Arg Substitution Research Articles

Protein engineering enables Serratia marcescens nuclease a to hydrolyze nucleic acids under high-salt conditions.

For the purpose of removing nucleotide impurities, Serratia marcescens Nuclease A (SmNucA) is widespread used in biopharmaceutical manufacturing, such as production of adeno-associated viral vector, one of the leading gene delivery platforms that features low immunogenicity. However, such utilization of wild-type SmNucA is limited in saline environment. Depending on downstream process development, the ionic strength can be as high as 500 mM, at which the potency of wild-type SmNucA plunges. We herein design an SmNucA variant with four Lys mutations, namely HighSalt NucA, to improve nucleic acids binding under high-salt conditions. Km determination and molecular dynamics (MD) simulation implies a new catalytical mechanism adopted by HighSalt NucA and another mutant that harbors four Arg substitutions at the same sites. We thereby conclude that basic-residue mutations on the SmNucA surface stabilize the local conformation in close proximity to the substrate-binding cleft at saline concentration of 500 mM. In addition to Lys and Arg mutations, saturation mutagenesis further indicated that certain hydrophobic residues (Ala, Val, Trp, Tyr) and polar residues (Ser, Thr, Asn, Gln) also render SmNucA salt-tolerant, albeit to differing extent. In contrast to activity loss of the wild-type with 400-500 mM NaCl, HighSalt NucA maintains broad substrate specificity even under these extreme conditions, which expands its application prospect in the removal of nucleic acid impurities during process development.

Predicting the pathogenicity of missense variants based on protein instability to support diagnosis of patients with novel variants of ARSL

Rare diseases are estimated to affect 3.5%–5.9% of the population worldwide and are difficult to diagnose. Genome analysis is useful for diagnosis. However, since some variants, especially missense variants, are also difficult to interpret, tools to accurately predict the effect of missense variants are very important and needed. Here we developed a method, “VarMeter”, to predict whether a missense variant is damaging based on Gibbs free energy and solvent-accessible surface area calculated from the AlphaFold 3D protein model. We applied this method to the whole-exome sequencing data of 900 individuals with rare or undiagnosed disease in our in-house database, and identified four who were hemizygous for missense variants of arylsulfatase L (ARSL; known as the genetic cause of chondrodysplasia punctata 1, CPDX1). Two individuals had a novel Ser89 to Asn (Ser89Asn) or Arg469 to Trp (Arg469Trp) substitution, respectively predicted as “damaging” or “benign”; the other two had an Arg111 to His (Arg111His) or Gly117 to Arg (Gly117Arg) substitution, respectively predicted as “damaging” or “possibly damaging” and previously reported in patients showing clinical manifestations of CDPX1. Expression and analysis of the missense variant proteins showed that the predicted pathogenic variants (Ser89Asn, Arg111His, and Gly117Arg) had complete loss of sulfatase activity and reduced protease resistance due to destabilization of protein structure, while the predicted benign variant (Arg469Trp) had activity and protease resistance comparable to those of wild-type ARSL. The individual with the novel pathogenic Ser89Asn variant exhibited characteristics of CDPX1, while the individual with the benign Arg469Trp variant exhibited no such characteristics. These findings demonstrate that VarMeter may be used to predict the deleteriousness of variants found in genome sequencing data and thereby support disease diagnosis.

