Mutation Of Aspartate Research Articles

A-031 Development of novel ELISAs for total GDF-15 and H-specific GDF-15

Abstract Background To develop sensitive and specific ELISAs for the quantitative measurement of total human GDF-15 and H-specific (H202D mutant) GDF-15 in human serum, and other biological fluids. Growth/differentiation Factor 15 (GDF-15) is a divergent member of the TGF-β superfamily of growth factors. It is encoded in humans by a gene in chromosome 19. The human GDF-15 gene encodes for a protein of 308 amino acid residues which consists of a signal sequence (residues 1-29), pro-domain (30-194), and mature growth factor domain (195-308). The molecular weight of a mature GDF-15 dimer is 25 kDa. Approximately 25% of humans have a missense polymorphism in the GDF-15 gene resulting in mutation of histidine 202 to aspartate (histidine 6 in the mature domain), close to the N-terminus of the mature growth factor. This variant is associated with phenotypes in prostate cancer, hyperemesis gravidarum, (severe morning sickness in pregnancy), and rheumatoid arthritis. Methods Highly specific and reproducible total GDF-15 (AL-1014-r) and H-specific GDF-15 (AL-1018-r) ELISAs have been developed using specific monoclonal antibodies to help estimate the total and mutant (DD) concentration in serum in the respective assays. The ELISAs use 10 uL sample volume and are calibrated to recombinant human GDF-15 from R & D Systems (Biotechne, USA). These ELISAs were validated for their specificity using HH, DD, HD recombinant preparations and their circulating levels in male, female, 1st, and 2nd-trimester serum samples. Results The Total GDF-15 ELISA assay detects total GDF-15 including histidine 202 to aspartate mutation (HH, HD & DD variant) in the mature domain in equimolar proportions. The H-specific GDF-15 assay is specific to histidine at 202aa in the GDF-15 sequence and does not detect histidine 202 to aspartate mutation (DD variant). Median Levels of total GDF-15 in healthy males (n=18), females (n=17), 1st trimester (n=20), and 2nd trimester (n=20) were 847.9 pg/mL, 1182.4 pg/mL, 12759.7 pg/mL, and 12951.2 pg/mL. Median Levels of H-specific GDF-15 in healthy males (n=18), females (n=17), 1st trimester (n=20), and 2nd trimester (n=20) were 723.2 pg/mL, 641.3 pg/mL, 8381.4 pg/mL and 4652.1 pg/mL. Method comparison between total GDF-15 and commercial GDF-15 assay using serum samples (HH, wild type) in the range of 400-2500 pg/mL yielded a slope of 0.98 (r=0.97). The HD and DD mutant samples in the total assay recovered at twice the concentrations of the commercial assay. The total and intact assays are highly reproducible with the total coefficient of variation less than 7%. Conclusions Use of total GDF-15 assay in combination with the GDF-15 H-specific assay (DD nondetectable) ELISA can accurately estimate the mutant (DD) concentration in serum. This will enhance the knowledge of the GDF-15 measurements in the literature as the commercial assays were compromised for its immunoreactivity to the HD DD genotypes which is highly prevalent.

First case of evolved herbicide resistance in the holoparasite sunflower broomrape, Orobanche cumana Wallr.

The development and commercialisation of sunflower varieties tolerant to acetolactate synthase (ALS)-inhibiting herbicides some 20 years ago provided farmers with an alternative method for the cost-effective control of Orobanche cumana. In 2020, however, two independent sunflower broomrape populations from Drama (GR-DRA) and Orestiada (GR-ORE), Greece, were reported to be heavily infested with O. cumana after application of the ALS-inhibiting herbicide imazamox. Here we have investigated the race of GR-DRA and GR-ORE and determined the basis of resistance to imazamox in the two Greek O. cumana samples. Using a set of five diagnostic sunflower varieties characterised by different resistant genes with respect to O. cumana infestation, we have clearly established that the GR-ORE and GR-DRA populations belong to the invasive broomrape races G and G+, respectively. Live underground tubercles and emerged shoots were identified at the recommended field rate of imazamox for GR-DRA and GR-ORE but not for two other standard sensitive populations in a whole plant dose response test using two different herbicide-tolerant sunflower hybrids as hosts. Sequencing of the ALS gene identified an alanine 205 to aspartate mutation in all GR-ORE samples. Most GR-DRA tubercles were characterised by a second serine 653 to asparagine ALS mutation whilst a few GR-DRA individuals contained the A205D mutation. Mutations at ALS codons 205 and 653 are known to impact on the binding and efficacy of imazamox and other imidazolinone herbicides. The knowledge generated here will be important for tracking and managing broomrape resistance to ALS-inhibiting herbicides in sunflower growing regions.

