptosis-related proteins, including ASC, caspase-1, IL-1β, IL-18, GSDMD and lactic dehydrogenase (LDH), were significantly increased in KD compared with healthy controls (HCs). Moreover, western blot analysis showed that the expression of GSDMD and mature IL-1β was notably elevated in KD sera. In vitro, exposure of human umbilical vein endothelial cells (HUVECs) to KD sera-treated THP1 cells resulted in the activation of NLRP3 inflammasome and subsequent pyroptosis induction, as evidenced by elevated expression of caspase-1, GSDMD, cleaved p30 form of GSDMD, IL-1β and IL-18, and increased LDH release and TUNEL and propidium iodide (PI)-positive cells. Furthermore, our results showed that NLRP3-dependent endothelial cell pyroptosis was activated by HMGB1/RAGE/cathepsin B signaling. These findings were also recapitulated in a mouse model of KD induced by Candida albicans cell wall extracts (CAWS). Together, our findings suggest that endothelial cell pyroptosis may play a significant role in coronary endothelial damage in KD, providing novel evidence that further elucidates its pathophysiology.

We recently found that Sodium butyrate (NaB) possesses anti-inflammatory effects in diabetic nephropathy (DN) mouse model and in high-glucose induced mouse glomerular mesangial cells. Pyroptosis is a programmed cell death accompanied with the release of pro-inflammatory factors. Gasdermin D (GSDMD) is a novel discovered pivotal executive protein of pyroptosis, which can be cleaved by inflammatory caspases. The aim of our study is to verify if NaB have some effects against high-glucose induces pyroptosis in renal Glomerular endothelial cells (GECs). For this aim, human GECs were cultured and exposed to high-glucose. Exogenous NaB, caspase 1 inhibitor Ac-YVAD-CMK (A-Y-C) or knockdown GSDMD by siRNA were used. We found high glucose could increase Propidium Iodide (PI) positive cells and elevate release of lactate dehydrogenase (LDH), Interleukin 1 beta (IL-1β) and Interleukin 18 (IL-18); protein levels of GSDMD, GSDMD N-terminal domain (GSDMD-N) and cleaved-caspase-1 were also elevated. Effect of NaB on LDH release and PI positive cells was further enhanced by inhibiting caspase 1-GSDMD. In addition, high glucose-induced nuclear factor kappa-B (NF-κB)/NF-κB inhibitor α (IκB-α) signaling pathway was reversed by NaB or A-Y-C administration. In conclusion, NaB could ameliorate high-glucose induced GECs via caspase1-GSDMD canonical pyroptosis pathway; and NF-κB/IκB-α signaling pathway was involved in it., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The multiple genetic approaches available for molecular diagnosis of human diseases have made possible to identify an increasing number of pathogenic genetic changes, particularly with the advent of next generation sequencing (NGS) technologies. However, the main challenge lies in the interpretation of their functional impact, which has resulted in the widespread use of animal models. We describe here the functional modelling of seven BBS loci variants, most of them novel, in zebrafish embryos to validate their in silico prediction of pathogenicity. We show that target knockdown (KD) of known BBS (BBS1, BB5 or BBS6) loci leads to developmental defects commonly associated with ciliopathies, as previously described. These KD pleiotropic phenotypes were rescued by co-injecting human wild type (WT) loci sequence but not with the equivalent mutated mRNAs, providing evidence of the pathogenic effect of these BBS changes. Furthermore, direct assessment of cilia located in Kupffer's vesicle (KV) showed a reduction of ciliary length associated with all the studied variants, thus confirming a deleterious effect. Taken together, our results seem to prove the pathogenicity of the already classified and unclassified new BBS variants, as well as highlight the usefulness of zebrafish as an animal model for in vivo assays in human ciliopathies.

Chloroplastic Cpn60 proteins are type I chaperonins comprising of Cpn60α and Cpn60β subunits. Arabidopsis genome contains six entries in Cpn60 family, out of which two are for Cpn60α subunit and four for Cpn60β subunit. We noted that the cpn60β4 knockout mutant plants (T-DNA insertion salk_064887 line) differed from the wild type Col-0 plants in the developmental programming. cpn60β4 mutant plants showed early seed germination. Radical emergence, hypocotyl emergence and cotyledons opening were faster in cpn60β4 mutant plants than WT. Importantly, cpn60β4 mutant plants showed early-flowering phenotype. The number of flowers and siliques as well as weight of the seeds were higher in cpn60β4 mutant plants as compared to Col-0 plants. These effects were reverted to wild type like growth and developmental patterns when genomic fragment of Arabidopsis encompassing Cpn60β4 gene was complemented in the mutant background. The overexpression of Cpn60β4 gene using CaMV35 promoter in wild type background (OE-Cpn60β4) delayed the floral transition as against wild type plants. The plastid division were affected in cpn60β4 mutant plants compared to Col-0. The results of this study suggest that Cpn60β4 plays important role(s) in chloroplast development and is a key factor in plant growth, development and flowering in Arabidopsis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Schwannomas are peripheral nerve sheath tumors associated with three genetically distinct disease entities, namely sporadic schwannoma, neurofibromatosis type-2, and schwannomatosis. Schwannomas are associated with severe disability and in cases lead to death. The primary treatment is operative resection that itself can cause neurologic damage and is at times contra-indicated due to tumor location. Given their homogenous Schwann-lineage cellular composition, schwannomas are appealing targets for gene therapy. In the present study, we have generated an adeno-associated serotype 1 virus (AAV1)-based vector delivering N-terminal of the pyroptotic gene Gasdermin-D; (GSDMDNterm) under the control of the Schwann-cell specific promoter, P0. we have demonstrated that AAV1-P0-GSDMDNterm injection into intra-sciatic schwannomas reduces the growth of these tumors and resolves tumor-associated pain without causing neurologic damage. This AAV1-P0-GSDMDNterm vector holds promise for clinical treatment of schwannomas via direct intra-tumoral injection.

Caspase-4, the cytosolic LPS sensor, and gasdermin D, its downstream effector, constitute the non-canonical inflammasome, which drives inflammatory responses during Gram-negative bacterial infections. It remains unclear whether other proteins regulate cytosolic LPS sensing, particularly in human cells. Here, we conduct a genome-wide CRISPR/Cas9 screen in a human monocyte cell line to identify genes controlling cytosolic LPS-mediated pyroptosis. We find that the transcription factor, IRF2, is required for pyroptosis following cytosolic LPS delivery and functions by directly regulating caspase-4 levels in human monocytes and iPSC-derived monocytes. CASP4, GSDMD, and IRF2 are the only genes identified with high significance in this screen highlighting the simplicity of the non-canonical inflammasome. Upon IFN-γ priming, IRF1 induction compensates IRF2 deficiency, leading to robust caspase-4 expression. Deficiency in IRF2 results in dampened inflammasome responses upon infection with Gram-negative bacteria. This study emphasizes the central role of IRF family members as specific regulators of the non-canonical inflammasome., (© 2019 The Authors.)

GSDMD is a recently discovered pyroptosis executioner in monocytes whose N-terminal domain can insert into the inner leaflet of cell membranes and form extensive pores. However, the function of GSDMD in other biological systems remains unclear. In this study, we showed that the expression of GSDMD was consistently correlated with CD8 + T cell markers in The Cancer Genome Atlas (TCGA) cohorts. GSDMD cleavage increased in OT-1 cytotoxic T lymphocytes (CTLs) and human activated CD8 + T cells. Colocalization of GSDMD with granzyme B was observed in the proximity of immune synapses, and GSDMD deficiency reduced the cytolytic capacity of CD8 + T cells. Overall, our study highlights a function, to our knowledge previously unknown, for GSDMD in CTLs and demonstrated that GSDMD is required for an optimal CTL response to cancer cells., (Copyright © 2019. Published by Elsevier B.V.)

