Discovery Of Novel Functions Research Articles

CRISPR-Cas9-Mediated Genome Editing in Paenibacillus polymyxa.

In recent years, the clustered regularly interspaced palindromic repeats-Cas (CRISPR-Cas) technology has become the method of choice for precision genome editing in many organisms due to its simplicity and efficacy. Multiplex genome editing, point mutations, and large genomic modifications are attractive features of the CRISPR-Cas9 system. These applications facilitate both the ease and velocity of genetic manipulations and the discovery of novel functions. In this protocol chapter, we describe the use of a CRISPR-Cas9 system for multiplex integration and deletion modifications, and deletions of large genomic regions by the use of a single guide RNA (sgRNA), and, finally, targeted point mutation modifications in Paenibacillus polymyxa.

Isolation, identification, degradation mechanism and exploration of active enzymes in the ochratoxin A degrading strain Acinetobacter pittii AP19

Ochratoxin A (OTA) is a polyketide mycotoxin that commonly contaminates agricultural products and causes significant economic losses. In this study, the efficient OTA-degrading strain AP19 was isolated from vineyard soil and was identified as Acinetobacter pittii. Compared with growth in nutrient broth supplemented with OTA (OTA-NB), strain AP19 grew faster in nutrient broth (NB), but the ability of the resulting cell lysates to remove OTA was weaker. After cultivation in NB, the cell lysate of strain AP19 was able to remove 100% of 1 mg/L OTA within 18 h. The cell lysate fraction > 30 kDa degraded 100% of OTA within 12 h, while the fractions < 30 kDa were practically unable to degrade OTA. Further anion exchange chromatography of the > 30 kDa fraction yielded two peaks exhibiting significant OTA degradation activity. The degradation product was identified as OTα. Amino acid metabolism exhibited major transcriptional trends in the response of AP19 to OTA. The dacC gene encoding carboxypeptidase was identified as one of the contributors to OTA degradation. Soil samples inoculated with strain AP19 showed significant OTA degradation. These results provide significant insights into the discovery of novel functions in A. pittii, as well as its potential as an OTA decomposer.

HAI-1 is required for the novel role of FGFBP1 in maintenance of cell morphology and F-actin rearrangement in human keratinocytes.

Formation and maintenance of skin barrier function require tightly controlled membrane-associated proteolysis, in which the integral membrane Kunitz-type serine protease inhibitor, HAI-1, functions as the primary inhibitor of the membrane-associated serine proteases, matriptase and prostasin. Previously, HAI-1 loss in HaCaT human keratinocytes resulted in an expected increase in prostasin proteolysis but a paradoxical decrease in matriptase proteolysis. The paradoxical decrease in shed active matriptase is further investigated in this study with an unexpected discovery of novel functions of fibroblast growth factor-binding protein 1 (FGFBP1), which acts as an extracellular ligand that can rapidly elicit F-actin rearrangement and subsequently affect the morphology of human keratinocytes. This novel growth factor-like function is in stark contrast to the canonical activity of this protein through interactions with FGFs for its pathophysiological functions. This discovery began with the observation that HAI-1 KO HaCaT cells lose the characteristic cobblestone morphology of the parental cells and exhibit aberrant F-actin formation along with altered subcellular targeting of matriptase and HAI-2. The alterations in cell morphology and F-actin status caused by targeted HAI-1 deletion can be restored by treatment with conditioned medium from parental HaCaT cells, in which FGFBP1 was identified by tandem mass spectrometry. Recombinant FGFBP1 down to 1ng/ml was able to revert the changes caused by HAI-1 loss. Our study reveals a novel function of FGFBP1 in the maintenance of keratinocyte morphology, which depends on HAI-1.

Topoisomerase 2β and DNA topology during B cell development.

