Unlocking Tn3-family transposase activity in vitro unveils an asymetric pathway for transposome assembly

The Tn3 family is a widespread group of replicative transposons that are notorious for their contribution to the dissemination of antibiotic resistance and the emergence of multiresistant pathogens worldwide. The TnpA transposase of these elements catalyzes DNA breakage and rejoining reactions required for transposition. It also is responsible for target immunity, a phenomenon that prevents multiple insertions of the transposon into the same genomic region. However, the molecular mechanisms whereby TnpA acts in both processes remain unknown. Here, we have developed sensitive biochemical assays for the TnpA transposase of the Tn3-family transposon Tn4430 and used these assays to characterize previously isolated TnpA mutants that are selectively affected in immunity. Compared with wild-type TnpA, these mutants exhibit deregulated activities. They spontaneously assemble a unique asymmetric synaptic complex in which one TnpA molecule simultaneously binds two transposon ends. In this complex, TnpA is in an activated state competent for DNA cleavage and strand transfer. Wild-type TnpA can form this complex only on precleaved ends mimicking the initial step of transposition. The data suggest that transposition is controlled at an early stage of transpososome assembly, before DNA cleavage, and that mutations affecting immunity have unlocked TnpA by stabilizing the protein in a monomeric activated synaptic configuration. We propose an asymmetric pathway for coupling active transpososome assembly with proper target recruitment and discuss this model with respect to possible immunity mechanisms.

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Regulatory Potential of bHLH-Type Transcription Factors on the Road to Rubber Biosynthesis in Hevea brasiliensis

Plants ◽

10.3390/plants9060674 ◽

2020 ◽

Vol 9 (6) ◽

pp. 674

Author(s):

Tomoko Yamaguchi ◽

Yukio Kurihara ◽

Yuko Makita ◽

Emiko Okubo-Kurihara ◽

Ami Kageyama ◽

...

Keyword(s):

Cell Differentiation ◽

Natural Rubber ◽

Hevea Brasiliensis ◽

Time Course ◽

Molecular Mechanisms ◽

Cultured Cells ◽

Early Stage ◽

Rubber Biosynthesis ◽

On The Road

Natural rubber is the main component of latex obtained from laticifer cells of Hevea brasiliensis. For improving rubber yield, it is essential to understand the genetic molecular mechanisms responsible for laticifer differentiation and rubber biosynthesis. Jasmonate enhances both secondary laticifer differentiation and rubber biosynthesis. Here, we carried out time-course RNA-seq analysis in suspension-cultured cells treated with methyljasmonic acid (MeJA) to characterize the gene expression profile. Gene Ontology (GO) analysis showed that the term “cell differentiation” was enriched in upregulated genes at 24 h after treatment, but inversely, the term was enriched in downregulated genes at 5 days, indicating that MeJA could induce cell differentiation at an early stage of the response. Jasmonate signaling is activated by MYC2, a basic helix–loop–helix (bHLH)-type transcription factor (TF). The aim of this work was to find any links between transcriptomic changes after MeJA application and regulation by TFs. Using an in vitro binding assay, we traced candidate genes throughout the whole genome that were targeted by four bHLH TFs: Hb_MYC2-1, Hb_MYC2-2, Hb_bHLH1, and Hb_bHLH2. The latter two are highly expressed in laticifer cells. Their physical binding sites were found in the promoter regions of a variety of other TF genes, which are differentially expressed upon MeJA exposure, and rubber biogenesis-related genes including SRPP1 and REF3. These studies suggest the possibilities that Hb_MYC2-1 and Hb_MYC2-2 regulate cell differentiation and that Hb_bHLH1 and Hb_bHLH2 promote rubber biosynthesis. We expect that our findings will help to increase natural rubber yield through genetic control in the future.

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Molecular Mechanisms of Anti-Melanogenic Gedunin Derived from Neem Tree (Azadirachta indica) Using B16F10 Mouse Melanoma Cells and Early-Stage Zebrafish

Plants ◽

10.3390/plants10020330 ◽

2021 ◽

Vol 10 (2) ◽

pp. 330

Author(s):

Hwang-Ju Jeon ◽

Kyeongnam Kim ◽

Chaeeun Kim ◽

Myoung-Jin Kim ◽

Tae-Oh Kim ◽

...

