Human Gene Product Research Articles

Capturing heart valve development with Gene Ontology.

Introduction: The normal development of all heart valves requires highly coordinated signaling pathways and downstream mediators. While genomic variants can be responsible for congenital valve disease, environmental factors can also play a role. Later in life valve calcification is a leading cause of aortic valve stenosis, a progressive disease that may lead to heart failure. Current research into the causes of both congenital valve diseases and valve calcification is using a variety of high-throughput methodologies, including transcriptomics, proteomics and genomics. High quality genetic data from biological knowledge bases are essential to facilitate analyses and interpretation of these high-throughput datasets. The Gene Ontology (GO, http://geneontology.org/) is a major bioinformatics resource used to interpret these datasets, as it provides structured, computable knowledge describing the role of gene products across all organisms. The UCL Functional Gene Annotation team focuses on GO annotation of human gene products. Having identified that the GO annotations included in transcriptomic, proteomic and genomic data did not provide sufficient descriptive information about heart valve development, we initiated a focused project to address this issue. Methods: This project prioritized 138 proteins for GO annotation, which led to the curation of 100 peer-reviewed articles and the creation of 400 heart valve development-relevant GO annotations. Results: While the focus of this project was heart valve development, around 600 of the 1000 annotations created described the broader cellular role of these proteins, including those describing aortic valve morphogenesis, BMP signaling and endocardial cushion development. Our functional enrichment analysis of the 28 proteins known to have a role in bicuspid aortic valve disease confirmed that this annotation project has led to an improved interpretation of a heart valve genetic dataset. Discussion: To address the needs of the heart valve research community this project has provided GO annotations to describe the specific roles of key proteins involved in heart valve development. The breadth of GO annotations created by this project will benefit many of those seeking to interpret a wide range of cardiovascular genomic, transcriptomic, proteomic and metabolomic datasets.

The Human Cytomegalovirus Latency-Associated Gene Product Latency Unique Natural Antigen Regulates Latent Gene Expression.

Human cytomegalovirus (HCMV) infection can lead to either lytic or latent infection, which is dependent on the regulation of the viral major immediate early promoter (MIEP). Suppression of the MIEP is a pre-requisite for latency and is driven by repressive epigenetic modifications at the MIEP during latent infection. However, other viral genes are expressed during latency and this is correlated with activatory epigenetic modifications at latent gene promoters. Yet the molecular basis of the differential regulation of latent and lytic gene expression by epigenetics is unclear. LUNA, a latent viral transcript, has been suggested to be important for HCMV latency and has also been shown to be important for efficient reactivation likely through its known deSUMOylase activity. Intriguingly, we and others have also observed that LUNA enhances latency-associated expression of the viral UL138 gene. Here, we show that in the absence of LUNA, the expression of multiple latency-associated transcripts is reduced during latent infection, which is correlated with a lack of activatory marks at their promoters. Interestingly, we also show that LUNA interacts with the hematopoietic transcription factor GATA-2, which has previously been shown to bind to a number of latency-associated gene promoters, and that this interaction is dependent on the deSUMOylase domain of LUNA. Finally, we show that the deSUMOylase activity of LUNA is required for the establishment and/or maintenance of an open chromatin configuration around latency-associated gene promoters. As such, LUNA plays a key role in efficient latency-associated viral gene expression and carriage of viral genome during latent carriage.

The Antiviral Activity of Equine Mx1 against Thogoto Virus Is Determined by the Molecular Structure of Its Viral Specificity Region.

