Negative Regulatory Mechanism Research Articles

Abstract PO-046: Combining PARP inhibition with radiation to sensitize homologous recombination proficient pancreatic cancer to immunotherapy

Abstract Pancreatic cancer is notoriously resistant to most cancer therapeutics including immunotherapy. We have previously shown that inhibition of the DNA damage response (DDR) enhances radiation-induced Type I interferon (T1IFN) and sensitizes pancreatic cancers to immunotherapy. In this study, we investigated the efficacy of radiation in combination with the poly (ADP-ribose) polymerase (PARP)inhibitor olaparib and programmed death ligand 1 (PD-L1) immune checkpoint blockade in otherwise resistant homologous recombination (HR) proficient pancreatic cancer. Initial studies demonstrated, in contrast to olaparib alone, that the combination of olaparib and radiation induced innate immune signaling in HR proficient pancreatic cancer cells marked by increases in pTBK1, T1IFN and pSTAT1. In animal tumor models, we found that the therapeutic efficacy of olaparib and radiation was greater in immune competent versus immune deficient hosts. In addition to the positive immune effects of olaparib and radiation, we also observed increased expression of PD-L1, a negative immune regulatory mechanism that likely restrains T1IFN-mediated innate immunity. We therefore went on to test the combined efficacy of PD-L1 blocking antibody with olaparib and radiation in syngeneic pancreatic tumor models. We found that monotherapy was ineffective in controlling tumor growth while doublet therapy with olaparib and radiation produced an intermediate tumor growth response. Importantly, maximal tumor growth inhibition was achieved following combined treatment with olaparib, radiation, and anti-PD-L1 which was significant relative to doublet therapy with olaparib-radiation or anti-PD-L1-radiation, and associated with a 20% complete response rate. Given that advances in therapy of pancreatic cancer require improvements in both local and systemic disease control, we examined the efficacy of combined therapy against tumors outside of the radiation field. In addition to the effects on tumors in the radiation field, we found that combined therapy with olaparib, radiation and anti-PD-L1 significantly delayed the growth of contralateral tumors outside of the radiation field, suggesting an adaptive immune response. Taken together, these studies illustrate the potential efficacy of combined therapy with olaparib, radiation, and anti-PD-L1 on both local and systemic pancreatic cancer and support our proposed clinical trial in patients with pancreatic cancer. Citation Format: Qiang Zhang, Long Jiang, Yawen Zheng, Theodore Lawrence, Vaibhav Sahai, Michael D. Green, Meredith Morgan. Combining PARP inhibition with radiation to sensitize homologous recombination proficient pancreatic cancer to immunotherapy [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-046.

Immune suppressive activity of myeloid-derived suppressor cells in cancer requires inactivation of the type I interferon pathway

Myeloid-derived suppressor cells (MDSC) are pathologically activated neutrophils and monocytes with potent immune suppressive activity. These cells play an important role in accelerating tumor progression and undermining the efficacy of anti-cancer therapies. The natural mechanisms limiting MDSC activity are not well understood. Here, we present evidence that type I interferons (IFN1) receptor signaling serves as a universal mechanism that restricts acquisition of suppressive activity by these cells. Downregulation of the IFNAR1 chain of this receptor is found in MDSC from cancer patients and mouse tumor models. The decrease in IFNAR1 depends on the activation of the p38 protein kinase and is required for activation of the immune suppressive phenotype. Whereas deletion of IFNAR1 is not sufficient to convert neutrophils and monocytes to MDSC, genetic stabilization of IFNAR1 in tumor bearing mice undermines suppressive activity of MDSC and has potent antitumor effect. Stabilizing IFNAR1 using inhibitor of p38 combined with the interferon induction therapy elicits a robust anti-tumor effect. Thus, negative regulatory mechanisms of MDSC function can be exploited therapeutically.

MicroRNA-induced translational control of antiviral immunity by the cap-binding protein 4EHP

Type I interferons (IFNs) are critical cytokines in the host defense against invading pathogens. Sustained production of IFNs, however, is detrimental to the host, as it provokes autoimmune diseases. Thus, the expression of IFNs is tightly controlled. We report that the mRNA 5' cap-binding protein 4EHP plays a key role in regulating type I IFN concomitant with controlling virus replication, both invitro and invivo. Mechanistically, 4EHP suppresses IFN-β production by effecting the miR-34a-induced translational silencing of Ifnb1 mRNA. miR-34a is upregulated by both RNA virus infection and IFN-β induction, prompting a negative feedback regulatory mechanism that represses IFN-β expression via 4EHP. These findings demonstrate the direct involvement of 4EHP in virus-induced host response, underscoring a critical translational silencing mechanism mediated by 4EHP and miR-34a to impede sustained IFN production. This study highlights an intrinsic regulatory function for miRNA and the translation machinery in maintaining host homeostasis.

