Analysis of Nonhomologous End Joining and Homologous Recombination Efficiency in HEK-293T Cells using GFP Based Reporter Systems.

DNA double-strand breaks (DSBs) represent the most perilous DNA lesions, capable of inducing substantial genetic information loss and cellular demise. In response, cells employ two primary mechanisms for DSB repair: nonhomologous end joining (NHEJ) and homologous recombination (HR). Quantifying the efficiency of NHEJ and HR separately is crucial for exploring the relevant mechanisms and factors associated with each. The NHEJ assay and HR assay are established methods used to measure the efficiency of their respective repair pathways. These methods rely on meticulously designed plasmids containing a disrupted green fluorescent protein (GFP) gene with recognition sites for endonuclease I-SceI, which induces DSBs. Here, we describe the extrachromosomal NHEJ assay and HR assay, which involve co-transfecting HEK-293T cells with EJ5-GFP/DR-GFP plasmids, an I-SceI expressing plasmid, and an mCherry expressing plasmid. Quantitative results of NHEJ and HR efficiency are obtained by calculating the ratio of GFP-positive cells to mCherry-positive cells, as counted by flow cytometry. In contrast to chromosomally integrated assays, these extrachromosomal assays are more suitable for conducting comparative investigations involving multiple established stable cell lines.

FAM21 interacts with Ku to promote the localization of WASH to DNA double strand break sites.

Cytoplasmic FAM21 works as a guiding protein in Wiskott-Aldrich Syndrome Protein and SCAR Homolog (WASH) complex by linking WASH complex to endosomes through its interaction with retromer. Recently, we have reported that nuclear WASH localizes to DNA double strand break (DSB) sites to promote DNA repair through non-homologous end-joining (NHEJ). However, whether FAM21, the close partner of WASH, is involved in the nuclear WASH localization and DNA repair remains to be clarified. Here, we show that FAM21 interacts with Ku and the interaction between C-terminal FAM21 and Ku is essential for its recruitment to DSB sites. Moreover, FAM21 depletion led to decreases in WASH recruitment to damaged DNA and repair capacity upon DNA damage. Taken together, these results reveal that FAM21 promotes DNA repair by orchestrating the recruitment of WASH to DSB sites, providing a mechanistic insight into WASH-dependent DNA DSB repair.

Fermented Chinese Herbal Medicine Promoted Growth Performance, Intestinal Health, and Regulated Bacterial Microbiota of Weaned Piglets.

To investigate the effects of fermented Chinese herbal medicine on growth performance, diarrhea rate, nutrient digestibility, and intestinal health of weaned piglets, and to provide the theoretical basis for applying fermented Chinese herbal medicines to weaned piglet production, a total of 162 weaned and castrated piglets at 25 days of age (Duroc × Landrace × Yorkshire, half male and half female) with an initial body weight of 7.77 ± 0.03 kg were randomly divided into the following three groups according to the principle of similar body weight: basal diet (CON) group, basal diet + 3 kg/t fermented Chinese herbal medicine (LFHM) group, and basal diet + 5 g/kg fermented Chinese herbal medicine (HFHM) group. Each group underwent six replicates and there were nine piglets in each replicate. The experiment lasted 24 days, i.e., 3 days for preliminary feeding, and 21 days for the experiment. From Day 1 of the experiment, the piglets were observed and recorded for diarrhea each day. As compared with the CON group, the results indicated: Following the addition of fermented Chinese herbal medicine, the piglets in the LFHM and HFHM groups increased final weight (FW); average daily feed intake (ADFI); average daily gain (ADG) (p < 0.01); apparent digestibility of crude protein (CP) (p < 0.05); as well as chymotrypsin, α-amylase, and lipase activities (p < 0.01). In addition, α-amylase activity in the LFHM group was higher than that in the HFHM group (p < 0.05); chymotrypsin activity in the LFHM group was lower than that in the HFHM group (p < 0.05); as compared with the CON group, the LFHM and the HFHM increased villus height (VH) and crypt depth (CD) in piglet jejunum; isovaleric acid concentration with the HFHM was higher than those with the CON and the LFHM (p < 0.05), but butyrate concentration with the HFFM was lower than those with the CON and the LFHM (p < 0.05). The high-throughput 16S rRNA sequencing of intestinal microbiota results showed that the LFHM and the HFHM affected the microbial α diversity index in weaned piglet colon (p < 0.01). In conclusion, fermented Chinese herbs can improve the growth performance of weaned piglets by promoting the secretion of intestinal digestive enzymes, changing intestinal microbial diversity, regulating the contents of intestinal short chain fatty acids (SCFAs), promoting intestinal health, and improving nutrients digestibility.