Mammalian myxovirus resistance (Mx) proteins are interferon-induced, large dynamin-like GTPases with a broad antiviral spectrum. Here, we analyzed the antiviral activity of selected mammalian Mx1 proteins against Thogoto virus (THOV). Of those, equine Mx1 (eqMx1) showed antiviral activity comparable to that of the human MX1 gene product, designated huMxA, whereas most Mx1 proteins were antivirally inactive. We previously demonstrated that the flexible loop L4 protruding from the stalk domain of huMxA, and especially the phenylalanine at position 561 (F561), determines its antiviral specificity against THOV (P. S. Mitchell, C. Patzina, M. Emerman, O. Haller, et al., Cell Host Microbe 12:598-604, 2012, https://doi.org/10.1016/j.chom.2012.09.005). However, despite the similar antiviral activity against THOV, the loop L4 sequence of eqMx1 substantially differs from the one of huMxA. Mutational analysis of eqMx1 L4 identified a tryptophan (W562) and the adjacent glycine (G563) as critical antiviral determinants against THOV, whereas the neighboring residues could be exchanged for nonpolar alanines without affecting the antiviral activity. Further mutational analyses revealed that a single bulky residue at position 562 and the adjacent tiny residue G563 were sufficient for antiviral activity. Moreover, this minimal set of L4 amino acids transferred anti-THOV activity to the otherwise inactive bovine Mx1 (boMx1) protein. Taken together, our data suggest a fairly simple architecture of the antiviral loop L4 that could serve as a mutational hot spot in an evolutionary arms race between Mx-escaping viral variants and their hosts. IMPORTANCE Most mammals encode two paralogs of the interferon-induced Mx proteins: Mx1, with antiviral activity largely against RNA viruses, like orthomyxoviruses and bunyaviruses; and Mx2, which is antivirally active against HIV-1 and herpesviruses. The human Mx1 protein, also called huMxA, is the best-characterized example of mammalian Mx1 proteins and was recently shown to prevent zoonotic virus transmissions. To evaluate the antiviral activity of other mammalian Mx1 proteins, we used Thogoto virus, a tick-transmitted orthomyxovirus, which is efficiently blocked by huMxA. Interestingly, we detected antiviral activity only with equine Mx1 (eqMx1) but not with other nonprimate Mx1 proteins. Detailed functional analysis of eqMx1 identified amino acid residues in the unstructured loop L4 of the stalk domain critical for antiviral activity. The structural insights of the present study explain the unique position of eqMx1 antiviral activity within the collection of nonhuman mammalian Mx1 proteins.

Double administration of self-complementary AAV9NDUFS4 prevents Leigh disease in Ndufs4-/- mice.

Leigh disease, or subacute necrotizing encephalomyelopathy, a genetically heterogeneous condition consistently characterized by defective mitochondrial bioenergetics, is the most common oxidative-phosphorylation related disease in infancy. Both neurological signs and pathological lesions of Leigh disease are mimicked by the ablation of the mouse mitochondrial respiratory chain subunit Ndufs4−/−, which is part of, and crucial for, normal Complex I activity and assembly, particularly in the brains of both children and mice. We previously conveyed the human NDUFS4 gene to the mouse brain using either single-stranded adeno-associated viral 9 recombinant vectors or the PHP.B adeno-associated viral vector. Both these approaches significantly prolonged the lifespan of the Ndufs4−/− mouse model but the extension of the survival was limited to a few weeks by the former approach, whereas the latter was applicable to a limited number of mouse strains, but not to primates. Here, we exploited the recent development of new, self-complementary adeno-associated viral 9 vectors, in which the transcription rate of the recombinant gene is markedly increased compared with the single-stranded adeno-associated viral 9 and can be applied to all mammals, including humans. Either single intra-vascular or double intra-vascular and intra-cerebro-ventricular injections were performed at post-natal Day 1. The first strategy ubiquitously conveyed the human NDUFS4 gene product in Ndufs4−/− mice, doubling the lifespan from 45 to ≈100 days after birth, when the mice developed rapidly progressive neurological failure. However, the double, contemporary intra-vascular and intra-cerebroventricular administration of self-complementary-adeno-associated viral NDUFS4 prolonged healthy lifespan up to 9 months of age. These mice were well and active at euthanization, at 6, 7, 8 and 9 months of age, to investigate the brain and other organs post-mortem. Robust expression of hNDUFS4 was detected in different cerebral areas preserving normal morphology and restoring Complex I activity and assembly. Our results warrant further investigation on the translatability of self-complementary-adeno-associated viral 9 NDUFS4-based therapy in the prodromal phase of the disease in mice and eventually humans.

