TaxaTarget: Fast, Sensitive, and Precise Classification of Microeukaryotes in Metagenomic Data

BackgroundMicrobial eukaryotes are nearly ubiquitous in microbiomes on Earth and contribute to many integral ecological functions. Metagenomics is a proven tool for studying the microbial diversity, functions, and ecology of microbiomes, but has been underutilized for microeukaryotes due to the computational challenges they present. For taxonomic classification, the use of a eukaryotic marker gene database can improve the computational efficiency, precision and sensitivity. However, state-of-the-art tools which use marker gene databases implement universal thresholds for classification rather than dynamically learning the thresholds from the database structure, impacting the accuracy of the classification process.ResultsHere we introduce taxaTarget, a method for the taxonomic classification of microeukaryotes in metagenomic data. Using a database of eukaryotic marker genes and a supervised learning approach for training, we learned the discriminatory power and classification thresholds for each 20 amino acid region of each marker gene in our database. This approach provided improved sensitivity and precision compared to other state-of-the-art approaches, with rapid runtimes and low memory usage. Additionally, taxaTarget was better able to detect the presence of multiple closely related species as well as species with no representative sequences in the database. One of the greatest challenges faced during the development of taxaTarget was the general sparsity of available sequences for microeukaryotes. Several algorithms were implemented, including threshold padding, which effectively handled the missing training data and reduced classification errors. Using taxaTarget on metagenomes from human fecal microbiomes, a broader range of genera were detected, including multiple parasites that the other tested tools missed.ConclusionData-driven methods for learning classification thresholds from the structure of an input database can provide granular information about the discriminatory power of the sequences and improve the sensitivity and precision of classification. These methods will help facilitate a more comprehensive analysis of metagenomic data and expand our knowledge about the diverse eukaryotes in microbial communities.

A Novel Actinobacterial Cutinase Containing a Non-Catalytic Polymer-Binding Domain

The single putative cutinase-encoding gene from the genome of Kineococcus radiotolerans SRS30216 was cloned and expressed in Escherichia coli as a secreted fusion protein, designated YebF-KrCUT, where YebF is the extracellular carrier protein. The 294-amino acid sequence of KrCUT is unique among currently characterized cutinases by having a C-terminal extension that consists of a short (Pro-Thr)-rich linker and a 55-amino-acid region resembling the substrate binding domain of poly(hydroxybutyrate) (PHB) depolymerases. Phylogenetically, KrCUT takes a unique position among known cutinases and cutinase-like proteins of bacterial and fungal origin. A modeled structure of KrCUT, although displaying a typical α/ß hydrolase fold, shows some unique loops close to the catalytic site. The 39-kDa YebF-KrCUT fusion protein and a truncated variant thereof were purified to electrophoretic homogeneity and functionally characterized. The melting temperatures ( T m ) of KrCUT and its variant KrCUT206 devoid of the putative PHB-binding domain were established to be very similar at 50–51°C. Cutinase activity was confirmed by the appearance of characteristic cutin components, C 16 and C 18 hydroxyl fatty acids, in the mass chromatograms following incubation of KrCUT with apple cutin as substrate. KrCUT also efficiently degraded synthetic polyesters such as polycaprolactone and poly(1,3-propylene adipate). Although incapable of PHB depolymerization, KrCUT could efficiently bind PHB, confirming the predicted characteristic of the C-terminal region. KrCUT also potentiated the activity of pectate lyase in the degradation of pectin from hemp fibres. This synergistic effect is relevant to the enzyme retting process of natural fibres. IMPORTANCE. To date only a limited number of cutinases have been isolated and characterized from nature, the majority being sourced from phytopathogenic fungi and thermophilic bacteria. The significance of our research relates to the identification and characterization of a unique member of microbial cutinases, of name KrCUT, that was derived from the genome of the Gram-positive Kineococcus radiotolerans SRS30216, a highly radiation-resistant actinobacterium. Given the wide-ranging importance of cutinases in applications such as the degradation of natural and synthetic polymers, in the textile industry, in laundry detergents, or in biocatalysis (e.g., transesterification reactions), our results could foster new research leading to broader biotechnological impacts. This study also demonstrated that genome mining or prospecting is a viable means to discover novel biocatalysts as environmentally friendly and biotechnological tool.

