Journal articles on the topic 'Protein variants'
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Gai, Nan, Therese Uniacke-Lowe, Jonathan O’Regan, Hope Faulkner, and Alan L. Kelly. "Effect of Protein Genotypes on Physicochemical Properties and Protein Functionality of Bovine Milk: A Review." Foods 10, no. 10 (October 11, 2021): 2409. http://dx.doi.org/10.3390/foods10102409.
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Milk protein comprises caseins (CNs) and whey proteins, each of which has different genetic variants. Several studies have reported the frequencies of these genetic variants and the effects of variants on milk physicochemical properties and functionality. For example, the C variant and the BC haplotype of αS1-casein (αS1-CN), β-casein (β-CN) B and A1 variants, and κ-casein (κ-CN) B variant, are favourable for rennet coagulation, as well as the B variant of β-lactoglobulin (β-lg). κ-CN is reported to be the only protein influencing acid gel formation, with the AA variant contributing to a firmer acid curd. For heat stability, κ-CN B variant improves the heat resistance of milk at natural pH, and the order of heat stability between phenotypes is BB > AB > AA. The A2 variant of β-CN is more efficient in emulsion formation, but the emulsion stability is lower than the A1 and B variants. Foaming properties of milk with β-lg variant B are better than A, but the differences between β-CN A1 and A2 variants are controversial. Genetic variants of milk proteins also influence milk yield, composition, quality and processability; thus, study of such relationships offers guidance for the selection of targeted genetic variants.
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Laddach, Anna, Joseph Chi Fung Ng, and Franca Fraternali. "Pathogenic missense protein variants affect different functional pathways and proteomic features than healthy population variants." PLOS Biology 19, no. 4 (April 28, 2021): e3001207. http://dx.doi.org/10.1371/journal.pbio.3001207.
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Missense variants are present amongst the healthy population, but some of them are causative of human diseases. A classification of variants associated with “healthy” or “diseased” states is therefore not always straightforward. A deeper understanding of the nature of missense variants in health and disease, the cellular processes they may affect, and the general molecular principles which underlie these differences is essential to offer mechanistic explanations of the true impact of pathogenic variants. Here, we have formalised a statistical framework which enables robust probabilistic quantification of variant enrichment across full-length proteins, their domains, and 3D structure-defined regions. Using this framework, we validate and extend previously reported trends of variant enrichment in different protein structural regions (surface/core/interface). By examining the association of variant enrichment with available functional pathways and transcriptomic and proteomic (protein half-life, thermal stability, abundance) data, we have mined a rich set of molecular features which distinguish between pathogenic and population variants: Pathogenic variants mainly affect proteins involved in cell proliferation and nucleotide processing and are enriched in more abundant proteins. Additionally, rare population variants display features closer to common than pathogenic variants. We validate the association between these molecular features and variant pathogenicity by comparing against existing in silico variant impact annotations. This study provides molecular details into how different proteins exhibit resilience and/or sensitivity towards missense variants and provides the rationale to prioritise variant-enriched proteins and protein domains for therapeutic targeting and development. The ZoomVar database, which we created for this study, is available at fraternalilab.kcl.ac.uk/ZoomVar. It allows users to programmatically annotate missense variants with protein structural information and to calculate variant enrichment in different protein structural regions.
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Alfaro-Chávez, Ana L., Jian-Wei Liu, Joanne L. Porter, Adrian Goldman, and David L. Ollis. "Improving on nature’s shortcomings: evolving a lipase for increased lipolytic activity, expression and thermostability." Protein Engineering, Design and Selection 32, no. 1 (January 2019): 13–24. http://dx.doi.org/10.1093/protein/gzz024.
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Abstract An enzyme must be soluble, stable, active and easy to produce to be useful in industrial applications. Not all enzymes possess these attributes. We set out to determine how many changes are required to convert an enzyme with poor properties into one that has useful properties. Lipase Lip3 from Drosophila melanogaster had been previously optimised for expression in Escherichia coli. The expression levels were good, but Lip3 was mainly insoluble with poor activity. Directed evolution was used to identify variants with enhanced activity along with improved solubility. Five variants and the wild-type (wt) enzyme were purified and characterised. The yield of the wt enzyme was just 2.2 mg/L of culture, while a variant, produced under the same conditions, gave 351 mg. The improvement of activity of the best variant was 200 times higher than that of the wt when the crude lysates were analysed using pNP-C8, but with purified protein, the improvement observed was 1.5 times higher. This means that most of the increase of activity is due to increase in solubility and stability. All the purified variants showed increased thermal stability compared with the wt enzyme that had a T1/2 of 37°C, while the mutant with P291L of 42.2°C and the mutant R7_47D with five mutations had a value of 52.9°C, corresponding to an improvement of 16°C. The improved variants had between five and nine changes compared with the wt enzyme. There were four changes that were found in all 30 final round variants for which sequences were obtained; three of these changes were found in the substrate-binding domain.
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Larsen, Ole Halfdan, Alisa D. Kjaergaard, Anne-Mette Hvas, and Peter H. Nissen. "Genetic Variants in the Protein S (PROS1) Gene and Protein S Deficiency in a Danish Population." TH Open 05, no. 04 (October 2021): e479-e488. http://dx.doi.org/10.1055/s-0041-1736636.
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AbstractProtein S (PS) deficiency is a risk factor for venous thromboembolism (VTE) and can be caused by variants of the gene encoding PS (PROS1). This study aimed to evaluate the clinical value of molecular analysis of the PROS1 gene in PS-deficient participants. We performed Sanger sequencing of the coding region of the PROS1 gene and multiplex ligation-dependent probe amplification to exclude large structural rearrangements. Free PS was measured by a particle-enhanced immunoassay, while PS activity was assessed by a clotting method.A total of 87 PS-deficient participants and family members were included. In 22 index participants, we identified 13 PROS1 coding variants. Five variants were novel. In 21 index participants, no coding sequence variants or structural rearrangements were identified. The free PS level was lower in index participants carrying a PROS1 variant compared with index participants with no variant (0.51 [0.32–0.61] vs. 0.62 [0.57–0.73] × 103 IU/L; p < 0.05). The p.(Thr78Met) variant was associated with only slightly decreased free PS levels (0.59 [0.53–0.66] × 103 IU/L) compared with the p.(Glu390Lys) variant (0.27 [0.24–0.37] × 103 IU/L, p < 0.01). The frequency of VTE in participants with a coding PROS1 variant was 43 and 17% in the group with normal PROS1 gene (p = 0.05).In conclusion, we report 13 PROS1 coding variants including five novel variants. PS levels differ by PROS1 variant and the frequency of VTE was higher when a coding PROS1 variant was present. Hence, molecular analysis of the PROS1 gene may add clinical value in the diagnostic work-up of PS deficiency.
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Chang, Glenn T. G., Bart H. A. Maas, Hans K. Ploos van Amstel, Pieter H. Reitsma, Rogier M. Bertina, and Bonno N. Bouma. "Studies of the Interaction between Human Protein S and Human C4b-Binding Protein Using Deletion Variants of Recombinant Human Protein S." Thrombosis and Haemostasis 71, no. 04 (1994): 461–67. http://dx.doi.org/10.1055/s-0038-1642461.
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SummaryHuman protein S interacts noncovalently with human C4b-binding protein (C4BP). We have studied this interaction using deletion variants of recombinant human protein S. Two deletion variants were constructed by restriction enzyme digestion and in vitro site-specific mutagenesis of the human protein S cDNA. The variants were stably expressed in Cl27 cells. Recombinant proteins were purified using Fast Flow Q anion-exchange chromatography. The activated protein C (APC) cofactor activity, C4BP binding properties and reactivity to different monoclonal antibodies against human protein S were examined. The first variant (E variant), which has a deletion of the third epidermal growth factor (EGF)-like domain (deletion of exon VII, corresponding to amino acid residues ASP-160 to Asp-202) expresses normal APC cofactor • activity in a plasma system. This activity was inhibited by the addition of purified C4BP. The second variant (L variant), which has a deletion of the C-terminal loop of the sex hormone binding globulin (SHBG)- like domain (deletion of exon XV, corresponding to amino acid residues Asp-583 to Ser-635) also expresses normal APC cofactor activity in plasma. This activity could only be partially inhibited by the addition of purified C4BP.Binding of the recombinant proteins to C4BP was studied in a system using purified proteins. The E variant binds to C4BP with the same affinity similar as recombinant wild type protein S (apparent Kd ∼ 10−10 M). The L variant, however, shows a markedly reduced affinity for binding to C4BP (apparent Kd ∼ 10−7 M).Three different Ca2+-independent monoclonal antibodies (S5, S12, S17) against protein S, which do not interfere with the APC cofactor activity and C4BP binding of protein S, were used to screen the deletion variants for possible conformational changes. Two of these showed the same affinity for the E and L variant as for wild type recombinant protein S. The third, S12, which recognizes an epitope in the vicinity of ser-460, reacts normally with the E variant but has a strongly reduced affinity for the L variant, although the presence of the epitope could be clearly demonstrated by immunoblotting under denaturing conditions.This suggests that the deletion of the C-terminal loop induces a conformational change in protein S which affects the epitope for S12. Therefore although our results indicate that the C-terminal loop of the SHBG-like domain of human protein S is involved in the interaction with C4BP, we cannot exclude the possibility that the deletion of the C-terminal loop induces a conformational change that results in a loss of binding affinity for C4BP elsewhere in the protein S molecule.
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FOLSOM, JAMES P., and JOSEPH F. FRANK. "Proteomic Analysis of a Hypochlorous Acid–Tolerant Listeria monocytogenes Cultural Variant Exhibiting Enhanced Biofilm Production." Journal of Food Protection 70, no. 5 (May 1, 2007): 1129–36. http://dx.doi.org/10.4315/0362-028x-70.5.1129.
