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The ligand-sensing transcription factor Nurr1 emerges as a promising therapeutic target for neurodegenerative pathologies but Nurr1 ligands for functional studies and therapeutic validation are lacking. Here pronounced Nurr1 modulation by statins for which clinically relevant neuroprotective effects are demonstrated, is reported. Several statins directly affect Nurr1 activity in cellular and cell-free settings with low micromolar to sub-micromolar potencies. Simvastatin as example exhibits anti-inflammatory effects in astrocytes, which are abrogated by Nurr1 knockdown. Differential gene expression analysis in native and Nurr1-silenced cells reveals strong proinflammatory effects of Nurr1 knockdown while simvastatin treatment induces several neuroprotective mechanisms via Nurr1 involving changes in inflammatory, metabolic and cell cycle gene expression. Further in vitro evaluation confirms reduced inflammatory response, improved glucose metabolism, and cell cycle inhibition of simvastatin-treated neuronal cells. These findings suggest Nurr1 involvement in the well-documented but mechanistically elusive neuroprotection by statins.
Thermally stable and highly conductive SAMs on Ag substrate — the impact of the anchoring group
(2021)
Self-assembled monolayers (SAMs) on metal substrates are an important part of modern interfacial chemistry and nanotechnology. The robustness of SAMs strongly depends on their thermal stability, which, together with electric conductivity, crucial for their applications in molecular/organic electronics. In this context, using a multidisciplinary approach, the structure, stability, and conductivity properties of conjugated aromatic SAMs featuring the naphthalene backbone and S, Se, or COO group, mediating bonding to the Ag substrate are addressed. Whereas thermal stability of these SAMs exhibits a strong dependence on anchoring group, their conductivity is similar, which is rationalized by tentative model considering redistribution of charge density along the molecular framework. The thermal stability of model naphthalenethiol SAM, emphasized by desorption energy of ≈1.69 eV, is better than that of typical N-heterocyclic carbene (NHC) monolayers considered currently as the most stable SAMs on metal substrates. However, in contrast to NHC SAMs, which are highly insulating, the naphtalene-based SAM, with S, Se or COO anchoring groups, are highly conductive, even in comparison with analogous oligophenyl SAMs (by a factor of 10). A unique combination of the ultimate thermal stability and superior conductivity for the naphthalenethiol SAM on Ag makes it highly attractive for applications.
Alzheimer’s disease (AD) is characterized by the deposition of aggregated species of amyloid beta (Aβ) in the brain, which leads to progressive cognitive deficits and dementia. Aβ is generated by the successive cleavage of the amyloid precursor protein (APP), first by β-site APP cleaving enzyme 1 (BACE1) and subsequently by the γ-secretase complex. Those conditions which enhace or reduce its clearance predispose to Aβ aggregation and the development of AD. In vitro studies have demonstrated that Aβ assemblies spark a feed-forward loop heightening Aβ production. However, the underlying mechanism remains unknown. Here, we show that oligomers and fibrils of Aβ enhance colocalization and physical interaction of APP and BACE1 in recycling endosomes of human neurons derived from induced pluripotent stem cells and other cell types, which leads to exacerbated amyloidogenic processing of APP and intracellular accumulation of Aβ42. In cells that are overexpressing the mutant forms of APP which are unable to bind Aβ or to activate Go protein, we have found that treatment with aggregated Aβ fails to increase colocalization of APP with BACE1 indicating that Aβ-APP/Go signaling is involved in this process. Moreover, inhibition of Gβγ subunit signaling with βARKct or gallein prevents Aβ-dependent interaction of APP and BACE1 in endosomes, β-processing of APP, and intracellular accumulation of Aβ42. Collectively, our findings uncover a signaling mechanism leading to a feed-forward loop of amyloidogenesis that might contribute to Aβ pathology in the early stages of AD and suggest that gallein could have therapeutic potential.