Contribution of Model Organisms to Investigating the Far-Reaching Consequences of PRPP Metabolism on Human Health and Well-Being.

Phosphoribosyl pyrophosphate synthetase (PRS EC 2.7.6.1) is a rate-limiting enzyme that irreversibly catalyzes the formation of phosphoribosyl pyrophosphate (PRPP) from ribose-5-phosphate and adenosine triphosphate (ATP). This key metabolite is required for the synthesis of purine and pyrimidine nucleotides, the two aromatic amino acids histidine and tryptophan, the cofactors nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+), all of which are essential for various life processes. Despite its ubiquity and essential nature across the plant and animal kingdoms, PRPP synthetase displays species-specific characteristics regarding the number of gene copies and architecture permitting interaction with other areas of cellular metabolism. The impact of mutated PRS genes in the model eukaryote Saccharomyces cerevisiae on cell signalling and metabolism may be relevant to the human neuropathies associated with PRPS mutations. Human PRPS1 and PRPS2 gene products are implicated in drug resistance associated with recurrent acute lymphoblastic leukaemia and progression of colorectal cancer and hepatocellular carcinoma. The investigation of PRPP metabolism in accepted model organisms, e.g., yeast and zebrafish, has the potential to reveal novel drug targets for treating at least some of the diseases, often characterized by overlapping symptoms, such as Arts syndrome and respiratory infections, and uncover the significance and relevance of human PRPS in disease diagnosis, management, and treatment.

Upregulated Ly-6 gene expression is associated with poor overall survival in uterine corpus endometrial carcinoma patients

Abstract The human Ly-6 (hLy-6) gene family has recently gained interest for its possible role in tumor progression. High expression of hLy-6 genes is associated with poor overall survival in pancreatic ductal adenocarcinoma patients. To expand upon this work, we have carried out in silico analyses of all known hLy-6 gene expression in other cancers using TNMplot which shows differential gene expression in cancer tissues. We have also analyzed the patient survival by Kaplan-Meier plotter after mining the TCGA database. We report that upregulated expression of several hLy-6 genes is associated with poor cancer patient survival in many other cancer types, especially uterine corpus endometrial carcinoma (UCEC). Importantly, the expression of a number of hLy-6 genes is elevated in UCEC tissue when compared to the expression in normal uterine tissue. Further analysis of tumor-specific expression of the hLy-6 gene family is needed to uncover the function of Ly-6 proteins as well as the signaling pathways these proteins trigger that endow tumor survival and poor patient prognosis. Human Ly-6 gene products can potentially be used as biomarkers for cancer detection and as tumor associated antigens to identify high-risk cancer patients and appropriately design and/or calibrate cancer treatments for such patients.

Establishment and evaluation of a quadruple quantitative real-time PCR assay for simultaneous detection of human coronavirus subtypes

BackgroundThe newly discovered severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and four seasonal human coronaviruses (HCoVs) (HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1) still circulate worldwide. The early clinical symptoms of SARS-CoV-2 and seasonal HCoV infections are similar, so rapid and accurate identification of the subtypes of HCoVs is crucial for early diagnosis, early treatment, prevention and control of these infections. However, current multiplex molecular diagnostic techniques for HCoV subtypes including SARS-CoV-2 are limited.MethodsWe designed primers and probes specific for the S and N genes of SARS-CoV-2, the N gene of severe acute respiratory syndrome coronavirus (SARS-CoV), and the ORF1ab gene of four seasonal HCoVs, as well as the human B2M gene product. We developed and optimized a quadruple quantitative real-time PCR assay (qq-PCR) for simultaneous detection of SARS-CoV-2, SARS-CoV and four seasonal HCoVs. This assay was further tested for specificity and sensitivity, and validated using 184 clinical samples.ResultsThe limit of detection of the qq-PCR assay was in the range 2.5 × 101 to 6.5 × 101 copies/μL for each gene and no cross-reactivity with other common respiratory viruses was observed. The intra-assay and inter-assay coefficients of variation were 0.5–2%. The qq-PCR assay had a 91.9% sensitivity and 100.0% specificity for SARS-CoV-2 and a 95.7% sensitivity and 100% specificity for seasonal HCoVs, using the approved commercial kits as the reference. Compared to the commercial kits, total detection consistency was 98.4% (181/184) for SARS-CoV-2 and 98.6% (142/144) for seasonal HCoVs.ConclusionWith the advantages of sensitivity, specificity, rapid detection, cost-effectiveness, and convenience, this qq-PCR assay has potential for clinical use for rapid discrimination between SARS-CoV-2, SARS-CoV and seasonal HCoVs.

