Deletion Of Glycine Research Articles

Identification of major histocompatibility complex genotypes associated with resistance to an amphibian emerging infectious disease.

Amphibian chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd), emerged from Asia and spread globally. By comparing functional MHC IIß1 alleles from an Asian Bd-resistant anuran species (Bufo gargarizans) with those of an Australasian Bd-susceptible species (Litoria caerulea), we identified MHC genotypes with a glycine deletion (G90β1) and adjacent motifs in the deepest pathogen-derived peptide-binding groove associated with Bd resistance. Every Bd-resistant individual, but no susceptible individuals, possessed at least one allele encoding the variant. We detected trans-species polymorphism at the end of the MHC IIβ1 sequences in both species. The G90β1 deletion was encoded by different alleles in the two species, suggesting it may have evolved independently in the two species rather than having been derived from a common ancestor. It is possible that MHC adaptations to chytridiomycosis evolved independently among amphibian species. These results are consistent with a scenario by which MHC adaptations that confer resistance to the pathogen have evolved by convergent evolution. Immunogenetic studies such as this are critical to ongoing conservation efforts.

Reducing Hinge Flexibility of CAR-T Cells Prolongs Survival In Vivo With Low Cytokines Release.

Chimeric antigen receptor (CAR)-modified T cells targeting CD19 demonstrate unparalleled responses in B cell malignancies. However, high tumor burden limits clinical efficacy and increases the risk of cytokine release syndrome and neurotoxicity, which is associated with over-activation of the CAR-T cells. The hinge domain plays an important role in the function of CAR-T cells. We hypothesized that deletion of glycine, an amino acid with good flexibility, may reduce the flexibility of the hinge region, thereby mitigating CAR-T cell over-activation. This study involved generating a novel CAR by deletion of two consecutive glycine residues in the CD8 hinge domain of second-generation (2nd) CAR, thereafter named 2nd-GG CAR. The 2nd-GG CAR-T cells showed similar efficacy of CAR expression but lower hinge flexibility, and its protein affinity to CD19 protein was lower than that of 2nd CAR-T cells. Compared to the 2nd CAR-T cells, 2nd-GG CAR-T cells reduced proinflammatory cytokine secretion without diminishing the specific cytotoxicity toward tumor cells in vitro. Furthermore, 2nd-GG CAR-T cells prolonged overall survival in an immunodeficient mouse model bearing NALM-6 when tumor burden was high. This study demonstrated that a lower-flexibility of CD8α hinge improved survival under high tumor burden and reduced proinflammatory cytokines in preclinical studies. While there is potential for improved safety and efficacy, yet this needs validation with clinical trials.

Emergence of a KPC Variant Conferring Resistance to Ceftazidime-Avibactam in a Widespread ST11 Carbapenem-Resistant Klebsiella pneumoniae Clone in China.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) infection poses a great threat to public health worldwide, and KPC-2-producing strains are the main factors responsible for resistance to carbapenems in China. Ceftazidime/avibactam (CZA) is a novel β-lactam/β-lactamase inhibitor combination with good activity against KPC-2 carbapenemase and is becoming the most important option for treating KPC-producing CRKP infection. Here, we report the emergence of a novel KPC-2 variant, designated KPC-74, produced by K. pneumoniae strain KP55, that conferred CZA resistance in a patient after CZA exposure. The novel blaKPC–74 variant showed a deletion of 6 nucleotides at positions 712–717 compared with blaKPC–2, and this deletion resulted in the consequent deletion of glycine and valine at positions 239 and 240. Antimicrobial susceptibility testing showed that KP55 presents multidrug resistance, including resistance to CZA and ertapenem, but is susceptible to imipenem, meropenem, and colistin. The blaKPC–74 gene was located on a plasmid, as determined by S1-nuclease pulsed-field gel electrophoresis followed by southern blotting, and confirmed to be 133,766 bp in length by whole-genome sequencing on both the Illumina and MinION platforms. The CZA resistance phenotype of the novel KPC variant was confirmed by both transformation of the blaKPC–74-harboring plasmid and a blaKPC–74 gene cloning assay, showing a 64-fold higher CZA minimum inhibitory concentration (MIC) than the recipient strains. The G239_V240del observed in KPC-74 was outside the omega-loop region but was still close to the active site Ser70 and omega-loop in the protein tertiary structure. The enzyme kinetic parameters and IC50 values further indicated that the hydrolytic activity of the KPC-74 enzyme against ceftazidime was potentiated twofold and that the affinity between KPC-74 and avibactam was alleviated 17-fold compared with that of the KPC-2 allele. This CZA resistance mediated by KPC-74 could be selected after CZA therapy and evolved to be more diverse and heterogeneous. Surveillance of CZA resistance is urgently needed in clinical settings.