Gram-negative Tripartite Resistance Nodulation and cell Division (RND) superfamily efflux pumps confer various functions, including multidrug and bile salt resistance, quorum-sensing, virulence and can influence the rate of mutations on the chromosome. Multidrug RND efflux systems are often characterized by a wide substrate specificity. Similarly to many other RND efflux pump systems, AcrAD-TolC confers resistance toward SDS, novobiocin and deoxycholate. In contrast to the other pumps, however, it in addition confers resistance against aminoglycosides and dianionic β-lactams, such as sulbenicillin, aztreonam and carbenicillin. Here, we could show that AcrD from Salmonella typhimurium confers resistance toward several hitherto unreported AcrD substrates such as temocillin, dicloxacillin, cefazolin and fusidic acid. In order to address the molecular determinants of the S. typhimurium AcrD substrate specificity, we conducted substitution analyses in the putative access and deep binding pockets and in the TM1/TM2 groove region. The variants were tested in E. coli ΔacrBΔacrD against β-lactams oxacillin, carbenicillin, aztreonam and temocillin. Deep binding pocket variants N136A, D276A and Y327A; access pocket variant R625A; and variants with substitutions in the groove region between TM1 and TM2 conferred a sensitive phenotype and might, therefore, be involved in anionic β-lactam export. In contrast, lower susceptibilities were observed for E. coli cells harbouring deep binding pocket variants T139A, D176A, S180A, F609A, T611A and F627A and the TM1/TM2 groove variant I337A. This study provides the first insights of side chains involved in drug binding and transport for AcrD from S. typhimurium.
Human serum albumin (HSA) nanoparticles represent a promising tool for targeted drug delivery to tumor cells. The coupling of the antibody trastuzumab to nanoparticles uses the capability of human epidermal growth factor receptor 2 (HER2)-positive cells to incorporate agents linked to HER2. In our present study, we developed targeted nanoparticles loaded with antisense oligonucleotides (ASOs) against polo-like kinase 1 (Plk1). We evaluated the receptor-mediated uptake into HER2-positive and -negative breast cancer and murine cell lines. We performed quantitative real-time PCR and Western blot analyses to monitor the impact on Plk1 expression in HER2-positive breast cancer cells. Antibody-conjugated nanoparticles showed a specific targeting to HER2-overexpressing cells with cellular uptake by receptor-mediated endocytosis and a release into HER2-positive BT-474 cells. We observed a significant reduction of Plk1 mRNA and protein expression and increased activation of Caspase 3/7. Thus, this is the first report about ASO-loaded HSA nanoparticles, where an impact on gene expression could be observed. The data provide the basis for the further development of carrier systems for Plk1-specific ASOs to reduce off-target effects evoked by systemically administered ASOs and to achieve a better penetration into primary and metastatic target cells. Treatment of tumors using trastuzumab-conjugated ASO-loaded HSA nanoparticles could be a promising approach to reach this goal.
To evade the host's immune response, herpes simplex virus employs the immediate early gene product ICP47 (IE12) to suppress antigen presentation to cytotoxic T-lymphocytes by inhibition of the ATP-binding cassette transporter associated with antigen processing (TAP). ICP47 is a membrane-associated protein adopting an alpha-helical conformation. Its active domain was mapped to residues 3-34 and shown to encode all functional properties of the full-length protein. The active domain of ICP47 was reconstituted into oriented phospholipid bilayers and studied by proton-decoupled 15N and 2H solid-state NMR spectroscopy. In phospholipid bilayers, the protein adopts a helix-loop-helix structure, where the average tilt angle of the helices relative to the membrane surface is approximately 15 degrees (+/- 7 degrees ). The alignment of both structured domains exhibits a mosaic spread of approximately 10 degrees . A flexible dynamic loop encompassing residues 17 and 18 separates the two helices. Refinement of the experimental data indicates that helix 1 inserts more deeply into the membrane. These novel insights into the structure of ICP47 represent an important step toward a molecular understanding of the immune evasion mechanism of herpes simplex virus and are instrumental for the design of new therapeutics.
Through its role in intron cleavage, tRNA splicing endonuclease (TSEN) plays a critical function in the maturation of intron-containing pre-tRNAs. The catalytic mechanism and core requirement for this process is conserved between archaea and eukaryotes, but for decades, it has been known that eukaryotic TSENs have evolved additional modes of RNA recognition, which have remained poorly understood. Recent research identified new roles for eukaryotic TSEN, including processing or degradation of additional RNA substrates, and determined the first structures of pre-tRNA-bound human TSEN complexes. These recent discoveries have changed our understanding of how the eukaryotic TSEN targets and recognizes substrates. Here, we review these recent discoveries, their implications, and the new questions raised by these findings.