SAT145 Familial Renal Glycosuria - A Differential Diagnosis For Hypoglycemia

Abstract Disclosure: S. Syed: None. S. Naqvi: None. L. Menon: None. J. Holthoff: None. A.R. Kasireddy: None. S. Alshami: None. Background: Familial renal glycosuria can be an uncommon cause of hypoglycemia. We present the case of a woman who was referred to the endocrinology clinic for further evaluation of hypoglycemia and was found to have familial renal glycosuria due to a novel mutation in the solute carrier family 5 member 2 (SCL5A2) gene. Case presentation: A 24 year-old woman with past medical history of rheumatoid arthritis, hyperlipidemia and renal glycosuria presented for persistent hypoglycemia. She first started noticing these episodes four years ago and they have increased over the last year. These events occur in the early hours of the morning or overnight despite a high carbohydrate meal at bedtime. She had the following labs: fasting C-peptide 1.04 (0.9-4.3 ng/ml), insulin 4.1 (2.4-21.8 microU/ml), fasting glucose 84 (70-110 mg/dl) and HbA1c 4.8%. DEXCOM download showed hypoglycemic episodes 2-3 hours after meals. On urinalysis, glucose was >1000 mg/dl and a 24 hour urine collection for glucose was 16.8 g/24 hours. She was first diagnosed with asymptomatic glycosuria during her first pregnancy 7 years ago but the cause was never investigated. After seeing endocrinology, she was referred to nephrology who ordered further workup. Initial markers of a complete Fanconi syndrome were normal: urine pH 5.5, magnesium and phosphorus were normal. Genetic testing was done and the patient was found to have a missense mutation in the SCL5A2 gene which results in a single amino acid substitution of Arg to Cys at codon 60 in exon 2 and encodes the SGLT2 protein. On review of literature, this particular mutation has never been reported and the inheritance pattern is likely to be autosomal dominant since she had a heterozygous mutation. The hypoglycemia was noted to be reactive and occurred after meals. She was advised to avoid simple sugars which can precipitate reactive hypoglycemia within a couple of hours of eating foods with a high glycemic index. She also has a bicornuate uterus and history of recurrent urinary tract infections. Out of her 4 children, two have omphalocele and genetic analysis showed that they are recessive carriers for Smith-Lemi-Optiz syndrome. One of those children suffered from hypoglycemia following birth with prolonged NICU stay. No urinalysis or genetic analysis is available for that child. Her third child was born premature and had hydronephrosis. Conclusion: In literature, a total of 86 mutations of the SLC5A2 gene have been reported to be associated with familial renal glycosuria. There are case reports on some of these mutations also causing hypoglycemia. Our case highlights that renal glycosuria should be considered as a differential when evaluating patients with hypoglycemia since these may be correlated. The patient had a novel mutation causing glycosuria, hypoglycemia and could be associated with congenital malformations. More studies on this association are needed. Presentation: Saturday, June 17, 2023

Identification of novel homozygous missense and deletion mutations manifesting oligospermia infertility in Kashmiri population.

Human male infertility has a lot of known molecular components that have an accurate diagnosis, such as Y chromosome deletion and monogenic causes. Only 4% of all infertile males are diagnosed with genetic causes, while 60-70% of infertile men remain without an accurate diagnosis and are classified as unexplained. Oligospermia is a major cause of human male infertility. Its etiology and pathogenesis are linked to genetic abnormalities. The majority of genetic causes related to human male infertility remain unclear. Generally, we found a significant association between the specific type of disease and gender (p= 0.003), and the regression value (R2 ) for this association was 0.75. Association of the type of disease with body mass index was not significant (p= 0.34). There was no statistically significant difference (p= 0.40) among disease types with patients occupations. All explored mutations are listed for primary and secondary infertility in relation to the oligospermia condition. p.Arg286X is the outcome of a mismatch mutation in which the nucleotide change resulted in the substitution of Arg (arginine) amino acid with X (any amino acid) at position 286 in the Hyal3 gene of primary infertile patients having oligospermia. In primary infertile patients with the p.Arg286X mutation, a frameshift deletion mutation was also found just after the 25 nucleotide sequences of the Hyal3 genes of the second mutated exon. This deletion mutation was only detected in patients with primary infertility and was not found in people with secondary infertility or healthy controls. The other mutations in secondary infertile patients with oligospermia were: p.Lys168Ser, replacement of lysine (Lys) with serine (Ser) at position 168; p.Lys168The, replacement of lysine (Lys) with threonine (The) at position 168; p.His113X, substitution of histidine (His) with an unknown amino acid (X) at position 113; p.Pro162X, substitution of proline (Pro) with an unknown amino acid (X) at position 162; and p.Phe157X, phenylalanine (Phe) substitution with an unknown amino acid (X) at position 157. This study clarifies the site of novel mismatch and frameshift deletion mutations in the Hyal3 gene in primary infertile oligospermia patients.

Functional Delineation of a Protein-Membrane Interaction Hotspot Site on the HIV-1 Neutralizing Antibody 10E8.

