Background Transthyretin amyloidosis (ATTR) is a degenerative disease affecting the heart and other organs. Transthyretin (TTR) aggregation is a driver of ATTR pathology, but the mechanism is poorly understood. We used proteomics and tissue clearing technology on wild‐type (WT) human cardiac (WT/WT) and V122I human cardiac (V122I/WT) tissue to better understand TTR cardiomyopathy. Methods Flash‐frozen cardiac tissue slices from human subjects with end‐stage WT‐TTR cardiomyopathy, end‐stage V122I TTR cardiomyopathy, and an age‐matched control were used. Fibrils and tissue proteomes were extracted and assessed by bottom‐up proteomics. Tissue clearing was performed using a lauryl sulfate–based lipid removal strategy. Slices were stained using indirect immunofluorescence against targets identified by proteomics. TTR deposits were imaged by antibody and AmyTracker 480 staining. Structures of ATTR fibrils were characterized using cryogenic electron microscopy. Results Proteomic analysis revealed high abundance of TTR, proteins associated with amyloid fibrils, as well as angiogenic, hemostatic, and complement cascade–associated proteins. Three‐dimensional imaging revealed loss of normal microvascular architecture, regions of hypervascularization and hypovascularization, and microvascular obstruction by capillary thrombosis. ATTR fibrils adopted the spearhead fold and were decorated with collagen VI, an extracellular matrix component. Conclusions Based on our imaging and proteomic data, we hypothesize that ATTR cardiomyopathy is a microangiopathy driven by capillary bed thromboinflammation and dysregulated angiogenic revascularization. In this model, increased capillary permeability exposes components of the vascular basement membrane to misfolded TTR. These components promote aggregation and stabilize amyloid fibrils. Congestion of the vascular basement membrane prevents appropriate revascularization, reducing cardiac exertional capacity over time, leading to heart failure.

Background:Transthyretin amyloidosis (ATTR) is a progressive, degenerative disease affecting the heart and other organ systems, as well as the peripheral, autonomic, and central nervous systems. Although pharmacological and genetic evidence establishes aggregation as a driver of ATTR pathology, the mechanism by which aggregation compromises post-mitotic tissue function is poorly understood. We utilized bottom-up proteomics on wild-type (WT) human cardiac (WT/WT genotype) and V122I human cardiac (V122I/WT genotype) tissue, combined with tissue clearing technology to create an optically transparent tissue architecture to visualize three-dimensional relationships, to better understand TTR cardiomyopathy (CM). Methods:Flash-frozen 0.5 mm cardiac tissue slices from human subjects with end-stage WT-TTR CM, end-stage V122I CM, and slices from an age-matched human control were used for these experiments. Fibril extraction from diseased tissue followed published protocols. Strong denaturant-mediated proteome tissue extraction on samples from each subject facilitated bottom-up proteomics by using liquid chromatography (LC)-mass spectrometry (MS)/MS. Tissue clearing was performed on 0.5 mm cardiac slices utilizing a lauryl sulfate-based lipid removal strategy. Slices were stained using indirect immunofluorescence with antibodies to protein targets identified by proteomics. We used an antibody to non-native TTR and AmyTracker 480 (an oligothiophene dye that binds to amyloid fibrils) to image TTR deposits. ATTR fibrils were characterized structurally using cryogenic electron microscopy (cryo-EM) followed by helical reconstruction. Results:Proteomic cardiac analysis afforded high spectral counts for transthyretin (TTR) and proteins typically associated with amyloid fibrils, e.g. serum amyloid P (APCS). Fibril and cardiac homogenate proteomics revealed high levels of angiogenic and hemostatic proteins, including those composing the complement and coagulation cascades. 3D imaging revealed loss of normal microvascular architecture in CM samples with regions of hyper- and hypovascularization. Microvascular obstruction by capillary thrombosis was also observed in CM. ATTR fibrils adopted the common spearhead fold and were decorated with collagen VI (COLVI), an extracellular matrix component. Conclusions:We hypothesize that ATTR CM is a microangiopathy driven by capillary bed thrombo-inflammation and dysregulated angiogenic revascularization. Phenotypic convergence of WT ATTR CM and V122I ATTR CM was observed via proteomics, 3D imaging, and ex vivo fibril characterization by cryo-EM. We provide evidence of capillary thrombosis in ex vivo ATTR CM tissue. Vasodilation and increased capillary permeability expose components of the vascular basement membrane (VBM) to misfolded TTR. These components are known to promote TTR aggregation and stabilize amyloid fibrils in the extracellular space. Congestion of the VBM prevents appropriate revascularization, reducing cardiac exertional capacity over time, leading to heart failure. Our ATTR CM heart tissue proteomics data shows significant overlap with the proteomic profiles of human AD brain tissues, revealing key amyloid, coagulation, complement, and angiogenesis proteins being changed in amyloidoses.