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Following exposure of Listeria monocytogenes Scott A (SA) to hypochlorous acid, rough colony variants were identified that were tolerant of hypochlorous acid and produced increased amounts of biofilm. A derivative of one of these variants was smooth, produced even more biofilm, and exhibited greater biofilm chlorine resistance. The objective of this research was to compare the protein expression of a cultural variant to SA and to identify proteins that might be associated with biofilm production and chlorine tolerance. Suspension chlorine tolerance for several cultural variants (SAR, SAR5, and SBS) was determined by exposure to 60 to 120 ppm of hypochlorous acid for 5 min. Hypochlorous acid tolerance of biofilms was determined after growing biofilms on stainless steel and then exposing them to 200 ppm of hypochlorous acid for 5 min. All cultural variants were able to survive 120 ppm of hypochlorous acid in suspension. There was little difference in the hypochlorous acid tolerance of the cultural variant planktonic cells. The cultural variants produced greater amounts of biofilm than the common form of L. monocytogenes and were more tolerant of hypochlorous acid. The SBS variant was selected for proteomic comparison because it was the variant that produced the most biofilm and was the most tolerant of hypochlorous acid when grown as a biofilm. Protein expression of planktonic and biofilm cells of SBS was compared to SA by two-dimensional difference gel electrophoresis. The 50s ribosomal protein, L10, was down-regulated in biofilm SBS. Other proteins down-regulated in planktonic SBS were the peroxide resistance protein (Dpr) and a sugar-binding protein (LMO0181). This sugar-binding protein was also up-regulated in biofilm SBS. One protein spot down-regulated in planktonic SBS contained both 50s ribosomal protein L7/L12 and an unknown protein (LMO1888).
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Overweg, Karin, Chris D. Pericone, Gerridina G. C. Verhoef, Jeffrey N. Weiser, Hugo D. Meiring, Ad P. J. M. De Jong, Ronald De Groot, and Peter W. M. Hermans. "Differential Protein Expression in Phenotypic Variants of Streptococcus pneumoniae." Infection and Immunity 68, no. 8 (August 1, 2000): 4604–10. http://dx.doi.org/10.1128/iai.68.8.4604-4610.2000.
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ABSTRACT Streptococcus pneumoniae undergoes spontaneous phase variation resulting in opaque and transparent colony forms. Differences in colony opacity correlate with differences in virulence: the transparent variants are more capable of colonizing the nasopharynx, whereas the opaque variants show increased virulence during systemic infections. To gain insight into the pathogenesis of pneumococcal disease at the molecular level, protein expression patterns of the phenotypic variants of two pneumococcal strains were compared by high-resolution two-dimensional protein electrophoresis. In comparison with transparent variants, the opaque variants reduced the expression of two proteins and overexpressed one protein. The proteins were identified by mass spectrometric analysis. The protein overexpressed in the opaque phenotype revealed significant homology to elongation factor Ts of Helicobacter pylori. One of the two proteins that were underexpressed in the opaque variants revealed significant homology to the proteinase maturation protein PrtM ofLactocobacillus paracasei, a member of the family of peptidyl-prolyl cis/trans isomerases. A consensus lipoprotein signal sequence suggests that the putative proteinase maturation protein A, designated PpmA, is located at the surface of the pneumococcus and may play a role in the maturation of surface or secreted proteins. The second underexpressed protein was identified as pyruvate oxidase, SpxB. The lower SpxB expression in opaque variants most probably explains the reduced production of hydrogen peroxide, a reaction product of SpxB, in this variant. Since aspxB-defective pneumococcal mutant has decreased ability to colonize the nasopharynx (B. Spellerberg, D. R. Cundell, J. Sandros, B. J. Pearce, I. Idanpaan-Heikkila, C. Rosenow, and H. R. Masure, 1996. Mol. Microbiol. 19:803–813, 1996), our data suggest that SpxB plays an important role in enhancing the ability of transparent variants to efficiently colonize the nasopharynx.
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Thorne, Lucy G., Mehdi Bouhaddou, Ann-Kathrin Reuschl, Lorena Zuliani-Alvarez, Ben Polacco, Adrian Pelin, Jyoti Batra, et al. "Evolution of enhanced innate immune evasion by SARS-CoV-2." Nature 602, no. 7897 (December 23, 2021): 487–95. http://dx.doi.org/10.1038/s41586-021-04352-y.
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AbstractThe emergence of SARS-CoV-2 variants of concern suggests viral adaptation to enhance human-to-human transmission1,2. Although much effort has focused on the characterization of changes in the spike protein in variants of concern, mutations outside of spike are likely to contribute to adaptation. Here, using unbiased abundance proteomics, phosphoproteomics, RNA sequencing and viral replication assays, we show that isolates of the Alpha (B.1.1.7) variant3 suppress innate immune responses in airway epithelial cells more effectively than first-wave isolates. We found that the Alpha variant has markedly increased subgenomic RNA and protein levels of the nucleocapsid protein (N), Orf9b and Orf6—all known innate immune antagonists. Expression of Orf9b alone suppressed the innate immune response through interaction with TOM70, a mitochondrial protein that is required for activation of the RNA-sensing adaptor MAVS. Moreover, the activity of Orf9b and its association with TOM70 was regulated by phosphorylation. We propose that more effective innate immune suppression, through enhanced expression of specific viral antagonist proteins, increases the likelihood of successful transmission of the Alpha variant, and may increase in vivo replication and duration of infection4. The importance of mutations outside the spike coding region in the adaptation of SARS-CoV-2 to humans is underscored by the observation that similar mutations exist in the N and Orf9b regulatory regions of the Delta and Omicron variants.
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Soorajkumar, Anjana, Ebrahim Alakraf, Mohammed Uddin, Stefan S. Du Plessis, Alawi Alsheikh-Ali, and Richard K. Kandasamy. "Computational Analysis of Short Linear Motifs in the Spike Protein of SARS-CoV-2 Variants Provides Possible Clues into the Immune Hijack and Evasion Mechanisms of Omicron Variant." International Journal of Molecular Sciences 23, no. 15 (August 8, 2022): 8822. http://dx.doi.org/10.3390/ijms23158822.
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Short linear motifs (SLiMs) are short linear sequences that can mediate protein–protein interaction. Mimicking eukaryotic SLiMs to compete with extra- or intracellular binding partners, or to sequester host proteins is the crucial strategy of viruses to pervert the host system. Evolved proteins in viruses facilitate minimal protein–protein interactions that significantly affect intracellular signaling networks. Unfortunately, very little information about SARS-CoV-2 SLiMs is known, especially across SARS-CoV-2 variants. Through the ELM database-based sequence analysis of spike proteins from all the major SARS-CoV-2 variants, we identified four overriding SLiMs in the SARS-CoV-2 Omicron variant, namely, LIG_TRFH_1, LIG_REV1ctd_RIR_1, LIG_CaM_NSCaTE_8, and MOD_LATS_1. These SLiMs are highly likely to interfere with various immune functions, interact with host intracellular proteins, regulate cellular pathways, and lubricate viral infection and transmission. These cellular interactions possibly serve as potential therapeutic targets for these variants, and this approach can be further exploited to combat emerging SARS-CoV-2 variants.
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Moghaddar, Mehrnoosh, Ramtin Radman, and Ian Macreadie. "Severity, Pathogenicity and Transmissibility of Delta and Lambda Variants of SARS-CoV-2, Toxicity of Spike Protein and Possibilities for Future Prevention of COVID-19." Microorganisms 9, no. 10 (October 18, 2021): 2167. http://dx.doi.org/10.3390/microorganisms9102167.
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The World Health Organization reports that SARS-CoV-2 has infected over 220 million people and claimed over 4.7 million lives globally. While there are new effective vaccines, the differences in behavior of variants are causing challenges in vaccine development or treatment. Here, we discuss Delta, a variant of concern, and Lambda, a variant of interest. They demonstrate high infectivity and are less responsive to the immune response in vaccinated individuals. In this review, we briefly summarize the reason for infectivity and the severity of the novel variants. Delta and Lambda variants exhibit more changes in NSPs proteins and the S protein, compared to the original Wuhan strain. Lambda also has numerous amino acid substitutions in NSPs and S proteins, plus a deletion in the NTD of S protein, leading to partial escape from neutralizing antibodies (NAbs) in vaccinated individuals. We discuss the role of furin protease and the ACE2 receptor in virus infection, hotspot mutations in the S protein, the toxicity of the S protein and the increased pathogenicity of Delta and Lambda variants. We discuss future therapeutic strategies, including those based on high stability of epitopes, conservation of the N protein and the novel intracellular antibody receptor, tripartite-motif protein 21 (TRIM21) recognized by antibodies against the N protein.
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Tang, Ziyang. "A Study on the Relationship between the 3-D Structure of Spike Proteins and Infectiousness of SARS-CoV-2 Delta Variant." Highlights in Science, Engineering and Technology 8 (August 17, 2022): 169–77. http://dx.doi.org/10.54097/hset.v8i.1124.