Backbone 1H, 15N, and 13C resonance assignments of the Phafin2 pleckstrin homology domain.

Phafin2 is a peripheral protein that triggers cellular signaling from endosomal and lysosomal compartments. The specific subcellular localization of Phafin2 is mediated by the presence of a tandem of phosphatidylinositol 3-phosphate (PtdIns3P)-binding domains, the pleckstrin homology (PH) and the Fab-1, YOTB, Vac1, and EEA1 (FYVE) domains. The requirement for both domains for binding to PtdIns3P still remains unclear. To understand the molecular interactions of the Phafin2 PH domain in detail, we report its nearly complete 1H, 15N, and 13C backbone resonance assignments.

High mobility group box 1 regulates gastric cancer cell proliferation and migration via RAGE-mTOR/ERK feedback loop.

Gastric cancer (GC) is a common malignancy tumour in China. Despite various therapeutic approaches to improve the survival rate of GC patients, the effectiveness of currently available treatments remains unsatisfactory. High mobility group box 1 (HMGB1) is reported to play a role in tumour development. However, the molecular mechanisms involved in HMGB1-mediated regulation of proliferation and migration of GC cells remain unclear. In the present study, we demonstrated that HMGB1 is highly expressed in GC cells and tissue. In HGC-27 GC cells, HMGB1 overexpression or HMGB1 RNA interference both demonstrated that HMGB1 could promote GC cell proliferation and migration. Investigation of the underlying molecular mechanisms revealed that HMGB1 enhanced cyclins expression, induced epithelial-to-mesenchymal transition and matrix metalloproteinase (MMPs) expression and promoted RAGE expression as well as RAGE-mediated activation of Akt/mTOR/P70S6K and ERK/P90RSK/CREB signalling pathways. We also found that inhibition of ERK and mTOR using specific inhibitors reduced recombinant human HMGB1-induced RAGE expression, suggesting that the RAGE-mTOR/ERK positive feedback loop is involved in HMGB1-induced GC cell proliferation and migration. Our study highlights a novel mechanism by which HMGB1 promotes GC cell proliferation and migration via RAGE-mediated Akt-mTOR and ERK-CREB signalling pathways which also involves the RAGE-mTOR/ERK feedback loop. These findings indicate that HMGB1 is a potential therapeutic target for GC.

Structural, in silico, and functional analysis of a Disabled-2-derived peptide for recognition of sulfatides.

Disabled-2 (Dab2) is an adaptor protein that regulates the extent of platelet aggregation by two mechanisms. In the first mechanism, Dab2 intracellularly downregulates the integrin αIIbß3 receptor, converting it to a low affinity state for adhesion and aggregation processes. In the second mechanism, Dab2 is released extracellularly and interacts with the pro-aggregatory mediators, the integrin αIIbß3 receptor and sulfatides, blocking their association to fibrinogen and P-selectin, respectively. Our previous research indicated that a 35-amino acid region within Dab2, which we refer to as the sulfatide-binding peptide (SBP), contains two potential sulfatide-binding motifs represented by two consecutive polybasic regions. Using molecular docking, nuclear magnetic resonance, lipid-binding assays, and surface plasmon resonance, this work identifies the critical Dab2 residues within SBP that are responsible for sulfatide binding. Molecular docking suggested that a hydrophilic region, primarily mediated by R42, is responsible for interaction with the sulfatide headgroup, whereas the C-terminal polybasic region contributes to interactions with acyl chains. Furthermore, we demonstrated that, in Dab2 SBP, R42 significantly contributes to the inhibition of platelet P-selectin surface expression. The Dab2 SBP residues that interact with sulfatides resemble those described for sphingolipid-binding in other proteins, suggesting that sulfatide-binding proteins share common binding mechanisms.

