Interdisciplinary Research Center of Biology and Chemistry

ISG15 (ISG15 ubiquitin-like modifier), a ubiquitin-like protein, is one of the major type I IFN (interferon) effector systems. ISG15 can be conjugated to target proteins (ISGylation) via the stepwise action of E1, E2, and E3 enzymes. Conjugated ISG15 can be removed (deISGylated) from target proteins by USP18 (ubiquitin-specific peptidase 18). Here we investigated the role of deISGylation by USP18 in regulating autophagy and EGFR degradation in cells treated with type I IFNs. We show that type I IFN induced expression of ISG15 leads to ISGylation of BECN1 at Lys117, as well as Lys263, Lys265, and Lys266 which competes with Lys63 ubiquitination of BECN1. We demonstrate that ISGylation of BECN1 at Lys117, as well as Lys263, Lys265, and Lys266 serve an important role in negative regulation of intracellular processes including autophagy and EGFR degradation that are critically dependent upon the activity of class III PtdIns 3-kinase. Our studies provide fundamental new mechanistic insights into the innate immunity response implemented by type I IFNs.

The autophagy receptor CALCOCO2/NDP52 functions as a bridging adaptor and plays an essential role in the selective autophagic degradation of invading pathogens by specifically recognizing ubiquitin-coated intracellular pathogens and subsequently targeting them to the autophagic machinery; thereby it is required for innate immune defense against a range of infectious pathogens in mammals. However, the mechanistic basis underlying CALCOCO2-mediated specific recognition of ubiqutinated pathogens is still unknown. Here, using biochemical and structural analyses, we demonstrated that the cargo-binding region of CALCOCO2 contains a dynamic unconventional zinc finger as well as a C2H2-type zinc-finger, and only the C2H2-type zinc finger specifically recognizes mono-ubiquitin or poly-ubiquitin chains. In addition to elucidating the specific ubiquitin recognition mechanism of CALCOCO2, the structure of the CALCOCO2 C2H2-type zinc finger in complex with mono-ubiquitin also uncovers a unique zinc finger-binding mode for ubiquitin. Our findings provide mechanistic insight into how CALCOCO2 targets ubiquitin-decorated pathogens for autophagic degradations.

FYCO1 (FYVE and coiled-coil domain containing 1) functions as an autophagy adaptor in directly linking autophagosomes with the microtubule-based kinesin motor, and plays an essential role in the microtubule plus end-directed transport of autophagic vesicles. The specific association of FYCO1 with autophagosomes is mediated by its interaction with Atg8-family proteins decorated on the outer surface of autophagosome. However, the mechanistic basis governing the interaction between FYCO1 and Atg8-family proteins is largely unknown. Here, using biochemical and structural analyses, we demonstrated that FYCO1 contains a unique LC3-interacting region (LIR), which discriminately binds to mammalian Atg8 orthologs and preferentially binds to the MAP1LC3A and MAP1LC3B. In addition to uncovering the detailed molecular mechanism underlying the FYCO1 LIR and MAP1LC3A interaction, the determined FYCO1-LIR-MAP1LC3A complex structure also reveals a unique LIR binding mode for Atg8-family proteins, and demonstrates, first, the functional relevance of adjacent sequences C-terminal to the LIR core motif for binding to Atg8-family proteins. Taken together, our findings not only provide new mechanistic insight into FYCO1-mediated transport of autophagosomes, but also expand our understanding of the interaction modes between LIR motifs and Atg8-family proteins in general.

In eukaryotes, aberrant expression of transposable elements (TEs) is detrimental to the host genome. Piwi-interacting RNAs (piRNAs) of ∼23 to 30 nucleotides bound to PIWI clade Argonaute proteins silence transposons in a manner that is strictly dependent on their sequence complementarity. Hence, a key goal in understanding piRNA pathways is to determine mechanisms that modulate piRNA sequences. Here, we identify a protein-protein interaction between the 3'-to-5' exoribonuclease Nibbler (Nbr) and Piwi that links Nbr activity with piRNA pathways. We show that there is a delicate balance in the interplay between Nbr and Hen1, a methyltransferase involved in 2'-O-methylation at the 3' terminal nucleotides of piRNAs, thus connecting two genes with opposing activities in the biogenesis of piRNA 3' ends. With age, piRNAs become shorter and fewer in number, which is coupled with the derepression of select TEs. We demonstrate that activities of Nbr and Hen1 inherently contribute to TE silencing and age-dependent profiles of piRNAs. We propose that antagonistic roles of Nbr and Hen1 define a mechanism to modulate piRNA 3' ends.