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Since the outbreak of novel coronavirus pneumonia in Wuhan in 2019, the SARS-CoV-2 epidemic has become a hot topic. Over time, SARS-CoV-2 has evolved many variants. The diversity of the 3-D structure of the variant’s proteins resulted in the difference in the binding ability and infectious differences between different virus variants and human angiotensin-converting enzyme 2 (ACE2) receptors. In 2020, an evolutionary analysis of the Delta and Delta Plus variants of SARS-CoV-2 provided a three-dimensional model of the protein of the delta variant. However, it only focused on the delta variant and Delta plus variant themselves and did not compare the delta variant or delta plus variant with the original strain. It is hard to give a direct or apparent reason why the delta variant is more infectious and difficult to cure than the original strain. Therefore, this paper further compared the 3-D structures of homologous trimeric spike glycoproteins (S-proteins) and the receptor-binding domain between the SARS-COV-2 original strain and the SARS-COV-2 delta variant. By observing and analyzing the models of the above proteins in the PyMOL Molecular Graphics System, the reasons for the increase of infectivity of the delta variant can be interpreted in a direct way. This article also focuses on the data of the Indian cases from the JHU database to deeply analyze the relationship between the structure and transmission ability of the SARS-CoV-2 delta variant. Last but not least, the reproductive ability of SARS-CoV-2 can be reflected by the number of NAG (2-acetamido-2-deoxy-beta-D-glucopyranose). Through data analysis and protein structure research, we can better understand the characteristics of the binding of SARS-CoV-2 to the human receptor, thus providing a theoretical basis for accurately predicting virus variation. Through the comparative study of virus structure and infectiousness, this paper will provide a scientific basis for the relevant departments to improve epidemic prevention and improve the public's vigilance against virus variants.
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Eppinger, Erik, and Andreas Stolz. "Expansion of the substrate range of the gentisate 1,2-dioxygenase from Corynebacterium glutamicum for the conversion of monohydroxylated benzoates." Protein Engineering, Design and Selection 30, no. 1 (December 15, 2016): 57–65. http://dx.doi.org/10.1093/protein/gzw061.
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AbstractThe gentisate 1,2-dioxygenases (GDOs) from Corynebacterium glutamicum and various other organisms oxidatively cleave the aromatic nucleus of gentisate (2,5-dihydroxybenzoate), but are not able to convert salicylate (2-hydroxybenzoate). In contrast, the α-proteobacterium Pseudaminobacter salicylatoxidans synthesises an enzyme (‘salicylate dioxygenase’, SDO) which cleaves gentisate, but also (substituted) salicylate(s). Sequence comparisons showed that the SDO belongs to a group of GDOs mainly originating from Gram-positive bacteria which also include the GDO from C. glutamicum ATCC 13032. The combination of sequence comparisons with previously performed structural and mutational analyses of the SDO allowed to identify an amino acid residue (Ala112) which might prevent the oxidation of (substituted) salicylate(s) by the GDO from C. glutamicum. Therefore, the relevant mutation (Ala→Gly) was introduced into the GDO from C. glutamicum. The GDO variant obtained gained the ability to oxidise salicylate and several other monohydroxylated substrates. In order to screen a broader range of enzyme variants a chromogenic assay was developed which allowed the detection of bacterial colonies converting salicylate. The applicability of this test system was proven by screening a set of GDO variants obtained by saturation mutagenesis at different positions. This demonstrated that also GDO variants carrying the mutations Ala112→Ser, Ala112→Ile and Ala112→Asp converted salicylate.
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Zhang, Lujia, Ya Li, Litao Qin, Yu Wu, and Bo Lei. "Autosomal Recessive Retinitis Pigmentosa Associated with Three Novel REEP6 Variants in Chinese Population." Genes 12, no. 4 (April 7, 2021): 537. http://dx.doi.org/10.3390/genes12040537.
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Retinitis pigmentosa 77 is caused by mutations of REEP6 (MIM: 609346), which encodes a protein for the development of photoreceptors. Our study was to identify disease-causing variants in three Chinese families using targeted next-generation sequencing (NGS). Multiple lines of computational predictions combined with in vitro cellular experiments were applied to evaluate the pathogenicity of the newly found variants. Three novel variants in REEP6, including one missense variant, c.268G>C, one frameshift variant, c.468delC, and one splicing variant, c.598+1G>C, were found, while c.268G>C was detected in all probands. The three variants were classified as likely pathogenic by the American College of Medical Genetics and Genomics (ACMG). REEP6 variant proteins c.268G>C and c.468delC in cultured cells destabilized the REEP6 protein and induced intracellular inclusions. Our data suggested that REEP6 c.268G>C may be a recurrent causative variant in Chinese autosomal recessive retinitis pigmentosa patients.
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Sugano, Aki, Yutaka Takaoka, Haruyuki Kataguchi, Mika Ohta, Shigemi Kimura, Masatake Araki, Yoshitomo Morinaga, and Yoshihiro Yamamoto. "SARS-CoV-2 Omicron BA.2.75 Variant May Be Much More Infective than Preexisting Variants Based on In Silico Model." Microorganisms 10, no. 10 (October 21, 2022): 2090. http://dx.doi.org/10.3390/microorganisms10102090.
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Previously, we developed a mathematical model via molecular simulation analysis to predict the infectivity of six SARS-CoV-2 variants. In this report, we aimed to predict the relative risk of the recent new variants of SARS-CoV-2 based on our previous research. We subjected Omicron BA.4/5 and BA.2.75 variants of SARS-CoV-2 to the analysis to determine the evolutionary distance of the spike protein gene (S gene) of the variants from the Wuhan variant so as to appreciate the changes in the spike protein. We performed molecular docking simulation analyses of the spike proteins with human angiotensin-converting enzyme 2 (ACE2) to understand the docking affinities of these variants. We then compared the evolutionary distances and the docking affinities of these variants with those of the variants that we had analyzed in our previous research. As a result, BA.2.75 has both the highest docking affinity (ratio per Wuhan variant) and the longest evolutionary distance of the S gene from the Wuhan variant. These results suggest that BA.2.75 infection can spread farther than can infections of preexisting variants.
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Cho, Yunje, Wei Gu, Steve Wakins, Sam-Pin Lee, Tae-Rak Kim, John W. Brady, and Carl A. Batt. "Thermostable variants of bovine β-lactoglobulin." "Protein Engineering, Design and Selection" 7, no. 2 (1994): 263–83. http://dx.doi.org/10.1093/protein/7.2.263.
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Dikici, E., X. Qu, L. Rowe, L. Millner, C. Logue, S. K. Deo, M. Ensor, and S. Daunert. "Aequorin variants with improved bioluminescence properties." Protein Engineering Design and Selection 22, no. 4 (January 10, 2009): 243–48. http://dx.doi.org/10.1093/protein/gzn083.
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Lau, A. Y., and D. I. Chasman. "Functional classification of proteins and protein variants." Proceedings of the National Academy of Sciences 101, no. 17 (April 15, 2004): 6576–81. http://dx.doi.org/10.1073/pnas.0305043101.
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Fukuzawa, Atsushi, Daniel Koch, Sarah Grover, Martin Rees, and Mathias Gautel. "When is an obscurin variant pathogenic? The impact of Arg4344Gln and Arg4444Trp variants on protein–protein interactions and protein stability." Human Molecular Genetics 30, no. 12 (January 12, 2021): 1131–41. http://dx.doi.org/10.1093/hmg/ddab010.
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Abstract Obscurin is a giant muscle protein that connects the sarcomere with the sarcoplasmic reticulum, and has poorly understood structural and signalling functions. Increasingly, obscurin variants are implicated in the pathophysiology of cardiovascular diseases. The Arg4344Gln variant (R4344Q) in obscurin domain Ig58, initially discovered in a patient with hypertrophic cardiomyopathy, has been reported to reduce binding to titin domains Z8-Z9, impairing obscurin’s Z-disc localization. An R4344Q knock-in mouse developed a cardiomyopathy-like phenotype with abnormal Ca2+-handling and arrhythmias, which were attributed to an enhanced affinity of a putative interaction between obscurin Ig58 and phospholamban (PLN) due to the R4344Q variant. However, the R4344Q variant is found in 15% of African Americans, arguing against its pathogenicity. To resolve this apparent paradox, we quantified the influence of the R4344Q variant (alongside another potentially pathogenic variant: Arg4444Trp (R4444W)) on binding to titin Z8-Z9, novex-3 and PLN using pull-down assays and microscale thermophoresis and characterized the influence on domain stability using differential scanning fluorimetry. We found no changes in titin binding and thermostability for both variants and modestly increased affinities of PLN for R4344Q and R4444W. While we could not confirm the novex-3/obscurin interaction, the PLN/obscurin interaction relies on the transmembrane region of PLN and is not reproducible in mammalian cells, suggesting it is an in vitro artefact. Without clear clinical evidence for disease involvement, we advise against classifying these obscurin variants as pathogenic.
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Iqbal, Sumaiya, David Hoksza, Eduardo Pérez-Palma, Patrick May, Jakob B. Jespersen, Shehab S. Ahmed, Zaara T. Rifat, et al. "MISCAST: MIssense variant to protein StruCture Analysis web SuiTe." Nucleic Acids Research 48, W1 (May 13, 2020): W132—W139. http://dx.doi.org/10.1093/nar/gkaa361.
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Abstract Human genome sequencing efforts have greatly expanded, and a plethora of missense variants identified both in patients and in the general population is now publicly accessible. Interpretation of the molecular-level effect of missense variants, however, remains challenging and requires a particular investigation of amino acid substitutions in the context of protein structure and function. Answers to questions like ‘Is a variant perturbing a site involved in key macromolecular interactions and/or cellular signaling?’, or ‘Is a variant changing an amino acid located at the protein core or part of a cluster of known pathogenic mutations in 3D?’ are crucial. Motivated by these needs, we developed MISCAST (missense variant to protein structure analysis web suite; http://miscast.broadinstitute.org/). MISCAST is an interactive and user-friendly web server to visualize and analyze missense variants in protein sequence and structure space. Additionally, a comprehensive set of protein structural and functional features have been aggregated in MISCAST from multiple databases, and displayed on structures alongside the variants to provide users with the biological context of the variant location in an integrated platform. We further made the annotated data and protein structures readily downloadable from MISCAST to foster advanced offline analysis of missense variants by a wide biological community.
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Francisco-Velilla, Rosario, Azman Embarc-Buh, Francisco del Caño-Ochoa, Salvador Abellan, Marçal Vilar, Sara Alvarez, Alberto Fernandez-Jaen, et al. "Functional and structural deficiencies of Gemin5 variants associated with neurological disorders." Life Science Alliance 5, no. 7 (April 7, 2022): e202201403. http://dx.doi.org/10.26508/lsa.202201403.