A preliminary assessment of physical function in aged male mice with constitutive activation of the aryl hydrocarbon receptors in skeletal muscle

BACKGROUND: Aging involves a gradual deterioration of physical and physiological functions, ultimately leading to mortality. Unfortunately, there are no treatments available that successfully mitigate the age-associated decline in physical function. Recent studies have revealed a strong association between elevated levels of L-kynurenine (L-Kyn) and frailty, muscle weakness, and neuromuscular junction degeneration in humans and mice, however a causal relationship has not been tested. L-Kyn is a product of tryptophan catabolism that has been linked to motor neuron death, skeletal muscle atrophy, and mitochondrial dysfunction, all hallmarks of aging muscle. Interestingly, L-Kyn is an endogenous ligand of the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor widely known for its role in xenobiotic metabolism. Though both pro- and anti-aging effects of the AHR have been reported in the literature, there are several chronic diseases that involve chronic AHR activation and produce strikingly similar phenotypes to exacerbated aging/frailty. OBJECTIVE: The objective of this study was to determine of constitutive activation of the AHR has a causal role in the decline of physical function with aging. METHODS: Aged (16-mo.) C57BL6 male mice (N=48) were obtained from the NIA breeding colony and were randomized to interventions employing muscle-specific adeno-associated virus with the following three treatment groups: 1) a constitutively active AHR mutant was constructed by deleting the ligand binding domain (amino acids 277-418); 2) a transcriptionally deficient AHR mutant (arginine to aspartate mutation at amino acid 39); and an empty vector control. Treadmill endurance, forelimb grip strength, and maximal walking speed were measured at 24-mos. of age. Results: Treadmill endurance capacity in mice with constitutively AHR activation was 5.3% lower than mice treated with the empty vector ( P=0.25) and 12.1% lower than mice treated with the transcriptionally deficient AHR mutant ( P=0.09). Grip strength and walking speed were not significantly different between groups ( P>0.57 and 0.22 respectively) at this 24-mo. age. CONCLUSIONS: These preliminary results in aging male C57BL6 mice may indicate possible role of AHR activation in the decline of physical function. Continued enrollment to increase the sample size and obtaining measures at later ages when physical function declines more severely, as well as examining female mice will provide a more thorough analysis. Funding Supported by grants from the National Institutes of Health R01-AG076490 (Ryan & Hepple) and T32 AG062728 (Wimberly). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Strain-release alkylation of Asp12 enables mutant selective targeting of K-Ras-G12D.

K-Ras is the most commonly mutated oncogene in human cancer. The recently approved non-small cell lung cancer drugs sotorasib and adagrasib covalently capture an acquired cysteine in K-Ras-G12C mutation and lock it in a signaling-incompetent state. However, covalent inhibition of G12D, the most frequent K-Ras mutation particularly prevalent in pancreatic ductal adenocarcinoma, has remained elusive due to the lack of aspartate-targeting chemistry. Here we present a set of malolactone-based electrophiles that exploit ring strain to crosslink K-Ras-G12D at the mutant aspartate to form stable covalent complexes. Structural insights from X-ray crystallography and exploitation of the stereoelectronic requirements for attack of the electrophile allowed development of a substituted malolactone that resisted attack by aqueous buffer but rapidly crosslinked with the aspartate-12 of K-Ras in both GDP and GTP state. The GTP-state targeting allowed effective suppression of downstream signaling, and selective inhibition of K-Ras-G12D-driven cancer cell proliferation in vitro and xenograft growth in mice.

A tyrosine phospho-switch within the Longin domain of VAMP721 modulates SNARE functionality.