Antibody engagement with the membrane-proximal external region (MPER) of the envelope glycoprotein (Env) of HIV-1 constitutes a distinctive molecular recognition phenomenon, the full appreciation of which is crucial for understanding the mechanisms that underlie the broad neutralization of the virus. Recognition of the HIV-1 Env antigen seems to depend on two specific features developed by antibodies with MPER specificity: (i) a large cavity at the antigen-binding site that holds the epitope amphipathic helix; and (ii) a membrane-accommodating Fab surface that engages with viral phospholipids. Thus, besides the main Fab–peptide interaction, molecular recognition of MPER depends on semi-specific (electrostatic and hydrophobic) interactions with membranes and, reportedly, on specific binding to the phospholipid head groups. Here, based on available cryo-EM structures of Fab–Env complexes of the anti-MPER antibody 10E8, we sought to delineate the functional antibody–membrane interface using as the defining criterion the neutralization potency and binding affinity improvements induced by Arg substitutions. This rational, Arg-based mutagenesis strategy revealed the position-dependent contribution of electrostatic interactions upon inclusion of Arg-s at the CDR1, CDR2 or FR3 of the Fab light chain. Moreover, the contribution of the most effective Arg-s increased the potency enhancement induced by inclusion of a hydrophobic-at-interface Phe at position 100c of the heavy chain CDR3. In combination, the potency and affinity improvements by Arg residues delineated a protein–membrane interaction site, whose surface and position support a possible mechanism of action for 10E8-induced neutralization. Functional delineation of membrane-interacting patches could open new lines of research to optimize antibodies of therapeutic interest that target integral membrane epitopes.

A Novel Frame-Shift Mutation in SCNN1B Identified in a Chinese Family Characterized by Early-Onset Hypertension.

BackgroundLiddle syndrome is a form of monogenic hypertension caused by mutations in the three homologous subunits of the epithelial sodium channels (ENaCs), α, β, and γ. It is characterized by early-onset refractory hypertension, hypokalemia, low renin activity, and hypoaldosteronism. In this study, we report a novel frame-shift mutation in SCNN1B responsible for Liddle syndrome in a Chinese family.MethodsDNA samples were collected from all participants. Whole-exome sequencing was performed in the proband to detect possible causative variants. Sanger sequencing was then conducted in the other family members to verify the candidate variant, and in 100 patients with hypertension and 100 normotensive controls to exclude population genetic polymorphism.ResultsWe identified a novel frame-shift mutation (c.1691_1693delinsG) in SCNN1B that was responsible for Liddle syndrome in this family. This mutation leads to the substitution of Arg in place of Gln at codon site 564 and generates a new stop codon at 592, influencing the crucial PY motif and resulting in reduced inactivation of the ENaCs. Aside from the proband, eight family members carried the mutation. Intra-familial phenotypic heterogeneity was observed in the blood pressure and serum potassium levels. Amiloride therapy combined with a low sodium diet is effective to alleviate the symptoms of patients with Liddle syndrome.Conclusionc.1691_1693delinsG, a novel frame-shift mutation in the β subunit of ENaC, was identified in a Chinese family with Liddle syndrome by whole-exome sequencing. Phenotypic heterogeneity can make diagnosis of Liddle syndrome difficult on the basis of clinical or biochemical characteristics alone. Genetic analysis is a useful tool allowing timely and accurate diagnosis of Liddle syndrome and playing a guiding role in precise treatment of the disease.

Metabolism of non-steroidal anti-inflammatory drugs (NSAIDs) by Streptomyces griseolus CYP105A1 and its variants

In the field of drug development, technology for producing human metabolites at a low cost is required. In this study, we explored the possibility of using prokaryotic water-soluble cytochrome P450 (CYP) to produce human metabolites. Streptomyces griseolus CYP105A1 metabolizes various non-steroidal anti-inflammatory drugs (NSAIDs), including diclofenac, mefenamic acid, flufenamic acid, tolfenamic acid, meclofenamic acid, and ibuprofen. CYP105A1 showed 4′-hydroxylation activity towards diclofenac, mefenamic acid, flufenamic acid, tolfenamic acid, and meclofenamic acid. It should be noted that this reaction specificity was similar to that of human CYP2C9. In the case of mefenamic acid, another metabolite, 3′-hydroxymethyl mefenamic acid, was detected as a major metabolite. Substitution of Arg at position 73 with Ala in CYP105A1 dramatically reduced the hydroxylation activity toward diclofenac, flufenamic acid, and ibuprofen, indicating that Arg73 is essential for the hydroxylation of these substrates. In contrast, substitution of Arg84 with Ala remarkably increased the hydroxylation activity towards diclofenac, mefenamic acid, and flufenamic acid. Recombinant Rhodococcus erythrocyte cells expressing the CYP105A1 variant R84A/M239A showed complete conversion of diclofenac into 4′-hydroxydiclofenac. These results suggest the usefulness of recombinant R. erythropolis cells expressing actinomycete CYP, such as CYP105A1, for the production of human drug metabolites.