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Dysfunction of RNA-binding proteins is often linked to a wide range of human disease, particularly with neurological conditions. Gemin5 is a member of the survival of the motor neurons (SMN) complex, a ribosome-binding protein and a translation reprogramming factor. Recently, pathogenic mutations in Gemin5 have been reported, but the functional consequences of these variants remain elusive. Here, we report functional and structural deficiencies associated with compound heterozygosity variants within the Gemin5 gene found in patients with neurodevelopmental disorders. These clinical variants are located in key domains of Gemin5, the tetratricopeptide repeat (TPR)–like dimerization module and the noncanonical RNA-binding site 1 (RBS1). We show that the TPR-like variants disrupt protein dimerization, whereas the RBS1 variant confers protein instability. All mutants are defective in the interaction with protein networks involved in translation and RNA-driven pathways. Importantly, the TPR-like variants fail to associate with native ribosomes, hampering its involvement in translation control and establishing a functional difference with the wild-type protein. Our study provides insights into the molecular basis of disease associated with malfunction of the Gemin5 protein.
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Alfaro-Chávez, Ana L., Jian-Wei Liu, Bradley J. Stevenson, Adrian Goldman, and David L. Ollis. "Evolving a lipase for hydrolysis of natural triglycerides along with enhanced tolerance towards a protease and surfactants." Protein Engineering, Design and Selection 32, no. 3 (March 2019): 129–43. http://dx.doi.org/10.1093/protein/gzz023.
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Abstract In the accompanying paper, we described evolving a lipase to the point where variants were soluble, stable and capable of degrading C8 TAG and C8 esters. These variants were tested for their ability to survive in an environment that might be encountered in a washing machine. Unfortunately, they were inactivated both by treatment with a protease used in laundry detergents and by very low concentrations of sodium dodecyl sulfate (SDS). In addition, all the variants had very low levels of activity with triglycerides with long aliphatic chains and with naturally occurring oils, like olive oil. Directed evolution was used to select variants with enhanced properties. In the first 10 rounds of evolution, the primary screen was selected for variants capable of hydrolyzing olive oil whereas the secondary screen was selected for enhanced tolerance towards a protease and SDS. In the final six rounds of evolution, the primary and secondary screens identified variants that retained activity after treatment with SDS. Sixteen cycles of evolution gave variants with greatly enhanced lipolytic activity on substrates that had both long (C16 and C18) as well as short (C3 and C8) chains. We found variants that were stable for more than 3 hours in protease concentrations that rapidly degrade the wild-type enzyme. Enhanced tolerance towards SDS was found in variants that could break down naturally occurring lipid and resist protease attack. The amino acid changes that gave enhanced properties were concentrated in the cap domain responsible for substrate binding.
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Guo, Jing, Owen J. L. Rackham, Niina Sandholm, Bing He, Anne-May Österholm, Erkka Valo, Valma Harjutsalo, et al. "Whole-Genome Sequencing of Finnish Type 1 Diabetic Siblings Discordant for Kidney Disease Reveals DNA Variants associated with Diabetic Nephropathy." Journal of the American Society of Nephrology 31, no. 2 (January 9, 2020): 309–23. http://dx.doi.org/10.1681/asn.2019030289.
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BackgroundSeveral genetic susceptibility loci associated with diabetic nephropathy have been documented, but no causative variants implying novel pathogenetic mechanisms have been elucidated.MethodsWe carried out whole-genome sequencing of a discovery cohort of Finnish siblings with type 1 diabetes who were discordant for the presence (case) or absence (control) of diabetic nephropathy. Controls had diabetes without complications for 15–37 years. We analyzed and annotated variants at genome, gene, and single-nucleotide variant levels. We then replicated the associated variants, genes, and regions in a replication cohort from the Finnish Diabetic Nephropathy study that included 3531 unrelated Finns with type 1 diabetes.ResultsWe observed protein-altering variants and an enrichment of variants in regions associated with the presence or absence of diabetic nephropathy. The replication cohort confirmed variants in both regulatory and protein-coding regions. We also observed that diabetic nephropathy–associated variants, when clustered at the gene level, are enriched in a core protein-interaction network representing proteins essential for podocyte function. These genes include protein kinases (protein kinase C isoforms ε and ι) and protein tyrosine kinase 2.ConclusionsOur comprehensive analysis of a diabetic nephropathy cohort of siblings with type 1 diabetes who were discordant for kidney disease points to variants and genes that are potentially causative or protective for diabetic nephropathy. This includes variants in two isoforms of the protein kinase C family not previously linked to diabetic nephropathy, adding support to previous hypotheses that the protein kinase C family members play a role in diabetic nephropathy and might be attractive therapeutic targets.
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Mitra, Kakoli, Thomas A. Steitz, and Donald M. Engelman. "Rational design of `water-soluble' bacteriorhodopsin variants." Protein Engineering, Design and Selection 15, no. 6 (June 2002): 485–92. http://dx.doi.org/10.1093/protein/15.6.485.
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Nakagawa, Hiroshi, Ai Tamura, Kanako Wakabayashi, Kazuyuki Hoshijima, Masayuki Komada, Takashi Yoshida, Satoshi Kometani, Takayoshi Matsubara, Kenta Mikuriya, and Toshihisa Ishikawa. "Ubiquitin-mediated proteasomal degradation of non-synonymous SNP variants of human ABC transporter ABCG2." Biochemical Journal 411, no. 3 (April 14, 2008): 623–31. http://dx.doi.org/10.1042/bj20071229.
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Clinical relevance is implicated between the genetic polymorphisms of the ABC (ATP-binding cassette) transporter ABCG2 (ABC subfamily G, member 2) and the individual differences in drug response. We expressed a total of seven non-synonymous SNP (single nucleotide polymorphism) variants in Flp-In-293 cells by using the Flp (flippase) recombinase system. Of these, ABCG2 F208S and S441N variants were found to be expressed at markedly low levels, whereas their mRNA levels were equal to those of the other SNP variants and ABCG2 WT (wild-type). Interestingly, protein expression levels of the ABCG2 F208S and S441N variants increased 6- to 12-fold when Flp-In-293 cells were treated with MG132, a proteasome inhibitor. Immunoprecipitation followed by immunoblot analysis showed that the ABCG2 F208S and S441N variant proteins were endogenously ubiquitinated in Flp-In-293 cells, and treatment with MG132 significantly enhanced the level of these ubiquitinated variants. Immunofluorescence microscopy demonstrated that MG132 greatly affected the ABCG2 F208S and S441N variants in terms of both protein levels and intracellular distribution. Immunoblot analysis revealed that those variants were N-glycosylated; however, their oligosaccharides were immature compared with those present on ABCG2 WT. The ABCG2 F208S and S441N variant proteins do not appear to be processed in the Golgi apparatus, but undergo ubiquitin-mediated protein degradation in proteasomes, whereas ABCG2 WT is sorted to the plasma membrane and then degraded via the lysosomal pathway. The present study provides the first evidence that certain genetic polymorphisms can affect the protein stability of ABCG2. Control of proteasomal degradation of ABCG2 would provide a novel approach in cancer chemotherapy to circumvent multidrug resistance of human cancers.
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van Wijk, Stan W., Wei Su, Leonoor F. J. M. Wijdeveld, Kennedy S. Ramos, and Bianca J. J. M. Brundel. "Cytoskeletal Protein Variants Driving Atrial Fibrillation: Potential Mechanisms of Action." Cells 11, no. 3 (January 25, 2022): 416. http://dx.doi.org/10.3390/cells11030416.
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The most common clinical tachyarrhythmia, atrial fibrillation (AF), is present in 1–2% of the population. Although common risk factors, including hypertension, diabetes, and obesity, frequently underlie AF onset, it has been recognized that in 15% of the AF population, AF is familial. In these families, genome and exome sequencing techniques identified variants in the non-coding genome (i.e., variant regulatory elements), genes encoding ion channels, as well as genes encoding cytoskeletal (-associated) proteins. Cytoskeletal protein variants include variants in desmin, lamin A/C, titin, myosin heavy and light chain, junctophilin, nucleoporin, nesprin, and filamin C. These cytoskeletal protein variants have a strong association with the development of cardiomyopathy. Interestingly, AF onset is often represented as the initial manifestation of cardiac disease, sometimes even preceding cardiomyopathy by several years. Although emerging research findings reveal cytoskeletal protein variants to disrupt the cardiomyocyte structure and trigger DNA damage, exploration of the pathophysiological mechanisms of genetic AF is still in its infancy. In this review, we provide an overview of cytoskeletal (-associated) gene variants that relate to genetic AF and highlight potential pathophysiological pathways that drive this arrhythmia.
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Ghosh, Asish Kumar, Marco Kaiser, Md Maruf Ahmed Molla, Tasnim Nafisa, Mahmuda Yeasmin, Rifat Hossain Ratul, Md Mohiuddin Sharif, et al. "Molecular and Serological Characterization of the SARS-CoV-2 Delta Variant in Bangladesh in 2021." Viruses 13, no. 11 (November 19, 2021): 2310. http://dx.doi.org/10.3390/v13112310.