Plasmodesmata act as key elements in intercellular communication, coordinating processes related to plant growth, development, and responses to environmental stresses. While many of the developmental, biotic, and abiotic signals are primarily perceived at the plasma membrane (PM) by receptor proteins, plasmodesmata also cluster receptor-like activities; whether these two pathways interact is currently unknown. Here, we show that specific PM-located Leu-rich-repeat receptor-like-kinases, Qiān Shŏu kinase (QSK1) and inflorescence meristem kinase2, which under optimal growth conditions are absent from plasmodesmata, rapidly relocate and cluster to the pores in response to osmotic stress. This process is remarkably fast, is not a general feature of PM-associated proteins, and is independent of sterol and sphingolipid membrane composition. Focusing on QSK1, previously reported to be involved in stress responses, we show that relocalization in response to mannitol depends on QSK1 phosphorylation. Loss-of-function mutation in QSK1 results in delayed lateral root (LR) development, and the mutant is affected in the root response to mannitol stress. Callose-mediated plasmodesmata regulation is known to regulate LR development. We found that callose levels are reduced in the qsk1 mutant background with a root phenotype resembling ectopic expression of PdBG1, an enzyme that degrades callose at the pores. Both the LR and callose phenotypes can be complemented by expression of wild-type and phosphomimic QSK1 variants, but not by phosphodead QSK1 mutant, which fails to relocalize at plasmodesmata. Together, the data indicate that reorganization of receptor-like-kinases to plasmodesmata is important for the regulation of callose and LR development as part of the plant response to osmotic stress., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Serum deprivation response (SDPR) gene has been recently characterized as a gene signature marker or serving a tumor suppressor role in specific types of cancer. However, gene expression alterations of SDPR in various types of cancer and their relevance to clinical outcomes remain unclear. In the present study, SDPR expression was profiled using the Oncomine database, and SDPR downregulation was indicated in most types of cancer. In agreement with previously reported breast cancer cases, downregulation of SDPR was indicated to be significantly associated with poor survival in patients with lung cancer, glioma and sarcoma. To clarify why SDPR expression was altered in these types of cancer, both DNA methylation patterns and potential transcriptional factors of SDPR were analyzed. Altered DNA methylation levels of SDPR were found in 17/18 cancer types using MethHC. To the best of our knowledge, the present study for the first time, identified the CpG site (cg10082589) as one of the best survival methylation markers for lung adenocarcinoma, and the CpG site (cg07488576) for sarcoma using Methsurv. In addition, GATA binding protein 2 was identified as a potential transcription factor for SDPR, by integrating and analyzing both the co‑expressed genes and the potential transcription factor binding sites of SDPR. In the present study, the systematic analysis of SDPR provided insight for the underlying molecular mechanisms in cancer progression, revealing novel perspectives for the clinical prognostic evaluation of lung adenocarcinoma and sarcoma.

During meiotic recombination, homologue-templated repair of programmed DNA double-strand breaks (DSBs) produces relatively few crossovers and many difficult-to-detect non-crossovers. By intercrossing two diverged mouse subspecies over five generations and deep-sequencing 119 offspring, we detect thousands of crossover and non-crossover events genome-wide with unprecedented power and spatial resolution. We find that both crossovers and non-crossovers are strongly depleted at DSB hotspots where the DSB-positioning protein PRDM9 fails to bind to the unbroken homologous chromosome, revealing that PRDM9 also functions to promote homologue-templated repair. Our results show that complex non-crossovers are much rarer in mice than humans, consistent with complex events arising from accumulated non-programmed DNA damage. Unexpectedly, we also find that GC-biased gene conversion is restricted to non-crossover tracts containing only one mismatch. These results demonstrate that local genetic diversity profoundly alters meiotic repair pathway decisions via at least two distinct mechanisms, impacting genome evolution and Prdm9-related hybrid infertility.

IL-1β is a potent pro-inflammatory cytokine that promotes immunity and host defense, and its dysregulation is associated with immune pathology. Toxoplasma gondii infection of myeloid cells triggers the production and release of IL-1β; however, the mechanisms regulating this pathway, particularly in human immune cells, are incompletely understood. We have identified a novel pathway of T. gondii induction of IL-1β via a Syk-CARD9-NF-κB signaling axis in primary human peripheral blood monocytes. Syk was rapidly phosphorylated during T. gondii infection of primary monocytes, and inhibiting Syk with the pharmacological inhibitors R406 or entospletinib, or genetic ablation of Syk in THP-1 cells, reduced IL-1β release. Inhibition of Syk in primary cells or deletion of Syk in THP-1 cells decreased parasite-induced IL-1β transcripts and the production of pro-IL-1β. Furthermore, inhibition of PKCδ, CARD9/MALT-1 and IKK reduced p65 phosphorylation and pro-IL-1β production in T. gondii-infected primary monocytes, and genetic knockout of PKCδ or CARD9 in THP-1 cells also reduced pro-IL-1β protein levels and IL-1β release during T. gondii infection, indicating that Syk functions upstream of this NF-κB-dependent signaling pathway for IL-1β transcriptional activation. IL-1β release from T. gondii-infected primary human monocytes required the NLRP3-caspase-1 inflammasome, but interestingly, was independent of gasdermin D (GSDMD) cleavage and pyroptosis. Moreover, GSDMD knockout THP-1 cells released comparable amounts of IL-1β to wild-type THP-1 cells after T. gondii infection. Taken together, our data indicate that T. gondii induces a Syk-CARD9/MALT-1-NF-κB signaling pathway and activation of the NLRP3 inflammasome for the release of IL-1β in a cell death- and GSDMD-independent manner. This research expands our understanding of the molecular basis for human innate immune regulation of inflammation and host defense during parasite infection., Competing Interests: The authors have declared that no competing interests exist.