An Expanded Polyproline Domain Maintains Mutant Huntingtin Soluble in vivo and During Aging

Huntington’s disease is a dominantly inherited neurodegenerative disorder caused by the expansion of a CAG repeat, encoding for the amino acid glutamine (Q), present in the first exon of the protein huntingtin. Over the threshold of Q39 HTT exon 1 (HTTEx1) tends to misfold and aggregate into large intracellular structures, but whether these end-stage aggregates or their on-pathway intermediates are responsible for cytotoxicity is still debated. HTTEx1 can be separated into three domains: an N-terminal 17 amino acid region, the polyglutamine (polyQ) expansion and a C-terminal proline rich domain (PRD). Alongside the expanded polyQ, these flanking domains influence the aggregation propensity of HTTEx1: with the N17 initiating and promoting aggregation, and the PRD modulating it. In this study we focus on the first 11 amino acids of the PRD, a stretch of pure prolines, which are an evolutionary recent addition to the expanding polyQ region. We hypothesize that this proline region is expanding alongside the polyQ to counteract its ability to misfold and cause toxicity, and that expanding this proline region would be overall beneficial. We generated HTTEx1 mutants lacking both flanking domains singularly, missing the first 11 prolines of the PRD, or with this stretch of prolines expanded. We then followed their aggregation landscape in vitro with a battery of biochemical assays, and in vivo in novel models of C. elegans expressing the HTTEx1 mutants pan-neuronally. Employing fluorescence lifetime imaging we could observe the aggregation propensity of all HTTEx1 mutants during aging and correlate this with toxicity via various phenotypic assays. We found that the presence of an expanded proline stretch is beneficial in maintaining HTTEx1 soluble over time, regardless of polyQ length. However, the expanded prolines were only advantageous in promoting the survival and fitness of an organism carrying a pathogenic stretch of Q48 but were extremely deleterious to the nematode expressing a physiological stretch of Q23. Our results reveal the unique importance of the prolines which have and still are evolving alongside expanding glutamines to promote the function of HTTEx1 and avoid pathology.

Inhibition of interferon I induction by non-structural protein NSs of Puumala virus and other vole-associated orthohantaviruses: phenotypic plasticity of the protein and potential functional domains

The orthohantavirus Puumala virus (PUUV), which is transmitted by bank voles (Clethrionomys glareolus), and other vole-borne hantaviruses contain in their small (S) genome segment two overlapping open reading frames, coding for the nucleocapsid protein and the non-structural protein NSs, a putative type I interferon (IFN-I) antagonist. To investigate the role of NSs of PUUV and other orthohantaviruses, the expression pattern of recombinant NSs constructs and their ability to inhibit human IFN-I promoter activity were investigated. The NSs proteins of PUUV and related cricetid-borne orthohantaviruses showed strong inhibition of IFN-I promoter induction. We identified protein products originating from three and two methionine initiation codons in the NSs ORF of PUUV during transfection and infection, respectively. The three putative start codons are conserved in all PUUV strains analysed. Translation initiation at these start codons influenced the inhibitory activity of the NSs products, with the wild-type (wt) construct expressing two proteins starting at the first and second methionine and showing strong inhibition activity. Analysis of in vitro-generated variants and naturally occurring PUUV NSs proteins indicated that amino acid variation in the NSs protein is well tolerated, suggesting its phenotypic plasticity. The N-terminal 20-amino-acid region of the NSs protein was found to be associated with strong inhibition and to be highly vulnerable to amino acid exchanges and tag fusions. Infection studies using human, bank vole, and Vero E6 cells did not show obvious differences in the replication capacity of PUUV Sotkamo wt and a strain with a truncated NSs protein (NSs21Stop), showing that the lack of a full-length NSs might be compensated by its N-terminal peptide, as seen in transfection experiments. These results contribute to our understanding of virus-host interactions and highlight the importance of future innate immunity studies in reservoir hosts.