Abstract Cellular homeostasis requires tight coordination between metabolic and translational networks. Here we identify a direct molecular link between these processes through a cryo-EM structure of human cytosolic seryl-tRNA synthetase (SerRS) in complex with the NAD⁺-dependent deacetylase SIRT2. This interaction is promoted by the NAD⁺ metabolite ADP-ribose (ADPR), which acts as a molecular bridge between the two enzymes. Within the SIRT2 active site, ADPR engages SerRS residue K414 located in a flexible catalytic-domain loop. Acetylation of K414 is dispensable for binding. Functionally, complex formation inhibits SIRT2 deacetylase activity by blocking substrate access, while SIRT2 association suppresses SerRS aminoacylation activity by preventing tRNA binding. Thus, SerRS and SIRT2 mutually regulate each other, with ADPR enhancing while tRNA attenuating their interaction. Oxidative stress promotes this interaction via a PARP1-dependent pathway, revealing an ADPR-responsive regulatory module that couples metabolic state to translational output. This regulatory module is likely conserved across vertebrates.

Biomolecular condensates have key roles in regulating cellular processes. Yet, the relationship between atomic features and condensate function remains poorly understood. We studied this relationship using the polar organizing protein Z (PopZ). Here, we revealed hierarchical assembly of PopZ into a filamentous condensate by integrating cryo-electron tomography, biochemistry, single-molecule techniques and molecular dynamics simulations. The PopZ helical domain drives filamentation and condensation, while the disordered region inhibits them. Phase-dependent conformational changes prevent interfilament contacts in the dilute phase and expose client-binding sites in the dense phase. Perturbing filament formation in vitro alters the dynamics of scaffold and client proteins and the condensate's wetting behavior. In cells, perturbing either filament formation or the ability of filaments to condense impairs PopZ function and leads to growth phenotypes. These findings establish a multiscale framework linking molecular interactions and condensate ultrastructure to cellular function.

Supplementary Fig. 1. Golcadomide induced transition from CRBNopen to CRBNclosed for efficient recruitment and degradaion. Supplementary Fig. 2. Antiproliferative effect of golcadomide is mediated via CRBN-based IKZF1/3 degradation. Supplementary Fig. 3. Golcadomide is broadly active with potent cell autonomous antiproliferative activity. Supplementary Fig. 4. Antiproliferative effect of golcadomide is mediated via CRBN based IKZF1/3 degradation. Supplementary Fig. 5. Golcadomide demonstrated minimal broad cytotoxicity and reversible effects on neutrophil maturation. Supplementary Fig. 6. Golcadomide stimulated immune activation, reversed T cell exhaustion, and exhibited potent antitumor synergy with rituximab. Supplementary Fig. 7. Overview of different treatment conditions and technical replicates in the CRISPR screen. Supplementary Fig. 8. CRISPR screen identified genes and pathways that exacerbated or attenuated antiproliferative effects of golcadomide in SU-DHL-4 upon inactivation. Supplementary Fig. 9. KO of PRC2 complex components enhanced cell antiproliferation by golcadomide. Supplementary Fig. 10. Apoptosis induction is linked to the antiproliferative effects of golcadomide. Supplementary Fig. 11. NF-κB hyperactivation reduced golcadomide-induced DLBCL cell apoptosis. Supplementary Fig. 12. XPO1 inhibitor enhances antiproliferative effects of golcadomide. Supplementary Fig. 13. AMBRA1 knockout renders resistance to golcadomide via cyclin D3/CDK4/6/Rb axis in DLBCL. Supplementary Fig. 14. KCTD5 ameliorated the antiproliferative effects of golcadomide via GNG5. Supplementary Fig. 15. Chemical synthesis of golcadomide. Supplementary Fig. 16. Representative gating strategies for flow cytometry-based assays.