The C-terminal acidic motif of Phafin2 inhibits PH domain binding to phosphatidylinositol 3-phosphate.

Changes in membrane curvature are required to control the function of subcellular compartments; malfunctions of such processes are associated with a wide range of human diseases. Membrane remodeling often depends upon the presence of phosphoinositides, which recruit protein effectors for a variety of cellular functions. Phafin2 is a phosphatidylinositol 3-phosphate (PtdIns3P)-binding effector involved in endosomal and lysosomal membrane-associated signaling. Both the Phafin2 PH and the FYVE domains bind PtdIns3P, although their redundant function in the protein is unclear. Through a combination of lipid-binding assays, we found that, unlike the FYVE domain, recognition of the PH domain to PtdIns3P requires a lipid bilayer. Using site-directed mutagenesis and truncation constructs, we discovered that the Phafin2 FYVE domain is constitutive for PtdIns3P binding, whereas PH domain binding to PtdIns3P is autoinhibited by a conserved C-terminal acidic motif. These findings suggest that binding of the Phafin2 PH domain to PtdIns3P in membrane compartments occurs through a highly regulated mechanism. Potential mechanisms are discussed throughout this report.

Aloin Inhibits the Proliferation and Migration of Gastric Cancer Cells by Regulating NOX2-ROS-Mediated Pro-Survival Signal Pathways.

BACKGROUND: Aloin has been reported to have many pharmacological effects including anti-inflammatory, anti-oxidant and anti-tumour activities. However, the precise molecular mechanisms underlying the anti-tumour properties of aloin are yet to be elucidated. METHODS: HGC-27 and BGC-823 gastric cancer cells were treated with aloin. EdU and colony formation assays were used to detect the proliferation ability of cells. The migration of cells was detected using wound healing and transwell assays. Western blotting was used to detect the levels of cyclinD1, cyclin E1, MMPs, N-cadherin, E-cadherin and NOX2. The phosphorylation of Akt, mTOR, P70S6K, S6, Src, stat3 and IκBα were also detected by Western blotting. Flow cytometry was used to detect the cell cycle distribution.The location of p65 in cells was determined by using a confocal microscopy assay. The total amounts of ROS present in cells were measured using an ROS assay kit. RESULTS: Here, we found that aloin inhibited the proliferation and migration of HGC-27 and BGC-823 gastric cancer cells using a combination of EdU, colony formation, wound healing and transwell assays. Further investigations revealed that aloin decreased the protein expression levels of cyclin D1, N-cadherin, and the matrix metalloproteinases (MMP)-2 and MMP-9; increased E-cadherin expression in a dose-dependent manner; inhibited reactive oxygen species (ROS) generation; and mediated the activation of Akt-mTOR, signal transducer and activator of transcription-3 (Stat3), and NF-κB signalling pathways. Our results also indicated that aloin is able to attenuate the expression levels of the two regulatory proteins of nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2), p47phox and p22phox, but had no effect on the level of gp91phox. N-acetylcysteine treatment of gastric cancer cells inhibited ROS production and Akt-mTOR, Stat3, and IκBα phosphorylation. Taken together, our data suggest that aloin inhibits the proliferation and migration of gastric cancer cells by downregulating NOX2-ROS-mediated activation of the Akt-mTOR, Stat3, and NF-κB signalling pathways. CONCLUSION: Our findings suggest a potential role for aloin in the prevention of gastric cancer cell proliferation and migration and provide novel insights into the anti-cancer properties of aloin.