SARS-CoV-2 spike protein 13-mer subdomain corresponds to the drug-binding domain of glutamyl-propyl-tRNA synthetase 1 and is targetable by halofuginone

Since its emergence, SARS-CoV-2 has been the subject of intense investigation. Early sequence analysis identified a unique 13 amino acid region (13-mer) nested within the receptor-binding domain (RBD) of the spike protein that directly interacts with the ACE2 receptor. Blasting with the 13-mer identified a highly conserved segment in propyl-tRNA synthetase enzymes. Comparison with the human analogue, glutamyl-propyl-tRNA synthetase 1, showed a high level of identity with its drug binding domain, which is targeted by halofuginone, a drug recently shown to block SARS-CoV-2 infection in vitro. In silico experiments predicted a high affinity interaction between halofuginone and the 13-mer. In vitro addition of halofuginone effectively inhibited binding of recombinant S1 monomer to ACE2. Accordingly, it appears that halofuginone inhibits viral infection by preventing correct interactions between spike protein and ACE2. These findings indicate that viral entry can potentially be drug-targeted and support the application of halofuginone in mitigation of COVID-19.

SARS-CoV-2 spike protein 13-mer subdomain corresponds to the drug-binding domain of 3 glutamyl-propyl-tRNA synthetase 1 and is targetable by halofuginone

Since its emergence, SARS-CoV-2 has been the subject of intense investigation. Early sequence analysis identified a unique 13 amino acid region (13-mer) nested within the receptor-binding domain (RBD) of the spike protein that directly interacts with the ACE2 receptor. Blasting with the 13-mer identified a highly conserved segment in propyl-tRNA synthetase enzymes. Comparison with the human analogue, glutamyl-propyl-tRNA synthetase 1, showed a high level of identity with its drug binding domain, which is targeted by halofuginone, a drug recently shown to block SARS-CoV-2 infection in vitro. In silico experiments predicted a high affinity interaction between halofuginone and the 13-mer. In vitro addition of halofuginone effectively inhibited binding of recombinant S1 monomer to ACE2. Accordingly, it appears that halofuginone inhibits viral infection by preventing correct interactions between spike protein and ACE2. These findings indicate that viral entry can potentially be drug-targeted and support the application of halofuginone in mitigation of COVID-19.

Glyceraldehyde 3 phosphate dehydrogenase restricted in cytoplasmic location by viral GP5 facilitates PRRSV replication via its glycolytic activity

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important endemic swine pathogens, causing enormous losses in the global swine industry. Commercially available vaccines only partially prevent or counteract the virus infection and correlated losses. PRRSV replication mechanism has not been well understood. In this study, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was screened to bind with the viral major envelope glycoprotein 5 (GP5) after PRRSV infection. The interacting sites are located within a 13 amino acid region (93 105 aa) of GP5 and at Lys227 of GAPDH. Interestingly, viral GP5 restricts the translocation of GAPDH from the cytoplasm to the nucleus. Moreover, cytoplasmic GAPDH facilitates PRRSV replication by virtue of its glycolytic activity. The results suggest that PRRSV GP5 restricts GAPDH into the nucleus and exploits its glycolytic activity to stimulate virus replication. The data provide insight into the role of GAPDH in PRRSV replication and reveal a potential target for controlling viral infection. Importance PRRSV poses a severe economic threat to the pig industry. PRRSV GP5, the major viral envelope protein, plays an important role in viral infection, pathogenicity, and immunity. However, interactions between GP5 and host proteins have not yet been well studied. Here, we show that GAPDH interacts with GP5 through binding a 13-amino-acid sequence (93–105 aa) in GP5, while GP5 interacts with GAPDH at the K277 amino acid residue of GAPDH. We demonstrate that GP5 interacts with GAPDH in the cytoplasm during PPRSV infection, inhibiting GAPDH entry into the nucleus. PRRSV exploits the glycolytic activity of GAPDH to promote viral replication. These results enrich our understanding of PRRSV infection and pathogenesis, and open a new avenue for antiviral prevention and PRRSV treatment strategies.