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Novel SARS-CoV-2 variants are emerging at an alarming rate. The delta variant and other variants of concern (VoC) carry spike (S)-protein mutations, which have the potential to evade protective immunity, to trigger break-through infections after COVID-19 vaccination, and to propagate future waves of COVID-19 pandemic. To identify SARS CoV-2 variants in Bangladesh, patients who are RT-PCR-positive for COVID-19 infections in Dhaka were screened by a RT-PCR melting curve analysis for spike protein mutations. To assess the anti-SARS CoV-2 antibody responses, the levels of the anti-S -proteins IgA and IgG and the anti-N-protein IgG were measured by ELISA. Of a total of 36 RT-PCR positive samples (75%), 27 were identified as delta variants, with one carrying an additional Q677H mutation and two with single nucleotide substitutions at position 23029 (compared to Wuhan-Hu-1 reference NC 045512) in the genome sequence. Three (8.3%) were identified as beta variants, two (5.5%) were identified as alpha variants, three (8.3%) were identified as having a B.1.1.318 lineage, and one sample was identified as an eta variant (B.1.525) carrying an additional V687L mutation. The trend of higher viral load (lower Cp values) among delta variants than in the alpha and beta variants was of borderline statistical significance (p = 0.045). Prospective studies with larger Bangladeshi cohorts are warranted to confirm the emergence of S-protein mutations and their association with antibody response in natural infection and potential breakthrough in vaccinated subjects.
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von Bülow, Sören, Mateusz Sikora, Florian E. C. Blanc, Roberto Covino, and Gerhard Hummer. "Antibody accessibility determines location of spike surface mutations in SARS-CoV-2 variants." PLOS Computational Biology 19, no. 1 (January 24, 2023): e1010822. http://dx.doi.org/10.1371/journal.pcbi.1010822.
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The steady emergence of SARS-CoV-2 variants gives us a real-time view of the interplay between viral evolution and the host immune defense. The spike protein of SARS-CoV-2 is the primary target of antibodies. Here, we show that steric accessibility to antibodies provides a strong predictor of mutation activity in the spike protein of SARS-CoV-2 variants, including Omicron. We introduce an antibody accessibility score (AAS) that accounts for the steric shielding effect of glycans at the surface of spike. We find that high values of the AAS correlate strongly with the sites of mutations in the spike proteins of newly emerging SARS-CoV-2 variants. We use the AAS to assess the escapability of variant spike proteins, i.e., their ability to escape antibody-based immune responses. The high calculated escapability of the Omicron variant BA.5 with respect to both wild-type (WT) vaccination and BA.1 infection is consistent with its rapid spread despite high rates of vaccination and prior infection with earlier variants. We calculated the AAS from structural and molecular dynamics simulation data that were available early in the pandemic, in the spring of 2020. The AAS thus allows us to prospectively assess the ability of variant spike proteins to escape antibody-based immune responses and to pinpoint regions of expected mutation activity in future variants.
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Cagiada, Matteo, Kristoffer E. Johansson, Audrone Valanciute, Sofie V. Nielsen, Rasmus Hartmann-Petersen, Jun J. Yang, Douglas M. Fowler, Amelie Stein, and Kresten Lindorff-Larsen. "Understanding the Origins of Loss of Protein Function by Analyzing the Effects of Thousands of Variants on Activity and Abundance." Molecular Biology and Evolution 38, no. 8 (March 29, 2021): 3235–46. http://dx.doi.org/10.1093/molbev/msab095.
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Abstract Understanding and predicting how amino acid substitutions affect proteins are keys to our basic understanding of protein function and evolution. Amino acid changes may affect protein function in a number of ways including direct perturbations of activity or indirect effects on protein folding and stability. We have analyzed 6,749 experimentally determined variant effects from multiplexed assays on abundance and activity in two proteins (NUDT15 and PTEN) to quantify these effects and find that a third of the variants cause loss of function, and about half of loss-of-function variants also have low cellular abundance. We analyze the structural and mechanistic origins of loss of function and use the experimental data to find residues important for enzymatic activity. We performed computational analyses of protein stability and evolutionary conservation and show how we may predict positions where variants cause loss of activity or abundance. In this way, our results link thermodynamic stability and evolutionary conservation to experimental studies of different properties of protein fitness landscapes.
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Chang, Glenn T. G., Leonie Aaldering, Tilman M. Hackeng, Pieter H. Reitsma, Rogier M. Bertina, and Bonno N. Bouma. "Construction and Characterization of Thrombin-resistant Variants of Recombinant Human Protein S." Thrombosis and Haemostasis 72, no. 05 (1994): 693–97. http://dx.doi.org/10.1055/s-0038-1648944.
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SummaryProtein S is a vitamin K-dependent plasma protein that functions as a cofactor of activated protein C (APC) in the inactivation of coagulation factors Va and Villa.Protein S, migrates as a doublet on reduced SDS polyacrylamide gel electrophoresis. This heterogeneity in molecular weight has been explained by limited proteolysis of protein S. Human protein S contains at Arg-49, Arg-60 and Arg-70 three potential cleavage sites. Whether cleavage occurs at all three sites is not known. To study the role of these arginine residues in human protein S, we have replaced them by leucine or isoleucine. All seven possible variants were constructed: three variants with single mutations (R49L, R60L, R70I), three variants with double mutations (R49L/R60L, R60L/R70I, R49L/R70I) and one variant with a triple mutation (R49L/R60L/R70I). On reduced SDS polyacrylamide gels the single and double variants migrate as a doublet just like the wild type protein S. The triple variant migrates as a single band at a molecular weight corresponding to the upper band of the doublet. The upper band of the single and double variants but not of the triple variant could be converted into the lower band by thrombin treatment.All variants showed cofactor activity to APC in a clotting assay. After thrombin treatment, this cofactor activity was abolished for the single (R49L, R60L, R70I) and double variants (R49L/R60L, R60L/R70I, R49L/R70I), while the triple variant (R49L/R60L/R70I) tested at several concentrations, retained its cofactor activity completely, suggesting resistance to thrombin. This shows that thrombin can cleave at all three arginine sites and that cleavage at each of these sites results in the loss of APC cofactor activity. Finally, all variants bind to C4b-binding protein with an affinity similar as the wild type recombinant molecule.
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Soldatenkova, A. V., A. M. Kudryashova, N. F. Gavrilova, I. V. Yakovleva, O. V. Borisova, V. V. Sviridov, and N. A. Mikhailova. "Development of ELISA test for the quality control of Pseudomonas aeruginosa recombinant vaccine based on the hybrid recombinant protein." Medical Immunology (Russia) 22, no. 4 (August 7, 2020): 805–10. http://dx.doi.org/10.15789/1563-0625-doe-1906.
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A hybrid recombinant protein containing the amino acid sequences of the three most significant Pseudomonas aeruginosa antigens (membrane proteins OprF, OprI and toxoid aTox) was incorporated into a vaccine against Pseudomonas infection. Quality control of a hybrid recombinant protein and appropriate vaccine includes determination of authentity and completeness of adsorption upon aluminum hydroxide adjuvant. The aim of our study was to develop techniques of quality control for a vaccine based on the hybrid OprF-aToxOprI recombinant protein specific to P. aeruginosa. Hybridomas secreting specific monoclonal antibodies for OprF-aTox-OprI were derived from the fusion of myeloma cells and murine spleen cells immunized with recombinant proteins P. aeruginosa. To produce sufficient quantities of antibodies, the hybrid cells were in vivo cultured in BALB/c mice. Supernates and ascite liquids were chromatographically purified with immune sorbent. Conjugation of antibodies with horseradish peroxidase was carried out according to P.K.Nakane. The hybrid OprF-aTox-OprI recombinant protein was detected by the solid-phase ELISA, using a panel of monoclonal antibodies and conjugates of monoclonal antibodies with horseradish peroxidase. Monoclonal antibodies were specific for different OprF-aTox-OprI epitopes. Titration assays containing OprF-aTox-OprI protein at 78 ng/ml to 5000 ng/ml were used as quantitative standards for calibration curves.To identify the recombinant protein OprF-aTox-OprI, 55 variants of of MAb pairs were tested. Limits of quantitative detection served for selection of most sensitive and specific ELISA variants. The quantitative detection limit was calculated for all 11 ELISA variants. Two ELISA variants with the highest sensitivity were selected for quality control of the hybrid recombinant protein. The limits of quantitative detection were, respectively, 2.9 and 13.6 ng/ml (0.0058 and 0.027% of the estimated antigen content in the vaccine) for the first and second ELISA variants. The first variant included a pair of monoclonal antibodies specific for the OprF and OprI epitopes, the second variant represented aTox and OprI epitopes. Two variants of ELISA were developed to detect the hybrid recombinant OprF-aTox-OprI protein. The first variant allows to determine the protein amount and to evaluate completeness of its adsorption on aluminum hydroxide. To confirm authenticity of the protein, both methods must be used, since they can detect all three antigens (OprF, aTox and OprI) which are present in the fusion protein.
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Rabaan, Ali A., Shamsah H. Al-Ahmed, Hawra Albayat, Sara Alwarthan, Mashael Alhajri, Mustafa A. Najim, Bashayer M. AlShehail, et al. "Variants of SARS-CoV-2: Influences on the Vaccines’ Effectiveness and Possible Strategies to Overcome Their Consequences." Medicina 59, no. 3 (March 5, 2023): 507. http://dx.doi.org/10.3390/medicina59030507.
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The immune response elicited by the current COVID-19 vaccinations declines with time, especially among the immunocompromised population. Furthermore, the emergence of novel SARS-CoV-2 variants, particularly the Omicron variant, has raised serious concerns about the efficacy of currently available vaccines in protecting the most vulnerable people. Several studies have reported that vaccinated people get breakthrough infections amid COVID-19 cases. So far, five variants of concern (VOCs) have been reported, resulting in successive waves of infection. These variants have shown a variable amount of resistance towards the neutralising antibodies (nAbs) elicited either through natural infection or the vaccination. The spike (S) protein, membrane (M) protein, and envelope (E) protein on the viral surface envelope and the N-nucleocapsid protein in the core of the ribonucleoprotein are the major structural vaccine target proteins against COVID-19. Among these targets, S Protein has been extensively exploited to generate effective vaccines against COVID-19. Hence, amid the emergence of novel variants of SARS-CoV-2, we have discussed their impact on currently available vaccines. We have also discussed the potential roles of S Protein in the development of novel vaccination approaches to contain the negative consequences of the variants’ emergence and acquisition of mutations in the S Protein of SARS-CoV-2. Moreover, the implications of SARS-CoV-2’s structural proteins were also discussed in terms of their variable potential to elicit an effective amount of immune response.