Ferritin is the major iron storage molecule of vertebrates, which can be detected in serum under numerous conditions, including inflammatory, neurodegenerative, and malignant diseases. Given this character, serum ferritin is frequently used as a biomarker in clinical settings. How the ferritin secreted to the serum has attracted much attention. Although some studies have found ferritin was mediated via the endoplasmic reticulum (ER)-Golgi secretion pathway or secretory lysosomes trafficking pathway under normal conditions, the secretion pathway of ferritin under pathological conditions, especially in sepsis is not very clear. In this report, we adopt a murine sepsis model to study the secretion pathway of ferritin in sepsis. We demonstrated caspase-11-GSDMD pathway and associated pyroptosis are required for secretion of ferritin in vitro and in vivo in sepsis. Moreover, our work connects pyroptosis to serum ferritin secretion and suggests a passive release process of ferritin, enhancing our understanding of the mechanism of ferritin secretion., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The pyroptotic cell death effector gasdermin D (GSDMD) is required for murine models of hereditary inflammasome-driven, IL-1β-dependent, autoinflammatory disease, making it an attractive therapeutic target. However, the importance of GSDMD for more common conditions mediated by pathological IL-1β activation, such as gout, remain unclear. In this study, we address whether GSDMD and the recently described GSDMD inhibitor necrosulfonamide (NSA) contribute to monosodium urate (MSU) crystal-induced cell death, IL-1β release, and autoinflammation. We demonstrate that MSU crystals, the etiological agent of gout, rapidly activate GSDMD in murine macrophages. Despite this, the genetic deletion of GSDMD or the other lytic effector implicated in MSU crystal killing, mixed lineage kinase domain-like (MLKL), did not prevent MSU crystal-induced cell death. Consequently, GSDMD or MLKL loss did not hinder MSU crystal-mediated release of bioactive IL-1β. Consistent with in vitro findings, IL-1β induction and autoinflammation in MSU crystal-induced peritonitis was not reduced in GSDMD-deficient mice. Moreover, we show that the reported GSDMD inhibitor, NSA, blocks inflammasome priming and caspase-1 activation, thereby preventing pyroptosis independent of GSDMD targeting. The inhibition of cathepsins, widely implicated in particle-induced macrophage killing, also failed to prevent MSU crystal-mediated cell death. These findings 1) demonstrate that not all IL-1β-driven autoinflammatory conditions will benefit from the therapeutic targeting of GSDMD, 2) document a unique mechanism of MSU crystal-induced macrophage cell death not rescued by pan-cathepsin inhibition, and 3) show that NSA inhibits inflammasomes upstream of GSDMD to prevent pyroptotic cell death and IL-1β release., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Neutrophil granule serine proteases contribute to immune responses through cleavage of microbial toxins and structural proteins. They induce tissue damage and modulate inflammation if levels exceed their inhibitors. Here, we show that the intracellular protease inhibitors Serpinb1a and Serpinb6a contribute to monocyte and neutrophil survival in steady-state and inflammatory settings by inhibiting cathepsin G (CatG). Importantly, we found that CatG efficiently cleaved gasdermin D (GSDMD) to generate the signature N-terminal domain GSDMD-p30 known to induce pyroptosis. Yet GSDMD deletion did not rescue neutrophil survival in Sb1a.Sb6a -/- mice. Furthermore, Sb1a.Sb6a -/- mice released high levels of pro-inflammatory cytokines upon endotoxin challenge in vivo in a CatG-dependent manner. Canonical inflammasome activation in Sb1a.Sb6a -/- macrophages showed increased IL-1β release that was dependent on CatG and GSDMD. Together, our findings demonstrate that cytosolic serpins expressed in myeloid cells prevent cell death and regulate inflammatory responses by inhibiting CatG and alternative activation of GSDMD., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Gasdermin D (GsdmD) was recently identified as the executioner of pyroptotic inflammatory cell death, and is a substrate for caspases-1 and 11. GsdmD is detrimental in lethal endotoxemia but protective in bacterial sepsis. However, little is known about its role during noninfectious/sterile injuries. In this study, we examined the contribution of GsdmD using WT and GsdmD -/- mice in two models of noninfectious liver injury: hemorrhagic shock with resuscitation (HS/R) and acetaminophen (APAP) overdose. GsdmD -/- mice had significantly increased liver damage at 6 h after HS/R or APAP vs WT, shown by significantly elevated ALT level and extended areas of cell death in liver. Caspase-8, a mediator of multiple cell death pathways, was highly elevated in GsdmD -/- mice after injury. Significantly increased cleavage of caspase-8 and subsequent high levels of apoptosis were found in livers of GsdmD -/- mice after HS/R, a relatively mild ROS-induced liver injury. However, during more severe APAP-mediated ROS-induced liver injury, caspase-8 cleavage in GsdmD -/- liver was inhibited compared with WT, resulting in accumulation of pro-caspase-8 and increased levels of necroptosis. Our findings indicate a novel hepatoprotective role for GsdmD in noninfectious inflammation models via regulation of caspase-8 expression and downstream cell death pathways. The effects of GsdmD protection are likely injury specific and may also depend on injury severity and levels of ROS produced. These data suggest modulation of GsdmD/caspase-8 may be a novel therapeutic option in ROS-mediated liver injury.

Gasdermin D (GSDMD) cleavage by caspase-1 or caspase-11 inflammasomes triggers pyroptosis, a lytic form of cell death protective against intracellular bacteria. In this study, we examine the role of GSDMD in a mouse model of melioidosis. Gsdmd -/- mice were more susceptible than wild-type mice to intranasal infection with Burkholderia thailandensis Production of IL-18, but not IL-1β, was decreased in Gsdmd -/- infected mice. Despite lower IL-18, IFN-γ was produced in similar amounts in wild-type and Gsdmd -/- mice. In vitro, secretion of both IL-1β and IL-18 by macrophages or dendritic cells infected with B. thailandensis was dependent on GSDMD. Surprisingly, wild-type or GSDMD-deficient neutrophils secreted similar amounts of IL-1β, suggesting these cells may be the source of the GSDMD-independent IL-1β detected in vivo. Recombinant GSDMD was able to directly kill B. thailandensis in vitro upon processing by active caspase-1. Moreover, bacteria harvested from wild-type, but not Gsdmd -/- , macrophages were more susceptible to the microbicidal effect of hydrogen peroxide or human β-defensin-3. Finally, we provide evidence that pyroptosis of in vitro infected macrophages is directly microbicidal. Taken together, these results indicate that the protective action of GSDMD in melioidosis is primarily due to induction of pyroptosis and direct killing of bacteria rather than production of cytokines., (Copyright © 2019 by The American Association of Immunologists, Inc.)

miR-21 is aberrantly expressed, and plays a role in various types of tumors and many other diseases. However, the mechanism of miR-21 in LPS-induced septic shock is still unclear. In this study, we investigated the mechanism of miR-21 in LPS-induced pyroptosis and septic shock. Here, we show that miR-21 deficiency inhibited NLRP3, ASC, and caspase-1 expression, as well as inflammasome activation in myeloid cells from both mice and humans. We found that the NF-κB pathway was regulated by miR-21, and that A20 was a direct target of miR-21. Furthermore, miR-21 deficiency inhibited the ASC pyroptosome, which restrained caspase-1 activation and GSDMD cleavage, thereby preventing LPS-induced pyroptosis and septic shock. miR-21 deficiency resulted in an increase in A20, which led to decreased IL-1β production and caspase-1 activation. Caspase-1-mediated GSDMD cleavage was consequently decreased, which prevented pyroptosis in LPS-induced sepsis in mice. Our results demonstrate that miR-21 is a critical positive regulator of the NF-κB pathway and NLRP3 inflammasomes in pyroptosis and septic shock via A20. In addition, by analyzing published miRNA expression profiles in the Gene Expression Omnibus database, we found that the miR-21 levels in peripheral blood from patients with septic shock were elevated. Thus, miR-21 may serve as a potential treatment target in patients with septic shock.

Gasdermin-D (GSDMD) is cleaved by caspase-1, caspase-4, and caspase-11 in response to canonical and noncanonical inflammasome activation. Upon cleavage, GSDMD oligomerizes and forms plasma membrane pores, resulting in interleukin-1β (IL-1β) secretion, pyroptotic cell death, and inflammatory pathologies, including periodic fever syndromes and septic shock-a plague on modern medicine. Here, we showed that IRF2, a member of the interferon regulatory factor (IRF) family of transcription factors, was essential for the transcriptional activation of GSDMD. A forward genetic screen with N -ethyl- N -nitrosourea (ENU)-mutagenized mice linked IRF2 to inflammasome signaling. GSDMD expression was substantially attenuated in IRF2- deficient macrophages, endothelial cells, and multiple tissues, which corresponded with reduced IL-1β secretion and inhibited pyroptosis. Mechanistically, IRF2 bound to a previously uncharacterized but unique site within the GSDMD promoter to directly drive GSDMD transcription for the execution of pyroptosis. Disruption of this single IRF2-binding site abolished signaling by both the canonical and noncanonical inflammasomes. Together, our data illuminate a key transcriptional mechanism for expression of the gene encoding GSDMD, a critical mediator of inflammatory pathologies., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

We describe a large consanguineous pedigree from a remote area of Northern Pakistan, with a complex developmental disorder associated with wide-ranging symptoms, including mental retardation, speech and language impairment and other neurological, psychiatric, skeletal and cardiac abnormalities. We initially carried out a genetic study using the HumanCytoSNP-12 v2.1 Illumina gene chip on nine family members and identified a single region of homozygosity shared amongst four affected individuals on chromosome 7p22 (positions 3059377-5478971). We performed whole-exome sequencing on two affected individuals from two separate branches of the extended pedigree and identified a novel nonsynonymous homozygous mutation in exon 9 of the WIPI2 (WD-repeat protein interacting with phosphoinositide 2) gene at position 5265458 (c.G745A;pV249M). WIPI2 plays a critical role in autophagy, an evolutionary conserved cellular pathway implicated in a growing number of medical conditions. The mutation is situated in a highly conserved and critically important region of WIPI2, responsible for binding PI(3)P and PI(3,5)P2, an essential requirement for autophagy to proceed. The mutation is absent in all public databases, is predicted to be damaging and segregates with the disease phenotype. We performed functional studies in vitro to determine the potential effects of the mutation on downstream pathways leading to autophagosome assembly. Binding of the V231M mutant of WIPI2b to ATG16L1 (as well as ATG5-12) is significantly reduced in GFP pull-down experiments, and fibroblasts derived from the patients show reduced WIPI2 puncta, reduced LC3 lipidation and reduced autophagic flux., (© The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain.)