Keyword(s):

Skin Color ◽

Molecular Mechanisms ◽

Early Stage ◽

Melanoma Cells ◽

Ultraviolet Rays ◽

Melanin Production ◽

Zebrafish Model ◽

Mouse Melanoma

Melanogenesis represents a series of processes that produce melanin, a protective skin pigment (against ultraviolet rays), and determines human skin color. Chemicals reducing melanin production have always been in demand in the cosmetic market because of skincare interests, such as whitening. The main mechanism for inhibiting melanin production is the inhibition of tyrosinase (TYR), a key enzyme for melanogenesis. Here, we evaluated gedunin (Ged), a representative limonoid, for its anti-melanogenesis action. Melanin production in vitro was stimulated by alpha-melanocyte stimulating hormone (α-MSH) in B16F10 mouse melanoma cells. Ged reduced α-MSH-stimulated melanin production, inhibiting TYR activity and protein amount. We confirmed this result in vivo in a zebrafish model for melanogenesis. There was no sign of toxicity and malformation of zebrafish embryos during development in all treated concentrations. Ged reduced the number of produced zebrafish embryo pigment dots and melanin contents of embryos. The highly active concentration of Ged (100 µM) was much lower than the positive control, kojic acid (8 mM). Hence, Ged could be a fascinating candidate for anti-melanogenesis reagents.

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37DEVELOPMENTAL POTENTIAL OF CLONE CELLS IN MURINE CLONE-FERTILIZED AGGREGATION CHIMERAS

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab37 ◽

2004 ◽

Vol 16 (2) ◽

pp. 141

Author(s):

S. Eckardt ◽

N.A. Leu ◽

K.J. McLaughlin

Keyword(s):

Early Stage ◽

Cumulus Cell ◽

Blastocyst Stage ◽

Wild Type ◽

Cell Stage ◽

Developmental Potential ◽

Preimplantation Stage ◽

Transgenic Embryos ◽

Clone Cells

In both murine and porcine preimplantation stage clones, mosaicism in gene expression has been observed, indicating variation in transcription of some genes between cells of the individual clone (Boiani M et al., 2002 Genes Dev. 16, 1209–1219; Park KW et al., 2002 Biol. Reprod. 66, 1001–1005). This observation raises the question as to whether all blastomeres within one early-stage clone are equivalent, or whether there are differences in developmental potential. To address this, we aggregated preimplantation-stage clone embryos with fertilized embryos and assessed contribution of Oct4-GFP expressing cells of clone origin in blastocysts and in vitro outgrowths. In normal embryos, the Oct4-GFP transgene is expressed during preimplantation stages and reflects expression of Oct4 protein. Mouse cumulus cell clones were produced from cells transgenic for Oct4-GFP (Szabó PE et al., 2002 Mech. Dev. 115, 157–160) as described (Boiani M et al., 2002 Genes Dev. 16, 1209–1219). Four-cell-stage clones and synchronous fertilized non-transgenic embryos were aggregated in micro-wells after removal of the zona pellucida using acid Tyrode’s solution. Aggregates were cultured to the blastocyst stage in -MEM supplemented with bovine serum albumin (0.4% w/v). All control chimeras produced from four-cell-stage fertilized non-transgenic and Oct4-GFP transgenic embryos formed blastocysts, and 15 of 20 had GFP-expressing cells. The majority of clone-wild-type aggregates developed to the blastocyst stage (35/40); however, contribution of GFP-expressing cells was observed in fewer blastocysts compared to controls (12/35; P<0.05). Contribution of GFP expressing clone cells to the ICM varied between 30% and 100% of cells as determined by subjective evaluation of GFP fluorescence overlaying bright-field images. During in vitro outgrowth formation of synchronous aggregation chimeras of clone and wild-type embryos, maintenance of clone contribution to the ICM mound was observed, but at a lower frequency (12% v. 34% at the blastocyst stage). The results suggest that aggregation with fertilized cells does not provide benefit to clone blastomeres during preimplantation stages. Possibly, clone blastomeres may not be competitive with wild-type blastomeres, or are developmentally asynchronous, which will be tested using asynchronous chimeras.

