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Ferroptosis is a type of cell death caused by radical-driven lipid peroxidation, leading to membrane damage and rupture. Here we show that enzymatically produced sulfane sulfur (S 0 ) species, specifically hydropersulfides, scavenge endogenously generated free radicals and, thereby, suppress lipid peroxidation and ferroptosis. By providing sulfur for S 0 biosynthesis, cysteine can support ferroptosis resistance independently of the canonical GPX4 pathway. Our results further suggest that hydropersulfides terminate radical chain reactions through the formation and self-recombination of perthiyl radicals. The autocatalytic regeneration of hydropersulfides may explain why low micromolar concentrations of persulfides suffice to produce potent cytoprotective effects on a background of millimolar concentrations of glutathione. We propose that increased S 0 biosynthesis is an adaptive cellular response to radical-driven lipid peroxidation, potentially representing a primordial radical protection system. Enzymatically generated sulfane sulfur species called hydropersulfides terminate free radical chain reactions to prevent oxidative membrane damage and ferroptosis induction.

A fundamental feature of cellular plasma membranes (PMs) is an asymmetric lipid distribution between the bilayer leaflets. However, neither the detailed, comprehensive compositions of individual PM leaflets nor how these contribute to structural membrane asymmetries have been defined. We report the distinct lipidomes and biophysical properties of both monolayers in living mammalian PMs. Phospholipid unsaturation is dramatically asymmetric, with the cytoplasmic leaflet being approximately twofold more unsaturated than the exoplasmic leaflet. Atomistic simulations and spectroscopy of leaflet-selective fluorescent probes reveal that the outer PM leaflet is more packed and less diffusive than the inner leaflet, with this biophysical asymmetry maintained in the endocytic system. The structural asymmetry of the PM is reflected in the asymmetric structures of protein transmembrane domains. These structural asymmetries are conserved throughout Eukaryota, suggesting fundamental cellular design principles. Lipidomics across the bilayer membrane plus biophysical and fluorescence approaches find asymmetry in phospholipid unsaturation and localization of protein transmembrane domains based on their ability to pack within the different membrane leaflets.

The formation of biomolecular condensates mediated by a coupling of associative and segregative phase transitions plays a critical role in controlling diverse cellular functions in nature. This has inspired the use of phase transitions to design synthetic systems. While design rules of phase transitions have been established for many synthetic intrinsically disordered proteins, most efforts have focused on investigating their phase behaviors in a test tube. Here, we present a rational engineering approach to program the formation and physical properties of synthetic condensates to achieve intended cellular functions. We demonstrate this approach through targeted plasmid sequestration and transcription regulation in bacteria and modulation of a protein circuit in mammalian cells. Our approach lays the foundation for engineering designer condensates for synthetic biology applications. Dai et al. present a streamlined approach for the design and engineering of synthetic biomolecular condensates for controlling different cellular processes, such as gene flow, transcriptional regulation and modulation of protein circuits.

Key Points Fragment-based drug discovery (FBDD) is playing an increasingly important part in delivering candidates to the clinic. Compared with screens of lead- or drug-sized molecules, fragments allow for a more thorough search of chemical space and can lead to superior molecules. Fragments can provide fundamental insights into molecular recognition between proteins and ligands. Rigorous use of multiple biophysical techniques is essential to identify and validate fragments. Early and creative medicinal chemistry is needed to transform a low-affinity fragment into a lead molecule. FBDD can be integrated with other lead discovery methods to tackle difficult problems. Fragment-based methods have made substantial contributions to drug discovery in the past 20 years, particularly for challenging targets. Erlanson and colleagues discuss progress in the field, key aspects such as the design of fragment libraries and the choice of screening technique, and how current challenges in fragment-based drug discovery might be overcome. After 20 years of sometimes quiet growth, fragment-based drug discovery (FBDD) has become mainstream. More than 30 drug candidates derived from fragments have entered the clinic, with two approved and several more in advanced trials. FBDD has been widely applied in both academia and industry, as evidenced by the large number of papers from universities, non-profit research institutions, biotechnology companies and pharmaceutical companies. Moreover, FBDD draws on a diverse range of disciplines, from biochemistry and biophysics to computational and medicinal chemistry. As the promise of FBDD strategies becomes increasingly realized, now is an opportune time to draw lessons and point the way to the future. This Review briefly discusses how to design fragment libraries, how to select screening techniques and how to make the most of information gleaned from them. It also shows how concepts from FBDD have permeated and enhanced drug discovery efforts.

Study of the interactions established between the viral glycoproteins and their host receptors is of critical importance for a better understanding of virus entry into cells. The novel coronavirus SARS-CoV-2 entry into host cells is mediated by its spike glycoprotein (S-glycoprotein), and the angiotensin-converting enzyme 2 (ACE2) has been identified as a cellular receptor. Here, we use atomic force microscopy to investigate the mechanisms by which the S-glycoprotein binds to the ACE2 receptor. We demonstrate, both on model surfaces and on living cells, that the receptor binding domain (RBD) serves as the binding interface within the S-glycoprotein with the ACE2 receptor and extract the kinetic and thermodynamic properties of this binding pocket. Altogether, these results provide a picture of the established interaction on living cells. Finally, we test several binding inhibitor peptides targeting the virus early attachment stages, offering new perspectives in the treatment of the SARS-CoV-2 infection. SARS-CoV-2 spike protein binds host ACE2 for virus entry. Here, the authors determine kinetic and thermodynamic properties of this interaction using atomic force microscopy, develop peptides that inhibit binding and suggest existence of additional attachment factors.

