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Phytopathogenic fungi secrete effector proteins to promote virulence. The MAX (Magnaporthe Avrs and ToxB-like) effectors form a structurally conserved family despite significant sequence diversity. AVR-Pia, a MAX effector from the rice blast fungus Magnaporthe oryzae, is recognised by the model rice nucleotide-binding leucine-rich repeat (NLR) receptor pair OsRGA4/OsRGA5 via direct binding to a heavy metal-associated (HMA) integrated domain (ID) in OsRGA5. While the structural basis of AVR-Pia recognition is well defined, the role of this effector in promoting virulence has remained elusive. Here, we reveal that AVR-Pia specifically interacts with four previously uncharacterised rice HMA domain-containing proteins, three HMA Plant Proteins (OsHPP09, OsHPP10 and OsHPP11), and one HMA Isoprenylated Plant Protein (OsHIPP21). AVR-Pia binds these proteins in vitro and in planta, engaging their HMA domains with differential affinities. Notably, AVR-Pia binds OsHPP09-HMA with considerably higher affinity than the HMA-ID of OsRGA5. By solving the crystal structure of the AVR-Pia/OsHPP09-HMA complex, we identified additional molecular contacts at the interface which underpin high affinity binding. Importantly, the H(I)PPs identified as AVR-Pia interactors are distinct from those bound by the MAX effectors AVR-Pik and Pwl2, underscoring target specialisation within the MAX effector family. Further, structural analyses of the AVR-Pia/OsHPP09-HMA complex revealed a markedly different interface compared to other MAX effector/H(I)PP complexes. Finally, structure-guided mutagenesis of OsHPP09 identified a single residue that is critical for AVR-Pia binding. This work provides structural insight into how distinct MAX effectors exploit HMA domain-containing proteins and offers a foundation towards targeted modification of HMA domains to disrupt effector binding and enhance cereal resistance to blast disease.
Plants survive extreme environments through rapid chromatin reprogramming, yet the epigenetic marks that confer stress resilience remain poorly understood. Histone deacetylase HDA19 is a key epigenetic regulator in Arabidopsis, and hda19-deficient mutants display tolerance to multiple abiotic stresses, including drought, heat, and salinity. Using lysine acetylome profiling, we identified a noncanonical K27/K36 diacetylation mark on histone H3.3, among nine H3 variants, as a specific substrate of HDA19. Under salinity stress, this mark decreased in wild-type plants but increased in hda19 mutants, while other known H3 modifications were similarly affected in both genotypes. Mimicking constitutive diacetylation of H3.3K27/K36 through lysine-to-glutamine substitutions promoted accumulation of stress-responsive late embryogenesis abundant (LEA) proteins and conferred salinity tolerance in seedlings, phenocopying hda19 mutants. Furthermore, generating the lea7-1/lea29-1/rab18-1 triple mutant abolished hda19-dependent salinity tolerance, confirming the LEA proteins' role downstream of HDA19. Our findings demonstrate that H3.3K27/K36 diacetylation, modulated by HDA19, drives LEA protein accumulation and enables plants to withstand environmental stress, revealing a core mechanism of plant stress resilience.
Acidocalcisomes are evolutionarily conserved acidic organelles that are rich in cations and inorganic phosphate, primarily polyphosphates. In kinetoplastid parasites, acidocalcisomes and their polyphosphate content are essential for osmoregulation and environmental adaptation during host switching. In this organelle, polyphosphate is synthesised and transported to the lumen by the vacuolar transporter chaperone (VTC) complex. Interestingly, unlike yeast VTC, which has five components, only two have been observed in kinetoplastids: Vtc1, which contains only a transmembrane domain and Vtc4, which, in addition to a transmembrane domain, also consists of SPX and catalytic domains. In this study, we used proximity-dependent biotinylation (BioID) in Leishmania tarentolae to identify proteins located close to the VTC complex. The complex was found near several known acidocalcisomal proteins, including membrane-bound pyrophosphatase (mPPase), vacuolar-type H ⁺ -ATPase (V-H+-ATPase), Ca² ⁺ -transporting P-type ATPase (Ca2+-ATPase), zinc transporter (ZnT), and palmitoyl acyltransferase 2 (PAT2). Importantly, this approach revealed three novel VTC binding partners (VBPs) that colocalise and interact with the complex in acidocalcisomes, as confirmed by confocal microscopy, pulldown assays, and AlphaFold3 structural predictions. Together, our results expand the acidocalcisome interactome and suggest that the newly identified VBPs may contribute to the structural organisation and regulatory function of the VTC complex in phosphate homeostasis of kinetoplastid parasites.