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Targeting Human Immunodeficiency Virus (HIV) Type 2 Integrase Protein into HIV Type 1

Journal of Virology ◽

10.1128/jvi.73.10.8831-8836.1999 ◽

1999 ◽

Vol 73 (10) ◽

pp. 8831-8836 ◽

Cited By ~ 10

Author(s):

Hongmei Liu ◽

Xiaoyun Wu ◽

Hongling Xiao ◽

John C. Kappes

Keyword(s):

Human Immunodeficiency Virus ◽

Reverse Transcription ◽

Wild Type Virus ◽

Strand Transfer ◽

Wild Type ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Integrase (IN) is the only retroviral enzyme necessary for the integration of retroviral cDNA into the host cell’s chromosomes. The structure and function of IN is highly conserved. The human immunodeficiency virus type 2 (HIV-2) IN has been shown to efficiently support 3′ processing and strand transfer of HIV-1 DNA substrate in vitro. To determine whether HIV-2 IN protein (IN2) could substitute for HIV-1 IN function in vivo, we used HIV-1 Vpr to deliver the IN2 into IN mutant HIV-1 virions by expression intrans as a Vpr-IN fusion protein.Trans-complementation with IN2 markedly increased the infectivity of IN-minus HIV-1. Compared with the homologous trans-IN protein, infectivity was increased to a level of 16%. Since IN has been found to play a role in reverse transcription (Wu et al., J. Virol. 73:2126–2135, 1999), cells infected with IN2-complemented HIV-1 were analyzed for DNA products of reverse transcription. DNA levels of approximately 18% of that of wild type were detected. The homologous trans-IN protein restored the synthesis of viral cDNA to approximately 86% of that of wild-type virus. By complementing integration-defective HIV-1 IN mutant viruses, which were not impaired in cDNA synthesis, thetrans-IN2 protein was shown to support integration up to a level of 55% compared with that of the homologoustrans-IN protein. The delivery of heterologous IN protein into HIV-1 particles in trans offers a novel approach to understand IN protein function in vivo.

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Flow-cytometric microglial sorting coupled with quantitative proteomics identifies moesin as a highly-abundant microglial protein with relevance to Alzheimer’s disease

10.1101/802694 ◽

2019 ◽

Author(s):

Sruti Rayaprolu ◽

Tianwen Gao ◽

Hailian Xiao ◽

Supriya Ramesha ◽

Laura D. Weinstock ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Cell Sorting ◽

Ribosomal Proteins ◽

Molecular Mechanisms ◽

Synaptic Proteins ◽

Wild Type ◽

Proteomic Data ◽

Core Set

AbstractBackgroundProteomic characterization of microglia provides the most proximate assessment of functionally relevant molecular mechanisms of neuroinflammation. However, microglial proteomics studies have been limited by low cellular yield and contamination by non-microglial proteins using existing enrichment strategies.MethodsWe coupled magnetic-activated cell sorting (MACS) and fluorescence activated cell sorting (FACS) of microglia with tandem mass tag-mass spectrometry (TMT-MS) to obtain a highly-pure microglial proteome and identified a core set of highly-abundant microglial proteins in adult mouse brain. We interrogated existing human proteomic data for Alzheimer’s disease (AD) relevance of highly-abundant microglial proteins and performed immuno-histochemical and in-vitro validation studies.ResultsQuantitative multiplexed proteomics by TMT-MS of CD11b+ MACS-enriched (N = 5 mice) and FACS-isolated (N = 5 mice), from adult wild-type mice, identified 1,791 proteins. A total of 203 proteins were highly abundant in both datasets, representing a core-set of highly abundant microglial proteins. In addition, we found 953 differentially enriched proteins comparing MACS and FACS-based approaches, indicating significant differences between both strategies. The FACS-isolated microglia proteome was enriched with cytosolic, endoplasmic reticulum, and ribosomal proteins involved in protein metabolism and immune system functions, as well as an abundance of canonical microglial proteins. Conversely, the MACS-enriched microglia proteome was enriched with mitochondrial and synaptic proteins and higher abundance of neuronal, oligodendrocytic and astrocytic proteins. From the 203 consensus microglial proteins with high abundance in both datasets, we confirmed microglial expression of moesin (Msn) in wild-type and 5xFAD mouse brains as well as in human AD brains. Msn expression is nearly exclusively found in microglia that surround Aβ plaques in 5xFAD brains. In in-vitro primary microglial studies, Msn silencing by siRNA decreased Aβ phagocytosis and increased lipopolysaccharide-induced production of the pro-inflammatory cytokine, tumor necrosis factor (TNF). In network analysis of human brain proteomic data, Msn was a hub protein of an inflammatory co-expression module positively associated with AD neuropathological features and cognitive dysfunction.ConclusionsUsing FACS coupled with TMT-MS as the method of choice for microglial proteomics, we define a core set of highly-abundant adult microglial proteins. Among these, we validate Msn as highly-abundant in plaque-associated microglia with relevance to human AD.