[Calenduloside E inhibits lipopolysaccharide-induced inflammatory response by inhibiting activation of ROS-mediated JAK1-stat3 signaling pathway in RAW264.7 cells].

OBJECTIVE: To investigate the effect of calenduloside E on lipopolysaccharide (LPS)-induced inflammatory response in RAW264.7 cells and explore the underlying molecular mechanism. METHODS: CCK-8 assay was used to examine the effect of different concentrations of calenduloside E (0-30 µg/mL) on the viability of RAW264.7 cells. The release of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) in RAW264.7 cells in response to pretreatment with 6, 8, and 10 µg/mL calenduloside E for 2 h followed by stimulation with 100 ng/mL LPS was detected using enzyme-linked immunosorbent assay (ELISA). The expression levels of iNOS and COX-2 and the activation of JAK-stats, MAPKs and NF-кB signaling pathways in the treated cells were determined using Western blotting. A reactive oxygen species (ROS) detection kit was used to detect ROS production in the cells, and the nuclear translocation of the transcription factor stat3 was observed by laser confocal microscopy. RESULTS: Calenduloside E below 20 µg/mL did not significantly affect the viability of RAW264.7 cells. Calenduloside E dose-dependently decreased the expression levels of iNOS and COX-2 induced by LPS, inhibited LPS-induced release of TNF-α and IL-1ß, and suppressed LPS-induced JAK1-stat3 signaling pathway activation and stat3 nuclear translocation. Calenduloside E also significantly reduced ROS production induced by LPS in RAW264.7 cells. CONCLUSIONS: Calenduloside E inhibits LPS-induced inflammatory response by blocking ROS-mediated activation of JAK1-stat3 signaling pathway in RAW264.7 cells.

Preferential phosphatidylinositol 5-phosphate binding contributes to a destabilization of the VHS domain structure of Tom1.

Tom1 transports endosomal ubiquitinated proteins that are targeted for degradation in the lysosomal pathway. Infection of eukaryotic cells by Shigella flexneri boosts oxygen consumption and promotes the synthesis of phosphatidylinositol-5-phosphate (PtdIns5P), which triggers Tom1 translocation to signaling endosomes. Removing Tom1 from its cargo trafficking function hinders protein degradation in the host and, simultaneously, enables bacterial survival. Tom1 preferentially binds PtdIns5P via its VHS domain, but the effects of a reducing environment as well as PtdIns5P on the domain structure and function are unknown. Thermal denaturation studies demonstrate that, under reducing conditions, the monomeric Tom1 VHS domain switches from a three-state to a two-state transition behavior. PtdIns5P reduced thermostability, interhelical contacts, and conformational compaction of Tom1 VHS, suggesting that the phosphoinositide destabilizes the protein domain. Destabilization of Tom1 VHS structure was also observed with other phospholipids. Isothermal calorimetry data analysis indicates that, unlike ubiquitin, Tom1 VHS endothermically binds to PtdIns5P through two noncooperative binding sites, with its acyl chains playing a relevant role in the interaction. Altogether, these findings provide mechanistic insights about the recognition of PtdIns5P by the VHS domain that may explain how Tom1, when in a different VHS domain conformational state, interacts with downstream effectors under S. flexneri infection.

The molecular mechanisms of Aloin induce gastric cancer cells apoptosis by targeting High Mobility Group Box 1.

