Detection of microRNAs expression signatures in vitreous humor of intraocular tuberculosis.

BACKGROUND: MicroRNA (miRNA) expression analysis has been shown to provide them as biomarkers in several eye diseases and has a regulatory role in pathogenesis. However, miRNA expression analysis in the vitreous humor (VH) of intraocular tuberculosis (IOTB) is not studied. Thus, we aim to find miRNA expression signatures in the VH of IOTB patients to identify their regulatory role in disease pathogenesis and to find them as potential biomarkers for IOTB. METHODS AND RESULTS: First, we profiled miRNAs in VH of three IOTB and three Macular hole (MH) samples as controls through small-RNA deep sequencing using Illumina Platform. In-house bioinformatics analysis identified 81 dysregulated miRNAs in IOTB. Further validation in VH of IOTB (n = 15) compared to MH (n = 15) using Real-Time quantitative PCR (RT-qPCR) identified three significantly upregulated miRNAs, hsa-miR-150-5p, hsa-miR-26b-5p, and hsa-miR-21-5p. Based on the miRNA target prediction, functional network analysis, and RT-qPCR analysis of target genes, the three miRNAs downregulating WNT5A, PRKCA, MAP3K7, IL7, TGFB2, IL1A, PRKCB, TNFA, and TP53 genes involving MAPK signaling pathway, PI3K-AKT signaling pathway, WNT signaling pathway, Cell cycle, TGF-beta signaling pathway, Long-term potentiation, and Sphingolipid signaling pathways, have a potential role in disease pathogenesis. The ROC analysis of RT-qPCR data showed that hsa-miR-150-5p with AUC = 0.715, hsa-miR-21-5p with AUC = 0.789, and hsa-miR-26b-5p with AUC = 0.738; however, the combination of hsa-miR-21-5p and hsa-miR-26b-5p with AUC = 0.796 could serve as a potential biomarker for IOTB. CONCLUSIONS: This study provides the first report on miRNA expression signatures detected in VH for IOTB pathogenesis and also provides a potential biomarker for IOTB.

Dysregulated expression of microRNAs in aqueous humor from intraocular tuberculosis patients.

BACKGROUND: Systemic Mycobacterium tuberculosis (Mtb) infection alters microRNA's expression that controls cellular processes and modulates host defense mechanisms. However, the role of miRNAs in intraocular tuberculosis (IOTB) remains unknown. Therefore, this study aims to identify dysregulated miRNAs in the aqueous humor (AH) of patients with IOTB. METHODS: AH from intraocular tuberculosis patients (n = 2) and cataract controls (n = 2) were used for small RNA deep sequencing using HiSeq Illumina sequencing platform. Differentially expressed miRNAs and their targets were identified by the bioinformatics approach, and their regulatory functions were predicted by pathway enrichment analysis. The expression of selected miRNAs and their binding targets were further validated by real-time quantitative PCR (RT-qPCR). RESULTS: In total, we identified 56 differentially expressed miRNAs in the AH of intraocular tuberculosis (IOTB) patients compared to controls. We selected four significantly dysregulated miRNAs (miR-423-5p, miR-328-3p, miR-21-5p, and miR-16-5p) based on the RT-qPCR validation and predicted their gene targets. We developed a miRNA-targets regulatory network by combining pathways of interest and genes associated with TB. We identified that these four miRNAs might play an important role in IOTB pathogenesis via tuberculosis-associated pathways; PI3K-Akt signaling, autophagy and MAPK pathway. CONCLUSIONS: For the first time, this study identifies the dysregulation of four miRNAs in the AH of IOTB patients using the ultra-low input small-RNA sequencing approach. Further target prediction and validation identify the role of these miRNAs in tuberculosis pathogenesis via tuberculosis-related pathways. This study identifies miRNAs as potentially ideal biomarkers in the aqueous humor of IOTB patients.

Interface interactions between ßγ-crystallin domain and Ig-like domain render Ca2+ -binding site inoperative in abundant perithecial protein of Neurospora crassa.