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Aggregation of Aβ40/42 chains in the presence of cyclic neuropeptides investigated by molecular dynamics simulations

PLoS Computational Biology ◽

10.1371/journal.pcbi.1008771 ◽

2021 ◽

Vol 17 (3) ◽

pp. e1008771

Author(s):

Min Wu ◽

Lyudmyla Dorosh ◽

Gerold Schmitt-Ulms ◽

Holger Wille ◽

Maria Stepanova

Keyword(s):

Molecular Dynamics ◽

Molecular Dynamics Simulations ◽

Cyclic Peptides ◽

Molecular Mechanisms ◽

Surface Effect ◽

Early Stage ◽

Aβ Peptides ◽

Hydrophobic Residues ◽

Dynamics Simulations

Alzheimer’s disease is associated with the formation of toxic aggregates of amyloid beta (Aβ) peptides. Despite tremendous efforts, our understanding of the molecular mechanisms of aggregation, as well as cofactors that might influence it, remains incomplete. The small cyclic neuropeptide somatostatin-14 (SST14) was recently found to be the most selectively enriched protein in human frontal lobe extracts that binds Aβ42 aggregates. Furthermore, SST14’s presence was also found to promote the formation of toxic Aβ42 oligomers in vitro. In order to elucidate how SST14 influences the onset of Aβ oligomerization, we performed all-atom molecular dynamics simulations of model mixtures of Aβ42 or Aβ40 peptides with SST14 molecules and analyzed the structure and dynamics of early-stage aggregates. For comparison we also analyzed the aggregation of Aβ42 in the presence of arginine vasopressin (AVP), a different cyclic neuropeptide. We observed the formation of self-assembled aggregates containing the Aβ chains and small cyclic peptides in all mixtures of Aβ42–SST14, Aβ42–AVP, and Aβ40–SST14. The Aβ42–SST14 mixtures were found to develop compact, dynamically stable, but small aggregates with the highest exposure of hydrophobic residues to the solvent. Differences in the morphology and dynamics of aggregates that comprise SST14 or AVP appear to reflect distinct (1) regions of the Aβ chains they interact with; (2) the propensities to engage in hydrogen bonds with Aβ peptides; and (3) solvent exposures of hydrophilic and hydrophobic groups. The presence of SST14 was found to impede aggregation in the Aβ42–SST14 system despite a high hydrophobicity, producing a stronger “sticky surface” effect in the aggregates at the onset of Aβ42–SST14 oligomerization.