The final step in secretion is membrane fusion facilitated by SNARE proteins that reside in opposite membranes. The formation of a trans-SNARE complex between one R and three Q coiled-coiled SNARE domains drives the final approach of the membranes providing the mechanical energy for fusion. Biological control of this mechanism is exerted by additional domains within some SNAREs. For example, the N-terminal Longin domain (LD) of R-SNAREs (also called Vesicle-associated membrane proteins, VAMPs) can fold back onto the SNARE domain blocking interaction with other cognate SNAREs. The LD may also determine the subcellular localization via interaction with other trafficking-related proteins. Here, we provide cell-biological and genetic evidence that phosphorylation of the Tyrosine57 residue regulates the functionality of VAMP721. We found that an aspartate mutation mimics phosphorylation, leading to protein instability and subsequent degradation in lytic vacuoles. The mutant SNARE also fails to rescue the defects of vamp721vamp722 loss-of-function lines in spite of its wildtype-like localization within the secretory pathway and the ability to interact with cognate SNARE partners. Most importantly, it imposes a dominant negative phenotype interfering with root growth, normal secretion and cytokinesis in wildtype plants generating large aggregates that mainly contain secretory vesicles. Non-phosphorylatable VAMP721Y57F needs higher gene dosage to rescue double mutants in comparison to native VAMP721 underpinning that phosphorylation modulates SNARE function. We propose a model where short-lived phosphorylation of Y57 serves as a regulatory step to control VAMP721 activity, favoring its open state and interaction with cognate partners to ultimately drive membrane fusion.

Low concentrations of tricyclic antidepressants stimulate TRPC4 channel activity by acting as an opioid receptor ligand.

Traditionally prescribed for mood disorders, tricyclic antidepressants (TCAs) have shown promising therapeutic effects on chronic neuralgia and irritable bowel syndrome. However, the mechanism by which these atypical effects manifest is unclear. Among the proposed mechanisms is the well-known pain-related inhibitory G-protein coupled receptor, namely the opioid receptor (OR). Here, we confirmed that TCA indeed stimulates OR and regulates the gating of TRPC4, a downstream signaling of the Gi-pathway. In an ELISA to quantify the amount of intracellular cAMP, a downstream product of OR/Gi-pathway, treatment with amitriptyline (AMI) showed a decrease in [cAMP]i similar to that of the μOR agonist. Next, we explored the binding site of TCA by modeling the previously revealed ligand-bound structure of μOR. A conserved aspartate residue of ORs was predicted to participate in salt bridge interaction with the amine group of TCAs, and in aspartate-to-arginine mutation, AMI did not decrease the FRET-based binding efficiency between the ORs and Gαi2. As an alternative way to monitor the downstream signaling of Gi-pathway, we evaluated the functional activity of TRPC4 channel, as it is well known to be activated by Gαi. TCAs increased the TRPC4 current through ORs, and TCA-evoked TRPC4 activation was abolished by an inhibitor of Gαi2 or its dominant-negative mutant. As expected, TCA-evoked activation of TRPC4 was not observed in the aspartate mutants of OR. Taken together, OR could be proclaimed as a promising target among numerous binding partners of TCA, and TCA-evoked TRPC4 activation may help to explain the nonopioid analgesic effect of TCA.NEW & NOTEWORTHY Endogenous opioid systems modulate pain perception, but concerns about opioid-related substance misuse limit their use. This study has raised TRPC4 channel as a candidate target for alternative analgesics, tricyclic antidepressants (TCAs). TCAs have been shown to bind to and activate opioid receptors (ORs), leading to downstream signaling pathways involving TRPC4. The functional selectivity and biased agonism of TCA towards TRPC4 in dependence on OR may provide a better understanding of its efficacy or side effects.

Specific Mutations near the Amyloid Precursor Protein Cleavage Site Increase γ-Secretase Sensitivity and Modulate Amyloid-β Production.

