Proteasomes are fundamental for protein homeostasis and genome integrity and essential in spermatogenesis and fertilization. However, their presence, composition and role within the sperm nucleus are a subject of debate. Here we use in situ cryo-electron tomography in human sperm cells to elucidate the molecular architecture of nuclear proteasomes, which cluster in DNA-free, nuclear cavities within the sperm nucleus. We show that the main population of proteasomes consists of 20S core particles, with a smaller fraction of 20S capped by PA200 activator. Using single-particle cryo-electron microscopy of purified native human sperm proteasomes, we elucidate the features of the essential testis-specific subunit α4s, reporting the presence of a unique splice variant. We resolve a native peptide in the catalytic β2 subunit, providing insight into the proteolysis mechanism and PA200-mediated enhancement of trypsin activity. We show nuclear enrichment of proteasomes during sperm-cell differentiation in human testis tissue, with 20S and PA200 clustering following meiosis, at the spermatid stage. Our findings shed light on the organization and compositional diversity of nuclear proteasomes in human sperm cells, as well as their catalytic function.
Mitochondrial proteostasis depends on precise N-terminal processing of imported precursor proteins. Defects in this maturation step are implicated in disease, yet the functional impact in humans remains unclear. Here we show that the intermediate cleaving peptidase ICP55, which removes a single amino acid, acts as a key stabilizer of multimeric mitochondrial protein complexes. Using proteomics and complexome profiling, we identify over 100 human ICP55 substrates and demonstrate that loss of ICP55 triggers widespread destabilization of protein assemblies, with a global shift toward smaller subcomplexes. Thus, we uncover a conserved, post-translational mechanism that safeguards mitochondrial proteostasis by regulating complex integrity through a single amino-acid cleavage, and we reveal N-terminal proteoform control as an unexpected layer of organellar homeostasis.
Newly imported mitochondrial proteins are trimmed to their mature form by processing peptidases; for a subset, ICP55 removes a single further amino acid from the N terminus. Kücükköse et al. now identify the substrates of human ICP55, using the HUNTER method, and show that this single cleavage is required for the stability of mitochondrial protein complexes.
G protein-coupled receptors (GPCRs) regulate human physiology and are major drug targets. Although cryo-electron microscopy has accelerated GPCR structural biology, inactive-state structures remain difficult because current fusion-based strategies often require extensive experimental screening to identify rigid constructs suitable for high-resolution reconstruction. Here we introduce a universal pipeline that integrates an in silico fusion construct screening program, NOAH (nonexperimental, artificial-intelligence-assisted, high-throughput construct screening for structural analysis), with a de novo designed fusion protein, ARK1 (artificially designed fiducial marker). NOAH enabled structure determination of vasopressin V2 receptor bound to the antagonist tolvaptan or partial agonist OPC51803 and bradykinin B2 receptor bound to the antagonist icatibant, revealing receptor activation and inhibition mechanisms. Coupling NOAH to ARK1 improved the V2 receptor-tolvaptan map and enabled high-resolution structures of lysophosphatidic acid receptor 2 bound to Ki16425 and free fatty acid receptor 2 bound to GLPG0974. NOAH-ARK1 minimizes trial-and-error construct optimization and provides a broadly applicable route for GPCR structural analysis and drug discovery.
N-methyl-D-aspartate receptors (NMDARs) mediate excitatory signaling essential for synaptic plasticity and memory. Unlike GluN2-containing NMDARs, GluN3-containing receptors are activated solely by glycine and exhibit profound desensitization and paradoxical potentiation by GluN1-selective antagonists, including CGP-78608 (CGP). Although GluN3A-containing NMDARs regulate synapse pruning and excitotoxicity, and are associated with schizophrenia, autism and stroke, their native stoichiometry and gating mechanism are poorly defined. Here, using single-molecule pull-down analysis, we show that native GluN3A-containing receptors are diheteromeric assemblies. Cryogenic-electron microscopy analysis of GluN1-GluN3A receptors in antagonist-bound, preactive, active and desensitized states, augmented by electrophysiology and pharmacology experiments, show how glycine activates the receptor solely via GluN3A-dependent conformational changes, opening the gate with two-fold symmetry, and induces a roughly four-fold symmetric desensitized state. CGP-bound GluN1 restricts GluN3A rotation, promoting glycine-induced activation by blocking desensitization. These findings illuminate how CGP potentiates GluN1-GluN3A receptor activity, place the receptor gating mechanism on a structural foundation and define the molecular basis for pharmacological modulation.