Topoisomerase 2β (TOP2B) introduces transient double strand breaks in the DNA helix to remove supercoiling structures and unwind entangled DNA strains. Advances in genomic technologies have enabled the discovery of novel functions for TOP2B in processes such as releasing of the paused RNA polymerase II and maintaining the genome organization through DNA loop domains. Thus, TOP2B can regulate transcription directly by acting on transcription elongation and indirectly by controlling interactions between enhancer and promoter regions through genome folding. The identification of TOP2B mutations in humans unexpectedly revealed a unique role of TOP2B in B-cell progenitors. Here we discuss the functions of TOP2B and the mechanisms leading to the B-cell development defect in patients with TOP2B deficiency.

Discovery of oridonin as a novel agonist for BRS-3

BackgroundBombesin Receptor Subtype-3 (BRS-3, Bombesin-like receptor, BB3) is an orphan G-protein coupled receptor (GPCR). Recent studies have shown that BRS-3 played a vital role in glucose regulation, insulin secretion, and energy homeostasis. Therefore, discovering more novel exogenous ligands with diverse structures for BRS-3 will be of great importance for target validation and drug development. PurposeIn this study, we aim to discover new agonists of BRS-3 from our natural compound libraries, providing a new probe to study the function of BRS-3. Study designMultiple cell-based assays and in vivo experiments were performed to identify the new ligand. MethodsBRS-3 overexpression cells were coupled with FLIPR assay, homogeneous time-resolved fluorescence (HTRF) IP-ONE assay, dynamic mass redistribution (DMR) assay, β-arrestin2 recruitment assay, and western blot to determine receptor activation and downstream signaling events. To further validate the target of BRS-3, a series of in vitro and in vivo experiences were conducted, including glucose uptake, glucose transporter type 4 (GLUT4) transportation in C2C12, and oral glucose tolerance test (OGTT) in mice. ResultsWe discovered and identified oridonin as a novel small molecule agonist of BRS-3, with a moderate affinity (EC50 of 2.236 × 10−7 M in calcium mobilization assay), specificity, and subtype selectivity. Further in vitro and in vivo tests demonstrated that oridonin exerted beneficial effects in glucose homeostasis through activating BRS-3. ConclusionsOridonin, as the discovered new ligand of BRS-3, provides a valuable tool compound to investigate BRS-3′s function, especially for target validation in type 2 diabetes and obesity. Oridonin is promising as a lead compound in the treatment of metabolic disorders. Compared to the known agonists of BRS-3, we can take advantage of the multiple reported pharmacological activities of ODN as a natural product and assess whether these pharmacological activities are regulated by BRS-3. This may facilitate the discovery of novel functions of BRS-3.

Biodegradation of polystyrene by bacteria from the soil in common environments

Polystyrene (PS), a major plastic waste, is difficult to biodegrade due to its unique chemical structure that comprises phenyl moieties attached to long linear alkanes. In this study, we investigated the biodegradation of PS by mesophilic bacterial cultures obtained from various soils in common environments. Two new strains, Pseudomonas lini JNU01 and Acinetobacter johnsonii JNU01, were specifically enriched in non-carbonaceous nutrient medium, with PS as the only source of carbon. Their growth after culturing in basal media increased more than 3-fold in the presence of PS. Fourier transform infrared spectroscopy analysis, used to confirm the formation of hydroxyl groups and potentially additional chemical bond groups, showed an increase in the amount of oxidized PS samples. Moreover, field emission scanning electron microcopy analysis confirmed PS biodegradation by biofilms of the screened microbes. Water contact angle measurement additionally offered insights into the increased hydrophilic characteristics of PS films. Bioinformatics and transcriptional analysis of A. johnsonii JNU01 revealed alkane-1-monooxygenase (AlkB) to be involved in PS biodegradation, which was confirmed by the hydroxylation of PS using recombinant AlkB. These results provide significant insights into the discovery of novel functions of Pseudomonas sp. and Acinetobacter sp., as well as their potential as PS decomposers.

