<i>In Cellulo</i> Mapping of Subcellular Localized Bilirubin

Bilirubin (BR) is a <i>de novo</i> synthesized metabolite of human cells. However, subcellular localization of BR in the different organelles of human cells has been largely unknown. Here, utilizing UnaG as a genetically encoded fluorescent BR sensor, we report the existence of relatively BR-enriched and BR-depleted microspaces in various cellular organelles of live cells. Our studies indicate that (i) the cytoplasmic facing membrane of the endoplasmic reticulum (ER) and the nucleus are relatively BR-enriched spaces and (ii) mitochondrial intermembrane space and the ER lumen are relatively BR-depleted spaces. Thus, we demonstrate a relationship between such asymmetrical BR distribution in the ER membrane and the BR metabolic pathway. Furthermore, our results suggest plausible BR-transport and BR-regulating machineries in other cellular compartments, including the nucleus and mitochondria

Molecular Insights into Human Serum Albumin as a Receptor of Amyloid-β in the Extracellular Region

Regulation of amyloid-β (Aβ) aggregation by metal ions and proteins is essential for understanding the pathology of Alzheimer’s disease (AD). Human serum albumin (HSA), a regulator of metal and protein transportation, can modulate metal–Aβ interactions and Aβ aggregation in human fluid; however, the molecular mechanisms for such activities remain unclear. Herein, we report the molecular-level complexation between Zn­(II), Cu­(II), Aβ, and HSA, which is able to alter the aggregation and cytotoxicity of Aβ peptides and induce their cellular transportation. In addition, a single Aβ monomer-bound HSA is observed with the structural change of Aβ from a random coil to an α-helix. Small-angle X-ray scattering (SAXS) studies indicate that Aβ–HSA complexation causes no structural variation of HSA in solution. Conversely, ion mobility mass spectrometry (IM-MS) results present that Aβ prevents the shrinkage of the V-shaped groove of HSA in the gas phase. Consequently, for the first time, HSA is demonstrated to predominantly capture a single Aβ monomer at the groove using the phase transfer of a protein heterodimer from solution to the gas phase. Moreover, HSA sequesters Zn­(II) and Cu­(II) from Aβ while maintaining Aβ–HSA interaction. Therefore, HSA is capable of controlling metal-free and metal-bound Aβ aggregation and aiding the cellular transportation of Aβ via Aβ–HSA complexation. The overall results and observations regarding HSA, Aβ, and metal ions advance our knowledge of how protein–protein interactions associated with Aβ and metal ions could be linked to AD pathogenesis

Luminescent Properties of Ruthenium(II) Complexes with Sterically Expansive Ligands Bound to DNA Defects

A new family of ruthenium­(II) complexes with sterically expansive ligands for targeting DNA defects was prepared, and their luminescent responses to base pair mismatches and/or abasic sites were investigated. Design of the complexes sought to combine the mismatch specificity of sterically expansive metalloinsertors, such as [Rh­(bpy)₂(chrysi)]³⁺ (chrysi = chrysene-5,6-quinone diimine), and the light switch behavior of [Ru­(bpy)₂(dppz)]²⁺ (dppz = dipyrido­[3,2-<i>a</i>:2′,3′-<i>c</i>]­phenazine). In one approach, complexes bearing analogues of chrysi incorporating hydrogen-bonding functionality similar to dppz were synthesized. While the complexes show luminescence only at low temperatures (77 K), competition experiments with [Ru­(bpy)₂(dppz)]²⁺ at ambient temperatures reveal that the chrysi derivatives preferentially bind DNA mismatches. In another approach, various substituents were introduced onto the dppz ligand to increase its steric bulk for mismatch binding while maintaining planarity. Steady state luminescence and luminescence lifetime measurements reveal that these dppz derivative complexes behave as DNA “light switches” but that the selectivity in binding and luminescence with mismatched/abasic versus well-matched DNA is not high. In all cases, luminescence depends sensitively upon structural perturbations to the dppz ligand

Tailoring Hydrophobic Interactions between Probes and Amyloid‑β Peptides for Fluorescent Monitoring of Amyloid‑β Aggregation