The PB1 protein of influenza A virus inhibits the innate immune response by targeting MAVS for NBR1-mediated selective autophagic degradation.

Influenza A virus (IAV) has evolved various strategies to counteract the innate immune response using different viral proteins. However, the mechanism is not fully elucidated. In this study, we identified the PB1 protein of H7N9 virus as a new negative regulator of virus- or poly(I:C)-stimulated IFN induction and specifically interacted with and destabilized MAVS. A subsequent study revealed that PB1 promoted E3 ligase RNF5 to catalyze K27-linked polyubiquitination of MAVS at Lys362 and Lys461. Moreover, we found that PB1 preferentially associated with a selective autophagic receptor neighbor of BRCA1 (NBR1) that recognizes ubiquitinated MAVS and delivers it to autophagosomes for degradation. The degradation cascade mediated by PB1 facilitates H7N9 virus infection by blocking the RIG-I-MAVS-mediated innate signaling pathway. Taken together, these data uncover a negative regulatory mechanism involving the PB1-RNF5-MAVS-NBR1 axis and provide insights into an evasion strategy employed by influenza virus that involves selective autophagy and innate signaling pathways.

In vivo effects of olive oil and trans-fatty acids on miR-134, miR-132, miR-124-1, miR-9-3 and mTORC1 gene expression in a DMBA-treated mouse model.

Both the intake of beneficial olive oil and of harmful trans-fatty acids (TFAs) in consumed foods are of great significance in tumor biology. In our present study we examined the effects they exert on the expression patterns of miR-134, miR-132, miR-124-1, miR-9-3 and mTOR in the liver, spleen and kidney of mice treated with 7,12-dimethylbenz [a] anthracene (DMBA). Feeding of TFA-containing diet significantly increased the expression of all studied miRs and mTORC1 in all organs examined, except the expression of mTORC1 in the spleen and kidney. Diet containing olive oil significantly reduced the expression of miR-124-1, miR-9-3 and mTORC1 in the liver and spleen. In the kidney, apart from the mTORC1 gene, the expression of all miRs examined significantly decreased compared to the DMBA control. According to our results, the cell membrane protective, antioxidant, and anti-inflammatory effects of olive oil and the cell membrane damaging, inflammatory, and carcinogenic properties of TFA suggest negative feedback regulatory mechanisms. In contrast to our expectations, mTORC1 gene expression in the kidney has not been shown to be an appropriate biomarker–presumably, because the many complex effects that regulate mTOR expression may quench each other.

Cleavage of PrePL by Lon promotes growth and pathogenesis in Magnaporthe oryzae.

ATP-dependent Lon proteases function in bacterial pathogenesis by regulating the expression of the Type III secretion system; however, little is known about how Lon proteases regulate fungal pathogenesis. We previously investigated Lon-binding proteins involved in fungal pathogenesis that interact with PrePL, the smallest Magnaporthe oryzae Lon-binding protein. Here, we show that Lon cleaves PrePL and produces Pc, an extracellular 11-kDa isoform with catalase and peroxidase activity. The ΔPrePL loss-of-function strain showed stronger sporulation and accelerated disease development, suggesting a temporally specific negative regulatory mechanism controlled by PrePL in disease progression. Neither the truncated Pc, nor the full-length PrePL missing the Lon cleavage site complemented the ΔPrePL phenotype, suggesting that full-length PrePL and Pc both function in fungal development. PrePL targeted to the mitochondria undergoes hydrolysis by Lon to produce Pc, which accumulates in the fungal apoplast. Importantly, recombinant Pc induced plant defence responses and cell death after being infiltrated into selected plant leaves, indicating that it functions as an avirulence factor. This work thus reveals a novel pathogenic factor in the fungal Lon-mediated pathway. Additionally, our results provide new insight into the functions of a full-length protein and its cleaved isoform in fungal pathogenesis.