The Order-Disorder Continuum: Linking Predictions of Protein Structure and Disorder through Molecular Simulation

Intrinsically disordered proteins (IDPs) and intrinsically disordered regions within proteins (IDRs) serve an increasingly expansive list of biological functions, including regulation of transcription and translation, protein phosphorylation, cellular signal transduction, as well as mechanical roles. The strong link between protein function and disorder motivates a deeper fundamental characterization of IDPs and IDRs for discovering new functions and relevant mechanisms. We review recent advances in experimental techniques that have improved identification of disordered regions in proteins. Yet, experimentally curated disorder information still does not currently scale to the level of experimentally determined structural information in folded protein databases, and disorder predictors rely on several different binary definitions of disorder. To link secondary structure prediction algorithms developed for folded proteins and protein disorder predictors, we conduct molecular dynamics simulations on representative proteins from the Protein Data Bank, comparing secondary structure and disorder predictions with simulation results. We find that structure predictor performance from neural networks can be leveraged for the identification of highly dynamic regions within molecules, linked to disorder. Low accuracy structure predictions suggest a lack of static structure for regions that disorder predictors fail to identify. While disorder databases continue to expand, secondary structure predictors and molecular simulations can improve disorder predictor performance, which aids discovery of novel functions of IDPs and IDRs. These observations provide a platform for the development of new, integrated structural databases and fusion of prediction tools toward protein disorder characterization in health and disease.

R-BIND: An Interactive Database for Exploring and Developing RNA-Targeted Chemical Probes.

While the opportunities available for targeting RNA with small molecules have been widely appreciated, the challenges associated with achieving specific RNA recognition in biological systems have hindered progress and prevented many researchers from entering the field. To facilitate the discovery of RNA-targeted chemical probes and their subsequent applications, we curated the RNA-targeted BIoactive ligaNd Database (R-BIND). This collection contains an array of information on reported chemical probes that target non-rRNA and have biological activity, and analysis has led to the discovery of RNA-privileged properties. Herein, we developed an online platform to make this information freely available to the community, offering search options, a suite of tools for probe development, and an updated R-BIND data set with detailed experimental information for each probe. We repeated the previous cheminformatics analysis on the updated R-BIND list and found that the distinguishing physicochemical, structural, and spatial properties remained unchanged, despite an almost 50% increase in the database size. Further, we developed several user-friendly tools, including queries based on cheminformatic parameters, experimental details, functional groups, and substructures. In addition, a nearest neighbor algorithm can assess the similarity of user-uploaded molecules to R-BIND ligands. These tools and resources can be used to design small molecule libraries, optimize lead ligands, or select targets, probes, assays, and control experiments. Chemical probes are critical to the study and discovery of novel functions for RNA, and we expect this resource to greatly assist researchers in exploring and developing successful RNA-targeted probes.

Jeremy Baskin: A Life at the Interface between Chemical and Cell Biology

Operating at the interface of chemical biology, biochemistry, and cell biology, the Baskin Lab develops innovative methods for imaging and probing various classes of lipids and couples these methods to the discovery of novel functions for cellular lipids. Notably, the Baskin Lab has developed a method for imaging the production the signaling lipid phosphatidic acid by phospholipase D enzymes and is applying this method, termed IMPACT, to reveal new biological roles for this lipid second messenger. As well, the Baskin Lab focuses on identifying new roles for inositol-containing phospholipids, or phosphoinositides, in the regulation of developmental and oncogenic signaling by characterizing novel phosphoinositide-binding proteins that act as readers of the membrane bilayer 'phosphoinositide code'. Collectively, work in the Baskin Lab will elucidate mechanisms of fundamental biological processes and also contribute to the understanding of diseases caused by perturbations in lipid metabolism, including cancer, developmental disorders, and neurological diseases affecting the myelin sheath such as multiple sclerosis.

Genetic Knockdown and Pharmacologic Inhibition of Hypoxia-Inducible Factor (HIF) Hydroxylases.