Purpose: Aloin (ALO), a bioactive ingredient extracted from aloe vera, has anti-tumor effects. High Mobility Group Box 1 (HMGB1), a highly conserved nuclear DNA-binding protein, has been implicated in various cancer types. Highly expressed HMGB1 is closely associated with tumor cells apoptosis, proliferation and migration. We investigated the specific molecular mechanisms by which ALO-induced apoptosis by targeting HMGB1 in gastric cancer cells. Materials and methods: Human gastric cancer HGC-27 cells were treated with different doses of ALO (100, 200 and 400 µg/ml) for 24 h, after which DAPI staining was used to observe the nuclear morphology, Annexin V/PI double staining assay was used to determine the rate of apoptosis; Western blotting was used to detect the levels of PARP, pro-caspase3, HMGB1 and RAGE; nuclear translocation of HMGB1 was determined by conducting a nucleoplasm separation experiment. The Enzyme linked immunosorbent assay (ELISA) assay was used to detect release of HMGB1. The HGC-27 cells, transfected with HMGB1 shRNA plasmids, were stimulated with ALO for 24 h, after which a flow cytometry assay was used to detect the rate of apoptosis. HGC-27 cells were pre-treated with or without ALO and then stimulated with rhHMGB1, the phosphorylation of Akt, mTOR, P70S6K, S6, 4EBP1, ERK, P90RSK, cAMP regulatory element binding (CREB) were detected by Western blotting. Results: After different doses of ALO treatment, the nuclei showed morphological changes characteristic of apoptosis. Apoptotic rates were enhanced in a dose dependent manner. The level of cleaved PARP was enhanced and pro-caspase3, HMGB1 and RAGE levels were reduced, HMGB1 nuclear translocation and release were inhibited. The activation of rhHMGB1-induced Akt-mTOR-P70S6K and ERK-CREB signalling pathways was inhibited by ALO. Blocking these signalling pathways by special inhibitors and HMGB1 knockdown could enhance ALO-induced HGC-27 cell apoptosis. Conclusion: ALO- induced HGC-27 cell apoptosis by down-regulating expressions of HMGB1 and RAGE, inhibiting HMGB1 release and then suppressing rhHMGB1-induced activation of Akt-mTOR-P70S6K and ERK-P90RSK-CREB signalling pathways.

[Aloin induces apoptosis via regulating the activation of MAPKs signaling pathway in human gastric cancer cells in vitro].

OBJECTIVE: To investigate the effect of aloin on apoptosis of human gastric cancer cells and explore the molecular mechanism. METHODS: Gastric cancer MKN-28 and HGC-27 cells were cultured routinely in 1640 medium supplemented with 10% fetal bovine serum and 10% non-essential amino acids (for HGC-27 cells) and treated with different concentrations of aloin for different durations. The cell viability, cell nuclear morphology, and apoptotic rate of the cells were detected using CCK-8 assay, DAPI staining and AnnexinV-FITC/PI, respectively; Western blotting was used to detect the expression levels of PARP, procaspase 3 and the phosphorylation of p38, ERK and JNK. The cells were treated with specific inhibitors of p38, ERK and JNK, and the inhibitory effects on these pathways were detected with Western blotting; DAPI staining was used to detect the effects of inhibitors on apoptosis of gastric cancer cells. RESULTS: Aloin dose-dependently inhibited the viability and induced apoptosis of HGC-27 and MKN-28 cells. Alion treatment obvious enhanced the phosphorylation of p38 and JNK but decreased ERK phosphorylation in the cells. Blocking ERK activation with the ERK inhibitor obviously enhanced aloin-induced cell apoptosis, where inhibiting p38 and JNK activation partly reversed alion-induced apoptosis in the cells. CONCLUSIONS: Aloin induces apoptosis of human gastric cancer cells in vitro by activating p38 and JNK signaling pathways and inhibiting ERK signaling pathway.

Inducible HSP70 antagonizes cisplatin­induced cell apoptosis through inhibition of the MAPK signaling pathway in HGC­27 cells.