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Jallat, S., D. Carvallo, L. H. Tessier, D. Roecklin, C. Roitsch, F. Ogushi, R. G. Crystal, and M. Courtney. "Altered specificities of genetically engineered α1 antitrypsin variants." "Protein Engineering, Design and Selection" 1, no. 1 (1986): 29–35. http://dx.doi.org/10.1093/protein/1.1.29.
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Ali, Muhammad Zeeshan, Arshad Farid, Safeer Ahmad, Muhammad Muzammal, Mohammed Al Mohaini, Abdulkhaliq J. Alsalman, Maitham A. Al Hawaj, et al. "In Silico Analysis Identified Putative Pathogenic Missense nsSNPs in Human SLITRK1 Gene." Genes 13, no. 4 (April 11, 2022): 672. http://dx.doi.org/10.3390/genes13040672.
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Human DNA contains several variations, which can affect the structure and normal functioning of a protein. These variations could be single nucleotide polymorphisms (SNPs) or insertion-deletions (InDels). SNPs, as opposed to InDels, are more commonly present in DNA and may cause genetic disorders. In the current study, several bioinformatic tools were used to prioritize the pathogenic variants in the SLITRK1 gene. Out of all of the variants, 16 were commonly predicted to be pathogenic by these tools. All the variants had very low frequency, i.e., <0.0001 in the global population. The secondary structure of all filtered variants was predicted, but no structural change was observed at the site of variation in any variant. Protein stability analysis of these variants was then performed, which determined a decrease in protein stability of 10 of the variants. Amino acid conservation analysis revealed that all the amino acids were highly conserved, indicating their structural and functional importance. Protein 3D structure of wildtype SLITRK1 and all of its variants was predicted using I-TASSER, and the effect of variation on 3D structure of the protein was observed using the Missense3D tool, which presented the probable structural loss in three variants, i.e., Asn529Lys, Leu496Pro and Leu94Phe. The wildtype SLITRK1 protein and these three variants were independently docked with their close interactor protein PTPRD, and remarkable differences were observed in the docking sites of normal and variants, which will ultimately affect the functional activity of the SLITRK1 protein. Previous studies have shown that mutations in SLITRK1 are involved in Tourette syndrome. The present study may assist a molecular geneticist in interpreting the variant pathogenicity in research as well as diagnostic setup.
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Bhattacharjee, Maloyjo Joyraj, Jinn-Jy Lin, Chih-Yao Chang, Yu-Ting Chiou, Tian-Neng Li, Chia-Wei Tai, Tz-Fan Shiu, et al. "Identifying Primate ACE2 Variants That Confer Resistance to SARS-CoV-2." Molecular Biology and Evolution 38, no. 7 (March 1, 2021): 2715–31. http://dx.doi.org/10.1093/molbev/msab060.
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Abstract SARS-CoV-2 infects humans through the binding of viral S-protein (spike protein) to human angiotensin I converting enzyme 2 (ACE2). The structure of the ACE2-S-protein complex has been deciphered and we focused on the 27 ACE2 residues that bind to S-protein. From human sequence databases, we identified nine ACE2 variants at ACE2–S-protein binding sites. We used both experimental assays and protein structure analysis to evaluate the effect of each variant on the binding affinity of ACE2 to S-protein. We found one variant causing complete binding disruption, two and three variants, respectively, strongly and mildly reducing the binding affinity, and two variants strongly enhancing the binding affinity. We then collected the ACE2 gene sequences from 57 nonhuman primates. Among the 6 apes and 20 Old World monkeys (OWMs) studied, we found no new variants. In contrast, all 11 New World monkeys (NWMs) studied share four variants each causing a strong reduction in binding affinity, the Philippine tarsier also possesses three such variants, and 18 of the 19 prosimian species studied share one variant causing a strong reduction in binding affinity. Moreover, one OWM and three prosimian variants increased binding affinity by >50%. Based on these findings, we proposed that the common ancestor of primates was strongly resistant to and that of NWMs was completely resistant to SARS-CoV-2 and so is the Philippine tarsier, whereas apes and OWMs, like most humans, are susceptible. This study increases our understanding of the differences in susceptibility to SARS-CoV-2 infection among primates.
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Li, Jinying, Hongen Xu, Jianfeng Sun, Yongan Tian, Danhua Liu, Yaping Qin, Huanfei Liu, et al. "Missense Variant of Endoplasmic Reticulum Region of WFS1 Gene Causes Autosomal Dominant Hearing Loss without Syndromic Phenotype." BioMed Research International 2021 (March 4, 2021): 1–9. http://dx.doi.org/10.1155/2021/6624744.
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Objective. Genetic variants in the WFS1 gene can cause Wolfram syndrome (WS) or autosomal dominant nonsyndromic low-frequency hearing loss (HL). This study is aimed at investigating the molecular basis of HL in an affected Chinese family and the genotype-phenotype correlation of WFS1 variants. Methods. The clinical phenotype of the five-generation Chinese family was characterized using audiological examinations and pedigree analysis. Target exome sequencing of 129 known deafness genes and bioinformatics analysis were performed among six patients and four normal subjects to screen suspected pathogenic variants. We built a complete WFS1 protein model to assess the potential effects of the variant on protein structure. Results. A novel heterozygous pathogenic variant NM_006005.3 c.2020G>T (p.Gly674Trp) was identified in the WFS1 gene, located in the C-terminal domain of the wolframin protein. We further showed that HL-related WFS1 missense variants were mainly concentrated in the endoplasmic reticulum (ER) domain. In contrast, WS-related missense variants are randomly distributed throughout the protein. Conclusions. In this family, we identified a novel variant p.Gly674Trp of WFS1 as the primary pathogenic variant causing the low-frequency sensorineural HL, enriching the mutational spectrum of the WFS1 gene.
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Stickler, Marcia, Anita Reddy, Joanna M. Xiong, Melanie H. Wong, Yoshiko Akamatsu, Paul R. Hinton, and Fiona A. Harding. "Design, creation and in vitro testing of a reduced immunogenicity humanized anti-CD25 monoclonal antibody that retains functional activity." Protein Engineering, Design and Selection 32, no. 12 (December 2019): 543–54. http://dx.doi.org/10.1093/protein/gzaa017.
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Abstract Humanized and fully human sequence-derived therapeutic antibodies retain the capacity to induce anti-drug antibodies. Daclizumab (humanized version of the murine anti-Tac antibody; E.HAT) was selected for a proof of concept application of engineering approaches to reduce potential immunogenicity due to its demonstrated immunogenicity in the clinic. Reduced immunogenicity variants of E.HAT were created by identifying and modifying a CD4+ T cell epitope region in the VH region. Variant epitope region peptides were selected for their reduced capacity to induce CD4+ T cell proliferative responses in vitro. Variant antibody molecules were created, and CD25 affinity and potency were similar to the unmodified parent antibody. Fab fragments from the variant antibodies induced a lower frequency and magnitude of responses in human peripheral blood mononuclear cells proliferation tests. By the empirical selection of two amino acid mutations, fully functional humanized E.HAT antibodies with reduced potential to induce immune responses in vitro were created.
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Shah, Shrijal S., Herbert Lannon, Leny Dias, Jia-Yue Zhang, Seth L. Alper, Martin R. Pollak, and David J. Friedman. "APOL1 Kidney Risk Variants Induce Cell Death via Mitochondrial Translocation and Opening of the Mitochondrial Permeability Transition Pore." Journal of the American Society of Nephrology 30, no. 12 (September 26, 2019): 2355–68. http://dx.doi.org/10.1681/asn.2019020114.
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BackgroundGenetic Variants in Apolipoprotein L1 (APOL1) are associated with large increases in CKD rates among African Americans. Experiments in cell and mouse models suggest that these risk-related polymorphisms are toxic gain-of-function variants that cause kidney dysfunction, following a recessive mode of inheritance. Recent data in trypanosomes and in human cells indicate that such variants may cause toxicity through their effects on mitochondria.MethodsTo examine the molecular mechanisms underlying APOL1 risk variant–induced mitochondrial dysfunction, we generated tetracycline-inducible HEK293 T-REx cells stably expressing the APOL1 nonrisk G0 variant or APOL1 risk variants. Using these cells, we mapped the molecular pathway from mitochondrial import of APOL1 protein to APOL1-induced cell death with small interfering RNA knockdowns, pharmacologic inhibitors, blue native PAGE, mass spectrometry, and assessment of mitochondrial permeability transition pore function.ResultsWe found that the APOL1 G0 and risk variant proteins shared the same import pathway into the mitochondrial matrix. Once inside, G0 remained monomeric, whereas risk variant proteins were prone to forming higher-order oligomers. Both nonrisk G0 and risk variant proteins bound components of the mitochondrial permeability transition pore, but only risk variant proteins activated pore opening. Blocking mitochondrial import of APOL1 risk variants largely eliminated oligomer formation and also rescued toxicity.ConclusionsOur study illuminates important differences in the molecular behavior of APOL1 nonrisk and risk variants, and our observations suggest a mechanism that may explain the very different functional effects of these variants, despite the lack of consistently observed differences in trafficking patterns, intracellular localization, or binding partners. Variant-dependent differences in oligomerization pattern may underlie APOL1’s recessive, gain-of-function biology.
