ACS Chemical Biology最新文献

Accumulation of misfolded α-synuclein (α-Syn) leads to the formation of Lewy bodies and is a major hallmark of Parkinson's disease (PD). The accumulation of α-Syn involves several post-translational modifications. Recently, though, glycation of α-Syn (advanced glycation end products) and activation of the receptor for advanced glycation end products (RAGE) have been linked to neuroinflammation, which leads to oxidative stress and accumulation of α-Syn. The present study aims to detect the effect of glycated α-Syn (gly-α-Syn)-induced synucleinopathy and loss of dopaminergic (DAergic) neurons in the development of PD. We isolated, purified, and prepared glycated recombinant human α-Syn using d-ribose. Gly-α-Syn was characterized by SDS-PAGE, intact mass analysis, and bottom-up peptide sequence through LC-HRMS/MS. The aggregation propensity of gly-α-Syn has been verified by morphological and shape analysis through Bio-AFM. The gly-α-Syn (2 μg/μL) was injected stereotaxically in the substantia nigra (SN) of ICR mice (3-4 months) and compared with the normal α-Syn, d ribose, and Tris-HCl/artificial CSF groups. 56 days postsurgery (DPS), an immunohistochemical examination was conducted to investigate gly-α-Syn-induced α-Syn accumulation, neuroinflammation, and neurodegeneration. The glycation of α-Syn led to the expression of transglutaminase 2 (TGM2), an enzyme that cross-linked with AGEs and may have caused the accumulation of α-Syn. Significant RAGE activation was also observed in gly-α-Syn, which might have induced glial cell activation, resulting in oxidative stress and, ultimately, apoptosis of dopaminergic neurons. It is important to note that TGM2, phosphorylated α-Syn, RAGE expression, and glial cell activation were only found in the gly-α-Syn group and not in the other groups. This suggests that gly-α-Syn plays a major role in synucleinopathy, neuroinflammation, and neurodegeneration. Overall, the present study demonstrated glycation of α-Syn as one of the important age-associated post-translational modifications that are involved in the degeneration of dopaminergic neurons, at least in a subset of the diabetic patients susceptible to developing PD.

The nonribosomal cyclic peptides (NRcPs) rufomycins, produced by Streptomyces atratus, have attracted attention as antimycobacterials. Thus, there has been interest in engineering the corresponding biosynthetic pathway to produce novel derivatives. We have thus investigated the type I thioesterase (TE) of the NRPS RufT that catalyzes rufomycin peptide macrocyclization to understand its tolerance to changes in substrate peptide sequence. In contrast to our previously reported efficient cyclization chemistry, the recombinant RufT-TE domain and RufT-PCP-TE didomain, while tolerating some substrate structural changes, both produce high levels of hydrolyzed peptide. Closer analysis led to the identification of the natural product diketopiperazine rufomyazine in assays. The data indicate, with significant implications for rufomycin production, that RufT produces both cyclic and linear peptides. We propose that rufomyazine forms non-enzymatically from the linear peptide. In addition, it provides evidence for TE domains as gatekeepers in NRPS biosynthesis.

Cellular RNA labeling using light-up aptamers that bind to and activate fluorogenic molecules has gained interest in recent years as an alternative to protein-based RNA labeling approaches. Aptamer-based systems are genetically encodable and cover the entire visible spectrum. However, the inherently temporary nature of the noncovalent aptamer-fluorogen interaction limits the utility of these systems in that imaging does not withstand dye washout, and dye dissociation can compromise RNA tracking. We propose that these limitations can be averted through covalent RNA labeling. Here, we describe a photoaffinity approach in which the aptamer ligand is functionalized with a photoactivatable diazirine reactive group such that irradiation with UV light results in covalent attachment to the RNA of interest. In addition to the robustness of the covalent linkage, this approach benefits from the ability to achieve spatiotemporal control over RNA labeling. To demonstrate this approach, we incorporated a photoaffinity linker into malachite green and fused a single copy of the malachite green aptamer to a Cajal body-associated small nuclear RNA of interest as well as a cytoplasmic mRNA. We observed improved sensitivity for live cell imaging of the target RNA upon UV irradiation and demonstrated visualization of RNA dynamics over a time scale of minutes. The covalent attachment uniquely enables these time-resolved experiments, whereas in noncovalent approaches, the dye molecule can be transferred between different RNA molecules, compromising tracking. We envision future applications of this method for a wide range of investigations into the cellular localization, dynamics, and protein-binding properties of cellular RNAs.