Prediction of ceftriaxone MIC inNeisseria gonorrhoeae usingDNA microarray technology and regression analysis

Decreased susceptibility of Neisseria gonorrhoeae to extended-spectrum cephalosporins is a major concern. Elucidation of the phenotypic and genetic characteristics of such isolates is a priority task. We developed a method for predicting the N. gonorrhoeae ceftriaxone susceptibility level (MICcro) by identifying genetic determinants of resistance using low-density hydrogel microarrays and a regression equation. A training dataset, containing 5631 isolates from the Pathogenwatch database and 181 isolates obtained in the Russian Federation during 2018-19, was used to build a regression model. The regression equation was tested on 14 WHO reference strains. Ceftriaxone resistance determinants for the 448 evaluated clinical isolates collected in Russia were identified using microarray analysis, and MICcro values were calculated using the regression equation and compared with those measured by the serial dilution method. The regression equation for calculating MICcro values included 20 chromosomal resistance determinants. The greatest contributions to the increase in MICcro were shown to be PBP2: Ala-501→Pro, Ala-311→Val, Gly-545→Ser substitutions, Asp(345-346) insertion; and PorB: Gly-120→Arg substitution. The substitutions PBP2: Ala-501→Thr/Val, PorB: Gly-120→Asn/Asp/Lys and PBP1: Leu-421→Pro had weaker effects. For 94.4% of the isolates in the evaluation set, the predicted MICcro was within one doubling dilution of the experimentally determined MICcro. No ceftriaxone-resistant isolates were identified in the analysed samples from Russia, and no interpretative errors were detected in the MICcro calculations. The developed strategy for predicting ceftriaxone MIC can be used for the continuous surveillance of known and emerging resistant N. gonorrhoeae isolates.

A Systematic Study of the Stability, Safety, and Efficacy of the de novo Designed Antimicrobial Peptide PepD2 and Its Modified Derivatives Against Acinetobacter baumannii.

Searching for new antimicrobials is a pressing issue to conquer the emergence of multidrug-resistant (MDR) bacteria and fungi. Antimicrobial peptides (AMPs) usually have antimicrobial mechanisms different from those of traditional antibiotics and bring new hope in the discovery of new antimicrobials. In addition to antimicrobial activity, stability and target selectivity are important concerns to decide whether an antimicrobial peptide can be applied in vivo. Here, we used a simple de novo designed peptide, pepD2, which contains only three kinds of amino acid residues (W, K, L), as an example to evaluate how the residues and modifications affect the antimicrobial activity against Acinetobacter baumannii, stability in plasma, and toxicity to human HEK293 cells. We found that pepI2 with a Leu→Ile substitution can decrease the minimum bactericidal concentrations (MBC) against A. baumannii by one half (4 μg/mL). A D-form peptide, pepdD2, in which the D-enantiomers replaced the L-enantiomers of the Lys(K) and Leu(L) residues, extended the peptide half-life in plasma by more than 12-fold. PepD3 is 3-residue shorter than pepD2. Decreasing peptide length did not affect antimicrobial activity but increased the IC50 to HEK293 cells, thus increased the selectivity index (SI) between A. baumannii and HEK293 cells from 4.7 to 8.5. The chain length increase of the N-terminal acyl group and the Lys→Arg substitution greatly enhanced the hemolytic activity, hence those modifications are not good for clinical application. Unlike colistin, the action mechanism of our peptides relies on negatively charged lipids rather than lipopolysaccharides. Therefore, not only gram-negative bacteria but also gram-positive bacteria can be killed by our peptides.

Molecular Mechanisms of the Deregulation of Muscle Contraction Induced by the R90P Mutation in Tpm3.12 and the Weakening of This Effect by BDM and W7.

Point mutations in the genes encoding the skeletal muscle isoforms of tropomyosin can cause a range of muscle diseases. The amino acid substitution of Arg for Pro residue in the 90th position (R90P) in γ-tropomyosin (Tpm3.12) is associated with congenital fiber type disproportion and muscle weakness. The molecular mechanisms underlying muscle dysfunction in this disease remain unclear. Here, we observed that this mutation causes an abnormally high Ca2+-sensitivity of myofilaments in vitro and in muscle fibers. To determine the critical conformational changes that myosin, actin, and tropomyosin undergo during the ATPase cycle and the alterations in these changes caused by R90P replacement in Tpm3.12, we used polarized fluorimetry. It was shown that the R90P mutation inhibits the ability of tropomyosin to shift towards the outer domains of actin, which is accompanied by the almost complete depression of troponin’s ability to switch actin monomers off and to reduce the amount of the myosin heads weakly bound to F-actin at a low Ca2+. These changes in the behavior of tropomyosin and the troponin–tropomyosin complex, as well as in the balance of strongly and weakly bound myosin heads in the ATPase cycle may underlie the occurrence of both abnormally high Ca2+-sensitivity and muscle weakness. BDM, an inhibitor of myosin ATPase activity, and W7, a troponin C antagonist, restore the ability of tropomyosin for Ca2+-dependent movement and the ability of the troponin–tropomyosin complex to switch actin monomers off, demonstrating a weakening of the damaging effect of the R90P mutation on muscle contractility.