GPAT4-Generated Saturated LPAs Induce Lipotoxicity through Inhibition of Autophagy by Abnormal Formation of Omegasomes.

SummaryExcessive levels of saturated fatty acids are toxic to vascular smooth muscle cells (VSMCs). We previously reported that mice lacking VSMC-stearoyl-CoA desaturase (SCD), a major enzyme catalyzing the detoxification of saturated fatty acids, develop severe vascular calcification from the massive accumulation of lipid metabolites containing saturated fatty acids. However, the mechanism by which SCD deficiency causes vascular calcification is not completely understood. Here, we demonstrate that saturated fatty acids significantly inhibit autophagic flux in VSMCs, contributing to vascular calcification and apoptosis. Mechanistically, saturated fatty acids are accumulated as saturated lysophosphatidic acids (LPAs) (i.e. 1-stearoyl-LPA) possibly synthesized through the reaction of GPAT4 at the contact site between omegasomes and the MAM. The accumulation of saturated LPAs at the contact site causes abnormal formation of omegasomes, resulting in accumulation of autophagosomal precursor isolation membranes, leading to inhibition of autophagic flux. Thus, saturated LPAs are major metabolites mediating autophagy inhibition and vascular calcification.

Carfentrazone-ethyl resistance in an Amaranthus tuberculatus population is not mediated by amino acid alterations in the PPO2 protein.

To date, the only known mechanism conferring protoporphyrinogen IX oxidase (PPO)-inhibitor resistance in waterhemp (Amaranthus tuberculatus) is a glycine deletion in PPO2 (ΔG210), which results in cross-resistance to foliar PPO-inhibiting herbicides. However, a metabolism-based, HPPD-inhibitor resistant waterhemp population from Illinois (named SIR) was suspected of having a non-target site resistance (NTSR) mechanism due to its resistance to carfentrazone-ethyl (CE) but sensitivity to diphenylethers (DPEs). In greenhouse experiments, SIR sustained less injury than two PPO inhibitor-sensitive populations (WCS and SEN) after applying a field-use rate of CE, and after initial rapid necrosis, regrowth of SIR plants was comparable to a known PPO inhibitor-resistant population (ACR) possessing the ΔG210 mutation. Dose-response analysis determined 50% growth reduction rates in CE-resistant (SIR and ACR) and sensitive (SEN) waterhemp populations, which showed SIR was 30-fold resistant compared to SEN and two-fold more resistant than ACR. Deduced amino acid sequences derived from SIR PPX2 partial cDNAs did not contain the ΔG210 mutation found in ACR or other target-site mutations that confer PPO-inhibitor resistance previously reported in Palmer amaranth (Amaranthus palmeri). Although several SIR cDNAs contained amino acid substitutions, none were uniform among samples. Additionally, SIR plants treated with malathion and CE showed a significant reduction in biomass accumulation compared to CE alone. These results indicate robust CE resistance in SIR is not mediated by amino acid changes in the PPO2 protein, but instead resistance may be conferred through a NTSR mechanism such as enhanced herbicide metabolism.

Mechanisms driving the antibacterial and antibiofilm properties of Hp1404 and its analogue peptides against multidrug-resistant Pseudomonas aeruginosa

Hp1404, identified from the venom of the scorpion Heterometrus petersii, displays antimicrobial activity with cytotoxicity. Several synthetic peptides were designed based on the parent peptide Hp1404 to reduce cytotoxicity and improve activity (deletion of glycine and phenylalanine, substitution with leucine and lysine). The analogue peptides generated comprised 12 amino acids and displayed amphipathic α-helical structures, with higher hydrophobic moments and net positive charge than those of the Hp1404. The analogues showed less hemolytic and toxic effects toward mammalian cells than the Hp1404, especially Hp1404-T1e, which exhibited particularly potent antibacterial and antibiofilm activities against multidrug-resistant Pseudomonas aeruginosa (MRPA) strains. The analogue peptide Hp1404-T1e was more stable against salt and trypsin than the Hp1404. Hp1404’s mechanism of action involves binding to lipopolysaccharide (LPS), thereby killing bacteria through membrane disruption. Hp1404-T1e kills bacteria more rapidly than Hp1404 and not only seems to bind more strongly to LPS but may also be able to enter bacterial cells and interact with their DNA. Additionally, Hp1404-T1e can effectively kill bacteria in vivo. The results of this study indicate that Hp1404-T1e not only displays antimicrobial activity, but is also functional in physiological conditions, confirming its potential use as an effective therapeutic agent against MRPA.