Amyloid-β peptides (Aβs) are produced via cleavage of the transmembrane region of the amyloid precursor protein (APP) by γ-secretase and are responsible for Alzheimer's disease. Familial Alzheimer's disease (FAD) is associated with APP mutations that disrupt the cleavage reaction and increase the production of neurotoxic Aβs, i.e., Aβ42 and Aβ43. Study of the mutations that activate and restore the cleavage of FAD mutants is necessary to understand the mechanism of Aβ production. In this study, using a yeast reconstruction system, we revealed that one of the APP FAD mutations, T714I, severely reduced the cleavage, and identified secondary APP mutations that restored the cleavage of APP T714I. Some mutants were able to modulate Aβ production by changing the proportions of Aβ species when introduced into mammalian cells. Secondary mutations include proline and aspartate residues; proline mutations are thought to act through helical structural destabilization, while aspartate mutations are thought to promote interactions in the substrate binding pocket. Our results elucidate the APP cleavage mechanism and could facilitate drug discovery.

Biochemical analysis of a novel allele of the OsPPDKB gene associated with floury endosperm

Here we isolated and characterized floTR1, a new floury endosperm rice mutant. Whole genome sequence and analysis of a cleaved amplified polymorphic sequence marker found that the FloTR1 gene encodes a pyruvate orthophosphate dikinase B (OsPPDKB). The floTR1 mutant has a single G to A base substitution in the OsPPDKB gene, which results in a missense glycine to aspartate mutation. Although there was little change in the mRNA expression levels of OsPPDKB between floTR1 and the original variety (WT) plants, PPDK activity in the floTR1 developing endosperm was null. Recombinant enzyme analysis also found that aspartate replacement, as found in floTR1, resulted in the complete loss of PPDK activity in vitro. Analysis of other recombinant enzymes suggested that the spatial configuration such as size and direction of the side chain in the replaced amino acids predominantly affected its activity. The rice flour of floTR1 showed not only smaller particle size distribution and lower damaged starch content but also higher specific rice bread loaf volume than WT. Our data provides new insights into effects on OsPPDK activity caused by a SNP mutation and suggests applications for this new floury rice mutant.

The rate‐limiting enzyme in the CDP‐choline pathway is regulated by phosphorylation‐domain charge density

Eukaryotic cell membranes are primarily composed of phospholipids, the most abundant of which is phosphatidylcholine (PC). De novo synthesis of PC by the CDP‐choline pathway is under the control of the rate‐limiting enzyme CTP:phosphocholine cytidylyltransferase (CCT). The nuclear CCTa isoform is regulated by translocation to the inner nuclear membrane (INM) and nuclear lipid droplets (nLD) in response to lipid activators, such as oleate, or deficiency in PC content. CCTa association with the INM is mediated by a positively‐charged membrane‐binding M‐domain and a phosphorylated P‐domain containing 16 serine phospho‐sites. Mutation of all P‐domain serine residues to alanine (dephosphorylated mimic) enhanced association with nLDs in oleate‐treated U2OS cells, while mutation to glutamate (phosphorylated mimic) or mutation of M‐domain lysine residues ablated nLD association. However, it is unknown if phosphorylation regulation of CCTa association with the INM and nLDs involves specific serine residues or the net charge of the P‐domain. To address this question, we mutated 4 sets of 2‐3 serine residues in the P‐domain to alanine or aspartate. CCTa cDNAs expressing different combinations of these mutations were expressed in oleate‐treated U2OS cells and the frequency of nLD association was measured. The mutants were also expressed in CHO cells with a temperature‐sensitive CCTα isoform and INM translocation in response to oleate was determined. The greatest inhibitory effect on INM and nLD association was observed when all 4 sets of serine residues were mutated to aspartate and not by any one site, suggesting that net charge density of the P‐domain regulates affinity for the NE and nLD. Additionally, CCTa serine‐to‐alanine mutants expressed in U2OS cells were unstable compared to the corresponding aspartate mutants. We conclude that CCTa association with the INM and nLD is inhibited as overall P‐domain phosphorylation increases. In its active membrane‐associated, dephosphorylated state CCTa protein has decreased stability, a potently negative feedback mechanism to control PC synthesis. These results provide novel insight into how PC synthesis is regulated that will be essential for identifying kinases and phosphatases that act on CCTα.

The utility of the Ion Torrent PGM next generation sequencing for analysis of the most commonly mutated genes among patients with colorectal cancer in India.