Secretion of the stress hormone cortisol follows a circadian rhythm and is stimulated following stress exposure. Cortisol regulates the transcription of several genes, primarily through activation of the glucocorticoid receptor (GR). Previously, we showed an upregulation of PERIOD genes PER1 and PER3 after pharmacological/glucocorticoid challenge in vivo and in vitro. The current study aims to investigate the temporal association between unstimulated, diurnal cortisol secretion and the expression of selected GR-target genes (PER1, PER2, PER3, FKBP5, GILZ and SDPR) in vivo to determine the timing of the most pronounced coupling between cortisol and mRNA expression. Unstimulated plasma and saliva cortisol concentrations and gene expression levels in whole blood were measured every 15 min from early morning until 16:00 h in 18 healthy men. Time-lagged correlations of cortisol concentrations with mRNA expression levels were assessed allowing lags between -240 and + 240 min. Strong positive correlations at non-zero lags between cortisol levels and the expression of FKBP5 (plasma: r = 0.74 (CI = 0.65-0.81), p <  0.001, lag + 90 min; saliva: r = 0.71 (CI = 0.61-0.78), p <  0.001, lag + 75 min), and GILZ (plasma: r = 0.59 (CI = 0.46-0.69), p <  0.001, lag + 30 min; saliva r = 0.53 (CI = 0.41-0.63), p <  0.001, lag +15 min) were observed. Expressions of PERIOD genes and SDPR correlated only weakly with cortisol (all |r| < 0.25). Our findings demonstrate strong correlations between cortisol secretion and gene expression in humans under unstimulated conditions. The observed time-lags can guide future research aiming to characterize glucocorticoid-dependent gene expression in clinical samples with stress-related disorders., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Bardet-Biedl syndrome (BBS) is a rare genetically heterogeneous ciliopathy which accompanies retinitis pigmentosa (RP). However, the BBS5 mutation remains unclear in Iranians with BBS. The purpose of study is to evaluate genetic analyses of a BBS Iranian family using targetted exome sequencing (TES). A male 11-year-old proband and three related family members were recruited. Biochemical tests, electrocardiography and visual acuity testing, such as funduscopic, fundus photography (FP), optical coherence tomography (OCT), and standard electroretinography, were conducted. Molecular analysis and high-throughput DNA sequence analysis were performed. The proband was diagnosed with possible BBS based on the presence of three primary features and two secondary features. The TES analysis of the proband with BBS resulted in the identification of a novel, homozygous splicing variant c. 208+2T>C of the BBS5 gene (NM&#95;152384.2) in this Iranian BBS family. This variant was confirmed and was completely co-segregated with the disease in this family by Sanger sequencing. Thus, we report a novel, homozygous splicing site variant c.208+2T>C in the BBS5 gene for the first time in the Iranian family., (© 2019 The Author(s).)

The endoplasmic reticulum (ER) of cancer cells needs to adapt to the enhanced proteotoxic stress associated with the accumulation of unfolded, misfolded and transformation-associated proteins. One way by which tumors thrive in the context of ER stress is by promoting ER-Associated Degradation (ERAD), although the mechanisms are poorly understood. Here, we show that the Small p97/VCP Interacting Protein (SVIP), an endogenous inhibitor of ERAD, undergoes DNA hypermethylation-associated silencing in tumorigenesis to achieve this goal. SVIP exhibits tumor suppressor features and its recovery is associated with increased ER stress and growth inhibition. Proteomic and metabolomic analyses show that cancer cells with epigenetic loss of SVIP are depleted in mitochondrial enzymes and oxidative respiration activity. This phenotype is reverted upon SVIP restoration. The dependence of SVIP hypermethylated cancer cells on aerobic glycolysis and glucose was also associated with sensitivity to an inhibitor of the glucose transporter GLUT1. This could be relevant to the management of tumors carrying SVIP epigenetic loss, because these occur in high-risk patients who manifest poor clinical outcomes. Overall, our study provides insights into how epigenetics helps deal with ER stress and how SVIP epigenetic loss in cancer may be amenable to therapies that target glucose transporters.

Improving the performance of the key photosynthetic enzyme Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) by protein engineering is a critical strategy for increasing crop yields. The extensive chaperone requirement of plant Rubisco for folding and assembly has long been an impediment to this goal. Production of plant Rubisco in Escherichia coli requires the coexpression of the chloroplast chaperonin and four assembly factors. Here, we demonstrate that simultaneous expression of Rubisco and chaperones from a T7 promotor produces high levels of functional enzyme. Expressing the small subunit of Rubisco with a C-terminal hexahistidine-tag further improved assembly, resulting in a ~ 12-fold higher yield than the previously published procedure. The expression system described here provides a platform for the efficient production and engineering of plant Rubisco., (© 2019 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Pyroptosis, a form of programmed cell death (PCD), has garnered increasing attention as it relates to innate immunity and diseases. However, the involvement of pyroptosis in the mechanism by which lobaplatin acts against colorectal cancer (CRC) is unclear. Our study revealed that treatment with lobaplatin reduced the viability of HT-29 and HCT116 cells in a dose-dependent manner. Morphologically, HT-29 and HCT116 cells treated with lobaplatin exhibited microscopic features of cell swelling and large bubbles emerging from the plasma membrane, and transmission electron microscopy (TEM) revealed multiple pores in the membrane. GSDME, rather than GSDMD, was cleaved in lobaplatin-induced pyroptosis in HT-29 and HCT116 cells due to caspase-3 activation. Knocking out GSDME switched lobaplatin-induced cell death from pyroptosis to apoptosis but did not affect lobaplatin-mediated inhibition of growth and tumour formation of HT-29 and HCT116 cells in vivo and in vitro. Further investigation indicates that lobaplatin induced reactive oxygen species (ROS) elevation and JNK phosphorylation. NAC, a ROS scavenger, completely reversed the pyroptosis of lobaplatin-treated HT-29 and HCT116 and JNK phosphorylation. Activated JNK recruited Bax to mitochondria, and thereby stimulated cytochrome c release to cytosol, followed by caspase-3/-9 cleavage and pyroptosis induction. Therefore, in colon cancer cells, GSDME mediates lobaplatin-induced pyroptosis downstream of the ROS/JNK/Bax-mitochondrial apoptotic pathway and caspase-3/-9 activation. Our study indicated that GSDME-dependent pyroptosis is an unrecognized mechanism by which lobaplatin eradicates neoplastic cells, which may have important implications for the clinical application of anticancer therapeutics.

