Comprehensive mutagenesis to identify amino acid residues contributing to the difference in thermostability between two originally thermostable ancestral proteins

Further improvement of the thermostability of inherently thermostable proteins is an attractive challenge because more thermostable proteins are industrially more useful and serve as better scaffolds for protein engineering. To establish guidelines that can be applied for the rational design of hyperthermostable proteins, we compared the amino acid sequences of two ancestral nucleoside diphosphate kinases, Arc1 and Bac1, reconstructed in our previous study. Although Bac1 is a thermostable protein whose unfolding temperature is around 100°C, Arc1 is much more thermostable with an unfolding temperature of 114°C. However, only 12 out of 139 amino acids are different between the two sequences. In this study, one or a combination of amino acid(s) in Bac1 was/were substituted by a residue(s) found in Arc1 at the same position(s). The best mutant, which contained three amino acid substitutions (S108D, G116A and L120P substitutions), showed an unfolding temperature more than 10°C higher than that of Bac1. Furthermore, a combination of the other nine amino acid substitutions also led to improved thermostability of Bac1, although the effects of individual substitutions were small. Therefore, not only the sum of the contributions of individual amino acids, but also the synergistic effects of multiple amino acids are deeply involved in the stability of a hyperthermostable protein. Such insights will be helpful for future rational design of hyperthermostable proteins.

Deep learning based kcat prediction enables improved enzyme constrained model reconstruction

Enzyme turnover numbers (kcat values) are key parameters to understand cell metabolism, proteome allocation and physiological diversity, but experimentally measured kcat data are sparse and noisy. Here we provide a deep learning approach to predict kcat values for metabolic enzymes in a high-throughput manner with the input of substrate structures and protein sequences. Our approach can capture kcat changes for mutated enzymes and identify amino acid residues with great impact on kcat values. Furthermore, we applied the approach to predict genome scale kcat values for over 300 yeast species, demonstrating that the predicted kcat values are consistent with current evolutional understanding. Additionally, we designed an automatic pipeline using the predicted kcat values to parameterize enzyme-constrained genome scale metabolic models (ecGEMs) facilitated by a Bayesian approach, which outperformed the default ecGEMs in predicting phenotypes and proteomes and enabled to explain phenotype differences among yeast species. The deep learning kcat prediction approach and automatic ecGEM construction pipeline would thus be a valuable tool to uncover the global trend of enzyme kinetics and physiological diversity, and to further elucidate cell metabolism on a large scale.

Identification of L-Cysteinamide as a Potent Inhibitor of Tyrosinase-Mediated Dopachrome Formation and Eumelanin Synthesis

The purpose of this study is to identify amino acid derivatives with potent anti-eumelanogenic activity. First, we compared the effects of twenty different amidated amino acids on tyrosinase (TYR)-mediated dopachrome formation in vitro and melanin content in dark-pigmented human melanoma MNT-1 cells. The results showed that only L-cysteinamide inhibited TYR-mediated dopachrome formation in vitro and reduced the melanin content of cells. Next, the antimelanogenic effect of L-cysteinamide was compared to those of other thiol compounds (L-cysteine, N-acetyl L-cysteine, glutathione, L-cysteine ethyl ester, N-acetyl L-cysteinamide, and cysteamine) and positive controls with known antimelanogenic effects (kojic acid and β-arbutin). The results showed the unique properties of L-cysteinamide, which effectively reduces melanin content without causing cytotoxicity. L-Cysteinamide did not affect the mRNA and protein levels of TYR, tyrosinase-related protein 1, and dopachrome tautomerase in MNT-1 cells. L-Cysteinamide exhibited similar properties in normal human epidermal melanocytes (HEMs). Experiments using mushroom TYR suggest that L-cysteinamide at certain concentrations can inhibit eumelanin synthesis through a dual mechanism by inhibiting TYR-catalyzed dopaquinone synthesis and by diverting the synthesized dopaquinone to the formation of DOPA-cysteinamide conjugates rather than dopachrome. Finally, L-cysteinamide was shown to increase pheomelanin content while decreasing eumelanin and total melanin contents in MNT-1 cells. This study suggests that L-cysteinamide has an optimal structure that can effectively and safely inhibit eumelanin synthesis in MNT-1 cells and HEMs, and will be useful in controlling skin hyperpigmentation.