The requirement for the mutation analysis for Kirsten rat sarcoma viral oncogene (KRAS) in colorectal cancer (CRC) is rapidly increasing as it is a predictive biomarker and also, its absence signifies response to anti-epidermal growth factor receptor (anti-EGFR) antibody treatment. The aim of our study was to investigate the pathological diagnosis and distribution of KRAS mutations in colorectal cancer with the use of next generation sequencing platform (Ion Torrent). A total of 56 CRC samples were tested to identify the genetic mutations, especially KRAS using the primers which included ~2800 COSMIC mutations of 50 oncogenes. Ion Torrent personal genome machine (semiconductor-based sequencing) was used for the sequencing and analysis. Along with KRAS, other 49 genes were also studied for COSMIC mutations. KRAS mutation 25 (44.6%) had the highest frequency, followed by TP53 10 (17.9%) and PIK3CA mutation 4 (7.1%). Of all the KRAS mutations identified, mutations in codon 12 were most frequent followed by mutations in codon 13 and 61. The most frequent substitution was glycine to aspartate mutation in codon 12 (p.Gly12Asp) followed by glycine to valine (p.Gly12Val). Combinations of mutations were also studied. Our study revealed that seven cases (12.5%) had both KRAS and TP53 mutations (highest of all the combinations). The analysis of KRAS mutation frequency and its mutational subtype analysis in human CRCs by using semiconductor-based platform in routine clinical practices have been performed in Indian population. The findings were similar to earlier published reports from the Western literature.

CsKDO is a candidate gene regulating seed germination lethality in cucumber.

Seed germination plays an important role in the initial stage of plant growth. However, few related studies focused on lethality after seed germination in plants. In this study, we identified an Ethyl methanesulfonate (EMS) mutagenesis mutant Csleth with abnormal seed germination in cucumber (Cucumis sativus L.). The radicle of the Csleth mutant grew slowly and detached from the cotyledon until 14 d after sowing. Genetic analysis showed that the mutant phenotype of Csleth was controlled by a single recessive gene. MutMap+ and Kompetitive Allele Specific PCR (KASP) genotyping results demonstrated that Csa3G104930 encoding 3-deoxy-manno-octulosonate cytidylyltransferase (CsKDO) was the candidate gene of the Csleth mutant. The transition mutation of aspartate occurred in Csa3G104930 co-segregated with the phenotyping data. CsKDO was highly expressed in male flowers in wild type cucumbers. Subcellular localization results showed that CsKDO was located in the nucleus. Overall, these results suggest CsKDO regulates lethality during seed germination in cucumber.

GTP-State-Selective Cyclic Peptide Ligands of K-Ras(G12D) Block Its Interaction with Raf.

We report the identification of three cyclic peptide ligands of K-Ras(G12D) using an integrated in vitro translation–mRNA display selection platform. These cyclic peptides show preferential binding to the GTP-bound state of K-Ras(G12D) over the GDP-bound state and block Ras-Raf interaction. A co-crystal structure of peptide KD2 with K-Ras(G12D)·GppNHp reveals that this peptide binds in the Switch II groove region with concomitant opening of the Switch II loop and a 40° rotation of the α2 helix, and that a threonine residue (Thr10) on KD2 has direct access to the mutant aspartate (Asp12) on K-Ras. Replacing this threonine with non-natural amino acids afforded peptides with improved potency at inhibiting the interaction between Raf1-RBD and K-Ras(G12D) but not wildtype K-Ras. The union of G12D over wildtype selectivity and GTP state/GDP state selectivity is particularly desirable, considering that oncogenic K-Ras(G12D) exists predominantly in the GTP state in cancer cells, and wildtype K-Ras signaling is important for the maintenance of healthy cells.