Rat renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin 1beta (IL-1beta). We tested whether ligands of the peroxisome proliferator-activated receptor (PPARalpha) could influence the cytokine-induced expression of MMP-9. Different PPARalpha agonists dose-dependently inhibited the IL-1beta-triggered increase in gelatinolytic activity mainly by decreasing the MMP-9 steady-state mRNA levels. PPARalpha agonists on their own had no effects on MMP-9 mRNA levels and gelatinolytic activity. Surprisingly, the reduction of MMP-9 mRNA levels by PPARalpha activators contrasted with an amplification of cytokine-mediated MMP-9 gene promoter activity and mRNA expression. The potentiation of MMP-9 promoter activity functionally depends on an upstream peroxisome proliferator-responsive element-like binding site, which displayed an increased DNA binding of a PPARalpha immunopositive complex. In contrast, the IL-1beta-induced DNA-binding of nuclear factor kappaB was significantly impaired by PPARalpha agonists. Most interestingly, in the presence of an inducible nitric-oxide synthase (iNOS) inhibitor, the PPARalpha-mediated suppression switched to a strong amplification of IL-1beta-triggered MMP-9 mRNA expression. Concomitantly, activators of PPARalpha potentiated the cytokine-induced iNOS expression. Using actinomycin D, we found that NO, but not PPARalpha activators, strongly reduced the stability of MMP-9 mRNA. In contrast, the stability of MMP-9 protein was not affected by PPARalpha activators. In summary, our data suggest that the inhibitory effects of PPARalpha agonists on cytokine-induced MMP-9 expression are indirect and primarily due to a superinduction of iNOS with high levels of NO reducing the half-life of MMP-9 mRNA.
An efficient route for delivering specific proteins and peptides into neurons could greatly accelerate the development of therapies for various diseases, especially those involving intracellular defects such as Parkinson disease. Here we report the novel use of polybutylcyanoacrylate nanoparticles for delivery of intact, functional proteins into neurons and neuronal cell lines. Uptake of these particles is primarily dependent on endocytosis via the low density lipoprotein receptor. The nanoparticles are rapidly turned over and display minimal toxicity to cultured neurons. Delivery of three different functional cargo proteins is demonstrated. When primary neuronal cultures are treated with recombinant Escherichia coli beta-galactosidase as nanoparticle cargo, persistent enzyme activity is measured beyond the period of nanoparticle degradation. Delivery of the small GTPase rhoG induces neurite outgrowth and differentiation in PC12 cells. Finally, a monoclonal antibody directed against synuclein is capable of interacting with endogenous alpha-synuclein in cultured neurons following delivery via nanoparticles. Polybutylcyanoacrylate nanoparticles are thus useful for intracellular protein delivery in vitro and have potential as carriers of therapeutic proteins for treatment of neuronal disorders in vivo.
Nicotinamide adenine dinucleotide (NAD) serves as a cap-like structure on cellular RNAs (NAD-RNAs) in all domains of life including the bacterium Escherichia coli. NAD also acts as a key molecule in phage-host interactions, where bacterial immune systems deplete NAD to abort phage infection. Nevertheless, NAD-RNAs have not yet been identified during phage infections of bacteria and the mechanisms of their synthesis and degradation are unknown in this context. The T4 phage that specifically infects E. coli presents an important model to study phage infections, but a systematic analysis of the presence and dynamics of NAD-RNAs during T4 phage infection is lacking. Here, we investigate the presence of NAD-RNAs during T4 phage infection in a dual manner. By applying time-resolved NAD captureSeq, we identify NAD-capped host and phage transcripts and their dynamic regulation during phage infection. We provide evidence that NAD-RNAs are – as reported earlier – generated by the host RNA polymerase by initiating transcription with NAD at canonical transcription start sites. In addition, we characterize NudE.1 – a T4 phage-encoded Nudix hydrolase – as the first phage-encoded NAD-RNA decapping enzyme. T4 phages carrying inactive NudE.1 display a delayed lysis phenotype. This study investigates for the first time the dual epitranscriptome of a phage and its host, thereby introducing epitranscriptomics as an important field of phage research.