Diverse mechanisms have been described for selective enrichment of biomolecules in membrane-bound organelles, but less is known about mechanisms by which molecules are selectively incorporated into biomolecular assemblies such as condensates that lack surrounding membranes. The chemical environments within condensates may differ from those outside these bodies, and if these differed among various types of condensate, then the different solvation environments would provide a mechanism for selective distribution among these intracellular bodies. Here we use small molecule probes to show that different condensates have distinct chemical solvating properties and that selective partitioning of probes in condensates can be predicted with deep learning approaches. Our results demonstrate that different condensates harbor distinct chemical environments that influence the distribution of molecules, show that clues to condensate chemical grammar can be ascertained by machine learning and suggest approaches to facilitate development of small molecule therapeutics with optimal subcellular distribution and therapeutic benefit. Condensates have been proposed to create a distinct chemical solvating environment. In vitro condensate screens suggest that condensate chemical environments influence the intracellular distribution of small molecules.

Biomolecular condensate formation has been implicated in a host of biological processes and has found relevance in biology and disease. Understanding the physical principles and underlying characteristics of how these macromolecular assemblies form and are regulated has become a central focus of the field. In this Review, we introduce features of phase-separating biomolecules from a general physical viewpoint and highlight how molecular features, including affinity, valence and a competition between inter- and intramolecular contacts, affect phase separation. We then discuss sequence properties of proteins that serve to mediate intermolecular interactions. Finally, we review how the intracellular environment can affect structural and sequence determinants of proteins and modulate physical parameters of their phase transitions. The works reviewed highlight that a complex interplay exists between structure, sequence and environmental determinants in the formation of biomolecular condensates.This Review introduces molecular features of the phase-separating biomolecules and how they affect phase-separation behavior in a complex intracellular environment, highlighting a complex interplay between structure, sequence and environment in the phase-separation process.

Advances in sample preparation and computation analysis make FTIR of biological materials a rapidly expanding research area. Researchers from a number of universities have collaborated to provide procedures for FTIR analysis of biological samples. IR spectroscopy is an excellent method for biological analyses. It enables the nonperturbative, label-free extraction of biochemical information and images toward diagnosis and the assessment of cell functionality. Although not strictly microscopy in the conventional sense, it allows the construction of images of tissue or cell architecture by the passing of spectral data through a variety of computational algorithms. Because such images are constructed from fingerprint spectra, the notion is that they can be an objective reflection of the underlying health status of the analyzed sample. One of the major difficulties in the field has been determining a consensus on spectral pre-processing and data analysis. This manuscript brings together as coauthors some of the leaders in this field to allow the standardization of methods and procedures for adapting a multistage approach to a methodology that can be applied to a variety of cell biological questions or used within a clinical setting for disease screening or diagnosis. We describe a protocol for collecting IR spectra and images from biological samples (e.g., fixed cytology and tissue sections, live cells or biofluids) that assesses the instrumental options available, appropriate sample preparation, different sampling modes as well as important advances in spectral data acquisition. After acquisition, data processing consists of a sequence of steps including quality control, spectral pre-processing, feature extraction and classification of the supervised or unsupervised type. A typical experiment can be completed and analyzed within hours. Example results are presented on the use of IR spectra combined with multivariate data processing.

Alzheimer’s disease is a neurodegenerative disorder associated with the aberrant aggregation of the amyloid-β peptide. Although increasing evidence implicates cholesterol in the pathogenesis of Alzheimer’s disease, the detailed mechanistic link between this lipid molecule and the disease process remains to be fully established. To address this problem, we adopt a kinetics-based strategy that reveals a specific catalytic role of cholesterol in the aggregation of Aβ42 (the 42-residue form of the amyloid-β peptide). More specifically, we demonstrate that lipid membranes containing cholesterol promote Aβ42 aggregation by enhancing its primary nucleation rate by up to 20-fold through a heterogeneous nucleation pathway. We further show that this process occurs as a result of cooperativity in the interaction of multiple cholesterol molecules with Aβ42. These results identify a specific microscopic pathway by which cholesterol dramatically enhances the onset of Aβ42 aggregation, thereby helping rationalize the link between Alzheimer’s disease and the impairment of cholesterol homeostasis.

Microsatellite repeat expansions within genes contribute to a number of neurological diseases 1 , 2 . The accumulation of toxic proteins and RNA molecules with repetitive sequences, and/or sequestration of RNA-binding proteins by RNA molecules containing expanded repeats are thought to be important contributors to disease aetiology 3 – 9 . Here we reveal that the adenosine in CAG repeat RNA can be methylated to N 1 -methyladenosine (m 1 A) by TRMT61A, and that m 1 A can be demethylated by ALKBH3. We also observed that the m 1 A/adenosine ratio in CAG repeat RNA increases with repeat length, which is attributed to diminished expression of ALKBH3 elicited by the repeat RNA. Additionally, TDP-43 binds directly and strongly with m 1 A in RNA, which stimulates the cytoplasmic mis-localization and formation of gel-like aggregates of TDP-43, resembling the observations made for the protein in neurological diseases. Moreover, m 1 A in CAG repeat RNA contributes to CAG repeat expansion-induced neurodegeneration in Caenorhabditis elegans and Drosophila . In sum, our study offers a new paradigm of the mechanism through which nucleotide repeat expansion contributes to neurological diseases and reveals a novel pathological function of m 1 A in RNA. These findings may provide an important mechanistic basis for therapeutic intervention in neurodegenerative diseases emanating from CAG repeat expansion. TDP-43 binds to N 1 -methyladenosine on CAG repeat RNA, resulting in the formation of gel-like TDP-43 aggregates in the cytoplasm that resemble those observed in neurological disease pathology.