Unraveling the Molecular Mechanism of Recognition of Human Interferon-Stimulated Gene Product 15 by Coronavirus Papain-Like Proteases: A Multiscale Simulation Study.

The papain-like protease (PLpro) of the coronavirus (CoV) family plays an essential role in processing the viral polyprotein and immune evasion. Additional proteolytic activities of PLpro include deubiquitination and deISGylation, which can reverse the post-translational modification of cellular proteins conjugated with ubiquitin or (Ub) or Ub-like interferon-stimulated gene product 15 (ISG15). These activities regulate innate immune responses against viral infection. Thus, PLpro is a potential antiviral target. Here, we have described the structural and energetic basis of recognition of PLpro by the human ISG15 protein (hISG15) using atomistic molecular dynamics simulation across the CoV family, i.e., MERS-CoV (MCoV), SARS-CoV (SCoV), and SARS-CoV-2 (SCoV2). The cumulative simulation length for all trajectories was 32.0 μs. In the absence of the complete crystal structure of complexes, protein–protein docking was used. A mutation (R167E) was introduced across all three PLpro to study the effect of mutation on the protein–protein binding. Our study reveals that the apo-ISG15 protein remains closed while it adopts an open conformation when bound to PLpro, although the degree of openness varies across the CoV family. The binding free energy analysis suggests that hISG15 binds more strongly with SCoV2-PLpro compared to SCoV or MCoV. The intermolecular electrostatic interaction drives the hISG15-PLpro complexation. Our study showed that SCoV or MCoV-PLpro binds more strongly with the C-domain of hISG15, while SCoV2-PLpro binds more favorably the N-domain of hISG15. Overall, our study explains the molecular basis of differential deISGylating activities of PLpro among the CoV family and the specificity of SCoV2-PLpro toward hISG15.

Opinion: Independent third-party entities as a model for validation of commercial antibodies

A vast array of commercial antibodies covers a large percentage of human gene products, but determining which among them is most appropriate for any given application is challenging. This leads to use of non-specific antibodies that contributes to issues with reproducibility. It is our opinion that the community of scientists who use commercial antibodies in their biomedical research would benefit from third-party antibody characterization entities that use standardized operating procedures to assess and compare antibody performance. Ideally, such entities would follow the principles of open science, such that all antibodies against any given protein target would be tested in parallel, and all data generated released to the public domain without bias. Furthermore, there should be no financial incentive for the entity beyond cost-recovery. Such non-profit organizations, combined with other scientific efforts, could catalyse new discoveries by providing scientists with better validated antibody tools.

Immunogenicity and humanization of single-domain antibodies.

Single-domain antibodies (sdAbs), the autonomous variable domains of camelid and shark heavy-chain antibodies, have many desirable properties as components of biologic drugs. However, their sequences may increase the risk of immunogenicity and antidrug antibody (ADA) development in humans, and thus, sdAbs are routinely humanized during development. Here, we review and summarize the available evidence regarding the factors governing immunogenicity of sdAbs and our current state of knowledge of strategies to mitigate immunogenicity risks by humanization. While several sdAb properties, including high homology of camelid VH Hs with human IGHV3 gene products, favor low immunogenicity in humans, epitopes absent in the human repertoire including the exposed VH :VL interface may be intrinsically immunogenic. While most clinical trials have demonstrated minimal sdAb immunogenicity, two notable exceptions (the tetrameric DR5-specific VH H TAS266 and the TNFR1-specific VH GSK1995057) illustrate that special caution must be taken in identifying preexisting ADAs against highly potent sdAbs. Nonhuman sequence alone does not adequately explain sdAb immunogenicity, as some camelid VH Hs are nonimmunogenic while some fully human VH s elicit ADAs. The presence of preexisting ADAs directed against the exposed C-termini of some sdAbs in a significant proportion of individuals awaits a molecular explanation. Whether sdAb humanization reduces or promotes immunogenicity remains unclear: reduction of nonhuman sequence content at the expense of introducing low-level aggregation in humanized variants may be counterproductive. Further work will establish thresholds for VH H and VNAR humanization to maximize human sequence content while avoiding loss of binding affinity and/or immunogenicity resulting from aggregation or decreased stability.