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Su, Zhe, Yang Yang, Shengru Wang, Sen Zhao, Hengqiang Zhao, Xiaoxin Li, Yuchen Niu, et al. "The Mutational Landscape of PTK7 in Congenital Scoliosis and Adolescent Idiopathic Scoliosis." Genes 12, no. 11 (November 12, 2021): 1791. http://dx.doi.org/10.3390/genes12111791.
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Depletion of ptk7 is associated with both congenital scoliosis (CS) and adolescent idiopathic scoliosis (AIS) in zebrafish models. However, only one human variant of PTK7 has been reported previously in a patient with AIS. In this study, we systemically investigated the variant landscape of PTK7 in 583 patients with CS and 302 patients with AIS from the Deciphering Disorders Involving Scoliosis and COmorbidities (DISCO) study. We identified a total of four rare variants in CS and four variants in AIS, including one protein truncating variant (c.464_465delAC) in a patient with CS. We then explored the effects of these variants on protein expression and sub-cellular location. We confirmed that the c.464_465delAC variant causes loss-of-function (LoF) of PTK7. In addition, the c.353C>T and c.2290G>A variants identified in two patients with AIS led to reduced protein expression of PTK7 as compared to that of the wild type. In conclusion, LoF and hypomorphic variants are associated with CS and AIS, respectively.
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Rak, Alexandra, Nikolay Gorbunov, Valeria Kostevich, Alexey Sokolov, Polina Prokopenko, Larisa Rudenko, and Irina Isakova-Sivak. "Assessment of Immunogenic and Antigenic Properties of Recombinant Nucleocapsid Proteins of Five SARS-CoV-2 Variants in a Mouse Model." Viruses 15, no. 1 (January 13, 2023): 230. http://dx.doi.org/10.3390/v15010230.
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COVID-19 cases caused by new variants of highly mutable SARS-CoV-2 continue to be identified worldwide. Effective control of the spread of new variants can be achieved through targeting of conserved viral epitopes. In this regard, the SARS-CoV-2 nucleocapsid (N) protein, which is much more conserved than the evolutionarily influenced spike protein (S), is a suitable antigen. The recombinant N protein can be considered not only as a screening antigen but also as a basis for the development of next-generation COVID-19 vaccines, but little is known about induction of antibodies against the N protein via different SARS-CoV-2 variants. In addition, it is important to understand how antibodies produced against the antigen of one variant can react with the N proteins of other variants. Here, we used recombinant N proteins from five SARS-CoV-2 strains to investigate their immunogenicity and antigenicity in a mouse model and to obtain and characterize a panel of hybridoma-derived monoclonal anti-N antibodies. We also analyzed the variable epitopes of the N protein that are potentially involved in differential recognition of antiviral antibodies. These results will further deepen our knowledge of the cross-reactivity of the humoral immune response in COVID-19.
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Ramalingam, Satish, Gopalan Natarajan, Chris Schafer, Dharmalingam Subramaniam, Randal May, Ilangovan Ramachandran, Lurdes Queimado, Courtney W. Houchen, and Shrikant Anant. "Novel intestinal splice variants of RNA-binding protein CUGBP2: isoform-specific effects on mitotic catastrophe." American Journal of Physiology-Gastrointestinal and Liver Physiology 294, no. 4 (April 2008): G971—G981. http://dx.doi.org/10.1152/ajpgi.00540.2007.
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CUG triplet repeat-binding protein 2 (CUGBP2) is a RNA-binding protein that regulates mRNA translation and modulates apoptosis. Here, we report the identification of two splice variants (termed variants 2 and 3) in cultured human intestinal epithelial cells and in mouse gastrointestinal tract. The variants are generated from alternative upstream promoters resulting in the inclusion of additional NH2-terminal residues. Although variant 2 is the predominant isoform in normal intestine, its expression is reduced, whereas variant 1 is overexpressed following γ-irradiation. All three variants bind cyclooxygenase-2 (COX-2) mRNA. However, only variant 1 inhibits the translation of the endogenous COX-2 mRNA and a chimeric luciferase mRNA containing the COX-2 3′untranslated region. Furthermore, whereas variant 1 is predominantly nuclear, variants 2 and 3 are predominantly cytoplasmic. These data imply that the additional amino acids affect CUGBP2 function. Previous studies have demonstrated that variant 1 induces intestinal epithelial cells to undergo apoptosis. However, in contrast to variant 1, the two novel variants do not affect proliferation or apoptosis of HCT116 cells. In addition, only variant 1 induced G2/M cell cycle arrest, which was overcome by prostaglandin E2. Moreover, variant 1 increased cellular levels of phosphorylated p53 and Bax and decreased Bcl2. Caspase-3 and -9 were also activated, suggesting the initiation of the intrinsic apoptotic pathway. Furthermore, increased phosphorylation of checkpoint kinase (Chk)1 and Chk2 kinases and increased nuclear localization of Cdc2 and cyclin B1 suggested that cells were in mitotic transition. Taken together, these data demonstrate that cells expressing CUGBP2 variant 1 undergo apoptosis during mitosis, suggesting mitotic catastrophe.
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Vermeer, Mathilde C. S. C., Daniela Andrei, Luisa Marsili, J. Peter van Tintelen, Herman H. W. Silljé, Maarten P. van den Berg, Peter van der Meer, and Maria C. Bolling. "Towards a Better Understanding of Genotype–Phenotype Correlations and Therapeutic Targets for Cardiocutaneous Genes: The Importance of Functional Studies above Prediction." International Journal of Molecular Sciences 23, no. 18 (September 15, 2022): 10765. http://dx.doi.org/10.3390/ijms231810765.
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Genetic variants in gene-encoding proteins involved in cell–cell connecting structures, such as desmosomes and gap junctions, may cause a skin and/or cardiac phenotype, of which the combination is called cardiocutaneous syndrome. The cardiac phenotype is characterized by cardiomyopathy and/or arrhythmias, while the skin particularly displays phenotypes such as keratoderma, hair abnormalities and skin fragility. The reported variants associated with cardiocutaneous syndrome, in genes DSP, JUP, DSC2, KLHL24, GJA1, are classified by interpretation guidelines from the American College of Medical Genetics and Genomics. The genotype–phenotype correlation, however, remains poorly understood. By providing an overview of variants that are assessed for a functional protein pathology, we show that this number (n = 115) is low compared to the number of variants that are assessed by in silico algorithms (>5000). As expected, there is a mismatch between the prediction of variant pathogenicity and the prediction of the functional effect compared to the real functional evidence. Aiding to improve genotype–phenotype correlations, we separate variants into ‘protein reducing’ or ‘altered protein’ variants and provide general conclusions about the skin and heart phenotype involved. We conclude by stipulating that adequate prognoses can only be given, and targeted therapies can only be designed, upon full knowledge of the protein pathology through functional investigation.
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Hawkins-Hooker, Alex, Florence Depardieu, Sebastien Baur, Guillaume Couairon, Arthur Chen, and David Bikard. "Generating functional protein variants with variational autoencoders." PLOS Computational Biology 17, no. 2 (February 26, 2021): e1008736. http://dx.doi.org/10.1371/journal.pcbi.1008736.
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The vast expansion of protein sequence databases provides an opportunity for new protein design approaches which seek to learn the sequence-function relationship directly from natural sequence variation. Deep generative models trained on protein sequence data have been shown to learn biologically meaningful representations helpful for a variety of downstream tasks, but their potential for direct use in the design of novel proteins remains largely unexplored. Here we show that variational autoencoders trained on a dataset of almost 70000 luciferase-like oxidoreductases can be used to generate novel, functional variants of the luxA bacterial luciferase. We propose separate VAE models to work with aligned sequence input (MSA VAE) and raw sequence input (AR-VAE), and offer evidence that while both are able to reproduce patterns of amino acid usage characteristic of the family, the MSA VAE is better able to capture long-distance dependencies reflecting the influence of 3D structure. To confirm the practical utility of the models, we used them to generate variants of luxA whose luminescence activity was validated experimentally. We further showed that conditional variants of both models could be used to increase the solubility of luxA without disrupting function. Altogether 6/12 of the variants generated using the unconditional AR-VAE and 9/11 generated using the unconditional MSA VAE retained measurable luminescence, together with all 23 of the less distant variants generated by conditional versions of the models; the most distant functional variant contained 35 differences relative to the nearest training set sequence. These results demonstrate the feasibility of using deep generative models to explore the space of possible protein sequences and generate useful variants, providing a method complementary to rational design and directed evolution approaches.
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Golub, Maksym, Virginia Guillon, Guillaume Gotthard, Dominik Zeller, Nicolas Martinez, Tilo Seydel, Michael M. Koza, et al. "Dynamics of a family of cyan fluorescent proteins probed by incoherent neutron scattering." Journal of The Royal Society Interface 16, no. 152 (March 2019): 20180848. http://dx.doi.org/10.1098/rsif.2018.0848.
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Cyan fluorescent proteins (CFPs) are variants of green fluorescent proteins in which the central tyrosine of the chromophore has been replaced by a tryptophan. The increased bulk of the chromophore within a compact protein and the change in the positioning of atoms capable of hydrogen bonding have made it difficult to optimize their fluorescence properties, which took approximately 15 years between the availability of the first useable CFP, enhanced cyan fluorescent protein (ECFP), and that of a variant with almost perfect fluorescence efficiency, mTurquoise2. To understand the molecular bases of the progressive improvement in between these two CFPs, we have studied by incoherent neutron scattering the dynamics of five different variants exhibiting progressively increased fluorescence efficiency along the evolution pathway. Our results correlate well with the analysis of the previously determined X-ray crystallographic structures, which show an increase in flexibility between ECFP and the second variant, Cerulean, which is then hindered in the three later variants, SCFP3A (Super Cyan Fluorescent Protein 3A), mTurquoise and mTurquoise2. This confirms that increasing the rigidity of the direct environment of the fluorescent chromophore is not the sole parameter leading to brighter fluorescent proteins and that increased flexibility in some cases may be helpful.