Tau aggregation plays a crucial role in the development of Alzheimer's disease (AD). Developing specific techniques that can isolate pathogenic tau from brain tissue is important for understanding tauopathies and advancing targeted therapies. Here, we develop photoaffinity small molecular probes and a novel method for in situ tissue labeling and investigate their activity in interacting with tau in cells and AD patient brains. Based on the reported chemical structures of tau PET tracers, we designed and synthesized two tau-specific probes, namely, Tau-2 and Tau-4. After validation in cell, mouse model, and patient brain samples, our photolabeling results suggested that Tau-2 effectively labels soluble tau in cell and mouse models, while Tau-4 selectively binds high-molecular-weight tau aggregates in late-stage AD patient brain tissues. Proteomic analysis verified the specific isolation of pathogenic tau from AD brain samples. Collectively, these findings underscore the potential of our photoaffinity probes as powerful tools for investigating tau proteins and neurofibrillary tangles in neurodegenerative diseases.

Fungal secondary metabolites (SMs) are complex organic compounds comprising a variety of biological activities that are essential in medicine. These natural products can be found in various environments, with studies demonstrating the importance of studying marine-sourced fungi due to the increased potency of the compounds they produce. In this study, we sourced a Penicillium rubens YAP001 strain isolated from Exaiptasia diaphana and explored an avenue for the upregulation of its SMs by combining the one-strain-many-compounds (OSMAC) strategy with genetic manipulation of negative global regulator of secondary metabolism, mcrA. Here, we generated a mcrAΔ strain of marine P. rubens (YAP001), which led to the detection of sorbicillinoids, which is significant due to the prior discovery that these compounds illicit cytotoxic effects that have the potential as an anticancer agent. Specifically, we found that sorbicillin was not only upregulated but the mutant strain also produced the dimeric product, trichodimerol, which often exhibits stronger biological activities compared to sorbicillin. Furthermore, the reduced form of trichodimerol, dihydrotrichodimerol, was also detected in the mutant strain. This work suggests that genetic manipulation of global regulators in combination with the OSMAC method in filamentous fungi is a promising technique for upregulating pathways of interest for small-molecule drug discovery.

Fungal secondary metabolites (SMs) are complex organic compounds comprising a variety of biological activities that are essential in medicine. These natural products can be found in various environments, with studies demonstrating the importance of studying marine-sourced fungi due to the increased potency of the compounds they produce. In this study, we sourced a Penicillium rubens YAP001 strain isolated from Exaiptasia diaphana and explored an avenue for the upregulation of its SMs by combining the one-strain-many-compounds (OSMAC) strategy with genetic manipulation of negative global regulator of secondary metabolism, mcrA. Here, we generated a mcrAΔ strain of marine P. rubens (YAP001), which led to the detection of sorbicillinoids, which is significant due to the prior discovery that these compounds illicit cytotoxic effects that have the potential as an anticancer agent. Specifically, we found that sorbicillin was not only upregulated but the mutant strain also produced the dimeric product, trichodimerol, which often exhibits stronger biological activities compared to sorbicillin. Furthermore, the reduced form of trichodimerol, dihydrotrichodimerol, was also detected in the mutant strain. This work suggests that genetic manipulation of global regulators in combination with the OSMAC method in filamentous fungi is a promising technique for upregulating pathways of interest for small-molecule drug discovery.