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Herpes Simplex Virus DNA Cleavage and Packaging: Association of Multiple Forms of UL15-Encoded Proteins with B Capsids Requires at Least the UL6, UL17, and UL28 Genes

Journal of Virology ◽

10.1128/jvi.72.4.3045-3050.1998 ◽

1998 ◽

Vol 72 (4) ◽

pp. 3045-3050 ◽

Cited By ~ 47

Author(s):

Brandy Salmon ◽

Joel D. Baines

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Dna Cleavage ◽

Rabbit Antiserum ◽

Wild Type ◽

Encoded Proteins ◽

Type C ◽

Simplex Virus ◽

Hsv 1

ABSTRACT The UL15 gene of herpes simplex virus (HSV) is one of several genes required for the packaging of viral DNA into intranuclear B capsids to produce C capsids that become enveloped at the inner nuclear membrane. A rabbit antiserum directed against UL15-encoded protein recognized three proteins with apparent M rs of 79,000, 80,000, and 83,000 in highly purified B capsids. The 83,000-M rprotein was detected in type C capsids and comigrated with the product of a UL15 cDNA transcribed and translated in vitro. The 83,000- and 80,000-M r proteins were readily detected in purified virions. Inasmuch as (i) none of these proteins were detectable in capsids purified from cells infected with HSV-1(ΔUL15), a virus lacking an intact UL15 gene, and (ii) corresponding proteins in capsids purified from cells infected with a recombinant virus [HSV-1(R7244), containing a 20-codon tag at the 3′ end of UL15] were decreased in electrophoretic mobility relative to the wild-type proteins, we conclude that the proteins with apparent M rs of 83,000, 80,000, and 79,000 are products of UL15 with identical C termini. The 79,000-, 80,000-, and 83,000-M r proteins remained associated with B capsids in the presence of 0.5 M guanidine HCl and remained detectable in capsids treated with 2.0 M guanidine HCl and lacking proteins associated with the capsid core. These data, therefore, indicate that UL15-encoded proteins are integral components of B capsids. Only the 83,000-M r protein was detected in B capsids purified from cells infected with viruses lacking the UL6, UL17, or UL28 genes, which are required for DNA cleavage and packaging, suggesting that capsid association of the 80,000- and 79,000-M rproteins requires intact cleavage and packaging machinery. These data, therefore, indicate that capsid association of the 80,000- and 79,000-M r UL15-encoded proteins reflects a previously unrecognized step in the DNA cleavage and packaging reaction.

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Anti-Inflammatory Performance of Lactose-Modified Chitosan and Hyaluronic Acid Mixtures in an In Vitro Macrophage-Mediated Inflammation Osteoarthritis Model

Cells ◽

10.3390/cells9061328 ◽

2020 ◽

Vol 9 (6) ◽

pp. 1328 ◽

Cited By ~ 1

Author(s):

Elena Tarricone ◽

Elena Mattiuzzo ◽

Elisa Belluzzi ◽

Rossella Elia ◽

Andrea Benetti ◽

...

Keyword(s):

Hyaluronic Acid ◽

Inflammatory Mediators ◽

Early Stage ◽

Initial Step ◽

Ros Generation ◽

Chondrocyte Viability ◽

Modified Chitosan ◽

Activated Monocytes ◽

Inflammatory Molecules

The development and progression of osteoarthritis (OA) is associated with macrophage-mediated inflammation that generates a broad spectrum of cytokines and reactive oxygen species (ROS). This study investigates the effects of mid-MW hyaluronic acid (HA) in combination with a lactose-modified chitosan (CTL), on pro-inflammatory molecules and metalloproteinases (MMPs) expression, using an in vitro model of macrophage-mediated inflammation. Methods. To assess chondrocyte response to HA and CTL in the presence of macrophage derived inflammatory mediators, cells were exposed to the conditioned medium (CM) of U937 activated monocytes and changes in cell viability, pro-inflammatory mediators and MMPs expression or ROS generation were analysed. Results. CTL induced changes in chondrocyte viability that are reduced by the presence of HA. The CM of activated U937 monocytes (macrophages) significantly increased gene expression of pro-inflammatory molecules and MMPs and intracellular ROS generation in human chondrocyte cultures. HA, CTL and their combinations counteracted the oxidative damage and restored gene transcription for IL-1β, TNF-α, Gal-1, MMP-3 and MMP-13 to near baseline values. Conclusions. This study suggests that HA-CTL mixture attenuated macrophage-induced inflammation, inhibited MMPs expression and exhibited anti-oxidative effects. This evidence provides an initial step toward the development of an early stage OA therapeutic treatment