Two new PPX2 mutations associated with resistance to PPO-inhibiting herbicides in Amaranthus palmeri.

Resistance to herbicides that inhibit protoporphyrinogen oxidase (PPO) is a widespread and growing problem for weed managers across the midwestern and midsouthern United States. In Amaranthus spp., this resistance is known to be conferred by a glycine deletion at the 210th amino acid (ΔG210) in PPO2. Preliminary analysis indicated that the ΔG210 mutation did not fully account for observed resistance to PPO inhibitors in two Amaranthus palmeri populations from Tennessee and one from Arkansas. Sequencing PPX2 cDNA from six resistant plants uncovered two new mutations at the R98 site (R98G and R98M), a site previously found to endow PPO-inhibitor resistance in Ambrosia artemisiifolia. Sequencing of this region from additional plants sprayed with 264 g fomesafen ha-1 showed the presence of one or both R98 mutations in a subset of the resistant plants from all three populations. No plants sensitive to fomesafen contained either mutation. A derived cleaved amplified polymorphic sequence (dCAPS) assay to test for the presence of these mutations in A. palmeri was developed. Two new mutations of PPX2 (R98G, R98M) likely confer resistance to PPO-inhibitors in A. palmeri, and can be rapidly identified using a dCAPS assay. © 2017 Society of Chemical Industry.

An Unusual Cause of Elevated LFTs: Progressive Familial Intrahepatic Cholestasis

Introduction: A 40-year-old otherwise healthy male presented to the GI clinic for follow-up of abnormal LFTs detected on a routine evaluation. The AST was 57 U/L, ALT 88 U/L, and ALP 214 U/L. There was no history of alcohol consumption or blood transfusion. Testing for hepatitis A, B, C, Wilson’s disease, autoimmune hepatitis, hemochromatosis, and alpha-1 antitrypsin deficiency was negative. Ultrasound and CT of the abdomen were unremarkable. MRCP with MR elastography revealed normal liver contour, with mild dilatation of a portion of the right intrahepatic biliary ducts and a mean liver stiffness value of 3.25 kPa. A liver biopsy showed evidence of disrupted hepatic architecture with micronodular cirrhosis. The histology findings were thought to be due to focal liver injury, possibly in or near the location of his prior cholecystectomy. One year later, his AST was 110 U/L, ALT 130 U/L, ALP 374 U/L, and total bilirubin 1.1 mg/dL. The mean stiffness on MR was 5.62 kPa. At that time, the patient revealed that his brother had just been diagnosed with esophageal varices. Given the abrupt change in liver stiffness over 12 months and a family history of esophageal varices, congenital hepatic fibrosis was suspected. Both the patient and his brother then tested positive for a novel heterozygous mutation: c.3531_3533delACG variant that causes in-frame deletion of Glycine at position 1178 of the ABCB4 protein. Thus, the diagnosis of progressive familial intrahepatic cholestasis (PFIC) type 3 was confirmed. The patient was started on ursodeoxycholic acid (UDCA) and his subsequent LFTs gradually normalized. PFIC type 3 is an autosomal recessive disease caused by mutations in ABCB4 gene, which encodes the multidrug resistance - 3 (MDR3) gene, an ATP-binding cassette transporter. It acts as a phospholipid trans-locator involved in biliary phospholipid excretion. ABCB4 defects cause impaired excretion of phosphatidylcholine, an important component in formation of mixed micelles that protect the cell membrane of biliary epithelium from the detergent action of bile salts. Functional deficiency of ABCB4 solubilizes the cell membrane of the biliary epithelium inducing apoptosis. Diagnostic features include elevated serum bile salts, increased GGT, light microscopy findings of ductular proliferation, and inflammatory infiltrate in early stage, progressing to cirrhosis and cholestasis in later stages. Typically, PFIC type 3 is diagnosed in childhood or early adulthood. Rarely, it can also present much later in life, as in this patient. Manifestations are limited to the liver only. UDCA is helpful in about 50% and transplantation is required for hepatic failure.