mTORC1 and GSK3 play critical roles in early stages of (macro)autophagy, but how they regulate late steps of autophagy remains poorly understood. Here we show that mTORC1 and GSK3-TIP60 signaling converge to modulate autophagosome maturation through Pacer, an autophagy regulator that was identified in our recent study. Hepatocyte-specific Pacer knockout in mice results in impaired autophagy flux, glycogen and lipid accumulation, and liver fibrosis. Under nutrient-rich conditions, mTORC1 phosphorylates Pacer at serine157 to disrupt the association of Pacer with Stx17 and the HOPS complex and thus abolishes Pacer-mediated autophagosome maturation. Importantly, dephosphorylation of Pacer under nutrient-deprived conditions promotes TIP60-mediated Pacer acetylation, which facilitates HOPS complex recruitment and is required for autophagosome maturation and lipid droplet clearance. This work not only identifies Pacer as a regulator in hepatic autophagy and liver homeostasis in vivo but also reveals a signal integration mechanism involved in late stages of autophagy and lipid metabolism., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Arsenic is an environmental toxicant and human carcinogen. The liver is the main site of arsenic storage and metabolism. Exposure to excessive arsenic causes liver damage and release of pro-inflammatory factors, which in turn lead to liver fibrosis. Gasdermin D (GSDMD), a mediator of pyroptosis, has low expression in hepatic tumor cells. In L-02 cells, arsenite caused increases of GSDMD and cleaved caspase-1 levels and decreases of caspase-1 and miR-379-5p levels. It also promoted the release of IL-1β in a concentration- and time-dependent manner. Luciferase reporter assays showed that GSDMD was a direct target of miR-379-5p. In L-02 cells, the over-expression of miR-379-5p blocked the arsenite-induced increases of GSDMD levels and the release of IL-1β, effects that were reversed by up-regulation of GSDMD. LX-2 cells, cultured in the media from arsenite-treated L-02 cells, showed elevated levels of proliferating cell nuclear antigen (PCNA), collagen I, vimentin, and α-smooth muscle actin (α-SMA), which indicated activation of these cells. Activation of LX-2 cells by media from arsenite-treated L-02 cells was inhibited by IL-1β neutralizing antibody. The media from arsenite-treated L-02 cells transfected with an miR-379-5p mimic inhibited the activation of LX-2 cells, a process that was reversed by up-regulation of GSDMD and by co-treatment with human recombinant IL-1β. Chronic exposure to arsenite induced, in liver tissue of mice, morphological damage, collagen deposition, and activation of hepatic stellate cells (HSCs). In liver tissue of arsenite-exposed mice, the levels of miR-379-5p were lower, but the levels of GSDMD and cleaved caspase-1 were elevated, and in sera from arsenite-exposed mice, the IL-1β levels were elevated. These results indicate that, by elevating the secretion of IL-1β, miR-379-5p regulation of GSDMD is involved in arsenite-induced activation of HSCs and in hepatic fibrosis. This establishes a previously unknown molecular mechanism for arsenite-induced liver damage, inflammation, and fibrosis.

Prolonged parenchymal cell death leads to activation of fibrogenic cells and extracellular matrix accumulation and eventually liver fibrosis. Autophagy, a major catabolic process of intracellular degradation and recycling, participates in hepatic fibrosis. However, the precise role of autophagy in the pathogenesis of hepatic fibrosis is controversial. The present study aims to investigate the key role of small VCP/p97 interacting protein (SVIP) against CCl 4 -induced hepatic fibrosis via activating autophagy. Autophagy could be activated by SVIP in HepG2 cells, but starvation cannot increase SVIP expression in vitro and in vivo. Moreover, SVIP expression, in agreement with autophagic activity and the volume of lipid droplets, first increases and then decreases during the progression of liver fibrosis with CCl 4 treatment in vivo and in vivo. Further, overexpression of SVIP can protect HepG2 cells from the toxicity of CCl 4 , which could be enhanced by starvation. Finally, starvation keeps SVIP and autophagy at such high levels in the rat livers that markedly delays the progress of hepatic fibrosis. Probably, the protective effect of SVIP is associated with stabilizing nuclear factor (erythroid-derived 2)-related factor 2 (Nrf2) and transcription factor EB (TFEB). The current study provides insight into the biological role of SVIP and autophagy in regulating hepatic fibrosis, targeting SVIP might be a novel therapeutic strategy in the future.

The gasdermin (GSDM) family consists of gasdermin A (GSDMA), B (GSDMB), C (GSDMC), D (GSDMD), E or DNFA5 (GSDME), and DFNB59 in human. Expressed in the skin, gastrointestinal tract, and various immune cells, GSDMs mediate homeostasis and inflammation upon activation by caspases and unknown proteases. In particular, GSDMD is activated by inflammasome-activated caspases-1/-4/-5/-11 as well as a caspase-8-mediated pathway during Yersinia infection. These caspases cleave GSDMD to release its functional N-terminal fragment (GSDMD-NT) from its auto-inhibitory C-terminal fragment (GSDMD-CT). GSDMD-NTs bind to acid lipids in mammalian cell membranes and bacterial membranes, oligomerize, and insert into the membranes to form large transmembrane pores. Consequently, cellular contents including inflammatory cytokines are released and cells can undergo pyroptosis, a highly inflammatory form of cell death. In this chapter, we summarize recent research findings and present experimental procedures to obtain pure recombinant GSDMs for biochemical studies. We highlight a liposome-based assay that yields robust fluorescence signals for characterizing GSDM activities in vitro and may be applicable to other pore-forming proteins and ion channels in general., (© 2019 Elsevier Inc. All rights reserved.)

Pho regulon is a highly evolved and conserved mechanism across the microbes to fulfil their phosphate need. In this study, 52 proteobacteria genomes were analyzed for the presence of phosphorus acquisition genes, their pattern of arrangement and copy numbers. The diverse genetic architecture of the Pho regulon genes indicates the evolutionary challenge of nutrient limitation, particularly phosphorus, faced by bacteria in their environment. The incongruence between the Pho regulon proteins phylogeny and species phylogeny along with the presence of additional copies of pstS and pstB genes, having cross similarity with other genera, suggest the possibility of horizontal gene transfer event. The substitution rate analysis and multiple sequence alignment of the Pho regulon proteins were analyzed to gain additional insight into the evolution of the Pho regulon system. This comprehensive study confirms that genes perform the regulatory function (phoBR) were vertically inherited, whereas interestingly, genes whose product involved in direct interaction with the environment (pstS) acquired by horizontal gene transfer. The substantial amino acid substitutions in PstS most likely contribute to the successful adaptation of bacteria in different ecological condition dealing with different phosphorus availability. The findings decipher the intelligence of the bacteria which enable them to carry out the targeted alteration of genes to cope up with the environmental condition.

Phosphate is an essential component of cell functions, and the specific transport of phosphorus into a cell is mediated by phosphate-binding protein (PBP). The mechanism of PBP-phosphate recognition remains controversial: on the basis of similar binding affinities at acidic and basic pHs, it is believed that the hydrogen network in the binding site is flexible to adapt to different protonation states of phosphates. However, only hydrogen (1H) phosphate was observed in the sub-angstrom X-ray structures. To address this inconsistency, we performed molecular dynamics simulations using the AMOEBA polarizable force field. Structural and free energy data from simulations suggested that 1H phosphate was the preferred bound form at both pHs. The binding of dihydrogen (2H) phosphate disrupted the hydrogen-bond network in the PBP pocket, and the computed affinity was much weaker than that of 1H phosphate. Furthermore, we showed that the discrepancy in the studies described above is resolved if the interaction between phosphate and the buffer agent is taken into account. The calculated apparent binding affinities are in excellent agreement with experimental measurements. Our results suggest the high specificity of PBP for 1H phosphate and highlight the importance of the buffer solution for the binding of highly charged ligands.