The amyloid fibrils of α-synuclein (α-syn) are crucial in the pathology of Parkinson's disease (PD), with the intrinsically disordered region (IDR) of its C-terminal playing a key role in interacting with receptors like LAG3 and RAGE, facilitating pathological neuronal spread and inflammation. In this study, we identified Givinostat (GS) as an effective inhibitor that disrupts the interaction of α-syn fibrils with receptors such as LAG3 and RAGE through high-throughput screening. By exploring the structure-activity relationship and optimizing GS, we developed several lead compounds, including GSD-16-24. Utilizing solution-state and solid-state NMR, along with cryo-EM techniques, we demonstrated that GSD-16-24 binds directly to the C-terminal IDR of α-syn monomer and fibril, preventing the fibril from binding to the receptors. Furthermore, GSD-16-24 significantly inhibits the association of α-syn fibrils with membrane receptors, thereby reducing neuronal propagation and pro-inflammatory effects of α-syn fibrils. Our findings introduce a novel approach to mitigate the pathological effects of α-syn fibrils by targeting their IDR with small molecules, offering potential leads for the development of clinical drugs to treat PD.

An efficient protocol was developed to access 3-fluoro-2-hydroxy-2-substituted benzo[b]furans with Selectfluor™ as the fluorinating reagent in MeCN and water. By utilizing SOCl2/Py as the dehydrating agent, the compounds above were readily converted to 3-fluorinated, 2-substituted benzo[b]furans in high yields.

chitinase, and/or mussel foot proteins (Mfps). Incorporation of fusion domains did not disrupt the typical β-sheet structures, but indeed affected assembly rate, morphology, and stiffness of resultant fibrils. Consequently, the CsgA-fusion fibrils, particularly those containing three domains, were much shorter than the CsgA-only fibrils. Furthermore, the stiffness of the resultant fibrils was heavily affected by the structural feature of fusion domains, with β-sheet-containing domains tending to increase the Young's modulus while random coil domains decreasing the Young's modulus. In addition, fibrils containing CBD domains showed higher chitin-binding activity compared to their CBD-free counterparts. The CBD-CsgA-Mfp3 construct exhibited significantly lower binding activity than Mfp5-CsgA-CBD due to inappropriate folding of the CBD domain in the former construct, in agreement with results based upon molecular dynamics modeling. Our study provides new insights into the assembly and functional properties of designer amyloid proteins with increasing complex domain structures and lays the foundation for the future design of functional amyloid-based structures and molecular materials.

A copper-catalyzed C–N cross-coupling reaction using commercially available 1<italic>H</italic>-indazoles with hypervalent iodine reagents to afford biologically active 2-substituted-2<italic>H</italic>-indazole is described.

anticancer activity comparable to that of the mouse PD-L1 antibody in a colon carcinoma (CT26) model. Cyclic peptide antagonists of this sort may provide novel drug candidates for cancer immunotherapy.

Abstract Cristae membrane state plays a central role in regulating mitochondrial function and cellular metabolism. The protein Optic atrophy 1 (Opa1) is an important crista remodeler that exists as two forms in the mitochondrion, a membrane-anchored long form (l-Opa1) and a processed short form (s-Opa1). The mechanisms for how Opa1 influences cristae shape have remained unclear due to lack of native three-dimensional views of cristae. We perform in situ cryo-electron tomography of cryo-focused ion beam milled mouse embryonic fibroblasts with defined Opa1 states to understand how each form of Opa1 influences cristae architecture. In our tomograms, we observe a variety of cristae shapes with distinct trends dependent on s-Opa1:l-Opa1 balance. Increased l-Opa1 levels promote cristae stacking and elongated mitochondria while increased s-Opa1 levels correlated with irregular cristae packing and round mitochondria shape. Functional assays indicate a role for l-Opa1 in wild-type apoptotic and calcium handling responses, and compromised respiratory function under Opa1 imbalance. In summary, we provide three-dimensional visualization of cristae architecture to reveal relationships between mitochondrial ultrastructure and cellular function dependent on Opa1-mediated membrane remodeling. Highlights In situ ultrastructural characterization of mitochondrial cristae with different forms of Opa1. Mitochondria with predominantly l-Opa1 show crista stacking, longer cristae, reduced globular cristae and an absence of tubular cristae. Mitochondria with mostly s-Opa1 showed irregular cristae packing with wider cristae junctions and narrower cristae. l-Opa1 expressing cells with WT-like cristae junction properties, show wild-type apoptotic response and calcium handling. Imbalance in Opa1 processing show compromised respiratory function and an increase in amorphous cristae.

