A single amino acid substitution alters the vanillylamine synthesis activity of Capsicum pAMT.

Some Capsicum synthesize a unique pungent alkaloid called capsaicin in their fruits. In the synthetic pathway of capsaicin, vanillylamine is produced from vanillin in a reaction catalyzed by a putative aminotransferase (pAMT). Therefore, the capsaicinoids content in the fruits is thought to partially depend on the characteristics of pAMT. Comparing Yume-matsuri (yume), C. annuum variety, and red habanero (RH), C. chinense variety, the vanillylamine synthesis activity of the placental extract was higher in yume than in RH. When each recombinant pAMT (rpAMT) was generated using the Escherichia coli expression system and their activities were compared, yume rpAMT synthesized 14-fold more vanillylamine than RH rpAMT. The amino acid sequence of yume and RH pAMT deduced from the cDNAs revealed that only 7 of 459 residues differed. When a single amino acid residue-substituted rpAMT was generated in which the 56th amino acid was swapped with one other, the amount of vanillylamine synthesis of yume and RH rpAMTs was inverted. Furthermore, it was suggested that the 56th amino acid contributed to the affinity for the coenzyme pyridoxal phosphate. Differences in the vanillylamine synthesis activity of pAMT may also lead to differences in the amount of capsaicin synthesis that accumulates in the fruit. Since capsaicin is a compound with commercial value, this finding may provide new insights into the creation of a variety that can synthesize more capsaicin.

Site-specific glycosylation and single amino acid substitution dramatically reduced the immunogenicity of ß-lactoglobulin.

To reduce the immunogenicity of ß-lactoglobulin (BLG), we prepared recombinant BLG which has both site-specific glycosylation and single amino acid substitution (D28N/P126A), and expressed it in the methylotrophic yeast Pichia pastoris by fusion of the cDNA to the sequence coding for the α-factor signal peptide from Saccharomyces cerevisiae. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that the D28N/P126A was conjugated with a ∼4 kDa high-mannose chain. D28N/P126A retained ∼61% of the retinol-binding activity of BLG. Structural analyses by circular dichroism (CD) spectra, intrinsic fluorescence, and Enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies indicated that the surface structure of BLG was slightly changed by using protein engineering techniques, but D28N/P126A was covered by high-mannose chains and substituted amino acid without substantial disruption of native conformation. Antibody responses to the D28N/P126A considerably reduced in C57BL/6 mice. We conclude that inducing both site-specific glycosylation and single amino acid substitution simultaneously is an effective method to reduce the immunogenicity of BLG.

PPVED: A machine learning tool for predicting the effect of single amino acid substitution on protein function in plants.

Single amino acid substitution (SAAS) produces the most common variant of protein function change under physiological conditions. As the number of SAAS events in plants has increased exponentially, an effective prediction tool is required to help identify and distinguish functional SAASs from the whole genome as either potentially causal traits or as variants. Here, we constructed a plant SAAS database that stores 12 865 SAASs in 6172 proteins and developed a tool called Plant Protein Variation Effect Detector (PPVED) that predicts the effect of SAASs on protein function in plants. PPVED achieved an 87% predictive accuracy when applied to plant SAASs, an accuracy that was much higher than those from six human database software: SIFT, PROVEAN, PANTHER-PSEP, PhD-SNP, PolyPhen-2, and MutPred2. The predictive effect of six SAASs from three proteins in Arabidopsis and maize was validated with wet lab experiments, of which five substitution sites were accurately predicted. PPVED could facilitate the identification and characterization of genetic variants that explain observed phenotype variations in plants, contributing to solutions for challenges in functional genomics and systems biology. PPVED can be accessed under a CC-BY (4.0) license via http://www.ppved.org.cn.

Assessing computational predictions of the phenotypic effect of cystathionine-beta-synthase variants.

Accurate prediction of the impact of genomic variation on phenotype is a major goal of computational biology and an important contributor to personalized medicine. Computational predictions can lead to a better understanding of the mechanisms underlying genetic diseases, including cancer, but their adoption requires thorough and unbiased assessment. Cystathionine-beta-synthase (CBS) is an enzyme that catalyzes the first step of the transsulfuration pathway, from homocysteine to cystathionine, and in which variations are associated with human hyperhomocysteinemia and homocystinuria. We have created a computational challenge under the CAGI framework to evaluate how well different methods can predict the phenotypic effect(s) of CBS single amino acid substitutions using a blinded experimental data set. CAGI participants were asked to predict yeast growth based on the identity of the mutations. The performance of the methods was evaluated using several metrics. The CBS challenge highlighted the difficulty of predicting the phenotype of an ex vivo system in a model organism when classification models were trained on human disease data. We also discuss the variations in difficulty of prediction for known benign and deleterious variants, as well as identify methodological and experimental constraints with lessons to be learned for future challenges.