Since cumulus cells (CCs) play an undeniable role in oocyte maturation by producing and transferring important molecules to the oocyte, examining these cells can provide a broad view of the factors affecting oocyte competence. Therefore, the aim of this study was to investigate the expression of genes and proteins ZNF83, ACY-1, andSMC5 in CCs of unfertilized oocytes and compare it with fertilized oocytes to gain new insights into the relationship between CCs function and oocyte competence. Eighteen healthy female oocyte donors were included in this study. After obtaining the cumulus-oocyte complex and isolating the CCs, oocytes were injected and the embryos were followed and morphologically graded on day 3. Then the expression of genes and proteins ZNF83, ACY-1, andSMC5 in CCs of non-fertilized oocytes and CCs from fertilized oocytes was investigated and compared. The expression of ZNF83, ACY-1, and SMC5 at gene and protein levels was reduced in the non-fertilized group compared to the fertilized group (p<0.05). The results highlight CCs' critical role in supporting oocyte fertilization. These findings show the way for novel biomarkers and therapeutic strategies to improve IVF outcomes. More functional studies are needed to support the hypothesis of this study and explore the clinical applications of these markers in ART.
Fracture-related infection is a challenging aspect of orthopedic care, as it leads to prolonged inflammation and impaired bone healing. Although isovitexin is a naturally occurring flavonoid with anti-inflammatory properties, its effects on fracture-related infection remain unclear. A rat fracture infection model was established and randomly divided into four groups: control, model, Vancomycin, and isovitexin. Tissue staining was used to assess bone healing. In contrast, enzyme-linked immunosorbent assay and biochemical kits were used to measure the levels of inflammatory factors and bone metabolism-related indicators. The expression of proteins associated with osteogenesis and the Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway was examined using Western blot. When compared to the model group, isovitexin treatment more effectively reduced inflammatory cell infiltration in bone tissue and promoted callus formation than the Vancomycin group. In addition, isovitexin improved the imbalance of bone metabolism and promoted the expression of osteogenesis-related proteins Bone Morphogenetic Protein 2 (BMP2), Osteopontin (OPN), and Runt-related Transcription Factor 2 (RUNX2). It also decreased the levels of pro-inflammatory factors tumor necrosis factor (TNF)-α and interleukin (IL)-6, and increased the level of the anti-inflammatory factor IL-10. Mechanistic studies showed that isovitexin significantly inhibited the activation of the NF-κB signaling pathway. Isovitexin can promote bone metabolism and healing, suppress the NF-κB signaling pathway, and reduce the inflammatory response associated with fracture-related infection. This study suggests that isovitexin may be a viable therapy option for fracture-related infections.