Despite their unique advantages, the full potential of molecular probes for fluorescent monitoring of amyloid-β (Aβ) aggregates has not been fully exploited. This limited utility stems from the lack of knowledge about the hydrophobic interactions between the molecules of Aβ probes, as well as those between the probe and the Aβ aggregate. Herein, we report the first mechanistic study, which firmly establishes a structure–signaling relationship of fluorescent Aβ probes. We synthesized a series of five fluorescent Aβ probes based on an archetypal donor–acceptor–donor scaffold (denoted as <b>SN1</b>–<b>SN5</b>). The arylamino donor moieties were systematically varied to identify molecular factors that could influence the interactions between molecules of each probe and that could influence their fluorescence outcomes in conditions mimicking the biological milieu. Our probes displayed different responses to aggregates of Aβ, Aβ₄₀ and Aβ₄₂, two major isoforms found in Alzheimer’s disease: <b>SN2</b>, having pyrrolidine donors, showed noticeable ratiometric fluorescence responses (Δν = 797 cm^–1) to the Aβ₄₀ and Aβ₄₂ samples that contained oligomeric species, whereas <b>SN4</b>, having <i>N</i>-methylpiperazine donors, produced significant fluorescence turn-on signaling in response to Aβ aggregates, including oligomers, protofibrils, and fibrils (with turn-on ratios of 14 and 10 for Aβ₄₂ and Aβ₄₀, respectively). Mechanistic investigations were carried out by performing field-emission scanning electron microscopy, X-ray crystallography, UV–vis absorption spectroscopy, and steady-state and transient photoluminescence spectroscopy experiments. The studies revealed that the <b>SN</b> probes underwent preassembly prior to interacting with the Aβ species and that the preassembled structures depended profoundly on the subtle differences between the amino moieties of the different probes. Importantly, the studies demonstrated that the mode of fluorescence signaling (i.e., ratiometric response versus turn-on response) was primarily governed by stacking geometries within the probe preassemblies. Specifically, ratiometric fluorescence responses were observed for probes capable of forming J-assembly, whereas fluorescence turn-on responses were obtained for probes incapable of forming J-aggregates. This finding provides an important guideline to follow in future efforts at developing fluorescent probes for Aβ aggregation. We also conclude, on the basis of our study, that the rational design of such fluorescent probes should consider interactions between the probe molecules, as well as those between Aβ peptides and the probe molecule

Decoding the Parkinson’s Symphony: PARIS, Maestro of Transcriptional Regulation and Metal Coordination for Dopamine Release

Parkin interacting substrate (PARIS) is a pivotal transcriptional regulator in the brain that orchestrates the activity of various enzymes through its intricate interactions with biomolecules, including nucleic acids. Notably, the binding of PARIS to insulin response sequences (IRSs) triggers a cascade of events that results in the functional loss in the substantia nigra, which impairs dopamine release and, subsequently, exacerbates the relentless neurodegeneration. Here, we report the details of the interactions of PARIS with IRSs via classical zinc finger (ZF) domains in PARIS, namely, PARIS(ZF2–4). Our biophysical studies with purified PARIS(ZF2–4) elucidated the binding partner of PARIS, which generates specific interactions with the IRS1 (5′-TATTTTT, Kd = 38.9 ± 2.4 nM) that is positioned in the promoter region of peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α). Mutational and metal-substitution studies demonstrated that Zn(II)–PARIS(ZF2–4) could recognize its binding partner selectively. Overall, our work provides submolecular details regarding PARIS and shows that it is a transcriptional factor that regulates dopamine release. Thus, PARIS could be a crucial target for therapeutic applications

Regulatory Activities of Dopamine and Its Derivatives toward Metal-Free and Metal-Induced Amyloid‑β Aggregation, Oxidative Stress, and Inflammation in Alzheimer’s Disease