Apremilast regulates acute effects of ethanol and other GABAergic drugs via protein kinase A-dependent signaling

Phosphodiesterase type 4 (PDE4) inhibitors prevent hydrolysis of cyclic adenosine monophosphate and increase protein kinase A (PKA)-mediated phosphorylation. PDE4 inhibitors also regulate responses to ethanol and GABAergic drugs. We investigated mechanisms by which the PDE4 inhibitor, apremilast, regulates acute effects of ethanol and GABAergic drugs in male and female mice. Apremilast prolonged the sedative-hypnotic effects of gaboxadol, zolpidem, and propofol but did not alter etomidate effects, and unexpectedly shortened the sedative-hypnotic effects of diazepam. Apremilast prolonged rotarod ataxia induced by zolpidem, propofol, and loreclezole, shortened recovery from diazepam, but had no effect on ataxia induced by gaboxadol or etomidate. The PKA inhibitor H-89 blocked apremilast's ability to prolong the sedative-hypnotic effects of ethanol, gaboxadol, and propofol and to prolong ethanol- and propofol-induced ataxia. H-89 also blocked apremilast's ability to shorten the sedative-hypnotic and ataxic effects of diazepam. The β1-specific antagonist, salicylidene salicylhydrazide (SCS), produced faster recovery from ethanol- and diazepam-induced ataxia, but did not alter propofol- or etomidate-induced ataxia. SCS shortened the sedative-hypnotic effects of ethanol and diazepam but not of propofol. In Xenopus oocytes, a phosphomimetic (aspartate) mutation at the PKA phosphorylation site in β1 subunits decreased the maximal GABA current in receptors containing α1 or α3, but not α2 subunits. In contrast, phosphomimetic mutations at PKA sites in β3 subunits increased the maximal GABA current in receptors containing α1 or α2, but not α3 subunits. The GABA potency and allosteric modulation by ethanol, propofol, etomidate, zolpidem, flunitrazepam, or diazepam were not altered by these mutations. We propose a model whereby apremilast increases PKA-mediated phosphorylation of β1-and β3-containing GABAA receptors and selectively alters acute tolerance to ethanol and GABAergic drugs.

Mutation of Aspartate 238 in FAD Synthase Isoform 6 Increases the Specific Activity by Weakening the FAD Binding.

FAD synthase (FADS, or FMN:ATP adenylyl transferase) coded by the FLAD1 gene is the last enzyme in the pathway of FAD synthesis. The mitochondrial isoform 1 and the cytosolic isoform 2 are characterized by the following two domains: the C-terminal PAPS domain (FADSy) performing FAD synthesis and pyrophosphorolysis; the N-terminal molybdopterin-binding domain (FADHy) performing a Co++/K+-dependent FAD hydrolysis. Mutations in FLAD1 gene are responsible for riboflavin responsive and non-responsive multiple acyl-CoA dehydrogenases and combined respiratory chain deficiency. In patients harboring frameshift mutations, a shorter isoform (hFADS6) containing the sole FADSy domain is produced representing an emergency protein. With the aim to ameliorate its function we planned to obtain an engineered more efficient hFADS6. Thus, the D238A mutant, resembling the D181A FMNAT “supermutant” of C. glabrata, was overproduced and purified. Kinetic analysis of this enzyme highlighted a general increase of Km, while the kcat was two-fold higher than that of WT. The data suggest that the FAD synthesis rate can be increased. Additional modifications could be performed to further improve the synthesis of FAD. These results correlate with previous data produced in our laboratory, and point towards the following proposals (i) FAD release is the rate limiting step of the catalytic cycle and (ii) ATP and FMN binding sites are synergistically connected.

1864-P: p38α MAPK Mediates Glucagon-Induced Hepatic Glucose Production through Phosphorylation of Foxo1 at Ser273