In vivo modulation of a dominant‐negative variant in mouse models of von Willebrand disease type 2A

Unlabelled Box AbstractBackgroundTreatment options for patients suffering from von Willebrand disease (VWD) are limited. Von Willebrand factor (VWF) is a polymeric protein that undergoes regulated dimerization and subsequent multimerization during its biosynthesis. Numerous heterozygous variants within the VWF gene display a dominant‐negative effect and result in severe VWD. Previous studies have suggested that preventing the assembly of wild‐type and mutant heteropolymers using siRNAs may have beneficial effects on VWF phenotypes in vitro. ObjectivesTo study heterozygous dominant‐negative variants in vivo, we developed a mouse model of VWD‐type 2A and tested two independent strategies to modulate its detrimental effect. MethodsThe p.P1127_C1948delinsR deletion/variant, causing defective VWF multimerization, was expressed in mice as a model of VWD‐type 2A variant. Two corrective strategies were applied. For the first time in a mouse model of VWD, we applied siRNAs selectively inhibiting translation of the mutant transcripts and we combined the VWD‐type 2A deletion with the Cys to Arg substitution at position 2773, which is known to prevent dimerization. ResultsThe RNA silencing approach induced a modest but consistent improvement of the VWF multimer profile. However, due to incomplete efficiency, the dominant‐negative effect of the original variant could not be completely prevented. In contrast, the DNA approach resulted in increased antigen levels and restoration of a normal multimer profile. ConclusionsOur data showed that preventing the detrimental impact of dominant‐negative VWF variants by independent molecular mechanisms has beneficial consequences in vivo, in mouse models of dominant VWD.

The Role Of Amino Acid Arginine And Nitric Oxide System In Implementing Cardioprotective Effect Of Non-Opioid Analogue Of Leu-Enkephalin In Newborn Albino Rats After Intrauterine Hypoxia

Objective ― to evaluate the role of the amino acid arginine in the structure of the non-opioid analogue of leu-enkephalin (NALE) and the involvement of the nitric oxide system in the implementation of its cardioprotective effect in newborn albino rats subjected to intrauterine hypoxia. Material and Methods ― Pregnant female rats were subjected daily to 4-hour hypobaric hypoxia (oxygen partial pressure – 65 mm Hg) on days 15-19 of their gestation. The 7-day-old offspring of hypoxified female rats were examined. The progeny of intact animals served the control. We studied body and heart weights; activity of proliferative processes and autophagy in the myocardium of subendocardial parts of the left ventricle, expressed via the immunohistochemical detection of Ki-67 and Beclin-1 proteins, respectively; karyometric and nucleolometric indicators of cardiomyocytes (CMC); intensity of free radical processes in the tissues of the heart by chemiluminescence parameters. Correction of post-hypoxic changes in newborn rats was carried out by intraperitoneal injection of two peptides (Phe–D-Ala–Gly–Phe–Leu–Arg – non-opioid analogue of leu-enkephalin, or NALE, and Phe–D-Ala–Gly–Phe–Leu–Gly – G peptide) daily from day 2 through day 6 of their lives at a dose of 100 μg/kg. To assess the involvement of the nitric oxide system in the implementation of the NALE effects, the NO synthase inhibitor – N-nitro-L-arginine methyl ester (L-NAME) was additionally administered at a dose of 50 mg/kg. Results ― Intrauterine hypoxia led to a decrease in body weights of 7-day-old animals, an increase in the number of CMC expressing the Beclin-1 protein, reduction in the size of CMC nuclei, activation of free radial oxidation, and a decrease in antiradical protection in the heart tissues. The administration of NALE to newborn animals, subjected to intrauterine hypoxia (IUH), normalized their body weight and size of the CMC nuclei, and partially corrected changes in Beclin-1 expression and in chemiluminescence parameters. In 7-day-old animals, subjected to IUH and neonatal administration of NALE and L-NAME, a lower body weight was observed than in the control. Against the background of nitric oxide blockade, the antioxidant effect of NALE diminished, but the corrective effect of NALE on the karyometric index and Beclin-1 expression remained. G peptide, which differs from NALE by the substitution of the C-terminal amino acid Arg for the amino acid Gly, exhibited a corrective effect similar to NALE on the consequences of IUH. Conclusion ― Administration of NALE and G peptides to newborn albino rats after IUH has a pronounced cardioprotective effect. The mechanisms of the NALE peptide effects are, in part, associated with the activation of the NOS-NO system. However, the affinity of this peptide for opioid-like receptors may be of greater importance.