Reduced oxygen supply that does not satisfy tissue and cellular demand (hypoxia) regularly occurs both in health and disease. Hence, the capacity for cellular oxygen sensing is of vital importance for each cell to be able to alter its energy metabolism and promote adaptation to hypoxia. The hypoxia-inducible factor (HIF) prolyl hydroxylases 1-3 (PHD1-3) and the asparagine hydroxylase factor-inhibiting HIF (FIH) are the primary cellular oxygen sensors, which confer cellular oxygen-dependent sensitivity upon HIF as well as other hypoxia-sensitive pathways, such as nuclear factor κB (NF-κB). Studying these enzymes allows us to understand the oxygen-dependent regulation of cellular processes and has led to the development of several putative novel therapeutics, which are currently in clinical trials for the treatment of anemia associated with kidney disease. Pharmacologic inhibition and genetic knockdown are commonly established techniques in protein biochemistry and are used to investigate the activity and function of proteins. Here, we describe specific protocols for the knockdown and inhibition of the HIF prolyl hydroxylases 1-3 (PHD1-3) and the asparagine hydroxylase factor-inhibiting HIF (FIH) using RNA interference (RNAi) and hydroxylase inhibitors, respectively. These techniques are essential tools for the analysis of the function of the HIF hydroxylases, allowing the investigation and discovery of novel functions and substrates of these enzymes.

CagL from Helicobacter pylori has ADP-ribosylation activity and exerts partial protective efficacy in mice

Mono ADP-ribosyltransferases are a class of functionally conserved enzymes present in prokaryotic and eukaryotic organisms. In prokaryotes, mono ADP-ribose transfer enzymes often represent a family of exotoxins that display activity in a variety of bacteria responsible for causing disease in plants and animals. A bioinformatic approach has allowed us to identify that CagL gene from some Helicobacter pylori strains shares a sequence pattern with ADP-ribosylating toxins of the CT-group. In this manuscript we show that recombinant CagL from Shi470 is catalytically active showing ADP-ribosyltransferase, NAD-glycohydrolase, and auto-ADP-ribosylation activities. This is the first time that a catalytically active member of the ADP-ribosyltransferase family is identified in Helicobacter pylori. This observation may lead to the discovery of novel functions exerted by CagL in the pathogenesis of Helicobacter pylori. Indeed, we have shown that vaccination with CagL has protective efficacy in mice indicating that CagL may be considered as potential component of a Helicobacter pylori vaccine.

Novel functions of PXR in cardiometabolic disease

Cardiometabolic disease emerges as a worldwide epidemic and there is urgent need to understand the molecular mechanisms underlying this chronic disease. The chemical environment to which we are exposed has significantly changed in the past few decades and recent research has implicated its contribution to the development of many chronic human diseases. However, the mechanisms of how exposure to chemicals contributes to the development of cardiometabolic disease are poorly understood. Numerous chemicals have been identified as ligands for the pregnane X receptor (PXR), a nuclear receptor functioning as a xenobiotic sensor to coordinately regulate xenobiotic metabolism via transcriptional regulation of xenobiotic-detoxifying enzymes and transporters. In the past decade, the function of PXR in the regulation of xenobiotic metabolism has been extensively studied by many laboratories and the role of PXR as a xenobiotic sensor has been well-established. The identification of PXR as a xenobiotic sensor has provided an important tool for the study of new mechanisms through which xenobiotic exposure impacts human chronic diseases. Recent studies have revealed novel and unexpected roles of PXR in modulating obesity, insulin sensitivity, lipid homeostasis, atherogenesis, and vascular functions. These studies suggest that PXR signaling may contribute significantly to the pathophysiological effects of many known xenobiotics on cardiometabolic disease in humans. The discovery of novel functions of PXR in cardiometabolic disease not only contributes to our understanding of “gene–environment interactions” in predisposing individuals to chronic diseases but also provides strong evidence to inform future risk assessment for relevant chemicals. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie.