Cereblon (CRBN) is the target of thalidomide derivatives1 that achieve therapeutic efficacy against some haematologic neoplasias2-4 by recruiting neosubstrates for degradation5-7. Despite the intense investigation of orthosteric thalidomide derivatives, little is known about alternate binding sites on CRBN. Here we report an evolutionarily conserved cryptic allosteric binding site on CRBN. Small-molecule SB-405483 binds the allosteric site to cooperatively enhance the binding of orthosteric ligands and alter their neosubstrate degradation profiles. A survey of over 100 orthosteric ligands and their degradation targets reveals trends in the classes of compounds and neosubstrates in which degradation outcomes are enhanced or inhibited by SB-405483. Structural investigations provide a mechanistic basis for the effects of the allosteric ligand by shifting the conformational distribution of CRBNopen to a novel CRBNint and increasing the CRBNclosed state. The discovery of a cryptic allosteric binding site on CRBN that alters the functional effects of orthosteric ligands opens new directions with broad implications for improving the selectivity and efficacy of CRBN therapeutics.

Bacteriophages are ubiquitous in the environment and are part of the natural human microbiome. Despite their abundance, the role of the human phagome in health and disease remains poorly understood. Here, we identify phage tails in ex vivo amyloid extracts from patients with lysozyme amyloidosis (ALys) and light-chain amyloidosis (AL). Using cryo-EM analysis of the ALys dataset, automated model building, and database searches, we assigned the observed tubular assemblies to a phage tail tube protein (TTP). Although we cannot fully rule out the possibility of contamination, the presence of phage tails raises the question of whether they bind to and are co-purified with amyloid fibrils. These structures may provide further insight into the potential relationship between phage-derived assemblies and amyloid remodeling, with possible implications for future therapeutic strategies in human amyloidosis.

Abstract Cytochrome P450 3A4 (CYP3A4) metabolizes roughly half of all marketed drugs, and its inhibition can cause clinically significant drug-drug interactions. The enzyme accommodates chemically diverse ligands, making binding modes and metabolic outcomes difficult to predict. Previous X-ray crystallography efforts have leveraged a truncated construct without the N-terminal segment that tethers CYP3A4 to the membrane. Here we show that the same construct assembles into a symmetric trimer that can be resolved by cryo-EM and determine structures of both unliganded and ligand-bound CYP3A4. Multiple ligands are resolved with density consistent with several mutually exclusive conformations. Protein remodeling to reshape the binding pocket is concentrated in the F/G loop, which is poorly resolved and unmodeled in many X-ray structures. These features likely underlie the poor predictive performance of co-folding methods on this target. The routine use of cryo-EM to resolve CYP3A4 ligand-bound complexes will provide the ground truth data needed to make predictive models of drug metabolism useful in practice.

Transthyretin (TTR) is a kinetically stable protein in the bloodstream, cerebrospinal fluid, and in the eye, whose aggregation causes a prominent human amyloid disease, TTR amyloidosis (ATTR). Dissociation of the wild-type TTR tetramer into metastable dimers is rate-limiting for aggregation at acidic pH and unfolding in denaturant solutions at neutral pH. However, this “canonical dimer” pathway of denaturation is not the only one accessible under conditions possibly relevant to amyloid disease. At pH-values reached in the late endosome and lysosome (pH 4.0 to 5.0), as well as with perturbing mutations at neutral pH, a second denaturation pathway becomes accessible involving a more expanded transition state. This “alternative unfolding” pathway is evident via a characteristic switch to a steeper slope in the plot of the log-transformed unfolding rate constant vs. the urea concentration. Using mutations, we identify globally distributed locations in the protein that are sensitive to pathway-switching and correlate them with structural information. We show that flux along the alternative denaturation pathway becomes kinetically competitive in a subset of variants under mildly acidic conditions. A small-molecule kinetic stabilizer of TTR decreases flux along the canonical denaturation pathway, and exhibits reduced influence on denaturation by the alternative pathway. We present a “universal” plot allowing classification of TTR mutants to either pathway, and suggest a mechanism by which the two pathways operate. We speculate that the existence of an alternative unfolding pathway could allow for rapid protein degradation and turnover of kinetically stable TTR under acidic conditions in the autolysosome.

Amyloid-mediated proteotoxicity underlies over 50 diseases. Cryo-EM establishes direct links between filament morphologies and pathology, although microbial functional amyloids illustrate that this fold can evolve to serve physiological roles, unlike their pathogenic counterparts. Despite the growing popularity of the processing software cryoSPARC for single-particle analyses, RELION remains the dominant software platform for performing helical reconstruction of amyloid structures, highlighting an area for further development. Here, we present comprehensive processing guidelines for helical reconstruction of helical amyloids using cryoSPARC. Through systematic reprocessing and validation of publicly deposited datasets, we demonstrate the current capabilities and identify the key limitations, emphasizing the need for amyloid-specific parameter optimization within cryoSPARC workflows. Our findings showcase a potential for developing unsupervised processing workflows to meet the demanding throughput requirements of time-resolved in vitro studies and large-scale compound-screening initiatives, thereby accelerating therapeutic drug development. Ultimately, our goal is to shift the focus of amyloid cryo-EM from computationally intensive processing challenges towards addressing fundamental biological questions that enhance our capacity for treatment discovery.