Glucagon is involved in glucose homeostasis via triggering gluconeogenesis and glycogenolysis. In addition to activation of protein kinase A (PKA), p38α MAPK (p38) is activated by glucagon and promotes glucagon-induced hepatic glucose production. However, underlying mechanisms by which p38 mediates the effect of glucagon are not well elucidated. Previously, we established that glucagon increases the phosphorylation of Foxo1 at Ser273 via PKA to promote hepatic glucose production. In this study, we investigated the potential role of p38 in the action of glucagon by phosphorylation of Foxo1 at Ser273. Using the mouse primary hepatocytes, we showed that activation of p38 by glucagon promoted phosphorylation of Foxo1 at Ser273, which results in both increased stability and nuclear translocation of Foxo1. In vivo kinase assay shows that the active p38 enzyme increased the phosphorylation of Foxo1 at Ser273. Moreover, p38 promoted glucagon-induced PKA activity to increase the phosphorylation of Foxo1 at Ser273. Suppression of p38 by siRNA and SB203580 decreased hepatic glucose production by glucagon in control mouse primary hepatocytes; however, such an effect was diminished in the hepatocytes isolated from the Foxo1-S273D knock-in mice, where Foxo1-Ser273 was replaced by the aspartate mutation (D). In mice, we further showed that injection of p38 inhibitor SB203580 decreased the fasting blood glucose and hepatic glucose production in wild type control mice, while this effect was not observed in Foxo1-S273D mice. These results revealed the novel role of p38 in control of Foxo1 phosphorylation at Ser273 to regulate the action of glucagon in glucose homeostasis. Disclosure W. Yang: None. W. Ai: None. X. Li: None. Q. Pan: None. Z. Shen: None. Y. Chen: None. Y. Sun: None. S. Guo: None. Funding American Diabetes Association (1-15-CD-09 to S.G.); National Institutes of Health (R01DK095118)

Design of a Human Rhinovirus-14 3C Protease-Inducible Caspase-3.

The engineering of enzymes for the purpose of controlling their activity represents a valuable approach to address challenges in both fundamental and applied research. Here, we describe and compare different design strategies for the generation of a human rhinovirus-14 (HRV14) 3C protease-inducible caspase-3 (CASP3). We exemplify the application potential of the resulting protease by controlling the activity of a synthetic enzyme cascade, which represents an important motif for the design of artificial signal transduction networks. In addition, we use our engineered CASP3 to characterize the effect of aspartate mutations on enzymatic activity. Besides the identification of mutations that render the enzyme inactive, we find the CASP3-D192E mutant (aspartate-to-glutamate exchange at position 192) to be inaccessible for 3C protease-mediated cleavage. This indicates a structural change of CASP3 that goes beyond a slight misalignment of the catalytic triad. This study could inspire the design of additional engineered proteases that could be used to unravel fundamental research questions or to expand the collection of biological parts for the design of synthetic signaling pathways.

Myxoma virus M013 protein antagonizes NF-κB and inflammasome pathways via distinct structural motifs

Among the repertoire of immunoregulatory proteins encoded by myxoma virus, M013 is a viral homologue of the viral pyrin domain-only protein (vPOP) family. In myeloid cells, M013 protein has been shown to inhibit both the inflammasome and NF-κB signaling pathways by direct binding to ASC1 and NF-κB1, respectively. In this study, a three-dimensional homology model of the M013 pyrin domain (PYD) was built based on similarities to known PYD structures. A distinctive feature of the deduced surface electrostatic map of the M013 PYD is the presence of a negatively region consisting of numerous aspartate and glutamate residues in close proximity. Single-site mutations of aspartate and glutamate residues reveal their role in interactions with ASC-1. The biological significance of charge complementarity in the M013-ASC-1 interaction was further confirmed by functional assays of caspase-1 activation and subsequent secretion of cytokines. M013 also has a unique 33-residue C-terminal tail that follows the N-terminal PYD, and it is enriched in positively charged residues. Deletion of the tail of M013 significantly inhibited the interactions between M013 and NF-κB1, thus compromising the ability of the viral protein to suppress the secretion of pro-inflammatory cytokines. These results demonstrate that vPOP M013 exploits distinct structural motifs to regulate both the inflammasome and NF-κB pathways.

An Allosteric Inhibitory Site Conserved in the Ectodomain of P2X Receptor Channels.