The influence of enzyme EII of the cellobiose-phosphotransferase system on the virulence of Streptococcus agalactiae in Nile tilapia (Oreochromis niloticus)

Streptococcus agalactiae is the main pathogen of tilapia, causing huge economic losses to the tilapia aquaculture industry in China. To elucidate the role of the EII complexes of the cellobiose-phosphotransferase system (cel-PTS) in the regulation of S. agalactiae virulence, the △cel-EII mutants of a high virulence strain (THN0901) and a low virulence strain (TFJ0901) were constructed using homologous recombination. The results showed that after the deletion of cel-EII complexes, the biological characteristics of THN0901 and TFJ0901 were consistent, including their cellobiose metabolism. Their biofilm formation abilities were significantly reduced (P < 0.01), and their ability to adhere to tilapia brain cells and compete when mixed in vitro and in vivo were enhanced. The virulence and histopathological lesions caused by △cel-EII-TFJ0901 were significantly enhanced relative to those of TFJ0901, while △cel-EII-THN0901 showed no significant differences relative to THN0901. The histopathological lesions caused by △cel-EII-TFJ0901 tend to be serious, especially in the liver, spleen, and heart. The expression levels of manA and ascA/C of PTS, ciaR and dltR of two-component signal transduction systems (TCS), and pavA, bca and spb1 of virulence genes of △cel-EII-TFJ0901 were significantly upregulated compared with TFJ0901 (P < 0.01), whereas those of △cel-EII-THN0901 were significantly downregulated (P < 0.01), or somewhat downregulated, compared with THN0901. Taken together, cel-EII complexes may have a negative effect on the virulence of TFJ0901 by regulating the expression of virulence-related genes, but the negative regulation mechanism of the cel-EII complex has no obvious effect on the virulence of THN0901.

The PalWRKY77 transcription factor negatively regulates salt tolerance and abscisic acid signaling in Populus.

High salinity, one of the most widespread abiotic stresses, inhibits photosynthesis, reduces vegetation growth, blocks respiration and disrupts metabolism in plants. In order to survive their long-term lifecycle, trees, such as Populus species, recruit the abscisic acid (ABA) signaling pathway to adapt to a saline environment. However, the molecular mechanism behind the ABA-mediated salt stress response in woody plants remains elusive. We have isolated a WRKY transcription factor gene, PalWRKY77, from Populus alba var. pyramidalis (poplar), the expression of which is repressed by salt stress. PalWRKY77 decreases salt tolerance in poplar. Furthermore, PalWRKY77 negatively regulated ABA-responsive genes and relieved ABA-mediated growth inhibition, indicating that PalWRKY77 is a repressor of the ABA response. In vivo and in vitro assays revealed that PalWRKY77 targets the ABA- and salt-induced PalNAC002 and PalRD26 genes by binding to the W-boxes in their promoters. In addition, overexpression of both PalNAC002 and PalRD26 could elevate salt tolerance in transgenic poplars. These findings reveal a novel negative regulation mechanism for the ABA signaling pathway mediated by PalWRKY77 that results in more sensitivity to salt stress in poplar. This deepens our understanding of the complex responses of woody species to salt stress.

Canonical Wnt signaling regulates branching morphogenesis of submandibular gland by modulating levels of lama5.

Branching morphogenesis is a crucial developmental mechanism for the formation of the typical bush-like structure of the submandibular gland (SMG). However, the detailed mechanism underlying this process remains to be fully understood. Here, we have investigated whether cross-talk may exist between the Wnt/beta-catenin signaling pathway and lama5 during the branching process in SMG development. An embryonic mouse SMG organ culture model was established, and the validity of this model was confirmed. The roles and possible interactions of the Wnt/beta-catenin signaling pathway, FGF signaling, and lama5 in the branching process were investigated by morphogenesis assays and gene expression patterns. Here, we show that the E12 or E13 SMG organ culture model can be used as an ideal approach to study the process of branching morphogenesis. Our branching morphogenesis assay revealed that the epithelial branching process can be promoted when the canonical Wnt pathway is inhibited and significantly suppressed when the wnt pathway is over activated. Further experiments indicated that FGF signaling most likely acts upstream as a negative regulator of the canonical Wnt pathway during the branching process, whose effect could be partially reversed by Wnt3a. Finally, we show that Wnt/beta-catenin signaling regulates branching morphogenesis through Lama5. We conclude that the Wnt/beta-catenin signaling pathway acting downstream of FGF signaling can serve as a negative regulatory mechanism in the process of SMG branching morphogenesis through Lama5.