A structure-function study of C-terminal residues predicted to line the export channel in Salmonella Flagellin

BackgroundStructural studies of a Salmonella Typhimurium flagellin protein indicated that four polar or charged C-terminal amino acid residues line the inner channel of the flagellum. The hydrophilic character of these putative channel-lining residues was predicted to be essential to facilitate the transport of unfolded flagellin monomers during flagellar assembly. The structure-function relationship of these putative channel-lining residues was investigated by site-directed mutagenesis to examine effects of side chain polarity and size on flagella assembly and function. MethodsChannel-lining residue variants were generated using site-directed mutagenesis to substitute alanine and other residues to examine the effects of altered side-chain polarity on export and assembly. The export, in vivo motility function, and flagellar structure of variants was characterized by agar motility, video microscopy, immunofluorescence, and SDS-PAGE. ResultsAlanine substitution yielded decreased motility and flagellar assembly for three of the four residues. However, alanine substitution of residue Arg 494 did not alter export, although substitution with negatively charged glutamate decreased motility and flagellar filament length. Furthermore, many of the C-terminal mutations affected flagellar filament morphology and stability, often resulting in more tightly coiled and/or more brittle flagella than the wild type. ConclusionsThe four channel-lining C-terminal residues may facilitate monomer protein transport but also have structural roles in determining the stability and morphology of the flagellum. General significanceThese results provide further insight into the complex process of bacterial flagellin export and flagellar assembly and provide evidence of previously unknown structural functions for the four putative channel-lining residues.

Selective anticancer activity of synthetic peptides derived from the host defence peptide tritrpticin

Antimicrobial peptides (AMPs) constitute a diverse family of peptides with the ability to protect their host against microbial infections. In addition to their ability to kill microorganisms, several AMPs also exhibit selective cytotoxicity towards cancer cells and are collectively referred to as anticancer peptides (ACPs). Here a large library of AMPs, mainly derived from the porcine cathelicidin peptide, tritrpticin (VRRFPWWWPFLRR), were assessed for their anticancer activity against the Jurkat T cell leukemia line. These anticancer potencies were compared to the cytotoxicity of the peptides towards normal cells isolated from healthy donors, namely peripheral blood mononuclear cells (PBMCs) and red blood cells (RBCs; where hemolytic activity was assessed). Among the active tritrpticin derivatives, substitution of Arg by Lys enhanced the selectivity of the peptides towards Jurkat cells when compared to PBMCs. Additionally, the side chain length of the Lys residues was also optimized to further enhance the tritrpticin ACP selectivity at low concentrations. The mechanism of action of the peptides with high selectivity involved the permeabilization of the cytoplasmic membrane of Jurkat cells, without formation of apoptotic bodies. The incorporation of non-natural Lys-based cationic amino acids could provide a new strategy to improve the selectivity of other synthetic ACPs to enhance their potential for therapeutic use against leukemia cells.

Brevinin-2GHk from Sylvirana guentheri and the Design of Truncated Analogs Exhibiting the Enhancement of Antimicrobial Activity.

Brevinins are an important antimicrobial peptide (AMP) family discovered in the skin secretions of Ranidae frogs. The members demonstrate a typical C-terminal ranabox, as well as a diverse range of other structural characteristics. In this study, we identified a novel brevinin-2 peptide from the skin secretion of Sylvirana guentheri, via cloning transcripts, and identifying the expressed mature peptide, in the skin secretion. The confirmed amino acid sequence of the mature peptide was designated brevinin-2GHk (BR2GK). Moreover, as a previous study had demonstrated that the N-terminus of brevinin-2 is responsible for exerting antimicrobial activity, we also designed a series of truncated derivatives of BR2GK. The results show that the truncated derivatives exhibit significantly improved antimicrobial activity and cytotoxicity compared to the parent peptide, except a Pro14 substituted analog. The circular dichroism (CD) analysis of this analog revealed that it did not fold into a helical conformation in the presence of either lipopolysaccharides (LPS) or TFE, indicating that position 14 is involved in the formation of the α-helix. Furthermore, three more analogs with the substitutions of Ala, Lys and Arg at the position 14, respectively, revealed the influence on the membrane disruption potency on bacteria and mammalian cells by the structural changes at this position. Overall, the N-terminal 25-mer truncates demonstrated the potent antimicrobial activity with low cytotoxicity.