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Lezzerini, Marco, Marianna Penzo, Marie-Françoise O’Donohue, Carolina Marques dos Santos Vieira, Manon Saby, Hyung L. Elfrink, Illja J. Diets, et al. "Ribosomal protein gene RPL9 variants can differentially impair ribosome function and cellular metabolism." Nucleic Acids Research 48, no. 2 (December 4, 2019): 770–87. http://dx.doi.org/10.1093/nar/gkz1042.
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Abstract Variants in ribosomal protein (RP) genes drive Diamond-Blackfan anemia (DBA), a bone marrow failure syndrome that can also predispose individuals to cancer. Inherited and sporadic RP gene variants are also linked to a variety of phenotypes, including malignancy, in individuals with no anemia. Here we report an individual diagnosed with DBA carrying a variant in the 5′UTR of RPL9 (uL6). Additionally, we report two individuals from a family with multiple cancer incidences carrying a RPL9 missense variant. Analysis of cells from these individuals reveals that despite the variants both driving pre-rRNA processing defects and 80S monosome reduction, the downstream effects are remarkably different. Cells carrying the 5′UTR variant stabilize TP53 and impair the growth and differentiation of erythroid cells. In contrast, ribosomes incorporating the missense variant erroneously read through UAG and UGA stop codons of mRNAs. Metabolic profiles of cells carrying the 5′UTR variant reveal an increased metabolism of amino acids and a switch from glycolysis to gluconeogenesis while those of cells carrying the missense variant reveal a depletion of nucleotide pools. These findings indicate that variants in the same RP gene can drive similar ribosome biogenesis defects yet still have markedly different downstream consequences and clinical impacts.
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Taylor, Susan S., Maximilian Wallbott, Erik M. F. Machal, Kristoffer Søberg, Faihaa Ahmed, Jessica Bruystens, Lily Vu, et al. "PKA Cβ: a forgotten catalytic subunit of cAMP-dependent protein kinase opens new windows for PKA signaling and disease pathologies." Biochemical Journal 478, no. 11 (June 11, 2021): 2101–19. http://dx.doi.org/10.1042/bcj20200867.
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3′,5′-cyclic adenosine monophosphate (cAMP) dependent protein kinase or protein kinase A (PKA) has served as a prototype for the large family of protein kinases that are crucially important for signal transduction in eukaryotic cells. The PKA catalytic subunits are encoded by the two major genes PRKACA and PRKACB, respectively. The PRKACA gene encodes two known splice variants, the ubiquitously expressed Cα1 and the sperm-specifically expressed Cα2. In contrast, the PRKACB gene encodes several splice variants expressed in a highly cell and tissue-specific manner. The Cβ proteins are called Cβ1, Cβ2, Cβ3, Cβ4 and so-called abc variants of Cβ3 and Cβ4. Whereas Cβ1 is ubiquitously expressed, Cβ2 is enriched in immune cells and the Cβ3, Cβ4 and their abc variants are solely expressed in neuronal cells. All Cα and Cβ splice variants share a kinase-conserved catalytic core and a C-terminal tail encoded by exons 2 through 10 in the PRKACA and PRKACB genes, respectively. All Cα and Cβ splice variants with the exception of Cα1 and Cβ1 are hyper-variable at the N-terminus. Here, we will discuss how the PRKACA and PRKACB genes have developed as paralogs that encode distinct and functionally non-redundant proteins. The fact that Cα and Cβ splice variant mutations are associated with numerous diseases further opens new windows for PKA-induced disease pathologies.
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Matsuura, Yuichi, Yukinobu Tohya, Masami Mochizuki, Kozo Takase, and Takaaki Sugimura. "Identification of conformational neutralizing epitopes on the capsid protein of canine calicivirus." Journal of General Virology 82, no. 7 (July 1, 2001): 1695–702. http://dx.doi.org/10.1099/0022-1317-82-7-1695.
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Two neutralizing monoclonal antibodies (MAbs) against canine calicivirus (CaCV), which has a distinct antigenicity from feline calicivirus (FCV), were obtained. Both MAbs recognized conformational epitopes on the capsid protein of CaCV and were used to identify these epitopes. Neutralization-resistant variants of CaCV were selected in the presence of individual MAbs in a cell culture. Cross-neutralization tests using the variants indicated that the MAbs recognized functionally independent epitopes on the capsid protein. Recombinantly expressed ORF2 products (capsid precursors) of the variants showed no reactivity to the MAbs used for the selection, suggesting that the resistance was induced by a failing in binding of the MAbs to the variant capsid proteins. Several nucleotide changes resulting in amino acid substitutions in the capsid protein were found by sequence analysis. Reactivities of the MAbs to the revertant ORF2 products produced from each variant ORF2 by site-directed mutagenesis identified a single amino acid substitution in each variant capsid protein responsible for the failure of MAb binding. The amino acid residues related to forming the conformational neutralizing epitopes were located in regions equivalent to the 5′ and 3′ hypervariable regions of the FCV capsid protein, where antigenic sites were demonstrated in previous studies. The recombinant ORF2 products expressed in bacteria failed to induce neutralizing antibody, suggesting that neutralizing antibodies were only generated when properly folded capsid protein was used as an antigen. In CaCV, the conformational epitopes may play a more important role in neutralization than do linear epitopes.
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Mangrolia, Parth, Dennis T. Yang, and Regina M. Murphy. "Transthyretin variants with improved inhibition of β-amyloid aggregation." Protein Engineering Design and Selection 29, no. 6 (April 19, 2016): 209–18. http://dx.doi.org/10.1093/protein/gzw008.
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Nicora, Giovanna, Marco Salemi, Simone Marini, and Riccardo Bellazzi. "Predicting emerging SARS-CoV-2 variants of concern through a One Class dynamic anomaly detection algorithm." BMJ Health & Care Informatics Online 29, no. 1 (December 2022): e100643. http://dx.doi.org/10.1136/bmjhci-2022-100643.
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ObjectivesThe objective of this study is the implementation of an automatic procedure to weekly detect new SARS-CoV-2 variants and non-neutral variants (variants of concern (VOC) and variants of interest (VOI)).MethodsWe downloaded spike protein primary sequences from the public resource GISAID and we represented each sequence as k-mer counts. For each week since 1 July 2020, we evaluate if each sequence represents an anomaly based on a One Class support vector machine (SVM) classification algorithm trained on neutral protein sequences collected from February to June 2020.ResultsWe assess the ability of the One Class classifier to detect known VOC and VOI, such as Alpha, Delta or Omicron, ahead of their official classification by health authorities. In median, the classifier predicts a non-neutral variant as outlier 10 weeks before the official date of designation as VOC/VOI.DiscussionThe identification of non-neutral variants during a pandemic usually relies on indicators available during time, such as changing population size of a variant. Automatic variant surveillance systems based on protein sequences can enhance the fast identification of variants of potential concern.ConclusionMachine learning, and in particular One Class SVM classification, can support the detection of potentially VOC/VOI variants during an evolving pandemics.
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Bian, Xinchao, Guangying Cheng, Xinbo Sun, Hongkun Liu, Xiangmao Zhang, Yu Han, Bo Li, and Ning Li. "Two novel truncating variants in UBAP1 are responsible for hereditary spastic paraplegia." PLOS ONE 16, no. 6 (June 30, 2021): e0253871. http://dx.doi.org/10.1371/journal.pone.0253871.
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Hereditary spastic paraplegias (HSPs) are a group of rare neurodegenerative disorders. HSPs are complex disorders and are clinically and genetically heterogeneous. To date, more than 80 genes or genetic loci have been reported to be responsible for HSPs in a Mendelian-dependent manner. Most recently, ubiquitin-associated protein 1 (UBAP1) has been recognized to be involved in HSP. Here, we identified novel protein truncating variants in two families with pure form of HSP. A novel deletion (c.468_469delTG) in the UBAP1 gene was found in the first family, whereas a nonsense variant (c.512T>G) was ascertained in the second family. The variants were confirmed in all patients but were not detected in unaffected family members. The mutations resulted in truncated proteins of UBAP1. The variants did not result in different subcellular localizations in neuro-2a cells. However, each of the two variants impaired neurite outgrowth. Taken together, our findings expand the pathogenic spectrum of UBAP1 variants in HSP.
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French, Dorothy M., Terry F. McElwain, Travis C. McGuire, and Guy H. Palmer. "Expression of Anaplasma marginale Major Surface Protein 2 Variants during Persistent Cyclic Rickettsemia." Infection and Immunity 66, no. 3 (March 1, 1998): 1200–1207. http://dx.doi.org/10.1128/iai.66.3.1200-1207.1998.
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ABSTRACT Anaplasma marginale is an intraerythrocytic rickettsial pathogen of cattle in which infection persists for the life of the animal. Persistent A. marginale infection is characterized by repetitive rickettsemic cycles which we hypothesize reflect emergence of A. marginale antigenic variants. In this study, we determined whether variants of major surface protein 2 (MSP-2), a target of protective immunity encoded by a polymorphic multigene family, arise during persistent rickettsemia. By using a quantitative competitive PCR to identify rickettsemic cycles,msp-2 transcripts expressed in vivo were isolated from peak rickettsemia of sequential cycles. Cloning and sequencing ofmsp-2 cDNA revealed that genetic variants of MSP-2 emerge representing a minimum of four genetic variant types in each cycle during persistent infection. Two-color immunofluorescence using variant-specific antibody showed that emergence of MSP-2 variants resulted in expression of a minimum of three antigenic types of MSP-2 within one rickettsemic cycle. Therefore immune control of each cycle would require responses to an antigenically diverse A. marginale population. These findings demonstrate that polymorphic MSP-2 variants emerge during cyclic rickettsemia in persistent A. marginale infection and suggest that emergent variants play an important role in persistence.