Transthyretin (TTR) is a secreted protein associated with cardiac and other amyloid diseases via misfolding. We have previously shown that agitation of human TTR solutions at neutral pH results in aggregation and fibril formation. Here we report that agitation-induced aggregation of TTR from species with very different heart rates (Anna's hummingbird, hbTTR, and African elephant, aeTTR) differs from that of human TTR (huTTR). Aggregation of hbTTR is slow and favors formation of smaller, fibrillar aggregates, while aeTTR aggregation is rapid and favors larger, more amorphous particles. Spherical, early-stage oligomeric intermediates were found for all variants by mass photometry and electron microscopy. The slow aggregation of hbTTR matches its resistance to denaturation by 8 M urea. The widely different aggregation behavior exhibited by these naturally occurring TTR variants in response to mechanical agitation under close to physiological conditions provides insight into how small sequence differences can contribute to the evolutionary fitness of different animals.

The air-water interface (AWI) remains the primary barrier to routine high-resolution cryo-EM structure determination, driving protein adsorption, structural denaturation, and restricted particle orientations during vitrification. Here, we describe a simple and broadly applicable strategy to mitigate these effects using the mild non-ionic detergent n-decyl-β-D-maltopyranoside (DM). Addition of DM at low millimolar concentrations immediately prior to vitrification consistently suppresses AWI-driven artifacts, resulting in improved angular sampling, reduced structural damage, and enhanced reconstruction quality across diverse macromolecular systems. Using this approach, we obtained a high-resolution reconstruction of the 65 kDa Nucleophosmin 1 pentamer, a target previously limited by severe preferred orientation issues. We further show that DM promotes isotropic particle distributions for high-resolution reconstruction of hemagglutinin, transthyretin, as well as suppressing denaturation of aldolase while stabilizing its C-terminus. Our results indicate that DM effectively passivates deleterious air-water interface interactions without compromising particle integrity. These results establish DM as an effective additive for improving the robustness of single-particle cryo-EM sample preparation.

The human ClpXP complex (hClpXP) orchestrates mitochondrial protein quality control through targeted degradation of misfolded and unnecessary proteins. While bacterial ClpXP systems are well characterized, the assembly and regulation of human ClpXP remain poorly understood. In this study, we elucidate the complete assembly pathway of hClpXP through high-resolution cryo-electron microscopy (cryo-EM) structures. Our findings confirm that hClpP exists as a single-ring heptamer in isolation and reveal a previously undocumented initial assembly complex in which hexameric hClpX first engages with heptameric hClpP. We further demonstrate how this interaction drives substantial conformational rearrangements that facilitate the formation of tetradecameric hClpP within the fully assembled complex. Notably, we characterize a unique eukaryotic sequence in hClpX, termed the E-loop, which plays a critical role in stabilizing hexamer assembly and maintaining ATPase activity. Additionally, we show that peptide binding at the hClpP active site triggers further structural changes essential for achieving full proteolytic competence. Together, these structures provide unprecedented mechanistic insights into the stepwise assembly and activation of hClpXP, significantly advancing our understanding of this essential mitochondrial protein degradation machinery. Mitochondrial ClpXP maintains protein quality through targeted degradation. Here, the authors use cryo-EM to define the stepwise assembly of human ClpXP, identifying key intermediates and a unique E-loop element that regulates complex formation and proteolytic activation.