Biphasic spatiotemporal regulation of GRB2 dynamics by p52SHC for transient RAS activation.

RTK-RAS-MAPK systems are major signaling pathways for cell fate decisions. Among the several RTK species, it is known that the transient activation of ERK (MAPK) stimulates cell proliferation, whereas its sustained activation induces cell differentiation. In both instances however, RAS activation is transient, suggesting that the strict temporal regulation of its activity is critical in normal cells. RAS on the cytoplasmic side of the plasma membrane is activated by SOS through the recruitment of GRB2/SOS complex to the RTKs that are phosphorylated after stimulation with growth factors. The adaptor protein GRB2 recognizes phospho-RTKs both directly and indirectly via another adaptor protein, SHC. We here studied the regulation of GRB2 recruitment under the SHC pathway using single-molecule imaging and fluorescence correlation spectroscopy in living cells. We stimulated MCF7 cells with a differentiation factor, heregulin, and observed the translocation, complex formation, and phosphorylation of cell signaling molecules including GRB2 and SHC. Our results suggest a biphasic regulation of the GRB2/SOS-RAS pathway by SHC: At the early stage (<10 min) of stimulation, SHC increased the amplitude of RAS activity by increasing the association sites for the GRB2/SOS complex on the plasma membrane. At the later stage however, SHC suppressed RAS activation and sequestered GRB2 molecules from the membrane through the complex formation in the cytoplasm. The latter mechanism functions additively to other mechanisms of negative feedback regulation of RAS from MEK and/or ERK to complete the transient activation dynamics of RAS.

Binding of non-canonical peptidoglycan controls Vibrio cholerae broad spectrum racemase activity

Broad-spectrum amino acid racemases (Bsrs) enable bacteria to generate non-canonical D-amino acids (NCDAAs), whose roles and impact on microbial physiology, including modulation of cell wall structure and dissolution of biofilms, are just beginning to be appreciated. Here we used a diverse array of structural, biochemical and molecular simulation studies to define and characterize how BsrV is post-translationally regulated. We discovered that contrary to Vibrio cholerae alanine racemase AlrV highly compacted active site, BsrV’s is broader and can be occupied by cell wall stem peptides. We found that peptidoglycan peptides modified with NCDAAs are better stabilized by BsrV’s catalytic cavity and show better inhibitory capacity than canonical muropeptides. Notably, BsrV binding and inhibition can be recapitulated by undigested peptidoglycan sacculi as it exists in the cell. Docking simulations of BsrV binding the peptidoglycan polymer generate a model where the peptide stems are perfectly accommodated and stabilized within each of the dimeŕs active sites. Taking these biochemical and structural data together, we propose that inhibition of BsrV by peptidoglycan peptides underlies a negative regulatory mechanism to avoid excessive NCDAA production. Our results collectively open the door to use “à la carte” synthetic peptides as a tool to modulate DAAs production of Bsr enzymes.

Dimeric Structure of the Pseudokinase IRAK3 Suggests an Allosteric Mechanism for Negative Regulation

SummaryInterleukin-1 receptor associated kinases (IRAKs) are key players in innate immune signaling that mediate the host response to pathogens. In contrast to the active kinases IRAK1 and IRAK4, IRAK2 and IRAK3 are pseudokinases lacking catalytic activity and their functions are poorly understood. IRAK3 is thought to be a negative regulator of innate immune signaling and mutations in IRAK3 are associated with asthma and cancer. Here, we report the crystal structure of the human IRAK3 pseudokinase domain in a closed, pseudoactive conformation. IRAK3 dimerizes in a unique way through a head-to-head arrangement not observed in any other kinases. Multiple conserved cysteine residues imply a potential redox control of IRAK3 conformation and dimerization. By analyzing asthma-associated mutations, we identify an evolutionarily conserved surface on IRAK3 that could form an interaction interface with IRAK4, suggesting a model for the negative regulation of IRAK4 by IRAK3.