Recently identified palmitoylation of PD-L1 is essential for immune regulation. To elucidate the underlying molecular mechanism, we performed giant plasma membrane vesicle (GPMV) experiments, μs-scale all-atom molecular dynamics (MD) simulations, fluorescence resonance energy transfer (FRET) experiments, and immune killing experiments. GPMV experiments indicated that PD-L1 palmitoylation enhanced its lipid raft affinity. MD simulations revealed dramatically different membrane orientation states of PD-L1 in liquid-ordered (Lo, lipid raft) compared to liquid-disordered (Ld, nonraft) membrane environments, which was validated by FRET experiments. The Ld region promoted the “lie-down” orientation of PD-L1, which could inhibit its association with the PD-1 protein on immune cells and thus promote the immune killing of cancer cells. This hypothesis was supported by immune killing experiments using γδT cells as effector cells and NCI-H1299 lung cancer cells as target cells. In short, our study demonstrates that the palmitoylation affects PD-L1’s membrane localization and then membrane orientation, which thus regulates its binding with T cell PD-1 and the immune regulation. These observations may guide therapeutic strategies by explicating the regulation of immune checkpoint proteins by post-translational modifications and membrane environments.

All-solution-processed, high-performance red phosphorescent OLED developed from hydrogen-bonded supramolecular material.

An asymmetric vinylogous Michael reaction of 2-furanone dimers with α,β-unsaturated nitroolefins utilizing bifunctional thiourea as a catalyst has been developed, which provides an easy access to a wide range of chiral γ,γ-disubstituted butenolides.

Amyloid fibrils, defined by their cross-β architecture, are central to both disease and function, yet the molecular principles governing their formation remain incompletely understood. Ninjurin-1 (NINJ1), a membrane protein essential for plasma membrane rupture (PMR) during cell death, contains an N-terminal amphipathic α-helix. Here, we investigate a key peptide fragment of this region (residues 40–69, HE30) and uncover its membrane-disruptive activity, self-assembly, and structural transitions. Monomeric HE30 reorganizes lipids to induce membrane thinning while undergoing an environmentally responsive α-helix–to−β-sheet transition that drives amyloid fibril formation. Fibrils formed at physiological temperatures are predominantly nontwisted, but elevated temperatures induce left-handed twisted structures with variable pitches and lengths, and even result in high-order superhelical bundles. We further resolved the twisted fibril structures of HE30 by cryo-EM, revealing two distinct fibril polymorphs stabilized by both hydrophobic and electrostatic interactions. Consistently, salts inhibit HE30 fibrillation, emphasizing the role of electrostatic interactions in stabilizing fibrils. Moreover, acidic conditions (∼pH 4.4) promote fibril formation, whereas alkaline conditions lead to disassembly into α-helical monomers in a reversible manner. In situ AFM tracking reveals the asymmetric growth of fibrils, where one end elongates faster and the opposite end exhibits slower growth or complete inhibition. Functionally, HE30 fibrils are nontoxic and act as scaffolds for the temperature-controlled assembly of gold nanoparticle (AuNPs) superstructures. These findings not only advance our understanding of NINJ1-induced PMR but also provide a detailed structural basis for HE30 fibril formation via α-helix to β-sheet transitions and underscore their potential as building blocks for fibril-based biomaterials.

ABSTRACT Secreted proteins mediate intercellular communication throughout the lives of multicellular organisms. However, due to the lack of new technology for secreted protein capturing, the progress of “secretomics’’ lags behind. Here, we report a two-step secretome enrichment method (tsSEM) combining unnatural amino acid labeling and click chemistry-based biorthogonal reaction, which enables in vitro secretome profiling in the presence of serum. Using this novel method, we systematically investigated the secretome of human iPSCs-derived astrocytes (iAst) in different disease models and identified a panel of astrocyte-secreted proteins that are responsible for its non-cell autonomous toxicity under disease conditions. Furthermore, we validated two astrocytes-derived novel neurotrophic proteins, FAM3C and KITLG, which we identified from disease models, and found that they could boost neurite outgrowth, protect neurons, and promote neural progenitor proliferation. Our study highlights the utility of secretome profiling of iAst and demonstrates its applications in disease study and target identification and validation in drug development.