In vitro culture and structural differences in the major immunoreactive protein gp36 of geographically distant Ehrlichia canis isolates

Ehrlichia canis, the etiologic agent of canine ehrlichiosis, is an obligate intracytoplasmic Gram-negative tick-borne bacterium belonging to the Anaplasmataceae family. E. canis is distributed worldwide and can cause serious and fatal infections in dogs. Among strains of E. canis, the 16S rRNA gene DNA sequences are highly conserved. Using this gene to genetically differentiate isolates is therefore difficult. As an alternative, the gene gp36, which encodes for a major immunoreactive protein in E. canis, has been successfully used to characterize the genetic diversity of this pathogen. The present study describes the isolation and continuous propagation of a Spanish and 2 South African isolates of E. canis in IDE8 tick cells. Subsequently, canine DH82 cell cultures were infected using initial bodies obtained from infected IDE8 cultures. It was possible to mimic the life cycle of E. canis in vitro by transferring infection from tick cells to canine cells and back again. To characterize these E. canis strains at the molecular level, the 16S rRNA and gp36 genes were amplified by PCR, sequenced, and aligned with corresponding sequences available in GenBank. All 16S rRNA sequences amplified in this study were identical to previously reported E. canis strains. Maximum likelihood analysis based on the gp36 amino acid sequences showed that the South African and Spanish strains fall into 2 well-defined phylogenetic clusters amongst other E. canis strains. The members of these 2 phylogenetic clusters shared 2 unique molecular properties in the gp36 amino acid sequences: (i) deletion of glycine 117 and (ii) the presence of an additional putative N-linked glycosylation site. We further show correlation between the putative secondary structure and the theoretical isoelectric point (pI) of the gp36 amino acid sequences. A putative role of gp36 as an adhesin in E. canis is discussed. Overall, we report the successful in vitro culture of 3 new E. canis strains which present different molecular properties in their gp36 sequences.

In vivo imaging and quantitative monitoring of autophagic flux in tobacco BY-2 cells

Autophagy has been shown to play essential roles in the growth, development and survival of eukaryotic cells. However, simple methods for quantification and visualization of autophagic flux remain to be developed in living plant cells. Here, we analyzed the autophagic flux in transgenic tobacco BY-2 cell lines expressing fluorescence-tagged NtATG8a as a marker for autophagosome formation. Under sucrose-starved conditions, the number of punctate signals of YFP-NtATG8a increased, and the fluorescence intensity of the cytoplasm and nucleoplasm decreased. Conversely, these changes were not observed in BY-2 cells expressing a C-terminal glycine deletion mutant of the NtATG8a protein (NtATG8aΔG). To monitor the autophagic flux more easily, we generated a transgenic BY-2 cell line expressing NtATG8a fused to a pH-sensitive fluorescent tag, a tandem fusion of the acid-insensitive RFP and the acid-sensitive YFP. In sucrose-rich conditions, both fluorescent signals were detected in the cytoplasm and only weakly in the vacuole. In contrast, under sucrose-starved conditions, the fluorescence intensity of the cytoplasm decreased, and the RFP signal clearly increased in the vacuole, corresponding to the fusion of the autophagosome to the vacuole and translocation of ATG8 from the cytoplasm to the vacuole. Moreover, we introduce a novel simple easy way to monitor the autophagic flux non-invasively by only measuring the ratio of fluorescence of RFP and YFP in the cell suspension using a fluorescent image analyzer without microscopy. The present in vivo quantitative monitoring system for the autophagic flux offers a powerful tool for determining the physiological functions and molecular mechanisms of plant autophagy induced by environmental stimuli.

Biochemical and structural consequences of a glycine deletion in the α-8 helix of protoporphyrinogen oxidase

A rare Gly210 deletion in protoporphyrinogen oxidase (PPO) was recently discovered in herbicide-resistant Amaranthus tuberculatus. According to the published X-ray structure of Nicotiana tabacum PPO, Gly210 is adjacent to, not in, the PPO active site, so it is a matter of interest to determine why its deletion imparts resistance to herbicides. In our kinetic experiments, this deletion did not affect the affinity of protoporphyrinogen IX nor the FAD content, but decreased the catalytic efficiency of the enzyme. The suboptimal Kcat was compensated by a significant increase in the Kis for inhibitors and a switch in their interactions from competitive to mixed-type inhibition. In our protein modeling studies on herbicide-susceptible PPO and resistant PPO, we show that Gly210 plays a key role in the alphaL helix-capping motif at the C-terminus of the alpha-8 helix which helps to stabilize the helix. In molecular dynamics simulations, the deletion had significant architecture consequences, destabilizing the alpha-8 helix-capping region and unraveling the last turn of the helix, leading to enlargement of the active site cavity by approximately 50%. This seemingly innocuous deletion of Gly210 of the mitochondrial PPO imparts herbicide resistance to this dual-targeted protein without severely affecting its normal physiological function, which may explain why this unusual mutation was the favored evolutionary path for achieving resistance to PPO inhibitors.