P2X7 Variants in Oncogenesis.

The P2X7 receptor for extracellular ATP is a well-established mediator of tumoral development and progression both in solid cancers and hematological malignancies. The human P2X7 gene is highly polymorphic, and several splice variants of the receptor have been identified in time. P2X7 single-nucleotide polymorphisms (SNPs) have been broadly analyzed by studies relating them to pathologies as different as infectious, inflammatory, nervous, and bone diseases, among which cancer is included. Moreover, in the last years, an increasing number of reports concentrated on P2X7 splice variants’ different roles and their implications in pathological conditions, including oncogenesis. Here, we give an overview of established and recent literature demonstrating a role for human P2X7 gene products in oncological conditions, mainly focusing on current data emerging on P2X7 isoform B and nfP2X7. We explored the role of these and other genetic variants of P2X7 in cancer insurgence, dissemination, and progression, as well as the effect of chemotherapy on isoforms expression. The described literature strongly suggests that P2X7 variants are potential new biomarkers and therapeutical targets in oncological conditions and that their study in carcinogenesis deserves to be further pursued.

Japanese eel retinol dehydrogenases 11/12-like are 17-ketosteroid reductases involved in sex steroid synthesis

The synthesis of 11-ketotestosterone (11KT) and estradiol-17β (E2), which play important roles in the regulation of gametogenesis in teleost fishes, is catalyzed by several steroidogenic enzymes. In particular, 17β-hydroxysteroid dehydrogenases (Hsd17bs) with 17-ketosteroid reducing activity (17KSR activity) are essential enzymes in the formation of these sex steroid hormones in the gonads and other tissues. Retinol dehydrogenase 11 (RDH11) has been suggested to be a novel tentative HSD17B (HSD17B15) in humans for a decade, however no definitive proof has been provided yet. In this study, three cDNAs related to human RDH11 were isolated from Japanese eel testis and characterized. Sequence similarity and phylogenetic analyses revealed their close relationship to human rdh11 and rdh12 gene products and they were designated as rdh11/12-like 1, rdh11/12-like 2, and rdh11/12-like 3. Three recombinant Rdh11/12-like proteins expressed in HEK293T cells catalyzed the transformation of estrone into E2 and androstenedione into testosterone. Only Rdh11/12-like 1 catalyzed the conversion of 11-ketoandrostenedione into 11KT. Tissue-distribution analysis by quantitative real-time polymerase chain reaction revealed, in immature male Japanese eel, that rdh11/12-like 1 and rdh11/12-like 2 are predominantly expressed in testis and brain, while rdh11/12-like 3 is expressed ubiquitously. Moreover, we analyzed the effects of gonadotropins and 11KT on the expression of the three rdh11/12-like mRNAs in the immature testis. In vitro incubation of immature testes with various doses of recombinant Japanese eel follicle stimulating hormone, luteinizing hormone, and 11KT indicated that the expression of rdh11/12-like 1 mRNA, rdh11/12-like 2, and rdh11/12-like 3 did not change. These findings suggest that the three Rdh11/12-like proteins metabolize sex steroids. Rdh11/12-like 1 may be one of the enzymes with 17KSR activity involved in the production of 11KT in the testis.

Majority of alpha2,6-sialylated glycans in the adult mouse brain exist in O-glycans: SALSA-MS analysis for knockout mice of alpha2,6-sialyltransferase genes.