Microvascular inflammation (MVI) has long been considered a hallmark of antibody-mediated rejection (AMR). However, the 2022 Banff classification introduced isolated MVI (iMVI; MVI+/donor-specific antibody [DSA]-/C4d-) as a distinct phenotype. The biological characteristics and clinical significance of iMVI remain incompletely understood. Kidney transplant recipients who underwent allograft biopsy between 2013 and 2024 were classified into iMVI, AMR (MVI+/DSA+/C4d+), and acute tubular injury (ATI; MVI-/DSA-/C4d-). After propensity score matching, 80 patients (30 iMVI, 30 AMR, and 20 ATI) compromised the main cohort. An independent validation cohort (n = 88; 26 iMVI, 26 AMR, 26 T-cell-mediated rejection, 10 ATI) was additionally assembled. Serum proteomic profiling was performed using a proximity extension assay. iMVI exhibited a distinct proteomic profile enriched for innate and cytotoxic immune activation proteins, including tumor necrosis factor receptor superfamily member 9, programmed death-ligand 1, interleukin-15 receptor subunit alpha, fibroblast growth factor 19, C-X3-C motif chemokine ligand 1, C-C motif chemokine ligand 23, signaling lymphocytic activation molecule family member 1, and interleukin-10 receptor subunit beta with enrichment of natural killer cell differentiation and leukocyte migration pathway. In contrast, AMR showed higher expression of adaptive and humoral immune mediators, including interleukin-33, interleukin-2, TNF-related apoptosis-inducing ligand, and interleukin-2 receptor subunit beta with enrichment of immunoglobulin production and interferon-γ-related adaptive immune pathways. Protein risk scores showed strong discriminative performance in the main cohort (area under the curve, 0.856 for iMVI; 0.879 for AMR) and retained significant ability in the validation cohort (area under the curve, 0.708 and 0.689, respectively). Several iMVI-enriched proteins were significantly associated with increased risk of death-censored graft loss. iMVI is characterized by dominant innate and cytotoxic immune activation distinct from AMR and is associated with adverse graft outcomes, supporting its recognition as a clinically meaningful phenotype beyond conventional AMR.
In angiosperms, lipid transfer proteins (LTPs) perform multiple roles, including the shuttling of lipids between organelles and to/through the apoplast. In pennycress (Thlaspi arvense L.), the LIPID TRANSFER PROTEIN 6 (TaLTP6) was identified as highly expressed in developing embryos, especially in high-oil accessions. Ectopic expression of pennycress LTP6 (TaLTP6) in Nicotiana benthamiana and Arabidopsis thaliana leaf mesophyll cells induced the proliferation of cytoplasmic lipid droplets (LDs), suggesting a role in neutral lipid accumulation. GFP-tagged TaLTP6 localized predominantly to LDs and endoplasmic reticulum (ER)/LD contact sites, while its Arabidopsis homolog, AtLTP6, localized to the ER and apoplast. Domain-swapping experiments revealed that their N-terminal regions determined these subcellular localizations. Loss-of-function mutants of Arabidopsis ltp6 exhibited major disruptions in LD organization in mature embryos, characterized by large lipid aggregates, and reduced seed oil content. Proteomics analysis revealed mislocalization of LD- and ER-associated proteins in ltp6 mutants, suggesting impaired LD biogenesis. Further, Arabidopsis ltp6 seeds exhibited reduced mucilage extrusion and impaired germination, pointing to a secondary role for AtLTP6 in seed coat function. Complementation of Arabidopsis ltp6 with TaLTP6 restored LD morphology and seed oil levels, but did not rescue mucilage and germination defects, indicating functional divergence between the two homologs. We conclude that LTP6 plays a dual role in seeds: (1) participation in embryo lipid storage, and (2) contribution to seed coat integrity and germination. The embryo-specific expression of TaLTP6 in pennycress suggests that it retained its evolutionary role in lipid storage, but lost functions related to seed coat development and germination.
Macromolecular crowding can reshape the conformational landscapes of intrinsically disordered and marginally stable proteins linked to neurodegeneration, enriching aggregation-prone states and changing how nucleation begins. Crowding can also promote liquid-liquid phase separation, leading to condensates that concentrate proteins, reshape interaction networks, and in some cases promote liquid-to-solid transitions into amyloid assemblies. This crowding-liquid-liquid phase separation-aggregation continuum may help explain why dilute solution assays often fail to capture the mechanisms that operate in cells. Crowding-aware structural biology, including in-cell nuclear magnetic resonance, cryo-electron microscopy of condensates, single-molecule methods, and thermodynamic-kinetic modelling, will be important for resolving physiologically relevant intermediates. From a therapeutic perspective, targeting condensate properties and early oligomeric states, rather than focussing only on mature fibrils, may offer new ways to limit pathogenic aggregation.