Characterization of human frataxin missense variants in cancer tissues.

Human frataxin is an iron-binding protein involved in the mitochondrial iron-sulfur (Fe-S) clusters assembly, a process fundamental for the functional activity of mitochondrial proteins. Decreased level of frataxin expression is associated with the neurodegenerative disease Friedreich ataxia. Defective function of frataxin may cause defects in mitochondria, leading to increased tumorigenesis. Tumor-initiating cells show higher iron uptake, a decrease in iron storage and a reduced Fe-S clusters synthesis and utilization. In this study, we selected, from COSMIC database, the somatic human frataxin missense variants found in cancer tissues p.D104G, p.A107V, p.F109L, p.Y123S, p.S161I, p.W173C, p.S181F, and p.S202F to analyze the effect of the single amino acid substitutions on frataxin structure, function, and stability. The spectral properties, the thermodynamic and the kinetic stability, as well as the molecular dynamics of the frataxin missense variants found in cancer tissues point to local changes confined to the environment of the mutated residues. The global fold of the variants is not altered by the amino acid substitutions; however, some of the variants show a decreased stability and a decreased functional activity in comparison with that of the wild-type protein.

Single amino acid substitutions in recombinant plant-derived human α1-proteinase inhibitor confer enhanced stability and functional efficacy.

BACKGROUND: Human α1-proteinase inhibitor (α1-PI) is the most abundant serine protease inhibitor in the blood and the heterologous expression of recombinant α1-PI has great potential for possible therapeutic applications. However, stability and functional efficacy of the recombinant protein expressed in alternate hosts are of major concern. METHODS: Five variants of plant-expressed recombinant α1-PI protein were developed by incorporating single amino acid substitutions at specific sites, namely F51C, F51L, A70G, M358V and M374I. Purified recombinant α1-PI variants were analyzed for their expression, biological activity, oxidation-resistance, conformational and thermal stability by DAC-ELISA, porcine pancreatic elastase (PPE) inhibition assays, transverse urea gradient (TUG) gel electrophoresis, fluorescence spectroscopy and far-UV CD spectroscopy. RESULTS: Urea-induced unfolding of recombinant α1-PI variants revealed that the F51C mutation shifted the mid-point of transition from 1.4M to 4.3M, thus increasing the conformational stability close to the human plasma form, followed by F51L, A70G and M374I variants. The variants also exhibited enhanced stability for heat denaturation, and the size-reducing substitution at Phe51 slowed down the deactivation rate ~5-fold at 54°C. The M358V mutation at the active site of the protein did not significantly affect the conformational or thermal stability of the recombinant α1-PI but provided enhanced resistance to oxidative inactivation. CONCLUSIONS: Our results suggest that single amino acid substitutions resulted in improved stability and oxidation-resistance of the plant-derived recombinant α1-PI protein, without inflicting the inhibitory activity of the protein. GENERAL SIGNIFICANCE: Our results demonstrate the significance of engineered modifications in plant-derived recombinant α1-PI protein molecule for further therapeutic development.

Methylation mediated by an anthocyanin, O-methyltransferase, is involved in purple flower coloration in Paeonia.

Anthocyanins are major pigments in plants. Methylation plays a role in the diversity and stability of anthocyanins. However, the contribution of anthocyanin methylation to flower coloration is still unclear. We identified two homologous anthocyanin O-methyltransferase (AOMT) genes from purple-flowered (PsAOMT) and red-flowered (PtAOMT) Paeonia plants, and we performed functional analyses of the two genes in vitro and in vivo. The critical amino acids for AOMT catalytic activity were studied by site-directed mutagenesis. We showed that the recombinant proteins, PsAOMT and PtAOMT, had identical substrate preferences towards anthocyanins. The methylation activity of PsAOMT was 60 times higher than that of PtAOMT in vitro. Interestingly, this vast difference in catalytic activity appeared to result from a single amino acid residue substitution at position 87 (arginine to leucine). There were significant differences between the 35S::PsAOMT transgenic tobacco and control flowers in relation to their chromatic parameters, which further confirmed the function of PsAOMT in vivo. The expression levels of the two homologous AOMT genes were consistent with anthocyanin accumulation in petals. We conclude that AOMTs are responsible for the methylation of cyanidin glycosides in Paeonia plants and play an important role in purple coloration in Paeonia spp.

Reduced immunogenicity of ß-lactoglobulin by single amino acid substitution.