We describe a set of proteins in which a ßγ-crystallin domain pairs with an Ig-like domain, and which are confined to microbes, like bacteria, slime molds and fungi. DdCAD-1 (Ca2+ -dependent cell adhesion molecule-1) and abundant perithecial protein (APP) represent this class of molecules. Using the crystal structure of APP-NTD (N-terminal domain of APP), we describe its mode of Ca2+ binding and provide a generalized theme for correct identification of the Ca2+ -binding site within this class of molecules. As a common feature, one of the two Ca2+ -binding sites is non-functional in the ßγ-crystallin domains of these proteins. While APP-NTD binds Ca2+ with a micromolar affinity which is comparable to DdCAD-1, APP surprisingly does not bind Ca2+ . Crystal structures of APP and Ca2+ -bound APP-NTD reveal that the interface interactions in APP render its Ca2+ -binding site inoperative. Thus, heterodomain association provides a novel mode of Ca2+ -binding regulation in APP. Breaking the interface interactions (mutating Asp30Ala, Leu132Ala and Ile135Ala) or separation from the Ig-like domain removes the constraints upon the required conformational transition and enables the ßγ-crystallin domain to bind Ca2+ . In mechanistic detail, our work demonstrates an interdomain interface adapted to distinct functional niches in APP and its homolog DdCAD-1.

Secretagogin Is a Redox-Responsive Ca2+ Sensor.

Secretagogin (SCGN), a multifunctional, Ca2+ binding, regulatory protein, known to regulate insulin release, has recently been implicated in the control of stress-related corticotropin-releasing hormone (CRH) secretion. Localization of SCGN to multiple intracellular (such as cytosol, nucleus, and endoplasmic reticulum) and extracellular sites appears to provide multifunctional capabilities; however, the structural elements conferring such a widespread cellular distribution to SCGN remain unidentified. We report that the spatial and functional attributes of SCGN plausibly originate from the interplay between Ca2+ and its redox state. The mutation of selective Cys residues provides further insights into the origin and mode of redox responsiveness. In the reducing milieu, SCGN exhibits a higher affinity for Ca2+, and more stability than in the oxidizing environment, suggesting it is a redox-responsive Ca2+ sensor protein, which is further supported by its response to dithiothreitol (reducing stress) in MIN6 cells. Our data provide a biophysical and biochemical explanation for the diverse localization of SCGN in the cellular scenario and beyond the cell.

A Transition Metal-Binding, Trimeric ßγ-Crystallin from Methane-Producing Thermophilic Archaea, Methanosaeta thermophila.

ßγ-Crystallins are important constituents of the vertebrate eye lens, whereas in microbes, they are prevalent as Ca2+-binding proteins. In archaea, ßγ-crystallins are conspicuously confined to two methanogens, viz., Methanosaeta and Methanosarcina. One of these, i.e., M-crystallin from Methanosarcina acetivorans, has been shown to be a typical Ca2+-binding ßγ-crystallin. Here, with the aid of a high-resolution crystal structure and isothermal titration calorimetry, we report that "Methallin", a ßγ-crystallin from Methanosaeta thermophila, is a trimeric, transition metal-binding protein. It binds Fe, Ni, Co, or Zn ion with nanomolar affinity, which is consistent even at 55 °C, the optimal temperature for the methanogen's growth. At the center of the protein trimer, the metal ion is coordinated by six histidines, two from each protomer, leading to an octahedral geometry. Small-angle X-ray scattering analysis confirms that the trimer seen in the crystal lattice is a biological assembly; this assembly dissociates to monomers upon removal of the metal ion. The introduction of two histidines (S17H/S19H) into a homologous ßγ-crystallin, Clostrillin, allows it to bind nickel at the introduced site, though with micromolar affinity. However, because of the lack of a compatible interface, nickel binding could not induce trimerization, affirming that Methallin is a naturally occurring trimer for high-affinity transition metal binding. While ßγ-crystallins are known to bind Ca2+ and form homodimers and oligomers, the transition metal-binding, trimeric Methallin is a new paradigm for ßγ-crystallins. The distinct features of Methallin, such as nickel or iron binding, are also possible imprints of biogeochemical changes during the period of its origin.