Genetic characterisation of the influenza viruses circulating in Bulgaria during the 2019–2020 winter season

Influenza viruses have a high potential for genetic changes. The objectives of this study were to analyse influenza virus circulation in Bulgaria during the 2019/2020 season, to perform a phylogenetic and molecular analyses of the haemagglutinin (HA) and neuraminidase (NA) sequences of representative influenza strains, and to identify amino acid substitutions compared to the current vaccine strains. Seasonal influenza viruses A(H3N2), A(H1N1)pdm09 and B/Victoria-lineage were detected using a real-time RT-PCR in 323 (23.3%), 149 (10.7%) and 138 (9.9%) out of 1387 patient samples studied, respectively. The HA genes of A(H3N2) viruses analysed belonged to clades 3C.3a (21 strains) and 3C.2a (5 strains): subclades 3C.2a1b + T131K, 3C.2a1b + T135K-B and 3C.2a1b + T135K-A. The clade 3C.3a and subclade 3C.2a1b viruses carried 5 and 14–17 substitutions in HA, as well as 3 and 9 substitutions in NA, respectively, in comparison with the A/Kansas/14/2017 vaccine virus, including some substitutions in the HA antigenic sites A, B, C and E. All 21 A(H1N1)pdm09 viruses sequenced fell into 6B.1A5A subclade. Amino acid sequence analysis revealed the presence of 7–11 substitutions in HA, compared to the A/Brisbane/02/2018 vaccine virus, three of which occurred in antigenic site Sb, along with 6–9 changes at positions in NA. All 10 B/Victoria-lineage viruses sequenced belonged to clade 1A with a triple deletion in HA1 (genetic group 1A(Δ3)B) and carried 7 and 3 substitutions in HA and NA, respectively, with respect to the B/Colorado/06/2017 vaccine virus. The results of this study confirm the rapid evolution of influenza viruses and the need for continuous antigenic and genetic surveillance.

Amino Acid Residues Involved in Inhibition of Host Gene Expression by Influenza A/Brevig Mission/1/1918 PA-X

The influenza A virus (IAV) PA-X protein is a virulence factor that selectively degrades host mRNAs leading to protein shutoff. This function modulates host inflammation, antiviral responses, cell apoptosis, and pathogenesis. In this work we describe a novel approach based on the use of bacteria and plasmid encoding of the PA-X gene under the control of the bacteriophage T7 promoter to identify amino acid residues important for A/Brevig Mission/1/1918 H1N1 PA-X’s shutoff activity. Using this system, we have identified PA-X mutants encoding single or double amino acid changes, which diminish its host shutoff activity, as well as its ability to counteract interferon responses upon viral infection. This novel bacteria-based approach could be used for the identification of viral proteins that inhibit host gene expression as well as the amino acid residues responsible for inhibition of host gene expression.

Amino acid residues involved in inhibition of host gene expression by influenza A/Brevig Mission/1/1918 PA-X

Influenza A virus (IAV) PA-X protein is a virulence factor that selectively degrades host mRNAs leading to protein shutoff. This function modulates host inflammation, antiviral responses, cell apoptosis, and pathogenesis. In this work we describe a novel approach based on the use of bacteria and a plasmid encoding the PA-X gene under the control of the bacteriophage T7 promoter to identify amino acid residues important for A/Brevig Mission/1/1918 H1N1 PA-X’s shutoff activity. Using this system, we have identified PA-X mutants encoding single or double amino acid changes, which diminish its host shutoff activity, as well as its ability to counteract interferon responses upon viral infection. This novel bacteria-based approach could be used for the identification of viral proteins that inhibit host gene expression as well as the amino acid residues responsible for inhibition of host gene expression.

Identification of amino acid residues required for inhibition of host gene expression by influenza A/Viet Nam/1203/2004 H5N1 PA-X