Synergistic defects in pre-rRNA processing from mutations in the U3-specific protein Rrp9 and U3 snoRNA

U3 snoRNA and the associated Rrp9/U3-55K protein are essential for 18S rRNA production by the SSU-processome complex. U3 and Rrp9 are required for early pre-rRNA cleavages at sites A0, A1 and A2, but the mechanism remains unclear. Substitution of Arg 289 in Rrp9 to Ala (R289A) specifically reduced cleavage at sites A1 and A2. Surprisingly, R289 is located on the surface of the Rrp9 β-propeller structure opposite to U3 snoRNA. To understand this, we first characterized the protein-protein interaction network of Rrp9 within the SSU-processome. This identified a direct interaction between the Rrp9 β-propeller domain and Rrp36, the strength of which was reduced by the R289A substitution, implicating this interaction in the observed processing phenotype. The Rrp9 R289A mutation also showed strong synergistic negative interactions with mutations in U3 that destabilize the U3/pre-rRNA base-pair interactions or reduce the length of their linking segments. We propose that the Rrp9 β-propeller and U3/pre-rRNA binding cooperate in the structure or stability of the SSU-processome. Additionally, our analysis of U3 variants gave insights into the function of individual segments of the 5′-terminal 72-nt sequence of U3. We interpret these data in the light of recently reported SSU-processome structures.

Insights into an unusual Auxiliary Activity 9 family member lacking the histidine brace motif of lytic polysaccharide monooxygenases

Lytic polysaccharide monooxygenases (LPMOs) are redox-enzymes involved in biomass degradation. All characterized LPMOs possess an active site of two highly conserved histidine residues coordinating a copper ion (the histidine brace), which are essential for LPMO activity. However, some protein sequences that belong to the AA9 LPMO family display a natural N-terminal His to Arg substitution (Arg-AA9). These are found almost entirely in the phylogenetic fungal class Agaricomycetes, associated with wood decay, but no function has been demonstrated for any Arg-AA9. Through bioinformatics, transcriptomic, and proteomic analyses we present data, which suggest that Arg-AA9 proteins could have a hitherto unidentified role in fungal degradation of lignocellulosic biomass in conjunction with other secreted fungal enzymes. We present the first structure of an Arg-AA9, LsAA9B, a naturally occurring protein from Lentinus similis The LsAA9B structure reveals gross changes in the region equivalent to the canonical LPMO copper-binding site, whereas features implicated in carbohydrate binding in AA9 LPMOs have been maintained. We obtained a structure of LsAA9B with xylotetraose bound on the surface of the protein although with a considerably different binding mode compared with other AA9 complex structures. In addition, we have found indications of protein phosphorylation near the N-terminal Arg and the carbohydrate-binding site, for which the potential function is currently unknown. Our results are strong evidence that Arg-AA9s function markedly different from canonical AA9 LPMO, but nonetheless, may play a role in fungal conversion of lignocellulosic biomass.

Comprehensive analysis of variation of cadmium accumulation in rice and detection of a new weak allele of OsHMA3.

Excessive cadmium (Cd) accumulation in rice poses a potential threat to human health. Rice varieties vary in their Cd content, which depends mainly on root-to-shoot translocation of Cd. However, cultivars accumulating high Cd in the natural population have not been completely investigated. In this study, we analyzed the variation in Cd accumulation in a diverse panel of 529 rice cultivars. Only a small proportion (11 of 529) showed extremely high root-to-shoot Cd transfer rates, and in seven of these cultivars this was caused by two known OsHMA3 alleles. Using quantitative trait loci mapping, we identified a new OsHMA3 allele that was associated with high Cd accumulation in three of the remaining cultivars. Using heterologous expression in yeast and comparative analysis among different rice cultivars, we observed that this new allele was weak at both the transcriptional and protein levels compared with the functional OsHMA3 genotypes. The weak Cd transport activity was further demonstrated to be caused by a Gly to Arg substitution at position 512. Our study comprehensively analyzed the variation in root-to-shoot Cd translocation rates in cultivated rice and identified a new OsHMA3 allele that caused high Cd accumulation in a few rice cultivars.