Inducible heat shock protein 70 (HSP70; also known as HSPA1 or HSP72) is implicated in cancer. As a stress­inducible heat shock protein, HSP70 is highly expressed in a variety of cancers and correlates with metastasis, chemotherapy resistance and tumor prognosis. The present study demonstrated that suppression of HSP70 through the specific inhibitor pifithrin­µ or by HSP70 knockdown enhanced cisplatin­induced apoptosis in HGC­27 gastric cancer cells. By contrast, upregulation of HSP70 through transfection of a HSP70 overexpressing plasmid decreased cisplatin­induced HGC­27 cell apoptosis. In exploring the underlying molecular mechanisms, the present results revealed that HSP70 antagonized cisplatin­induced HGC­27 cell apoptosis by regulating the mitogen­activated protein kinase (MAPK) signaling pathway. In addition, suppressing the MAPK pathway enhanced cisplatin­induced HGC­27 cell apoptosis. Collectively, the present findings suggest that inhibition of HSP70 expression enhanced the sensitivity of HGC­27 cells to cisplatin via the MAPK signaling pathway, and that HSP70 may serve as a potential therapeutic target in gastric cancer.

Aloin suppresses lipopolysaccharide­induced inflammation by inhibiting JAK1­STAT1/3 activation and ROS production in RAW264.7 cells.

The anti­inflammatory effects of aloin, a bioactive ingredient extracted from Aloe vera, have been described previously. The present study aimed to assess these effects and explore the underlying molecular mechanisms. RAW264.7 cells were incubated with different doses of aloin (100, 150 and 200 µg/ml) and lipopolysaccharide (LPS; 100 ng/ml) for the indicated times. Then, inducible nitric oxide synthase (iNOS) and cyclooxygenase­2 expression levels were detected by western blot analysis and reverse transcription polymerase chain reaction (RT­PCR).The concentrations of inflammatory cytokines in the cell culture supernatant were determined by ELISA. Total nitric oxide (NO) assay and reactive oxygen species (ROS) kits were used to detect NO and ROS levels, respectively. Mitogen­activated protein kinase, nuclear factor κB and Janus kinase­signal transducer and activator of transcription (JAK­STAT) pathway activation were verified by western blot analysis. Confocal and nucleocytoplasmic separation experiments were used to detect STAT nuclear translocation. It was identified that aloin decreased the level of LPS­induced iNOS expression, inhibiting the release of interleukin (IL)­1ß, IL­6, tumour necrosis factor­α and NO dose­dependently. Mechanistically, aloin suppressed LPS­induced JAK1­STAT1/3 activation and STAT1/3 nuclear translocation. Additionally, LPS­induced ROS production was inhibited by aloin. Collectively, these data suggest that aloin attenuated LPS­induced inflammation by inhibiting ROS­mediated activation of the JAK1­STAT1/3 signalling pathway, thereby inhibiting the nuclear translocation of STAT1/3 in RAW264.7 cells. The present study provides an experimental basis for the clinical application of aloin in inflammatory­associated diseases.

Salidroside inhibits the proliferation and migration of gastric cancer cells via suppression of Src­associated signaling pathway activation and heat shock protein 70 expression.

Salidroside, an active ingredient extracted from the Rhodiola rosea plant, has potential anti­tumor effects. However, the effects of salidroside on gastric cancer cell proliferation and migration remain unclear. In the present study, the inhibitory effects of salidroside on gastric cancer cell proliferation, migration and invasion and the molecular mechanisms underlying these effects were investigated. The human gastric cancer cell line, BGC­823, was treated with different concentrations of salidroside (200, 400 and 600 µg/ml). Cell proliferation was determined with Cell Counting Kit­8 and colony formation assays, and the migration and invasion of cells was detected by a wound healing and Transwell assay, respectively. Western blotting was performed to detect the levels of N­cadherin, E­cadherin and heat shock protein (HSP)70. In addition, the phosphorylation of proto­oncogene tyrosine­protein kinase Src (Src), protein kinase B (Akt), mitogen activated protein kinase 1 (ERK), signal transducer and activator of transcription (STAT)3 and focal adhesion kinase 1 (FAK) was examined by western blotting. The levels of matrix metalloproteinase (MMP)­2 and MMP­9 were determined by enzyme­linked immunosorbent assay kits. Levels of reactive oxygen species (ROS) in cells were measured by a fluorescence plate reader with dichloro­dihydro­fluorescein diacetate. The results indicated that salidroside significantly suppressed cell proliferation and colony formation, inhibited cell migration and invasion, increased E­cadherin expression and decreased N­cadherin, MMP­2 and MMP­9 expression. Furthermore, salidroside suppressed ROS production and subsequently reduced the phosphorylation of Src, Akt, ERK and FAK. Salidroside also inhibited HSP70 expression, and HSP70 overexpression reversed the inhibitory effects of salidroside on BGC­823 cell proliferation, migration and invasion. In conclusion, the present study revealed that salidroside inhibited the proliferation, migration and invasion of BGC­823 cells by downregulating ROS­mediated Src­associated signaling pathway activation and HSP70 expression.