The endoplasmic reticulum (ER) transporter solute carrier family 33 member 1 (SLC33A1) has emerged as an attractive therapeutic target in etiologically diverse diseases, ranging from lung cancer to neurodegenerative disorders. Yet, no pharmacologic SLC33A1 modulators have been described. Here, we show that the small molecule IXA4, a highly selective activator of the adaptive IRE1/XBP1s signaling arm of the unfolded protein response (UPR), binds to SLC33A1 and inhibits its activity. Genetic depletion of SLC33A1 phenocopies the selective induction of IRE1/XBP1s signaling brought about by IXA4 treatment. Chemoproteomic analyses and cryo-electron microscopy show that IXA4 binds SLC33A1 within the central channel to inhibit transport of its substrate metabolite(s). Binding of IXA4 to SLC33A1 leads to the accumulation of oxidized glutathione within the ER, hyperoxidizing the ER lumen and inducing activation of adaptive IRE1/XBP1s signaling. Consistent with this function, we find that pharmacologic inhibition of SLC33A1 with IXA4 selectively reduces viability of KEAP1-deficient lung adenocarcinoma cells that have elevated levels of glutathione, mimicking the sensitivity of these cells to genetic deletion of SLC33A1. Our work demonstrates a new physiologic role of SLC33A1 in regulation of ER redox homeostasis and designates IXA4 as a pharmacologic inhibitor of SLC33A1 that can be used to evaluate the biological impact and therapeutic utility of SLC33A1 inhibition in homeostasis and in disease.

The E3 ligase complex SIFI silences the integrated stress response (ISR) by targeting stress-induced proteins for degradation. In the May 6th issue of Nature, Yang et al.1 revealed how this megadalton complex recognizes diverse substrates and coordinates ubiquitin chain formation. Their insights into the ISR shutdown mechanism suggest new avenues for modulating stress responses in neurodegenerative disease.

Single particle cryo-electron microscopy (cryo-EM) has revolutionized structural biology by enabling high-resolution determination of macromolecular structures. However, the field faces challenges in data management, processing workflow integration, and software extensibility. We present Magellon, an innovative cryo-EM software platform that addresses these challenges through a modern microservices architecture. Magellon consists of an extensible backend with a web-based front end that we call Magellon Viewer. Together, these combine high-performance computing capabilities with an intuitive user interface, enabling researchers to efficiently process and analyze cryo-EM data. The platform's distinguishing features include a plugin-based architecture, distributed processing capabilities, comprehensive monitoring systems, and a novel approach to data organization and visualization. A key philosophy of the approach is that the Magellon backend provides a platform that uses robust industry-standard libraries to orchestrate computational tasks while offering users and developers flexibility in selecting the computational resources for performing calculations. Magellon represents a significant advancement in cryo-EM software infrastructure, offering flexibility, scalability, and extensibility while maintaining ease of use.

AAA+ enzymes use energy from ATP hydrolysis to remodel diverse cellular targets. Structures of substrate-bound AAA+ complexes suggest that these enzymes employ a conserved hand-over-hand mechanism to thread substrates through their central pore. However, the fundamental aspects of the mechanisms governing motor function and substrate processing within specific AAA+ families remain unresolved. We used cryoelectron microscopy to structurally interrogate reaction intermediates from in vitro biochemical assays to inform the underlying regulatory mechanisms of the human mitochondrial AAA+ protease, LONP1. Our results demonstrate that substrate binding, rather than nucleotide binding, activates the assembly and allosterically regulates proteolytic activity. The N-terminal domain plays a critical role in this process and facilitates the initial stages of substrate selection and engagement. Moreover, structures of LONP1 actively degrading a substrate in the presence of ATP provide important context to the conventional understanding of the hand-over-hand translocation mechanism, suggesting that ATP hydrolysis is likely not limited to a single position in the right-handed spiral during the hand-over-hand translocation mechanism.

Biological reactions often involve macromolecules that undergo substrate-induced conformational changes in under a second, yet capturing these transient states remains challenging. While high-resolution structural techniques such as X-ray crystallography and cryo-electron microscopy (cryo-EM) have advanced our mechanistic understanding of protein-substrate interactions, traditional sample preparation methods are too slow to capture rapid biochemical events. Time-resolved cryo-EM has emerged as a promising approach to visualize structural dynamics on microsecond-to-millisecond timescales, but its widespread adoption has been limited by costly equipment and challenges in achieving rapid mixing, application, and vitrification of samples in a reproducible manner. To address these limitations, we developed "Mix-it-up" (MIU), a modified spray device designed for rapid on-grid mixing and vitrification of cryo-EM samples. By manually applying one sample onto the EM grid, blotting, and subsequently spraying the second sample, we achieve on-grid mixing with a vitrification delay of as low as ~120 milliseconds. We demonstrate MIU's time-resolved capabilities through high-resolution structure determination of mixed samples, pH-induced viral capsid contraction, and ligand-dependent complex formation. These findings establish MIU as a cost-effective, versatile tool for studying rapid biochemical processes and lay the groundwork for future applications to time-resolved cryo-EM.