Stomata are leaf pores that regulate CO 2 uptake and evapotranspirational water loss. By controlling CO 2 uptake, stomata impact on photosynthesis and dry matter accumulation. The regulation of evapotranspiration is equally important because it impacts on nutrient accumulation and leaf cooling and enables the plant to limit water loss during drought [1]. Our work centers on stomatal closure [2-6]. This involves loss of potassium from the guard cell by a two-step process. Salt is released across the plasma membrane via anion channels such as SLAC1 [7-9] and depolarization-activated channels such as GORK [10, 11], with the net result that cations and anions exit guard cells. However, this critically depends on K + release from the vacuole; with ∼160 and 100 mM K + in cytoplasm and vacuole of open guard cells [12], vacuolar K + efflux is driven by the negative tonoplast potential, and this expels K + from the vacuole via tonoplast K + channels like TPK1. In all, guard cell salt release leads to a loss of turgor that brings about stomatal closure. First, we show that the TPK1 vacuolar K + channel is important for abscisic acid (ABA)- and CO 2 -mediated stomatal closure. Second, we reveal that, during ABA- and CO 2 -mediated closure, TPK1 is phosphorylated and activated by the KIN7 receptor-like protein kinase (RLK), which co-expresses in the tonoplast and plasma membrane. The net result is K + release from the vacuole. Taken together, our work reveals new components involved in guard cell signaling and describes a new mechanism potentially involved in fine-tuning ABA- and CO 2 -induced stomatal closure., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Fluorescently labeled phosphate-binding proteins can be used as biomolecular tools to measure the release of inorganic phosphate (P i ) from enzymes in real time, enabling the detailed kinetic analysis of dephosphorylating enzymes using rapid-kinetics approaches. Previously reported methods to purify fluorescently labeled phosphate-binding proteins (PhoS) from Escherichia coli are laborious, and a simplified approach is needed. Here, we report the characterization of a cytosol-localized variant (A197C) of PhoS that allows a streamlined purification for subsequent covalent conjugation with a fluorescent dye. We show that export of PhoS into the periplasmic space is not required for the fluorescence-based detection of P i binding. Furthermore, we report the addition of a C-terminal His-tag, simplifying the purification of PhoS from the cytosol via Ni 2+ -affinity chromatography, yielding a fully functional fusion protein (HC PhoS A197C). We demonstrate the utility of fluorescently labeled HC PhoS A197C for rapid-kinetics applications by measuring, using stopped-flow, the P i release kinetics from LepA/EF4 following 70S ribosome-stimulated GTP hydrolysis. Altogether, the approach developed here allows for the high-yield and simplified in-house production of a P i detection system suitable for rapid-kinetics approaches with comparable sensitivity to the commercially available Phosphate Sensor., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The Caulobacter crescentus cell extension known as a stalk represents an unusual bacterial morphology. C. crescentus produces stalks under multiple nutrient conditions, but the length of the stalk is increased in response to phosphate starvation. However, the exact function of the stalk is not known, nor is it known how much stalk biogenesis or function is conserved with other stalked bacteria. Work presented here shows that many organisms in the Caulobacter genus and the next closest genus (Brevundimonas) generally do not synthesize stalks in the relatively-rich PYE growth medium, suggesting that the synthesis of a stalk under nutrient-rich conditions by C. crescentus may be the exception instead of the norm among its phylogenetic group. Brevundimonas subvibrioides can be induced to synthesize stalks by genetically mimicking phosphate starvation conditions, indicating stalk synthesis in this organism may be performed on an as-need basis. This mutation, however, does not appear to increase the incidence of holdfast synthesis. While B. subvibrioides stalks appear to be synthesized with the same polarity with respect to holdfast as C. crescentus stalks, evidence is presented that suggests B. subvibrioides may disassemble stalks when they are no longer needed. Many homologs of C. crescentus genes encoding stalk-associated proteins are absent in the B. subvibrioides genome, and B. subvibrioides PstA-GFP as well as C. crescentus StpX-GFP are able to enter the B. subvibrioides stalk compartment, calling into question the level of compartmentalization of the B. subvibrioides stalk. In summary, this work begins to address how much the C. crescentus model for this unusual morphological adaptation can be extended to related organisms.

One of the most abundant proteins in V. cholerae O1 cells grown under inorganic phosphate (Pi) limitation is PstS, the periplasmic Pi-binding component of the high-affinity Pi transport system Pst2 (PstSCAB), encoded in pst2 operon (pstS-pstC2-pstA2-pstB2). Besides its role in Pi uptake, Pst2 has been also associated with V. cholerae virulence. However, the mechanisms regulating pst2 expression and the non-stoichiometric production of the Pst2 components under Pi-limitation are unknown. A computational-experimental approach was used to elucidate the regulatory mechanisms behind pst2 expression in V. cholerae O1. Bioinformatics analysis of pst2 operon nucleotide sequence revealed start codons for pstS and pstC genes distinct from those originally annotated, a regulatory region upstream pstS containing potential PhoB-binding sites and a pstS-pstC intergenic region longer than predicted. Analysis of nucleotide sequence between pstS-pstC revealed inverted repeats able to form stem-loop structures followed by a potential RNAse E-cleavage site. Another putative RNase E recognition site was identified within the pstA-pstB intergenic sequence. In silico predictions of pst2 operon expression regulation were subsequently tested using cells grown under Pi limitation by promoter-lacZ fusion, gel electrophoresis mobility shift assay and quantitative RT-PCR. The experimental and in silico results matched very well and led us to propose a pst2 promoter sequence upstream of pstS gene distinct from the previously annotated. Furthermore, V. cholerae O1 pst2 operon transcription is PhoB-dependent and generates a polycistronic mRNA molecule that is rapidly processed into minor transcripts of distinct stabilities. The most stable was the pstS-encoding mRNA, which correlates with PstS higher levels relative to other Pst2 components in Pi-starved cells. The relatively higher stability of pstS and pstB transcripts seems to rely on the secondary structures at their 3' untranslated regions that are known to block 3'-5' exonucleolytic attacks., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Phosphate-binding proteins (PBPs) are key proteins that belong to the bacterial ABC-type phosphate transporters. PBPs are periplasmic (or membrane-anchored) proteins that capture phosphate anions from the environment and release them to the transmembrane transporter. Recent work has suggested that PBPs have evolved for high affinity as well as high selectivity. In particular, a short, unique hydrogen bond between the phosphate anion and an aspartate residue has been shown to be critical for selectivity, yet is not strictly conserved in PBPs. Here, the PBP from Polaromonas JS666 is focused on. Interestingly, this PBP is predicted to harbor different phosphate-binding residues to currently known PBPs. Here, it is shown that the PBP from Polaromonas JS666 is capable of binding phosphate, with a maximal binding activity at pH 8. Its structure is expected to reveal its binding-cleft configuration as well as its phosphate-binding mode. Here, the expression, purification, characterization, crystallization and X-ray diffraction data collection to 1.35 Å resolution of the PBP from Polaromonas JS666 are reported.

In Escherichia coli, the ATP-Binding Cassette transporter for phosphate is encoded by the pstSCAB operon. PstS is the periplasmic component responsible for affinity and specificity of the system and has also been related to a regulatory role and chemotaxis during depletion of phosphate. Xanthomonas citri has two phosphate-binding proteins: PstS and PhoX, which are differentially expressed under phosphate limitation. In this work, we focused on PhoX characterization and comparison with PstS. The PhoX three-dimensional structure was solved in a closed conformation with a phosphate engulfed in the binding site pocket between two domains. Comparison between PhoX and PstS revealed that they originated from gene duplication, but despite their similarities they show significant differences in the region that interacts with the permeases.