Eukaryotic sequence-specific transcription factors (TFs) must find their cognate DNA targets hidden in genomic chromatin amid an excess of nonspecific sequences and degenerate motifs. Although static TF interactions with nucleosomal targets have been elucidated, how TFs efficiently search for cognate sites within native gene-sized chromatin domains has been unclear. Here we used purified Saccharomyces cerevisiae HIS3 minichromosomes and single-molecule imaging to compare association and dissociation kinetics of transcription activator GCN4 on chromatin and naked genomic DNA. GCN4 displays widespread and stable off-target binding on bare DNA because of entrapment by degenerate sites and interactions with nonspecific DNA of increasing length, indicative of one-dimensional (1D) diffusion. Nucleosome organization on the minichromosome reduces promiscuous GCN4 residence times by obstructing TF association and restricting 1D target search within nucleosome-free regions. Furthermore, the intrinsically disordered GCN4 activation domain independently enhances targeting efficiency and specificity by accelerating association-dissociation kinetics in vitro and in living cells. Altogether, both nucleosome organization and activation domains independently suppress promiscuous GCN4 binding, which, if unchecked, may cause aberrant cryptic transcription known to occur upon chromatin disruptions.
Damage-associated molecular patterns (DAMPs) are endogenous danger signals. They can be preformed molecules released upon membrane rupture and stress-induced or newly generated factors arising during cell death. These signals link cellular demise to diverse host responses. Rather than passive by-products, DAMPs are actively mobilized through membrane-remodeling proteins, vesicular trafficking and metabolic regulation. Conformational changes, oligomerization and post-translational modifications shape their release and immunogenicity, as illustrated by redox-dependent DAMP states, pore-forming gasdermins and MLKL, and NINJ1-mediated membrane rupture. At the sensing interface, receptors such as TLR4, P2X7 and AGER, together with cytosolic STING1 pathways, translate DAMP recognition into downstream signaling through assembly-driven mechanisms. Cross-talk with metabolic pathways and membrane repair systems, including ESCRT-III and autophagy, further refines DAMP signaling dynamics. Here, we survey and contextualize recent literature to provide a structural and molecular framework for understanding how DAMPs encode immune outcomes and highlight opportunities for targeted therapeutic intervention.
Traditional models of biomolecular phase separation focus largely on equilibrium phase boundaries. But emerging biophysical and biochemical evidence indicates that metastable nanoclusters and non-equilibrium RNA dynamics are essential intermediate states that shape the formation, dynamics and functional regulation of cellular condensates.
The dominantly inherited Flemish mutation-an A692G substitution in the amyloid precursor protein, corresponding to an A21G change in amyloid-β (Aβ)-causes a rare, early-onset form of Alzheimer disease characterized by pronounced cerebral amyloid angiopathy and unusually large senile plaque cores. Here, we report cryo-electron microscopy structures of amyloid filaments extracted from the postmortem parietal lobes of two individuals representing the only two known Flemish pedigrees worldwide. Although tau paired helical filaments were present, the predominant filaments comprise Aβ40-A21G, assembled as two identical protofilaments (D1-V40) packed with two-start helical symmetry. Aβ40-A21G and wild-type Aβ42 filaments share a substructure preceding the substitution site (Y10-F19); however, loss of the methyl group at residue 21 gives rise to a distinct arrangement, termed the 'Flemish fold', which differs from all previously characterized Aβ folds and is defined by a unique hydrophobic interface. Using a cell-based assay, we find that this distinctive fold is associated with the vascular tropism characteristic of the Flemish variant. Together, our structural and cellular data define a familial Alzheimer-disease-associated amyloid fold and provide insight into the molecular basis of Flemish-type dementia and cerebral hemorrhage.
β-arrestins, pivotal regulators of G protein-coupled receptor (GPCR) signaling, assemble with hundreds of GPCRs. How this assembly rises to functionally distinct complexes in which β-arrestin engages the GPCR tail, core or both, remains a central question. Here employing single-molecule force spectroscopy and molecular dynamics simulations, we monitor assembly of β2-adrenergic receptor (β2AR)-β-arrestin2 (βarr2) tail, core and tail-core complexes in phospholipid membranes and dissect their mechanical and kinetic stabilities. We show that βarr2 engages the phosphorylated β2AR carboxy-terminus (C-tail) within milliseconds, much faster than the active receptor core. In addition, the phospholipid membrane contributes substantially to complex stability, with phosphatidylinositol 4,5-bisphosphate (PIP2) modulating stability and conformation. While PIP2 stabilizes the β2AR-βarr2 core, it precludes βarr2 from concomitantly binding the phosphorylated β2AR C-tail. βarr2 activation and PIP2 strengthen βarr2-membrane association through insertion of the C-edge and finger loop. These findings establish PIP2, alongside ligand binding and receptor phosphorylation, as a central determinant of β2AR-βarr2 complex assembly, offering mechanistic insight into the regulation of GPCR signaling.