Sialic acids are unique sugars with negative charge and exert various biological functions such as regulation of immune systems, maintenance of nerve tissues and expression of malignant properties of cancers. Alpha 2,6 sialylated N-glycans, one of representative sialylation forms, are synthesized by St6gal1 or St6gal2 gene products in humans and mice. Previously, it has been reported that St6gal1 gene is ubiquitously expressed in almost all tissues. On the other hand, St6gal2 gene is expressed mainly in the embryonic and perinatal stages of brain tissues. However, roles of St6gal2 gene have not been clarified. Expression profiles of N-glycans with terminal α2,6 sialic acid generated by St6gal gene products in the brain have never been directly studied. Using conventional lectin blotting and novel sialic acid linkage-specific alkylamidationmass spectrometry method (SALSA-MS), we investigated the function and expression of St6gal genes and profiles of their products in the adult mouse brain by establishing KO mice lacking St6gal1 gene, St6gal2 gene, or both of them (double knockout). Consequently, α2,6-sialylated N-glycans were scarcely detected in adult mouse brain tissues, and a majority of α2,6-sialylated glycans found in the mouse brain were O-linked glycans. The majority of these α2,6-sialylated O-glycans were shown to be disialyl-T antigen and sialyl-(6)T antigen by mass spectrometry analysis. Moreover, it was revealed that a few α2,6-sialylated N-glycans were produced by the action of St6gal1 gene, despite both St6gal1 and St6gal2 genes being expressed in the adult mouse brain. In the future, where and how sialylated O-linked glycoproteins function in the brain tissue remains to be clarified.

Angioimmunoblastic T-cell lymphoma-like lymphadenopathy in mice transgenic for human RHOA with p.Gly17Val mutation

ABSTRACT A missense mutation in RHOA encoding p.Gly17 Val has been reported to occur frequently in angioimmunoblastic T-cell lymphoma (AITL). Here, we describe a murine model which expresses the human RHOA mutant gene product in a T-cell specific manner and develops AITL-like symptoms. Most transgenic mice feature with latency one or two enlarged lymph nodes characterized by aberrant lymph node architecture, extensive lymphocytic infiltration, extrafollicular meshwork of follicular dendritic cells (FDC) and arborized endothelial venules. Importantly, we provide evidence for expansion of PD-1+ follicular helper T (Tfh) cells which are the neoplastic cells of AITL. In addition, we saw proliferation of B-cells leading to hypergammaglobulinemia and the presence of dominant T cell clonal populations. Transplantation of lymph node cells to immunocompromised mice partly recreated lymphadenopathy after a long latency and with low penetrance suggesting that cells have undergone partial transformation to a premalignant state. Transcriptomic profiling revealed that the gene expression pattern within affected lymph nodes of the mice closely resembles that of AITL patients with the identical RHOA p.Gly17 Val mutation. The murine model should, therefore, be useful in dissecting pathogenesis of AITL at the molecular level particularly for the cases with the RHOA p.Gly17Val mutation.

Development and Clinical Translation of Approved Gene Therapy Products for Genetic Disorders.

The field of gene therapy is striving more than ever to define a path to the clinic and the market. Twenty gene therapy products have already been approved and over two thousand human gene therapy clinical trials have been reported worldwide. These advances raise great hope to treat devastating rare and inherited diseases as well as incurable illnesses. Understanding of the precise pathomechanisms of diseases as well as the development of efficient and specific gene targeting and delivery tools are revolutionizing the global market. Currently, human cancers and monogenic disorders are indications number one. The elevated prevalence of genetic disorders and cancers, clear gene manipulation guidelines and increasing financial support for gene therapy in clinical trials are major trends. Gene therapy is presently starting to become commercially profitable as a number of gene and cell-based gene therapy products have entered the market and the clinic. This article reviews the history and development of twenty approved human gene and cell-based gene therapy products that have been approved up-to-now in clinic and markets of mainly North America, Europe and Asia.