The antagonistic interplay between canonical Wnt signalling and Dickkopf (Dkk) proteins is fundamental to tissue organisation, including stem cell differentiation and body-axis formation. Disruptions in this interaction are linked to various human diseases, yet the mechanisms by which β-catenin/Wnt-Dkk interactions give rise to robust spatial patterning remain unclear. A key model system for Wnt-driven pattern formation is the pre-bilaterian organism Hydra, where two ancestral Dkk proteins interact with Wnt signalling to self-organise the body axis. While Hydra patterning has been extensively studied within the activator-inhibitor framework, a model that directly integrates experimentally identified molecular components has been lacking. Here, we introduce a mathematical model incorporating both Dkk molecules and their experimentally established interactions with Wnt signalling. Numerical simulations and analytical results show that the Wnt-Dkk network alone is sufficient to drive de novo body-axis formation across a broad parameter range. The model provides a biologically grounded realisation of the general local activation-long-range inhibition (LALI) principle, in which effective local activation emerges from mutual inhibition rather than molecular self-activation. In contrast to previous Hydra models, it explicitly links experimentally characterised Wnt-Dkk interactions to pattern formation, accounts for the experimentally observed role of injury-induced activation, and exhibits robust behaviour under perturbations.
The biogenesis of multipass membrane proteins challenges the endoplasmic reticulum (ER) quality control, particularly when transmembrane segments contain polar or charged residues required for function. Fks1, the catalytic subunit of yeast β-(1,3)-glucan synthase, exemplifies this challenge because its large multipass transmembrane architecture must support glucan synthesis at the plasma membrane while also undergoing efficient biogenesis in the ER. Here, we investigate the cellular role of PBR1 (YNL181W), an essential gene whose role remains uncharacterized even though its predicted product has similarity to oxidoreductases. By integrating quantitative morphological profiling with global genetic interaction analysis, we found that PBR1 function converges on cell-wall biosynthesis and closely parallels that of FKS1. Partial loss of Pbr1 function caused temperature-sensitive growth defects but also impaired β-(1,3)-glucan synthesis, and weakened cell-wall integrity. Under these conditions, Fks1 failed to accumulate at the cell surface and, instead, accumulated in ER-associated compartments, where it exhibited reduced stability. Biochemical analyses revealed the accumulation of immature Fks1 species, including forms defective in glycosylation, consistent with compromised ER quality control. A spontaneous missense suppressor allele of FKS1 partially restored Fks1 stability and growth, supporting a functional relationship between the two proteins. Pbr1 is a cytosol-facing ER membrane protein that physically associates with Fks1, and structural modeling suggests that it adopts a Rossmann-like fold capable of binding pyridine nucleotides despite divergence from canonical catalytic motifs. Together, these findings identify Pbr1 as an ER-associated, chaperone-like factor required for the folding and maturation of Fks1.
Small extracellular vesicles (sEVs) are nanosized, lipid-bound particles that mediate intercellular communication through the transfer of proteins, lipids, and ribonucleic acids (RNAs). Isolation of sEVs from solid tissues such as skeletal muscle (SkM) and bone marrow (BM) remains challenging due to low yield and contamination from non-vesicular components. This protocol describes a reproducible workflow for isolating sEVs from mouse SkM and BM. SkM is enzymatically digested, whereas BM is processed directly and subsequently subjected to differential centrifugation and size-exclusion chromatography (SEC). For SkM, the void volume (~2.5 mL) is discarded, and the subsequent ~1.6 mL is collected as EV-enriched fractions, typically subdivided into 400 µL sequential fractions (F1-F4). For BM, a 700 µL void volume is discarded, followed by collection of an ~850 µL EV-enriched fraction, which can be subdivided into ~170 µL sequential fractions (F1-F5) and pooled based on EV marker enrichment. Using this approach, sEVs can be isolated from small tissue volumes (approximately 500 µL from a single quadriceps and 150 µL from pooled femur and tibia BM), yielding ~108 particles·mL-1·mg-1 tissue with particle diameters predominantly <200 nm. Vesicle integrity and purity are validated using transmission electron microscopy, nanoparticle tracking analysis, and Western blotting for canonical EV markers, with additional characterization by single-particle interferometric detection to quantify tetraspanin-defined vesicle subpopulations. This method enables reproducible isolation of high-purity sEVs from structurally complex tissues using minimal input material, supporting downstream molecular and functional analyses of tissue-derived vesicles.