Amyotrophic Lateral Sclerosis (ALS) is a lethal neurodegenerative disease that damages motor neurons in the central nervous system, causing progressive muscle weakness that ultimately leads to death. However, its underlying mechanisms still need to be fully understood, particularly the heterogeneity and similarity between various gene mutants during disease progression. In this study, we conducted temporal RNA-seq profiling in human induced pluripotent stem cells (hiPSCs) and iPSC-derived motor neurons (iMNs) carrying the C9orf72, FUS, TARDBP, and SOD1 mutations from both ALS patients and healthy individuals. We discovered dysregulated gene expression and alternative splicing (AS) throughout iMN development and maturation, and ALS iMNs display enrichment of cytoskeletal defects and synaptic alterations from premature stage to mature iMNs. Our findings indicate that synaptic gene dysfunction is the common molecular hallmark of fALS, which might result in neuronal susceptibility and progressive motor neuron degeneration. Analysis of upstream splicing factors revealed that differentially expressed RNA-binding proteins (RBPs) in ALS iMNs may cause abnormal AS events, suggesting the importance of studying RBP defects in ALS research. Overall, our research provides a comprehensive and valuable resource for gaining insights into the shared mechanisms of ALS pathogenesis during motor neuron development and maturation in iMN models.

This book presents Zero Theory, a self-contained scalar phase-field framework that seeks to derive the fundamental quantities of physics not as primitive assumptions, but as emergent consequences of phase structure, cyclic behavior, and topological constraints. The work begins with a foundational question: can physics be reconstructed from an entirely pre-geometric starting point, without assuming spacetime, metric structure, or predefined fundamental constants? Within this framework, the only primary entity is a phase field, denoted by φ(p), defined over an abstract label space in which distance, time, and classical geometry have not yet emerged. From this perspective, quantities such as length, time, Planck’s constant, electric charge, mass, and even field equations may arise as emergent layers generated through phase-cycle counting, 2π closure conditions, and topological organization. The theory introduces a single fundamental scale, ℓ₀, interpreted primarily as a translation scale between emergent regimes rather than as a freely adjustable parameter. This published version presents the central theoretical construction of the framework together with its mathematical formulation, conceptual interpretation, and discussion of the mechanisms through which effective physical quantities may emerge.

Time is one of the most fundamental yet conceptually subtle quantities in physics. While it appears as a parameter in many equations, its physical meaning depends on operational definitions involving clocks and spacetime geometry. This article presents a comprehensive conceptual and mathematical discussion of the nature of time in modern physics. We review the transition from Newtonian absolute time to relativistic spacetime, introduce proper time as the physically measured quantity along worldlines, discuss time dilation, analyze the thermodynamic arrow of time, and address common misconceptions. The goal is to clarify what time represents in physical theory and how it is measured in practice.

We present a concise guide to the eight-layer donut model, a parameter-free effective description exclusively for the electron. The model derives the electron rest mass from the closed-path quantization principle kL = 2πn, combined with a discrete ladder of length and time units anchored at the Planck scale. An eight-layer mirror-symmetric structure with capacities Ci proportional to powers of Ξ = 2/α yields a total wave count Stotal = 2(Ξ8 + 2Ξ4 + 2Ξ2 + 2Ξ + 1) ≈ 6.36718 × 1019. This exceeds half the Compton frequency of the electron by about 3%, corresponding to an inertial margin of 7.8 keV. The model predicts a narrow resonance in electron-photon scattering around 7.8 keV and a possible anomaly in gravitational redshift experiments. This paper serves as a roadmap to the accompanying block package (45 self-contained blocks), explaining the logical flow and guiding the reader through the core ideas, numerical results, and experimental consequences. Important clarification: This model is developed strictly for the electron. Any discussion of other particles in the accompanying blocks is purely speculative and intended only to suggest possible future directions; such extensions are not part of the core model and should be treated as informal remarks rather than predictions.

This work examines how environmental gravitational potentials may introduce systematic bias in cosmological redshift measurements. Within the framework of general relativity, clocks run slower in deeper gravitational potentials due to the lapse function. When comparing signals from distant emitters to local observers, this effect contributes a gravitational redshift component that is not accounted for in standard FLRW interpretations. We decompose the observed redshift into cosmological, peculiar, and environmental gravitational components, and estimate the magnitude of the environmental term using typical cluster-scale potentials ($\Phi/c^2 \sim 10^{-5}$). A simple statistical model linking environmental redshift to local galaxy density ($z_{env} = \alpha \rho_g$) is proposed, leading to testable predictions: correlation between supernova residuals and galaxy density, environmental dependence of $H_0$, and consistency between galaxy rotation curve potentials and large-scale structure. The framework introduces no new physics or modifications to Einstein's equations; it merely examines the operational consequences of comparing clocks in different gravitational environments. This perspective complements existing work on inhomogeneous cosmologies and backreaction, offering a distinct observational pathway to investigate environmental effects in precision cosmology.