TOM and TIM22 complexes facilitate the transport of carrier protein substrates into mitochondria. New work shows that, in human mitochondria, these two protein complexes form transient supercomplexes to facilitate protein biogenesis.
The ATR protein kinase preserves genomic integrity during DNA replication by controlling checkpoints needed for the orderly progression of S phase and for the responses to replication stress. ATR, with its obligate partner ATRIP, is activated by the TOPBP1 and ETAA1 proteins, which control different branches of ATR signaling. TOPBP1 is essential for induction of the S phase checkpoint in response to stalled replication forks, while ETAA1 is required for timely progression to mitosis from an unperturbed S phase. TOPBP1 and ETAA1 contain ATR-activating domains (AADs) of limited homology, but how they activate ATR has not yet been fully elucidated. Here we present the 3.0-Å cryo-EM structure of the human ATR-ATRIP complex bound to the TOPBP1 AAD, showing that TOPBP1 activates ATR by inducing a global conformational change that allosterically realigns active site residues in the kinase domain ~70 Å away. We also present the 3.3-Å structure of the ATR-ATRIP-ETAA1 AAD complex, which reveals a binding mode distinct from TOPBP1. Our data suggest that the distinct binding modes of TOPBP1 and ETAA1 contribute to the different cellular contexts and outcomes of ATR-ATRIP activation.
Metabolite carriers that control essential metabolite transport are imported into mitochondria through the TOM and TIM22 complexes. How TOM and TIM22 coordinate in human mitochondria has remained largely unknown. Here we show that human TOM and TIM22 assemble into a supercomplex that seamlessly couples carrier translocation across the outer and inner membranes, unlike in yeast where the two complexes appear to function separately. Cryo-electron microscopy structures of the human TOM-TIM22 supercomplex reveal unpaired carrier transmembrane segments traversing the TOM channel along a hydrophobic path and exiting through an unexpected lateral groove outside the channel. The membrane-bound small Tim subunits provide the substrate entry site for TIM22, while a membrane-exposed groove of TIM22 serves as the exit for carrier insertion into the inner membrane. These findings provide insights into the human carrier translocation pathway at molecular resolution and establish the TOM-TIM22 supercomplex as a central organizing unit of mitochondrial carrier import.
Ferroptosis is an iron-dependent form of regulated cell death driven by phospholipid peroxidation, and it has emerged as a potential therapeutic vulnerability of cancer. Here we identify the secretory phospholipase PLA2G2F (phospholipase A2 group IIF) as a ferroptosis suppressor in bladder cancer and elucidate its regulation and mechanism of action. PLA2G2F functions through an intracellular mechanism by localizing to the endoplasmic reticulum to inhibit ferroptosis. Our genetic and pharmacological analyses reveal that peroxisome proliferator-activated receptor γ (PPARG), a nuclear hormone receptor and transcription factor previously implicated in ferroptosis regulation, upregulates PLA2G2F and that PPARG-mediated ferroptosis resistance is largely dependent on PLA2G2F in bladder cancer. Further, lipidomic profiling suggests that PLA2G2F preferentially acts on ether-linked phospholipids containing polyunsaturated fatty acids, thereby reducing the pool of peroxidation-prone polyunsaturated fatty acid-containing phospholipids. Together, our findings establish PLA2G2F as an endoplasmic reticulum-resident ferroptosis suppressor regulated by PPARG and show that inhibiting PPARG signaling or PLA2G2F activity can sensitize bladder cancer cells to ferroptosis induction.
Human Argonaute2 (AGO2) binds siRNAs to target complementary mRNAs. Although guided–target complementary is required for slicing, there is a wide range of slicing efficacy. A study now shows the precise role guide–target geometry has in slicing efficacy.
The χ-conotoxins are venom-derived peptides that specifically target the noradrenaline transporter (also known as norepinephrine transporter, NET). Regulation of noradrenergic signaling by NET affects neurophysiological processes, including pain. Therefore, the χ-conotoxin MrIA and its synthetic analogs have been previously investigated for their analgesic activity. Here we describe the synthesis and pharmacological characterization of χ-AoIA, a peptide that selectively inhibits NET with a higher potency compared to MrIA in in vitro radiotracer flux assays. Furthermore, we resolved the structure of the human NET:χ-AoIA complex by cryogenic electron microscopy, which revealed an atypical binding mode consisting of both the central binding site and the outer vestibule of the transporter. Lastly, χ-AoIA displays antinociceptive efficacy in a model of inflammatory pain after subcutaneous administration in mice. Our results demonstrate the efficacy of χ-AoIA as a highly selective ligand of NET and provide a mechanistic basis for its potential development as a nonopioid analgesic.