Evolution began in water; it's not surprising that water had a profound influence on protein-ligand complex formation. However, water's contribution to binding is low, perhaps up to ±5 kJ/mol for displacement or recruitment. When affinity is factorized into enthalpy and entropy, a large scatter of both is observed, especially when both mutually compensate. Since this also applies to water, its impact can easily be overlooked and go unnoticed, especially when only affinity data are available. Nevertheless, water can enhance ligand affinity when a well-designed solvation shell assembles around the formed protein-ligand complex. Examples are discussed of pockets exhibiting varying water density in uncomplexed proteins, or transient pockets that only open with ligand binding. Are these pockets solvated before the ligand enters, or can the ligand penetrate without allowing water molecules to temporarily fill the pocket? Rules are needed to help medicinal chemists select the best optimization strategy while considering water's impact.
The ability to discriminate self from nonself is found throughout the tree of life and underlies a diverse range of processes, including immunity, mate choice, and cooperative social interactions. Self-/nonself-recognition also operates during development, for example, coordinating the formation of complex neural circuits. Despite this ubiquity, proteins that mediate self-/nonself-recognition are lineage-specific. Novel genetic interactions have arisen repeatedly and in a punctuated manner, in some cases even evolving de novo within the same lineages. This review examines the fundamental principles of self-/nonself-recognition across diverse taxa, focusing on the cellular and molecular mechanisms underlying discrimination, the generation and maintenance of novel recognition specificities, and how these systems have been modified over time. Together, these modifications provide a mechanistic foundation to address a central problem in evolutionary biology: the origins of vertebrate adaptive immunity, a system capable of unparalleled self-/nonself-discrimination.
Virtual screening has become an indispensable tool in modern structure-based drug discovery, enabling the identification of candidate molecules by computationally evaluating their potential to bind target proteins. The accuracy of such screenings critically depends on the quality of the target structures employed. Recent advances in protein structure prediction, particularly AlphaFold2, have revolutionized this field with unprecedented accuracy. However, AlphaFold2 models often exhibit limitations in local structural details, especially within binding pockets, which limit their utility for small molecule docking. In contrast, molecular dynamics simulations with accurate atomistic force fields can refine protein structures, but lack the ability to leverage the structural information provided by deep learning approaches. Here, we introduce bAIes, an integrative method that bridges this gap by combining physics-based force fields with data-driven predictions through Bayesian inference. Crucially, bAIes demonstrates a superior ability to discriminate between binders and nonbinders in virtual screening campaigns, outperforming both AlphaFold2 and molecular dynamics-refined models. By enhancing the usability of AlphaFold2 models without requiring extensive experimental or computational resources, bAIes offers a convenient solution to a longstanding challenge in structure-based drug design, potentially accelerating the early phases of drug discovery.
Identification of drug-target interactions (DTI) is an important and challenging task in drug discovery and development. Traditional methods generally require biological experiments, which are costly and time-consuming. Machine learning-based methods can rapidly predict DTI using only computer algorithmic models, allowing researchers to validate only the most promising interactions through biochemical experiments. This holds promise for effectively addressing the current challenges of lengthy development cycles and high costs in new drug development. However, it is difficult for the existing DTI prediction methods to learn complete and effective feature information from the compound and protein. Therefore, this work proposes a DTI prediction method based on the global self-attentive pooled graph neural network and protein pretraining model, called T-pGNN4DTI. On the one hand, T-pGNN4DTI uses a global self-attention pooled graph neural network to learn more meaningful features of the drug molecule by paying more attention to the information features of certain important atomic nodes of the molecular structure and ignoring some weakly relevant node information features. On the other hand, T-pGNN4DTI uses a pre-trained Transformer-based model to capture the semantic relationships of contexts in long sequences of proteins, which can learn more complete feature information. The results of comparing experiments on three benchmark datasets show that the performance of the proposed T-pGNN4DTI model is better than that of the existing DTI prediction methods, effectively improving the DTI prediction. It provides a new way of thinking to help solve the DTI-related problems.