A novel negative regulatory mechanism of Smurf2 in BMP/Smad signaling in bone

Transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) play important roles in bone metabolism. Smad ubiquitination regulatory factors (Smurfs) regulate TGF-β/BMP signaling via ubiquitination, resulting in degradation of signaling molecules to prevent excessive activation of TGF-β/BMP signaling. Though Smurf2 has been shown to negatively regulate TGF-β/Smad signaling, its involvement in BMP/Smad signaling in bone metabolism has not been thoroughly investigated. In the present study, we sought to evaluate the role of Smurf2 in BMP/Smad signaling in bone metabolism. Absorbable collagen sponges containing 3 μg of recombinant human BMP2 (rhBMP2) were implanted in the dorsal muscle pouches of wild type (WT) and Smurf2−/− mice. The rhBMP2-induced ectopic bone in Smurf2−/− mice showed greater bone mass, higher mineral apposition and bone formation rates, and greater osteoblast numbers than the ectopic bone in WT mice. In WT mice, the ectopic bone consisted of a thin discontinuous outer cortical shell and scant inner trabecular bone. In contrast, in Smurf2−/− mice, the induced bone consisted of a thick, continuous outer cortical shell and abundant inner trabecular bone. Additionally, rhBMP2-stimulated bone marrow stromal cells (BMSCs) from Smurf2−/− mice showed increased osteogenic differentiation. Smurf2 induced the ubiquitination of Smad1/5. BMP/Smad signaling was enhanced in Smurf2−/− BMSCs stimulated with rhBMP2, and the inhibition of BMP/Smad signaling suppressed osteogenic differentiation of these BMSCs. These findings demonstrate that Smurf2 negatively regulates BMP/Smad signaling, thereby identifying a new regulatory mechanism in bone metabolism.

A Screen for PKN3 Substrates Reveals an Activating Phosphorylation of ARHGAP18.

Protein kinase N3 (PKN3) is a serine/threonine kinase implicated in tumor progression of multiple cancer types, however, its substrates and effector proteins still remain largely understudied. In the present work we aimed to identify novel PKN3 substrates in a phosphoproteomic screen using analog sensitive PKN3. Among the identified putative substrates we selected ARHGAP18, a protein from RhoGAP family, for validation of the screen and further study. We confirmed that PKN3 can phosphorylate ARHGAP18 in vitro and we also characterized the interaction of the two proteins, which is mediated via the N-terminal part of ARHGAP18. We present strong evidence that PKN3-ARHGAP18 interaction is increased upon ARHGAP18 phosphorylation and that the phosphorylation of ARHGAP18 by PKN3 enhances its GAP domain activity and contributes to negative regulation of active RhoA. Taken together, we identified new set of potential PKN3 substrates and revealed a new negative feedback regulatory mechanism of Rho signaling mediated by PKN3-induced ARHGAP18 activation.

A Novel Transcript Isoform of TBK1 Negatively Regulates Type I IFN Production by Promoting Proteasomal Degradation of TBK1 and Lysosomal Degradation of IRF3.

TANK-binding kinase 1 (TBK1), an IKK-related serine/threonine kinase, is pivotal for the induction of antiviral type I interferon (IFN) by TLR and RLR signaling pathways. In a previous study, we demonstrated that TBK1 spliced isoforms (TBK1_tv1 and TBK1_tv2) from zebrafish were dominant negative regulators in the RLR antiviral pathway by targeting the functional TBK1–IRF3 complex formation. In this study, we show that the third TBK1 isoform (namely TBK1_tv3) inhibits zebrafish type I IFN production by promoting TBK1 and IRF3 degradation. First, ectopic expression of TBK1_tv3 suppresses poly(I:C)- and Spring viremia of carp virus-induced type I IFN response, and also inhibits the up-regulation of IFN promoter activities stimulated by RIG-I, MDA5, MAVS, TBK1, and IRF3. Second, TBK1_tv3 targets TBK1 and IRF3 to impair the formation of TBK1 dimer, TBK1–IRF3 complex, and IRF3 dimer. Notably, TBK1_tv3 promotes the degradation of TBK1 through the ubiquitin–proteasome pathway and the degradation of IRF3 through the lysosomal pathway. Further analysis demonstrates that TBK1_tv3 promotes the degradation of TBK1 for K48-linked ubiquitination by targeting the K251, K256, and K271 sites of TBK1. Collectively, our results suggest a novel TBK1 isoform-mediated negative regulation mechanism, which serves to balance the production of type I IFN and ISGs.

TBK1/IKKε Negatively Regulate LPS-Induced Neutrophil Necroptosis and Lung Inflammation.