Identification of Lipid Binding Modulators Using the Protein-Lipid Overlay Assay.

The protein-lipid overlay assay is an inexpensive, easy-to-implement, and high-throughput methodology that employs nitrocellulose membranes to immobilize lipids in order to rapid screen and identify protein-lipid interactions. In this chapter, we show how this methodology can identify potential modulators of protein-lipid interactions by screening water-soluble lipid competitors or even the introduction of pH changes during the binding assay to identify pH-dependent lipid binding events.

Structural, thermodynamic, and phosphatidylinositol 3-phosphate binding properties of Phafin2.

Phafin2 is a phosphatidylinositol 3-phosphate (PtdIns(3)P) binding protein involved in the regulation of endosomal cargo trafficking and lysosomal induction of autophagy. Binding of Phafin2 to PtdIns(3)P is mediated by both its PH and FYVE domains. However, there are no studies on the structural basis, conformational stability, and lipid interactions of Phafin2 to better understand how this protein participates in signaling at the surface of endomembrane compartments. Here, we show that human Phafin2 is a moderately elongated monomer of ∼28 kDa with an intensity-average hydrodynamic diameter of ∼7 nm. Circular dichroism (CD) analysis indicates that Phafin2 exhibits an α/ß structure and predicts ∼40% random coil content in the protein. Heteronuclear NMR data indicates that a unique conformation of Phafin2 is present in solution and dispersion of resonances suggests that the protein exhibits random coiled regions, in agreement with the CD data. Phafin2 is stable, displaying a melting temperature of 48.4°C. The folding-unfolding curves, obtained using urea- and guanidine hydrochloride-mediated denaturation, indicate that Phafin2 undergoes a two-state native-to-denatured transition. Analysis of these transitions shows that the free energy change for urea- and guanidine hydrochloride-induced Phafin2 denaturation in water is ∼4 kcal mol-1 . PtdIns(3)P binding to Phafin2 occurs with high affinity, triggering minor conformational changes in the protein. Taken together, these studies represent a platform for establishing the structural basis of Phafin2 molecular interactions and the role of the two potentially redundant PtdIns(3)P-binding domains of the protein in endomembrane compartments.

Membrane targeting of TIRAP is negatively regulated by phosphorylation in its phosphoinositide-binding motif.

Pathogen-activated Toll-like receptors (TLRs), such as TLR2 and TLR4, dimerize and move laterally across the plasma membrane to phosphatidylinositol (4,5)-bisphosphate-enriched domains. At these sites, TLRs interact with the TIR domain-containing adaptor protein (TIRAP), triggering a signaling cascade that leads to innate immune responses. Membrane recruitment of TIRAP is mediated by its phosphoinositide (PI)-binding motif (PBM). We show that TIRAP PBM transitions from a disordered to a helical conformation in the presence of either zwitterionic micelles or monodispersed PIs. TIRAP PBM bound PIs through basic and nonpolar residues with high affinity, favoring a more ordered structure. TIRAP is phosphorylated at Thr28 within its PBM, which leads to its ubiquitination and degradation. We demonstrate that phosphorylation distorts the helical structure of TIRAP PBM, reducing PI interactions and cell membrane targeting. Our study provides the basis for TIRAP membrane insertion and the mechanism by which it is removed from membranes to avoid sustained innate immune responses.