Post-transcriptional modifications on RNA play an important role in biological processes, but we lack an understanding of the molecular mechanisms underlying the function of many modifications. Here we characterize the distribution and dynamic regulation of 5-formylcytidine (f⁵C), a modification primarily found on tRNAs, across different cell lines, mouse tissues, and in response to environmental stress. We identify perturbation in bulk f⁵C levels using nucleoside LC-MS and quantify individual modification stoichiometry at the wobble base of mt-tRNA-Met and tRNA-Leu-CAA using nucleotide resolution f⁵C sequencing technology. Our studies show that f⁵C modifications on tRNAs are dynamic, and responsive to fluctuations in cellular iron levels and O₂ concentration. Further, we show using a translation reporter assay that decoding of Leu UUA codons is impaired in cells lacking f⁵C, implicating f⁵C(m)34 on tRNA-Leu-CAA in wobble decoding. Together, our work illuminates dynamic epitranscriptomic mechanisms regulating protein translation in response to environment.

Glioblastoma multiforme (GBM) is a WHO grade 4 glioma and the most common malignant primary brain tumor. Addressing the clinical management of GBM presents an exceptionally daunting and intricate challenge, particularly in overcoming the blood–brain barrier (BBB) to deliver effective therapies to the brain. Nanotechnology-based drug delivery systems have exhibited considerable promise in tackling this aggressive brain cancer. However, the BBB remains a key challenge in achieving effective brain delivery of nanocarriers. Here, we have optimized a lipid nanoparticle (LNP) formulation (C2) and modified the LNP with Angiopep-2 peptide, which exhibits the most significant improvements in blood–brain barrier penetration and brain accumulation (about 2.23% injection dose). Using the Ang-2-coupled C2 LNP formulation, we researched the therapeutic effect of Polo-like Kinase 1(PLK1)-targeted siRNA delivery to treat a mouse model of GBM. The optimized LNP formulation was demonstrated to significantly inhibit mouse GBM growth and extend the median survival of mice (2.18-fold). This work demonstrates the efficacy of a brain-targeted siRNA delivery system in GBM treatment. As the understanding of the role of RNAs in GBM deepens and innovative delivery methods are continually developed and refined, RNA-based therapies could emerge as a crucial breakthrough in the advancement of brain tumor treatment.

Notch plays critical roles in developmental processes and disease pathogenesis, which have led to numerous efforts to modulate its function with small molecules and antibodies. Here we present a nanobody inhibitor of Notch signaling derived from a synthetic phage-display library targeting the Notch negative regulatory region (NRR). The nanobody inhibits Notch signaling in a luciferase reporter assay with an IC₅₀ of about 5 μM and in a Notch-dependent hematopoietic progenitor cell differentiation assay, despite a modest 19 μM affinity for the Notch NRR. We addressed the low affinity by fusion to a mutant varient of the β-pore-forming toxin aerolysin, resulting in a significantly improved IC₅₀ for Notch inhibition. The nanobody-aerolysin fusion inhibits proliferation of T-ALL cell lines with efficacy similar to that of other Notch pathway inhibitors. Overall, this study reports the development of a Notch inhibitory antibody and demonstrates a proof-of-concept for a generalizable strategy to increase the efficacy and potency of low-affinity antibody binders.

Post-transcriptional modifications on RNA play an important role in biological processes, but we lack an understanding of the molecular mechanisms underlying the function of many modifications. Here we characterize the distribution and dynamic regulation of 5-formylcytidine (f⁵C), a modification primarily found on tRNAs, across different cell lines, mouse tissues, and in response to environmental stress. We identify perturbation in bulk f⁵C levels using nucleoside LC-MS and quantify individual modification stoichiometry at the wobble base of mt-tRNA-Met and tRNA-Leu-CAA using nucleotide resolution f⁵C sequencing technology. Our studies show that f⁵C modifications on tRNAs are dynamic, and responsive to fluctuations in cellular iron levels and O₂ concentration. Further, we show using a translation reporter assay that decoding of Leu UUA codons is impaired in cells lacking f⁵C, implicating f⁵C(m)34 on tRNA-Leu-CAA in wobble decoding. Together, our work illuminates dynamic epitranscriptomic mechanisms regulating protein translation in response to environment.