Escherichia coli YggS is a highly conserved pyridoxal 5'-phosphate (PLP)-binding protein whose biochemical function is currently unknown. A previous study with a yggS-deficient E. coli strain (ΔyggS) demonstrated that YggS controls l-Ile- and l-Val-metabolism by modulating 2-ketobutyrate (2-KB), l-2-aminobutyrate (l-2-AB), and/or coenzyme A (CoA) availability in a PLP-dependent fashion. In this study, we found that ΔyggS accumulates an unknown metabolite as judged by amino acid analyses. LC/MS and MS/MS analyses of the compound with propyl chloroformate derivatization, and co-chromatography analysis identified this compound as γ-l-glutamyl-l-2-aminobutyryl-glycine (ophthalmic acid), a glutathione (GSH) analogue in which the l-Cys moiety is replaced by l-2-AB. We also determine the metabolic consequence of the yggS mutation. Absence of YggS initially increases l-2-AB availability, and then causes ophthalmic acid accumulation and CoA limitation in the cell. The expression of a γ-glutamylcysteine synthetase and a glutathione synthetase in a ΔyggS background causes high-level accumulation of ophthalmic acid in the cells (∼1.2 nmol/mg cells) in a minimal synthetic medium. This opens the possibility of a first fermentative production of ophthalmic acid., (Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

The transcriptomic responses of bacteria to environmental stresses have been studied extensively, yet we know little about how the stressed cells respond to bacteriophage infection. Here, we conducted the first whole transcriptome sequencing (RNA-seq) study of stressed bacteria to phage infection by infecting the marine picocyanobacterium Prochlorococcus NATL2A with cyanomyovirus P-SSM2 under P limitation, a strong selective force in the oceans. Transcripts of the P-acquisition genes in the uninfected cells were enriched after P limitation, including the high-affinity phosphate-binding protein gene pstS. They were still enriched in the infected cells under P-limited conditions. In contrast, transcripts of adenosine triphosphate (ATP) synthase and ribosomal protein genes were depleted in the uninfected cells after P limitation but were enriched during phage infection of P-starved cells. Cyanophage P-SSM2 contains pstS, and pstS and its adjacent gene g247 of unknown function were the only phage genes that were enriched under P-limited conditions. We further found that the host pstS transcript number per cell decreased after infection, however, it was still much higher in the P-limited infected cells than that in the nutrient-replete cells. Moreover, phage pstS transcript number per cell was ∼ 20 times higher than the host copy, which may help maintain the host phosphate uptake rate during infection., (© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.)

A fluorescent reagentless biosensor for inorganic phosphate (Pi), based on the E. coli PstS phosphate binding protein, was redesigned to allow measurements of higher Pi concentrations and at low, substoichiometric concentrations of biosensor. This was achieved by weakening Pi binding of the previous biosensor, and different approaches are described that could enable this change in properties. The readout, providing response to the Pi concentration, is delivered by tetramethylrhodamine fluorescence. In addition to two cysteine mutations for rhodamine labeling at positions 17 and 197, the final variant had an I76G mutation in the hinge region between the two lobes that make up the protein. Upon Pi binding, the lobes rotate on this hinge and the mutation on the hinge lowers affinity ∼200-fold, with a dissociation constant now in the tens to hundreds micromolar range, depending on solution conditions. The signal change on Pi binding was up to 9-fold, depending on pH. The suitability of the biosensor for steady-state ATPase assays was demonstrated with low biosensor usage and its advantage in ability to cope with Pi contamination.

Xanthomonas citri subsp. citri (X. citri) is the causative agent of the citrus canker, a disease that affects several citrus plants in Brazil and across the world. Although many studies have demonstrated the importance of genes for infection and pathogenesis in this bacterium, there are no data related to phosphate uptake and assimilation pathways. To identify the proteins that are involved in the phosphate response, we performed a proteomic analysis of X. citri extracts after growth in three culture media with different phosphate concentrations. Using mass spectrometry and bioinformatics analysis, we showed that X. citri conserved orthologous genes from Pho regulon in Escherichia coli, including the two-component system PhoR/PhoB, ATP binding cassette (ABC transporter) Pst for phosphate uptake, and the alkaline phosphatase PhoA. Analysis performed under phosphate starvation provided evidence of the relevance of the Pst system for phosphate uptake, as well as both periplasmic binding proteins, PhoX and PstS, which were formed in high abundance. The results from this study are the first evidence of the Pho regulon activation in X. citri and bring new insights for studies related to the bacterial metabolism and physiology. Biological significance Using proteomics and bioinformatics analysis we showed for the first time that the phytopathogenic bacterium X. citri conserves a set of proteins that belong to the Pho regulon, which are induced during phosphate starvation. The most relevant in terms of conservation and up-regulation were the periplasmic-binding proteins PstS and PhoX from the ABC transporter PstSBAC for phosphate, the two-component system composed by PhoR/PhoB and the alkaline phosphatase PhoA., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Pseudomonas aeruginosa (PA) infections pose a serious threat to human health. PA is a leading cause of fatal lung infections in cystic fibrosis and immune-suppressed patients, of sepsis in burn victims and of nosocomial infections. An important element in PA virulence is its ability to establish biofilms that evade suppression by the host's immune system and antibiotics. PstS, a periplasmic subunit of the Pst phosphate-transport system of PA, plays a critical role in the establishment of biofilms. In some drug-resistant PA strains, PstS is secreted in large quantities from the bacteria, where it participates in the assembly of adhesion fibres that enhance bacterial virulence. In order to understand the dual function of PstS in biofilm formation and phosphate transport, the crystal structure of PA PstS was determined. Here, the overexpression in Escherichia coli and purification of PA PstS in the presence of phosphate are described. Two crystal forms were obtained using the vapour-diffusion method at 20°C and X-ray diffraction data were collected. The first crystal form belonged to the centred orthorhombic space group C222₁, with unit-cell parameters a=67.5, b=151.3, c=108.9 Å. Assuming the presence of a dimer in the asymmetric unit gives a crystal volume per protein weight (VM) of 2.09 Å3 Da(-1) and a solvent content of 41%. The second crystal form belonged to the primitive orthorhombic space group P2₁2₁2₁, with unit-cell parameters a=35.4, b=148.3, c=216.7 Å. Assuming the presence of a tetramer in the asymmetric unit gives a crystal volume per protein weight (VM) of 2.14 Å3 Da(-1) and a solvent content of 42.65%. A pseudo-translational symmetry is present in the P212121 crystal form which is consistent with a filamentous arrangement of PstS in the crystal lattice.

During wastewater treatment, phosphate removal is an important and challenging process; thus, diverse technologies, including those derived from biological means, have been devised for efficient phosphate removal. Although conventional biological methods are effective in decreasing wastewater phosphate levels to ~1 mg/L, long periods of microbial adaptation are required for effective phosphate removal, and the removal efficiency of these methods is relatively poor at lower phosphate concentrations. In the present work, we constructed a recombinant Escherichia coli with periplasmic-expressed phosphate-binding protein (PBP) and investigated its biological removal ability for low phosphate levels. We found that the PBP-expressing recombinant E. coli cells showed efficient (> 94 %) removal of phosphate at low concentrations (0.2-1.0 mg/L) in a treated cell mass-dependent manner. Collectively, we propose that our PBP-expressing recombinant whole-cell system could be successfully used during wastewater treatment for the biological removal of low concentrations of phosphate.

phoU mutants of bacteria are potentially useful for the removal of inorganic phosphate (Pi) from sewage because they can accumulate a large amounts of polyphosphate (polyP). However, the growth of phoU mutants is severely defective and is easily outgrown by revertant(s) that have lost the ability to accumulate polyP during growth in a nutrient-rich medium. We found that a pseudo-revertant, designated LAP[+], that appeared in a culture of an Escherichia coli phoU mutant that could accumulate polyP even after ten serial passages. Reduction in the expression of the Pi-specific transporter Pst in LAP[+] may contribute to relieving stresses such as excess Pi incorporation that could stimulate reversions. The discovery of a LAP[+] provides a clue to generate phoU mutants that accumulate polyP in a stable manner.