The study of functional heterogeneity in mesenchymal stem cells relies on the efficient isolation of specific subpopulations (e.g., podoplanin (PDPN)-positive cells). However, for most membrane protein targets, directly available commercial magnetic beads for sorting are limited. To address this, our laboratory has established and tested a standardized magnetic bead sorting protocol based on the indirect coupling principle of "phycoerythrin (PE)-labeled flow cytometry antibodies/anti-PE magnetic beads." Using PDPN-positive cells as a model target population, this protocol enriches a target cell fraction without relying on expensive flow cytometric cell sorters. Researchers can use compatible PE-labeled flow cytometry antibodies targeting accessible cell-surface proteins and combine them with commercially available anti-PE microbeads to establish a sorting workflow for selected targets after target-specific optimization. This approach avoids the lengthy lead times and high costs associated with customizing or ordering target-specific magnetic beads for each new target, making it particularly suitable for accessible surface markers that are rare or understudied. In the PDPN model system tested here, the protocol enabled enrichment of the target subpopulation with high post-sort PDPN positivity and preserved CCK-8-based proliferation/metabolic activity compared with unsorted MSCs.
Cardiac amyloidosis (CA) is characterized by extracellular protein deposition, generating ventricular hypertrophy, heart failure and arrhythmias. The main proteins involved are light chains (CA-AL) and transthyretin (CA-TTR). To describe clinical, imaging and biochemical characteristics of patients with CA, their prognosis and access to specific therapies for each etiology. Prospective multicenter registry of patients >50 years old with red flags for CA, such as left ventricular hypertrophy (septum >12 mm) associated to: restrictive filling; aortic stenosis; low voltage, pseudoinfarction pattern in electrocardiogram (ECG), heart failure with NT-proBNP >600 pg/ml, elevated troponins, carpal tunnel, polyneuropathy, compatible cardioresonance. 60 patients were included, 37 men, age 68±13 years; 36 AC-AL; 24 CA-TTR (10 patients CA-TTR mutated). The most frequent red flags were: strain pattern or global longitudinal deformation (97%), heart failure (95%), restrictive filling (72%), compatible cardioresonance (85%), pseudoinfarction in ECG (62%), elevated troponins (75%). CA-TTR patients were older, greater prevalence of atrial fibrillation and atrioventricular block; larger septal thickness (17.0±0.45 versus 14.7±2.5 mm) and left atrial volume (55±13 versus 42±8 ml/m2). CA-AL patients showed larger troponin elevation. There were no differences in systolic function, left ventricular deformation or NT-proBNP. There were no differences in mortality. CA-AL patients had greater access to specific therapies (87% versus 17%). Patients with CA presented moderate/severe hypertrophy, restrictive filling, atrial dilatation and biomarker elevation, suggestive of late diagnosis of CA. Mortality between groups was similar, with greater access to specific therapies in CA-AL.
The identification of novel noninvasive biomarkers remains a major challenge in the diagnosis of neurodegenerative diseases. Significant efforts focus on fluid biomarkers, including proteins, peptides, and miRNAs, detectable in blood plasma and peripheral blood cells. Here, we review recent findings on blood plasma and peripheral blood cells physical parameters in Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis emphasizing atomic force microscopy and calorimetry assay. Alterations in morphology, nanostructure, and stiffness of red blood cells and platelets, together with thermodynamic signatures of red blood cells and plasma, provide sensitive indicators of disease-related changes. These integrated biophysical parameters not only distinguish neurodegeneration from healthy states but also enable discrimination among different neurodegenerative disorders, highlighting their potential as minimally invasive diagnostic markers.