Cell necroptosis, a form of regulated inflammatory cell death, is one of the mechanisms that controls cell release of inflammatory mediators from innate immune cells, such as polymorphonuclear neutrophils (PMNs), and critically regulates the progress of inflammation. Cell necroptosis features receptor-interacting protein (RIPK) 1 activation and necroptosome formation. This leads to loss of plasma membrane integrity, the release of cell contents into the extracellular space, and subsequent increased inflammation. Here, we report an intra-PMN mechanism of negative regulation of necroptosis mediated through TBK1/IKKε. Using an in vivo mouse model of intratracheal injection (i.t.) of LPS and in vitro LPS stimulation of mouse PMN, we found that LPS-TLR4 signaling in PMNs activates and phosphorylates TBK1 and IKKε, which in turn suppress LPS-induced formation of the RIPK1-RIPK3-MLKL (necrosome) complex. TBK1 dysfunction by knockdown or inhibitor significantly increases the phosphorylation of RIPK1 (∼67%), RIPK3 (∼68%), and MLKL (∼50%) and promotes RIPK1-RIPK3 and RIPK3-MLKL interactions and increases PMN necroptosis (∼83%) in response to LPS, with subsequent augmented lung inflammation. These findings suggest that the LPS-TLR4-TBK1 axis serves as a negative regulator for PMN necroptosis and might be a therapeutic target for modulating PMN death and inflammation.

Polysialic acid and Siglec-E orchestrate negative feedback regulation of microglia activation

Polysialic acid (polySia) emerges as a novel regulator of microglia activity. We recently identified polysialylated proteins in the Golgi compartment of murine microglia that are released in response to inflammatory stimulation. Since exogenously added polySia is able to attenuate the inflammatory response, we proposed that the release of polysialylated proteins constitutes a mechanism for negative feedback regulation of microglia activation. Here, we demonstrate that translocation of polySia from the Golgi to the cell surface can be induced by calcium depletion of the Golgi compartment and that polysialylated proteins are continuously released for at least 24 h after the onset of inflammatory stimulation. The latter was unexpected, because polySia signals detected by immunocytochemistry are rapidly depleted. However, it indicates that the amount of released polySia is much higher than anticipated based on immunostaining. This may be crucial for microglial responses during traumatic brain injury (TBI), as we detected polySia signals in activated microglia around a stab wound in the adult mouse brain. In BV2 microglia, the putative polySia receptor Siglec-E is internalized during lipopolysaccharide (LPS)-induced activation and in response to polySia exposure, indicating interaction. Correspondingly, CRISPR/Cas9-mediated Siglec-E knockout prevents inhibition of pro inflammatory activation by exogenously added polySia and leads to a strong increase of the LPS response. A comparable increase of LPS-induced activation has been observed in microglia with abolished polySia synthesis. Together, these results indicate that the release of the microglia-intrinsic polySia pool, as implicated in TBI, inhibits the inflammatory response by acting as a trans-activating ligand of Siglec-E.

The pyrin inflammasome and the Yersinia effector interaction.

Pyrin is a cytosolic pattern-recognition receptor that normally functions as a guard to trigger capase-1 inflammasome assembly in response to bacterial toxins and effectors that inactivate RhoA. The MEFV gene encoding human pyrin is preferentially expressed in phagocytes. Key domains in pyrin include a pyrin domain (PYD), a linker region, and a B30.2 domain. Binding of ASC to pyrin by a PYD-PYD interaction triggers inflammasome assembly. Pyrin is held in an inactive conformation by negative regulation mechanisms to avoid premature inflammasome assembly. One mechanism of negative regulation involves phosphorylation of the linker by PRK kinase which in turn is positively regulated by active RhoA. The B30.2 domain also negatively regulates pyrin. Gain of function mutations in MEFV responsible for the autoinflammatory disease Familial Mediterranean Fever (FMF) map to exon 10 encoding the B30.2 domain. Insights into pyrin regulation have come from studies of several Yersinia effectors, which are injected into phagocytes and interact with the RhoA-PRK-pyrin axis during infection. Two effectors, YopE and YopT, inactivate RhoA to disrupt phagocytic signaling. To counteract an effector-triggered immune response, a third effector, YopM, binds to and inhibits pyrin by hijacking PRK and RSK and directing linker phosphorylation. Inhibition of pyrin by YopM is required for virulence of Yersinia pestis, the agent of plague. Recent results from infection studies with human phagocytes and mice producing pyrin B30.2 FMF variants show that gain of function MEFV mutations bypass inhibition by YopM. Population genetic data suggest that MEFV mutations were selected for in individuals of Mediterranean decent during historic plague pandemics. This review discusses current concepts of pyrin regulation and its interaction with Yersinia effectors.