Arsenate and phosphate are abundant on Earth and have striking similarities: nearly identical pK(a) values, similarly charged oxygen atoms, and thermochemical radii that differ by only 4% (ref. 3). Phosphate is indispensable and arsenate is toxic, but this extensive similarity raises the question whether arsenate may substitute for phosphate in certain niches. However, whether it is used or excluded, discriminating phosphate from arsenate is a paramount challenge. Enzymes that utilize phosphate, for example, have the same binding mode and kinetic parameters as arsenate, and the latter's presence therefore decouples metabolism. Can proteins discriminate between these two anions, and how would they do so? In particular, cellular phosphate uptake systems face a challenge in arsenate-rich environments. Here we describe a molecular mechanism for this process. We examined the periplasmic phosphate-binding proteins (PBPs) of the ABC-type transport system that mediates phosphate uptake into bacterial cells, including two PBPs from the arsenate-rich Mono Lake Halomonas strain GFAJ-1. All PBPs tested are capable of discriminating phosphate over arsenate at least 500-fold. The exception is one of the PBPs of GFAJ-1 that shows roughly 4,500-fold discrimination and its gene is highly expressed under phosphate-limiting conditions. Sub-ångström-resolution structures of Pseudomonas fluorescens PBP with both arsenate and phosphate show a unique mode of binding that mediates discrimination. An extensive network of dipole-anion interactions, and of repulsive interactions, results in the 4% larger arsenate distorting a unique low-barrier hydrogen bond. These features enable the phosphate transport system to bind phosphate selectively over arsenate (at least 10(3) excess) even in highly arsenate-rich environments.

Cyanobacteria blooms caused by species such as Microcystis have become commonplace in many freshwater ecosystems. Although phosphorus (P) typically limits the growth of freshwater phytoplankton populations, little is known regarding the molecular response of Microcystis to variation in P concentrations and sources. For this study, we examined genes involved in P acquisition in Microcystis including two high-affinity phosphate-binding proteins (pstS and sphX) and a putative alkaline phosphatase (phoX). Sequence analyses among ten clones of Microcystis aeruginosa and one clone of Microcystis wesenbergii indicates that these genes are present and conserved within the species, but perhaps not the genus, as phoX was not identified in M. wesenbergii. Experiments with clones of M. aeruginosa indicated that expression of these three genes was strongly upregulated (50- to 400-fold) under low inorganic P conditions and that the expression of phoX was correlated with alkaline phosphatase activity (p < 0.005). In contrast, cultures grown exclusively on high levels of organic phosphorus sources (adenosine 5'-monophosphate, β-glycerol phosphate, and D: -glucose-6-phosphate) or under nitrogen-limited conditions displayed neither high levels of gene expression nor alkaline phosphatase activity. Since Microcystis dominates phytoplankton assemblages in summer when levels of inorganic P (P(i)) are often low and/or dominate lakes with low P(i) and high organic P, our findings suggest this cyanobacterium may rely on pstS, sphX, and phoX to efficiently transport P(i) and exploit organic sources of P to form blooms.

Protein-ligand interactions are essential for nearly all biological processes, and yet the biophysical mechanism that enables potential binding partners to associate before specific binding occurs remains poorly understood. Fundamental questions include which factors influence the formation of protein-ligand encounter complexes, and whether designated association pathways exist. To address these questions, we developed a computational approach to systematically analyze the complete ensemble of association pathways. Here, we use this approach to study the binding of a phosphate ion to the Escherichia coli phosphate-binding protein. Various mutants of the protein are considered, and their effects on binding free-energy profiles, association rates, and association pathway distributions are quantified. The results reveal the existence of two anion attractors, i.e., regions that initially attract negatively charged particles and allow them to be efficiently screened for phosphate, which is subsequently specifically bound. Point mutations that affect the charge on these attractors modulate their attraction strength and speed up association to a factor of 10 of the diffusion limit, and thus change the association pathways of the phosphate ligand. It is demonstrated that a phosphate that prebinds to such an attractor neutralizes its attraction effect to the environment, making the simultaneous association of a second phosphate ion unlikely. This study suggests ways in which structural properties can be used to tune molecular association kinetics so as to optimize the efficiency of binding, and highlights the importance of kinetic properties., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

DAHP synthase (EC 4.1.2.15) is one of the key enzymes involved in aromatic amino acid biosynthesis in Escherichia coli. An approximately twofold decrease in DAHP synthase activity level was detected in the late growth phase of the L-phenylalanine (Phe)-producing E. coli strain, in which this enzyme encoded by aroG4 is resistant to feedback inhibition. An additional copy of aroG4 that is controlled by promoters of E. coli phoA or pstS genes was integrated into the chromosome of the Phe producer. The choice of promoter was based on the detected activation of the Pho regulon that occurs in response to the depletion of soluble inorganic orthophosphate (P(i)) in the medium, provided that the optical density of the Phe-producing culture did not exceed 70% of its maximum value. Pho-mediated aroG4 transcription increased both the accumulation of Phe and the level of DAHP synthase activity in the late stage of batch cultivation on glucose in P(i)-limited conditions. Disruption of rpoS led to the improved performance of a P(phoA)-aroG4 strain. The pstS promoter that is recognized by the σ(70)/σ(S)-associated core RNA polymerase resulted in the stable maintenance of DAHP synthase activity during long-drawn fed-batch cultivation of the RpoS(+) strain carrying the P(pstS)-aroG4.

The conventional enhanced biological phosphorus removal (EBPR) system often deteriorates at low chemical oxygen demand (COD) or under aeration conditions. A new approach that incorporates phosphate-eutrophic wastewater remediation was introduced through immobilization of an intracellular phosphate-binding protein (PBP) onto the surface of Pseudomonas putida or Escherichia coli, using the N-terminal anchor (InaQ-N) of a newly identified ice nucleation protein from Pseudomonas syringae. A green fluorescent protein-fusion protein was expressed and used to confirm surface localization. The PBP was then targeted to the surface of E. coli JM109 and P. putida AB92019. The engineered P. putida and E. coli microorganisms were capable of absolute biosorption of total phosphates at rates of 60 and 80 mg L(-1), respectively, over 5 h. In the recombinant P. putida cells, a surface-immobilized PBP fusion that had three tandemly repeated InaQ-Ns exhibited the maximum increment in phosphate biosorption, at sixfold compared with the control strain. Even heat-killed recombinant cells of either P. putida or E. coli retained substantial biosorptive activities. The current study demonstrates that the bacterial surface display of PBP should be considered as a strong contender for generating organisms capable of functioning in EBPR systems under low COD, resulting in improved removal of eutrophic phosphorus from wastewaters., (© 2009 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

PstS and DING proteins are members of a superfamily of secreted, high-affinity phosphate-binding proteins. Whereas microbial PstS have a well-defined role in phosphate ABC transporters, the physiological function of DING proteins, named after their DINGGG N termini, still needs to be determined. PstS and DING proteins co-exist in some Pseudomonas strains, to which they confer a highly adhesive and virulent phenotype. More than 30 DING proteins have now been purified, mostly from eukaryotes. They are often associated with infections or with dysregulation of cell proliferation. Consequently, eukaryotic DING proteins could also be involved in cell-cell communication or adherence. The ubiquitous presence in eukaryotes of proteins structurally and functionally related to bacterial virulence factors is intriguing, as is the absence of eukaryotic genes encoding DING proteins in databases. DING proteins in eukaryotes could originate from unidentified commensal or symbiotic bacteria and could contribute to essential functions. Alternatively, DING proteins could be encoded by eukaryotic genes sharing special features that prevent their cloning. Both hypotheses are discussed.