Synthetic biology approaches to fluorinated polyketides.

The catalytic diversity of living systems offers a broad range of opportunities for developing new methods to produce small molecule targets such as fuels, materials, and pharmaceuticals. In addition to providing cost-effective and renewable methods for large-scale commercial processes, the exploration of the unusual chemical phenotypes found in living organisms can also enable the expansion of chemical space for discovery of novel function by combining orthogonal attributes from both synthetic and biological chemistry. In this context, we have focused on the development of new fluorine chemistry using synthetic biology approaches. While fluorine has become an important feature in compounds of synthetic origin, the scope of biological fluorine chemistry in living systems is limited, with fewer than 20 organofluorine natural products identified to date. In order to expand the diversity of biosynthetically accessible organofluorines, we have begun to develop methods for the site-selective introduction of fluorine into complex natural products by engineering biosynthetic machinery to incorporate fluorinated building blocks. To gain insight into how both enzyme active sites and metabolic pathways can be evolved to manage and select for fluorinated compounds, we have studied one of the only characterized natural hosts for organofluorine biosynthesis, the soil microbe Streptomyces cattleya. This information provides a template for designing engineered organofluorine enzymes, pathways, and hosts and has allowed us to initiate construction of enzymatic and cellular pathways for the production of fluorinated polyketides.

Triggering receptor expressed on myeloid cells-1 (TREM-1): a new player in antiviral immunity?

The triggering receptor expressed on myeloid cells (TREM) family of protein receptors is rapidly emerging as a critical regulator of a diverse array of cellular functions, including amplification of inflammation. Although the ligand(s) for TREM have not yet been fully identified, circumstantial evidence indicates that danger- and pathogen-associated molecular patterns (DAMPs and PAMPs) can induce cytokine production via TREM-1 activation. The discovery of novel functions of TREM, such as regulation of T-cell proliferation and activation of antigen-presenting cells, suggests a larger role of TREM proteins in modulation of host immune responses to microbial pathogens, such as bacteria and fungi. However, the significance of TREM signaling in innate immunity to virus infections and the underlying mechanisms remain largely unclear. The nature and intensity of innate immune responses, specifically production of type I interferon and inflammatory cytokines is a crucial event in dictating recovery vs. adverse outcomes from virus infections. In this review, we highlight the emerging roles of TREM-1, including synergy with classical pathogen recognition receptors. Based on the literature using viral PAMPs and other infectious disease models, we further discuss how TREM-1 may influence host-virus interactions and viral pathogenesis. A deeper conceptual understanding of the mechanisms associated with pathogenic and/or protective functions of TREM-1 in antiviral immunity is essential to develop novel therapeutic strategies for the control of virus infection by modulating innate immune signaling.

Classification of intrinsically disordered regions and proteins.

1.1. Uncharacterized Protein Segments Are a Source of Functional Novelty Over the past decade, we have observed a massive increase in the amount of information describing protein sequences from a variety of organisms.1,2 While this may reflect the diversity in sequence space, and possibly also in function space,3 a large proportion of the sequences lacks any useful function annotation.4,5 Often these sequences are annotated as putative or hypothetical proteins, and for the majority their functions still remain unknown.6,7 Suggestions about potential protein function, primarily molecular function, often come from computational analysis of their sequences. For instance, homology detection allows for the transfer of information from well-characterized protein segments to those with similar sequences that lack annotation of molecular function.8−10 Other aspects of function, such as the biological processes proteins participate in, may come from genetic- and disease-association studies, expression and interaction network data, and comparative genomics approaches that investigate genomic context.11−17 Characterization of unannotated and uncharacterized protein segments is expected to lead to the discovery of novel functions as well as provide important insights into existing biological processes. In addition, it is likely to shed new light on molecular mechanisms of diseases that are not yet fully understood. Thus, uncharacterized protein segments are likely to be a large source of functional novelty relevant for discovering new biology.

