Function and Characterization of an Alanine Dehydrogenase Homolog From Nocardia seriolae

Fish nocardiosis is a chronic, systemic, granulomatous disease in aquaculture. Nocardia seriolae has been reported to be one of the main pathogenic bacteria of fish nocardiosis. There are few studies on the associated virulence factors and pathogenesis of N. seriolae. Alanine dehydrogenase (ALD), which may be a secreted protein, was discovered by analysis using bioinformatics methods throughout the whole genomic sequence of N. seriolae. Nevertheless, the roles of ALD and its homologs in the pathogenesis of N. seriolae are not demonstrated. In this study, the function of N. seriolae ALD (NsALD) was preliminarily investigated by gene cloning, host cell subcellular localization, secreted protein identification, and cell apoptosis detection. Identification of the extracellular products of N. seriolae via mass spectrometry (MS) analysis revealed that NsALD is a secreted protein. In addition, subcellular localization of NsALD-GFP recombinant protein in fathead minnow (FHM) cells showed that the strong green fluorescence co-localized with the mitochondria. Moreover, apoptosis assays demonstrated that the overexpression of NsALD induces apoptosis in FHM cells. This study may lay the foundation for further exploration of the function of NsALD and facilitate further understanding of the pathogenic mechanism and the associated virulence factors of N. seriolae.

Evolutionary Origin of Insulin-Degrading Enzyme and Its Subcellular Localization and Secretion Mechanism: A Study in Microglial Cells

The insulin-degrading enzyme (IDE) is a zinc-dependent metalloendopeptidase that belongs to the M16A metalloprotease family. IDE is markedly expressed in the brain, where it is particularly relevant due to its in vitro amyloid beta (Aβ)-degrading activity. The subcellular localization of IDE, a paramount aspect to understand how this enzyme can perform its proteolytic functions in vivo, remains highly controversial. In this work, we addressed IDE subcellular localization from an evolutionary perspective. Phylogenetic analyses based on protein sequence and gene and protein structure were performed. An in silico analysis of IDE signal peptide suggests an evolutionary shift in IDE exportation at the prokaryote/eukaryote divide. Subcellular localization experiments in microglia revealed that IDE is mostly cytosolic. Furthermore, IDE associates to membranes by their cytoplasmatic side and further partitions between raft and non-raft domains. When stimulated, microglia change into a secretory active state, produces numerous multivesicular bodies and IDE associates with their membranes. The subsequent inward budding of such membranes internalizes IDE in intraluminal vesicles, which later allows IDE to be exported outside the cells in small extracellular vesicles. We further demonstrate that such an IDE exportation mechanism is regulated by stimuli relevant for microglia in physiological conditions and upon aging and neurodegeneration.

Protein Subcellular Localization Based on Evolutionary Information and Segmented Distribution

The prediction of protein subcellular localization not only is important for the study of protein structure and function but also can facilitate the design and development of new drugs. In recent years, feature extraction methods based on protein evolution information have attracted much attention and made good progress. Based on the protein position-specific score matrix (PSSM) obtained by PSI-BLAST, PSSM-GSD method is proposed according to the data distribution characteristics. In order to reflect the protein sequence information as much as possible, AAO method, PSSM-AAO method, and PSSM-GSD method are fused together. Then, conditional entropy-based classifier chain algorithm and support vector machine are used to locate multilabel proteins. Finally, we test Gpos-mPLoc and Gneg-mPLoc datasets, considering the severe imbalance of data, and select SMOTE algorithm to expand a few sample; the experiment shows that the AAO + PSSM ∗ method in the paper achieved 83.1% and 86.8% overall accuracy, respectively. After experimental comparison of different methods, AAO + PSSM ∗ has good performance and can effectively predict protein subcellular location.

Genome-wide identification and expression analysis of CYP450 gene family in Cucumis sativus L.