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Cross-subunit catalysis and a new phenomenon of recessive resurrection in Escherichia coli RNase E

Nucleic Acids Research ◽

10.1093/nar/gkz1152 ◽

2019 ◽

Vol 48 (2) ◽

pp. 847-861 ◽

Cited By ~ 2

Author(s):

Nida Ali ◽

Jayaraman Gowrishankar

Keyword(s):

Escherichia Coli ◽

Active Site ◽

Rna Binding ◽

Initial Step ◽

Dominant Negative ◽

Rnase E ◽

Wild Type ◽

Catalytic Active Site

Abstract RNase E is a 472-kDa homo-tetrameric essential endoribonuclease involved in RNA processing and turnover in Escherichia coli. In its N-terminal half (NTH) is the catalytic active site, as also a substrate 5′-sensor pocket that renders enzyme activity maximal on 5′-monophosphorylated RNAs. The protein's non-catalytic C-terminal half (CTH) harbours RNA-binding motifs and serves as scaffold for a multiprotein degradosome complex, but is dispensable for viability. Here, we provide evidence that a full-length hetero-tetramer, composed of a mixture of wild-type and (recessive lethal) active-site mutant subunits, exhibits identical activity in vivo as the wild-type homo-tetramer itself (‘recessive resurrection’). When all of the cognate polypeptides lacked the CTH, the active-site mutant subunits were dominant negative. A pair of C-terminally truncated polypeptides, which were individually inactive because of additional mutations in their active site and 5′-sensor pocket respectively, exhibited catalytic function in combination, both in vivo and in vitro (i.e. intragenic or allelic complementation). Our results indicate that adjacent subunits within an oligomer are separately responsible for 5′-sensing and cleavage, and that RNA binding facilitates oligomerization. We propose also that the CTH mediates a rate-determining initial step for enzyme function, which is likely the binding and channelling of substrate for NTH’s endonucleolytic action.

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Making the cut(s): how Cas12a cleaves target and non-target DNA

Biochemical Society Transactions ◽

10.1042/bst20190564 ◽

2019 ◽

Vol 47 (5) ◽

pp. 1499-1510 ◽

Cited By ~ 5

Author(s):

Daan C. Swarts

Keyword(s):

Conformational Changes ◽

Dna Sequences ◽

Dna Cleavage ◽

Transcriptional Control ◽

Molecular Mechanisms ◽

Nucleic Acid Detection ◽

Target Dna ◽

Activated State ◽

Dna Strand ◽

Cis And Trans

Abstract CRISPR–Cas12a (previously named Cpf1) is a prokaryotic deoxyribonuclease that can be programmed with an RNA guide to target complementary DNA sequences. Upon binding of the target DNA, Cas12a induces a nick in each of the target DNA strands, yielding a double-stranded DNA break. In addition to inducing cis-cleavage of the targeted DNA, target DNA binding induces trans-cleavage of non-target DNA. As such, Cas12a–RNA guide complexes can provide sequence-specific immunity against invading nucleic acids such as bacteriophages and plasmids. Akin to CRISPR–Cas9, Cas12a has been repurposed as a genetic tool for programmable genome editing and transcriptional control in both prokaryotic and eukaryotic cells. In addition, its trans-cleavage activity has been applied for high-sensitivity nucleic acid detection. Despite the demonstrated value of Cas12a for these applications, the exact molecular mechanisms of both cis- and trans-cleavage of DNA were not completely understood. Recent studies have revealed mechanistic details of Cas12a-mediates DNA cleavage: base pairing of the RNA guide and the target DNA induces major conformational changes in Cas12a. These conformational changes render Cas12a in a catalytically activated state in which it acts as deoxyribonuclease. This deoxyribonuclease activity mediates cis-cleavage of the displaced target DNA strand first, and the RNA guide-bound target DNA strand second. As Cas12a remains in the catalytically activated state after cis-cleavage, it subsequently demonstrates trans-cleavage of non-target DNA. Here, I review the mechanistic details of Cas12a-mediated cis- and trans-cleavage of DNA. In addition, I discuss how bacteriophage-derived anti-CRISPR proteins can inhibit Cas12a activity.

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