To reduce the immunogenicity of ß-lactoglobulin (BLG), we prepared single amino acid substituted recombinant BLG mutants (BLG/P126A, BLG/V128D and BLG/D129A) in the methylotrophic yeast Pichia Pastris by fusion of the cDNA to the sequence coding for the α-factor signal peptide from Saccharomyces cerevisiae. Isoelectric points of single amino acid substituted BLGs were lower than that of native BLG. CD spectra indicated that the secondary structure of BLG had maintained native structure in single amino acid substituted BLGs. Fluorescence studies indicated that the conformation around Trp had not changed in single amino acid substituted BLGs. Anti-BLG antibody response was evaluated after immunization to C57BL/6 mice. Antibody response was reduced after immunization with BLG/P126A, BLG/V128D and BLG/D129A. And novel immunogenicity was not observed in the experiments. T cell proliferative response was evaluated in C57BL/6 mice, and it was clarified that BLG mutants also showed low response. Methods employed in this study was considered to be very effective to reduce immunogenicity of BLG.

MotSASi: Functional short linear motifs (SLiMs) prediction based on genomic single nucleotide variants and structural data.

Short linear motifs (SLiMs) are key to cell physiology mediating reversible protein-protein interactions. Precise identification of SLiMs remains a challenge, being the main drawback of most bioinformatic prediction tools, their low specificity (high number of false positives). An important, usually overlooked, aspect is the relation between SLiMs mutations and disease. The presence of variants in each residue position can be used to assess the relevance of the corresponding residue(s) for protein function, and its (in)tolerance to change. In the present work, we combined sequence variant information and structural analysis of the energetic impact of single amino acid substitution (SAS) in SLiM-Receptor complex structure, and showed that it improves prediction of true functional SLiMs. Our strategy is based on building a SAS tolerance matrix that shows, for each position, whether one of the possible 19 SAS is tolerated or not. Herein we present the MotSASi strategy and analyze in detail 3 SLiMs involved in intracellular protein trafficking (phospho-independent tyrosine-based motif (NPx[Y/F]), type 1 PDZ-binding motif ([S/T]x[V/I/L]COOH) and tryptophan-acidic motif ([L/M]xW[D/E])). Our results show that inclusion of variant and structure information improves both prediction of true SLiMs and rejection of false positives, while also allowing better classification of variants inside SLiMs, a result with a direct impact in clinical genomics.

Impact of Single Amino Acid Substitutions in Parkinsonism-Associated Deglycase-PARK7 and Their Association with Parkinson's Disease.

Parkinsonism-associated deglycase-PARK7/DJ-1 (PARK7) is a multifunctional protein having significant roles in inflammatory and immune disorders and cell protection against oxidative stress. Mutations in PARK7 may result in the onset and progression of a few neurodegenerative disorders such as Parkinson's disease. This study has analyzed the non-synonymous single nucleotide polymorphisms (nsSNPs) resulting in single amino acid substitutions in PARK7 to explore its disease-causing variants and their structural dysfunctions. Initially, we retrieved the mutational dataset of PARK7 from the Ensembl database and performed detailed analyses using sequence-based and structure-based approaches. The pathogenicity of the PARK7 was then performed to distinguish the destabilizing/deleterious variants. Aggregation propensity, noncovalent interactions, packing density, and solvent accessible surface area analyses were carried out on the selected pathogenic mutations. The SODA study suggested that mutations in PARK7 result in aggregation, inducing disordered helix and altering the strand propensity. The effect of mutations alters the number of hydrogen bonds and hydrophobic interactions in PARK7, as calculated from the Arpeggio server. The study indicated that the alteration in the hydrophobic contacts and frustration of the protein could alter the stability of the missense variants of the PARK7, which might result in disease progression. This study provides a detailed understanding of the destabilizing effects of single amino acid substitutions in PARK7.

Recent developments in isotope-aided NMR methods for supramolecular protein complexes -SAIL aromatic TROSY.

BACKGROUND: The structure-function relationships for large protein complexes at the atomic level would be comprehensively understood, if hitherto unexplored aromatic ring NMR signals became accessible in addition to the currently used backbone amide and side-chain methyl signals. METHODS: The 82 kDa malate synthase G (MSG) proteins, selectively labeled with Trp and Phe bearing relaxation optimized isotope-labeled rings, were prepared to investigate the optimal conditions for obtaining the aromatic TROSY spectra. RESULTS: The MSG proteins, selectively labeled with either [δ1,ε1,ε3,η2]-SAIL Trp or ζ-SAIL Phe, provided well-separated, narrow TROSY signals for the 12 Trp and 19 Phe residues in MSG. The signals were assigned sequence-specifically, using the set of single amino acid substitution mutants. The site-specific substitution of each Phe with Tyr or Leu induced substantial chemical shifts for the other aromatic ring signals, allowing us to identify the aromatic clusters in MSG, which were comparable to the structural domains proposed previously. CONCLUSIONS: We demonstrated that the aromatic ring 13CH pairs without directly bonded 13C and adjacent 1H spins provide surprisingly narrow TROSY signals, if the rings are surrounded by fully deuterated amino acids. The observed signals can be readily assigned by either the single amino acid substitution or the NOEs between the aromatic and methyl protons, if the methyl assignments are available. GENERAL SIGNIFICANCE: The method described here should be generally applicable for difficult targets, such as proteins in lipid bilayers or possibly in living cells, thus providing unprecedented opportunities to use these new probes in structural biology.