PA-X is a non-structural protein of influenza A virus (IAV), which is encoded by the polymerase acidic (PA) N-terminal region that contains a C-terminal +1 frameshifted sequence. IAV PA-X protein modulates virus-induced host innate immune responses and viral pathogenicity via suppression of host gene expression or cellular shutoff, through cellular mRNA cleavage. Highly pathogenic avian influenza viruses (HPAIV) of the H5N1 subtype naturally infect different avian species, they have an enormous economic impact in the poultry farming, and they also have zoonotic and pandemic potential, representing a risk to human public health. In the present study, we describe a novel bacteria-based approach to identify amino acid residues in the PA-X protein of the HPAIV A/Viet Nam/1203/2004 H5N1 that are important for its ability to inhibit host protein expression or cellular shutoff activity. Identified PA-X mutants displayed a reduced shutoff activity as compared to that of the wild-type (WT) A/Viet Nam/1203/2004 H5N1 PA-X protein. Notably, this new bacteria-based screening allowed us to identify amino acid residues widely distributed over the entire N-terminal region of PA-X. Furthermore, we found that some of the residues affecting A/Viet Nam/1203/2004 H5N1 PA-X host shutoff activity also affect PA polymerase activity in a minigenome assay. This information could be used for the rational design of new and more effective compounds with antiviral activity against IAV. Moreover, our results demonstrate the feasibility of using this bacteria-based approach to identify amino acid residues important for the activity of viral proteins to inhibit host gene expression. IMPORTANCE Highly pathogenic avian influenza viruses (HPAIV) continue to pose a huge threat to global animal and human health. Despite of the limited genome size of Influenza A virus (IAV), the virus encodes eight main viral structural proteins and multiple accessory non-structural proteins, depending on the IAV type, subtype or strain. One of the IAV accessory proteins, PA-X, is encoded by the polymerase acidic (PA) protein and is involved in pathogenicity through the modulation of IAV-induced host inflammatory and innate immune responses. However, the molecular mechanism(s) of IAV PA-X regulation of the host immune response is not well understood. In this work, we used, for the first time, a bacteria-based approach for the identification of amino acids important for the ability of IAV PA-X to induce host shutoff activity and describe novel residues relevant for its ability to inhibit host gene expression, and their contribution in PA polymerase activity.

The impact of viral mutations on recognition by SARS-CoV-2 specific T-cells

We identify amino acid variants within dominant SARS-CoV-2 T-cell epitopes by interrogating global sequence data. Several variants within nucleocapsid and ORF3a epitopes have arisen independently in multiple lineages and result in loss of recognition by epitope-specific T-cells assessed by IFN-γ and cytotoxic killing assays. These data demonstrate the potential for T-cell evasion and highlight the need for ongoing surveillance for variants capable of escaping T-cell as well as humoral immunity.

Structural bioinformatics predicts that the Retinitis Pigmentosa-28 protein of unknown function FAM161A is a homologue of the microtubule nucleation factor Tpx2

Background: FAM161A is a microtubule-associated protein conserved widely across eukaryotes, which is mutated in the inherited blinding disease Retinitis Pigmentosa-28. FAM161A is also a centrosomal protein, being a core component of a complex that forms an internal skeleton of centrioles. Despite these observations about the importance of FAM161A, current techniques used to examine its sequence reveal no homologies to other proteins. Methods: Sequence profiles derived from multiple sequence alignments of FAM161A homologues were constructed by PSI-BLAST and HHblits, and then used by the profile-profile search tool HHsearch, implemented online as HHpred, to identify homologues. These in turn were used to create profiles for reverse searches and pair-wise searches. Multiple sequence alignments were also used to identify amino acid usage in functional elements. Results: FAM161A has a single homologue: the targeting protein for Xenopus kinesin-like protein-2 (Tpx2), which is a strong hit across more than 200 residues. Tpx2 is also a microtubule-associated protein, and it has been shown previously by a cryo-EM molecular structure to nucleate microtubules through two small elements: an extended loop and a short helix. The homology between FAM161A and Tpx2 includes these elements, as FAM161A has three copies of the loop, and one helix that has many, but not all, properties of the one in Tpx2. Conclusions: FAM161A and ­its homologues are predicted to be a previously unknown variant of Tpx2, and hence bind microtubules in the same way. This prediction allows precise, testable molecular models to be made of FAM161A-microtubule complexes.

Highly conserved extracellular residues mediate interactions between pore-forming and regulatory subunits of the yeast Ca2+ channel related to the animal VGCC/NALCN family

Yeasts and fungi generate Ca2+ signals in response to environmental stresses through Ca2+ channels essentially composed of Cch1 and Mid1. Cch1 is homologous to the pore-forming α1 subunit of animal voltage-gated Ca2+ channels (VGCCs) and sodium leak channels nonselective (NALCNs), whereas Mid1 is a membrane-associated protein similar to the regulatory α2/δ subunit of VGCCs and the regulatory subunit of NALCNs. Although the physiological roles of Cch1/Mid1 channels are known, their molecular regulation remains elusive, including subunit interactions regulating channel functionality. Herein, we identify amino acid residues involved in interactions between the pore-forming Cch1 subunit and the essential regulatory Mid1 subunit of Saccharomyces cerevisiae. In vitro mutagenesis followed by functional assays and co-immunoprecipitation experiments reveal that three residues present in a specific extracellular loop in the repeat III region of Cch1 are required for interaction with Mid1, and that one essential Mid1 residue is required for interaction with Cch1. Importantly, these residues are necessary for Ca2+ channel activity and are highly conserved in fungal and animal counterparts. We discuss that this unique subunit interaction-based regulatory mechanism for Cch1 differs from that of VGCCs/NALCNs.