A catecholamine neurotransmitter, dopamine (<b>DA</b>), is suggested to be linked to the pathology of dementia; however, the involvement of <b>DA</b> and its structural analogues in the pathogenesis of Alzheimer’s disease (AD), the most common form of dementia, composed of multiple pathogenic factors has not been clear. Herein, we report that <b>DA</b> and its rationally designed structural derivatives (<b>1</b>–<b>6</b>) based on <b>DA</b>’s oxidative transformation are able to modulate multiple pathological elements found in AD [i.e., metal ions, metal-free amyloid-β (Aβ), metal-bound Aβ (metal–Aβ), and reactive oxygen species (ROS)], with demonstration of detailed molecular-level mechanisms. Our multidisciplinary studies validate that the protective effects of <b>DA</b> and its derivatives on Aβ aggregation and Aβ-mediated toxicity are induced by their oxidative transformation with concomitant ROS generation under aerobic conditions. In particular, <b>DA</b> and the derivatives (i.e., <b>3</b> and <b>4</b>) show their noticeable anti-amyloidogenic ability toward metal-free Aβ and/or metal–Aβ, verified to occur via their oxidative transformation that facilitates Aβ oxidation. Moreover, in primary pan-microglial marker (CD11b)-positive cells, the major producers of inflammatory mediators in the brain, <b>DA</b> and its derivatives significantly diminish inflammation and oxidative stress triggered by lipopolysaccharides and Aβ through the reduced induction of inflammatory mediators as well as upregulated expression of heme oxygenase-1, the enzyme responsible for production of antioxidants. Collectively, we illuminate how <b>DA</b> and its derivatives could prevent multiple pathological features found in AD. The overall studies could advance our understanding regarding distinct roles of neurotransmitters in AD and identify key interactions for alleviation of AD pathology

Development of Bifunctional Stilbene Derivatives for Targeting and Modulating Metal-Amyloid-β Species

Amyloid-β (Aβ) peptides and their metal-associated aggregated states have been implicated in the pathogenesis of Alzheimer’s disease (AD). Although the etiology of AD remains uncertain, understanding the role of metal-Aβ species could provide insights into the onset and development of the disease. To unravel this, bifunctional small molecules that can specifically target and modulate metal-Aβ species have been developed, which could serve as suitable chemical tools for investigating metal-Aβ-associated events in AD. Through a rational structure-based design principle involving the incorporation of a metal binding site into the structure of an Aβ interacting molecule, we devised stilbene derivatives (<b>L1-a</b> and <b>L1-b</b>) and demonstrated their reactivity toward metal-Aβ species. In particular, the dual functions of compounds with different structural features (e.g., with or without a dimethylamino group) were explored by UV–vis, X-ray crystallography, high-resolution 2D NMR, and docking studies. Enhanced bifunctionality of compounds provided greater effects on metal-induced Aβ aggregation and neurotoxicity in vitro and in living cells. Mechanistic investigations of the reaction of <b>L1-a</b> and <b>L1-b</b> with Zn²⁺-Aβ species by UV–vis and 2D NMR suggest that metal chelation with ligand and/or metal–ligand interaction with the Aβ peptide may be driving factors for the observed modulation of metal-Aβ aggregation pathways. Overall, the studies presented herein demonstrate the importance of a structure-interaction-reactivity relationship for designing small molecules to target metal-Aβ species allowing for the modulation of metal-induced Aβ reactivity and neurotoxicity

Tuning Structures and Properties for Developing Novel Chemical Tools toward Distinct Pathogenic Elements in Alzheimer’s Disease

Multiple pathogenic factors [e.g., amyloid-β (Aβ), metal ions, metal-bound Aβ (metal–Aβ), reactive oxygen species (ROS)] are found in the brain of patients with Alzheimer’s disease (AD). In order to elucidate the roles of pathological elements in AD, chemical tools able to regulate their activities would be valuable. Due to the complicated link among multiple pathological factors, however, it has been challenging to invent such chemical tools. Herein, we report novel small molecules as chemical tools toward modulation of single or multiple target(s), designed via a rational structure-property-directed strategy. The chemical properties (e.g., oxidation potentials) of our molecules and their coverage of reactivities toward the pathological targets were successfully differentiated through a minor structural variation [i.e., replacement of one nitrogen (N) or sulfur (S) donor atom in the framework]. Among our compounds (<b>1</b>–<b>3</b>), <b>1</b> with the lowest oxidation potential is able to noticeably modify the aggregation of both metal-free Aβ and metal–Aβ, as well as scavenge free radicals. Compound <b>2</b> with the moderate oxidation potential significantly alters the aggregation of Cu­(II)–Aβ₄₂. The hardly oxidizable compound, <b>3</b>, relative to <b>1</b> and <b>2</b>, indicates no noticeable interactions with all pathogenic factors, including metal-free Aβ, metal–Aβ, and free radicals. Overall, our studies demonstrate that the design of small molecules as chemical tools able to control distinct pathological components could be achieved via fine-tuning of structures and properties