Molecular underpinnings of integrin binding to collagen-mimetic peptides containing vascular Ehlers–Danlos syndrome–associated substitutions

Collagens carry out critical extracellular matrix (ECM) functions by interacting with numerous cell receptors and ECM components. Single glycine substitutions in collagen III, which predominates in vascular walls, result in vascular Ehlers-Danlos syndrome (vEDS), leading to arterial, uterine, and intestinal rupture and an average life expectancy of <50 years. Collagen interactions with integrin α2β1 are vital for platelet adhesion and activation; however, how these interactions are impacted by vEDS-associated mutations and by specific amino acid substitutions is unclear. Here, we designed collagen-mimetic peptides (CMPs) with previously reported Gly → Xaa (Xaa = Ala, Arg, or Val) vEDS substitutions within a high-affinity integrin α2β1-binding motif, GROGER. We used these peptides to investigate, at atomic-level resolution, how these amino acid substitutions affect the collagen III-integrin α2β1 interaction. Using a multitiered approach combining biological adhesion assays, CD, NMR, and molecular dynamics (MD) simulations, we found that these substitutions differentially impede human mesenchymal stem cell spreading and integrin α2-inserted (α2I) domain binding to the CMPs and were associated with triple-helix destabilization. Although an Ala substitution locally destabilized hydrogen bonding and enhanced mobility, it did not significantly reduce the CMP-integrin interactions. MD simulations suggested that bulkier Gly → Xaa substitutions differentially disrupt the CMP-α2I interaction. The Gly → Arg substitution destabilized CMP-α2I side-chain interactions, and the Gly → Val change broke the essential Mg2+ coordination. The relationship between the loss of functional binding and the type of vEDS substitution provides a foundation for developing potential therapies for managing collagen disorders.

The Influence of β1‐Adrenergic Receptor Genotype of Renal Sodium Handling and Blood Pressure Response to Angiotensin Receptor Blockers in Hypertension Patients

IntroductionThe β1‐adrenergic receptors (ADRB1) are found primarily in the heart and the juxtaglomerular cells of the kidneys. Within the kidneys, ADRB1 promote the release of renin and the activation of the renin‐angiotensin‐aldosterone system (RAAS). The RAAS plays a primary role in the regulation of renal Na+ handling and blood pressure (BP). Specifically, within the RAAS, aldosterone preferentially increases renal tubular luminal Na+ transport via increasing apical Na+ permeability. As a result, systolic and diastolic blood pressures are impacted by RAAS activity. Additionally, baseline renin levels predict Na+ sensitivity. Multiple polymorphisms of the ADRB1 have been identified as including a glycine (Gly) for arginine (Arg) substitution at amino acid 389. The Gly389 polymorphism is associated with a lower risk of hypertension and increased plasma renin levels compared to the Arg389 polymorphism. The influence of ADRB1 genotype on renin release and blood pressure suggests the functional role it may play in renal Na+ handling.PurposeThe purpose of this study was to identify the influence of ADRB1 genotype on renal Na+ handling and response to angiotensin receptor blocker (ARB) therapy in hypertension patients.Methods158 patients with controlled hypertension on a prescribed ARB completed one study visit consisting of a buccal swab collection and a medical chart review for objective BP history. Buccal swabs were analyzed for ADRB1 389 genotype (Gly389Gly = 14, Gly389Arg = 69, and Arg389Arg = 75). The relationship between genotype and Δ systolic blood pressure (SBP), Δ diastolic blood pressure (DBP), Δ mean arterial blood pressure (MAP), and the average SBP, DBP, and MAP for the last year was assessed.ResultsThere were no differences between genotype groups for age, height, weight, or BMI. There were no differences between genotype groups for ΔSBP, ΔDBP, ΔMAP (ΔSBP = −29.29 ± 2.91, −34.36 ± 3.59, −32.30 ± 3.21, ΔDBP = −16.43 ± 2.48, −11.89 ± 3.84, −13.71 ± 2.72, ΔMAP = −20.71 ± 2.22, −16.91 ± 3.71, −16.83 ± 2.81 for Gly389Gly, Gly389Arg, and Arg389Arg respectively). Additionally, there was no differences in one‐year average for DBP and MAP (DBP = 79.17 ± 2.65, 77.97 ± 0.99, 76.98 ± 0.93, MAP = 98.22 ± 2.85, 94.12 ± 0.93, 94.32 ± 0.83 for Gly389Gly, Gly389Arg, and Arg389Arg respectively). There was a statistically significant difference in one‐year average for SBP between genotype groups (136.32 ± 3.62, 126.67 ± 1.35, 128.98 ± 1.15 for Gly389Gly, Gly389Arg, and Arg389Arg respectively)ConclusionsThe present study suggests ADRB1 389 genotype influences BP response to ARB therapy in hypertension patients, particularly the one‐year average of SBP.Support or Funding InformationGeneticure Inc.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.