Marine and coastal fungi experience intense environmental variability, yet the genomic features associated with tolerance to such conditions remain unclear. From 56 fungal isolates collected along the Lailai rocky shore in northern Taiwan, we selected the coastal isolate Annulohypoxylon annulatoides RYS0019 for phenotypic and genomic investigation because of its prevalence and distinctive stress-response profile. Compared with five bark-derived conspecific strains, RYS0019 showed distinct growth and recovery dynamics under salinity, temperature, and UV-associated stress treatments. We generated a high-quality 41.8 Mbp de novo genome assembly with 11,523 predicted proteins and compared it with 15 other Hypoxylaceae genomes. Across Annulohypoxylon genomes, we identified variably sized and dispersed AT-rich isochores that are repeat-enriched and gene-poor. Despite variation in AT content, core gene content and Pfam domain profiles remained broadly conserved. Most AT-rich isochores were embedded within syntenically conserved regions and showed limited positional conservation across species, supporting recurrent, lineage-specific formation or expansion after species divergence. These regions also exhibit several sequence and structural features consistent with scaffold/matrix attachment regions (S/MARs), raising the possibility that they influence higher-order genome organisation or context-dependent regulation. Together, our findings identify repeat-rich genome architecture as a dynamic feature of Annulohypoxylon genome evolution and provide a framework for testing how such regions may contribute to fungal environmental flexibility.
Prion diseases can mimic Alzheimer disease (AD) at presentation. Alzheimer's Association AD diagnostic criteria suggest that a single abnormal highly specific plasma biomarker (including p-tau217) is sufficient for a biological diagnosis. We investigated the performance of AD plasma biomarkers in distinguishing AD and prion diseases. We examined plasma biomarker data from patients with prion disease from a prospective cohort study recruited through the UK National Prion Clinic. Prion diseases were diagnosed clinically or with autopsy confirmation, and AD was diagnosed clinically with CSF biomarker confirmation. Plasma p-tau217, p-tau181, Aβ42/40 ratio, brain-derived tau (BD-tau), neurofilament light chain (NfL), and glial fibrillary acid protein (GFAP) were measured using Simoa. Median biomarker values in different groups were compared with Kruskal-Wallis test, and area under the receiver operating characteristic curve was used to compare accuracy in distinguishing prion diseases from sporadic AD (sAD). Lumipulse p-tau217 and NfL were measured in a validation study in a different laboratory. In the main study, we analyzed 345 samples from 278 individuals (mean age 58 [SD 13.5], 48.2% female), including 204 with prion diseases (121 sporadic Creutzfeldt-Jakob disease [CJD], 11 iatrogenic CJD, 9 variant CJD, 47 slow-progressing inherited prion disease (IPD) and 16 fast-progressing IPD), 33 with AD, and 41 healthy controls. For discriminating prion disease without AD copathology from sAD, none of p-tau217 (area under the curve [AUC] [95% CI] 0.605 [0.486-0.724]), p-tau181 (AUC 0.554 [0.446-0.661]), or GFAP (AUC 0.514 [0.389-0.640]) performed well. Aβ42/40 discriminated moderately (AUC 0.770 [0.684-0.856]). NfL/p-tau217 ratio (AUC 0.996 [0.987-1.000]), NfL (AUC 0.988 [0.974-1.000]), BD-tau/p-tau217 ratio (AUC 0.963 [0.929-0.996]), and BD-tau (AUC 0.934 [0.890-0.978]) discriminated very well. In an independent validation study, consecutive samples were analyzed from 32 patients with sAD and 35 patients with sporadic Creutzfeldt-Jakob disease (mean age 65.0 [SD 6.4], 56.7% female). NfL/p-tau217 again discriminated almost perfectly (AUC 0.986 [95% CI 0.966-1.000]). Plasma p-tau217 and p-tau181 are increased in both AD and prion diseases (regardless of burden of AD copathology). Diagnosing AD with a single abnormal p-tau plasma biomarker risks misdiagnosing prion diseases as AD. Plasma NfL/p-tau217 discriminates near-perfectly and could act as a flag to suspect prion diseases where this is a diagnostic possibility. This study provides Class II evidence that plasma NfL/p-tau217 discriminates patients with CJD from those with AD.