Rules for designing protein fold switches and their implications for the folding code

We have engineered switches between the three most common small folds, 3a, 4b+a, and a/b plait, referred to here as A, B, and S, respectively. Mutations were introduced into the natural S protein until sequences were created that have a stable S-fold in their longer (~90 amino acid) form and have an alternative fold (either A or B) in their shorter (56 amino acid) form. Five sequence pairs were designed and key structures were determined using NMR spectroscopy. Each protein pair is 100% identical in the 56 amino acid region of overlap. Several rules for engineering switches emerged. First, designing one sequence with good native state interactions in two folds requires care but is feasible. Once this condition is met, fold populations are determined by the stability of the embedded A- or B-fold relative to the S-fold and the conformational propensities of the ends that are generated in the switch to the embedded fold. If the stabilities of the embedded fold and the longer fold are similar, conformation is highly sensitive to mutation so that even a single amino acid substitution can radically shift the population to the alternative fold. The results provide insight into why dimorphic sequences can be engineered and sometimes exist in nature, while most natural protein sequences populate single folds. Proteins may evolve toward unique folds because dimorphic sequences generate interactions that destabilize and can produce aberrant functions. Thus two-state behavior may result from nature's negative design rather than being an inherent property of the folding code.

Interaction of Peptides Containing CRAC Motifs with Lipids in Membranes of Various Composition

The lateral distribution of integral and peripheral proteins, as well as lipids in the plasma membranes of mammalian cells is extremely heterogeneous. It is believed that various lipid-protein domains are formed in membranes. Domains enriched in sphingomyelin and cholesterol are called rafts. It is assumed that the distribution of proteins into rafts is largely related to the presence in their primary sequence of a specific amino acid region called the CRAC motif, which is responsible for cholesterol binding. In this work, the interaction of two peptides containing CRAC motifs in their structure with membranes of different compositions was studied by means of molecular dynamics. It has been shown that the average number of lipid molecules in contact with each peptide is proportional to the mole fraction of lipid in the membrane. The predominant interaction of peptides with cholesterol was not observed. In addition, cholesterol did not form long-lived contacts with any amino acid or amino acid sequence. We suppose that in some cases the predominant lateral distribution of peptides and proteins containing CRAC motifs into rafts may be due to amphipathicity of the CRAC motif rather than due to specific strong binding of cholesterol.

Bora phosphorylation substitutes in trans for T-loop phosphorylation in Aurora A to promote mitotic entry

Polo-like kinase 1 (Plk1) is instrumental for mitotic entry and progression. Plk1 is activated by phosphorylation on a conserved residue Thr210 in its activation segment by the Aurora A kinase (AURKA), a reaction that critically requires the co-factor Bora phosphorylated by a CyclinA/B-Cdk1 kinase. Here we show that phospho-Bora is a direct activator of AURKA kinase activity. We localize the key determinants of phospho-Bora function to a 100 amino acid region encompassing two short Tpx2-like motifs and a phosphoSerine-Proline motif at Serine 112, through which Bora binds AURKA. The latter substitutes in trans for the Thr288 phospho-regulatory site of AURKA, which is essential for an active conformation of the kinase domain. We demonstrate the importance of these determinants for Bora function in mitotic entry both in Xenopus egg extracts and in human cells. Our findings unveil the activation mechanism of AURKA that is critical for mitotic entry.