Repeated unpredictable stress and social isolation induce chronic HPA axis dysfunction and persistent abnormal fear memory

The lack of progress in the psychopharmacological treatment of stress-related disorders such as PTSD is an ongoing crisis due to its negative socioeconomic implications. Current PTSD pharmacotherapy relies on a few FDA approved medications used primarily for depression which offer only symptomatic relief and show limited efficacy. As the population of PTSD patients is growing, the identification of effective etiology-based treatments for the condition is a high priority. This requires an in-depth understanding of the neurobiological and behavioral outcomes of stress in translationally relevant animal models.In this study, we use neuroendocrine, biochemical and behavioral measures to assess the HPA axis function and fear-memory deficits in a mouse model of chronic stress. The chronic stress procedures involved exposure to 21 days of repeated unpredictable stress (RUS), including predator stress, restraint and foot shock, followed by chronic social isolation. We show that mice exposed to our stress paradigm demonstrate exaggerated fear memory recall and blunted HPA axis functionality at one month after RUS. Our neuroendocrinal testing suggests that the attenuated stress response in our model may be related to an alteration in the adrenal MC2 receptor reactivity. While there was no noticeable change in pituitary negative feedback regulation mechanisms, CRH and phosphorylated Glucocorticoid receptors levels were altered in the hypothalamus. We also show that chronic supplementation with a peripheral glucocorticoid receptor agonist (low-dose dexamethasone) after RUS partially restores a number of stress-related behavioral deficits in the RUS model. This suggests a direct relationship between HPA axis function and behavior in our model. Our findings emphasize the importance of the adrenal receptors as a target for HPA axis dysfunction in stress and fear-related disorders.

Induction of Autophagy by Pterostilbene Contributes to the Prevention of Renal Fibrosis via Attenuating NLRP3 Inflammasome Activation and Epithelial-Mesenchymal Transition.

Chronic kidney disease (CKD) is recognized as a global public health problem. NLRP3 inflammasome activation has been characterized to mediate diverse aspect mechanisms of CKD through regulation of proinflammatory cytokines, tubulointerstitial injury, glomerular diseases, renal inflammation, and fibrosis pathways. Autophagy is a characterized negative regulation mechanism in the regulation of the NLRP3 inflammasome, which is now recognized as the key regulator in the pathogenesis of inflammation and fibrosis in CKD. Thus, autophagy is undoubtedly an attractive target for developing new renal protective treatments of kidney disease via its potential effects in regulation of inflammasome. However, there is no clinical useful agent targeting the autophagy pathway for patients with renal diseases. Pterostilbene (PT, trans-3,5-dimethoxy-4-hydroxystilbene) is a natural analog of resveratrol that has various health benefits including autophagy inducing effects. Accordingly, we aim to investigate underlying mechanisms of preventive and therapeutic effects of PT by reducing NLRP3 inflammasome activation and fibrosis through autophagy-inducing effects. The renal protective effects of PT were evaluated by potassium oxonate (PO)-induced hyperuricemia and high adenine diet-induced CKD models. The autophagy induction mechanisms and anti-fibrosis effects of PT by down-regulation of NLRP3 inflammasome are investigated by using immortalized rat kidney proximal tubular epithelial NRK-52E cells. To determine the role of autophagy induction in the alleviating of NLRP3 inflammasome activation and epithelial-mesenchymal transition (EMT), NRK-52E with Atg5 knockdown [NRK-Atg5-(2)] cells were applied in the study. The results indicated that PT significantly reduces serum uric acid levels, liver xanthine oxidase activity, collagen accumulation, macrophage recruitment, and renal fibrosis in CKD models. At the molecular levels, pretreatment with PT downregulating TGF-β-triggered NLRP3 inflammasome activation, and subsequent EMT in NRK-52E cells. After blockage of autophagy by treatment of Atg5 shRNA, PT loss of its ability to prevent NLRP3 inflammasome activation and EMT. Taken together, we suggested the renal protective effects of PT in urate nephropathy and proved that PT induces autophagy leading to restraining TGF-β-mediated NLRP3 inflammasome activation and EMT. This study is also the first one to provide a clinical potential application of PT for a better management of CKD through its autophagy inducing effects.