Novel CBWD Gene Products Augment Mammalian Zinc Homeostasis

The CBWD genes (coding for COBW domain‐containing proteins) was previously identified through an in silico search of the human genome for the presence of a zinc‐responsive element known as Zinc Transcriptional Regulatory Element (ZTRE)1. The human CBWD gene family consist of six isoforms with different chromosomal and cytogenetic locations and exhibit a remarkable nucleotide and amino acid sequence conservation. The prokaryotic homologues of CBWD proteins belonging to the GOG0523 family of proteins have been reported to have roles in zinc homeostasis2, however, no known role has yet been documented for the mammalian types. The discovery that the zinc‐sensitive element (ZTRE) occurred in multiple copies of the CBWD genes was intriguing and triggered further investigations into a likely role for this gene family in mammalian cellular zinc handling and/or trafficking. Moreover, we reported a protein that acts at the ZTRE site to mediate regulation of zinc homeostatic genes and found CBWD genes to be a prominent feature3. Using Caco‐2 cells as in vitro model of human enterocytes, we demonstrate the ability of CBWD genes to respond to changes in zinc content at both transcriptional and post‐transcriptional levels evidenced by decreased transcript and overexpressed protein abundance in response to increased extracellular zinc concentration. We also show that increased expression of CBWD protein through a transgene extended cellular tolerance to extreme windows of zinc availability, demonstrating the potential of the human CBWD gene products to augment cellular zinc buffering. This potential buffering ability of CBWD gene products may be effected through cellular zinc redistribution evidenced by a less punctate zinc distribution in cells made to overexpress CBWD protein compared to control cells. Furthermore, overexpression of COBW protein attenuated the responsiveness of a zinc‐sensitive metallothionein promoter, indicating an action consistent with a role in zinc trafficking or buffering. These findings give plausible evidence in support of the involvement of the mammalian CBWD gene products in cellular zinc homeostasis.Support or Funding InformationThis work was supported by the Nigerian Tertiary Education Trust Fund through Benue State University, Makurdi, Benue State NigeriaThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Using the drug-protein interactome to identify anti-ageing compounds for humans.

Advancing age is the dominant risk factor for most of the major killer diseases in developed countries. Hence, ameliorating the effects of ageing may prevent multiple diseases simultaneously. Drugs licensed for human use against specific diseases have proved to be effective in extending lifespan and healthspan in animal models, suggesting that there is scope for drug repurposing in humans. New bioinformatic methods to identify and prioritise potential anti-ageing compounds for humans are therefore of interest. In this study, we first used drug-protein interaction information, to rank 1,147 drugs by their likelihood of targeting ageing-related gene products in humans. Among 19 statistically significant drugs, 6 have already been shown to have pro-longevity properties in animal models (p < 0.001). Using the targets of each drug, we established their association with ageing at multiple levels of biological action including pathways, functions and protein interactions. Finally, combining all the data, we calculated a ranked list of drugs that identified tanespimycin, an inhibitor of HSP-90, as the top-ranked novel anti-ageing candidate. We experimentally validated the pro-longevity effect of tanespimycin through its HSP-90 target in Caenorhabditis elegans.

DASHR 2.0: integrated database of human small non-coding RNA genes and mature products

MotivationSmall non-coding RNAs (sncRNAs, <100 nts) are highly abundant RNAs that regulate diverse and often tissue-specific cellular processes by associating with transcription factor complexes or binding to mRNAs. While thousands of sncRNA genes exist in the human genome, no single resource provides searchable, unified annotation, expression and processing information for full sncRNA transcripts and mature RNA products derived from these larger RNAs.ResultsOur goal is to establish a complete catalog of annotation, expression, processing, conservation, tissue-specificity and other biological features for all human sncRNA genes and mature products derived from all major RNA classes. DASHR (Database of small human non-coding RNAs) v2.0 database is the first that integrates human sncRNA gene and mature products profiles obtained from multiple RNA-seq protocols. Altogether, 185 tissues/cell types and sncRNA annotations and >800 curated experiments from ENCODE and GEO/SRA across multiple RNA-seq protocols for both GRCh38/hg38 and GRCh37/hg19 assemblies are integrated in DASHR. Moreover, DASHR is the first to contain both known and novel, previously un-annotated sncRNA loci identified by unsupervised segmentation (13 times more loci with 1 678 800 total). Additionally, DASHR v2.0 adds >3 200 000 annotations for non-small RNA genes and other genomic features (long-noncoding RNAs, mRNAs, promoters, repeats). Furthermore, DASHR v2.0 introduces an enhanced user interface, interactive experiment-by-locus table view, sncRNA locus sorting and filtering by biological features. All annotation and expression information directly downloadable and accessible as UCSC genome browser tracks.Availability and implementationDASHR v2.0 is freely available at https://lisanwanglab.org/DASHRv2.Supplementary information Supplementary data are available at Bioinformatics online.