Tuning Reactivity of Diphenylpropynone Derivatives with Metal-Associated Amyloid‑β Species via Structural Modifications

A diphenylpropynone derivative, <b>DPP2</b>, has been recently demonstrated to target metal-associated amyloid-β (metal–Aβ) species implicated in Alzheimer’s disease (AD). <b>DPP2</b> was shown to interact with metal–Aβ species and subsequently control Aβ aggregation (reactivity) in vitro; however, its cytotoxicity has limited further biological applications. In order to improve reactivity toward Aβ species and lower cytotoxicity, along with gaining an understanding of a structure-reactivity-cytotoxicity relationship, we designed, prepared, and characterized a series of small molecules (<b>C1</b>/<b>C2</b>, <b>P1</b>/<b>P2</b>, and <b>PA1</b>/<b>PA2</b>) as structurally modified <b>DPP2</b> analogues. A similar metal binding site to that of <b>DPP2</b> was contained in these compounds while their structures were varied to afford different interactions and reactivities with metal ions, Aβ species, and metal–Aβ species. Distinct reactivities of our chemical family toward in vitro Aβ aggregation in the absence and presence of metal ions were observed. Among our chemical series, the compound (<b>C2</b>) with a relatively rigid backbone and a dimethylamino group was observed to noticeably regulate both metal-free and metal-mediated Aβ aggregation to different extents. Using our compounds, cell viability was significantly improved, compared to that with <b>DPP2</b>. Lastly, modifications on the <b>DPP</b> framework maintained the structural properties for potential blood-brain barrier (BBB) permeability. Overall, our studies demonstrated that structural variations adjacent to the metal binding site of <b>DPP2</b> could govern different metal binding properties, interactions with Aβ and metal–Aβ species, reactivity toward metal-free and metal-induced Aβ aggregation, and cytotoxicity of the compounds, establishing a structure-reactivity-cytotoxicity relationship. This information could help gain insight into structural optimization for developing nontoxic chemical reagents toward targeting metal–Aβ species and modulating their reactivity in biological systems

Reactivity of Diphenylpropynone Derivatives Toward Metal-Associated Amyloid‑β Species

In Alzheimer’s disease (AD), metal-associated amyloid-β (metal–Aβ) species have been suggested to be involved in neurotoxicity; however, their role in disease development is still unclear. To elucidate this aspect, chemical reagents have been developed as valuable tools for targeting metal–Aβ species, modulating the interaction between the metal and Aβ, and subsequently altering metal–Aβ reactivity. Herein, we report the design, preparation, characterization, and reactivity of two diphenylpropynone derivatives (<b>DPP1</b> and <b>DPP2</b>) composed of structural moieties for metal chelation and Aβ interaction (bifunctionality). The interactions of these compounds with metal ions and Aβ species were confirmed by UV–vis, NMR, mass spectrometry, and docking studies. The effects of these bifunctional molecules on the control of in vitro metal-free and metal-induced Aβ aggregation were investigated and monitored by gel electrophoresis and transmission electron microscopy (TEM). Both <b>DPP1</b> and <b>DPP2</b> showed reactivity toward metal–Aβ species over metal-free Aβ species to different extents. In particular, <b>DPP2</b>, which contains a dimethylamino group, exhibited greater reactivity with metal–Aβ species than <b>DPP1</b>, suggesting a structure-reactivity relationship. Overall, our studies present a new bifunctional scaffold that could be utilized to develop chemical reagents for investigating metal–Aβ species in AD