Single amino acid substitution in LC-CDR1 induces Russell body phenotype that attenuates cellular protein synthesis through eIF2α phosphorylation and thereby downregulates IgG secretion despite operational secretory pathway traffic.

Amino acid sequence differences in the variable region of immunoglobulin (Ig) cause wide variations in secretion outputs. To address how a primary sequence difference comes to modulate Ig secretion, we investigated the biosynthetic process of 2 human IgG2κ monoclonal antibodies (mAbs) that differ only by one amino acid in the light chain complementarity-determining region 1 while showing ∼20-fold variance in secretion titer. Although poorly secreted, the lower-secreting mAb of the 2 was by no means defective in terms of its folding stability, antigen binding, and in vitro biologic activity. However, upon overexpression in HEK293 cells, the low-secreting mAb revealed a high propensity to aggregate into enlarged globular structures called Russell bodies (RBs) in the endoplasmic reticulum. While Golgi morphology was affected by the formation of RBs, secretory pathway membrane traffic remained operational in those cells. Importantly, cellular protein synthesis was severely suppressed in RB-positive cells through the phosphorylation of eIF2α. PERK-dependent signaling was implicated in this event, given the upregulation and nuclear accumulation of downstream effectors such as ATF4 and CHOP. These findings illustrated that the underlining process of poor Ig secretion in RB-positive cells was due to downregulation of Ig synthesis instead of a disruption or blockade of secretory pathway trafficking. Therefore, RB formation signifies an end of active Ig production at the protein translation level. Consequently, depending on how soon and how severely an antibody-expressing cell develops the RB phenotype, the productive window of Ig secretion can vary widely among the cells expressing different mAbs.

Ectopic expression of EbFIE from apomictic Eulaliopsis binata in rice results in pleiotropic phenotypes likely due to interaction with OsCLF.

FERTILIZATION INDEPENDENT ENDOSPERM (FIE) is a core component of PcG complexes and functions in plant phase transition and seed generation. However, understanding in its function of apomictic monocot plants remains blank. Here an FIE homology EbFIE, has been isolated from apomictic Graminae species Eulaliopsis binata. EbFIE shares higher homology to OsFIE2 than OsFIE1, and has been classified into the monocot FIE2 clade. In addition, the broad expression pattern of EbFIE is also similar to OsFIE2. While, ectopic expression of EbFIE in rice resulted in pleiotropic phenotypes similar to that of OsFIE1 over-expressing plants. Meanwhile, EbFIE could bind OsCLF in vitro as OsFIE1 but different with OsFIE2. Molecular models comparison indicated that both EbFIE and OsFIE1 had a smaller E(z) protein binding groove than OsFIE2. Further site-directed mutagenesis analysis revealed that single amino acid substitution of I194F in OsFIE2 could improve its OsCLF binding capacity. Taken together, our results suggested that EbFIE was a conserved FIE homolog belonging to monocot FIE2 clade, but due to the similarity in protein conformation with FIE1, EbFIE might play a broad role in vegetative and reproductive development regulation by interaction with CLF homolog.

News from the protein mutability landscape.

Some mutations of protein residues matter more than others, and these are often conserved evolutionarily. The explosion of deep sequencing and genotyping increasingly requires the distinction between effect and neutral variants. The simplest approach predicts all mutations of conserved residues to have an effect; however, this works poorly, at best. Many computational tools that are optimized to predict the impact of point mutations provide more detail. Here, we expand the perspective from the view of single variants to the level of sketching the entire mutability landscape. This landscape is defined by the impact of substituting every residue at each position in a protein by each of the 19 non-native amino acids. We review some of the powerful conclusions about protein function, stability and their robustness to mutation that can be drawn from such an analysis. Large-scale experimental and computational mutagenesis experiments are increasingly furthering our understanding of protein function and of the genotype-phenotype associations. We also discuss how these can be used to improve predictions of protein function and pathogenicity of missense variants.