S38151 [ p-guanidinobenzoyl-[Des-Gly 10]-MCH(7-17)] is a potent and selective antagonist at the MCH 1 receptor and has anti-feeding properties in vivo

Structure–activity relationships studies have established the minimal sequence of melanin-concentrating hormone (MCH) that retains full agonist potency at the MCH 1, to be the dodecapeptide MCH(6-17). The α-amino function is not required for activity since arginine 6 can be replaced by p-guanidinobenzoyl, further improving activity. We report that the deletion of glycine in this short potent agonist (EC 50 3.4 nM) turns it into a potent and new MCH 1 antagonist (S38151, K B 4.3 nM in the [ 35S]-GTPγS binding assay), which is selective versus MCH 2. A compared Ala-scan of the agonist and antagonist sequences reveals major differences in the residues that are mandatory for affinity, including arginine 11 and tyrosine 13 for the agonist and leucine 9 for the antagonist, whereas methionine 8 was necessary for both agonist and antagonist activities. A complete molecular study of the antagonist behavior is described in the present report, with a particular focus on the description of several analogues, attempting to find structure–activity relationships. Finally, S38151 antagonizes food intake when injected intra-cerebroventricularly in the rat. This is in agreement with the in vitro data and with our previous demonstration of a good correlation between in vitro and in vivo data on MCH 1 agonists.

β‐Defensin 3: a novel and unexpected key that unlocks the melanocortin 1 receptor

β‐Defensin 3: a novel and unexpected key that unlocks the melanocortin 1 receptor

The alpha-synuclein mutation E46K promotes aggregation in cultured cells

Parkinson’s disease (PD) is characterized by the polymerization of wild-type (WT) or mutant alpha-synuclein (AS) into aggregates and fibrils, which are observed as Lewy bodies (LBs) and Lewy neurites (LNs) in PD patients. However, inability to demonstrate aggregation in many cell culture systems is a major drawback for effective in vitro modeling of AS aggregation. Utilizing PCR-based cloning approach, we generated A30P, A53T, and the recently reported E46K encoding mutation in the KTKEGV repeat region of AS gene. While cloning E46K mutant, a glycine deletion mutation (E46KΔG) adjacent to the intended lysine mutation was serendipitously generated. Expression of mutant constructs and green fluorescent protein (GFP)-tagged mutant constructs in catecholaminergic SH-SY5Y (5Y) cells revealed 40% of AS-E46KΔG and 18% of AS-E46K transfected cells formed aggregates as compared to 12% in AS-A53T, 6% in AS-WT, and 2% in AS-A30P transfected cells. Western blot analysis demonstrated the formation of high molecular weight AS aggregates. Electron microscopic analysis of 5Y cells expressing the E46K and E46KΔG mutants demonstrated two distinct kinds of inclusions: Type I, which showed dense granular profile; and Type II, which were largely membranous vacuolar inclusions without granular material. These two inclusions are reminiscent of Lewy bodies and pale bodies observed in PD postmortem brain samples. Our results demonstrate that mutations in 4th KTKEGV repeat lead to higher propensity of aggregation of AS compared to other mutants.

Characterization of collagen-like peptides containing interruptions in the repeating Gly-X-Y sequence.

Glycine is found as every third residue along the entire length of triple helices in fibrillar collagens, but the triple-helix regions of nonfibrillar collagens and other proteins usually contain one or more interruptions in this repeating pattern. A set of four peptides was designed to model the effect of interruptions in the (Gly-X-Y)n repeating pattern on triple-helix formation, stability, and folding. Into the middle of the stable triple-helical peptide (Pro-Hyp-Gly)10, an interruption was introduced representing one of the four possible categories: a glycine deletion, a deletion of a hydroxyproline (Y position), an alanine insertion, or a glycine to alanine substitution. As shown by sedimentation equilibrium, NMR, and CD studies, the introduction of an interruption still allowed formation of trimers in solution, but with marked decrease in stability. The degree of destabilization and the thermodynamic basis for the loss of stability depended on the type of interruption. The glycine substitution and alanine insertion were the least disruptive, followed by the hydroxyproline deletion, with the glycine deletion being the most destabilizing. Our results suggest that the breaks in these peptides affect both the triple-helical conformation and the monomer conformation. These studies provide a basis for considering the structural and functional consequences of different kinds of interruptions in collagen.