The cytochrome P450 (CYP450) gene family plays a vital role in basic metabolism and enhances plant resistance to stress and pests. However, little information is available on the genome-wide characterization and evolutionary relationship of the CYP450 gene family in Cucumis sativus L. In the present study, a genome-wide bioinformatics analysis was performed, including gene structure, conserved motif, cis-acting promoter element, evolutionary analysis, collinearity, subcellular localization, and expression profile. The gene expression profile of CYP450 was verified using transcriptome sequencing and quantitative reverse transcription polymerase chain reaction. A total of 165 P450 genes were identified in the cucumber genome. These genes were classified into eight subfamilies and unevenly distributed on seven chromosomes. Subcellular localization predicted that most of P450 genes were located in chloroplasts and a few were located on the plasma membrane. CYP450 genes were differentially expressed in different tissues and in response to salicylic acid (SA) treatment. The sizes of all cucumber P450 proteins ranged from 317 to 1,056 aa, the theoretical isoelectric points ranged from 5.05 to 10.31, and the molecular weights ranged from 36,095 to 121,403 KD. This study provides a theoretical basis for further research on the biological functions of the P450 gene in cucumber plants.

Deciphering the tRNA-derived small RNAs: origin, development, and future

Transfer RNA (tRNA)-derived small RNAs (tsRNAs), a novel category of small noncoding RNAs, are enzymatically cleaved from tRNAs. Previous reports have shed some light on the roles of tsRNAs in the development of human diseases. However, our knowledge about tsRNAs is still relatively lacking. In this paper, we review the biogenesis, classification, subcellular localization as well as action mechanism of tsRNAs, and discuss the association between chemical modifications of tRNAs and the production and functions of tsRNAs. Furthermore, using immunity, metabolism, and malignancy as examples, we summarize the molecular mechanisms of tsRNAs in diseases and evaluate the potential of tsRNAs as new biomarkers and therapeutic targets. At the same time, we compile and introduce several resource databases that are currently publicly available for analyzing tsRNAs. Finally, we discuss the challenges associated with research in this field and future directions.

Tonoplast-Localized Theanine Transporter CsCAT2 May Mediate Theanine Storage in the Root of Tea Plants (Camellia sinensis L.)

Theanine is the component endowing tea infusion with “umami” taste and antidepression benefits. Theanine is primarily synthesized and stored in root in winter and is transported via vascular tissues to the new shoot in spring. However, the mechanism underlying theanine storage in the root of tea plants remains largely unknown. Cationic amino acid transporter 2 (CsCAT2) in tea plants is homologous to glutamine permease 1 (GNP1), the specific glutamine transporter in yeast. In this study, we identified CsCAT2 as an H+-dependent theanine transporter with medium affinity for theanine. The result of subcellular localization showed that CsCAT2 was a tonoplast-localized transporter. Importantly, CsCAT2 highly expressed in the root in winter during theanine storage and reduced its expression in the root during theanine transport from root-to-shoot in spring. In addition, CsCAT2 expression in the roots of 5 varieties at four time points during December and April was significant negatively correlated with the capacity of theanine root-to-shoot movement. Taken together, these results suggested that CsCAT2 may mediate theanine storage in the vacuole of root cells and may negatively modulate theanine transport from root to shoot.

AGIA Tag System for Ultrastructural Protein Localization Analysis in Blood-Stage Plasmodium falciparum

Precise subcellular localization of proteins is the key to elucidating the physiological role of these molecules in malaria parasite development, understanding of pathogenesis, and protective immunity. In Plasmodium falciparum, however, detection of proteins in the blood-stage parasites is greatly hampered by the lack of versatile protein tags which can intrinsically label such molecules. Thus, in this study, to develop a novel system that can be used to evaluate subcellular localization of known and novel proteins, we assessed the application of AGIA tag, consisting of 9 amino acids (EEAAGIARP), in P. falciparum blood-stage parasites. Specifically, AGIA-tagged ring-infected erythrocyte surface antigen (RESA-AGIA) was episomally expressed in P. falciparum 3D7 strain. The RESA-AGIA protein was detected by Western blotting and immunofluorescence assay (IFA) using recombinant rabbit anti-AGIA tag monoclonal antibody (mAb) with a high signal/noise ratio. Similarly, AGIA-tagged multidrug resistance protein 1 (MDR1-AGIA), as an example of polyptic transmembrane protein, was endogenously expressed and detected by Western blotting and IFA with anti-AGIA tag mAb. Immunoelectron microscopy of the RESA-AGIA transfected merozoites revealed that mouse anti-RESA and the rabbit anti-AGIA mAb signals could definitively co-localize to the dense granules. Put together, this study demonstrates AGIA tag/anti-AGIA rabbit mAb system as a potentially useful tool for elucidating the subcellular localization of new and understudied proteins in blood-stage malaria parasites at the nanometer-level resolution.