Human MxB Protein Is a Pan-herpesvirus Restriction Factor.

Herpesvirus infections are highly prevalent in the human population and persist for life. They are often acquired subclinically but potentially progress to life-threatening diseases in immunocompromised individuals. The interferon system is indispensable for the control of herpesviral replication. However, the responsible antiviral effector mechanisms are not well characterized. The type I interferon-induced, human myxovirus resistance 2 (MX2) gene product MxB, a dynamin-like large GTPase, has recently been identified as a potent inhibitor of HIV-1. We now show that MxB also interferes with an early step of herpesvirus replication, affecting alpha-, beta-, and gammaherpesviruses before or at the time of immediate early gene expression. Defined MxB mutants influencing GTP binding and hydrolysis revealed that the effector mechanism against herpesviruses is thoroughly different from that against HIV-1. Overall, our findings demonstrate that MxB serves as a broadly acting intracellular restriction factor that controls the establishment of not only retrovirus but also herpesvirus infection of all three subfamilies.IMPORTANCE Human herpesviruses pose a constant threat to human health. Reactivation of persisting herpesvirus infections, particularly in immunocompromised individuals and the elderly, can cause severe diseases, such as zoster, pneumonia, encephalitis, or cancer. The interferon system is relevant for the control of herpesvirus replication as exemplified by fatal disease outcomes in patients with primary immunodeficiencies. Here, we describe the interferon-induced, human MX2 gene product MxB as an efficient restriction factor of alpha-, beta-, and gammaherpesviruses. MxB has previously been described as an inhibitor of HIV-1. Importantly, our mutational analyses of MxB reveal an antiviral mechanism of herpesvirus restriction distinct from that against HIV-1. Thus, the dynamin-like MxB GTPase serves as a broadly acting intracellular restriction factor that controls retrovirus as well as herpesvirus infections.

Validation of an Automated Patch-Clamp Screening Assay on Human Kir2.1 Cardiac Ion Channels

The Kir2.1 potassium channel underlies the IK1 current which is a main contributor to the diastolic resting potential of cardiac myocytes. These currents are also involved in the shaping of the initial depolarization and final repolarization of the action potential (AP). Drug-induced disturbances of Kir2.1 function can increase the duration of the AP and produce dangerous arrhythmias. Here we have developed a functional cell-based assay on an automated electrophysiology platform to complete a panel of pre-clinical cardiosafety tests for early discovery compounds. CHO cells stably expressing the human KCNJ2 gene product under the control of a tetracycline-inducible promoter were recorded on a QPatch HTX platform. Series of incrementing voltage steps from a −20 mV holding potential elicited large, non-inactivating negative inward currents at membrane potentials below the K+-equilibrium potential, with strong rectification above. Enhancing the external [K+]o concentration resulted in greater inward and outward currents, and a cross-over of the outward currents on the I-V curves. The zero-current potential shifted by 56 ± 6 mV per ten-fold variation of [K+]o, in good agreement with the Nernst equation prediction. Addition of millimolar concentrations of Ba2+or Cs+ dose- and/or voltage-dependently blocked the inward currents as described for native IK1 currents. Pharmacological studies using a step-ramp voltage protocol allowing stable recordings for 30 min demonstrated inhibitory activity in the low micro-molar range for the methoxybenzyl-methanamide Kir-inhibitor ML133 and the pentamidine-derivative PA-6, although these compounds did not exhibit selectivity over hERG channels as claimed.