Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis.

Identification and annotation of functional residues are fundamental questions in protein sequence analysis. Sequence and structure conservation provides valuable information to tackle these questions. It is, however, limited by the incomplete sampling of sequence space in natural evolution. Moreover, proteins often have multiple functions, with overlapping sequences that present challenges to accurate annotation of the exact functions of individual residues by conservation-based methods. Using the influenza A virus PB1 protein as an example, we developed a method to systematically identify and annotate functional residues. We used saturation mutagenesis and high-throughput sequencing to measure the replication capacity of single nucleotide mutations across the entire PB1 protein. After predicting protein stability upon mutations, we identified functional PB1 residues that are essential for viral replication. To further annotate the functional residues important to the canonical or noncanonical functions of viral RNA-dependent RNA polymerase (vRdRp), we performed a homologous-structure analysis with 16 different vRdRp structures. We achieved high sensitivity in annotating the known canonical polymerase functional residues. Moreover, we identified a cluster of noncanonical functional residues located in the loop region of the PB1 β-ribbon. We further demonstrated that these residues were important for PB1 protein nuclear import through the interaction with Ran-binding protein 5. In summary, we developed a systematic and sensitive method to identify and annotate functional residues that are not restrained by sequence conservation. Importantly, this method is generally applicable to other proteins about which homologous-structure information is available.ImportanceTo fully comprehend the diverse functions of a protein, it is essential to understand the functionality of individual residues. Current methods are highly dependent on evolutionary sequence conservation, which is usually limited by sampling size. Sequence conservation-based methods are further confounded by structural constraints and multifunctionality of proteins. Here we present a method that can systematically identify and annotate functional residues of a given protein. We used a high-throughput functional profiling platform to identify essential residues. Coupling it with homologous-structure comparison, we were able to annotate multiple functions of proteins. We demonstrated the method with the PB1 protein of influenza A virus and identified novel functional residues in addition to its canonical function as an RNA-dependent RNA polymerase. Not limited to virology, this method is generally applicable to other proteins that can be functionally selected and about which homologous-structure information is available

Phylogenetic Analyses of Plastid-Originated Proteins Imply Universal Endosymbiosis in Ancestors of Animals and Fungi

We searched and analyzed cyanobacteria-originated metazoa/fungi proteins (COPs) by phylogenetic analyses. Analysis of them showed that for millions of years universal plastid endosymbiosis and gene transfer occurred in ancestors of metazoa/fungi, and some transferred fragments have been reserved until now even in modern mammals. Most eukaryotes contained plastids in the ancient era, and some of them lost them later. Functions of homologues in cyanobacterial genomes and eukaryotic genomes are in consensus, and most are involved in the organic compound metabolism. With emergence of organelles and subcellular structure in eukaryotic cells, the locations of these proteins diversified. Furthermore, some novel functions were endowed for COPs, especially in vertebrates

Involvement of Indoleamine 2,3-Dioxygenase in Impairing Tumor-Infiltrating CD8+ T-Cell Functions in Esophageal Squamous Cell Carcinoma

The indoleamine 2,3-dioxygenase-(IDO-) mediated microenvironment plays an important role in tumor immune escape. However, the inhibitory effects of IDO on the CD8+ tumour-infiltrating lymphocytes (CD8+ TILs) in esophageal squamous cell carcinoma (ESCC) have not been clarified yet. Here, we found that the level of IDO expression in ESCC tumor specimens correlated with a reduction in the number of CD8+ TILs. Patients with high IDO expression and a low number of CD8+ TILs had significantly impaired overall survival time. IDO expression and functional enzyme activity in ESCC cell lines could be induced by IFNγ. When exposed to the milieu generated by IDO-expressing Eca109 cells, the CD8+ TILs were suppressed in proliferation, and their cytolytic functions against target tumor cells were lost. These results suggested that impairing CD8+ TIL functions by IDO expressed in ESCC possibly contributed to the finding that patients with higher IDO expression have more aggressive disease progression and shorter overall survival time

6-Amino-3-methyl-4-(3-nitro­phen­yl)-1-phenyl-1H,4H-pyrano[2,3-c]pyrazole-5-carbonitrile

The title compound, C20H15N5O3, was synthesized by the one-pot reaction of a four-component reaction protocol in aqueous medium. The pyrano[2,3-c]pyrazole system is essentially planar, with a maximum deviation of 0.026 (2) Å. The 3-nitro­phenyl and phenyl rings make dihedral angles of 81.11 (5) and 13.36 (1)°, respectively, with the mean plane of the pyrano[2,3-c]pyrazole ring. The crystal structure is stabilized by N—H⋯N hydrogen bonds, which form infinite chain propagating along the c axis and by N—H⋯O hydrogen bonds, which form infinite chains propagating along the a axis. There are also N—O⋯N—C dipole–dipole inter­actions along the a axis with an O⋯N distance of 3.061 (3) Å, which is shorter than that of the N—H⋯O hydrogen bond [3.196 (3) Å]

Meta-analysis of antiviral protection of white spot syndrome virus vaccine to the shrimp

Currently, white spot syndrome virus (WSSV) is one of the most serious pathogens that impacts shrimp farming around the world. A WSSV vaccine provides a significant protective benefit to the host shrimp. Although various types of vaccines against WSSV have emerged, the immune effects among them were not compared, and it remains unclear which type of vaccine has the strongest protective effect. Meanwhile, due to the lack of effective routes of administration and immunization programs, WSSV vaccines have been greatly limited in the actual shrimp farming. To answer these questions, this study conducted a comprehensive meta-analysis over dozens of studies and compared all types WSSV vaccines, which include sub-unit protein vaccines, whole virus inactivated vaccines, DNA vaccines and RNA-based vaccines. The results showed that the RNA-based vaccine had the highest protection rate over the other three types of vaccines. Among the various sub-unit protein vaccines, VP26 vaccine had the best protective effects than other sub-unit protein vaccines. Moreover, this study demonstrated that vaccines expressed in eukaryotic hosts had higher protection rates than that of prokaryotic systems. Among the three immunization modes (oral administration, immersion and injection) used in monovalent protein vaccines, oral administration had the highest protection rate. In natural conditions, shrimp are mostly infected by the virus orally. These results provide a guide for exploration of a novel WSSV vaccine and help facilitate the application of WSSV vaccines in shrimp farming

When uncertainty meets life: The effect of animacy on probability expression

Everyone faces uncertainty on a daily basis. Two kinds of probability expressions, verbal and numerical, have been used to characterize the uncertainty that we face. Because our cognitive concept of living things differs from that of non-living things, and distinguishing cognitive concepts might have linguistic markers, we designed four studies to test whether people use different probability expressions when faced with animate or inanimate uncertainty. We found that verbal probability is the preferred way to express animate uncertainty, whereas numerical probability is the preferred way to express inanimate uncertainty. The “verbal-animate” and “numerical-inanimate” associations were robust enough to persist when tested with forced-choice response patterns regardless of the information (e.g., equally likely outcomes, frequencies, or personal beliefs) used to construct probabilities of events. When the response pattern was changed to free-responses, the associations were evident unless the subjects were asked to write their own probability predictions for vague uncertainty. Given that the world around us consists of both animate (i.e., living) and inanimate (i.e., non-living) things, “verbal-animate” and “numerical-inanimate” associations may play a major role in risk communication and may otherwise be useful for practitioners and consultants