P2X receptors constitute a gene family of cation channels gated by extracellular ATP. They mediate fast ionotropic purinergic signaling in neurons and non-excitable cell types in vertebrates. The highly calcium-permeable P2X4 subtype has been shown to play a significant role in cardiovascular physiology, inflammatory responses and neuro-immune communication. We previously reported the discovery of a P2X4-selective antagonist, the small organic compound BX430, with submicromolar potency for human P2X4 receptors and marked species-dependence (Ase et al., 2015). The present study investigates the molecular basis of P2X4 inhibition by the non-competitive blocker BX430 using a structural and functional approach relying on mutagenesis and electrophysiology. We provide evidence for the critical contribution of a single hydrophobic residue located in the ectodomain of P2X4 channel subunits, Ile312 in human P2X4, which determines blockade by BX430. We also show that the nature of this extracellular residue in various vertebrate P2X4 orthologs underlies their specific sensitivity or resistance to the inhibitory effects of BX430. Taking advantage of high-resolution crystallographic data available on zebrafish P2X4, we used molecular dynamics simulation to model the docking of BX430 on an allosteric binding site around Ile315 (zebrafish numbering) in the ectodomain of P2X4. We also observed that the only substitution I312D (human numbering) that renders P2X4 silent by itself has also a profound silencing effect on all other P2X subtypes tested when introduced at homologous positions. The generic impact of this aspartate mutation on P2X function indicates that the pre-TM2 subregion involved is conserved functionally and defines a novel allosteric inhibitory site present in all P2X receptor channels. This conserved structure-channel activity relationship might be exploited for the rational design of potent P2X subtype-selective antagonists of therapeutic value.

Point Mutation of Aquaporin 4 Impairs its Binding in Neuromyelitis Optica

Aquaporin 4 (AQP4) is the principal aquaporin expressed in astrocytes throughout the central nervous system. An autoantibody, IgG, that binds to AQP4 leads to a rare disease called neuromyelitis optica (NMO), which is characterized by paralysis and loss of vision. Current treatments for NMO are few and involve non‐specific immunosuppression. The Summit Country Day School MSOE Center for BioMolecular Modeling MAPS Team used 3D modeling and printing technology to examine structure‐function relationships of the AQP4 epitope for NMO‐IgG. Understanding the molecular structure of the AQP4 epitope is an important step in explaining its binding to NMO‐IgG. NMO‐IgG primarily interacts with the accessible AQP4 extracellular loops A, C, and E. These loops change their conformation when AQP4 arrange into orthogonal arrays of particles, thus enabling attachment of NMO‐IgG and facilitating complement binding. Studies on the point mutation of aspartate in position 69 (D69) revealed conformational changes such as 1) reorientation of threonine in position 62, 2) weakened hydrogen bond interaction between leucine in position 53 and threonine in position 56, 3) increased mobility of loop A, and 4) disruption of epitope reorganization of AQP4. Overall, D69 substitution and the subsequent conformational changes in loop A leads to impairment of binding between AQP4 and the NMO‐IgG. Determining the role of extracellular loop A between and within AQP4 monomers will aid in understanding how it contributes to antigen recognition. Prevention of the binding of AQP4 by NMO‐IgG is an important strategy for developing targeted therapy in NMO.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mutated Channelrhodopsins with Increased Sodium and Calcium Permeability

(1) Background: After the discovery and application of Chlamydomonas reinhardtii channelrhodopsins, the optogenetic toolbox has been greatly expanded with engineered and newly discovered natural channelrhodopsins. However, channelrhodopsins of higher Ca2+ conductance or more specific ion permeability are in demand. (2) Methods: In this study, we mutated the conserved aspartate of the transmembrane helix 4 (TM4) within Chronos and PsChR and compared them with published ChR2 aspartate mutants. (3) Results: We found that the ChR2 D156H mutant (XXM) showed enhanced Na+ and Ca2+ conductance, which was not noticed before, while the D156C mutation (XXL) influenced the Na+ and Ca2+ conductance only slightly. The aspartate to histidine and cysteine mutations of Chronos and PsChR also influenced their photocurrent, ion permeability, kinetics, and light sensitivity. Most interestingly, PsChR D139H showed a much-improved photocurrent, compared to wild type, and even higher Na+ selectivity to H+ than XXM. PsChR D139H also showed a strongly enhanced Ca2+ conductance, more than two-fold that of the CatCh. (4) Conclusions: We found that mutating the aspartate of the TM4 influences the ion selectivity of channelrhodopsins. With the large photocurrent and enhanced Na+ selectivity and Ca2+ conductance, XXM and PsChR D139H are promising powerful optogenetic tools, especially for Ca2+ manipulation.