Antibodies and Their Receptors: Different Potential Roles in Mucosal Defense

Over recent years it has become increasingly apparent that mucosal antibodies are not only restricted to the IgM and IgA isotypes, but that also other isotypes and particularly IgG can be found in significant quantities at some mucosal surfaces, such as in the genital tract. Their role is more complex than traditionally believed with, among other things, the discovery of novel function of mucosal immunoglobulin receptors. A thorough knowledge in the source and function and mucosal immunoglobulins is particularly important in development of vaccines providing mucosal immunity, and also in the current climate of microbicide development, to combat major world health issues such as HIV. We present here a comprehensive review of human antibody mediated mucosal immunity.

Chemical Genetics: Budding Yeast as a Platform for Drug Discovery and Mapping of Genetic Pathways

The budding yeast Saccharomyces cerevisiae is a widely used model organism, and yeast genetic methods are powerful tools for discovery of novel functions of genes. Recent advancements in chemical-genetics and chemical-genomics have opened new avenues for development of clinically relevant drug treatments. Systematic mapping of genetic networks by high-throughput chemical-genetic screens have given extensive insight in connections between genetic pathways. Here, I review some of the recent developments in chemical-genetic techniques in budding yeast.

Proton channels in algae: reasons to be excited

A fundamental requirement of all eukaryotes is the ability to translocate protons across membranes. This is critical in bioenergetics, for compartmentalized metabolism, and to regulate intracellular pH (pH(i)) within a range that is compatible with cellular metabolism. Plants, animals, and algae utilize specialized transport machinery for membrane energization and pH homeostasis that reflects the prevailing ionic conditions in which they evolved. The recent characterization of H(+)-permeable channels in marine and freshwater algae has led to the discovery of novel functions for these transport proteins in both cellular pH homeostasis and sensory biology. Here we review the potential implications for understanding the origins and evolution of membrane excitability and the phytoplankton-based marine ecosystem responses to ocean acidification.

Innate Natural Killer Cell Phenotype and Function During HIV-1 Infection: Potential Avenues for Modulation

Recent developments in the field of innate immunity and the discovery of novel functions of natural killer (NK) cells have opened up potential new avenues for therapeutic intervention in HIV-1 infection. NK cells inhibit virus entry into CD4+T cells, kill infected CD4+T cells, help the maturation of antigen-specific T-cell responses and promote physiological repair processes throughout the body. NK cells are numerically and functionally compromised during chronic HIV-1 infection, with reduced ability to kill infected cells and to produce regulatory cytokines. HAART induces a recovery in cell number and in some functions of blood NK cells, whilst others remain suppressed. This article reviews the potential of supplementary cytokines, hormones and therapeutic vaccines to reconstitute NK cell functions in people with HIV-1 infection receiving HAART.

Genome-Wide Transcript Analysis of Bradyrhizobium japonicum Bacteroids in Soybean Root Nodules

The transcriptome of endosymbiotic Bradyrhizobium japonicum bacteroids was assessed, using RNA extracted from determinate soybean root nodules. Results were compared with the transcript profiles of B. japonicum cells grown in either aerobic or microaerobic culture. Microoxia is a known trigger for the induction of symbiotically relevant genes. In fact, one third of the genes induced in bacteroids at day 21 after inoculation are congruent with those up-regulated in culture by a decreased oxygen concentration. The other induced genes, however, may be regulated by cues other than oxygen limitation. Both groups of genes provide a rich source for the possible discovery of novel functions related to symbiosis. Samples taken at different timepoints in nodule development have led to the distinction of genes expressed early and late in bacteroids. The experimental approach applied here is also useful for B. japonicum mutant analyses. As an example, we compared the transcriptome of wild-type bacteroids with that of bacteroids formed by a mutant defective in the RNA polymerase transcription factor sigma54. This led to a collection of hitherto unrecognized B. japonicum genes potentially transcribed in planta in a sigma54-dependent manner.